Structure and function of gap junction proteins: role of gap junction proteins in embryonic heart development.

PubMed

Ahir, Bhavesh K; Pratten, Margaret K

2014-01-01

Intercellular (cell-to-cell) communication is a crucial and complex mechanism during embryonic heart development. In the cardiovascular system, the beating of the heart is a dynamic and key regulatory process, which is functionally regulated by the coordinated spread of electrical activity through heart muscle cells. Heart tissues are composed of individual cells, each bearing specialized cell surface membrane structures called gap junctions that permit the intercellular exchange of ions and low molecular weight molecules. Gap junction channels are essential in normal heart function and they assist in the mediated spread of electrical impulses that stimulate synchronized contraction (via an electrical syncytium) of cardiac tissues. This present review describes the current knowledge of gap junction biology. In the first part, we summarise some relevant biochemical and physiological properties of gap junction proteins, including their structure and function. In the second part, we review the current evidence demonstrating the role of gap junction proteins in embryonic development with particular reference to those involved in embryonic heart development. Genetics and transgenic animal studies of gap junction protein function in embryonic heart development are considered and the alteration/disruption of gap junction intercellular communication which may lead to abnormal heart development is also 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.

Functional assessment of gap junctions in monolayer and three-dimensional cultures of human tendon cells using fluorescence recovery after photobleaching

PubMed Central

Kuzma-Kuzniarska, Maria; Yapp, Clarence; Pearson-Jones, Thomas W.; Jones, Andrew K.; Hulley, Philippa A.

2014-01-01

Abstract. Gap junction-mediated intercellular communication influences a variety of cellular activities. In tendons, gap junctions modulate collagen production, are involved in strain-induced cell death, and are involved in the response to mechanical stimulation. The aim of the present study was to investigate gap junction-mediated intercellular communication in healthy human tendon-derived cells using fluorescence recovery after photobleaching (FRAP). The FRAP is a noninvasive technique that allows quantitative measurement of gap junction function in living cells. It is based on diffusion-dependent redistribution of a gap junction-permeable fluorescent dye. Using FRAP, we showed that human tenocytes form functional gap junctions in monolayer and three-dimensional (3-D) collagen I culture. Fluorescently labeled tenocytes following photobleaching rapidly reacquired the fluorescent dye from neighboring cells, while HeLa cells, which do not communicate by gap junctions, remained bleached. Furthermore, both 18 β-glycyrrhetinic acid and carbenoxolone, standard inhibitors of gap junction activity, impaired fluorescence recovery in tendon cells. In both monolayer and 3-D cultures, intercellular communication in isolated cells was significantly decreased when compared with cells forming many cell-to-cell contacts. In this study, we used FRAP as a tool to quantify and experimentally manipulate the function of gap junctions in human tenocytes in both two-dimensional (2-D) and 3-D cultures. PMID:24390370

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.

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.

An intercellular polyamine transfer via gap junctions regulates proliferation and response to stress in epithelial cells

PubMed Central

Desforges, Bénédicte; Curmi, Patrick A.; Bounedjah, Ouissame; Nakib, Samir; Hamon, Loic; De Bandt, Jean-Pascal; Pastré, David

2013-01-01

In the organism, quiescent epithelial cells have the potential to resume cycling as a result of various stimuli, including wound healing or oxidative stress. Because quiescent cells have a low polyamine level, resuming their growth requires an increase of their intracellular polyamine levels via de novo polyamine synthesis or their uptake from plasma. Another alternative, explored here, is an intercellular exchange with polyamine-rich cycling cells via gap junctions. We show that polyamines promote gap junction communication between proliferating cells by promoting dynamical microtubule plus ends at the cell periphery and thus allow polyamine exchange between cells. In this way, cycling cells favor regrowth in adjacent cells deprived of polyamines. In addition, intercellular interactions mediated by polyamines can coordinate the translational response to oxidative stress through the formation of stress granules. Some putative in vivo consequences of polyamine-mediated intercellular interactions are also discussed regarding cancer invasiveness and tissue regeneration. PMID:23515223

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.

Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

NASA Technical Reports Server (NTRS)

Jorgensen, Niklas Rye; Teilmann, Stefan Cuoni; Henriksen, Zanne; Civitelli, Roberto; Sorensen, Ole Helmer; Steinberg, Thomas H.

2003-01-01

The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting in "slow" ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal of extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx43 (UMR/Cx43) we confirmed that nifedipine sensitivity of ICW required Cx43 expression. In human osteoblastic cells, gap junction-dependent ICW also required activation of L-type calcium channels and influx of extracellular calcium.

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.

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

Kinetic Measurements Reveal Enhanced Protein-Protein Interactions at Intercellular Junctions

PubMed Central

Shashikanth, Nitesh; Kisting, Meridith A.; Leckband, Deborah E.

2016-01-01

The binding properties of adhesion proteins are typically quantified from measurements with soluble fragments, under conditions that differ radically from the confined microenvironment of membrane bound proteins in adhesion zones. Using classical cadherin as a model adhesion protein, we tested the postulate that confinement within quasi two-dimensional intercellular gaps exposes weak protein interactions that are not detected in solution binding assays. Micropipette-based measurements of cadherin-mediated, cell-cell binding kinetics identified a unique kinetic signature that reflects both adhesive (trans) bonds between cadherins on opposing cells and lateral (cis) interactions between cadherins on the same cell. In solution, proposed lateral interactions were not detected, even at high cadherin concentrations. Mutations postulated to disrupt lateral cadherin association altered the kinetic signatures, but did not affect the adhesive (trans) binding affinity. Perturbed kinetics further coincided with altered cadherin distributions at junctions, wound healing dynamics, and paracellular permeability. Intercellular binding kinetics thus revealed cadherin interactions that occur within confined, intermembrane gaps but not in solution. Findings further demonstrate the impact of these revealed interactions on the organization and function of intercellular junctions. PMID:27009566

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.

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.

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 Sleep-inducing Lipid Oleamide Deconvolutes Gap Junction Communication and Calcium Wave Transmission in Glial Cells

PubMed Central

Guan, Xiaojun; Cravatt, Benjamin F.; Ehring, George R.; Hall, James E.; Boger, Dale L.; Lerner, Richard A.; Gilula, Norton B.

1997-01-01

Oleamide is a sleep-inducing lipid originally isolated from the cerebrospinal fluid of sleep-deprived cats. Oleamide was found to potently and selectively inactivate gap junction–mediated communication between rat glial cells. In contrast, oleamide had no effect on mechanically stimulated calcium wave transmission in this same cell type. Other chemical compounds traditionally used as inhibitors of gap junctional communication, like heptanol and 18β-glycyrrhetinic acid, blocked not only gap junctional communication but also intercellular calcium signaling. Given the central role for intercellular small molecule and electrical signaling in central nervous system function, oleamide- induced inactivation of glial cell gap junction channels may serve to regulate communication between brain cells, and in doing so, may influence higher order neuronal events like sleep induction. PMID:9412472

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

Astroglial Metabolic Networks Sustain Hippocampal Synaptic Transmission

NASA Astrophysics Data System (ADS)

Rouach, Nathalie; Koulakoff, Annette; Abudara, Veronica; Willecke, Klaus; Giaume, Christian

2008-12-01

Astrocytes provide metabolic substrates to neurons in an activity-dependent manner. However, the molecular mechanisms involved in this function, as well as its role in synaptic transmission, remain unclear. Here, we show that the gap-junction subunit proteins connexin 43 and 30 allow intercellular trafficking of glucose and its metabolites through astroglial networks. This trafficking is regulated by glutamatergic synaptic activity mediated by AMPA receptors. In the absence of extracellular glucose, the delivery of glucose or lactate to astrocytes sustains glutamatergic synaptic transmission and epileptiform activity only when they are connected by gap junctions. These results indicate that astroglial gap junctions provide an activity-dependent intercellular pathway for the delivery of energetic metabolites from blood vessels to distal neurons.

Astroglial metabolic networks sustain hippocampal synaptic transmission.

PubMed

Rouach, Nathalie; Koulakoff, Annette; Abudara, Veronica; Willecke, Klaus; Giaume, Christian

2008-12-05

Astrocytes provide metabolic substrates to neurons in an activity-dependent manner. However, the molecular mechanisms involved in this function, as well as its role in synaptic transmission, remain unclear. Here, we show that the gap-junction subunit proteins connexin 43 and 30 allow intercellular trafficking of glucose and its metabolites through astroglial networks. This trafficking is regulated by glutamatergic synaptic activity mediated by AMPA receptors. In the absence of extracellular glucose, the delivery of glucose or lactate to astrocytes sustains glutamatergic synaptic transmission and epileptiform activity only when they are connected by gap junctions. These results indicate that astroglial gap junctions provide an activity-dependent intercellular pathway for the delivery of energetic metabolites from blood vessels to distal neurons.

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

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

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

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

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

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

Gap-junction-mediated communication in human periodontal ligament cells.

PubMed

Kato, R; Ishihara, Y; Kawanabe, N; Sumiyoshi, K; Yoshikawa, Y; Nakamura, M; Imai, Y; Yanagita, T; Fukushima, H; Kamioka, H; Takano-Yamamoto, T; Yamashiro, T

2013-07-01

Periodontal tissue homeostasis depends on a complex cellular network that conveys cell-cell communication. Gap junctions (GJs), one of the intercellular communication systems, are found between adjacent human periodontal ligament (hPDL) cells; however, the functional GJ coupling between hPDL cells has not yet been elucidated. In this study, we investigated functional gap-junction-mediated intercellular communication in isolated primary hPDL cells. SEM images indicated that the cells were in contact with each other via dendritic processes, and also showed high anti-connexin43 (Cx43) immunoreactivity on these processes. Gap-junctional intercellular communication (GJIC) among hPDL cells was assessed by fluorescence recovery after a photobleaching (FRAP) analysis, which exhibited dye coupling between hPDL cells, and was remarkably down-regulated when the cells were treated with a GJ blocker. Additionally, we examined GJs under hypoxic stress. The fluorescence recovery and expression levels of Cx43 decreased time-dependently under the hypoxic condition. Exposure to GJ inhibitor or hypoxia increased RANKL expression, and decreased OPG expression. This study shows that GJIC is responsible for hPDL cells and that its activity is reduced under hypoxia. This is consistent with the possible role of hPDL cells in regulating the biochemical reactions in response to changes in the hypoxic environment.

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.

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.

Intercellular nanotubes: insights from imaging studies and beyond

PubMed Central

Hurtig, Johan; Chiu, Daniel T.; Önfelt, Björn

2017-01-01

Cell-cell communication is critical to the development, maintenance, and function of multicellular organisms. Classical mechanisms for intercellular communication include secretion of molecules into the extracellular space and transport of small molecules through gap junctions. Recent reports suggest that cells also can communicate over long distances via a network of transient intercellular nanotubes. Such nanotubes have been shown to mediate intercellular transfer of organelles as well as membrane components and cytoplasmic molecules. Moreover, intercellular nanotubes have been observed in vivo and have been shown to enhance the transmission of pathogens such as human immunodeficiency virus (HIV)-1 and prions in vitro. These studies indicate that intercellular nanotubes may play a role both in normal physiology and in disease. PMID:20166114

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.

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

Rescue of Notch signaling in cells incapable of GDP-L-fucose synthesis by gap junction transfer of GDP-L-fucose in Drosophila.

PubMed

Ayukawa, Tomonori; Matsumoto, Kenjiroo; Ishikawa, Hiroyuki O; Ishio, Akira; Yamakawa, Tomoko; Aoyama, Naoki; Suzuki, Takuya; Matsuno, Kenji

2012-09-18

Notch (N) is a transmembrane receptor that mediates cell-cell interactions to determine many cell-fate decisions. N contains EGF-like repeats, many of which have an O-fucose glycan modification that regulates N-ligand binding. This modification requires GDP-L-fucose as a donor of fucose. The GDP-L-fucose biosynthetic pathways are well understood, including the de novo pathway, which depends on GDP-mannose 4,6 dehydratase (Gmd) and GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase/4-reductase (Gmer). However, the potential for intercellularly supplied GDP-L-fucose and the molecular basis of such transportation have not been explored in depth. To address these points, we studied the genetic effects of mutating Gmd and Gmer on fucose modifications in Drosophila. We found that these mutants functioned cell-nonautonomously, and that GDP-L-fucose was supplied intercellularly through gap junctions composed of Innexin-2. GDP-L-fucose was not supplied through body fluids from different isolated organs, indicating that the intercellular distribution of GDP-L-fucose is restricted within a given organ. Moreover, the gap junction-mediated supply of GDP-L-fucose was sufficient to support the fucosylation of N-glycans and the O-fucosylation of the N EGF-like repeats. Our results indicate that intercellular delivery is a metabolic pathway for nucleotide sugars in live animals under certain circumstances.

Homotypic gap junctional communication associated with metastasis increases suppression increases with PKA kinase activity and is unaffected by P13K inhibition

USDA-ARS?s Scientific Manuscript database

Loss of gap junctional intercellular communication (GJIC) between cancer cells is a common characteristic of malignant transformation. This communication is mediated by connexin proteins that make up the functional units of gap junctions. Connexins are highly regulated at the protein level and phosp...

The tight junction protein ZO-2 and Janus kinase 1 mediate intercellular communications in vascular smooth muscle cells

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

Tkachuk, Natalia; Tkachuk, Sergey; Patecki, Margret

2011-07-08

Highlights: {yields} The tight junction protein ZO-2 associates with Jak1 in vascular smooth muscle cells via ZO-2 N-terminal fragment. {yields} Jak1 mediates ZO-2 tyrosine phosphorylation and ZO-2 localization to the sites of homotypic intercellular contacts. {yields} The urokinase receptor uPAR regulates ZO-2/Jak1 functional association. {yields} The ZO-2/Jak1/uPAR signaling complex is required for vascular smooth muscle cells functional network formation. -- Abstract: Recent evidence points to a multifunctional role of ZO-2, the tight junction protein of the MAGUK (membrane-associated guanylate kinase-like) family. Though ZO-2 has been found in cell types lacking tight junction structures, such as vascular smooth muscle cells (VSMC),more » little is known about ZO-2 function in these cells. We provide evidence that ZO-2 mediates specific homotypic cell-to-cell contacts between VSMC. Using mass spectrometry we found that ZO-2 is associated with the non-receptor tyrosine kinase Jak1. By generating specific ZO-2 constructs we further found that the N-terminal fragment of ZO-2 molecule is responsible for this interaction. Adenovirus-based expression of Jak1 inactive mutant demonstrated that Jak1 mediates ZO-2 tyrosine phosphorylation. By means of RNA silencing, expression of Jak1 mutant form and fluorescently labeled ZO-2 fusion protein we further specified that active Jak1, but not Jak1 inactive mutant, mediates ZO-2 localization to the sites of intercellular contacts. We identified the urokinase receptor uPAR as a pre-requisite for these cellular events. Functional requirement of the revealed signaling complex for VSMC network formation was confirmed in experiments using Matrigel and in contraction assay. Our findings imply involvement of the ZO-2 tight junction independent signaling complex containing Jak1 and uPAR in VSMC intercellular communications. This mechanism may contribute to vascular remodeling in occlusive cardiovascular diseases and in arteriogenesis.« less

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

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

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.

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.

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

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.

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

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.

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.

Connexin 32 and its derived homotypic gap junctional intercellular communication inhibit the migration and invasion of transfected HeLa cells via enhancement of intercellular adhesion.

PubMed

Yang, Jie; Liu, Bing; Wang, Qin; Yuan, Dongdong; Hong, Xiaoting; Yang, Yan; Tao, Liang

2011-01-01

The effects of connexin (Cx) and its derived homotypic gap junctional intercellular communication (GJIC) between tumor cells on the invasion of metastatic cancers and the underlying mechanisms remain unclear. In this study, we investigated the influence of Cx32 and the homotypic GJIC mediated by this Cx on the migration, invasion and intercellular adhesion of transfected HeLa cells. The expression of Cx32 significantly increased cell adhesion and inhibited migration and invasion. The inhibition of GJIC by oleamide, a widely used GJIC inhibitor, reduced the enhanced adhesion and partly reversed the decreased migration and invasion that had been induced by Cx32 expression. Blockage of the p38 and extracellular signal-regulated kinase 1 and 2 mitogen-activated protein kinase (ERK1/2 MAPKs) pathways using their specific inhibitors attenuated the effects of Cx32, but not those of GJIC, on cell adhesion, migration and invasion. These results indicate that the homotypic GJIC mediated by Cx32, as well as the Cx itself, inhibit cell migration and invasion, most likely through the elevation of intercellular adhesion. The suppressive effect of Cx32 on the migration and invasion of cancer cells, but not that of its derived homotypic GJIC, partly depends on the activation of the p38 and the ERK1/2 MAPKs pathways.

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

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.

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.

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.

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.

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

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.

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

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

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.

Pannexin-1 channels show distinct morphology and no gap junction characteristics in mammalian cells.

PubMed

Beckmann, Anja; Grissmer, Alexander; Krause, Elmar; Tschernig, Thomas; Meier, Carola

2016-03-01

Pannexins (Panx) are proteins with a similar membrane topology to connexins, the integral membrane protein of gap junctions. Panx1 channels are generally of major importance in a large number of system and cellular processes and their function has been thoroughly characterized. In contrast, little is known about channel structure and subcellular distribution. We therefore determine the subcellular localization of Panx1 channels in cultured cells and aim at the identification of channel morphology in vitro. Using freeze-fracture replica immunolabeling on EYFP-Panx1-overexpressing HEK 293 cells, large particles were identified in plasma membranes, which were immunogold-labeled using either GFP or Panx1 antibodies. There was no labeling or particles in the nuclear membranes of these cells, pointing to plasma membrane localization of Panx1-EYFP channels. The assembly of particles was irregular, this being in contrast to the regular pattern of gap junctions. The fact that no counterparts were identified on apposing cells, which would have been indicative of intercellular signaling, supported the idea of Panx1 channels within one membrane. Control cells (transfected with EYFP only, non-transfected) were devoid of both particles and immunogold labeling. Altogether, this study provides the first demonstration of Panx1 channel morphology and assembly in intact cells. The identification of Panx1 channels as large particles within the plasma membrane provides the knowledge required to enable recognition of Panx1 channels in tissues in future studies. Thus, these results open up new avenues for the detailed analysis of the subcellular localization of Panx1 and of its nearest neighbors such as purinergic receptors in vivo.

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.

An electrostatic mechanism for Ca2+-mediated regulation of gap junction channels

PubMed Central

Bennett, Brad C.; Purdy, Michael D.; Baker, Kent A.; Acharya, Chayan; McIntire, William E.; Stevens, Raymond C.; Zhang, Qinghai; Harris, Andrew L.; Abagyan, Ruben; Yeager, Mark

2016-01-01

Gap junction channels mediate intercellular signalling that is crucial in tissue development, homeostasis and pathologic states such as cardiac arrhythmias, cancer and trauma. To explore the mechanism by which Ca2+ blocks intercellular communication during tissue injury, we determined the X-ray crystal structures of the human Cx26 gap junction channel with and without bound Ca2+. The two structures were nearly identical, ruling out both a large-scale structural change and a local steric constriction of the pore. Ca2+ coordination sites reside at the interfaces between adjacent subunits, near the entrance to the extracellular gap, where local, side chain conformational rearrangements enable Ca2+chelation. Computational analysis revealed that Ca2+-binding generates a positive electrostatic barrier that substantially inhibits permeation of cations such as K+ into the pore. Our results provide structural evidence for a unique mechanism of channel regulation: ionic conduction block via an electrostatic barrier rather than steric occlusion of the channel pore. PMID:26753910

[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.

Serotonin passes through myoendothelial gap junctions to promote pulmonary arterial smooth muscle cell differentiation.

PubMed

Gairhe, Salina; Bauer, Natalie N; Gebb, Sarah A; McMurtry, Ivan F

2012-11-01

Myoendothelial gap junctional signaling mediates pulmonary arterial endothelial cell (PAEC)-induced activation of latent TGF-β and differentiation of cocultured pulmonary arterial smooth muscle cells (PASMCs), but the nature of the signal passing from PAECs to PASMCs through the gap junctions is unknown. Because PAECs but not PASMCs synthesize serotonin, and serotonin can pass through gap junctions, we hypothesized that the monoamine is the intercellular signal. We aimed to determine whether PAEC-derived serotonin mediates PAEC-induced myoendothelial gap junction-dependent activation of TGF-β signaling and differentiation of PASMCs. Rat PAECs and PASMCs were monocultured or cocultured with (touch) or without (no-touch) direct cell-cell contact. In all cases, tryptophan hydroxylase 1 (Tph1) transcripts were expressed predominantly in PAECs. Serotonin was detected by immunostaining in both PAECs and PASMCs in PAEC/PASMC touch coculture but was not found in PASMCs in either PAEC/PASMC no-touch coculture or in PASMC/PASMC touch coculture. Furthermore, inhibition of gap junctions but not of the serotonin transporter in PAEC/PASMC touch coculture prevented serotonin transfer from PAECs to PASMCs. Inhibition of serotonin synthesis pharmacologically or by small interfering RNAs to Tph1 in PAECs inhibited the PAEC-induced activation of TGF-β signaling and differentiation of PASMCs. We concluded that serotonin synthesized by PAECs is transferred through myoendothelial gap junctions into PASMCs, where it activates TGF-β signaling and induces a more differentiated phenotype. This finding suggests a novel role of gap junction-mediated intercellular serotonin signaling in regulation of PASMC phenotype.

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.

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

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

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.

Myosin IXa Regulates Epithelial Differentiation and Its Deficiency Results in Hydrocephalus

PubMed Central

Abouhamed, Marouan; Grobe, Kay; Leefa Chong San, Isabelle V.; Thelen, Sabine; Honnert, Ulrike; Balda, Maria S.; Matter, Karl

2009-01-01

The ependymal multiciliated epithelium in the brain restricts the cerebrospinal fluid to the cerebral ventricles and regulates its flow. We report here that mice deficient for myosin IXa (Myo9a), an actin-dependent motor molecule with a Rho GTPase–activating (GAP) domain, develop severe hydrocephalus with stenosis and closure of the ventral caudal 3rd ventricle and the aqueduct. Myo9a is expressed in maturing ependymal epithelial cells, and its absence leads to impaired maturation of ependymal cells. The Myo9a deficiency further resulted in a distorted ependyma due to irregular epithelial cell morphology and altered organization of intercellular junctions. Ependymal cells occasionally delaminated, forming multilayered structures that bridged the CSF-filled ventricular space. Hydrocephalus formation could be significantly attenuated by the inhibition of the Rho-effector Rho-kinase (ROCK). Administration of ROCK-inhibitor restored maturation of ependymal cells, but not the morphological distortions of the ependyma. Similarly, down-regulation of Myo9a by siRNA in Caco-2 adenocarcinoma cells increased Rho-signaling and induced alterations in differentiation, cell morphology, junction assembly, junctional signaling, and gene expression. Our results demonstrate that Myo9a is a critical regulator of Rho-dependent and -independent signaling mechanisms that guide epithelial differentiation. Moreover, Rho-kinases may represent a new target for therapeutic intervention in some forms of hydrocephalus. PMID:19828736

Directed Evolution to Engineer Monobody for FRET Biosensor Assembly and Imaging at Live-Cell Surface.

