Adherens junction turnover: regulating adhesion through cadherin endocytosis, degradation, and recycling

PubMed Central

Nanes, Benjamin A.; Kowalczyk, Andrew P.

2014-01-01

Adherens junctions are important mediators of intercellular adhesion, but they are not static structures. They are regularly formed, broken, and rearranged in a variety of situations, requiring changes in the amount of cadherins, the main adhesion molecule in adherens junctions, present at the cell surface. Thus, endocytosis, degradation, and recycling of cadherins are crucial for dynamic regulation of adherens junctions and control of intercellular adhesion. In this chapter, we review the involvement of cadherin endocytosis in development and disease. We discuss the various endocytic pathways available to cadherins, the adaptors involved, and the sorting of internalized cadherin for recycling or lysosomal degradation. In addition, we review the regulatory pathways controlling cadherin endocytosis and degradation, including regulation of cadherin endocytosis by catenins, cadherin ubiquitination, and growth factor receptor signaling pathways. Lastly, we discuss the proteolytic cleavage of cadherins at the plasma membrane. PMID:22674073

Re-establishment of gap junctional intercellular communication (GJIC) between human endometrial carcinomas by prostaglandin E(2).

PubMed

Schlemmer, Scott R; Kaufman, David G

2012-12-01

Reduced intercellular communication via gap junctions is correlated with carcinogenesis. Gap junctional intercellular communication (GJIC), between normal human endometrial epithelial cells is enhanced when endometrial stromal cells were present in culture. This enhancement of GJIC between normal epithelial cells also occurs when they are cultured in medium conditioned by stromal cells. This observation indicated that a soluble compound (or compounds) produced and secreted by stromal cells mediates GJIC in epithelial cells. Previous studies have shown that endometrial stromal cells release prostaglandin E(2) (PGE(2)) and prostaglandin F(2α) (PGF(2α)) under physiological conditions. When we evaluated the response of normal endometrial epithelial cells to various concentrations of PGE(2,) we found enhanced GJIC with 1nM PGE(2). This is a smaller increase in GJIC than that induced by medium conditioned by stromal cells. When the extracellular concentration of PGE(2) was measured after incubation with stromal cells, it was found to be similar to the concentrations showing maximal GJIC between the normal epithelial cells. When indomethacin was used to inhibit prostaglandin synthesis by stromal cells, GJIC was reduced but not eliminated between normal endometrial epithelial cells. These observations suggest that although PGE(2) secreted by stromal cells is an important mediator of GJIC between the epithelial cells, it is not the sole mediator. Transformed endometrial epithelial cells did not demonstrate GJIC even in the presence of stromal cells. However, we were able to re-establish GJIC in transformed epithelial cells when we added PGE(2) to the cells. Our findings show that PGE(2) may serve as an intercellular mediator between stromal and epithelial cells that regulates GJIC in normal and malignant epithelial cells. This suggests that maintenance of GJIC by preserving or replacing PGE(2) secretion by endometrial stromal cells may have the potential to suppress carcinogenesis in endometrial epithelial cells. Copyright © 2012 Elsevier Inc. All rights reserved.

Physiological and physiopathological aspects of connexins and communicating gap junctions in spermatogenesis

PubMed Central

Pointis, Georges; Gilleron, Jérome; Carette, Diane; Segretain, Dominique

2010-01-01

Spermatogenesis is a highly regulated process of germ cell proliferation and differentiation, starting from spermatogonia to spermatocytes and giving rise to spermatids, the future spermatozoa. In addition to endocrine regulation, testicular cell–cell interactions are essential for spermatogenesis. This precise control is mediated through paracrine/autocrine pathways, direct intercellular contacts and through intercellular communication channels, consisting of gap junctions and their constitutive proteins, the connexins. Gap junctions are localized between adjacent Leydig cells, between Sertoli cells and between Sertoli cells and specific germ cells. This review focuses on the distribution of connexins within the seminiferous epithelium, their participation in gap junction channel formation, the control of their expression and the physiological relevance of these junctions in both the Sertoli–Sertoli cell functional synchronization and the Sertoli–germ cell dialogue. In this review, we also discuss the potential implication of disrupted connexin in testis cancer, since impaired expression of connexin has been described as a typical feature of tumoral proliferation. PMID:20403873

Variations in gap junctional intercellular communication and connexin expression in fibroblasts derived from keloid and hypertrophic scars.

PubMed

Lu, Feng; Gao, JianHua; Ogawa, Rei; Hyakusoku, Hiko

2007-03-01

Expression of connexins and other constituent proteins of gap junctions along with gap junctional intercellular communication are involved in cellular development and differentiation processes. In addition, an increasing number of hereditary skin disorders appear to be linked to connexins. Therefore, in this report, the authors studied in vitro gap junctional intercellular communication function and connexin expression in fibroblasts derived from keloid and hypertrophic scar patients. Fibroblasts harvested from each of six keloid and hypertrophic scar patients were used for this study. Gap junctional intercellular communication function was investigated using the gap fluorescence recovery after photobleaching method, and expression of connexin proteins was studied using quantitative confocal microscopic analyses. Compared with normal skin, a decreased level of gap junctional intercellular communication was seen in fibroblasts derived from hypertrophic scar tissue, whereas an extremely low gap junctional intercellular communication level was detected in fibroblasts derived from keloid tissue. We also detected little connexin 43 (Cx43) protein localized in fibroblasts derived from keloids. Moreover, Cx43 protein levels were much lower in fibroblasts derived from hypertrophic scars than in those derived from normal skin. The authors' data suggest that the loss of gap junctional intercellular communication and connexin expression may affect intercellular recognition and thus break the proliferation and apoptosis balance in fibroblasts derived from keloid and hypertrophic scar tissue.

Drosophila Shaking-B protein forms gap junctions in paired Xenopus oocytes.

PubMed

Phelan, P; Stebbings, L A; Baines, R A; Bacon, J P; Davies, J A; Ford, C

1998-01-08

In most multicellular organisms direct cell-cell communication is mediated by the intercellular channels of gap junctions. These channels allow the exchange of ions and molecules that are believed to be essential for cell signalling during development and in some differentiated tissues. Proteins called connexins, which are products of a multigene family, are the structural components of vertebrate gap junctions. Surprisingly, molecular homologues of the connexins have not been described in any invertebrate. A separate gene family, which includes the Drosophila genes shaking-B and l(1)ogre, and the Caenorhabditis elegans genes unc-7 and eat-5, encodes transmembrane proteins with a predicted structure similar to that of the connexins. shaking-B and eat-5 are required for the formation of functional gap junctions. To test directly whether Shaking-B is a channel protein, we expressed it in paired Xenopus oocytes. Here we show that Shaking-B localizes to the membrane, and that its presence induces the formation of functional intercellular channels. To our knowledge, this is the first structural component of an invertebrate gap junction to be characterized.

Dioscin augments HSV-tk-mediated suicide gene therapy for melanoma by promoting connexin-based intercellular communication

PubMed Central

Li, Bin; Wu, Yingya; Liu, Xijuan; Tan, Yuhui; Du, Biaoyan

2017-01-01

Suicide gene therapy is a promising strategy against melanoma. However, the low efficiency of the gene transfer technique can limit its application. Our preliminary data showed that dioscin, a glucoside saponin, could upregulate the expression of connexins Cx26 and Cx43, major components of gap junctions, in melanoma cells. We hypothesized that dioscin may increase the bystander effect of herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) through increasing the formation of gap junctions. Further analysis showed that dioscin indeed could increase the gap junctional intercellular communication in B16 melanoma cells, resulting in more efficient GCV-induced bystander killing in B16tk cells. By contrast, overexpression of dominant negative Cx43 impaired the cell-cell communication of B16 cells and subsequently weakened the bystander effect of HSV-tk/GCV gene therapy. In vivo, combination treatment with dioscin and GCV of tumor-bearing mice with 30% positive B16tk cells and 70% wild-type B16 cells caused a significant reduction in tumor volume and weight compared to treatment with GCV or dioscin alone. Taken together, these results demonstrated that dioscin could augment the bystander effect of the HSV-tk/GCV system through increasing connexin-mediated gap junction coupling. PMID:27903977

IL-4 and IL-13 Compromise the Sinonasal Epithelial Barrier and Perturb Intercellular Junction Protein Expression

PubMed Central

Wise, Sarah K.; Laury, Adrienne M.; Katz, Elizabeth H.; Den Beste, Kyle A.; Parkos, Charles A.; Nusrat, Asma

2014-01-01

Introduction Altered expression of epithelial intercellular junction proteins has been observed in sinonasal biopsies from nasal polyps and epithelial layers cultured from nasal polyp patients. These alterations comprise a “leaky” epithelial barrier phenotype. We hypothesize that Th2 cytokines IL-4 and IL-13 modulate epithelial junction proteins thereby contributing to the leaky epithelial barrier. Methods Differentiated primary sinonasal epithelial layers cultured at the air-liquid interface were exposed to IL-4, IL-13, and controls for 24 hours at 37°C. Epithelial resistance measurements were taken every 4 hours during cytokine exposure. Western blot and immunofluorescence staining/confocal microscopy were used to assess changes in a panel of tight and adherens junction proteins. Western blot densitometry was quantified with image analysis. Results IL-4 and IL-13 exposure resulted in a mean decrease in transepithelial resistance at 24 hours to 51.6% (n=6) and 68.6% (n=8) of baseline, respectively. Tight junction protein JAM-A expression decreased 42.2% with IL-4 exposure (n=9) and 37.5% with IL-13 exposure (n=9). Adherens junction protein E-cadherin expression decreased 35.3% with IL-4 exposure (n=9) and 32.9% with IL-13 exposure (n=9). Tight junction protein claudin-2 showed more variability but had a trend toward higher expression with Th2 cytokine exposure. There were no appreciable changes in claudin-1, occludin, or ZO-1 with IL-4 or IL-13 exposure. Conclusion Sinonasal epithelial exposure to Th2 cytokines IL-4 and IL-13 results in alterations in intercellular junction proteins, reflecting increased epithelial permeability. Such changes may explain some of the phenotypic manifestations of Th2-mediated sinonasal disease, such as edema, nasal discharge, and environmental reactivity. PMID:24510479

Simvastatin-induced up-regulation of gap junctions composed of connexin 43 sensitize Leydig tumor cells to etoposide: an involvement of PKC pathway.

PubMed

Wang, Lingzhi; Fu, Yanni; Peng, Jianxin; Wu, Dengpan; Yu, Meiling; Xu, Chengfang; Wang, Qin; Tao, Liang

2013-10-04

Some of lipophilic statins have been reported to enhance toxicities induced by antineoplastic agents but the underling mechanism is unclear. The authors investigated the involvement of Cx43-mediated gap junction intercellular communication (GJIC) in the effect of simvastatin on the cellular toxicity induced by etoposide in this study. The results showed that a major component of the cytotoxicity of therapeutic levels of etoposide is mediated by gap junctions composed of connexin 43(Cx43) and simvastatin at the dosage which does not induce cytotoxicity enhances etoposide toxicity by increasing gap junction coupling. The augmentative effect of simvastatin on GJIC was related to the inhibition of PKC-mediated Cx43 phosphorylation at ser368 and subsequent enhancement of Cx43 membrane location induced by the agent. The present study suggests the possibility that upregulation of gap junctions may be utilized to increase the efficacy of anticancer chemotherapies. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Interacting Network of the Gap Junction (GJ) Protein Connexin43 (Cx43) is Modulated by Ischemia and Reperfusion in the Heart.

PubMed

Martins-Marques, Tania; Anjo, Sandra Isabel; Pereira, Paulo; Manadas, Bruno; Girão, Henrique

2015-11-01

The coordinated and synchronized cardiac muscle contraction relies on an efficient gap junction-mediated intercellular communication (GJIC) between cardiomyocytes, which involves the rapid anisotropic impulse propagation through connexin (Cx)-containing channels, namely of Cx43, the most abundant Cx in the heart. Expectedly, disturbing mechanisms that affect channel activity, localization and turnover of Cx43 have been implicated in several cardiomyopathies, such as myocardial ischemia. Besides gap junction-mediated intercellular communication, Cx43 has been associated with channel-independent functions, including modulation of cell adhesion, differentiation, proliferation and gene transcription. It has been suggested that the role played by Cx43 is dictated by the nature of the proteins that interact with Cx43. Therefore, the characterization of the Cx43-interacting network and its dynamics is vital to understand not only the molecular mechanisms underlying pathological malfunction of gap junction-mediated intercellular communication, but also to unveil novel and unanticipated biological functions of Cx43. In the present report, we applied a quantitative SWATH-MS approach to characterize the Cx43 interactome in rat hearts subjected to ischemia and ischemia-reperfusion. Our results demonstrate that, in the heart, Cx43 interacts with proteins related with various biological processes such as metabolism, signaling and trafficking. The interaction of Cx43 with proteins involved in gene transcription strengthens the emerging concept that Cx43 has a role in gene expression regulation. Importantly, our data shows that the interactome of Cx43 (Connexome) is differentially modulated in diseased hearts. Overall, the characterization of Cx43-interacting network may contribute to the establishment of new therapeutic targets to modulate cardiac function in physiological and pathological conditions. Data are available via ProteomeXchange with identifier PXD002331. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Interleukin-4 and interleukin-13 compromise the sinonasal epithelial barrier and perturb intercellular junction protein expression.

PubMed

Wise, Sarah K; Laury, Adrienne M; Katz, Elizabeth H; Den Beste, Kyle A; Parkos, Charles A; Nusrat, Asma

2014-05-01

Altered expression of epithelial intercellular junction proteins has been observed in sinonasal biopsies from nasal polyps and epithelial layers cultured from nasal polyp patients. These alterations comprise a "leaky" epithelial barrier phenotype. We hypothesize that T helper 2 (Th2) cytokines interleukin (IL)-4 and IL-13 modulate epithelial junction proteins, thereby contributing to the leaky epithelial barrier. Differentiated primary sinonasal epithelial layers cultured at the air-liquid interface were exposed to IL-4, IL-13, and controls for 24 hours at 37°C. Epithelial resistance measurements were taken every 4 hours during cytokine exposure. Western blot and immunofluorescence staining/confocal microscopy were used to assess changes in a panel of tight and adherens junction proteins. Western blot densitometry was quantified with image analysis. IL-4 and IL-13 exposure resulted in a mean decrease in transepithelial resistance at 24 hours to 51.6% (n = 6) and 68.6% (n = 8) of baseline, respectively. Tight junction protein junctional adhesion molecule-A (JAM-A) expression decreased 42.2% with IL-4 exposure (n = 9) and 37.5% with IL-13 exposure (n = 9). Adherens junction protein E-cadherin expression decreased 35.3% with IL-4 exposure (n = 9) and 32.9% with IL-13 exposure (n = 9). Tight junction protein claudin-2 showed more variability but had a trend toward higher expression with Th2 cytokine exposure. There were no appreciable changes in claudin-1, occludin, or zonula occludens-1 (ZO-1) with IL-4 or IL-13 exposure. Sinonasal epithelial exposure to Th2 cytokines IL-4 and IL-13 results in alterations in intercellular junction proteins, reflecting increased epithelial permeability. Such changes may explain some of the phenotypic manifestations of Th2-mediated sinonasal disease, such as edema, nasal discharge, and environmental reactivity. © 2014 ARS-AAOA, LLC.

Decursin inhibits VEGF-mediated inner blood-retinal barrier breakdown by suppression of VEGFR-2 activation.

PubMed

Kim, Jin Hyoung; Kim, Jeong Hun; Lee, You Mie; Ahn, Eun-Mi; Kim, Kyu-Won; Yu, Young Suk

2009-09-01

The blood-retinal barrier (BRB) is essential for the normal structural and functional integrity of the retina, whose breakdown could cause the serious vision loss. Vascular endothelial growth factor (VEGF), as a permeable factor, induces alteration of tight junction proteins to result in BRB breakdown. Herein, we demonstrated that decursin inhibits VEGF-mediated inner BRB breakdown through suppression of VEGFR-2 signaling pathway. In retinal endothelial cells, decursin inhibited VEGF-mediated hyperpermeability. Decursin prevented VEGF-mediated loss of tight junction proteins including zonula occludens-1 (ZO-1), ZO-2, and occludin in retinal endothelial cells, which was also supported by restoration of tight junction proteins in intercellular junction. In addition, decursin significantly inhibited VEGF-mediated vascular leakage from retinal vessels, which was accompanied by prevention of loss of tight junction proteins in retinal vessels. Decursin significantly suppressed VEGF-induced VEGFR-2 phosphrylation that consequently led to inhibition of extracellular signal-regulated kinase (ERK) 1/2 activation. Moreover, decursin induced no cytotoxicity to retinal endothelial cells and no retinal toxicity under therapeutic concentrations. Therefore, our results suggest that decursin prevents VEGF-mediated BRB breakdown through blocking of loss of tight junction proteins, which might be regulated by suppression of VEGFR-2 activation. As a novel inhibitor to BRB breakdown, decursin could be applied to variable retinopathies with BRB breakdown.

JAM-C regulates tight junctions and integrin-mediated cell adhesion and migration.

PubMed

Mandicourt, Guillaume; Iden, Sandra; Ebnet, Klaus; Aurrand-Lions, Michel; Imhof, Beat A

2007-01-19

Junctional Adhesion Molecules (JAMs) have been described as major components of tight junctions in endothelial and epithelial cells. Tight junctions are crucial for the establishment and maintenance of cell polarity. During tumor development, they are remodeled, enabling neoplastic cells to escape from constraints imposed by intercellular junctions and to adopt a migratory behavior. Using a carcinoma cell line we tested whether JAM-C could affect tight junctions and migratory properties of tumor cells. We show that transfection of JAM-C improves the tight junctional barrier in tumor cells devoid of JAM-C expression. This is dependent on serine 281 in the cytoplasmic tail of JAM-C because serine mutation into alanine abolishes the specific localization of JAM-C in tight junctions and establishment of cell polarity. More importantly, the same mutation stimulates integrin-mediated cell migration and adhesion via the modulation of beta1 and beta3 integrin activation. These results highlight an unexpected function for JAM-C in controlling epithelial cell conversion from a static, polarized state to a pro-migratory phenotype.

Gap Junction Intercellular Communication: A Review of a Potential Platform to Modulate Craniofacial Tissue Engineering

PubMed Central

Rossello, Ricardo A.; Kohn, David H.

2009-01-01

Defects in craniofacial tissues, resulting from trauma, congenital abnormalities, oncologic resection or progressive deforming diseases, may result in aesthetic deformity, pain and reduced function. Restoring the structure, function and aesthetics of craniofacial tissues represents a substantial clinical problem in need of new solutions. More biologically-interactive biomaterials could potentially improve the treatment of craniofacial defects, and an understanding of developmental processes may help identify strategies and materials that can be used in tissue engineering. One such strategy that can potentially advance tissue engineering is cell–cell communication. Gap junction intercellular communication is the most direct way of achieving such signaling. Gap junction communication through connexin-mediated junctions, in particular connexin 43 (Cx43), plays a major role bone development. Given the important role of Cx43 in controlling development and differentiation, especially in bone cells, controlling the expression of Cx43 may provide control over cell-to-cell communication and may help overcome some of the challenges in craniofacial tissue engineering. Following a review of gap junctions in bone cells, the ability to enhance cell–cell communication and osteogenic differentiation via control of gap junctions is discussed, as is the potential utility of this approach in craniofacial tissue engineering. PMID:18481782

Structural and functional diversity of cadherin at the adherens junction

PubMed Central

2011-01-01

Adhesion between cells is essential to the evolution of multicellularity. Indeed, morphogenesis in animals requires firm but flexible intercellular adhesions that are mediated by subcellular structures like the adherens junction (AJ). A key component of AJs is classical cadherins, a group of transmembrane proteins that maintain dynamic cell–cell associations in many animal species. An evolutionary reconstruction of cadherin structure and function provides a comprehensive framework with which to appreciate the diversity of morphogenetic mechanisms in animals. PMID:21708975

Contractility in type III cochlear fibrocytes is dependent on non-muscle myosin II and intercellular gap junctional coupling.

PubMed

Kelly, John J; Forge, Andrew; Jagger, Daniel J

2012-08-01

The cochlear spiral ligament is a connective tissue that plays diverse roles in normal hearing. Spiral ligament fibrocytes are classified into functional sub-types that are proposed to carry out specialized roles in fluid homeostasis, the mediation of inflammatory responses to trauma, and the fine tuning of cochlear mechanics. We derived a secondary sub-culture from guinea pig spiral ligament, in which the cells expressed protein markers of type III or "tension" fibrocytes, including non-muscle myosin II (nmII), α-smooth muscle actin (αsma), vimentin, connexin43 (cx43), and aquaporin-1. The cells formed extensive stress fibers containing αsma, which were also associated intimately with nmII expression, and the cells displayed the mechanically contractile phenotype predicted by earlier modeling studies. cx43 immunofluorescence was evident within intercellular plaques, and the cells were coupled via dye-permeable gap junctions. Coupling was blocked by meclofenamic acid (MFA), an inhibitor of cx43-containing channels. The contraction of collagen lattice gels mediated by the cells could be prevented reversibly by blebbistatin, an inhibitor of nmII function. MFA also reduced the gel contraction, suggesting that intercellular coupling modulates contractility. The results demonstrate that these cells can impart nmII-dependent contractile force on a collagenous substrate, and support the hypothesis that type III fibrocytes regulate tension in the spiral ligament-basilar membrane complex, thereby determining auditory sensitivity.

Diabetes Increases Cryoinjury Size with Associated Effects on Cx43 Gap Junction Function and Phosphorylation in the Mouse Heart.

PubMed

Palatinus, Joseph A; Gourdie, Robert G

2016-01-01

Diabetic patients develop larger myocardial infarctions and have an increased risk of death following a heart attack. The poor response to myocardial injury in the diabetic heart is likely related to the many metabolic derangements from diabetes that create a poor substrate in general for wound healing, response to injury and infection. Studies in rodents have implicated a role for the gap junction protein connexin 43 (Cx43) in regulating the injury response in diabetic skin wounds. In this study, we sought to determine whether diabetes alters Cx43 molecular interactions or intracellular communication in the cryoinjured STZ type I diabetic mouse heart. We found that epicardial cryoinjury size is increased in diabetic mice and this increase is prevented by preinjury insulin administration. Consistent with these findings, we found that intercellular coupling via gap junctions is decreased after insulin administration in diabetic and nondiabetic mice. This decrease in coupling is associated with a concomitant increase in phosphorylation of Cx43 at serine 368, a residue known to decrease channel conductance. Taken together, our results suggest that insulin regulates both gap junction-mediated intercellular communication and injury propagation in the mouse heart.

Drosophila wing imaginal discs respond to mechanical injury via slow InsP3R-mediated intercellular calcium waves

NASA Astrophysics Data System (ADS)

Restrepo, Simon; Basler, Konrad

2016-08-01

Calcium signalling is a highly versatile cellular communication system that modulates basic functions such as cell contractility, essential steps of animal development such as fertilization and higher-order processes such as memory. We probed the function of calcium signalling in Drosophila wing imaginal discs through a combination of ex vivo and in vivo imaging and genetic analysis. Here we discover that wing discs display slow, long-range intercellular calcium waves (ICWs) when mechanically stressed in vivo or cultured ex vivo. These slow imaginal disc intercellular calcium waves (SIDICs) are mediated by the inositol-3-phosphate receptor, the endoplasmic reticulum (ER) calcium pump SERCA and the key gap junction component Inx2. The knockdown of genes required for SIDIC formation and propagation negatively affects wing disc recovery after mechanical injury. Our results reveal a role for ICWs in wing disc homoeostasis and highlight the utility of the wing disc as a model for calcium signalling studies.

INHIBITION OF GAP JUNCTIONAL INTERCELLULAR COMMUNICATION BY PERFLUORINATED COMPOUNDS IN RAT LIVER AND DOLPHIN KIDNEY EPITHELIAL CELL LINES IN VITRO AND SPRAGUE-DAWLEY RATS IN VIVO

EPA Science Inventory

Abstract

Gap Junctional Intercellular Communication (GJIC) is the major pathway of intercellular signal transduction, and is, thus, important for normal cell growth and function. Recent studies have revealed a global distribution of some perfluorinated organic compounds e...

TLR2 mediates gap junctional intercellular communication through connexin-43 in intestinal epithelial barrier injury.

PubMed

Ey, Birgit; Eyking, Annette; Gerken, Guido; Podolsky, Daniel K; Cario, Elke

2009-08-14

Gap junctional intercellular communication (GJIC) coordinates cellular functions essential for sustaining tissue homeostasis; yet its regulation in the intestine is not well understood. Here, we identify a novel physiological link between Toll-like receptor (TLR) 2 and GJIC through modulation of Connexin-43 (Cx43) during acute and chronic inflammatory injury of the intestinal epithelial cell (IEC) barrier. Data from in vitro studies reveal that TLR2 activation modulates Cx43 synthesis and increases GJIC via Cx43 during IEC injury. The ulcerative colitis-associated TLR2-R753Q mutant targets Cx43 for increased proteasomal degradation, impairing TLR2-mediated GJIC during intestinal epithelial wounding. In vivo studies using mucosal RNA interference show that TLR2-mediated mucosal healing depends functionally on intestinal epithelial Cx43 during acute inflammatory stress-induced damage. Mice deficient in TLR2 exhibit IEC-specific alterations in Cx43, whereas administration of a TLR2 agonist protects GJIC by blocking accumulation of Cx43 and its hyperphosphorylation at Ser368 to prevent spontaneous chronic colitis in MDR1alpha-deficient mice. Finally, adding the TLR2 agonist to three-dimensional intestinal mucosa-like cultures of human biopsies preserves intestinal epithelial Cx43 integrity and polarization ex vivo. In conclusion, Cx43 plays an important role in innate immune control of commensal-mediated intestinal epithelial wound repair.

TLR2 Mediates Gap Junctional Intercellular Communication through Connexin-43 in Intestinal Epithelial Barrier Injury*

PubMed Central

Ey, Birgit; Eyking, Annette; Gerken, Guido; Podolsky, Daniel K.; Cario, Elke

2009-01-01

Gap junctional intercellular communication (GJIC) coordinates cellular functions essential for sustaining tissue homeostasis; yet its regulation in the intestine is not well understood. Here, we identify a novel physiological link between Toll-like receptor (TLR) 2 and GJIC through modulation of Connexin-43 (Cx43) during acute and chronic inflammatory injury of the intestinal epithelial cell (IEC) barrier. Data from in vitro studies reveal that TLR2 activation modulates Cx43 synthesis and increases GJIC via Cx43 during IEC injury. The ulcerative colitis-associated TLR2-R753Q mutant targets Cx43 for increased proteasomal degradation, impairing TLR2-mediated GJIC during intestinal epithelial wounding. In vivo studies using mucosal RNA interference show that TLR2-mediated mucosal healing depends functionally on intestinal epithelial Cx43 during acute inflammatory stress-induced damage. Mice deficient in TLR2 exhibit IEC-specific alterations in Cx43, whereas administration of a TLR2 agonist protects GJIC by blocking accumulation of Cx43 and its hyperphosphorylation at Ser368 to prevent spontaneous chronic colitis in MDR1α-deficient mice. Finally, adding the TLR2 agonist to three-dimensional intestinal mucosa-like cultures of human biopsies preserves intestinal epithelial Cx43 integrity and polarization ex vivo. In conclusion, Cx43 plays an important role in innate immune control of commensal-mediated intestinal epithelial wound repair. PMID:19528242

Increase of gap junction activities in SW480 human colorectal cancer cells.

PubMed

Bigelow, Kristina; Nguyen, Thu A

2014-07-09

Colorectal cancer is one of the most common cancers in the United States with an early detection rate of only 39%. Colorectal cancer cells along with other cancer cells exhibit many deficiencies in cell-to-cell communication, particularly gap junctional intercellular communication (GJIC). GJIC has been reported to diminish as cancer cells progress. Gap junctions are intercellular channels composed of connexin proteins, which mediate the direct passage of small molecules from one cell to the next. They are involved in the regulation of the cell cycle, cell differentiation, and cell signaling. Since the regulation of gap junctions is lost in colorectal cancer cells, the goal of this study is to determine the effect of GJIC restoration in colorectal cancer cells. Gap Junction Activity Assay and protein analysis were performed to evaluate the effects of overexpression of connexin 43 (Cx43) and treatment of PQ1, a small molecule, on GJIC. Overexpression of Cx43 in SW480 colorectal cancer cells causes a 6-fold increase of gap junction activity compared to control. This suggests that overexpressing Cx43 can restore GJIC. Furthermore, small molecule like PQ1 directly targeting gap junction channel was used to increase GJIC. Gap junction enhancers, PQ1, at 200 nM showed a 4-fold increase of gap junction activity in SW480 cells. A shift from the P0 to the P2 isoform of Cx43 was seen after 1 hour treatment with 200 nM PQ1. Overexpression of Cx43 and treatment of PQ1 can directly increase gap junction activity. The findings provide an important implication in which restoration of gap junction activity can be targeted for drug development.

Structural basis for PECAM-1 homophilic binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Paddock, C.; Zhou, D.; Lertkiatmongkol, P.

2015-12-23

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kDa member of the immunoglobulin gene superfamily (IgSF) that is present on the surface of circulating platelets and leukocytes, and highly expressed at the junctions of confluent endothelial cell monolayers. PECAM-1–mediated homophilic interactions, known to be mediated by its 2 amino-terminal immunoglobulin homology domains, are essential for concentrating PECAM-1 at endothelial cell intercellular junctions, where it functions to facilitate diapedesis, maintain vascular integrity, and transmit survival signals into the cell. Given the importance of PECAM-1–mediated homophilic interactions in mediating each of these cell physiological events, and to reveal the nature and orientationmore » of the PECAM-1–PECAM-1 homophilic-binding interface, we undertook studies aimed at determining the crystal structure of the PECAM-1 homophilic-binding domain, which is composed of amino-terminal immunoglobulin homology domains 1 and 2 (IgD1 and IgD2). The crystal structure revealed that both IgD1 and IgD2 exhibit a classical IgSF fold, having a β-sandwich topology formed by 2 sheets of antiparallel β strands stabilized by the hallmark disulfide bond between the B and F strands. Interestingly, despite previous assignment to the C2 class of immunoglobulin-like domains, the structure of IgD1 reveals that it actually belongs to the I2 set of IgSF folds. Both IgD1 and IgD2 participate importantly in the formation of the trans homophilic-binding interface, with a total buried interface area of >2300 Å 2. These and other unique structural features of PECAM-1 allow for the development of an atomic-level model of the interactions that PECAM-1 forms during assembly of endothelial cell intercellular junctions.« less

Effects of Intercellular Junction Protein Expression on Intracellular Ice Formation in Mouse Insulinoma Cells

PubMed Central

Higgins, Adam Z.; Karlsson, Jens O.M.

2013-01-01

The development of cryopreservation procedures for tissues has proven to be difficult in part because cells within tissue are more susceptible to intracellular ice formation (IIF) than are isolated cells. In particular, previous studies suggest that cell-cell interactions increase the likelihood of IIF by enabling propagation of ice between neighboring cells, a process thought to be mediated by gap junction channels. In this study, we investigated the effects of cell-cell interactions on IIF using three genetically modified strains of the mouse insulinoma cell line MIN6, each of which expressed key intercellular junction proteins (connexin-36, E-cadherin, and occludin) at different levels. High-speed video cryomicroscopy was used to visualize the freezing process in pairs of adherent cells, revealing that the initial IIF event in a given cell pair was correlated with a hitherto unrecognized precursor phenomenon: penetration of extracellular ice into paracellular spaces at the cell-cell interface. Such paracellular ice penetration occurred in the majority of cell pairs observed, and typically preceded and colocalized with the IIF initiation events. Paracellular ice penetration was generally not observed at temperatures >−5.65°C, which is consistent with a penetration mechanism via defects in tight-junction barriers at the cell-cell interface. Although the maximum temperature of paracellular penetration was similar for all four cell strains, genetically modified cells exhibited a significantly higher frequency of ice penetration and a higher mean IIF temperature than did wild-type cells. A four-state Markov chain model was used to quantify the rate constants of the paracellular ice penetration process, the penetration-associated IIF initiation process, and the intercellular ice propagation process. In the initial stages of freezing (>−15°C), junction protein expression appeared to only have a modest effect on the kinetics of propagative IIF, and even cell strains lacking the gap junction protein connexin-36 exhibited nonnegligible ice propagation rates. PMID:24209845

Connexin43 synthesis, phosphorylation, and degradation in regulation of transient inhibition of gap junction intercellular communication by the phorbol ester TPA in rat liver epithelial cells.

PubMed

Rivedal, Edgar; Leithe, Edward

2005-01-15

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces transient inhibition of gap junction intercellular communication (GJIC) in several cell types. The initial block in GJIC has been attributed to protein kinase C (PKC) mediated phosphorylation of connexin gap junction proteins, including connexin43 (Cx43). Restoration of GJIC, associated with normalization of the Cx43 phosphorylation status, has been ascribed to different events, including dephosphorylation of Cx43 and de novo synthesis of Cx43 or other, non-gap junctional, proteins. The data presented suggest that restoration of GJIC during continuous TPA exposure in normal and transformed rat liver epithelial cells is dependent on synthesis of Cx43 protein, as well as the transport of already synthesized Cx43 from intracellular pools to the plasma membrane. Reactivation of inactivated Cx43 by dephosphorylation does not appear to be involved in the recovery of GJIC. Both PKC and MAP kinase is involved in TPA-induced degradation of Cx43 and inhibition of GJIC. We show that coincubation of TPA with the protein synthesis inhibitor cycloheximide or the transcription inhibitor actinomycin D results in synergistic enhancement of the level of activated ERK1/2. Together, the present data highlight Cx43 degradation and synthesis as critical determinants in TPA-induced modifications of cell-cell communication via gap junctions.

Epidermal growth factor receptor and integrins control force-dependent vinculin recruitment to E-cadherin junctions.

PubMed

Sehgal, Poonam; Kong, Xinyu; Wu, Jun; Sunyer, Raimon; Trepat, Xavier; Leckband, Deborah

2018-03-20

This study reports novel findings that link E-cadherin (also known as CDH1)-mediated force-transduction signaling to vinculin targeting to intercellular junctions via epidermal growth factor receptor (EGFR) and integrins. These results build on previous findings that demonstrated that mechanically perturbed E-cadherin receptors activate phosphoinositide 3-kinase and downstream integrins in an EGFR-dependent manner. Results of this study show that this EGFR-mediated kinase cascade controls the force-dependent recruitment of vinculin to stressed E-cadherin complexes - a key early signature of cadherin-based mechanotransduction. Vinculin targeting requires its phosphorylation at tyrosine 822 by Abl family kinases (hereafter Abl), but the origin of force-dependent Abl activation had not been identified. We now present evidence that integrin activation, which is downstream of EGFR signaling, controls Abl activation, thus linking E-cadherin to Abl through a mechanosensitive signaling network. These findings place EGFR and integrins at the center of a positive-feedback loop, through which force-activated E-cadherin signals regulate vinculin recruitment to cadherin complexes in response to increased intercellular tension.This article has an associated First Person interview with the first author of the paper. © 2018. Published by The Company of Biologists Ltd.

A Co-operative Regulation of Neuronal Excitability by UNC-7 Innexin and NCA/NALCN Leak Channel

PubMed Central

2011-01-01

Gap junctions mediate the electrical coupling and intercellular communication between neighboring cells. Some gap junction proteins, namely connexins and pannexins in vertebrates, and innexins in invertebrates, may also function as hemichannels. A conserved NCA/Dmα1U/NALCN family cation leak channel regulates the excitability and activity of vertebrate and invertebrate neurons. In the present study, we describe a genetic and functional interaction between the innexin UNC-7 and the cation leak channel NCA in Caenorhabditis elegans neurons. While the loss of the neuronal NCA channel function leads to a reduced evoked postsynaptic current at neuromuscular junctions, a simultaneous loss of the UNC-7 function restores the evoked response. The expression of UNC-7 in neurons reverts the effect of the unc-7 mutation; moreover, the expression of UNC-7 mutant proteins that are predicted to be unable to form gap junctions also reverts this effect, suggesting that UNC-7 innexin regulates neuronal activity, in part, through gap junction-independent functions. We propose that, in addition to gap junction-mediated functions, UNC-7 innexin may also form hemichannels to regulate C. elegans' neuronal activity cooperatively with the NCA family leak channels. PMID:21489288

Effect of sound on gap-junction-based intercellular signaling: Calcium waves under acoustic irradiation.

PubMed

Deymier, P A; Swinteck, N; Runge, K; Deymier-Black, A; Hoying, J B

2015-01-01

We present a previously unrecognized effect of sound waves on gap-junction-based intercellular signaling such as in biological tissues composed of endothelial cells. We suggest that sound irradiation may, through temporal and spatial modulation of cell-to-cell conductance, create intercellular calcium waves with unidirectional signal propagation associated with nonconventional topologies. Nonreciprocity in calcium wave propagation induced by sound wave irradiation is demonstrated in the case of a linear and a nonlinear reaction-diffusion model. This demonstration should be applicable to other types of gap-junction-based intercellular signals, and it is thought that it should be of help in interpreting a broad range of biological phenomena associated with the beneficial therapeutic effects of sound irradiation and possibly the harmful effects of sound waves on health.

A transwell assay that excludes exosomes for assessment of tunneling nanotube-mediated intercellular communication.

PubMed

Thayanithy, Venugopal; O'Hare, Patrick; Wong, Phillip; Zhao, Xianda; Steer, Clifford J; Subramanian, Subbaya; Lou, Emil

2017-11-13

Tunneling nanotubes (TNTs) are naturally-occurring filamentous actin-based membranous extensions that form across a wide spectrum of mammalian cell types to facilitate long-range intercellular communication. Valid assays are needed to accurately assess the downstream effects of TNT-mediated transfer of cellular signals in vitro. We recently reported a modified transwell assay system designed to test the effects of intercellular transfer of a therapeutic oncolytic virus, and viral-activated drugs, between cells via TNTs. The objective of the current study was to demonstrate validation of this in vitro approach as a new method for effectively excluding diffusible forms of long- and close-range intercellular transfer of intracytoplasmic cargo, including exosomes/microvesicles and gap junctions in order to isolate TNT-selective cell communication. We designed several steps to effectively reduce or eliminate diffusion and long-range transfer via these extracellular vesicles, and used Nanoparticle Tracking Analysis to quantify exosomes following implementation of these steps. The experimental approach outlined here effectively reduced exosome trafficking by >95%; further use of heparin to block exosome uptake by putative recipient cells further impeded transfer of these extracellular vesicles. This validated assay incorporates several steps that can be taken to quantifiably control for extracellular vesicles in order to perform studies focused on TNT-selective communication.

Low Connexin Channel-Dependent Intercellular Communication in Human Adult Hematopoietic Progenitor/Stem Cells: Probing Mechanisms of Autologous Stem Cell Therapy

PubMed Central

Yang, Jian; Darley, Richard L; Hallett, Maurice; Evans, W Howard

2009-01-01

Human bone marrow is a clinical source of autologous progenitor stem cells showing promise for cardiac repair following ischemic insult. Functional improvements following delivery of adult bone marrow CD34+ cells into heart tissue may require metabolic/electrical communication between participating cells. Since connexin43 (Cx43) channels are implicated in cardiogenesis and provide intercellular connectivity in the heart, the authors analyzed the expression of 20 connexins (Cx) in CD34+ cells and in monocytes and granulocytes in bone marrow and spinal cord. Reverse transcriptase-polymerase chain reaction (RT-PCR) detected only low expression of Cx43 and Cx37. Very low level dye coupling was detected by flow cytometry between CD34+ cells and other Cx43 expressing cells, including HL-1 cardiac cells, and was not inhibited by specific gap junction inhibitors. The results indicate that CD34+ cells are unlikely to communicate via gap junctions and the authors conclude that use of CD34+ cells to repair damaged hearts is unlikely to involve gap junctions. The results concur with the hypothesis that bone marrow cells elicit improved cardiac function through release of undefined paracrine mediators. PMID:20298144

Gap junction blockage promotes cadmium-induced apoptosis in BRL 3A derived from Buffalo rat liver cells.

PubMed

Hu, Di; Zou, Hui; Han, Tao; Xie, Junze; Dai, Nannan; Zhuo, Liling; Gu, Jianhong; Bian, Jianchun; Yuan, Yan; Liu, Xuezhong; Liu, Zongping

2016-03-01

Gap junctions mediate direct communication between cells; however, toxicological cascade triggered by nonessential metals can abrogate cellular signaling mediated by gap junctions. Although cadmium (Cd) is known to induce apoptosis in organs and tissues, the mechanisms that underlie gap junction activity in Cd-induced apoptosis in BRL 3A rat liver cells has yet to be established. In this study, we showed that Cd treatment decreased the cell index (a measure of cellular electrical impedance) in BRL 3A cells. Mechanistically, we found that Cd exposure decreased expression of connexin 43 (Cx43), increased expression of p-Cx43 and elevated intracellular free Ca(2+) concentration, corresponding to a decrease in gap junctional intercellular communication. Gap junction blockage pretreatment with 18β-glycyrrhizic acid (GA) promoted Cd-induced apoptosis, involving changes in expression of Bax, Bcl-2, caspase-3 and the mitochondrial transmembrane electrical potential (Δψm). Additionally, GA was found to enhance ERK and p38 activation during Cd-induced activation of mitogen-activated protein kinases, but had no significant effect on JNK activation. Our results indicated the apoptosis-related proteins and the ERK and p38 signaling pathways may participate in gap junction blockage promoting Cd-induced apoptosis in BRL 3A cells.

Gap junctional intercellular communication dysfunction mediates the cognitive impairment induced by cerebral ischemia-reperfusion injury: PI3K/Akt pathway involved.

PubMed

Zhou, Shujun; Fang, Zheng; Wang, Gui; Wu, Song

2017-01-01

Cerebral ischemia/reperfusion (I/R) injury causes hippocampal apoptosis and cognitive impairment, and the dysfunction of gap junction intercellular communication (GJIC) may contribute to the cognitive impairment. We aim to examine the impact of cerebral I/R injury on cognitive impairment, the role of GJIC dysfunction in the rat hippocampus and the involvement of the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) pathway. Rats were subjected to a cerebral I/R procedure and underwent cognitive assessment with the novel object recognition and Morris Water Maze tasks. The distance of Lucifer Yellow dye transfer and the Cx43 protein were examined to measure GJIC. Neural apoptosis was assessed with the terminal deoxynucleotide-transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) method. After rats received inhibitors of the PI3K/Akt pathway, GJIC and cognitive ability were measured again. GJIC promotion by ZP123 significantly reversed cognitive impairment and hippocampal apoptosis induced by cerebral I/R, while the inhibition of GJIC by octanol significantly facilitated cognitive impairment and hippocampal apoptosis. The phosphorylation of Akt was enhanced by cerebral I/R and octanol but inhibited by ZP123. The inhibition of the PI3K/Akt pathway significantly suppressed GJIC and cognitive impairment. The PI3K/Akt pathway is involved in cognitive impairment caused by gap junctional communication dysfunction in the rat hippocampus after ischemia-reperfusion injury.

Gap junction intercellular communication mediated by connexin43 in astrocytes is essential for their resistance to oxidative stress.

PubMed

Le, Hoa T; Sin, Wun Chey; Lozinsky, Shannon; Bechberger, John; Vega, José Luis; Guo, Xu Qiu; Sáez, Juan C; Naus, Christian C

2014-01-17

Oxidative stress induced by reactive oxygen species (ROS) is associated with various neurological disorders including aging, neurodegenerative diseases, as well as traumatic and ischemic insults. Astrocytes have an important role in the anti-oxidative defense in the brain. The gap junction protein connexin43 (Cx43) forms intercellular channels as well as hemichannels in astrocytes. In the present study, we investigated the contribution of Cx43 to astrocytic death induced by the ROS hydrogen peroxide (H2O2) and the mechanism by which Cx43 exerts its effects. Lack of Cx43 expression or blockage of Cx43 channels resulted in increased ROS-induced astrocytic death, supporting a cell protective effect of functional Cx43 channels. H2O2 transiently increased hemichannel activity, but reduced gap junction intercellular communication (GJIC). GJIC in wild-type astrocytes recovered after 7 h, but was absent in Cx43 knock-out astrocytes. Blockage of Cx43 hemichannels incompletely inhibited H2O2-induced hemichannel activity, indicating the presence of other hemichannel proteins. Panx1, which is predicted to be a major hemichannel contributor in astrocytes, did not appear to have any cell protective effect from H2O2 insults. Our data suggest that GJIC is important for Cx43-mediated ROS resistance. In contrast to hypoxia/reoxygenation, H2O2 treatment decreased the ratio of the hypophosphorylated isoform to total Cx43 level. Cx43 has been reported to promote astrocytic death induced by hypoxia/reoxygenation. We therefore speculate the increase in Cx43 dephosphorylation may account for the facilitation of astrocytic death. Our findings suggest that the role of Cx43 in response to cellular stress is dependent on the activation of signaling pathways leading to alteration of Cx43 phosphorylation states.

Intercellular Ca2+ Waves: Mechanisms and Function

PubMed Central

Sanderson, Michael J.

2012-01-01

Intercellular calcium (Ca2+) waves (ICWs) represent the propagation of increases in intracellular Ca2+ through a syncytium of cells and appear to be a fundamental mechanism for coordinating multicellular responses. ICWs occur in a wide diversity of cells and have been extensively studied in vitro. More recent studies focus on ICWs in vivo. ICWs are triggered by a variety of stimuli and involve the release of Ca2+ from internal stores. The propagation of ICWs predominately involves cell communication with internal messengers moving via gap junctions or extracellular messengers mediating paracrine signaling. ICWs appear to be important in both normal physiology as well as pathophysiological processes in a variety of organs and tissues including brain, liver, retina, cochlea, and vascular tissue. We review here the mechanisms of initiation and propagation of ICWs, the key intra- and extracellular messengers (inositol 1,4,5-trisphosphate and ATP) mediating ICWs, and the proposed physiological functions of ICWs. PMID:22811430

Gap junction-mediated intercellular communication in the immune system.

PubMed

Neijssen, Joost; Pang, Baoxu; Neefjes, Jacques

2007-01-01

Immune cells are usually considered non-attached blood cells, which would exclude the formation of gap junctions. This is a misconception since many immune cells express connexin 43 (Cx43) and other connexins and are often residing in tissue. The role of gap junctions is largely ignored by immunologists as is the immune system in the field of gap junction research. Here, the current knowledge of the distribution of connexins and the function of gap junctions in the immune system is discussed. Gap junctions appear to play many roles in antibody productions and specific immune responses and may be important in sensing danger in tissue by the immune system. Gap junctions not only transfer electrical and metabolical but also immunological information in the form of peptides for a process called cross-presentation. This is essential for proper immune responses to viruses and possibly tumours. Until now only 40 research papers on gap junctions in the immune system appeared and this will almost certainly expand with the increased mutual interest between the fields of immunology and gap junction research.

Gap junction coupling is required for tumor cell migration through lymphatic endothelium.

PubMed

Karpinich, Natalie O; Caron, Kathleen M

2015-05-01

The lymphatic vasculature is a well-established conduit for metastasis, but the mechanisms by which tumor cells interact with lymphatic endothelial cells (LECs) to facilitate escape remain poorly understood. Elevated levels of the lymphangiogenic peptide adrenomedullin are found in many tumors, and we previously characterized that its expression is necessary for lymphatic vessel growth within both tumors and sentinel lymph nodes and for distant metastasis. This study used a tumor cell-LEC coculture system to identify a series of adrenomedullin-induced events that facilitated transendothelial migration of the tumor cells through a lymphatic monolayer. High levels of adrenomedullin expression enhanced adhesion of tumor cells to LECs, and further analysis revealed that adrenomedullin promoted gap junction coupling between LECs as evidenced by spread of Lucifer yellow dye. Adrenomedullin also enhanced heterocellular gap junction coupling as demonstrated by Calcein dye transfer from tumor cells into LECs. This connexin-mediated gap junction intercellular communication was necessary for tumor cells to undergo transendothelial migration because pharmacological blockade of this heterocellular communication prevented the ability of tumor cells to transmigrate through the lymphatic monolayer. In addition, treatment of LECs with adrenomedullin caused nuclear translocation of β-catenin, a component of endothelial cell junctions, causing an increase in transcription of the downstream target gene C-MYC. Importantly, blockade of gap junction intercellular communication prevented β-catenin nuclear translocation. Our findings indicate that maintenance of cell-cell communication is necessary to facilitate a cascade of events that lead to tumor cell migration through the lymphatic endothelium. © 2015 American Heart Association, Inc.

Tanshinone IIA Increases the Bystander Effect of Herpes Simplex Virus Thymidine Kinase/Ganciclovir Gene Therapy via Enhanced Gap Junctional Intercellular Communication

PubMed Central

Liu, Xijuan; Wu, Yingya; Du, Biaoyan; Li, Jiefen; Zhou, Jing; Li, Jingjing; Tan, Yuhui

2013-01-01

The bystander effect is an intriguing phenomenon by which adjacent cells become sensitized to drug treatment during gene therapy with herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV). This effect is reported to be mediated by gap junctional intercellular communication (GJIC), and therefore, we postulated that upregulation of genes that facilitate GJIC may enhance the HSV-tk/GCV bystander effect. Previous findings have shown Tanshinone IIA (Tan IIA), a chemical substance derived from a Chinese medicine herb, promotes the upregulation of the connexins Cx26 and Cx43 in B16 cells. Because gap junctions are formed by connexins, we hypothesized that Tan IIA might increase GJIC. Our results show that Tan IIA increased GJIC in B16 melanoma cells, leading to more efficient GCV-induced bystander killing in cells stably expressing HSV-tk. Additionally, in vivo experiments demonstrated that tumors in mice with 10% HSV-tk positive B16 cells and 90% wild-type B16 cells became smaller following treatment with the combination of GCV and Tan IIA as compared to GCV or Tan IIA alone. These data demonstrate that Tan IIA can augment the bystander effect of HSV-tk/GCV system through increased gap junction coupling, which adds strength to the promising strategy that develops connexins inducer to potentiate the effects of suicide gene therapy. PMID:23861780

Irsogladine maleate regulates gap junctional intercellular communication-dependent epithelial barrier in human nasal epithelial cells.

PubMed

Miyata, Ryo; Nomura, Kazuaki; Kakuki, Takuya; Takano, Ken-Ichi; Kohno, Takayuki; Konno, Takumi; Sawada, Norimasa; Himi, Tetsuo; Kojima, Takashi

2015-04-01

The airway epithelium of the human nasal mucosa acts as the first physical barrier that protects against inhaled substances and pathogens. Irsogladine maleate (IM) is an enhancer of gastric mucosal protective factors via upregulation of gap junctional intercellular communication (GJIC). GJIC is thought to participate in the formation of functional tight junctions. However, the effects of IM on GJIC and the epithelial barrier in human nasal epithelial cells (HNECs) remain unknown. To investigate the effects of IM on GJIC and the tight junctional barrier in HNECs, primary cultures of HNECs transfected with human telomerase reverse transcriptase (hTERT-HNECs) were treated with IM and the GJIC inhibitors oleamide and 18β-GA. Some cells were pretreated with IM before treatment with TLR3 ligand poly(I:C) to examine whether IM prevented the changes via TLR3-mediated signal pathways. In hTERT-HNECs, GJIC blockers reduced the expression of tight junction molecules claudin-1, -4, -7, occludin, tricellulin, and JAM-A. IM induced GJIC activity and enhanced the expression of claudin-1, -4, and JAM-A at the protein and mRNA levels with an increase of barrier function. GJIC blockers prevented the increase of the tight junction proteins induced by IM. Furthermore, IM prevented the reduction of JAM-A but not induction of IL-8 and TNF-α induced by poly(I:C). In conclusion, IM can maintain the GJIC-dependent tight junctional barrier via regulation of GJIC in upper airway nasal epithelium. Therefore, it is possible that IM may be useful as a nasal spray to prevent the disruption of the epithelial barrier by viral infections and exposure to allergens in human nasal mucosa.

Homeostasis in the vertebrate lens: mechanisms of solute exchange

PubMed Central

Dahm, Ralf; van Marle, Jan; Quinlan, Roy A.; Prescott, Alan R.; Vrensen, Gijs F. J. M.

2011-01-01

The eye lens is avascular, deriving nutrients from the aqueous and vitreous humours. It is, however, unclear which mechanisms mediate the transfer of solutes between these humours and the lens' fibre cells (FCs). In this review, we integrate the published data with the previously unpublished ultrastructural, dye loading and magnetic resonance imaging results. The picture emerging is that solute transfer between the humours and the fibre mass is determined by four processes: (i) paracellular transport of ions, water and small molecules along the intercellular spaces between epithelial and FCs, driven by Na+-leak conductance; (ii) membrane transport of such solutes from the intercellular spaces into the fibre cytoplasm by specific carriers and transporters; (iii) gap-junctional coupling mediating solute flux between superficial and deeper fibres, Na+/K+-ATPase-driven efflux of waste products in the equator, and electrical coupling of fibres; and (iv) transcellular transfer via caveoli and coated vesicles for the uptake of macromolecules and cholesterol. There is evidence that the Na+-driven influx of solutes occurs via paracellular and membrane transport and the Na+/K+-ATPase-driven efflux of waste products via gap junctions. This micro-circulation is likely restricted to the superficial cortex and nearly absent beyond the zone of organelle loss, forming a solute exchange barrier in the lens. PMID:21402585

Cleavage of transmembrane junction proteins and their role in regulating epithelial homeostasis

PubMed Central

Nava, Porfirio; Kamekura, Ryuta; Nusrat, Asma

2013-01-01

Epithelial tissues form a selective barrier that separates the external environment from the internal tissue milieu. Single epithelial cells are densely packed and associate via distinct intercellular junctions. Intercellular junction proteins not only control barrier properties of the epithelium but also play an important role in regulating epithelial homeostasis that encompasses cell proliferation, migration, differentiation and regulated shedding. Recent studies have revealed that several proteases target epithelial junction proteins during physiological maturation as well as in pathologic states such as inflammation and cancer. This review discusses mechanisms and biological consequences of transmembrane junction protein cleavage. The influence of junction protein cleavage products on pathogenesis of inflammation and cancer is discussed. PMID:24665393

MicroRNA Intercellular Transfer and Bioelectrical Regulation of Model Multicellular Ensembles by the Gap Junction Connectivity.

PubMed

Cervera, Javier; Meseguer, Salvador; Mafe, Salvador

2017-08-17

We have studied theoretically the microRNA (miRNA) intercellular transfer through voltage-gated gap junctions in terms of a biophysically grounded system of coupled differential equations. Instead of modeling a specific system, we use a general approach describing the interplay between the genetic mechanisms and the single-cell electric potentials. The dynamics of the multicellular ensemble are simulated under different conditions including spatially inhomogeneous transcription rates and local intercellular transfer of miRNAs. These processes result in spatiotemporal changes of miRNA, mRNA, and ion channel protein concentrations that eventually modify the bioelectrical states of small multicellular domains because of the ensemble average nature of the electrical potential. The simulations allow a qualitative understanding of the context-dependent nature of the effects observed when specific signaling molecules are transferred through gap junctions. The results suggest that an efficient miRNA intercellular transfer could permit the spatiotemporal control of small cellular domains by the conversion of single-cell genetic and bioelectric states into multicellular states regulated by the gap junction interconnectivity.

Intercellular communication via gap junctions affected by mechanical load in the bovine annulus fibrosus.

PubMed

Desrochers, Jane; Duncan, Neil A

2014-01-01

Cells in the intervertebral disc, as in other connective tissues including tendon, ligament and bone, form interconnected cellular networks that are linked via functional gap junctions. These cellular networks may be necessary to affect a coordinated response to mechanical and environmental stimuli. Using confocal microscopy with fluorescence recovery after photobleaching methods, we explored the in situ strain environment of the outer annulus of an intact bovine disc and the effect of high-level flexion on gap junction signalling. The in situ strain environment in the extracellular matrix of the outer annulus under high flexion load was observed to be non-uniform with the extensive cellular processes remaining crimped sometimes at flexion angles greater than 25°. A significant transient disruption of intercellular communication via functional gap junctions was measured after 10 and 20 min under high flexion load. This study illustrates that in healthy annulus fibrosus tissue, high mechanical loads can impede the functioning of the gap junctions. Future studies will explore more complex loading conditions to determine whether losses in intercellular communication can be permanent and whether gap junctions in aged and degenerated tissues become more susceptible to load. The current research suggests that cellular structures such as gap junctions and intercellular networks, as well as other cell-cell and cell-matrix interconnections, need to be considered in computational models in order to fully understand how macroscale mechanical signals are transmitted across scales to the microscale and ultimately into a cellular biosynthetic response in collagenous tissues.

Release of Applied Mechanical Loading Stimulates Intercellular Calcium Waves in Drosophila Wing Discs.

PubMed

Narciso, Cody E; Contento, Nicholas M; Storey, Thomas J; Hoelzle, David J; Zartman, Jeremiah J

2017-07-25

Mechanical forces are critical but poorly understood inputs for organogenesis and wound healing. Calcium ions (Ca 2+ ) are critical second messengers in cells for integrating environmental and mechanical cues, but the regulation of Ca 2+ signaling is poorly understood in developing epithelial tissues. Here we report a chip-based regulated environment for microorgans that enables systematic investigations of the crosstalk between an organ's mechanical stress environment and biochemical signaling under genetic and chemical perturbations. This method enabled us to define the essential conditions for generating organ-scale intercellular Ca 2+ waves in Drosophila wing discs that are also observed in vivo during organ development. We discovered that mechanically induced intercellular Ca 2+ waves require fly extract growth serum as a chemical stimulus. Using the chip-based regulated environment for microorgans, we demonstrate that not the initial application but instead the release of mechanical loading is sufficient, but not necessary, to initiate intercellular Ca 2+ waves. The Ca 2+ response depends on the prestress intercellular Ca 2+ activity and not on the magnitude or duration of the mechanical stimulation applied. Mechanically induced intercellular Ca 2+ waves rely on IP 3 R-mediated Ca 2+ -induced Ca 2+ release and propagation through gap junctions. Thus, intercellular Ca 2+ waves in developing epithelia may be a consequence of stress dissipation during organ growth. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Redox-mediated regulation of connexin proteins; focus on nitric oxide.

PubMed

García, Isaac E; Sánchez, Helmuth A; Martínez, Agustín D; Retamal, Mauricio A

2018-01-01

Connexins are membrane proteins that form hemichannels and gap junction channels at the plasma membrane. Through these channels connexins participate in autocrine and paracrine intercellular communication. Connexin-based channels are tightly regulated by membrane potential, phosphorylation, pH, redox potential, and divalent cations, among others, and the imbalance of this regulation have been linked to many acquired and genetic diseases. Concerning the redox potential regulation, the nitric oxide (NO) has been described as a modulator of the hemichannels and gap junction channels properties. However, how NO regulates these channels is not well understood. In this mini-review, we summarize the current knowledge about the effects of redox potential focused in NO on the trafficking, formation and functional properties of hemichannels and gap junction channels. Copyright © 2017 Elsevier B.V. All rights reserved.

Gap junctions and connexin hemichannels in the regulation of haemostasis and thrombosis.

PubMed

Vaiyapuri, Sakthivel; Flora, Gagan D; Gibbins, Jonathan M

2015-06-01

Platelets are involved in the maintenance of haemostasis but their inappropriate activation leads to thrombosis, a principal trigger for heart attack and ischaemic stroke. Although platelets circulate in isolation, upon activation they accumulate or aggregate together to form a thrombus, where they function in a co-ordinated manner to prevent loss of blood and control wound repair. Previous report (1) indicates that the stability and functions of a thrombus are maintained through sustained, contact-dependent signalling between platelets. Given the role of gap junctions in the co-ordination of tissue responses, it was hypothesized that gap junctions may be present within a thrombus and mediate intercellular communication between platelets. Therefore studies were performed to explore the presence and functions of connexins in platelets. In this brief review, the roles of hemichannels and gap junctions in the control of thrombosis and haemostasis and the future directions for this research will be discussed.

Relative Roles of Gap Junction Channels and Cytoplasm in Cell-to-Cell Diffusion of Fluorescent Tracers

NASA Astrophysics Data System (ADS)

Safranyos, Richard G. A.; Caveney, Stanley; Miller, James G.; Petersen, Nils O.

1987-04-01

Intercellular (tissue) diffusion of molecules requires cytoplasmic diffusion and diffusion through gap junctional (or cell-to-cell) channels. The rates of tissue and cytoplasmic diffusion of fluorescent tracers, expressed as an effective diffusion coefficient, De, and a cytoplasmic diffusion coefficient, Dcyt, have been measured among the developing epidermal cells of a larval beetle, Tenebrio molitor L., to determine the contribution of the junctional channels to intercellular diffusion. Tracer diffusion was measured by injecting fluorescent tracers into cells and quantitating the rate of subsequent spread into adjacent cells. Cytoplasmic diffusion was determined by fluorescence photobleaching. These experiments show that gap junctional channels constitute approximately 70-80% of the total cell-to-cell resistance to the diffusion of organic tracers at high concentrations in this tissue. At low concentrations, however, the binding of tracer to cytoplasm slows down the cytoplasmic diffusion, which may limit intercellular diffusion.

Ischemic preconditioning protects against gap junctional uncoupling in cardiac myofibroblasts.

PubMed

Sundset, Rune; Cooper, Marie; Mikalsen, Svein-Ole; Ytrehus, Kirsti

2004-01-01

Ischemic preconditioning increases the heart's tolerance to a subsequent longer ischemic period. The purpose of this study was to investigate the role of gap junction communication in simulated preconditioning in cultured neonatal rat cardiac myofibroblasts. Gap junctional intercellular communication was assessed by Lucifer yellow dye transfer. Preconditioning preserved intercellular coupling after prolonged ischemia. An initial reduction in coupling in response to the preconditioning stimulus was also observed. This may protect neighboring cells from damaging substances produced during subsequent regional ischemia in vivo, and may preserve gap junctional communication required for enhanced functional recovery during subsequent reperfusion.

Propofol inhibits gap junctions by attenuating sevoflurane-induced cytotoxicity against rat liver cells in vitro.

PubMed

Huang, Fei; Li, Shangrong; Gan, Xiaoliang; Wang, Ren; Chen, Zhonggang

2014-04-01

Liver abnormalities are seen in a small proportion of patients following anaesthesia with sevoflurane. To investigate whether the cytotoxicity of sevoflurane against rat liver cells was mediated by gap junction intercellular communications, and the effect of propofol on sevoflurane-induced cytotoxicity. Experimental study. The study was carried out in the central laboratory of The Third Affiliated Hospital, Sun Yat-sen University. BRL-3A rat liver cells. Immortal rat liver cells BRL-3A were grown at low and high density. Colony-forming assays were performed to determine clonogenic growth of these cells. To investigate the effect of oleamide and propofol on gap junction function, we measured fluorescence transmission between cells using parachute dye-coupling assays. Immunoblotting assays were performed to determine connexin32 and connexin43 expression. Our colony formation assays revealed that, in low-density culture, sevoflurane caused no apparent inhibition of clonogenic growth of BRL-3A cells. In high-density culture, 2.2 to 4.4% sevoflurane markedly inhibited clonogenic growth of BRL-3A cells with 67.6 (0.34)% and 61.2 (0.17)% of the cells being viable, respectively (P = 0.003 vs. low-density culture), suggesting cell density dependency of sevoflurane-induced cytotoxicity. Our colony formation assays revealed that propofol markedly attenuated the suppression by sevoflurane of the clonogenic growth of BRL-3A cells (viability: propofol and sevoflurane, 91.5 (0.014)% vs. sevoflurane, 56.6 (0.019)%; P <0.01). Blocking gap junctions with 10 μmol l oleamide significantly attenuated 4.4% sevoflurane-induced suppression with a viability of 83.6 ± 0.138% (oleamide and sevoflurane vs. sevoflurane, P < 0.01). Immunoblotting assays further showed that propofol (3.2 μg ml) markedly reduced CX32 levels and significantly inhibited gap junctional intercellular communications as revealed by parachute dye-coupling assays. Values are mean (SD). This study provides the first direct evidence that sevoflurane-induced cytotoxicity, which is mediated through gap junctions, is attenuated by propofol, possibly by its action on Cx32 homomeric or heteromeric complexes.

Intercellular Diffusion of a Fluorescent Sucrose Analog via the Septal Junctions in a Filamentous Cyanobacterium

PubMed Central

Nürnberg, Dennis J.; Mariscal, Vicente; Bornikoel, Jan; Nieves-Morión, Mercedes; Krauß, Norbert; Herrero, Antonia

2015-01-01

ABSTRACT Many filamentous cyanobacteria produce specialized nitrogen-fixing cells called heterocysts, which are located at semiregular intervals along the filament with about 10 to 20 photosynthetic vegetative cells in between. Nitrogen fixation in these complex multicellular bacteria depends on metabolite exchange between the two cell types, with the heterocysts supplying combined-nitrogen compounds but dependent on the vegetative cells for photosynthetically produced carbon compounds. Here, we used a fluorescent tracer to probe intercellular metabolite exchange in the filamentous heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. We show that esculin, a fluorescent sucrose analog, is incorporated by a sucrose import system into the cytoplasm of Anabaena cells. The cytoplasmic esculin is rapidly and reversibly exchanged across vegetative-vegetative and vegetative-heterocyst cell junctions. Our measurements reveal the kinetics of esculin exchange and also show that intercellular metabolic communication is lost in a significant fraction of older heterocysts. SepJ, FraC, and FraD are proteins located at the intercellular septa and are suggested to form structures analogous to gap junctions. We show that a ΔsepJ ΔfraC ΔfraD triple mutant shows an altered septum structure with thinner septa but a denser peptidoglycan layer. Intercellular diffusion of esculin and fluorescein derivatives is impaired in this mutant, which also shows a greatly reduced frequency of nanopores in the intercellular septal cross walls. These findings suggest that FraC, FraD, and SepJ are important for the formation of junctional structures that constitute the major pathway for feeding heterocysts with sucrose. PMID:25784700

Subsets of ATP-sensitive potassium channel (KATP) inhibitors increase gap junctional intercellular communication in metastatic cancer cell lines independent of SUR expression

USDA-ARS?s Scientific Manuscript database

Gap junctional intercellular communication (GJIC) is a process whereby cells share molecules and nutrients with each other by physical contact through cell membrane pores. In tumor cells, GJIC is often altered, suggesting that this process may be important in the context of cancer. Certain ion chan...

Analysis of gap junctional intercellular communications using a dielectrophoresis-based microchip.

PubMed

Tellez-Gabriel, Marta; Charrier, Céline; Brounais-Le Royer, Bénédicte; Mullard, Mathilde; Brown, Hannah K; Verrecchia, Franck; Heymann, Dominique

2017-03-01

Gap junctions are transmembrane structures that directly connect the cytoplasm of adjacent cells, making intercellular communications possible. It has been shown that the behaviour of several tumours - such as bone tumours - is related to gap junction intercellular communications (GJIC). Several methodologies are available for studying GJIC, based on measuring different parameters that are useful for multiple applications, such as the study of carcinogenesis for example. These methods nevertheless have several limitations. The present manuscript describes the setting up of a dielectrophoresis (DEP)-based lab-on-a-chip platform for the real-time study of Gap Junctional Intercellular Communication between osteosarcoma cells and the main cells accessible to their microenvironment. We conclude that using the DEParray technology for the GJIC assessment has several advantages comparing to current techniques. This methodology is less harmful for cells integrity; cells can be recovered after interaction to make further molecular analysis; it is possible to study GJIC in real time; we can promote cell interactions using up to five different populations. The setting up of this new methodology overcomes several difficulties to perform experiments for solving questions about GJIC process that we are not able to do with current technics. Copyright © 2017 Elsevier GmbH. All rights reserved.

Smad ubiquitination regulatory factor-2 controls gap junction intercellular communication by modulating endocytosis and degradation of connexin43.

PubMed

Fykerud, Tone Aase; Kjenseth, Ane; Schink, Kay Oliver; Sirnes, Solveig; Bruun, Jarle; Omori, Yasufumi; Brech, Andreas; Rivedal, Edgar; Leithe, Edward

2012-09-01

Gap junctions consist of arrays of intercellular channels that enable adjacent cells to communicate both electrically and metabolically. Gap junction channels are made of a family of integral membrane proteins called connexins, of which the best-studied member is connexin43. Gap junctions are dynamic plasma membrane domains, and connexin43 has a high turnover rate in most tissue types. However, the mechanisms involved in the regulation of connexin43 endocytosis and transport to lysosomes are still poorly understood. Here, we demonstrate by live-cell imaging analysis that treatment of cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) induces endocytosis of subdomains of connexin43 gap junctions. The internalized, connexin43-enriched vesicles were found to fuse with early endosomes, which was followed by transport of connexin43 to the lumen of early endosomes. The HECT E3 ubiquitin ligase smad ubiquitination regulatory factor-2 (Smurf2) was found to be recruited to connexin43 gap junctions in response to TPA treatment. Depletion of Smurf2 by small interfering RNA resulted in enhanced levels of connexin43 gap junctions between adjacent cells and increased gap junction intercellular communication. Smurf2 depletion also counteracted the TPA-induced endocytosis and degradation of connexin43. Collectively, these data identify Smurf2 as a novel regulator of connexin43 gap junctions.

HIV-1 gp120 Glycoprotein Interacting with Dendritic Cell-specific Intercellular Adhesion Molecule 3-grabbing Non-integrin (DC-SIGN) Down-Regulates Tight Junction Proteins to Disrupt the Blood Retinal Barrier and Increase Its Permeability.

PubMed

Qian, Yi-Wen; Li, Chuan; Jiang, Ai-Ping; Ge, Shengfang; Gu, Ping; Fan, Xianqun; Li, Tai-Sheng; Jin, Xia; Wang, Jian-Hua; Wang, Zhi-Liang

2016-10-28

Approximately 70% of HIV-1 infected patients acquire ocular opportunistic infections and manifest eye disorders during the course of their illness. The mechanisms by which pathogens invade the ocular site, however, are unclear. Under normal circumstances, vascular endothelium and retinal pigment epithelium (RPE), which possess a well developed tight junction complex, form the blood-retinal barrier (BRB) to prevent pathogen invasion. We hypothesize that disruption of the BRB allows pathogen entry into ocular sites. The hypothesis was tested using in vitro models. We discovered that human RPE cells could bind to either HIV-1 gp120 glycoproteins or HIV-1 viral particles. Furthermore, the binding was mediated by dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) expressed on RPE cells. Upon gp120 binding to DC-SIGN, cellular NF-κB signaling was triggered, leading to the induction of matrix metalloproteinases, which subsequently degraded tight junction proteins and disrupted the BRB integrity. DC-SIGN knockdown or prior blocking with a specific antibody abolished gp120-induced matrix metalloproteinase expression and reduced the degradation of tight junction proteins. This study elucidates a novel mechanism by which HIV, type 1 invades ocular tissues and provides additional insights into the translocation or invasion process of ocular complication-associated pathogens. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

TC-PTP directly interacts with connexin43 to regulate gap junction intercellular communication

PubMed Central

Li, Hanjun; Spagnol, Gaelle; Naslavsky, Naava; Caplan, Steve; Sorgen, Paul L.

2014-01-01

ABSTRACT Protein kinases have long been reported to regulate connexins; however, little is known about the involvement of phosphatases in the modulation of intercellular communication through gap junctions and the subsequent downstream effects on cellular processes. Here, we identify an interaction between the T-cell protein tyrosine phosphatase (TC-PTP, officially known as PTPN2) and the carboxyl terminus of connexin43 (Cx43, officially known as GJA1). Two cell lines, normal rat kidney (NRK) cells endogenously expressing Cx43 and an NRK-derived cell line expressing v-Src with temperature-sensitive activity, were used to demonstrate that EGF and v-Src stimulation, respectively, induced TC-PTP to colocalize with Cx43 at the plasma membrane. Cell biology experiments using phospho-specific antibodies and biophysical assays demonstrated that the interaction is direct and that TC-PTP dephosphorylates Cx43 residues Y247 and Y265, but does not affect v-Src. Transfection of TC-PTP also indirectly led to the dephosphorylation of Cx43 S368, by inactivating PKCα and PKCδ, with no effect on the phosphorylation of S279 and S282 (MAPK-dependent phosphorylation sites). Dephosphorylation maintained Cx43 gap junctions at the plaque and partially reversed the channel closure caused by v-Src-mediated phosphorylation of Cx43. Understanding dephosphorylation, along with the well-documented roles of Cx43 phosphorylation, might eventually lead to methods to modulate the regulation of gap junction channels, with potential benefits for human health. PMID:24849651

Neisseria meningitidis colonization of the brain endothelium and cerebrospinal fluid invasion.

PubMed

Miller, Florence; Lécuyer, Hervé; Join-Lambert, Olivier; Bourdoulous, Sandrine; Marullo, Stefano; Nassif, Xavier; Coureuil, Mathieu

2013-04-01

The brain and meningeal spaces are protected from bacterial invasion by the blood-brain barrier, formed by specialized endothelial cells and tight intercellular junctional complexes. However, once in the bloodstream, Neisseria meningitidis crosses this barrier in about 60% of the cases. This highlights the particular efficacy with which N. meningitidis targets the brain vascular cell wall. The first step of central nervous system invasion is the direct interaction between bacteria and endothelial cells. This step is mediated by the type IV pili, which induce a remodelling of the endothelial monolayer, leading to the opening of the intercellular space. In this review, strategies used by the bacteria to survive in the bloodstream, to colonize the brain vasculature and to cross the blood-brain barrier will be discussed. © 2012 Blackwell Publishing Ltd.

Sulforaphane counteracts aggressiveness of pancreatic cancer driven by dysregulated Cx43-mediated gap junctional intercellular communication

PubMed Central

Zhang, Yiyao; Isayev, Orkhan; Heilmann, Katharina; Schoensiegel, Frank; Liu, Li; Nessling, Michelle; Richter, Karsten; Labsch, Sabrina; Nwaeburu, Clifford C.; Mattern, Juergen; Gladkich, Jury; Giese, Nathalia; Werner, Jens; Schemmer, Peter; Gross, Wolfgang; Gebhard, Martha M.; Gerhauser, Clarissa; Schaefer, Michael; Herr, Ingrid

2014-01-01

The extreme aggressiveness of pancreatic ductal adenocarcinoma (PDA) has been associated with blocked gap junctional intercellular communication (GJIC) and the presence of cancer stem cells (CSCs). We examined whether disturbed GJIC is responsible for a CSC phenotype in established and primary cancer cells and patient tissue of PDA using interdisciplinary methods based in physiology, cell and molecular biology, histology and epigenetics. Flux of fluorescent dyes and gemcitabine through gap junctions (GJs) was intact in less aggressive cells but not in highly malignant cells with morphological dysfunctional GJs. Among several connexins, only Cx43 was expressed on the cell surface of less aggressive and GJIC-competent cells, whereas Cx43 surface expression was absent in highly malignant, E-cadherin-negative and GJIC-incompetent cells. The levels of total Cx43 protein and Cx43 phosphorylated at Ser368 and Ser279/282 were high in normal tissue but low to absent in malignant tissue. si-RNA-mediated inhibition of Cx43 expression in GJIC-competent cells prevented GJIC and induced colony formation and the expression of stem cell-related factors. The bioactive substance sulforaphane enhanced Cx43 and E-cadherin levels, inhibited the CSC markers c-Met and CD133, improved the functional morphology of GJs and enhanced GJIC. Sulforaphane altered the phosphorylation of several kinases and their substrates and inhibition of GSK3, JNK and PKC prevented sulforaphane-induced CX43 expression. The sulforaphane-mediated expression of Cx43 was not correlated with enhanced Cx43 RNA expression, acetylated histone binding and Cx43 promoter de-methylation, suggesting that posttranslational phosphorylation is the dominant regulatory mechanism. Together, the absence of Cx43 prevents GJIC and enhances aggressiveness, whereas sulforaphane counteracts this process, and our findings highlight dietary co-treatment as a viable treatment option for PDA. PMID:24742583

Sulforaphane counteracts aggressiveness of pancreatic cancer driven by dysregulated Cx43-mediated gap junctional intercellular communication.

PubMed

Forster, Tobias; Rausch, Vanessa; Zhang, Yiyao; Isayev, Orkhan; Heilmann, Katharina; Schoensiegel, Frank; Liu, Li; Nessling, Michelle; Richter, Karsten; Labsch, Sabrina; Nwaeburu, Clifford C; Mattern, Juergen; Gladkich, Jury; Giese, Nathalia; Werner, Jens; Schemmer, Peter; Gross, Wolfgang; Gebhard, Martha M; Gerhauser, Clarissa; Schaefer, Michael; Herr, Ingrid

2014-03-30

The extreme aggressiveness of pancreatic ductal adenocarcinoma (PDA) has been associated with blocked gap junctional intercellular communication (GJIC) and the presence of cancer stem cells (CSCs). We examined whether disturbed GJIC is responsible for a CSC phenotype in established and primary cancer cells and patient tissue of PDA using interdisciplinary methods based in physiology, cell and molecular biology, histology and epigenetics. Flux of fluorescent dyes and gemcitabine through gap junctions (GJs) was intact in less aggressive cells but not in highly malignant cells with morphological dysfunctional GJs. Among several connexins, only Cx43 was expressed on the cell surface of less aggressive and GJIC-competent cells, whereas Cx43 surface expression was absent in highly malignant, E-cadherin-negative and GJIC-incompetent cells. The levels of total Cx43 protein and Cx43 phosphorylated at Ser368 and Ser279/282 were high in normal tissue but low to absent in malignant tissue. si-RNA-mediated inhibition of Cx43 expression in GJIC-competent cells prevented GJIC and induced colony formation and the expression of stem cell-related factors. The bioactive substance sulforaphane enhanced Cx43 and E-cadherin levels, inhibited the CSC markers c-Met and CD133, improved the functional morphology of GJs and enhanced GJIC. Sulforaphane altered the phosphorylation of several kinases and their substrates and inhibition of GSK3, JNK and PKC prevented sulforaphane-induced CX43 expression. The sulforaphane-mediated expression of Cx43 was not correlated with enhanced Cx43 RNA expression, acetylated histone binding and Cx43 promoter de-methylation, suggesting that posttranslational phosphorylation is the dominant regulatory mechanism. Together, the absence of Cx43 prevents GJIC and enhances aggressiveness, whereas sulforaphane counteracts this process, and our findings highlight dietary co-treatment as a viable treatment option for PDA.

Tight junctions and human diseases.

PubMed

Sawada, Norimasa; Murata, Masaki; Kikuchi, Keisuke; Osanai, Makoto; Tobioka, Hirotoshi; Kojima, Takashi; Chiba, Hideki

2003-09-01

Tight junctions are intercellular junctions adjacent to the apical end of the lateral membrane surface. They have two functions, the barrier (or gate) function and the fence function. The barrier function of tight junctions regulates the passage of ions, water, and various macromolecules, even of cancer cells, through paracellular spaces. The barrier function is thus relevant to edema, jaundice, diarrhea, and blood-borne metastasis. On the other hand, the fence function maintains cell polarity. In other words, tight junctions work as a fence to prevent intermixing of molecules in the apical membrane with those in the lateral membrane. This function is deeply involved in cancer cell biology, in terms of loss of cell polarity. Of the proteins comprising tight junctions, integral membrane proteins occludin, claudins, and JAMs have been recently discovered. Of these molecules, claudins are exclusively responsible for the formation of tight-junction strands and are connected with the actin cytoskeleton mediated by ZO-1. Thus, both functions of tight junctions are dependent on the integrity of the actin cytoskeleton as well as ATP. Mutations in the claudin14 and the claudin16 genes result in hereditary deafness and hereditary hypomagnesemia, respectively. Some pathogenic bacteria and viruses target and affect the tight-junction function, leading to diseases. In this review, the relationship between tight junctions and human diseases is summarized.

Effects of topical steroids on tight junction proteins and spongiosis in esophageal epithelia of patients with eosinophilic esophagitis.

PubMed

Katzka, David A; Tadi, Ravikanth; Smyrk, Thomas C; Katarya, Eesha; Sharma, Anamay; Geno, Deborah M; Camilleri, Michael; Iyer, Prasad G; Alexander, Jeffrey A; Buttar, Navtej S

2014-11-01

The allergic response associated with eosinophilic esophagitis (EoE) occurs when food antigens permeate tight junction-mediated epithelial dilated intercellular spaces. We assessed whether levels of tight junction proteins correlate with the dilation of intercellular spaces (spongiosis) and the effects of topical steroids on these parameters. We assessed esophageal biopsy samples from 10 patients with active EoE treated with topical fluticasone, 10 untreated patients, and 10 patients without esophageal disease (controls) for degree of spongiosis. Immunohistochemical assays were used to determine the levels of the tight junction proteins filaggrin, zonula occludens (ZO)-1, ZO-2, ZO-3, and claudin-1. Histology and immunohistochemistry results were assessed blindly, with levels of tight junction proteins and degree of spongiosis rated on scales of 0 to 3. The mean degrees of spongiosis in untreated and treated patients with EoE were 1.3 and 0.4, respectively (P = .016). Esophageal epithelia did not stain significantly for ZO-1 or ZO-2. Filaggrin was observed in a predominant cytoplasmic pattern, compared with the cytoplasmic and membranous patterns of ZO-3 and claudin-1. In biopsy specimens from patients with active EoE, the mean staining intensities for filaggrin, ZO-3, and claudin-1 were 1.6, 1.4, and 0.7, respectively. In biopsy specimens from patients treated with fluticasone, levels of filaggrin, ZO-3, and claudin-1 were 2.8 (P = .002 compared with untreated patients), 1.7 (P = .46 compared with untreated patients), and 1.3 (P = .25 compared with untreated patients), respectively. The correlation between the level of filaggrin and the degree of spongiosis was r = 0.23, and between ZO-3 staining and the degree of spongiosis was r = .016 (P = .001 for filaggrin vs ZO-3 staining). Filaggrin, ZO-3, and claudin-1 (but not ZO-1 or ZO-2) are detected in the esophageal mucosa of patients with EoE treated with steroids and individuals without esophageal disease. Without treatment, spongiosis increases, corresponding with reduced levels of filaggrin, ZO-3, and claudin-1. Loss of tight junction regulators and dilation of intercellular spaces appear to be involved in the pathophysiology of EoE and could be targets for treatment. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

Cell contact as an independent factor modulating cardiac myocyte hypertrophy and survival in long-term primary culture

NASA Technical Reports Server (NTRS)

Clark, W. A.; Decker, M. L.; Behnke-Barclay, M.; Janes, D. M.; Decker, R. S.

1998-01-01

Cardiac myocytes maintained in cell culture develop hypertrophy both in response to mechanical loading as well as to receptor-mediated signaling mechanisms. However, it has been shown that the hypertrophic response to these stimuli may be modulated through effects of intercellular contact achieved by maintaining cells at different plating densities. In this study, we show that the myocyte plating density affects not only the hypertrophic response and features of the differentiated phenotype of isolated adult myocytes, but also plays a significant role influencing myocyte survival in vitro. The native rod-shaped phenotype of freshly isolated adult myocytes persists in an environment which minimizes myocyte attachment and spreading on the substratum. However, these conditions are not optimal for long-term maintenance of cultured adult cardiac myocytes. Conditions which promote myocyte attachment and spreading on the substratum, on the other hand, also promote the re-establishment of new intercellular contacts between myocytes. These contacts appear to play a significant role in the development of spontaneous activity, which enhances the redevelopment of highly differentiated contractile, junctional, and sarcoplasmic reticulum structures in the cultured adult cardiomyocyte. Although it has previously been shown that adult cardiac myocytes are typically quiescent in culture, the addition of beta-adrenergic agonists stimulates beating and myocyte hypertrophy, and thereby serves to increase the level of intercellular contact as well. However, in densely-plated cultures with intrinsically high levels of intercellular contact, spontaneous contractile activity develops without the addition of beta-adrenergic agonists. In this study, we compare the function, morphology, and natural history of adult feline cardiomyocytes which have been maintained in cultures with different levels of intercellular contact, with and without the addition of beta-adrenergic agonists. Intercellular contact, communication, and transmission of contractile forces between myocytes appears to play a primary role in remodeling the 2-dimensional cell layer into a parallel alignment of elongated myocytes with highly developed intercalated disk-like junctions. This highly differentiated state is very stable, and cultures which achieve this state exhibit significantly greater longevity than more sparsely plated myocytes. These myocytes typically continue beating, and survive from 6 to more than 12 weeks in culture. When this level of contact and differentiation are not achieved, even among beta-adrenergic stimulated myocytes, contractile activity is not sustained, myofibrils atrophy, there is little or no development of junctional complexes, and the period of myocyte viability is typically no more than 5 weeks in vitro.

Destruction of the hepatocyte junction by intercellular invasion of Leptospira causes jaundice in a hamster model of Weil's disease

PubMed Central

Miyahara, Satoshi; Saito, Mitsumasa; Kanemaru, Takaaki; Villanueva, Sharon Y A M; Gloriani, Nina G; Yoshida, Shin-ichi

2014-01-01

Weil's disease, the most severe form of leptospirosis, is characterized by jaundice, haemorrhage and renal failure. The mechanisms of jaundice caused by pathogenic Leptospira remain unclear. We therefore aimed to elucidate the mechanisms by integrating histopathological changes with serum biochemical abnormalities during the development of jaundice in a hamster model of Weil's disease. In this work, we obtained three-dimensional images of infected hamster livers using scanning electron microscope together with freeze-cracking and cross-cutting methods for sample preparation. The images displayed the corkscrew-shaped bacteria, which infiltrated the Disse's space, migrated between hepatocytes, detached the intercellular junctions and disrupted the bile canaliculi. Destruction of bile canaliculi coincided with the elevation of conjugated bilirubin, aspartate transaminase and alkaline phosphatase levels in serum, whereas serum alanine transaminase and γ-glutamyl transpeptidase levels increased slightly, but not significantly. We also found in ex vivo experiments that pathogenic, but not non-pathogenic leptospires, tend to adhere to the perijunctional region of hepatocyte couplets isolated from hamsters and initiate invasion of the intercellular junction within 1 h after co-incubation. Our results suggest that pathogenic leptospires invade the intercellular junctions of host hepatocytes, and this invasion contributes in the disruption of the junction. Subsequently, bile leaks from bile canaliculi and jaundice occurs immediately. Our findings revealed not only a novel pathogenicity of leptospires, but also a novel mechanism of jaundice induced by bacterial infection. PMID:24945433

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy.

PubMed

Lemcke, Heiko; Voronina, Natalia; Steinhoff, Gustav; David, Robert

2017-06-19

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Treponema pallidum Invades Intercellular Junctions of Endothelial Cell Monolayers

NASA Astrophysics Data System (ADS)

Thomas, D. Denee; Navab, Mahamad; Haake, David A.; Fogelman, Alan M.; Miller, James N.; Lovett, Michael A.

1988-05-01

The pathogenesis of syphilis reflects invasive properties of Treponema pallidum, but the actual mode of tissue invasion is unknown. We have found two in vitro parallels of treponemal invasiveness. We tested whether motile T. pallidum could invade host cells by determining the fate of radiolabeled motile organisms added to a HeLa cell monolayer; 26% of treponemes associated with the monolayer in a trypsin-resistant niche, presumably between the monolayer and the surface to which it adhered, but did not attain intracellularity. Attachment of T. pallidum to cultured human and rabbit aortic and human umbilical vein endothelial cells was 2-fold greater than to HeLa cells. We added T. pallidum to aortic endothelial cells grown on membrane filters under conditions in which tight intercellular junctions had formed. T. pallidum was able to pass through the endothelial cell monolayers without altering tight junctions, as measured by electrical resistance. In contrast, heat-killed T. pallidum and the nonpathogen Treponema phagedenis biotype Reiter failed to penetrate the monolayer. Transmission electron micrographs of sections of the monolayer showed T. pallidum in intercellular junctions. Our in vitro observations suggest that these highly motile spirochetes may leave the circulation by invading the junctions between endothelial cells.

The Peptidoglycan-Binding Protein SjcF1 Influences Septal Junction Function and Channel Formation in the Filamentous Cyanobacterium Anabaena.

PubMed

Rudolf, Mareike; Tetik, Nalan; Ramos-León, Félix; Flinner, Nadine; Ngo, Giang; Stevanovic, Mara; Burnat, Mireia; Pernil, Rafael; Flores, Enrique; Schleiff, Enrico

2015-06-30

Filamentous, heterocyst-forming cyanobacteria exchange nutrients and regulators between cells for diazotrophic growth. Two alternative modes of exchange have been discussed involving transport either through the periplasm or through septal junctions linking adjacent cells. Septal junctions and channels in the septal peptidoglycan are likely filled with septal junction complexes. While possible proteinaceous factors involved in septal junction formation, SepJ (FraG), FraC, and FraD, have been identified, little is known about peptidoglycan channel formation and septal junction complex anchoring to the peptidoglycan. We describe a factor, SjcF1, involved in regulation of septal junction channel formation in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. SjcF1 interacts with the peptidoglycan layer through two peptidoglycan-binding domains and is localized throughout the cell periphery but at higher levels in the intercellular septa. A strain with an insertion in sjcF1 was not affected in peptidoglycan synthesis but showed an altered morphology of the septal peptidoglycan channels, which were significantly wider in the mutant than in the wild type. The mutant was impaired in intercellular exchange of a fluorescent probe to a similar extent as a sepJ deletion mutant. SjcF1 additionally bears an SH3 domain for protein-protein interactions. SH3 binding domains were identified in SepJ and FraC, and evidence for interaction of SjcF1 with both SepJ and FraC was obtained. SjcF1 represents a novel protein involved in structuring the peptidoglycan layer, which links peptidoglycan channel formation to septal junction complex function in multicellular cyanobacteria. Nonetheless, based on its subcellular distribution, this might not be the only function of SjcF1. Cell-cell communication is central not only for eukaryotic but also for multicellular prokaryotic systems. Principles of intercellular communication are well established for eukaryotes, but the mechanisms and components involved in bacteria are just emerging. Filamentous heterocyst-forming cyanobacteria behave as multicellular organisms and represent an excellent model to study prokaryotic cell-cell communication. A path for intercellular metabolite exchange appears to involve transfer through molecular structures termed septal junctions. They are reminiscent of metazoan gap junctions that directly link adjacent cells. In cyanobacteria, such structures need to traverse the peptidoglycan layers in the intercellular septa of the filament. Here we describe a factor involved in the formation of channels across the septal peptidoglycan layers, thus contributing to the multicellular behavior of these organisms. Copyright © 2015 Rudolf et al.