PubMed

Limsakul, Praopim; Peng, Qin; Wu, Yiqian; Allen, Molly E; Liang, Jing; Remacle, Albert G; Lopez, Tyler; Ge, Xin; Kay, Brian K; Zhao, Huimin; Strongin, Alex Y; Yang, Xiang-Lei; Lu, Shaoying; Wang, Yingxiao

2018-04-19

Monitoring enzymatic activities at the cell surface is challenging due to the poor efficiency of transport and membrane integration of fluorescence resonance energy transfer (FRET)-based biosensors. Therefore, we developed a hybrid biosensor with separate donor and acceptor that assemble in situ. The directed evolution and sequence-function analysis technologies were integrated to engineer a monobody variant (PEbody) that binds to R-phycoerythrin (R-PE) dye. PEbody was used for visualizing the dynamic formation/separation of intercellular junctions. We further fused PEbody with the enhanced CFP and an enzyme-specific peptide at the extracellular surface to create a hybrid FRET biosensor upon R-PE capture for monitoring membrane-type-1 matrix metalloproteinase (MT1-MMP) activities. This biosensor revealed asymmetric distribution of MT1-MMP activities, which were high and low at loose and stable cell-cell contacts, respectively. Therefore, directed evolution and rational design are promising tools to engineer molecular binders and hybrid FRET biosensors for monitoring molecular regulations at the surface of living cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

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.

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.

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

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

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.

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.

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.

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.

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.

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.

Quantifying cadherin mechanotransduction machinery assembly/disassembly dynamics using fluorescence covariance analysis.

PubMed

Vedula, Pavan; Cruz, Lissette A; Gutierrez, Natasha; Davis, Justin; Ayee, Brian; Abramczyk, Rachel; Rodriguez, Alexis J

2016-06-30

Quantifying multi-molecular complex assembly in specific cytoplasmic compartments is crucial to understand how cells use assembly/disassembly of these complexes to control function. Currently, biophysical methods like Fluorescence Resonance Energy Transfer and Fluorescence Correlation Spectroscopy provide quantitative measurements of direct protein-protein interactions, while traditional biochemical approaches such as sub-cellular fractionation and immunoprecipitation remain the main approaches used to study multi-protein complex assembly/disassembly dynamics. In this article, we validate and quantify multi-protein adherens junction complex assembly in situ using light microscopy and Fluorescence Covariance Analysis. Utilizing specific fluorescently-labeled protein pairs, we quantified various stages of adherens junction complex assembly, the multiprotein complex regulating epithelial tissue structure and function following de novo cell-cell contact. We demonstrate: minimal cadherin-catenin complex assembly in the perinuclear cytoplasm and subsequent localization to the cell-cell contact zone, assembly of adherens junction complexes, acto-myosin tension-mediated anchoring, and adherens junction maturation following de novo cell-cell contact. Finally applying Fluorescence Covariance Analysis in live cells expressing fluorescently tagged adherens junction complex proteins, we also quantified adherens junction complex assembly dynamics during epithelial monolayer formation.

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.

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

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

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.

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.

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

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

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.

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.

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.

A WAVE2–Arp2/3 actin nucleator apparatus supports junctional tension at the epithelial zonula adherens

PubMed Central

Verma, Suzie; Han, Siew Ping; Michael, Magdalene; Gomez, Guillermo A.; Yang, Zhe; Teasdale, Rohan D.; Ratheesh, Aparna; Kovacs, Eva M.; Ali, Radiya G.; Yap, Alpha S.

2012-01-01

The epithelial zonula adherens (ZA) is a specialized adhesive junction where actin dynamics and myosin-driven contractility coincide. The junctional cytoskeleton is enriched in myosin II, which generates contractile force to support junctional tension. It is also enriched in dynamic actin filaments, which are replenished by ongoing actin assembly. In this study we sought to pursue the relationship between actin assembly and junctional contractility. We demonstrate that WAVE2–Arp2/3 is a major nucleator of actin assembly at the ZA and likely acts in response to junctional Rac signaling. Furthermore, WAVE2–Arp2/3 is necessary for junctional integrity and contractile tension at the ZA. Maneuvers that disrupt the function of either WAVE2 or Arp2/3 reduced junctional tension and compromised the ability of cells to buffer side-to-side forces acting on the ZA. WAVE2–Arp2/3 disruption depleted junctions of both myosin IIA and IIB, suggesting that dynamic actin assembly may support junctional tension by facilitating the local recruitment of myosin. PMID:23051739

A WAVE2-Arp2/3 actin nucleator apparatus supports junctional tension at the epithelial zonula adherens.

PubMed

Verma, Suzie; Han, Siew Ping; Michael, Magdalene; Gomez, Guillermo A; Yang, Zhe; Teasdale, Rohan D; Ratheesh, Aparna; Kovacs, Eva M; Ali, Radiya G; Yap, Alpha S

2012-12-01

The epithelial zonula adherens (ZA) is a specialized adhesive junction where actin dynamics and myosin-driven contractility coincide. The junctional cytoskeleton is enriched in myosin II, which generates contractile force to support junctional tension. It is also enriched in dynamic actin filaments, which are replenished by ongoing actin assembly. In this study we sought to pursue the relationship between actin assembly and junctional contractility. We demonstrate that WAVE2-Arp2/3 is a major nucleator of actin assembly at the ZA and likely acts in response to junctional Rac signaling. Furthermore, WAVE2-Arp2/3 is necessary for junctional integrity and contractile tension at the ZA. Maneuvers that disrupt the function of either WAVE2 or Arp2/3 reduced junctional tension and compromised the ability of cells to buffer side-to-side forces acting on the ZA. WAVE2-Arp2/3 disruption depleted junctions of both myosin IIA and IIB, suggesting that dynamic actin assembly may support junctional tension by facilitating the local recruitment of myosin.

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.

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

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

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.

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.

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.

Analyzing phorbol ester effects on gap junctional communication: a dramatic inhibition of assembly

PubMed Central

1994-01-01

The effect of 12-O-tetradeconylphorbol-13-acetate (TPA) on gap junction assembly between Novikoff hepatoma cells was examined. Cells were dissociated with EDTA to single cells and then reaggregated to form new junctions. When TPA (25 nM) was added to the cells at the onset of the 60-min reaggregation, dye transfer was detected at only 0.6% of the cell-cell interfaces compared to 72% for the untreated control and 74% for 4-alpha TPA, an inactive isomer of TPA. Freeze-fracture electron microscopy of reaggregated control cells showed interfaces containing an average of more than 600 aggregated intramembranous gap junction particles, while TPA-treated cells had no gap junctions. However, Lucifer yellow dye transfer between nondissociated cells via gap junctions was unaffected by 60 min of TPA treatment. Therefore, TPA dramatically inhibited gap junction assembly but did not alter channel gating nor enhance disassembly of preexisting gap junction structures. Short term TPA treatment (< 30 min) increased phosphorylation of the gap junction protein molecular weight of 43,000 (Cx43), but did not change the cellular level of Cx43. Cell surface biotinylation experiments suggested that TPA did not substantially reduce the plasma membrane concentration of Cx43. Therefore, the simple presence of Cx43 in the plasma membrane is not sufficient for gap junction assembly, and protein kinase C probably exerts an effect on assembly of gap junctions at the plasma membrane level. PMID:7806568

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

Intrinsically disordered proteins aggregate at fungal cell-to-cell channels and regulate intercellular connectivity.

PubMed

Lai, Julian; Koh, Chuan Hock; Tjota, Monika; Pieuchot, Laurent; Raman, Vignesh; Chandrababu, Karthik Balakrishna; Yang, Daiwen; Wong, Limsoon; Jedd, Gregory

2012-09-25

Like animals and plants, multicellular fungi possess cell-to-cell channels (septal pores) that allow intercellular communication and transport. Here, using a combination of MS of Woronin body-associated proteins and a bioinformatics approach that identifies related proteins based on composition and character, we identify 17 septal pore-associated (SPA) proteins that localize to the septal pore in rings and pore-centered foci. SPA proteins are not homologous at the primary sequence level but share overall physical properties with intrinsically disordered proteins. Some SPA proteins form aggregates at the septal pore, and in vitro assembly assays suggest aggregation through a nonamyloidal mechanism involving mainly α-helical and disordered structures. SPA loss-of-function phenotypes include excessive septation, septal pore degeneration, and uncontrolled Woronin body activation. Together, our data identify the septal pore as a complex subcellular compartment and focal point for the assembly of unstructured proteins controlling diverse aspects of intercellular connectivity.

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.

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

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.

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.

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.

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.

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.

A Key Claudin Extracellular Loop Domain is Critical for Epithelial Barrier Integrity

PubMed Central

Mrsny, Randall J.; Brown, G. Thomas; Gerner-Smidt, Kirsten; Buret, Andre G.; Meddings, Jon B.; Quan, Clifford; Koval, Michael; Nusrat, Asma

2008-01-01

Intercellular tight junctions (TJs) regulate epithelial barrier properties. Claudins are major structural constituents of TJs and belong to a large family of tetra-spanning membrane proteins that have two predicted extracellular loops (ELs). Given that claudin-1 is widely expressed in epithelia, we further defined the role of its EL domains in determining TJ function. The effects of several claudin-1 EL mimetic peptides on epithelial barrier structure and function were examined. Incubation of model human intestinal epithelial cells with a 27-amino acid peptide corresponding to a portion of the first EL domain (Cldn-153–80) reversibly interfered with epithelial barrier function by inducing the rearrangement of key TJ proteins: occludin, claudin-1, junctional adhesion molecule-A, and zonula occludens-1. Cldn-153–80 associated with both claudin-1 and occludin, suggesting both the direct interference with the ability of these proteins to assemble into functional TJs and their close interaction under physiological conditions. These effects were specific for Cldn-153–80, because peptides corresponding to other claudin-1 EL domains failed to influence TJ function. Furthermore, the oral administration of Cldn-153–80 to rats increased paracellular gastric permeability. Thus, the identification of a critical claudin-1 EL motif, Cldn-153–80, capable of regulating TJ structure and function, offers a useful adjunct to treatments that require drug delivery across an epithelial barrier. PMID:18349130

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.

Intrinsically disordered proteins aggregate at fungal cell-to-cell channels and regulate intercellular connectivity

PubMed Central

Lai, Julian; Koh, Chuan Hock; Tjota, Monika; Pieuchot, Laurent; Raman, Vignesh; Chandrababu, Karthik Balakrishna; Yang, Daiwen; Wong, Limsoon; Jedd, Gregory

2012-01-01

Like animals and plants, multicellular fungi possess cell-to-cell channels (septal pores) that allow intercellular communication and transport. Here, using a combination of MS of Woronin body-associated proteins and a bioinformatics approach that identifies related proteins based on composition and character, we identify 17 septal pore-associated (SPA) proteins that localize to the septal pore in rings and pore-centered foci. SPA proteins are not homologous at the primary sequence level but share overall physical properties with intrinsically disordered proteins. Some SPA proteins form aggregates at the septal pore, and in vitro assembly assays suggest aggregation through a nonamyloidal mechanism involving mainly α-helical and disordered structures. SPA loss-of-function phenotypes include excessive septation, septal pore degeneration, and uncontrolled Woronin body activation. Together, our data identify the septal pore as a complex subcellular compartment and focal point for the assembly of unstructured proteins controlling diverse aspects of intercellular connectivity. PMID:22955885

Phosphorylation on Ser-279 and Ser-282 of connexin43 regulates endocytosis and gap junction assembly in pancreatic cancer cells

PubMed Central

Johnson, Kristen E.; Mitra, Shalini; Katoch, Parul; Kelsey, Linda S.; Johnson, Keith R.; Mehta, Parmender P.

2013-01-01

The molecular mechanisms regulating the assembly of connexins (Cxs) into gap junctions are poorly understood. Using human pancreatic tumor cell lines BxPC3 and Capan-1, which express Cx26 and Cx43, we show that, upon arrival at the cell surface, the assembly of Cx43 is impaired. Connexin43 fails to assemble, because it is internalized by clathrin-mediated endocytosis. Assembly is restored upon expressing a sorting-motif mutant of Cx43, which does not interact with the AP2 complex, and by expressing mutants that cannot be phosphorylated on Ser-279 and Ser-282. The mutants restore assembly by preventing clathrin-mediated endocytosis of Cx43. Our results also document that the sorting-motif mutant is assembled into gap junctions in cells in which the expression of endogenous Cx43 has been knocked down. Remarkably, Cx43 mutants that cannot be phosphorylated on Ser-279 or Ser-282 are assembled into gap junctions only when connexons are composed of Cx43 forms that can be phosphorylated on these serines and forms in which phosphorylation on these serines is abolished. Based on the subcellular fate of Cx43 in single and contacting cells, our results document that the endocytic itinerary of Cx43 is altered upon cell–cell contact, which causes Cx43 to traffic by EEA1-negative endosomes en route to lysosomes. Our results further show that gap-junctional plaques formed of a sorting motif–deficient mutant of Cx43, which is unable to be internalized by the clathrin-mediated pathway, are predominantly endocytosed in the form of annular junctions. Thus the differential phosphorylation of Cx43 on Ser-279 and Ser-282 is fine-tuned to control Cx43’s endocytosis and assembly into gap junctions. PMID:23363606

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

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

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.

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.

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.

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

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.

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.

Regulation of tight junction assembly and epithelial morphogenesis by the heat shock protein Apg-2

PubMed Central

Aijaz, Saima; Sanchez-Heras, Elena; Balda, Maria S; Matter, Karl

2007-01-01

Background Tight junctions are required for epithelial barrier formation and participate in the regulation of signalling mechanisms that control proliferation and differentiation. ZO-1 is a tight junction-associated adaptor protein that regulates gene expression, junction assembly and epithelial morphogenesis. We have previously demonstrated that the heat shock protein Apg-2 binds ZO-1 and thereby regulates its role in cell proliferation. Here, we addressed the question whether Apg-2 is also important for junction formation and epithelial morphogenesis. Results We demonstrate that depletion of Apg-2 by RNAi in MDCK cells did not prevent formation of functional tight junctions. Similar to ZO-1, however, reduced expression of Apg-2 retarded de novo junction assembly if analysed in a Ca-switch model. Formation of functional junctions, as monitored by measuring transepithelial electrical resistance, and recruitment of tight and adherens junction markers were retarded. If cultured in three dimensional extracellular matrix gels, Apg-2 depleted cells, as previously shown for ZO-1 depleted cells, did not form hollow polarised cysts but poorly organised, irregular structures. Conclusion Our data indicate that Apg-2 regulates junction assembly and is required for normal epithelial morphogenesis in a three-dimensional culture system, suggesting that Apg-2 is an important regulator of epithelial differentiation. As the observed phenotypes are similar to those previously described for ZO-1 depleted cells and depletion of Apg-2 retards junctional recruitment of ZO-1, regulation of ZO-1 is likely to be an important functional role for Apg-2 during epithelial differentiation. PMID:18028534

Regulation of tight junction assembly and epithelial morphogenesis by the heat shock protein Apg-2.

PubMed

Aijaz, Saima; Sanchez-Heras, Elena; Balda, Maria S; Matter, Karl

2007-11-20

Tight junctions are required for epithelial barrier formation and participate in the regulation of signalling mechanisms that control proliferation and differentiation. ZO-1 is a tight junction-associated adaptor protein that regulates gene expression, junction assembly and epithelial morphogenesis. We have previously demonstrated that the heat shock protein Apg-2 binds ZO-1 and thereby regulates its role in cell proliferation. Here, we addressed the question whether Apg-2 is also important for junction formation and epithelial morphogenesis. We demonstrate that depletion of Apg-2 by RNAi in MDCK cells did not prevent formation of functional tight junctions. Similar to ZO-1, however, reduced expression of Apg-2 retarded de novo junction assembly if analysed in a Ca-switch model. Formation of functional junctions, as monitored by measuring transepithelial electrical resistance, and recruitment of tight and adherens junction markers were retarded. If cultured in three dimensional extracellular matrix gels, Apg-2 depleted cells, as previously shown for ZO-1 depleted cells, did not form hollow polarised cysts but poorly organised, irregular structures. Our data indicate that Apg-2 regulates junction assembly and is required for normal epithelial morphogenesis in a three-dimensional culture system, suggesting that Apg-2 is an important regulator of epithelial differentiation. As the observed phenotypes are similar to those previously described for ZO-1 depleted cells and depletion of Apg-2 retards junctional recruitment of ZO-1, regulation of ZO-1 is likely to be an important functional role for Apg-2 during epithelial differentiation.

New insights into desmosome regulation and pemphigus blistering as a desmosome-remodeling disease.

PubMed

Kitajima, Yasuo

2013-01-01

Desmosomes in keratinocytes are the most important intercellular adhering junctions that provide structural strength for the epidermis. These junctions are connected directly with desmosomal cadherin proteins. Desmosomal cadherins are divided into four desmogleins (Dsgs), Dsg1-4, and three desmocollins (Dscs), Dsc1-3, all of which are involved in desmosomal adhesion by homo- and/or heterophilic binding between Dsgs and Dscs in a Ca(2+)-dependent manner. Cadherins are present on the cell surface and anchor keratin intermediate filaments (KIFs) to their inner cytoplasmic surface to generate an intracellular KIF-skeletal scaffold through several associate proteins, including plakoglobin, plakophillin, and desmoplakins. As such, the desmosomal contacts between adjacent cells generate an intercellular KIF scaffold throughout the whole epidermal sheet. However, despite these critical roles in maintaining epidermal adhesion and integrity, desmosomes are not static structures. Rather, they are dynamic units that undergo regular remodeling, i.e., assembly and disassembly, to allow for cell migration within the epidermis in response to outside-in signaling during epidermal differentiation. Recently, two cell-cell adhesion states controlled by desmosomes have been recognized, including "stable hyperadhesion (Ca(2+)-independent)" and "dynamic weak-adhesion (Ca(2+)-dependent)" conditions. These conditions are mutually reversible through cell signaling events involving protein kinase C (PKC) and epidermal growth factor receptor. Pemphigus vulgaris (PV) is an autoimmune bullous disease caused by anti-Dsg3 antibodies. Binding of these antibodies to Dsg3 causes endocytosis of Dsg3 from the cell surface and results in the specific depletion of Dsg3 from desmosomes, an event linked to acantholysis in the epidermis. This binding of anti-Dsg3 antibody to Dsg3 in epidermal keratinocytes activates PKC, to generate the "weak-adhesion (Ca(2+)-dependent)" state of desmosomes. The weak-adhesion desmosomes appear to be the susceptible desmosomal state and a prerequisite for Dsg3 depletion from desmosomes, pivotal and specific events leading to PV blistering. These observations allow us to propose a concept for pemphigus blistering disorders as a "desmosome-remodeling impairment disease" involving a mechanism of Dsg3 nonassembly and depletion from desmosomes through PV immunoglobulin G-activated intracellular signaling events. Copyright © 2012. Published by Elsevier B.V.

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.

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.

Enhancing the intestinal absorption of low molecular weight chondroitin sulfate by conjugation with α-linolenic acid and the transport mechanism of the conjugates.

PubMed

Xiao, Yuliang; Li, Pingli; Cheng, Yanna; Zhang, Xinke; Sheng, Juzheng; Wang, Decai; Li, Juan; Zhang, Qian; Zhong, Chuanqing; Cao, Rui; Wang, Fengshan

2014-04-25

The purpose of this report was to demonstrate the effect of amphiphilic polysaccharides-based self-assembling micelles on enhancing the oral absorption of low molecular weight chondroitin sulfate (LMCS) in vitro and in vivo, and identify the transepithelial transport mechanism of LMCS micelles across the intestinal barrier. α-Linolenic acid-low molecular weight chondroitin sulfate polymers(α-LNA-LMCS) were successfully synthesized, and characterized by FTIR, (1)HNMR, TGA/DSC, TEM, laser light scattering and zeta potential. The significant oral absorption enhancement and elimination half-life (t₁/₂) extension of LNA-LMCS2 in rats were evidenced by intragastric administration in comparison with CS and LMCS. Caco-2 transport studies demonstrated that the apparent permeability coefficient (Papp) of LNA-LMCS2 was significantly higher than that of CS and LMCS (p<0.001), and no significant effects on the overall integrity of the monolayer were observed during the transport process. In addition, α-LNA-LMCS micelles accumulated around the cell membrane and intercellular space observed by confocal laser scanning microscope (CLSM). Furthermore, evident alterations in the F-actin cytoskeleton were detected by CLSM observation following the treatment of the cell monolayers with α-LNA-LMCS micelles, which further certified the capacity of α-LNA-LMCS micelles to open the intercellular tight junctions rather than disrupt the overall integrity of the monolayer. Therefore, LNA-LMCS2 with low cytotoxicity and high bioavailability might be a promising substitute for CS in clinical use, such as treating osteoarthritis, atherosclerosis, etc. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

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.

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

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.

Assembly of the Escherichia coli RuvABC resolvasome directs the orientation of Holliday junction resolution

PubMed Central

van Gool, Alain J.; Hajibagheri, Nasser M.A.; Stasiak, Andrzej; West, Stephen C.

1999-01-01

Genetic recombination can lead to the formation of intermediates in which DNA molecules are linked by Holliday junctions. Movement of a junction along DNA, by a process known as branch migration, leads to heteroduplex formation, whereas resolution of a junction completes the recombination process. Holliday junctions can be resolved in either of two ways, yielding products in which there has, or has not, been an exchange of flanking markers. The ratio of these products is thought to be determined by the frequency with which the two isomeric forms (conformers) of the Holliday junction are cleaved. Recent studies with enzymes that process Holliday junctions in Escherichia coli, the RuvABC proteins, however, indicate that protein binding causes the junction to adopt an open square-planar configuration. Within such a structure, DNA isomerization can have little role in determining the orientation of resolution. To determine the role that junction-specific protein assembly has in determining resolution bias, a defined in vitro system was developed in which we were able to direct the assembly of the RuvABC resolvasome. We found that the bias toward resolution in one orientation or the other was determined simply by the way in which the Ruv proteins were positioned on the junction. Additionally, we provide evidence that supports current models on RuvABC action in which Holliday junction resolution occurs as the resolvasome promotes branch migration. PMID:10421637

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.

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.

Kinase programs spatiotemporally regulate gap junction assembly and disassembly: effects on wound repair

PubMed Central

Solan, Joell L.; Lampe, Paul D.

2016-01-01

Gap junctions are highly ordered plasma membrane domains that are constantly assembled, remodeled and turned over due to the short half-life of connexins, the integral membrane proteins that form gap junctions. Connexin 43 (Cx43), by far the most widely expressed connexin, is phosphorylated at multiple serine residues in the cytoplasmic, C-terminal region allowing for exquisite cellular control over gap junctional communication. This is evident during epidermal wounding where spatiotemporal changes in connexin expression occur as cells are instructed whether to die, proliferate or migrate to promote repair. Early gap junctional communication is required for initiation of keratinocyte migration, but accelerated Cx43 turnover is also critical for proper wound healing at later stages. These events are controlled via a "kinase program" where sequential phosphorylation of Cx43 leads to reductions in Cx43’s half-life and significant depletion of gap junctions from the plasma membrane within several hours. The complex regulation of gap junction assembly and turnover affords several steps where intervention might speed wound healing. PMID:26706150

Kinase programs spatiotemporally regulate gap junction assembly and disassembly: Effects on wound repair.