Intercellular diffusion of a fluorescent sucrose analog via the septal junctions in a filamentous cyanobacterium.

PubMed

Nürnberg, Dennis J; Mariscal, Vicente; Bornikoel, Jan; Nieves-Morión, Mercedes; Krauß, Norbert; Herrero, Antonia; Maldener, Iris; Flores, Enrique; Mullineaux, Conrad W

2015-03-17

Many filamentous cyanobacteria produce specialized nitrogen-fixing cells called heterocysts, which are located at semiregular intervals along the filament with about 10 to 20 photosynthetic vegetative cells in between. Nitrogen fixation in these complex multicellular bacteria depends on metabolite exchange between the two cell types, with the heterocysts supplying combined-nitrogen compounds but dependent on the vegetative cells for photosynthetically produced carbon compounds. Here, we used a fluorescent tracer to probe intercellular metabolite exchange in the filamentous heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. We show that esculin, a fluorescent sucrose analog, is incorporated by a sucrose import system into the cytoplasm of Anabaena cells. The cytoplasmic esculin is rapidly and reversibly exchanged across vegetative-vegetative and vegetative-heterocyst cell junctions. Our measurements reveal the kinetics of esculin exchange and also show that intercellular metabolic communication is lost in a significant fraction of older heterocysts. SepJ, FraC, and FraD are proteins located at the intercellular septa and are suggested to form structures analogous to gap junctions. We show that a ΔsepJ ΔfraC ΔfraD triple mutant shows an altered septum structure with thinner septa but a denser peptidoglycan layer. Intercellular diffusion of esculin and fluorescein derivatives is impaired in this mutant, which also shows a greatly reduced frequency of nanopores in the intercellular septal cross walls. These findings suggest that FraC, FraD, and SepJ are important for the formation of junctional structures that constitute the major pathway for feeding heterocysts with sucrose. Anabaena and its relatives are filamentous cyanobacteria that exhibit a sophisticated form of prokaryotic multicellularity, with the formation of differentiated cell types, including normal photosynthetic cells and specialized nitrogen-fixing cells called heterocysts. The question of how heterocysts communicate and exchange metabolites with other cells in the filament is key to understanding this form of bacterial multicellularity. Here we provide the first information on the intercellular exchange of a physiologically important molecule, sucrose. We show that a fluorescent sucrose analog can be imported into the Anabaena cytoplasm by a sucrose import system. Once in the cytoplasm, it is rapidly and reversibly exchanged among all of the cells in the filament by diffusion across the septal junctions. Photosynthetically produced sucrose likely follows the same route from cytoplasm to cytoplasm. We identify some of the septal proteins involved in sucrose exchange, and our results indicate that these proteins form structures functionally analogous to metazoan gap junctions. Copyright © 2015 Nürnberg et al.

Molecular cell biology and physiology of solute transport

PubMed Central

Caplan, Michael J.; Seo-Mayer, Patricia; Zhang, Li

2010-01-01

Purpose of review An enormous body of research has been focused on exploring the mechanisms through which epithelial cells establish their characteristic polarity. It is clear that under normal circumstances cell–cell contacts mediated by the calcium-dependent adhesion proteins of the intercellular adhesion junctions are required to initiate complete polarization. Furthermore, formation of the tight, or occluding, junctions that limit paracellular permeability has long been thought to help to establish polarity by preventing the diffusion of membrane proteins between the two plasmalemmal domains. This review will discuss several selected kinases and protein complexes and highlight their relevance to transporting epithelial cell polarization. Recent findings Recent work has shed new light on the roles of junctional complexes in establishing and maintaining epithelial cell polarity. In addition, work from several laboratories, suggests that the formation of these junctions is tied to processes that regulate cellular energy metabolism. Summary Junctional complexes and energy sensing kinases constitute a novel class of machinery whose capacity to generate and modulate epithelial cell polarity is likely to have wide ranging and important physiological ramifications. PMID:18695392

Group A Streptococcus tissue invasion by CD44-mediated cell signalling

NASA Astrophysics Data System (ADS)

Cywes, Colette; Wessels, Michael R.

2001-12-01

Streptococcus pyogenes (also known as group A Streptococcus, GAS), the agent of streptococcal sore throat and invasive soft-tissue infections, attaches to human pharyngeal or skin epithelial cells through specific recognition of its hyaluronic acid capsular polysaccharide by the hyaluronic-acid-binding protein CD44 (refs 1, 2). Because ligation of CD44 by hyaluronic acid can induce epithelial cell movement on extracellular matrix, we investigated whether molecular mimicry by the GAS hyaluronic acid capsule might induce similar cellular responses. Here we show that CD44-dependent GAS binding to polarized monolayers of human keratinocytes induced marked cytoskeletal rearrangements manifested by membrane ruffling and disruption of intercellular junctions. Transduction of the signal induced by GAS binding to CD44 on the keratinocyte surface involved Rac1 and the cytoskeleton linker protein ezrin, as well as tyrosine phosphorylation of cellular proteins. Studies of bacterial translocation in two models of human skin indicated that cell signalling triggered by interaction of the GAS capsule with CD44 opened intercellular junctions and promoted tissue penetration by GAS through a paracellular route. These results support a model of host cytoskeleton manipulation and tissue invasion by an extracellular bacterial pathogen.

Articular chondrocyte network mediated by gap junctions: role in metabolic cartilage homeostasis.

PubMed

Mayan, Maria D; Gago-Fuentes, Raquel; Carpintero-Fernandez, Paula; Fernandez-Puente, Patricia; Filgueira-Fernandez, Purificacion; Goyanes, Noa; Valiunas, Virginijus; Brink, Peter R; Goldberg, Gary S; Blanco, Francisco J

2015-01-01

This study investigated whether chondrocytes within the cartilage matrix have the capacity to communicate through intercellular connections mediated by voltage-gated gap junction (GJ) channels. Frozen cartilage samples were used for immunofluorescence and immunohistochemistry assays. Samples were embedded in cacodylate buffer before dehydration for scanning electron microscopy. Co-immunoprecipitation experiments and mass spectrometry (MS) were performed to identify proteins that interact with the C-terminal end of Cx43. GJ communication was studied through in situ electroporation, electrophysiology and dye injection experiments. A transwell layered culture system and MS were used to identify and quantify transferred amino acids. Microscopic images revealed the presence of multiple cellular projections connecting chondrocytes within the matrix. These projections were between 5 and 150 µm in length. MS data analysis indicated that the C-terminus of Cx43 interacts with several cytoskeletal proteins implicated in Cx trafficking and GJ assembly, including α-tubulin and β-tubulin, actin, and vinculin. Electrophysiology experiments demonstrated that 12-mer oligonucleotides could be transferred between chondrocytes within 12 min after injection. Glucose was homogeneously distributed within 22 and 35 min. No transfer was detected when glucose was electroporated into A549 cells, which have no GJs. Transwell layered culture systems coupled with MS analysis revealed connexins can mediate the transfer of L-lysine and L-arginine between chondrocytes. This study reveals that intercellular connections between chondrocytes contain GJs that play a key role in cell-cell communication and a metabolic function by exchange of nutrients including glucose and essential amino acids. A three-dimensional cellular network mediated through GJs might mediate metabolic and physiological homeostasis to maintain cartilage tissue. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Destruction of the hepatocyte junction by intercellular invasion of Leptospira causes jaundice in a hamster model of Weil's disease.

PubMed

Miyahara, Satoshi; Saito, Mitsumasa; Kanemaru, Takaaki; Villanueva, Sharon Y A M; Gloriani, Nina G; Yoshida, Shin-ichi

2014-08-01

Weil's disease, the most severe form of leptospirosis, is characterized by jaundice, haemorrhage and renal failure. The mechanisms of jaundice caused by pathogenic Leptospira remain unclear. We therefore aimed to elucidate the mechanisms by integrating histopathological changes with serum biochemical abnormalities during the development of jaundice in a hamster model of Weil's disease. In this work, we obtained three-dimensional images of infected hamster livers using scanning electron microscope together with freeze-cracking and cross-cutting methods for sample preparation. The images displayed the corkscrew-shaped bacteria, which infiltrated the Disse's space, migrated between hepatocytes, detached the intercellular junctions and disrupted the bile canaliculi. Destruction of bile canaliculi coincided with the elevation of conjugated bilirubin, aspartate transaminase and alkaline phosphatase levels in serum, whereas serum alanine transaminase and γ-glutamyl transpeptidase levels increased slightly, but not significantly. We also found in ex vivo experiments that pathogenic, but not non-pathogenic leptospires, tend to adhere to the perijunctional region of hepatocyte couplets isolated from hamsters and initiate invasion of the intercellular junction within 1 h after co-incubation. Our results suggest that pathogenic leptospires invade the intercellular junctions of host hepatocytes, and this invasion contributes in the disruption of the junction. Subsequently, bile leaks from bile canaliculi and jaundice occurs immediately. Our findings revealed not only a novel pathogenicity of leptospires, but also a novel mechanism of jaundice induced by bacterial infection. © 2014 The Authors. International Journal of Experimental Pathology © 2014 International Journal of Experimental Pathology.

Inhibition of gap junction intercellular communication is involved in silica nanoparticles-induced H9c2 cardiomyocytes apoptosis via the mitochondrial pathway.

PubMed

Du, Zhong-Jun; Cui, Guan-Qun; Zhang, Juan; Liu, Xiao-Mei; Zhang, Zhi-Hu; Jia, Qiang; Ng, Jack C; Peng, Cheng; Bo, Cun-Xiang; Shao, Hua

2017-01-01

Gap junction intercellular communication (GJIC) between cardiomyocytes is essential for synchronous heart contraction and relies on connexin-containing channels. Connexin 43 (Cx43) is a major component involved in GJIC in heart tissue, and its abnormal expression is closely associated with various cardiac diseases. Silica nanoparticles (SNPs) are known to induce cardiovascular toxicity. However, the mechanisms through which GJIC plays a role in cardiomyocytes apoptosis induced by SNPs remain unknown. The aim of the present study is to determine whether SNPs-decreased GJIC promotes apoptosis in rat cardiomyocytes cell line (H9c2 cells) via the mitochondrial pathway using CCK-8 Kit, scrape-loading dye transfer technique, Annexin V/PI double-staining assays, and Western blot analysis. The results showed that SNPs elicited cytotoxicity in H9c2 cells in a time- and concentration-dependent manner. SNPs also reduced GJIC in H9c2 cells in a concentration-dependent manner through downregulation of Cx43 and upregulation of P-Cx43. Inhibition of gap junctions by gap junction blocker carbenoxolone disodium resulted in decreased survival and increased apoptosis, whereas enhancement of the gap junctions by retinoic acid led to enhanced survival but decreased apoptosis. Furthermore, SNPs-induced apoptosis through the disrupted functional gap junction was correlated with abnormal expressions of the proteins involved in the mitochondrial pathway-related apoptosis such as Bcl-2/Bax, cytochrome C, Caspase-9, and Caspase-3. Taken together, our results provide the first evidence that SNPs-decreased GJIC promotes apoptosis in cardiomyocytes via the mitochondrial pathway. In addition, downregulation of GJIC by SNPs in cardiomyocytes is mediated through downregulation of Cx43 and upregulation of P-Cx43. These results suggest that in rat cardiomyocytes cell line, GJIC plays a protective role in SNPs-induced apoptosis and that GJIC may be one of the targets for SNPs-induced biological effects.

Intercellular Calcium Waves in HeLa Cells Expressing GFP-labeled Connexin 43, 32, or 26

PubMed Central

Paemeleire, Koen; Martin, Patricia E. M.; Coleman, Sharon L.; Fogarty, Kevin E.; Carrington, Walter A.; Leybaert, Luc; Tuft, Richard A.; Evans, W. Howard; Sanderson, Michael J.

2000-01-01

This study was undertaken to obtain direct evidence for the involvement of gap junctions in the propagation of intercellular Ca2+ waves. Gap junction-deficient HeLa cells were transfected with plasmids encoding for green fluorescent protein (GFP) fused to the cytoplasmic carboxyl termini of connexin 43 (Cx43), 32 (Cx32), or 26 (Cx26). The subsequently expressed GFP-labeled gap junctions rendered the cells dye- and electrically coupled and were detected at the plasma membranes at points of contact between adjacent cells. To correlate the distribution of gap junctions with the changes in [Ca2+]i associated with Ca2+ waves and the distribution of the endoplasmic reticulum (ER), cells were loaded with fluorescent Ca2+-sensitive (fluo-3 and fura-2) and ER membrane (ER-Tracker) dyes. Digital high-speed microscopy was used to collect a series of image slices from which the three-dimensional distribution of the gap junctions and ER were reconstructed. Subsequently, intercellular Ca2+ waves were induced in these cells by mechanical stimulation with or without extracellular apyrase, an ATP-degrading enzyme. In untransfected HeLa cells and in the absence of apyrase, cell-to-cell propagating [Ca2+]i changes were characterized by initiating Ca2+ puffs associated with the perinuclear ER. By contrast, in Cx–GFP-transfected cells and in the presence of apyrase, [Ca2+]i changes were propagated without initiating perinuclear Ca2+ puffs and were communicated between cells at the sites of the Cx–GFP gap junctions. The efficiency of Cx expression determined the extent of Ca2+ wave propagation. These results demonstrate that intercellular Ca2+ waves may be propagated simultaneously via an extracellular pathway and an intracellular pathway through gap junctions and that one form of communication may mask the other. PMID:10793154

Structural basis for the selective permeability of channels made of communicating junction proteins

PubMed Central

Ek-Vitorin, Jose F.; Burt, Janis M.

2012-01-01

The open state(s) of gap junction channels is evident from their permeation by small ions in response to an applied intercellular (transjunctional/transchannel) voltage gradient. That an open channel allows variable amounts of current to transit from cell-to-cell in the face of a constant intercellular voltage difference indicates channel open/closing can be complete or partial. The physiological significance of such open state options is, arguably, the main concern of junctional regulation. Because gap junctions are permeable to many substances, it is sensible to inquire whether and how each open state influences the intercellular diffusion of molecules as valuable as, but less readily detected than current-carrying ions. Presumably, structural changes perceived as shifts in channel conductivity would significantly alter the transjunctional diffusion of molecules whose limiting diameter approximates the pore’s limiting diameter. Moreover, changes in junctional permeability to some molecules might occur without evident changes in conductivity, either at macroscopic or single channel level. Open gap junction channels allow the exchange of cytoplasmic permeants between contacting cells by simple diffusion. The identity of such permeants, and the functional circumstances and consequences of their junctional exchange presently constitute the most urgent (and demanding) themes of the field. Here, we consider the necessity for regulating this exchange, the possible mechanism(s) and structural elements likely involved in such regulation, and how regulatory phenomena could be perceived as changes in chemical vs. electrical coupling; an overall reflection on our collective knowledge of junctional communication is then applied to suggest new avenues of research. PMID:22342665

The Structure of the Human Vaginal Stratum Corneum and its Role in Immune Defense

PubMed Central

Anderson, Deborah J.; Marathe, Jai; Pudney, Jeffrey

2014-01-01

The superficial layers of the human vaginal epithelium, which form an interface between host and environment, are comprised of dead flattened cells that have undergone a terminal cell differentiation program called cornification. This entails extrusion of nuclei and intercellular organelles, and the depletion of functional DNA and RNA precluding the synthesis of new proteins. As a consequence, the terminally differentiated cells do not maintain robust intercellular junctions and have a diminished capacity to actively respond to microbial exposure, yet the vaginal stratum corneum (SC) mounts an effective defense against invasive microbial infections. The vaginal SC in reproductive aged women is comprised of loosely connected glycogen-filled cells which are permeable to bacterial and viral microbes as well as molecular and cellular mediators of immune defense. We propose here that the vaginal SC provides a unique microenvironment that maintains vaginal health by fostering endogenous lactobacillii and retaining critical mediators of acquired and innate immunity. A better understanding of the molecular and physicochemical properties of the vaginal SC could promote the design of more effective topical drugs and microbicides. PMID:24661416

Pseudorabies Virus US3-Induced Tunneling Nanotubes Contain Stabilized Microtubules, Interact with Neighboring Cells via Cadherins, and Allow Intercellular Molecular Communication

PubMed Central

Jansens, Robert J. J.; Van den Broeck, Wim; De Pelsmaeker, Steffi; Lamote, Jochen A. S.; Van Waesberghe, Cliff; Couck, Liesbeth

2017-01-01

ABSTRACT Tunneling nanotubes (TNTs) are long bridge-like structures that connect eukaryotic cells and mediate intercellular communication. We found earlier that the conserved alphaherpesvirus US3 protein kinase induces long cell projections that contact distant cells and promote intercellular virus spread. In this report, we show that the US3-induced cell projections constitute TNTs. In addition, we report that US3-induced TNTs mediate intercellular transport of information (e.g., green fluorescent protein [GFP]) in the absence of other viral proteins. US3-induced TNTs are remarkably stable compared to most TNTs described in the literature. In line with this, US3-induced TNTs were found to contain stabilized (acetylated and detyrosinated) microtubules. Transmission electron microscopy showed that virus particles are individually transported in membrane-bound vesicles in US3-induced TNTs and are released along the TNT and at the contact area between a TNT and the adjacent cell. Contact between US3-induced TNTs and acceptor cells is very stable, which correlated with a marked enrichment in adherens junction components beta-catenin and E-cadherin at the contact area. These data provide new structural insights into US3-induced TNTs and how they may contribute to intercellular communication and alphaherpesvirus spread. IMPORTANCE Tunneling nanotubes (TNT) represent an important and yet still poorly understood mode of long-distance intercellular communication. We and others reported earlier that the conserved alphaherpesvirus US3 protein kinase induces long cellular protrusions in infected and transfected cells. Here, we show that US3-induced cell projections constitute TNTs, based on structural properties and transport of biomolecules. In addition, we report on different particular characteristics of US3-induced TNTs that help to explain their remarkable stability compared to physiological TNTs. In addition, transmission electron microscopy assays indicate that, in infected cells, virions travel in the US3-induced TNTs in membranous transport vesicles and leave the TNT via exocytosis. These data generate new fundamental insights into the biology of (US3-induced) TNTs and into how they may contribute to intercellular virus spread and communication. PMID:28747498

Structural basis for the selective permeability of channels made of communicating junction proteins.

PubMed

Ek-Vitorin, Jose F; Burt, Janis M

2013-01-01

The open state(s) of gap junction channels is evident from their permeation by small ions in response to an applied intercellular (transjunctional/transchannel) voltage gradient. That an open channel allows variable amounts of current to transit from cell-to-cell in the face of a constant intercellular voltage difference indicates channel open/closing can be complete or partial. The physiological significance of such open state options is, arguably, the main concern of junctional regulation. Because gap junctions are permeable to many substances, it is sensible to inquire whether and how each open state influences the intercellular diffusion of molecules as valuable as, but less readily detected than current-carrying ions. Presumably, structural changes perceived as shifts in channel conductivity would significantly alter the transjunctional diffusion of molecules whose limiting diameter approximates the pore's limiting diameter. Moreover, changes in junctional permeability to some molecules might occur without evident changes in conductivity, either at macroscopic or single channel level. Open gap junction channels allow the exchange of cytoplasmic permeants between contacting cells by simple diffusion. The identity of such permeants, and the functional circumstances and consequences of their junctional exchange presently constitute the most urgent (and demanding) themes of the field. Here, we consider the necessity for regulating this exchange, the possible mechanism(s) and structural elements likely involved in such regulation, and how regulatory phenomena could be perceived as changes in chemical vs. electrical coupling; an overall reflection on our collective knowledge of junctional communication is then applied to suggest new avenues of research. This article is part of a Special Issue entitled: The Communicating junctions, roles and dysfunctions. Copyright © 2012 Elsevier B.V. All rights reserved.

Regulation of Endothelial Adherens Junctions by Tyrosine Phosphorylation

PubMed Central

Adam, Alejandro Pablo

2015-01-01

Endothelial cells form a semipermeable, regulated barrier that limits the passage of fluid, small molecules, and leukocytes between the bloodstream and the surrounding tissues. The adherens junction, a major mechanism of intercellular adhesion, is comprised of transmembrane cadherins forming homotypic interactions between adjacent cells and associated cytoplasmic catenins linking the cadherins to the cytoskeleton. Inflammatory conditions promote the disassembly of the adherens junction and a loss of intercellular adhesion, creating openings or gaps in the endothelium through which small molecules diffuse and leukocytes transmigrate. Tyrosine kinase signaling has emerged as a central regulator of the inflammatory response, partly through direct phosphorylation and dephosphorylation of the adherens junction components. This review discusses the findings that support and those that argue against a direct effect of cadherin and catenin phosphorylation in the disassembly of the adherens junction. Recent findings indicate a complex interaction between kinases, phosphatases, and the adherens junction components that allow a fine regulation of the endothelial permeability to small molecules, leukocyte migration, and barrier resealing. PMID:26556953

Gap Junction Regulation of Vascular Tone: Implications of Modulatory Intercellular Communication During Gestation

PubMed Central

Ampey, Bryan C.; Morschauser, Timothy J.; Lampe, Paul D.

2017-01-01

In the vasculature, gap junctions (GJ) play a multifaceted role by serving as direct conduits for cell–cell intercellular communication via the facilitated diffusion of signaling molecules. GJs are essential for the control of gene expression and coordinated vascular development in addition to vascular function. The coupling of endothelial cells to each other, as well as with vascular smooth muscle cells via GJs, plays a relevant role in the control of vasomotor tone, tissue perfusion and arterial blood pressure. The regulation of cell-signaling is paramount to cardiovascular adaptations of pregnancy. Pregnancy requires highly developed cell-to-cell coupling, which is affected partly through the formation of intercellular GJs by Cx43, a gap junction protein, within adjacent cell membranes to help facilitate the increase of uterine blood flow (UBF) in order to ensure adequate perfusion for nutrient and oxygen delivery to the placenta and thus the fetus. One mode of communication that plays a critical role in regulating Cx43 is the release of endothelial-derived vasodilators such as prostacyclin (PGI2) and nitric oxide (NO) and their respective signaling mechanisms involving second messengers (cAMP and cGMP, respectively) that are likely to be important in maintaining UBF. Therefore, the assertion we present in this review is that GJs play an integral if not a central role in maintaining UBF by controlling rises in vasodilators (PGI2 and NO) via cyclic nucleotides. In this review, we discuss: (1) GJ structure and regulation; (2) second messenger regulation of GJ phosphorylation and formation; (3) pregnancy-induced changes in cell-signaling; and (4) the role of uterine arterial endothelial GJs during gestation. These topics integrate the current knowledge of this scientific field with interpretations and hypotheses regarding the vascular effects that are mediated by GJs and their relationship with vasodilatory vascular adaptations required for modulating the dramatic physiological rises in uteroplacental perfusion and blood flow observed during normal pregnancy. PMID:25015806

Intercellular communication in sensory ganglia by purinergic receptors and gap junctions: implications for chronic pain.

PubMed

Hanani, Menachem

2012-12-03

Peripheral injury can cause abnormal activity in sensory neurons, which is a major factor in chronic pain. Recent work has shown that injury induces major changes not only in sensory neurons but also in the main type of glial cells in sensory ganglia-satellite glial cells (SGCs), and that interactions between sensory neurons and SGCs contribute to neuronal activity in pain models. The main functional changes observed in SGCs after injury are an increased gap junction-mediated coupling among these cells, and augmented sensitivity to ATP. There is evidence that the augmented gap junctions contribute to neuronal hyperexcitability in pain models, but the mechanism underlying this effect is not known. The changes in SGCs described above have been found following a wide range of injuries (both axotomy and inflammation) in somatic, orofacial and visceral regions, and therefore appear to be a general feature in chronic pain. We have found that in cultures of sensory ganglia calcium signals can spread from an SGC to neighboring cells by calcium waves, which are mediated by gap junctions and ATP acting on purinergic P2 receptors. A model is proposed to explain how augmented gap junctions and greater sensitivity to ATP can combine to produce enhanced calcium waves, which can lead to neuronal excitation. Thus this simple scheme can account for several major changes in sensory ganglia that are common to a great variety of pain models. Copyright © 2012 Elsevier B.V. All rights reserved.

Neuronal Target Identification Requires AHA-1-Mediated Fine-Tuning of Wnt Signaling in C. elegans

PubMed Central

Zhang, Jingyan; Li, Xia; Jevince, Angela R.; Guan, Liying; Wang, Jiaming; Hall, David H.; Huang, Xun; Ding, Mei

2013-01-01

Electrical synaptic transmission through gap junctions is a vital mode of intercellular communication in the nervous system. The mechanism by which reciprocal target cells find each other during the formation of gap junctions, however, is poorly understood. Here we show that gap junctions are formed between BDU interneurons and PLM mechanoreceptors in C. elegans and the connectivity of BDU with PLM is influenced by Wnt signaling. We further identified two PAS-bHLH family transcription factors, AHA-1 and AHR-1, which function cell-autonomously within BDU and PLM to facilitate the target identification process. aha-1 and ahr-1 act genetically upstream of cam-1. CAM-1, a membrane-bound receptor tyrosine kinase, is present on both BDU and PLM cells and likely serves as a Wnt antagonist. By binding to a cis-regulatory element in the cam-1 promoter, AHA-1 enhances cam-1 transcription. Our study reveals a Wnt-dependent fine-tuning mechanism that is crucial for mutual target cell identification during the formation of gap junction connections. PMID:23825972

Inter-Cellular Exchange of Cellular Components via VE-Cadherin-Dependent Trans-Endocytosis

PubMed Central

Sakurai, Takashi; Woolls, Melissa J.; Jin, Suk-Won

2014-01-01

Cell-cell communications typically involve receptor-mediated signaling initiated by soluble or cell-bound ligands. Here, we report a unique mode of endocytosis: proteins originating from cell-cell junctions and cytosolic cellular components from the neighboring cell are internalized, leading to direct exchange of cellular components between two adjacent endothelial cells. VE-cadherins form transcellular bridges between two endothelial cells that are the basis of adherence junctions. At such adherens junction sites, we observed the movement of the entire VE-cadherin molecule from one endothelial cell into the other with junctional and cytoplasmic components. This phenomenon, here termed trans-endocytosis, requires the establishment of a VE-cadherin homodimer in trans with internalization proceeding in a Rac1-, and actomyosin-dependent manner. Importantly, the trans-endocytosis is not dependent on any known endocytic pathway including clathrin-dependent endocytosis, macropinocytosis or phagocytosis. This novel form of cell-cell communications, leading to a direct exchange of cellular components, was observed in 2D and 3D-cultured endothelial cells as well as in the developing zebrafish vasculature. PMID:24603875

The formation of ordered nanoclusters controls cadherin anchoring to actin and cell–cell contact fluidity

PubMed Central

Strale, Pierre-Olivier; Duchesne, Laurence; Peyret, Grégoire; Montel, Lorraine; Nguyen, Thao; Png, Evelyn; Tampé, Robert; Troyanovsky, Sergey; Hénon, Sylvie; Ladoux, Benoit

2015-01-01

Oligomerization of cadherins could provide the stability to ensure tissue cohesion. Cadherins mediate cell–cell adhesion by forming trans-interactions. They form cis-interactions whose role could be essential to stabilize intercellular junctions by shifting cadherin clusters from a fluid to an ordered phase. However, no evidence has been provided so far for cadherin oligomerization in cellulo and for its impact on cell–cell contact stability. Visualizing single cadherins within cell membrane at a nanometric resolution, we show that E-cadherins arrange in ordered clusters, providing the first demonstration of the existence of oligomeric cadherins at cell–cell contacts. Studying the consequences of the disruption of the cis-interface, we show that it is not essential for adherens junction formation. Its disruption, however, increased the mobility of junctional E-cadherin. This destabilization strongly affected E-cadherin anchoring to actin and cell–cell rearrangement during collective cell migration, indicating that the formation of oligomeric clusters controls the anchoring of cadherin to actin and cell–cell contact fluidity. PMID:26195669

Gap Junction Intercellular Communication Mediates Ammonia-Induced Neurotoxicity.

PubMed

Bobermin, Larissa Daniele; Arús, Bernardo Assein; Leite, Marina Concli; Souza, Diogo Onofre; Gonçalves, Carlos-Alberto; Quincozes-Santos, André

2016-02-01

Astrocytes are important brain targets of ammonia, a neurotoxin implicated in the development of hepatic encephalopathy. During hyperammonemia, the pivotal role of astrocytes in brain function and homeostasis is impaired. These cells are abundantly interconnected by gap junctions (GJ), which are intercellular channels that allow the exchange of signaling molecules and metabolites. This communication may also increase cellular vulnerability during injuries, while GJ uncoupling could limit the extension of a lesion. Therefore, the current study was performed to investigate whether astrocyte coupling through GJ contributes to ammonia-induced cytotoxicity. We found that carbenoxolone (CBX), an effective GJ blocker, prevented the following effects induced by ammonia in astrocyte primary cultures: (1) decrease in cell viability and membrane integrity; (2) increase in reactive oxygen species production; (3) decrease in GSH intracellular levels; (4) GS activity; (5) pro-inflammatory cytokine release. On the other hand, CBX had no effect on C6 astroglial cells, which are poorly coupled via GJ. To our knowledge, this study provides the first evidence that GJ play a role in ammonia-induced cytotoxicity. Although more studies in vivo are required to confirm our hypothesis, our data suggest that GJ communication between astrocytes may transmit damage signals and excitotoxic components from unhealthy to normal cells, thereby contributing to the propagation of the neurotoxicity of ammonia.

Extract from the Zooxanthellate Jellyfish Cotylorhiza tuberculata Modulates Gap Junction Intercellular Communication in Human Cell Cultures

PubMed Central

Leone, Antonella; Lecci, Raffaella Marina; Durante, Miriana; Piraino, Stefano

2013-01-01

On a global scale, jellyfish populations in coastal marine ecosystems exhibit increasing trends of abundance. High-density outbreaks may directly or indirectly affect human economical and recreational activities, as well as public health. As the interest in biology of marine jellyfish grows, a number of jellyfish metabolites with healthy potential, such as anticancer or antioxidant activities, is increasingly reported. In this study, the Mediterranean “fried egg jellyfish” Cotylorhiza tuberculata (Macri, 1778) has been targeted in the search forputative valuable bioactive compounds. A medusa extract was obtained, fractionated, characterized by HPLC, GC-MS and SDS-PAGE and assayed for its biological activity on breast cancer cells (MCF-7) and human epidermal keratinocytes (HEKa). The composition of the jellyfish extract included photosynthetic pigments, valuable ω-3 and ω-6 fatty acids, and polypeptides derived either from jellyfish tissues and their algal symbionts. Extract fractions showed antioxidant activity and the ability to affect cell viability and intercellular communication mediated by gap junctions (GJIC) differentially in MCF-7and HEKa cells. A significantly higher cytotoxicity and GJIC enhancement in MCF-7 compared to HEKa cells was recorded. A putative action mechanism for the anticancer bioactivity through the modulation of GJIC has been hypothesized and its nutraceutical and pharmaceutical potential was discussed. PMID:23697954

Extract from the zooxanthellate jellyfish Cotylorhiza tuberculata modulates gap junction intercellular communication in human cell cultures.

PubMed

Leone, Antonella; Lecci, Raffaella Marina; Durante, Miriana; Piraino, Stefano

2013-05-22

On a global scale, jellyfish populations in coastal marine ecosystems exhibit increasing trends of abundance. High-density outbreaks may directly or indirectly affect human economical and recreational activities, as well as public health. As the interest in biology of marine jellyfish grows, a number of jellyfish metabolites with healthy potential, such as anticancer or antioxidant activities, is increasingly reported. In this study, the Mediterranean "fried egg jellyfish" Cotylorhiza tuberculata (Macri, 1778) has been targeted in the search forputative valuable bioactive compounds. A medusa extract was obtained, fractionated, characterized by HPLC, GC-MS and SDS-PAGE and assayed for its biological activity on breast cancer cells (MCF-7) and human epidermal keratinocytes (HEKa). The composition of the jellyfish extract included photosynthetic pigments, valuable ω-3 and ω-6 fatty acids, and polypeptides derived either from jellyfish tissues and their algal symbionts. Extract fractions showed antioxidant activity and the ability to affect cell viability and intercellular communication mediated by gap junctions (GJIC) differentially in MCF-7 and HEKa cells. A significantly higher cytotoxicity and GJIC enhancement in MCF-7 compared to HEKa cells was recorded. A putative action mechanism for the anticancer bioactivity through the modulation of GJIC has been hypothesized and its nutraceutical and pharmaceutical potential was discussed.

The role of gap junctions in megakaryocyte-mediated osteoblast proliferation and differentiation.

PubMed

Ciovacco, Wendy A; Goldberg, Carolyn G; Taylor, Amanda F; Lemieux, Justin M; Horowitz, Mark C; Donahue, Henry J; Kacena, Melissa A

2009-01-01

Gap junctions (GJs) are membrane-spanning channels that facilitate intercellular communication by allowing small signaling molecules (e.g. calcium ions, inositol phosphates, and cyclic nucleotides) to pass from cell to cell. Over the past two decades, many studies have described a role for GJ intercellular communication (GJIC) in the proliferation and differentiation of many cells, including bone cells. Recently, we reported that megakaryocytes (MKs) enhance osteoblast (OB) proliferation by a juxtacrine signaling mechanism. Here we determine whether this response is facilitated by GJIC. First we demonstrate that MKs express connexin 43 (Cx43), the predominant GJ protein expressed by bone cells, including OBs. Next, we provide data showing that MKs can communicate with OBs via GJIC, and that the addition of two distinct GJ uncouplers, 18alpha-glycyrrhetinic acid (alphaGA) or oleamide, inhibits this communication. We then demonstrate that inhibiting MK-mediated GJIC further enhances the ability of MKs to stimulate OB proliferation. Finally, we show that while culturing MKs with OBs reduces gene expression of several differentiation markers/matrix proteins (type I collagen, osteocalcin, and alkaline phosphatase), reduces alkaline phosphatase enzymatic activity, and decreases mineralization in OBs, blocking GJIC does not result in MK-induced reductions in OB gene expression, enzymatic levels, or mineralized nodule formation. Overall, these data provide evidence that GJIC between MKs and OBs is functional, and that inhibiting GJIC in MK-OB cultures enhances OB proliferation without apparently altering differentiation when compared to similarly treated OB cultures. Thus, these observations regarding MK-OB GJIC inhibition may provide insight regarding potential novel targets for anabolic bone formation.

The Role of Gap Junctions in Megakaryocyte-Mediated Osteoblast Proliferation and Differentiation

PubMed Central

Ciovacco, Wendy A.; Goldberg, Carolyn G.; Taylor, Amanda F.; Lemieux, Justin M.; Horowitz, Mark C.; Donahue, Henry J.; Kacena, Melissa A.

2009-01-01

Gap junctions (GJs) are membrane-spanning channels that facilitate intercellular communication by allowing small signaling molecules (e.g. calcium ions, inositol phosphates, and cyclic nucleotides) to pass from cell to cell. Over the past two decades, many studies have described a role for GJ intercellular communication (GJIC) in the proliferation and differentiation of many cells, including bone cells. Recently, we reported that megakaryocytes (MKs) enhance osteoblast (OB) proliferation by a juxtacrine signaling mechanism. Here we determine whether that response is facilitated by GJIC. First we demonstrate that MKs express connexin 43 (Cx43), the predominant GJ protein expressed by bone cells, including OBs. Next, we provide data showing that MKs can communicate with OBs via GJIC, and that the addition of two distinct GJ uncouplers, 18α-glycyrrhetinic acid (αGA) or oleamide, inhibits this communication. We then demonstrate that inhibiting MK-mediated GJIC further enhances the ability of MK to stimulate OB proliferation. Finally, we show that while culturing MKs with OBs reduces gene expression of several differentiation markers/matrix proteins (type I collagen, osteocalcin, and alkaline phosphatase), reduces alkaline phosphatase enzymatic activity, and decreases mineralization in OBs, blocking GJIC does not result in MK-induced reductions in OB gene expression, enzymatic levels, or mineralized nodule formation. Overall, these data provide evidence that GJIC between MKs and OBs is functional, and that inhibiting GJIC in MK-OB cultures enhances OB proliferation without apparently altering differentiation when compared to similarly treated OB cultures. Thus, these observations regarding MK-OB GJIC inhibition may provide insight regarding potential novel targets for anabolic bone formation. PMID:18848655

Low-molecular-weight hyaluronan (LMW-HA) accelerates lymph node metastasis of melanoma cells by inducing disruption of lymphatic intercellular adhesion.

PubMed

Du, Yan; Cao, Manlin; Liu, Yiwen; He, Yiqing; Yang, Cuixia; Wu, Man; Zhang, Guoliang; Gao, Feng

2016-01-01

Endothelial integrity defects initiate lymphatic metastasis of tumor cells. Low-molecular-weight hyaluronan (LMW-HA) derived from plasma and interstitial fluid was reported to be associated with tumor lymphatic metastasis. In addition, LMW-HA was proved to disrupt lymphatic vessel endothelium integrity, thus promoting lymphatic metastasis of tumor cells. Until now, there are few reports on how LMW-HA modulates lymphatic endothelial cells adhesion junctions and affects cancer cells metastasizing into lymph vessels. The aim of our study is to unravel the novel mechanism of LMW-HA in mediating tumor lymphatic metastasis. Here, we employed a melanoma metastasis model to investigate whether LMW-HA facilitates tumor cells transferring from foci to remote lymph nodes by disrupting the lymphatic endothelial integrity. Our data indicate that LMW-HA significantly induces metastasis of melanoma cells to lymph nodes and accelerates interstitial-lymphatic flow in vivo . Further experiments show that increased migration of melanoma cells across human dermal lymphatic endothelial cell (HDLEC) monolayers is accompanied by impaired lymphatic endothelial barrier function and increased permeability. The mechanism study reveals that VE-cadherin-β-catenin pathway and relevant signals are involved in modulating the interactions between endothelial cells and that a significant inhibition of lymphatic endothelium disruption is observed when antibodies to the LMW-HA receptor (LYVE-1) are present. Thus, our findings demonstrate a disruptive effect of LMW-HA on lymphatic endothelium continuity which leads to a promotion on melanoma lymphatic metastasis and also suggest a cellular signaling mechanism associated with VE-cadherin-mediated lymphatic intercellular junctions.

Synaptopodin couples epithelial contractility to α-actinin-4–dependent junction maturation

PubMed Central

Kannan, Nivetha

2015-01-01

The epithelial junction experiences mechanical force exerted by endogenous actomyosin activities and from interactions with neighboring cells. We hypothesize that tension generated at cell–cell adhesive contacts contributes to the maturation and assembly of the junctional complex. To test our hypothesis, we used a hydraulic apparatus that can apply mechanical force to intercellular junction in a confluent monolayer of cells. We found that mechanical force induces α-actinin-4 and actin accumulation at the cell junction in a time- and tension-dependent manner during junction development. Intercellular tension also induces α-actinin-4–dependent recruitment of vinculin to the cell junction. In addition, we have identified a tension-sensitive upstream regulator of α-actinin-4 as synaptopodin. Synaptopodin forms a complex containing α-actinin-4 and β-catenin and interacts with myosin II, indicating that it can physically link adhesion molecules to the cellular contractile apparatus. Synaptopodin depletion prevents junctional accumulation of α-actinin-4, vinculin, and actin. Knockdown of synaptopodin and α-actinin-4 decreases the strength of cell–cell adhesion, reduces the monolayer permeability barrier, and compromises cellular contractility. Our findings underscore the complexity of junction development and implicate a control process via tension-induced sequential incorporation of junctional components. PMID:26504173

Trpm7 Protein Contributes to Intercellular Junction Formation in Mouse Urothelium*

PubMed Central

Watanabe, Masaki; Suzuki, Yoshiro; Uchida, Kunitoshi; Miyazaki, Naoyuki; Murata, Kazuyoshi; Matsumoto, Seiji; Kakizaki, Hidehiro; Tominaga, Makoto

2015-01-01

Trpm7 is a divalent cation-permeable channel that has been reported to be involved in magnesium homeostasis as well as cellular adhesion and migration. We generated urothelium-specific Trpm7 knock-out (KO) mice to reveal the function of Trpm7 in vivo. A Trpm7 KO was induced by tamoxifen and was confirmed by genomic PCR and immunohistochemistry. By using patch clamp recordings in primary urothelial cells, we observed that Mg2+-inhibitable cation currents as well as acid-inducible currents were significantly smaller in Trpm7 KO urothelial cells than in cells from control mice. Assessment of voiding behavior indicated a significantly smaller voided volume in Trpm7 KO mice (mean voided volume 0.28 ± 0.08 g in KO mice and 0.36 ± 0.04 g in control mice, p < 0.05, n = 6–8). Histological analysis showed partial but substantial edema in the submucosal layer of Trpm7 KO mice, most likely due to inflammation. The expression of proinflammatory cytokines TNF-α and IL-1β was significantly higher in Trpm7 KO bladders than in controls. In transmission electron microscopic analysis, immature intercellular junctions were observed in Trpm7 KO urothelium but not in control mice. These results suggest that Trpm7 is involved in the formation of intercellular junctions in mouse urothelium. Immature intercellular junctions in Trpm7 knock-out mice might lead to a disruption of barrier function resulting in inflammation and hypersensitive bladder afferent nerves that may affect voiding behavior in vivo. PMID:26504086

Trpm7 Protein Contributes to Intercellular Junction Formation in Mouse Urothelium.

PubMed

Watanabe, Masaki; Suzuki, Yoshiro; Uchida, Kunitoshi; Miyazaki, Naoyuki; Murata, Kazuyoshi; Matsumoto, Seiji; Kakizaki, Hidehiro; Tominaga, Makoto

2015-12-11

Trpm7 is a divalent cation-permeable channel that has been reported to be involved in magnesium homeostasis as well as cellular adhesion and migration. We generated urothelium-specific Trpm7 knock-out (KO) mice to reveal the function of Trpm7 in vivo. A Trpm7 KO was induced by tamoxifen and was confirmed by genomic PCR and immunohistochemistry. By using patch clamp recordings in primary urothelial cells, we observed that Mg(2+)-inhibitable cation currents as well as acid-inducible currents were significantly smaller in Trpm7 KO urothelial cells than in cells from control mice. Assessment of voiding behavior indicated a significantly smaller voided volume in Trpm7 KO mice (mean voided volume 0.28 ± 0.08 g in KO mice and 0.36 ± 0.04 g in control mice, p < 0.05, n = 6-8). Histological analysis showed partial but substantial edema in the submucosal layer of Trpm7 KO mice, most likely due to inflammation. The expression of proinflammatory cytokines TNF-α and IL-1β was significantly higher in Trpm7 KO bladders than in controls. In transmission electron microscopic analysis, immature intercellular junctions were observed in Trpm7 KO urothelium but not in control mice. These results suggest that Trpm7 is involved in the formation of intercellular junctions in mouse urothelium. Immature intercellular junctions in Trpm7 knock-out mice might lead to a disruption of barrier function resulting in inflammation and hypersensitive bladder afferent nerves that may affect voiding behavior in vivo. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Holding Tight: Cell Junctions and Cancer Spread.

PubMed

Knights, Alexander J; Funnell, Alister P W; Crossley, Merlin; Pearson, Richard C M

2012-01-01

Cell junctions are sites of intercellular adhesion that maintain the integrity of epithelial tissue and regulate signalling between cells. These adhesive junctions are comprised of protein complexes that serve to establish an intercellular cytoskeletal network for anchoring cells, in addition to regulating cell polarity, molecular transport and communication. The expression of cell adhesion molecules is tightly controlled and their downregulation is essential for epithelial-mesenchymal transition (EMT), a process that facilitates the generation of morphologically and functionally diverse cell types during embryogenesis. The characteristics of EMT are a loss of cell adhesion and increased cellular mobility. Hence, in addition to its normal role in development, dysregulated EMT has been linked to cancer progression and metastasis, the process whereby primary tumors migrate to invasive secondary sites in the body. This paper will review the current understanding of cell junctions and their role in cancer, with reference to the abnormal regulation of junction protein genes. The potential use of cell junction molecules as diagnostic and prognostic markers will also be discussed, as well as possible therapies for adhesive dysregulation.

A novel adhering junction in the apical ciliary apparatus of the rotifer Brachionus plicatilis (Rotifera, Monogononta).

PubMed

Dallai, R; Lupetti, P; Lane, N J

1996-10-01

Cultures of the rotifer Brachionus plicatilis were examined with regard to their interepithelial junctions after infiltration with the extracellular tracer lanthanum, freeze-fracturing or quick-freeze deep-etching. The lateral borders between ciliated cells have an unusual apical adhering junction. This apical part of their intercellular cleft looks desmosome-like, but it is characterized by unusual intramembranous E-face clusters of particles. Deep-etching reveals that these are packed together in short rows which lie parallel to one another in orderly arrays. The true membrane surface in these areas features filaments in the form of short ribbons; these are produced by projections, possibly part of the glycocalyx, emerging from the membranes, between which the electron-dense tracer lanthanum permeates. These projections appear to overlap with each other in the centre of the intercellular cleft; this would provide a particularly flexible adaptation to maintain cell-cell contact and coordination as a consequence. The filamentous ribbons may be held in position by the intramembranous particle arrays since both have a similar size and distribution. These contacts are quite different from desmosomes and appear to represent a distinct new category of adhesive cell-cell junction. Beneath these novel structures, conventional pleated septate junctions are found, exhibiting the undulating intercellular ribbons typical of this junctional type, as well as the usual parallel alignments of intramembranous rows of EF grooves and PF particles. Below these are found gap junctions as close-packed plaques of intramembranous particles on either the P-face or E-face. After freeze-fracturing, the complementary fracture face to the particles shows pits, usually on the P-face, arrayed with a very precise hexagonal pattern.

Terbinafine inhibits gap junctional intercellular communication

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Ju Yeun, E-mail: whitewndus@naver.com

Terbinafine is an antifungal agent that selectively inhibits fungal sterol synthesis by blocking squalene epoxidase. We evaluated the effect of terbinafine on gap junctional intercellular communication (GJIC). Fluorescence recovery after photobleaching (FRAP) and I-YFP GJIC assays revealed that terbinafine inhibits GJIC in a reversible and dose-dependent manner in FRT-Cx43 and LN215 cells. Treatment with terbinafine did not affect Cx43 phosphorylation status or intracellular Ca{sup 2+} concentration, well-known action mechanisms of various GJIC blockers. While a structurally related chemical, naftifine, attenuated GJIC, epigallocatechin gallate, another potent squalene epoxidase inhibitor with a different structure, did not. These results suggest that terbinafine inhibitsmore » GJIC with a so far unknown mechanism of action. - Highlights: • In vitro pharmacological studies were performed on FRT-Cx43 and LN215 cells. • Terbinafine inhibits gap junctional intercellular communication in both cell lines. • The inhibitory effect of terbinafine is reversible and dose-dependent. • Treatment of terbinafine does not alter Cx43 phosphorylation or cytosolic Ca{sup 2+} concentration. • Inhibition of squalene epoxidase is not involved in this new effect of terbinafine.« less

In vitro early changes in intercellular junctions by treatment with a chemical carcinogen.

PubMed

Tachikawa, T; Kohno, Y; Matsui, Y; Yoshiki, S

1986-06-01

To examine early intercellular junction changes caused by treatment with 9,10-dimethyl-1,2-benzanthracene (DMBA), rat lingual epithelium was cultivated in isolation and observed by electrophysiological, freeze-fracture and whole-mount electron microscopy. Electrophysiological measurements showed a transient decrease in membrane potential of -10.2 mV 6 h after the treatment. It returned to almost the same level as that of the control group 1 day later. Six hours after treatment, input resistance decreased rapidly to 5.3 M omega but increased to 18.0 M omega 12 h after treatment. Transient reduction of input resistance and membrane potential occurred prior to the decrease in the coupling ratio 6 h after treatment with DMBA. In freeze-fracture replicas, the number of gap junctions decreased by approximately 45% of the control value 6 h after treatment with DMBA. At 12 h and thereafter, the number and area of gap junctions subsequently decreased by 60-80% of the control value. Alterations in the number and area of desmosomes were similar to those of the gap junctions. The formation of epithelial cytoskeletons, partially devoid of the 2-4 and 5-8 nm filaments was also observed. A decrease in the density of filament networks beneath the plasma membranes was especially apparent. Treatment with a carcinogen brought about morphological cellular changes as early as 6 h after treatment, and such early changes might trigger metabolic cellular abnormalities. Affected cells appear to move away from normal cells in a process of repeated destruction and revision of intercellular junctions, and cytoskeletons.

Patterning of wound-induced intercellular Ca2+ flashes in a developing epithelium

NASA Astrophysics Data System (ADS)

Narciso, Cody; Wu, Qinfeng; Brodskiy, Pavel; Garston, George; Baker, Ruth; Fletcher, Alexander; Zartman, Jeremiah

2015-10-01

Differential mechanical force distributions are increasingly recognized to provide important feedback into the control of an organ’s final size and shape. As a second messenger that integrates and relays mechanical information to the cell, calcium ions (Ca2+) are a prime candidate for providing important information on both the overall mechanical state of the tissue and resulting behavior at the individual-cell level during development. Still, how the spatiotemporal properties of Ca2+ transients reflect the underlying mechanical characteristics of tissues is still poorly understood. Here we use an established model system of an epithelial tissue, the Drosophila wing imaginal disc, to investigate how tissue properties impact the propagation of Ca2+ transients induced by laser ablation. The resulting intercellular Ca2+ flash is found to be mediated by inositol 1,4,5-trisphosphate and depends on gap junction communication. Further, we find that intercellular Ca2+ transients show spatially non-uniform characteristics across the proximal-distal axis of the larval wing imaginal disc, which exhibit a gradient in cell size and anisotropy. A computational model of Ca2+ transients is employed to identify the principle factors explaining the spatiotemporal patterning dynamics of intercellular Ca2+ flashes. The relative Ca2+ flash anisotropy is principally explained by local cell shape anisotropy. Further, Ca2+ velocities are relatively uniform throughout the wing disc, irrespective of cell size or anisotropy. This can be explained by the opposing effects of cell diameter and cell elongation on intercellular Ca2+ propagation. Thus, intercellular Ca2+ transients follow lines of mechanical tension at velocities that are largely independent of tissue heterogeneity and reflect the mechanical state of the underlying tissue.

Role of connexin43 and ATP in long-range bystander radiation damage and oncogenesis in vivo.

PubMed

Mancuso, M; Pasquali, E; Leonardi, S; Rebessi, S; Tanori, M; Giardullo, P; Borra, F; Pazzaglia, S; Naus, C C; Di Majo, V; Saran, A

2011-11-10

Ionizing radiation is a genotoxic agent and human carcinogen. Recent work has questioned long-held dogmas by showing that cancer-associated genetic alterations occur in cells and tissues not directly exposed to radiation, questioning the robustness of the current system of radiation risk assessment. In vitro, diverse mechanisms involving secreted soluble factors, gap junction intercellular communication (GJIC) and oxidative metabolism are proposed to mediate these indirect effects. In vivo, the mechanisms behind long-range 'bystander' responses remain largely unknown. Here, we investigate the role of GJIC in propagating radiation stress signals in vivo, and in mediating radiation-associated bystander tumorigenesis in mouse central nervous system using a mouse model in which intercellular communication is downregulated by targeted deletion of the connexin43 (Cx43) gene. We show that GJIC is critical for transmission of oncogenic radiation damage to the non-targeted cerebellum, and that a mechanism involving adenosine triphosphate release and upregulation of Cx43, the major GJIC constituent, regulates transduction of oncogenic damage to unirradiated tissues in vivo. Our data provide a novel hypothesis for transduction of distant bystander effects and suggest that the highly branched nervous system, similar to the vascular network, has an important role.

A recessive mutation in the DSP gene linked to cardiomyopathy, skin fragility and hair defects impairs the binding of desmoplakin to epidermal keratins and the muscle-specific intermediate filament desmin.

PubMed

Favre, B; Begré, N; Borradori, L

2018-06-07

Desmoplakin (DSP) is a cytolinker of the plakin family. It mediates the connection of intermediate filaments (IFs) to desmosomes, intercellular adhesion junctions. The carboxyl (C)-terminal tail of DSP binds to IFs, while its amino-terminal part interacts with the armadillo proteins plakophilins and plakoglobin that in turn associate with the desmosomal cadherin desmogleins and desmocollins 1 . This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

The status of intercellular junctions in established lens epithelial cell lines

PubMed Central

Dave, Alpana; Craig, Jamie E.

2012-01-01

Purpose Cataract is the major cause of vision-related disability worldwide. Mutations in the crystallin genes are the most common known cause of inherited congenital cataract. Mutations in the genes associated with intercellular contacts, such as Nance-Horan Syndrome (NHS) and Ephrin type A receptor-2 (EPHA2), are other recognized causes of congenital cataract. The EPHA2 gene has been also associated with age-related cataract, suggesting that intercellular junctions are important in not only lens development, but also in maintaining lens transparency. The purpose of this study was to analyze the expression and localization of the key cell junction and cytoskeletal proteins, and of NHS and EPHA2, in established lens epithelial cell lines to determine their suitability as model epithelial systems for the functional investigation of genes involved in intercellular contacts and implicated in cataract. Methods The expression and subcellular localization of occludin and zona occludens protein-1 (ZO-1), which are associated with tight junctions; E-cadherin, which is associated with adherence junctions; and the cytoskeletal actin were analyzed in monolayers of a human lens epithelial cell line (SRA 01/04) and a mouse lens epithelial cell line (αTN4). In addition, the expression and subcellular localization of the NHS and EPHA2 proteins were analyzed in these cell lines. Protein or mRNA expression was respectively determined by western blotting or reverse transcription-polymerase chain reaction (RT–PCR), and localization was determined by immunofluorescence labeling. Results Human SRA 01/04 and mouse αTN4 lens epithelial cells expressed either the proteins of interest or their encoding mRNA. Occludin, ZO-1, and NHS proteins localized to the cellular periphery, whereas E-cadherin, actin, and EPHA2 localized in the cytoplasm in these cell lines. Conclusions The human SRA 01/04 and mouse αTN4 lens epithelial cells express the key junctional proteins. The localization patterns of these proteins suggest that these cell lines form tight junctions but do not form E-cadherin-based adherence junctions. These data further indicate that the regulatory role of NHS in actin remodeling, suggested in another study, is cell type dependent. In conclusion, the SRA 01/04 and αTN4 lens epithelial cell lines model some characteristics of an epithelium. PMID:23288986

The status of intercellular junctions in established lens epithelial cell lines.

PubMed

Dave, Alpana; Craig, Jamie E; Sharma, Shiwani

2012-01-01

Cataract is the major cause of vision-related disability worldwide. Mutations in the crystallin genes are the most common known cause of inherited congenital cataract. Mutations in the genes associated with intercellular contacts, such as Nance-Horan Syndrome (NHS) and Ephrin type A receptor-2 (EPHA2), are other recognized causes of congenital cataract. The EPHA2 gene has been also associated with age-related cataract, suggesting that intercellular junctions are important in not only lens development, but also in maintaining lens transparency. The purpose of this study was to analyze the expression and localization of the key cell junction and cytoskeletal proteins, and of NHS and EPHA2, in established lens epithelial cell lines to determine their suitability as model epithelial systems for the functional investigation of genes involved in intercellular contacts and implicated in cataract. The expression and subcellular localization of occludin and zona occludens protein-1 (ZO-1), which are associated with tight junctions; E-cadherin, which is associated with adherence junctions; and the cytoskeletal actin were analyzed in monolayers of a human lens epithelial cell line (SRA 01/04) and a mouse lens epithelial cell line (αTN4). In addition, the expression and subcellular localization of the NHS and EPHA2 proteins were analyzed in these cell lines. Protein or mRNA expression was respectively determined by western blotting or reverse transcription-polymerase chain reaction (RT-PCR), and localization was determined by immunofluorescence labeling. Human SRA 01/04 and mouse αTN4 lens epithelial cells expressed either the proteins of interest or their encoding mRNA. Occludin, ZO-1, and NHS proteins localized to the cellular periphery, whereas E-cadherin, actin, and EPHA2 localized in the cytoplasm in these cell lines. The human SRA 01/04 and mouse αTN4 lens epithelial cells express the key junctional proteins. The localization patterns of these proteins suggest that these cell lines form tight junctions but do not form E-cadherin-based adherence junctions. These data further indicate that the regulatory role of NHS in actin remodeling, suggested in another study, is cell type dependent. In conclusion, the SRA 01/04 and αTN4 lens epithelial cell lines model some characteristics of an epithelium.

Transfected connexin45 alters gap junction permeability in cells expressing endogenous connexin43

PubMed Central

1995-01-01

Many cells express multiple connexins, the gap junction proteins that interconnect the cytosol of adjacent cells. Connexin43 (Cx43) channels allow intercellular transfer of Lucifer Yellow (LY, MW = 443 D), while connexin45 (Cx45) channels do not. We transfected full-length or truncated chicken Cx45 into a rat osteosarcoma cell line ROS-17/2.8, which expresses endogenous Cx43. Both forms of Cx45 were expressed at high levels and colocalized with Cx43 at plasma membrane junctions. Cells transfected with full-length Cx45 (ROS/Cx45) and cells transfected with Cx45 missing the 37 carboxyl-terminal amino acids (ROS/Cx45tr) showed 30-60% of the gap junctional conductance exhibited by ROS cells. Intercellular transfer of three negatively charged fluorescent reporter molecules was examined. In ROS cells, microinjected LY was transferred to an average of 11.2 cells/injected cell, while dye transfer between ROS/Cx45 cells was reduced to 3.9 transfer between ROS/Cx45 cells was reduced to 3.9 cells. In contrast, ROS/Cx45tr cells transferred LY to > 20 cells. Transfer of calcein (MW = 623 D) was also reduced by approximately 50% in ROS/Cx45 cells, but passage of hydroxycoumarin carboxylic acid (HCCA; MW = 206 D) was only reduced by 35% as compared to ROS cells. Thus, introduction of Cx45 altered intercellular coupling between cells expressing Cx43, most likely the result of direct interaction between Cx43 and Cx45. Transfection of Cx45tr and Cx45 had different effects in ROS cells, consistent with a role of the carboxyl-terminal domain of Cx45 in determining gap junction permeability or interactions between connexins. These data suggest that coexpression of multiple connexins may enable cells to achieve forms of intercellular communication that cannot be attained by expression of a single connexin. PMID:7642714

Two Classes of Gap Junction Channels Mediate Soma-Germline Interactions Essential for Germline Proliferation and Gametogenesis in Caenorhabditis elegans

PubMed Central

Starich, Todd A.; Hall, David H.; Greenstein, David

2014-01-01

In all animals examined, somatic cells of the gonad control multiple biological processes essential for germline development. Gap junction channels, composed of connexins in vertebrates and innexins in invertebrates, permit direct intercellular communication between cells and frequently form between somatic gonadal cells and germ cells. Gap junctions comprise hexameric hemichannels in apposing cells that dock to form channels for the exchange of small molecules. Here we report essential roles for two classes of gap junction channels, composed of five innexin proteins, in supporting the proliferation of germline stem cells and gametogenesis in the nematode Caenorhabditis elegans. Transmission electron microscopy of freeze-fracture replicas and fluorescence microscopy show that gap junctions between somatic cells and germ cells are more extensive than previously appreciated and are found throughout the gonad. One class of gap junctions, composed of INX-8 and INX-9 in the soma and INX-14 and INX-21 in the germ line, is required for the proliferation and differentiation of germline stem cells. Genetic epistasis experiments establish a role for these gap junction channels in germline proliferation independent of the glp-1/Notch pathway. A second class of gap junctions, composed of somatic INX-8 and INX-9 and germline INX-14 and INX-22, is required for the negative regulation of oocyte meiotic maturation. Rescue of gap junction channel formation in the stem cell niche rescues germline proliferation and uncovers a later channel requirement for embryonic viability. This analysis reveals gap junctions as a central organizing feature of many soma–germline interactions in C. elegans. PMID:25195067

Crk1/2-dependent signaling is necessary for podocyte foot process spreading in mouse models of glomerular disease

PubMed Central

George, Britta; Verma, Rakesh; Soofi, Abdulsalam A.; Garg, Puneet; Zhang, Jidong; Park, Tae-Ju; Giardino, Laura; Ryzhova, Larisa; Johnstone, Duncan B.; Wong, Hetty; Nihalani, Deepak; Salant, David J.; Hanks, Steven K.; Curran, Tom; Rastaldi, Maria Pia; Holzman, Lawrence B.

2012-01-01

The morphology of healthy podocyte foot processes is necessary for maintaining the characteristics of the kidney filtration barrier. In most forms of glomerular disease, abnormal filter barrier function results when podocytes undergo foot process spreading and retraction by remodeling their cytoskeletal architecture and intercellular junctions during a process known as effacement. The cell adhesion protein nephrin is necessary for establishing the morphology of the kidney podocyte in development by transducing from the specialized podocyte intercellular junction phosphorylation-mediated signals that regulate cytoskeletal dynamics. The present studies extend our understanding of nephrin function by showing that nephrin activation in cultured podocytes induced actin dynamics necessary for lamellipodial protrusion. This process required a PI3K-, Cas-, and Crk1/2-dependent signaling mechanism distinct from the previously described nephrin-Nck1/2 pathway necessary for assembly and polymerization of actin filaments. Our present findings also support the hypothesis that mechanisms governing lamellipodial protrusion in culture are similar to those used in vivo during foot process effacement in a subset of glomerular diseases. In mice, podocyte-specific deletion of Crk1/2 prevented foot process effacement in one model of podocyte injury and attenuated foot process effacement and associated proteinuria in a delayed fashion in a second model. In humans, focal adhesion kinase and Cas phosphorylation — markers of focal adhesion complex–mediated Crk-dependent signaling — was induced in minimal change disease and membranous nephropathy, but not focal segmental glomerulosclerosis. Together, these observations suggest that activation of a Cas-Crk1/2–dependent complex is necessary for foot process effacement observed in distinct subsets of human glomerular diseases. PMID:22251701

Actin cable dynamics and Rho/Rock orchestrate a polarized cytoskeletal architecture in the early steps of assembling a stratified epithelium.

PubMed

Vaezi, Alec; Bauer, Christoph; Vasioukhin, Valeri; Fuchs, Elaine

2002-09-01

To enable stratification and barrier function, the epidermis must permit self-renewal while maintaining adhesive connections. By generating K14-GFP-actin mice to monitor actin dynamics in cultured primary keratinocytes, we uncovered a role for the actin cytoskeleton in establishing cellular organization. During epidermal sheet formation, a polarized network of nascent intercellular junctions and radial actin cables assemble in the apical plane of the monolayer. These actin fibers anchor to a central actin-myosin network, creating a tension-based plane of cytoskeleton across the apical surface of the sheet. Movement of the sheet surface relative to its base expands the zone of intercellular overlap, catalyzing new sites for nascent intercellular junctions. This polarized cytoskeleton is dependent upon alpha-catenin, Rho, and Rock, and its regulation may be important for wound healing and/or stratification, where coordinated tissue movements are involved.

Epithelial Permeability Alterations in an In Vitro Air-Liquid Interface Model of Allergic Fungal Rhinosinusitis

PubMed Central

Den Beste, Kyle A.; Hoddeson, Elizabeth K.; Parkos, Charles A.; Nusrat, Asma; Wise, Sarah K.

2012-01-01

Background Chronic rhinosinusitis (CRS) is an inflammatory upper-airway disease with numerous etiologies. Patients with a characteristic subtype of CRS, allergic fungal rhinosinusitis (AFRS), display increased expression of Th2 cytokines and antigen-specific IgE. Various sinonasal inflammatory conditions are associated with alterations in epithelial barrier function. The aim of this study was to compare epithelial permeability and intercellular junctional protein expression amongst cultured primary sinonasal cells from AFRS patients versus non-inflammatory controls. Methods Epithelial cells isolated from paranasal sinus mucosa of AFRS and non-inflammatory control patients were grown to confluence on permeable supports and transitioned to air-liquid interface (ALI). Trans-epithelial resistance (TER) was measured with a horizontal Ussing chamber to characterize the functional permeability of each cell type. After TER recordings were complete, a panel of intercellular junctional proteins was assessed by Western blot and immunofluorescence labeling followed by confocal microscopy. Results After 12 samples were measured from each group, we observed a 41% mean decrease in TER in AFRS cells (296±89 ohms × cm2) compared to control (503±134 ohms × cm2, P=0.006). TER deficits observed in AFRS were associated with decreased expression of the tight junction proteins occludin and Junctional Adhesion Molecule-A (JAM-A), and increased expression of a leaky tight junction protein claudin-2. Conclusions Cultured sinonasal epithelium from AFRS patients displayed increased epithelial permeability and altered expression of intercellular junctional proteins. Given that these cells were not incubated with inflammatory cytokines in vitro, the cultured AFRS epithelial alterations may represent a retained modification in protein expression from the in vivo phenotype. PMID:22927233

Septal Junctions in Filamentous Heterocyst-Forming Cyanobacteria.

PubMed

Flores, Enrique; Herrero, Antonia; Forchhammer, Karl; Maldener, Iris

2016-02-01

In the filaments of heterocyst-forming cyanobacteria, septal junctions that traverse the septal peptidoglycan join adjacent cells, allowing intercellular communication. Perforations in the septal peptidoglycan have been observed, and proteins involved in the formation of such perforations and putative protein components of the septal junctions have been identified, but their relationships are debated. Copyright © 2015 Elsevier Ltd. All rights reserved.

Expression of connexin 43 mRNA and protein in developing follicles of prepubertal porcine ovaries

USGS Publications Warehouse

Melton, C.M.; Zaunbrecher, G.M.; Yoshizaki, G.; Patio, R.; Whisnant, S.; Rendon, A.; Lee, V.H.

2001-01-01

A major form of cell-cell communication is mediated by gap junctions, aggregations of intercellular channels composed of connexins (Cxs), which are responsible for exchange of low molecular weight (< 1200 Da) cytosolic materials. These channels are a growing family of related proteins. This study was designed to determine the ontogeny of connexin 43 (Cx43) during early stages of follicular development in prepubertal porcine ovaries. A partial-length (412 base) cDNA clone was obtained from mature porcine ovaries and determined to have 98% identity with published porcine Cx43. Northern blot analysis demonstrated a 4.3-kb mRNA in total RNA isolated from prepubertal and adult porcine ovaries. In-situ hybridization revealed that Cx43 mRNA was detectable in granulosa cells of primary follicles but undetectable in dormant primordial follicles. The intensity of the signal increased with follicular growth and was greatest in the large antral follicles. Immunohistochemical evaluation indicated that Cx43 protein expression correlated with the presence of Cx43 mRNA. These results indicate that substantial amounts of Cx43 are first expressed in granulosa cells following activation of follicular development and that this expression increases throughout follicular growth and maturation. These findings suggest an association between the enhancement of intercellular gap-junctional communication and onset of follicular growth. ?? 2001 Elsevier Science Inc. All rights reserved.

Possible Involvement of Tight Junctions, Extracellular Matrix and Nuclear Receptors in Epithelial Differentiation

PubMed Central

Ichikawa-Tomikawa, Naoki; Sugimoto, Kotaro; Satohisa, Seiro; Nishiura, Keisuke; Chiba, Hideki

2011-01-01

Tight junctions are intercellular junctions localized at the most apical end of the lateral plasma membrane. They consist of four kinds of transmembrane proteins (occludin, claudins, junctional adhesion molecules, and tricellulin) and huge numbers of scaffolding proteins and contribute to the paracellular barrier and fence function. The mutation and deletion of these proteins impair the functions of tight junctions and cause various human diseases. In this paper, we provide an overview of recent studies on transmembrane proteins of tight junctions and highlight the functional significance of tight junctions, extracellular matrix, and nuclear receptors in epithelial differentiation. PMID:22162632

Establishment of cell-cell junctions depends on the oligomeric states of VE-cadherin

PubMed Central

Bibert, Stéphanie; Ayari, Hélène; Riveline, Daniel; Concord, Evelyne; Hermant, Bastien; Vernet, Thierry; Gulino-Debrac, Danièle

2008-01-01

Specifically expressed at intercellular adherens junctions of endothelial cells, VE-cadherin is a receptor that exhibits particular self-association properties. Indeed, in vitro studies demonstrated that the extracellular part of VE-cadherin elaborates Ca++-dependent hexameric structures. We hypothesized that this assembly could be at the basis of a new cadherin-mediated cell-cell adhesion mechanism. To verify this assumption, we first demonstrated that VE-cadherin can elaborate hexamers at the cell surface of confluent endothelial cells. Second, mutations were introduced within the extracellular part of VE-cadherin to destabilize the hexamer. Following an in vitro screening, three mutants were selected, among which, one is able to elaborate only dimers. The selected mutations were expressed as C-terminal Green Fluorescent Protein fusions in CHO cells. Despite their capacity to elaborate nascent cell-cell contacts, the mutants seem to be rapidly degraded and or internalized. Altogether, our results suggest that the formation of VE-cadherin hexamers protects this receptor and might allow the elaboration of mature endothelial cell-cell junctions. PMID:18343874

Exocytosis of vacuolar apical compartment (VAC): a cell-cell contact controlled mechanism for the establishment of the apical plasma membrane domain in epithelial cells

PubMed Central

1988-01-01

The vacuolar apical compartment (VAC) is an organelle found in Madin- Darby canine kidney (MDCK) cells with incomplete intercellular contacts by incubation in 5 microM Ca++ and in cells without contacts (single cells in subconfluent culture); characteristically, it displays apical biochemical markers and microvilli and excludes basolateral markers (Vega-Salas, D. E., P. J. I. Salas, and E. Rodriguez-Boulan. 1987. J. Cell Biol. 104:1249-1259). The apical surface of cells kept under these culture conditions is immature, with reduced numbers of microvilli and decreased levels of an apical biochemical marker (184 kD), which is, however, still highly polarized (Vega-Salas, D. E., P. J. I. Salas, D. Gundersen, and E. Rodriguez-Boulan. 1987. J. Cell Biol. 104:905-916). We describe here the morphological stages of VAC exocytosis which ultimately lead to the establishment of a differentiated apical domain. Addition of 1.8 mM Ca++ to monolayers developed in 5 microM Ca++ causes the rapid (20-40 min) fusion of VACs with the plasma membrane and their accessibility to external antibodies, as demonstrated by immunofluorescence, immunoperoxidase EM, and RIA with antibodies against the 184-kD apical plasma membrane marker. Exocytosis occurs towards areas of cell-cell contact in the developing lateral surface where they form intercellular pockets; fusion images are always observed immediately adjacent to the incomplete junctional bands detected by the ZO-1 antibody (Stevenson, B. R., J. D. Siliciano, M. S. Mooseker, and D. A. Goodenough. 1986. J. Cell Biol. 103:755-766). Blocks of newly incorporated VAC microvilli and 184-kD protein progressively move from intercellular ("primitive" lateral) spaces towards the microvilli-poor free cell surface. The definitive lateral domain is sealed behind these blocks by the growing tight junctional fence. These results demonstrate a fundamental role of cell-cell contact-mediated VAC exocytosis in the establishment of epithelial surface polarity. Because isolated stages (intercellular pockets) of the stereotyped sequence of events triggered by the establishment of intercellular contacts in MDCK cells have been reported during normal differentiation of intestine epithelium (Colony, P. C., and M. R. Neutra. 1983. Dev. Biol. 97:349-363), we speculate that the mechanism we describe here plays an important role in the establishment of epithelial cell polarity in vivo. PMID:3053735

Intercellular junctions between palisade nerve endings and outer root sheath cells of rat vellus hairs.

PubMed

Kaidoh, T; Inoué, T

2000-05-15

Hair follicles have a longitudinal set of sensory nerve endings called palisade nerve endings (PN). We examined the junctional structures between the PN and outer root sheath (ORS) cells of hair follicles in the rat external ear. Transmission electron microscopy of serial thin sections showed that the processes of the ORS cells penetrated the basal lamina of the hair follicle, forming intercellular junctions with the PN (PN-ORS junctions). Two types of junctions were found: junctions between nerve endings and ORS cells (N-ORS junctions) and those between Schwann cell processes and ORS cells (S-ORS junctions). The N-ORS junctions had two subtypes: 1) a short process or small eminence of the ORS cell was attached to the nerve ending (type I); or 2) a process of the ORS cell was invaginated into the nerve ending (type II). The S-ORS junctions also had two subtypes: 1) a short process or small eminence of the ORS cell was abutted on the Schwann cell process (type I); or 2) a process of the ORS cell was invaginated into the Schwann cell process (type II). Vesicles, coated pits, coated vesicles, and endosomes were sometimes seen in nerve endings, Schwann cells, and ORS cells near the junctions. Computer-aided reconstruction of the serial thin sections displayed the three-dimensional structure of these junctions. These results suggested that the PN-ORS junctions provided direct relationships between the PN and ORS in at least four different patterns. The discovery of these junctions shows the PN-ORS relationship to be closer than previously realized. We speculate that these junctions may have roles in attachment of the PN to the ORS, contributing to increases in the sensitivity of the PN, and in chemical signaling between the PN and ORS.

Cell-specific expression of connexins and evidence of restricted gap junctional coupling between glial cells and between neurons.

PubMed

Rash, J E; Yasumura, T; Dudek, F E; Nagy, J I

2001-03-15

The transmembrane connexin proteins of gap junctions link extracellularly to form channels for cell-to-cell exchange of ions and small molecules. Two primary hypotheses of gap junction coupling in the CNS are the following: (1) generalized coupling occurs between neurons and glia, with some connexins expressed in both neurons and glia, and (2) intercellular junctional coupling is restricted to specific coupling partners, with different connexins expressed in each cell type. There is consensus that gap junctions link neurons to neurons and astrocytes to oligodendrocytes, ependymocytes, and other astrocytes. However, unresolved are the existence and degree to which gap junctions occur between oligodendrocytes, between oligodendrocytes and neurons, and between astrocytes and neurons. Using light microscopic immunocytochemistry and freeze-fracture replica immunogold labeling of adult rat CNS, we investigated whether four of the best-characterized CNS connexins are each present in one or more cell types, whether oligodendrocytes also share gap junctions with other oligodendrocytes or with neurons, and whether astrocytes share gap junctions with neurons. Connexin32 (Cx32) was found only in gap junctions of oligodendrocyte plasma membranes, Cx30 and Cx43 were found only in astrocyte membranes, and Cx36 was only in neurons. Oligodendrocytes shared intercellular gap junctions only with astrocytes, with each oligodendrocyte isolated from other oligodendrocytes except via astrocyte intermediaries. Finally, neurons shared gap junctions only with other neurons and not with glial cells. Thus, the different cell types of the CNS express different connexins, which define separate pathways for neuronal versus glial gap junctional communication.

Connexin-43 upregulation in micrometastases and tumor vasculature and its role in tumor cell attachment to pulmonary endothelium

PubMed Central

Elzarrad, M Khair; Haroon, Abu; Willecke, Klaus; Dobrowolski, Radoslaw; Gillespie, Mark N; Al-Mehdi, Abu-Bakr

2008-01-01

Background The modulation of gap junctional communication between tumor cells and between tumor and vascular endothelial cells during tumorigenesis and metastasis is complex. The notion of a role for loss of gap junctional intercellular communication in tumorigenesis and metastasis has been controversial. While some of the stages of tumorigenesis and metastasis, such as uncontrolled cell division and cellular detachment, would necessitate the loss of intercellular junctions, other stages, such as intravasation, endothelial attachment, and vascularization, likely require increased cell-cell contact. We hypothesized that, in this multi-stage scheme, connexin-43 is centrally involved as a cell adhesion molecule mediating metastatic tumor attachment to the pulmonary endothelium. Methods Tumor cell attachment to pulmonary vasculature, tumor growth, and connexin-43 expression was studied in metastatic lung tumor sections obtained after tail-vein injection into nude mice of syngeneic breast cancer cell lines, overexpressing wild type connexin-43 or dominant-negatively mutated connexin-43 proteins. High-resolution immunofluorescence microscopy and Western blot analysis was performed using a connexin-43 monoclonal antibody. Calcein Orange Red AM dye transfer by fluorescence imaging was used to evaluate the gap junction function. Results Adhesion of breast cancer cells to the pulmonary endothelium increased with cancer cells overexpressing connexin-43 and markedly decreased with cells expressing dominant-negative connexin-43. Upregulation of connexin-43 was observed in tumor cell-endothelial cell contact areas in vitro and in vivo, and in areas of intratumor blood vessels and in micrometastatic foci. Conclusion Connexin-43 facilitates metastatic 'homing' by increasing adhesion of cancer cells to the lung endothelial cells. The marked upregulation of connexin-43 in tumor cell-endothelial cell contact areas, whether in preexisting 'homing' vessels or in newly formed tumor vessels, suggests that connexin-43 can serve as a potential marker of micrometastases and tumor vasculature and that it may play a role in the early incorporation of endothelial cells into small tumors as seeds for vasculogenesis. PMID:18647409

Connexin-43 upregulation in micrometastases and tumor vasculature and its role in tumor cell attachment to pulmonary endothelium.

PubMed

Elzarrad, M Khair; Haroon, Abu; Willecke, Klaus; Dobrowolski, Radoslaw; Gillespie, Mark N; Al-Mehdi, Abu-Bakr

2008-07-22

The modulation of gap junctional communication between tumor cells and between tumor and vascular endothelial cells during tumorigenesis and metastasis is complex. The notion of a role for loss of gap junctional intercellular communication in tumorigenesis and metastasis has been controversial. While some of the stages of tumorigenesis and metastasis, such as uncontrolled cell division and cellular detachment, would necessitate the loss of intercellular junctions, other stages, such as intravasation, endothelial attachment, and vascularization, likely require increased cell-cell contact. We hypothesized that, in this multi-stage scheme, connexin-43 is centrally involved as a cell adhesion molecule mediating metastatic tumor attachment to the pulmonary endothelium. Tumor cell attachment to pulmonary vasculature, tumor growth, and connexin-43 expression was studied in metastatic lung tumor sections obtained after tail-vein injection into nude mice of syngeneic breast cancer cell lines, overexpressing wild type connexin-43 or dominant-negatively mutated connexin-43 proteins. High-resolution immunofluorescence microscopy and Western blot analysis was performed using a connexin-43 monoclonal antibody. Calcein Orange Red AM dye transfer by fluorescence imaging was used to evaluate the gap junction function. Adhesion of breast cancer cells to the pulmonary endothelium increased with cancer cells overexpressing connexin-43 and markedly decreased with cells expressing dominant-negative connexin-43. Upregulation of connexin-43 was observed in tumor cell-endothelial cell contact areas in vitro and in vivo, and in areas of intratumor blood vessels and in micrometastatic foci. Connexin-43 facilitates metastatic 'homing' by increasing adhesion of cancer cells to the lung endothelial cells. The marked upregulation of connexin-43 in tumor cell-endothelial cell contact areas, whether in preexisting 'homing' vessels or in newly formed tumor vessels, suggests that connexin-43 can serve as a potential marker of micrometastases and tumor vasculature and that it may play a role in the early incorporation of endothelial cells into small tumors as seeds for vasculogenesis.

Regulation of gap junction function and Connexin 43 expression by cytochrome P450 oxidoreductase (CYPOR)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Polusani, Srikanth R.; Kar, Rekha; Riquelme, Manuel A.

2011-08-05

Highlights: {yields} Humans with severe forms of cytochrome P450 oxidoreductase (CYPOR) mutations show bone defects as observed in Antley-Bixler Syndrome. {yields} First report showing knockdown of CYPOR in osteoblasts decreased Connexin 43 (Cx43) protein levels. Cx43 is known to play an important role in bone modeling. {yields} Knockdown of CYPOR decreased Gap Junctional Intercellular Communication and hemichannel activity. {yields} Knockdown of CYPOR decreased Cx43 in mouse primary calvarial osteoblasts. {yields} Decreased Cx43 expression was observed at the transcriptional level. -- Abstract: Cytochrome P450 oxidoreductase (CYPOR) is a microsomal electron-transferring enzyme containing both FAD and FMN as co-factors, which provides themore » reducing equivalents to various redox partners, such as cytochromes P450 (CYPs), heme oxygenase (HO), cytochrome b{sub 5} and squalene monooxygenase. Human patients with severe forms of CYPOR mutation show bone defects such as cranio- and humeroradial synostoses and long bone fractures, known as Antley-Bixler-like Syndrome (ABS). To elucidate the role of CYPOR in bone, we knocked-down CYPOR in multiple osteoblast cell lines using RNAi technology. In this study, knock-down of CYPOR decreased the expression of Connexin 43 (Cx43), known to play a critical role in bone formation, modeling, and remodeling. Knock-down of CYPOR also decreased Gap Junction Intercellular Communication (GJIC) and hemichannel activity. Promoter luciferase assays revealed that the decrease in expression of Cx43 in CYPOR knock-down cells was due to transcriptional repression. Primary osteoblasts isolated from bone specific Por knock-down mice calvariae confirmed the findings in the cell lines. Taken together, our study provides novel insights into the regulation of gap junction function by CYPOR and suggests that Cx43 may play an important role(s) in CYPOR-mediated bone defects seen in patients.« less

Regulation of germ cell development by intercellular signaling in the mammalian ovarian follicle.

PubMed

Clarke, Hugh J

2018-01-01

Prior to ovulation, the mammalian oocyte undergoes a process of differentiation within the ovarian follicle that confers on it the ability to give rise to an embryo. Differentiation comprises two phases-growth, during which the oocyte increases more than 100-fold in volume as it accumulates macromolecules and organelles that will sustain early embryogenesis; and meiotic maturation, during which the oocyte executes the first meiotic division and prepares for the second division. Entry of an oocyte into the growth phase appears to be triggered when the adjacent granulosa cells produce specific growth factors. As the oocyte grows, it elaborates a thick extracellular coat termed the zona pellucida. Nonetheless, cytoplasmic extensions of the adjacent granulosa cells, termed transzonal projections (TZPs), enable them to maintain contact-dependent communication with the oocyte. Through gap junctions located where the TZP tips meet the oocyte membrane, they provide the oocyte with products that sustain its metabolic activity and signals that regulate its differentiation. Conversely, the oocyte secretes diffusible growth factors that regulate proliferation and differentiation of the granulosa cells. Gap junction-permeable products of the granulosa cells prevent precocious initiation of meiotic maturation, and the gap junctions also enable oocyte maturation to begin in response to hormonal signals received by the granulosa cells. Development of the oocyte or the somatic compartment may also be regulated by extracellular vesicles newly identified in follicular fluid and at TZP tips, which could mediate intercellular transfer of macromolecules. Oocyte differentiation thus depends on continuous signaling interactions with the somatic cells of the follicle. WIREs Dev Biol 2018, 7:e294. doi: 10.1002/wdev.294 This article is categorized under: Gene Expression and Transcriptional Hierarchies > Cellular Differentiation Signaling Pathways > Cell Fate Signaling Early Embryonic Development > Gametogenesis. © 2017 Wiley Periodicals, Inc.

Gap Junctions

PubMed Central

Nielsen, Morten Schak; Axelsen, Lene Nygaard; Sorgen, Paul L.; Verma, Vandana; Delmar, Mario; Holstein-Rathlou, Niels-Henrik

2013-01-01

Gap junctions are essential to the function of multicellular animals, which require a high degree of coordination between cells. In vertebrates, gap junctions comprise connexins and currently 21 connexins are known in humans. The functions of gap junctions are highly diverse and include exchange of metabolites and electrical signals between cells, as well as functions, which are apparently unrelated to intercellular communication. Given the diversity of gap junction physiology, regulation of gap junction activity is complex. The structure of the various connexins is known to some extent; and structural rearrangements and intramolecular interactions are important for regulation of channel function. Intercellular coupling is further regulated by the number and activity of channels present in gap junctional plaques. The number of connexins in cell-cell channels is regulated by controlling transcription, translation, trafficking, and degradation; and all of these processes are under strict control. Once in the membrane, channel activity is determined by the conductive properties of the connexin involved, which can be regulated by voltage and chemical gating, as well as a large number of posttranslational modifications. The aim of the present article is to review our current knowledge on the structure, regulation, function, and pharmacology of gap junctions. This will be supported by examples of how different connexins and their regulation act in concert to achieve appropriate physiological control, and how disturbances of connexin function can lead to disease. © 2012 American Physiological Society. Compr Physiol 2:1981-2035, 2012. PMID:23723031

A molecular imaging analysis of C×43 association with Cdo during skeletal myoblast differentiation

NASA Astrophysics Data System (ADS)

Nosi, Daniele; Mercatelli, Raffaella; Chellini, Flaminia; Soria, Silvia; Pini, Alessandro; Formigli, Lucia; Quercioli, Franco

2014-02-01

Cell-to-cell contacts are crucial for cell differentiation. The promyogenic cell surface protein, Cdo, functions as a component of multiprotein clusters to mediate cell adhesion signaling. Connexin43, the main connexin forming gap junctions, also plays a key role in myogenesis. At least part of its effects are independent of the intercellular channel function, but the mechanisms underlying are unknown. Here, using multiple optical approaches, we provided the first evidence that Cx43 physically interacts with Cdo to form dynamic complexes during myoblast differentiation, offering clues for considering this interaction a structural basis of the channel-independent function of Cx43.

Role of gap junction intercellular communication in testicular leydig cell apoptosis induced by oxaliplatin via the mitochondrial pathway.

PubMed

Tong, Xuhui; Han, Xi; Yu, Binbin; Yu, Meiling; Jiang, Guojun; Ji, Jie; Dong, Shuying

2015-01-01

Platinum agents are widely used in the chemotherapy of testicular cancer. However, adverse reactions and resistance to such agents have limited their application in antineoplastic treatment. The aim of the present study was to determine the role of gap junction intercellular communication (GJIC) composed of Cx43 on oxaliplatin‑induced survival/apoptosis in mouse leydig normal and cancer cells using MTT, Annexin V/PI double staining assays and western blot analysis. The results showed that GJIC exerted opposite effects on the mouse leydig cancer (I-10) and normal (TM3) cell apoptosis induced by oxaliplatin. In leydig cancer cells, survival of cells exposed to oxaliplatin was substantially reduced when gap junctions formed as compared to no gap junctions. Pharmacological inhibition of gap junctions by oleamide and 18-α-glycyrrhetinic acid resulted in enhanced survival/decreased apoptosis while enhancement of gap junctions by retinoic acid led to decreased survival/increased apoptosis. These effects occurred only in high‑density cultures (gap junction formed), while the pharmacological modulations had no effects when there was no opportunity for gap junction formation. Notably, GJIC played an opposite (protective) role in normal leydig cells survival/apoptosis following exposure to oxaliplatin. Furthermore, this converse oxaliplatin‑inducing apoptosis exerted through the functional gap junction was correlated with the mitochondrial pathway‑related protein Bcl-2/Bax and caspase‑3/9. These results suggested that in testicular leydig normal/cancer cells, GJIC plays an opposite role in oxaliplatin‑induced apoptosis via the mitochondrial pathway.

Two classes of gap junction channels mediate soma-germline interactions essential for germline proliferation and gametogenesis in Caenorhabditis elegans.

PubMed

Starich, Todd A; Hall, David H; Greenstein, David

2014-11-01

In all animals examined, somatic cells of the gonad control multiple biological processes essential for germline development. Gap junction channels, composed of connexins in vertebrates and innexins in invertebrates, permit direct intercellular communication between cells and frequently form between somatic gonadal cells and germ cells. Gap junctions comprise hexameric hemichannels in apposing cells that dock to form channels for the exchange of small molecules. Here we report essential roles for two classes of gap junction channels, composed of five innexin proteins, in supporting the proliferation of germline stem cells and gametogenesis in the nematode Caenorhabditis elegans. Transmission electron microscopy of freeze-fracture replicas and fluorescence microscopy show that gap junctions between somatic cells and germ cells are more extensive than previously appreciated and are found throughout the gonad. One class of gap junctions, composed of INX-8 and INX-9 in the soma and INX-14 and INX-21 in the germ line, is required for the proliferation and differentiation of germline stem cells. Genetic epistasis experiments establish a role for these gap junction channels in germline proliferation independent of the glp-1/Notch pathway. A second class of gap junctions, composed of somatic INX-8 and INX-9 and germline INX-14 and INX-22, is required for the negative regulation of oocyte meiotic maturation. Rescue of gap junction channel formation in the stem cell niche rescues germline proliferation and uncovers a later channel requirement for embryonic viability. This analysis reveals gap junctions as a central organizing feature of many soma-germline interactions in C. elegans. Copyright © 2014 by the Genetics Society of America.

Regulation of gap junctional charge selectivity in cells coexpressing connexin 40 and connexin 43.