PubMed

Solan, Joell L; Lampe, Paul D

2016-02-01

Gap junctions are highly ordered plasma membrane domains that are constantly assembled, remodeled and turned over due to the short half-life of connexins, the integral membrane proteins that form gap junctions. Connexin 43 (Cx43), by far the most widely expressed connexin, is phosphorylated at multiple serine residues in the cytoplasmic, C-terminal region allowing for exquisite cellular control over gap junctional communication. This is evident during epidermal wounding where spatiotemporal changes in connexin expression occur as cells are instructed whether to die, proliferate or migrate to promote repair. Early gap junctional communication is required for initiation of keratinocyte migration, but accelerated Cx43 turnover is also critical for proper wound healing at later stages. These events are controlled via a "kinase program" where sequential phosphorylation of Cx43 leads to reductions in Cx43's half-life and significant depletion of gap junctions from the plasma membrane within several hours. The complex regulation of gap junction assembly and turnover affords several steps where intervention might speed wound healing. Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

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.

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

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.

Tailoring the vapor-liquid-solid growth toward the self-assembly of GaAs nanowire junctions.

PubMed

Dai, Xing; Dayeh, Shadi A; Veeramuthu, Vaithianathan; Larrue, Alexandre; Wang, Jian; Su, Haibin; Soci, Cesare

2011-11-09

New insights into understanding and controlling the intriguing phenomena of spontaneous merging (kissing) and the self-assembly of monolithic Y- and T-junctions is demonstrated in the metal-organic chemical vapor deposition growth of GaAs nanowires. High-resolution transmission electron microscopy for determining polar facets was coupled to electrostatic-mechanical modeling and position-controlled synthesis to identify nanowire diameter, length, and pitch, leading to junction formation. When nanowire patterns are designed so that the electrostatic energy resulting from the interaction of polar surfaces exceeds the mechanical energy required to bend the nanowires to the point of contact, their fusion can lead to the self-assembly of monolithic junctions. Understanding and controlling this phenomenon is a great asset for the realization of dense arrays of vertical nanowire devices and opens up new ways toward the large scale integration of nanowire quantum junctions or nanowire intracellular probes.

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

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

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.

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

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.

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.

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.

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.

The Carboxyl Tail of Connexin32 Regulates Gap Junction Assembly in Human Prostate and Pancreatic Cancer Cells*

PubMed Central

Katoch, Parul; Mitra, Shalini; Ray, Anuttoma; Kelsey, Linda; Roberts, Brett J.; Wahl, James K.; Johnson, Keith R.; Mehta, Parmender P.

2015-01-01

Connexins, the constituent proteins of gap junctions, are transmembrane proteins. A connexin (Cx) traverses the membrane four times and has one intracellular and two extracellular loops with the amino and carboxyl termini facing the cytoplasm. The transmembrane and the extracellular loop domains are highly conserved among different Cxs, whereas the carboxyl termini, often called the cytoplasmic tails, are highly divergent. We have explored the role of the cytoplasmic tail of Cx32, a Cx expressed in polarized and differentiated cells, in regulating gap junction assembly. Our results demonstrate that compared with the full-length Cx32, the cytoplasmic tail-deleted Cx32 is assembled into small gap junctions in human pancreatic and prostatic cancer cells. Our results further document that the expression of the full-length Cx32 in cells, which express the tail-deleted Cx32, increases the size of gap junctions, whereas the expression of the tail-deleted Cx32 in cells, which express the full-length Cx32, has the opposite effect. Moreover, we show that the tail is required for the clustering of cell-cell channels and that in cells expressing the tail-deleted Cx32, the expression of cell surface-targeted cytoplasmic tail alone is sufficient to enhance the size of gap junctions. Our live-cell imaging data further demonstrate that gap junctions formed of the tail-deleted Cx32 are highly mobile compared with those formed of full-length Cx32. Our results suggest that the cytoplasmic tail of Cx32 is not required to initiate the assembly of gap junctions but for their subsequent growth and stability. Our findings suggest that the cytoplasmic tail of Cx32 may be involved in regulating the permeability of gap junctions by regulating their size. PMID:25548281

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.

Aberrant Cx43 Expression and Mislocalization in Metastatic Human Melanomas.

PubMed

Alaga, Katanya C; Crawford, Melissa; Dagnino, Lina; Laird, Dale W

2017-01-01

At present, it is unclear if melanocytes contain Cx43 gap junctions and whether Cx43 expression is regulated in melanoma onset and progression. To this end, we cultured pure populations of mouse melanocytes and found that they had no detectable Cx43 and exhibited an inability for dye transfer indicating they were devoid of functional gap junctions. Given the evidence that melanomas acquire the expression of other connexin isoforms during tumor progression, we assessed if Cx43 was also expressed and assembled into gap junctions at any stage of human melanoma onset and progression to distant metastases. Nearly all primary melanomas within the epidermis lacked Cx43. In contrast, nodal metastases expressed low levels of Cx43 which was markedly higher in distant metastases that had invaded vital organs. Importantly, in all stages of melanoma progression, Cx43 could be detected in intracellular compartments but was rarely assembled into gap junctions indicative of functional gap junction channels. Overall, these studies suggest that melanocytes do not form Cx43 homocellular gap junctions and even though Cx43 levels increase during melanoma progression, this connexin rarely assembles into gap junction structures.

Aberrant Cx43 Expression and Mislocalization in Metastatic Human Melanomas

PubMed Central

Alaga, Katanya C.; Crawford, Melissa; Dagnino, Lina; Laird, Dale W.

2017-01-01

At present, it is unclear if melanocytes contain Cx43 gap junctions and whether Cx43 expression is regulated in melanoma onset and progression. To this end, we cultured pure populations of mouse melanocytes and found that they had no detectable Cx43 and exhibited an inability for dye transfer indicating they were devoid of functional gap junctions. Given the evidence that melanomas acquire the expression of other connexin isoforms during tumor progression, we assessed if Cx43 was also expressed and assembled into gap junctions at any stage of human melanoma onset and progression to distant metastases. Nearly all primary melanomas within the epidermis lacked Cx43. In contrast, nodal metastases expressed low levels of Cx43 which was markedly higher in distant metastases that had invaded vital organs. Importantly, in all stages of melanoma progression, Cx43 could be detected in intracellular compartments but was rarely assembled into gap junctions indicative of functional gap junction channels. Overall, these studies suggest that melanocytes do not form Cx43 homocellular gap junctions and even though Cx43 levels increase during melanoma progression, this connexin rarely assembles into gap junction structures. PMID:28607585

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.

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

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.

Magnetic assembly of 3D cell clusters: visualizing the formation of an engineered tissue.

PubMed

Ghosh, S; Kumar, S R P; Puri, I K; Elankumaran, S

2016-02-01

Contactless magnetic assembly of cells into 3D clusters has been proposed as a novel means for 3D tissue culture that eliminates the need for artificial scaffolds. However, thus far its efficacy has only been studied by comparing expression levels of generic proteins. Here, it has been evaluated by visualizing the evolution of cell clusters assembled by magnetic forces, to examine their resemblance to in vivo tissues. Cells were labeled with magnetic nanoparticles, then assembled into 3D clusters using magnetic force. Scanning electron microscopy was used to image intercellular interactions and morphological features of the clusters. When cells were held together by magnetic forces for a single day, they formed intercellular contacts through extracellular fibers. These kept the clusters intact once the magnetic forces were removed, thus serving the primary function of scaffolds. The cells self-organized into constructs consistent with the corresponding tissues in vivo. Epithelial cells formed sheets while fibroblasts formed spheroids and exhibited position-dependent morphological heterogeneity. Cells on the periphery of a cluster were flattened while those within were spheroidal, a well-known characteristic of connective tissues in vivo. Cells assembled by magnetic forces presented visual features representative of their in vivo states but largely absent in monolayers. This established the efficacy of contactless assembly as a means to fabricate in vitro tissue models. © 2016 John Wiley & Sons Ltd.

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

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

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.

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.

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.

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

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: 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.

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

A NEUTRONIC REACTOR

DOEpatents

Luebke, E.A.; Vandenberg, L.B.

1959-09-01

A nuclear reactor for producing thermoelectric power is described. The reactor core comprises a series of thermoelectric assemblies, each assembly including fissionable fuel as an active element to form a hot junction and a thermocouple. The assemblies are disposed parallel to each other to form spaces and means are included for Introducing an electrically conductive coolant between the assemblies to form cold junctions of the thermocouples. An electromotive force is developed across the entire series of the thermoelectric assemblies due to fission heat generated in the fuel causing a current to flow perpendicular to the flow of coolant and is distributed to a load outside of the reactor by means of bus bars electrically connected to the outermost thermoelectric assembly.

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

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.

The carboxyl tail of connexin32 regulates gap junction assembly in human prostate and pancreatic cancer cells.

PubMed

Katoch, Parul; Mitra, Shalini; Ray, Anuttoma; Kelsey, Linda; Roberts, Brett J; Wahl, James K; Johnson, Keith R; Mehta, Parmender P

2015-02-20

Connexins, the constituent proteins of gap junctions, are transmembrane proteins. A connexin (Cx) traverses the membrane four times and has one intracellular and two extracellular loops with the amino and carboxyl termini facing the cytoplasm. The transmembrane and the extracellular loop domains are highly conserved among different Cxs, whereas the carboxyl termini, often called the cytoplasmic tails, are highly divergent. We have explored the role of the cytoplasmic tail of Cx32, a Cx expressed in polarized and differentiated cells, in regulating gap junction assembly. Our results demonstrate that compared with the full-length Cx32, the cytoplasmic tail-deleted Cx32 is assembled into small gap junctions in human pancreatic and prostatic cancer cells. Our results further document that the expression of the full-length Cx32 in cells, which express the tail-deleted Cx32, increases the size of gap junctions, whereas the expression of the tail-deleted Cx32 in cells, which express the full-length Cx32, has the opposite effect. Moreover, we show that the tail is required for the clustering of cell-cell channels and that in cells expressing the tail-deleted Cx32, the expression of cell surface-targeted cytoplasmic tail alone is sufficient to enhance the size of gap junctions. Our live-cell imaging data further demonstrate that gap junctions formed of the tail-deleted Cx32 are highly mobile compared with those formed of full-length Cx32. Our results suggest that the cytoplasmic tail of Cx32 is not required to initiate the assembly of gap junctions but for their subsequent growth and stability. Our findings suggest that the cytoplasmic tail of Cx32 may be involved in regulating the permeability of gap junctions by regulating their size. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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

Epithelial organization and cyst lumen expansion require efficient Sec13-Sec31-driven secretion.

PubMed

Townley, Anna K; Schmidt, Katy; Hodgson, Lorna; Stephens, David J

2012-02-01

Epithelial morphogenesis is directed by interactions with the underlying extracellular matrix. Secretion of collagen and other matrix components requires efficient coat complex II (COPII) vesicle formation at the endoplasmic reticulum. Here, we show that suppression of the outer layer COPII component, Sec13, in zebrafish embryos results in a disorganized gut epithelium. In human intestinal epithelial cells (Caco-2), Sec13 depletion causes defective epithelial polarity and organization on permeable supports. Defects are seen in the ability of cells to adhere to the substrate, form a monolayer and form intercellular junctions. When embedded in a three-dimensional matrix, Sec13-depleted Caco-2 cells form cysts but, unlike controls, are defective in lumen expansion. Incorporation of primary fibroblasts within the three-dimensional culture substantially restores normal morphogenesis. We conclude that efficient COPII-dependent secretion, notably assembly of Sec13-Sec31, is required to drive epithelial morphogenesis in both two- and three-dimensional cultures in vitro, as well as in vivo. Our results provide insight into the role of COPII in epithelial morphogenesis and have implications for the interpretation of epithelial polarity and organization assays in cell culture.

Epithelial organization and cyst lumen expansion require efficient Sec13–Sec31-driven secretion

PubMed Central

Townley, Anna K.; Schmidt, Katy; Hodgson, Lorna; Stephens, David J.

2012-01-01

Epithelial morphogenesis is directed by interactions with the underlying extracellular matrix. Secretion of collagen and other matrix components requires efficient coat complex II (COPII) vesicle formation at the endoplasmic reticulum. Here, we show that suppression of the outer layer COPII component, Sec13, in zebrafish embryos results in a disorganized gut epithelium. In human intestinal epithelial cells (Caco-2), Sec13 depletion causes defective epithelial polarity and organization on permeable supports. Defects are seen in the ability of cells to adhere to the substrate, form a monolayer and form intercellular junctions. When embedded in a three-dimensional matrix, Sec13-depleted Caco-2 cells form cysts but, unlike controls, are defective in lumen expansion. Incorporation of primary fibroblasts within the three-dimensional culture substantially restores normal morphogenesis. We conclude that efficient COPII-dependent secretion, notably assembly of Sec13–Sec31, is required to drive epithelial morphogenesis in both two- and three-dimensional cultures in vitro, as well as in vivo. Our results provide insight into the role of COPII in epithelial morphogenesis and have implications for the interpretation of epithelial polarity and organization assays in cell culture. PMID:22331354

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.

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

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.

Thermoelectric generator with hinged assembly for fins

DOEpatents

Purdy, David L.; Shapiro, Zalman M.; Hursen, Thomas F.; Maurer, Gerould W.

1976-11-02

A cylindrical casing has a central shielded capsule of radioisotope fuel. A plurality of thermonuclear modules are axially arranged with their hot junctions resiliently pressed toward the shield and with their cold junctions adjacent a transition member having fins radiating heat to the environment. For each module, the assembly of transition member and fins is hinged to the casing for swinging to permit access to and removal of such module. A ceramic plate having gold layers on opposite faces prevents diffusion bonding of the hot junction to the shield.

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

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

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.

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

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.

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.

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

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

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

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.

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

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.

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.

Creating Sub-50 Nm Nanofluidic Junctions in PDMS Microfluidic Chip via Self-Assembly Process of Colloidal Particles

PubMed Central

Wei, Xi; Syed, Abeer; Mao, Pan; Han, Jongyoon; Song, Yong-Ak

2016-01-01

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its transparency. Due to the softness of the PDMS material, however, it is challenging to use PDMS for building nanochannels. The channels tend to collapse easily during plasma bonding. In this paper, we present an evaporation-driven self-assembly method of silica colloidal nanoparticles to create nanofluidic junctions with sub-50 nm pores between two microchannels. The pore size as well as the surface charge of the nanofluidic junction is tunable simply by changing the colloidal silica bead size and surface functionalization outside of the assembled microfluidic device in a vial before the self-assembly process. Using the self-assembly of nanoparticles with a bead size of 300 nm, 500 nm, and 900 nm, it was possible to fabricate a porous membrane with a pore size of ~45 nm, ~75 nm and ~135 nm, respectively. Under electrical potential, this nanoporous membrane initiated ion concentration polarization (ICP) acting as a cation-selective membrane to concentrate DNA by ~1,700 times within 15 min. This non-lithographic nanofabrication process opens up a new opportunity to build a tunable nanofluidic junction for the study of nanoscale transport processes of ions and molecules inside a PDMS microfluidic chip. PMID:27023724

Charge splitters and charge transport junctions based on guanine quadruplexes

NASA Astrophysics Data System (ADS)

Sha, Ruojie; Xiang, Limin; Liu, Chaoren; Balaeff, Alexander; Zhang, Yuqi; Zhang, Peng; Li, Yueqi; Beratan, David N.; Tao, Nongjian; Seeman, Nadrian C.

2018-04-01

Self-assembling circuit elements, such as current splitters or combiners at the molecular scale, require the design of building blocks with three or more terminals. A promising material for such building blocks is DNA, wherein multiple strands can self-assemble into multi-ended junctions, and nucleobase stacks can transport charge over long distances. However, nucleobase stacking is often disrupted at junction points, hindering electric charge transport between the two terminals of the junction. Here, we show that a guanine-quadruplex (G4) motif can be used as a connector element for a multi-ended DNA junction. By attaching specific terminal groups to the motif, we demonstrate that charges can enter the structure from one terminal at one end of a three-way G4 motif, and can exit from one of two terminals at the other end with minimal carrier transport attenuation. Moreover, we study four-way G4 junction structures by performing theoretical calculations to assist in the design and optimization of these connectors.

Inspirations on Virus Replication and Cell-to-Cell Movement from Studies Examining the Cytopathology Induced by Lettuce infectious yellows virus in Plant Cells

PubMed Central

Qiao, Wenjie; Medina, Vicente; Falk, Bryce W.

2017-01-01

Lettuce infectious yellows virus (LIYV) is the type member of the genus Crinivirus in the family Closteroviridae. Like many other positive-strand RNA viruses, LIYV infections induce a number of cytopathic changes in plant cells, of which the two most characteristic are: Beet yellows virus-type inclusion bodies composed of vesicles derived from cytoplasmic membranes; and conical plasmalemma deposits (PLDs) located at the plasmalemma over plasmodesmata pit fields. The former are not only found in various closterovirus infections, but similar structures are known as ‘viral factories’ or viroplasms in cells infected with diverse types of animal and plant viruses. These are generally sites of virus replication, virion assembly and in some cases are involved in cell-to-cell transport. By contrast, PLDs induced by the LIYV-encoded P26 non-virion protein are not involved in replication but are speculated to have roles in virus intercellular movement. These deposits often harbor LIYV virions arranged to be perpendicular to the plasma membrane over plasmodesmata, and our recent studies show that P26 is required for LIYV systemic plant infection. The functional mechanism of how LIYV P26 facilitates intercellular movement remains unclear, however, research on other plant viruses provides some insights on the possible ways of viral intercellular movement through targeting and modifying plasmodesmata via interactions between plant cellular components and viral-encoded factors. In summary, beginning with LIYV, we review the studies that have uncovered the biological determinants giving rise to these cytopathological effects and their importance in viral replication, virion assembly and intercellular movement during the plant infection by closteroviruses, and compare these findings with those for other positive-strand RNA viruses. PMID:29021801

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.

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.

Rapid construction of mechanically- confined multi- cellular structures using dendrimeric intercellular linker.

PubMed

Mo, Xuejun; Li, Qiushi; Yi Lui, Lena Wai; Zheng, Baixue; Kang, Chiang Huen; Nugraha, Bramasta; Yue, Zhilian; Jia, Rui Rui; Fu, Hong Xia; Choudhury, Deepak; Arooz, Talha; Yan, Jie; Lim, Chwee Teck; Shen, Shali; Hong Tan, Choon; Yu, Hanry

2010-10-01

Tissue constructs that mimic the in vivo cell-cell and cell-matrix interactions are especially useful for applications involving the cell- dense and matrix- poor internal organs. Rapid and precise arrangement of cells into functional tissue constructs remains a challenge in tissue engineering. We demonstrate rapid assembly of C3A cells into multi- cell structures using a dendrimeric intercellular linker. The linker is composed of oleyl- polyethylene glycol (PEG) derivatives conjugated to a 16 arms- polypropylenimine hexadecaamine (DAB) dendrimer. The positively charged multivalent dendrimer concentrates the linker onto the negatively charged cell surface to facilitate efficient insertion of the hydrophobic oleyl groups into the cellular membrane. Bringing linker- treated cells into close proximity to each other via mechanical means such as centrifugation and micromanipulation enables their rapid assembly into multi- cellular structures within minutes. The cells exhibit high levels of viability, proliferation, three- dimensional (3D) cell morphology and other functions in the constructs. We constructed defined multi- cellular structures such as rings, sheets or branching rods that can serve as potential tissue building blocks to be further assembled into complex 3D tissue constructs for biomedical applications. 2010 Elsevier Ltd. All rights reserved.

Gap junctions are essential for murine primordial follicle assembly immediately before birth.

PubMed

Teng, Zhen; Wang, Chao; Wang, Yijing; Huang, Kun; Xiang, Xi; Niu, Wanbao; Feng, Lizhao; Zhao, Lihua; Yan, Hao; Zhang, Hua

2016-02-01

The reserve of primordial follicles determines the reproductive ability of the female mammal over its reproductive life. The primordial follicle is composed of two types of cells: oocytes and surrounding pre-granulosa cells. However, the underlying mechanism regulating primordial follicle assembly is largely undefined. In this study, we found that gap junction communication (GJC) established between the ovarian cells in the perinatal mouse ovary may be involved in the process. First, gap junction structures between the oocyte and surrounding pre-granulosa cells appear at about 19.0 dpc (days post coitum). As many as 12 gap junction-related genes are upregulated at birth, implying that a complex communication may exist between ovarian cells, because specifically silencing the genes of individual gap junction proteins, such as Gja1, Gja4 or both, has no influence on primordial follicle assembly. On the other hand, non-specific blockers of GJC, such as carbenoxolone (CBX) and 18α-glycyrrhetinic acid (AGA), significantly inhibit mouse primordial follicle assembly. We proved that the temporal window for establishment of GJC in the fetal ovary is from 19.5 dpc to 1 dpp (days postpartum). In addition, the expression of ovarian somatic cell (OSC)-specific genes, such as Notch2, Foxl2 and Irx3, was negatively affected by GJC blockers, whereas oocyte-related genes, such as Ybx2, Nobox and Sohlh1, were hardly affected, implying that the establishment of GJC during this period may be more important to OSCs than to oocytes. In summary, our results indicated that GJC involves in the mouse primordial follicle assembly process at a specific temporal window that needs Notch signaling cross-talking. © 2016 Society for Reproduction and Fertility.

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

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

Connexins in Prostate Cancer Initiation and Progression

DTIC Science & Technology

2013-11-01

the Golgi Apparatus for Cargo Transport Prior to Complete Assembly. Mol.Biol.Cell, 17, 4105-4117. 79. Hunziker,W. and Geuze,H. (2011...tumor growth by inducing the assembly of other junctional and signaling complexes? Wild type connexins which form functional gap junctions and mutant...and influences the function of two other important proteins that have been shown to prevent the spread of cancer cells from prostate to distant organs

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

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.

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.

A molecular tug-of-war: Global plant proteome changes during viral infection

USDA-ARS?s Scientific Manuscript database

Plant pathogenic viruses cause a number of economically important diseases in food, fuel, and fiber crops worldwide. As obligate parasites with highly reduced genomes, viruses rely heavily on their hosts for replication, assembly, intra- and intercellular movement, and attraction of vectors for disp...

Lattice-free prediction of three-dimensional structure of programmed DNA assemblies

PubMed Central

Pan, Keyao; Kim, Do-Nyun; Zhang, Fei; Adendorff, Matthew R.; Yan, Hao; Bathe, Mark

2014-01-01

DNA can be programmed to self-assemble into high molecular weight 3D assemblies with precise nanometer-scale structural features. Although numerous sequence design strategies exist to realize these assemblies in solution, there is currently no computational framework to predict their 3D structures on the basis of programmed underlying multi-way junction topologies constrained by DNA duplexes. Here, we introduce such an approach and apply it to assemblies designed using the canonical immobile four-way junction. The procedure is used to predict the 3D structure of high molecular weight planar and spherical ring-like origami objects, a tile-based sheet-like ribbon, and a 3D crystalline tensegrity motif, in quantitative agreement with experiments. Our framework provides a new approach to predict programmed nucleic acid 3D structure on the basis of prescribed secondary structure motifs, with possible application to the design of such assemblies for use in biomolecular and materials science. PMID:25470497

Test-beds for molecular electronics: metal-molecules-metal junctions based on Hg electrodes.