PubMed

Heyman, Nathanael S; Kurjiaka, David T; Ek Vitorin, Jose F; Burt, Janis M

2009-07-01

Expression of connexin 40 (Cx40) and Cx43 in cardiovascular tissues varies as a function of age, injury, and development with unknown consequences on the selectivity of junctional communication and its acute regulation. We investigated the PKC-dependent regulation of charge selectivity in junctions composed of Cx43, Cx40, or both by simultaneous assessment of junctional permeance rate constants (B(dye)) for dyes of similar size but opposite charge, N,N,N-trimethyl-2-[methyl-(7-nitro-2,1,3-benzoxadiol-4-yl)amino]ethanaminium (NBD-M-TMA; +1) and Alexa 350 (-1). The ratio of dye rate constants (B(NBD-M-TMA)/B(Alexa 350)) indicated that Cx40 junctions are cation selective (10.7 +/- 0.5), whereas Cx43 junction are nonselective (1.22 +/- 0.14). In coexpressing cells, a broad range of junctional selectivities was observed with mean cation selectivity increasing as the Cx40 to Cx43 expression ratio increased. PKC activation reduced or eliminated dye permeability of Cx43 junctions without altering their charge selectivity, had no effect on either permeability or charge selectivity of Cx40 junctions, and significantly increased the cation selectivity of junctions formed by coexpressing cells (approaching charge selectivity of Cx40 junctions). Junctions composed of Cx43 truncated at residue 257 (Cx43tr) were also not charge selective, but when Cx43tr was coexpressed with Cx40, a broad range of junctional selectivities that was unaffected by PKC activation was observed. Thus, whereas the charge selectivities of homomeric/homotypic Cx43 and Cx40 junctions appear invariant, the selectivities of junctions formed by cells coexpressing Cx40 and Cx43 vary considerably, reflecting both their relative expression levels and phosphorylation-dependent regulation. Such regulation could represent a mechanism by which coexpressing cells such as vascular endothelium and atrial cells regulate acutely the selective intercellular communication mediated by their gap junctions.

Expression pattern of adhesion molecules in junctional epithelium differs from that in other gingival epithelia.

PubMed

Hatakeyama, S; Yaegashi, T; Oikawa, Y; Fujiwara, H; Mikami, T; Takeda, Y; Satoh, M

2006-08-01

The gingival epithelium is the physiologically important interface between the bacterially colonized gingival sulcus and periodontal soft and mineralized connective tissues, requiring protection from exposure to bacteria and their products. However, of the three epithelia comprising the gingival epithelium, the junctional epithelium has much wider intercellular spaces than the sulcular epithelium and oral gingival epithelium. Hence, the aim of the present study was to characterize the cell adhesion structure in the junctional epithelium compared with the other two epithelia. Gingival epithelia excised at therapeutic flap surgery from patients with periodontitis were examined for expression of adhesion molecules by immunofluorescence. In the oral gingival epithelium and sulcular epithelium, but not in the junctional epithelium, desmoglein 1 and 2 in cell-cell contact sites were more abundant in the upper than the suprabasal layers. E-cadherin, the main transmembranous molecule of adherens junctions, was present in spinous layers of the oral gingival epithelium and sulcular epithelium, but was scarce in the junctional epithelium. In contrast, desmoglein 3 and P-cadherin were present in all layers of the junctional epithelium as well as the oral gingival epithelium and sulcular epithelium. Connexin 43 was clearly localized to spinous layers of the oral gingival epithelium, sulcular epithelium and parts of the junctional epithelium. Claudin-1 and occludin were expressed in the cell membranes of a few superficial layers of the oral gingival epithelium. These findings indicated that the junctional epithelium contains only a few desmosomes, composed of only desmoglein 3; adherens junctions are probably absent because of defective E-cadherin. Thus, the anchoring junctions connecting junctional epithelium cells are lax, causing widened intercellular spaces. In contrast, the oral gingival epithelium, which has a few tight junctions, functions as a barrier.

The alpha2-adrenoreceptor agonist dexmedetomidine protects against lipopolysaccharide-induced apoptosis via inhibition of gap junctions in lung fibroblasts.

PubMed

Zhang, Yuan; Tan, Xiaoming; Xue, Lianfang

2018-01-01

The α2-adrenoceptor inducer dexmedetomidine protects against acute lung injury (ALI), but the mechanism of this effect is largely unknown. The present study investigated the effect of dexmedetomidine on apoptosis induced by lipopolysaccharide (LPS) and the relationship between this effect and gap junction intercellular communication in human lung fibroblast cell line. Flow cytometry was used to detect apoptosis induced by LPS. Parachute dye coupling assay was used to measure gap junction function, and western blot analysis was used to determine the expression levels of connexin43 (Cx43). The results revealed that exposure of human lung fibroblast cell line to LPS for 24 h increased the apoptosis, and pretreatment of dexmedetomidine and 18α-GA significantly reduced LPS-induced apoptosis. Dexmedetomidine exposure for 1 h inhibited gap junction function mainly via a decrease in Cx43 protein levels in human lung fibroblast cell line. These results demonstrated that the inhibition of gap junction intercellular communication by dexmedetomidine affected the LPS-induced apoptosis through inhibition of gap junction function by reducing Cx43 protein levels. The present study provides evidence of a novel mechanism underlying the effects of analgesics in counteracting ALI. Copyright © 2017 Elsevier Inc. All rights reserved.

Does rat granulation tissue maturation involve gap junction communications?

PubMed

Au, Katherine; Ehrlich, H Paul

2007-07-01

Wound healing, a coordinated process, proceeds by sequential changes in cell differentiation and terminates with the deposition of a new connective tissue matrix, a scar. Initially, there is the migratory fibroblast, followed by the proliferative fibroblast, then the synthetic fibroblast, which transforms into the myofibroblast, and finally the apoptotic fibroblast. Gap junction intercellular communications are proposed to coordinate the stringent control of fibroblast phenotypic changes. Does added oleamide, a natural fatty acid that blocks gap junction intercellular communications, alter the phenotypic progression of wound fibroblasts? Pairs of polyvinyl alcohol sponges attached to Alzet pumps, which constantly pumped either oleamide or vehicle solvent, were implanted subcutaneously into three rats. On day 8, implants were harvested and evaluated histologically and biochemically. The capsule of oleamide-treated sponge contained closely packed fibroblasts with little connective tissue between them. The birefringence intensity of that connective tissue was reduced, indicating a reduced density of collagen fiber bundles. Myofibroblasts, identified immunohistologically by alpha-smooth muscle actin-stained stress fibers, were reduced in oleamide-treated implants. Western blot analysis showing less alpha-smooth muscle actin confirmed the reduced density of myofibroblasts. It appears that oleamide retards the progression of wound repair, where less connective tissue is deposited, the collagen is less organized, and the appearance of myofibroblasts is impaired. These findings support the hypothesis that gap junction intercellular communications between wound fibroblasts in granulation tissue play a role in the progression of repair and the maturation of granulation tissue into scar.

Innexin-3 forms connexin-like intercellular channels.

PubMed

Landesman, Y; White, T W; Starich, T A; Shaw, J E; Goodenough, D A; Paul, D L

1999-07-01

Innexins comprise a large family of genes that are believed to encode invertebrate gap junction channel-forming proteins. However, only two Drosophila innexins have been directly tested for the ability to form intercellular channels and only one of those was active. Here we tested the ability of Caenorhabditis elegans family members INX-3 and EAT-5 to form intercellular channels between paired Xenopus oocytes. We show that expression of INX-3 but not EAT-5, induces electrical coupling between the oocyte pairs. In addition, analysis of INX-3 voltage and pH gating reveals a striking degree of conservation in the functional properties of connexin and innnexin channels. These data strongly support the idea that innexin genes encode intercellular channels.

Role of the gut endoderm in relaying left-right patterning in mice.

PubMed

Viotti, Manuel; Niu, Lei; Shi, Song-Hai; Hadjantonakis, Anna-Katerina

2012-01-01

Establishment of left-right (LR) asymmetry occurs after gastrulation commences and utilizes a conserved cascade of events. In the mouse, LR symmetry is broken at a midline structure, the node, and involves signal relay to the lateral plate, where it results in asymmetric organ morphogenesis. How information transmits from the node to the distantly situated lateral plate remains unclear. Noting that embryos lacking Sox17 exhibit defects in both gut endoderm formation and LR patterning, we investigated a potential connection between these two processes. We observed an endoderm-specific absence of the critical gap junction component, Connexin43 (Cx43), in Sox17 mutants. Iontophoretic dye injection experiments revealed planar gap junction coupling across the gut endoderm in wild-type but not Sox17 mutant embryos. They also revealed uncoupling of left and right sides of the gut endoderm in an isolated domain of gap junction intercellular communication at the midline, which in principle could function as a barrier to communication between the left and right sides of the embryo. The role for gap junction communication in LR patterning was confirmed by pharmacological inhibition, which molecularly recapitulated the mutant phenotype. Collectively, our data demonstrate that Cx43-mediated communication across gap junctions within the gut endoderm serves as a mechanism for information relay between node and lateral plate in a process that is critical for the establishment of LR asymmetry in mice.

Improving cardiac gap junction communication as a new antiarrhythmic mechanism: the action of antiarrhythmic peptides.

PubMed

Dhein, Stefan; Hagen, Anja; Jozwiak, Joanna; Dietze, Anna; Garbade, Jens; Barten, Markus; Kostelka, Martin; Mohr, Friedrich-Wilhelm

2010-03-01

Co-ordinated electrical activation of the heart is maintained by intercellular coupling of cardiomyocytes via gap junctional channels located in the intercalated disks. These channels consist of two hexameric hemichannels, docked to each other, provided by either of the adjacent cells. Thus, a complete gap junction channel is made from 12 protein subunits, the connexins. While 21 isoforms of connexins are presently known, cardiomyocytes typically are coupled by Cx43 (most abundant), Cx40 or Cx45. Some years ago, antiarrhythmic peptides were discovered and synthesised, which were shown to increase macroscopic gap junction conductance (electrical coupling) and enhance dye transfer (metabolic coupling). The lead substance of these peptides is AAP10 (H-Gly-Ala-Gly-Hyp-Pro-Tyr-CONH(2)), a peptide with a horseshoe-like spatial structure as became evident from two-dimensional nuclear magnetic resonance studies. A stable D: -amino-acid derivative of AAP10, rotigaptide, as well as a non-peptide analogue, gap-134, has been developed in recent years. Antiarrhythmic peptides act on Cx43 and Cx45 gap junctions but not on Cx40 channels. AAP10 has been shown to enhance intercellular communication in rat, rabbit and human cardiomyocytes. Antiarrhythmic peptides are effective against ventricular tachyarrhythmias, such as late ischaemic (type IB) ventricular fibrillation, CaCl(2) or aconitine-induced arrhythmia. Interestingly, the effect of antiarrhythmic peptides is higher in partially uncoupled cells and was shown to be related to maintained Cx43 phosphorylation, while arrhythmogenic conditions like ischaemia result in Cx43 dephosphorylation and intercellular decoupling. It is still a matter of debate whether these drugs also act against atrial fibrillation. The present review outlines the development of this group of peptides and derivatives, their mode of action and molecular mechanisms, and discusses their possible therapeutic potential.

Rescue of perfluorooctanesulfonate (PFOS)-mediated Sertoli cell injury by overexpression of gap junction protein connexin 43

NASA Astrophysics Data System (ADS)

Li, Nan; Mruk, Dolores D.; Chen, Haiqi; Wong, Chris K. C.; Lee, Will M.; Cheng, C. Yan

2016-07-01

Perfluorooctanesulfonate (PFOS) is an environmental toxicant used in developing countries, including China, as a stain repellent for clothing, carpets and draperies, but it has been banned in the U.S. and Canada since the late 2000s. PFOS perturbed the Sertoli cell tight junction (TJ)-permeability barrier, causing disruption of actin microfilaments in cell cytosol, perturbing the localization of cell junction proteins (e.g., occluden-ZO-1, N-cadherin-ß-catenin). These changes destabilized Sertoli cell blood-testis barrier (BTB) integrity. These findings suggest that human exposure to PFOS might induce BTB dysfunction and infertility. Interestingly, PFOS-induced Sertoli cell injury associated with a down-regulation of the gap junction (GJ) protein connexin43 (Cx43). We next investigated if overexpression of Cx43 in Sertoli cells could rescue the PFOS-induced cell injury. Indeed, overexpression of Cx43 in Sertoli cells with an established TJ-barrier blocked the disruption in PFOS-induced GJ-intercellular communication, resulting in the re-organization of actin microfilaments, which rendered them similar to those in control cells. Furthermore, cell adhesion proteins that utilized F-actin for attachment became properly distributed at the cell-cell interface, resealing the disrupted TJ-barrier. In summary, Cx43 is a good target that might be used to manage PFOS-induced reproductive dysfunction.

Reversible Opening of Intercellular Junctions of Intestinal Epithelial and Brain Endothelial Cells With Tight Junction Modulator Peptides.

PubMed

Bocsik, Alexandra; Walter, Fruzsina R; Gyebrovszki, Andrea; Fülöp, Lívia; Blasig, Ingolf; Dabrowski, Sebastian; Ötvös, Ferenc; Tóth, András; Rákhely, Gábor; Veszelka, Szilvia; Vastag, Monika; Szabó-Révész, Piroska; Deli, Mária A

2016-02-01

The intercellular junctions restrict the free passage of hydrophilic compounds through the paracellular clefts. Reversible opening of the tight junctions of biological barriers is investigated as one of the ways to increase drug delivery to the systemic circulation or the central nervous system. Six peptides, ADT-6, HAV-6, C-CPE, 7-mer (FDFWITP, PN-78), AT-1002, and PN-159, acting on different integral membrane and linker junctional proteins were tested on Caco-2 intestinal epithelial cell line and a coculture model of the blood-brain barrier. All peptides tested in nontoxic concentrations showed a reversible tight junctions modulating effect and were effective to open the paracellular pathway for the marker molecules fluorescein and albumin. The change in the structure of cell-cell junctions was verified by immunostaining for occludin, claudin-4,-5, ZO-1, β-catenin, and E-cadherin. Expression levels of occludin and claudins were measured in both models. We could demonstrate a selectivity of C-CPE, ADT-6, and HAV-6 peptides for epithelial cells and 7-mer and AT-1002 peptides for brain endothelial cells. PN-159 was the most effective modulator of junctional permeability in both models possibly acting via claudin-1 and -5. Our results indicate that these peptides can be effectively and selectively used as potential pharmaceutical excipients to improve drug delivery across biological barriers. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Quantification of gap junction selectivity.

PubMed

Ek-Vitorín, Jose F; Burt, Janis M

2005-12-01

Gap junctions, which are essential for functional coordination and homeostasis within tissues, permit the direct intercellular exchange of small molecules. The abundance and diversity of this exchange depends on the number and selectivity of the comprising channels and on the transjunctional gradient for and chemical character of the permeant molecules. Limited knowledge of functionally significant permeants and poor detectability of those few that are known have made it difficult to define channel selectivity. Presented herein is a multifaceted approach to the quantification of gap junction selectivity that includes determination of the rate constant for intercellular diffusion of a fluorescent probe (k2-DYE) and junctional conductance (gj) for each junction studied, such that the selective permeability (k2-DYE/gj) for dyes with differing chemical characteristics or junctions with differing connexin (Cx) compositions (or treatment conditions) can be compared. In addition, selective permeability can be correlated using single-channel conductance when this parameter is also measured. Our measurement strategy is capable of detecting 1) rate constants and selective permeabilities that differ across three orders of magnitude and 2) acute changes in that rate constant. Using this strategy, we have shown that 1) the selective permeability of Cx43 junctions to a small cationic dye varied across two orders of magnitude, consistent with the hypothesis that the various channel configurations adopted by Cx43 display different selective permeabilities; and 2) the selective permeability of Cx37 vs. Cx43 junctions was consistently and significantly lower.

Intercellular communication in the immune system: differential expression of connexin40 and 43, and perturbation of gap junction channel functions in peripheral blood and tonsil human lymphocyte subpopulations

PubMed Central

Oviedo‐orta, E; Hoy, T; Evans, W H

2000-01-01

The distribution and function of connexins (integral membrane proteins assembled into gap junction intercellular communication channels) were studied in human lymphocyte subpopulations. The expression of mRNA encoding connexins in peripheral blood and tonsil‐derived T, B and natural killer (NK) lymphocytes was examined. Connexin43 (Cx43) mRNA was expressed in peripheral blood and tonsil lymphocytes, but Cx40 mRNA expression was confined to tonsil‐derived T and B lymphocytes; Cx26, Cx32, Cx37 and Cx45 were not detected by reverse transcription–polymerase chain reaction (RT–PCR). Western blot analysis also demonstrated the presence of Cx40 and Cx43 proteins in T and B lymphocytes in a manner coincidental to the mRNA detection. Stimulation in vitro of T and B lymphocytes with phytohaemagglutinin (PHA) and lipopolysaccharide (LPS), respectively, increased Cx40 and Cx43 protein expression. Flow cytometric analysis, using antibodies to extracellular loop amino acid sequences of connexins, confirmed the surface expression of connexins in all lymphocyte subpopulations. Assembly of connexins into gap junctions providing direct intercellular channels linking attached lymphocytes was demonstrated by using a dye transfer technique. The exchange of dye between lymphocytes was inhibited by a connexin extracellular loop mimetic peptide and α‐glycyrrhetinic acid, two reagents that restrict intercellular communication across gap junctions. Dye coupling occurred between homologous and heterologous co‐cultures of T and B lymphocytes, and was not influenced by their stimulation with PHA and LPS. The connexin mimetic peptide caused a significant decrease in the in vitro synthesis of immunoglobulin M (IgM) by T‐ and B‐lymphocyte co‐cultured populations in the presence or absence of stimulation by PHA. The results identify connexins as important cell surface components that modulate immune processes. PMID:10792506

Measurement of Single Channel Currents from Cardiac Gap Junctions

NASA Astrophysics Data System (ADS)

Veenstra, Richard D.; Dehaan, Robert L.

1986-08-01

Cardiac gap junctions consist of arrays of integral membrane proteins joined across the intercellular cleft at points of cell-to-cell contact. These junctional proteins are thought to form pores through which ions can diffuse from cytosol to cytosol. By monitoring whole-cell currents in pairs of embryonic heart cells with two independent patch-clamp circuits, the properties of single gap junction channels have been investigated. These channels had a conductance of about 165 picosiemens and underwent spontaneous openings and closings that were independent of voltage. Channel activity and macroscopic junctional conductance were both decreased by the uncoupling agent 1-octanol.

‘Gap Junctions and Cancer: Communicating for 50 Years’

PubMed Central

Aasen, Trond; Mesnil, Marc; Naus, Christian C.; Lampe, Paul D.; Laird, Dale W.

2017-01-01

Fifty years ago, tumour cells were found to lack electrical coupling, leading to the hypothesis that loss of direct intercellular communication is commonly associated with cancer onset and progression. Subsequent studies linked this phenomenon to gap junctions composed of connexin proteins. While many studies support the notion that connexins are tumour suppressors, recent evidence suggests that, in some tumour types, they may facilitate specific stages of tumour progression through both junctional and non-junctional signalling pathways. This Timeline article highlights the milestones connecting gap junctions to cancer, and underscores important unanswered questions, controversies and therapeutic opportunities in the field. PMID:27782134

Adipocytes in both brown and white adipose tissue of adult mice are functionally connected via gap junctions: implications for Chagas disease.

PubMed

Burke, Shoshana; Nagajyothi, Fnu; Thi, Mia M; Hanani, Menachem; Scherer, Philipp E; Tanowitz, Herbert B; Spray, David C

2014-11-01

Adipose tissue serves as a host reservoir for the protozoan Trypanosoma cruzi, the causative organism in Chagas disease. Gap junctions interconnect cells of most tissues, serving to synchronize cell activities including secretion in glandular tissue, and we have previously demonstrated that gap junctions are altered in various tissues and cells infected with T. cruzi. Herein, we examined the gap junction protein connexin 43 (Cx43) expression in infected adipose tissues. Adipose tissue is the largest endocrine organ of the body and is also involved in other physiological functions. In mammals, it is primarily composed of white adipocytes. Although gap junctions are a prominent feature of brown adipocytes, they have not been explored extensively in white adipocytes, especially in the setting of infection. Thus, we examined functional coupling in both white and brown adipocytes in mice. Injection of electrical current or the dye Lucifer Yellow into adipocytes within fat tissue spread to adjacent cells, which was reduced by treatment with agents known to block gap junctions. Moreover, Cx43 was detected in both brown and white fat tissue. At thirty and ninety days post-infection, Cx43 was downregulated in brown adipocytes and upregulated in white adipocytes. Gap junction-mediated intercellular communication likely contributes to hormone secretion and other functions in white adipose tissue and to nonshivering thermogenesis in brown fat, and modulation of the coupling by T. cruzi infection is expected to impact these functions. Copyright © 2014. Published by Elsevier Masson SAS.

Polarized release of T-cell-receptor-enriched microvesicles at the immunological synapse.

PubMed

Choudhuri, Kaushik; Llodrá, Jaime; Roth, Eric W; Tsai, Jones; Gordo, Susana; Wucherpfennig, Kai W; Kam, Lance C; Stokes, David L; Dustin, Michael L

2014-03-06

The recognition events that mediate adaptive cellular immunity and regulate antibody responses depend on intercellular contacts between T cells and antigen-presenting cells (APCs). T-cell signalling is initiated at these contacts when surface-expressed T-cell receptors (TCRs) recognize peptide fragments (antigens) of pathogens bound to major histocompatibility complex molecules (pMHC) on APCs. This, along with engagement of adhesion receptors, leads to the formation of a specialized junction between T cells and APCs, known as the immunological synapse, which mediates efficient delivery of effector molecules and intercellular signals across the synaptic cleft. T-cell recognition of pMHC and the adhesion ligand intercellular adhesion molecule-1 (ICAM-1) on supported planar bilayers recapitulates the domain organization of the immunological synapse, which is characterized by central accumulation of TCRs, adjacent to a secretory domain, both surrounded by an adhesive ring. Although accumulation of TCRs at the immunological synapse centre correlates with T-cell function, this domain is itself largely devoid of TCR signalling activity, and is characterized by an unexplained immobilization of TCR-pMHC complexes relative to the highly dynamic immunological synapse periphery. Here we show that centrally accumulated TCRs are located on the surface of extracellular microvesicles that bud at the immunological synapse centre. Tumour susceptibility gene 101 (TSG101) sorts TCRs for inclusion in microvesicles, whereas vacuolar protein sorting 4 (VPS4) mediates scission of microvesicles from the T-cell plasma membrane. The human immunodeficiency virus polyprotein Gag co-opts this process for budding of virus-like particles. B cells bearing cognate pMHC receive TCRs from T cells and initiate intracellular signals in response to isolated synaptic microvesicles. We conclude that the immunological synapse orchestrates TCR sorting and release in extracellular microvesicles. These microvesicles deliver transcellular signals across antigen-dependent synapses by engaging cognate pMHC on APCs.

Polarized release of T-cell-receptor-enriched microvesicles at the immunological synapse

NASA Astrophysics Data System (ADS)

Choudhuri, Kaushik; Llodrá, Jaime; Roth, Eric W.; Tsai, Jones; Gordo, Susana; Wucherpfennig, Kai W.; Kam, Lance C.; Stokes, David L.; Dustin, Michael L.

2014-03-01

The recognition events that mediate adaptive cellular immunity and regulate antibody responses depend on intercellular contacts between T cells and antigen-presenting cells (APCs). T-cell signalling is initiated at these contacts when surface-expressed T-cell receptors (TCRs) recognize peptide fragments (antigens) of pathogens bound to major histocompatibility complex molecules (pMHC) on APCs. This, along with engagement of adhesion receptors, leads to the formation of a specialized junction between T cells and APCs, known as the immunological synapse, which mediates efficient delivery of effector molecules and intercellular signals across the synaptic cleft. T-cell recognition of pMHC and the adhesion ligand intercellular adhesion molecule-1 (ICAM-1) on supported planar bilayers recapitulates the domain organization of the immunological synapse, which is characterized by central accumulation of TCRs, adjacent to a secretory domain, both surrounded by an adhesive ring. Although accumulation of TCRs at the immunological synapse centre correlates with T-cell function, this domain is itself largely devoid of TCR signalling activity, and is characterized by an unexplained immobilization of TCR-pMHC complexes relative to the highly dynamic immunological synapse periphery. Here we show that centrally accumulated TCRs are located on the surface of extracellular microvesicles that bud at the immunological synapse centre. Tumour susceptibility gene 101 (TSG101) sorts TCRs for inclusion in microvesicles, whereas vacuolar protein sorting 4 (VPS4) mediates scission of microvesicles from the T-cell plasma membrane. The human immunodeficiency virus polyprotein Gag co-opts this process for budding of virus-like particles. B cells bearing cognate pMHC receive TCRs from T cells and initiate intracellular signals in response to isolated synaptic microvesicles. We conclude that the immunological synapse orchestrates TCR sorting and release in extracellular microvesicles. These microvesicles deliver transcellular signals across antigen-dependent synapses by engaging cognate pMHC on APCs.

Terbinafine inhibits gap junctional intercellular communication.

PubMed

Lee, Ju Yeun; Yoon, Sei Mee; Choi, Eun Ju; Lee, Jinu

2016-09-15

Terbinafine is an antifungal agent that selectively inhibits fungal sterol synthesis by blocking squalene epoxidase. We evaluated the effect of terbinafine on gap junctional intercellular communication (GJIC). Fluorescence recovery after photobleaching (FRAP) and I-YFP GJIC assays revealed that terbinafine inhibits GJIC in a reversible and dose-dependent manner in FRT-Cx43 and LN215 cells. Treatment with terbinafine did not affect Cx43 phosphorylation status or intracellular Ca(2+) concentration, well-known action mechanisms of various GJIC blockers. While a structurally related chemical, naftifine, attenuated GJIC, epigallocatechin gallate, another potent squalene epoxidase inhibitor with a different structure, did not. These results suggest that terbinafine inhibits GJIC with a so far unknown mechanism of action. Copyright © 2016 Elsevier Inc. All rights reserved.

UNC-1 regulates gap junctions important to locomotion in C. elegans.

PubMed

Chen, Bojun; Liu, Qiang; Ge, Qian; Xie, Jia; Wang, Zhao-Wen

2007-08-07

In C. elegans, loss-of-function (lf) mutations of the stomatin-like protein (SLP) UNC-1 and the innexin UNC-9 inhibit locomotion [1, 2] and modulate sensitivity to volatile anesthetics [3, 4]. It was unknown why unc-1(lf) and unc-9(lf) mutants have similar phenotypes. We tested the hypothesis that UNC-1 is a regulator of gap junctions formed by UNC-9. Analyses of junctional currents between body-wall muscle cells showed that electrical coupling was inhibited to a similar degree in unc-1(lf), unc-9(lf), and unc-1(lf);unc-9(lf) double mutants, suggesting that UNC-1 and UNC-9 function together. Expression of Punc-1::DsRED2 and Punc-9::GFP transcriptional fusions suggests that unc-1 and unc-9 are coexpressed in neurons and body-wall muscle cells. Immunohistochemistry showed that UNC-1 and UNC-9 colocalized at intercellular junctions and that unc-1(lf) did not alter UNC-9 expression or subcellular localization. Bimolecular fluorescence complementation (BiFC) assays suggest that UNC-1 and UNC-9 are physically very close at intercellular junctions. Targeted rescue experiments suggest that UNC-9 and UNC-1 function predominantly in neurons to control locomotion. Thus, in addition to the recently reported function of regulating mechanosensitive ion channels [5, 6], SLPs might have a novel function of regulating gap junctions.

Lovastatin inhibits gap junctional communication in cultured aortic smooth muscle cells.

PubMed

Shen, Jing; Wang, Li-Hong; Zheng, Liang-Rong; Zhu, Jian-Hua; Hu, Shen-Jiang

2010-09-01

Gap junctions, which serve as intercellular channels that allow the passage of ions and other small molecules between neighboring cells, play an important role in vital functions, including the regulation of cell growth, differentiation, and development. Statins, the 3-hydroxy-3-methylglutaryl-coenzymeA (HMG-CoA) reductase inhibitors, have been shown to inhibit the migration and proliferation of smooth muscle cells (SMCs) leading to an antiproliferative effect. Recent studies have shown that statins can reduce gap junction protein connexin43 (Cx43) expression both in vivo and in vitro. However, little work has been done on the effects of statins on gap junctional intercellular communication (GJIC). We hypothesized in this study that lovastatin inhibits vascular smooth muscle cells (VSMCs) migration through the inhibition of the GJIC. Rat aortic SMCs (RASMCs) were exposed to lovastatin. Vascular smooth muscle cells migration was then assessed with a Transwell migration assay. Gap junctional intercellular communication was determined by using fluorescence recovery after photobleaching (FRAP) analysis, which was performed with a laser-scanning confocal microscope. The migration of the cultured RASMCs were detected by Transwell system. Cell migration was dose-dependently inhibited with lovastatin. Compared with that in the control (110 ± 26), the number of migrated SMCs was significantly reduced to 72 ± 24 (P < .05), 62 ± 18 (P < .01), and 58 ± 19 (P < .01) at the concentration of 0.4, 2, and 10 umol/L, per field. The rate of fluorescence recovery (R) at 5 minutes after photobleaching was adopted as the functional index of GJIC. The R- value of cells exposed to lovastatin 10 umol/L for 48 hours was 24.38% ± 4.84%, whereas the cells in the control group had an R- value of 36.11% ± 10.53%, demonstrating that the GJIC of RASMCs was significantly inhibited by lovastatin (P < .01). Smaller concentrations of lovastatin 0.08 umol/L did not change gap junction coupling (P > .05). These results suggest that lovastatin inhibits migration in a dose-dependent manner by attenuating JIC. Suppression of gap junction function could add another explanation of statin-induced antiproliferative effect.

Heme Oxygenase-1 Protects Corexit 9500A-Induced Respiratory Epithelial Injury across Species

PubMed Central

Oliva, Octavio M.; Karki, Suman; Surolia, Ranu; Wang, Zheng; Watson, R. Douglas; Thannickal, Victor J.; Powell, Mickie; Watts, Stephen; Kulkarni, Tejaswini; Batra, Hitesh; Bolisetty, Subhashini; Agarwal, Anupam; Antony, Veena B.

2015-01-01

The effects of Corexit 9500A (CE) on respiratory epithelial surfaces of terrestrial mammals and marine animals are largely unknown. This study investigated the role of CE-induced heme oxygenase-1 (HO-1), a cytoprotective enzyme with anti-apoptotic and antioxidant activity, in human bronchial airway epithelium and the gills of exposed aquatic animals. We evaluated CE-mediated alterations in human airway epithelial cells, mice lungs and gills from zebrafish and blue crabs. Our results demonstrated that CE induced an increase in gill epithelial edema and human epithelial monolayer permeability, suggesting an acute injury caused by CE exposure. CE induced the expression of HO-1 as well as C-reactive protein (CRP) and NADPH oxidase 4 (NOX4), which are associated with ROS production. Importantly, CE induced caspase-3 activation and subsequent apoptosis of epithelial cells. The expression of the intercellular junctional proteins, such as tight junction proteins occludin, zonula occludens (ZO-1), ZO-2 and adherens junctional proteins E-cadherin and Focal Adhesion Kinase (FAK), were remarkably inhibited by CE, suggesting that these proteins are involved in CE-induced increased permeability and subsequent apoptosis. The cytoskeletal protein F-actin was also disrupted by CE. Treatment with carbon monoxide releasing molecule-2 (CORM-2) significantly inhibited CE-induced ROS production, while the addition of HO-1 inhibitor, significantly increased CE-induced ROS production and apoptosis, suggesting a protective role of HO-1 or its reaction product, CO, in CE-induced apoptosis. Using HO-1 knockout mice, we further demonstrated that HO-1 protected against CE-induced inflammation and cellular apoptosis and corrected CE-mediated inhibition of E-cadherin and FAK. These observations suggest that CE activates CRP and NOX4-mediated ROS production, alters permeability by inhibition of junctional proteins, and leads to caspase-3 dependent apoptosis of epithelial cells, while HO-1 and its reaction products protect against oxidative stress and apoptosis. PMID:25835394

Connexin-Mediated Functional and Metabolic Coupling Between Astrocytes and Neurons.

PubMed

Mayorquin, Lady C; Rodriguez, Andrea V; Sutachan, Jhon-Jairo; Albarracín, Sonia L

2018-01-01

The central nervous system (CNS) requires sophisticated regulation of neuronal activity. This modulation is partly accomplished by non-neuronal cells, characterized by the presence of transmembrane gap junctions (GJs) and hemichannels (HCs). This allows small molecule diffusion to guarantee neuronal synaptic activity and plasticity. Astrocytes are metabolically and functionally coupled to neurons by the uptake, binding and recycling of neurotransmitters. In addition, astrocytes release metabolites, such as glutamate, glutamine, D-serine, adenosine triphosphate (ATP) and lactate, regulating synaptic activity and plasticity by pre- and postsynaptic mechanisms. Uncoupling neuroglial communication leads to alterations in synaptic transmission that can be detrimental to neuronal circuit function and behavior. Therefore, understanding the pathways and mechanisms involved in this intercellular communication is fundamental for the search of new targets that can be used for several neurological disease treatments. This review will focus on molecular mechanisms mediating physiological and pathological coupling between astrocytes and neurons through GJs and HCs.

The fine structure of the rectal pads of Zorotypus caudelli Karny (Zoraptera, Insecta).

PubMed

Dallai, R; Mercati, D; Mashimo, Y; Machida, R; Beutel, R G

2016-07-01

The rectal pads of a species of the controversial polyneopteran order Zoraptera were examined using histological sections and TEM micrographs. Six pads are present along the thin rectal epithelium. Each pad consists of a few large principal cells surrounded by flattened junctional cells, which extend also beneath the principal cells. The cells are lined by a thin apical cuticle. No basal cells and no cavity have been observed beneath the pad. Principal cells have a regular layer of apical microvilli and are joined by intercellular septate junctions, which are interrupted by short dilatations of the intercellular space. At these levels the two adjacent plasma membranes are joined by short zonulae adhaerentes. In the cytoplasm, a rich system of strict associations between lateral plasma membranes and mitochondria forms scalariform junctions. Rectal pads share ultrastructural features with similar excretory organs of several neopteran groups, in particular with Blattodea (roaches and termites) and Thysanoptera, and are involved in fluid reabsorption and ion regulation. Copyright © 2016 Elsevier Ltd. All rights reserved.

[Effects of Chinese herbal compound for supplementing qi and activating blood circulation on actin, Cx43 expressions and gap junctional intercellular communication functions of myocardial cells in patients with Coxsackie virus B 3 viral myocarditis].

PubMed

Zhang, Ming-xue; He, Wei; Gu, Ping

2010-08-01

To observe the effect of Chinese herbal compound for supplementing qi and activating blood circulation (CHC) on the gap junctional intercellular communication (GJIC) function of myocardial cells in patients with Coxsackie virus B 3 (CVB3) viral myocarditis. Expressions of actin and connexin43 (Cx43) in myocardial cells of patients arranged in three groups (the normal control group, the viral infected group and the CHC treated group) were detected by immunohistochemical method; the fluorescence photobleaching recovery rate of cells was detected by laser scanning confocal microscope. As compared with the viral infected group, the expressions of actin and Cx43 were increased and the GJIC function was improved in the CHC treated group. CHC could antagonize viral injury on skeleton protein, and repair the structure of gap junction channel to improve the GJIC function of myocardial cells after being attacked by CVB3.

Applying 3D-FRAP microscopy to analyse gap junction-dependent shuttling of small antisense RNAs between cardiomyocytes.

PubMed

Lemcke, Heiko; Peukert, Janine; Voronina, Natalia; Skorska, Anna; Steinhoff, Gustav; David, Robert

2016-09-01

Small antisense RNAs like miRNA and siRNA are of crucial importance in cardiac physiology, pathology and, moreover, can be applied as therapeutic agents for the treatment of cardiovascular diseases. Identification of novel strategies for miRNA/siRNA therapy requires a comprehensive understanding of the underlying mechanisms. Emerging data suggest that small RNAs are transferred between cells via gap junctions and provoke gene regulatory effects in the recipient cell. To elucidate the role of miRNA/siRNA as signalling molecules, suitable tools are required that will allow the analysis of these small RNAs at the cellular level. In the present study, we applied 3 dimensional fluorescence recovery after photo bleaching microscopy (3D-FRAP) to visualise and quantify the gap junctional exchange of small RNAs between neonatal cardiomyocytes in real time. Cardiomyocytes were transfected with labelled miRNA and subjected to FRAP microscopy. Interestingly, we observed recovery rates of 21% already after 13min, indicating strong intercellular shuttling of miRNA, which was significantly reduced when connexin43 was knocked down. Flow cytometry analysis confirmed our FRAP results. Furthermore, using an EGFP/siRNA reporter construct we demonstrated that the intercellular transfer does not affect proper functioning of small RNAs, leading to marker gene silencing in the recipient cell. Our results show that 3D-FRAP microscopy is a straightforward, non-invasive live cell imaging technique to evaluate the GJ-dependent shuttling of small RNAs with high spatio-temporal resolution. Moreover, the data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation where small RNAs act as signalling molecules within the intercellular network. Copyright © 2016 Elsevier Ltd. All rights reserved.

Nitric oxide-mediated bystander signal transduction induced by heavy-ion microbeam irradiation

NASA Astrophysics Data System (ADS)

Tomita, Masanori; Matsumoto, Hideki; Funayama, Tomoo; Yokota, Yuichiro; Otsuka, Kensuke; Maeda, Munetoshi; Kobayashi, Yasuhiko

2015-07-01

In general, a radiation-induced bystander response is known to be a cellular response induced in non-irradiated cells after receiving bystander signaling factors released from directly irradiated cells within a cell population. Bystander responses induced by high-linear energy transfer (LET) heavy ions at low fluence are an important health problem for astronauts in space. Bystander responses are mediated via physical cell-cell contact, such as gap-junction intercellular communication (GJIC) and/or diffusive factors released into the medium in cell culture conditions. Nitric oxide (NO) is a well-known major initiator/mediator of intercellular signaling within culture medium during bystander responses. In this study, we investigated the NO-mediated bystander signal transduction induced by high-LET argon (Ar)-ion microbeam irradiation of normal human fibroblasts. Foci formation by DNA double-strand break repair proteins was induced in non-irradiated cells, which were co-cultured with those irradiated by high-LET Ar-ion microbeams in the same culture plate. Foci formation was suppressed significantly by pretreatment with an NO scavenger. Furthermore, NO-mediated reproductive cell death was also induced in bystander cells. Phosphorylation of NF-κB and Akt were induced during NO-mediated bystander signaling in the irradiated and bystander cells. However, the activation of these proteins depended on the incubation time after irradiation. The accumulation of cyclooxygenase-2 (COX-2), a downstream target of NO and NF-κB, was observed in the bystander cells 6 h after irradiation but not in the directly irradiated cells. Our findings suggest that Akt- and NF-κB-dependent signaling pathways involving COX-2 play important roles in NO-mediated high-LET heavy-ion-induced bystander responses. In addition, COX-2 may be used as a molecular marker of high-LET heavy-ion-induced bystander cells to distinguish them from directly irradiated cells, although this may depend on the time after irradiation.

Anthrax edema toxin disrupts distinct steps in Rab11-dependent junctional transport

PubMed Central

Guichard, Annabel; Jain, Prashant; Moayeri, Mahtab; Cruz-Moreno, Beatriz; Leppla, Stephen H.; Nizet, Victor

2017-01-01

Various bacterial toxins circumvent host defenses through overproduction of cAMP. In a previous study, we showed that edema factor (EF), an adenylate cyclase from Bacillus anthracis, disrupts endocytic recycling mediated by the small GTPase Rab11. As a result, cargo proteins such as cadherins fail to reach inter-cellular junctions. In the present study, we provide further mechanistic dissection of Rab11 inhibition by EF using a combination of Drosophila and mammalian systems. EF blocks Rab11 trafficking after the GTP-loading step, preventing a constitutively active form of Rab11 from delivering cargo vesicles to the plasma membrane. Both of the primary cAMP effector pathways -PKA and Epac/Rap1- contribute to inhibition of Rab11-mediated trafficking, but act at distinct steps of the delivery process. PKA acts early, preventing Rab11 from associating with its effectors Rip11 and Sec15. In contrast, Epac functions subsequently via the small GTPase Rap1 to block fusion of recycling endosomes with the plasma membrane, and appears to be the primary effector of EF toxicity in this process. Similarly, experiments conducted in mammalian systems reveal that Epac, but not PKA, mediates the activity of EF both in cell culture and in vivo. The small GTPase Arf6, which initiates endocytic retrieval of cell adhesion components, also contributes to junctional homeostasis by counteracting Rab11-dependent delivery of cargo proteins at sites of cell-cell contact. These studies have potentially significant practical implications, since chemical inhibition of either Arf6 or Epac blocks the effect of EF in cell culture and in vivo, opening new potential therapeutic avenues for treating symptoms caused by cAMP-inducing toxins or related barrier-disrupting pathologies. PMID:28945820

Anthrax edema toxin disrupts distinct steps in Rab11-dependent junctional transport.

PubMed

Guichard, Annabel; Jain, Prashant; Moayeri, Mahtab; Schwartz, Ruth; Chin, Stephen; Zhu, Lin; Cruz-Moreno, Beatriz; Liu, Janet Z; Aguilar, Bernice; Hollands, Andrew; Leppla, Stephen H; Nizet, Victor; Bier, Ethan

2017-09-01

Various bacterial toxins circumvent host defenses through overproduction of cAMP. In a previous study, we showed that edema factor (EF), an adenylate cyclase from Bacillus anthracis, disrupts endocytic recycling mediated by the small GTPase Rab11. As a result, cargo proteins such as cadherins fail to reach inter-cellular junctions. In the present study, we provide further mechanistic dissection of Rab11 inhibition by EF using a combination of Drosophila and mammalian systems. EF blocks Rab11 trafficking after the GTP-loading step, preventing a constitutively active form of Rab11 from delivering cargo vesicles to the plasma membrane. Both of the primary cAMP effector pathways -PKA and Epac/Rap1- contribute to inhibition of Rab11-mediated trafficking, but act at distinct steps of the delivery process. PKA acts early, preventing Rab11 from associating with its effectors Rip11 and Sec15. In contrast, Epac functions subsequently via the small GTPase Rap1 to block fusion of recycling endosomes with the plasma membrane, and appears to be the primary effector of EF toxicity in this process. Similarly, experiments conducted in mammalian systems reveal that Epac, but not PKA, mediates the activity of EF both in cell culture and in vivo. The small GTPase Arf6, which initiates endocytic retrieval of cell adhesion components, also contributes to junctional homeostasis by counteracting Rab11-dependent delivery of cargo proteins at sites of cell-cell contact. These studies have potentially significant practical implications, since chemical inhibition of either Arf6 or Epac blocks the effect of EF in cell culture and in vivo, opening new potential therapeutic avenues for treating symptoms caused by cAMP-inducing toxins or related barrier-disrupting pathologies.

Overexpression of SepJ alters septal morphology and heterocyst pattern regulated by diffusible signals in Anabaena.

PubMed

Mariscal, Vicente; Nürnberg, Dennis J; Herrero, Antonia; Mullineaux, Conrad W; Flores, Enrique

2016-09-01

Filamentous, N2 -fixing, heterocyst-forming cyanobacteria grow as chains of cells that are connected by septal junctions. In the model organism Anabaena sp. strain PCC 7120, the septal protein SepJ is required for filament integrity, normal intercellular molecular exchange, heterocyst differentiation, and diazotrophic growth. An Anabaena strain overexpressing SepJ made wider septa between vegetative cells than the wild type, which correlated with a more spread location of SepJ in the septa as observed with a SepJ-GFP fusion, and contained an increased number of nanopores, the septal peptidoglycan perforations that likely accommodate septal junctions. The septa between heterocysts and vegetative cells, which are narrow in wild-type Anabaena, were notably enlarged in the SepJ-overexpressing mutant. Intercellular molecular exchange tested with fluorescent tracers was increased for the SepJ-overexpressing strain specifically in the case of calcein transfer between vegetative cells and heterocysts. These results support an association between calcein transfer, SepJ-related septal junctions, and septal peptidoglycan nanopores. Under nitrogen deprivation, the SepJ-overexpressing strain produced an increased number of contiguous heterocysts but a decreased percentage of total heterocysts. These effects were lost or altered in patS and hetN mutant backgrounds, supporting a role of SepJ in the intercellular transfer of regulatory signals for heterocyst differentiation. © 2016 John Wiley & Sons Ltd.

A history of gap junction structure: hexagonal arrays to atomic resolution.

PubMed

Grosely, Rosslyn; Sorgen, Paul L

2013-02-01

Gap junctions are specialized membrane structures that provide an intercellular pathway for the propagation and/or amplification of signaling cascades responsible for impulse propagation, cell growth, and development. Prior to the identification of the proteins that comprise gap junctions, elucidation of channel structure began with initial observations of a hexagonal nexus connecting apposed cellular membranes. Concomitant with technological advancements spanning over 50 years, atomic resolution structures are now available detailing channel architecture and the cytoplasmic domains that have helped to define mechanisms governing the regulation of gap junctions. Highlighted in this review are the seminal structural studies that have led to our current understanding of gap junction biology.

Active properties of living tissues lead to size-dependent dewetting

NASA Astrophysics Data System (ADS)

Perez-Gonzalez, Carlos; Alert, Ricard; Blanch-Mercader, Carles; Gomez-Gonzalez, Manuel; Casademunt, Jaume; Trepat, Xavier

Key biological processes such as cancer and development are characterized by drastic transitions from 2D to a 3D geometry. These rearrangements have been classically studied as a wetting problem. According to this theory, wettability of a substrate by an epithelium is determined by the competition between cell-cell and cell-substrate adhesion energies. In contrast, we found that, far from a passive process, tissue dewetting is an active process driven by tissue internal forces. Experimentally, we reproduced epithelial dewetting by promoting a progressive formation of intercellular junctions in a monolayer of epithelial cells. Interestingly, the formation of intercellular junctions produces an increase in cell contractility, with the subsequent increase in traction and intercellular stress. At a certain time, tissue tension overcomes cell-substrate maximum adhesion and the monolayer spontaneously dewets the substrate. We developed an active polar fluid model, finding both theoretically and experimentally that critical contractility to promote wetting-dewetting transition depends on cell-substrate adhesion and, unexpectedly, on tissue size. As a whole, this work generalizes wetting theory to living tissues, unveiling unprecedented properties due to their unique active nature.

Twist1-positive epithelial cells retain adhesive and proliferative capacity throughout dissemination

PubMed Central

Shamir, Eliah R.; Coutinho, Kester; Georgess, Dan; Auer, Manfred

2016-01-01

ABSTRACT Dissemination is the process by which cells detach and migrate away from a multicellular tissue. The epithelial-to-mesenchymal transition (EMT) conceptualizes dissemination in a stepwise fashion, with downregulation of E-cadherin leading to loss of intercellular junctions, induction of motility, and then escape from the epithelium. This gain of migratory activity is proposed to be mutually exclusive with proliferation. We previously developed a dissemination assay based on inducible expression of the transcription factor Twist1 and here utilize it to characterize the timing and dynamics of intercellular adhesion, proliferation and migration during dissemination. Surprisingly, Twist1+ epithelium displayed extensive intercellular junctions, and Twist1– luminal epithelial cells could still adhere to disseminating Twist1+ cells. Although proteolysis and proliferation were both observed throughout dissemination, neither was absolutely required. Finally, Twist1+ cells exhibited a hybrid migration mode; their morphology and nuclear deformation were characteristic of amoeboid cells, whereas their dynamic protrusive activity, pericellular proteolysis and migration speeds were more typical of mesenchymal cells. Our data reveal that epithelial cells can disseminate while retaining competence to adhere and proliferate. PMID:27402962

Inhibition of gap junction currents by the abused solvent toluene.

PubMed

Del Re, Angelo M; Woodward, John J

2005-05-09

Abused inhalants are a large class of compounds that are inhaled for their intoxicating and mood altering effects. They include chemicals with known therapeutic uses such as anesthetic gases as well as volatile organic solvents like toluene that are found in paint thinners and adhesives. Because of their widespread commercial use and availability, inhalants are often among the first drugs that children encounter and use of these compounds is often associated with adverse acute and long-term consequences. The cellular and molecular sites of action for abused inhalants is not well known although recent studies report that toluene and other organic solvents alter the activity of specific ligand- and voltage-gated ion channels that regulate cellular excitability. As part of an ongoing effort to define molecular sites of action for abused inhalants, this study examined the effect of toluene on the function of gap junction proteins endogenously expressed in human embryonic kidney (HEK 293) cells. Gap junctions allow cell-to-cell electrical communication as well as passage of small molecular weight substances and are critical for synchronizing cellular activity in certain tissues. Gap junction currents in HEK 293 cells were measured during brief voltage steps using patch-clamp electrophysiology and were blocked by known gap junction blockers confirming expression of connexin proteins in these cells. Toluene dose-dependently inhibited these conductances with threshold effects appearing at approximately 0.4 mM and near complete inhibition occurring at concentrations of 1 mM and higher. The estimated EC50 value for toluene inhibition of gap junction currents in HEK 293 cells was 0.57 mM. The results of these studies suggest that volatile solvents including toluene may produce some of their effects by disrupting inter-cellular communication mediated by gap junction proteins.

Rescue of perfluorooctanesulfonate (PFOS)-mediated Sertoli cell injury by overexpression of gap junction protein connexin 43

PubMed Central

Li, Nan; Mruk, Dolores D.; Chen, Haiqi; Wong, Chris K. C.; Lee, Will M.; Cheng, C. Yan

2016-01-01

Perfluorooctanesulfonate (PFOS) is an environmental toxicant used in developing countries, including China, as a stain repellent for clothing, carpets and draperies, but it has been banned in the U.S. and Canada since the late 2000s. PFOS perturbed the Sertoli cell tight junction (TJ)-permeability barrier, causing disruption of actin microfilaments in cell cytosol, perturbing the localization of cell junction proteins (e.g., occluden-ZO-1, N-cadherin-ß-catenin). These changes destabilized Sertoli cell blood-testis barrier (BTB) integrity. These findings suggest that human exposure to PFOS might induce BTB dysfunction and infertility. Interestingly, PFOS-induced Sertoli cell injury associated with a down-regulation of the gap junction (GJ) protein connexin43 (Cx43). We next investigated if overexpression of Cx43 in Sertoli cells could rescue the PFOS-induced cell injury. Indeed, overexpression of Cx43 in Sertoli cells with an established TJ-barrier blocked the disruption in PFOS-induced GJ-intercellular communication, resulting in the re-organization of actin microfilaments, which rendered them similar to those in control cells. Furthermore, cell adhesion proteins that utilized F-actin for attachment became properly distributed at the cell-cell interface, resealing the disrupted TJ-barrier. In summary, Cx43 is a good target that might be used to manage PFOS-induced reproductive dysfunction. PMID:27436542

Rapid Changes in Connexin-43 in Response to Genotoxic Stress Stabilize Cell–Cell Communication in Corneal Endothelium

PubMed Central

Roh, Danny S.

2011-01-01

Purpose. To determine how corneal endothelial (CE) cells respond to acute genotoxic stress through changes in connexin-43 (Cx43) and gap junction intercellular communication (GJIC). Methods. Cultured bovine CE cells were exposed to mitomycin C or other DNA-damaging agents. Changes in the levels, stability, binding partners, and trafficking of Cx43 were assessed by Western blot analysis and immunostaining. Live-cell imaging of a Cx43–green fluorescent protein (GFP) fusion protein was used to evaluate internalization of cell surface Cx43. Dye transfer and fluorescent recovery after photobleaching (FRAP) assessed GJIC. Results. After genotoxic stress, Cx43 accumulated in large gap junction plaques, had reduced zonula occludens-1 binding, and displayed increased stability. Live-cell imaging of Cx43–GFP plaques in stressed CE cells revealed reduced gap junction internalization and degradation compared to control cells. Mitomycin C enhanced transport of Cx43 from the endoplasmic reticulum to the cell surface and formation of gap junction plaques. Mitomycin C treatment also protected GJIC from disruption after cytokine treatment. Discussion. These results show a novel CE cell response to genotoxic stress mediated by marked and rapid changes in Cx43 and GJIC. This stabilization of cell–cell communication may be an important early adaptation to acute stressors encountered by CE. PMID:21666237

SLO BK Potassium Channels Couple Gap Junctions to Inhibition of Calcium Signaling in Olfactory Neuron Diversification.

PubMed

Alqadah, Amel; Hsieh, Yi-Wen; Schumacher, Jennifer A; Wang, Xiaohong; Merrill, Sean A; Millington, Grethel; Bayne, Brittany; Jorgensen, Erik M; Chuang, Chiou-Fen

2016-01-01

The C. elegans AWC olfactory neuron pair communicates to specify asymmetric subtypes AWCOFF and AWCON in a stochastic manner. Intercellular communication between AWC and other neurons in a transient NSY-5 gap junction network antagonizes voltage-activated calcium channels, UNC-2 (CaV2) and EGL-19 (CaV1), in the AWCON cell, but how calcium signaling is downregulated by NSY-5 is only partly understood. Here, we show that voltage- and calcium-activated SLO BK potassium channels mediate gap junction signaling to inhibit calcium pathways for asymmetric AWC differentiation. Activation of vertebrate SLO-1 channels causes transient membrane hyperpolarization, which makes it an important negative feedback system for calcium entry through voltage-activated calcium channels. Consistent with the physiological roles of SLO-1, our genetic results suggest that slo-1 BK channels act downstream of NSY-5 gap junctions to inhibit calcium channel-mediated signaling in the specification of AWCON. We also show for the first time that slo-2 BK channels are important for AWC asymmetry and act redundantly with slo-1 to inhibit calcium signaling. In addition, nsy-5-dependent asymmetric expression of slo-1 and slo-2 in the AWCON neuron is necessary and sufficient for AWC asymmetry. SLO-1 and SLO-2 localize close to UNC-2 and EGL-19 in AWC, suggesting a role of possible functional coupling between SLO BK channels and voltage-activated calcium channels in AWC asymmetry. Furthermore, slo-1 and slo-2 regulate the localization of synaptic markers, UNC-2 and RAB-3, in AWC neurons to control AWC asymmetry. We also identify the requirement of bkip-1, which encodes a previously identified auxiliary subunit of SLO-1, for slo-1 and slo-2 function in AWC asymmetry. Together, these results provide an unprecedented molecular link between gap junctions and calcium pathways for terminal differentiation of olfactory neurons.

Connexin36 localization to pinealocytes in the pineal gland of mouse and rat.

PubMed

Wang, S G; Tsao, D D; Vanderpool, K G; Yasumura, T; Rash, J E; Nagy, J I

2017-06-01

Several cell types in the pineal gland are known to establish intercellular gap junctions, but the connexin constituents of those junctions have not been fully characterized. Specifically, the expression of connexin36 (Cx36) protein and mRNA has been examined in the pineal, but the identity of cells that produce Cx36 and that form Cx36-containing gap junctions has not been determined. We used immunofluorescence and freeze fracture replica immunogold labelling (FRIL) of Cx36 to investigate the cellular and subcellular localization of Cx36 in the pineal gland of adult mouse and rat. Immunofluorescence labelling of Cx36 was visualized exclusively as puncta or short immunopositive strands that were distributed throughout the pineal, and which were absent in pineal sections from Cx36 null mice. By double immunofluorescence labelling, Cx36 was localized to tryptophan hydroxylase-positive and 5-hydroxytryptamine-positive pinealocyte cell bodies and their large initial processes, including at intersections of those processes and at sites displaying a confluence of processes. Labelling for the cell junction marker zonula occludens-1 (ZO-1) either overlapped or was closely associated with labelling for Cx36. Pinealocytes thus form Cx36-containing gap junctions that also incorporate the scaffolding protein ZO-1. FRIL revealed labelling of Cx36 at ultrastructurally defined gap junctions between pinealocytes, most of which was at gap junctions having reticular, ribbon or string configurations. The results suggest that the endocrine functions of pinealocytes and their secretion of melatonin is supported by their intercellular communication via Cx36-containing gap junctions, which may now be tested by the use of Cx36 null mice. © 2017 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Plasmodium falciparum Adhesion on Human Brain Microvascular Endothelial Cells Involves Transmigration-Like Cup Formation and Induces Opening of Intercellular Junctions

PubMed Central

Jambou, Ronan; Combes, Valery; Jambou, Marie-Jose; Weksler, Babeth B.; Couraud, Pierre-Olivier; Grau, Georges E.

2010-01-01

Cerebral malaria, a major cause of death during malaria infection, is characterised by the sequestration of infected red blood cells (IRBC) in brain microvessels. Most of the molecules implicated in the adhesion of IRBC on endothelial cells (EC) are already described; however, the structure of the IRBC/EC junction and the impact of this adhesion on the EC are poorly understood. We analysed this interaction using human brain microvascular EC monolayers co-cultured with IRBC. Our study demonstrates the transfer of material from the IRBC to the brain EC plasma membrane in a trogocytosis-like process, followed by a TNF-enhanced IRBC engulfing process. Upon IRBC/EC binding, parasite antigens are transferred to early endosomes in the EC, in a cytoskeleton-dependent process. This is associated with the opening of the intercellular junctions. The transfer of IRBC antigens can thus transform EC into a target for the immune response and contribute to the profound EC alterations, including peri-vascular oedema, associated with cerebral malaria. PMID:20686652

Anticonvulsant effects of mefloquine on generalized tonic-clonic seizures induced by two acute models in rats.

PubMed

Franco-Pérez, Javier; Ballesteros-Zebadúa, Paola; Manjarrez-Marmolejo, Joaquín

2015-03-01

Mefloquine can cross the blood-brain barrier and block the gap junction intercellular communication in the brain. Enhanced electrical coupling mediated by gap junctions is an underlying mechanism involved in the generation and maintenance of seizures. For this reason, the aim of this study was to analyze the effects of the systemic administration of mefloquine on tonic-clonic seizures induced by two acute models such as pentylenetetrazole and maximal electroshock. All the control rats presented generalized tonic-clonic seizures after the administration of pentylenetetrazole. However, the incidence of seizures induced by pentylenetetrazole significantly decreased in the groups administered systematically with 40 and 80 mg/kg of mefloquine. In the control group, none of the rats survived after the generalized tonic-clonic seizures induced by pentylenetetrazole, but survival was improved by mefloquine. Besides, mefloquine significantly modified the total spectral power as well as the duration, amplitude and frequency of the epileptiform activity induced by pentylenetetrazole. For the maximal electroshock model, mefloquine did not change the occurrence of tonic hindlimb extension. However, this gap junction blocker significantly decreased the duration of the tonic hindlimb extension induced by the acute electroshock. These data suggest that mefloquine at low doses might be eliciting some anticonvulsant effects when is systemically administered to rats.

Ca2+-dependent localization of integrin-linked kinase to cell junctions in differentiating keratinocytes.

PubMed

Vespa, Alisa; Darmon, Alison J; Turner, Christopher E; D'Souza, Sudhir J A; Dagnino, Lina

2003-03-28

Integrin complexes are necessary for proper proliferation and differentiation of epidermal keratinocytes. Differentiation of these cells is accompanied by down-regulation of integrins and focal adhesions as well as formation of intercellular adherens junctions through E-cadherin homodimerization. A central component of integrin adhesion complexes is integrin-linked kinase (ILK), which can induce loss of E-cadherin expression and epithelial-mesenchymal transformation when ectopically expressed in intestinal and mammary epithelia. In cultured primary mouse keratinocytes, we find that ILK protein levels are independent of integrin expression and signaling, since they remain constant during Ca(2+)-induced differentiation. In contrast, keratinocyte differentiation is accompanied by marked reduction in kinase activity in ILK immunoprecipitates and altered ILK subcellular distribution. Specifically, ILK distributes in close apposition to actin fibers along intercellular junctions in differentiated but not in undifferentiated keratinocytes. ILK localization to cell-cell borders occurs independently of integrin signaling and requires Ca(2+) as well as an intact actin cytoskeleton. Further, and in contrast to what is observed in other epithelial cells, ILK overexpression in differentiated keratinocytes does not promote E-cadherin down-regulation and epithelial-mesenchymal transition. Thus, novel tissue-specific mechanisms control the formation of ILK complexes associated with cell-cell junctions in differentiating murine epidermal keratinocytes.

Integrin-Linked Kinase Is Indispensable for Keratinocyte Differentiation and Epidermal Barrier Function.

PubMed

Sayedyahossein, Samar; Rudkouskaya, Alena; Leclerc, Valerie; Dagnino, Lina

2016-02-01

A functional permeability barrier is essential to prevent the passage of water and electrolytes, macromolecules, and pathogens through the epidermis. This is accomplished in terminally differentiated keratinocytes through formation of a cornified envelope and the assembly of tight intercellular junctions. Integrin-linked kinase (ILK) is a scaffold protein essential for hair follicle morphogenesis and epidermal attachment to the basement membrane. However, the biological functions of ILK in differentiated keratinocytes remain poorly understood. Furthermore, whether ILK is implicated in keratinocyte differentiation and intercellular junction formation has remained an unresolved issue. Here we describe a pivotal role for ILK in keratinocyte differentiation responses to increased extracellular Ca(2+), regulation of adherens and tight junction assembly, and the formation of an outside-in permeability barrier toward macromolecules. In the absence of ILK, the calcium sensing receptor, E-cadherin, and ZO-1 fail to translocate to the cell membrane, through mechanisms that involve abnormalities in microtubules and in RhoA activation. In situ, ILK-deficient epidermis exhibits reduced tight junction formation and increased outside-in permeability to a dextran tracer, indicating reduced barrier properties toward macromolecules. Therefore, ILK is an essential component of keratinocyte differentiation programs that contribute to epidermal integrity and the establishment of its barrier properties. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Morphological examination of the effects of defibrotide on experimentally induced bladder injury and its relation to interstitial cystitis.

PubMed

Aydin, H; Ercan, F; Cetinel, S; San, T

2001-08-01

This morphological study aims to investigate the effects of defibrotide, a deoxyribonucleic acid derivative drug with cytoprotective, immunosuppressive and vasorelaxant effects, on protamine sulfate induced bladder injury. Wistar albino female rats were catheterized and intravesically infused with phosphate buffered solution (control group) or, either protamine sulfate (bladder injury group) or protamine sulfate+defibrotide (bladder injury+defibrotide group) dissolved in phosphate buffered solution. The morphology of the urinary bladder was investigated using light and electron microscopy. The number of mast cells in the mucosa, mucosal alterations, intercellular junctions, surface topography and the glycosaminoglycan (GAG) layer as well as microvillus formation on the luminal surface were evaluated. In the bladder injury group, ulcerated areas, irregularity of the GAG layer, increased number of mast cells, vacuole formation, dilated perinuclear cistern, formation of pleomorphic and uniform microvilli and dilatations in the intercellular spaces in the urothelium were observed. In the bladder injury+defibrotide group a relatively normal urothelial topography, GAG layer and a few mast cells in the mucosa, some dilatations between the intercellular areas, less uniform microvilli, regular perinuclear cistern and tight junctions were observed. These results show that defibrotide can inhibit PS induced bladder damage.

Clustering and negative feedback by endocytosis in planar cell polarity signaling is modulated by ubiquitinylation of prickle.

PubMed

Cho, Bomsoo; Pierre-Louis, Gandhy; Sagner, Andreas; Eaton, Suzanne; Axelrod, Jeffrey D

2015-05-01

The core components of the planar cell polarity (PCP) signaling system, including both transmembrane and peripheral membrane associated proteins, form asymmetric complexes that bridge apical intercellular junctions. While these can assemble in either orientation, coordinated cell polarization requires the enrichment of complexes of a given orientation at specific junctions. This might occur by both positive and negative feedback between oppositely oriented complexes, and requires the peripheral membrane associated PCP components. However, the molecular mechanisms underlying feedback are not understood. We find that the E3 ubiquitin ligase complex Cullin1(Cul1)/SkpA/Supernumerary limbs(Slimb) regulates the stability of one of the peripheral membrane components, Prickle (Pk). Excess Pk disrupts PCP feedback and prevents asymmetry. We show that Pk participates in negative feedback by mediating internalization of PCP complexes containing the transmembrane components Van Gogh (Vang) and Flamingo (Fmi), and that internalization is activated by oppositely oriented complexes within clusters. Pk also participates in positive feedback through an unknown mechanism promoting clustering. Our results therefore identify a molecular mechanism underlying generation of asymmetry in PCP signaling.

Sparse short-distance connections enhance calcium wave propagation in a 3D model of astrocyte networks

PubMed Central

Lallouette, Jules; De Pittà, Maurizio; Ben-Jacob, Eshel; Berry, Hugues

2014-01-01

Traditionally, astrocytes have been considered to couple via gap-junctions into a syncytium with only rudimentary spatial organization. However, this view is challenged by growing experimental evidence that astrocytes organize as a proper gap-junction mediated network with more complex region-dependent properties. On the other hand, the propagation range of intercellular calcium waves (ICW) within astrocyte populations is as well highly variable, depending on the brain region considered. This suggests that the variability of the topology of gap-junction couplings could play a role in the variability of the ICW propagation range. Since this hypothesis is very difficult to investigate with current experimental approaches, we explore it here using a biophysically realistic model of three-dimensional astrocyte networks in which we varied the topology of the astrocyte network, while keeping intracellular properties and spatial cell distribution and density constant. Computer simulations of the model suggest that changing the topology of the network is indeed sufficient to reproduce the distinct ranges of ICW propagation reported experimentally. Unexpectedly, our simulations also predict that sparse connectivity and restriction of gap-junction couplings to short distances should favor propagation while long–distance or dense connectivity should impair it. Altogether, our results provide support to recent experimental findings that point toward a significant functional role of the organization of gap-junction couplings into proper astroglial networks. Dynamic control of this topology by neurons and signaling molecules could thus constitute a new type of regulation of neuron-glia and glia-glia interactions. PMID:24795613

Lipopolysaccharide effects on the proliferation of NRK52E cells via alternations in gap-junction function.

PubMed

Hei, Ziqing; Zhang, Ailan; Wei, Jing; Gan, Xiaoliang; Wang, Yanling; Luo, Gangjian; Li, Xiaoyun

2012-07-01

Gap junctions regulate proper kidney function by facilitating intercellular communication, vascular conduction, and tubular purinergic signaling. However, no clear relationship has been described between gap-junction function and acute kidney injury induced by the endotoxin lipopolysaccharide (LPS). Normal rat kidney epithelial cells (NRK52E cells) were seeded at high and low densities to promote or impede gap-junction formation, respectively, and establish distinctive levels of intercellular communication in culture. Cells were then challenged with LPS at various concentrations (10-1,000 ng/mL). LPS-induced formation and function of gap junctions were assessed by measuring changes in cell proliferation and colony-forming rates, fluorescent dye transmission to adjacent cells, expression levels of connexin43, and repositioning of confluent cells in response to the gap junction inhibitor oleamide or agonist retinoic acid. The cell proliferation rate and colony-forming rate of high- and low-density NRK52E cells were decreased upon LPS challenge, in a dose-dependent manner. The colony-forming rate of confluent high-density cells was significantly lower than that of low-density cells. Oleamide treatment raised the LPS-induced colony-forming rate of high-density cells, whereas retinoic acid decreased the rate. Neither oleamide nor retinoic acid significantly affected the LPS-induced colony-forming rate of low-density cells. Fluorescence transmission of high-density cells was reduced by LPS challenge, in a dose-dependent manner, but inclusion of retinoic acid increased the LPS-induced transmission of fluorescence. LPS challenge of either high- or low-density NRK52E cells resulted in down-regulated connexin43 expression. Gap-junction function plays an important role in concentration-dependent cytotoxic effect of LPS on normal rat kidney cells in vitro.

Prostate cancer cells specifically reorganize epithelial cell-fibroblast communication through proteoglycan and junction pathways.

PubMed

Suhovskih, Anastasia V; Kashuba, Vladimir I; Klein, George; Grigorieva, Elvira V

2017-01-02

Microenvironment and stromal fibroblasts are able to inhibit tumor cell proliferation both through secreted signaling molecules and direct cell-cell interactions but molecular mechanisms of these effects remain unclear. In this study, we investigated a role of cell-cell contact-related molecules (protein ECM components, proteoglycans (PGs) and junction-related molecules) in intercellular communications between the human TERT immortalized fibroblasts (BjTERT fibroblasts) and normal (PNT2) or cancer (LNCaP, PC3, DU145) prostate epithelial cells. It was shown that BjTERT-PNT2 cell coculture resulted in significant decrease of both BjTERT and PNT2 proliferation rates and reorganization of transcriptional activity of cell-cell contact-related genes in both cell types. Immunocytochemical staining revealed redistribution of DCN and LUM in PNT2 cells and significant increase of SDC1 at the intercellular contact zones between BjTERT and PNT2 cells, suggesting active involvement of the PGs in cell-cell contacts and contact inhibition of cell proliferation. Unlike to PNT2 cells, PC3 cells did not respond to BjTERT in terms of PGs expression, moderately increased transcriptional activity of junctions-related genes (especially tight junction) and failed to establish PC3-BjTERT contacts. At the same time, PC3 cells significantly down-regulated junctions-related genes (especially focal adhesions and adherens junctions) in BjTERT fibroblasts resulting in visible preference for homotypic PC3-PC3 over heterotypic PC3-BjTERT contacts and autonomous growth of PC3 clones. Taken together, the results demonstrate that an instructing role of fibroblasts to normal prostate epithelial cells is revoked by cancer cells through deregulation of proteoglycans and junction molecules expression and overall disorganization of fibroblast-cancer cell communication.

Dedifferentiation, Proliferation, and Redifferentiation of Adult Mammalian Cardiomyocytes After Ischemic Injury.

PubMed

Wang, Wei Eric; Li, Liangpeng; Xia, Xuewei; Fu, Wenbin; Liao, Qiao; Lan, Cong; Yang, Dezhong; Chen, Hongmei; Yue, Rongchuan; Zeng, Cindy; Zhou, Lin; Zhou, Bin; Duan, Dayue Darrel; Chen, Xiongwen; Houser, Steven R; Zeng, Chunyu

2017-08-29

Adult mammalian hearts have a limited ability to generate new cardiomyocytes. Proliferation of existing adult cardiomyocytes (ACMs) is a potential source of new cardiomyocytes. Understanding the fundamental biology of ACM proliferation could be of great clinical significance for treating myocardial infarction (MI). We aim to understand the process and regulation of ACM proliferation and its role in new cardiomyocyte formation of post-MI mouse hearts. β-Actin-green fluorescent protein transgenic mice and fate-mapping Myh6-MerCreMer-tdTomato/lacZ mice were used to trace the fate of ACMs. In a coculture system with neonatal rat ventricular myocytes, ACM proliferation was documented with clear evidence of cytokinesis observed with time-lapse imaging. Cardiomyocyte proliferation in the adult mouse post-MI heart was detected by cell cycle markers and 5-ethynyl-2-deoxyuridine incorporation analysis. Echocardiography was used to measure cardiac function, and histology was performed to determine infarction size. In vitro, mononucleated and bi/multinucleated ACMs were able to proliferate at a similar rate (7.0%) in the coculture. Dedifferentiation proceeded ACM proliferation, which was followed by redifferentiation. Redifferentiation was essential to endow the daughter cells with cardiomyocyte contractile function. Intercellular propagation of Ca 2+ from contracting neonatal rat ventricular myocytes into ACM daughter cells was required to activate the Ca 2+ -dependent calcineurin-nuclear factor of activated T-cell signaling pathway to induce ACM redifferentiation. The properties of neonatal rat ventricular myocyte Ca 2+ transients influenced the rate of ACM redifferentiation. Hypoxia impaired the function of gap junctions by dephosphorylating its component protein connexin 43, the major mediator of intercellular Ca 2+ propagation between cardiomyocytes, thereby impairing ACM redifferentiation. In vivo, ACM proliferation was found primarily in the MI border zone. An ischemia-resistant connexin 43 mutant enhanced the redifferentiation of ACM-derived new cardiomyocytes after MI and improved cardiac function. Mature ACMs can reenter the cell cycle and form new cardiomyocytes through a 3-step process: dedifferentiation, proliferation, and redifferentiation. Intercellular Ca 2+ signal from neighboring functioning cardiomyocytes through gap junctions induces the redifferentiation process. This novel mechanism contributes to new cardiomyocyte formation in post-MI hearts in mammals. © 2017 American Heart Association, Inc.

Cytotoxic effect of the Her-2/Her-1 inhibitor PKI-166 on renal cancer cells expressing the connexin 32 gene.

PubMed

Fujimoto, Eriko; Yano, Tomohiro; Sato, Hiromi; Hagiwara, Kiyokazu; Yamasaki, Hiroshi; Shirai, Sumiko; Fukumoto, Keiko; Hagiwara, Hiromi; Negishi, Etsuko; Ueno, Koichi

2005-02-01

We have reported that connexin (Cx) 32 acts as a tumor suppressor gene in renal cancer cells partly due to Her-2 inactivation. Here, we determined if a Her-2/Her-1 inhibitor (PKI-166) can enhance the tumor-suppressive effect of Cx32 in Caki-2 cells from human renal cell carcinoma. The expression of Cx32 in Caki-2 cells was required for PKI-166-induced cytotoxic effect at lower doses. The cyctotoxicity was dependent on the occurrence of apoptosis and partly mediated by Cx32-driven gap junction intercellular communications. These results suggest that PKI-166 further supports the tumor-suppressive effect of the Cx32 gene in renal cancer cells through the induction of apoptosis.

Cell-derived microparticles: new targets in the therapeutic management of disease.

PubMed

Roseblade, Ariane; Luk, Frederick; Rawling, Tristan; Ung, Alison; Grau, Georges E R; Bebawy, Mary

2013-01-01

Intercellular communication is essential to maintain vital physiological activities and to regulate the organism's phenotype. There are a number of ways in which cells communicate with one another. This can occur via autocrine signaling, endocrine signaling or by the transfer of molecular mediators across gap junctions. More recently communication via microvesicular shedding has gained important recognition as a significant pathway by which cells can coordinate the spread and dominance of selective traits within a population. Through this communication apparatus, cells can now acquire and secure a survival advantage, particularly in the context of malignant disease. This review aims to highlight some of the functions and implications of microparticles in physiology of various disease states, and present a novel therapeutic strategy through the regulation of microparticle production.

Intestinal epithelial barrier function and tight junction proteins with heat and exercise

PubMed Central

Zuhl, Micah N.; Moseley, Pope L.

2015-01-01

A single layer of enterocytes and tight junctions (intercellular multiprotein complexes) form the intestinal epithelial barrier that controls transport of molecules through transcellular and paracellular pathways. A dysfunctional or “leaky” intestinal tight junction barrier allows augmented permeation of luminal antigens, endotoxins, and bacteria into the blood stream. Various substances and conditions have been shown to affect the maintenance of the intestinal epithelial tight junction barrier. The primary focus of the present review is to analyze the effects of exertional or nonexertional (passive hyperthermia) heat stress on tight junction barrier function in in vitro and in vivo (animals and humans) models. Our secondary focus is to review changes in tight junction proteins in response to exercise or hyperthermic conditions. Finally, we discuss some pharmacological or nutritional interventions that may affect the cellular mechanisms involved in maintaining homeostasis of the intestinal epithelial tight junction barrier during heat stress or exercise. PMID:26359485

Intestinal epithelial barrier function and tight junction proteins with heat and exercise.

PubMed

Dokladny, Karol; Zuhl, Micah N; Moseley, Pope L

2016-03-15

A single layer of enterocytes and tight junctions (intercellular multiprotein complexes) form the intestinal epithelial barrier that controls transport of molecules through transcellular and paracellular pathways. A dysfunctional or "leaky" intestinal tight junction barrier allows augmented permeation of luminal antigens, endotoxins, and bacteria into the blood stream. Various substances and conditions have been shown to affect the maintenance of the intestinal epithelial tight junction barrier. The primary focus of the present review is to analyze the effects of exertional or nonexertional (passive hyperthermia) heat stress on tight junction barrier function in in vitro and in vivo (animals and humans) models. Our secondary focus is to review changes in tight junction proteins in response to exercise or hyperthermic conditions. Finally, we discuss some pharmacological or nutritional interventions that may affect the cellular mechanisms involved in maintaining homeostasis of the intestinal epithelial tight junction barrier during heat stress or exercise. Copyright © 2016 the American Physiological Society.

The macrophage cytoskeleton acts as a contact sensor upon interaction with Entamoeba histolytica to trigger IL-1β secretion

PubMed Central

Moreau, France; Gorman, Hayley

2017-01-01

Entamoeba histolytica (Eh) is the causative agent of amebiasis, one of the major causes of dysentery-related morbidity worldwide. Recent studies have underlined the importance of the intercellular junction between Eh and host cells as a determinant in the pathogenesis of amebiasis. Despite the fact that direct contact and ligation between Eh surface Gal-lectin and EhCP-A5 with macrophage α5β1 integrin are absolute requirements for NLRP3 inflammasome activation and IL-1β release, many other undefined molecular events and downstream signaling occur at the interface of Eh and macrophage. In this study, we investigated the molecular events at the intercellular junction that lead to recognition of Eh through modulation of the macrophage cytoskeleton. Upon Eh contact with macrophages key cytoskeletal-associated proteins were rapidly post-translationally modified only with live Eh but not with soluble Eh proteins or fragments. Eh ligation with macrophages rapidly activated caspase-6 dependent cleavage of the cytoskeletal proteins talin, Pyk2 and paxillin and caused robust release of the pro-inflammatory cytokine, IL-1β. Macrophage cytoskeletal cleavages were dependent on Eh cysteine proteinases EhCP-A1 and EhCP-A4 but not EhCP-A5 based on pharmacological blockade of Eh enzyme inhibitors and EhCP-A5 deficient parasites. These results unravel a model where the intercellular junction between macrophages and Eh form an area of highly interacting proteins that implicate the macrophage cytoskeleton as a sensor for Eh contact that leads downstream to subsequent inflammatory immune responses. PMID:28837696

Mammary collective cell migration involves transient loss of epithelial features and individual cell migration within the epithelium

PubMed Central

Ewald, Andrew J.; Huebner, Robert J.; Palsdottir, Hildur; Lee, Jessie K.; Perez, Melissa J.; Jorgens, Danielle M.; Tauscher, Andrew N.; Cheung, Kevin J.; Werb, Zena; Auer, Manfred

2012-01-01

Normal mammary morphogenesis involves transitions between simple and multilayered epithelial organizations. We used electron microscopy and molecular markers to determine whether intercellular junctions and apico-basal polarity were maintained in the multilayered epithelium. We found that multilayered elongating ducts had polarized apical and basal tissue surfaces both in three-dimensional culture and in vivo. However, individual cells were only polarized on surfaces in contact with the lumen or extracellular matrix. The basolateral marker scribble and the apical marker atypical protein kinase C zeta localized to all interior cell membranes, whereas PAR3 displayed a cytoplasmic localization, suggesting that the apico-basal polarity was incomplete. Despite membrane localization of E-cadherin and β-catenin, we did not observe a defined zonula adherens connecting interior cells. Instead, interior cells were connected through desmosomes and exhibited complex interdigitating membrane protrusions. Single-cell labeling revealed that individual cells were both protrusive and migratory within the epithelial multilayer. Inhibition of Rho kinase (ROCK) further reduced intercellular adhesion on apical and lateral surfaces but did not disrupt basal tissue organization. Following morphogenesis, segregated membrane domains were re-established and junctional complexes re-formed. We observed similar epithelial organization during mammary morphogenesis in organotypic culture and in vivo. We conclude that mammary epithelial morphogenesis involves a reversible, spatially limited, reduction in polarity and intercellular junctions and active individualistic cell migration. Our data suggest that reductions in polarity and adhesion during breast cancer progression might reflect partial recapitulation of a normal developmental program. PMID:22344263

ASC Induces Apoptosis via Activation of Caspase-9 by Enhancing Gap Junction-Mediated Intercellular Communication

PubMed Central

Hida, Shigeaki; Fujii, Chifumi; Taniguchi, Shun’ichiro; Ito, Kensuke; Matsumura, Tomio; Okada, Nagisa; Sakaizawa, Takashi; Kobayashi, Akira; Takeoka, Michiko; Miyagawa, Shin-ichi

2017-01-01

ASC (apoptosis-associated speck-like protein containing a CARD) is a key adaptor molecule of inflammasomes that mediates inflammatory and apoptotic signals. Aberrant methylation-induced silencing of ASC has been observed in a variety of cancer cells, thus implicating ASC in tumor suppression, although this role remains incompletely defined especially in the context of closely neighboring cell proliferation. As ASC has been confirmed to be silenced by abnormal methylation in HT1080 fibrosarcoma cells as well, this cell line was investigated to characterize the precise role and mechanism of ASC in tumor progression. The effects of ASC were examined using in vitro cell cultures based on comparisons between low and high cell density conditions as well as in a xenograft murine model. ASC overexpression was established by insertion of the ASC gene into pcDNA3 and pMX-IRES-GFP vectors, the latter being packed into a retrovirus and subjected to reproducible competitive assays using parental cells as an internal control, for evaluation of cell viability. p21 and p53 were silenced using shRNA. Cell viability was suppressed in ASC-expressing transfectants as compared with control cells at high cell density conditions in in vitro culture and colony formation assays and in in vivo ectopic tumor formation trials. This suppression was not detected in low cell density conditions. Furthermore, remarkable progression of apoptosis was observed in ASC-introduced cells at a high cell density, but not at a low one. ASC-dependent apoptosis was mediated not by p21, p53, or caspase-1, but rather by cleavage of caspase-9 as well as by suppression of the NF-κB-related X-linked inhibitor-of-apoptosis protein. Caspase-9 cleavage was observed to be dependent on gap junction formation. The remarkable effect of ASC on the induction of apoptosis through caspase-9 and gap junctions revealed in this study may lead to promising new approaches in anticancer therapy. PMID:28056049

Transient suppression of gap junctional intercellular communication after exposure to 100-nanosecond pulsed electric fields.

PubMed

Steuer, Anna; Schmidt, Anke; Labohá, Petra; Babica, Pavel; Kolb, Juergen F

2016-12-01

Gap junctional intercellular communication (GJIC) is an important mechanism that is involved and affected in many diseases and injuries. So far, the effect of nanosecond pulsed electric fields (nsPEFs) on the communication between cells was not investigated. An in vitro approach is presented with rat liver epithelial WB-F344 cells grown and exposed in a monolayer. In order to observe sub-lethal effects, cells were exposed to pulsed electric fields with a duration of 100ns and amplitudes between 10 and 20kV/cm. GJIC strongly decreased within 15min after treatment but recovered within 24h. Gene expression of Cx43 was significantly decreased and associated with a reduced total amount of Cx43 protein. In addition, MAP kinases p38 and Erk1/2, involved in Cx43 phosphorylation, were activated and Cx43 became hyperphosphorylated. Immunofluorescent staining of Cx43 displayed the disassembly of gap junctions. Further, a reorganization of the actin cytoskeleton was observed whereas tight junction protein ZO-1 was not significantly affected. All effects were field- and time-dependent and most pronounced within 30 to 60min after treatment. A better understanding of a possible manipulation of GJIC by nsPEFs might eventually offer a possibility to develop and improve treatments. Copyright © 2016 Elsevier B.V. All rights reserved.

Occludin confers adhesiveness when expressed in fibroblasts.

PubMed

Van Itallie, C M; Anderson, J M

1997-05-01

Occludin is an integral membrane protein specifically associated with tight junctions. Previous studies suggest it is likely to function in forming the intercellular seal. In the present study, we expressed occludin under an inducible promotor in occludin-null fibroblasts to determine whether this protein confers intercellular adhesion. When human occludin is stably expressed in NRK and Rat-1 fibroblasts, which lack endogenous occludin and tight junctions but do have well developed ZO-1-containing adherens-like junctions, occludin colocalizes with ZO-1 to points of cell-cell contact. In contrast, L-cell fibroblasts which lack cadherin-based adherens junctions, target neither ZO-1 nor occludin to sites of cell contact. Occludin-induced adhesion was next quantified using a suspended cell assay. In NRK and Rat-1 cells, occludin expression induces adhesion in the absence of calcium, thus independent of cadherin-cadherin contacts. In contrast, L-cells are nonadhesive in this assay and show no increase in adhesion after induction of occludin expression. Binding of an antibody to the first of the putative extracellular loops of occludin confirmed that this sequence was exposed on the cell surface, and synthetic peptides containing the amino acid sequence of this loop inhibit adhesion induced by occludin expression. These results suggest that the extracellular surface of occludin is directly involved in cell-cell adhesion and the ability to confer adhesiveness correlates with the ability to colocalize with its cytoplasmic binding protein, ZO-1.

Remodelling of cellular excitation (reaction) and intercellular coupling (diffusion) by chronic atrial fibrillation represented by a reaction-diffusion system

NASA Astrophysics Data System (ADS)

Zhang, Henggui; Garratt, Clifford J.; Kharche, Sanjay; Holden, Arun V.

2009-06-01

Human atrial tissue is an excitable system, in which myocytes are excitable elements, and cell-to-cell electrotonic interactions are via diffusive interactions of cell membrane potentials. We developed a family of excitable system models for human atrium at cellular, tissue and anatomical levels for both normal and chronic atrial fibrillation (AF) conditions. The effects of AF-induced remodelling of cell membrane ionic channels (reaction kinetics) and intercellular gap junctional coupling (diffusion) on atrial excitability, conduction of excitation waves and dynamics of re-entrant excitation waves are quantified. Both ionic channel and gap junctional coupling remodelling have rate dependent effects on atrial propagation. Membrane channel conductance remodelling allows the propagation of activity at higher rates than those sustained in normal tissue or in tissue with gap junctional remodelling alone. Membrane channel conductance remodelling is essential for the propagation of activity at rates higher than 300/min as seen in AF. Spatially heterogeneous gap junction coupling remodelling increased the risk of conduction block, an essential factor for the genesis of re-entry. In 2D and 3D anatomical models, the dynamical behaviours of re-entrant excitation waves are also altered by membrane channel modelling. This study provides insights to understand the pro-arrhythmic effects of AF-induced reaction and diffusion remodelling in atrial tissue.

Tunneling nanotubes: A versatile target for cancer therapy.

PubMed

Sahoo, Pragyaparamita; Jena, Soumya Ranjan; Samanta, Luna

2017-11-29

Currently Cancer is the leading cause of death worldwide. Malignancy or cancer is a class of diseases characterized by uncontrolled cell growth that eventually invade other tissues and dvelop secondary malignant growth at other sites by metastasis. Intercellular communication plays a major in cancer, particularly in the process of cell proliferation and coordination which in turn leads to tumor invasion, metastasis and development of resistance to therapy. Cells communicate among themselves in a variety of ways, namely, i) via gap junctions with adjacent cells, ii) via exosomes with nearby cells and iii) via chemical messengers with distant cells. Besides, cell - cell connection by tunneling nanotubes (TnTs) is recently gaining importance where intercellular components are transferred between cells. In general cell organelles like Golgi vesicle and mitochondria; and biomolecules like nucleic acids and proteins are transferred through these TnTs. These TnTs are long cytoplasmic extensions made up of actin that function as intercellular bridge and connect a wide verity of cell types. Malignant cells form TnTs with either another malignant cells or cells of the surrounding tumor matrix. These TnTs help in the process of initiation of tumor formation, its organization and propagation. The current review focuses on the role of TnTs mediated cell – cell signaling in cancer micro-environment. Drugs that inhibit TnT-formation such as metformin and everolimus can be targeted towards TnTs in the management of cancer growth, proliferation, tumor invasion and metastasis. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Acute Slices of Mice Testis Seminiferous Tubules Unveil Spontaneous and Synchronous Ca2+ Oscillations in Germ Cell Clusters1

PubMed Central

Sánchez-Cárdenas, Claudia; Guerrero, Adán; Treviño, Claudia Lydia; Hernández-Cruz, Arturo; Darszon, Alberto

2012-01-01

ABSTRACT Spermatogenic cell differentiation involves changes in the concentration of cytoplasmic Ca2+ ([Ca2+]i); however, very few studies exist on [Ca2+]i dynamics in these cells. Other tissues display Ca2+ oscillations involving multicellular functional arrangements. These phenomena have been studied in acute slice preparations that preserve tissue architecture and intercellular communications. Here we report the implementation of intracellular Ca2+ imaging in a sliced seminiferous tubule (SST) preparation to visualize [Ca2+]i changes of living germ cells in situ within the SST preparation. Ca2+ imaging revealed that a subpopulation of male germ cells display spontaneous [Ca2+]i fluctuations resulting from Ca2+ entry possibly throughout CaV3 channels. These [Ca2+]i fluctuation patterns are also present in single acutely dissociated germ cells, but they differ from those recorded from germ cells in the SST preparation. Often, spontaneous Ca2+ fluctuations of spermatogenic cells in the SST occur synchronously, so that clusters of cells can display Ca2+ oscillations for at least 10 min. Synchronous Ca2+ oscillations could be mediated by intercellular communication via gap junctions, although intercellular bridges could also be involved. We also observed an increase in [Ca2+]i after testosterone application, suggesting the presence of functional Sertoli cells in the SST. In summary, we believe that the SST preparation is suitable to explore the physiology of spermatogenic cells in their natural environment, within the seminiferous tubules, in particular Ca2+ signaling phenomena, functional cell-cell communication, and multicellular functional arrangements. PMID:22914313

Distribution of cardiac sodium channels in clusters potentiates ephaptic interactions in the intercalated disc.