PubMed

Simeone, Felice Carlo; Rampi, Maria Anita

2010-01-01

Junctions based on mesoscopic Hg electrodes are used to characterize the electrical properties of the organic molecules organized in self-assembled monolayers (SAMs). The junctions M-SAM//SAM-Hg are formed by one electrode based on metals (M) such as Hg, Ag, Au, covered by a SAM, and by a second electrode always formed by a Hg drop carrying also a SAM. The electrodes, brought together by using a micromanipulator, sandwich SAMs of different nature at the contact area (approximately = 0.7 microm2). The high versatility of the system allows a series of both electrical and electrochemical junctions to be assembled and characterized: (i) The compliant nature of the Hg electrodes allows incorporation into the junction and measurement of the electrical behavior of a large number of molecular systems and correlation of their electronic structure to the electrical behavior; (ii) by functionalizing both electrodes with SAMs exposing different functional groups, X and Y, it is possible to compare the rate of electron transfer through different X...Y molecular interactions; (iii) when the junction incorporates one of the electrode formed by a semitransparent film of Au, it allows electrical measurements under irradiation of the sandwiched SAMs. In this case the junction behaves as a photoswitch; iv) incorporation of redox centres with low lying, easily reachable energy levels, provides electron stations as indicated by the hopping mechanism dominating the current flow; (v) electrochemical junctions incorporating redox centres by both covalent and electrostatic interactions permit control of the potential of the electrodes with respect to that of the redox state by means of an external reference electrode. Both these junctions show an electrical behavior similar to that of conventional diodes, even though the mechanism generating the current flow is different. These systems, demonstrating high mechanical stability and reproducibility, easy assembly, and a wide variety of produced results, are convenient test-beds for molecular electronics and represent a useful complement to physics-based experimental methods.

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

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.

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.

Creating Sub-50 nm Nanofluidic Junctions in PDMS Microchip via Self-Assembly Process of Colloidal Silica Beads for Electrokinetic Concentration of Biomolecules

PubMed Central

Syed, A.; Mangano, L.; Mao, P.; Han, J.

2014-01-01

In this work we describe a novel and simple self-assembly of colloidal silica beads to create nanofluidic junction between two microchannels. The nanoporous membrane was used to induce ion concentration polarization inside the microchannel and this electrokinetic preconcentration system allowed rapid concentration of DNA samples by ∼1700 times and protein samples by ∼100 times within 5 minutes. PMID:25254651

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.

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.

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

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

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.

Charge transport in vertically aligned, self-assembled peptide nanotube junctions.

PubMed

Mizrahi, Mordechay; Zakrassov, Alexander; Lerner-Yardeni, Jenny; Ashkenasy, Nurit

2012-01-21

The self-assembly propensity of peptides has been extensively utilized in recent years for the formation of supramolecular nanostructures. In particular, the self-assembly of peptides into fibrils and nanotubes makes them promising building blocks for electronic and electro-optic applications. However, the mechanisms of charge transfer in these wire-like structures, especially in ambient conditions, are not yet fully understood. We describe here a layer-by-layer deposition methodology of short self-assembled cyclic peptide nanotubes, which results in vertically oriented nanotubes on gold substrates. Using this novel deposition methodology, we have fabricated molecular junctions with a conductive atomic force microscopy tip as a second electrode. Studies of the junctions' current-voltage characteristics as a function of the nanotube length revealed an efficient charge transfer in these supramolecular structures, with a low current attenuation constant of 0.1 Å(-1), which indicate that electron transfer is dominated by hopping. Moreover, the threshold voltage to field-emission dominated transport was found to increase with peptide length in a manner that depends on the nature of the contact with the electrodes. The flexibility in the design of the peptide monomers and the ability to control their sequential order over the nanotube by means of the layer-by-layer assembly process, which is demonstrated in this work, can be used to engineer the electronic properties of self-assembled peptide nanotubes toward device applications.

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

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.

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.

AII amacrine cells discriminate between heterocellular and homocellular locations when assembling connexin36-containing gap junctions

PubMed Central

Meyer, Arndt; Hilgen, Gerrit; Dorgau, Birthe; Sammler, Esther M.; Weiler, Reto; Monyer, Hannah; Dedek, Karin; Hormuzdi, Sheriar G.

2014-01-01

ABSTRACT Electrical synapses (gap junctions) rapidly transmit signals between neurons and are composed of connexins. In neurons, connexin36 (Cx36) is the most abundant isoform; however, the mechanisms underlying formation of Cx36-containing electrical synapses are unknown. We focus on homocellular and heterocellular gap junctions formed by an AII amacrine cell, a key interneuron found in all mammalian retinas. In mice lacking native Cx36 but expressing a variant tagged with enhanced green fluorescent protein at the C-terminus (KO-Cx36-EGFP), heterocellular gap junctions formed between AII cells and ON cone bipolar cells are fully functional, whereas homocellular gap junctions between two AII cells are not formed. A tracer injected into an AII amacrine cell spreads into ON cone bipolar cells but is excluded from other AII cells. Reconstruction of Cx36–EGFP clusters on an AII cell in the KO-Cx36-EGFP genotype confirmed that the number, but not average size, of the clusters is reduced – as expected for AII cells lacking a subset of electrical synapses. Our studies indicate that some neurons exhibit at least two discriminatory mechanisms for assembling Cx36. We suggest that employing different gap-junction-forming mechanisms could provide the means for a cell to regulate its gap junctions in a target-cell-specific manner, even if these junctions contain the same connexin. PMID:24463820

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.

Development and fabrication of a solar cell junction processing system

NASA Technical Reports Server (NTRS)

Bunker, S.

1981-01-01

A solar cell junction processing system was developed and fabricated. A pulsed electron beam for the four inch wafers is being assembled and tested, wafers were successfully pulsed, and solar cells fabricated. Assembly of the transport locks is completed. The transport was operated successfully but not with sufficient reproducibility. An experiment test facility to examine potential scaleup problems associated with the proposed ion implanter design was constructed and operated. Cells were implanted and found to have efficiency identical to the normal Spire implant process.

Self-Assembly of Hierarchical DNA Nanotube Architectures with Well-Defined Geometries.

PubMed

Jorgenson, Tyler D; Mohammed, Abdul M; Agrawal, Deepak K; Schulman, Rebecca

2017-02-28

An essential motif for the assembly of biological materials such as actin at the scale of hundreds of nanometers and beyond is a network of one-dimensional fibers with well-defined geometry. Here, we demonstrate the programmed organization of DNA filaments into micron-scale architectures where component filaments are oriented at preprogrammed angles. We assemble L-, T-, and Y-shaped DNA origami junctions that nucleate two or three micron length DNA nanotubes at high yields. The angles between the nanotubes mirror the angles between the templates on the junctions, demonstrating that nanoscale structures can control precisely how micron-scale architectures form. The ability to precisely program filament orientation could allow the assembly of complex filament architectures in two and three dimensions, including circuit structures, bundles, and extended materials.

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.

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

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

Perispeckles are major assembly sites for the exon junction core complex

PubMed Central

Daguenet, Elisabeth; Baguet, Aurélie; Degot, Sébastien; Schmidt, Ute; Alpy, Fabien; Wendling, Corinne; Spiegelhalter, Coralie; Kessler, Pascal; Rio, Marie-Christine; Le Hir, Hervé; Bertrand, Edouard; Tomasetto, Catherine

2012-01-01

The exon junction complex (EJC) is loaded onto mRNAs as a consequence of splicing and regulates multiple posttranscriptional events. MLN51, Magoh, Y14, and eIF4A3 form a highly stable EJC core, but where this tetrameric complex is assembled in the cell remains unclear. Here we show that EJC factors are enriched in domains that we term perispeckles and are visible as doughnuts around nuclear speckles. Fluorescence resonance energy transfer analyses and EJC assembly mutants show that perispeckles do not store free subunits, but instead are enriched for assembled cores. At the ultrastructural level, perispeckles are distinct from interchromatin granule clusters that may function as storage sites for splicing factors and intermingle with perichromatin fibrils, where nascent RNAs and active RNA Pol II are present. These results support a model in which perispeckles are major assembly sites for the tetrameric EJC core. This subnuclear territory thus represents an intermediate region important for mRNA maturation, between transcription sites and splicing factor reservoirs and assembly sites. PMID:22419818

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

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.

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

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.

Coordination of Septate Junctions Assembly and Completion of Cytokinesis in Proliferative Epithelial Tissues.

PubMed

Daniel, Emeline; Daudé, Marion; Kolotuev, Irina; Charish, Kristi; Auld, Vanessa; Le Borgne, Roland

2018-05-07

How permeability barrier function is maintained when epithelial cells divide is largely unknown. Here, we have investigated how the bicellular septate junctions (BSJs) and tricellular septate junctions (TSJs) are remodeled throughout completion of cytokinesis in Drosophila epithelia. We report that, following cytokinetic ring constriction, the midbody assembles, matures within SJs, and is displaced basally in two phases. In a first slow phase, the neighboring cells remain connected to the dividing cells by means of SJ-containing membrane protrusions pointing to the maturing midbody. Fluorescence recovery after photobleaching (FRAP) experiments revealed that SJs within the membrane protrusions correspond to the old SJs that were present prior to cytokinesis. In contrast, new SJs are assembled below the adherens junctions and spread basally to build a new belt of SJs in a manner analogous to a conveyor belt. Loss of function of a core BSJ component, the Na+/K+-ATPase pump Nervana 2 subunit, revealed that the apical-to-basal spread of BSJs drives the basal displacement of the midbody. In contrast, loss of the TSJ protein Bark beetle indicated that remodeling of TSJs is rate limiting and slowed down midbody migration. In the second phase, once the belt of SJs is assembled, the basal displacement of the midbody is accelerated and ultimately leads to abscission. This last step is temporally uncoupled from the remodeling of SJs. We propose that cytokinesis in epithelia involves the coordinated polarized assembly and remodeling of SJs both in the dividing cell and its neighbors to ensure the maintenance of permeability barrier integrity in proliferative epithelia. Copyright © 2018 Elsevier Ltd. All rights reserved.

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.

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.

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.

A Single-Level Tunnel Model to Account for Electrical Transport through Single Molecule- and Self-Assembled Monolayer-based Junctions

PubMed Central

Garrigues, Alvar R.; Yuan, Li; Wang, Lejia; Mucciolo, Eduardo R.; Thompon, Damien; del Barco, Enrique; Nijhuis, Christian A.

2016-01-01

We present a theoretical analysis aimed at understanding electrical conduction in molecular tunnel junctions. We focus on discussing the validity of coherent versus incoherent theoretical formulations for single-level tunneling to explain experimental results obtained under a wide range of experimental conditions, including measurements in individual molecules connecting the leads of electromigrated single-electron transistors and junctions of self-assembled monolayers (SAM) of molecules sandwiched between two macroscopic contacts. We show that the restriction of transport through a single level in solid state junctions (no solvent) makes coherent and incoherent tunneling formalisms indistinguishable when only one level participates in transport. Similar to Marcus relaxation processes in wet electrochemistry, the thermal broadening of the Fermi distribution describing the electronic occupation energies in the electrodes accounts for the exponential dependence of the tunneling current on temperature. We demonstrate that a single-level tunnel model satisfactorily explains experimental results obtained in three different molecular junctions (both single-molecule and SAM-based) formed by ferrocene-based molecules. Among other things, we use the model to map the electrostatic potential profile in EGaIn-based SAM junctions in which the ferrocene unit is placed at different positions within the molecule, and we find that electrical screening gives rise to a strongly non-linear profile across the junction. PMID:27216489

Dimerization of the SP1 Region of HIV-1 Gag Induces a Helical Conformation and Association into Helical Bundles: Implications for Particle Assembly.

PubMed

Datta, Siddhartha A K; Clark, Patrick K; Fan, Lixin; Ma, Buyong; Harvin, Demetria P; Sowder, Raymond C; Nussinov, Ruth; Wang, Yun-Xing; Rein, Alan

2016-02-15

HIV-1 immature particle (virus-like particle [VLP]) assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We previously investigated the role of SP1, a "spacer" between CA and NC, in VLP assembly. We found that small changes in SP1 drastically disrupt assembly and that a peptide representing the sequence around the CA-SP1 junction is helical at high but not low concentrations. We suggested that by virtue of such a concentration-dependent change, this region could act as a molecular switch to activate HIV-1 Gag for VLP assembly. A leucine zipper domain can replace NC in Gag and still lead to the efficient assembly of VLPs. We find that SP1 mutants also disrupt assembly by these Gag-Zip proteins and have now studied a small fragment of this Gag-Zip protein, i.e., the CA-SP1 junction region fused to a leucine zipper. Dimerization of the zipper places SP1 at a high local concentration, even at low total concentrations. In this context, the CA-SP1 junction region spontaneously adopts a helical conformation, and the proteins associate into tetramers. Tetramerization requires residues from both CA and SP1. The data suggest that once this region becomes helical, its propensity to self-associate could contribute to Gag-Gag interactions and thus to particle assembly. There is complete congruence between CA/SP1 sequences that promote tetramerization when fused to zippers and those that permit the proper assembly of full-length Gag; thus, equivalent interactions apparently participate in VLP assembly and in SP1-Zip tetramerization. Assembly of HIV-1 Gag into virus-like particles (VLPs) appears to require an interaction with nucleic acid, but replacement of its principal nucleic acid-binding domain with a dimerizing leucine zipper domain leads to the assembly of RNA-free VLPs. It has not been clear how dimerization triggers assembly. Results here show that the SP1 region spontaneously switches to a helical state when fused to a leucine zipper and that these helical molecules further associate into tetramers, mediated by interactions between hydrophobic faces of the helices. Thus, the correct juxtaposition of the SP1 region makes it "association competent." Residues from both capsid and SP1 contribute to tetramerization, while mutations disrupting proper assembly in Gag also prevent tetramerization. Thus, this region is part of an associating interface within Gag, and its intermolecular interactions evidently help stabilize the immature Gag lattice. These interactions are disrupted by proteolysis of the CA-SP1 junction during virus maturation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Dimerization of the SP1 Region of HIV-1 Gag Induces a Helical Conformation and Association into Helical Bundles: Implications for Particle Assembly

PubMed Central

Clark, Patrick K.; Fan, Lixin; Ma, Buyong; Harvin, Demetria P.; Sowder, Raymond C.; Nussinov, Ruth; Wang, Yun-Xing

2015-01-01

ABSTRACT HIV-1 immature particle (virus-like particle [VLP]) assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We previously investigated the role of SP1, a “spacer” between CA and NC, in VLP assembly. We found that small changes in SP1 drastically disrupt assembly and that a peptide representing the sequence around the CA-SP1 junction is helical at high but not low concentrations. We suggested that by virtue of such a concentration-dependent change, this region could act as a molecular switch to activate HIV-1 Gag for VLP assembly. A leucine zipper domain can replace NC in Gag and still lead to the efficient assembly of VLPs. We find that SP1 mutants also disrupt assembly by these Gag-Zip proteins and have now studied a small fragment of this Gag-Zip protein, i.e., the CA-SP1 junction region fused to a leucine zipper. Dimerization of the zipper places SP1 at a high local concentration, even at low total concentrations. In this context, the CA-SP1 junction region spontaneously adopts a helical conformation, and the proteins associate into tetramers. Tetramerization requires residues from both CA and SP1. The data suggest that once this region becomes helical, its propensity to self-associate could contribute to Gag-Gag interactions and thus to particle assembly. There is complete congruence between CA/SP1 sequences that promote tetramerization when fused to zippers and those that permit the proper assembly of full-length Gag; thus, equivalent interactions apparently participate in VLP assembly and in SP1-Zip tetramerization. IMPORTANCE Assembly of HIV-1 Gag into virus-like particles (VLPs) appears to require an interaction with nucleic acid, but replacement of its principal nucleic acid-binding domain with a dimerizing leucine zipper domain leads to the assembly of RNA-free VLPs. It has not been clear how dimerization triggers assembly. Results here show that the SP1 region spontaneously switches to a helical state when fused to a leucine zipper and that these helical molecules further associate into tetramers, mediated by interactions between hydrophobic faces of the helices. Thus, the correct juxtaposition of the SP1 region makes it “association competent.” Residues from both capsid and SP1 contribute to tetramerization, while mutations disrupting proper assembly in Gag also prevent tetramerization. Thus, this region is part of an associating interface within Gag, and its intermolecular interactions evidently help stabilize the immature Gag lattice. These interactions are disrupted by proteolysis of the CA-SP1 junction during virus maturation. PMID:26637452

Supramolecular Systems and Chemical Reactions in Single-Molecule Break Junctions.

PubMed

Li, Xiaohui; Hu, Duan; Tan, Zhibing; Bai, Jie; Xiao, Zongyuan; Yang, Yang; Shi, Jia; Hong, Wenjing

2017-04-01

The major challenges of molecular electronics are the understanding and manipulation of the electron transport through the single-molecule junction. With the single-molecule break junction techniques, including scanning tunneling microscope break junction technique and mechanically controllable break junction technique, the charge transport through various single-molecule and supramolecular junctions has been studied during the dynamic fabrication and continuous characterization of molecular junctions. This review starts from the charge transport characterization of supramolecular junctions through a variety of noncovalent interactions, such as hydrogen bond, π-π interaction, and electrostatic force. We further review the recent progress in constructing highly conductive molecular junctions via chemical reactions, the response of molecular junctions to external stimuli, as well as the application of break junction techniques in controlling and monitoring chemical reactions in situ. We suggest that beyond the measurement of single molecular conductance, the single-molecule break junction techniques provide a promising access to study molecular assembly and chemical reactions at the single-molecule scale.

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.

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

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.

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

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.

14. INTERIOR VIEW TO THE SOUTH OF ROOM 136, COLD ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

14. INTERIOR VIEW TO THE SOUTH OF ROOM 136, COLD ASSEMBLY BAY NO. 2. - Nevada Test Site, Reactor Maintenance Assembly & Dissassembly Facility, Area 25, Jackass Flats, Junction of Roads F & G, Mercury, Nye County, NV

13. INTERIOR VIEW TO THE SOUTHEAST OF ROOM 101, COLD ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

13. INTERIOR VIEW TO THE SOUTHEAST OF ROOM 101, COLD ASSEMBLY BAY NO. 1. - Nevada Test Site, Reactor Maintenance Assembly & Dissassembly Facility, Area 25, Jackass Flats, Junction of Roads F & G, Mercury, Nye County, NV

35. OUTLET WORKS: GATE HOIST ASSEMBLY. Sheet 44, August 20, ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

35. OUTLET WORKS: GATE HOIST ASSEMBLY. Sheet 44, August 20, 1938. File no. SA 121/84(?). - Prado Dam, Outlet Works, Santa Ana River near junction of State Highways 71 & 91, Corona, Riverside County, CA

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.

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.

Systems and assemblies for transferring high power laser energy through a rotating junction

DOEpatents

Norton, Ryan J.; McKay, Ryan P.; Fraze, Jason D.; Rinzler, Charles C.; Grubb, Daryl L.; Faircloth, Brian O.; Zediker, Mark S.

2016-01-26

There are provided high power laser devices and systems for transmitting a high power laser beam across a rotating assembly, including optical slip rings and optical rotational coupling assemblies. These devices can transmit the laser beam through the rotation zone in free space or within a fiber.

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.

Rectified tunneling current response of bio-functionalized metal-bridge-metal junctions.

PubMed

Liu, Yaqing; Offenhäusser, Andreas; Mayer, Dirk

2010-01-15

Biomolecular bridged nanostructures allow direct electrical addressing of electroactive biomolecules, which is of interest for the development of bioelectronic and biosensing hybrid junctions. In the present paper, the electroactive biomolecule microperoxidase-11 (MP-11) was integrated into metal-bridge-metal (MBM) junctions assembled from a scanning tunneling microscope (STM) setup. Before immobilization of MP-11, the Au working electrode was first modified by a self-assembled monolayer of 1-undecanethiol (UDT). A symmetric and potential independent response of current-bias voltage (I(t)/V(b)) was observed for the Au (substrate)/UDT/Au (tip) junction. However, the I(t)/V(b) characteristics became potential dependent and asymmetrical after binding of MP-11 between the electrodes of the junction. The rectification ratio of the asymmetric current response varies with gate electrode modulation. A resonant tunneling process between metal electrode and MP-11 enhances the tunneling current and is responsible for the observed rectification. Our investigations demonstrated that functional building blocks of proteins can be reassembled into new conceptual devices with operation modes deviating from their native function, which could prove highly useful in the design of future biosensors and bioelectronic devices. Copyright 2009 Elsevier B.V. All rights reserved.

Degradation of connexins and gap junctions

PubMed Central

Falk, Matthias M.; Kells, Rachael M.; Berthoud, Viviana M.

2014-01-01

Connexin proteins are short-lived within the cell, whether present in the secretory pathway or in gap junction plaques. Their levels can be modulated by their rate of degradation. Connexins, at different stages of assembly, are degraded through the proteasomal, endo-/lysosomal, and phago-/lysosomal pathways. In this review, we summarize the current knowledge about connexin and gap junction degradation including the signals and protein-protein interactions that participate in their targeting for degradation. PMID:24486527

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.

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

4. EXTERIOR VIEW TO THE EAST OF THE WEST ELEVATION ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

4. EXTERIOR VIEW TO THE EAST OF THE WEST ELEVATION OF THE COLD ASSEMBLY AREA. - Nevada Test Site, Reactor Maintenance Assembly & Dissassembly Facility, Area 25, Jackass Flats, Junction of Roads F & G, Mercury, Nye County, NV

Tuning the Rectification Ratio by Changing the Electronic Nature (Open-Shell and Closed-Shell) in Donor-Acceptor Self-Assembled Monolayers.

PubMed

Souto, Manuel; Yuan, Li; Morales, Dayana C; Jiang, Li; Ratera, Imma; Nijhuis, Christian A; Veciana, Jaume

2017-03-29

This Communication describes the mechanism of charge transport across self-assembled monolayers (SAMs) of two donor-acceptor systems consisting of a polychlorotriphenylmethyl (PTM) electron-acceptor moiety linked to an electron-donor ferrocene (Fc) unit supported by ultraflat template-stripped Au and contacted by a eutectic alloy of gallium and indium top contacts. The electronic and supramolecular structures of these SAMs were well characterized. The PTM unit can be switched between the nonradical and radical forms, which influences the rectification behavior of the junction. Junctions with nonradical units rectify currents via the highest occupied molecular orbital (HOMO) with a rectification ratio R = 99, but junctions with radical units have a new accessible state, a single-unoccupied molecular orbital (SUMO), which turns rectification off and drops R to 6.