PubMed

Hichri, Echrak; Abriel, Hugues; Kucera, Jan P

2018-02-15

It has been proposed that ephaptic conduction, relying on interactions between the sodium (Na + ) current and the extracellular potential in intercalated discs, might contribute to cardiac conduction when gap junctional coupling is reduced, but this mechanism is still controversial. In intercalated discs, Na + channels form clusters near gap junction plaques, but the functional significance of these clusters has never been evaluated. In HEK cells expressing cardiac Na + channels, we show that restricting the extracellular space modulates the Na + current, as predicted by corresponding simulations accounting for ephaptic effects. In a high-resolution model of the intercalated disc, clusters of Na + channels that face each other across the intercellular cleft facilitate ephaptic impulse transmission when gap junctional coupling is reduced. Thus, our simulations reveal a functional role for the clustering of Na + channels in intercalated discs, and suggest that rearrangement of these clusters in disease may influence cardiac conduction. It has been proposed that ephaptic interactions in intercalated discs, mediated by extracellular potentials, contribute to cardiac impulse propagation when gap junctional coupling is reduced. However, experiments demonstrating ephaptic effects on the cardiac Na + current (I Na ) are scarce. Furthermore, Na + channels form clusters around gap junction plaques, but the electrophysiological significance of these clusters has never been investigated. In patch clamp experiments with HEK cells stably expressing human Na v 1.5 channels, we examined how restricting the extracellular space modulates I Na elicited by an activation protocol. In parallel, we developed a high-resolution computer model of the intercalated disc to investigate how the distribution of Na + channels influences ephaptic interactions. Approaching the HEK cells to a non-conducting obstacle always increased peak I Na at step potentials near the threshold of I Na activation and decreased peak I Na at step potentials far above threshold (7 cells, P = 0.0156, Wilcoxon signed rank test). These effects were consistent with corresponding control simulations with a uniform Na + channel distribution. In the intercalated disc computer model, redistributing the Na + channels into a central cluster of the disc potentiated ephaptic effects. Moreover, ephaptic impulse transmission from one cell to another was facilitated by clusters of Na + channels facing each other across the intercellular cleft when gap junctional coupling was reduced. In conclusion, our proof-of-principle experiments demonstrate that confining the extracellular space modulates cardiac I Na , and our simulations reveal the functional role of the aggregation of Na + channels in the perinexus. These findings highlight novel concepts in the physiology of cardiac excitation. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

Nonlinear Gap Junctions Enable Long-Distance Propagation of Pulsating Calcium Waves in Astrocyte Networks

PubMed Central

Goldberg, Mati; De Pittà, Maurizio; Volman, Vladislav; Berry, Hugues; Ben-Jacob, Eshel

2010-01-01

A new paradigm has recently emerged in brain science whereby communications between glial cells and neuron-glia interactions should be considered together with neurons and their networks to understand higher brain functions. In particular, astrocytes, the main type of glial cells in the cortex, have been shown to communicate with neurons and with each other. They are thought to form a gap-junction-coupled syncytium supporting cell-cell communication via propagating Ca2+ waves. An identified mode of propagation is based on cytoplasm-to-cytoplasm transport of inositol trisphosphate (IP3) through gap junctions that locally trigger Ca2+ pulses via IP3-dependent Ca2+-induced Ca2+ release. It is, however, currently unknown whether this intracellular route is able to support the propagation of long-distance regenerative Ca2+ waves or is restricted to short-distance signaling. Furthermore, the influence of the intracellular signaling dynamics on intercellular propagation remains to be understood. In this work, we propose a model of the gap-junctional route for intercellular Ca2+ wave propagation in astrocytes. Our model yields two major predictions. First, we show that long-distance regenerative signaling requires nonlinear coupling in the gap junctions. Second, we show that even with nonlinear gap junctions, long-distance regenerative signaling is favored when the internal Ca2+ dynamics implements frequency modulation-encoding oscillations with pulsating dynamics, while amplitude modulation-encoding dynamics tends to restrict the propagation range. As a result, spatially heterogeneous molecular properties and/or weak couplings are shown to give rise to rich spatiotemporal dynamics that support complex propagation behaviors. These results shed new light on the mechanisms implicated in the propagation of Ca2+ waves across astrocytes and the precise conditions under which glial cells may participate in information processing in the brain. PMID:20865153

Gap junctional intercellular communication is required to maintain embryonic stem cells in a non-differentiated and proliferative state.

PubMed

Todorova, Mariana G; Soria, Bernat; Quesada, Ivan

2008-02-01

Pluripotent embryonic stem (ES) cells are capable of maintaining a self-renewal state and have the potential to differentiate into derivatives of all three embryonic germ layers. Despite their importance in cell therapy and developmental biology, the mechanisms whereby ES cells remain in a proliferative and pluripotent state are still not fully understood. Here we establish a critical role of gap junctional intercellular communication (GJIC) and connexin43 (Cx43) in both processes. Pharmacological blockers of GJIC and Cx43 down-regulation by small interfering RNA (siRNA) caused a profound inhibitory effect on GJIC, as evidenced by experiments of fluorescence recovery after photobleaching. This deficient intercellular communication in ES cells induced a loss of their pluripotent state, which was manifested in morphological changes, a decrease in alkaline phosphatase activity, Oct-3/4 and Nanog expression, as well as an up-regulation of several differentiation markers. A decrease in the proliferation rate was also detected. Under these conditions, the formation of embryoid bodies from mouse ES cells was impaired, although this inhibition was reversible upon restoration of GJIC. Our findings define a major function of GJIC in the regulation of self-renewal and maintenance of pluripotency in ES cells. (c) 2007 Wiley-Liss, Inc.

The Importance of Connexin Hemichannels During Chondroprogenitor Cell Differentiation in Hydrogel Versus Microtissue Culture Models

PubMed Central

Schrobback, Karsten; Klein, Travis Jacob

2015-01-01

Appropriate selection of scaffold architecture is a key challenge in cartilage tissue engineering. Gap junction-mediated intercellular contacts play important roles in precartilage condensation of mesenchymal cells. However, scaffold architecture could potentially restrict cell–cell communication and differentiation. This is particularly important when choosing the appropriate culture platform as well as scaffold-based strategy for clinical translation, that is, hydrogel or microtissues, for investigating differentiation of chondroprogenitor cells in cartilage tissue engineering. We, therefore, studied the influence of gap junction-mediated cell–cell communication on chondrogenesis of bone marrow-derived mesenchymal stromal cells (BM-MSCs) and articular chondrocytes. Expanded human chondrocytes and BM-MSCs were either (re-) differentiated in micromass cell pellets or encapsulated as isolated cells in alginate hydrogels. Samples were treated with and without the gap junction inhibitor 18-α glycyrrhetinic acid (18αGCA). DNA and glycosaminoglycan (GAG) content and gene expression levels (collagen I/II/X, aggrecan, and connexin 43) were quantified at various time points. Protein localization was determined using immunofluorescence, and adenosine-5′-triphosphate (ATP) was measured in conditioned media. While GAG/DNA was higher in alginate compared with pellets for chondrocytes, there were no differences in chondrogenic gene expression between culture models. Gap junction blocking reduced collagen II and extracellular ATP in all chondrocyte cultures and in BM-MSC hydrogels. However, differentiation capacity was not abolished completely by 18αGCA. Connexin 43 levels were high throughout chondrocyte cultures and peaked only later during BM-MSC differentiation, consistent with the delayed response of BM-MSCs to 18αGCA. Alginate hydrogels and microtissues are equally suited culture platforms for the chondrogenic (re-)differentiation of expanded human articular chondrocytes and BM-MSCs. Therefore, reducing direct cell–cell contacts does not affect in vitro chondrogenesis. However, blocking gap junctions compromises cell differentiation, pointing to a prominent role for hemichannel function in this process. Therefore, scaffold design strategies that promote an increasing distance between single chondroprogenitor cells do not restrict their differentiation potential in tissue-engineered constructs. PMID:25693425

The importance of connexin hemichannels during chondroprogenitor cell differentiation in hydrogel versus microtissue culture models.

PubMed

Schrobback, Karsten; Klein, Travis Jacob; Woodfield, Tim B F

2015-06-01

Appropriate selection of scaffold architecture is a key challenge in cartilage tissue engineering. Gap junction-mediated intercellular contacts play important roles in precartilage condensation of mesenchymal cells. However, scaffold architecture could potentially restrict cell-cell communication and differentiation. This is particularly important when choosing the appropriate culture platform as well as scaffold-based strategy for clinical translation, that is, hydrogel or microtissues, for investigating differentiation of chondroprogenitor cells in cartilage tissue engineering. We, therefore, studied the influence of gap junction-mediated cell-cell communication on chondrogenesis of bone marrow-derived mesenchymal stromal cells (BM-MSCs) and articular chondrocytes. Expanded human chondrocytes and BM-MSCs were either (re-) differentiated in micromass cell pellets or encapsulated as isolated cells in alginate hydrogels. Samples were treated with and without the gap junction inhibitor 18-α glycyrrhetinic acid (18αGCA). DNA and glycosaminoglycan (GAG) content and gene expression levels (collagen I/II/X, aggrecan, and connexin 43) were quantified at various time points. Protein localization was determined using immunofluorescence, and adenosine-5'-triphosphate (ATP) was measured in conditioned media. While GAG/DNA was higher in alginate compared with pellets for chondrocytes, there were no differences in chondrogenic gene expression between culture models. Gap junction blocking reduced collagen II and extracellular ATP in all chondrocyte cultures and in BM-MSC hydrogels. However, differentiation capacity was not abolished completely by 18αGCA. Connexin 43 levels were high throughout chondrocyte cultures and peaked only later during BM-MSC differentiation, consistent with the delayed response of BM-MSCs to 18αGCA. Alginate hydrogels and microtissues are equally suited culture platforms for the chondrogenic (re-)differentiation of expanded human articular chondrocytes and BM-MSCs. Therefore, reducing direct cell-cell contacts does not affect in vitro chondrogenesis. However, blocking gap junctions compromises cell differentiation, pointing to a prominent role for hemichannel function in this process. Therefore, scaffold design strategies that promote an increasing distance between single chondroprogenitor cells do not restrict their differentiation potential in tissue-engineered constructs.

Extremely Low-Frequency Electromagnetic Fields Affect Myogenic Processes in C2C12 Myoblasts: Role of Gap-Junction-Mediated Intercellular Communication

PubMed Central

Rovetta, Francesca; Boniotti, Jennifer; Mazzoleni, Giovanna

2017-01-01

Extremely low-frequency electromagnetic fields (ELF-EMFs) can interact with biological systems. Although they are successfully used as therapeutic agents in physiatrics and rehabilitative practice, they might represent environmental pollutants and pose a risk to human health. Due to the lack of evidence of their mechanism of action, the effects of ELF-EMFs on differentiation processes in skeletal muscle were investigated. C2C12 myoblasts were exposed to ELF-EMFs generated by a solenoid. The effects of ELF-EMFs on cell viability and on growth and differentiation rates were studied using colorimetric and vital dye assays, cytomorphology, and molecular analysis of MyoD and myogenin expression, respectively. The establishment of functional gap junctions was investigated analyzing connexin 43 expression levels and measuring cell permeability, using microinjection/dye-transfer assays. The ELF-EMFs did not affect C2C12 myoblast viability or proliferation rate. Conversely, at ELF-EMF intensity in the mT range, the myogenic process was accelerated, through increased expression of MyoD, myogenin, and connexin 43. The increase in gap-junction function suggests promoting cell fusion and myotube differentiation. These data provide the first evidence of the mechanism through which ELF-EMFs may provide therapeutic benefits and can resolve, at least in part, some conditions of muscle dysfunction. PMID:28607928

Real-time TIRF observation of vinculin recruitment to stretched α-catenin by AFM.

PubMed

Maki, Koichiro; Han, Sung-Woong; Hirano, Yoshinori; Yonemura, Shigenobu; Hakoshima, Toshio; Adachi, Taiji

2018-01-25

Adherens junctions (AJs) adaptively change their intensities in response to intercellular tension; therefore, they integrate tension generated by individual cells to drive multicellular dynamics, such as morphogenetic change in embryos. Under intercellular tension, α-catenin, which is a component protein of AJs, acts as a mechano-chemical transducer to recruit vinculin to promote actin remodeling. Although in vivo and in vitro studies have suggested that α-catenin-mediated mechanotransduction is a dynamic molecular process, which involves a conformational change of α-catenin under tension to expose a cryptic vinculin binding site, there are no suitable experimental methods to directly explore the process. Therefore, in this study, we developed a novel system by combining atomic force microscopy (AFM) and total internal reflection fluorescence (TIRF). In this system, α-catenin molecules (residues 276-634; the mechano-sensitive M 1 -M 3 domain), modified on coverslips, were stretched by AFM and their recruitment of Alexa-labeled full-length vinculin molecules, dissolved in solution, were observed simultaneously, in real time, using TIRF. We applied a physiologically possible range of tensions and extensions to α-catenin and directly observed its vinculin recruitment. Our new system could be used in the fields of mechanobiology and biophysics to explore functions of proteins under tension by coupling biomechanical and biochemical information.

Nanoparticles can cause DNA damage across a cellular barrier

NASA Astrophysics Data System (ADS)

Bhabra, Gevdeep; Sood, Aman; Fisher, Brenton; Cartwright, Laura; Saunders, Margaret; Evans, William Howard; Surprenant, Annmarie; Lopez-Castejon, Gloria; Mann, Stephen; Davis, Sean A.; Hails, Lauren A.; Ingham, Eileen; Verkade, Paul; Lane, Jon; Heesom, Kate; Newson, Roger; Case, Charles Patrick

2009-12-01

The increasing use of nanoparticles in medicine has raised concerns over their ability to gain access to privileged sites in the body. Here, we show that cobalt-chromium nanoparticles (29.5 +/- 6.3 nm in diameter) can damage human fibroblast cells across an intact cellular barrier without having to cross the barrier. The damage is mediated by a novel mechanism involving transmission of purine nucleotides (such as ATP) and intercellular signalling within the barrier through connexin gap junctions or hemichannels and pannexin channels. The outcome, which includes DNA damage without significant cell death, is different from that observed in cells subjected to direct exposure to nanoparticles. Our results suggest the importance of indirect effects when evaluating the safety of nanoparticles. The potential damage to tissues located behind cellular barriers needs to be considered when using nanoparticles for targeting diseased states.

Activation of protein kinase C and disruption of endothelial monolayer integrity by sodium arsenite-Potential mechanism in the development of atherosclerosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pereira, Flavia E.; Coffin, J. Douglas; Beall, Howard D.

2007-04-15

Arsenic exposure has been shown to exacerbate atherosclerosis, beginning with activation of the endothelium that lines the vessel wall. Endothelial barrier integrity is maintained by proteins of the adherens junction (AJ) such as vascular endothelial cadherin (VE-cadherin) and {beta}-catenin and their association with the actin cytoskeleton. In the present study, human aortic endothelial cells (HAECs) were exposed to 1, 5 and 10 {mu}M sodium arsenite [As(III)] for 1, 6, 12 and 24 h, and the effects on endothelial barrier integrity were determined. Immunofluorescence studies revealed formation of actin stress fibers and non-uniform VE-cadherin and {beta}-catenin staining at cell-cell junctions thatmore » were concentration- and time-dependent. Intercellular gaps were observed with a measured increase in endothelial permeability. In addition, concentration-dependent increases in tyrosine phosphorylation (PY) of {beta}-catenin and activation of protein kinase C{alpha} (PKC{alpha}) were observed. Inhibition of PKC{alpha} restored VE-cadherin and {beta}-catenin staining at cell-cell junctions and abolished the As(III)-induced formation of actin stress fibers and intercellular gaps. Endothelial permeability and PY of {beta}-catenin were also reduced to basal levels. These results demonstrate that As(III) induces activation of PKC{alpha}, which leads to increased PY of {beta}-catenin downstream of PKC{alpha} activation. Phosphorylation of {beta}-catenin plausibly severs the association of VE-cadherin and {beta}-catenin, which along with formation of actin stress fibers, results in intercellular gap formation and increased endothelial permeability. To the best of our knowledge, this is the first report demonstrating that As(III) causes a loss of endothelial monolayer integrity, which potentially could contribute to the development of atherosclerosis.« less

The Human “Cochlear Battery” – Claudin-11 Barrier and Ion Transport Proteins in the Lateral Wall of the Cochlea

PubMed Central

Liu, Wei; Schrott-Fischer, Annelies; Glueckert, Rudolf; Benav, Heval; Rask-Andersen, Helge

2017-01-01

Background: The cochlea produces an electric field potential essential for hair cell transduction and hearing. This biological “battery” is situated in the lateral wall of the cochlea and contains molecular machinery that secretes and recycles K+ ions. Its functioning depends on junctional proteins that restrict the para-cellular escape of ions. The tight junction protein Claudin-11 has been found to be one of the major constituents of this barrier that maintains ion gradients (Gow et al., 2004; Kitajiri et al., 2004a). We are the first to elucidate the human Claudin-11 framework and the associated ion transport machinery using super-resolution fluorescence illumination microscopy (SR-SIM). Methods: Archival cochleae obtained during meningioma surgery were used for SR-SIM together with transmission electron microscopy after ethical consent. Results: Claudin-11-expressing cells formed parallel tight junction lamellae that insulated the epithelial syncytium of the stria vascularis and extended to the suprastrial region. Intercellular gap junctions were found between the barrier cells and fibrocytes. Conclusion: Transmission electron microscopy, confocal microscopy and SR-SIM revealed exclusive cell specialization in the various subdomains of the lateral wall of the human cochlea. The Claudin-11-expressing cells exhibited both conductor and isolator characteristics, and these micro-porous separators may selectively mediate the movement of charged units to the intrastrial space in a manner that is analogous to a conventional electrochemical “battery.” The function and relevance of this battery for the development of inner ear disease are discussed. PMID:28848383

Extracellular domains play different roles in gap junction formation and docking compatibility.

PubMed

Bai, Donglin; Wang, Ao Hong

2014-02-15

GJ (gap junction) channels mediate direct intercellular communication and play an important role in many physiological processes. Six connexins oligomerize to form a hemichannel and two hemichannels dock together end-to-end to form a GJ channel. Connexin extracellular domains (E1 and E2) have been shown to be important for the docking, but the molecular mechanisms behind the docking and formation of GJ channels are not clear. Recent developments in atomic GJ structure and functional studies on a series of connexin mutants revealed that E1 and E2 are likely to play different roles in the docking. Non-covalent interactions at the docking interface, including hydrogen bonds, are predicted to form between interdocked extracellular domains. Protein sequence alignment analysis on the docking compatible/incompatible connexins indicate that the E1 domain is important for the formation of the GJ channel and the E2 domain is important in the docking compatibility in heterotypic channels. Interestingly, the hydrogen-bond forming or equivalent residues in both E1 and E2 domains are mutational hot spots for connexin-linked human diseases. Understanding the molecular mechanisms of GJ docking can assist us to develop novel strategies in rescuing the disease-linked connexin mutants.

Ultrastructural changes of the capillaries of the cat iris in experimental neuroparalytic keratitis.

PubMed

Saari, M; Huhtala, A; Johansson, G

1975-01-01

In order to study the morphological basis of the increased permeability of the capillaries of the iris in neuroparalytic keratitis the ophthalmic division of the trigeminal nerve in the cat was denervated using a stereotactic method. The homolateral iris was studied by electron microscopy three days after denervation. Abnormally large pinocytotic vacuoles were observed in the endothelial cells of the iris capillaries and the intercellular junctions of the endothelial cells showed widened inter-cellular space and macula occludens. These ultrastructural changes may explain the protein leakage into the anterior chamber in neuroparalytic keratitis.

The inner CSF–brain barrier: developmentally controlled access to the brain via intercellular junctions

PubMed Central

Whish, Sophie; Dziegielewska, Katarzyna M.; Møllgård, Kjeld; Noor, Natassya M.; Liddelow, Shane A.; Habgood, Mark D.; Richardson, Samantha J.; Saunders, Norman R.

2015-01-01

In the adult the interface between the cerebrospinal fluid and the brain is lined by the ependymal cells, which are joined by gap junctions. These intercellular connections do not provide a diffusional restrain between the two compartments. However, during development this interface, initially consisting of neuroepithelial cells and later radial glial cells, is characterized by “strap” junctions, which limit the exchange of different sized molecules between cerebrospinal fluid and the brain parenchyma. Here we provide a systematic study of permeability properties of this inner cerebrospinal fluid-brain barrier during mouse development from embryonic day, E17 until adult. Results show that at fetal stages exchange across this barrier is restricted to the smallest molecules (286Da) and the diffusional restraint is progressively removed as the brain develops. By postnatal day P20, molecules the size of plasma proteins (70 kDa) diffuse freely. Transcriptomic analysis of junctional proteins present in the cerebrospinal fluid-brain interface showed expression of adherens junctional proteins, actins, cadherins and catenins changing in a development manner consistent with the observed changes in the permeability studies. Gap junction proteins were only identified in the adult as was claudin-11. Immunohistochemistry was used to localize at the cellular level some of the adherens junctional proteins of genes identified from transcriptomic analysis. N-cadherin, β - and α-catenin immunoreactivity was detected outlining the inner CSF-brain interface from E16; most of these markers were not present in the adult ependyma. Claudin-5 was present in the apical-most part of radial glial cells and in endothelial cells in embryos, but only in endothelial cells including plexus endothelial cells in adults. Claudin-11 was only immunopositive in the adult, consistent with results obtained from transcriptomic analysis. These results provide information about physiological, molecular and morphological-related permeability changes occurring at the inner cerebrospinal fluid-brain barrier during brain development. PMID:25729345

Bioavailability and efficacy of a gap junction enhancer (PQ7) in a mouse mammary tumor model.

PubMed

Shishido, Stephanie N; Prasain, Keshar; Beck, Amanda; Nguyen, Thi D T; Hua, Duy H; Nguyen, Thu Annelise

2013-01-01

The loss of gap junctional intercellular communication is characteristic of neoplastic cells, suggesting that the restoration with a gap junction enhancer may be a new therapeutic treatment option with less detrimental effects than traditional antineoplastic drugs. A gap junction enhancer, 6-methoxy-8-[(2-furanylmethyl) amino]-4-methyl-5-(3-trifluoromethylphenyloxy) quinoline (PQ7), on the normal tissue was evaluated in healthy C57BL/6J mice in a systemic drug distribution study. Immunoblot analysis of the vital organs indicates a reduction in Cx43 expression in PQ7-treated animals with no observable change in morphology. Next the transgenic strain FVB/N-Tg(MMTV-PyVT) 634Mul/J (also known as PyVT) was used as a spontaneous mammary tumor mouse model to determine the biological and histological effects of PQ7 on tumorigenesis and metastasis at three stages of development: Pre tumor, Early tumor, and Late tumor formation. PQ7 was assessed to have a low toxicity through intraperitoneal administration, with the majority of the compound being detected in the heart, liver, and lungs six hours post injection. The treatment of tumor bearing animals with PQ7 had a 98% reduction in tumor growth, while also decreasing the total tumor burden compared to control mice during the Pre stage of development. PQ7 treatment increased Cx43 expression in the neoplastic tissue during Pre-tumor formation; however, this effect was not observed in Late stage tumor formation. This study shows that the gap junction enhancer, PQ7, has low toxicity to normal tissue in healthy C57BL/6J mice, while having clinical efficacy in the treatment of spontaneous mammary tumors of PyVT mice. Additionally, gap junctional intercellular communication and neoplastic cellular growth are shown to be inversely related, while treatment with PQ7 inhibits tumor growth through targeting gap junction expression.

Berberine potentizes apoptosis induced by X-rays irradiation probably through modulation of gap junctions.

PubMed

Liu, Bing; Wang, Qin; Yuan, Dong-dong; Hong, Xiao-ting; Tao, Liang

2011-04-01

Clinical combination of some traditional Chinese medical herbs, including berberine, with irradiation is demonstrated to improve efficacy of tumor radiotherapy, yet the mechanisms for such effect remain largely unknown. The present study investigated the effect of berberine on apoptosis induced by X-rays irradiation and the relation between this effect and gap junction intercellular communication (GJIC). The role of gap junctions in the modulation of X-rays irradiation-induced apoptosis was explored by manipulation of connexin (Cx) expression, and gap junction function, using oleamide, a GJIC inhibitor, and berberine. In transfected HeLa cells, Cx32 expression increased apoptosis induced by X-rays irradiation, while inhibition of gap junction by oleamide reduced the irradiation responses, indicating the dependence of X-rays irradiation-induced apoptosis on GJIC. Berberine, at the concentrations without cytotoxicity, enhanced apoptosis induced by irradiation only in the presence of functional gap junctions. These results suggest that berberine potentizes cell apoptosis induced by X-rays irradiation, probably through enhancement of gap junction activity.

On the self-association potential of transmembrane tight junction proteins.

PubMed

Blasig, I E; Winkler, L; Lassowski, B; Mueller, S L; Zuleger, N; Krause, E; Krause, G; Gast, K; Kolbe, M; Piontek, J

2006-02-01

Tight junctions seal intercellular clefts via membrane-related strands, hence, maintaining important organ functions. We investigated the self-association of strand-forming transmembrane tight junction proteins. The regulatory tight junction protein occludin was differently tagged and cotransfected in eucaryotic cells. These occludins colocalized within the plasma membrane of the same cell, coprecipitated and exhibited fluorescence resonance energy transfer. Differently tagged strand-forming claudin-5 also colocalized in the plasma membrane of the same cell and showed fluorescence resonance energy transfer. This demonstrates self-association in intact cells both of occludin and claudin-5 in one plasma membrane. In search of dimerizing regions of occludin, dimerization of its cytosolic C-terminal coiledcoil domain was identified. In claudin-5, the second extracellular loop was detected as a dimer. Since the transmembrane junctional adhesion molecule also is known to dimerize, the assumption that homodimerization of transmembrane tight junction proteins may serve as a common structural feature in tight junction assembly is supported.

RESPONSE TO KLAUNIG, J.E. ET AL, EPIGENETIC MECHANISMS OF CHEMICAL CARCINOGENESIS

EPA Science Inventory

The article by Klaunig et al. is a comprehensive review of the general principles underlying the induction of tumors by epigenetic mechanisms. The review describes the roles of cell proliferation, loss of apoptotic function, gap junctional intercellular communication, P450 induct...

Cotton fibre cross-section properties

USDA-ARS?s Scientific Manuscript database

From a structural perspective the cotton fibre is a singularly discrete, elongated plant cell with no junctions or inter-cellular boundaries. Its form in nature is essentially unadulterated from the field to the spinning mill where its cross-section properties, as for any textile fibre, are central ...

Comparison of ultrastructure, tight junction-related protein expression and barrier function of human corneal epithelial cells cultivated on amniotic membrane with and without air-lifting.

PubMed

Ban, Yuriko; Cooper, Leanne J; Fullwood, Nigel J; Nakamura, Takahiro; Tsuzuki, Masakatsu; Koizumi, Noriko; Dota, Atsuyoshi; Mochida, Chikako; Kinoshita, Shigeru

2003-06-01

To evaluate the usefulness of the air-lifting technique for culturing corneal limbal epithelial cells on amniotic membrane (AM) for use in ocular surface reconstruction. A cultured sheet that has a good barrier function should be better for this purpose. In corneal epithelium, tight junctions (TJ) play a vital role in the barrier function. The TJ complex includes the integral transmembrane proteins occludin and the claudins, and some membrane-associated proteins such as ZO-1. In this paper, we investigated the barrier function and the expression of TJ related proteins. Corneal limbal epithelium obtained from donor corneas and cultivated on acellular AM was divided into two groups. These were the non-air-lifting (Non-AL) group, which was continuously submerged in medium, and the air-lifting (AL) group, which was submerged in medium for 3 weeks, then exposed to air by lowering the medium level. Morphology and the permeability to horseradish peroxidase (HRP) were determined by electron microscopy. Tight junction (TJ)-related protein and mRNA expression changes were assessed by immunoblotting and reverse transcription-polymerase chain reaction. The cultures of both groups formed 4-5-layer-thick, well-stratified epithelium. The AL cultures had tightly packed epithelial cells with all the HRP/diaminobenzidine (DAB) reaction product accumulated on the apical surface of the superficial cells. The Non-AL culture, by contrast, had more loosely packed epithelial cells with larger intercellular spaces. The HRP/DAB reaction product penetrated the intercellular space to a depth of 3-4 cell layers. Statistically, there was a significant difference in intercellular spaces and desmosome count in the superficial cells between the groups. With AL, TJ-related proteins localized at the apical portion of the lateral membrane. TJ-related protein and mRNA amounts were not changed by AL while claudin subtype expression became more consistent and closer to that of in vivo corneal epithelium. The AL technique reduces intercellular spaces in the superficial cells and promotes the formation of the barrier function. It is useful in culturing corneal epithelial cells for use in ocular surface reconstruction.

'Til Eph do us part': intercellular signaling via Eph receptors and ephrin ligands guides cerebral cortical development from birth through maturation.

PubMed

North, Hilary A; Clifford, Meredith A; Donoghue, Maria J

2013-08-01

Eph receptors, the largest family of surface-bound receptor tyrosine kinases and their ligands, the ephrins, mediate a wide variety of cellular interactions in most organ systems throughout both development and maturity. In the forming cerebral cortex, Eph family members are broadly and dynamically expressed in particular sets of cortical cells at discrete times. Here, we review the known functions of Eph-mediated intercellular signaling in the generation of progenitors, the migration of maturing cells, the differentiation of neurons, the formation of functional connections, and the choice between life and death during corticogenesis. In synthesizing these results, we posit a signaling paradigm in which cortical cells maintain a life history of Eph-mediated intercellular interactions that guides subsequent cellular decision-making.

Integrin-linked kinase and ELMO2 modulate recycling endosomes in keratinocytes.

PubMed

Ho, Ernest; Ivanova, Iordanka A; Dagnino, Lina

2016-12-01

The formation of tight cell-cell junctions is essential in the epidermis for its barrier properties. In this tissue, keratinocytes follow a differentiation program tightly associated with their movement from the innermost basal to the outer suprabasal layers, and with changes in their cell-cell adhesion profile. Intercellular adhesion in keratinocytes is mediated through cell-cell contacts, including E-cadherin-based adherens junctions. Although the mechanisms that mediate E-cadherin delivery to the plasma membrane have been widely studied in simple epithelia, this process is less well understood in the stratified epidermis. In this study, we have investigated the role of Engulfment and Cell Motility 2 (ELMO2) and integrin-linked kinase (ILK) in the positioning of E-cadherin-containing recycling endosomes during establishment of cell-cell contacts in differentiating keratinocytes. We now show that induction of keratinocyte differentiation by Ca 2+ is accompanied by localization of ELMO2 and ILK to Rab4- and Rab11a-containing recycling endosomes. The positioning of long-loop Rab11a-positive endosomes at areas adjacent to cell-cell contacts is disrupted in ELMO2- or ILK-deficient keratinocytes, and is associated with impaired localization of E-cadherin to cell borders. Our studies show a previously unrecognized role for ELMO2 and ILK in modulation of endosomal positioning, which may play key roles in epidermal sheet maintenance and permeability barrier function. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular mechanisms regulating formation, trafficking and processing of annular gap junctions.

PubMed

Falk, Matthias M; Bell, Cheryl L; Kells Andrews, Rachael M; Murray, Sandra A

2016-05-24

Internalization of gap junction plaques results in the formation of annular gap junction vesicles. The factors that regulate the coordinated internalization of the gap junction plaques to form annular gap junction vesicles, and the subsequent events involved in annular gap junction processing have only relatively recently been investigated in detail. However it is becoming clear that while annular gap junction vesicles have been demonstrated to be degraded by autophagosomal and endo-lysosomal pathways, they undergo a number of additional processing events. Here, we characterize the morphology of the annular gap junction vesicle and review the current knowledge of the processes involved in their formation, fission, fusion, and degradation. In addition, we address the possibility for connexin protein recycling back to the plasma membrane to contribute to gap junction formation and intercellular communication. Information on gap junction plaque removal from the plasma membrane and the subsequent processing of annular gap junction vesicles is critical to our understanding of cell-cell communication as it relates to events regulating development, cell homeostasis, unstable proliferation of cancer cells, wound healing, changes in the ischemic heart, and many other physiological and pathological cellular phenomena.

Alternation in the gap-junctional intercellular communication capacity during the maturation of osteocytes in the embryonic chick calvaria.

PubMed

Wang, Ziyi; Odagaki, Naoya; Tanaka, Tomoyo; Hashimoto, Mana; Nakamura, Masahiro; Hayano, Satoru; Ishihara, Yoshihito; Kawanabe, Noriaki; Kamioka, Hiroshi

2016-10-01

The intercellular network of cell-cell communication among osteocytes is mediated by gap junctions. Gap junctional intercellular communication (GJIC) is thought to play an important role in the integration and synchronization of bone remodeling. To further understand the mechanism of bone development it is important to quantify the difference in the GJIC capacity of young and developmentally mature osteocytes. We first established an embryonic chick calvaria growth model to show the growth of the calvaria in embryos at 13 to 21days of age. We then applied a fluorescence recovery after photobleaching (FRAP) technique to compare the difference in the GJIC capacity of young osteocytes with that of developmentally mature osteocytes. Finally, we quantified the dye (Calcein) diffusion from the FRAP data using a mathematic model of simple diffusion which was also used to identify simple diffusion GJIC pattern cells (fitted model) and accelerated diffusion GJIC pattern cells (non-fitted model). The relationship between the longest medial-lateral length of the calvaria (frontal bone) and the embryonic age fit a logarithmic growth model: length=5.144×ln(day)-11.340. The morphometric data during osteocyte differentiation showed that the cellular body becomes more spindle-shaped and that the cell body volume decreased by approximately 22% with an increase in the length of the processes between the cells. However, there were no significant differences in the cellular body surface area or in the distance between the mass centres of the cells. The dye-displacement rate in young osteocytes was significantly higher than that in developmentally mature osteocytes: dye displacement only occurred in 26.88% of the developmentally mature osteocytes, while it occurred in 64.38% of the young osteocytes. Additionally, in all recovered osteocytes, 36% of the developmentally mature osteocytes comprised non-fitted model cells while 53.19% of the young osteocytes were the non-fitted model, which indicates the active transduction of dye molecules. However, there were no statistically significant differences between the young and developmentally mature osteocytes with regard to the diffusion coefficient, permeability coefficient, or permeance of the osteocyte processes, which were 3.93±3.77 (×10(-8)cm(2)/s), 5.12±4.56 (×10(-5)cm(2)/s) and 2.99±2.47 (×10(-13)cm(2)/s) (mean±SD), respectively. These experiments comprehensively quantified the GJIC capacity in the embryonic chick calvaria and indicated that the cell-cell communication capacity of the osteocytes in the embryonic chick calvaria was related to their development. Copyright © 2016 Elsevier Inc. All rights reserved.

MELATONIN ENHANCES JUNCTIONAL TRANSFER IN NORMAL C3H/1OT1/2 CELLS

EPA Science Inventory

There is strong evidence that pineal melatonin is involved in controlling neoplastic processes. e have reported that physiological, but not pharmacological or subphysiological, concentrations of melatonin enhance intercellular communication in normal C3H/1OT1/2 fibroblasts. ap ju...

Increase in gap junctional intercellular communication by high molecular weight hyaluronic acid associated with fibroblast growth factor 2 and keratinocyte growth factor production in normal human dermal fibroblasts.

PubMed

Park, Jeong Ung; Tsuchiya, Toshie

2002-07-01

The effects of different molecular weights of hyaluronic acid (HA), a major component of extracellular matrix, on gap junctional intercellular communication (GJIC) in normal human dermal fibroblasts (NHDF cells) were investigated. NHDF cells were cultured for 4 days with different molecular weights of HA and then the extent of GJIC was assessed by the scrape-loading dye transfer method, using Lucifer yellow. The area of dye transfer was greater in the dishes coated with HA than in those to which HA was added. Thus, NHDF cells cultured on surfaces coated with high molecular weight (HMW) HA (MW, 800 kDa) showed greatly enhanced GJIC. Furthermore, another aim of this study was to evaluate the effects of different molecular weights of HA on the production of FGF-2 and KGF, because both are important cytokines produced by NHDF cells. When FGF-2 and KGF cultured levels of cell extracts and media were determined by ELISA, both levels were significantly enhanced when cells were grown on plates coated with HMW HA. This finding indicated that the function of gap junction channels in NHDF cells grown on plates coated with HMW HA may promote the biosynthesis of growth factors such as FGF-2 and KGF.

Evolution of Microbial Quorum Sensing to Human Global Quorum Sensing: An Insight into How Gap Junctional Intercellular Communication Might Be Linked to the Global Metabolic Disease Crisis.

PubMed

Trosko, James E

2016-06-15

The first anaerobic organism extracted energy for survival and reproduction from its source of nutrients, with the genetic means to ensure protection of its individual genome but also its species survival. While it had a means to communicate with its community via simple secreted molecules ("quorum sensing"), the eventual shift to an aerobic environment led to multi-cellular metazoan organisms, with evolutionary-selected genes to form extracellular matrices, stem cells, stem cell niches, and a family of gap junction or "connexin" genes. These germinal and somatic stem cells responded to extracellular signals that triggered intra-cellular signaling to regulate specific genes out of the total genome. These extra-cellular induced intra-cellular signals also modulated gap junctional intercellular communication (GJIC) in order to regulate the new cellular functions of symmetrical and asymmetrical cell division, cell differentiation, modes of cell death, and senescence. Within the hierarchical and cybernetic concepts, differentiated by neurons organized in the brain of the Homo sapiens, the conscious mind led to language, abstract ideas, technology, myth-making, scientific reasoning, and moral decision-making, i.e., the creation of culture. Over thousands of years, this has created the current collision between biological and cultural evolution, leading to the global "metabolic disease" crisis.

Electric Stimulus Opens Intercellular Spaces in Skin*

PubMed Central

Hama, Susumu; Kimura, Yuki; Mikami, Aya; Shiota, Kanako; Toyoda, Mao; Tamura, Atsushi; Nagasaki, Yukio; Kanamura, Kiyoshi; Kajimoto, Kazuaki; Kogure, Kentaro

2014-01-01

Iontophoresis is a technology for transdermal delivery of ionic small medicines by faint electricity. Since iontophoresis can noninvasively deliver charged molecules into the skin, this technology could be a useful administration method that may enhance patient comfort. Previously, we succeeded in the transdermal penetration of positively charged liposomes (diameters: 200–400 nm) encapsulating insulin by iontophoresis (Kajimoto, K., Yamamoto, M., Watanabe, M., Kigasawa, K., Kanamura, K., Harashima, H., and Kogure, K. (2011) Int. J. Pharm. 403, 57–65). However, the mechanism by which these liposomes penetrated the skin was difficult to define based on general knowledge of principles such as electro-repulsion and electro-osmosis. In the present study, we confirmed that rigid nanoparticles could penetrate into the epidermis by iontophoresis. We further found that levels of the gap junction protein connexin 43 protein significantly decreased after faint electric stimulus (ES) treatment, although occludin, CLD-4, and ZO-1 levels were unchanged. Moreover, connexin 43 phosphorylation and filamentous actin depolymerization in vivo and in vitro were observed when permeation of charged liposomes through intercellular spaces was induced by ES. Ca2+ inflow into cells was promoted by ES with charged liposomes, while a protein kinase C inhibitor prevented ES-induced permeation of macromolecules. Consequently, we demonstrate that ES treatment with charged liposomes induced dissociation of intercellular junctions via cell signaling pathways. These findings suggest that ES could be used to regulate skin physiology. PMID:24318878

Distinct moieties underlie biphasic H+ gating of connexin43 channels, producing a pH optimum for intercellular communication

PubMed Central

Garciarena, Carolina D.; Malik, Akif; Swietach, Pawel; Moreno, Alonso P.; Vaughan-Jones, Richard D.

2018-01-01

Most mammalian cells can intercommunicate via connexin-assembled, gap-junctional channels. To regulate signal transmission, connexin (Cx) channel permeability must respond dynamically to physiological and pathophysiological stimuli. One key stimulus is intracellular pH (pHi), which is modulated by a tissue’s metabolic and perfusion status. Our understanding of the molecular mechanism of H+ gating of Cx43 channels—the major isoform in the heart and brain—is incomplete. To interrogate the effects of acidic and alkaline pHi on Cx43 channels, we combined voltage-clamp electrophysiology with pHi imaging and photolytic H+ uncaging, performed over a range of pHi values. We demonstrate that Cx43 channels expressed in HeLa or N2a cell pairs are gated biphasically by pHi via a process that consists of activation by H+ ions at alkaline pHi and inhibition at more acidic pHi. For Cx43 channel–mediated solute/ion transmission, the ensemble of these effects produces a pHi optimum, near resting pHi. By using Cx43 mutants, we demonstrate that alkaline gating involves cysteine residues of the C terminus and is independent of motifs previously implicated in acidic gating. Thus, we present a molecular mechanism by which cytoplasmic acid–base chemistry fine tunes intercellular communication and establishes conditions for the optimal transmission of solutes and signals in tissues, such as the heart and brain.—Garciarena, C. D., Malik, A., Swietach, P., Moreno, A. P., Vaughan-Jones, R. D. Distinct moieties underlie biphasic H+ gating of connexin43 channels, producing a pH optimum for intercellular communication. PMID:29183963

Extracellular Vesicles, Tunneling Nanotubes, and Cellular Interplay: Synergies and Missing Links

PubMed Central

Nawaz, Muhammad; Fatima, Farah

2017-01-01

The process of intercellular communication seems to have been a highly conserved evolutionary process. Higher eukaryotes use several means of intercellular communication to address both the changing physiological demands of the body and to fight against diseases. In recent years, there has been an increasing interest in understanding how cell-derived nanovesicles, known as extracellular vesicles (EVs), can function as normal paracrine mediators of intercellular communication, but can also elicit disease progression and may be used for innovative therapies. Over the last decade, a large body of evidence has accumulated to show that cells use cytoplasmic extensions comprising open-ended channels called tunneling nanotubes (TNTs) to connect cells at a long distance and facilitate the exchange of cytoplasmic material. TNTs are a different means of communication to classical gap junctions or cell fusions; since they are characterized by long distance bridging that transfers cytoplasmic organelles and intracellular vesicles between cells and represent the process of heteroplasmy. The role of EVs in cell communication is relatively well-understood, but how TNTs fit into this process is just emerging. The aim of this review is to describe the relationship between TNTs and EVs, and to discuss the synergies between these two crucial processes in the context of normal cellular cross-talk, physiological roles, modulation of immune responses, development of diseases, and their combinatory effects in tissue repair. At the present time this review appears to be the first summary of the implications of the overlapping roles of TNTs and EVs. We believe that a better appreciation of these parallel processes will improve our understanding on how these nanoscale conduits can be utilized as novel tools for targeted therapies. PMID:28770210

Collective cell migration of thyroid carcinoma cells: a beneficial ability to override unfavourable substrates.

PubMed

Lobastova, Liudmila; Kraus, Dominik; Glassmann, Alexander; Khan, Dilaware; Steinhäuser, Christian; Wolff, Christina; Veit, Nadine; Winter, Jochen; Probstmeier, Rainer

2017-02-01

Tumor cell invasion and metastasis are life threatening events. Invasive tumor cells tend to migrate as collective sheets. In the present in vitro study we aimed to (i) assess whether collective tumor cells gain benefits in their migratory potential compared to single cells and (ii) to identify its putative underlying molecular mechanisms. The migratory potential of single and collective carcinoma cells was assessed using video time lapse microscopy and cell migration assays in the absence and presence of seven potential gap junction inhibitors or the Rac1 inhibitor Z62954982. The perturbation of gap junctions was assessed using a dye diffusion assay. In addition, LDH-based cytotoxicity and RT-PCR-based expression analyses were performed. Whereas single breast, cervix and thyroid carcinoma cells were virtually immobile on unfavourable plastic surfaces, we found that they gained pronounced migratory capacities as collectives under comparable conditions. Thyroid carcinoma cells, that were studied in more detail, were found to express specific subsets of connexins and to form active gap junctions as revealed by dye diffusion analysis. Although all potential gap junction blockers suppressed intercellular dye diffusion in at least one of the cell lines tested, only two of them were found to inhibit collective cell migration and none of them to inhibit single cell migration. In the presence of the Rac1 inhibitor Z62954982 collective migration, but not single cell migration, was found to be reduced up to 20 %. Our data indicate that collective migration enables tumor cells to cross otherwise unfavourable substrate areas. This capacity seems to be independent of intercellular communication via gap junctions, whereas Rac1-dependent intracellular signalling seems to be essential.

Asymmetric homotypic interactions of the atypical cadherin Flamingo mediate intercellular polarity signaling

PubMed Central

Chen, Wei-Shen; Antic, Dragana; Matis, Maja; Logan, Catriona Y.; Povelones, Michael; Anderson, Graham; Nusse, Roel; Axelrod, Jeffrey D.

2008-01-01

Acquisition of planar cell polarity (PCP) in epithelia involves intercellular communication, during which cells align their polarity with that of their neighbors. The transmembrane proteins Frizzled (Fz) and Van Gogh (Vang) are essential components of the intercellular communication mechanism, as loss of either strongly perturbs the polarity of neighboring cells. How Fz and Vang communicate polarity information between neighboring cells is poorly understood. The atypical cadherin, Flamingo (Fmi), is implicated in this process, yet whether Fmi acts permissively as a scaffold, or instructively as a signal is unclear. Here, we provide evidence that Fmi functions instructively to mediate Fz-Vang intercellular signal relay, recruiting Fz and Vang to opposite sides of cell boundaries. We propose that two functional forms of Fmi, one of which is induced by and physically interacts with Fz, form cadherin homodimers that signal bidirectionally and asymmetrically, instructing unequal responses in adjacent cell membranes to establish molecular asymmetry. PMID:18555784

A Study of Intercellular Spaces in the Rabbit Jejunum during Acute Volume Expansion and after Treatment with Cholera Toxin

PubMed Central

DiBona, Donald R.; Chen, Lincoln C.; Sharp, Geoffrey W. G.

1974-01-01

The effects of acute volume expansion and of intraluminal administration of cholera toxin have been examined in rabbit jejunum. Acute volume expansion was shown to reverse the normal reabsorptive flux of water and cause significant fluid secretion. Phase and electronmicroscopic examination of the jejunal epithelium showed that marked distension of the intercellular spaces had occurred. Examination of the jejunal epithelium after treatment with cholera toxin showed that, in association with high rates of fluid secretion, the intercellular spaces were extremely small and lateral membranes of adjacent cells were in close apposition to one another. Thus the mechanisms of fluid secretion in these two situations would appear to be quite different. The secretion associated with volume expansion, and accompanied by a rise in venous pressure and bullous deformations of terminal junctions, could well be due to hydrostatic pressure applied through intercellular channels. The secretion of cholera appears to be unrelated to hydrostatic pressure and is more likely due to body-to-lumen active ion transport. Images PMID:4596506

Cell-to-cell communication in plants, animals, and fungi: a comparative review.

PubMed

Bloemendal, Sandra; Kück, Ulrich

2013-01-01

Cell-to-cell communication is a prerequisite for differentiation and development in multicellular organisms. This communication has to be tightly regulated to ensure that cellular components such as organelles, macromolecules, hormones, or viruses leave the cell in a precisely organized way. During evolution, plants, animals, and fungi have developed similar ways of responding to this biological challenge. For example, in higher plants, plasmodesmata connect adjacent cells and allow communication to regulate differentiation and development. In animals, two main general structures that enable short- and long-range intercellular communication are known, namely gap junctions and tunneling nanotubes, respectively. Finally, filamentous fungi have also developed specialized structures called septal pores that allow intercellular communication via cytoplasmic flow. This review summarizes the underlying mechanisms for intercellular communication in these three eukaryotic groups and discusses its consequences for the regulation of differentiation and developmental processes.

Cellular level robotic surgery: Nanodissection of intermediate filaments in live keratinocytes.

PubMed

Yang, Ruiguo; Song, Bo; Sun, Zhiyong; Lai, King Wai Chiu; Fung, Carmen Kar Man; Patterson, Kevin C; Seiffert-Sinha, Kristina; Sinha, Animesh A; Xi, Ning

2015-01-01

We present the nanosurgery on the cytoskeleton of live cells using AFM based nanorobotics to achieve adhesiolysis and mimic the effect of pathophysiological modulation of intercellular adhesion. Nanosurgery successfully severs the intermediate filament bundles and disrupts cell-cell adhesion similar to the desmosomal protein disassembly in autoimmune disease, or the cationic modulation of desmosome formation. Our nanomechanical analysis revealed that adhesion loss results in a decrease in cellular stiffness in both cases of biochemical modulation of the desmosome junctions and mechanical disruption of intercellular adhesion, supporting the notion that intercellular adhesion through intermediate filaments anchors the cell structure as focal adhesion does and that intermediate filaments are integral components in cell mechanical integrity. The surgical process could potentially help reveal the mechanism of autoimmune pathology-induced cell-cell adhesion loss as well as its related pathways that lead to cell apoptosis. Copyright © 2015 Elsevier Inc. All rights reserved.

Cell-to-cell communication in plants, animals, and fungi: a comparative review

NASA Astrophysics Data System (ADS)

Bloemendal, Sandra; Kück, Ulrich

2013-01-01

Cell-to-cell communication is a prerequisite for differentiation and development in multicellular organisms. This communication has to be tightly regulated to ensure that cellular components such as organelles, macromolecules, hormones, or viruses leave the cell in a precisely organized way. During evolution, plants, animals, and fungi have developed similar ways of responding to this biological challenge. For example, in higher plants, plasmodesmata connect adjacent cells and allow communication to regulate differentiation and development. In animals, two main general structures that enable short- and long-range intercellular communication are known, namely gap junctions and tunneling nanotubes, respectively. Finally, filamentous fungi have also developed specialized structures called septal pores that allow intercellular communication via cytoplasmic flow. This review summarizes the underlying mechanisms for intercellular communication in these three eukaryotic groups and discusses its consequences for the regulation of differentiation and developmental processes.

Propofol depresses cisplatin cytotoxicity via the inhibition of gap junctions.

PubMed

Zhang, Yuan; Wang, Xiyan; Wang, Qin; Ge, Hui; Tao, Liang

2016-06-01

The general anesthetic, propofol, affects chemotherapeutic activity, however, the mechanism underlying its effects remains to be fully elucidated. Our previous study showed that tramadol and flurbiprofen depressed the cytotoxicity of cisplatin via the inhibition of gap junction (GJ) intercellular communication (GJIC) in connexin (Cx)32 HeLa cells. The present study investigated whether the effects of propofol on the cytotoxicity of cisplatin were mediated by GJ in U87 glioma cells and Cx26‑transfected HeLa cells. Standard colony formation assay was used to determine the cytotoxicity of cisplatin. Parachute dye coupling assay was used to measure GJ function, and western blot analysis was used to determine the expression levels of Cx32. The results revealed that exposure of the U87 glioma cells and the Cx26-transfected HeLa cells to cisplatin for 1 h reduced clonogenic survival in low density cultures (without GJs) and high density cultures (with GJs). However, the toxic effect was higher in the high density culture. In addition, pretreatment of the cells with propofol significantly reduced cisplatin‑induced cytotoxicity, but only in the presence of functional GJs. Furthermore, propofol significantly inhibited dye coupling through junctional channels, and a long duration of exposure of the cells to propofol downregulated the expression levels of Cx43 and Cx26. These results demonstrated that the inhibition of GJIC by propofol affected the therapeutic efficacy of chemotherapeutic drugs. The present study provides evidence of a novel mechanism underlying the effects of analgesics in counteracting chemotherapeutic efficiency.

Gap junction-mediated calcium waves define communication networks among murine postnatal neural progenitor cells.

PubMed

Lacar, Benjamin; Young, Stephanie Z; Platel, Jean-Claude; Bordey, Angélique

2011-12-01

In the postnatal neurogenic niche, two populations of astrocyte-like cells (B cells) persist, one acting as neural progenitor cells (NPCs, B1 cells) and one forming a structural boundary between the neurogenic niche and the striatum (B2 cells, niche astrocytes). Despite being viewed as two distinct entities, we found that B1 and B2 cells express the gap junction protein connexin 43 and display functional coupling involving 50-60 cells. Using neonatal electroporation to label slowly cycling radial glia-derived B1 cells, which send a basal process onto blood vessels, we further confirmed dye coupling between NPCs. To assess the functionality of the coupling, we used calcium imaging in a preparation preserving the three-dimensional architecture of the subventricular zone. Intercellular calcium waves were observed among B cells. These waves travelled bidirectionally between B1 and B2 cells and propagated on blood vessels. Inter-B-cell calcium waves were absent in the presence of a gap junction blocker but persisted with purinergic receptor blockers. These findings show that privileged microdomains of communication networks exist among NPCs and niche astrocytes. Such functional coupling between these two cell types suggests that niche astrocytes do not merely have a structural role, but may play an active role in shaping the behavior of NPCs. © 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

An In Vitro Model of the Blood-Brain Barrier: Naegleria fowleri Affects the Tight Junction Proteins and Activates the Microvascular Endothelial Cells.

PubMed

Coronado-Velázquez, Daniel; Betanzos, Abigail; Serrano-Luna, Jesús; Shibayama, Mineko

2018-04-14

Naegleria fowleri causes a fatal disease known as primary amoebic meningoencephalitis. This condition is characterized by an acute inflammation that originates from the free passage of peripheral blood cells to the central nervous system through the alteration of the blood-brain barrier. In this work, we established models of the infection in rats and in a primary culture of endothelial cells from rat brains with the aim of evaluating the activation and the alterations of these cells by N. fowleri. We proved that the rat develops the infection similar to the mouse model. We also found that amoebic cysteine proteases produced by the trophozoites and the conditioned medium induced cytopathic effect in the endothelial cells. In addition, N. fowleri can decrease the transendothelial electrical resistance by triggering the destabilization of the tight junction proteins claudin-5, occludin, and ZO-1 in a time-dependent manner. Furthermore, N. fowleri induced the expression of VCAM-1 and ICAM-1 and the production of IL-8, IL-1β, TNF-α, and IL-6 as well as nitric oxide. We conclude that N. fowleri damaged the blood-brain barrier model by disrupting the intercellular junctions and induced the presence of inflammatory mediators by allowing the access of inflammatory cells to the olfactory bulbs. © 2018 The Author(s) Journal of Eukaryotic Microbiology © 2018 International Society of Protistologists.

Endothelial connexin 32 regulates tissue factor expression induced by inflammatory stimulation and direct cell-cell interaction with activated cells.

PubMed

Okamoto, Takayuki; Akita, Nobuyuki; Hayashi, Tatsuya; Shimaoka, Motomu; Suzuki, Koji

2014-10-01

Endothelial cell (EC) interacts with adjacent EC through gap junction, and abnormal expression or function of Cxs is associated with cardiovascular diseases. In patients with endothelial dysfunction, the up-regulation of tissue factor (TF) expression promotes the pathogenic activation of blood coagulation, however the relationship between gap junctions and TF expression in ECs remains uncharacterized. ECs express the gap junction (GJ) proteins connexin32 (Cx32), Cx37, Cx40 and Cx43. We investigated the role of endothelial gap junctions, particularly Cx32, in modulating TF expression during vascular inflammation. Human umbilical vein endothelial cells (HUVECs) were stimulated with tumor necrosis factor-α (TNF-α) and TF activity was assessed in the presence of GJ blockers and an inhibitory anti-Cx32 monoclonal antibody. Treatment with GJ blockers and anti-Cx32 monoclonal antibody enhanced the TNF-α-induced TF activity and mRNA expression in HUVECs. TNF-α-activated effector HUVECs or mouse MS-1 cells were co-cultured with non-stimulated acceptor HUVECs and TF expression in acceptor HUVECs was detected. Effector EC induced TF expression in adjacent acceptor HUVECs through direct cell-cell interaction. Cell-cell interaction induced TF expression was reduced by anti-intercellular adhesion molecule-1 (ICAM1) monoclonal antibody. Soluble ICAM1-Fc fusion protein promotes TF expression. GJ blockers and anti-Cx32 monoclonal antibody enhanced TF expression induced by cell-cell interaction and ICAM1-Fc treatment. Blockade of endothelial Cx32 increased TF expression induced by TNF-α stimulation and cell-cell interaction which was at least partly dependent upon ICAM1. These results suggest that direct Cx32-mediated interaction modulates TF expression in ECs during vascular inflammation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Interactive effects of inflammatory cytokine and abundant low-molecular-weight PAHs on inhibition of gap junctional intercellular communication, disruption of cell proliferation control, and the AhR-dependent transcription.

PubMed

Kabátková, Markéta; Svobodová, Jana; Pěnčíková, Kateřina; Mohatad, Dilshad Shaik; Šmerdová, Lenka; Kozubík, Alois; Machala, Miroslav; Vondráček, Jan

2015-01-05

Polycyclic aromatic hydrocarbons (PAHs) with lower molecular weight exhibit lesser genotoxicity and carcinogenicity than highly carcinogenic PAHs with a higher number of benzene rings. Nevertheless, they elicit specific effects linked with tumor promotion, such as acute inhibition of gap junctional intercellular communication (GJIC). Although inflammatory reaction may alter bioactivation and toxicity of carcinogenic PAHs, little is known about the impact of pro-inflammatory cytokines on toxic effects of the low-molecular-weight PAHs. Here, we investigated the impact of a pro-inflammatory cytokine, tumor necrosis factor-α (TNF-α), on the effects associated with tumor promotion and with induction of the aryl hydrocarbon receptor (AhR)-dependent gene expression in rat liver epithelial cells. We found that a prolonged incubation with TNF-α induced a down-regulation of GJIC, associated with reduced expression of connexin 43 (Cx43), a major connexin isoform found in liver epithelial cells. The Cx43 down-regulation was partly mediated by the activity of the mitogen-activated protein (MAP) p38 kinase. Independently of GJIC modulation, or p38 activation, TNF-α potentiated the AhR-dependent proliferative effect of a model low-molecular-weight PAH, fluoranthene, on contact-inhibited cells. In contrast, this pro-inflammatory cytokine repressed the fluoranthene-induced expression of a majority of model AhR gene targets, such as Cyp1a1, Ahrr or Tiparp. The results of the present study indicate that inflammatory reaction may differentially modulate various toxic effects of low-molecular-weight PAHs; the exposure to pro-inflammatory cytokines may both strengthen (inhibition of GJIC, disruption of contact inhibition) and repress (expression of a majority of AhR-dependent genes) their impact on toxic endpoints associated with carcinogenesis. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 5: intercellular junctions and contacts between germs cells and Sertoli cells and their regulatory interactions, testicular cholesterol, and genes/proteins associated with more than one germ cell generation.

PubMed

Hermo, Louis; Pelletier, R-Marc; Cyr, Daniel G; Smith, Charles E

2010-04-01

In the testis, cell adhesion and junctional molecules permit specific interactions and intracellular communication between germ and Sertoli cells and apposed Sertoli cells. Among the many adhesion family of proteins, NCAM, nectin and nectin-like, catenins, and cadherens will be discussed, along with gap junctions between germ and Sertoli cells and the many members of the connexin family. The blood-testis barrier separates the haploid spermatids from blood borne elements. In the barrier, the intercellular junctions consist of many proteins such as occludin, tricellulin, and claudins. Changes in the expression of cell adhesion molecules are also an essential part of the mechanism that allows germ cells to move from the basal compartment of the seminiferous tubule to the adluminal compartment thus crossing the blood-testis barrier and well-defined proteins have been shown to assist in this process. Several structural components show interactions between germ cells to Sertoli cells such as the ectoplasmic specialization which are more closely related to Sertoli cells and tubulobulbar complexes that are processes of elongating spermatids embedded into Sertoli cells. Germ cells also modify several Sertoli functions and this also appears to be the case for residual bodies. Cholesterol plays a significant role during spermatogenesis and is essential for germ cell development. Lastly, we list genes/proteins that are expressed not only in any one specific generation of germ cells but across more than one generation. Copyright 2009 Wiley-Liss, Inc.

Herpes simplex virus glycoprotein D relocates nectin-1 from intercellular contacts

PubMed Central

Bhargava, Arjun K.; Rothlauf, Paul W.; Krummenacher, Claude

2016-01-01

Herpes simplex virus (HSV) uses the cell adhesion molecule nectin-1 as a receptor to enter neurons and epithelial cells. The viral glycoprotein D (gD) is used as a non-canonical ligand for nectin-1. The gD binding site on nectin-1 overlaps with a functional adhesive site involved in nectin-nectin homophilic trans-interaction. Consequently, when nectin-1 is engaged with a cellular ligand at cell junctions, the gD binding site is occupied. Here we report that HSV gD is able to disrupt intercellular homophilic trans-interaction of nectin-1 and induce a rapid redistribution of nectin-1 from cell junctions. This movement does not require the receptor’s interaction with the actin-binding adaptor afadin. Interaction of nectin-1 with afadin is also dispensable for virion surfing along nectin-1-rich filopodia. Cells seeded on gD-coated surfaces also fail to accumulate nectin-1 at cell contact. These data indicate that HSV gD affects nectin-1 locally through direct interaction and more globally through signaling. PMID:27723487

Ultrafast single molecule technique for the study of force dependent kinetics and conformational changes of actin-protein interaction involved in mechanotransduction

NASA Astrophysics Data System (ADS)

Sergides, M.; Arbore, C.; Pavone, F. S.; Capitanio, M.

2018-02-01

Mechanical signals occurring at the interface between cell membrane and extracellular matrix and at intercellular junctions trigger biochemical signals that are fundamental for cell growth, development and regulation. Adaptor proteins, which link the cell membrane to the actin cytoskeleton, seem to partake in this process of mechanotransduction. In particular, catenins play a key role in intercellular junctions, where they act as a bridge between the cell membrane and actin. Studies suggest that α-catenin contains a domain that normally masks vinculin binding sites, which can become accessible after a conformational change induced by an external force. Here we demonstrate a single-molecule technique for investigating actin-protein interactions at different forces (up to 17 pN) with adequate temporal resolution (sub-ms). This system is based on the ultrafast force-clamp spectroscopy technique that has been recently developed by our group and is adapted to study and measure force-dependent kinetics of the catenin-actin interaction, as well as the amplitude of the expected conformational changes such as force-induced protein unfolding.

Herpes simplex virus glycoprotein D relocates nectin-1 from intercellular contacts.

PubMed

Bhargava, Arjun K; Rothlauf, Paul W; Krummenacher, Claude

2016-12-01

Herpes simplex virus (HSV) uses the cell adhesion molecule nectin-1 as a receptor to enter neurons and epithelial cells. The viral glycoprotein D (gD) is used as a non-canonical ligand for nectin-1. The gD binding site on nectin-1 overlaps with a functional adhesive site involved in nectin-nectin homophilic trans-interaction. Consequently, when nectin-1 is engaged with a cellular ligand at cell junctions, the gD binding site is occupied. Here we report that HSV gD is able to disrupt intercellular homophilic trans-interaction of nectin-1 and induce a rapid redistribution of nectin-1 from cell junctions. This movement does not require the receptor's interaction with the actin-binding adaptor afadin. Interaction of nectin-1 with afadin is also dispensable for virion surfing along nectin-1-rich filopodia. Cells seeded on gD-coated surfaces also fail to accumulate nectin-1 at cell contact. These data indicate that HSV gD affects nectin-1 locally through direct interaction and more globally through signaling. Copyright © 2016 Elsevier Inc. All rights reserved.

Effects of multipurpose solutions (MPS) for hydrogel contact lenses on gap-junctional intercellular communication (GJIC) in rabbit corneal keratocytes.

PubMed

Sumide, Taizo; Tsuchiya, Toshie

2003-02-15

To ensure the effects of multipurpose solutions (MPS) for hydrogel contact lenses on the cornea, the inhibitory activity of three types of MPS on corneal cells has been evaluated with the use of scrape loading and dye transfer assay (SLDT assay) and Western blotting on rabbit corneal keratocytes (RC4). In SLDT assay, MPS-A and poloxamine showed dose-dependent inhibitory activity, suggesting the inhibitory action of MPS-A and poloxamine to gap junctional intercellular communication (GJIC) in the tested cells. Moreover, after treatment with MPS-A, the GJIC was initially inhibited within 4 h, and thereafter gradually turned to an approximately 60% level of the initial value. When MPS-A was removed from the incubation media after exposure of the cells, the recovery of GJIC was time dependent and returned to approximately initial levels at 8 h. Complete recovery was established after approximately 24 h. These findings suggested that the inhibitory action of MPS-A on corneal keratocytes was reversible. This inhibition was accompanied by a decrease in the quantity of connexin-43, which is a major protein constituting the gap junctional channel of these cells, and its change in the phosphorylation state. Taken together, it was suggested that MPS-A interacts with connexin-43, inducing an inhibitory action on GJIC. (c) 2002 Wiley Periodicals, Inc.

A Novel N14Y Mutation in Connexin26 in Keratitis-Ichthyosis-Deafness Syndrome

PubMed Central

Arita, Ken; Akiyama, Masashi; Aizawa, Tomoyasu; Umetsu, Yoshitaka; Segawa, Ikuo; Goto, Maki; Sawamura, Daisuke; Demura, Makoto; Kawano, Keiichi; Shimizu, Hiroshi

2006-01-01

Connexins (Cxs) are transmembranous proteins that connect adjacent cells via channels known as gap junctions. The N-terminal 21 amino acids of Cx26 are located at the cytoplasmic side of the channel pore and are thought to be essential for the regulation of channel selectivity. We have found a novel mutation, N14Y, in the N-terminal domain of Cx26 in a case of keratitis-ichthyosis-deafness syndrome. Reduced gap junctional intercellular communication was observed in the patient’s keratinocytes by the dye transfer assay using scrape-loading methods. The effect of this mutation on molecular structure was investigated using synthetic N-terminal peptides from both wild-type and mutated Cx26. Two-dimensional 1H nuclear magnetic resonance and circular dichroism measurements demonstrated that the secondary structures of these two model peptides are similar to each other. However, several novel nuclear Overhauser effect signals appeared in the N14Y mutant, and the secondary structure of the mutant peptide was more susceptible to induction of 2,2,2-trifluoroethanol than wild type. Thus, it is likely that the N14Y mutation induces a change in local structural flexibility of the N-terminal domain, which is important for exerting the activity of the channel function, resulting in impaired gap junctional intercellular communication. PMID:16877344

Evolution of Microbial Quorum Sensing to Human Global Quorum Sensing: An Insight into How Gap Junctional Intercellular Communication Might Be Linked to the Global Metabolic Disease Crisis

PubMed Central

Trosko, James E.

2016-01-01

The first anaerobic organism extracted energy for survival and reproduction from its source of nutrients, with the genetic means to ensure protection of its individual genome but also its species survival. While it had a means to communicate with its community via simple secreted molecules (“quorum sensing”), the eventual shift to an aerobic environment led to multi-cellular metazoan organisms, with evolutionary-selected genes to form extracellular matrices, stem cells, stem cell niches, and a family of gap junction or “connexin” genes. These germinal and somatic stem cells responded to extracellular signals that triggered intra-cellular signaling to regulate specific genes out of the total genome. These extra-cellular induced intra-cellular signals also modulated gap junctional intercellular communication (GJIC) in order to regulate the new cellular functions of symmetrical and asymmetrical cell division, cell differentiation, modes of cell death, and senescence. Within the hierarchical and cybernetic concepts, differentiated by neurons organized in the brain of the Homo sapiens, the conscious mind led to language, abstract ideas, technology, myth-making, scientific reasoning, and moral decision–making, i.e., the creation of culture. Over thousands of years, this has created the current collision between biological and cultural evolution, leading to the global “metabolic disease” crisis. PMID:27314399

Regulation of intestinal permeability: The role of proteases

PubMed Central

Van Spaendonk, Hanne; Ceuleers, Hannah; Witters, Leonie; Patteet, Eveline; Joossens, Jurgen; Augustyns, Koen; Lambeir, Anne-Marie; De Meester, Ingrid; De Man, Joris G; De Winter, Benedicte Y

2017-01-01

The gastrointestinal barrier is - with approximately 400 m2 - the human body’s largest surface separating the external environment from the internal milieu. This barrier serves a dual function: permitting the absorption of nutrients, water and electrolytes on the one hand, while limiting host contact with noxious luminal antigens on the other hand. To maintain this selective barrier, junction protein complexes seal the intercellular space between adjacent epithelial cells and regulate the paracellular transport. Increased intestinal permeability is associated with and suggested as a player in the pathophysiology of various gastrointestinal and extra-intestinal diseases such as inflammatory bowel disease, celiac disease and type 1 diabetes. The gastrointestinal tract is exposed to high levels of endogenous and exogenous proteases, both in the lumen and in the mucosa. There is increasing evidence to suggest that a dysregulation of the protease/antiprotease balance in the gut contributes to epithelial damage and increased permeability. Excessive proteolysis leads to direct cleavage of intercellular junction proteins, or to opening of the junction proteins via activation of protease activated receptors. In addition, proteases regulate the activity and availability of cytokines and growth factors, which are also known modulators of intestinal permeability. This review aims at outlining the mechanisms by which proteases alter the intestinal permeability. More knowledge on the role of proteases in mucosal homeostasis and gastrointestinal barrier function will definitely contribute to the identification of new therapeutic targets for permeability-related diseases. PMID:28405139

Perfluorooctanesulfonate (PFOS) Perturbs Male Rat Sertoli Cell Blood-Testis Barrier Function by Affecting F-Actin Organization via p-FAK-Tyr407: An in Vitro Study

PubMed Central

Wan, Hin-Ting; Mruk, Dolores D.; Wong, Chris K. C.

2014-01-01

Environmental toxicants such as perfluorooctanesulfonate (PFOS) have been implicated in male reproductive dysfunction, including reduced sperm count and semen quality, in humans. However, the underlying mechanism(s) remains unknown. Herein PFOS at 10–20 μM (∼5–10 μg/mL) was found to be more potent than bisphenol A (100 μM) in perturbing the blood-testis barrier (BTB) function by disrupting the Sertoli cell tight junction-permeability barrier without detectable cytotoxicity. We also delineated the underlying molecular mechanism by which PFOS perturbed Sertoli cell BTB function using an in vitro model that mimics the BTB in vivo. First, PFOS perturbed F-actin organization in Sertoli cells, causing truncation of actin filaments at the BTB. Thus, the actin-based cytoskeleton was no longer capable of supporting the distribution and/or localization of actin-regulatory and adhesion proteins at the cell-cell interface necessary to maintain BTB integrity. Second, PFOS was found to perturb inter-Sertoli cell gap junction (GJ) communication based on a dye-transfer assay by down-regulating the expression of connexin-43, a GJ integral membrane protein. Third, phosphorylated focal adhesion kinase (FAK)-Tyr407 was found to protect the BTB from the destructive effects of PFOS as shown in a study via an overexpression of an FAK Y407E phosphomimetic mutant. Also, transfection of Sertoli cells with an FAK-specific microRNA, miR-135b, to knock down the expression of phosphorylated FAK-Tyr407 was found to worsen PFOS-mediated Sertoli cell tight junction disruption. In summary, PFOS-induced BTB disruption is mediated by down-regulating phosphorylated FAK-Tyr407 and connexin-43, which in turn perturbed F-actin organization and GJ-based intercellular communication, leading to mislocalization of actin-regulatory and adhesion proteins at the BTB. PMID:24169556

The functional interrelationship between gap junctions and fenestrae in endothelial cells of the liver organoid.

PubMed

Saito, Masaya; Matsuura, Tomokazu; Nagatsuma, Keisuke; Tanaka, Ken; Maehashi, Haruka; Shimizu, Keiko; Hataba, Yoshiaki; Kato, Fumitaka; Kashimori, Isao; Tajiri, Hisao; Braet, Filip

2007-06-01

Functional intact liver organoid can be reconstructed in a radial-flow bioreactor when human hepatocellular carcinoma (FLC-5), mouse immortalized sinusoidal endothelial M1 (SEC) and A7 (HSC) hepatic stellate cell lines are cocultured. The structural and functional characteristics of the reconstructed organoid closely resemble the in vivo liver situation. Previous liver organoid studies indicated that cell-to-cell communications might be an important factor for the functional and structural integrity of the reconstructed organoid, including the expression of fenestrae. Therefore, we examined the possible relationship between functional intact gap junctional intercellular communication (GJIC) and fenestrae dynamics in M1-SEC cells. The fine morphology of liver organoid was studied in the presence of (1) irsogladine maleate (IM), (2) oleamide and (3) oleamide followed by IM treatment. Fine ultrastructural changes were studied by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) and compared with control liver organoid data. TEM revealed that oleamide affected the integrity of cell-to-cell contacts predominantly in FLC-5 hepatocytes. SEM observation showed the presence of fenestrae on M1-SEC cells; however, oleamide inhibited fenestrae expression on the surface of endothelial cells. Interestingly, fenestrae reappeared when IM was added after initial oleamide exposure. GJIC mediates the number of fenestrae in endothelial cells of the liver organoid.

Immunohistochemical localization of thrombomodulin in the stratified epithelium of the rat is restricted to the keratinizing epidermis.

PubMed

Daimon, T; Nakano, M

1999-12-01

The expression and function of thrombomodulin (TM), an endothelial cofactor protein for thrombin-mediated protein C activation, in the epithelium are not fully characterized. This report describes the distribution and localization of TM in the various types of epithelia in the rat by light and electron microscopic immunocytochemistry. TM showed a limited distribution and was expressed by the keratinizing stratified epithelia of the skin, tongue, and esophagus, but was not present on the non-keratinizing epithelia of the vagina, ureter, trachea, stomach, or gut. An identical pattern of TM expression was seen in mucocutaneous junctions, transitional zones from a non-keratinizing stratified epithelium to a keratinizing epithelium at the edge of the eyelid and in the anal canal. As the keratinization of the stratified epithelia proceeded, the staining intensity increased in the transitional zones. Within the keratinizing stratified epithelia, TM staining was limited to the keratinocytes of the spinous layer, the spinous cells. The subcellular localization of TM on the spinous cells was restricted to the plasma membrane facing the intercellular spaces. TM was not detectable on the desmosomes or the two membranes making up the junction, presumably the nexus. The functional significance of TM in keratinizing epithelia is discussed.

Intercellular communications-redox interactions in radiation toxicity; potential targets for radiation mitigation.

PubMed

Farhood, Bagher; Goradel, Nasser Hashemi; Mortezaee, Keywan; Khanlarkhani, Neda; Salehi, Ensieh; Nashtaei, Maryam Shabani; Shabeeb, Dheyauldeen; Musa, Ahmed Eleojo; Fallah, Hengameh; Najafi, Masoud

2018-06-17

Nowadays, using ionizing radiation (IR) is necessary for clinical, agricultural, nuclear energy or industrial applications. Accidental exposure to IR after a radiation terror or disaster poses a threat to human. In contrast to the old dogma of radiation toxicity, several experiments during the last two recent decades have revealed that intercellular signaling and communications play a key role in this procedure. Elevated level of cytokines and other intercellular signals increase oxidative damage and inflammatory responses via reduction/oxidation interactions (redox system). Intercellular signals induce production of free radicals and inflammatory mediators by some intermediate enzymes such as cyclooxygenase-2 (COX-2), nitric oxide synthase (NOS), NADPH oxidase, and also via triggering mitochondrial ROS. Furthermore, these signals facilitate cell to cell contact and increasing cell toxicity via cohort effect. Nitric oxide is a free radical with ability to act as an intercellular signal that induce DNA damage and changes in some signaling pathways in irradiated as well as non-irradiated adjacent cells. Targeting of these mediators by some anti-inflammatory agents or via antioxidants such as mitochondrial ROS scavengers opens a window to mitigate radiation toxicity after an accidental exposure. Experiments which have been done so far suggests that some cytokines such as IL-1β, TNF-α, TGF-β, IL-4 and IL-13 are some interesting targets that depend on irradiated organs and may help mitigate radiation toxicity. Moreover, animal experiments in recent years indicated that targeting of toll like receptors (TLRs) may be more useful for radioprotection and mitigation. In this review, we aimed to describe the role of intercellular interactions in oxidative injury, inflammation, cell death and killing effects of IR. Moreover, we described evidence on potential mitigation of radiation injury via targeting of these mediators.

Connexins: Synthesis, Post-Translational Modifications, and Trafficking in Health and Disease

PubMed Central

Vidal-Brime, Laia; Lynn, K. Sabrina

2018-01-01

Connexins are tetraspan transmembrane proteins that form gap junctions and facilitate direct intercellular communication, a critical feature for the development, function, and homeostasis of tissues and organs. In addition, a growing number of gap junction-independent functions are being ascribed to these proteins. The connexin gene family is under extensive regulation at the transcriptional and post-transcriptional level, and undergoes numerous modifications at the protein level, including phosphorylation, which ultimately affects their trafficking, stability, and function. Here, we summarize these key regulatory events, with emphasis on how these affect connexin multifunctionality in health and disease. PMID:29701678

Low-temperature and conventional scanning electron microscopy of human urothelial neoplasms.

PubMed

Hopkins, D M; Morris, J A; Oates, K; Huddart, H; Staff, W G

1989-05-01

The appearance of neoplastic human urothelium viewed by low-temperature scanning electron microscopy (LTSEM) and conventional scanning electron microscopy (CSEM) was compared. Fixed, dehydrated neoplastic cells viewed by CSEM had well-defined, often raised cell junctions; no intercellular gaps; and varying degrees of pleomorphic surface microvilli. The frozen hydrated material viewed by LTSEM, however, was quite different. The cells had a flat or dimpled surface, but no microvilli. There were labyrinthine lateral processes which interdigitated with those of adjacent cells and outlined large intercellular gaps. The process of fixation and dehydration will inevitably distort cell contours and on theoretical grounds, the images of frozen hydrated material should more closely resemble the in vivo appearance.

Flavonoids (apigenin, tangeretin) counteract tumor promoter-induced inhibition of intercellular communication of rat liver epithelial cells.

PubMed

Chaumontet, C; Droumaguet, C; Bex, V; Heberden, C; Gaillard-Sanchez, I; Martel, P

1997-03-19

We have shown previously that two flavonoids, apigenin and tangeretin, enhance gap junctional intercellular communication (GJIC) in rat liver epithelial cells, named REL cells. Here, we show that these two flavones also antagonize the inhibition of GJIC induced by tumor promoters like 12-O-tetradecanoyl-phorbol-acetate (TPA) and 3,5,di-tertio-butyl-4-hydroxytoluene (BHT). Their preventive effect is rapid. It does not seem to involve any change of the amount of the connexin expressed in REL cells, connexin 43 (Cx 43), and in its phosphorylation state. Other flavonoids tested including naringenin, myricetin, catechin and chrysin did not enhance GJIC nor counteract TPA-induced inhibition of GJIC.

Calcium spikes, waves and oscillations in a large, patterned epithelial tissue

PubMed Central

Balaji, Ramya; Bielmeier, Christina; Harz, Hartmann; Bates, Jack; Stadler, Cornelia; Hildebrand, Alexander; Classen, Anne-Kathrin

2017-01-01

While calcium signaling in excitable cells, such as muscle or neurons, is extensively characterized, calcium signaling in epithelial tissues is little understood. Specifically, the range of intercellular calcium signaling patterns elicited by tightly coupled epithelial cells and their function in the regulation of epithelial characteristics are little explored. We found that in Drosophila imaginal discs, a widely studied epithelial model organ, complex spatiotemporal calcium dynamics occur. We describe patterns that include intercellular waves traversing large tissue domains in striking oscillatory patterns as well as spikes confined to local domains of neighboring cells. The spatiotemporal characteristics of intercellular waves and oscillations arise as emergent properties of calcium mobilization within a sheet of gap-junction coupled cells and are influenced by cell size and environmental history. While the in vivo function of spikes, waves and oscillations requires further characterization, our genetic experiments suggest that core calcium signaling components guide actomyosin organization. Our study thus suggests a possible role for calcium signaling in epithelia but importantly, introduces a model epithelium enabling the dissection of cellular mechanisms supporting the initiation, transmission and regeneration of long-range intercellular calcium waves and the emergence of oscillations in a highly coupled multicellular sheet. PMID:28218282

Three-dimensional modeling and quantitative analysis of gap junction distributions in cardiac tissue.

PubMed

Lackey, Daniel P; Carruth, Eric D; Lasher, Richard A; Boenisch, Jan; Sachse, Frank B; Hitchcock, Robert W

2011-11-01

Gap junctions play a fundamental role in intercellular communication in cardiac tissue. Various types of heart disease including hypertrophy and ischemia are associated with alterations of the spatial arrangement of gap junctions. Previous studies applied two-dimensional optical and electron-microscopy to visualize gap junction arrangements. In normal cardiomyocytes, gap junctions were primarily found at cell ends, but can be found also in more central regions. In this study, we extended these approaches toward three-dimensional reconstruction of gap junction distributions based on high-resolution scanning confocal microscopy and image processing. We developed methods for quantitative characterization of gap junction distributions based on analysis of intensity profiles along the principal axes of myocytes. The analyses characterized gap junction polarization at cell ends and higher-order statistical image moments of intensity profiles. The methodology was tested in rat ventricular myocardium. Our analysis yielded novel quantitative data on gap junction distributions. In particular, the analysis demonstrated that the distributions exhibit significant variability with respect to polarization, skewness, and kurtosis. We suggest that this methodology provides a quantitative alternative to current approaches based on visual inspection, with applications in particular in characterization of engineered and diseased myocardium. Furthermore, we propose that these data provide improved input for computational modeling of cardiac conduction.

Dysfunction in gap junction intercellular communication induces aberrant behavior of the inner cell mass and frequent collapses of expanded blastocysts in mouse embryos.

PubMed

Togashi, Kazue; Kumagai, Jin; Sato, Emiko; Shirasawa, Hiromitsu; Shimoda, Yuki; Makino, Kenichi; Sato, Wataru; Kumazawa, Yukiyo; Omori, Yasufumi; Terada, Yukihiro

2015-06-01

We investigated the role of gap junctions (GJs) in embryological differentiation, and observed the morphological behavior of the inner cell mass (ICM) by time-lapse movie observation (TLM) with gap junction inhibitors (GJis). ICR mouse embryos were exposed to two types of GJis in CZB medium: oleamide (0 to 50 μM) and 1-heptanol (0 to 10 mM). We compared the rate of blastocyst formation at embryonic day 4.5 (E4.5) with E5.5. We also observed and evaluated the times from the second cleavage to each embryonic developing stage by TLM. We investigated embryonic distribution of DNA, Nanog protein, and Connexin 43 protein with immunofluorescent staining. In the comparison of E4.5 with E5.5, inhibition of gap junction intercellular communication (GJIC) delayed embryonic blastocyst formation. The times from the second cleavage to blastocyst formation were significantly extended in the GJi-treated embryos (control vs with oleamide, 2224 ± 179 min vs 2354 ± 278 min, p = 0.013). Morphological differences were traced in control versus GJi-treated embryos until the hatching stage. Oleamide induced frequent severe collapses of expanded blastocysts (77.4 % versus 26.3 %, p = 0.0001) and aberrant ICM divisions connected to sticky strands (74.3 % versus 5.3 %, p = 0.0001). Immunofluorescent staining indicated Nanog-positive cells were distributed in each divided ICM. GJIC plays an important role in blastocyst formation, collapses of expanded blastocysts, and the ICM construction in mouse embryos.

Mammalian target of rapamycin complex (mTOR) pathway modulates blood-testis barrier (BTB) function through F-actin organization and gap junction

PubMed Central

Li, Nan; Cheng, C. Yan

2016-01-01

mTOR (mammalian target of rapamycin) is one of the most important signaling molecules in mammalian cells which regulates an array of cellular events, ranging from cell metabolism to cell proliferation. Based on the association of mTOR with the core component proteins, such as Raptor or Rictor, mTOR can become the mTORC1 (mammalian target of rapamycin complex 1) or mTORC2, respectively. Studies have shown that during the epithelial cycle of spermatogenesis, mTORC1 promotes remodeling and restructuring of the blood-testis barrier (BTB) in vitro and in vivo, making the Sertoli cell tight junction (TJ)-permeability barrier “leaky”; whereas mTORC2 promotes BTB integrity, making the Sertoli cell TJ-barrier “tighter”. These contrasting effects, coupled with the spatiotemporal expression of the core signaling proteins at the BTB that confer the respective functions of mTORC1 vs. mTORC2 thus provide a unique mechanism to modulate BTB dynamics, allowing or disallowing the transport of biomolecules and also preleptotene spermatocytes across the immunological barrier. More importantly, studies have shown that these changes to BTB dynamics conferred by mTORC1 and mTORC2 are mediated by changes in the organization of the actin microfilament networks at the BTB, and involve gap junction (GJ) intercellular communication. Since GJ has recently been shown to be crucial to reboot spermatogenesis and meiosis following toxicant-induced aspermatogenesis, these findings thus provide new insightful information regarding the integration of mTOR and GJ to regulate spermatogenesis. PMID:26957088

The extracellular matrix controls gap junction protein expression and function in postnatal hippocampal neural progenitor cells

PubMed Central

Imbeault, Sophie; Gauvin, Lianne G; Toeg, Hadi D; Pettit, Alexandra; Sorbara, Catherine D; Migahed, Lamiaa; DesRoches, Rebecca; Menzies, A Sheila; Nishii, Kiyomasa; Paul, David L; Simon, Alexander M; Bennett, Steffany AL

2009-01-01

Background Gap junction protein and extracellular matrix signalling systems act in concert to influence developmental specification of neural stem and progenitor cells. It is not known how these two signalling systems interact. Here, we examined the role of ECM components in regulating connexin expression and function in postnatal hippocampal progenitor cells. Results We found that Cx26, Cx29, Cx30, Cx37, Cx40, Cx43, Cx45, and Cx47 mRNA and protein but only Cx32 and Cx36 mRNA are detected in distinct neural progenitor cell populations cultured in the absence of exogenous ECM. Multipotential Type 1 cells express Cx26, Cx30, and Cx43 protein. Their Type 2a progeny but not Type 2b and 3 neuronally committed progenitor cells additionally express Cx37, Cx40, and Cx45. Cx29 and Cx47 protein is detected in early oligodendrocyte progenitors and mature oligodendrocytes respectively. Engagement with a laminin substrate markedly increases Cx26 protein expression, decreases Cx40, Cx43, Cx45, and Cx47 protein expression, and alters subcellular localization of Cx30. These changes are associated with decreased neurogenesis. Further, laminin elicits the appearance of Cx32 protein in early oligodendrocyte progenitors and Cx36 protein in immature neurons. These changes impact upon functional connexin-mediated hemichannel activity but not gap junctional intercellular communication. Conclusion Together, these findings demonstrate a new role for extracellular matrix-cell interaction, specifically laminin, in the regulation of intrinsic connexin expression and function in postnatal neural progenitor cells. PMID:19236721

Spatio-temporal regulation of connexin43 phosphorylation and gap junction dynamics.

PubMed

Solan, Joell L; Lampe, Paul D

2018-01-01

Gap junctions are specialized membrane domains containing tens to thousands of intercellular channels. These channels permit exchange of small molecules (<1000Da) including ions, amino acids, nucleotides, metabolites and secondary messengers (e.g., calcium, glucose, cAMP, cGMP, IP 3 ) between cells. The common reductionist view of these structures is that they are composed entirely of integral membrane proteins encoded by the 21 member connexin human gene family. However, it is clear that the normal physiological function of this structure requires interaction and regulation by a variety of proteins, especially kinases. Phosphorylation is capable of directly modulating connexin channel function but the most dramatic effects on gap junction activity occur via the organization of the gap junction structures themselves. This is a direct result of the short half-life of the primary gap junction protein, connexin, which requires them to be constantly assembled, remodeled and turned over. The biological consequences of this remodeling are well illustrated during cardiac ischemia, a process wherein gap junctions are disassembled and remodeled resulting in arrhythmia and ultimately heart failure. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve. Copyright © 2017 Elsevier B.V. All rights reserved.

Oleamide derivatives are prototypical anti-metastasis drugs that act by inhibiting Connexin 26.

PubMed

Nojima, Hiroshi; Ohba, Yusuke; Kita, Yasuyuki

2007-09-01

Despite considerable research, metastasis remains a major challenge in the clinical management of cancer. Recent reports show that abnormally augmented expression of Cx26 is responsible for the enhanced spontaneous metastasis of mouse BL6 melanoma cells. The function of Cx26 appears to be responsible for this phenotype since exogenous expression of a dominant-negative form of Cx26 and oleamide derivatives called MI-18 and MI-22 that specifically inhibit Cx26-mediated gap junction-mediated intercellular communications (GJIC) prevent the spontaneous metastasis of BL6 cells. As expected from their structural similarity to oleic acid (the major component of olive oil), both MI-18 and MI-22 are safe drugs; nonetheless, they are potent inhibitors of the spontaneous metastasis of BL6 mouse melanoma cells. Thus, they are a novel prototype of an anti-metastasis drug that has minimal side effects. While the primary tumors do not necessarily show strong Cx26-immunostaining signals, pronounced Cx26 expression is detected in the highly invasive tumor regions; it is also more frequently observed in metastasized tumors. Thus, Cx26 expression may be useful as a prognostic tool that can predict the existence of highly metastatic cancer cells in clinical samples.

Junctional and nonjunctional effects of heptanol and glycyrrhetinic acid derivates in rat mesenteric small arteries

PubMed Central

Matchkov, Vladimir V; Rahman, Awahan; Peng, Hongli; Nilsson, Holger; Aalkjær, Christian

2004-01-01

Heptanol, 18α-glycyrrhetinic acid (18αGA) and 18β-glycyrrhetinic acid (18βGA) are known blockers of gap junctions, and are often used in vascular studies. However, actions unrelated to gap junction block have been repeatedly suggested in the literature for these compounds. We report here the findings from a comprehensive study of these compounds in the arterial wall. Rat isolated mesenteric small arteries were studied with respect to isometric tension (myography), [Ca2+]i (Ca2+-sensitive dyes), membrane potential and – as a measure of intercellular coupling – input resistance (sharp intracellular glass electrodes). Also, membrane currents (patch-clamp) were measured in isolated smooth muscle cells (SMCs). Confocal imaging was used for visualisation of [Ca2+]i events in single SMCs in the arterial wall. Heptanol (150 μM) activated potassium currents, hyperpolarised the membrane, inhibited the Ca2+ current, and reduced [Ca2+]i and tension, but had little effect on input resistance. Only at concentrations above 200 μM did heptanol elevate input resistance, desynchronise SMCs and abolish vasomotion. 18βGA (30 μM) not only increased input resistance and desynchronised SMCs but also had nonjunctional effects on membrane currents. 18αGA (100 μM) had no significant effects on tension, [Ca2+]i, total membrane current and synchronisation in vascular smooth muscle. We conclude that in mesenteric small arteries, heptanol and 18βGA have important nonjunctional effects at concentrations where they have little or no effect on intercellular communication. Thus, the effects of heptanol and 18βGA on vascular function cannot be interpreted as being caused only by effects on gap junctions. 18αGA apparently does not block communication between SMCs in these arteries, although an effect on myoendothelial gap junctions cannot be excluded. PMID:15210581

Disruption of gap junctional intercellular communication by antibiotic gentamicin is associated with aberrant localization of occludin, N-cadherin, connexin 43, and vimentin in SerW3 Sertoli cells in vitro.

PubMed

Bekheet, Souad H M; Stahlmann, Ralf

2009-09-01

Spermatogenesis is a very complex process by which male germ cells differentiate into mature spermatozoa. The sophisticated communication network that controls spermatogenesis can be derailed so that dysfunction of one cell type propagates to all types as a cascade. This accounts for the particular vulnerability of the testis to environmental factors such as drugs and xenobiotics. Sertoli cells play an important role in protecting developing germ cells by forming a physiological barrier, limiting exposure to potentially toxic substrates, or conversely, facilitating uptake of xenobiotics within the testis. In this study, cells from the rat Sertoli line (SerW3) were incubated for 3, 6 and 9 subsequent days in serum free DMEM (SFDM) composed of DMEM supplemented with three different concentrations of antibiotic gentamicin (10, 30, and 100 μg). The effect of the three different concentrations of this antibiotic was determined on Sertoli cell-cell interaction through impaired expression of their constitutive tight junction proteins as early targets for different toxicants in vitro by immunochemistry analysis. The Sertoli SerW3 cell line illustrated the cytotoxicity of GS, as the intercellular junction proteins such as occludin, N-cadherin, connexin 43, and vimentin were delocalized from the membrane to the cytoplasmic compartment during exposure to the antibiotic. This study underlines the potential deleterious effects of the routine use of antibiotics during continuous cell culture.

Ultrastructural demonstration of Cx43 gap junctions in induced pluripotent stem cells from human cord blood.

PubMed

Beckmann, Anja; Schubert, Madline; Hainz, Nadine; Haase, Alexandra; Martin, Ulrich; Tschernig, Thomas; Meier, Carola

2016-11-01

Gap junction proteins are essential for direct intercellular communication but also influence cellular differentiation and migration. The expression of various connexin gap junction proteins has been demonstrated in embryonic stem cells, with Cx43 being the most intensely studied. As Cx43 is the most prominent gap junction protein in the heart, cardiomyocyte-differentiated stem cells have been studied intensely. To date, however, little is known about the expression and the subcellular distribution of Cx43 in undifferentiated stem cells or about the structural arrangement of channels. We, therefore, here investigate expression of Cx43 in undifferentiated human cord-blood-derived induced pluripotent stem cells (hCBiPS2). For this purpose, we carried out quantitative real-time PCR and immunohistochemistry. For analysis of Cx43 ultrastructure and protein assembly, we performed freeze-fracture replica immunogold labeling (FRIL). Cx43 expression was detected at mRNA and protein level in hCBIPS2 cells. For the first time, ultrastructural data are presented on gap junction morphology in induced pluripotent stem (iPS) cells from cord blood: Our FRIL and electron microscopical analysis revealed the occurrence of gap junction plaques in undifferentiated iPS cells. In addition, these gap junctions were shown to contain the gap junction protein Cx43.

Cell-cell bioelectrical interactions and local heterogeneities in genetic networks: a model for the stabilization of single-cell states and multicellular oscillations.

PubMed

Cervera, Javier; Manzanares, José A; Mafe, Salvador

2018-04-04

Genetic networks operate in the presence of local heterogeneities in single-cell transcription and translation rates. Bioelectrical networks and spatio-temporal maps of cell electric potentials can influence multicellular ensembles. Could cell-cell bioelectrical interactions mediated by intercellular gap junctions contribute to the stabilization of multicellular states against local genetic heterogeneities? We theoretically analyze this question on the basis of two well-established experimental facts: (i) the membrane potential is a reliable read-out of the single-cell electrical state and (ii) when the cells are coupled together, their individual cell potentials can be influenced by ensemble-averaged electrical potentials. We propose a minimal biophysical model for the coupling between genetic and bioelectrical networks that associates the local changes occurring in the transcription and translation rates of an ion channel protein with abnormally low (depolarized) cell potentials. We then analyze the conditions under which the depolarization of a small region (patch) in a multicellular ensemble can be reverted by its bioelectrical coupling with the (normally polarized) neighboring cells. We show also that the coupling between genetic and bioelectric networks of non-excitable cells, modulated by average electric potentials at the multicellular ensemble level, can produce oscillatory phenomena. The simulations show the importance of single-cell potentials characteristic of polarized and depolarized states, the relative sizes of the abnormally polarized patch and the rest of the normally polarized ensemble, and intercellular coupling.

Ultrastructural study of the human neurohypophysis. III. Vascular and perivascular structures.

PubMed

Seyama, S; Pearl, G S; Takei, Y

1980-01-01

The vascular and perivascular regions of the human neurohypophysis were studied electron microscopically. The abluminal basement membrane, perivascular space, luminal basement membrane and endothelium are interposed between the neural parenchyma and the blood stream. The capillaries are fenestrated, with pores measuring 30 to 50 nm in diameter. The perivascular and intercellular spaces form prominent networks that penetrate between rows of neurohypophysial parenchymal cells. The perivascular space contains pericytes, histiocytes, fibroblasts and mast cells, with ultrastructural features typical of each cell type. No transitional forms between histiocytes and pericytes were observed. A schema for the extracellular flow of neurohypophysial hormones through the sinusoidal and perivascular spaces is proposed, suggesting an important role for the pituicytes and their intercellular junctions in the control of hormone release.

The histopathological effect of intracameral ropivacaine in different concentrations on corneal endothelium.

PubMed

Caça, Ihsan; Kavak, Vatan; Unlü, Kaan; Ari, Seyhmus; Nergis, Yusuf; Take, Gülnür

2006-01-01

We evaluated the histopathological changes occurring in corneal endothelium after intracameral injection ropivacaine into rats. Intracamerally administered ropivacaine in 1, 0.5, and 0.1% concentrations resulted in impairment of hexagonal structure of corneal endothelial cells and intercellular junctions, destruction of microvilli on the cell surface, roughness of cell borders, picnotic nucleus, diffuse vacuolization, and crystalysis in mitochondria.

Apigenin and tangeretin enhance gap junctional intercellular communication in rat liver epithelial cells.

PubMed

Chaumontet, C; Bex, V; Gaillard-Sanchez, I; Seillan-Heberden, C; Suschetet, M; Martel, P

1994-10-01

Two flavones, apigenin and tangeretin, were studied for their ability to modulate gap junctional intercellular communication (GJIC) in the rat liver epithelial cell line REL. Their cytotoxicity was first determined by cell density and neutral red uptake assays: neither apigenin nor tangeretin are cytotoxic at 10 and 25 microM, the concentrations used in our experiments. We then studied GJIC using the dye transfer assay and we observed that both apigenin and tangeretin enhance it, the maximum stimulation (x 1.7-1.8) being achieved at 25 microM for 24 h. When the dye transfer was enhanced, the amount of connexin 43 increased, which was demonstrated by Western blot and immunofluorescence analysis. For apigenin only, Northern blot analysis showed an accumulation of connexin 43 mRNA. In addition, the incubation of REL cells with the two compounds, for 1 or 24 h, prevented the inhibition of dye transfer by 12-O-tetradecanoylphorbol-13-acetate (1 or 10 ng/ml). The enhancement of GJIC by apigenin could be one of the major mechanisms responsible for apigenin's anti-tumour promoting action in vivo. As for tangeretin, its capacity to enhance GJIC completes its potential protective properties towards the post-initiation process.

Differentiation of breast cancer cells in vitro is promoted by the concurrent influence of myoepithelial cells and relaxin.

PubMed Central

Bani, D.; Riva, A.; Bigazzi, M.; Bani Sacchi, T.

1994-01-01

Our previous studies showed that relaxin promotes differentiation of MCF-7 breast adenocarcinoma cells. In the current investigation, we aimed to elucidate whether the effect of the hormone is potentiated when MCF-7 cells are grown together with myoepithelial cells, thus creating a microenvironment reminiscent of the organised tissue architecture of the mammary parenchyma in vivo. The findings obtained reveal that most MCF-7 cells cultured alone have an undifferentiated, blast-like phenotype, only a minority showing a more differentiated phenotype with more organelles and rudimentary intercellular junctions. When co-cultured with myoepithelial cells more MCF-7 cells acquire ultrastructural features consistent with a more differentiated phenotype, such as a rich organellular complement, apical microvilli and intercellular junctions. When relaxin was added to the co-cultures, the ultrastructural signs of differentiation could be observed in even more MCF-7 cells and became more pronounced than in the absence of the hormone, judged by the appearance of a clear-cut polarisation of cytoplasmic organelles, an almost continuous coat of apical microvilli and numerous intracellular pseudolumina. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7947095

Cell line specific modulation of connexin43 expression after exposure to ionizing radiation.

PubMed

Banaz-Yaşar, Ferya; Tischka, Rabea; Iliakis, George; Winterhager, Elke; Gellhaus, Alexandra

2005-01-01

Gap junctional intercellular communication plays a significant role in mediating radiation-induced bystander effects. However, the level of Cx43 itself is influenced by ionizing radiation, which could modify the bystander effect. Here we have investigated several cell lines for the modulation of Cx43 expression 24 h after irradiation with 5 Gy X-rays. The mouse endothelial cell line bEnd3 revealed a significantly elevated level of Cx43 already 15 min after exposure to X-rays, whereas human hybrid endothelial cells (EA.hy926) exhibited a transient downregulation of Cx43 mRNA. No obvious changes in the communication properties of the different cell lines could be observed after irradiation. The communication-deficient malignant human trophoblast cell line Jeg3 stably transfected with Cx43 did not reveal any induction of endogenous nor alteration in the exogenous Cx43 transcript level upon exposure to 5 Gy. Taken together, our data show a cell line specific modulation of Cx43 expression after exposure to X-rays.

New functions for alpha-catenins in health and disease: from cancer to heart regeneration.

PubMed

Vite, Alexia; Li, Jifen; Radice, Glenn L

2015-06-01

Strong cell-cell adhesion mediated by adherens junctions is dependent on anchoring the transmembrane cadherin molecule to the underlying actin cytoskeleton. To do this, the cadherin cytoplasmic domain interacts with catenin proteins, which include α-catenin that binds directly to filamentous actin. Originally thought to be a static structure, the connection between the cadherin/catenin adhesion complex and the actin cytoskeleton is now considered to be dynamic and responsive to both intercellular and intracellular signals. Alpha-catenins are mechanosensing proteins that undergo conformational change in response to cytoskeletal tension thus modifying the linkage between the cadherin and the actin cytoskeleton. There are three α-catenin isoforms expressed in mouse and human: αE-catenin (CTNNA1), αN-catenin (CTNNA2) and αT-catenin (CTNNA3). This review summarizes recent progress in understanding the in vivo function(s) of α-catenins in tissue morphogenesis, homeostasis and disease. The role of α-catenin in the regulation of cellular proliferation will be discussed in the context of cancer and regeneration.

Control of the neurovascular coupling by nitric oxide-dependent regulation of astrocytic Ca2+ signaling

PubMed Central

Muñoz, Manuel F.; Puebla, Mariela; Figueroa, Xavier F.

2015-01-01

Neuronal activity must be tightly coordinated with blood flow to keep proper brain function, which is achieved by a mechanism known as neurovascular coupling. Then, an increase in synaptic activity leads to a dilation of local parenchymal arterioles that matches the enhanced metabolic demand. Neurovascular coupling is orchestrated by astrocytes. These glial cells are located between neurons and the microvasculature, with the astrocytic endfeet ensheathing the vessels, which allows fine intercellular communication. The neurotransmitters released during neuronal activity reach astrocytic receptors and trigger a Ca2+ signaling that propagates to the endfeet, activating the release of vasoactive factors and arteriolar dilation. The astrocyte Ca2+ signaling is coordinated by gap junction channels and hemichannels formed by connexins (Cx43 and Cx30) and channels formed by pannexins (Panx-1). The neuronal activity-initiated Ca2+ waves are propagated among neighboring astrocytes directly via gap junctions or through ATP release via connexin hemichannels or pannexin channels. In addition, Ca2+ entry via connexin hemichannels or pannexin channels may participate in the regulation of the astrocyte signaling-mediated neurovascular coupling. Interestingly, nitric oxide (NO) can activate connexin hemichannel by S-nitrosylation and the Ca2+-dependent NO-synthesizing enzymes endothelial NO synthase (eNOS) and neuronal NOS (nNOS) are expressed in astrocytes. Therefore, the astrocytic Ca2+ signaling triggered in neurovascular coupling may activate NO production, which, in turn, may lead to Ca2+ influx through hemichannel activation. Furthermore, NO release from the hemichannels located at astrocytic endfeet may contribute to the vasodilation of parenchymal arterioles. In this review, we discuss the mechanisms involved in the regulation of the astrocytic Ca2+ signaling that mediates neurovascular coupling, with a special emphasis in the possible participation of NO in this process. PMID:25805969

Crosstalk between reticular adherens junctions and platelet endothelial cell adhesion molecule-1 regulates endothelial barrier function.

PubMed

Fernández-Martín, Laura; Marcos-Ramiro, Beatriz; Bigarella, Carolina L; Graupera, Mariona; Cain, Robert J; Reglero-Real, Natalia; Jiménez, Anaïs; Cernuda-Morollón, Eva; Correas, Isabel; Cox, Susan; Ridley, Anne J; Millán, Jaime

2012-08-01

Endothelial cells provide a barrier between the blood and tissues, which is reduced during inflammation to allow selective passage of molecules and cells. Adherens junctions (AJ) play a central role in regulating this barrier. We aim to investigate the role of a distinctive 3-dimensional reticular network of AJ found in the endothelium. In endothelial AJ, vascular endothelial-cadherin recruits the cytoplasmic proteins β-catenin and p120-catenin. β-catenin binds to α-catenin, which links AJ to actin filaments. AJ are usually described as linear structures along the actin-rich intercellular contacts. Here, we show that these AJ components can also be organized in reticular domains that contain low levels of actin. Reticular AJ are localized in areas where neighboring cells overlap and encompass the cell adhesion receptor platelet endothelial cell adhesion molecule-1 (PECAM-1). Superresolution microscopy revealed that PECAM-1 forms discrete structures distinct from and distributed along AJ, within the voids of reticular domains. Inflammatory tumor necrosis factor-α increases permeability by mechanisms that are independent of actomyosin-mediated tension and remain incompletely understood. Reticular AJ, but not actin-rich linear AJ, were disorganized by tumor necrosis factor-α. This correlated with PECAM-1 dispersal from cell borders. PECAM-1 inhibition with blocking antibodies or small interfering RNA specifically disrupted reticular AJ, leaving linear AJ intact. This disruption recapitulated typical tumor necrosis factor-α-induced alterations of barrier function, including increased β-catenin phosphorylation, without altering the actomyosin cytoskeleton. We propose that reticular AJ act coordinately with PECAM-1 to maintain endothelial barrier function in regions of low actomyosin-mediated tension. Selective disruption of reticular AJ contributes to permeability increase in response to tumor necrosis factor-α.

Gap junctional communication modulates gene transcription by altering the recruitment of Sp1 and Sp3 to connexin-response elements in osteoblast promoters

NASA Technical Reports Server (NTRS)

Stains, Joseph P.; Lecanda, Fernando; Screen, Joanne; Towler, Dwight A.; Civitelli, Roberto

2003-01-01

Loss-of-function mutations of gap junction proteins, connexins, represent a mechanism of disease in a variety of tissues. We have shown that recessive (gene deletion) or dominant (connexin45 overexpression) disruption of connexin43 function results in osteoblast dysfunction and abnormal expression of osteoblast genes, including down-regulation of osteocalcin transcription. To elucidate the molecular mechanisms of gap junction-sensitive transcriptional regulation, we systematically analyzed the rat osteocalcin promoter for sensitivity to gap junctional intercellular communication. We identified an Sp1/Sp3 containing complex that assembles on a minimal element in the -70 to -57 region of the osteocalcin promoter in a gap junction-dependent manner. This CT-rich connexin-response element is necessary and sufficient to confer gap junction sensitivity to the osteocalcin proximal promoter. Repression of osteocalcin transcription occurs as a result of displacement of the stimulatory Sp1 by the inhibitory Sp3 on the promoter when gap junctional communication is perturbed. Modulation of Sp1/Sp3 recruitment also occurs on the collagen Ialpha1 promoter and translates into gap junction-sensitive transcriptional control of collagen Ialpha1 gene expression. Thus, regulation of Sp1/Sp3 recruitment to the promoter may represent a potential general mechanism for transcriptional control of target genes by signals passing through gap junctions.

Hexadecameric structure of an invertebrate gap junction channel.

PubMed

Oshima, Atsunori; Matsuzawa, Tomohiro; Murata, Kazuyoshi; Tani, Kazutoshi; Fujiyoshi, Yoshinori

2016-03-27

Innexins are invertebrate-specific gap junction proteins with four transmembrane helices. These proteins oligomerize to constitute intercellular channels that allow for the passage of small signaling molecules associated with neural and muscular electrical activity. In contrast to the large number of structural and functional studies of connexin gap junction channels, few structural studies of recombinant innexin channels are reported. Here we show the three-dimensional structure of two-dimensionally crystallized Caenorhabditis elegans innexin-6 (INX-6) gap junction channels. The N-terminal deleted INX-6 proteins are crystallized in lipid bilayers. The three-dimensional reconstruction determined by cryo-electron crystallography reveals that a single INX-6 gap junction channel comprises 16 subunits, a hexadecamer, in contrast to chordate connexin channels, which comprise 12 subunits. The channel pore diameters at the cytoplasmic entrance and extracellular gap region are larger than those of connexin26. Two bulb densities are observed in each hemichannel, one in the pore and the other at the cytoplasmic side of the hemichannel in the channel pore pathway. These findings imply a structural diversity of gap junction channels among multicellular organisms. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Gap junctions in cells of the immune system: structure, regulation and possible functional roles.

PubMed

Sáez, J C; Brañes, M C; Corvalán, L A; Eugenín, E A; González, H; Martínez, A D; Palisson, F

2000-04-01

Gap junction channels are sites of cytoplasmic communication between contacting cells. In vertebrates, they consist of protein subunits denoted connexins (Cxs) which are encoded by a gene family. According to their Cx composition, gap junction channels show different gating and permeability properties that define which ions and small molecules permeate them. Differences in Cx primary sequences suggest that channels composed of different Cxs are regulated differentially by intracellular pathways under specific physiological conditions. Functional roles of gap junction channels could be defined by the relative importance of permeant substances, resulting in coordination of electrical and/or metabolic cellular responses. Cells of the native and specific immune systems establish transient homo- and heterocellular contacts at various steps of the immune response. Morphological and functional studies reported during the last three decades have revealed that many intercellular contacts between cells in the immune response present gap junctions or "gap junction-like" structures. Partial characterization of the molecular composition of some of these plasma membrane structures and regulatory mechanisms that control them have been published recently. Studies designed to elucidate their physiological roles suggest that they might permit coordination of cellular events which favor the effective and timely response of the immune system.

Cell-to-cell diffusion of glucose in the mammalian heart is disrupted by high glucose. Implications for the diabetic heart.

PubMed

De Mello, Walmor C

2015-06-10

The cell-to-cell diffusion of glucose in heart cell pairs isolated from the left ventricle of adult Wistar Kyoto rats was investigated. For this, fluorescent glucose was dialyzed into one cell of the pair using the whole cell clamp technique, and its diffusion from cell-to-cell was investigated by measuring the fluorescence in the dialyzed as well as in non-dialyzed cell as a function of time. The results indicated that: 1) glucose flows easily from cell-to-cell through gap junctions; 2) high glucose solution (25 mM) disrupted chemical communication between cardiac cells and abolished the intercellular diffusion of glucose; 3) the effect of high glucose solution on the cell-to-cell diffusion of glucose was drastically reduced by Bis-1 (10(-9)M) which is a PKC inhibitor; 4) intracellular dialysis of Ang II (100 nM) or increment of intracellular calcium concentration (10(-8)M) also inhibited the intercellular diffusion of glucose; 5) high glucose enhances oxidative stress in heart cells; 6) calculation of gap junction permeability (Pj) (cm/s) indicated a value of 0.74±0.08×10(-4) cm/s (5 animals) for the controls and 0.4±0.001×10(-5) cm/s; n=35 (5 animals) (P<0.05) for cells incubated with high glucose solution for 24h; 7) measurements of Pj for cell pairs treated with high glucose plus Bis-1 (10(-9)M) revealed no significant change of Pj (P>0.05); 8) increase of intracellular Ca(2+) concentration (10(-8)M) drastically decreased Pj (Pj=0.3±0.003×10(-5) cm/s). Conclusions indicate that: 1) glucose flows from cell-to-cell in the heart through gap junctions; 2) high glucose (25 mM) inhibited the intercellular diffusion of glucose-an effect significantly reduced by PKC inhibition; 3) high intracellular Ca(2+) concentration abolished the cell-to-cell diffusion of glucose; 4) intracellular Ang II (100 nM) inhibited the intercellular diffusion of glucose indicating that intracrine Ang II, in part activated by high glucose, severely impairs the exchange of glucose between cardiac myocytes. These observations support the view that the intracrine renin angiotensin system is a modulator of chemical communication in the heart. The implications of these findings for the diabetic heart were discussed. Copyright © 2015 Elsevier Inc. All rights reserved.

Specific Glucoside Transporters Influence Septal Structure and Function in the Filamentous, Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120.

PubMed

Nieves-Morión, Mercedes; Lechno-Yossef, Sigal; López-Igual, Rocío; Frías, José E; Mariscal, Vicente; Nürnberg, Dennis J; Mullineaux, Conrad W; Wolk, C Peter; Flores, Enrique

2017-04-01

When deprived of combined nitrogen, some filamentous cyanobacteria contain two cell types: vegetative cells that fix CO 2 through oxygenic photosynthesis and heterocysts that are specialized in N 2 fixation. In the diazotrophic filament, the vegetative cells provide the heterocysts with reduced carbon (mainly in the form of sucrose) and heterocysts provide the vegetative cells with combined nitrogen. Septal junctions traverse peptidoglycan through structures known as nanopores and appear to mediate intercellular molecular transfer that can be traced with fluorescent markers, including the sucrose analog esculin (a coumarin glucoside) that is incorporated into the cells. Uptake of esculin by the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 was inhibited by the α-glucosides sucrose and maltose. Analysis of Anabaena mutants identified components of three glucoside transporters that move esculin into the cells: GlsC (Alr4781) and GlsP (All0261) are an ATP-binding subunit and a permease subunit of two different ABC transporters, respectively, and HepP (All1711) is a major facilitator superfamily (MFS) protein that was shown previously to be involved in formation of the heterocyst envelope. Transfer of fluorescent markers (especially calcein) between vegetative cells of Anabaena was impaired by mutation of glucoside transporter genes. GlsP and HepP interact in bacterial two-hybrid assays with the septal junction-related protein SepJ, and GlsC was found to be necessary for the formation of a normal number of septal peptidoglycan nanopores and for normal subcellular localization of SepJ. Therefore, beyond their possible role in nutrient uptake in Anabaena , glucoside transporters influence the structure and function of septal junctions. IMPORTANCE Heterocyst-forming cyanobacteria have the ability to perform oxygenic photosynthesis and to assimilate atmospheric CO 2 and N 2 These organisms grow as filaments that fix these gases specifically in vegetative cells and heterocysts, respectively. For the filaments to grow, these types of cells exchange nutrients, including sucrose, which serves as a source of reducing power and of carbon skeletons for the heterocysts. Movement of sucrose between cells in the filament takes place through septal junctions and has been traced with a fluorescent sucrose analog, esculin, that can be taken up by the cells. Here, we identified α-glucoside transporters of Anabaena that mediate uptake of esculin and, notably, influence septal structure and the function of septal junctions. Copyright © 2017 American Society for Microbiology.

Specific Glucoside Transporters Influence Septal Structure and Function in the Filamentous, Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120

PubMed Central

Nieves-Morión, Mercedes; Lechno-Yossef, Sigal; López-Igual, Rocío; Frías, José E.; Mariscal, Vicente; Nürnberg, Dennis J.; Mullineaux, Conrad W.; Wolk, C. Peter

2017-01-01

ABSTRACT When deprived of combined nitrogen, some filamentous cyanobacteria contain two cell types: vegetative cells that fix CO2 through oxygenic photosynthesis and heterocysts that are specialized in N2 fixation. In the diazotrophic filament, the vegetative cells provide the heterocysts with reduced carbon (mainly in the form of sucrose) and heterocysts provide the vegetative cells with combined nitrogen. Septal junctions traverse peptidoglycan through structures known as nanopores and appear to mediate intercellular molecular transfer that can be traced with fluorescent markers, including the sucrose analog esculin (a coumarin glucoside) that is incorporated into the cells. Uptake of esculin by the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 was inhibited by the α-glucosides sucrose and maltose. Analysis of Anabaena mutants identified components of three glucoside transporters that move esculin into the cells: GlsC (Alr4781) and GlsP (All0261) are an ATP-binding subunit and a permease subunit of two different ABC transporters, respectively, and HepP (All1711) is a major facilitator superfamily (MFS) protein that was shown previously to be involved in formation of the heterocyst envelope. Transfer of fluorescent markers (especially calcein) between vegetative cells of Anabaena was impaired by mutation of glucoside transporter genes. GlsP and HepP interact in bacterial two-hybrid assays with the septal junction-related protein SepJ, and GlsC was found to be necessary for the formation of a normal number of septal peptidoglycan nanopores and for normal subcellular localization of SepJ. Therefore, beyond their possible role in nutrient uptake in Anabaena, glucoside transporters influence the structure and function of septal junctions. IMPORTANCE Heterocyst-forming cyanobacteria have the ability to perform oxygenic photosynthesis and to assimilate atmospheric CO2 and N2. These organisms grow as filaments that fix these gases specifically in vegetative cells and heterocysts, respectively. For the filaments to grow, these types of cells exchange nutrients, including sucrose, which serves as a source of reducing power and of carbon skeletons for the heterocysts. Movement of sucrose between cells in the filament takes place through septal junctions and has been traced with a fluorescent sucrose analog, esculin, that can be taken up by the cells. Here, we identified α-glucoside transporters of Anabaena that mediate uptake of esculin and, notably, influence septal structure and the function of septal junctions. PMID:28096449

Exploiting the Gastric Epithelial Barrier: Helicobacter pylori's Attack on Tight and Adherens Junctions.

PubMed

Backert, Steffen; Schmidt, Thomas P; Harrer, Aileen; Wessler, Silja

2017-01-01

Highly organized intercellular tight and adherens junctions are crucial structural components for establishing and maintenance of epithelial barrier functions, which control the microbiota and protect against intruding pathogens in humans. Alterations in these complexes represent key events in the development and progression of multiple infectious diseases as well as various cancers. The gastric pathogen Helicobacter pylori exerts an amazing set of strategies to manipulate these epithelial cell-to-cell junctions, which are implicated in changing cell polarity, migration and invasive growth as well as pro-inflammatory and proliferative responses. This chapter focuses on the H. pylori pathogenicity factors VacA, CagA, HtrA and urease, and how they can induce host cell signaling involved in altering cell-to-cell permeability. We propose a stepwise model for how H. pylori targets components of tight and adherens junctions in order to disrupt the gastric epithelial cell layer, giving fresh insights into the pathogenesis of this important bacterium.

Integrin β4 Signaling Promotes Mammary Tumor Cell Adhesion to Brain Microvascular Endothelium by Inducing ErbB2-mediated Secretion of VEGF

PubMed Central

Fan, Jie; Cai, Bin; Zeng, Min; Hao, Yanyan

2015-01-01

Prior studies have indicated that the β4 integrin promotes mammary tumor invasion and metastasis by combining with ErbB2 and amplifying its signaling capacity. However, the effector pathways and cellular functions by which the β4 integrin exerts these effects are incompletely understood. To examine if β4 signaling plays a role during mammary tumor cell adhesion to microvascular endothelium, we have examined ErbB2-transformed mammary tumor cells expressing either a wild-type (WT) or a signaling-defective form of β4 (1355T). We report that WT cells adhere to brain microvascular endothelium in vitro to a significantly larger extent as compared to 1355T cells. Interestingly, integrin β4 signaling does not exert a direct effect on adhesion to the endothelium or the underlying basement membrane. Rather, it enhances ErbB2-dependent expression of VEGF by tumor cells. VEGF in turn disrupts the tight and adherens junctions of endothelial monolayers, enabling the exposure of underlying basement membrane and increasing the adhesion of tumor cells to the intercellular junctions of endothelium. Inhibition of ErbB2 on tumor cells or the VEGFR-2 on endothelial cells suppresses mammary tumor cell adhesion to microvascular endothelium. Our results indicate that β4 signaling regulates VEGF expression by the mammary tumor cells thereby enhancing their adhesion to microvascular endothelium. PMID:21556948

Viral Interactions with PDZ Domain-Containing Proteins-An Oncogenic Trait?

PubMed

James, Claire D; Roberts, Sally

2016-01-18

Many of the human viruses with oncogenic capabilities, either in their natural host or in experimental systems (hepatitis B and C, human T cell leukaemia virus type 1, Kaposi sarcoma herpesvirus, human immunodeficiency virus, high-risk human papillomaviruses and adenovirus type 9), encode in their limited genome the ability to target cellular proteins containing PSD95/ DLG/ZO-1 (PDZ) interaction modules. In many cases (but not always), the viruses have evolved to bind the PDZ domains using the same short linear peptide motifs found in host protein-PDZ interactions, and in some cases regulate the interactions in a similar fashion by phosphorylation. What is striking is that the diverse viruses target a common subset of PDZ proteins that are intimately involved in controlling cell polarity and the structure and function of intercellular junctions, including tight junctions. Cell polarity is fundamental to the control of cell proliferation and cell survival and disruption of polarity and the signal transduction pathways involved is a key event in tumourigenesis. This review focuses on the oncogenic viruses and the role of targeting PDZ proteins in the virus life cycle and the contribution of virus-PDZ protein interactions to virus-mediated oncogenesis. We highlight how many of the viral associations with PDZ proteins lead to deregulation of PI3K/AKT signalling, benefitting virus replication but as a consequence also contributing to oncogenesis.

Gap Junctions Are Involved in the Rescue of CFTR-Dependent Chloride Efflux by Amniotic Mesenchymal Stem Cells in Coculture with Cystic Fibrosis CFBE41o- Cells.

PubMed

Carbone, Annalucia; Zefferino, Roberto; Beccia, Elisa; Casavola, Valeria; Castellani, Stefano; Di Gioia, Sante; Giannone, Valentina; Seia, Manuela; Angiolillo, Antonella; Colombo, Carla; Favia, Maria; Conese, Massimo

2018-01-01

We previously found that human amniotic mesenchymal stem cells (hAMSCs) in coculture with CF immortalised airway epithelial cells (CFBE41o- line, CFBE) on Transwell® filters acquired an epithelial phenotype and led to the expression of a mature and functional CFTR protein. In order to explore the role of gap junction- (GJ-) mediated intercellular communication (GJIC) in this rescue, cocultures (hAMSC : CFBE, 1 : 5 ratio) were studied for the formation of GJIC, before and after silencing connexin 43 (Cx43), a major component of GJs. Functional GJs in cocultures were inhibited when the expression of the Cx43 protein was downregulated. Transfection of cocultures with siRNA against Cx43 resulted in the absence of specific CFTR signal on the apical membrane and reduction in the mature form of CFTR (band C), and in parallel, the CFTR-dependent chloride channel activity was significantly decreased. Cx43 downregulation determined also a decrease in transepithelial resistance and an increase in paracellular permeability as compared with control cocultures, implying that GJIC may regulate CFTR expression and function that in turn modulate airway epithelium tightness. These results indicate that GJIC is involved in the correction of CFTR chloride channel activity upon the acquisition of an epithelial phenotype by hAMSCs in coculture with CF cells.

Evolution of the vertebrate claudin gene family: insights from a basal vertebrate, the sea lamprey.

PubMed

Mukendi, Christian; Dean, Nicholas; Lala, Rushil; Smith, Jeramiah; Bronner, Marianne E; Nikitina, Natalya V

2016-01-01

Claudins are major constituents of tight junctions, contributing both to their intercellular sealing and selective permeability properties. While claudins and claudin-like molecules are present in some invertebrates, the association of claudins with tight junctions has been conclusively documented only in vertebrates. Here we report the sequencing, phylogenetic analysis and comprehensive spatiotemporal expression analysis of the entire claudin gene family in the basal extant vertebrate, the sea lamprey. Our results demonstrate that clear orthologues to about half of all mammalian claudins are present in the lamprey, suggesting that at least one round of whole genome duplication contributed to the diversification of this gene family. Expression analysis revealed that claudins are expressed in discrete and specific domains, many of which represent vertebrate-specific innovations, such as in cranial ectodermal placodes and the neural crest; whereas others represent structures characteristic of chordates, e.g. pronephros, notochord, somites, endostyle and pharyngeal arches. By comparing the embryonic expression of claudins in the lamprey to that of other vertebrates, we found that ancestral expression patterns were often preserved in higher vertebrates. Morpholino mediated loss of Cldn3b demonstrated a functional role for this protein in placode and pharyngeal arch morphogenesis. Taken together, our data provide novel insights into the origins and evolution of the claudin gene family and the significance of claudin proteins in the evolution of vertebrates.

Disruption of gap junctions attenuates aminoglycoside-elicited renal tubular cell injury.

PubMed

Yao, Jian; Huang, Tao; Fang, Xin; Chi, Yuan; Zhu, Ying; Wan, Yigang; Matsue, Hiroyuki; Kitamura, Masanori

2010-08-01

Gap junctions play important roles in the regulation of cell phenotype and in determining cell survival after various insults. Here, we investigated the role of gap junctions in aminoglycoside-induced injury to renal tubular cells. Two tubular epithelial cell lines NRK-E52 and LLC-PK1 were compared for gap junction protein expression and function by immunofluorescent staining, Western blot and dye transfer assay. Cell viability after exposure to aminoglycosides was evaluated by WST assay. Gap junctions were modulated by transfection of the gap junction protein, connexin 43 (Cx43), use of Cx43 siRNA and gap junction inhibitors. NRK-E52 cells expressed abundant Cx43 and were functionally coupled by gap junctional intercellular communication (GJIC). Exposure of NRK-E52 cells to aminoglycosides, G418 and hygromycin, increased Cx43 phosphorylation and GJIC. The aminoglycosides also decreased cell viability that was prevented by gap junction inhibitors and Cx43 siRNA. LLC-PK1 cells were gap junction-deficient and resistant to aminoglycoside-induced cytotoxicity. Over-expression of a wild-type Cx43 converted LLC-PK1 cells to a drug-sensitive phenotype. The gap junction inhibitor alpha-glycyrrhetinic acid (alpha-GA) activated Akt in NRK-E52 cells. Inhibition of the Akt pathway enhanced cell toxicity to G418 and abolished the protective effects of alpha-GA. In addition, gentamycin-elicited cytotoxicity in NRK-E52 cells was also significantly attenuated by alpha-GA. Gap junctions contributed to the cytotoxic effects of aminoglycosides. Modulation of gap junctions could be a promising approach for prevention and treatment of aminoglycoside-induced renal tubular cell injury.

Intercellular and systemic spread of RNA and RNAi in plants.

PubMed

Nazim Uddin, Mohammad; Kim, Jae-Yean

2013-01-01

Plants possess dynamic networks of intercellular communication that are crucial for plant development and physiology. In plants, intercellular communication involves a combination of ligand-receptor-based apoplasmic signaling, and plasmodesmata and phloem-mediated symplasmic signaling. The intercellular trafficking of macromolecules, including RNAs and proteins, has emerged as a novel mechanism of intercellular communication in plants. Various forms of regulatory RNAs move over distinct cellular boundaries through plasmodesmata and phloem. This plant-specific, non-cell-autonomous RNA trafficking network is also involved in development, nutrient homeostasis, gene silencing, pathogen defense, and many other physiological processes. However, the mechanism underlying macromolecular trafficking in plants remains poorly understood. Current progress made in RNA trafficking research and its biological relevance to plant development will be summarized. Diverse plant regulatory mechanisms of cell-to-cell and systemic long-distance transport of RNAs, including mRNAs, viral RNAs, and small RNAs, will also be discussed. Copyright © 2013 John Wiley & Sons, Ltd.

Microtubule-assisted altered trafficking of astrocytic gap junction protein connexin 43 is associated with depletion of connexin 47 during mouse hepatitis virus infection.

PubMed

Basu, Rahul; Bose, Abhishek; Thomas, Deepthi; Das Sarma, Jayasri

2017-09-08

Gap junctions (GJs) are important for maintenance of CNS homeostasis. GJ proteins, connexin 43 (Cx43) and connexin 47 (Cx47), play a crucial role in production and maintenance of CNS myelin. Cx43 is mainly expressed by astrocytes in the CNS and forms gap junction intercellular communications between astrocytes-astrocytes (Cx43-Cx43) and between astrocytes-oligodendrocytes (Cx43-Cx47). Mutations of these connexin (Cx) proteins cause dysmyelinating diseases in humans. Previously, it has been shown that Cx43 localization and expression is altered due to mouse hepatitis virus (MHV)-A59 infection both in vivo and in vitro ; however, its mechanism and association with loss of myelin protein was not elaborated. Thus, we explored potential mechanisms by which MHV-A59 infection alters Cx43 localization and examined the effects of viral infection on Cx47 expression and its association with loss of the myelin marker proteolipid protein. Immunofluorescence and total internal reflection fluorescence microscopy confirmed that MHV-A59 used microtubules (MTs) as a conduit to reach the cell surface and restricted MT-mediated Cx43 delivery to the cell membrane. Co-immunoprecipitation experiments demonstrated that Cx43-β-tubulin molecular interaction was depleted due to protein-protein interaction between viral particles and MTs. During acute MHV-A59 infection, oligodendrocytic Cx47, which is mainly stabilized by Cx43 in vivo , was down-regulated, and its characteristic staining remained disrupted even at chronic phase. The loss of Cx47 was associated with loss of proteolipid protein at the chronic stage of MHV-A59 infection. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Modulation of connexin expression and gap junction communication in astrocytes by the gram-positive bacterium S. aureus.

PubMed

Esen, Nilufer; Shuffield, Debbie; Syed, Mohsin M D; Kielian, Tammy

2007-01-01

Gap junctions establish direct intercellular conduits between adjacent cells and are formed by the hexameric organization of protein subunits called connexins (Cx). It is unknown whether the proinflammatory milieu that ensues during CNS infection with S. aureus, one of the main etiologic agents of brain abscess in humans, is capable of eliciting regional changes in astrocyte homocellular gap junction communication (GJC) and, by extension, influencing neuron homeostasis at sites distant from the primary focus of infection. Here we investigated the effects of S. aureus and its cell wall product peptidoglycan (PGN) on Cx43, Cx30, and Cx26 expression, the main Cx isoforms found in astrocytes. Both bacterial stimuli led to a time-dependent decrease in Cx43 and Cx30 expression; however, Cx26 levels were elevated following bacterial exposure. Functional examination of dye coupling, as revealed by single-cell microinjections of Lucifer yellow, demonstrated that both S. aureus and PGN inhibited astrocyte GJC. Inhibition of protein synthesis with cyclohexamide (CHX) revealed that S. aureus directly modulates, in part, Cx43 and Cx30 expression, whereas Cx26 levels appear to be regulated by a factor(s) that requires de novo protein production; however, CHX did not alter the inhibitory effects of S. aureus on astrocyte GJC. The p38 MAPK inhibitor SB202190 was capable of partially restoring the S. aureus-mediated decrease in astrocyte GJC to that of unstimulated cells, suggesting the involvement of p38 MAPK-dependent pathway(s). These findings could have important implications for limiting the long-term detrimental effects of abscess formation in the brain which may include seizures and cognitive deficits. Copyright 2006 Wiley-Liss, Inc.

Intercellular ice propagation: experimental evidence for ice growth through membrane pores.

PubMed Central

Acker, J P; Elliott, J A; McGann, L E

2001-01-01

Propagation of intracellular ice between cells significantly increases the prevalence of intracellular ice in confluent monolayers and tissues. It has been proposed that gap junctions facilitate ice propagation between cells. This study develops an equation for capillary freezing-point depression to determine the effect of temperature on the equilibrium radius of an ice crystal sufficiently small to grow through gap junctions. Convection cryomicroscopy and video image analysis were used to examine the incidence and pattern of intracellular ice formation (IIF) in the confluent monolayers of cell lines that do (MDCK) and do not (V-79W) form gap junctions. The effect of gap junctions on intracellular ice propagation was strongly temperature-dependent. For cells with gap junctions, IIF occurred in a directed wave-like pattern in 100% of the cells below -3 degrees C. At temperatures above -3 degrees C, there was a marked drop in the incidence of IIF, with isolated individual cells initially freezing randomly throughout the sample. This random pattern of IIF was also observed in the V-79W monolayers and in MDCK monolayers treated to prevent gap junction formation. The significant change in the low temperature behavior of confluent MDCK monolayers at -3 degrees C is likely the result of the inhibition of gap junction-facilitated ice propagation, and supports the theory that gap junctions facilitate ice nucleation between cells. PMID:11509353

Changes in intestinal tight junction permeability associated with industrial food additives explain the rising incidence of autoimmune disease.

PubMed

Lerner, Aaron; Matthias, Torsten

2015-06-01

The incidence of autoimmune diseases is increasing along with the expansion of industrial food processing and food additive consumption. The intestinal epithelial barrier, with its intercellular tight junction, controls the equilibrium between tolerance and immunity to non-self-antigens. As a result, particular attention is being placed on the role of tight junction dysfunction in the pathogenesis of AD. Tight junction leakage is enhanced by many luminal components, commonly used industrial food additives being some of them. Glucose, salt, emulsifiers, organic solvents, gluten, microbial transglutaminase, and nanoparticles are extensively and increasingly used by the food industry, claim the manufacturers, to improve the qualities of food. However, all of the aforementioned additives increase intestinal permeability by breaching the integrity of tight junction paracellular transfer. In fact, tight junction dysfunction is common in multiple autoimmune diseases and the central part played by the tight junction in autoimmune diseases pathogenesis is extensively described. It is hypothesized that commonly used industrial food additives abrogate human epithelial barrier function, thus, increasing intestinal permeability through the opened tight junction, resulting in entry of foreign immunogenic antigens and activation of the autoimmune cascade. Future research on food additives exposure-intestinal permeability-autoimmunity interplay will enhance our knowledge of the common mechanisms associated with autoimmune progression. Copyright © 2015. Published by Elsevier B.V.

Methoxychlor and Vinclozolin Induce Rapid Changes in Intercellular and Intracellular Signaling in Liver Progenitor Cells

PubMed Central

Babica, Pavel; Zurabian, Rimma; Kumar, Esha R.; Chopra, Rajus; Mianecki, Maxwell J.; Park, Joon-Suk; Jaša, Libor; Trosko, James E.; Upham, Brad L.

2016-01-01

Methoxychlor (MXC) and vinclozolin (VIN) are well-recognized endocrine disrupting chemicals known to alter epigenetic regulations and transgenerational inheritance; however, non-endocrine disruption endpoints are also important. Thus, we determined the effects of MXC and VIN on the dysregulation of gap junctional intercellular communication (GJIC) and activation of mitogen-activated protein kinases (MAPKs) in WB-F344 rat liver epithelial cells. Both chemicals induced a rapid dysregulation of GJIC at non-cytotoxic doses, with 30 min EC50 values for GJIC inhibition being 10 µM for MXC and 126 µM for VIN. MXC inhibited GJIC for at least 24 h, while VIN effects were transient and GJIC recovered after 4 h. VIN induced rapid hyperphosphorylation and internalization of gap junction protein connexin43, and both chemicals also activated MAPK ERK1/2 and p38. Effects on GJIC were not prevented by MEK1/2 inhibitor, but by an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), resveratrol, and in the case of VIN, also, by a p38 inhibitor. Estrogen (ER) and androgen receptor (AR) modulators (estradiol, ICI 182,780, HPTE, testosterone, flutamide, VIN M2) did not attenuate MXC or VIN effects on GJIC. Our data also indicate that the effects were elicited by the parental compounds of MXC and VIN. Our study provides new evidence that MXC and VIN dysregulate GJIC via mechanisms involving rapid activation of PC-PLC occurring independently of ER- or AR-dependent genomic signaling. Such alterations of rapid intercellular and intracellular signaling events involved in regulations of gene expression, tissue development, function and homeostasis, could also contribute to transgenerational epigenetic effects of endocrine disruptors. PMID:27413106

The gap junction inhibitor 2-aminoethoxy-diphenyl-borate protects against acetaminophen hepatotoxicity by inhibiting cytochrome P450 enzymes and c-jun N-terminal kinase activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du, Kuo; Williams, C. David; McGill, Mitchell R.

2013-12-15

Acetaminophen (APAP) hepatotoxicity is the leading cause of acute liver failure in the US. Although many aspects of the mechanism are known, recent publications suggest that gap junctions composed of connexin32 function as critical intercellular communication channels which transfer cytotoxic mediators into neighboring hepatocytes and aggravate liver injury. However, these studies did not consider off-target effects of reagents used in these experiments, especially the gap junction inhibitor 2-aminoethoxy-diphenyl-borate (2-APB). In order to assess the mechanisms of protection of 2-APB in vivo, male C56Bl/6 mice were treated with 400 mg/kg APAP to cause extensive liver injury. This injury was prevented whenmore » animals were co-treated with 20 mg/kg 2-APB and was attenuated when 2-APB was administered 1.5 h after APAP. However, the protection was completely lost when 2-APB was given 4–6 h after APAP. Measurement of protein adducts and c-jun-N-terminal kinase (JNK) activation indicated that 2-APB reduced both protein binding and JNK activation, which correlated with hepatoprotection. Although some of the protection was due to the solvent dimethyl sulfoxide (DMSO), in vitro experiments clearly demonstrated that 2-APB directly inhibits cytochrome P450 activities. In addition, JNK activation induced by phorone and tert-butylhydroperoxide in vivo was inhibited by 2-APB. The effects against APAP toxicity in vivo were reproduced in primary cultured hepatocytes without use of DMSO and in the absence of functional gap junctions. We conclude that the protective effect of 2-APB was caused by inhibition of metabolic activation of APAP and inhibition of the JNK signaling pathway and not by blocking connexin32-based gap junctions. - Highlights: • 2-APB protected against APAP-induced liver injury in mice in vivo and in vitro • 2-APB protected by inhibiting APAP metabolic activation and JNK signaling pathway • DMSO inhibited APAP metabolic activation as the solvent of 2-APB • Off-target effects of connexin32 gene knock-out mice need to be considered.« less

Intercellular signaling via cyclic GMP diffusion through gap junctions restarts meiosis in mouse ovarian follicles.

PubMed

Shuhaibar, Leia C; Egbert, Jeremy R; Norris, Rachael P; Lampe, Paul D; Nikolaev, Viacheslav O; Thunemann, Martin; Wen, Lai; Feil, Robert; Jaffe, Laurinda A

2015-04-28

Meiosis in mammalian oocytes is paused until luteinizing hormone (LH) activates receptors in the mural granulosa cells of the ovarian follicle. Prior work has established the central role of cyclic GMP (cGMP) from the granulosa cells in maintaining meiotic arrest, but it is not clear how binding of LH to receptors that are located up to 10 cell layers away from the oocyte lowers oocyte cGMP and restarts meiosis. Here, by visualizing intercellular trafficking of cGMP in real-time in live follicles from mice expressing a FRET sensor, we show that diffusion of cGMP through gap junctions is responsible not only for maintaining meiotic arrest, but also for rapid transmission of the signal that reinitiates meiosis from the follicle surface to the oocyte. Before LH exposure, the cGMP concentration throughout the follicle is at a uniformly high level of ∼2-4 μM. Then, within 1 min of LH application, cGMP begins to decrease in the peripheral granulosa cells. As a consequence, cGMP from the oocyte diffuses into the sink provided by the large granulosa cell volume, such that by 20 min the cGMP concentration in the follicle is uniformly low, ∼100 nM. The decrease in cGMP in the oocyte relieves the inhibition of the meiotic cell cycle. This direct demonstration that a physiological signal initiated by a stimulus in one region of an intact tissue can travel across many layers of cells via cyclic nucleotide diffusion through gap junctions could provide a general mechanism for diverse cellular processes.

Bi-directional gap junction-mediated soma-germline communication is essential for spermatogenesis.

PubMed

Smendziuk, Christopher M; Messenberg, Anat; Vogl, A Wayne; Tanentzapf, Guy

2015-08-01

Soma-germline interactions play conserved essential roles in regulating cell proliferation, differentiation, patterning and homeostasis in the gonad. In the Drosophila testis, secreted signalling molecules of the JAK-STAT, Hedgehog, BMP and EGF pathways are used to mediate soma-germline communication. Here, we demonstrate that gap junctions may also mediate direct, bi-directional signalling between the soma and germ line. When gap junctions between the soma and germ line are disrupted, germline differentiation is blocked and germline stem cells are not maintained. In the soma, gap junctions are required to regulate proliferation and differentiation. Localization and RNAi-mediated knockdown studies reveal that gap junctions in the fly testis are heterotypic channels containing Zpg (Inx4) and Inx2 on the germ line and the soma side, respectively. Overall, our results show that bi-directional gap junction-mediated signalling is essential to coordinate the soma and germ line to ensure proper spermatogenesis in Drosophila. Moreover, we show that stem cell maintenance and differentiation in the testis are directed by gap junction-derived cues. © 2015. Published by The Company of Biologists Ltd.

Oleamide derivatives suppress the spontaneous metastasis by inhibiting connexin 26.

PubMed

Ohba, Yusuke; Kanao, Yukiko; Morita, Nobuyoshi; Fujii, Eri; Hohrai, Mai; Takatsuji, Mayuko; Hirose, Hideki; Miura, Daisaku; Watari, Akihiro; Yutsudo, Masuo; Zhao, Hanjun; Yabuta, Norikazu; Ito, Akihiko; Kita, Yasuyuki; Nojima, Hiroshi

2007-07-01

We previously reported that overexpressing connexin 26 (Cx26) enhances the spontaneous metastasis of mouse BL6 melanoma cells. In contrast, daily intraperitoneal injections of an oleamide derivative named MI-18 potently inhibits the spontaneous metastasis of BL6 cells. In the present study, we chemically synthesized a novel oleamide derivative named MI-22 and found that it also efficiently suppressed the spontaneous metastasis of BL6 cells. Both MI-18 and MI-22 inhibited the gap junction-mediated intercellular communications (GJIC) that are formed between HeLa cells by the ectopic expression of the hCx26 and hCx32 human connexin subtypes; however, they had no effect on GJIC mediated by hCx40, hCx43 or hCx45. Fluorescently labeled MI-18 primarily localized not only at plasma membrane but also at Golgi/endosome. This suggests that this oleamide derivative may also act on the Cx26 molecules that accumulate in the Golgi/endosome because of their overexpression. Notably, neither derivative had a cytotoxic effect on HeLa cells when they were added into the tissue culture medium. Taken together, we propose that the MI-18 and MI-22 oleamide derivatives may serve as prototypes for novel and clinically important anticancer drugs.

Disruption of gap junctions attenuates aminoglycoside-elicited renal tubular cell injury

PubMed Central

Yao, Jian; Huang, Tao; Fang, Xin; Chi, Yuan; Zhu, Ying; Wan, Yigang; Matsue, Hiroyuki; Kitamura, Masanori

2010-01-01

BACKGROUND AND PURPOSE Gap junctions play important roles in the regulation of cell phenotype and in determining cell survival after various insults. Here, we investigated the role of gap junctions in aminoglycoside-induced injury to renal tubular cells. EXPERIMENTAL APPROACH Two tubular epithelial cell lines NRK-E52 and LLC-PK1 were compared for gap junction protein expression and function by immunofluorescent staining, Western blot and dye transfer assay. Cell viability after exposure to aminoglycosides was evaluated by WST assay. Gap junctions were modulated by transfection of the gap junction protein, connexin 43 (Cx43), use of Cx43 siRNA and gap junction inhibitors. KEY RESULTS NRK-E52 cells expressed abundant Cx43 and were functionally coupled by gap junctional intercellular communication (GJIC). Exposure of NRK-E52 cells to aminoglycosides, G418 and hygromycin, increased Cx43 phosphorylation and GJIC. The aminoglycosides also decreased cell viability that was prevented by gap junction inhibitors and Cx43 siRNA. LLC-PK1 cells were gap junction-deficient and resistant to aminoglycoside-induced cytotoxicity. Over-expression of a wild-type Cx43 converted LLC-PK1 cells to a drug-sensitive phenotype. The gap junction inhibitor α-glycyrrhetinic acid (α-GA) activated Akt in NRK-E52 cells. Inhibition of the Akt pathway enhanced cell toxicity to G418 and abolished the protective effects of α-GA. In addition, gentamycin-elicited cytotoxicity in NRK-E52 cells was also significantly attenuated by α-GA. CONCLUSION AND IMPLICATIONS Gap junctions contributed to the cytotoxic effects of aminoglycosides. Modulation of gap junctions could be a promising approach for prevention and treatment of aminoglycoside-induced renal tubular cell injury. PMID:20649601

Interactions of Entamoeba Histolytica with Host Cells in the Gut Mucosa,

DTIC Science & Technology

1977-01-01

amebae were observed close to the cecal epithelium, the apposing epithelial cell was found to have protruded in the lumen and making contact with the... amebae . Such cells often had become detached from their basal lamina and intercellular tight junctions, affording spaces through which amebae invaded the...mucosa. Other epithelial cells in close proximity to amebae showed degenerative changes characterized by swelling of mitochondria and endoplasmic

Red paprika (Capsicum annuum L.) and its main carotenoids, capsanthin and β-carotene, prevent hydrogen peroxide-induced inhibition of gap-junction intercellular communication.

PubMed

Kim, Ji-Sun; Lee, Woo-Moon; Rhee, Han Cheol; Kim, Suna

2016-07-25

This study was conducted to investigate the protective effect of red paprika extract (RPE) and its main carotenoids, namely, capsanthin (CST) and β-carotene (BCT), on the H2O2-induced inhibition of gap-junction intercellular communication (GJIC) in WB-F344 rat liver epithelial cells (WB cells). We found that pre-treatment with RPE, CST and BCT protected WB cells from H2O2-induced inhibition of GJIC. RPE, CST and BCT not only recovered connexin 43 (Cx43) mRNA expression but also prevented phosphorylation of Cx43 protein by H2O2 treatment. RPE attenuated the phosphorylation of ERK, p38 and JNK, whereas pre-treatment with CST and BCT only attenuated the phosphorylation of ERK and p38 and did not affect JNK in H2O2-treated WB cells. RPE, CST and BCT significantly suppressed the formation of reactive oxygen species (ROS) in H2O2-treated cells compared to untreated WB cells. These results suggest that dietary intake of red paprika might be helpful for lowering the risk of diseases caused by oxidative stress. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Calreticulin attenuated microwave radiation-induced human microvascular endothelial cell injury through promoting actin acetylation and polymerization.

PubMed

Xu, Feifei; Wang, You; Tao, Tianqi; Song, Dandan; Liu, Xiuhua

2017-01-01

Recent work reveals that actin acetylation modification has been linked to different normal and disease processes and the effects associated with metabolic and environmental stressors. Herein, we highlight the effects of calreticulin on actin acetylation and cell injury induced by microwave radiation in human microvascular endothelial cell (HMEC). HMEC injury was induced by high-power microwave of different power density (10, 30, 60, 100 mW/cm 2 , for 6 min) with or without exogenous recombinant calreticulin. The cell injury was assessed by lactate dehydrogenase (LDH) activity and Cell Counting Kit-8 in culture medium, migration ability, intercellular junction, and cytoskeleton staining in HMEC. Western blotting analysis was used to detected calreticulin expression in cytosol and nucleus and acetylation of globular actin (G-actin). We found that HMEC injury was induced by microwave radiation in a dose-dependent manner. Pretreatment HMEC with calreticulin suppressed microwave radiation-induced LDH leakage and increased cell viability and improved microwave radiation-induced decrease in migration, intercellular junction, and cytoskeleton. Meanwhile, pretreatment HMEC with exogenous calreticulin upregulated the histone acetyltransferase activity and the acetylation level of G-actin and increased the fibrous actin (F-actin)/G-actin ratio. We conclude that exogenous calreticulin protects HMEC against microwave radiation-induced injury through promoting actin acetylation and polymerization.

Inhibition of gap-junctional intercellular communication and activation of mitogen-activated protein kinases by cyanobacterial extracts--indications of novel tumor-promoting cyanotoxins?

PubMed

Bláha, Ludĕk; Babica, Pavel; Hilscherová, Klára; Upham, Brad L

2010-01-01

Toxicity and liver tumor promotion of cyanotoxins microcystins have been extensively studied. However, recent studies document that other metabolites present in the complex cyanobacterial water blooms may also have adverse health effects. In this study we used rat liver epithelial stem-like cells (WB-F344) to examine the effects of cyanobacterial extracts on two established markers of tumor promotion, inhibition of gap-junctional intercellular communication (GJIC) and activation of mitogen-activated protein kinases (MAPKs) - ERK1/2. Extracts of cyanobacteria (laboratory cultures of Microcystis aeruginosa and Aphanizomenon flos-aquae and water blooms dominated by these species) inhibited GJIC and activated MAPKs in a dose-dependent manner (effective concentrations ranging 0.5-5mgd.w./mL). Effects were independent of the microcystin content and the strongest responses were elicited by the extracts of Aphanizomenon sp. Neither pure microcystin-LR nor cylindrospermopsin inhibited GJIC or activated MAPKs. Modulations of GJIC and MAPKs appeared to be specific to cyanobacterial extracts since extracts from green alga Chlamydomonas reinhardtii, heterotrophic bacterium Klebsiella terrigena, and isolated bacterial lipopolysaccharides had no comparable effects. Our study provides the first evidence on the existence of unknown cyanobacterial toxic metabolites that affect in vitro biomarkers of tumor promotion, i.e. inhibition of GJIC and activation of MAPKs.

Gap junctions between CA3 pyramidal cells contribute to network synchronization in neonatal hippocampus.

PubMed

Molchanova, Svetlana M; Huupponen, Johanna; Lauri, Sari E; Taira, Tomi

2016-08-01

Direct electrical coupling between neurons through gap junctions is prominent during development, when synaptic connectivity is scarce, providing the additional intercellular connectivity. However, functional studies of gap junctions are hampered by the unspecificity of pharmacological tools available. Here we have investigated gap-junctional coupling between CA3 pyramidal cells in neonatal hippocampus and its contribution to early network activity. Four different gap junction inhibitors, including the general blocker carbenoxolone, decreased the frequency of network activity bursts in CA3 area of hippocampus of P3-6 rats, suggesting the involvement of electrical connections in the generation of spontaneous network activity. In CA3 pyramidal cells, spikelets evoked by local stimulation of stratum oriens, were inhibited by carbenoxolone, but not by inhibitors of glutamatergic and GABAergic synaptic transmission, signifying the presence of electrical connectivity through axo-axonic gap junctions. Carbenoxolone also decreased the success rate of firing antidromic action potentials in response to stimulation, and changed the pattern of spontaneous action potential firing of CA3 pyramidal cells. Altogether, these data suggest that electrical coupling of CA3 pyramidal cells contribute to the generation of the early network events in neonatal hippocampus by modulating their firing pattern and synchronization. Copyright © 2016 Elsevier Ltd. All rights reserved.

Gap Junctions and Connexin Hemichannels Underpin Haemostasis and Thrombosis

PubMed Central

Vaiyapuri, Sakthivel; Jones, Chris I.; Sasikumar, Parvathy; Moraes, Leonardo A.; Munger, Stephanie J.; Wright, Joy R.; Ali, Marfoua S.; Sage, Tanya; Kaiser, William J.; Tucker, Katherine L.; Stain, Christopher J.; Bye, Alexander P.; Jones, Sarah; Oviedo-Orta, Ernesto; Simon, Alexander M.; Mahaut-Smith, Martyn P.; Gibbins, Jonathan M.

2012-01-01

Background Connexins are a widespread family of membrane proteins that assemble into hexameric hemichannels, also known as connexons. Connexons regulate membrane permeability in individual cells or couple between adjacent cells to form gap junctions and thereby provide a pathway for regulated intercellular communication. We have now examined the role of connexins in platelets, blood cells that circulate in isolation, but upon tissue injury adhere to each other and the vessel wall to prevent blood loss and facilitate wound repair. Methods and Results We report the presence of connexins in platelets, notably connexin37, and that the formation of gap junctions within platelet thrombi is required for the control of clot retraction. Inhibition of connexin function modulated a range of platelet functional responses prior to platelet-platelet contact, and reduced laser induced thrombosis in vivo in mice. Deletion of the Cx37 gene (Gja4) in transgenic mice reduced platelet aggregation, fibrinogen binding, granule secretion and clot retraction indicating an important role for Cx37 hemichannels and gap junctions in platelet thrombus function. Conclusions Together, these data demonstrate that platelet gap junctions and hemichannels underpin the control of haemostasis and thrombosis and represent potential therapeutic targets. PMID:22528526

[Gap junctions: A new therapeutic target in major depressive disorder?].

PubMed

Sarrouilhe, D; Dejean, C

2015-11-01

Major depressive disorder is a multifactorial chronic and debilitating mood disease with high lifetime prevalence and is associated with excess mortality, especially from cardiovascular diseases and through suicide. The treatments of this disease with tricyclic antidepressants and monoamine oxidase inhibitors are poorly tolerated and those that selectively target serotonin and norepinephrine re-uptake are not effective in all patients, showing the need to find new therapeutic targets. Post-mortem studies of brains from patients with major depressive disorders described a reduced expression of the gap junction-forming membrane proteins connexin 30 and connexin 43 in the prefrontal cortex and the locus coeruleus. The use of chronic unpredictable stress, a rodent model of depression, suggests that astrocytic gap junction dysfunction contributes to the pathophysiology of major depressive disorder. Chronic treatments of rats with fluoxetine and of rat cultured cortical astrocytes with amitriptyline support the hypothesis that the upregulation of gap junctional intercellular communication between brain astrocytes could be a novel mechanism for the therapeutic effect of antidepressants. In conclusion, astrocytic gap junctions are emerging as a new potential therapeutic target for the treatment of patients with major depressive disorder. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

The canonical WNT2 pathway and FSH interact to regulate gap junction assembly in mouse granulosa cells.

PubMed

Wang, Hong-Xing; Gillio-Meina, Carolina; Chen, Shuli; Gong, Xiang-Qun; Li, Tony Y; Bai, Donglin; Kidder, Gerald M

2013-08-01

WNTs are extracellular signaling molecules that exert their actions through receptors of the frizzled (FZD) family. Previous work indicated that WNT2 regulates cell proliferation in mouse granulosa cells acting through CTNNB1 (beta-catenin), a key component in canonical WNT signaling. In other cells, WNT signaling has been shown to regulate expression of connexin43 (CX43), a gap junction protein, as well as gap junction assembly. Since previous work demonstrated that CX43 is also essential in ovarian follicle development, the objective of this study was to determine if WNT2 regulates CX43 expression and/or gap-junctional intercellular communication (GJIC) in granulosa cells. WNT2 knockdown via siRNA markedly reduced CX43 expression and GJIC. CX43 expression, the extent of CX43-containing gap junction membrane, and GJIC were also reduced by CTNNB1 transient knockdown. CTNNB1 is mainly localized to the membranes between granulosa cells but disappeared from this location after WNT2 knockdown. Furthermore, CTNNB1 knockdown interfered with the ability of follicle-stimulating hormone (FSH) to promote the mobilization of CX43 into gap junctions. We propose that the WNT2/CTNNB1 pathway regulates CX43 expression and GJIC in granulosa cells by modulating CTNNB1 stability and localization in adherens junctions, and that this is essential for FSH stimulation of GJIC.

The E3 ubiquitin ligase NEDD4 induces endocytosis and lysosomal sorting of connexin 43 to promote loss of gap junctions.

PubMed

Totland, Max Z; Bergsland, Christian H; Fykerud, Tone A; Knudsen, Lars M; Rasmussen, Nikoline L; Eide, Peter W; Yohannes, Zeremariam; Sørensen, Vigdis; Brech, Andreas; Lothe, Ragnhild A; Leithe, Edward

2017-09-01

Intercellular communication via gap junctions has an important role in controlling cell growth and in maintaining tissue homeostasis. Connexin 43 (Cx43; also known as GJA1) is the most abundantly expressed gap junction channel protein in humans and acts as a tumor suppressor in multiple tissue types. Cx43 is often dysregulated at the post-translational level during cancer development, resulting in loss of gap junctions. However, the molecular basis underlying the aberrant regulation of Cx43 in cancer cells has remained elusive. Here, we demonstrate that the oncogenic E3 ubiquitin ligase NEDD4 regulates the Cx43 protein level in HeLa cells, both under basal conditions and in response to protein kinase C activation. Furthermore, overexpression of NEDD4, but not a catalytically inactive form of NEDD4, was found to result in nearly complete loss of gap junctions and increased lysosomal degradation of Cx43 in both HeLa and C33A cervical carcinoma cells. Collectively, the data provide new insights into the molecular basis underlying the regulation of gap junction size and represent the first evidence that an oncogenic E3 ubiquitin ligase promotes loss of gap junctions and Cx43 degradation in human carcinoma cells. © 2017. Published by The Company of Biologists Ltd.

Thrombin Induces Inositol Trisphosphate-Mediated Spatially Extensive Responses in Lung Microvessels.

PubMed

Escue, Rachel; Kandasamy, Kathirvel; Parthasarathi, Kaushik

2017-04-01

Activation of plasma membrane receptors initiates compartmentalized second messenger signaling. Whether this compartmentalization facilitates the preferential intercellular diffusion of specific second messengers is unclear. Toward this, the receptor-mediated agonist, thrombin, was instilled into microvessels in a restricted region of isolated blood-perfused mouse lungs. Subsequently, the thrombin-induced increase in endothelial F-actin was determined using confocal fluorescence microscopy. Increased F-actin was evident in microvessels directly treated with thrombin and in those located in adjoining thrombin-free regions. This increase was abrogated by inhibiting inositol trisphosphate-mediated calcium release with Xestospongin C (XeC). XeC also inhibited the thrombin-induced increase in the amplitude of endothelial cytosolic Ca 2+ oscillations. Instillation of thrombin and XeC into adjacent restricted regions increased F-actin in microvessels in the thrombin-treated and adjacent regions but not in those in the XeC-treated region. Thus, inositol trisphosphate, and not calcium, diffused interendothelially to the spatially remote thrombin-free microvessels. Thus, activation of plasma membrane receptors increased the ambit of inflammatory responses via a second messenger different from that used by stimuli that induce cell-wide increases in second messengers. Thrombin however failed to induce the spatially extensive response in microvessels of mice lacking endothelial connexin43, suggesting a role for connexin43 gap junctions. Compartmental second messenger signaling and interendothelial communication define the specific second messenger involved in exacerbating proinflammatory responses to receptor-mediated agonists. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Coronary Heart Disease Alters Intercellular Communication by Modifying Microparticle-Mediated MicroRNA Transport

PubMed Central

Finn, Nnenna A.; Eapen, Danny; Manocha, Pankaj; Kassem, Hatem Al; Lassegue, Bernard; Ghasemzadeh, Nima; Quyyumi, Arshed; Searles, Charles D.

2013-01-01

Coronary heart disease (CHD) is characterized by abnormal intercellular communication and circulating microRNAs (miRNAs) are likely involved in this process. Here, we show that CHD was associated with changes in the transport of circulating miRNA, particularly decreased miRNA enrichment in microparticles (MPs). Additionally, MPs from CHD patients were less efficient at transferring miRNA to cultured HUVECs, which correlated with their diminished capacity to bind developmental endothelial locus-1 (Del-1). In summary, CHD was associated with distinct changes in circulating miRNA transport and these changes may contribute to the abnormal intercellular communication that underlies CHD initiation and progression. PMID:24042051

Stress- and Rho-activated ZO-1–associated nucleic acid binding protein binding to p21 mRNA mediates stabilization, translation, and cell survival

PubMed Central

Nie, Mei; Balda, Maria S.; Matter, Karl

2012-01-01

A central component of the cellular stress response is p21WAF1/CIP1, which regulates cell proliferation, survival, and differentiation. Inflammation and cell stress often up-regulate p21 posttranscriptionally by regulatory mechanisms that are poorly understood. ZO-1–associated nucleic acid binding protein (ZONAB)/DbpA is a Y-box transcription factor that is regulated by components of intercellular junctions that are affected by cytokines and tissue damage. We therefore asked whether ZONAB activation is part of the cellular stress response. Here, we demonstrate that ZONAB promotes cell survival in response to proinflammatory, hyperosmotic, and cytotoxic stress and that stress-induced ZONAB activation involves the Rho regulator GEF-H1. Unexpectedly, stress-induced ZONAB activation does not stimulate ZONAB’s activity as a transcription factor but leads to the posttranscriptional up-regulation of p21 protein and mRNA. Up-regulation is mediated by ZONAB binding to specific sites in the 3′-untranslated region of the p21 mRNA, resulting in mRNA stabilization and enhanced translation. Binding of ZONAB to mRNA is activated by GEF-H1 via Rho stimulation and also mediates Ras-induced p21 expression. We thus identify a unique type of stress and Rho signaling activated pathway that drives mRNA stabilization and translation and links the cellular stress response to p21 expression and cell survival. PMID:22711822

Gap Junction Intercellular Communication in Bone Marrow Failure

DTIC Science & Technology

2012-10-01

enzyme systems, making individuals with these syndromes highly sensitive to DNA-damaging events. However, researchers suspect that modifier genes or...associated with a single gene defect. A major example of the progress in this area is Fanconi Anemia (FA), where mutations in up to 15 different...proteins have been associated to this disease, being FA-A the most frequent (1, 2). Single mutated genes in the DNA repair or ribosome biogenesis of HSC

Regulation of Glutathione in a Rat Diploid Hepatic Epithelial Cell Line

DTIC Science & Technology

1990-06-01

supporting the contention that they are not pre-neoplastic (60). Metabolic cooperation by gap- junctional intercellular communication has been demonstrated...counted. The resulting population statistics allowed calculation and display of cycle-specific cell characteristics and compartment transit times (see...was repeated in chinese hamster V79 cells to see if the effect is idiosyncratic. It is not - V79 cells respond to CYC in the same fashion as WB344(s) if

A Dominant Loss-of-Function GJA1 (Cx43) Mutant Impairs Parturition in the Mouse1

PubMed Central

Tong, Dan; Lu, Xuerong; Wang, Hong-Xing; Plante, Isabelle; Lui, Ed; Laird, Dale W.; Bai, Donglin; Kidder, Gerald M.

2009-01-01

Expression of GJA1 (commonly known as connexin43 or Cx43), a major myometrial gap junction protein, is upregulated before the onset of delivery, suggesting an essential role for Cx43-mediated gap junctional intercellular communication (GJIC) in normal uterine contraction during parturition. To determine how a disease-linked Cx43 mutation affects myometrial function, we studied a mutant mouse model carrying an autosomal dominant mutation (Gja1Jrt) in the gene encoding Cx43 that displays features of the human genetic disease oculodentodigital dysplasia. We found that Cx43 level, specifically the phosphorylated species of the protein, is significantly reduced in the myometrium of the mutant mice (Gja1Jrt/+), as revealed by Western blotting and immunostaining. Patch-clamp electrophysiological measurements demonstrated that coupling between myometrial smooth muscle cells is reduced to <15% of wild-type, indicating that the mutant protein acts dominantly on its wild-type counterpart. The phosphorylated species of Cx43 in the mutant myometrium failed to increase prior to parturition as well as in response to exogenous estrogen. Correspondingly, in vitro experiments with uterine strips revealed weaker contraction of the mutant myometrium and reduced responsiveness to oxytocin, providing an explanation for the prolonged gestation and presence of suffocated fetuses in the uteri that were observed in some of the mutant mice. We conclude that the Gja1Jrt mutation has a dominant-negative effect on Cx43 function in the myometrium, severely reducing GJIC, leading to impaired parturition. PMID:19176884

A correlation of long term effects and radiation quality in the progeny of bystander cells after microbeam radiations: The experimental study of radiotherapy for cancer risk mitigation

NASA Astrophysics Data System (ADS)

Autsavapromporn, N.; Konishi, T.; Liu, C.; Plante, I.; Funayama, T.; Usami, N.; Azzam, EI; Suzuki, M.

2017-06-01

The goal of this study is to investigate the role of radiation quality and gap junction intercellular communication (GJIC) in the propagation of delayed stressful effects in the progeny of bystander human skin fibroblasts cultures (NB1RGB). Briefly, confluent NB1RGB cells in the presence and absence of gap junction inhibitor (AGA) were exposed to ionizing radiation (IR) with a different linear energy transfer (LET) either 5.35 keV X rays (LET ∼6 keV/μm) or 18.3 MeV/u carbon (LET ∼103 keV/μm) microbeam radiations. Following 20 populations post-irradiation, the progeny of bystander NB1RGB cells were harvested and assayed for several of biological endpoints. Our results showed that expression of stressful effects in the progeny of bystander cells is dependent on LET. The progeny of bystander cells exposed to low-LET X rays showed the persistence of oxidative stress and it was correlated with the increased mutant fraction. Such effect were not observed after high-LET carbon ions. Interestingly, inhibition of GJIC mitigated the toxic effects in the progeny of bystander cells. Together, the results contribute to the understanding of the fundamental radiation biology relating to the high-LET carbon ions to mitigate cancer risk after radiotherapy. Furthermore, GJIC be considered as a critical mediator in the bystander mutagenic effect.

150(th) anniversary series: Desmosomes and autoimmune disease, perspective of dynamic desmosome remodeling and its impairments in pemphigus.

PubMed

Kitajima, Yasuo

2014-12-01

Desmosomes are the most important intercellular adhering junctions that adhere two adjacent keratinocytes directly with desmosomal cadherins, that is, desmogleins (Dsgs) and desmocollins, forming an epidermal sheet. Recently, two cell-cell adhesion states of desmosomes, that is, "stable hyper-adhesion" and "dynamic weak-adhesion" conditions have been recognized. They are mutually reversible through cell signaling events involving protein kinase C (PKC), Src and epidermal growth factor receptor (EGFR) during Ca(2+)-switching and wound healing. This remodeling is impaired in pemphigus vulgaris (PV, an autoimmune blistering disease), caused by anti-Dsg3 antibodies. The antibody binding to Dsg3 activates PKC, Src and EGFR, linked to generation of dynamic weak-adhesion desmosomes, followed by p38MAPK-mediated endocytosis of Dsg3, resulting in the specific depletion of Dsg3 from desmosomes and acantholysis. A variety of pemphigus outside-in signaling may explain different clinical (non-inflammatory, inflammatory, and necrolytic) types of pemphigus. Pemphigus could be referred to a "desmosome-remodeling disease involving pemphigus IgG-activated outside-in signaling events".

Fluorescence recovery after photobleaching reveals regulation and distribution of connexin36 gap junction coupling within mouse islets of Langerhans

PubMed Central

Farnsworth, Nikki L; Hemmati, Alireza; Pozzoli, Marina; Benninger, Richard K P

2014-01-01

The pancreatic islets are central to the maintenance of glucose homeostasis through insulin secretion. Glucose-stimulated insulin secretion is tightly linked to electrical activity in β cells within the islet. Gap junctions, composed of connexin36 (Cx36), form intercellular channels between β cells, synchronizing electrical activity and insulin secretion. Loss of gap junction coupling leads to altered insulin secretion dynamics and disrupted glucose homeostasis. Gap junction coupling is known to be disrupted in mouse models of pre-diabetes. Although approaches to measure gap junction coupling have been devised, they either lack cell specificity, suitable quantification of coupling or spatial resolution, or are invasive. The purpose of this study was to develop fluorescence recovery after photobleaching (FRAP) as a technique to accurately and robustly measure gap junction coupling in the islet. The cationic dye Rhodamine 123 was used with FRAP to quantify dye diffusion between islet β cells as a measure of Cx36 gap junction coupling. Measurements in islets with reduced Cx36 verified the accuracy of this technique in distinguishing between distinct levels of gap junction coupling. Analysis of individual cells revealed that the distribution of coupling across the islet is highly heterogeneous. Analysis of several modulators of gap junction coupling revealed glucose- and cAMP-dependent modulation of gap junction coupling in islets. Finally, FRAP was used to determine cell population specific coupling, where no functional gap junction coupling was observed between α cells and β cells in the islet. The results of this study show FRAP to be a robust technique which provides the cellular resolution to quantify the distribution and regulation of Cx36 gap junction coupling in specific cell populations within the islet. Future studies utilizing this technique may elucidate the role of gap junction coupling in the progression of diabetes and identify mechanisms of gap junction regulation for potential therapies. PMID:25172942

Fluorescence recovery after photobleaching reveals regulation and distribution of connexin36 gap junction coupling within mouse islets of Langerhans.

PubMed

Farnsworth, Nikki L; Hemmati, Alireza; Pozzoli, Marina; Benninger, Richard K P

2014-10-15

The pancreatic islets are central to the maintenance of glucose homeostasis through insulin secretion. Glucose‐stimulated insulin secretion is tightly linked to electrical activity in β cells within the islet. Gap junctions, composed of connexin36 (Cx36), form intercellular channels between β cells, synchronizing electrical activity and insulin secretion. Loss of gap junction coupling leads to altered insulin secretion dynamics and disrupted glucose homeostasis. Gap junction coupling is known to be disrupted in mouse models of pre‐diabetes. Although approaches to measure gap junction coupling have been devised, they either lack cell specificity, suitable quantification of coupling or spatial resolution, or are invasive. The purpose of this study was to develop fluorescence recovery after photobleaching (FRAP) as a technique to accurately and robustly measure gap junction coupling in the islet. The cationic dye Rhodamine 123 was used with FRAP to quantify dye diffusion between islet β cells as a measure of Cx36 gap junction coupling. Measurements in islets with reduced Cx36 verified the accuracy of this technique in distinguishing between distinct levels of gap junction coupling. Analysis of individual cells revealed that the distribution of coupling across the islet is highly heterogeneous. Analysis of several modulators of gap junction coupling revealed glucose‐ and cAMP‐dependent modulation of gap junction coupling in islets. Finally, FRAP was used to determine cell population specific coupling, where no functional gap junction coupling was observed between α cells and β cells in the islet. The results of this study show FRAP to be a robust technique which provides the cellular resolution to quantify the distribution and regulation of Cx36 gap junction coupling in specific cell populations within the islet. Future studies utilizing this technique may elucidate the role of gap junction coupling in the progression of diabetes and identify mechanisms of gap junction regulation for potential therapies.

Heterotypic gap junctions at glutamatergic mixed synapses are abundant in goldfish brain

PubMed Central

Rash, John E.; Kamasawa, Naomi; Vanderpool, Kimberly G.; Yasumura, Thomas; O'Brien, John; Nannapaneni, Srikant; Pereda, Alberto E.; Nagy, James I.

2014-01-01

Gap junctions provide for direct intercellular electrical and metabolic coupling. The abundance of gap junctions at “large myelinated club ending” synapses on Mauthner cells of the teleost brain provided a convenient model to correlate anatomical and physiological properties of electrical synapses. There, presynaptic action potentials were found to evoke short-latency electrical “pre-potentials” immediately preceding their accompanying glutamate-induced depolarizations, making these the first unambiguously identified “mixed” (i.e., chemical plus electrical) synapses in the vertebrate CNS. We recently showed that gap junctions at these synapses exhibit asymmetric electrical resistance (i.e., electrical rectification), which we correlated with total molecular asymmetry of connexin composition in their apposing gap junction hemiplaques, with Cx35 restricted to axon terminal hemiplaques and Cx34.7 restricted to apposing Mauthner cell plasma membranes. We now show that similarly heterotypic neuronal gap junctions are abundant throughout goldfish brain, with labeling exclusively for Cx35 in presynaptic hemiplaques and exclusively for Cx34.7 in postsynaptic hemiplaques. Moreover, the vast majority of these asymmetric gap junctions occur at glutamatergic axon terminals. The widespread distribution of heterotypic gap junctions at glutamatergic mixed synapses throughout goldfish brain and spinal cord implies that pre- vs. postsynaptic asymmetry at electrical synapses evolved early in the chordate lineage. We propose that the advantages of the molecular and functional asymmetry of connexins at electrical synapses that are so prominently expressed in the teleost CNS are unlikely to have been abandoned in higher vertebrates. However, to create asymmetric coupling in mammals, where most gap junctions are composed of Cx36 on both sides, would require some other mechanism, such as differential phosphorylation of connexins on opposite sides of the same gap junction or on asymmetric differences in the complement of their scaffolding and regulatory proteins. PMID:25451276

Dilated intercellular spaces as a marker of GERD.

PubMed

Orlando, Lori A; Orlando, Roy C

2009-06-01

Gastroesophageal reflux disease (GERD) is typically heralded by the substernal burning pain of heartburn. On endoscopic examination, about one third of GERD subjects with heartburn have erosive disease, and the remainder have nonerosive reflux disease (NERD). Unlike patients with erosive disease, those with NERD (approximately 50%) often do not respond to therapy with proton pump inhibitors (PPIs), raising the question of whether they have NERD and, if they do, whether the cause of their symptoms is similar to those who respond to PPIs. Recently, biopsies established that subjects with heartburn and PPI-responsive NERD, like those with erosive esophagitis, have lesions within the esophageal epithelium known as dilated intercellular space (DIS). In this article, we discuss the physicochemical basis for DIS in acid-injured esophageal epithelium and its significance in GERD. Although DIS is not pathognomic of GERD, it is a marker of a break in the epithelial (junctional) barrier reflecting an increase in paracellular permeability.

The gap junction channel protein connexin 43 is covalently modified and regulated by SUMOylation.

PubMed

Kjenseth, Ane; Fykerud, Tone A; Sirnes, Solveig; Bruun, Jarle; Yohannes, Zeremariam; Kolberg, Matthias; Omori, Yasufumi; Rivedal, Edgar; Leithe, Edward

2012-05-04

SUMOylation is a posttranslational modification in which a member of the small ubiquitin-like modifier (SUMO) family of proteins is conjugated to lysine residues in specific target proteins. Most known SUMOylation target proteins are located in the nucleus, but there is increasing evidence that SUMO may also be a key determinant of many extranuclear processes. Gap junctions consist of arrays of intercellular channels that provide direct transfer of ions and small molecules between adjacent cells. Gap junction channels are formed by integral membrane proteins called connexins, of which the best-studied isoform is connexin 43 (Cx43). Here we show that Cx43 is posttranslationally modified by SUMOylation. The data suggest that the SUMO system regulates the Cx43 protein level and the level of functional Cx43 gap junctions at the plasma membrane. Cx43 was found to be modified by SUMO-1, -2, and -3. Evidence is provided that the membrane-proximal lysines at positions 144 and 237, located in the Cx43 intracellular loop and C-terminal tail, respectively, act as SUMO conjugation sites. Mutations of lysine 144 or lysine 237 resulted in reduced Cx43 SUMOylation and reduced Cx43 protein and gap junction levels. Altogether, these data identify Cx43 as a SUMOylation target protein and represent the first evidence that gap junctions are regulated by the SUMO system.

CLASP2 interacts with p120-catenin and governs microtubule dynamics at adherens junctions

PubMed Central

Shahbazi, Marta N.; Megias, Diego; Epifano, Carolina; Akhmanova, Anna; Gundersen, Gregg G.; Fuchs, Elaine

2013-01-01

Classical cadherins and their connections with microtubules (MTs) are emerging as important determinants of cell adhesion. However, the functional relevance of such interactions and the molecular players that contribute to tissue architecture are still emerging. In this paper, we report that the MT plus end–binding protein CLASP2 localizes to adherens junctions (AJs) via direct interaction with p120-catenin (p120) in primary basal mouse keratinocytes. Reductions in the levels of p120 or CLASP2 decreased the localization of the other protein to cell–cell contacts and altered AJ dynamics and stability. These features were accompanied by decreased MT density and altered MT dynamics at intercellular junction sites. Interestingly, CLASP2 was enriched at the cortex of basal progenitor keratinocytes, in close localization to p120. Our findings suggest the existence of a new mechanism of MT targeting to AJs with potential functional implications in the maintenance of proper cell–cell adhesion in epidermal stem cells. PMID:24368809

p120 catenin associates with kinesin and facilitates the transport of cadherin–catenin complexes to intercellular junctions

PubMed Central

Chen, Xinyu; Kojima, Shin-ichiro; Borisy, Gary G.; Green, Kathleen J.

2003-01-01

p120 catenin (p120) is a component of adherens junctions and has been implicated in regulating cadherin-based cell adhesion as well as the activity of Rho small GTPases, but its exact roles in cell–cell adhesion are unclear. Using time-lapse imaging, we show that p120-GFP associates with vesicles and exhibits unidirectional movements along microtubules. Furthermore, p120 forms a complex with kinesin heavy chain through the p120 NH2-terminal head domain. Overexpression of p120, but not an NH2-terminal deletion mutant deficient in kinesin binding, recruits endogenous kinesin to N-cadherin. Disruption of the interaction between N-cadherin and p120, or the interaction between p120 and kinesin, leads to a delayed accumulation of N-cadherin at cell–cell contacts during calcium-initiated junction reassembly. Our analyses identify a novel role of p120 in promoting cell surface trafficking of cadherins via association and recruitment of kinesin. PMID:14610057

Coronary heart disease alters intercellular communication by modifying microparticle-mediated microRNA transport.

PubMed

Finn, Nnenna A; Eapen, Danny; Manocha, Pankaj; Al Kassem, Hatem; Lassegue, Bernard; Ghasemzadeh, Nima; Quyyumi, Arshed; Searles, Charles D

2013-11-01

Coronary heart disease (CHD) is characterized by abnormal intercellular communication and circulating microRNAs (miRNAs) are likely involved in this process. Here, we show that CHD was associated with changes in the transport of circulating miRNA, particularly decreased miRNA enrichment in microparticles (MPs). Additionally, MPs from CHD patients were less efficient at transferring miRNA to cultured HUVECs, which correlated with their diminished capacity to bind developmental endothelial locus-1 (Del-1). In summary, CHD was associated with distinct changes in circulating miRNA transport and these changes may contribute to the abnormal intercellular communication that underlies CHD initiation and progression. Published by Elsevier B.V.

Soyasaponins prevent H₂O₂-induced inhibition of gap junctional intercellular communication by scavenging reactive oxygen species in rat liver cells.

PubMed

Chen, Jiading; Sun, Suxia; Zha, Dingsheng; Wu, Jiguo; Mao, Limei; Deng, Hong; Chu, Xinwei; Luo, Haiji; Zha, Longying

2014-01-01

It appears to be more practical and effective to prevent carcinogenesis by targeting the tumor promotion stage. Gap junctional intercellular communication (GJIC) is strongly involved in carcinogenesis, especially the tumor promotion stage. Considerable interest has been focused on the chemoprevention activities of soyasaponin (SS), which are major phytochemicals found in soybeans and soy products. However, less is known about the preventive effects of SS (especially SS with different chemical structures) against tumor promoter-induced inhibition of GJIC. We investigated the protective effects of SS-A1, SS-A2, and SS-I against hydrogen peroxide (H2O2)-induced GJIC inhibition and reactive oxygen species (ROS) production in Buffalo rat liver (BRL) cells. The present results clearly show for the first time that SS-A1, SS-A2, and SS-I prevent the H2O2-induced GJIC inhibition by scavenging ROS in BRL cells in a dose-dependent manner at the concentration range of from 25 to 100 μg/mL. Soyasaponins attenuated the H2O2-induced ROS through potentiating the activities of superoxide dismutase and glutathione peroxidase. This may be an important mechanism by which SS protects against tumor promotion. In addition, various chemical structures of SS appear to exhibit different protective abilities against GJIC inhibition. This may partly attribute to their differences in ROS-scavenging activities.