Magnetic Tunnel Junctions Based On Alkanethiol Self Assembled Monolayers

NASA Astrophysics Data System (ADS)

Delprat, Sophie; Quinard, Benoit; Galbiati, Marta; Mattera, Michele; Manas-Valero, Samuel; Forment-Aliaga, Alicia; Tatay, Sergio; Deranlot, Cyrile; Collin, Sophie; Bouzehouane, Karim; Mattana, Richard; Seneor, Pierre; Petroff, Frederic

Molecular spintronics has opened novel and exciting functionalities for spintronics devices. Among them, it was shown that spin dependent hybridization at metal/molecule interfaces could lead to radical tailoring of spintronics properties. In this direction Self-Assembled Monolayers (SAMs) appear to be a very promising candidate with their impressive molecular scale crafting properties. Despite all the promising possibilities, up to now less than a handful of experiments on SAMs as spin-dependent tunnel barriers have been reported at low temperatures, but already showing potential. Towards room temperature spin signal, we studied magnetic tunnel junctions based on alkanethiol and conventional ferromagnets such as Co,NiFe for which we developed a process to recover the ferromagnet from oxidiation. We will present NiFe/SAMs/Co molecular magnetic tunnel junctions with magnetoresistance effects up to 10% observed at 300K.

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

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

Fabrication and characterization of graphene/molecule/graphene vertical junctions with aryl alkane monolayers

NASA Astrophysics Data System (ADS)

Jeong, Inho; Song, Hyunwook

2017-11-01

In this study, we fabricated and characterized graphene/molecule/graphene (GMG) vertical junctions with aryl alkane monolayers. The constituent molecules were chemically self-assembled via electrophilic diazonium reactions into a monolayer on the graphene bottom electrode, while the other end physically contacted the graphene top electrode. A full understanding of the transport properties of molecular junctions is a key step in the realization of molecular-scale electronic devices and requires detailed microscopic characterization of the junction's active region. Using a multiprobe approach combining a variety of transport techniques, we elucidated the transport mechanisms and electronic structure of the GMG junctions, including temperature- and length-variable transport measurements, and transition voltage spectroscopy. These results provide criteria to establish a valid molecular junction and to determine the most probable transport characteristics of the GMG junctions.

10. INTERIOR VIEW TO THE NORTH OF THE HALLWAY WITHIN ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

10. INTERIOR VIEW TO THE NORTH OF THE HALLWAY WITHIN THE ADMINISTRATION PORTION OF THE COLD ASSEMBLY AREA. - Nevada Test Site, Reactor Maintenance Assembly & Dissassembly Facility, Area 25, Jackass Flats, Junction of Roads F & G, Mercury, Nye County, NV

15. INTERIOR VIEW TO THE EAST OF ROOM 102, A ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

15. INTERIOR VIEW TO THE EAST OF ROOM 102, A MACHINE SHOP ADJACENT TO ASSEMBLY BAY NO. 1. - Nevada Test Site, Reactor Maintenance Assembly & Dissassembly Facility, Area 25, Jackass Flats, Junction of Roads F & G, Mercury, Nye County, NV

Epithelial junctions, cytoskeleton, and polarity.

PubMed

Pásti, Gabriella; Labouesse, Michel

2014-11-04

A distinctive feature of polarized epithelial cells is their specialized junctions, which contribute to cell integrity and provide platforms to orchestrate cell shape changes. This chapter discusses the composition, assembly and remodeling of C. elegans cell-cell (CeAJ) and hemidesmosome-like cell-extracellular matrix junctions (CeHD), proteins that anchor the cytoskeleton, and mechanisms involved in establishing epithelial polarity. Major recent progress in this area has come from the analysis of mechanisms that maintain cell polarity, which involve lipids and trafficking, and on the impact of mechanical forces on junction remodeling. This chapter focuses on cellular, rather than developmental, aspects of epithelial cells.

Desmoplakin II expression is not restricted to stratified epithelia.

PubMed

Angst, B D; Nilles, L A; Green, K J

1990-10-01

Desmosomes are major intercellular junctions found in association with intermediate filaments in epithelial, cardiac and arachnoidal tissue. Desmoplakins I and II (DPI and II) are highly related proteins localized in the innermost part of the desmosomal plaque and are candidates for linking intermediate filaments (IF) to the desmosomal complex. While investigators agree that DPI is present in all epithelia, they disagree on the distribution of DPII. Some have reported DPII to be restricted to stratified tissue and have furthermore suggested that the expression of DPII may be linked to stratification. We have compared the expression of DPI and II at the mRNA and protein levels in cell lines derived from simple, transitional and stratified epithelia. Northern blot analysis revealed DPI and II mRNA to be present in all cell lines as well as simple and stratified epithelial tissues. However, DPII mRNA could not be detected in cardiac muscle tissue. Immunoblotting and immunoprecipitation demonstrated the presence of DPI and II in all cell lines at the whole-cell protein level as well as in association with cytoskeletal fractions. Immunofluorescence staining was used to correlate the biochemical findings with the localization of DPI and II. While most cell lines exhibited typical intercellular and in many cases cytoplasmic DP staining, T24 cells exhibited predominantly diffuse and dotty cytoplasmic staining. In addition, we investigated whether changes in DPI and II expression occurred following calcium-induced cell contact formation and stratification in the human pharyngeal cell line, FaDu. No significant changes in mRNA or whole-cell protein levels were observed during a period of 5 days following the calcium switch. However, immunoblotting revealed a significant increase in DPI and II levels in the insoluble protein pool during desmosome formation. These observations indicated a possible recruitment of soluble DPI/II into an insoluble pool after induction of desmosome assembly by the calcium switch, consistent with earlier reports for MDCK cells. In summary, our results suggest that the expression of DPII is not strictly linked to stratification or differentiation; however, the apparent absence of DPII mRNA from cardiac muscle suggests it may not be a constituent of all desmosomes.

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

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

Controlling destructive quantum interference in tunneling junctions comprising self-assembled monolayers via bond topology and functional groups† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc00165k

PubMed Central

Zhang, Yanxi; Ye, Gang; Soni, Saurabh; Qiu, Xinkai; Krijger, Theodorus L.; Jonkman, Harry T.; Carlotti, Marco; Sauter, Eric; Zharnikov, Michael

2018-01-01

Quantum interference effects (QI) are of interest in nano-scale devices based on molecular tunneling junctions because they can affect conductance exponentially through minor structural changes. However, their utilization requires the prediction and deterministic control over the position and magnitude of QI features, which remains a significant challenge. In this context, we designed and synthesized three benzodithiophenes based molecular wires; one linearly-conjugated, one cross-conjugated and one cross-conjugated quinone. Using eutectic Ga–In (EGaIn) and CP-AFM, we compared them to a well-known anthraquinone in molecular junctions comprising self-assembled monolayers (SAMs). By combining density functional theory and transition voltage spectroscopy, we show that the presence of an interference feature and its position can be controlled independently by manipulating bond topology and electronegativity. This is the first study to separate these two parameters experimentally, demonstrating that the conductance of a tunneling junction depends on the position and depth of a QI feature, both of which can be controlled synthetically. PMID:29896382

Structural and thermodynamic analysis of modified nucleosides in self-assembled DNA cross-tiles.

PubMed

Hakker, Lauren; Marchi, Alexandria N; Harris, Kimberly A; LaBean, Thomas H; Agris, Paul F

2014-01-01

DNA Holliday junctions are important natural strand-exchange structures that form during homologous recombination. Immobile four-arm junctions, analogs to Holliday junctions, have been designed to self-assemble into cross-tile structures by maximizing Watson-Crick base pairing and fixed crossover points. The cross-tiles, self-assembled from base pair recognition between designed single-stranded DNAs, form higher order lattice structures through cohesion of self-associating sticky ends. These cross-tiles have 16 unpaired nucleosides in the central loop at the junction of the four duplex stems. The importance of the centralized unpaired nucleosides to the structure's thermodynamic stability and self-assembly is unknown. Cross-tile DNA nanostructures were designed and constructed from nine single-stranded DNAs with four shell strands, four arms, and a central loop containing 16 unpaired bases. The 16 unpaired bases were either 2'-deoxyribothymidines, 2'-O-methylribouridines, or abasic 1',2'-dideoxyribonucleosides. Thermodynamic profiles and structural base-stacking contributions were assessed using UV absorption spectroscopy during thermal denaturation and circular dichroism spectroscopy, respectively, and the resulting structures were observed by atomic force microscopy. There were surprisingly significant changes in the thermodynamic and structural properties of lattice formation as a result of altering only the 16 unpaired, centralized nucleosides. The 16 unpaired 2'-O-methyluridines were stabilizing and produced uniform tubular structures. In contrast, the abasic nucleosides were destabilizing producing a mixture of structures. These results strongly indicate the importance of a small number of centrally located unpaired nucleosides within the structures. Since minor modifications lead to palpable changes in lattice formation, DNA cross-tiles present an easily manipulated structure convenient for applications in biomedical and biosensing devices.

Specific Cx43 phosphorylation events regulate gap junction turnover in vivo

PubMed Central

Solan, Joell L.; Lampe, Paul D.

2014-01-01

Gap junctions, composed of proteins from the connexin gene family, are highly dynamic structures that are regulated by kinase-mediated signaling pathways and interactions with other proteins. Phosphorylation of Connexin43 (Cx43) at different sites controls gap junction assembly, gap junction size and gap junction turnover. Here we present a model describing how Akt, mitogen activated protein kinase (MAPK) and src kinase coordinate to regulate rapid turnover of gap junctions. Specifically, Akt phosphorylates Cx43 at S373 eliminating interaction with zona occludens-1 (ZO-1) allowing gap junctions to enlarge. Then MAPK and src phosphorylate Cx43 to initiate turnover. We integrate published data with new data to test and refine this model. Finally, we propose that differential coordination of kinase activation and Cx43 phosphorylation controls the specific routes of disassembly, e.g., annular junction formation or gap junctions can potentially “unzip” and be internalized/endocytosed into the cell that produced each connexin. PMID:24508467

16. INTERIOR VIEW TO THE SOUTHEAST OF ROOM 137, A ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

16. INTERIOR VIEW TO THE SOUTHEAST OF ROOM 137, A REACTOR CONTROL LAB ADJACENT TO ASSEMBLY BAY NO. 2. - Nevada Test Site, Reactor Maintenance Assembly & Dissassembly Facility, Area 25, Jackass Flats, Junction of Roads F & G, Mercury, Nye County, NV

20. INTERIOR VIEW TO THE EAST OF THE ACCESS RAMP ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

20. INTERIOR VIEW TO THE EAST OF THE ACCESS RAMP TO THE HOT DISASSEMBLY AREA FROM THE COLD ASSEMBLY AREA. - Nevada Test Site, Reactor Maintenance Assembly & Dissassembly Facility, Area 25, Jackass Flats, Junction of Roads F & G, Mercury, Nye County, NV

Neural-like computing with populations of superparamagnetic basis functions.

PubMed

Mizrahi, Alice; Hirtzlin, Tifenn; Fukushima, Akio; Kubota, Hitoshi; Yuasa, Shinji; Grollier, Julie; Querlioz, Damien

2018-04-18

In neuroscience, population coding theory demonstrates that neural assemblies can achieve fault-tolerant information processing. Mapped to nanoelectronics, this strategy could allow for reliable computing with scaled-down, noisy, imperfect devices. Doing so requires that the population components form a set of basis functions in terms of their response functions to inputs, offering a physical substrate for computing. Such a population can be implemented with CMOS technology, but the corresponding circuits have high area or energy requirements. Here, we show that nanoscale magnetic tunnel junctions can instead be assembled to meet these requirements. We demonstrate experimentally that a population of nine junctions can implement a basis set of functions, providing the data to achieve, for example, the generation of cursive letters. We design hybrid magnetic-CMOS systems based on interlinked populations of junctions and show that they can learn to realize non-linear variability-resilient transformations with a low imprint area and low power.

Azobenzenes as light-controlled molecular electronic switches in nanoscale metal-molecule-metal junctions.

PubMed

Mativetsky, Jeffrey M; Pace, Giuseppina; Elbing, Mark; Rampi, Maria A; Mayor, Marcel; Samorì, Paolo

2008-07-23

Conductance switching associated with the photoisomerization of azobenzene-based (Azo) molecules was observed in nanoscopic metal-molecule-metal junctions. The junctions were formed by using a conducting atomic force microscope (C-AFM) approach, where a metallic AFM tip was used to electrically contact a gold-supported Azo self-assembled monolayer. The measured 30-fold increase in conductance is consistent with the expected decrease in tunneling barrier length resulting from the conformational change of the Azo molecule.

The spread of prion-like proteins by lysosomes and tunneling nanotubes: Implications for neurodegenerative diseases.

PubMed

Victoria, Guiliana Soraya; Zurzolo, Chiara

2017-09-04

Progression of pathology in neurodegenerative diseases is hypothesized to be a non-cell-autonomous process that may be mediated by the productive spreading of prion-like protein aggregates from a "donor cell" that is the source of misfolded aggregates to an "acceptor cell" in which misfolding is propagated by conversion of the normal protein. Although the proteins involved in the various diseases are unrelated, common pathways appear to be used for their intercellular propagation and spreading. Here, we summarize recent evidence of the molecular mechanisms relevant for the intercellular trafficking of protein aggregates involved in prion, Alzheimer's, Huntington's, and Parkinson's diseases. We focus in particular on the common roles that lysosomes and tunneling nanotubes play in the formation and spreading of prion-like assemblies. © 2017 Victoria and Zurzolo.

The spread of prion-like proteins by lysosomes and tunneling nanotubes: Implications for neurodegenerative diseases

PubMed Central

Victoria, Guiliana Soraya

2017-01-01

Progression of pathology in neurodegenerative diseases is hypothesized to be a non–cell-autonomous process that may be mediated by the productive spreading of prion-like protein aggregates from a “donor cell” that is the source of misfolded aggregates to an “acceptor cell” in which misfolding is propagated by conversion of the normal protein. Although the proteins involved in the various diseases are unrelated, common pathways appear to be used for their intercellular propagation and spreading. Here, we summarize recent evidence of the molecular mechanisms relevant for the intercellular trafficking of protein aggregates involved in prion, Alzheimer’s, Huntington’s, and Parkinson’s diseases. We focus in particular on the common roles that lysosomes and tunneling nanotubes play in the formation and spreading of prion-like assemblies. PMID:28724527

17. INTERIOR VIEW TO THE EAST OF ROOM 215, A ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

17. INTERIOR VIEW TO THE EAST OF ROOM 215, A SECOND FLOOR OFFICE ABOVE ROOM 137 IN THE COLD ASSEMBLY AREA. - Nevada Test Site, Reactor Maintenance Assembly & Dissassembly Facility, Area 25, Jackass Flats, Junction of Roads F & G, Mercury, Nye County, NV

Automated array assembly, phase 2

NASA Technical Reports Server (NTRS)

Carbajal, B. G.

1979-01-01

Tasks of scaling up the tandem junction cell (TJC) from 2 cm x 2 cm to 6.2 cm and the assembly of several modules using these large area TJC's are described. The scale-up of the TJC was based on using the existing process and doing the necessary design activities to increase the cell area to an acceptably large area. The design was carried out using available device models. The design was verified and sample large area TJCs were fabricated. Mechanical and process problems occurred causing a schedule slippage that resulted in contract expiration before enough large-area TJCs were fabricated to populate the sample tandem junction modules (TJM). A TJM design was carried out in which the module interconnects served to augment the current collecting buses on the cell. No sample TJMs were assembled due to a shortage of large-area TJCs.

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.

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.

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.

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.

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.

An EMMPRIN-γ-catenin-Nm23 complex drives ATP production and actomyosin contractility at endothelial junctions.

PubMed

Moreno, Vanessa; Gonzalo, Pilar; Gómez-Escudero, Jesús; Pollán, Ángela; Acín-Pérez, Rebeca; Breckenridge, Mark; Yáñez-Mó, María; Barreiro, Olga; Orsenigo, Fabrizio; Kadomatsu, Kenji; Chen, Christopher S; Enríquez, José A; Dejana, Elisabetta; Sánchez-Madrid, Francisco; Arroyo, Alicia G

2014-09-01

Cell-cell adhesions are important sites through which cells experience and resist forces. In endothelial cells, these forces regulate junction dynamics and determine endothelial barrier strength. We identify the Ig superfamily member EMMPRIN (also known as basigin) as a coordinator of forces at endothelial junctions. EMMPRIN localization at junctions correlates with endothelial junction strength in different mouse vascular beds. Accordingly, EMMPRIN-deficient mice show altered junctions and increased junction permeability. Lack of EMMPRIN alters the localization and function of VE-cadherin (also known as cadherin-5) by decreasing both actomyosin contractility and tugging forces at endothelial cell junctions. EMMPRIN ensures proper actomyosin-driven maturation of competent endothelial junctions by forming a molecular complex with γ-catenin (also known as junction plakoglobin) and Nm23 (also known as NME1), a nucleoside diphosphate kinase, thereby locally providing ATP to fuel the actomyosin machinery. These results provide a novel mechanism for the regulation of actomyosin contractility at endothelial junctions and might have broader implications in biological contexts such as angiogenesis, collective migration and tissue morphogenesis by coupling compartmentalized energy production to junction assembly. © 2014. Published by The Company of Biologists Ltd.

An EMMPRIN–γ-catenin–Nm23 complex drives ATP production and actomyosin contractility at endothelial junctions

PubMed Central

Moreno, Vanessa; Gonzalo, Pilar; Gómez-Escudero, Jesús; Pollán, Ángela; Acín-Pérez, Rebeca; Breckenridge, Mark; Yáñez-Mó, María; Barreiro, Olga; Orsenigo, Fabrizio; Kadomatsu, Kenji; Chen, Christopher S.; Enríquez, José A.; Dejana, Elisabetta; Sánchez-Madrid, Francisco; Arroyo, Alicia G.

2014-01-01

ABSTRACT Cell–cell adhesions are important sites through which cells experience and resist forces. In endothelial cells, these forces regulate junction dynamics and determine endothelial barrier strength. We identify the Ig superfamily member EMMPRIN (also known as basigin) as a coordinator of forces at endothelial junctions. EMMPRIN localization at junctions correlates with endothelial junction strength in different mouse vascular beds. Accordingly, EMMPRIN-deficient mice show altered junctions and increased junction permeability. Lack of EMMPRIN alters the localization and function of VE-cadherin (also known as cadherin-5) by decreasing both actomyosin contractility and tugging forces at endothelial cell junctions. EMMPRIN ensures proper actomyosin-driven maturation of competent endothelial junctions by forming a molecular complex with γ-catenin (also known as junction plakoglobin) and Nm23 (also known as NME1), a nucleoside diphosphate kinase, thereby locally providing ATP to fuel the actomyosin machinery. These results provide a novel mechanism for the regulation of actomyosin contractility at endothelial junctions and might have broader implications in biological contexts such as angiogenesis, collective migration and tissue morphogenesis by coupling compartmentalized energy production to junction assembly. PMID:24994937

Protein phosphatase 2A associates with and regulates atypical PKC and the epithelial tight junction complex

PubMed Central

Nunbhakdi-Craig, Viyada; Machleidt, Thomas; Ogris, Egon; Bellotto, Dennis; White, Charles L.; Sontag, Estelle

2002-01-01

Tight junctions (TJs) play a crucial role in the establishment of cell polarity and regulation of paracellular permeability in epithelia. Here, we show that upon calcium-induced junction biogenesis in Madin-Darby canine kidney cells, ABαC, a major protein phosphatase (PP)2A holoenzyme, is recruited to the apical membrane where it interacts with the TJ complex. Enhanced PP2A activity induces dephosphorylation of the TJ proteins, ZO-1, occludin, and claudin-1, and is associated with increased paracellular permeability. Expression of PP2A catalytic subunit severely prevents TJ assembly. Conversely, inhibition of PP2A by okadaic acid promotes the phosphorylation and recruitment of ZO-1, occludin, and claudin-1 to the TJ during junctional biogenesis. PP2A negatively regulates TJ assembly without appreciably affecting the organization of F-actin and E-cadherin. Significantly, inhibition of atypical PKC (aPKC) blocks the calcium- and serum-independent membrane redistribution of TJ proteins induced by okadaic acid. Indeed, PP2A associates with and critically regulates the activity and distribution of aPKC during TJ formation. Thus, we provide the first evidence for calcium-dependent targeting of PP2A in epithelial cells, we identify PP2A as the first serine/threonine phosphatase associated with the multiprotein TJ complex, and we unveil a novel role for PP2A in the regulation of epithelial aPKC and TJ assembly and function. PMID:12196510

Quantitative cell polarity imaging defines leader-to-follower transitions during collective migration and the key role of microtubule-dependent adherens junction formation.

PubMed

Revenu, Céline; Streichan, Sebastian; Donà, Erika; Lecaudey, Virginie; Hufnagel, Lars; Gilmour, Darren

2014-03-01

The directed migration of cell collectives drives the formation of complex organ systems. A characteristic feature of many migrating collectives is a 'tissue-scale' polarity, whereby 'leader' cells at the edge of the tissue guide trailing 'followers' that become assembled into polarised epithelial tissues en route. Here, we combine quantitative imaging and perturbation approaches to investigate epithelial cell state transitions during collective migration and organogenesis, using the zebrafish lateral line primordium as an in vivo model. A readout of three-dimensional cell polarity, based on centrosomal-nucleus axes, allows the transition from migrating leaders to assembled followers to be quantitatively resolved for the first time in vivo. Using live reporters and a novel fluorescent protein timer approach, we investigate changes in cell-cell adhesion underlying this transition by monitoring cadherin receptor localisation and stability. This reveals that while cadherin 2 is expressed across the entire tissue, functional apical junctions are first assembled in the transition zone and become progressively more stable across the leader-follower axis of the tissue. Perturbation experiments demonstrate that the formation of these apical adherens junctions requires dynamic microtubules. However, once stabilised, adherens junction maintenance is microtubule independent. Combined, these data identify a mechanism for regulating leader-to-follower transitions within migrating collectives, based on the relocation and stabilisation of cadherins, and reveal a key role for dynamic microtubules in this process.

Regulation of neuronal axon specification by glia-neuron gap junctions in C. elegans.

PubMed

Meng, Lingfeng; Zhang, Albert; Jin, Yishi; Yan, Dong

2016-10-21

Axon specification is a critical step in neuronal development, and the function of glial cells in this process is not fully understood. Here, we show that C. elegans GLR glial cells regulate axon specification of their nearby GABAergic RME neurons through GLR-RME gap junctions. Disruption of GLR-RME gap junctions causes misaccumulation of axonal markers in non-axonal neurites of RME neurons and converts microtubules in those neurites to form an axon-like assembly. We further uncover that GLR-RME gap junctions regulate RME axon specification through activation of the CDK-5 pathway in a calcium-dependent manner, involving a calpain clp-4 . Therefore, our study reveals the function of glia-neuron gap junctions in neuronal axon specification and shows that calcium originated from glial cells can regulate neuronal intracellular pathways through gap junctions.

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

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

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.

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

5. EXTERIOR VIEW TO THE SOUTHEAST OF THE NORTH AND ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

5. EXTERIOR VIEW TO THE SOUTHEAST OF THE NORTH AND WEST ELEVATIONS, WITH THE COLD ASSEMBLY AREA TO THE RIGHT AND THE HOT DISASSEMBLY AREA TO THE LEFT. - Nevada Test Site, Reactor Maintenance Assembly & Dissassembly Facility, Area 25, Jackass Flats, Junction of Roads F & G, Mercury, Nye County, NV

3. EXTERIOR VIEW TO THE NORTH OF THE SOUTH ELEVATION ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

3. EXTERIOR VIEW TO THE NORTH OF THE SOUTH ELEVATION OF THE ADMINISTRATION AREA IN THE COLD ASSEMBLY AREA, WITH THE MAIN ENTRANCE 'KENNEDY DOORS' IN THE MIDDLE. - Nevada Test Site, Reactor Maintenance Assembly & Dissassembly Facility, Area 25, Jackass Flats, Junction of Roads F & G, Mercury, Nye County, NV

Trestle #1, detail of spacer and bolt assembly with tie ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

Trestle #1, detail of spacer and bolt assembly with tie and guard timber, southwest end of deck. View to east-northeast - Promontory Route Railroad Trestles, S.P. Trestle 779.91, One mile southwest of junction of State Highway 83 and Blue Creek, Corinne, Box Elder County, UT

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

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.