The desmosome and pemphigus

PubMed Central

2008-01-01

Desmosomes are patch-like intercellular adhering junctions (“maculae adherentes”), which, in concert with the related adherens junctions, provide the mechanical strength to intercellular adhesion. Therefore, it is not surprising that desmosomes are abundant in tissues subjected to significant mechanical stress such as stratified epithelia and myocardium. Desmosomal adhesion is based on the Ca2+-dependent, homo- and heterophilic transinteraction of cadherin-type adhesion molecules. Desmosomal cadherins are anchored to the intermediate filament cytoskeleton by adaptor proteins of the armadillo and plakin families. Desmosomes are dynamic structures subjected to regulation and are therefore targets of signalling pathways, which control their molecular composition and adhesive properties. Moreover, evidence is emerging that desmosomal components themselves take part in outside-in signalling under physiologic and pathologic conditions. Disturbed desmosomal adhesion contributes to the pathogenesis of a number of diseases such as pemphigus, which is caused by autoantibodies against desmosomal cadherins. Beside pemphigus, desmosome-associated diseases are caused by other mechanisms such as genetic defects or bacterial toxins. Because most of these diseases affect the skin, desmosomes are interesting not only for cell biologists who are inspired by their complex structure and molecular composition, but also for clinical physicians who are confronted with patients suffering from severe blistering skin diseases such as pemphigus. To develop disease-specific therapeutic approaches, more insights into the molecular composition and regulation of desmosomes are required. PMID:18386043

Monitoring gap junctional communication in astrocytes from acute adult mouse brain slices using the gap-FRAP technique.

PubMed

Yi, Chenju; Teillon, Jérémy; Koulakoff, Annette; Berry, Hugues; Giaume, Christian

2018-06-01

Intercellular communication through gap junction channels plays a key role in cellular homeostasis and in synchronizing physiological functions, a feature that is modified in number of pathological situations. In the brain, astrocytes are the cell population that expresses the highest amount of gap junction proteins, named connexins. Several techniques have been used to assess the level of gap junctional communication in astrocytes, but so far they remain very difficult to apply in adult brain tissue. Here, using specific loading of astrocytes with sulforhodamine 101, we adapted the gap-FRAP (Fluorescence Recovery After Photobleaching) to acute hippocampal slices from 9 month-old adult mice. We show that gap junctional communication monitored in astrocytes with this technique was inhibited either by pharmacological treatment with a gap junctional blocker or in mice lacking the two main astroglial connexins, while a partial inhibition was measured when only one connexin was knocked-out. We validate this approach using a mathematical model of sulforhodamine 101 diffusion in an elementary astroglial network and a quantitative analysis of the exponential fits to the fluorescence recovery curves. Consequently, we consider that the adaptation of the gap-FRAP technique to acute brain slices from adult mice provides an easy going and valuable approach that allows overpassing this age-dependent obstacle and will facilitate the investigation of gap junctional communication in adult healthy or pathological brain. Copyright © 2018 Elsevier B.V. All rights reserved.

A structural and functional comparison of gap junction channels composed of connexins and innexins

PubMed Central

Williams, Jamal B.

2016-01-01

ABSTRACT Methods such as electron microscopy and electrophysiology led to the understanding that gap junctions were dense arrays of channels connecting the intracellular environments within almost all animal tissues. The characteristics of gap junctions were remarkably similar in preparations from phylogenetically diverse animals such as cnidarians and chordates. Although few studies directly compared them, minor differences were noted between gap junctions of vertebrates and invertebrates. For instance, a slightly wider gap was noted between cells of invertebrates and the spacing between invertebrate channels was generally greater. Connexins were identified as the structural component of vertebrate junctions in the 1980s and innexins as the structural component of pre‐chordate junctions in the 1990s. Despite a lack of similarity in gene sequence, connexins and innexins are remarkably similar. Innexins and connexins have the same membrane topology and form intercellular channels that play a variety of tissue‐ and temporally specific roles. Both protein types oligomerize to form large aqueous channels that allow the passage of ions and small metabolites and are regulated by factors such as pH, calcium, and voltage. Much more is currently known about the structure, function, and structure–function relationships of connexins. However, the innexin field is expanding. Greater knowledge of innexin channels will permit more detailed comparisons with their connexin‐based counterparts, and provide insight into the ubiquitous yet specific roles of gap junctions. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 522–547, 2017 PMID:27582044

A structural and functional comparison of gap junction channels composed of connexins and innexins.

PubMed

Skerrett, I Martha; Williams, Jamal B

2017-05-01

Methods such as electron microscopy and electrophysiology led to the understanding that gap junctions were dense arrays of channels connecting the intracellular environments within almost all animal tissues. The characteristics of gap junctions were remarkably similar in preparations from phylogenetically diverse animals such as cnidarians and chordates. Although few studies directly compared them, minor differences were noted between gap junctions of vertebrates and invertebrates. For instance, a slightly wider gap was noted between cells of invertebrates and the spacing between invertebrate channels was generally greater. Connexins were identified as the structural component of vertebrate junctions in the 1980s and innexins as the structural component of pre-chordate junctions in the 1990s. Despite a lack of similarity in gene sequence, connexins and innexins are remarkably similar. Innexins and connexins have the same membrane topology and form intercellular channels that play a variety of tissue- and temporally specific roles. Both protein types oligomerize to form large aqueous channels that allow the passage of ions and small metabolites and are regulated by factors such as pH, calcium, and voltage. Much more is currently known about the structure, function, and structure-function relationships of connexins. However, the innexin field is expanding. Greater knowledge of innexin channels will permit more detailed comparisons with their connexin-based counterparts, and provide insight into the ubiquitous yet specific roles of gap junctions. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 522-547, 2017. © 2016 The Authors Developmental Neurobiology Published by Wiley Periodicals, Inc.

Actin-interacting protein 1 controls assembly and permeability of intestinal epithelial apical junctions

PubMed Central

Baranwal, Somesh

2015-01-01

Adherens junctions (AJs) and tight junctions (TJs) are crucial regulators of the integrity and restitution of the intestinal epithelial barrier. The structure and function of epithelial junctions depend on their association with the cortical actin cytoskeleton that, in polarized epithelial cells, is represented by a prominent perijunctional actomyosin belt. The assembly and stability of the perijunctional cytoskeleton is controlled by constant turnover (disassembly and reassembly) of actin filaments. Actin-interacting protein (Aip) 1 is an emerging regulator of the actin cytoskeleton, playing a critical role in filament disassembly. In this study, we examined the roles of Aip1 in regulating the structure and remodeling of AJs and TJs in human intestinal epithelium. Aip1 was enriched at apical junctions in polarized human intestinal epithelial cells and normal mouse colonic mucosa. Knockdown of Aip1 by RNA interference increased the paracellular permeability of epithelial cell monolayers, decreased recruitment of AJ/TJ proteins to steady-state intercellular contacts, and attenuated junctional reassembly in a calcium-switch model. The observed defects of AJ/TJ structure and functions were accompanied by abnormal organization and dynamics of the perijunctional F-actin cytoskeleton. Moreover, loss of Aip1 impaired the apico-basal polarity of intestinal epithelial cell monolayers and inhibited formation of polarized epithelial cysts in 3-D Matrigel. Our findings demonstrate a previously unanticipated role of Aip1 in regulating the structure and remodeling of intestinal epithelial junctions and early steps of epithelial morphogenesis. PMID:25792565

Actin-interacting protein 1 controls assembly and permeability of intestinal epithelial apical junctions.

PubMed

Lechuga, Susana; Baranwal, Somesh; Ivanov, Andrei I

2015-05-01

Adherens junctions (AJs) and tight junctions (TJs) are crucial regulators of the integrity and restitution of the intestinal epithelial barrier. The structure and function of epithelial junctions depend on their association with the cortical actin cytoskeleton that, in polarized epithelial cells, is represented by a prominent perijunctional actomyosin belt. The assembly and stability of the perijunctional cytoskeleton is controlled by constant turnover (disassembly and reassembly) of actin filaments. Actin-interacting protein (Aip) 1 is an emerging regulator of the actin cytoskeleton, playing a critical role in filament disassembly. In this study, we examined the roles of Aip1 in regulating the structure and remodeling of AJs and TJs in human intestinal epithelium. Aip1 was enriched at apical junctions in polarized human intestinal epithelial cells and normal mouse colonic mucosa. Knockdown of Aip1 by RNA interference increased the paracellular permeability of epithelial cell monolayers, decreased recruitment of AJ/TJ proteins to steady-state intercellular contacts, and attenuated junctional reassembly in a calcium-switch model. The observed defects of AJ/TJ structure and functions were accompanied by abnormal organization and dynamics of the perijunctional F-actin cytoskeleton. Moreover, loss of Aip1 impaired the apico-basal polarity of intestinal epithelial cell monolayers and inhibited formation of polarized epithelial cysts in 3-D Matrigel. Our findings demonstrate a previously unanticipated role of Aip1 in regulating the structure and remodeling of intestinal epithelial junctions and early steps of epithelial morphogenesis. Copyright © 2015 the American Physiological Society.

Physiological Role of Gap-Junctional Hemichannels

PubMed Central

Quist, Arjan Pieter; Rhee, Seung Keun; Lin, Hai; Lal, Ratneshwar

2000-01-01

Hemichannels in the overlapping regions of apposing cells plasma membranes join to form gap junctions and provide an intercellular communication pathway. Hemichannels are also present in the nonjunctional regions of individual cells and their activity is gated by several agents, including calcium. However, their physiological roles are unknown. Using techniques of atomic force microscopy (AFM), fluorescent dye uptake assay, and laser confocal immunofluorescence imaging, we have examined the extracellular calcium-dependent modulation of cell volume. In response to a change in the extracellular physiological calcium concentration (1.8 to ≤1.6 mM) in an otherwise isosmotic condition, real-time AFM imaging revealed a significant and reversible increase in the volume of cells expressing gap-junctional proteins (connexins). Volume change did not occur in cells that were not expressing connexins. However, after the transient or stable transfection of connexin43, volume change did occur. The volume increase was accompanied by cytochalasin D-sensitive higher cell stiffness, which helped maintain cell integrity. These cellular physical changes were prevented by gap-junctional blockers, oleamide and β-glycyrrhetinic acid, or were reversed by returning extracellular calcium to the normal level. We conclude that nongap-junctional hemichannels regulate cell volume in response to the change in extracellular physiological calcium in an otherwise isosmotic situation. PMID:10704454

Junctional complexes in the inner cyst tissue of the cysticercoid of Hymenolepis diminuta (Cestoda).

PubMed

Richards, K S; Arme, C

1983-10-01

The inner cyst tissue development is anteriad and centripetal. The cells produce lamellar extensions which assume parallel alignment. The first contact points (approximately 4 days post-infection) establish heptalaminar (gap) junctions. Lamellar attenuation results in a decreased intercellular space, and at 5-6 days pentalaminar junctions (with fused outer plasmalemma leaflets to give an electron-dense, approximately 3 nm wide O-O line) occur. This is the first maturation (M1) stage. The O-O lines are permeable to lanthanum, and evidence of their possible transformation from heptalaminar junctions is presented. Continued lamellar attenuation, associated with scolex retraction and subsequent growth, results in cytoplasmic occlusion and contact between the inner leaflets of the same lamella. The resultant electron-dense I-I line is approximately 3 nm wide; the O-O line is now less electron-dense and thinner (approximately 2 nm). This final maturation (M2) stage, resembling vertebrate myelin, occurs over limited areas; closely adjacent regions either remaining at the M1 stage, or not displaying junctional complexes. Since in vivo and in vitro excystment can occur before the M2 stage, the inner cyst tissue is not considered to be protective against the definitive host. That the tissue may function in limiting nutrient flow, thus regulating the size of the presumptive adult, is discussed.

The role of exosomes in hepatitis, liver cirrhosis and hepatocellular carcinoma.

PubMed

Shen, Jiliang; Huang, Chiung-Kuei; Yu, Hong; Shen, Bo; Zhang, Yaping; Liang, Yuelong; Li, Zheyong; Feng, Xu; Zhao, Jie; Duan, Lian; Cai, Xiujun

2017-05-01

Exosomes are small vesicles that were initially thought to be a mechanism for discarding unneeded membrane proteins from reticulocytes. Their mediation of intercellular communication appears to be associated with several biological functions. Current studies have shown that most mammalian cells undergo the process of exosome formation and utilize exosome-mediated cell communication. Exosomes contain various microRNAs, mRNAs and proteins. They have been reported to mediate multiple functions, such as antigen presentation, immune escape and tumour progression. This concise review highlights the findings regarding the roles of exosomes in liver diseases, particularly hepatitis B, hepatitis C, liver cirrhosis and hepatocellular carcinoma. However, further elucidation of the contributions of exosomes to intercellular information transmission is needed. The potential medical applications of exosomes in liver diseases seem practical and will depend on the ingenuity of future investigators and their insights into exosome-mediated biological processes. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

An update on minding the gap in cancer.

PubMed

Mesnil, Marc; Aasen, Trond; Boucher, Jonathan; Chépied, Amandine; Cronier, Laurent; Defamie, Norah; Kameritsch, Petra; Laird, Dale W; Lampe, Paul D; Lathia, Justin D; Leithe, Edward; Mehta, Parmender P; Monvoisin, Arnaud; Pogoda, Kristin; Sin, Wun-Chey; Tabernero, Arantxa; Yamasaki, Hiroshi; Yeh, Elizabeth S; Dagli, Maria Lucia Zaidan; Naus, Christian C

2018-01-01

This article is a report of the "International Colloquium on Gap junctions: 50Years of Impact on Cancer" that was held 8-9 September 2016, at the Amphitheater "Pôle Biologie Santé" of the University of Poitiers (Poitiers, France). The colloquium was organized by M Mesnil (Université de Poitiers, Poitiers, France) and C Naus (University of British Columbia, Vancouver, Canada) to celebrate the 50th anniversary of the seminal work published in 1966 by Loewenstein and Kanno [Intercellular communication and the control of tissue growth: lack of communication between cancer cells, Nature, 116 (1966) 1248-1249] which initiated studies on the involvement of gap junctions in carcinogenesis. During the colloquium, 15 participants presented reviews or research updates in the field which are summarized below. Copyright © 2017 Elsevier B.V. All rights reserved.

Streptococcus pyogenes translocates across an epithelial barrier.

PubMed

Sumitomo, Tomoko

2017-01-01

Streptococcus pyogenes is a β-hemolytic organism responsible for a wide variety of human diseases that commonly occur as self-limiting purulent diseases of the pharynx and skin. Although the occurrence of invasive infections by S. pyogenes is rare, mortality rates remain high even with progressive medical therapy. As a prerequisite for causing the severe invasive disease, S. pyogenes must invade underlying sterile tissues by translocating across the epithelial barrier. In this study, streptolysin S and SpeB were identified as the novel factors that facilitate bacterial translocation via degradation of intercellular junctions. Furthermore, we found that S. pyogenes exploits host plasminogen for acceleration of bacterial invasion into deeper tissues via tricellular tight junctions. Here, I would like to show our study on bacterial translocation across the epithelial barrier through paracellular route.

Proteomic overview and perspectives of the radiation-induced bystander effects.

PubMed

Chevalier, François; Hamdi, Dounia Houria; Saintigny, Yannick; Lefaix, Jean-Louis

2015-01-01

Radiation proteomics is a recent, promising and powerful tool to identify protein markers of direct and indirect consequences of ionizing radiation. The main challenges of modern radiobiology is to predict radio-sensitivity of patients and radio-resistance of tumor to be treated, but considerable evidences are now available regarding the significance of a bystander effect at low and high doses. This "radiation-induced bystander effect" (RIBE) is defined as the biological responses of non-irradiated cells that received signals from neighboring irradiated cells. Such intercellular signal is no more considered as a minor side-effect of radiotherapy in surrounding healthy tissue and its occurrence should be considered in adapting radiotherapy protocols, to limit the risk for radiation-induced secondary cancer. There is no consensus on a precise designation of RIBE, which involves a number of distinct signal-mediated effects within or outside the irradiated volume. Indeed, several cellular mechanisms were proposed, including the secretion of soluble factors by irradiated cells in the extracellular matrix, or the direct communication between irradiated and neighboring non-irradiated cells via gap junctions. This phenomenon is observed in a context of major local inflammation, linked with a global imbalance of oxidative metabolism which makes its analysis challenging using in vitro model systems. In this review article, the authors first define the radiation-induced bystander effect as a function of radiation type, in vitro analysis protocols, and cell type. In a second time, the authors present the current status of protein biomarkers and proteomic-based findings and discuss the capacities, limits and perspectives of such global approaches to explore these complex intercellular mechanisms. Copyright © 2014 Elsevier B.V. All rights reserved.

Methoxychlor and Vinclozolin Induce Rapid Changes in Intercellular and Intracellular Signaling in Liver Progenitor Cells.

PubMed

Babica, Pavel; Zurabian, Rimma; Kumar, Esha R; Chopra, Rajus; Mianecki, Maxwell J; Park, Joon-Suk; Jaša, Libor; Trosko, James E; Upham, Brad L

2016-09-01

Methoxychlor (MXC) and vinclozolin (VIN) are well-recognized endocrine disrupting chemicals known to alter epigenetic regulations and transgenerational inheritance; however, non-endocrine disruption endpoints are also important. Thus, we determined the effects of MXC and VIN on the dysregulation of gap junctional intercellular communication (GJIC) and activation of mitogen-activated protein kinases (MAPKs) in WB-F344 rat liver epithelial cells. Both chemicals induced a rapid dysregulation of GJIC at non-cytotoxic doses, with 30 min EC50 values for GJIC inhibition being 10 µM for MXC and 126 µM for VIN. MXC inhibited GJIC for at least 24 h, while VIN effects were transient and GJIC recovered after 4 h. VIN induced rapid hyperphosphorylation and internalization of gap junction protein connexin43, and both chemicals also activated MAPK ERK1/2 and p38. Effects on GJIC were not prevented by MEK1/2 inhibitor, but by an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), resveratrol, and in the case of VIN, also, by a p38 inhibitor. Estrogen (ER) and androgen receptor (AR) modulators (estradiol, ICI 182,780, HPTE, testosterone, flutamide, VIN M2) did not attenuate MXC or VIN effects on GJIC. Our data also indicate that the effects were elicited by the parental compounds of MXC and VIN. Our study provides new evidence that MXC and VIN dysregulate GJIC via mechanisms involving rapid activation of PC-PLC occurring independently of ER- or AR-dependent genomic signaling. Such alterations of rapid intercellular and intracellular signaling events involved in regulations of gene expression, tissue development, function and homeostasis, could also contribute to transgenerational epigenetic effects of endocrine disruptors. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Angelica archengelica extract induced perturbation of rat skin and tight junctional protein (ZO-1) of HaCaT cells

PubMed Central

Kaushal, N.; Naz, S.; Tiwary, AK.

2011-01-01

Background and purpose of the study Herbal enhancers compared to the synthetic ones have shown less toxis effects. Coumarins have been shown at concentrations inhibiting phospoliphase C-Y (Phc-Y) are able to enhance tight junction (TJ) permeability due to hyperpoalation of Zonolous Occludense-1 (ZO-1) proteins. The purpose of this study was to evaluate the influence of ethanolic extract of Angelica archengelica (AA-E) which contain coumarin on permeation of repaglinide across rat epidermis and on the tight junction plaque protein ZO-1 in HaCaT cells. Methods Transepidermal water loss (TEWL) from the rat skin treated with different concentrations of AA-E was assessed by Tewameter. Scanning and Transmission Electron Microscopy (TEM) on were performed on AA-E treated rat skin portions. The possibility of AA-E influence on the architecture of tight junctions by adverse effect on the cytoplasmic ZO-1 in HaCaT cells was investigated. Finally, the systemic delivery of repaglinide from the optimized transdermal formulation was investigated in rats. Results The permeation of repaglinide across excised rat epidermis was 7-fold higher in the presence of AA-E (5% w/v) as compared to propylene glycol:ethanol (7:3) mixture. The extract was found to perturb the lipid microconstituents in both excised and viable rat skin, although, the effect was less intense in the later. The enhanced permeation of repaglinide across rat epidermis excised after treatment with AA-E (5% w/v) for different periods was in concordance with the high TEWL values of similarly treated viable rat skin. Further, the observed increase in intercellular space, disordering of lipid structure and corneocyte detachment indicated considerable effect on the ultrastructure of rat epidermis. Treatment of HaCaT cell line with AA-E (0.16% w/v) for 6 hrs influenced ZO-1 as evidenced by reduced immunofluorescence of anti-TJP1 (ZO-1) antibody in Confocal Laser Scanning Microscopy studies (CLSM) studies. The plasma concentration of repaglinide from transdermal formulation was maintained higher and for longer time as compared to oral administration of repaglinide. Major conclusion Results suggest the overwhelming influence of Angelica archengelica in enhancing the percutaneous permeation of repaglinide to be mediated through perturbation of skin lipids and tight junction protein (ZO-1). PMID:22615634

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rivedal, Edgar; Leithe, Edward

Benzene is used at large volumes in many different human activities. Hematotoxicity and cancer-causation as a result of benzene exposure was recognized many years ago, but the mechanisms involved remain unclear. Aberrant regulation of gap junction intercellular communication (GJIC) has been linked to both cancer induction and interference with normal hematopoietic development. We have previously suggested that inhibition of GJIC may play a role in benzene toxicity since benzene metabolites were found to block GJIC, the ring-opened trans,trans-muconaldehyde (MUC) being the most potent metabolite. In the present work we have studied the molecular mechanisms underlying the MUC-induced inhibition of gapmore » junctional communication. We show that MUC induces cross-linking of the gap junction protein connexin43 and that this is likely to be responsible for the induced inhibition of GJIC, as well as the loss of connexin43 observed in Western blots. We also show that glutaraldehyde possesses similar effects as MUC, and we compare the effects to that of formaldehyde. The fact that glutaraldehyde and formaldehyde have been associated with induction of leukemia as well as disturbance of hematopoiesis, strengthens the possible link between the effect of MUC on gap junctions, and the toxic effects of benzene.« less

Intercellular ultrafast Ca2+ wave in vascular smooth muscle cells: numerical and experimental study

NASA Astrophysics Data System (ADS)

Quijano, J. C.; Raynaud, F.; Nguyen, D.; Piacentini, N.; Meister, J. J.

2016-08-01

Vascular smooth muscle cells exhibit intercellular Ca2+ waves in response to local mechanical or KCl stimulation. Recently, a new type of intercellular Ca2+ wave was observed in vitro in a linear arrangement of smooth muscle cells. The intercellular wave was denominated ultrafast Ca2+ wave and it was suggested to be the result of the interplay between membrane potential and Ca2+ dynamics which depended on influx of extracellular Ca2+, cell membrane depolarization and its intercel- lular propagation. In the present study we measured experimentally the conduction velocity of the membrane depolarization and performed simulations of the ultrafast Ca2+ wave along coupled smooth muscle cells. Numerical results reproduced a wide spectrum of experimental observations, including Ca2+ wave velocity, electrotonic membrane depolarization along the network, effects of inhibitors and independence of the Ca2+ wave speed on the intracellular stores. The numerical data also provided new physiological insights suggesting ranges of crucial model parameters that may be altered experimentally and that could significantly affect wave kinetics allowing the modulation of the wave characteristics experimentally. Numerical and experimental results supported the hypothesis that the propagation of membrane depolarization acts as an intercellular messenger mediating intercellular ultrafast Ca2+ waves in smooth muscle cells.

Deciphering the potential efficacy of acetyl-L-carnitine (ALCAR) in maintaining connexin-mediated lenticular homeostasis

PubMed Central

Muralidharan, Arumugam Ramachandran; Leema, George; Annadurai, Thangaraj; Anitha, Thirugnanasambandhar Sivasubramanian; Thomas, Philip A.

2012-01-01

Purpose To determine the putative role of acetyl-L-carnitine (ALCAR) in maintaining normal intercellular communication in the lens through connexin. Methods In the present study, Wistar rat pups were divided into 3 groups of eight each. On postpartum day ten, Group I rat pups received an intraperitoneal injection (50 µl) of 0.89% saline. Rats in Groups II and III received a subcutaneous injection (50 µl) of sodium selenite (19 µmol/kg bodyweight); Group III rat pups also received an intraperitoneal injection of ALCAR (200 mg/kg bodyweight) once daily on postpartum days 9–14. Both eyes of each pup were examined from day 16 up to postpartum day 30. Alterations in the mean activity of the channel pumps, calcium-ATPase and sodium/potassium-ATPase, were determined. The expression of genes encoding key lenticular gap junctions (connexin 46 and connexin 50) and a channel pump (plasma membrane Ca2+-ATPase [PMCA1]) was evaluated by reverse transcription-PCR. Immunoblot analysis was also performed to confirm the differential expression of key lenticular connexin proteins. In addition, bioinformatics analysis was performed to determine the interacting residues of the connexin proteins with ALCAR. Results Significantly lower mean activities of Ca2+-ATPase and Na+/K+ -ATPase were observed in the lenses of Group II rats than those in Group I rat lenses. However, the observed mean activities of Ca2+-ATPase and Na+/K+-ATPase in Group III rat lenses were significantly higher than those in Group II rat lenses. The mean mRNA transcript levels of the connexin 46 and connexin 50 genes were significantly lower, while the mean levels of PMCA1 gene transcripts were significantly higher, in Group II rat lenses than in Group I rat lenses. Immunoblot analysis also confirmed the altered expression of connexin proteins in lysates of whole lenses of Group II rats. However, the expression of connexin 46 and connexin 50 proteins in lenses from group III rats was essentially similar to that noted in lenses from normal (Group I) rats. Hydrogen bond-interaction between ALCAR and amino acid residues at the functional domain regions of connexin 46 and connexin 50 proteins was also demonstrated through bioinformatics tools. Conclusions The results suggest that ALCAR plays a key role in maintaining lenticular homeostasis by promoting gap junctional intercellular communication. PMID:22876134

Deciphering the potential efficacy of acetyl-L-carnitine (ALCAR) in maintaining connexin-mediated lenticular homeostasis.

PubMed

Muralidharan, Arumugam Ramachandran; Leema, George; Annadurai, Thangaraj; Anitha, Thirugnanasambandhar Sivasubramanian; Thomas, Philip A; Geraldine, Pitchairaj

2012-01-01

To determine the putative role of acetyl-L-carnitine (ALCAR) in maintaining normal intercellular communication in the lens through connexin. In the present study, Wistar rat pups were divided into 3 groups of eight each. On postpartum day ten, Group I rat pups received an intraperitoneal injection (50 µl) of 0.89% saline. Rats in Groups II and III received a subcutaneous injection (50 µl) of sodium selenite (19 µmol/kg bodyweight); Group III rat pups also received an intraperitoneal injection of ALCAR (200 mg/kg bodyweight) once daily on postpartum days 9-14. Both eyes of each pup were examined from day 16 up to postpartum day 30. Alterations in the mean activity of the channel pumps, calcium-ATPase and sodium/potassium-ATPase, were determined. The expression of genes encoding key lenticular gap junctions (connexin 46 and connexin 50) and a channel pump (plasma membrane Ca(2+)-ATPase [PMCA1]) was evaluated by reverse transcription-PCR. Immunoblot analysis was also performed to confirm the differential expression of key lenticular connexin proteins. In addition, bioinformatics analysis was performed to determine the interacting residues of the connexin proteins with ALCAR. Significantly lower mean activities of Ca(2+)-ATPase and Na(+)/K(+) -ATPase were observed in the lenses of Group II rats than those in Group I rat lenses. However, the observed mean activities of Ca(2+)-ATPase and Na(+)/K(+)-ATPase in Group III rat lenses were significantly higher than those in Group II rat lenses. The mean mRNA transcript levels of the connexin 46 and connexin 50 genes were significantly lower, while the mean levels of PMCA1 gene transcripts were significantly higher, in Group II rat lenses than in Group I rat lenses. Immunoblot analysis also confirmed the altered expression of connexin proteins in lysates of whole lenses of Group II rats. However, the expression of connexin 46 and connexin 50 proteins in lenses from group III rats was essentially similar to that noted in lenses from normal (Group I) rats. Hydrogen bond-interaction between ALCAR and amino acid residues at the functional domain regions of connexin 46 and connexin 50 proteins was also demonstrated through bioinformatics tools. The results suggest that ALCAR plays a key role in maintaining lenticular homeostasis by promoting gap junctional intercellular communication.

Cytochemical Changes in Hepatocytes of Rats with Endotoxemia and Sepsis: Localization of Fibronectin, Calcium, and Enzymes

DTIC Science & Technology

1988-01-01

rate was deposited predominantly on the outer surfaces of in the pathogn~nesis of endotoxernia and septic shock. The the RER of hepatocytes. in additron...es; and (c) tin it- the basal (perisinusoidal) surfaces and in the cisternae G-6-Pase activity. LPS treatment also leads to reduced num- of tough...by Kupffer cells (Cook et al., 1985’ Gut et al., sue fibronectin results in widening intercellular junctions anci in- 198: ~nkaa ndIwsk,18

Targeted p120-catenin ablation disrupts dental enamel development.

PubMed

Bartlett, John D; Dobeck, Justine M; Tye, Coralee E; Perez-Moreno, Mirna; Stokes, Nicole; Reynolds, Albert B; Fuchs, Elaine; Skobe, Ziedonis

2010-09-16

Dental enamel development occurs in stages. The ameloblast cell layer is adjacent to, and is responsible for, enamel formation. When rodent pre-ameloblasts become tall columnar secretory-stage ameloblasts, they secrete enamel matrix proteins, and the ameloblasts start moving in rows that slide by one another. This movement is necessary to form the characteristic decussating enamel prism pattern. Thus, a dynamic system of intercellular interactions is required for proper enamel development. Cadherins are components of the adherens junction (AJ), and they span the cell membrane to mediate attachment to adjacent cells. p120 stabilizes cadherins by preventing their internalization and degradation. So, we asked if p120-mediated cadherin stability is important for dental enamel formation. Targeted p120 ablation in the mouse enamel organ had a striking effect. Secretory stage ameloblasts detached from surrounding tissues, lost polarity, flattened, and ameloblast E- and N-cadherin expression became undetectable by immunostaining. The enamel itself was poorly mineralized and appeared to be composed of a thin layer of merged spheres that abraded from the tooth. Significantly, p120 mosaic mouse teeth were capable of forming normal enamel demonstrating that the enamel defects were not a secondary effect of p120 ablation. Surprisingly, blood-filled sinusoids developed in random locations around the developing teeth. This has not been observed in other p120-ablated tissues and may be due to altered p120-mediated cell signaling. These data reveal a critical role for p120 in tooth and dental enamel development and are consistent with p120 directing the attachment and detachment of the secretory stage ameloblasts as they move in rows.

Adenovirus Vector E4 Gene Regulates Connexin 40 and 43 Expression in Endothelial Cells via PKA and PI3K Signal Pathways

PubMed Central

Zhang, Fan; Cheng, Joseph; Lam, George; Jin, David K.; Vincent, Loïc; Hackett, Neil R.; Wang, Shiyang; Young, Lauren M.; Hempstead, Barbara; Crystal, Ronald G.; Rafii, Shahin

2010-01-01

Connexins (Cxs) provide a means for intercellular communication and play important roles in the pathophysiology of vascular cardiac diseases. Infection of endothelial cells (ECs) with first-generation E1/E3-deleted E4+ adenovirus (AdE4+) selectively modulates the survival and angiogenic potential of ECs by as of yet unrecognized mechanisms. We show here that AdE4+ vectors potentiate Cx expression in ECs in vitro and in mouse heart tissue. Infection of ECs with AdE4+, but not AdE4−, resulted in a time- and dose-dependent induction of junctional Cx40 expression and suppression of Cx43 protein and mRNA expression. Treatment of ECs with PKA inhibitor H89 or PI3K inhibitor LY294002 prevented the AdE4+-mediated regulation of Cx40 and Cx43 that was associated with diminished AdE4+-mediated survival of ECs. Moreover, both PKA activity and cAMP-response element (CRE)-binding activity were enhanced by treatment of ECs with AdE4+. However, there is no causal evidence of a cross-talk between the 2 modulatory pathways, PKA and PI3K. Remarkably, Cx40 immunostaining was markedly increased and Cx43 was decreased in the heart tissue of mice treated with intratracheal AdE4+. Taken together, these results suggest that AdE4+ may play an important role in the regulation of Cx expression in ECs, and that these effects are mediated by both the PKA/CREB and PI3K signaling pathways. PMID:15831817

The glomerular epithelial cell anti-adhesin podocalyxin associates with the actin cytoskeleton through interactions with ezrin.

PubMed

Orlando, R A; Takeda, T; Zak, B; Schmieder, S; Benoit, V M; McQuistan, T; Furthmayr, H; Farquhar, M G

2001-08-01

During development, renal glomerular epithelial cells (podocytes) undergo extensive morphologic changes necessary for creation of the glomerular filtration apparatus. These changes include formation of interdigitating foot processes, replacement of tight junctions with slit diaphragms, and the concomitant opening of intercellular urinary spaces. It was postulated previously and confirmed recently that podocalyxin, a sialomucin, plays a major role in maintaining the urinary space open by virtue of the physicochemical properties of its highly negatively charged ectodomain. This study examined whether the highly conserved cytoplasmic tail of podocalyxin also contributes to the unique organization of podocytes by interacting with the cytoskeletal network found in their cell bodies and foot processes. By immunocytochemistry, it was shown that podocalyxin and the actin binding protein ezrin are co-expressed in podocytes and co-localize along the apical plasma membrane, where they form a co-immunoprecipitable complex. Selective detergent extraction followed by differential centrifugation revealed that some of the podocalyxin cosediments with actin filaments. Moreover, its sedimentation is dependent on polymerized actin and is mediated by complex formation with ezrin. Once formed, podocalyxin/ezrin complexes are very stable, because they are insensitive to actin depolymerization or inactivation of Rho kinase, which is known to be necessary for regulation of ezrin and to mediate Rho-dependent actin organization. These data indicate that in podocytes, podocalyxin is complexed with ezrin, which mediates its link to the actin cytoskeleton. Thus, in addition to its ectodomain, the cytoplasmic tail of podocalyxin also likely contributes to maintaining the unique podocyte morphology.

Tropomyosin-related kinase C (TrkC) enhances podocyte migration by ERK-mediated WAVE2 activation.

PubMed

Gromnitza, Sascha; Lepa, Carolin; Weide, Thomas; Schwab, Albrecht; Pavenstädt, Hermann; George, Britta

2018-03-01

Podocyte malfunction is central to glomerular diseases and is marked by defective podocyte intercellular junctions and actin cytoskeletal dynamics. Podocytes share many morphologic features with neurons, so that similar sets of proteins appear to regulate cell process formation. One such protein is the tropomyosin-related kinase C (TrkC). TrkC deficiency in mice leads to proteinuria as a surrogate of defective kidney filter function. Activation of endogenous TrkC by its ligand neurotrophin-3 resulted in increased podocyte migration-a surrogate of podocyte actin dynamics in vivo. Employing a mutagenesis approach, we found that the Src homologous and collagen-like (Shc) binding site Tyr 516 within the TrkC cytoplasmic domain was necessary for TrkC-induced migration of podocytes. TrkC activation led to a mobility shift of Wiskott-Aldrich syndrome family verprolin-homologous protein (WAVE)-2 which is known to orchestrate Arp2/3 activation and actin polymerization. Chemical inactivation of Erk or mutagenesis of 2 of 4 known Erk target sites within WAVE2, Thr 346 and Ser 351 , abolished the TrkC-induced WAVE2 mobility shift. Knockdown of WAVE2 by shRNA abolished TrkC-induced podocyte migration. In summary, TrkC signals to the podocyte actin cytoskeleton to induce migration by phosphorylating WAVE2 Erk dependently. This signaling mechanism may be important for TrkC-mediated cytoskeletal dynamics in podocyte disease.-Gromnitza, S., Lepa, C., Weide, T., Schwab, A., Pavenstädt, H., George, B. Tropomyosin-related kinase C (TrkC) enhances podocyte migration by ERK-mediated WAVE2 activation.

Extracellular vesicle communication pathways as regulatory targets of oncogenic transformation.

PubMed

Choi, Dongsic; Lee, Tae Hoon; Spinelli, Cristiana; Chennakrishnaiah, Shilpa; D'Asti, Esterina; Rak, Janusz

2017-07-01

Pathogenesis of human cancers bridges intracellular oncogenic driver events and their impact on intercellular communication. Among multiple mediators of this 'pathological connectivity' the role of extracellular vesicles (EVs) and their subsets (exosomes, ectosomes, oncosomes) is of particular interest for several reasons. The release of EVs from cancer cells represents a unique mechanism of regulated expulsion of bioactive molecules, a process that also mediates cell-to-cell transfer of lipids, proteins, and nucleic acids. Biological effects of these processes have been implicated in several aspects of cancer-related pathology, including tumour growth, invasion, angiogenesis, metastasis, immunity and thrombosis. Notably, the emerging evidence suggests that oncogenic mutations may impact several aspects of EV-mediated cell-cell communication including: (i) EV release rate and protein content; (ii) molecular composition of cancer EVs; (iii) the inclusion of oncogenic and mutant macromolecules in the EV cargo; (iv) EV-mediated release of genomic DNA; (v) deregulation of mechanisms responsible for EV biogenesis (vesiculome) and (vi) mechanisms of EV uptake by cancer cells. Intriguingly, EV-mediated intercellular transfer of mutant and oncogenic molecules between subpopulations of cancer cells, their indolent counterparts and stroma may exert profound biological effects that often resemble (but are not tantamount to) oncogenic transformation, including changes in cell growth, clonogenicity and angiogenic phenotype, or cause cell stress and death. However, several biological barriers likely curtail a permanent horizontal transformation of normal cells through EV-mediated mechanisms. The ongoing analysis and targeting of EV-mediated intercellular communication pathways can be viewed as a new therapeutic paradigm in cancer, while the analysis of oncogenic cargo contained in EVs released from cancer cells into biofluids is being developed for clinical use as a biomarker and companion diagnostics. Indeed, studies are underway to further explore the multiple links between molecular causality in cancer and various aspects of cellular vesiculation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Connexin Channel Permeability to Cytoplasmic Molecules

PubMed Central

Harris, Andrew L.

2007-01-01

Connexin channels are known to be permeable to a variety of cytoplasmic molecules. The first observation of second messenger junctional permeability, made ∼30 years ago, sparked broad interest in gap junction channels as mediators of intercellular molecular signaling. Since then, much has been learned about the diversity of connexin channels with regard to isoform diversity, tissue and developmental distribution, modes of channel regulation, assembly and expression, biochemical modification and permeability, all of which appear to be dynamically regulated. This information has expanded the potential roles of connexin channels in development, physiology and disease, and made their elucidation much more complex - 30 years ago such an orchestra of junctional dynamics was unanticipated. Only recently, however, have investigators been able to directly address, in this more complex framework, the key issue: What specific biological molecules, second messengers and others, are able to permeate the various types of connexin channels, and how well? An important related issue, given the ever-growing list of connexin-related pathologies, is how these permeabilities are altered by disease-causing connexin mutations. Together, many studies show that a variety of cytoplasmic molecules can permeate the different types of connexin channels. A few studies reveal differences in permeation by different molecules through a particular type of connexin channel, and differences in permeation by a particular molecule through different types of connexin channels. This article describes and evaluates the various methods used to obtain these data, presents an annotated compilation of the results, and discusses the findings in the context of what can be inferred about mechanism of selectivity and potential relevance to signaling. The data strongly suggest that highly specific interactions take place between connexin pores and specific biological molecular permeants, and that those interactions determine which cytoplasmic molecules can permeate and how well. At this time, the nature of those interactions is unclear. One hopes that with more detailed permeability and structural information, the specific molecular mechanisms of the selectivity can be elucidated. PMID:17470375

Enhanced effect of gap junction uncouplers on macroscopic electrical properties of reperfused myocardium

PubMed Central

Rodriguez-Sinovas, Antonio; García-Dorado, David; Ruiz-Meana, Marisol; Soler-Soler, Jordi

2004-01-01

Transient inhibition of gap junction (GJ)-mediated communication with heptanol during myocardial reperfusion limits infarct size. However, inhibition of cell coupling in normal myocardium may be arrhythmogenic. The purpose of this study was to test the hypothesis that the consequences of GJ inhibition may be magnified in reperfused myocardium compared with normal tissue, thus allowing the inhibition of GJs in reperfused tissue while only minimally modifying overall macroscopic cell coupling in normal myocardium. Concentration–response curves were defined for the effects of heptanol, 18α-glycyrrhetinic acid, halothane, and palmitoleic acid on conduction velocity, tissue electrical impedance, developed tension and lactate dehydrogenase (LDH) release in normoxically perfused rat hearts (n = 17). Concentrations lacking significant effects on tissue impedance were added during the initial 15 min of reperfusion in hearts submitted to 60 min (n = 43) or 30 min (n = 35) of ischaemia. These concentrations markedly increased myocardial electrical impedance (resistivity and phase angle) in myocardium reperfused after either 30 or 60 min of ischaemia, and reduced reperfusion-induced LDH release after 1 h of ischaemia by 83.6, 57.9, 51.7 and 52.5% for heptanol, 18α-glycyrrhetinic acid, halothane and palmitoleic acid, respectively. LDH release was minimal in hearts submitted to 30 min of ischaemia, independently of group allocation. In conclusion, the present results strongly support the hypothesis that intercellular communication in postischaemic myocardium may be effectively reduced by concentrations of GJ inhibitors affecting only minimally overall electrical impedance in normal myocardium. Reduction of cell coupling during initial reperfusion was consistently associated with attenuated lethal reperfusion injury. PMID:15218064

Paracellular transport in the collecting duct

PubMed Central

Hou, Jianghui

2016-01-01

Purpose of review The paracellular pathway through the tight junction provides an important route for chloride reabsorption in the collecting duct of the kidney. This review describes recent findings of how defects in paracellular chloride permeation pathway may cause kidney diseases and how such a pathway may be regulated to maintain normal chloride homeostasis. Recent findings The tight junction in the collecting duct expresses two important claudin genes – claudin-4 and claudin-8. Transgenic knockout of either claudin gene causes hypotension, hypochloremia, and metabolic alkalosis in experimental animals. The claudin-4 mediated chloride permeability can be regulated by a protease endogenously expressed by the collecting duct cell – Cap1. Cap1 regulates the intercellular interaction of claudin-4 and its membrane stability. KLHL3, previously identified as a causal gene for Gordon’s syndrome, also known as pseudohypoaldosteronism II (PHA-II), directly interacts with claudin-8 and regulates its ubiquitination and degradation. The dominant PHA-II mutation (R528H) in KLHL3 abolishes claudin-8 binding, ubiquitination, and degradation. Summary The paracellular chloride permeation pathway in the kidney is an important but understudied area in nephrology. It plays vital roles in renal salt handling and regulation of extracellular fluid volume and blood pressure. Two claudin proteins – claudin-4 and claudin-8 contribute to the function of this paracellular pathway. Deletion of either claudin protein from the collecting duct causes renal chloride reabsorption defects and low blood pressure. Claudins can be regulated on post-translational levels by several mechanisms involving protease and ubiquitin ligase. Deregulation of claudins may cause human hypertension as exemplified in the Gordon’s syndrome. PMID:27490784

Gap junction-dependent homolog avoidance in the developing CNS.

PubMed

Baker, Michael W; Yazdani, Neema; Macagno, Eduardo R

2013-10-16

Oppositely directed projections of some homologous neurons in the developing CNS of the medicinal leech (Hirudo verbana), such as the AP cells, undergo a form of contact-dependent homolog avoidance. Embryonic APs extend axons within the connective nerve toward adjacent ganglia, in which they meet and form gap junctions (GJs) with the oppositely directed axons of their segmental homologs, stop growing, and are later permanently retracted (Wolszon et al., 1994a,b). However, early deletion of an AP neuron leads to resumed growth and permanent maintenance of the projections of neighboring APs. Here we test the hypothesis that a GJ-based signaling mechanism is responsible for this instance of homolog avoidance. We demonstrate that selective knockdown of GJ gene Hve-inx1 expression in single embryonic APs, by expressing a short-hairpin interfering RNA, leads to continued growth of the projections of the cell toward, into, and beyond adjacent ganglia. Moreover, the projections of the APs in adjacent ganglia also resume growth, mimicking their responses to cell deletion. Continued growth was also observed when two different INX1 mutant transgenes that abolish dye coupling between APs were expressed. These include a mutant transgene that effectively downregulates all GJ plaques that include the INX1 protein and a closed channel INX1 mutant that retains the adhesive cellular binding characteristic of INX1 GJs but not the open channel pore function. Our results add GJ intercellular communication to the list of molecular signaling mechanisms that can act as mediators of growth-inhibiting cell-cell interactions that define the topography of neuronal arbors.

Astrocyte Sodium Signalling and Panglial Spread of Sodium Signals in Brain White Matter.

PubMed

Moshrefi-Ravasdjani, Behrouz; Hammel, Evelyn L; Kafitz, Karl W; Rose, Christine R

2017-09-01

In brain grey matter, excitatory synaptic transmission activates glutamate uptake into astrocytes, inducing sodium signals which propagate into neighboring astrocytes through gap junctions. These sodium signals have been suggested to serve an important role in neuro-metabolic coupling. So far, it is unknown if astrocytes in white matter-that is in brain regions devoid of synapses-are also able to undergo such intra- and intercellular sodium signalling. In the present study, we have addressed this question by performing quantitative sodium imaging in acute tissue slices of mouse corpus callosum. Focal application of glutamate induced sodium transients in SR101-positive astrocytes. These were largely unaltered in the presence of ionotropic glutamate receptors blockers, but strongly dampened upon pharmacological inhibition of glutamate uptake. Sodium signals induced in individual astrocytes readily spread into neighboring SR101-positive cells with peak amplitudes decaying monoexponentially with distance from the stimulated cell. In addition, spread of sodium was largely unaltered during pharmacological inhibition of purinergic and glutamate receptors, indicating gap junction-mediated, passive diffusion of sodium between astrocytes. Using cell-type-specific, transgenic reporter mice, we found that sodium signals also propagated, albeit less effectively, from astrocytes to neighboring oligodendrocytes and NG2 cells. Again, panglial spread was unaltered with purinergic and glutamate receptors blocked. Taken together, our results demonstrate that activation of sodium-dependent glutamate transporters induces sodium signals in white matter astrocytes, which spread within the astrocyte syncytium. In addition, we found a panglial passage of sodium signals from astrocytes to NG2 cells and oligodendrocytes, indicating functional coupling between these macroglial cells in white matter.

β-Catenin Serves as a Clutch between Low and High Intercellular E-Cadherin Bond Strengths

PubMed Central

Bajpai, Saumendra; Feng, Yunfeng; Wirtz, Denis; Longmore, Gregory D.

2013-01-01

A wide range of invasive pathological outcomes originate from the loss of epithelial phenotype and involve either loss of function or downregulation of transmembrane adhesive receptor complexes, including Ecadherin (Ecad) and binding partners β-catenin and α-catenin at adherens junctions. Cellular pathways regulating wild-type β-catenin level, or direct mutations in β-catenin that affect the turnover of the protein have been shown to contribute to cancer development, through induction of uncontrolled proliferation of transformed tumor cells, particularly in colon cancer. Using single-molecule force spectroscopy, we show that depletion of β-catenin or the prominent cancer-related S45 deletion mutation in β-catenin present in human colon cancers both weaken tumor intercellular Ecad/Ecad bond strength and diminishes the capacity of specific extracellular matrix proteins—including collagen I, collagen IV, and laminin V—to modulate intercellular Ecad/Ecad bond strength through α-catenin and the kinase activity of glycogen synthase kinase 3 (GSK-3β). Thus, in addition to regulating tumor cell proliferation, cancer-related mutations in β-catenin can influence tumor progression by weakening the adhesion of tumor cells to one another through reduced individual Ecad/Ecad bond strength and cellular adhesion to specific components of the extracellular matrix and the basement membrane. PMID:24268141

Symplastic Transport of Carboxyfluorescein in Staminal Hairs of Setcreasea purpurea Is Diffusive and Includes Loss to the Vacuole.

PubMed

Tucker, J E; Mauzerall, D; Tucker, E B

1989-07-01

The kinetics of symplastic transport in staminal hairs of Setcreasea purpurea was studied. The tip cell of a staminal hair was microinjected with carboxyfluorescein (CF) and the symplastic transport of this CF was videotaped and the digital data analyzed to produce kinetic curves. Using a finite difference equation for diffusion between cells and for loss of dye into the vacuole, kinetic curves were calculated and fitted to the observed data. These curves were matched with data from actual microinjection experiments by adjusting K (the coefficient of intercellular junction diffusion) and L (the coefficient of intracellular loss) until a minimum in the least squares difference between the curves was obtained. (a) Symplastic transport of CF was governed by diffusion through intercellular pores (plasmodesmata) and intracellular loss. Diffusion within the cell cytoplasm was never limiting. (b) Each cell and its plasmodesmata must be considered as its own diffusion system. Therefore, a diffusion coefficient cannot be calculated for an entire chain of cells. (c) The movement through plasmodesmata in either direction was the same since the data are fit by a diffusion equation. (d) Diffusion through the intercellular pores was estimated to be slower than diffusion through similar pores filled with water.

Symplastic Transport of Carboxyfluorescein in Staminal Hairs of Setcreasea purpurea Is Diffusive and Includes Loss to the Vacuole 1

PubMed Central

Tucker, Joseph E.; Mauzerall, David; Tucker, Edward B.

1989-01-01

The kinetics of symplastic transport in staminal hairs of Setcreasea purpurea was studied. The tip cell of a staminal hair was microinjected with carboxyfluorescein (CF) and the symplastic transport of this CF was videotaped and the digital data analyzed to produce kinetic curves. Using a finite difference equation for diffusion between cells and for loss of dye into the vacuole, kinetic curves were calculated and fitted to the observed data. These curves were matched with data from actual microinjection experiments by adjusting K (the coefficient of intercellular junction diffusion) and L (the coefficient of intracellular loss) until a minimum in the least squares difference between the curves was obtained. (a) Symplastic transport of CF was governed by diffusion through intercellular pores (plasmodesmata) and intracellular loss. Diffusion within the cell cytoplasm was never limiting. (b) Each cell and its plasmodesmata must be considered as its own diffusion system. Therefore, a diffusion coefficient cannot be calculated for an entire chain of cells. (c) The movement through plasmodesmata in either direction was the same since the data are fit by a diffusion equation. (d) Diffusion through the intercellular pores was estimated to be slower than diffusion through similar pores filled with water. PMID:16666864

Connexin 39.9 Protein Is Necessary for Coordinated Activation of Slow-twitch Muscle and Normal Behavior in Zebrafish*

PubMed Central

Hirata, Hiromi; Wen, Hua; Kawakami, Yu; Naganawa, Yuriko; Ogino, Kazutoyo; Yamada, Kenta; Saint-Amant, Louis; Low, Sean E.; Cui, Wilson W.; Zhou, Weibin; Sprague, Shawn M.; Asakawa, Kazuhide; Muto, Akira; Kawakami, Koichi; Kuwada, John Y.

2012-01-01

In many tissues and organs, connexin proteins assemble between neighboring cells to form gap junctions. These gap junctions facilitate direct intercellular communication between adjoining cells, allowing for the transmission of both chemical and electrical signals. In rodents, gap junctions are found in differentiating myoblasts and are important for myogenesis. Although gap junctions were once believed to be absent from differentiated skeletal muscle in mammals, recent studies in teleosts revealed that differentiated muscle does express connexins and is electrically coupled, at least at the larval stage. These findings raised questions regarding the functional significance of gap junctions in differentiated muscle. Our analysis of gap junctions in muscle began with the isolation of a zebrafish motor mutant that displayed weak coiling at day 1 of development, a behavior known to be driven by slow-twitch muscle (slow muscle). We identified a missense mutation in the gene encoding Connexin 39.9. In situ hybridization found connexin 39.9 to be expressed by slow muscle. Paired muscle recordings uncovered that wild-type slow muscles are electrically coupled, whereas mutant slow muscles are not. The further examination of cellular activity revealed aberrant, arrhythmic touch-evoked Ca2+ transients in mutant slow muscle and a reduction in the number of muscle fibers contracting in response to touch in mutants. These results indicate that Connexin 39.9 facilitates the spreading of neuronal inputs, which is irregular during motor development, beyond the muscle cells and that gap junctions play an essential role in the efficient recruitment of slow muscle fibers. PMID:22075003

Non-invasive microfluidic gap junction assay.

PubMed

Chen, Sisi; Lee, Luke P

2010-03-01

Gap junctions are protein channels between cells that allow direct electrical and metabolic coupling via the exchange of biomolecules and ions. Their expression, though ubiquitous in most mammalian cell types, is especially important for the proper functioning of cardiac and neuronal systems. Many existing methods for studying gap junction communication suffer from either unquantifiable data or difficulty of use. Here, we measure the extent of dye spread and effective diffusivities through gap junction connected cells using a quantitative microfluidic cell biology platform. After loading dye by hydrodynamic focusing of calcein/AM, dye transfer dynamics into neighboring, unexposed cells can be monitored via timelapse fluorescent microscopy. By using a selective microfluidic dye loading over a confluent layer of cells, we found that high expression of gap junctions in C6 cells transmits calcein across the monolayer with an effective diffusivity of 3.4 x 10(-13) m(2)/s, which are highly coupled by Cx43. We also found that the gap junction blocker 18alpha-GA works poorly in the presence of serum even at high concentrations (50 microM); however, it is highly effective down to 2.5 microM in the absence of serum. Furthermore, when the drug is washed out, dye spread resumes rapidly within 1 min for all doses, indicating the drug does not affect transcriptional regulation of connexins in these Cx43+ cells, in contrast to previous studies. This integrated microfluidic platform enables the in situ monitoring of gap junction communication, yielding dynamic information about intercellular molecular transfer and pharmacological inhibition and recovery.

Macrophage conditioned medium induced cellular network formation in MCF-7 cells through enhanced tunneling nanotube formation and tunneling nanotube mediated release of viable cytoplasmic fragments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Patheja, Pooja, E-mail: pooja.patheja8@gmail.com; Homi Bhabha National Institute, Training School Complex, Anushaktinagar, Mumbai 400094, Maharashtra; Sahu, Khageswar

Infiltrating macrophages in tumor microenvironment, through their secreted cytokines and growth factors, regulate several processes of cancer progression such as cancer cell survival, proliferation, invasion, metastasis and angiogenesis. Recently, intercellular cytoplasmic bridges between cancer cells referred as tunneling nanotubes (TNTs) have been recognized as novel mode of intercellular communication between cancer cells. In this study, we investigated the effect of inflammatory mediators present in conditioned medium derived from macrophages on the formation of TNTs in breast adenocarcinoma cells MCF-7. Results show that treatment with macrophage conditioned medium (MφCM) not only enhanced TNT formation between cells but also stimulated the releasemore » of independently migrating viable cytoplasmic fragments, referred to as microplasts, from MCF-7 cells. Time lapse microscopy revealed that microplasts were released from parent cancer cells in extracellular space through formation of TNT-like structures. Mitochondria, vesicles and cytoplasm could be transferred from parent cell body to microplasts through connecting TNTs. The microplasts could also be resorbed into the parent cell body by retraction of the connecting TNTs. Microplast formation inhibited in presence cell migration inhibitor, cytochalasin-B. Notably by utilizing migratory machinery within microplasts, distantly located MCF-7 cells formed several TNT based intercellular connections, leading to formation of physically connected network of cells. Together, these results demonstrate novel role of TNTs in microplast formation, novel modes of TNT formation mediated by microplasts and stimulatory effect of MφCM on cellular network formation in MCF-7 cells mediated through enhanced TNT and microplast formation.« less

Desmosomes: A light microscopic and ultrastructural analysis of desmosomes in odontogenic cysts.

PubMed

Raju, Pratima; Wadhwan, Vijay; Chaudhary, Minal S

2014-01-01

Desmosomes together with adherens junctions represent the major adhesive cell-cell junctions of epithelial cells. Any damage to these junctions leads to loss of structural balance. The present study was designed to analyze the desmosomal junctions in different odontogenic cysts and compare them with their corresponding hematoxylin and eosin (H and E)   stained sections. Ten cases each of odontogenic keratocyst (OKC), dentigerous cysts (DCs), radicular cysts (RCs) and normal mucosa were stained with hematoxylin and eosin. Scanning electron microscopy (SEM) analysis of the sections was then carried out of all the sections. The area of interest on H and E stained section was marked and this marking was later superimposed onto the corresponding unstained sections and were subjected to SEM analysis. OKC at ×1000 magnification showed many prominent desmosomes. However, an increase in the intercellular space was also noted. SEM analysis demonstrated similar findings with the presence of many desmosomes, though they were seen to be damaged and fragile. H and E stained DC under oil immersion did not show any prominent desmosomes. SEM analysis of the same confirmed the observation and very minimal number were seen with a very condense arrangement of the epithelial cells. RC at ×1000 magnification revealed plenty of desmosomes, which were again confirmed by SEM. The number and quality of desmosomal junctions in all the cysts has a role in the clinical behavior of the cyst.

Elevated Src family kinase activity stabilizes E-cadherin-based junctions and collective movement of head and neck squamous cell carcinomas

PubMed Central

Veracini, Laurence; Grall, Dominique; Schaub, Sébastien; Divonne, Stéphanie Beghelli-de la Forest; Etienne-Grimaldi, Marie-Christine; Milano, Gérard; Bozec, Alexandre; Babin, Emmanuel; Sudaka, Anne; Thariat, Juliette; Van Obberghen-Schilling, Ellen

2015-01-01

EGF receptor (EGFR) overexpression is thought to drive head and neck carcinogenesis however clinical responses to EGFR-targeting agents have been modest and alternate targets are actively sought to improve results. Src family kinases (SFKs), reported to act downstream of EGFR are among the alternative targets for which increased expression or activity in epithelial tumors is commonly associated to the dissolution of E-cadherin-based junctions and acquisition of a mesenchymal-like phenotype. Robust expression of total and activated Src was observed in advanced stage head and neck tumors (N=60) and in head and neck squamous cell carcinoma lines. In cultured cancer cells Src co-localized with E-cadherin in cell-cell junctions and its phosphorylation on Y419 was both constitutive and independent of EGFR activation. Selective inhibition of SFKs with SU6656 delocalized E-cadherin and disrupted cellular junctions without affecting E-cadherin expression and this effect was phenocopied by knockdown of Src or Yes. These findings reveal an EGFR-independent role for SFKs in the maintenance of intercellular junctions, which likely contributes to the cohesive invasion E-cadherin-positive cells in advanced tumors. Further, they highlight the need for a deeper comprehension of molecular pathways that drive collective cell invasion, in absence of mesenchymal transition, in order to combat tumor spread. PMID:25779657

Connexin Type and Fluorescent Protein Fusion Tag Determine Structural Stability of Gap Junction Plaques.

PubMed

Stout, Randy F; Snapp, Erik Lee; Spray, David C

2015-09-25

Gap junctions (GJs) are made up of plaques of laterally clustered intercellular channels and the membranes in which the channels are embedded. Arrangement of channels within a plaque determines subcellular distribution of connexin binding partners and sites of intercellular signaling. Here, we report the discovery that some connexin types form plaque structures with strikingly different degrees of fluidity in the arrangement of the GJ channel subcomponents of the GJ plaque. We uncovered this property of GJs by applying fluorescence recovery after photobleaching to GJs formed from connexins fused with fluorescent protein tags. We found that connexin 26 (Cx26) and Cx30 GJs readily diffuse within the plaque structures, whereas Cx43 GJs remain persistently immobile for more than 2 min after bleaching. The cytoplasmic C terminus of Cx43 was required for stability of Cx43 plaque arrangement. We provide evidence that these qualitative differences in GJ arrangement stability reflect endogenous characteristics, with the caveat that the sizes of the GJs examined were necessarily large for these measurements. We also uncovered an unrecognized effect of non-monomerized fluorescent protein on the dynamically arranged GJs and the organization of plaques composed of multiple connexin types. Together, these findings redefine our understanding of the GJ plaque structure and should be considered in future studies using fluorescent protein tags to probe dynamics of highly ordered protein complexes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural Interaction Between GFP-Labeled Diazotrophic Endophytic Bacterium Herbaspirillum seropedicae RAM10 and Pineapple Plantlets ‘VitóRia’

PubMed Central

Estrela Borges Baldotto, Lílian; Lopes Olivares, Fábio; Bressan-Smith, Ricardo

2011-01-01

The events involved in the structural interaction between the diazotrophic endophytic bacterium Herbaspirillum seropedicae, strain RAM10, labeled with green fluorescent protein, and pineapple plantlets ‘Vitória’ were evaluated by means of bright-field and fluorescence microscopy, combined with scanning electron microscopy for 28 days after inoculation. After 6 hours of inoculation, H. seropedicae was already adhered to the roots, colonizing mainly root hair surface and bases, followed by epidermal cell wall junctions. Bacteria adherence in the initial periods occurred mainly in the form of solitary cells and small aggregates with pleomorphic cells. Bacteria infection of root tissue occurred through the cavities caused by the disruption of epidermal cells during the emergence of lateral roots and the endophytic establishment by the colonization of intercellular spaces of the cortical parenchyma. Moreover, within 1 day after inoculation the bacteria were colonizing the shoots. In this region, the preferred sites of epiphytic colonization were epidermal cell wall junctions, peltate scutiform trichomes and non-glandular trichomes. Subsequently, the bacteria occupied the outer periclinal walls of epidermal cells and stomata. The penetration into the shoot occurred passively through stoma aperture followed by the endophytic establishment on the substomatal chambers and spread to the intercellular spaces of spongy chlorenchyma. After 21 days of inoculation, bacterial biofilm were seen at the root hair base and on epidermal cell wall surface of root and leaf, also confirming the epiphytic nature of H. seropedicae. PMID:24031612

Structural Interaction Between GFP-Labeled Diazotrophic Endophytic Bacterium Herbaspirillum seropedicae RAM10 and Pineapple Plantlets 'VitóRia'.

PubMed

Estrela Borges Baldotto, Lílian; Lopes Olivares, Fábio; Bressan-Smith, Ricardo

2011-01-01

The events involved in the structural interaction between the diazotrophic endophytic bacterium Herbaspirillum seropedicae, strain RAM10, labeled with green fluorescent protein, and pineapple plantlets 'Vitória' were evaluated by means of bright-field and fluorescence microscopy, combined with scanning electron microscopy for 28 days after inoculation. After 6 hours of inoculation, H. seropedicae was already adhered to the roots, colonizing mainly root hair surface and bases, followed by epidermal cell wall junctions. Bacteria adherence in the initial periods occurred mainly in the form of solitary cells and small aggregates with pleomorphic cells. Bacteria infection of root tissue occurred through the cavities caused by the disruption of epidermal cells during the emergence of lateral roots and the endophytic establishment by the colonization of intercellular spaces of the cortical parenchyma. Moreover, within 1 day after inoculation the bacteria were colonizing the shoots. In this region, the preferred sites of epiphytic colonization were epidermal cell wall junctions, peltate scutiform trichomes and non-glandular trichomes. Subsequently, the bacteria occupied the outer periclinal walls of epidermal cells and stomata. The penetration into the shoot occurred passively through stoma aperture followed by the endophytic establishment on the substomatal chambers and spread to the intercellular spaces of spongy chlorenchyma. After 21 days of inoculation, bacterial biofilm were seen at the root hair base and on epidermal cell wall surface of root and leaf, also confirming the epiphytic nature of H. seropedicae.

Connexin Communication Compartments and Wound Repair in Epithelial Tissue.

PubMed

Chanson, Marc; Watanabe, Masakatsu; O'Shaughnessy, Erin M; Zoso, Alice; Martin, Patricia E

2018-05-03

Epithelial tissues line the lumen of tracts and ducts connecting to the external environment. They are critical in forming an interface between the internal and external environment and, following assault from environmental factors and pathogens, they must rapidly repair to maintain cellular homeostasis. These tissue networks, that range from a single cell layer, such as in airway epithelium, to highly stratified and differentiated epithelial surfaces, such as the epidermis, are held together by a junctional nexus of proteins including adherens, tight and gap junctions, often forming unique and localised communication compartments activated for localised tissue repair. This review focuses on the dynamic changes that occur in connexins, the constituent proteins of the intercellular gap junction channel, during wound-healing processes and in localised inflammation, with an emphasis on the lung and skin. Current developments in targeting connexins as corrective therapies to improve wound closure and resolve localised inflammation are also discussed. Finally, we consider the emergence of the zebrafish as a concerted whole-animal model to study, visualise and track the events of wound repair and regeneration in real-time living model systems.

Structural organization of intercellular channels II. Amino terminal domain of the connexins: sequence, functional roles, and structure.

PubMed

Beyer, Eric C; Lipkind, Gregory M; Kyle, John W; Berthoud, Viviana M

2012-08-01

The amino terminal domain (NT) of the connexins consists of their first 22-23 amino acids. Site-directed mutagenesis studies have demonstrated that NT amino acids are determinants of gap junction channel properties including unitary conductance, permeability/selectivity, and gating in response to transjunctional voltage. The importance of this region has also been emphasized by the identification of multiple disease-associated connexin mutants affecting amino acid residues in the NT region. The first part of the NT is α-helical. The structure of the Cx26 gap junction channel shows that the NT α-helix localizes within the channel, and lines the wall of the pore. Interactions of the amino acid residues in the NT with those in the transmembrane helices may be critical for holding the channel open. The predicted sites of these interactions and the applicability of the Cx26 structure to the NT of other connexins are considered. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics. Copyright © 2011. Published by Elsevier B.V.

Crystal structure of the Haemophilus influenzae Hap adhesin reveals an intercellular oligomerization mechanism for bacterial aggregation

PubMed Central

Meng, Guoyu; Spahich, Nicole; Kenjale, Roma; Waksman, Gabriel; St Geme, Joseph W

2011-01-01

Bacterial biofilms are complex microbial communities that are common in nature and are being recognized increasingly as an important determinant of bacterial virulence. However, the structural determinants of bacterial aggregation and eventual biofilm formation have been poorly defined. In Gram-negative bacteria, a major subgroup of extracellular proteins called self-associating autotransporters (SAATs) can mediate cell–cell adhesion and facilitate biofilm formation. In this study, we used the Haemophilus influenzae Hap autotransporter as a prototype SAAT to understand how bacteria associate with each other. The crystal structure of the H. influenzae HapS passenger domain (harbouring the SAAT domain) was determined to 2.2 Å by X-ray crystallography, revealing an unprecedented intercellular oligomerization mechanism for cell–cell interaction. The C-terminal SAAT domain folds into a triangular-prism-like structure that can mediate Hap–Hap dimerization and higher degrees of multimerization through its F1–F2 edge and F2 face. The intercellular multimerization can give rise to massive buried surfaces that are required for overcoming the repulsive force between cells, leading to bacterial cell–cell interaction and formation of complex microcolonies. PMID:21841773

Exposure to Lipopolysaccharide and/or Unconjugated Bilirubin Impair the Integrity and Function of Brain Microvascular Endothelial Cells

PubMed Central

Cardoso, Filipa L.; Kittel, Ágnes; Veszelka, Szilvia; Palmela, Inês; Tóth, Andrea; Brites, Dora; Deli, Mária A.; Brito, Maria A.

2012-01-01

Background Sepsis and jaundice are common conditions in newborns that can lead to brain damage. Though lipopolysaccharide (LPS) is known to alter the integrity of the blood-brain barrier (BBB), little is known on the effects of unconjugated bilirubin (UCB) and even less on the joint effects of UCB and LPS on brain microvascular endothelial cells (BMEC). Methodology/Principal Findings Monolayers of primary rat BMEC were treated with 1 µg/ml LPS and/or 50 µM UCB, in the presence of 100 µM human serum albumin, for 4 or 24 h. Co-cultures of BMEC with astroglial cells, a more complex BBB model, were used in selected experiments. LPS led to apoptosis and UCB induced both apoptotic and necrotic-like cell death. LPS and UCB led to inhibition of P-glycoprotein and activation of matrix metalloproteinases-2 and -9 in mono-cultures. Transmission electron microscopy evidenced apoptotic bodies, as well as damaged mitochondria and rough endoplasmic reticulum in BMEC by either insult. Shorter cell contacts and increased caveolae-like invaginations were noticeable in LPS-treated cells and loss of intercellular junctions was observed upon treatment with UCB. Both compounds triggered impairment of endothelial permeability and transendothelial electrical resistance both in mono- and co-cultures. The functional changes were confirmed by alterations in immunostaining for junctional proteins β-catenin, ZO-1 and claudin-5. Enlargement of intercellular spaces, and redistribution of junctional proteins were found in BMEC after exposure to LPS and UCB. Conclusions LPS and/or UCB exert direct toxic effects on BMEC, with distinct temporal profiles and mechanisms of action. Therefore, the impairment of brain endothelial integrity upon exposure to these neurotoxins may favor their access to the brain, thus increasing the risk of injury and requiring adequate clinical management of sepsis and jaundice in the neonatal period. PMID:22586454

Overexpression of connexin 43 reduces melanoma proliferative and metastatic capacity

PubMed Central

Tittarelli, A; Guerrero, I; Tempio, F; Gleisner, M A; Avalos, I; Sabanegh, S; Ortíz, C; Michea, L; López, M N; Mendoza-Naranjo, A; Salazar-Onfray, F

2015-01-01

Background: Alterations in connexin 43 (Cx43) expression and/or gap junction (GJ)-mediated intercellular communication are implicated in cancer pathogenesis. Herein, we have investigated the role of Cx43 in melanoma cell proliferation and apoptosis sensitivity in vitro, as well as metastatic capability and tumour growth in vivo. Methods: Connexin 43 expression levels, GJ coupling and proliferation rates were analysed in four different human melanoma cell lines. Furthermore, tumour growth and lung metastasis of high compared with low Cx43-expressing FMS cells were evaluated in vivo using a melanoma xenograft model. Results: Specific inhibition of Cx43 channel activity accelerated melanoma cell proliferation, whereas overexpression of Cx43 increased GJ coupling and reduced cell growth. Moreover, Cx43 overexpression in FMS cells increased basal and tumour necrosis factor-α-induced apoptosis and resulted in decreased melanoma tumour growth and lower number and size of metastatic foci in vivo. Conclusions: Our findings reveal an important role for Cx43 in intrinsically controlling melanoma growth, death and metastasis, and emphasise the potential use of compounds that selectively enhance Cx43 expression on melanoma in the future chemotherapy and/or immunotherapy protocols. PMID:26135897

Increased frequency of spontaneous neoplastic transformation in progeny of bystander cells from cultures exposed to densely ionizing radiation.

PubMed

Buonanno, Manuela; de Toledo, Sonia M; Azzam, Edouard I

2011-01-01

An increased risk of carcinogenesis caused by exposure to space radiation during prolonged space travel is a limiting factor for human space exploration. Typically, astronauts are exposed to low fluences of ionizing particles that target only a few cells in a tissue at any one time. The propagation of stressful effects from irradiated to neighboring bystander cells and their transmission to progeny cells would be of importance in estimates of the health risks of exposure to space radiation. With relevance to the risk of carcinogenesis, we investigated, in model C3H 10T½ mouse embryo fibroblasts (MEFs), modulation of the spontaneous frequency of neoplastic transformation in the progeny of bystander MEFs that had been in co-culture 10 population doublings earlier with MEFs exposed to moderate doses of densely ionizing iron ions (1 GeV/nucleon) or sparsely ionizing protons (1 GeV). An increase (P<0.05) in neoplastic transformation frequency, likely mediated by intercellular communication through gap junctions, was observed in the progeny of bystander cells that had been in co-culture with cells irradiated with iron ions, but not with protons.

An UPF3-based nonsense-mediated decay in Paramecium.

PubMed

Contreras, Julia; Begley, Victoria; Macias, Sandra; Villalobo, Eduardo

2014-12-01

Nonsense-mediated decay recognises mRNAs containing premature termination codons. One of its components, UPF3, is a molecular link bridging through its binding to the exon junction complex nonsense-mediated decay and splicing. In protists UPF3 has not been identified yet. We report that Paramecium tetraurelia bears an UPF3 gene and that it has a role in nonsense-mediated decay. Interestingly, the identified UPF3 has not conserved the essential amino acids required to bind the exon junction complex. Though, our data indicates that this ciliate bears genes coding for core proteins of the exon junction complex. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Homo sapiens Systemic RNA Interference-defective-1 Transmembrane Family Member 1 (SIDT1) Protein Mediates Contact-dependent Small RNA Transfer and MicroRNA-21-driven Chemoresistance*

PubMed Central

Elhassan, Mohamed O.; Christie, Jennifer; Duxbury, Mark S.

2012-01-01

Locally initiated RNA interference (RNAi) has the potential for spatial propagation, inducing posttranscriptional gene silencing in distant cells. In Caenorhabditis elegans, systemic RNAi requires a phylogenetically conserved transmembrane channel, SID-1. Here, we show that a human SID-1 orthologue, SIDT1, facilitates rapid, contact-dependent, bidirectional small RNA transfer between human cells, resulting in target-specific non-cell-autonomous RNAi. Intercellular small RNA transfer can be both homotypic and heterotypic. We show SIDT1-mediated intercellular transfer of microRNA-21 to be a driver of resistance to the nucleoside analog gemcitabine in human adenocarcinoma cells. Documentation of a SIDT1-dependent small RNA transfer mechanism and the associated phenotypic effects on chemoresistance in human cancer cells raises the possibility that conserved systemic RNAi pathways contribute to the acquisition of drug resistance. Mediators of non-cell-autonomous RNAi may be tractable targets for novel therapies aimed at improving the efficacy of current cytotoxic agents. PMID:22174421

Eye lens membrane junctional microdomains: a comparison between healthy and pathological cases

NASA Astrophysics Data System (ADS)

Buzhynskyy, Nikolay; Sens, Pierre; Behar-Cohen, Francine; Scheuring, Simon

2011-08-01

The eye lens is a transparent tissue constituted of tightly packed fiber cells. To maintain homeostasis and transparency of the lens, the circulation of water, ions and metabolites is required. Junctional microdomains connect the lens cells and ensure both tight cell-to-cell adhesion and intercellular flow of fluids through a microcirculation system. Here, we overview membrane morphology and tissue functional requirements of the mammalian lens. Atomic force microscopy (AFM) has opened up the possibility of visualizing the junctional microdomains at unprecedented submolecular resolution, revealing the supramolecular assembly of lens-specific aquaporin-0 (AQP0) and connexins (Cx). We compare the membrane protein assembly in healthy lenses with senile and diabetes-II cataract cases and novel data of the lens membranes from a congenital cataract. In the healthy case, AQP0s form characteristic square arrays confined by connexons. In the cases of senile and diabetes-II cataract patients, connexons were degraded, leading to malformation of AQP0 arrays and breakdown of the microcirculation system. In the congenital cataract, connexons are present, indicating probable non-membranous grounds for lens opacification. Further, we discuss the energetic aspects of the membrane organization in junctional microdomains. The AFM hence becomes a biomedical nano-imaging tool for the analysis of single-membrane protein supramolecular association in healthy and pathological membranes.

The Septate Junction Protein Caspr is Required for Structural Support and Retention of KCNQ4 at Calyceal Synapses of Vestibular Hair Cells

PubMed Central

Sousa, Aurea D.; Andrade, Leonardo R.; Salles, Felipe T.; Pillai, Anilkumar M.; Buttermore, Elizabeth; Bhat, Manzoor A.; Kachar, Bechara

2009-01-01

The afferent innervation contacting the type I hair cells of the vestibular sensory epithelia form distinct calyceal synapses. The apposed pre- and post-synaptic membranes at this large area of synaptic contact are kept at a remarkably regular distance. Here, we show by freeze-fracture electron microscopy that a patterned alignment of proteins at the calyceal membrane resembles a type of intercellular junction that is rare in vertebrates, the septate junction (SJ). We found that a core molecular component of SJs, Caspr, colocalizes with the K+ channel KCNQ4 at the post-synaptic membranes of these calyceal synapses. Immunolabeling and ultrastructural analyses of Caspr knockout mice reveal that, in the absence of Caspr, the separation between the membranes of the hair cells and the afferent neurons is conspicuously irregular and often increased by an order of magnitude. In these mutants, KCNQ4 fails to cluster at the post-synaptic membrane and appears diffused along the entire calyceal membrane. Our results indicate that a septate-like junction provides structural support to calyceal synaptic contact with the vestibular hair cell, and that Caspr is required for the recruitment or retention of KCNQ4 at these synapses. PMID:19279247

Cellular Interaction of Integrin α3β1 with Laminin 5 Promotes Gap Junctional Communication

PubMed Central

Lampe, Paul D.; Nguyen, Beth P.; Gil, Susana; Usui, Marcia; Olerud, John; Takada, Yoshikazu; Carter, William G.

1998-01-01

Wounding of skin activates epidermal cell migration over exposed dermal collagen and fibronectin and over laminin 5 secreted into the provisional basement membrane. Gap junctional intercellular communication (GJIC) has been proposed to integrate the individual motile cells into a synchronized colony. We found that outgrowths of human keratinocytes in wounds or epibole cultures display parallel changes in the expression of laminin 5, integrin α3β1, E-cadherin, and the gap junctional protein connexin 43. Adhesion of keratinocytes on laminin 5, collagen, and fibronectin was found to differentially regulate GJIC. When keratinocytes were adhered on laminin 5, both structural (assembly of connexin 43 in gap junctions) and functional (dye transfer) assays showed a two- to threefold increase compared with collagen and five- to eightfold over fibronectin. Based on studies with immobilized integrin antibody and integrin-transfected Chinese hamster ovary cells, the interaction of integrin α3β1 with laminin 5 was sufficient to promote GJIC. Mapping of intermediate steps in the pathway linking α3β1–laminin 5 interactions to GJIC indicated that protein trafficking and Rho signaling were both required. We suggest that adhesion of epithelial cells to laminin 5 in the basement membrane via α3β1 promotes GJIC that integrates individual cells into synchronized epiboles. PMID:9852164

Phylogenetic and bioinformatic analysis of gap junction-related proteins, innexins, pannexins and connexins.

PubMed

Fushiki, Daisuke; Hamada, Yasuo; Yoshimura, Ryoichi; Endo, Yasuhisa

2010-04-01

All multi-cellular animals, including hydra, insects and vertebrates, develop gap junctions, which communicate directly with neighboring cells. Gap junctions consist of protein families called connexins in vertebrates and innexins in invertebrates. Connexins and innexins have no homology in their amino acid sequence, but both are thought to have some similar characteristics, such as a tetra-membrane-spanning structure, formation of a channel by hexamer, and transmission of small molecules (e.g. ions) to neighboring cells. Pannexins were recently identified as a homolog of innexins in vertebrate genomes. Although pannexins are thought to share the function of intercellular communication with connexins and innexins, there is little information about the relationship among these three protein families of gap junctions. We phylgenetically and bioinformatically examined these protein families and other tetra-membrane-spanning proteins using a database and three analytical softwares. The clades formed by pannexin families do not belong to the species classification but do to paralogs of each member of pannexins. Amino acid sequences of pannexins are closely related to those of innexins but less to those of connexins. These data suggest that innexins and pannexins have a common origin, but the relationship between innexins/pannexins and connexins is as slight as that of other tetra-membrane-spanning members.

INTEGRIN-MEDIATED CELL ATTACHMENT SHOWS TIME-DEPENDENT UPREGULATION OF GAP JUNCTION COMMUNICATION.

EPA Science Inventory

Integrin-mediated Cell Attachment Shows Time-Dependent Upregulation of Gap Junction
Communication

Rachel Grindstaff and Carl Blackman, National Health & Environmental Effects Research
Laboratory, Office of Research and Development, US EPA, Research Triang...

PGE2 maintains self-renewal of human adult stem cells via EP2-mediated autocrine signaling and its production is regulated by cell-to-cell contact.

PubMed

Lee, Byung-Chul; Kim, Hyung-Sik; Shin, Tae-Hoon; Kang, Insung; Lee, Jin Young; Kim, Jae-Jun; Kang, Hyun Kyoung; Seo, Yoojin; Lee, Seunghee; Yu, Kyung-Rok; Choi, Soon Won; Kang, Kyung-Sun

2016-05-27

Mesenchymal stem cells (MSCs) possess unique immunomodulatory abilities. Many studies have elucidated the clinical efficacy and underlying mechanisms of MSCs in immune disorders. Although immunoregulatory factors, such as Prostaglandin E2 (PGE2), and their mechanisms of action on immune cells have been revealed, their effects on MSCs and regulation of their production by the culture environment are less clear. Therefore, we investigated the autocrine effect of PGE2 on human adult stem cells from cord blood or adipose tissue, and the regulation of its production by cell-to-cell contact, followed by the determination of its immunomodulatory properties. MSCs were treated with specific inhibitors to suppress PGE2 secretion, and proliferation was assessed. PGE2 exerted an autocrine regulatory function in MSCs by triggering E-Prostanoid (EP) 2 receptor. Inhibiting PGE2 production led to growth arrest, whereas addition of MSC-derived PGE2 restored proliferation. The level of PGE2 production from an equivalent number of MSCs was down-regulated via gap junctional intercellular communication. This cell contact-mediated decrease in PGE2 secretion down-regulated the suppressive effect of MSCs on immune cells. In conclusion, PGE2 produced by MSCs contributes to maintenance of self-renewal capacity through EP2 in an autocrine manner, and PGE2 secretion is down-regulated by cell-to-cell contact, attenuating its immunomodulatory potency.

Glial response to polyglutamine-mediated stress

PubMed Central

Vig, Parminder J.S.; Shao, Qingmei; Lopez, Maripar E

2009-01-01

Neurodegenerative trinucleotide (CAG) repeat disorders are caused by the expansion of polyglutamine tracts within the disease proteins. Some of these proteins have an unknown function. How does expanded polyglutamine cause target neurons to degenerate, is not clear. Recent evidence suggests that intercellular miscommunication may contribute to polyglutamine pathogenesis in CAG repeat disorders. Polyglutamine induced degeneration of the target neuron can be mediated via glia-neuron interactions. Here we hypothesize during neurodegenerative process the failure of cell: cell interactions have more severe consequences than alterations in intracellular neuron biology. We further believe that bidirectional communication between neurons and glia are prerequisite for the normal development and function of either cell-type. Understanding intercellular signaling mechanisms such as glial trophic factors and their receptors, cell adhesion or other well-defined signaling molecules provide opportunities for developing potential therapies. PMID:20046986

Critical role of gap junction communication, calcium and nitric oxide signaling in bystander responses to focal photodynamic injury.

PubMed

Calì, Bianca; Ceolin, Stefano; Ceriani, Federico; Bortolozzi, Mario; Agnellini, Andrielly H R; Zorzi, Veronica; Predonzani, Andrea; Bronte, Vincenzo; Molon, Barbara; Mammano, Fabio

2015-04-30

Ionizing and nonionizing radiation affect not only directly targeted cells but also surrounding "bystander" cells. The underlying mechanisms and therapeutic role of bystander responses remain incompletely defined. Here we show that photosentizer activation in a single cell triggers apoptosis in bystander cancer cells, which are electrically coupled by gap junction channels and support the propagation of a Ca2+ wave initiated in the irradiated cell. The latter also acts as source of nitric oxide (NO) that diffuses to bystander cells, in which NO levels are further increased by a mechanism compatible with Ca(2+)-dependent enzymatic production. We detected similar signals in tumors grown in dorsal skinfold chambers applied to live mice. Pharmacological blockade of connexin channels significantly reduced the extent of apoptosis in bystander cells, consistent with a critical role played by intercellular communication, Ca2+ and NO in the bystander effects triggered by photodynamic therapy.

Hemichannel composition and electrical synaptic transmission: molecular diversity and its implications for electrical rectification

PubMed Central

Palacios-Prado, Nicolás; Huetteroth, Wolf; Pereda, Alberto E.

2014-01-01

Unapposed hemichannels (HCs) formed by hexamers of gap junction proteins are now known to be involved in various cellular processes under both physiological and pathological conditions. On the other hand, less is known regarding how differences in the molecular composition of HCs impact electrical synaptic transmission between neurons when they form intercellular heterotypic gap junctions (GJs). Here we review data indicating that molecular differences between apposed HCs at electrical synapses are generally associated with rectification of electrical transmission. Furthermore, this association has been observed at both innexin and connexin (Cx) based electrical synapses. We discuss the possible molecular mechanisms underlying electrical rectification, as well as the potential contribution of intracellular soluble factors to this phenomenon. We conclude that asymmetries in molecular composition and sensitivity to cellular factors of each contributing hemichannel can profoundly influence the transmission of electrical signals, endowing electrical synapses with more complex functional properties. PMID:25360082

Endothelial cell regulation of leukocyte infiltration in inflammatory tissues

PubMed Central

Mantovani, A.; Introna, M.; Dejana, E.

1995-01-01

Endothelial cells play an important, active role in the onset and regulation of inflammatory and immune reactions. Through the production of chemokines they attract leukocytes and activate their adhesive receptors. This leads to the anchorage of leukocytes to the adhesive molecules expressed on the endothelial surface. Leukocyte adhesion to endothelial cells is frequently followed by their extravasation. The mechanisms which regulate the passage of leukocytes through endothelial clefts remain to be clarified. Many indirect data suggest that leukocytes might transfer signals to endothelial cells both through the release of active agents and adhesion to the endothelial cell surface. Adhesive molecules (such as PECAM) on the endothelial cell surface might also ‘direct’ leukocytes through the intercellular junction by haptotaxis. The information available on the molecular structure and functional properties of endothelial chemokines, adhesive molecules or junction organization is still fragmentary. Further work is needed to clarify how they interplay in regulating leukocyte infiltration into tissues. PMID:18475659

Regulation of gap junction channels and hemichannels by phosphorylation and redox changes: a revision.

PubMed

Pogoda, Kristin; Kameritsch, Petra; Retamal, Mauricio A; Vega, José L

2016-05-24

Post-translational modifications of connexins play an important role in the regulation of gap junction and hemichannel permeability. The prerequisite for the formation of functional gap junction channels is the assembly of connexin proteins into hemichannels and their insertion into the membrane. Hemichannels can affect cellular processes by enabling the passage of signaling molecules between the intracellular and extracellular space. For the intercellular communication hemichannels from one cell have to dock to its counterparts on the opposing membrane of an adjacent cell to allow the transmission of signals via gap junctions from one cell to the other. The controlled opening of hemichannels and gating properties of complete gap junctions can be regulated via post-translational modifications of connexins. Not only channel gating, but also connexin trafficking and assembly into hemichannels can be affected by post-translational changes. Recent investigations have shown that connexins can be modified by phosphorylation/dephosphorylation, redox-related changes including effects of nitric oxide (NO), hydrogen sulfide (H2S) or carbon monoxide (CO), acetylation, methylation or ubiquitination. Most of the connexin isoforms are known to be phosphorylated, e.g. Cx43, one of the most studied connexin at all, has 21 reported phosphorylation sites. In this review, we provide an overview about the current knowledge and relevant research of responsible kinases, connexin phosphorylation sites and reported effects on gap junction and hemichannel regulation. Regarding the effects of oxidants we discuss the role of NO in different cell types and tissues and recent studies about modifications of connexins by CO and H2S.

CAVEOLIN-1 REGULATES HIV-1 TAT-INDUCED ALTERATIONS OF TIGHT JUNCTION PROTEIN EXPRESSION VIA MODULATION OF THE RAS SIGNALING

PubMed Central

Zhong, Yu; Smart, Eric J.; Weksler, Babette; Couraud, Pierre-Olivier; Hennig, Bernhard; Toborek, Michal

2009-01-01

The blood-brain barrier (BBB) is the critical structure for preventing HIV trafficking into the brain. Specific HIV proteins, such as Tat protein, can contribute to the dysfunction of tight junctions at the BBB and HIV entry into the brain. Tat is released by HIV-1 infected cells and can interact with a variety of cell surface receptors activating several signal transduction pathways, including those localized in caveolae. The present study focused on the mechanisms of Tat-induced caveolae-associated Ras signaling at the level of the BBB. Treatment with Tat activated the Ras pathway in human brain microvascular endothelial cells (HBMEC). However, caveolin-1 silencing markedly attenuated these effects. Because the integrity of the brain endothelium is regulated by intercellular tight junctions, these structural elements of the BBB were also evaluated in the present study. Exposure to Tat diminished the expression of several tight junction proteins, namely, occludin, zonula occludens (ZO)-1, and ZO-2 in the caveolar fraction of HBMEC. These effects were effectively protected by pharmacological inhibition of the Ras signaling and by silencing of caveolin-1. The present data indicate the importance of caveolae-associated signaling in the disruption of tight junctions upon Tat exposure. They also demonstrate that caveolin-1 may constitute an early and critical modulator that controls signaling pathways leading to the disruption of tight junction proteins. Thus, caveolin-1 may provide an effective target to protect against Tat-induced HBMEC dysfunction and the disruption of the BBB in HIV-1-infected patients. PMID:18667611

Macrophage conditioned medium induced cellular network formation in MCF-7 cells through enhanced tunneling nanotube formation and tunneling nanotube mediated release of viable cytoplasmic fragments.

PubMed

Patheja, Pooja; Sahu, Khageswar

2017-06-15

Infiltrating macrophages in tumor microenvironment, through their secreted cytokines and growth factors, regulate several processes of cancer progression such as cancer cell survival, proliferation, invasion, metastasis and angiogenesis. Recently, intercellular cytoplasmic bridges between cancer cells referred as tunneling nanotubes (TNTs) have been recognized as novel mode of intercellular communication between cancer cells. In this study, we investigated the effect of inflammatory mediators present in conditioned medium derived from macrophages on the formation of TNTs in breast adenocarcinoma cells MCF-7. Results show that treatment with macrophage conditioned medium (MɸCM) not only enhanced TNT formation between cells but also stimulated the release of independently migrating viable cytoplasmic fragments, referred to as microplasts, from MCF-7 cells. Time lapse microscopy revealed that microplasts were released from parent cancer cells in extracellular space through formation of TNT-like structures. Mitochondria, vesicles and cytoplasm could be transferred from parent cell body to microplasts through connecting TNTs. The microplasts could also be resorbed into the parent cell body by retraction of the connecting TNTs. Microplast formation inhibited in presence cell migration inhibitor, cytochalasin-B. Notably by utilizing migratory machinery within microplasts, distantly located MCF-7 cells formed several TNT based intercellular connections, leading to formation of physically connected network of cells. Together, these results demonstrate novel role of TNTs in microplast formation, novel modes of TNT formation mediated by microplasts and stimulatory effect of MɸCM on cellular network formation in MCF-7 cells mediated through enhanced TNT and microplast formation. Copyright © 2017 Elsevier Inc. All rights reserved.