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

Anterograde Jelly belly ligand to Alk receptor signaling at developing synapses is regulated by Mind the gap.

PubMed

Rohrbough, Jeffrey; Broadie, Kendal

2010-10-01

Bidirectional trans-synaptic signals induce synaptogenesis and regulate subsequent synaptic maturation. Presynaptically secreted Mind the gap (Mtg) molds the synaptic cleft extracellular matrix, leading us to hypothesize that Mtg functions to generate the intercellular environment required for efficient signaling. We show in Drosophila that secreted Jelly belly (Jeb) and its receptor tyrosine kinase Anaplastic lymphoma kinase (Alk) are localized to developing synapses. Jeb localizes to punctate aggregates in central synaptic neuropil and neuromuscular junction (NMJ) presynaptic terminals. Secreted Jeb and Mtg accumulate and colocalize extracellularly in surrounding synaptic boutons. Alk concentrates in postsynaptic domains, consistent with an anterograde, trans-synaptic Jeb-Alk signaling pathway at developing synapses. Jeb synaptic expression is increased in Alk mutants, consistent with a requirement for Alk receptor function in Jeb uptake. In mtg null mutants, Alk NMJ synaptic levels are reduced and Jeb expression is dramatically increased. NMJ synapse morphology and molecular assembly appear largely normal in jeb and Alk mutants, but larvae exhibit greatly reduced movement, suggesting impaired functional synaptic development. jeb mutant movement is significantly rescued by neuronal Jeb expression. jeb and Alk mutants display normal NMJ postsynaptic responses, but a near loss of patterned, activity-dependent NMJ transmission driven by central excitatory output. We conclude that Jeb-Alk expression and anterograde trans-synaptic signaling are modulated by Mtg and play a key role in establishing functional synaptic connectivity in the developing motor circuit.

Photovoltaic module kit including connector assembly for non-penetrating array installation

DOEpatents

Botkin, Jonathan [El Cerrito, CA; Graves, Simon [Berkeley, CA; Danning, Matt [Oakland, CA; Culligan, Matthew [Berkeley, CA

2011-11-22

A PV module kit for non-penetrating rooftop installation, including a plurality of PV modules and a plurality of connectors. Each of the PV modules includes a PV laminate and a frame forming a mounting region assembled thereto. The connectors include a male connector having a male fastener extending from a head, and a female connector having a female fastener assembled within a head. The heads are entirely formed of plastic. The kit provides a mounted array state including a junction at which the mounting region of at least two of the PV modules are aligned and interconnected by engagement of the male connector with the female connector. The so-formed junction is substantially electrically insulated. The plurality of connectors can further include a spacer connector including a head forming a bore sized to slidably receive the male fastener, with all of the connector heads being identical.

Photovoltaic module kit including connector assembly for non-penetrating array installation

DOEpatents

Botkin, Jonathan; Graves, Simon; Danning, Matt; Culligan, Matthew

2012-10-23

A PV module kit for non-penetrating rooftop installation, including a plurality of PV modules and a plurality of connectors. Each of the PV modules includes a PV laminate and a frame forming a mounting region assembled thereto. The connectors include a male connector having a male fastener extending from a head, and a female connector having a female fastener assembled within a head. The heads are entirely formed of plastic. The kit provides a mounted array state including a junction at which the mounting region of at least two of the PV modules are aligned and interconnected by engagement of the male connector with the female connector. The so-formed junction is substantially electrically insulated. The plurality of connectors can further include a spacer connector including a head forming a bore sized to slidably receive the male fastener, with all of the connector heads being identical.

Single gate p-n junctions in graphene-ferroelectric devices

NASA Astrophysics Data System (ADS)

Hinnefeld, J. Henry; Xu, Ruijuan; Rogers, Steven; Pandya, Shishir; Shim, Moonsub; Martin, Lane W.; Mason, Nadya

2016-05-01

Graphene's linear dispersion relation and the attendant implications for bipolar electronics applications have motivated a range of experimental efforts aimed at producing p-n junctions in graphene. Here we report electrical transport measurements of graphene p-n junctions formed via simple modifications to a PbZr0.2Ti0.8O3 substrate, combined with a self-assembled layer of ambient environmental dopants. We show that the substrate configuration controls the local doping region, and that the p-n junction behavior can be controlled with a single gate. Finally, we show that the ferroelectric substrate induces a hysteresis in the environmental doping which can be utilized to activate and deactivate the doping, yielding an "on-demand" p-n junction in graphene controlled by a single, universal backgate.

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.

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

Venkadesh, S.; Mandal, P.K.; Gautham, N., E-mail: n_gautham@hotmail.com

Highlights: {yields} This is the first crystal structure of a four-way junction with sticky ends. {yields} Four junction structures bind to each other and form a rhombic cavity. {yields} Each rhombus binds to others to form 'infinite' 2D tiles. {yields} This is an example of bottom-up fabrication of a DNA nano-lattice. -- Abstract: We report here the crystal structure of the partially self-complementary decameric sequence d(CGGCGGCCGC), which self assembles to form a four-way junction with sticky ends. Each junction binds to four others through Watson-Crick base pairing at the sticky ends to form a rhombic structure. The rhombuses bind tomore » each other and form two dimensional tiles. The tiles stack to form the crystal. The crystal diffracted in the space group P1 to a resolution of 2.5 A. The junction has the anti-parallel stacked-X conformation like other junction structures, though the formation of the rhombic net noticeably alters the details of the junction geometry.« less

Gap junction plasticity as a mechanism to regulate network-wide oscillations

PubMed Central

Nicola, Wilten; Clopath, Claudia

2018-01-01

Cortical oscillations are thought to be involved in many cognitive functions and processes. Several mechanisms have been proposed to regulate oscillations. One prominent but understudied mechanism is gap junction coupling. Gap junctions are ubiquitous in cortex between GABAergic interneurons. Moreover, recent experiments indicate their strength can be modified in an activity-dependent manner, similar to chemical synapses. We hypothesized that activity-dependent gap junction plasticity acts as a mechanism to regulate oscillations in the cortex. We developed a computational model of gap junction plasticity in a recurrent cortical network based on recent experimental findings. We showed that gap junction plasticity can serve as a homeostatic mechanism for oscillations by maintaining a tight balance between two network states: asynchronous irregular activity and synchronized oscillations. This homeostatic mechanism allows for robust communication between neuronal assemblies through two different mechanisms: transient oscillations and frequency modulation. This implies a direct functional role for gap junction plasticity in information transmission in cortex. PMID:29529034

Pronounced Environmental Effects on Injection Currents in EGaIn Tunneling Junctions Comprising Self-Assembled Monolayers.

PubMed

Carlotti, Marco; Degen, Maarten; Zhang, Yanxi; Chiechi, Ryan C

2016-09-15

Large-area tunneling junctions using eutectic Ga-In (EGaIn) as a top contact have proven to be a robust, reproducible, and technologically relevant platform for molecular electronics. Thus far, the majority of studies have focused on saturated molecules with backbones consisting mainly of alkanes in which the frontier orbitals are either highly localized or energetically inaccessible. We show that self-assembled monolayers of wire-like oligophenyleneethynylenes (OPEs), which are fully conjugated, only exhibit length-dependent tunneling behavior in a low-O 2 environment. We attribute this unexpected behavior to the sensitivity of injection current on environment. We conclude that, contrary to previous reports, the self-limiting layer of Ga 2 O 3 strongly influences transport properties and that the effect is related to the wetting behavior of the electrode. This result sheds light on the nature of the electrode-molecule interface and suggests that adhesive forces play a significant role in tunneling charge-transport in large-area molecular junctions.

Pronounced Environmental Effects on Injection Currents in EGaIn Tunneling Junctions Comprising Self-Assembled Monolayers

PubMed Central

2016-01-01

Large-area tunneling junctions using eutectic Ga–In (EGaIn) as a top contact have proven to be a robust, reproducible, and technologically relevant platform for molecular electronics. Thus far, the majority of studies have focused on saturated molecules with backbones consisting mainly of alkanes in which the frontier orbitals are either highly localized or energetically inaccessible. We show that self-assembled monolayers of wire-like oligophenyleneethynylenes (OPEs), which are fully conjugated, only exhibit length-dependent tunneling behavior in a low-O2 environment. We attribute this unexpected behavior to the sensitivity of injection current on environment. We conclude that, contrary to previous reports, the self-limiting layer of Ga2O3 strongly influences transport properties and that the effect is related to the wetting behavior of the electrode. This result sheds light on the nature of the electrode–molecule interface and suggests that adhesive forces play a significant role in tunneling charge-transport in large-area molecular junctions. PMID:27738488

Structural and rectifying junction properties of self-assembled ZnO nanoparticles in polystyrene diblock copolymers on (1 0 0)Si substrates

NASA Astrophysics Data System (ADS)

Ali, H. A.; Iliadis, A. A.; Martinez-Miranda, L. J.; Lee, U.

2006-06-01

The structural and electronic transport properties of self-assembled ZnO nanoparticles in polystyrene-acrylic acid, [PS] m/[PAA] n, diblock copolymer on p-type (1 0 0)Si substrates are reported for the first time. Four different block repeat unit ratios ( m/ n) of 159/63, 139/17,106/17, and 106/4, were examined in order to correlate the physical parameters (size, density) of the nanoparticles with the copolymer block lengths m and n. We established that the self-assembled ZnO nanoparticle average size increased linearly with minority block length n, while the average density decreased exponentially with majority block length m. Average size varied from 20 nm to 250 nm and average density from 3.5 × 10 7 cm -2 to 1 × 10 10 cm -2, depending on copolymer parameters. X-ray diffraction studies showed the particles to have a wurtzite crystal structure with the (1 0 0) being the dominant orientation. Room temperature current-voltage characteristics measured for an Al/ZnO-nanocomposite/Si structure exhibited rectifying junction properties and indicated the formation of Al/ZnO-nanocomposite Schottky type junction with a barrier height of 0.7 V.

1. EXTERIOR VIEW TO THE NORTH OF THE SOUTH ELEVATIONS ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

1. EXTERIOR VIEW TO THE NORTH OF THE SOUTH ELEVATIONS OF THE R-MAD FACILITY WITH THE COLD ASSEMBLY AREA ON THE LEFT AND THE HOT DISASSEMBLY AREA TO THE RIGHT. - Nevada Test Site, Reactor Maintenance Assembly & Dissassembly Facility, Area 25, Jackass Flats, Junction of Roads F & G, Mercury, Nye County, NV

Installation and Assembly, Electrical Ground Support Equipment (GSE), Specification for

NASA Technical Reports Server (NTRS)

Denson, Erik C.

2014-01-01

This specification covers the general workmanship requirements and procedures for the complete installation and assembly of electrical ground support equipment (EGSE) such as terminal distributors, junction boxes, conduit and fittings, cable trays and accessories, interconnecting cables (including routing requirements), motor-control equipment, and necessary hardware as specified by the applicable contract and drawings.

Transmission, Development, and Plasticity of Synapses

PubMed Central

Harris, Kathryn P.

2015-01-01

Chemical synapses are sites of contact and information transfer between a neuron and its partner cell. Each synapse is a specialized junction, where the presynaptic cell assembles machinery for the release of neurotransmitter, and the postsynaptic cell assembles components to receive and integrate this signal. Synapses also exhibit plasticity, during which synaptic function and/or structure are modified in response to activity. With a robust panel of genetic, imaging, and electrophysiology approaches, and strong evolutionary conservation of molecular components, Drosophila has emerged as an essential model system for investigating the mechanisms underlying synaptic assembly, function, and plasticity. We will discuss techniques for studying synapses in Drosophila, with a focus on the larval neuromuscular junction (NMJ), a well-established model glutamatergic synapse. Vesicle fusion, which underlies synaptic release of neurotransmitters, has been well characterized at this synapse. In addition, studies of synaptic assembly and organization of active zones and postsynaptic densities have revealed pathways that coordinate those events across the synaptic cleft. We will also review modes of synaptic growth and plasticity at the fly NMJ, and discuss how pre- and postsynaptic cells communicate to regulate plasticity in response to activity. PMID:26447126

PLEKHA7 Recruits PDZD11 to Adherens Junctions to Stabilize Nectins.

PubMed

Guerrera, Diego; Shah, Jimit; Vasileva, Ekaterina; Sluysmans, Sophie; Méan, Isabelle; Jond, Lionel; Poser, Ina; Mann, Matthias; Hyman, Anthony A; Citi, Sandra

2016-05-20

PLEKHA7 is a junctional protein implicated in stabilization of the cadherin protein complex, hypertension, cardiac contractility, glaucoma, microRNA processing, and susceptibility to bacterial toxins. To gain insight into the molecular basis for the functions of PLEKHA7, we looked for new PLEKHA7 interactors. Here, we report the identification of PDZ domain-containing protein 11 (PDZD11) as a new interactor of PLEKHA7 by yeast two-hybrid screening and by mass spectrometry analysis of PLEKHA7 immunoprecipitates. We show that PDZD11 (17 kDa) is expressed in epithelial and endothelial cells, where it forms a complex with PLEKHA7, as determined by co-immunoprecipitation analysis. The N-terminal Trp-Trp (WW) domain of PLEKHA7 interacts directly with the N-terminal 44 amino acids of PDZD11, as shown by GST-pulldown assays. Immunofluorescence analysis shows that PDZD11 is localized at adherens junctions in a PLEKHA7-dependent manner, because its junctional localization is abolished by knock-out of PLEKHA7, and is rescued by re-expression of exogenous PLEKHA7. The junctional recruitment of nectin-1 and nectin-3 and their protein levels are decreased via proteasome-mediated degradation in epithelial cells where either PDZD11 or PLEKHA7 have been knocked-out. PDZD11 forms a complex with nectin-1 and nectin-3, and its PDZ domain interacts directly with the PDZ-binding motif of nectin-1. PDZD11 is required for the efficient assembly of apical junctions of epithelial cells at early time points in the calcium-switch model. These results show that the PLEKHA7-PDZD11 complex stabilizes nectins to promote efficient early junction assembly and uncover a new molecular mechanism through which PLEKHA7 recruits PDZ-binding membrane proteins to epithelial adherens junctions. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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 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

Single gate p-n junctions in graphene-ferroelectric devices

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

Hinnefeld, J. Henry; Mason, Nadya, E-mail: nadya@illinois.edu; Xu, Ruijuan

Graphene's linear dispersion relation and the attendant implications for bipolar electronics applications have motivated a range of experimental efforts aimed at producing p-n junctions in graphene. Here we report electrical transport measurements of graphene p-n junctions formed via simple modifications to a PbZr{sub 0.2}Ti{sub 0.8}O{sub 3} substrate, combined with a self-assembled layer of ambient environmental dopants. We show that the substrate configuration controls the local doping region, and that the p-n junction behavior can be controlled with a single gate. Finally, we show that the ferroelectric substrate induces a hysteresis in the environmental doping which can be utilized to activatemore » and deactivate the doping, yielding an “on-demand” p-n junction in graphene controlled by a single, universal backgate.« less

Study on iron disilicide thermoelectric generator

NASA Astrophysics Data System (ADS)

Anderson, Gerald

1987-11-01

The first objective of the experimental work is to verify the characteristics of commercially available material. The Japanese company Komatsu Electronics Ltd., sells U-Shaped couples of FeSi2. Twenty-four couples are now in our laboratory. Each leg of the couple is made of one type (N or P) of material and the junction is placed directly into a flame. Being almost impossible to measure the hot junction temperature in the flame and to evaluate the heat flux going through the material between hot and cold junctions, we have designed an experimental assembly, suited to measure these values. The main problem is to obtain a good thermal contact for the hot junctions. If not, there is an important temperature drop between the hot source and the hot junction of the couple leading to wrong values of the characteristics.

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

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.

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.

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

Dendrobium chrysotoxum Lindl. Alleviates Diabetic Retinopathy by Preventing Retinal Inflammation and Tight Junction Protein Decrease

PubMed Central

Yu, Zengyang; Gong, Chenyuan; Lu, Bin; Yang, Li; Sheng, Yuchen; Ji, Lili; Wang, Zhengtao

2015-01-01

Diabetic retinopathy (DR) is a serious complication of diabetes mellitus. This study aimed to observe the alleviation of the ethanol extract of Dendrobium chrysotoxum Lindl. (DC), a traditional Chinese herbal medicine, on DR and its engaged mechanism. After DC (30 or 300 mg/kg) was orally administrated, the breakdown of blood retinal barrier (BRB) in streptozotocin- (STZ-) induced diabetic rats was attenuated by DC. Decreased retinal mRNA expression of tight junction proteins (including occludin and claudin-1) in diabetic rats was also reversed by DC. Western blot analysis and retinal immunofluorescence staining results further confirmed that DC reversed the decreased expression of occludin and claudin-1 proteins in diabetic rats. DC reduced the increased retinal mRNA expressions of intercellular adhesion molecule-1 (ICAM-1), tumor necrosis factor α (TNFα), interleukin- (IL-) 6, and IL-1β in diabetic rats. In addition, DC alleviated the increased 1 and phosphorylated p65, IκB, and IκB kinase (IKK) in diabetic rats. DC also reduced the increased serum levels of TNFα, interferon-γ (IFN-γ), IL-6, IL-1β, IL-8, IL-12, IL-2, IL-3, and IL-10 in diabetic rats. Therefore, DC can alleviate DR by inhibiting retinal inflammation and preventing the decrease of tight junction proteins, such as occludin and claudin-1. PMID:25685822

Investigation of Proposed Process Sequence for the Array Automated Assembly Task, Phase 2. [low cost silicon solar array fabrication

NASA Technical Reports Server (NTRS)

Mardesich, N.; Garcia, A.; Bunyan, S.; Pepe, A.

1979-01-01

The technological readiness of the proposed process sequence was reviewed. Process steps evaluated include: (1) plasma etching to establish a standard surface; (2) forming junctions by diffusion from an N-type polymeric spray-on source; (3) forming a p+ back contact by firing a screen printed aluminum paste; (4) forming screen printed front contacts after cleaning the back aluminum and removing the diffusion oxide; (5) cleaning the junction by a laser scribe operation; (6) forming an antireflection coating by baking a polymeric spray-on film; (7) ultrasonically tin padding the cells; and (8) assembling cell strings into solar circuits using ethylene vinyl acetate as an encapsulant and laminating medium.

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

Visualizing the effect of dynamin inhibition on annular gap vesicle formation and fission

PubMed Central

Nickel, Beth; Boller, Marie; Schneider, Kimberly; Shakespeare, Teresa; Gay, Vernon; Murray, Sandra A.

2013-01-01

Summary Although gap junction plaque assembly has been extensively studied, mechanisms involved in plaque disassembly are not well understood. Disassembly involves an internalization process in which annular gap junction vesicles are formed. These vesicles undergo fission, but the molecular machinery needed for these fissions has not been described. The mechanoenzyme dynamin has been previously demonstrated to play a role in gap junction plaque internalization. To investigate the role of dynamin in annular gap junction vesicle fission, immunocytochemical, time-lapse and transmission electron microscopy were used to analyze SW-13 adrenocortical cells in culture. Dynamin was demonstrated to colocalize with gap junction plaques and vesicles. Dynamin inhibition, by siRNA knockdown or treatment with the dynamin GTPase inhibitor dynasore, increased the number and size of gap junction ‘buds’ suspended from the gap junction plaques. Buds, in control populations, were frequently released to form annular gap junction vesicles. In dynamin-inhibited populations, the buds were larger and infrequently released and thus fewer annular gap junction vesicles were formed. In addition, the number of annular gap junction vesicle fissions per hour was reduced in the dynamin-inhibited populations. We believe this to be the first report addressing the details of annular gap junction vesicle fissions and demonstrating a role of dynamin in this process. This information is crucial for elucidating the relationship between gap junctions, membrane regulation and cell behavior. PMID:23591819

Visualizing the effect of dynamin inhibition on annular gap vesicle formation and fission.

PubMed

Nickel, Beth; Boller, Marie; Schneider, Kimberly; Shakespeare, Teresa; Gay, Vernon; Murray, Sandra A

2013-06-15

Although gap junction plaque assembly has been extensively studied, mechanisms involved in plaque disassembly are not well understood. Disassembly involves an internalization process in which annular gap junction vesicles are formed. These vesicles undergo fission, but the molecular machinery needed for these fissions has not been described. The mechanoenzyme dynamin has been previously demonstrated to play a role in gap junction plaque internalization. To investigate the role of dynamin in annular gap junction vesicle fission, immunocytochemical, time-lapse and transmission electron microscopy were used to analyze SW-13 adrenocortical cells in culture. Dynamin was demonstrated to colocalize with gap junction plaques and vesicles. Dynamin inhibition, by siRNA knockdown or treatment with the dynamin GTPase inhibitor dynasore, increased the number and size of gap junction 'buds' suspended from the gap junction plaques. Buds, in control populations, were frequently released to form annular gap junction vesicles. In dynamin-inhibited populations, the buds were larger and infrequently released and thus fewer annular gap junction vesicles were formed. In addition, the number of annular gap junction vesicle fissions per hour was reduced in the dynamin-inhibited populations. We believe this to be the first report addressing the details of annular gap junction vesicle fissions and demonstrating a role of dynamin in this process. This information is crucial for elucidating the relationship between gap junctions, membrane regulation and cell behavior.

YAP/TAZ regulates sprouting angiogenesis and vascular barrier maturation

PubMed Central

Kim, Yoo Hyung; Kim, Jaeryung; Park, Do Young; Bae, Hosung; Lee, Da-Hye; Kim, Kyun Hoo; Hong, Seon Pyo; Jang, Seung Pil; Kwon, Young-Guen; Lim, Dae-Sik

2017-01-01

Angiogenesis is a multistep process that requires coordinated migration, proliferation, and junction formation of vascular endothelial cells (ECs) to form new vessel branches in response to growth stimuli. Major intracellular signaling pathways that regulate angiogenesis have been well elucidated, but key transcriptional regulators that mediate these signaling pathways and control EC behaviors are only beginning to be understood. Here, we show that YAP/TAZ, a transcriptional coactivator that acts as an end effector of Hippo signaling, is critical for sprouting angiogenesis and vascular barrier formation and maturation. In mice, endothelial-specific deletion of Yap/Taz led to blunted-end, aneurysm-like tip ECs with fewer and dysmorphic filopodia at the vascular front, a hyper-pruned vascular network, reduced and disarranged distributions of tight and adherens junction proteins, disrupted barrier integrity, subsequent hemorrhage in growing retina and brain vessels, and reduced pathological choroidal neovascularization. Mechanistically, YAP/TAZ activates actin cytoskeleton remodeling, an important component of filopodia formation and junction assembly. Moreover, YAP/TAZ coordinates EC proliferation and metabolic activity by upregulating MYC signaling. Overall, these results show that YAP/TAZ plays multifaceted roles for EC behaviors, proliferation, junction assembly, and metabolism in sprouting angiogenesis and barrier formation and maturation and could be a potential therapeutic target for treating neovascular diseases. PMID:28805663

Gap junctions between accessory medulla neurons appear to synchronize circadian clock cells of the cockroach Leucophaea maderae.

PubMed

Schneider, Nils-Lasse; Stengl, Monika

2006-03-01

The temporal organization of physiological and behavioral states is controlled by circadian clocks in apparently all eukaryotic organisms. In the cockroach Leucophaea maderae lesion and transplantation studies located the circadian pacemaker in the accessory medulla (AMe). The AMe is densely innervated by gamma-aminobutyric acid (GABA)-immunoreactive and peptidergic neurons, among them the pigment-dispersing factor immunoreactive circadian pacemaker candidates. The large majority of cells of the cockroach AMe spike regularly and synchronously in the gamma frequency range of 25-70 Hz as a result of synaptic and nonsynaptic coupling. Although GABAergic coupling forms assemblies of phase-locked cells, in the absence of synaptic release the cells remain synchronized but fire now at a stable phase difference. To determine whether these coupling mechanisms of AMe neurons, which are independent of synaptic release, are based on electrical synapses between the circadian pacemaker cells the gap-junction blockers halothane, octanol, and carbenoxolone were used in the presence and absence of synaptic transmission. Here, we show that different populations of AMe neurons appear to be coupled by gap junctions to maintain synchrony at a stable phase difference. This synchronization by gap junctions is a prerequisite to phase-locked assembly formation by synaptic interactions and to synchronous gamma-type action potential oscillations within the circadian clock.

Homophilic and heterophilic polycystin 1 interactions regulate E-cadherin recruitment and junction assembly in MDCK cells

PubMed Central

Streets, Andrew J.; Wagner, Bart E.; Harris, Peter C.; Ward, Christopher J.; Ong, Albert C. M.

2009-01-01

Summary Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited human renal disease and is caused by mutations in two genes, PKD1 (85%) and PKD2 (15%). Cyst epithelial cells are characterised by a complex cellular phenotype including changes in proliferation, apoptosis, basement membrane composition and apicobasal polarity. Since polycystin 1 (PC1), the PKD1 protein, has been located in the basolateral membrane of kidney epithelial cells, we hypothesised that it might have a key role in mediating or stabilising cell-cell interactions. In non-ciliated L929 cells, stable or transient surface expression of the PC1 extracellular domain was sufficient to confer an adhesive phenotype and stimulate junction formation. In MDCK cells, we found that PC1 was recruited to the lateral membranes coincident with E-cadherin within 30 minutes after a `calcium switch'. Recruitment of both proteins was significantly delayed when cells were treated with a PC1 blocking antibody raised to the PKD domains. Finally, PC1 and E-cadherin could be coimmunoprecipitated together from MDCK cells. We conclude that PC1 has a key role in initiating junction formation via initial homophilic interactions and facilitates junction assembly and the establishment of apicobasal polarity by E-cadherin recruitment. PMID:19351715

Human metapneumovirus Induces Reorganization of the Actin Cytoskeleton for Direct Cell-to-Cell Spread

PubMed Central

El Najjar, Farah; Cifuentes-Muñoz, Nicolás; Zhu, Haining; Buchholz, Ursula J.; Moncman, Carole L.; Dutch, Rebecca Ellis

2016-01-01

Paramyxovirus spread generally involves assembly of individual viral particles which then infect target cells. We show that infection of human bronchial airway cells with human metapneumovirus (HMPV), a recently identified paramyxovirus which causes significant respiratory disease, results in formation of intercellular extensions and extensive networks of branched cell-associated filaments. Formation of these structures is dependent on actin, but not microtubule, polymerization. Interestingly, using a co-culture assay we show that conditions which block regular infection by HMPV particles, including addition of neutralizing antibodies or removal of cell surface heparan sulfate, did not prevent viral spread from infected to new target cells. In contrast, inhibition of actin polymerization or alterations to Rho GTPase signaling pathways significantly decreased cell-to-cell spread. Furthermore, viral proteins and viral RNA were detected in intercellular extensions, suggesting direct transfer of viral genetic material to new target cells. While roles for paramyxovirus matrix and fusion proteins in membrane deformation have been previously demonstrated, we show that the HMPV phosphoprotein extensively co-localized with actin and induced formation of cellular extensions when transiently expressed, supporting a new model in which a paramyxovirus phosphoprotein is a key player in assembly and spread. Our results reveal a novel mechanism for HMPV direct cell-to-cell spread and provide insights into dissemination of respiratory viruses. PMID:27683250

Expression and in vitro regulation of integrins by normal human urothelial cells.

PubMed

Southgate, J; Kennedy, W; Hutton, K A; Trejdosiewicz, L K

1995-08-01

Integrins are thought to be essential adhesion receptors for the maintenance of tissue histioarchitecture. The purpose of this study was to determine integrin expression patterns in the human stratified transitional epithelium of the urinary tract (urothelium). In situ expression patterns were compared with in vitro expression, using a normal cell culture model system in which the effects of cell stratification can be studied independently of differentiation. By immunohistological criteria, the urothelia of bladder, ureter and renal pelvis expressed alpha 2 beta 1 and alpha 3 beta 1 integrins in all layers at intercellular junctions, and cytoplasmically in the lower strata. By contrast, alpha 6 beta 4 and occasionally alpha v beta 4 were expressed only by basal cells and localised to the basal lamina. These expression patterns were unaltered in specimens where an inflammatory cell infiltrate was present. In long-term cultures of normal urothelial cells maintained in a low-Ca++ serum-free medium, the monolayer cultures expressed alpha 2 beta 1, alpha 3 beta 1 and alpha 5 beta 1 integrins at intercellular junctions and in cytoplasmic inclusions, whereas alpha 6 beta 4 was distributed in a random pattern over the substratum. Increasing exogenous Ca++ concentrations induced cell stratification and desmosome formation, but not cytodifferentiation. Under these conditions, alpha 6 beta 4 became cell-, rather than substratum-associated, localising particularly to filopodia and lamellipodia. Quantitation of integrin expression by flow cytometry confirmed increased surface expression of alpha 6 beta 4 in high Ca++ media, and also of alpha 3 and alpha 5, but not alpha 2, subunits. These results suggest that alpha 2 beta 1 and alpha 3 beta 1 integrins, although differentially regulated, are mainly involved in homotypic cell-cell interactions and the maintenance of a stratified morphology, whereas alpha 6 beta 4 is the principal integrin involved in substratum adhesion.

Increase in gap-junctional intercellular communications (GJIC) of normal human dermal fibroblasts (NHDF) on surfaces coated with high-molecular-weight hyaluronic acid (HMW HA).

PubMed

Park, Jeong Ung; Tsuchiya, Toshie

2002-06-15

Normal human dermal fibroblast (NHDF) cells were used to detect differences in gap-junctional intercellular communication (GJIC) by hyaluronic acid (HA), a linear polymer built from repeating disaccharide units that consist of N-acetyl-D-glucosamine (GlcNa) and D-glucuronic acid (GlcA) linked by a beta 1-4 glycosidic bond. The NHDF cells were cultured with different molecular weights (MW) of HA for 4 days. The rates of cell attachment in dishes coated with high-molecular-weight (HMW; 310 kDa or 800 kDa) HA at 2 mg/dish were significantly reduced at an early time point compared with low-molecular-weight (LMW; 4.8 kDa or 48 kDa) HA with the same coating amounts. HA-coated surfaces were observed by atomic force microscopy (AFM) under air and showed that HA molecules ran parallel in the dish coated with LMW HA and had an aggregated island structure in the dish coated with HMW HA surfaces. The cell functions of GJIC were assayed by a scrape-loading dye transfer (SLDT) method using a dye solution of Lucifer yellow. Promotion of the dye transfer was clearly obtained in the cell monolayer grown on the surface coated with HMW HA. These results suggest that HMW HA promotes the function of GJIC in NHDF cells. In contrast, when HMW HA was added to the monolayer of NHDF cells, the functions of GJIC clearly were lowered in comparison with the cells grown in the control dish or with those grown on the surface of HMW HA. Therefore it is concluded that the MW size of HA and its application method are important factors for generating biocompatible tissue-engineered products because of the manner in which the GJIC participates in cell differentiation and cell growth rate. Copyright 2002 Wiley Periodicals, Inc. J Biomed Mater Res 60: 541-547, 2002

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.

Estrogenic compounds inhibit gap junctional intercellular communication in mouse Leydig TM3 cells

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

Iwase, Yumiko; Fukata, Hideki; Mori, Chisato

2006-05-01

Some estrogenic compounds are reported to cause testicular disorders in humans and/or experimental animals by direct action on Leydig cells. In carcinogenesis and normal development, gap junctional intercellular communication (GJIC) plays an essential role in maintaining homeostasis. In this study, we examine the effects of diethylstilbestrol (DES, a synthetic estrogen), 17{beta}-estradiol (E{sub 2}, a natural estrogen), and genistein (GEN, a phytoestrogen) on GJIC between mouse Leydig TM3 cells using Lucifer yellow microinjection. The three compounds tested produced GJIC inhibition in the TM3 cells after 24 h. Gradually, 10 {mu}M DES began to inhibit GJIC for 24 h and this effectmore » was observed until 72 h. On the other hand, both 20 {mu}M E{sub 2} and 25 {mu}M GEN rapidly inhibited GJIC in 6 h and 2 h, respectively. The effects continued until 24 h, but weakened by 72 h. Furthermore, a combined effect at {mu}M level between DES and E{sub 2} on GJIC inhibition was observed, but not between GEN and E{sub 2}. DES and E{sub 2} showed GJIC inhibition at low dose levels (nearly physiological estrogen levels) after 72 h, but GEN did not. DES-induced GJIC inhibition at 10 pM and 10 {mu}M was completely counteracted by ICI 182,780 (ICl), an estrogen receptor antagonist. On the other hand, the inhibitory effects on GJIC with E{sub 2} (10 pM and 20 {mu}M) and GEN (25 {mu}M) were partially blocked by ICI or calphostin C, a protein kinase C (PKC) inhibitor, and were completely blocked by the combination of ICI and calphostin C. These results demonstrate that DES inhibits GJIC between Leydig cells via the estrogen receptor (ER), and that E{sub 2} and GEN inhibit GJIC via ER and PKC. These estrogenic compounds may have different individual nongenotoxic mechanism including PKC pathway on testicular carcinogenesis or development.« less

Intercellular adhesion molecules (ICAMs) and spermatogenesis

PubMed Central

Xiao, Xiang; Mruk, Dolores D.; Cheng, C. Yan

2013-01-01

BACKGROUND During the seminiferous epithelial cycle, restructuring takes places at the Sertoli–Sertoli and Sertoli–germ cell interface to accommodate spermatogonia/spermatogonial stem cell renewal via mitosis, cell cycle progression and meiosis, spermiogenesis and spermiation since developing germ cells, in particular spermatids, move ‘up and down’ the seminiferous epithelium. Furthermore, preleptotene spermatocytes differentiated from type B spermatogonia residing at the basal compartment must traverse the blood–testis barrier (BTB) to enter the adluminal compartment to prepare for meiosis at Stage VIII of the epithelial cycle, a process also accompanied by the release of sperm at spermiation. These cellular events that take place at the opposite ends of the epithelium are co-ordinated by a functional axis designated the apical ectoplasmic specialization (ES)—BTB—basement membrane. However, the regulatory molecules that co-ordinate cellular events in this axis are not known. METHODS Literature was searched at http://www.pubmed.org and http://scholar.google.com to identify published findings regarding intercellular adhesion molecules (ICAMs) and the regulation of this axis. RESULTS Members of the ICAM family, namely ICAM-1 and ICAM-2, and the biologically active soluble ICAM-1 (sICAM-1) are the likely regulatory molecules that co-ordinate these events. sICAM-1 and ICAM-1 have antagonistic effects on the Sertoli cell tight junction-permeability barrier, involved in Sertoli cell BTB restructuring, whereas ICAM-2 is restricted to the apical ES, regulating spermatid adhesion during the epithelial cycle. Studies in other epithelia/endothelia on the role of the ICAM family in regulating cell movement are discussed and this information has been evaluated and integrated into studies of these proteins in the testis to create a hypothetical model, depicting how ICAMs regulate junction restructuring events during spermatogenesis. CONCLUSIONS ICAMs are crucial regulatory molecules of spermatogenesis. The proposed hypothetical model serves as a framework in designing functional experiments for future studies. PMID:23287428

Gap junction disorders of myelinating cells.

PubMed

Kleopa, Kleopas A; Orthmann-Murphy, Jennifer; Sargiannidou, Irene

2010-01-01

Gap junctions (GJs) are channels that allow the diffusion of ions and small molecules across apposed cell membranes. In peripheral nerves, Schwann cells express the GJ proteins connexin32 (Cx32) and Cx29, which have distinct localizations. Cx32 forms GJs through non-compact myelin areas, whereas Cx29 forms hemichannels in the innermost layers of myelin apposing axonal Shaker-type K+ channels. In the CNS, rodent oligodendrocytes express Cx47, Cx32 and Cx29. Cx47 is expressed by all types of oligodendrocytes both in the white and grey matter and forms GJs on cell bodies and proximal processes, as well as most of the intercellular channels with astrocytes. Cx32 is expressed mostly by white matter oligodendrocytes and is localized in the myelin sheath of large diameter fibers. Cx29, and its human ortholog Cx31.3, appear to be restricted to oligodendrocytes that myelinate small caliber fibers, likely forming hemichannels. The importance of intercellular and intracellular GJs in myelinating cells are demonstrated by human disorders resulting from mutations affecting GJ proteins. The X-linked Charcot Marie Tooth disease (CMT1X) is caused by hundreds of mutations affecting Cx32. Patients with CMT1X present mainly with a progressive peripheral neuropathy, which may be accompanied by CNS myelin dysfunction. Mutations in Cx47 may cause a devastating leukodystrophy called Pelizaeus-Merzbacher-like disease or a milder spastic paraplegia. In addition, CNS demyelination may be caused by defects in genes expressing astrocytic GJ proteins, which are essential for oligodendrocytes. Findings from in vitro and in vivo models of these disorders developed over the last decade indicate that most mutations cause loss of function and an inability of the mutant connexins to form functional GJs. Here we review the clinical, genetic, and neurobiological aspects of GJ disorders affecting the PNS and CNS myelinating cells.

Zinc enhances intestinal epithelial barrier function through the PI3K/AKT/mTOR signaling pathway in Caco-2 cells.

PubMed

Shao, Yuxin; Wolf, Patricia G; Guo, Shuangshuang; Guo, Yuming; Gaskins, H Rex; Zhang, Bingkun

2017-05-01

Zinc plays an important role in maintaining intestinal barrier function as well as modulating cellular signaling recognition and protein kinase activities. The phosphatidylinositol 3-kinase (PI3K) cascade has been demonstrated to affect intercellular integrity and tight junction (TJ) proteins. The current study investigated the hypothesis that zinc regulates intestinal intercellular junction integrity through the PI3K/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway. A transwell model of Caco-2 cell was incubated with 0, 50 and 100 μM of zinc at various time points. Transepithelial electrical resistance (TEER), paracellular permeability, TJ proteins, cell proliferation, differentiation and cell damage were measured. Compared with controls, 50 and 100 μM of zinc increased cell growth at 6, 12 and 24 h and the expression of proliferating cell nuclear antigen at 24 h. Zinc (100 μM) significantly elevated TEER at 6-24 h and reduced TJ permeability at 24 h, accompanied by the up-regulation of alkaline phosphatase (AP) activity and zonula occludens (ZO)-1 expression. In addition, zinc (100 μM) affected the PI3K/AKT/mTOR pathway by stimulating phosphorylation of AKT and the downstream target mTOR. Inhibition of PI3K signaling by LY294002 counteracted zinc promotion, as shown by a decrease in AP activity, TEER, the abundance of ZO-1 and phosphorylation of AKT and mTOR. Additionally, TJ permeability and the expression of caspase-3 and LC3II (markers of cell damage) were increased by addition of PI3K inhibitor. In conclusion, the activation of PI3K/AKT/mTOR signaling by zinc is involved in improving intestinal barrier function by enhancing cell differentiation and expression of TJ protein ZO-1. Copyright © 2017 Elsevier Inc. All rights reserved.

Development of the endolymphatic sac and duct in the Japanese red-bellied newt, Cynops pyrrhogaster

NASA Technical Reports Server (NTRS)

Gao, W.; Wiederhold, M. L.; Harrison, J. L.

1998-01-01

The development and maturation of the endolymphatic sac (ES) and duct (ED) were studied in the newt Cynops pyrrhogaster. The ES first appears as an oval capsule at the dorsal-medial tip of the otic vesicle at stage 39, about 11 days after oviposition. The ES consists of polymorphous epithelial cells with a minimum of cytoplasm. The intercellular space (IS) between the epithelial cells is narrow and has a smooth surface. At stage 44, the size of the ES increases as many vacuoles in the IS become filled. At stage 46, 18 days after oviposition, the ES elongates markedly and a slit-like lumen is found in the ES. The epithelium contains a few cell organelles which are scattered in the cytoplasm. The vacuoles in the IS are fused, which expands the IS. Two days later (stage 48), floccular material (endolymph) is present in the expanded lumen. The IS dilates and has a wide and irregular appearance. At stage 50, approximately 26 days after oviposition, the ES extends and expands significantly and crystals (otoconia) can now be seen in the widened lumen of the ES. The cytoplasm of the cuboidal epithelial cells contains an abundance of vesicles surrounded by ribosomes and Golgi complexes. Intercellular digitations are formed in the expanded IS. At stage 54, the ES forms a large bellow-like pouch. Numerous otoconia accumulate in the lumen. Free floating cells and cell debris can be seen in the lumen at this stage. The epithelial cells contain numerous cytoplasmic organelles which are evenly distributed in the cytoplasm. Granules are found in the apical and lateral cytoplasm. The IS is loose and displays a labyrinthine appearance. The primitive ED first appears as a connection between the ES and the saccule but no lumen is present inside at stage 39. At stage 46, a narrow lumen is formed in the ED, which corresponds to the formation of the ES lumen. At stage 50, as the ED extends, floccular material is seen in the lumen. At stage 54, the ED bears numerous microvilli on its luminal surface. Otoconia and endolymph are present in the ED. Tight junctions between the epithelial cells are formed at stage 46. A fully developed intercellular junctional complex is produced at stage 54. Based on the development of the ES and ED, the maturation of function of the ES and ED are discussed.

Analysis of trafficking, stability and function of human connexin 26 gap junction channels with deafness-causing mutations in the fourth transmembrane helix.

PubMed

Ambrosi, Cinzia; Walker, Amy E; Depriest, Adam D; Cone, Angela C; Lu, Connie; Badger, John; Skerrett, I Martha; Sosinsky, Gina E

2013-01-01

Human Connexin26 gene mutations cause hearing loss. These hereditary mutations are the leading cause of childhood deafness worldwide. Mutations in gap junction proteins (connexins) can impair intercellular communication by eliminating protein synthesis, mis-trafficking, or inducing channels that fail to dock or have aberrant function. We previously identified a new class of mutants that form non-functional gap junction channels and hemichannels (connexons) by disrupting packing and inter-helix interactions. Here we analyzed fourteen point mutations in the fourth transmembrane helix of connexin26 (Cx26) that cause non-syndromic hearing loss. Eight mutations caused mis-trafficking (K188R, F191L, V198M, S199F, G200R, I203K, L205P, T208P). Of the remaining six that formed gap junctions in mammalian cells, M195T and A197S formed stable hemichannels after isolation with a baculovirus/Sf9 protein purification system, while C202F, I203T, L205V and N206S formed hemichannels with varying degrees of instability. The function of all six gap junction-forming mutants was further assessed through measurement of dye coupling in mammalian cells and junctional conductance in paired Xenopus oocytes. Dye coupling between cell pairs was reduced by varying degrees for all six mutants. In homotypic oocyte pairings, only A197S induced measurable conductance. In heterotypic pairings with wild-type Cx26, five of the six mutants formed functional gap junction channels, albeit with reduced efficiency. None of the mutants displayed significant alterations in sensitivity to transjunctional voltage or induced conductive hemichannels in single oocytes. Intra-hemichannel interactions between mutant and wild-type proteins were assessed in rescue experiments using baculovirus expression in Sf9 insect cells. Of the four unstable mutations (C202F, I203T, L205V, N206S) only C202F and N206S formed stable hemichannels when co-expressed with wild-type Cx26. Stable M195T hemichannels displayed an increased tendency to aggregate. Thus, mutations in TM4 cause a range of phenotypes of dysfunctional gap junction channels that are discussed within the context of the X-ray crystallographic structure.

Disruption of the epithelial barrier during intestinal inflammation: Quest for new molecules and mechanisms.

PubMed

Lechuga, Susana; Ivanov, Andrei I

2017-07-01

The intestinal epithelium forms a key protective barrier that separates internal organs from the harmful environment of the gut lumen. Increased permeability of the gut barrier is a common manifestation of different inflammatory disorders contributing to the severity of disease. Barrier permeability is controlled by epithelial adherens junctions and tight junctions. Junctional assembly and integrity depend on fundamental homeostatic processes such as cell differentiation, rearrangements of the cytoskeleton, and vesicle trafficking. Alterations of intestinal epithelial homeostasis during mucosal inflammation may impair structure and remodeling of apical junctions, resulting in increased permeability of the gut barrier. In this review, we summarize recent advances in our understanding of how altered epithelial homeostasis affects the structure and function of adherens junctions and tight junctions in the inflamed gut. Specifically, we focus on the transcription reprogramming of the cell, alterations in the actin cytoskeleton, and junctional endocytosis and exocytosis. We pay special attention to knockout mouse model studies and discuss the relevance of these mechanisms to human gastrointestinal disorders. Copyright © 2017 Elsevier B.V. All rights reserved.

Growth and assembly of cobalt oxide nanoparticle rings at liquid nanodroplets with solid junction.

PubMed

Zhou, Yilong; Powers, Alexander S; Zhang, Xiaowei; Xu, Tao; Bustillo, Karen; Sun, Litao; Zheng, Haimei

2017-09-28

Using liquid cell TEM, we imaged the formation of CoO nanoparticle rings. Nanoparticles nucleated and grew tracing the perimeter of droplets sitting on the SiN x solid substrate, and finally formed necklace-like rings. By tracking single nanoparticle trajectories during the ring formation and an estimation of the forces between droplets and nanoparticles using a simplified model, we found the junction of liquid nanodroplets with a solid substrate is the attractive site for CoO nanoparticles. Coalescing droplets were capable of pushing nanoparticles to the perimeter of the new droplet and nanoparticles on top of the droplets rolled off toward the perimeter. We propose that the curved surface morphology of the droplets created a force gradient that contributed to the assembly of nanoparticles at the droplet perimeter. Revealing the dynamics of nanoparticle movements and the interactions of nanoparticles with the liquid nanodroplet provides insights on developing novel self-assembly strategies for building precisely defined nanostructures on solid substrates.

All solution-processed lead halide perovskite-BiVO4 tandem assembly for photolytic solar fuels production.