Critical role of ATP-induced ATP release for Ca2+ signaling in nonsensory cell networks of the developing cochlea

PubMed Central

Ceriani, Federico; Pozzan, Tullio; Mammano, Fabio

2016-01-01

Spatially and temporally coordinated variations of the cytosolic free calcium concentration ([Ca2+]c) play a crucial role in a variety of tissues. In the developing sensory epithelium of the mammalian cochlea, elevation of extracellular adenosine trisphosphate concentration ([ATP]e) triggers [Ca2+]c oscillations and propagation of intercellular inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ waves. What remains uncertain is the relative contribution of gap junction channels and connexin hemichannels to these fundamental mechanisms, defects in which impair hearing acquisition. Another related open question is whether [Ca2+]c oscillations require oscillations of the cytosolic IP3 concentration ([IP3]c) in this system. To address these issues, we performed Ca2+ imaging experiments in the lesser epithelial ridge of the mouse cochlea around postnatal day 5 and constructed a computational model in quantitative adherence to experimental data. Our results indicate that [Ca2+]c oscillations are governed by Hopf-type bifurcations within the experimental range of [ATP]e and do not require [IP3]c oscillations. The model replicates accurately the spatial extent and propagation speed of intercellular Ca2+ waves and predicts that ATP-induced ATP release is the primary mechanism underlying intercellular propagation of Ca2+ signals. The model also uncovers a discontinuous transition from propagating regimes (intercellular Ca2+ wave speed > 11 μm⋅s−1) to propagation failure (speed = 0), which occurs upon lowering the maximal ATP release rate below a minimal threshold value. The approach presented here overcomes major limitations due to lack of specific connexin channel inhibitors and can be extended to other coupled cellular systems. PMID:27807138

Targeted p120-Catenin Ablation Disrupts Dental Enamel Development

PubMed Central

Bartlett, John D.; Dobeck, Justine M.; Tye, Coralee E.; Perez-Moreno, Mirna; Stokes, Nicole; Reynolds, Albert B.; Fuchs, Elaine; Skobe, Ziedonis

2010-01-01

Dental enamel development occurs in stages. The ameloblast cell layer is adjacent to, and is responsible for, enamel formation. When rodent pre-ameloblasts become tall columnar secretory-stage ameloblasts, they secrete enamel matrix proteins, and the ameloblasts start moving in rows that slide by one another. This movement is necessary to form the characteristic decussating enamel prism pattern. Thus, a dynamic system of intercellular interactions is required for proper enamel development. Cadherins are components of the adherens junction (AJ), and they span the cell membrane to mediate attachment to adjacent cells. p120 stabilizes cadherins by preventing their internalization and degradation. So, we asked if p120-mediated cadherin stability is important for dental enamel formation. Targeted p120 ablation in the mouse enamel organ had a striking effect. Secretory stage ameloblasts detached from surrounding tissues, lost polarity, flattened, and ameloblast E- and N-cadherin expression became undetectable by immunostaining. The enamel itself was poorly mineralized and appeared to be composed of a thin layer of merged spheres that abraded from the tooth. Significantly, p120 mosaic mouse teeth were capable of forming normal enamel demonstrating that the enamel defects were not a secondary effect of p120 ablation. Surprisingly, blood-filled sinusoids developed in random locations around the developing teeth. This has not been observed in other p120-ablated tissues and may be due to altered p120-mediated cell signaling. These data reveal a critical role for p120 in tooth and dental enamel development and are consistent with p120 directing the attachment and detachment of the secretory stage ameloblasts as they move in rows. PMID:20862276

β-Catenin serves as a clutch between low and high intercellular E-cadherin bond strengths.

PubMed

Bajpai, Saumendra; Feng, Yunfeng; Wirtz, Denis; Longmore, Gregory D

2013-11-19

A wide range of invasive pathological outcomes originate from the loss of epithelial phenotype and involve either loss of function or downregulation of transmembrane adhesive receptor complexes, including Ecadherin (Ecad) and binding partners β-catenin and α-catenin at adherens junctions. Cellular pathways regulating wild-type β-catenin level, or direct mutations in β-catenin that affect the turnover of the protein have been shown to contribute to cancer development, through induction of uncontrolled proliferation of transformed tumor cells, particularly in colon cancer. Using single-molecule force spectroscopy, we show that depletion of β-catenin or the prominent cancer-related S45 deletion mutation in β-catenin present in human colon cancers both weaken tumor intercellular Ecad/Ecad bond strength and diminishes the capacity of specific extracellular matrix proteins-including collagen I, collagen IV, and laminin V-to modulate intercellular Ecad/Ecad bond strength through α-catenin and the kinase activity of glycogen synthase kinase 3 (GSK-3β). Thus, in addition to regulating tumor cell proliferation, cancer-related mutations in β-catenin can influence tumor progression by weakening the adhesion of tumor cells to one another through reduced individual Ecad/Ecad bond strength and cellular adhesion to specific components of the extracellular matrix and the basement membrane. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Pathogenetic role of the deafness-related M34T mutation of Cx26

PubMed Central

Bicego, Massimiliano; Beltramello, Martina; Melchionda, Salvatore; Carella, Massimo; Piazza, Valeria; Zelante, Leopoldo; Bukauskas, Feliksas F.; Arslan, Edoardo; Cama, Elona; Pantano, Sergio; Bruzzone, Roberto; D’Andrea, Paola; Mammano, Fabio

2010-01-01

Mutations in the GJB2 gene, which encodes the gap junction protein connexin26 (Cx26), are the major cause of genetic non-syndromic hearing loss. The role of the allelic variant M34T in causing hereditary deafness remains controversial. By combining genetic, clinical, biochemical, electrophysiological and structural modeling studies, we have re-assessed the pathogenetic role of the M34T mutation. Genetic and audiological data indicate that the majority of heterozygous carriers and all five compound heterozygotes exhibited an impaired auditory function. Functional expression in transiently transfected HeLa cells showed that, although M34T was correctly synthesized and targeted to the plasma membrane, it inefficiently formed intercellular channels that displayed an abnormal electrical behavior and retained only 11% of the unitary conductance of the wild-type protein (HCx26wt). Moreover, M34T channels failed to support the intercellular diffusion of Lucifer Yellow and the spreading of mechanically induced intercellular Ca2+ waves. When co-expressed together with HCx26wt, M34T exerted dominant-negative effects on cell–cell coupling. Our findings are consistent with a structural model, predicting that the mutation leads to a constriction of the channel pore. These data support the view that M34T is a pathological variant of Cx26 associated with hearing impairment. PMID:16849369

Intercellular transfer along the trichomes of the invasive terminal heterocyst forming cyanobacterium Cylindrospermopsis raciborskii CS-505.

PubMed

Plominsky, Álvaro M; Delherbe, Nathalie; Mandakovic, Dinka; Riquelme, Brenda; González, Karen; Bergman, Birgitta; Mariscal, Vicente; Vásquez, Mónica

2015-03-01

Cylindrospermopsis raciborskii CS-505 is an invasive freshwater filamentous cyanobacterium that when grown diazotrophically may develop trichomes of up to 100 vegetative cells while differentiating only two end heterocysts, the sole sites for their N2-fixation process. We examined the diazotrophic growth and intercellular transfer mechanisms in C. raciborskii CS-505. Subjecting cultures to a combined-nitrogen-free medium to elicit N2 fixation, the trichome length remained unaffected while growth rates decreased. The structures and proteins for intercellular communication showed that while a continuous periplasmic space was apparent along the trichomes, the putative septal junction sepJ gene is divided into two open reading frames and lacks several transmembrane domains unlike the situation in Anabaena, differentiating a 5-fold higher frequency of heterocysts. FRAP analyses also showed that the dyes calcein and 5-CFDA were taken up by heterocysts and vegetative cells, and that the transfer from heterocysts and 'terminal' vegetative cells showed considerably higher transfer rates than that from vegetative cells located in the middle of the trichomes. The data suggest that C. raciborskii CS-505 compensates its low-frequency heterocyst phenotype by a highly efficient transfer of the fixed nitrogen towards cells in distal parts of the trichomes (growing rapidly) while cells in central parts suffers (slow growth). © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The Integrated Role of Wnt/β-Catenin, N-Glycosylation, and E-Cadherin-Mediated Adhesion in Network Dynamics

PubMed Central

Vargas, Diego A.; Sun, Meng; Sadykov, Khikmet; Kukuruzinska, Maria A.; Zaman, Muhammad H.

2016-01-01

The cellular network composed of the evolutionarily conserved metabolic pathways of protein N-glycosylation, Wnt/β-catenin signaling pathway, and E-cadherin-mediated cell-cell adhesion plays pivotal roles in determining the balance between cell proliferation and intercellular adhesion during development and in maintaining homeostasis in differentiated tissues. These pathways share a highly conserved regulatory molecule, β-catenin, which functions as both a structural component of E-cadherin junctions and as a co-transcriptional activator of the Wnt/β-catenin signaling pathway, whose target is the N-glycosylation-regulating gene, DPAGT1. Whereas these pathways have been studied independently, little is known about the dynamics of their interaction. Here we present the first numerical model of this network in MDCK cells. Since the network comprises a large number of molecules with varying cell context and time-dependent levels of expression, it can give rise to a wide range of plausible cellular states that are difficult to track. Using known kinetic parameters for individual reactions in the component pathways, we have developed a theoretical framework and gained new insights into cellular regulation of the network. Specifically, we developed a mathematical model to quantify the fold-change in concentration of any molecule included in the mathematical representation of the network in response to a simulated activation of the Wnt/ β-catenin pathway with Wnt3a under different conditions. We quantified the importance of protein N-glycosylation and synthesis of the DPAGT1 encoded enzyme, GPT, in determining the abundance of cytoplasmic β-catenin. We confirmed the role of axin in β-catenin degradation. Finally, our data suggest that cell-cell adhesion is insensitive to E-cadherin recycling in the cell. We validate the model by inhibiting β-catenin-mediated activation of DPAGT1 expression and predicting changes in cytoplasmic β-catenin concentration and stability of E-cadherin junctions in response to DPAGT1 inhibition. We show the impact of pathway dysregulation through measurements of cell migration in scratch-wound assays. Collectively, our results highlight the importance of numerical analyses of cellular networks dynamics to gain insights into physiological processes and potential design of therapeutic strategies to prevent epithelial cell invasion in cancer. PMID:27427963

Epithelial cytoskeletal framework and nuclear matrix-intermediate filament scaffold: three-dimensional organization and protein composition.

PubMed

Fey, E G; Wan, K M; Penman, S

1984-06-01

Madin-Darby canine kidney (MDCK) cells grow as differentiated, epithelial colonies that display tissue-like organization. We examined the structural elements underlying the colony morphology in situ using three consecutive extractions that produce well-defined fractions for both microscopy and biochemical analysis. First, soluble proteins and phospholipid were removed with Triton X-100 in a physiological buffer. The resulting skeletal framework retained nuclei, dense cytoplasmic filament networks, intercellular junctional complexes, and apical microvillar structures. Scanning electron microscopy showed that the apical cell morphology is largely unaltered by detergent extraction. Residual desmosomes, as can be seen in thin sections, were also well-preserved. The skeletal framework was visualized in three dimensions as an unembedded whole mount that revealed the filament networks that were masked in Epon-embedded thin sections of the same preparation. The topography of cytoskeletal filaments was relatively constant throughout the epithelial sheet, particularly across intercellular borders. This ordering of epithelial skeletal filaments across contiguous cell boundaries was in sharp contrast to the more independent organization of networks in autonomous cells such as fibroblasts. Further extraction removed the proteins of the salt-labile cytoskeleton and the chromatin as separate fractions, and left the nuclear matrix-intermediate filament (NM-IF) scaffold. The NM-IF contained only 5% of total cellular protein, but whole mount transmission electron microscopy and immunofluorescence showed that this scaffold was organized as in the intact epithelium. Immunoblots demonstrate that vimentin, cytokeratins, desmosomal proteins, and a 52,000-mol-wt nuclear matrix protein were found almost exclusively in the NM-IF scaffold. Vimentin was largely perinuclear while the cytokeratins were localized at the cell borders. The 52,000-mol-wt nuclear matrix protein was confined to the chromatin-depleted matrix and the desmosomal proteins were observed in punctate polygonal arrays at intercellular junctions. The filaments of the NM-IF were seen to be interconnected, via the desmosomes, over the entire epithelial colony. The differentiated epithelial morphology was reflected in both the cytoskeletal framework and the NM-IF scaffold.

Epithelial cytoskeletal framework and nuclear matrix-intermediate filament scaffold: three-dimensional organization and protein composition

PubMed Central

1984-01-01

Madin-Darby canine kidney (MDCK) cells grow as differentiated, epithelial colonies that display tissue-like organization. We examined the structural elements underlying the colony morphology in situ using three consecutive extractions that produce well-defined fractions for both microscopy and biochemical analysis. First, soluble proteins and phospholipid were removed with Triton X-100 in a physiological buffer. The resulting skeletal framework retained nuclei, dense cytoplasmic filament networks, intercellular junctional complexes, and apical microvillar structures. Scanning electron microscopy showed that the apical cell morphology is largely unaltered by detergent extraction. Residual desmosomes, as can be seen in thin sections, were also well- preserved. The skeletal framework was visualized in three dimensions as an unembedded whole mount that revealed the filament networks that were masked in Epon-embedded thin sections of the same preparation. The topography of cytoskeletal filaments was relatively constant throughout the epithelial sheet, particularly across intercellular borders. This ordering of epithelial skeletal filaments across contiguous cell boundaries was in sharp contrast to the more independent organization of networks in autonomous cells such as fibroblasts. Further extraction removed the proteins of the salt-labile cytoskeleton and the chromatin as separate fractions, and left the nuclear matrix-intermediate filament (NM-IF) scaffold. The NM-IF contained only 5% of total cellular protein, but whole mount transmission electron microscopy and immunofluorescence showed that this scaffold was organized as in the intact epithelium. Immunoblots demonstrate that vimentin, cytokeratins, desmosomal proteins, and a 52,000-mol-wt nuclear matrix protein were found almost exclusively in the NM-IF scaffold. Vimentin was largely perinuclear while the cytokeratins were localized at the cell borders. The 52,000-mol-wt nuclear matrix protein was confined to the chromatin- depleted matrix and the desmosomal proteins were observed in punctate polygonal arrays at intercellular junctions. The filaments of the NM-IF were seen to be interconnected, via the desmosomes, over the entire epithelial colony. The differentiated epithelial morphology was reflected in both the cytoskeletal framework and the NM-IF scaffold. PMID:6202700

Epithelial-mesenchymal transition abolishes the susceptibility of polarized epithelial cell lines to measles virus.

PubMed

Shirogane, Yuta; Takeda, Makoto; Tahara, Maino; Ikegame, Satoshi; Nakamura, Takanori; Yanagi, Yusuke

2010-07-02

Measles virus (MV), an enveloped negative-strand RNA virus, remains a major cause of morbidity and mortality in developing countries. MV predominantly infects immune cells by using signaling lymphocyte activation molecule (SLAM; also called CD150) as a receptor, but it also infects polarized epithelial cells, forming tight junctions in a SLAM-independent manner. Although the ability of MV to infect polarized epithelial cells is thought to be important for its transmission, the epithelial cell receptor for MV has not been identified. A transcriptional repressor, Snail, induces epithelial-mesenchymal transition (EMT), in which epithelial cells lose epithelial cell phenotypes, such as adherens and tight junctions. In this study, EMT was induced by expressing Snail in a lung adenocarcinoma cell line, II-18, which is highly susceptible to wild-type MV. Snail-expressing II-18 cells lost adherens and tight junctions. Microarray analysis confirmed the induction of EMT in II-18 cells and suggested a novel function of Snail in protein degradation and distribution. Importantly, wild-type MV no longer entered EMT-induced II-18 cells, suggesting that the epithelial cell receptor is down-regulated by the induction of EMT. Other polarized cell lines, NCI-H358 and HT-29, also lost susceptibility to wild-type MV when EMT was induced. However, the complete formation of tight junctions rather reduced MV entry into HT-29 cells. Taken together, these data suggest that the unidentified epithelial cell receptor for MV is involved in the formation of epithelial intercellular junctions.

Ontogeny of the collar cord: neurulation in the hemichordate Saccoglossus kowalevskii.

PubMed

Kaul, Sabrina; Stach, Thomas

2010-10-01

The chordate body plan is characterized by a central notochord, a pharynx perforated by gill pores, and a dorsal central nervous system. Despite progress in recent years, the evolutionary origin of each of theses characters remains controversial. In the case of the nervous system, two contradictory hypotheses exist. In the first, the chordate nervous system is derived directly from a diffuse nerve net; whereas, the second proposes that a centralized nervous system is found in hemichordates and, therefore, predates chordate evolution. Here, we document the ontogeny of the collar cord of the enteropneust Saccoglossus kowalevskii using transmission electron microscopy and 3D-reconstruction based on completely serially sectioned stages. We demonstrate that the collar cord develops from a middorsal neural plate that is closed in a posterior to anterior direction. Transversely oriented ependymal cells possessing myofilaments mediate this morphogenetic process and surround the remnants of the neural canal in juveniles. A mid-dorsal glandular complex is present in the collar. The collar cord in juveniles is clearly separated into a dorsal saddle-like region of somata and a ventral neuropil. We characterize two cell types in the somata region, giant neurons and ependymal cells. Giant neurons connect via a peculiar cell junction that seems to function in intercellular communication. Synaptic junctions containing different vesicle types are present in the neuropil. These findings support the hypotheses that the collar cord constitutes a centralized element of the nervous system and that the morphogenetic process in the ontogeny of the collar cord is homologous to neurulation in chordates. Moreover, we suggest that these similarities are indicative of a close phylogenetic relationship between enteropneusts and chordates. ©2010 Wiley-Liss, Inc.

Direct Ca2+-dependent Heterophilic Interaction between Desmosomal Cadherins, Desmoglein and Desmocollin, Contributes to Cell–Cell Adhesion

PubMed Central

Chitaev, Nikolai A.; Troyanovsky, Sergey M.

1997-01-01

Human fibrosarcoma cells, HT-1080, feature extensive adherens junctions, lack mature desmosomes, and express a single known desmosomal protein, Desmoglein 2 (Dsg2). Transfection of these cells with bovine Desmocollin 1a (Dsc1a) caused dramatic changes in the subcellular distribution of endogenous Dsg2. Both cadherins clustered in the areas of the adherens junctions, whereas only a minor portion of Dsg2 was seen in these areas in the parental cells. Deletion mapping showed that intact extracellular cadherin-like repeats of Dsc1a (Arg1-Thr170) are required for the translocation of Dsg2. Deletion of the intracellular C-domain that mediates the interaction of Dsc1a with plakoglobin, or the CSI region that is involved in the binding to desmoplakin, had no effect. Coimmunoprecipitation experiments of cell lysates stably expressing Dsc1a with anti-Dsc or -Dsg antibodies demonstrate that the desmosomal cadherins, Dsg2 and Dsc1a, are involved in a direct Ca2+-dependent interaction. This conclusion was further supported by the results of solid phase binding experiments. These showed that the Dsc1a fragment containing cadherin-like repeats 1 and 2 binds directly to the extracellular portion of Dsg in a Ca2+-dependent manner. The contribution of the Dsg/ Dsc interaction to cell–cell adhesion was tested by coculturing HT-1080 cells expressing Dsc1a with HT-1080 cells lacking Dsc but expressing myc-tagged plakoglobin (MPg). In the latter cells, MPg and the endogenous Dsg form stable complexes. The observed specific coimmunoprecipitation of MPg by anti-Dsc antibodies in coculture indicates that an intercellular interaction between Dsc1 and Dsg is involved in cell–cell adhesion. PMID:9214392

Up-Regulation of Connexin43 in Glomerular Podocytes in Response to Injury

PubMed Central

Yaoita, Eishin; Yao, Jian; Yoshida, Yutaka; Morioka, Tetsuo; Nameta, Masaaki; Takata, Takuma; Kamiie, Jun-ichi; Fujinaka, Hidehiko; Oite, Takashi; Yamamoto, Tadashi

2002-01-01

Podocyte injury or podocyte loss in the renal glomerulus has been proposed as the crucial mechanism in the development of focal segmental glomerulosclerosis. However, it is poorly understood how podocytes respond to injury. In this study, glomerular expression of connexin43 (Cx43) gap junction protein was examined at both protein and transcript levels in an experimental model of podocyte injury, puromycin aminonucleoside (PAN) nephrosis. A striking increase in the number of immunoreactive dots with anti-Cx43 antibody was demonstrated along the glomerular capillary wall in the early to nephrotic stage of PAN nephrosis. The conspicuous change was not detected in the other areas including the mesangium and Bowman’s capsule. Immunoelectron microscopy showed that the immunogold particles for Cx43 along the capillary wall were localized predominantly at the cell-cell contact sites of podocytes. Consistently, Western blotting and ribonuclease protection assay revealed a distinct increase of Cx43 protein, phosphorylation, and transcript in glomeruli during PAN nephrosis. The changes were detected by 6 hours after PAN injection. These findings indicate that the increase of Cx43 expression is one of the earliest responses that have ever been reported in podocyte injury. To show the presence of functional gap junctional intercellular communication (GJIC) in podocytes, GJIC was assessed in podocytes in the primary culture by transfer of fluorescent dye, Lucifer yellow, after a single-cell microinjection. Diffusion of the dye into adjacent cells was observed frequently in the cultured podocytes, but scarcely in cultured parietal epithelial cells of Bowman’s capsule, which was compatible with their Cx43 staining. Thus, it is concluded that Cx43-mediated GJIC is present between podocytes, suggesting that podocytes may respond to injury as an integrated epithelium on a glomerulus rather than individually as a separate cell. PMID:12414508

Mouse Mast Cell Protease 4 and 5 Mediate Epidermal Injury Through Disruption of Tight Junctions

PubMed Central

Bankova, Lora G.; Lezcano, Cecilia; Pejler, Gunnar; Stevens, Richard L.; Murphy, George F.; Austen, K. Frank; Gurish, Michael F.

2014-01-01

We previously established a mast cell (MC)-dependent thermal injury model in mice with ulceration and scar formation that depended on non-redundant functions of mouse MC protease 4 (mMCP4) and mMCP5. We hypothesized that MC activation is an early event and now find by histology that exocytosis of granule contents occurred by 2 min after thermal injury in wild type (WT) C57BL/6 mice and in the mMCP4- or mMCP5-deficient mice. The degranulation was equivalent for MCs in the dermis and hypodermis of all three strains, but only the WT mice showed an appreciable increase in epidermal thickness. There was no loss of total MCs, partially degranulated plus intact, over the 4 h of observation. By electron microscopy, MCs in all strains showed early zonal degranulation at 30 s with marked progression in magnitude by 120 s and no mitochondrial injury or cellular necrosis. Concomitantly there was an increase in intercellular spaces indicative of tight junction (TJ) disruption in WT mice but not in the mMCP4- or mMCP5-deficient strains. The desmosomes were intact in all strains. Immunodetection of the TJ protein claudin 4 in WT and mMCP5-deficient mice indicated a significant reduction after scald injury while mMCP4−/− mice showed no significant changes. Taken together, these findings reveal that a second degree burn injury can initiate an immediate novel zonal degranulation of MCs throughout all skin layers and a disruption of the epidermal TJs dependent on the non-redundant presence of mMCP4 and mMCP5. PMID:24523504

Neurological manifestations of oculodentodigital dysplasia: a Cx43 channelopathy of the central nervous system?

PubMed Central

De Bock, Marijke; Kerrebrouck, Marianne; Wang, Nan; Leybaert, Luc

2013-01-01

The coordination of tissue function is mediated by gap junctions (GJs) that enable direct cell–cell transfer of metabolic and electric signals. GJs are formed by connexins of which Cx43 is most widespread in the human body. In the brain, Cx43 GJs are mostly found in astroglia where they coordinate the propagation of Ca2+ waves, spatial K+ buffering, and distribution of glucose. Beyond its role in direct intercellular communication, Cx43 also forms unapposed, non-junctional hemichannels in the plasma membrane of glial cells. These allow the passage of several neuro- and gliotransmitters that may, combined with downstream paracrine signaling, complement direct GJ communication among glial cells and sustain glial-neuronal signaling. Mutations in the GJA1 gene encoding Cx43 have been identified in a rare, mostly autosomal dominant syndrome called oculodentodigital dysplasia (ODDD). ODDD patients display a pleiotropic phenotype reflected by eye, hand, teeth, and foot abnormalities, as well as craniofacial and bone malformations. Remarkably, neurological symptoms such as dysarthria, neurogenic bladder (manifested as urinary incontinence), spasticity or muscle weakness, ataxia, and epilepsy are other prominent features observed in ODDD patients. Over 10 mutations detected in patients diagnosed with neurological disorders are associated with altered functionality of Cx43 GJs/hemichannels, but the link between ODDD-related abnormal channel activities and neurologic phenotype is still elusive. Here, we present an overview on the nature of the mutants conveying structural and functional changes of Cx43 channels and discuss available evidence for aberrant Cx43 GJ and hemichannel function. In a final step, we examine the possibilities of how channel dysfunction may lead to some of the neurological manifestations of ODDD. PMID:24133447

Effects of 18beta-glycyrrhetinic acid on the junctional complex and steroidogenesis in rat adrenocortical cells.

PubMed

Huang, Shih-Horng; Wu, Jiahn-Chun; Hwang, Ra-Der; Yeo, Hui-Lin; Wang, Seu-Mei

2003-09-01

Cellular junctions play important roles in cell differentiation, signal transduction, and cell function. This study investigated their function in steroid secretion by adrenal cells. Immunofluorescence staining revealed the presence of gap junctions and adherens junctions between adrenal cells. The major gap junction protein, connexin43, was seen as a linear dotted pattern of the typical gap junction plaques, in contrast to alpha-, beta-, and gamma-catenin, which were seen as continuous, linear staining of cell-cell adherens junction. Treatment with 18beta-glycyrrhetinic acid, a gap junction inhibitor, reduced the immunoreactivity of these proteins in a time- and dose-dependent manner, and caused the gap junction and adherens junction to separate longitudinally from the cell-cell contact sites, indicating the structural interdependency of these two junctions. Interestingly, 18beta-glycyrrhetinic acid stimulated a two- to three-fold increase in steroid production in these adrenal cells lacking intact cell junctions. These data raise the question of the necessity for cell communication for the endocrine function of adrenal cells. Pharmacological analyses indicated that the steroidogenic effect of 18beta-glycyrrhetinic acid was partially mediated by extracellular signal-related kinase and calcium/calmodulin-dependent kinase, a pathway distinct from the protein kinase A signaling pathway already known to mediate steroidogenesis in adrenal cells. Copyright 2003 Wiley-Liss, Inc.

Gap junctions modulate glioma invasion by direct transfer of microRNA.

PubMed

Hong, Xiaoting; Sin, Wun Chey; Harris, Andrew L; Naus, Christian C

2015-06-20

The invasiveness of high-grade glioma is the primary reason for poor survival following treatment. Interaction between glioma cells and surrounding astrocytes are crucial to invasion. We investigated the role of gap junction mediated miRNA transfer in this context. By manipulating gap junctions with a gap junction inhibitor, siRNAs, and a dominant negative connexin mutant, we showed that functional glioma-glioma gap junctions suppress glioma invasion while glioma-astrocyte and astrocyte-astrocyte gap junctions promote it in an in vitro transwell invasion assay. After demonstrating that glioma-astrocyte gap junctions are permeable to microRNA, we compared the microRNA profiles of astrocytes before and after co-culture with glioma cells, identifying specific microRNAs as candidates for transfer through gap junctions from glioma cells to astrocytes. Further analysis showed that transfer of miR-5096 from glioma cells to astrocytes is through gap junctions; this transfer is responsible, in part, for the pro-invasive effect. Our results establish a role for glioma-astrocyte gap junction mediated microRNA signaling in modulation of glioma invasive behavior, and that gap junction coupling among astrocytes magnifies the pro-invasive signaling. Our findings reveal the potential for therapeutic interventions based on abolishing alteration of stromal cells by tumor cells via manipulation of microRNA and gap junction channel activity.

Gap junctions modulate glioma invasion by direct transfer of microRNA

PubMed Central

Hong, Xiaoting; Sin, Wun Chey; Harris, Andrew L.; Naus, Christian C.

2015-01-01

The invasiveness of high-grade glioma is the primary reason for poor survival following treatment. Interaction between glioma cells and surrounding astrocytes are crucial to invasion. We investigated the role of gap junction mediated miRNA transfer in this context. By manipulating gap junctions with a gap junction inhibitor, siRNAs, and a dominant negative connexin mutant, we showed that functional glioma-glioma gap junctions suppress glioma invasion while glioma-astrocyte and astrocyte-astrocyte gap junctions promote it in an in vitro transwell invasion assay. After demonstrating that glioma-astrocyte gap junctions are permeable to microRNA, we compared the microRNA profiles of astrocytes before and after co-culture with glioma cells, identifying specific microRNAs as candidates for transfer through gap junctions from glioma cells to astrocytes. Further analysis showed that transfer of miR-5096 from glioma cells to astrocytes is through gap junctions; this transfer is responsible, in part, for the pro-invasive effect. Our results establish a role for glioma-astrocyte gap junction mediated microRNA signaling in modulation of glioma invasive behavior, and that gap junction coupling among astrocytes magnifies the pro-invasive signaling. Our findings reveal the potential for therapeutic interventions based on abolishing alteration of stromal cells by tumor cells via manipulation of microRNA and gap junction channel activity. PMID:25978028

Intercellular and intracellular signalling systems that globally control the expression of virulence genes in plant pathogenic bacteria.

PubMed

Ham, Jong Hyun

2013-04-01

Plant pathogenic bacteria utilize complex signalling systems to control the expression of virulence genes at the cellular level and within populations. Quorum sensing (QS), an important intercellular communication mechanism, is mediated by different types of small molecules, including N-acyl homoserine lactones (AHLs), fatty acids and small proteins. AHL-mediated signalling systems dependent on the LuxI and LuxR family proteins play critical roles in the virulence of a wide range of Gram-negative plant pathogenic bacteria belonging to the Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria. Xanthomonas spp. and Xylella fastidiosa, members of the Gammaproteobacteria, however, possess QS systems that are mediated by fatty acid-type diffusible signal factors (DSFs). Recent studies have demonstrated that Ax21, a 194-amino-acid protein in Xanthomonas oryzae pv. oryzae, plays dual functions in activating a rice innate immune pathway through binding to the rice XA21 pattern recognition receptor and in regulating bacterial virulence and biofilm formation as a QS signal molecule. In xanthomonads, DSF-mediated QS systems are connected with the signalling pathways mediated by cyclic diguanosine monophosphate (c-di-GMP), which functions as a second messenger for the control of virulence gene expression in these bacterial pathogens. © 2012 BSPP AND BLACKWELL PUBLISHING LTD.

Exosomes Mediate Intercellular Transmission of Porcine Reproductive and Respiratory Syndrome Virus.

PubMed

Wang, Ting; Fang, Liurong; Zhao, Fuwei; Wang, Dang; Xiao, Shaobo

2018-02-15

Exosomes are small membrane-enclosed vesicles produced by various cells and actively released into the extracellular space. They participate in intercellular communication and transfer of biologically active proteins, lipids, and nucleic acids. Accumulating evidence suggests that exosomes derived from cells infected by some viruses selectively encapsulate viral proteins, genetic materials, or even virions to mediate cell-to-cell communication and/or virus transmission. Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has been devastating the global swine industry since the late 1980s. Recent studies have shown that major proteins secreted from PRRSV-infected cells are exosomal proteins and that the serum-derived exosomes from PRRSV-infected pigs contain viral proteins. However, the role of exosomes in PRRSV infection remains unclear. In this study, purified exosomes isolated from PRRSV-infected cells were shown with reverse transcription-PCR and mass spectrometry to contain viral genomic RNA and partial viral proteins. Furthermore, exosomes from PRRSV-infected cells established productive infection in both PRRSV-susceptible and -nonsusceptible cells. More importantly, exosome-mediated infection was not completely blocked by PRRSV-specific neutralizing antibodies. In summary, this study demonstrated that exosomes can mediate PRRSV transmission and are even resistant to antibody neutralization, identifying a potential immune evasion mechanism utilized by PRRSV. IMPORTANCE Exosomes have recently been characterized as bioactive vesicles that function to promote intercellular communication. The exosomes from virally infected cells containing altered compositions confer numerous novel functionalities. A study of the secretome of cells infected with PRRSV indicated that the exosomal pathway is strongly activated by PRRSV infection. Here, we demonstrate that PRRSV can utilize host exosomes to infect naive healthy cells. Furthermore, exosome-mediated viral transmission is largely resistant to PRRSV-specific neutralizing antibodies. Our study provides novel insights into an alternative mechanism of PRRSV transmission that can compromise the host's anti-PRRSV immune response. Copyright © 2018 American Society for Microbiology.

The Molecular Basis of Communication between Cells.

ERIC Educational Resources Information Center

Snyder, Solomon H.

1985-01-01

Chemical messengers mediate long-range hormonal communication and short-range neural communication between cells. Background information on peptides, steroids, neuropeptides, and specialized enzymes is given. Investigations reveal that the two systems have many common intercellular messenger molecules. (DH)

Cysteine-rich peptides (CRPs) mediate diverse aspects of cell-cell communication in plant reproduction and development.

PubMed

Marshall, Eleanor; Costa, Liliana M; Gutierrez-Marcos, Jose

2011-03-01

Cell-cell communication in plants is essential for the correct co-ordination of reproduction, growth, and development. Studies to dissect this mode of communication have previously focussed primarily on the action of plant hormones as mediators of intercellular signalling. In animals, peptide signalling is a well-documented intercellular communication system, however, relatively little is known about this system in plants. In recent years, numerous reports have emerged about small, secreted peptides controlling different aspects of plant reproduction. Interestingly, most of these peptides are cysteine-rich, and there is convincing evidence suggesting multiple roles for related cysteine-rich peptides (CRPs) as signalling factors in developmental patterning as well as during plant pathogen responses and symbiosis. In this review, we discuss how CRPs are emerging as key signalling factors in regulating multiple aspects of vegetative growth and reproductive development in plants.

Tunneling nanotubes spread fibrillar α-synuclein by intercellular trafficking of lysosomes.

PubMed

Abounit, Saïda; Bousset, Luc; Loria, Frida; Zhu, Seng; de Chaumont, Fabrice; Pieri, Laura; Olivo-Marin, Jean-Christophe; Melki, Ronald; Zurzolo, Chiara

2016-10-04

Synucleinopathies such as Parkinson's disease are characterized by the pathological deposition of misfolded α-synuclein aggregates into inclusions throughout the central and peripheral nervous system. Mounting evidence suggests that intercellular propagation of α-synuclein aggregates may contribute to the neuropathology; however, the mechanism by which spread occurs is not fully understood. By using quantitative fluorescence microscopy with co-cultured neurons, here we show that α-synuclein fibrils efficiently transfer from donor to acceptor cells through tunneling nanotubes (TNTs) inside lysosomal vesicles. Following transfer through TNTs, α-synuclein fibrils are able to seed soluble α-synuclein aggregation in the cytosol of acceptor cells. We propose that donor cells overloaded with α-synuclein aggregates in lysosomes dispose of this material by hijacking TNT-mediated intercellular trafficking. Our findings thus reveal a possible novel role of TNTs and lysosomes in the progression of synucleinopathies. © 2016 The Authors.

Adrenocortical Gap Junctions and Their Functions

PubMed Central

Bell, Cheryl L.; Murray, Sandra A.

2016-01-01

Adrenal cortical steroidogenesis and proliferation are thought to be modulated by gap junction-mediated direct cell–cell communication of regulatory molecules between cells. Such communication is regulated by the number of gap junction channels between contacting cells, the rate at which information flows between these channels, and the rate of channel turnover. Knowledge of the factors regulating gap junction-mediated communication and the turnover process are critical to an understanding of adrenal cortical cell functions, including development, hormonal response to adrenocorticotropin, and neoplastic dedifferentiation. Here, we review what is known about gap junctions in the adrenal gland, with particular attention to their role in adrenocortical cell steroidogenesis and proliferation. Information and insight gained from electrophysiological, molecular biological, and imaging (immunocytochemical, freeze fracture, transmission electron microscopic, and live cell) techniques will be provided. PMID:27445985

Mechanisms that determine the internal environment of the developing brain: a transcriptomic, functional and ultrastructural approach.

PubMed

Liddelow, Shane A; Dziegielewska, Katarzyna M; Ek, C Joakim; Habgood, Mark D; Bauer, Hannelore; Bauer, Hans-Christian; Lindsay, Helen; Wakefield, Matthew J; Strazielle, Nathalie; Kratzer, Ingrid; Møllgård, Kjeld; Ghersi-Egea, Jean-François; Saunders, Norman R

2013-01-01

We provide comprehensive identification of embryonic (E15) and adult rat lateral ventricular choroid plexus transcriptome, with focus on junction-associated proteins, ionic influx transporters and channels. Additionally, these data are related to new structural and previously published permeability studies. Results reveal that most genes associated with intercellular junctions are expressed at similar levels at both ages. In total, 32 molecules known to be associated with brain barrier interfaces were identified. Nine claudins showed unaltered expression, while two claudins (6 and 8) were expressed at higher levels in the embryo. Expression levels for most cytoplasmic/regulatory adaptors (10 of 12) were similar at the two ages. A few junctional genes displayed lower expression in embryos, including 5 claudins, occludin and one junctional adhesion molecule. Three gap junction genes were enriched in the embryo. The functional effectiveness of these junctions was assessed using blood-delivered water-soluble tracers at both the light and electron microscopic level: embryo and adult junctions halted movement of both 286Da and 3kDa molecules into the cerebrospinal fluid (CSF). The molecular identities of many ion channel and transporter genes previously reported as important for CSF formation and secretion in the adult were demonstrated in the embryonic choroid plexus (and validated with immunohistochemistry of protein products), but with some major age-related differences in expression. In addition, a large number of previously unidentified ion channel and transporter genes were identified for the first time in plexus epithelium. These results, in addition to data obtained from electron microscopical and physiological permeability experiments in immature brains, indicate that exchange between blood and CSF is mainly transcellular, as well-formed tight junctions restrict movement of small water-soluble molecules from early in development. These data strongly indicate the brain develops within a well-protected internal environment and the exchange between the blood, brain and CSF is transcellular and not through incomplete barriers.

Fine structure of the ependyma and intercellular junctions in the area postrema of the rat.

PubMed

Gotow, T; Hashimoto, P H

1979-09-03

Ependymal cells and their junctional complexes in the area postrema of the rat were studied in detail by tracer experiments using horseradish peroxidase (HRP) and colloidal lanthanum and by freeze-etch techniques, in addition to routine electron microscopy. The ependyma of the area postrema is characterized as flattened cells possessing very few cilia, a moderate amount of microvilli, a well-developed Golgi apparatus and rough endoplasmic reticulum. Numerous vesicles or tubular formations with internal dense content were found to accumulate in the basal processes of ependymal cells; the basal process makes contact with the perivascular basal lamina. It is suggested that the dense material in the tubulovesicular formations is synthesized within the ependymal cell and discharged into the perivascular space. The apical junctions between adjacent ependymal cells display very close apposition, with a gap of 2--3 nm, but no fusion of adjacent plasma membranes; they thus represent a transitional form between the zonulae adhaerentes present in the ordinary mural ependyma and the zonulae occludentes in the choroidal epithelium. A direct intercommunication between the ventricular cerebrospinal fluid (CSF) and the blood vascular system indicates that a region exists lacking a blood-ventricular CSF barrier.

Branched actin networks push against each other at adherens junctions to maintain cell-cell adhesion.

PubMed

Efimova, Nadia; Svitkina, Tatyana M

2018-05-07

Adherens junctions (AJs) are mechanosensitive cadherin-based intercellular adhesions that interact with the actin cytoskeleton and carry most of the mechanical load at cell-cell junctions. Both Arp2/3 complex-dependent actin polymerization generating pushing force and nonmuscle myosin II (NMII)-dependent contraction producing pulling force are necessary for AJ morphogenesis. Which actin system directly interacts with AJs is unknown. Using platinum replica electron microscopy of endothelial cells, we show that vascular endothelial (VE)-cadherin colocalizes with Arp2/3 complex-positive actin networks at different AJ types and is positioned at the interface between two oppositely oriented branched networks from adjacent cells. In contrast, actin-NMII bundles are located more distally from the VE-cadherin-rich zone. After Arp2/3 complex inhibition, linear AJs split, leaving gaps between cells with detergent-insoluble VE-cadherin transiently associated with the gap edges. After NMII inhibition, VE-cadherin is lost from gap edges. We propose that the actin cytoskeleton at AJs acts as a dynamic push-pull system, wherein pushing forces maintain extracellular VE-cadherin transinteraction and pulling forces stabilize intracellular adhesion complexes. © 2018 Efimova and Svitkina.

EMP-1 is a junctional protein in a liver stem cell line and in the liver.

PubMed

Lee, Hsuan-Shu; Sherley, James L; Chen, Jeremy J W; Chiu, Chien-Chang; Chiou, Ling-Ling; Liang, Ja-Der; Yang, Pan-Chyr; Huang, Guan-Tarn; Sheu, Jin-Chuan

2005-09-09

In an attempt to discover cell markers for liver stem cells, a cDNA microarray analysis was carried out to compare the gene expression profiles between an adult liver stem cell line, Lig-8, and mature hepatocytes. Several genes in the categories of extracellular matrix, cell membrane, cell adhesion, transcription factor, signal molecule, transporter, and metabolic enzyme were shown to be differentially expressed in Lig-8 cells. Among them, epithelial membrane protein (EMP)-1 has been previously implicated with stem cell phenotypes. Antiserum to EMP-1 was produced to localize its expression. On monolayers of Lig-8 cells, EMP-1 was expressed along the intercellular border. In the liver harboring proliferating oval cells, the liver progenitors, EMP-1 was localized as ribbon bands, a staining pattern for epithelial junctions, all the way through bile duct epithelia, oval cell ductules, and into peri-hepatocytic regions. These peri-hepatocytic regions were proved to be bile canaliculi by co-localization of EMP-1 and dipeptidyl peptidase IV, an enzyme located on bile canaliculi. This report is the first to indicate EMP-1 to be a junctional protein in the liver.

The biological significance of brain barrier mechanisms: help or hindrance in drug delivery to the central nervous system?

PubMed Central

Saunders, Norman R.; Habgood, Mark D.; Møllgård, Kjeld; Dziegielewska, Katarzyna M.

2016-01-01

Barrier mechanisms in the brain are important for its normal functioning and development. Stability of the brain’s internal environment, particularly with respect to its ionic composition, is a prerequisite for the fundamental basis of its function, namely transmission of nerve impulses. In addition, the appropriate and controlled supply of a wide range of nutrients such as glucose, amino acids, monocarboxylates, and vitamins is also essential for normal development and function. These are all cellular functions across the interfaces that separate the brain from the rest of the internal environment of the body. An essential morphological component of all but one of the barriers is the presence of specialized intercellular tight junctions between the cells comprising the interface: endothelial cells in the blood-brain barrier itself, cells of the arachnoid membrane, choroid plexus epithelial cells, and tanycytes (specialized glial cells) in the circumventricular organs. In the ependyma lining the cerebral ventricles in the adult brain, the cells are joined by gap junctions, which are not restrictive for intercellular movement of molecules. But in the developing brain, the forerunners of these cells form the neuroepithelium, which restricts exchange of all but the smallest molecules between cerebrospinal fluid and brain interstitial fluid because of the presence of strap junctions between the cells. The intercellular junctions in all these interfaces are the physical basis for their barrier properties. In the blood-brain barrier proper, this is combined with a paucity of vesicular transport that is a characteristic of other vascular beds. Without such a diffusional restrain, the cellular transport mechanisms in the barrier interfaces would be ineffective. Superimposed on these physical structures are physiological mechanisms as the cells of the interfaces contain various metabolic transporters and efflux pumps, often ATP-binding cassette (ABC) transporters, that provide an important component of the barrier functions by either preventing entry of or expelling numerous molecules including toxins, drugs, and other xenobiotics. In this review, we summarize these influx and efflux mechanisms in normal developing and adult brain, as well as indicating their likely involvement in a wide range of neuropathologies. There have been extensive attempts to overcome the barrier mechanisms that prevent the entry of many drugs of therapeutic potential into the brain. We outline those that have been tried and discuss why they may so far have been largely unsuccessful. Currently, a promising approach appears to be focal, reversible disruption of the blood-brain barrier using focused ultrasound, but more work is required to evaluate the method before it can be tried in patients. Overall, our view is that much more fundamental knowledge of barrier mechanisms and development of new experimental methods will be required before drug targeting to the brain is likely to be a successful endeavor. In addition, such studies, if applied to brain pathologies such as stroke, trauma, or multiple sclerosis, will aid in defining the contribution of brain barrier pathology to these conditions, either causative or secondary. PMID:26998242

An epidermal plakin that integrates actin and microtubule networks at cellular junctions.

PubMed

Karakesisoglou, I; Yang, Y; Fuchs, E

2000-04-03

Plakins are cytoskeletal linker proteins initially thought to interact exclusively with intermediate filaments (IFs), but recently were found to associate additionally with actin and microtubule networks. Here, we report on ACF7, a mammalian orthologue of the Drosophila kakapo plakin genetically involved in epidermal-muscle adhesion and neuromuscular junctions. While ACF7/kakapo is divergent from other plakins in its IF-binding domain, it has at least one actin (K(d) = 0.35 microM) and one microtubule (K(d) approximately 6 microM) binding domain. Similar to its fly counterpart, ACF7 is expressed in the epidermis. In well spread epidermal keratinocytes, ACF7 discontinuously decorates the cytoskeleton at the cell periphery, including microtubules (MTs) and actin filaments (AFs) that are aligned in parallel converging at focal contacts. Upon calcium induction of intercellular adhesion, ACF7 and the cytoskeleton reorganize at cell-cell borders but with different kinetics from adherens junctions and desmosomes. Treatments with cytoskeletal depolymerizing drugs reveal that ACF7's cytoskeletal association is dependent upon the microtubule network, but ACF7 also appears to stabilize actin at sites where microtubules and microfilaments meet. We posit that ACF7 may function in microtubule dynamics to facilitate actin-microtubule interactions at the cell periphery and to couple the microtubule network to cellular junctions. These attributes provide a clear explanation for the kakapo mutant phenotype in flies.

Cell-to-cell contact dependence and junctional protein content are correlated with in vivo maturation of pancreatic beta cells.

PubMed

Santos-Silva, Junia Carolina; Carvalho, Carolina Prado de França; de Oliveira, Ricardo Beltrame; Boschero, Antonio Carlos; Collares-Buzato, Carla Beatriz

2012-07-01

In this study, we investigated the cellular distribution of junctional proteins and the dependence on cell-cell contacts of pancreatic beta cells during animal development. Fetus and newborn rat islets, which display a relatively poor insulin secretory response to glucose, present an immature morphology and cytoarchitecture when compared with young and adult islets that are responsive to glucose. At the perinatal stage, beta cells display a low junctional content of neural cell adhesion molecule (N-CAM), α- and β-catenins, ZO-1, and F-actin, while a differential distribution of N-CAM and Pan-cadherin was seen in beta cells and nonbeta cells only from young and adult islets. In the absence of intercellular contacts, the glucose-stimulated insulin secretion was completely blocked in adult beta cells, but after reaggregation they partially reestablished the secretory response to glucose. By contrast, neonatal beta cells were poorly responsive to sugar, regardless of whether they were arranged as intact islets or as isolated cells. Interestingly, after 10 days of culturing, neonatal beta cells, known to display increased junctional protein content in vitro, became responsive to glucose and concomitantly dependent on cell-cell contacts. Therefore, our data suggest that the developmental acquisition of an adult-like insulin secretory pattern is paralleled by a dependence on direct cell-cell interactions.

A non-cell-autonomous role for Ras signaling in C. elegans neuroblast delamination

PubMed Central

Parry, Jean M.; Sundaram, Meera V.

2014-01-01

Receptor tyrosine kinase (RTK) signaling through Ras influences many aspects of normal cell behavior, including epithelial-to-mesenchymal transition, and aberrant signaling promotes both tumorigenesis and metastasis. Although many such effects are cell-autonomous, here we show a non-cell-autonomous role for RTK-Ras signaling in the delamination of a neuroblast from an epithelial organ. The C. elegans renal-like excretory organ is initially composed of three unicellular epithelial tubes, namely the canal, duct and G1 pore cells; however, the G1 cell later delaminates from the excretory system to become a neuroblast and is replaced by the G2 cell. G1 delamination and G2 intercalation involve cytoskeletal remodeling, interconversion of autocellular and intercellular junctions and migration over a luminal extracellular matrix, followed by G1 junction loss. LET-23/EGFR and SOS-1, an exchange factor for Ras, are required for G1 junction loss but not for initial cytoskeletal or junction remodeling. Surprisingly, expression of activated LET-60/Ras in the neighboring duct cell, but not in the G1 or G2 cells, is sufficient to rescue sos-1 delamination defects, revealing that Ras acts non-cell-autonomously to permit G1 delamination. We suggest that, similarly, oncogenic mutations in cells within a tumor might help create a microenvironment that is permissive for other cells to detach and ultimately metastasize. PMID:25371363

Myosin VI facilitates connexin 43 gap junction accretion.

PubMed

Waxse, Bennett J; Sengupta, Prabuddha; Hesketh, Geoffrey G; Lippincott-Schwartz, Jennifer; Buss, Folma

2017-03-01

In this study, we demonstrate myosin VI enrichment at Cx43 (also known as GJA1)-containing gap junctions (GJs) in heart tissue, primary cardiomyocytes and cell culture models. In primary cardiac tissue and in fibroblasts from the myosin VI-null mouse as well as in tissue culture cells transfected with siRNA against myosin VI, we observe reduced GJ plaque size with a concomitant reduction in intercellular communication, as shown by fluorescence recovery after photobleaching (FRAP) and a new method of selective calcein administration. Analysis of the molecular role of myosin VI in Cx43 trafficking indicates that myosin VI is dispensable for the delivery of Cx43 to the cell surface and connexon movement in the plasma membrane. Furthermore, we cannot corroborate clathrin or Dab2 localization at gap junctions and we do not observe a function for the myosin-VI-Dab2 complex in clathrin-dependent endocytosis of annular gap junctions. Instead, we found that myosin VI was localized at the edge of Cx43 plaques by using total internal reflection fluorescence (TIRF) microscopy and use FRAP to identify a plaque accretion defect as the primary manifestation of myosin VI loss in Cx43 homeostasis. A fuller understanding of this derangement may explain the cardiomyopathy or gliosis associated with the loss of myosin VI. © 2017. Published by The Company of Biologists Ltd.

Cadmium disorganises the scaffolding of gap and tight junction proteins in the hepatic cell line WIF B9.

PubMed

Boucherie, Sylviane; Decaens, Catherine; Verbavatz, Jean-Marc; Grosse, Brigitte; Erard, Marie; Merola, Fabienne; Cassio, Doris; Combettes, Laurent

2013-12-01

Hepatocytes, which perform the main functions of the liver, are particularly vulnerable to toxic agents such as cadmium, an environmental pollutant. To identify the molecular targets for cadmium in hepatocytes, we have studied the effects of CdCl2 on the hybrid cell line WIF-B9 that exhibits stable structural and functional hepatocytic polarity. We showed that the toxicity of CdCl2 (1 µM, 24 h) resulted in a reduction in direct intercellular communication (via gap junctions) and in an increase in paracellular permeability (decrease in the sealing of tight junctions). These effects were not related to changes in the expression of the key proteins involved, Cx32 and claudin 2, the first being constitutive of gap junctions and the second of tight junctions in this cell line. Using immunofluorescence experiments, we observed a change in the location of Cx32 and claudin 2: these two proteins were less often found in the tight junction network that closes the bile canaliculi (BC). In control cells, 'Proximity Ligation Assay' (PLA Duolink®) has confirmed in situ that molecules of claudin 2 and Cx32 are very close to each other at the BC (probably less than 16 nm). This was no longer the case after treatment with CdCl2 . Localisation of occludin and Cx32 relative to each other was not modified by CdCl2 , but CdCl2 increased the PLA signal between molecules of JAM-A and Cx32. Finally, examination of freeze-fracture replicas obtained from cultures treated with CdCl2 showed the disruption of the network of tight junctions and the depletion or the disintegration of the junctional plaques associated with tight junctions. This study demonstrates in situ the changes induced by cadmium on the organisation of cell-cell junctions and points out the importance of the association Cx32/claudin 2 for the maintenance of normal hepatocyte functions. © 2013 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

Gap junction blockade induces apoptosis in human endometrial stromal cells.

PubMed

Yu, Jie; Berga, Sarah L; Zou, Wei; Sun, He-Ying; Johnston-MacAnanny, Erika; Yalcinkaya, Tamer; Sidell, Neil; Bagchi, Indrani C; Bagchi, Milan K; Taylor, Robert N

2014-07-01

One of the most dynamic adult human tissues is the endometrium. Through coordinated, cyclical proliferation, differentiation, leukocyte recruitment, apoptosis, and desquamation, the uterine lining is expanded and shed monthly, unless pregnancy is established. Errors in these steps potentially cause endometrial dysfunction, abnormal uterine bleeding, failed embryonic implantation, infertility, or endometrial carcinoma. Our prior studies showed that gap junctions comprised of Gap junction alpha-1 (GJA1) protein, also known as connexin 43 (CX43), subunits are critical to endometrial stromal cell differentiation. The current studies were undertaken to explore the mechanism of endometrial dysfunction when gap junction intercellular communication (GJIC) is disrupted. Gap junction blockade by two distinct GJIC inhibitors, 18α-glycyrrhetinic acid (AGA) and octanol (OcOH), suppressed proliferation and induced apoptosis in endometrial stromal cells, as manifested by reduced biomarkers of cell viability, increased TUNEL staining, caspase-3 activation, sub-G1 chromosomal DNA complement, as well as shortened telomere length. Unexpectedly, we also observed that the chemical inhibitors blocked CX43 gene expression. Moreover, when endometrial stromal cells were induced to undergo hormonal decidualization, following a 7-day exposure to 10 nM 17β-estradiol + 100 nM progesterone + 0.5 mM dibutyryl cAMP, characteristic epithelioid changes in cell shape and secretion of prolactin were blunted in the presence of AGA or OcOH, recapitulating effects of RNA interference of CX43. Our findings indicate that endometrial stromal cell proliferation and maintenance of decidualized endometrial function are GJIC-dependent, and that disruption of gap junctions induces endometrial stromal cell apoptosis. These observations may have important implications for several common clinical endometrial pathologies. © 2014 Wiley Periodicals, Inc.

Regulation of osteoclastogenesis by gap junction communication.

PubMed

Matemba, Stephen F; Lie, Anita; Ransjö, Maria

2006-10-01

Receptor activator of NF-kappaB ligand (RANKL) is crucial in osteoclastogenesis but signaling events involved in osteoclast differentiation are far from complete and other signals may play a role in osteoclastogenesis. A more direct pathway for cellular crosstalk is provided by gap junction intercellular channel, which allows adjacent cells to exchange second messengers, ions, and cellular metabolites. Here we have investigated the role of gap junction communication in osteoclastogenesis in mouse bone marrow cultures. Immunoreactive sites for the gap junction protein connexin 43 (Cx43) were detected in the marrow stromal cells and in mature osteoclasts. Carbenoxolone (CBX) functionally blocked gap junction communication as demonstrated by a scrape loading Lucifer Yellow dye transfer technique. CBX caused a dose-dependent inhibition (significant > or = 90 microM) of the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells formed in 7- to 8-day marrow cultures stimulated by parathyroid hormone (PTH; 10 nM) or forskolin (FSK; 1 microM). Furthermore, CBX (100 microM) significantly inhibited prostaglandin E2 (PGE2; 10 microM) and 1,25(OH)2-vitamin D3 stimulated osteoclast differentiation in the mouse bone marrow cultures. Consequently, quantitative real-time polymerase chain reaction (PCR) analysis demonstrated that CBX downregulated the expression of osteoclast phenotypic markers, but without having any significant effects on RANK, RANKL, and osteoprotegerin (OPG) mRNA expression. However, the results demonstrated that CBX significantly inhibits RANKL-stimulated (100 ng/ml) osteoclastogenesis in the mouse bone marrow cultures. Taken together, our results suggests that gap junctional diffusion of messenger molecules interacts with signaling pathways downstream RANKL in osteoclast differentiation. Further studies are required to define the precise mechanisms and molecular targets involved. Copyright 2006 Wiley-Liss, Inc.

Neuronal gap junctions play a role in the secondary neuronal death following controlled cortical impact.

PubMed

Belousov, Andrei B; Wang, Yongfu; Song, Ji-Hoon; Denisova, Janna V; Berman, Nancy E; Fontes, Joseph D

2012-08-22

In the mammalian CNS, excessive release of glutamate and overactivation of glutamate receptors are responsible for the secondary (delayed) neuronal death following neuronal injury, including ischemia, traumatic brain injury (TBI) and epilepsy. Recent studies in mice showed a critical role for neuronal gap junctions in NMDA receptor-mediated excitotoxicity and ischemia-mediated neuronal death. Here, using controlled cortical impact (CCI) in adult mice, as a model of TBI, and Fluoro-Jade B staining for analysis of neuronal death, we set to determine whether neuronal gap junctions play a role in the CCI-mediated secondary neuronal death. We report that 24h post-CCI, substantial neuronal death is detected in a number of brain regions outside the injury core, including the striatum. The striatal neuronal death is reduced both in wild-type mice by systemic administration of mefloquine (a relatively selective blocker of neuronal gap junctions) and in knockout mice lacking connexin 36 (neuronal gap junction protein). It is also reduced by inactivation of group II metabotropic glutamate receptors (with LY341495) which, as reported previously, control the rapid increase in neuronal gap junction coupling following different types of neuronal injury. The results suggest that neuronal gap junctions play a critical role in the CCI-induced secondary neuronal death. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Ultrastructure of myocardial widened Z bands and endocardial cells in two teleostean species.

PubMed

Leknes, I L

1981-01-01

Widened myocardial Z bands and endocardial cells are described in two teleostean species Cichlasoma meeki and Corydoras aeneus. Widened Z bands containing mainly amorphous and electron-dense material were seen in a number of myocardial cells. Further, similar material may occur in large amounts beneath the sarcolemma and at intercellular junctions. Occasionally, we observed continuity between the latter material and that in expanded Z bands. In C. meeki the ventricular endocardial layer consists of two structurally different cell types, whereas in C. aeneus only one cell type was seen. The functional aspects of widened Z bands are discussed.

Mechanically induced intercellular calcium communication in confined endothelial structures.

PubMed

Junkin, Michael; Lu, Yi; Long, Juexuan; Deymier, Pierre A; Hoying, James B; Wong, Pak Kin

2013-03-01

Calcium signaling in the diverse vascular structures is regulated by a wide range of mechanical and biochemical factors to maintain essential physiological functions of the vasculature. To properly transmit information, the intercellular calcium communication mechanism must be robust against various conditions in the cellular microenvironment. Using plasma lithography geometric confinement, we investigate mechanically induced calcium wave propagation in networks of human umbilical vein endothelial cells organized. Endothelial cell networks with confined architectures were stimulated at the single cell level, including using capacitive force probes. Calcium wave propagation in the network was observed using fluorescence calcium imaging. We show that mechanically induced calcium signaling in the endothelial networks is dynamically regulated against a wide range of probing forces and repeated stimulations. The calcium wave is able to propagate consistently in various dimensions from monolayers to individual cell chains, and in different topologies from linear patterns to cell junctions. Our results reveal that calcium signaling provides a robust mechanism for cell-cell communication in networks of endothelial cells despite the diversity of the microenvironmental inputs and complexity of vascular structures. Copyright © 2012 Elsevier Ltd. All rights reserved.

Increase in the adhesion molecule P-selectin in endothelium overlying atherosclerotic plaques. Coexpression with intercellular adhesion molecule-1.

PubMed Central

Johnson-Tidey, R. R.; McGregor, J. L.; Taylor, P. R.; Poston, R. N.

1994-01-01

P-selectin (GMP-140) is an adhesion molecule present within endothelial cells that is rapidly translocated to the cell membrane upon activation, where it mediates endothelial-leukocyte interactions. Immunohistochemical analysis of human atherosclerotic plaques has shown strong expression of P-selectin by the endothelium overlying active atherosclerotic plaques. P-selectin is not, however, detected in normal arterial endothelium or in endothelium overlying inactive fibrous plaques. Color image analysis was used to quantitate the degree of P-selectin expression in the endothelium and demonstrates a statistically significant increase in P-selectin expression by atherosclerotic endothelial cells. Double immunofluorescence shows that some of this P-selectin is expressed on the luminal surface of the endothelial cells. Previous work has demonstrated a significant up-regulation in the expression of the intercellular adhesion molecule-1 in atherosclerotic endothelium and a study on the expression of intercellular adhesion molecule-1 and P-selectin in atherosclerosis shows a highly positive correlation. These results suggest that the selective and cooperative expression of P-selectin and intercellular adhesion molecule-1 may be involved in the recruitment of monocytes into sites of atherosclerosis. Images Figure 1 Figure 3 Figure 4 Figure 5 PMID:7513951

Calcium Channels and Oxidative Stress Mediate a Synergistic Disruption of Tight Junctions by Ethanol and Acetaldehyde in Caco-2 Cell Monolayers.

PubMed

Samak, Geetha; Gangwar, Ruchika; Meena, Avtar S; Rao, Roshan G; Shukla, Pradeep K; Manda, Bhargavi; Narayanan, Damodaran; Jaggar, Jonathan H; Rao, RadhaKrishna

2016-12-13

Ethanol is metabolized into acetaldehyde in most tissues. In this study, we investigated the synergistic effect of ethanol and acetaldehyde on the tight junction integrity in Caco-2 cell monolayers. Expression of alcohol dehydrogenase sensitized Caco-2 cells to ethanol-induced tight junction disruption and barrier dysfunction, whereas aldehyde dehydrogenase attenuated acetaldehyde-induced tight junction disruption. Ethanol up to 150 mM did not affect tight junction integrity or barrier function, but it dose-dependently increased acetaldehyde-mediated tight junction disruption and barrier dysfunction. Src kinase and MLCK inhibitors blocked this synergistic effect of ethanol and acetaldehyde on tight junction. Ethanol and acetaldehyde caused a rapid and synergistic elevation of intracellular calcium. Calcium depletion by BAPTA or Ca 2+ -free medium blocked ethanol and acetaldehyde-induced barrier dysfunction and tight junction disruption. Diltiazem and selective knockdown of TRPV6 or Ca V 1.3 channels, by shRNA blocked ethanol and acetaldehyde-induced tight junction disruption and barrier dysfunction. Ethanol and acetaldehyde induced a rapid and synergistic increase in reactive oxygen species by a calcium-dependent mechanism. N-acetyl-L-cysteine and cyclosporine A, blocked ethanol and acetaldehyde-induced barrier dysfunction and tight junction disruption. These results demonstrate that ethanol and acetaldehyde synergistically disrupt tight junctions by a mechanism involving calcium, oxidative stress, Src kinase and MLCK.

Polypetide signaling molecules in plant development

USDA-ARS?s Scientific Manuscript database

Intercellular communication mediated by small signaling molecules is a key mechanism for coordinating plant growth and development. In the past few years, polypeptide signals have been shown to play prominent roles in processes as diverse as shoot and root meristem maintenance, vascular differentiat...

Traction force dynamics predict gap formation in activated endothelium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Valent, Erik T.; Nieuw Amerongen, Geerten P. van; Hinsbergh, Victor W.M. van

In many pathological conditions the endothelium becomes activated and dysfunctional, resulting in hyperpermeability and plasma leakage. No specific therapies are available yet to control endothelial barrier function, which is regulated by inter-endothelial junctions and the generation of acto-myosin-based contractile forces in the context of cell-cell and cell-matrix interactions. However, the spatiotemporal distribution and stimulus-induced reorganization of these integral forces remain largely unknown. Traction force microscopy of human endothelial monolayers was used to visualize contractile forces in resting cells and during thrombin-induced hyperpermeability. Simultaneously, information about endothelial monolayer integrity, adherens junctions and cytoskeletal proteins (F-actin) were captured. This revealed a heterogeneousmore » distribution of traction forces, with nuclear areas showing lower and cell-cell junctions higher traction forces than the whole-monolayer average. Moreover, junctional forces were asymmetrically distributed among neighboring cells. Force vector orientation analysis showed a good correlation with the alignment of F-actin and revealed contractile forces in newly formed filopodia and lamellipodia-like protrusions within the monolayer. Finally, unstable areas, showing high force fluctuations within the monolayer were prone to form inter-endothelial gaps upon stimulation with thrombin. To conclude, contractile traction forces are heterogeneously distributed within endothelial monolayers and force instability, rather than force magnitude, predicts the stimulus-induced formation of intercellular gaps. - Highlights: • Endothelial monolayers exert dynamic- and heterogeneous traction forces. • High traction forces correlate with junctional areas and the F-actin cytoskeleton. • Newly formed inter-endothelial gaps are characterized by opposing traction forces. • Force stability is a key feature controlling endothelial permeability.« less

Cues to Opening Mechanisms From in Silico Electric Field Excitation of Cx26 Hemichannel and in Vitro Mutagenesis Studies in HeLa Transfectans

PubMed Central

Zonta, Francesco; Buratto, Damiano; Crispino, Giulia; Carrer, Andrea; Bruno, Francesca; Yang, Guang; Mammano, Fabio; Pantano, Sergio

2018-01-01

Connexin channels play numerous essential roles in virtually every organ by mediating solute exchange between adjacent cells, or between cytoplasm and extracellular milieu. Our understanding of the structure-function relationship of connexin channels relies on X-ray crystallographic data for human connexin 26 (hCx26) intercellular gap junction channels. Comparison of experimental data and molecular dynamics simulations suggests that the published structures represent neither fully-open nor closed configurations. To facilitate the search for alternative stable configurations, we developed a coarse grained (CG) molecular model of the hCx26 hemichannel and studied its responses to external electric fields. When challenged by a field of 0.06 V/nm, the hemichannel relaxed toward a novel configuration characterized by a widened pore and an increased bending of the second transmembrane helix (TM2) at the level of the conserved Pro87. A point mutation that inhibited such transition in our simulations impeded hemichannel opening in electrophysiology and dye uptake experiments conducted on HeLa tranfectants. These results suggest that the hCx26 hemichannel uses a global degree of freedom to transit between different configuration states, which may be shared among the whole connexin family. PMID:29904340

Neurobeachin is required postsynaptically for electrical and chemical synapse formation

PubMed Central

Miller, Adam C.; Voelker, Lisa H.; Shah, Arish N.; Moens, Cecilia B.

2014-01-01

Summary Background Neural networks and their function are defined by synapses, which are adhesions specialized for intercellular communication that can be either chemical or electrical. At chemical synapses transmission between neurons is mediated by neurotransmitters, while at electrical synapses direct ionic and metabolic coupling occurs via gap junctions between neurons. The molecular pathways required for electrical synaptogenesis are not well understood and whether they share mechanisms of formation with chemical synapses is not clear. Results Here, using a forward genetic screen in zebrafish we find that the autism-associated gene neurobeachin (nbea), which encodes a BEACH-domain containing protein implicated in endomembrane trafficking, is required for both electrical and chemical synapse formation. Additionally, we find that nbea is dispensable for axonal formation and early dendritic outgrowth, but is required to maintain dendritic complexity. These synaptic and morphological defects correlate with deficiencies in behavioral performance. Using chimeric animals in which individually identifiable neurons are either mutant or wildtype we find that Nbea is necessary and sufficient autonomously in the postsynaptic neuron for both synapse formation and dendritic arborization. Conclusions Our data identify a surprising link between electrical and chemical synapse formation and show that Nbea acts as a critical regulator in the postsynaptic neuron for the coordination of dendritic morphology with synaptogenesis. PMID:25484298

Regulation of Connexin-Based Channels by Fatty Acids

PubMed Central

Puebla, Carlos; Retamal, Mauricio A.; Acuña, Rodrigo; Sáez, Juan C.

2017-01-01

In this mini-review, we briefly summarize the current knowledge about the effects of fatty acids (FAs) on connexin-based channels, as well as discuss the limited information about the impact FAs may have on pannexins (Panxs). FAs regulate diverse cellular functions, some of which are explained by changes in the activity of channels constituted by connexins (Cxs) or Panxs, which are known to play critical roles in maintaining the functional integrity of diverse organs and tissues. Cxs are transmembrane proteins that oligomerize into hexamers to form hemichannels (HCs), which in turn can assemble into dodecamers to form gap junction channels (GJCs). While GJCs communicate the cytoplasm of contacting cells, HCs serve as pathways for the exchange of ions and small molecules between the intra and extracellular milieu. Panxs, as well as Cx HCs, form channels at the plasma membrane that enable the interchange of molecules between the intra and extracellular spaces. Both Cx- and Panx-based channels are controlled by several post-translational modifications. However, the mechanism of action of FAs on these channels has not been described in detail. It has been shown however that FAs frequently decrease GJC-mediated cell-cell communication. The opposite effect also has been described for HC or Panx-dependent intercellular communication, where, the acute FA effect can be reversed upon washout. Additionally, changes in GJCs mediated by FAs have been associated with post-translational modifications (e.g., phosphorylation), and seem to be directly related to chemical properties of FAs (e.g., length of carbon chain and/or degree of saturation), but this possible link remains poorly understood. PMID:28174541

Autocrine Regulation of UVA-Induced IL-6 Production via Release of ATP and Activation of P2Y Receptors

PubMed Central

Kawano, Ayumi; Kadomatsu, Remi; Ono, Miyu; Kojima, Shuji; Tsukimoto, Mitsutoshi; Sakamoto, Hikaru

2015-01-01

Extracellular nucleotides, such as ATP, are released from cells in response to various stimuli and act as intercellular signaling molecules through activation of P2 receptors. Exposure to the ultraviolet radiation A (UVA) component of sunlight causes molecular and cellular damage, and in this study, we investigated the involvement of extracellular nucleotides and P2 receptors in the UVA-induced cellular response. Human keratinocyte-derived HaCaT cells were irradiated with a single dose of UVA (2.5 J/cm2), and ATP release and interleukin (IL)-6 production were measured. ATP was released from cells in response to UVA irradiation, and the release was blocked by pretreatment with inhibitors of gap junction hemichannels or P2X7 receptor antagonist. IL-6 production was increased after UVA irradiation, and this increase was inhibited by ecto-nucleotidase or by antagonists of P2Y11 or P2Y13 receptor. These results suggest that UVA-induced IL-6 production is mediated by release of ATP through hemichannels and P2X7 receptor, followed by activation of P2Y11 and P2Y13 receptors. Interestingly, P2Y11 and P2Y13 were associated with the same pattern of IL-6 production, though they trigger different intracellular signaling cascades: Ca2+-dependent and PI3K-dependent, respectively. Thus, IL-6 production in response to UVA-induced ATP release involves at least two distinct pathways, mediated by activation of P2Y11 and P2Y13 receptors. PMID:26030257

[The biological role of exosomes in bone remodeling and bone diseases.

PubMed

Urabe, Fumihiko; Yoshioka, Yusuke; Ochiya, Takahiro

Exosomes are about 100nm membrane vesicles, and released from almost all cell types. They carry and transfer a wide variety of molecules, such as mRNAs, microRNAs, proteins, and lipids, as modulators of intercellular communication. Various studies have shown that this exosome-mediated intercellular communication lead to proliferation, invasion and metastasis of cancer cells. In addition to that, emerging data suggest that exosomes are also involved in physiological processes of bone remodeling and bone diseases. Increasing understanding of the working mechanism of exosomes will provide us with new therapeutic and diagnostic opportunities. Here we summarize the current research on exosomes in bone remodeling and bone diseases.

Changes in corneal biometry and the associated histology in rhesus monkeys wearing orthokeratology contact lenses.

PubMed

Ding, Hui; Pu, Aijun; He, Hong; Xie, Ruo Zhong; Yang, Jun; Liao, Aiping; Gao, Shaohui; Zhong, Xingwu

2012-08-01

This study compared the effect of orthokeratology (OK) lens wear on corneal topography and microstructure between the 1-night and 30-night wearers to predict any adverse responses of the eyes to long-term OK lens wear. Nine rhesus monkeys with one eye wearing an OK lens and the fellow eye wearing a rigid gas permeable (RGP) lens were used in this study. The lenses were worn for 11 hours, with the measurement of corneal curvature and thickness and axial components of the eye at 0 night (n = 9), 1 night (n = 9), and 30 nights (n = 6). Histology was performed at 1 and 30 nights of lens wear. Corneal surface power in the 3-mm central region was reduced in the OK eye when compared with the RGP eye (P < 0.05). Central corneal thickness was only reduced in 30 nights of OK lens wear (P < 0.05). Under light microscopy, the 1-night OK or RGP eyes showed normal morphology in all layers of the cornea. The 30-night OK eyes showed a thinned central corneal epithelium and a thickened but less stratified paracentral corneal epithelium when compared with the 30-night RGP eyes. Under electron microscopy, intercellular junctions of corneal epithelium in the 30-night OK eyes were much looser than those in the 1-night OK eyes. However, the density and morphology of hemidesmosomes were similar between the OK and RGP eyes at 30 nights of lens wear. Overnight OK can change the corneal curvature through anterior corneal remodeling, resulting in an effective correction of myopia. However, a decrease in intercellular junctions during long-term wear may compromise the functions of the corneal epithelium as a border mechanical barrier.

Local Oxidative Stress Expansion through Endothelial Cells – A Key Role for Gap Junction Intercellular Communication

PubMed Central

Feine, Ilan; Pinkas, Iddo; Salomon, Yoram; Scherz, Avigdor

2012-01-01

Background Major circulation pathologies are initiated by oxidative insult expansion from a few injured endothelial cells to distal sites; this possibly involves mechanisms that are important to understanding circulation physiology and designing therapeutic management of myocardial pathologies. We tested the hypothesis that a localized oxidative insult of endothelial cells (ECs) propagates through gap junction inter-cellular communication (GJIC). Methodology/Principal Findings Cultures comprising the bEnd.3 cell line, that have been established and recognized as suitable for examining communication among ECs, were used to study the propagation of a localized oxidative insult to remote cells. Spatially confined near infrared illumination of parental or genetically modified bEnd.3 cultures, pretreated with the photosensitizer WST11, generated O2•− and •OH radicals in the illuminated cells. Time-lapse fluorescence microscopy, utilizing various markers, and other methods, were used to monitor the response of non-illuminated bystander and remote cells. Functional GJIC among ECs was shown to be mandatory for oxidative insult propagation, comprising de-novo generation of reactive oxygen and nitrogen species (ROS and RNS, respectively), activation and nuclear translocation of c-Jun N-terminal kinase, followed by massive apoptosis in all bystander cells adjacent to the primarily injured ECs. The oxidative insult propagated through GJIC for many hours, over hundreds of microns from the primary photogeneration site. This wave is shown to be limited by intracellular ROS scavenging, chemical GJIC inhibition or genetic manipulation of connexin 43 (a key component of GJIC). Conclusion/Significance Localized oxidative insults propagate through GJIC between ECs, while stimulating de-novo generation of ROS and RNS in bystander cells, thereby driving the insult's expansion. PMID:22911831

Intercellular Communication by Exosome-Derived microRNAs in Cancer

PubMed Central

Hannafon, Bethany N.; Ding, Wei-Qun

2013-01-01

The development of human cancers is a multistep process in which normal cells acquire characteristics that ultimately lead to their conversion into cancer cells. Many obstacles must be overcome for this process to occur; of these obstacles, is the ability to survive an inhospitable microenvironment. It is recognized that the intercommunication between tumor cells and their surrounding microenvironment is essential to overcoming this obstacle and for the tumor to progress, metastasize and establish itself at distant sites. Exosomes are membrane-derived vesicles that have recently been recognized as important mediators of intercellular communication, as they carry lipids, proteins, mRNAs and microRNAs that can be transferred to a recipient cell via fusion of the exosome with the target cell membrane. In the context of cancer cells, this process entails the transfer of cancer-promoting cellular contents to surrounding cells within the tumor microenvironment or into the circulation to act at distant sites, thereby enabling cancer progression. In this process, the transfer of exosomal microRNAs to a recipient cell where they can regulate target gene expression is of particular interest, both in understanding the basic biology of cancer progression and for the development of therapeutic approaches. This review discusses the exosome-mediated intercellular communication via microRNAs within the tumor microenvironment in human cancers, with a particular focus on breast cancer exosomes. PMID:23839094

Glucose Transporters are Abundant in Cells with "Occluding" Junctions at the Blood-Eye Barriers

NASA Astrophysics Data System (ADS)

Harik, Sami I.; Kalaria, Rajesh N.; Whitney, Paul M.; Andersson, Lars; Lundahl, Per; Ledbetter, Steven R.; Perry, George

1990-06-01

We studied the distribution of the "erythroid/brain" glucose transporter protein in the human and rat eye by immunocytochemistry with monoclonal and polyclonal antibodies to the C terminus of the human erythrocyte glucose transporter. We found intense immunocytochemical staining in the endothelium of microvessels of the retina, optic nerve, and iris but not in microvessels of the choroid, ciliary body, sclera, and other retro-orbital tissues. In addition, we found marked immunocytochemical staining of retinal pigment epithelium, ciliary body epithelium, and posterior epithelium of the iris. The common feature of all those endothelial and epithelial cells that stained intensely for the glucose transporter is the presence of "occluding" intercellular junctions, which constitute the anatomical bases of the blood-eye barriers. We propose that a high density of the glucose transporter is a biochemical concomitant of epithelial and endothelial cells with barrier characteristics, at least in tissues that have a high metabolic requirement for glucose.

Tight Junction Proteins and Oxidative Stress in Heavy Metals-Induced Nephrotoxicity

PubMed Central

Reyes, José L.; Molina-Jijón, Eduardo; Rodríguez-Muñoz, Rafael; Bautista-García, Pablo; Debray-García, Yazmin; Namorado, María del Carmen

2013-01-01

Kidney is a target organ for heavy metals. They accumulate in several segments of the nephron and cause profound alterations in morphology and function. Acute intoxication frequently causes acute renal failure. The effects of chronic exposure have not been fully disclosed. In recent years increasing awareness of the consequences of their presence in the kidney has evolved. In this review we focus on the alterations induced by heavy metals on the intercellular junctions of the kidney. We describe that in addition to the proximal tubule, which has been recognized as the main site of accumulation and injury, other segments of the nephron, such as glomeruli, vessels, and distal nephron, show also deleterious effects. We also emphasize the participation of oxidative stress as a relevant component of the renal damage induced by heavy metals and the beneficial effect that some antioxidant drugs, such as vitamin A (all-trans-retinoic acid) and vitamin E (α-tocopherol), depict on the morphological and functional alterations induced by heavy metals. PMID:23710457

Tick-borne encephalitis virus infects human brain microvascular endothelial cells without compromising blood-brain barrier integrity.

PubMed

Palus, Martin; Vancova, Marie; Sirmarova, Jana; Elsterova, Jana; Perner, Jan; Ruzek, Daniel

2017-07-01

Alteration of the blood-brain barrier (BBB) is a hallmark of tick-borne encephalitis (TBE), a life-threating human viral neuroinfection. However, the mechanism of BBB breakdown during TBE, as well as TBE virus (TBEV) entry into the brain is unclear. Here, primary human microvascular endothelial cells (HBMECs) were infected with TBEV to study interactions with the BBB. Although the number of infected cells was relatively low in culture (<5%), the infection was persistent with high TBEV yields (>10 6 pfu/ml). Infection did not induce any significant changes in the expression of key tight junction proteins or upregulate the expression of cell adhesion molecules, and did not alter the highly organized intercellular junctions between HBMECs. In an in vitro BBB model, the virus crossed the BBB via a transcellular pathway without compromising the integrity of the cell monolayer. The results indicate that HBMECs may support TBEV entry into the brain without altering BBB integrity. Copyright © 2017 Elsevier Inc. All rights reserved.

Targeting neuronal gap junctions in mouse retina offers neuroprotection in glaucoma

PubMed Central

Kumar, Sandeep; Ramakrishnan, Hariharasubramanian; Roy, Kaushambi; Viswanathan, Suresh; Bloomfield, Stewart A.

2017-01-01

The progressive death of retinal ganglion cells and resulting visual deficits are hallmarks of glaucoma, but the underlying mechanisms remain unclear. In many neurodegenerative diseases, cell death induced by primary insult is followed by a wave of secondary loss. Gap junctions (GJs), intercellular channels composed of subunit connexins, can play a major role in secondary cell death by forming conduits through which toxic molecules from dying cells pass to and injure coupled neighbors. Here we have shown that pharmacological blockade of GJs or genetic ablation of connexin 36 (Cx36) subunits, which are highly expressed by retinal neurons, markedly reduced loss of neurons and optic nerve axons in a mouse model of glaucoma. Further, functional parameters that are negatively affected in glaucoma, including the electroretinogram, visual evoked potential, visual spatial acuity, and contrast sensitivity, were maintained at control levels when Cx36 was ablated. Neuronal GJs may thus represent potential therapeutic targets to prevent the progressive neurodegeneration and visual impairment associated with glaucoma. PMID:28604388

Trafficking Highways to the Intercalated Disc: New Insights Unlocking the Specificity of Connexin 43 Localization

PubMed Central

Zhang, Shan-Shan; Shaw, Robin M.

2016-01-01

With each heartbeat, billions of cardiomyocytes work in concert to propagate the electrical excitation needed to effectively circulate blood. Regulated expression and timely delivery of connexin proteins to form gap junctions at the specialized cell – cell contact region, known as the intercalated disc, is essential to ventricular cardiomyocyte coupling. We focus this review on several regulatory mechanisms that have been recently found to govern the lifecycle of connexin 43 (Cx43), the short-lived and most abundantly expressed connexin in cardiac ventricular muscle. The Cx43 lifecycle begins with gene expression, followed by oligomerization into hexameric channels, and then cytoskeletal-based transport toward the disc region. Once delivered, hemichannels interact with resident disc proteins and are organized to effect intercellular coupling. We highlight recent studies exploring regulation of Cx43 localization to the intercalated disc, with emphasis on alternatively translated Cx43 isoforms and cytoskeletal transport machinery that together regulate Cx43 gap junction coupling between cardiomyocytes. PMID:24460200

Critical role of gap junction communication, calcium and nitric oxide signaling in bystander responses to focal photodynamic injury

PubMed Central

Calì, Bianca; Ceolin, Stefano; Ceriani, Federico; Bortolozzi, Mario; Agnellini, Andrielly H.R.; Zorzi, Veronica; Predonzani, Andrea; Bronte, Vincenzo

2015-01-01

Ionizing and nonionizing radiation affect not only directly targeted cells but also surrounding “bystander” cells. The underlying mechanisms and therapeutic role of bystander responses remain incompletely defined. Here we show that photosentizer activation in a single cell triggers apoptosis in bystander cancer cells, which are electrically coupled by gap junction channels and support the propagation of a Ca2+ wave initiated in the irradiated cell. The latter also acts as source of nitric oxide (NO) that diffuses to bystander cells, in which NO levels are further increased by a mechanism compatible with Ca2+-dependent enzymatic production. We detected similar signals in tumors grown in dorsal skinfold chambers applied to live mice. Pharmacological blockade of connexin channels significantly reduced the extent of apoptosis in bystander cells, consistent with a critical role played by intercellular communication, Ca2+ and NO in the bystander effects triggered by photodynamic therapy. PMID:25868859

Mechanical signaling coordinates the embryonic heart

NASA Astrophysics Data System (ADS)

Chiou, Kevin; Rocks, Jason; Prosser, Benjamin; Discher, Dennis; Liu, Andrea

The heart is an active material which relies on robust signaling mechanisms between cells in order to produce well-timed, coordinated beats. Heart tissue is composed primarily of active heart muscle cells (cardiomyocytes) embedded in a passive extracellular matrix. During a heartbeat, cardiomyocyte contractions are coordinated across the heart to form a wavefront that propagates through the tissue to pump blood. In the adult heart, this contractile wave is coordinated via intercellular electrical signaling.Here we present theoretical and experimental evidence for mechanical coordination of embryonic heartbeats. We model cardiomyocytes as mechanically excitable Eshelby inclusions embedded in an overdamped elastic-fluid biphasic medium. For physiological parameters, this model replicates recent experimental measurements of the contractile wavefront which are not captured by electrical signaling models. We additionally challenge our model by pharmacologically blocking gap junctions, inhibiting electrical signaling between myocytes. We find that while adult hearts stop beating almost immediately after gap junctions are blocked, embryonic hearts continue beating even at significantly higher concentrations, providing strong support for a mechanical signaling mechanism.

Cadherin Switch during EMT in Neural Crest Cells Leads to Contact Inhibition of Locomotion via Repolarization of Forces.

PubMed

Scarpa, Elena; Szabó, András; Bibonne, Anne; Theveneau, Eric; Parsons, Maddy; Mayor, Roberto

2015-08-24

Contact inhibition of locomotion (CIL) is the process through which cells move away from each other after cell-cell contact, and it contributes to malignant invasion and developmental migration. Various cell types exhibit CIL, whereas others remain in contact after collision and may form stable junctions. To investigate what determines this differential behavior, we study neural crest cells, a migratory stem cell population whose invasiveness has been likened to cancer metastasis. By comparing pre-migratory and migratory neural crest cells, we show that the switch from E- to N-cadherin during EMT is essential for acquisition of CIL behavior. Loss of E-cadherin leads to repolarization of protrusions, via p120 and Rac1, resulting in a redistribution of forces from intercellular tension to cell-matrix adhesions, which break down the cadherin junction. These data provide insight into the balance of physical forces that contributes to CIL in cells in vivo. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Cadherin Switch during EMT in Neural Crest Cells Leads to Contact Inhibition of Locomotion via Repolarization of Forces

PubMed Central

Scarpa, Elena; Szabó, András; Bibonne, Anne; Theveneau, Eric; Parsons, Maddy; Mayor, Roberto

2015-01-01

Summary Contact inhibition of locomotion (CIL) is the process through which cells move away from each other after cell-cell contact, and it contributes to malignant invasion and developmental migration. Various cell types exhibit CIL, whereas others remain in contact after collision and may form stable junctions. To investigate what determines this differential behavior, we study neural crest cells, a migratory stem cell population whose invasiveness has been likened to cancer metastasis. By comparing pre-migratory and migratory neural crest cells, we show that the switch from E- to N-cadherin during EMT is essential for acquisition of CIL behavior. Loss of E-cadherin leads to repolarization of protrusions, via p120 and Rac1, resulting in a redistribution of forces from intercellular tension to cell-matrix adhesions, which break down the cadherin junction. These data provide insight into the balance of physical forces that contributes to CIL in cells in vivo. PMID:26235046

[Inhibitory effect of Mig-7 silencing by retrovirus-mediated shRNA on vasculogenic mimicry, invasion and metastasis of human hepatocellular carcinoma cells in vitro].

PubMed

Qu, Bo; Sheng, Guan-Nan; Yu, Fei; Chen, Guan-Nan; Lv, Qi; Mao, Zhong-Peng; Guo, Long; Lv, Yi

2016-11-20

To explore the inhibitory effect of migration-inducing gene 7 (Mig-7) gene silencing induced by retroviral-mediated small hairpin RNA (shRNA) on vasculogenic mimicry (VM), invasion and metastasis of human hepatocellular carcinoma (HCC) cells in vitro. Two target sequences (Mig-7 shRNA-1 and Mig-7 shRNA-2) and one negative control sequence (Mig-7 shRNA-N) were synthesized. The recombinant retroviral vectors carrying Mig-7 shRNA were constructed, and HCC cell line MHCC-97H were transfected with Mig-7 shRNA-1, Mig-7 shRNA-2, Mig-7 shRNA-N, or the empty vector, or treated with 125 µg/mL recombinant human endostatin (ES). Mig-7 expression in the treated cells was detected using semi-quantitative PCR and Western blotting. The inhibitory effect of Mig-7 silencing on VM formation was investigated in a 3-dimensional cell culture system; the changes in cell adhesion, invasion and migration were assessed with intercellular adhesion assay, Transwell invasion assay and Transwell migration assay, respectively. The expression of Mig-7 at both mRNA and protein levels decreased significantly, VM formation, invasion and metastasis were suppressed, while intercellular adhesion increased significantly in MHCC-97H cells in Mig-7 shRNA-1 and Mig-7 shRNA-2 groups (P<0.05); such changes were not observed in cells transfected with Mig-7 shRNA-N or the empty vector, nor in cells treated with ES. Mig-7 silencing by retroviral-mediated shRNA significantly inhibits VM formation, invasion and metastasis and increases the intercellular adhesion of the HCC cells, while ES does not have such inhibitory effects.

Bioglass promotes wound healing by affecting gap junction connexin 43 mediated endothelial cell behavior.