PubMed

Chen, Yong-Siou; Manser, Joseph S; Kamat, Prashant V

2015-01-21

The quest for economic, large-scale hydrogen production has motivated the search for new materials and device designs capable of splitting water using only energy from the sun. Here we introduce an all solution-processed tandem water splitting assembly composed of a BiVO4 photoanode and a single-junction CH3NH3PbI3 hybrid perovskite solar cell. This unique configuration allows efficient solar photon management, with the metal oxide photoanode selectively harvesting high energy visible photons, and the underlying perovskite solar cell capturing lower energy visible-near IR wavelengths in a single-pass excitation. Operating without external bias under standard AM 1.5G illumination, the photoanode-photovoltaic architecture, in conjunction with an earth-abundant cobalt phosphate catalyst, exhibits a solar-to-hydrogen conversion efficiency of 2.5% at neutral pH. The design of low-cost tandem water splitting assemblies employing single-junction hybrid perovskite materials establishes a potentially promising new frontier for solar water splitting research.

Microtubules and epithem-cell morphogenesis in hydathodes of Pilea cadierei.

PubMed

Galatis, B

1988-12-01

When cell divisions have ceased, the epithem of the hydathodes of Pilea cadierei Gagnep. et Guill. consists of small polyhedral cells exhibiting a meristematic appearance, and completely lacks intercellular spaces. The cortical microtubules in epithem cells exhibit a unique organization: they are not scattered along the whole wall surface but form groups lying at some distance from each other. In sections, from two to eight groups of microtubules can be observed, each lining a wall region averaging between 0.5 and 1.5 μm in length. These groups represent sections of microtubule bundles girdling a major part or the whole of the cell periphery. They are connected to one another by anastomoses, forming a microtubular reticulum. The assembly of microtubule bundles is followed by the appearance of distinct local thickenings in the adjacent wall areas. The cellulose microfibrils in the thickenings are deposited in parallel to the underlying microtubules. Gradually, the vacuolating epithem cells undergo swelling, except for the areas bounded by the wall thickenings. Since the latter, and actually their constituent bundles of cellulose microfibrils, cannot extend in length the differential cell growth results in schizogenous formation of intercellular spaces between contiguous cell walls at their thickened regions. The spaces then broaden and merge to become an extensive intercellular space system. As a result of the above processes, the epithem cells become constricted and finally deeply lobed. The observations show that (i) the cortical microtubules are intimately involved in the morphogenesis of the epithem cells and (ii) the initiation and development of the epithem intercellular spaces is a phenomenon directly related to cell morphogenesis and therefore to the cortical microtubule cytoskeleton. The sites of initiation of these spaces are highly predictable.

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.

Ultrastructure of the external gill epithelium of the axolotl, Ambystoma mexicanum with reference to ionic transport.

PubMed

Jarial, M S; Wilkins, J H

2003-10-01

The ultrastructure of the external gill epithelium of the axolotl, Ambystoma mexicanum, has been examined using conventional transmission electron microscopy to elucidate its role in ionic transport. Four cell types are identified in the gill filament and primary gill bar epithelium. These are granular, ciliated, Leydig and basal cells. A fifth cell type, the flat mitochondria-rich cell is only found in the gill bar epithelium. The predominant granular cells display microvilli at their surface and their cytoplasm contains abundant mitochondria, rough endoplasmic reticulum, Golgi complexes, vesicles and PAS+ secretory granules that are extruded at the surface, which along with secretions from the Leydig cells form a mucous coat. The granular cells are joined apically by junctional complexes consisting of zonulae occludens, zonulae adherens and desmosomes. The lateral membranes of granular cells enclose large intercellular spaces that are closed at the apical ends but remain open at the basal ends adjoining capillaries. In AgNO3-treated axolotl, the gills become darkly stained, the silver grains penetrate apical membranes and appear in the cytoplasm, accumulating near the lateral membranes and also enter the intercellular spaces. These findings are consistent with the dual role of the gill epithelium in mucus production and active ionic transport.

Islands of spatially discordant APD alternans underlie arrhythmogenesis by promoting electrotonic dyssynchrony in models of fibrotic rat ventricular myocardium

NASA Astrophysics Data System (ADS)

Majumder, Rupamanjari; Engels, Marc C.; de Vries, Antoine A. F.; Panfilov, Alexander V.; Pijnappels, Daniël A.

2016-04-01

Fibrosis and altered gap junctional coupling are key features of ventricular remodelling and are associated with abnormal electrical impulse generation and propagation. Such abnormalities predispose to reentrant electrical activity in the heart. In the absence of tissue heterogeneity, high-frequency impulse generation can also induce dynamic electrical instabilities leading to reentrant arrhythmias. However, because of the complexity and stochastic nature of such arrhythmias, the combined effects of tissue heterogeneity and dynamical instabilities in these arrhythmias have not been explored in detail. Here, arrhythmogenesis was studied using in vitro and in silico monolayer models of neonatal rat ventricular tissue with 30% randomly distributed cardiac myofibroblasts and systematically lowered intercellular coupling achieved in vitro through graded knockdown of connexin43 expression. Arrhythmia incidence and complexity increased with decreasing intercellular coupling efficiency. This coincided with the onset of a specialized type of spatially discordant action potential duration alternans characterized by island-like areas of opposite alternans phase, which positively correlated with the degree of connexinx43 knockdown and arrhythmia complexity. At higher myofibroblast densities, more of these islands were formed and reentrant arrhythmias were more easily induced. This is the first study exploring the combinatorial effects of myocardial fibrosis and dynamic electrical instabilities on reentrant arrhythmia initiation and complexity.

Dominant de novo DSP mutations cause erythrokeratodermia-cardiomyopathy syndrome

PubMed Central

Boyden, Lynn M.; Kam, Chen Y.; Hernández-Martín, Angela; Zhou, Jing; Craiglow, Brittany G.; Sidbury, Robert; Mathes, Erin F.; Maguiness, Sheilagh M.; Crumrine, Debra A.; Williams, Mary L.; Hu, Ronghua; Lifton, Richard P.; Elias, Peter M.; Green, Kathleen J.; Choate, Keith A.

2016-01-01

Disorders of keratinization (DOK) show marked genotypic and phenotypic heterogeneity. In most cases, disease is primarily cutaneous, and further clinical evaluation is therefore rarely pursued. We have identified subjects with a novel DOK featuring erythrokeratodermia and initially-asymptomatic, progressive, potentially fatal cardiomyopathy, a finding not previously associated with erythrokeratodermia. We show that de novo missense mutations clustered tightly within a single spectrin repeat of DSP cause this novel cardio-cutaneous disorder, which we term erythrokeratodermia-cardiomyopathy (EKC) syndrome. We demonstrate that DSP mutations in our EKC syndrome subjects affect localization of desmosomal proteins and connexin 43 in the skin, and result in desmosome aggregation, widening of intercellular spaces, and lipid secretory defects. DSP encodes desmoplakin, a primary component of desmosomes, intercellular adhesion junctions most abundant in the epidermis and heart. Though mutations in DSP are known to cause other disorders, our cohort features the unique clinical finding of severe whole-body erythrokeratodermia, with distinct effects on localization of desmosomal proteins and connexin 43. These findings add a severe, previously undescribed syndrome featuring erythrokeratodermia and cardiomyopathy to the spectrum of disease caused by mutation in DSP, and identify a specific region of the protein critical to the pathobiology of EKC syndrome and to DSP function in the heart and skin. PMID:26604139

ELECTRON MICROSCOPY OF ABSORPTION OF TRACER MATERIALS BY TOAD URINARY BLADDER EPITHELIUM

PubMed Central

Choi, Jae Kwon

1965-01-01

The absorption of Thorotrast and saccharated iron oxide by the epithelium of the toad urinary bladder was studied by electron microscopy. Whether the toads were hydrated, dehydrated, or given Pitressin, no significant differences in transport of colloidal particles by epithelial cells were observed. This implies that these physiological factors had little effect on the transport of the tracer particles. Tracer particles were encountered in three types of epithelial cells which line the bladder lumen, but most frequently in the mitochondria-rich cells. Tracer materials were incorporated into the cytoplasm of epithelial cells after being adsorbed to the coating layer covering the luminal surface of the cells. In the intermediate stage (1 to 3 hours after introducing tracer) particles were present in small vesicles, tubules, and multivesicular bodies. In the later stages (up to 65 hours), the particles were more commonly seen to be densely packed within large membrane-bounded bodies which were often found near the Golgi region. These large bodies probably were formed by the fusion of small vesicles. Irrespective of the stages of absorption, no particles were found in the intercellular spaces or in the submucosa. Particles apparently did not penetrate the intercellular spaces of the epithelium beyond the level of the tight junction. PMID:14287173

Mixed Monolayers of Spiropyrans Maximize Tunneling Conductance Switching by Photoisomerization at the Molecule–Electrode Interface in EGaIn Junctions

PubMed Central

2016-01-01

This paper describes the photoinduced switching of conductance in tunneling junctions comprising self-assembled monolayers of a spiropyran moiety using eutectic Ga–In top contacts. Despite separation of the spiropyran unit from the electrode by a long alkyl ester chain, we observe an increase in the current density J of a factor of 35 at 1 V when the closed form is irradiated with UV light to induce the ring-opening reaction, one of the highest switching ratios reported for junctions incorporating self-assembled monolayers. The magnitude of switching of hexanethiol mixed monolayers was higher than that of pure spiropyran monolayers. The first switching event recovers 100% of the initial value of J and in the mixed-monolayers subsequent dampening is not the result of degradation of the monolayer. The observation of increased conductivity is supported by zero-bias DFT calculations showing a change in the localization of the density of states near the Fermi level as well as by simulated transmission spectra revealing positive resonances that broaden and shift toward the Fermi level in the open form. PMID:27602432

Shingle system

DOEpatents

Dinwoodie, Thomas L [Piedmont, CA

2008-02-12

A barrier, such as a PV module, is secured to a base by a support to create a shingle assembly with a venting region defined between the barrier and base for temperature regulation. Water resistant junctions may be formed between the bases of adjacent shingle assemblies of an array of shingle assemblies. The base may include an insulation layer underlying a water barrier. The base may also include a waterproofing element; the width and height of the barrier may be shorter than the width and height of the waterproofing element.

Solar cell circuit and method for manufacturing solar cells

NASA Technical Reports Server (NTRS)

Mardesich, Nick (Inventor)

2010-01-01

The invention is a novel manufacturing method for making multi-junction solar cell circuits that addresses current problems associated with such circuits by allowing the formation of integral diodes in the cells and allows for a large number of circuits to readily be placed on a single silicon wafer substrate. The standard Ge wafer used as the base for multi-junction solar cells is replaced with a thinner layer of Ge or a II-V semiconductor material on a silicon/silicon dioxide substrate. This allows high-voltage cells with multiple multi-junction circuits to be manufactured on a single wafer, resulting in less array assembly mass and simplified power management.

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

Amino acid sequence of bovine muzzle epithelial desmocollin derived from cloned cDNA: a novel subtype of desmosomal cadherins.

PubMed

Koch, P J; Goldschmidt, M D; Walsh, M J; Zimbelmann, R; Schmelz, M; Franke, W W

1991-05-01

Desmosomes are cell-type-specific intercellular junctions found in epithelium, myocardium and certain other tissues. They consist of assemblies of molecules involved in the adhesion of specific cell types and in the anchorage of cell-type-specific cytoskeletal elements, the intermediate-size filaments, to the plasma membrane. To explore the individual desmosomal components and their functions we have isolated DNA clones encoding the desmosomal glycoprotein, desmocollin, using antibodies and a cDNA expression library from bovine muzzle epithelium. The cDNA-deduced amino-acid sequence of desmocollin (presently we cannot decide to which of the two desmocollins, DC I or DC II, this clone relates) defines a polypeptide with a calculated molecular weight of 85,000, with a single candidate sequence of 24 amino acids sufficiently long for a transmembrane arrangement, and an extracellular aminoterminal portion of 561 amino acid residues, compared to a cytoplasmic part of only 176 amino acids. Amino acid sequence comparisons have revealed that desmocollin is highly homologous to members of the cadherin family of cell adhesion molecules, including the previously sequenced desmoglein, another desmosome-specific cadherin. Using riboprobes derived from cDNAs for Northern-blot analyses, we have identified an mRNA of approximately 6 kb in stratified epithelia such as muzzle epithelium and tongue mucosa but not in two epithelial cell culture lines containing desmosomes and desmoplakins. The difference may indicate drastic differences in mRNA concentration or the existence of cell-type-specific desmocollin subforms. The molecular topology of desmocollin(s) is discussed in relation to possible functions of the individual molecular domains.

Connexin-dependent gap junction enhancement is involved in the synergistic effect of sorafenib and all-trans retinoic acid on HCC growth inhibition.

PubMed

Yang, Yan; Qin, Shu-Kui; Wu, Qiong; Wang, Zi-Shu; Zheng, Rong-Sheng; Tong, Xu-Hui; Liu, Hao; Tao, Liang; He, Xian-Di

2014-02-01

Increasing gap junction activity in tumor cells provides a target by which to enhance antineoplastic therapies. Previously, several naturally occurring agents, including all-trans retinoic acid (ATRA) have been demonstrated to increase gap junctional intercellular communication (GJIC) in a number of types of cancer cells. In the present study, we investigated in vitro whether ATRA modulates the response of human hepatocellular carcinoma (HCC) cells to sorafenib, the only proven oral drug for advanced HCC, and the underlying mechanisms. HepG2 and SMMC-7721 cells were treated with sorafenib and/or ATRA, and cell proliferation and apoptosis were analyzed; the role of GJIC was also explored. We found that ATRA, at non-toxic concentrations, enhanced sorafenib-induced growth inhibition in both HCC cell lines, and this effect was abolished by two GJIC inhibitors, 18-α-GA and oleamide. Whereas lower concentrations of sorafenib (5 µM) or ATRA (0.1 or 10 µM) alone modestly induced GJIC activity, the combination of sorafenib plus ATRA resulted in a strong enhancement of GJIC. However, the action paradigm differed in the HepG2 and SMMC-7721 cells, with the dominant effect of GJIC dependent on the cell-specific connexin increase in protein amounts and relocalization. RT-PCR assay further revealed a transcriptional modification of the key structural connexin in the two cell lines. Thus, a connexin-dependent gap junction enhancement may play a central role in ATRA plus sorafenib synergy in inhibiting HCC cell growth. Since both agents are available for human use, the combination treatment represents a future profitable strategy for the treatment of advanced HCC.

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.

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.

Lightweight IMM PV Flexible Blanket Assembly

NASA Technical Reports Server (NTRS)

Spence, Brian

2015-01-01

Deployable Space Systems (DSS) has developed an inverted metamorphic multijunction (IMM) photovoltaic (PV) integrated modular blanket assembly (IMBA) that can be rolled or z-folded. This IMM PV IMBA technology enables a revolutionary flexible PV blanket assembly that provides high specific power, exceptional stowed packaging efficiency, and high-voltage operation capability. DSS's technology also accommodates standard third-generation triple junction (ZTJ) PV device technologies to provide significantly improved performance over the current state of the art. This SBIR project demonstrated prototype, flight-like IMM PV IMBA panel assemblies specifically developed, designed, and optimized for NASA's high-voltage solar array missions.

Investigation of the mechanism for penetration of low density lipoprotein into the arterial wall

NASA Astrophysics Data System (ADS)

Glukhova, O. E.; Zyktin, A. A.; Slepchenkov, M. M.

2018-02-01

Currently, the pathology of the cardiovascular system is an extremely urgent problem of fundamental and clinical medicine. These diseases are caused, mainly, by atherosclerotic changes in the wall of blood vessels. The predominant role in the development of atherosclerosis is attributed to the penetration of various kinds of lipoproteins into the arterial intima. In this paper, we in silico investigated the dynamics of the penetration of low density lipoprotein (LDL) through the intercellular gap using molecular modeling methods. The simulation was carried out in the GROMACS software package using a coarse-grained MARTINI model. During investigation we carried out the LDL self-assembly for the first time. The coarse-grained model of LDL was collected from the following molecules: POPC (phosphatidylcholine) - 630 molecules, LPC (lysophosphatidylcholine) - 80 molecules CHOL (cholesterol) - 600 molecules CHYO (cholesteryl oleate) - 1600 molecules TOG (glycerol trioleate) 180 Molecules. The coarse-grained model of the intercellular endothelial gap was based on a model of lipid bilayer consisting of DPPC phospholipids and cholesterol in a percentage ratio of 70% and 30%, respectively. Based on the obtained results, we can predict the mechanism of LDL diffusion. Lipoproteins can be deformed so as to pass through narrow gaps. Our investigations open the way for the research of the behavior dynamics of LDL moving with the blood flow rate when interacting with the intercellular gaps of the endothelial layer of the vessel inner wall.

Processing of the VP1/2A junction is not necessary for production of foot-and-mouth disease virus empty capsids and infectious viruses: characterization of "self-tagged" particles.

PubMed

Gullberg, Maria; Polacek, Charlotta; Bøtner, Anette; Belsham, Graham J

2013-11-01

The foot-and-mouth disease virus (FMDV) capsid protein precursor, P1-2A, is cleaved by 3C(pro) to generate VP0, VP3, VP1, and the peptide 2A. The capsid proteins self-assemble into empty capsid particles or viruses which do not contain 2A. In a cell culture-adapted strain of FMDV (O1 Manisa [Lindholm]), three different amino acid substitutions (E83K, S134C, and K210E) were identified within the VP1 region of the P1-2A precursor compared to the field strain (wild type [wt]). Expression of the O1 Manisa P1-2A (wt or with the S134C substitution in VP1) plus 3C(pro), using a transient expression system, resulted in efficient capsid protein production and self-assembly of empty capsid particles. Removal of the 2A peptide from the capsid protein precursor had no effect on capsid protein processing or particle assembly. However, modification of E83K alone abrogated particle assembly with no apparent effect on protein processing. Interestingly, the K210E substitution, close to the VP1/2A junction, completely blocked processing by 3C(pro) at this cleavage site, but efficient assembly of "self-tagged" empty capsid particles, containing the uncleaved VP1-2A, was observed. These self-tagged particles behaved like the unmodified empty capsids in antigen enzyme-linked immunosorbent assays and integrin receptor binding assays. Furthermore, mutant viruses with uncleaved VP1-2A could be rescued in cells from full-length FMDV RNA transcripts encoding the K210E substitution in VP1. Thus, cleavage of the VP1/2A junction is not essential for virus viability. The production of such engineered self-tagged empty capsid particles may facilitate their purification for use as diagnostic reagents and vaccines.

Ultrasonic wrench produces leaktight connections

NASA Technical Reports Server (NTRS)

Blaise, H. T.; Maropis, N.

1967-01-01

Ultrasonic wrench system produces leaktight seals in flared tubing connections. It induces a flexural vibration mode in the coupling nut. The system consists of a frequency converter, a junction box, and wrench assembly.

Polarity Proteins as Regulators of Cell Junction Complexes: Implications for Breast Cancer

PubMed Central

Bazzoun, Dana; Lelièvre, Sophie; Talhouk, Rabih

2013-01-01

The epithelium of multicellular organisms possesses a well-defined architecture, referred to as polarity that coordinates the regulation of essential cell features. Polarity proteins are intimately linked to the protein complexes that make the tight, adherens and gap junctions; they contribute to the proper localization and assembly of these cell-cell junctions within cells and consequently to functional tissue organization. The establishment of cell-cell junctions and polarity are both implicated in the regulation of epithelial modifications in normal and cancer situations. Uncovering the mechanisms through which cell-cell junctions and epithelial polarization are established and how their interaction with the microenvironment direct cell and tissue organization has opened new venues for the development of cancer therapies. In this review, we focus on the breast epithelium to highlight how polarity and cell-cell junction proteins interact together in normal and cancerous contexts to regulate major cellular mechanisms such as migration. The impact of these proteins on epigenetic mechanisms responsible for resetting cells towards oncogenesis is discussed in light of increasing evidence that tissue polarity modulates chromatin function. Finally, we give an overview of recent breast cancer therapies that target proteins involved in cell-cell junctions. PMID:23458609

Intercellular communications within the rat anterior pituitary. XVI: postnatal changes of distribution of S-100 protein positive cells, connexin 43 and LH-RH positive sites in the pars tuberalis of the rat pituitary gland. An immunohistochemical and electron microscopic study.

PubMed

Wada, Ikuo; Sakuma, Eisuke; Shirasawa, Nobuyuki; Wakabayashi, Kenjiro; Otsuka, Takanobu; Hattori, Kazuki; Yashiro, Takashi; Herbert, Damon C; Soji, Tsuyoshi

2014-02-01

The architecture of luteinizing hormone-releasing hormone (LH-RH) nerve ends and the S-100 protein containing folliculo-stellate cells forming gap junctions in the pars tuberalis is basically important in understanding the regulation of the hormone producing mechanism of anterior pituitary glands. In this study, intact male rats 5-60 days old were prepared for immunohistochemistry and electron microscopy. From immunostained sections, the S-100 containing cells in pars tuberalis were first detected on day 30 and increased in number to day 60; this was parallel to the immunohistochemical staining of gap junction protein, connexin 43. LH-RH positive sites were clearly observed on just behind the optic chiasm and on the root of pituitary stalk on day 30. On day 60, the width of layer increased, while follicles and gap junctions were frequently observed between agranular cells in 10 or more layers of pars tuberalis. In the present study, we investigated the sexual maturation of the anterior pituitary glands through the postnatal development of S-100 positive cells, connexin 43 and LH-RH nerves. It is suggested that the folliculo-stellate cell system including the LH-RH neurons in the pars tuberalis participates in the control of LH secretion along with the portal vein system. Copyright © 2013 Elsevier Ltd. All rights reserved.

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.

A high-grain diet alters the omasal epithelial structure and expression of tight junction proteins in a goat model.

PubMed

Liu, Jun-Hua; Xu, Ting-Ting; Zhu, Wei-Yun; Mao, Sheng-Yong

2014-07-01

The omasal epithelial barrier plays important roles in maintaining nutrient absorption and immune homeostasis in ruminants. However, little information is currently available about the changes in omasal epithelial barrier function at the structural and molecular levels during feeding of a high-grain (HG) diet. Ten male goats were randomly assigned to two groups, fed either a hay diet (0% grain; n = 5) or HG diet (65% grain; n = 5). Changes in omasal epithelial structure and expression of tight junction (TJ) proteins were determined via electron microscopy and Western blot analysis. After 7 weeks on each diet, omasal contents in the HG group showed significantly lower pH (P <0.001) and significantly higher concentrations of free lipopolysaccharides (LPS; P = 0.001) than the hay group. The goats fed a HG diet showed profound alterations in omasal epithelial structure and TJ proteins, corresponding to depression of thickness of total epithelia, stratum granulosum, and the sum of the stratum spinosum and stratum basale, marked epithelial cellular damage, erosion of intercellular junctions and down-regulation in expression of the TJ proteins, claudin-4 and occludin. The study demonstrates that feeding a HG diet is associated with omasal epithelial cellular damage and changes in expression of TJ proteins. These research findings provide an insight into the possible significance of diet on the omasal epithelial barrier in ruminants. Copyright © 2014 Elsevier Ltd. All rights reserved.

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.