PubMed

Li, Haiyan; He, Jin; Yu, Hongfei; Green, Colin R; Chang, Jiang

2016-04-01

It is well known that gap junctions play an important role in wound healing, and bioactive glass (BG) has been shown to help healing when applied as a wound dressing. However, the effects of BG on gap junctional communication between cells involved in wound healing is not well understood. We hypothesized that BG may be able to affect gap junction mediated cell behavior to enhance wound healing. Therefore, we set out to investigate the effects of BG on gap junction related behavior of endothelial cells in order to elucidate the mechanisms through which BG is operating. In in vitro studies, BG ion extracts prevented death of human umbilical vein endothelial cells (HUVEC) following hypoxia in a dose dependent manner, possibly through connexin hemichannel modulation. In addition, BG showed stimulatory effects on gap junction communication between HUVECs and upregulated connexin43 (Cx43) expression. Furthermore, BG prompted expression of vascular endothelial growth factor and basic fibroblast growth factor as well as their receptors, and vascular endothelial cadherin in HUVECs, all of which are beneficial for vascularization. In vivo wound healing results showed that the wound closure of full-thickness excisional wounds of rats was accelerated by BG with reduced inflammation during initial stages of healing and stimulated angiogenesis during the proliferation stage. Therefore, BG can stimulate wound healing through affecting gap junctions and gap junction related endothelial cell behaviors, including prevention of endothelial cell death following hypoxia, stimulation of gap junction communication and upregulation of critical vascular growth factors, which contributes to the enhancement of angiogenesis in the wound bed and finally to accelerate wound healing. Although many studies have reported that BG stimulates angiogenesis and wound healing, this work reveals the relationship between BG and gap junction connexin 43 mediated endothelial cell behavior and elucidates one of the possible mechanisms through which BG stimulates wound healing. Copyright © 2016 Elsevier Ltd. All rights reserved.

A mechanism enhancing macromolecule transport through paracellular spaces induced by Poly-L-Arginine: Poly-L-Arginine induces the internalization of tight junction proteins via clathrin-mediated endocytosis.

PubMed

Yamaki, Tsutomu; Kamiya, Yusuke; Ohtake, Kazuo; Uchida, Masaki; Seki, Toshinobu; Ueda, Hideo; Kobayashi, Jun; Morimoto, Yasunori; Natsume, Hideshi

2014-09-01

Poly-L-arginine (PLA) enhances the paracellular permeability of the Caco-2 cell monolayer to hydrophilic macromolecules by disappearance of tight junction (TJ) proteins from cell-cell junctions. However, the mechanism of the disappearance of TJ proteins in response to PLA has been unclear. In this study, we investigated the mechanism of disappearance of TJ proteins from cell-cell junctions after the application of PLA to Caco-2 cell monolayers. The membrane conductance (Gt), FITC-dextran (FD-4) permeability, and localization of TJ proteins were examined after the treatment of Caco-2 cell monolayers with PLA in the presence of various endocytosis inhibitors. In addition, the localization of endosome marker proteins was also observed. Clathrin-mediated endocytosis inhibitors suppressed the increase in Gt and Papp of FD-4 induced by PLA, and also significantly suppressed the disappearance of TJ proteins induced by PLA. Furthermore, occludin, one of the TJ proteins, colocalized with early endosome and recycling endosomes after the internalization of occludin induced by PLA, and then was recycled to the cell-cell junctions. PLA induced the transient internalization of TJ proteins in cell-cell junctions via clathrin-mediated endocytosis, subsequently increasing the permeability of the Caco-2 cell monolayer to FD-4 via a paracellular route.

siRNA-based Analysis of the Abrogation of the Protective Function of Membrane-associated Catalase of Tumor Cells.

PubMed

Bauer, Georg

2017-02-01

Tumor cells, in contrast to non-malignant cells, show sustained expression of membrane-associated NADPH oxidase-1 and therefore generate extracellular superoxide anions and their dismutation product H 2 O 2 In order to prevent intercellular reactive oxygen species/reactive nitrogen species (ROS/RNS)-dependent apoptosis-inducing signaling, tumor cells need to express membrane-associated catalase that interferes with HOCl and nitric oxide/peroxynitrite signaling. Catalase is attached to tumor cells through the activity of transglutaminase-2 and is prevented from superoxide anion-dependent inhibition through coexpression of membrane-associated superoxide dismutase. Therefore, specific inhibition of membrane-associated catalase should reactivate intercellular ROS/RNS-dependent apoptosis-inducing signaling. These processes are analyzed here through small interfering RNA-mediated knockdown of essential signaling compounds. This allows to establish a rather comprehensive picture of intercellular ROS/RNS signaling that may be instrumental for future therapeutic approaches. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Tunneling nanotubes promote intercellular mitochondria transfer followed by increased invasiveness in bladder cancer cells

PubMed Central

Lu, Jinjin; Zheng, Xiufen; Li, Fan; Yu, Yang; Chen, Zhong; Liu, Zheng; Wang, Zhihua; Xu, Hua; Yang, Weimin

2017-01-01

Intercellular transfer of organelles via tunneling nanotubes (TNTs) is a novel means of cell-to-cell communication. Here we demonstrate the existence of TNTs between co-cultured RT4 and T24 bladder cancer cells using light microscopy, fluorescence imaging, and scanning electron microscopy (SEM). Spontaneous unidirectional transfer of mitochondria from T24 to RT4 cells was detected using fluorescence imaging and flow cytometry. The distribution of mitochondria migrated from T24 cells was in good agreement with the original mitochondria in RT4 cells, which may imply mitochondrial fusion. We detected cytoskeleton reconstruction in RT4-Mito-T24 cells by observing F-actin redistribution. Akt, mTOR, and their downstream mediators were activated and increased. The resultant increase in the invasiveness of bladder cancer cells was detected in vitro and in vivo. These data indicate that TNTs promote intercellular mitochondrial transfer between heterogeneous cells, followed by an increase in the invasiveness of bladder cancer cells. PMID:28107184

Intercellular adhesion molecule-1 augments myoblast adhesion and fusion through homophilic trans-interactions.

PubMed

Pizza, Francis X; Martin, Ryan A; Springer, Evan M; Leffler, Maxwell S; Woelmer, Bryce R; Recker, Isaac J; Leaman, Douglas W

2017-07-11

The overall objective of the study was to identify mechanisms through which intercellular adhesion molecule-1 (ICAM-1) augments the adhesive and fusogenic properties of myogenic cells. Hypotheses were tested using cultured myoblasts and fibroblasts, which do not constitutively express ICAM-1, and myoblasts and fibroblasts forced to express full length ICAM-1 or a truncated form lacking the cytoplasmic domain of ICAM-1. ICAM-1 mediated myoblast adhesion and fusion were quantified using novel assays and cell mixing experiments. We report that ICAM-1 augments myoblast adhesion to myoblasts and myotubes through homophilic trans-interactions. Such adhesive interactions enhanced levels of active Rac in adherent and fusing myoblasts, as well as triggered lamellipodia, spreading, and fusion of myoblasts through the signaling function of the cytoplasmic domain of ICAM-1. Rac inhibition negated ICAM-1 mediated lamellipodia, spreading, and fusion of myoblasts. The fusogenic property of ICAM-1-ICAM-1 interactions was restricted to myogenic cells, as forced expression of ICAM-1 by fibroblasts did not augment their fusion to ICAM-1+ myoblasts/myotubes. We conclude that ICAM-1 augments myoblast adhesion and fusion through its ability to self-associate and initiate Rac-mediated remodeling of the actin cytoskeleton.

EFFECTS OF TRICHLOROETHYLENE AND ITS METABOLITES ON RODENT HEPATOCYTE INTERCELLULAR COMMUNICATION

EPA Science Inventory

Chronic exposure to trichloroethylene (TCE) results in hepatocellular cancer in mice but not rats. The induction of hepatic tumors by TCE appears to be mediated through nongenotoxic or tumor promotion mechanisms. One cellular effect exhibited by a number of nongentoxic carcinogen...

The heat-shock protein Apg-2 binds to the tight junction protein ZO-1 and regulates transcriptional activity of ZONAB.

PubMed

Tsapara, Anna; Matter, Karl; Balda, Maria S

2006-03-01

The tight junction adaptor protein ZO-1 regulates intracellular signaling and cell proliferation. Its Src homology 3 (SH3) domain is required for the regulation of proliferation and binds to the Y-box transcription factor ZO-1-associated nucleic acid binding protein (ZONAB). Binding of ZO-1 to ZONAB results in cytoplasmic sequestration and hence inhibition of ZONAB's transcriptional activity. Here, we identify a new binding partner of the SH3 domain that modulates ZO-1-ZONAB signaling. Expression screening of a cDNA library with a fusion protein containing the SH3 domain yielded a cDNA coding for Apg-2, a member of the heat-shock protein 110 (Hsp 110) subfamily of Hsp70 heat-shock proteins, which is overexpressed in carcinomas. Regulated depletion of Apg-2 in Madin-Darby canine kidney cells inhibits G(1)/S phase progression. Apg-2 coimmunoprecipitates with ZO-1 and partially localizes to intercellular junctions. Junctional recruitment and coimmunoprecipitation with ZO-1 are stimulated by heat shock. Apg-2 competes with ZONAB for binding to the SH3 domain in vitro and regulates ZONAB's transcriptional activity in reporter gene assays. Our data hence support a model in which Apg-2 regulates ZONAB function by competing for binding to the SH3 domain of ZO-1 and suggest that Apg-2 functions as a regulator of ZO-1-ZONAB signaling in epithelial cells in response to cellular stress.

The Heat-Shock Protein Apg-2 Binds to the Tight Junction Protein ZO-1 and Regulates Transcriptional Activity of ZONAB

PubMed Central

Tsapara, Anna; Matter, Karl; Balda, Maria S.

2006-01-01

The tight junction adaptor protein ZO-1 regulates intracellular signaling and cell proliferation. Its Src homology 3 (SH3) domain is required for the regulation of proliferation and binds to the Y-box transcription factor ZO-1-associated nucleic acid binding protein (ZONAB). Binding of ZO-1 to ZONAB results in cytoplasmic sequestration and hence inhibition of ZONAB's transcriptional activity. Here, we identify a new binding partner of the SH3 domain that modulates ZO-1–ZONAB signaling. Expression screening of a cDNA library with a fusion protein containing the SH3 domain yielded a cDNA coding for Apg-2, a member of the heat-shock protein 110 (Hsp 110) subfamily of Hsp70 heat-shock proteins, which is overexpressed in carcinomas. Regulated depletion of Apg-2 in Madin-Darby canine kidney cells inhibits G1/S phase progression. Apg-2 coimmunoprecipitates with ZO-1 and partially localizes to intercellular junctions. Junctional recruitment and coimmunoprecipitation with ZO-1 are stimulated by heat shock. Apg-2 competes with ZONAB for binding to the SH3 domain in vitro and regulates ZONAB's transcriptional activity in reporter gene assays. Our data hence support a model in which Apg-2 regulates ZONAB function by competing for binding to the SH3 domain of ZO-1 and suggest that Apg-2 functions as a regulator of ZO-1–ZONAB signaling in epithelial cells in response to cellular stress. PMID:16407410

The distribution and functional properties of Pelizaeus-Merzbacher-like disease-linked Cx47 mutations on Cx47/Cx47 homotypic and Cx47/Cx43 heterotypic gap junctions.

PubMed

Kim, Mi Seong; Gloor, Gregory B; Bai, Donglin

2013-06-01

GJs (gap junctions) allow direct intercellular communication, and consist of Cxs (connexins). In the mammalian central nervous system, oligodendrocytes express Cx47, Cx32 and Cx29, whereas astrocytes express Cx43, Cx30 and Cx26. Homotypic Cx47/Cx47 GJs couple oligodendrocytes, and heterotypic Cx47/Cx43 channels are the primary GJs at oligodendrocyte/astrocyte junctions. Interestingly, autosomal recessive mutations in the gene GJC2 encoding Cx47 have been linked to a central hypomyelinating disease termed PMLD (Pelizaeus-Merzbacher-like disease). The aim of the present study was to determine the cellular distribution and functional properties of PMLD-associated Cx47 mutants (I46M, G149S, G236R, G236S, M286T and T398I). Expressing GFP (green fluorescent protein)-tagged mutant versions of Cx47 in gap-junction-deficient model cells revealed that these mutants were detected at the cell-cell interface similar to that observed for wild-type Cx47. Furthermore, four of the six mutants showed no electrical coupling in both Cx47/Cx47 and Cx47/Cx43 GJ channels. These results suggest that most of the PMLD-linked Cx47 mutants disrupt Cx47/Cx47 and Cx47/Cx43 GJ function in the glial network, which may play a role in leading to PMLD symptoms.

The Role of Gap Junction Communication and Oxidative Stress in the Propagation of Toxic Effects among High-Dose α-Particle-Irradiated Human Cells

PubMed Central

Autsavapromporn, Narongchai; de Toledo, Sonia M.; Little, John B.; Jay-Gerin, Jean-Paul; Harris, Andrew L.; Azzam, Edouard I.

2011-01-01

We investigated the roles of gap junction communication and oxidative stress in modulating potentially lethal damage repair in human fibroblast cultures exposed to doses of α particles or γ rays that targeted all cells in the cultures. As expected, α particles were more effective than γ rays at inducing cell killing; further, holding γ-irradiated cells in the confluent state for several hours after irradiation promoted increased survival and decreased chromosomal damage. However, maintaining α-particle-irradiated cells in the confluent state for various times prior to subculture resulted in increased rather than decreased lethality and was associated with persistent DNA damage and increased protein oxidation and lipid peroxidation. Inhibiting gap junction communication with 18-α-glycyrrhetinic acid or by knockdown of connexin43, a constitutive protein of junctional channels in these cells, protected against the toxic effects in α-particle-irradiated cell cultures during confluent holding. Upregulation of antioxidant defense by ectopic overexpression of glutathione peroxidase protected against cell killing by α particles when cells were analyzed shortly after exposure. However, it did not attenuate the decrease in survival during confluent holding. Together, these findings indicate that the damaging effect of α particles results in oxidative stress, and the toxic effects in the hours after irradiation are amplified by intercellular communication, but the communicated molecule(s) is unlikely to be a substrate of glutathione peroxidase. PMID:21388278

Connexins and Pannexins in Vascular Function and Disease.

PubMed

Molica, Filippo; Figueroa, Xavier F; Kwak, Brenda R; Isakson, Brant E; Gibbins, Jonathan M

2018-06-05

Connexins (Cxs) and pannexins (Panxs) are ubiquitous membrane channel forming proteins that are critically involved in many aspects of vascular physiology and pathology. The permeation of ions and small metabolites through Panx channels, Cx hemichannels and gap junction channels confers a crucial role to these proteins in intercellular communication and in maintaining tissue homeostasis. This review provides an overview of current knowledge with respect to the pathophysiological role of these channels in large arteries, the microcirculation, veins, the lymphatic system and platelet function. The essential nature of these membrane proteins in vascular homeostasis is further emphasized by the pathologies that are linked to mutations and polymorphisms in Cx and Panx genes.

Using a Large Scale Computational Model to Study the Effect of Longitudinal and Radial Electrical Coupling in the Cochlea

NASA Astrophysics Data System (ADS)

Mistrík, Pavel; Ashmore, Jonathan

2009-02-01

We describe a large scale computational model of electrical current flow in the cochlea which is constructed by a flexible Modified Nodal Analysis algorithm to incorporate electrical components representing hair cells and the intercellular radial and longitudinal current flow. The model is used as a laboratory to study the effects of changing longitudinal gap junctional coupling, and shows the way in which cochlear microphonic spreads and tuning is affected. The process for incorporating mechanical longitudinal coupling and feedback is described. We find a difference in tuning and attenuation depending on whether longitudinal or radial couplings are altered.

Lymphatic endothelial cell line (CH3) from a recurrent retroperitoneal lymphangioma.

PubMed

Way, D; Hendrix, M; Witte, M; Witte, C; Nagle, R; Davis, J

1987-09-01

An endothelial cell line derived from a massive recurrent chyle-containing retroperitoneal lymphangioma was isolated in monolayer culture. Scanning and transmission electron microscopy and immunohistochemistry confirmed a close resemblance to blood vascular endothelium with typical cobblestone morphology, positive immunofluorescence staining for endothelial marker Factor VIII-associated antigen and fibronectin, and prominent Weibel-Palade bodies. The endothelial cells also exhibited other ultrastructural features characteristic of lymphatic endothelium, including sparse microvillous surface projections, overlapping intercellular junctions, and abundant intermediate filaments. This endothelial cell line represents a new source of proliferating lymphatic endothelium for future study, including structural and functional comparison to blood vascular endothelium.

Dynamics between actin and the VE-cadherin/catenin complex

PubMed Central

Abu Taha, Abdallah; Schnittler, Hans-J

2014-01-01

Endothelial adherens junctions are critical for physiological and pathological processes such as differentiation, maintenance of entire monolayer integrity, and the remodeling. The endothelial-specific VE-cadherin/catenin complex provides the backbone of adherens junctions and acts in close interaction with actin filaments and actin/myosin-mediated contractility to fulfill the junction demands. The functional connection between the cadherin/catenin complex and actin filaments might be either directly through α-catenins, or indirectly e.g., via linker proteins such as vinculin, p120ctn, α-actinin, or EPLIN. However, both junction integrity and dynamic remodeling have to be contemporarily coordinated. The actin-related protein complex ARP2/3 and its activating molecules, such as N-WASP and WAVE, have been shown to regulate the lammellipodia-mediated formation of cell junctions in both epithelium and endothelium. Recent reports now demonstrate a novel aspect of the ARP2/3 complex and the nucleating-promoting factors in the maintenance of endothelial barrier function and junction remodeling of established endothelial cell junctions. Those mechanisms open novel possibilities; not only in fulfilling physiological demands but obtained information may be of critical importance in pathologies such as wound healing, angiogenesis, inflammation, and cell diapedesis. PMID:24621569

Clostridium sordellii lethal toxin kills mice by inducing a major increase in lung vascular permeability.

PubMed

Geny, Blandine; Khun, Huot; Fitting, Catherine; Zarantonelli, Leticia; Mazuet, Christelle; Cayet, Nadège; Szatanik, Marek; Prevost, Marie-Christine; Cavaillon, Jean-Marc; Huerre, Michel; Popoff, Michel R

2007-03-01

When intraperitoneally injected into Swiss mice, Clostridium sordellii lethal toxin reproduces the fatal toxic shock syndrome observed in humans and animals after natural infection. This animal model was used to study the mechanism of lethal toxin-induced death. Histopathological and biochemical analyses identified lung and heart as preferential organs targeted by lethal toxin. Massive extravasation of blood fluid in the thoracic cage, resulting from an increase in lung vascular permeability, generated profound modifications such as animal dehydration, increase in hematocrit, hypoxia, and finally, cardiorespiratory failure. Vascular permeability increase induced by lethal toxin resulted from modifications of lung endothelial cells as evidenced by electron microscopy. Immunohistochemical analysis demonstrated that VE-cadherin, a protein participating in intercellular adherens junctions, was redistributed from membrane to cytosol in lung endothelial cells. No major sign of lethal toxin-induced inflammation was observed that could participate in the toxic shock syndrome. The main effect of the lethal toxin is the glucosylation-dependent inactivation of small GTPases, in particular Rac, which is involved in actin polymerization occurring in vivo in lungs leading to E-cadherin junction destabilization. We conclude that the cells most susceptible to lethal toxin are lung vascular endothelial cells, the adherens junctions of which were altered after intoxication.

Clostridium sordellii Lethal Toxin Kills Mice by Inducing a Major Increase in Lung Vascular Permeability

PubMed Central

Geny, Blandine; Khun, Huot; Fitting, Catherine; Zarantonelli, Leticia; Mazuet, Christelle; Cayet, Nadège; Szatanik, Marek; Prevost, Marie-Christine; Cavaillon, Jean-Marc; Huerre, Michel; Popoff, Michel R.

2007-01-01

When intraperitoneally injected into Swiss mice, Clostridium sordellii lethal toxin reproduces the fatal toxic shock syndrome observed in humans and animals after natural infection. This animal model was used to study the mechanism of lethal toxin-induced death. Histopathological and biochemical analyses identified lung and heart as preferential organs targeted by lethal toxin. Massive extravasation of blood fluid in the thoracic cage, resulting from an increase in lung vascular permeability, generated profound modifications such as animal dehydration, increase in hematocrit, hypoxia, and finally, cardiorespiratory failure. Vascular permeability increase induced by lethal toxin resulted from modifications of lung endothelial cells as evidenced by electron microscopy. Immunohistochemical analysis demonstrated that VE-cadherin, a protein participating in intercellular adherens junctions, was redistributed from membrane to cytosol in lung endothelial cells. No major sign of lethal toxin-induced inflammation was observed that could participate in the toxic shock syndrome. The main effect of the lethal toxin is the glucosylation-dependent inactivation of small GTPases, in particular Rac, which is involved in actin polymerization occurring in vivo in lungs leading to E-cadherin junction destabilization. We conclude that the cells most susceptible to lethal toxin are lung vascular endothelial cells, the adherens junctions of which were altered after intoxication. PMID:17322384

Endocochlear potential generation is associated with intercellular communication in the stria vascularis: structural analysis in the viable dominant spotting mouse mutant.

PubMed

Carlisle, L; Steel, K; Forge, A

1990-11-01

Deafness in the viable dominant spotting mouse mutant is due to a primary defect of the stria vascularis which results in absence of the positive endocochlear potential in scala media. Endocochlear potentials were measured and the structure of stria vascularis of mutants with potentials close to zero was compared with that in normal littermate controls by use of morphometric methods. The stria vascularis was significantly thinner in mutants. Marginal cells were not significantly different from controls in terms of volume density or intramembrane particle density but the network density of tight junctions was significantly reduced in the mutants. A virtual absence of gap junctions between basal cells and marginal or intermediate cells was observed, but intramembrane particle density and junctional complexes between adjacent basal cells were not different from controls. The volume density of basal cells was significantly greater in mutants. Intermediate cells accounted for a significantly smaller volume density of the stria vascularis in mutants and had a lower density of intramembrane particles than controls. Melanocytes were not identified in the stria vascularis of mutants. These results suggest that communication between marginal, intermediate and basal cells might be important to the normal function of the stria vascularis.

Active cell-matrix coupling regulates cellular force landscapes of cohesive epithelial monolayers

NASA Astrophysics Data System (ADS)

Zhao, Tiankai; Zhang, Yao; Wei, Qiong; Shi, Xuechen; Zhao, Peng; Chen, Long-Qing; Zhang, Sulin

2018-03-01

Epithelial cells can assemble into cohesive monolayers with rich morphologies on substrates due to competition between elastic, edge, and interfacial effects. Here we present a molecularly based thermodynamic model, integrating monolayer and substrate elasticity, and force-mediated focal adhesion formation, to elucidate the active biochemical regulation over the cellular force landscapes in cohesive epithelial monolayers, corroborated by microscopy and immunofluorescence studies. The predicted extracellular traction and intercellular tension are both monolayer size and substrate stiffness dependent, suggestive of cross-talks between intercellular and extracellular activities. Our model sets a firm ground toward a versatile computational framework to uncover the molecular origins of morphogenesis and disease in multicellular epithelia.

Intracellular cytoskeleton and junction proteins of endothelial cells in the porcine iris microvasculature.

PubMed

Yang, Hongfang; Yu, Paula K; Cringle, Stephen J; Sun, Xinghuai; Yu, Dao-Yi

2015-11-01

Recently we reported studies of the iris microvasculature and its endothelial cells using intra-luminal micro-perfusion, fixation, and silver staining, suggesting that the iris vascular endothelium may be crucial for maintaining homeostasis in the ocular anterior segment. Here we present information regarding the intracellular structure and cell junctions of the iris endothelium. Thirty-seven porcine eyes were used for this study. The temporal long posterior ciliary artery was cannulated to assess the iris microvascular network and its endothelium using intra-luminal micro-perfusion, fixation, and staining with phalloidin for intracellular cytoskeleton f-actin, and with antibodies against claudin-5 and VE-cadherin for junction proteins. Nuclei were counterstained with Hoechst. The iris was flat-mounted for confocal imaging. The iris microvasculature was studied for its distribution, branch orders and endothelial morphometrics with endothelial cell length measured for each vessel order. Our results showed that morphometrics of the iris microvasculature was comparable with our previous silver staining. Abundant stress fibres and peripheral border staining were seen within the endothelial cells in larger arteries. An obvious decrease in cytoplasmic stress fibres was evident further downstream in the smaller arterioles, and they tended to be absent from capillaries and veins. Endothelial intercellular junctions throughout the iris vasculature were VE-cadherin and claudin-5 immuno-positive, indicating the presence of both adherent junctions and tight junctions between vascular endothelial cells throughout the iris microvasculature. Unevenness of claudin-5 staining was noted along the endothelial cell borders in almost every order of vessels, especially in veins and small arterioles. Our results suggest that significant heterogeneity of intracellular structure and junction proteins is present in different orders of the iris vasculature in addition to vascular diameter and shape of the endothelia. Detailed information of the topography and intracellular structure and junction proteins of the endothelium of the iris microvasculature combined with unique structural features of the iris may help us to further understand the physiological and pathogenic roles of the iris vasculature in relevant ocular diseases. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effects of cadmium on intercellular junctions in a renal epithelial cell line grown on permeable membrane supports

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prozialeck, W.C.; Niewenhuis, R.J.

1991-03-11

Recent findings from the authors laboratories have shown that Cd{sup 2+} has relatively specific damaging effects on adhering and occluding junctions in the established porcine renal epithelial cell line, LLC-PK{sub 1}. The present studies were undertaken in order to further characterize the junction-perturbing effects of Cd{sup 2+} in polarized monolayers of LLC-PK{sub 1} cells, and to begin to identify the mechanisms underlying these effects. LLC-PK{sub 1} cells were grown to confluency on Millicell HA chambers and exposed to Cd{sup 2+} in polarized monolayers of LLC-PK{sub 1} cells, and to begin to identify the mechanisms underlying these effects. LLC-PK{sub 1} cellsmore » were grown to confluency on Millicell HA chambers an exposed to Cd{sup 2+} by adding CdCl{sub 2} to the solutions on either side of the cell monolayer. The integrity of cell-cell junctions was assessed by monitoring the transepithelial electrical resistance. The results showed that exposure to Cd{sup 2+} caused a pronounced decrease in transepithelial resistance without causing the cells to detach from the Millicell membrane. This decrease in resistance occurred more quickly and was much more pronounced when Cd{sup 2+} was added to the basolateral surface rather than the apical surface. Furthermore, the effects of Cd{sup 2+} were greatly reduced when excess Ca{sup 2+} was present in the medium. These results suggest that Cd{sup 2+} was present in the medium. These results suggest that Cd{sup 2+} may disrupt cell-cell junctions by interacting with Ca{sup 2+} binding sites or Ca{sup 2+} channels that are oriented toward the basolateral cell surface.« less

Connexin36 vs. connexin32, "miniature" neuronal gap junctions, and limited electrotonic coupling in rodent suprachiasmatic nucleus.

PubMed

Rash, J E; Olson, C O; Pouliot, W A; Davidson, K G V; Yasumura, T; Furman, C S; Royer, S; Kamasawa, N; Nagy, J I; Dudek, F E

2007-10-26

Suprachiasmatic nucleus (SCN) neurons generate circadian rhythms, and these neurons normally exhibit loosely-synchronized action potentials. Although electrotonic coupling has long been proposed to mediate this neuronal synchrony, ultrastructural studies have failed to detect gap junctions between SCN neurons. Nevertheless, it has been proposed that neuronal gap junctions exist in the SCN; that they consist of connexin32 or, alternatively, connexin36; and that connexin36 knockout eliminates neuronal coupling between SCN neurons and disrupts circadian rhythms. We used confocal immunofluorescence microscopy and freeze-fracture replica immunogold labeling to examine the distributions of connexin30, connexin32, connexin36, and connexin43 in rat and mouse SCN and used whole-cell recordings to re-assess electrotonic and tracer coupling. Connexin32-immunofluorescent puncta were essentially absent in SCN but connexin36 was relatively abundant. Fifteen neuronal gap junctions were identified ultrastructurally, all of which contained connexin36 but not connexin32, whereas nearby oligodendrocyte gap junctions contained connexin32. In adult SCN, one neuronal gap junction was >600 connexons, whereas 75% were smaller than 50 connexons, which may be below the limit of detectability by fluorescence microscopy and thin-section electron microscopy. Whole-cell recordings in hypothalamic slices revealed tracer coupling with neurobiotin in <5% of SCN neurons, and paired recordings (>40 pairs) did not reveal obvious electrotonic coupling or synchronized action potentials, consistent with few neurons possessing large gap junctions. However, most neurons had partial spikes or spikelets (often <1 mV), which remained after QX-314 [N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium bromide] had blocked sodium-mediated action potentials within the recorded neuron, consistent with spikelet transmission via small gap junctions. Thus, a few "miniature" gap junctions on most SCN neurons appear to mediate weak electrotonic coupling between limited numbers of neuron pairs, thus accounting for frequent detection of partial spikes and hypothetically providing the basis for "loose" electrical or metabolic synchronization of electrical activity commonly observed in SCN neuronal populations during circadian rhythms.

Toll-like receptor 4 increases intestinal permeability through up-regulation of membrane PKC activity in alcoholic steatohepatitis.

PubMed

Li, Xin; Wang, Chen; Nie, Jiao; Lv, Dong; Wang, Tianyi; Xu, Youqing

2013-09-01

Intestinal hyperpermeability is a causal factor for the development of alcoholic endotoxemia and steatohepatitis. However, the mechanisms governing this link remain unknown. The purpose of this study was to determine whether toll-like receptor 4 (TLR4) is involved in ethanol's deleterious effects on the intestinal barrier. Caco-2 cells were incubated in vitro with 1-10% ethanol. The results indicated that ethanol had a dose-dependent effect in increasing TLR4 expression and intercellular permeability. Then the effects of TLR4 on protein kinase C (PKC) and the intercellular junction protein occludin were assessed with and without pretreatment with a TLR4 inhibitor. The results indicated that TLR4 increased nonspecific PKC activity and reduced the expression of phosphorylated occludin in the membrane, which increased intercellular permeability. These effects were prevented by pretreatment with TLR4 mAb. Wild-type C57BL/6 mice were fed an ethanol or isocaloric liquid diet for 6 weeks. Hepatitis was diagnosed by the presence of an associated elevated blood endotoxin level. Chronic ethanol treatment significantly elevated blood endotoxin levels, intestinal permeability, and the expression of TLR4 in the ileum and colon. Moreover, ethanol exposure reduced the distribution of phosphorylated occludin in the intestinal epithelium because of PKC activation. In conclusion, chronic ethanol exposure induces a high response of TLR4 to lipopolysaccharide (LPS), and TLR4 increases intestinal permeability through down-regulation of phosphorylated occludin expression in the intestinal epithelial barrier, accompanied by membrane PKC hyperactivity. Copyright © 2013 Elsevier Inc. All rights reserved.

Evolution of disorder in Mediator complex and its functional relevance

PubMed Central

Nagulapalli, Malini; Maji, Sourobh; Dwivedi, Nidhi; Dahiya, Pradeep; Thakur, Jitendra K.

2016-01-01

Mediator, an important component of eukaryotic transcriptional machinery, is a huge multisubunit complex. Though the complex is known to be conserved across all the eukaryotic kingdoms, the evolutionary topology of its subunits has never been studied. In this study, we profiled disorder in the Mediator subunits of 146 eukaryotes belonging to three kingdoms viz., metazoans, plants and fungi, and attempted to find correlation between the evolution of Mediator complex and its disorder. Our analysis suggests that disorder in Mediator complex have played a crucial role in the evolutionary diversification of complexity of eukaryotic organisms. Conserved intrinsic disordered regions (IDRs) were identified in only six subunits in the three kingdoms whereas unique patterns of IDRs were identified in other Mediator subunits. Acquisition of novel molecular recognition features (MoRFs) through evolution of new subunits or through elongation of the existing subunits was evident in metazoans and plants. A new concept of ‘junction-MoRF’ has been introduced. Evolutionary link between CBP and Med15 has been provided which explain the evolution of extended-IDR in CBP from Med15 KIX-IDR junction-MoRF suggesting role of junction-MoRF in evolution and modulation of protein–protein interaction repertoire. This study can be informative and helpful in understanding the conserved and flexible nature of Mediator complex across eukaryotic kingdoms. PMID:26590257

Induction of Cell Scattering by Expression of β1 Integrins in β1-Deficient Epithelial Cells Requires Activation of Members of the Rho Family of Gtpases and Downregulation of Cadherin and Catenin Function

PubMed Central

Gimond, Clotilde; van der Flier, Arjan; van Delft, Sanne; Brakebusch, Cord; Kuikman, Ingrid; Collard, John G.; Fässler, Reinhard; Sonnenberg, Arnoud

1999-01-01

Adhesion receptors, which connect cells to each other and to the surrounding extracellular matrix (ECM), play a crucial role in the control of tissue structure and of morphogenesis. In this work, we have studied how intercellular adhesion molecules and β1 integrins influence each other using two different β1-null cell lines, epithelial GE11 and fibroblast-like GD25 cells. Expression of β1A or the cytoplasmic splice variant β1D, induced the disruption of intercellular adherens junctions and cell scattering in both GE11 and GD25 cells. In GE11 cells, the morphological change correlated with the redistribution of zonula occluden (ZO)-1 from tight junctions to adherens junctions at high cell confluency. In addition, the expression of β1 integrins caused a dramatic reorganization of the actin cytoskeleton and of focal contacts. Interaction of β1 integrins with their respective ligands was required for a complete morphological transition towards the spindle-shaped fibroblast-like phenotype. The expression of an interleukin-2 receptor (IL2R)-β1A chimera and its incorporation into focal adhesions also induced the disruption of cadherin-based adhesions and the reorganization of ECM–cell contacts, but failed to promote cell migration on fibronectin, in contrast to full-length β1A. This indicates that the disruption of cell–cell adhesion is not simply the consequence of the stimulated cell migration. Expression of β1 integrins in GE11 cells resulted in a decrease in cadherin and α-catenin protein levels accompanied by their redistribution from the cytoskeleton-associated fraction to the detergent-soluble fraction. Regulation of α-catenin protein levels by β1 integrins is likely to play a role in the morphological transition, since overexpression of α-catenin in GE11 cells before β1 prevented the disruption of intercellular adhesions and cell scattering. In addition, using biochemical activity assays for Rho-like GTPases, we show that the expression of β1A, β1D, or IL2R-β1A in GE11 or GD25 cells triggers activation of both RhoA and Rac1, but not of Cdc42. Moreover, dominant negative Rac1 (N17Rac1) inhibited the disruption of cell–cell adhesions when expressed before β1. However, all three GTPases might be involved in the morphological transition, since expression of either N19RhoA, N17Rac1, or N17Cdc42 reversed cell scattering and partially restored cadherin-based adhesions in GE11-β1A cells. Our results indicate that β1 integrins regulate the polarity and motility of epithelial cells by the induction of intracellular molecular events involving a downregulation of α-catenin function and the activation of the Rho-like G proteins Rac1 and RhoA. PMID:10601344

Intracellular Ca2+ release mediates cationic but not anionic poly(amidoamine) (PAMAM) dendrimer-induced tight junction modulation.

PubMed

Avaritt, Brittany R; Swaan, Peter W

2014-09-01

Poly(amidoamine) (PAMAM) dendrimers show great promise for utilization as oral drug delivery vehicles. These polymers are capable of traversing epithelial barriers, and have been shown to translocate by both transcellular and paracellular routes. While many proof-of-concept studies have shown that PAMAM dendrimers improve intestinal transport, little information exists on the mechanisms of paracellular transport, specifically dendrimer-induced tight junction modulation. Using anionic G3.5 and cationic G4 PAMAM dendrimers with known absorption enhancers, we investigated tight junction modulation in Caco-2 monolayers by visualization and mannitol permeability and compared dendrimer-mediated tight junction modulation to that of established permeation enhancers. [(14)C]-Mannitol permeability in the presence and absence of phospholipase C-dependent signaling pathway inhibitors was also examined and indicated that this pathway may mediate dendrimer-induced changes in permeability. Differences between G3.5 and G4 in tight junction protein staining and permeability with inhibitors were evident, suggesting divergent mechanisms were responsible for tight junction modulation. These dissimilarities are further intimated by the intracellular calcium release caused by G4 but not G3.5. Based on our results, it is apparent that the underlying mechanisms of dendrimer permeability are complex, and the complexities are likely a result of the density and sign of the surface charges of PAMAM dendrimers. The results of this study will have implications on the future use of PAMAM dendrimers for oral drug delivery.

Effects of lead intoxication on intercellular junctions and biochemical alterations of the renal proximal tubule cells.

PubMed

Navarro-Moreno, L G; Quintanar-Escorza, M A; González, S; Mondragón, R; Cerbón-Solorzáno, J; Valdés, J; Calderón-Salinas, J V

2009-10-01

Lead intoxication is a worldwide health problem which frequently affects the kidney. In this work, we studied the effects of chronic lead intoxication (500 ppm of Pb in drinking water during seven months) on the structure, function and biochemical properties of rat proximal tubule cells. Lead-exposed animals showed increased lead concentration in kidney, reduction of calcium and amino acids uptake, oxidative damage and glucosuria, proteinuria, hematuria and reduced urinary pH. These biochemical and physiological alterations were related to striking morphological modifications in the structure of tubule epithelial cells and in the morphology of their mitochondria, nuclei, lysosomes, basal and apical membranes. Interestingly, in addition to the nuclei, inclusion bodies were found in the cytoplasm and in mitochondria. The epithelial cell structure modifications included an early loss of the apical microvillae, followed by a decrement of the luminal space and the respective apposition and proximity of apical membranes, resulting in the formation of atypical intercellular contacts and adhesion structures. Similar but less marked alterations were observed in subacute lead intoxication as well. Our work contributes in the understanding of the physiopathology of lead intoxication on the structure of renal tubular epithelial cell-cell contacts in vivo.

Cx43-hemichannel function and regulation in physiology and pathophysiology: insights from the bovine corneal endothelial cell system and beyond

PubMed Central

D'hondt, Catheleyne; Iyyathurai, Jegan; Himpens, Bernard; Leybaert, Luc; Bultynck, Geert

2014-01-01

Intercellular communication in primary bovine corneal endothelial cells (BCECs) is mainly driven by the release of extracellular ATP through Cx43 hemichannels. Studying the characteristics of Ca2+-wave propagation in BCECs, an important form of intercellular communication, in response to physiological signaling events has led to the discovery of important insights in the functional properties and regulation of native Cx43 hemichannels. Together with ectopic expression models for Cx43 hemichannels and truncated/mutated Cx43 versions, it became very clear that loop/tail interactions play a key role in controlling the activity of Cx43 hemichannels. Interestingly, the negative regulation of Cx43 hemichannels by enhanced actin/myosin contractility seems to impinge upon loss of these loop/tail interactions essential for opening Cx43 hemichannels. Finally, these molecular insights have spurred the development of novel peptide tools that can selectively inhibit Cx43 hemichannels, but neither Cx43 gap junctions nor hemichannels formed by other Cx isoforms. These tools now set the stage to hunt for novel physiological functions for Cx43 hemichannels in primary cells and tissues and to tackle disease conditions associated with excessive, pathological Cx43-hemichannel openings. PMID:25309448

Subepithelial corneal fibrosis partially due to epithelial-mesenchymal transition of ocular surface epithelium

PubMed Central

Kawashima, Motoko; Higa, Kazunari; Satake, Yoshiyuki; Omoto, Masahiro; Tsubota, Kazuo; Shimmura, Shigeto; Shimazaki, Jun

2010-01-01

Purpose To determine whether epithelial-mesenchymal transition is involved in the development of corneal subepithelial fibrosis (pannus). Methods Frozen samples of pannus tissue removed from human corneas with a diagnosis of total limbal stem cell deficiency were characterized by immunostaining for both epithelial and mesenchymal markers. We selected transformation-related protein 63 (p63) and pancytokeratin as epithelial markers and vimentin and α-smooth muscle actin (α-SMA) as mesenchymal markers. Immunostaining for β-catenin and E-cadherin was performed to determine wingless-Int (Wnt)-pathway activation. RT–PCR analysis was also performed on epithelial tissue obtained from pannus samples after dispase digestion. Results Immunohistochemistry revealed strong nuclear expression of p63 and weak intercellular expression of E-cadherin in epithelial basal cells of pannus tissue. Furthermore, translocation of β-catenin from intercellular junctions to the nucleus and cytoplasm was also observed. Double-positive cells for both p63 and α-SMA were observed in the subepithelial stroma of pannus tissue, which was supported by RT–PCR and cytospin analysis. Conclusions Epithelial-mesenchymal transition may be partially involved in the development of subepithelial corneal fibrosis due to total limbal stem cell deficiency. PMID:21179238

E-cadherin suppression accelerates squamous cell carcinoma progression in three-dimensional, human tissue constructs.

PubMed

Margulis, Alexander; Zhang, Weitian; Alt-Holland, Addy; Crawford, Howard C; Fusenig, Norbert E; Garlick, Jonathan A

2005-03-01

We studied the link between loss of E-cadherin-mediated adhesion and acquisition of malignant properties in three-dimensional, human tissue constructs that mimicked the initial stages of squamous cell cancer progression. Suppression of E-cadherin expression in early-stage, skin-derived tumor cells (HaCaT-II-4) was induced by cytoplasmic sequestration of beta-catenin upon stable expression of a dominant-negative E-cadherin fusion protein (H-2Kd-Ecad). In monolayer cultures, expression of H-2Kd-Ecad resulted in decreased levels of E-cadherin, redistribution of beta-catenin to the cytoplasm, and complete loss of intercellular adhesion when compared with control II-4 cells. This was accompanied by a 7-fold decrease in beta-catenin-mediated transcription and a 12-fold increase in cell migration. In three-dimensional constructs, E-cadherin-deficient tissues showed disruption of architecture, loss of adherens junctional proteins from cell contacts, and focal tumor cell invasion. Invasion was linked to activation of matrix metalloproteinase (MMP)-mediated degradation of basement membrane in H-2Kd-Ecad-expressing tissue constructs that was blocked by MMP inhibition (GM6001). Quantitative reverse transcription-PCR showed a 2.5-fold increase in MMP-2 and an 8-fold increase in MMP-9 in cells expressing the H-2Kd-Ecad fusion protein when compared with controls, and gel zymography showed increased MMP protein levels. Following surface transplantation of three-dimensional tissues, suppression of E-cadherin expression greatly accelerated tumorigenesis in vivo by inducing a switch to high-grade carcinomas that resulted in a 5-fold increase in tumor size after 4 weeks. Suppression of E-cadherin expression and loss of its function fundamentally modified squamous cell carcinoma progression by activating a highly invasive, aggressive tumor phenotype, whereas maintenance of E-cadherin prevented invasion in vitro and limited tumor progression in vivo.

Radiation-induced genomic instability and bystander effects: related inflammatory-type responses to radiation-induced stress and injury? A review.

PubMed

Lorimore, S A; Wright, E G

2003-01-01

To review studies of radiation responses in the haemopoietic system in the context of radiation-induced genomic instability, bystander effects and inflammatory-type processes. There is considerable evidence that cells that themselves are not exposed to ionizing radiation but are the progeny of cells irradiated many cell divisions previously may express a high frequency of gene mutations, chromosomal aberrations and cell death. These effects are collectively known as radiation-induced genomic instability. A second untargeted effect results in non-irradiated cells exhibiting responses typically associated with direct radiation exposure but occurs as a consequence of contact with irradiated cells or by receiving soluble signals from irradiated cells. These effects are collectively known as radiation-induced bystander effects. Reported effects include increases or decreases in damage-inducible and stress-related proteins; increases or decreases in reactive oxygen species, cell death or cell proliferation, and induction of mutations and chromosome aberrations. This array of responses is reminiscent of effects mediated by cytokines and other similar regulatory factors that may involve, but do not necessarily require, gap junction-mediated transfer, have multiple inducers and a variety of context-dependent consequences in different cell systems. That chromosomal instability in haemopoietic cells can be induced by an indirect bystander-type mechanism both in vitro and in vivo provides a potential link between these two untargeted effects and there are radiation responses in vivo consistent with the microenvironment contributing secondary cell damage as a consequence of an inflammatory-type response to radiation-induced injury. Intercellular signalling, production of cytokines and free radicals are features of inflammatory responses that have the potential for both bystander-mediated and persisting damage as well as for conferring a predisposition to malignancy. The induction of bystander effects and instabilities may reflect interrelated aspects of a non-specific inflammatory-type response to radiation-induced stress and injury and be involved in a variety of the pathological consequences of radiation exposures.

Electrical transmission between mammalian neurons is supported by a small fraction of gap junction channels.

PubMed

Curti, Sebastian; Hoge, Gregory; Nagy, James I; Pereda, Alberto E

2012-06-01

Electrical synapses formed by gap junctions between neurons create networks of electrically coupled neurons in the mammalian brain, where these networks have been found to play important functional roles. In most cases, interneuronal gap junctions occur at remote dendro-dendritic contacts, making difficult accurate characterization of their physiological properties and correlation of these properties with their anatomical and morphological features of the gap junctions. In the mesencephalic trigeminal (MesV) nucleus where neurons are readily accessible for paired electrophysiological recordings in brain stem slices, our recent data indicate that electrical transmission between MesV neurons is mediated by connexin36 (Cx36)-containing gap junctions located at somato-somatic contacts. We here review evidence indicating that electrical transmission between these neurons is supported by a very small fraction of the gap junction channels present at cell-cell contacts. Acquisition of this evidence was enabled by the unprecedented experimental access of electrical synapses between MesV neurons, which allowed estimation of the average number of open channels mediating electrical coupling in relation to the average number of gap junction channels present at these contacts. Our results indicate that only a small proportion of channels (~0.1 %) appear to be conductive. On the basis of similarities with other preparations, we postulate that this phenomenon might constitute a general property of vertebrate electrical synapses, reflecting essential aspects of gap junction function and maintenance.

Gap junction connexins in female reproductive organs: implications for women's reproductive health.

PubMed

Winterhager, Elke; Kidder, Gerald M

2015-01-01

Connexins comprise a family of ~20 proteins that form intercellular membrane channels (gap junction channels) providing a direct route for metabolites and signalling molecules to pass between cells. This review provides a critical analysis of the evidence for essential roles of individual connexins in female reproductive function, highlighting implications for women's reproductive health. No systematic review has been carried out. Published literature from the past 35 years was surveyed for research related to connexin involvement in development and function of the female reproductive system. Because of the demonstrated utility of genetic manipulation for elucidating connexin functions in various organs, much of the cited information comes from research with genetically modified mice. In some cases, a distinction is drawn between connexin functions clearly related to the formation of gap junction channels and those possibly linked to non-channel roles. Based on work with mice, several connexins are known to be required for female reproductive functions. Loss of connexin43 (CX43) causes an oocyte deficiency, and follicles lacking or expressing less CX43 in granulosa cells exhibit reduced growth, impairing fertility. CX43 is also expressed in human cumulus cells and, in the context of IVF, has been correlated with pregnancy outcome, suggesting that this connexin may be a determinant of oocyte and embryo quality in women. Loss of CX37, which exclusively connects oocytes with granulosa cells in the mouse, caused oocytes to cease growing without acquiring meiotic competence. Blocking of CX26 channels in the uterine epithelium disrupted implantation whereas loss or reduction of CX43 expression in the uterine stroma impaired decidualization and vascularization in mouse and human. Several connexins are important in placentation and, in the human, CX43 is a key regulator of the fusogenic pathway from the cytotrophoblast to the syncytiotrophoblast, ensuring placental growth. CX40, which characterizes the extravillous trophoblast (EVT), supports proliferation of the proximal EVTs while preventing them from differentiating into the invasive pathway. Furthermore, women with recurrent early pregnancy loss as well as those with endometriosis exhibit reduced levels of CX43 in their decidua. The antimalaria drug mefloquine, which blocks gap junction function, is responsible for increased risk of early pregnancy loss and stillbirth, probably due to inhibition of intercellular communication in the decidua or between trophoblast layers followed by an impairment of placental growth. Gap junctions also play a critical role in regulating uterine blood flow, contributing to the adaptive response to pregnancy. Given that reproductive impairment can result from connexin mutations in mice, it is advised that women suffering from somatic disease symptoms associated with connexin gene mutations be additionally tested for impacts on reproductive function. Better knowledge of these essential connexin functions in human female reproductive organs is important for safeguarding women's reproductive health. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Low Level Pro-inflammatory Cytokines Decrease Connexin36 Gap Junction Coupling in Mouse and Human Islets through Nitric Oxide-mediated Protein Kinase Cδ*

PubMed Central

Farnsworth, Nikki L.; Walter, Rachelle L.; Hemmati, Alireza; Westacott, Matthew J.; Benninger, Richard K. P.

2016-01-01

Pro-inflammatory cytokines contribute to the decline in islet function during the development of diabetes. Cytokines can disrupt insulin secretion and calcium dynamics; however, the mechanisms underlying this are poorly understood. Connexin36 gap junctions coordinate glucose-induced calcium oscillations and pulsatile insulin secretion across the islet. Loss of gap junction coupling disrupts these dynamics, similar to that observed during the development of diabetes. This study investigates the mechanisms by which pro-inflammatory cytokines mediate gap junction coupling. Specifically, as cytokine-induced NO can activate PKCδ, we aimed to understand the role of PKCδ in modulating cytokine-induced changes in gap junction coupling. Isolated mouse and human islets were treated with varying levels of a cytokine mixture containing TNF-α, IL-1β, and IFN-γ. Islet dysfunction was measured by insulin secretion, calcium dynamics, and gap junction coupling. Modulators of PKCδ and NO were applied to determine their respective roles in modulating gap junction coupling. High levels of cytokines caused cell death and decreased insulin secretion. Low levels of cytokine treatment disrupted calcium dynamics and decreased gap junction coupling, in the absence of disruptions to insulin secretion. Decreases in gap junction coupling were dependent on NO-regulated PKCδ, and altered membrane organization of connexin36. This study defines several mechanisms underlying the disruption to gap junction coupling under conditions associated with the development of diabetes. These mechanisms will allow for greater understanding of islet dysfunction and suggest ways to ameliorate this dysfunction during the development of diabetes. PMID:26668311

Ultraselective electrochemiluminescence biosensor based on locked nucleic acid modified toehold-mediated strand displacement reaction and junction-probe.

PubMed

Zhang, Xi; Zhang, Jing; Wu, Dongzhi; Liu, Zhijing; Cai, Shuxian; Chen, Mei; Zhao, Yanping; Li, Chunyan; Yang, Huanghao; Chen, Jinghua

2014-12-07

Locked nucleic acid (LNA) is applied in toehold-mediated strand displacement reaction (TMSDR) to develop a junction-probe electrochemiluminescence (ECL) biosensor for single-nucleotide polymorphism (SNP) detection in the BRCA1 gene related to breast cancer. More than 65-fold signal difference can be observed with perfectly matched target sequence to single-base mismatched sequence under the same conditions, indicating good selectivity of the ECL biosensor.

Con-nectin axons and dendrites.

PubMed

Beaudoin, Gerard M J

2006-07-03

Unlike adherens junctions, synapses are asymmetric connections, usually between axons and dendrites, that rely on various cell adhesion molecules for structural stability and function. Two cell types of adhesion molecules found at adherens junctions, cadherins and nectins, are thought to mediate homophilic interaction between neighboring cells. In this issue, Togashi et al. (see p. 141) demonstrate that the differential localization of two heterophilic interacting nectins mediates the selective attraction of axons and dendrites in cooperation with cadherins.

Calcium/Ask1/MKK7/JNK2/c-Src signalling cascade mediates disruption of intestinal epithelial tight junctions by dextran sulfate sodium.

PubMed

Samak, Geetha; Chaudhry, Kamaljit K; Gangwar, Ruchika; Narayanan, Damodaran; Jaggar, Jonathan H; Rao, RadhaKrishna

2015-02-01

Disruption of intestinal epithelial tight junctions is an important event in the pathogenesis of ulcerative colitis. Dextran sodium sulfate (DSS) induces colitis in mice with symptoms similar to ulcerative colitis. However, the mechanism of DSS-induced colitis is unknown. We investigated the mechanism of DSS-induced disruption of intestinal epithelial tight junctions and barrier dysfunction in Caco-2 cell monolayers in vitro and mouse colon in vivo. DSS treatment resulted in disruption of tight junctions, adherens junctions and actin cytoskeleton leading to barrier dysfunction in Caco-2 cell monolayers. DSS induced a rapid activation of c-Jun N-terminal kinase (JNK), and the inhibition or knockdown of JNK2 attenuated DSS-induced tight junction disruption and barrier dysfunction. In mice, DSS administration for 4 days caused redistribution of tight junction and adherens junction proteins from the epithelial junctions, which was blocked by JNK inhibitor. In Caco-2 cell monolayers, DSS increased intracellular Ca(2+) concentration, and depletion of intracellular Ca(2+) by 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester) (BAPTA/AM) or thapsigargin attenuated DSS-induced JNK activation, tight junction disruption and barrier dysfunction. Knockdown of apoptosis signal-regulated kinase 1 (Ask1) or MKK7 blocked DSS-induced tight junction disruption and barrier dysfunction. DSS activated c-Src by a Ca2+ and JNK-dependent mechanism. Inhibition of Src kinase activity or knockdown of c-Src blocked DSS-induced tight junction disruption and barrier dysfunction. DSS increased tyrosine phosphorylation of occludin, zonula occludens-1 (ZO-1), E-cadherin and β-catenin. SP600125 abrogated DSS-induced tyrosine phosphorylation of junctional proteins. Recombinant JNK2 induced threonine phosphorylation and auto-phosphorylation of c-Src. The present study demonstrates that Ca(2+)/Ask1/MKK7/JNK2/cSrc signalling cascade mediates DSS-induced tight junction disruption and barrier dysfunction.

Mannosyl Glycodendritic Structure Inhibits DC-SIGN-Mediated Ebola Virus Infection in cis and in trans

PubMed Central

Lasala, Fátima; Arce, Eva; Otero, Joaquín R.; Rojo, Javier; Delgado, Rafael

2003-01-01

We have designed a glycodendritic structure, BH30sucMan, that blocks the interaction between dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and Ebola virus (EBOV) envelope. BH30sucMan inhibits DC-SIGN-mediated EBOV infection at nanomolar concentrations. BH30sucMan may counteract important steps of the infective process of EBOV and, potentially, of microorganisms shown to exploit DC-SIGN for cell entry and infection. PMID:14638512

Receptor Complex Mediated Regulation of Symplastic Traffic.

PubMed

Stahl, Yvonne; Faulkner, Christine

2016-05-01

Plant receptor kinases (RKs) and receptor proteins (RPs) are involved in a plethora of cellular processes, including developmental decisions and immune responses. There is increasing evidence that plasmodesmata (PD)-localized RKs and RPs act as nexuses that perceive extracellular signals and convey them into intra- and intercellular responses by regulating the exchange of molecules through PD. How RK/RP complexes regulate the specific and nonspecific traffic of molecules through PD, and how these receptors are specifically targeted to PD, have been elusive but underpin comprehensive understanding of the function and regulation of the symplast. In this review we gather the current knowledge of RK/RP complex function at PD and how they might regulate intercellular traffic. Copyright © 2015 Elsevier Ltd. All rights reserved.

Enzyme-activated intracellular drug delivery with tubule clay nanoformulation

PubMed Central

Dzamukova, Maria R.; Naumenko, Ekaterina A.; Lvov, Yuri M.; Fakhrullin, Rawil F.

2015-01-01

Fabrication of stimuli-triggered drug delivery vehicle s is an important milestone in treating cancer. Here we demonstrate the selective anticancer drug delivery into human cells with biocompatible 50-nm diameter halloysite nanotube carriers. Physically-adsorbed dextrin end stoppers secure the intercellular release of brilliant green. Drug-loaded nanotubes penetrate through the cellular membranes and their uptake efficiency depends on the cells growth rate. Intercellular glycosyl hydrolases-mediated decomposition of the dextrin tube-end stoppers triggers the release of the lumen-loaded brilliant green, which allowed for preferable elimination of human lung carcinoma cells (А549) as compared with hepatoma cells (Hep3b). The enzyme-activated intracellular delivery of brilliant green using dextrin-coated halloysite nanotubes is a promising platform for anticancer treatment. PMID:25976444

Differential identity of Filopodia and Tunneling Nanotubes revealed by the opposite functions of actin regulatory complexes.

PubMed

Delage, Elise; Cervantes, Diégo Cordero; Pénard, Esthel; Schmitt, Christine; Syan, Sylvie; Disanza, Andrea; Scita, Giorgio; Zurzolo, Chiara

2016-12-23

Tunneling Nanotubes (TNTs) are actin enriched filopodia-like protrusions that play a pivotal role in long-range intercellular communication. Different pathogens use TNT-like structures as "freeways" to propagate across cells. TNTs are also implicated in cancer and neurodegenerative diseases, making them promising therapeutic targets. Understanding the mechanism of their formation, and their relation with filopodia is of fundamental importance to uncover their physiological function, particularly since filopodia, differently from TNTs, are not able to mediate transfer of cargo between distant cells. Here we studied different regulatory complexes of actin, which play a role in the formation of both these structures. We demonstrate that the filopodia-promoting CDC42/IRSp53/VASP network negatively regulates TNT formation and impairs TNT-mediated intercellular vesicle transfer. Conversely, elevation of Eps8, an actin regulatory protein that inhibits the extension of filopodia in neurons, increases TNT formation. Notably, Eps8-mediated TNT induction requires Eps8 bundling but not its capping activity. Thus, despite their structural similarities, filopodia and TNTs form through distinct molecular mechanisms. Our results further suggest that a switch in the molecular composition in common actin regulatory complexes is critical in driving the formation of either type of membrane protrusion.

A derivative of oleamide potently inhibits the spontaneous metastasis of mouse melanoma BL6 cells.

PubMed

Ito, Akihiko; Morita, Nobuyoshi; Miura, Daisaku; Koma, Yu-Ichiro; Kataoka, Tatsuki R; Yamasaki, Hiroshi; Kitamura, Yukihiko; Kita, Yasuyuki; Nojima, Hiroshi

2004-10-01

We reported previously that the abnormally augmented expression of connexin 26 (Cx26) is responsible for the enhanced spontaneous metastasis of mouse BL6 melanoma cells, and that the exogenous expression of a dominant negative form of Cx26 inhibits the spontaneous metastasis of BL6. Here we show that daily intraperitoneal (i.p.) injections of oleamide, a sleep-inducing lipid hormone, weakly inhibited the spontaneous metastasis of BL6 cells. To obtain a more effective reagent, 19 oleamide derivatives were chemically synthesized and tested for their ability to inhibit the gap junction-mediated intercellular communications (GJIC) that are formed between HeLa cells by the ectopic expression of Cx26 or Cx43. One of these, denoted metastasis inhibitor-18 (MI-18), inhibited the GJIC formed by Cx26 as well as oleamide but unlike oleamide, which is a non-selective inhibitor of connexin, it did not inhibit the GJIC formed by Cx43. Daily i.p. injections of MI-18 potently blocked the spontaneous metastasis of BL6 cells down to 15% of that in the untreated control mice. MI-18 was safe because even after >7 weeks of daily injections, the survival rate of the mice was 93%. We propose that MI-18 may serve as a novel and clinically important prototype of a potent inhibitor of spontaneous metastasis.

Cell–cell communication enhances the capacity of cell ensembles to sense shallow gradients during morphogenesis

PubMed Central

Ellison, David; Mugler, Andrew; Brennan, Matthew D.; Lee, Sung Hoon; Huebner, Robert J.; Shamir, Eliah R.; Woo, Laura A.; Kim, Joseph; Amar, Patrick; Nemenman, Ilya; Ewald, Andrew J.; Levchenko, Andre

2016-01-01

Collective cell responses to exogenous cues depend on cell–cell interactions. In principle, these can result in enhanced sensitivity to weak and noisy stimuli. However, this has not yet been shown experimentally, and little is known about how multicellular signal processing modulates single-cell sensitivity to extracellular signaling inputs, including those guiding complex changes in the tissue form and function. Here we explored whether cell–cell communication can enhance the ability of cell ensembles to sense and respond to weak gradients of chemotactic cues. Using a combination of experiments with mammary epithelial cells and mathematical modeling, we find that multicellular sensing enables detection of and response to shallow epidermal growth factor (EGF) gradients that are undetectable by single cells. However, the advantage of this type of gradient sensing is limited by the noisiness of the signaling relay, necessary to integrate spatially distributed ligand concentration information. We calculate the fundamental sensory limits imposed by this communication noise and combine them with the experimental data to estimate the effective size of multicellular sensory groups involved in gradient sensing. Functional experiments strongly implicated intercellular communication through gap junctions and calcium release from intracellular stores as mediators of collective gradient sensing. The resulting integrative analysis provides a framework for understanding the advantages and limitations of sensory information processing by relays of chemically coupled cells. PMID:26792522

Specific membrane lipid composition is important for plasmodesmata function in Arabidopsis.

PubMed

Grison, Magali S; Brocard, Lysiane; Fouillen, Laetitia; Nicolas, William; Wewer, Vera; Dörmann, Peter; Nacir, Houda; Benitez-Alfonso, Yoselin; Claverol, Stéphane; Germain, Véronique; Boutté, Yohann; Mongrand, Sébastien; Bayer, Emmanuelle M

2015-04-01

Plasmodesmata (PD) are nano-sized membrane-lined channels controlling intercellular communication in plants. Although progress has been made in identifying PD proteins, the role played by major membrane constituents, such as the lipids, in defining specialized membrane domains in PD remains unknown. Through a rigorous isolation of "native" PD membrane fractions and comparative mass spectrometry-based analysis, we demonstrate that lipids are laterally segregated along the plasma membrane (PM) at the PD cell-to-cell junction in Arabidopsis thaliana. Remarkably, our results show that PD membranes display enrichment in sterols and sphingolipids with very long chain saturated fatty acids when compared with the bulk of the PM. Intriguingly, this lipid profile is reminiscent of detergent-insoluble membrane microdomains, although our approach is valuably detergent-free. Modulation of the overall sterol composition of young dividing cells reversibly impaired the PD localization of the glycosylphosphatidylinositol-anchored proteins Plasmodesmata Callose Binding 1 and the β-1,3-glucanase PdBG2 and altered callose-mediated PD permeability. Altogether, this study not only provides a comprehensive analysis of the lipid constituents of PD but also identifies a role for sterols in modulating cell-to-cell connectivity, possibly by establishing and maintaining the positional specificity of callose-modifying glycosylphosphatidylinositol proteins at PD. Our work emphasizes the importance of lipids in defining PD membranes. © 2015 American Society of Plant Biologists. All rights reserved.

Specific Membrane Lipid Composition Is Important for Plasmodesmata Function in Arabidopsis

PubMed Central

Grison, Magali S.; Brocard, Lysiane; Fouillen, Laetitia; Nicolas, William; Wewer, Vera; Dörmann, Peter; Nacir, Houda; Benitez-Alfonso, Yoselin; Claverol, Stéphane; Germain, Véronique; Boutté, Yohann; Mongrand, Sébastien; Bayer, Emmanuelle M.

2015-01-01

Plasmodesmata (PD) are nano-sized membrane-lined channels controlling intercellular communication in plants. Although progress has been made in identifying PD proteins, the role played by major membrane constituents, such as the lipids, in defining specialized membrane domains in PD remains unknown. Through a rigorous isolation of “native” PD membrane fractions and comparative mass spectrometry-based analysis, we demonstrate that lipids are laterally segregated along the plasma membrane (PM) at the PD cell-to-cell junction in Arabidopsis thaliana. Remarkably, our results show that PD membranes display enrichment in sterols and sphingolipids with very long chain saturated fatty acids when compared with the bulk of the PM. Intriguingly, this lipid profile is reminiscent of detergent-insoluble membrane microdomains, although our approach is valuably detergent-free. Modulation of the overall sterol composition of young dividing cells reversibly impaired the PD localization of the glycosylphosphatidylinositol-anchored proteins Plasmodesmata Callose Binding 1 and the β-1,3-glucanase PdBG2 and altered callose-mediated PD permeability. Altogether, this study not only provides a comprehensive analysis of the lipid constituents of PD but also identifies a role for sterols in modulating cell-to-cell connectivity, possibly by establishing and maintaining the positional specificity of callose-modifying glycosylphosphatidylinositol proteins at PD. Our work emphasizes the importance of lipids in defining PD membranes. PMID:25818623

Role of vitamin D on gut microbiota in cystic fibrosis.

PubMed

Kanhere, Mansi; Chassaing, Benoit; Gewirtz, Andrew T; Tangpricha, Vin

2018-01-01

This review explores the potential for vitamin D to favorably alter the gut microbiota, given emerging evidence of the role of vitamin D in controlling mucosal inflammation in the gut. It will focus on cystic fibrosis (CF) patients, a population with both vitamin D deficiency due to gut malabsorption and an altered gut microbiota composition. Recent evidence shows that vitamin D acts to maintain the integrity of the gut mucosal barrier by enhancement of intercellular junctions that control mucosal permeability and reduction of pro-inflammatory cytokines such as IL-8. In addition, vitamin D receptor-mediated signaling has been shown to inhibit inflammation-induced apoptosis of intestinal epithelial cells. As a result of these effects on the intestinal mucosa, maintenance of sufficient vitamin D status may be essential for the development of a healthy gut microbiota, particularly in conditions defined by chronic mucosal inflammation such as CF. We hypothesize here that high dose vitamin D may be used to favorably manipulate the aberrant mucosa seen in patients with CF. This may result in improved clinical outcomes in association with a low inflammatory environment that allows beneficial bacteria to outcompete opportunistic pathogens. Current evidence is sparse but encouraging, and additional evidence is needed to establish vitamin D as a therapeutic approach for gut microbiota modification. Copyright © 2016 Elsevier Ltd. All rights reserved.

Dilated intercellular spaces and chronic cough as an extra-oesophageal manifestation of gastrooesophageal reflux disease.

PubMed

Orlando, Roy C

2011-06-01

Chronic cough is one of the extra-oesophageal manifestations of gastrooesophageal reflux disease (GORD). It is presumed to occur either directly by microaspiration of acidic gastric contents into the airway or indirectly by a reflex triggered by contact of acidic refluxates with the oesophageal epithelium in GORD. How contact of the oesophageal epithelium with acidic refluxates promotes sensitization for chronic cough is unknown, but like heartburn, which is a necessary accompaniment, it requires acid activation of nociceptors within the oesophageal mucosa. Dilated intercellular spaces within the oesophageal epithelium, a reflection of an increase in paracellular permeability, is a histopathologic feature of both erosive and non-erosive forms of GORD. Since it correlates with the symptom of heartburn, it is hypothesized herein that the increase in paracellular permeability to acid reflected by dilated intercellular spaces in oesophageal epithelium also serves as mediator of the signals that produce the reflex-induced sensitization for cough--a sensitization that can occur centrally within the medullary Nucleus Tractus Solitarius or peripherally within the tracheobronchial tree. Copyright © 2010 Elsevier Ltd. All rights reserved.

Gap Junctional Coupling is Essential for Epithelial Repair in the Avian Cochlea

PubMed Central

Nickel, Regina; Forge, Andrew

2014-01-01

The loss of auditory hair cells triggers repair responses within the population of nonsensory supporting cells. When hair cells are irreversibly lost from the mammalian cochlea, supporting cells expand to fill the resulting lesions in the sensory epithelium, an initial repair process that is dependent on gap junctional intercellular communication (GJIC). In the chicken cochlea (the basilar papilla or BP), dying hair cells are extruded from the epithelium and supporting cells expand to fill the lesions and then replace hair cells via mitotic and/or conversion mechanisms. Here, we investigated the involvement of GJIC in the initial epithelial repair process in the aminoglycoside-damaged BP. Gentamicin-induced hair cell loss was associated with a decrease of chicken connexin43 (cCx43) immunofluorescence, yet cCx30-labeled gap junction plaques remained. Fluorescence recovery after photobleaching experiments confirmed that the GJIC remained robust in gentamicin-damaged explants, but regionally asymmetric coupling was no longer evident. Dye injections in slice preparations from undamaged BP explants identified cell types with characteristic morphologies along the neural-abneural axis, but these were electrophysiologically indistinct. In gentamicin-damaged BP, supporting cells expanded to fill space formerly occupied by hair cells and displayed more variable electrophysiological phenotypes. When GJIC was inhibited during the aminoglycoside damage paradigm, the epithelial repair response halted. Dying hair cells were retained within the sensory epithelium and supporting cells remained unexpanded. These observations suggest that repair of the auditory epithelium shares common mechanisms across vertebrate species and emphasize the importance of functional gap junctions in maintaining a homeostatic environment permissive for subsequent hair cell regeneration. PMID:25429127

[The Functional Role of Exosomes in Cancer Biology and Their Potential as Biomarkers and Therapeutic Targets of Cancer].

PubMed

Naito, Yutaka; Yoshioka, Yusuke; Ochiya, Takahiro

2015-06-01

Intercellular communication plays an important role in the regulation of various cellular events. In particular, cancer cells and the surrounding cells communicate with each other, and this intercellular communication triggers cancer initiation and progression through the secretion of molecules, including growth factors and cytokines. Recent advances in cancer biology have indicated that small membrane vesicles, termed exosomes, also serve as regulatory agents in intercellular communications. Exosomes contain functional cellular components, including proteins and microRNAs (miRNAs), and they transfer these components to recipient cells. This exosome-mediated intercellular communication leads to increased growth, invasion, and metastasis of cancer. Thus, researchers regard exosomes as important cues to understanding the molecular mechanisms of cancer biology. Indeed, several lines of evidence have demonstrated that exosomes can explain multiple aspects of cancer biology. In addition, increasing evidence suggests that exosomes and their specific molecules are also attractive for use as biomarkers and therapeutic targets in cancer. Recent reports showed the efficacy of a novel diagnosis by detecting component molecules of cancer-derived exosomes, including miRNAs and membrane proteins. Furthermore, clinical trials that test the application of exosomes for cancer therapy have already been reported. From these points of view, we will summarize experimental data that support the role of exosomes in cancer progression and the potential of exosomes for use in novel diagnostic and therapeutic approaches for cancer.

Evolution of disorder in Mediator complex and its functional relevance.

PubMed

Nagulapalli, Malini; Maji, Sourobh; Dwivedi, Nidhi; Dahiya, Pradeep; Thakur, Jitendra K

2016-02-29

Mediator, an important component of eukaryotic transcriptional machinery, is a huge multisubunit complex. Though the complex is known to be conserved across all the eukaryotic kingdoms, the evolutionary topology of its subunits has never been studied. In this study, we profiled disorder in the Mediator subunits of 146 eukaryotes belonging to three kingdoms viz., metazoans, plants and fungi, and attempted to find correlation between the evolution of Mediator complex and its disorder. Our analysis suggests that disorder in Mediator complex have played a crucial role in the evolutionary diversification of complexity of eukaryotic organisms. Conserved intrinsic disordered regions (IDRs) were identified in only six subunits in the three kingdoms whereas unique patterns of IDRs were identified in other Mediator subunits. Acquisition of novel molecular recognition features (MoRFs) through evolution of new subunits or through elongation of the existing subunits was evident in metazoans and plants. A new concept of 'junction-MoRF' has been introduced. Evolutionary link between CBP and Med15 has been provided which explain the evolution of extended-IDR in CBP from Med15 KIX-IDR junction-MoRF suggesting role of junction-MoRF in evolution and modulation of protein-protein interaction repertoire. This study can be informative and helpful in understanding the conserved and flexible nature of Mediator complex across eukaryotic kingdoms. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Ultrastructural study of the semicircular canal cells of the frog Rana esculenta.

PubMed

Oudar, O; Ferrary, E; Feldmann, G

1988-03-01

The ultrastructure of the nonsensory cells (dark cells, transitional cells, and undifferentiated cells) of the frog semicircular canal was studied by using transmission electron microscopy in an attempt to correlate the structure with the functions of these epithelial cells. All the nonsensory cells were linked by tight junctions and desmosomes; this suggested that there is little paracellular ionic transport from perilymph to endolymph. In the dark cell epithelium, the apical intercellular spaces were dilated; in the basal part, numerous basolateral plasma membrane infoldings, containing mitochondria, delimited electron-lucent spaces. The undifferentiated cells and the transitional cells were devoid of any basal membrane infolding. Surrounding the semicircular canal, very flattened and interdigitated mesothelial cells constituted a thin multilayer tissue which limited the perilymphatic space. The morphological aspect of the dark cells suggests that they may play a role in the secretion and/or in the reabsorption of endolymph, which bathes the apical pole of these cells. The undifferentiated and transitional cells can play a role in the maintenance of the endolymphatic ionic composition because of their apical tight junctions and desmosomes.

Single and collective cell migration: the mechanics of adhesions

PubMed Central

De Pascalis, Chiara; Etienne-Manneville, Sandrine

2017-01-01

Chemical and physical properties of the environment control cell proliferation, differentiation, or apoptosis in the long term. However, to be able to move and migrate through a complex three-dimensional environment, cells must quickly adapt in the short term to the physical properties of their surroundings. Interactions with the extracellular matrix (ECM) occur through focal adhesions or hemidesmosomes via the engagement of integrins with fibrillar ECM proteins. Cells also interact with their neighbors, and this involves various types of intercellular adhesive structures such as tight junctions, cadherin-based adherens junctions, and desmosomes. Mechanobiology studies have shown that cell–ECM and cell–cell adhesions participate in mechanosensing to transduce mechanical cues into biochemical signals and conversely are responsible for the transmission of intracellular forces to the extracellular environment. As they migrate, cells use these adhesive structures to probe their surroundings, adapt their mechanical properties, and exert the appropriate forces required for their movements. The focus of this review is to give an overview of recent developments showing the bidirectional relationship between the physical properties of the environment and the cell mechanical responses during single and collective cell migration. PMID:28684609

GSK3- and PRMT-1–dependent modifications of desmoplakin control desmoplakin–cytoskeleton dynamics

PubMed Central

Albrecht, Lauren V.; Zhang, Lichao; Shabanowitz, Jeffrey; Purevjav, Enkhsaikhan; Towbin, Jeffrey A.; Hunt, Donald F.

2015-01-01

Intermediate filament (IF) attachment to intercellular junctions is required for skin and heart integrity, but how the strength and dynamics of this attachment are modulated during normal and pathological remodeling is poorly understood. We show that glycogen synthase kinase 3 (GSK3) and protein arginine methyltransferase 1 (PRMT-1) cooperate to orchestrate a series of posttranslational modifications on the IF-anchoring protein desmoplakin (DP) that play an essential role in coordinating cytoskeletal dynamics and cellular adhesion. Front-end electron transfer dissociation mass spectrometry analyses of DP revealed six novel serine phosphorylation sites dependent on GSK3 signaling and four novel arginine methylation sites including R2834, the mutation of which has been associated with arrhythmogenic cardiomyopathy (AC). Inhibition of GSK3 or PRMT-1 or overexpression of the AC-associated mutant R2834H enhanced DP–IF associations and delayed junction assembly. R2834H blocked the GSK3 phosphorylation cascade and reduced DP–GSK3 interactions in cultured keratinocytes and in the hearts of transgenic R2834H DP mice. Interference with this regulatory machinery may contribute to skin and heart diseases. PMID:25733715

cGAS-Mediated Innate Immunity Spreads Intercellularly through HIV-1 Env-Induced Membrane Fusion Sites.

PubMed

Xu, Shuting; Ducroux, Aurélie; Ponnurangam, Aparna; Vieyres, Gabrielle; Franz, Sergej; Müsken, Mathias; Zillinger, Thomas; Malassa, Angelina; Ewald, Ellen; Hornung, Veit; Barchet, Winfried; Häussler, Susanne; Pietschmann, Thomas; Goffinet, Christine

2016-10-12

Upon sensing cytoplasmic retroviral DNA in infected cells, cyclic GMP-AMP (cGAMP) synthase (cGAS) produces the cyclic dinucleotide cGAMP, which activates STING to trigger a type I interferon (IFN) response. We find that membrane fusion-inducing contact between donor cells expressing the HIV envelope (Env) and primary macrophages endogenously expressing the HIV receptor CD4 and coreceptor enable intercellular transfer of cGAMP. This cGAMP exchange results in STING-dependent antiviral IFN responses in target macrophages and protection from HIV infection. Furthermore, under conditions allowing cell-to-cell transmission of HIV-1, infected primary T cells, but not cell-free virions, deliver cGAMP to autologous macrophages through HIV-1 Env and CD4/coreceptor-mediated membrane fusion sites and induce a STING-dependent, but cGAS-independent, IFN response in target cells. Collectively, these findings identify an infection-specific mode of horizontal transfer of cGAMP between primary immune cells that may boost antiviral responses, particularly in infected tissues in which cell-to-cell transmission of virions exceeds cell-free infection. Copyright © 2016 Elsevier Inc. All rights reserved.

Effects of lactadherin on plasma D-lactic acid and small intestinal MUC2 and claudin-1 expression levels in rats with rotavirus-induced diarrhea

PubMed Central

XU, RUI; LEI, YI-HUI; SHI, JUN; ZHOU, YI-JUN; CHEN, YING-WEI; HE, ZHEN-JUAN

2016-01-01

The aim of the present study was to investigate the effects of lactadherin on plasma D-lactic acid and small intestinal mucin (MUC) 2 and claudin-1 expression levels in rats with diarrhea induced by rotavirus (RV) infection. A total of 75 seven-day-old healthy Sprague-Dawley rats were randomly divided into the following five groups: Control (C), RV infection (RVI), lactadherin before rotavirus infection (LBRI), lactadherin after rotavirus infection (LARI), and blank (B). On day 4 of artificial feeding, the rats in groups RVI, LBRI and LARI were intragastric administered 1×106 PFU RV; whereas the rats in groups C and B were intragastrically administered an equal volume of maintenance solution from the RV supernatant and normal saline, respectively. In the LBRI and LARI groups, rats received daily intragastric administration of 0.25 mg lactadherin for three days prior to and following infection with RV, respectively. The course of diarrheal symptoms was observed in each group and samples were collected on days 1, 4, and 7 post-infection in order to determine the mucosal morphology, plasma D-lactic acid levels and the expression levels of MUC2 and the intracellular junction protein, claudin-1, in the small intestine. On day 4 post-infection, the rats in group RVI demonstrated severely damaged small intestines and typical diarrheal characteristics, as detected by light microscopy; whereas rats in groups LBRI and LARI demonstrated intact small intestinal villi with partial vacuolation of epithelial cells and changes in the position of their nuclei. Electron microscopy demonstrated that the rats in the RVI group had sparse, shortened, disordered intestinal microvilli and widened intercellular junctions; whereas those in groups LBRI and LARI had long intestinal microvilli sparser compared with groups B and C and slightly widened intercellular junctions. Plasma D-lactic acid levels were increased in groups RVI, LBRI and LARI, as compared with groups B and C, and the greatest levels were detected in the RVI group on days 1, 4 and 7 post-infection. In addition to maintaining intestinal permeability, lactadherin enhanced the expression levels of MUC2 and reduced the expression of claudin-1; therefore, further protecting the intestinal epithelial barrier, which may contribute to the prevention and treatment of diarrhea induced by infection with RV. PMID:26998017

Effects of lactadherin on plasma D-lactic acid and small intestinal MUC2 and claudin-1 expression levels in rats with rotavirus-induced diarrhea.

PubMed

Xu, Rui; Lei, Yi-Hui; Shi, Jun; Zhou, Yi-Jun; Chen, Ying-Wei; He, Zhen-Juan

2016-03-01

The aim of the present study was to investigate the effects of lactadherin on plasma D-lactic acid and small intestinal mucin (MUC) 2 and claudin-1 expression levels in rats with diarrhea induced by rotavirus (RV) infection. A total of 75 seven-day-old healthy Sprague-Dawley rats were randomly divided into the following five groups: Control (C), RV infection (RVI), lactadherin before rotavirus infection (LBRI), lactadherin after rotavirus infection (LARI), and blank (B). On day 4 of artificial feeding, the rats in groups RVI, LBRI and LARI were intragastric administered 1×106 PFU RV; whereas the rats in groups C and B were intragastrically administered an equal volume of maintenance solution from the RV supernatant and normal saline, respectively. In the LBRI and LARI groups, rats received daily intragastric administration of 0.25 mg lactadherin for three days prior to and following infection with RV, respectively. The course of diarrheal symptoms was observed in each group and samples were collected on days 1, 4, and 7 post-infection in order to determine the mucosal morphology, plasma D-lactic acid levels and the expression levels of MUC2 and the intracellular junction protein, claudin-1, in the small intestine. On day 4 post-infection, the rats in group RVI demonstrated severely damaged small intestines and typical diarrheal characteristics, as detected by light microscopy; whereas rats in groups LBRI and LARI demonstrated intact small intestinal villi with partial vacuolation of epithelial cells and changes in the position of their nuclei. Electron microscopy demonstrated that the rats in the RVI group had sparse, shortened, disordered intestinal microvilli and widened intercellular junctions; whereas those in groups LBRI and LARI had long intestinal microvilli sparser compared with groups B and C and slightly widened intercellular junctions. Plasma D-lactic acid levels were increased in groups RVI, LBRI and LARI, as compared with groups B and C, and the greatest levels were detected in the RVI group on days 1, 4 and 7 post-infection. In addition to maintaining intestinal permeability, lactadherin enhanced the expression levels of MUC2 and reduced the expression of claudin-1; therefore, further protecting the intestinal epithelial barrier, which may contribute to the prevention and treatment of diarrhea induced by infection with RV.

NHERF1 and CFTR restore tight junction organisation and function in cystic fibrosis airway epithelial cells: role of ezrin and the RhoA/ROCK pathway.

PubMed

Castellani, Stefano; Guerra, Lorenzo; Favia, Maria; Di Gioia, Sante; Casavola, Valeria; Conese, Massimo

2012-11-01

Tight junctions (TJs) restrict the transit of ions and molecules through the paracellular route and act as a barrier to regulate access of inflammatory cells into the airway lumen. The pathophysiology of cystic fibrosis (CF) lung disease is characterised by abnormal ion and fluid transport across the epithelium and polymorphonuclear (PMN) leukocyte-dominated inflammatory response. Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) is a protein involved in PKA-dependent activation of CFTR by interacting with CFTR via its PDZ domains and with ezrin via its C-terminal domain. We have previously found that the NHERF1-overexpression dependent rescue CFTR-dependent chloride secretion is due to the re-organisation of the actin cytoskeleton network induced by the formation of the multiprotein complex NHERF1-RhoA-ezrin-actin. In this context, we here studied whether NHERF1 and CFTR are involved in the organisation and function of TJs. F508del CFBE41o⁻ monolayers presented nuclear localisation of zonula occludens (ZO-1) and occludin as well as disorganisation of claudin 1 and junction-associated adhesion molecule 1 as compared with wild-type 16HBE14o⁻ monolayers, paralleled by increased permeability to dextrans and PMN transmigration. Overexpression of either NHERF1 or CFTR in CFBE41o⁻ cells rescued TJ proteins to their proper intercellular location and decreased permeability and PMN transmigration, while this effect was not achieved by overexpressing either NHERF1 deprived of ezrin-binding domain. Further, expression of a phospho-dead ezrin mutant, T567A, increased permeability in both 16HBE14o⁻ cells and in a CFBE clone stably overexpressing NHERF1 (CFBE/sNHERF1), whereas a constitutively active form of ezrin, T567D, achieved the opposite effect in CFBE41o⁻ cells. A dominant-negative form of RhoA (RhoA-N19) also disrupted ZO-1 localisation at the intercellular contacts dislodging it to the nucleus and increased permeability in CFBE/sNHERF1. The inhibitor Y27632 of Rho kinase (ROCK) increased permeability as well. Overall, these data suggest a significant role for the multiprotein complex CFTR-NHERF1-ezrin-actin in maintaining TJ organisation and barrier function, and suggest that the RhoA/ROCK pathway is involved.

Three distinct suppressors of RNA silencing encoded by a 20-kb viral RNA genome

NASA Astrophysics Data System (ADS)

Lu, Rui; Folimonov, Alexey; Shintaku, Michael; Li, Wan-Xiang; Falk, Bryce W.; Dawson, William O.; Ding, Shou-Wei

2004-11-01

Viral infection in both plant and invertebrate hosts requires a virus-encoded function to block the RNA silencing antiviral defense. Here, we report the identification and characterization of three distinct suppressors of RNA silencing encoded by the 20-kb plus-strand RNA genome of citrus tristeza virus (CTV). When introduced by genetic crosses into plants carrying a silencing transgene, both p20 and p23, but not coat protein (CP), restored expression of the transgene. Although none of the CTV proteins prevented DNA methylation of the transgene, export of the silencing signal (capable of mediating intercellular silencing spread) was detected only from the F1 plants expressing p23 and not from the CP- or p20-expressing F1 plants, demonstrating suppression of intercellular silencing by CP and p20 but not by p23. Thus, intracellular and intercellular silencing are each targeted by a CTV protein, whereas the third, p20, inhibits silencing at both levels. Notably, CP suppresses intercellular silencing without interfering with intracellular silencing. The novel property of CP suggests a mechanism distinct to p20 and all of the other viral suppressors known to interfere with intercellular silencing and that this class of viral suppressors may not be consistently identified by Agrobacterium coinfiltration because it also induces RNA silencing against the infiltrated suppressor transgene. Our analyses reveal a sophisticated viral counter-defense strategy that targets the silencing antiviral pathway at multiple steps and may be essential for protecting CTV with such a large RNA genome from antiviral silencing in the perennial tree host. RNA interference | citrus tristeza virus | virus synergy | antiviral immunity

Gap junction- and hemichannel-independent actions of connexins.

PubMed

Jiang, Jean X; Gu, Sumin

2005-06-10

Connexins have been known to be the protein building blocks of gap junctions and mediate cell-cell communication. In contrast to the conventional dogma, recent evidence suggests that in addition to forming gap junction channels, connexins possess gap junction-independent functions. One important gap junction-independent function for connexins is to serve as the major functional component for hemichannels, the un-apposed halves of gap junctions. Hemichannels, as independent functional units, play roles that are different from that of gap junctions in the cell. The other functions of connexins appear to be gap junction- and hemichannel-independent. Published studies implicate the latter functions of connexins in cell growth, differentiation, tumorigenicity, injury, and apoptosis, although the mechanistic aspects of these actions remain largely unknown. In this review, gap junction- and hemichannel-independent functions of connexins are summarized, and the molecular mechanisms underlying these connexin functions are speculated and discussed.

Death of Neurons following Injury Requires Conductive Neuronal Gap Junction Channels but Not a Specific Connexin

PubMed Central

Fontes, Joseph D.; Ramsey, Jon; Polk, Jeremy M; Koop, Andre; Denisova, Janna V.; Belousov, Andrei B.

2015-01-01

Pharmacological blockade or genetic knockout of neuronal connexin 36 (Cx36)-containing gap junctions reduces neuronal death caused by ischemia, traumatic brain injury and NMDA receptor (NMDAR)-mediated excitotoxicity. However, whether Cx36 gap junctions contribute to neuronal death via channel-dependent or channel-independent mechanism remains an open question. To address this, we manipulated connexin protein expression via lentiviral transduction of mouse neuronal cortical cultures and analyzed neuronal death twenty-four hours following administration of NMDA (a model of NMDAR excitotoxicity) or oxygen-glucose deprivation (a model of ischemic injury). In cultures prepared from wild-type mice, over-expression and knockdown of Cx36-containing gap junctions augmented and prevented, respectively, neuronal death from NMDAR-mediated excitotoxicity and ischemia. In cultures obtained form from Cx36 knockout mice, re-expression of functional gap junction channels, containing either neuronal Cx36 or non-neuronal Cx43 or Cx31, resulted in increased neuronal death following insult. In contrast, the expression of communication-deficient gap junctions (containing mutated connexins) did not have this effect. Finally, the absence of ethidium bromide uptake in non-transduced wild-type neurons two hours following NMDAR excitotoxicity or ischemia suggested the absence of active endogenous hemichannels in those neurons. Taken together, these results suggest a role for neuronal gap junctions in cell death via a connexin type-independent mechanism that likely relies on channel activities of gap junctional complexes among neurons. A possible contribution of gap junction channel-permeable death signals in neuronal death is discussed. PMID:26017008

Bioelectrical coupling in multicellular domains regulated by gap junctions: A conceptual approach.

PubMed

Cervera, Javier; Pietak, Alexis; Levin, Michael; Mafe, Salvador

2018-04-21

We review the basic concepts involved in bioelectrically-coupled multicellular domains, focusing on the role of membrane potentials (V mem ). In the first model, single-cell V mem is modulated by two generic polarizing and depolarizing ion channels, while intercellular coupling is implemented via voltage-gated gap junctions. Biochemical and bioelectrical signals are integrated via a feedback loop between V mem and the transcription and translation of a protein forming an ion channel. The effective rate constants depend on the single-cell V mem because these potentials modulate the local concentrations of signaling molecules and ions. This electrochemically-based idealization of the complex biophysical problem suggests that the spatio-temporal map of single-cell potentials can influence downstream patterning processes by means of the voltage-gated gap junction interconnectivity, much as in the case of electronic devices where the control of electric potentials and currents allows the local modulation of the circuitry to achieve full functionality. An alternative theoretical approach, the BioElectrical Tissue Simulation Engine (BETSE), is also presented. The BETSE modeling environment utilizes finite volume techniques to simulate bioelectric states from the perspective of ion concentrations and fluxes. This model has been successfully applied to make predictions and explain experimental observations in a variety of embryonic, regenerative, and oncogenic contexts. Copyright © 2018 Elsevier B.V. All rights reserved.