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Sample records for adherens junction proteins

  1. Protein zero is necessary for E-cadherin-mediated adherens junction formation in Schwann cells.

    PubMed

    Menichella, D M; Arroyo, E J; Awatramani, R; Xu, T; Baron, P; Vallat, J M; Balsamo, J; Lilien, J; Scarlato, G; Kamholz, J; Scherer, S S; Shy, M E

    2001-12-01

    Protein Zero (P0), the major structural protein in the peripheral nervous system (PNS) myelin, acts as a homotypic adhesion molecule and is thought to mediate compaction of adjacent wraps of myelin membrane. E-Cadherin, a calcium-dependent adhesion molecule, is also expressed in myelinating Schwann cells in the PNS and is involved in forming adherens junctions between adjacent loops of membrane at the paranode. To determine the relationship, if any, between P0-mediated and cadherin-mediated adhesion during myelination, we investigated the expression of E-cadherin and its binding partner, beta-catenin, in sciatic nerve of mice lacking P0 (P0(-/-)). We find that in P0(-/-) peripheral myelin neither E-cadherin nor beta-catenin are localized to paranodes, but are instead found in small puncta throughout the Schwann cell. In addition, only occasional, often rudimentary, adherens junctions are formed. Analysis of E-cadherin and beta-catenin expression during nerve development demonstrates that E-cadherin and beta-catenin are localized to the paranodal region after the onset of myelin compaction. Interestingly, axoglial junction formation is normal in P0(-/-) nerve. Taken together, these data demonstrate that P0 is necessary for the formation of adherens junctions but not axoglial junctions in myelinating Schwann cells. PMID:11749037

  2. The protein tyrosine phosphatase Pez is a major phosphatase of adherens junctions and dephosphorylates beta-catenin.

    PubMed

    Wadham, Carol; Gamble, Jennifer R; Vadas, Mathew A; Khew-Goodall, Yeesim

    2003-06-01

    Cell-cell adhesion regulates processes important in embryonal development, normal physiology, and cancer progression. It is regulated by various mechanisms including tyrosine phosphorylation. We have previously shown that the protein tyrosine phosphatase Pez is concentrated at intercellular junctions in confluent, quiescent monolayers but is nuclear in cells lacking cell-cell contacts. We show here with an epithelial cell model that Pez localizes to the adherens junctions in confluent monolayers. A truncation mutant lacking the catalytic domain acts as a dominant negative mutant to upregulate tyrosine phosphorylation at adherens junctions. We identified beta-catenin, a component of adherens junctions, as a substrate of Pez by a "substrate trapping" approach and by in vitro dephosphorylation with recombinant Pez. Consistent with this, ectopic expression of the dominant negative mutant caused an increase in tyrosine phosphorylation of beta-catenin, demonstrating that Pez regulates the level of tyrosine phosphorylation of adherens junction proteins, including beta-catenin. Increased tyrosine phosphorylation of adherens junction proteins has been shown to decrease cell-cell adhesion, promoting cell migration as a result. Accordingly, the dominant negative Pez mutant enhanced cell motility in an in vitro "wound" assay. This suggests that Pez is also a regulator of cell motility, most likely through its action on cell-cell adhesion. PMID:12808048

  3. Adherens junction proteins are expressed in collagen corneal equivalents produced in vitro with human cells

    PubMed Central

    Deschambeault, Alexandre; Carrier, Patrick; Germain, Lucie

    2014-01-01

    Purpose To test whether adherens junction proteins are present in the epithelium and the endothelium of corneal equivalents. Methods Corneal cell types were harvested from human eyes and grown separately. Stromal equivalents were constructed by seeding fibroblasts into a collagen gel on which epithelial and endothelial cells were added on each side. Alternatively, bovine endothelial cells were used. At maturity, sections of stromal equivalents were processed for Masson's trichrome or indirect immunofluorescence using antibodies against pan-, N-, or E-cadherins or α- or β-catenins. Alternatively, stromal equivalents were dissected, to separate the proteins from the epithelium, endothelium, and stroma with sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Western blots of the transferred proteins exposed to these primary antibodies were detected with chemiluminescence. Native corneas were processed similarly. Results Three or four layers of epithelial cells reminiscent of the native cornea (basal cuboidal and superficial flatter cells) lay over a stromal construct containing fibroblastic cells under which an endothelium is present. Western blots and indirect immunofluorescence revealed that, similarly to the native cornea, the epithelium reacted positively to antibodies against catenins (α and β) and E-cadherin. The endothelium of corneal constructs, whether of human or bovine origin, reacted mildly to catenins and N-cadherin. Conclusions This collagen-based corneal equivalent simulated the native cornea. Cells from the epithelial and endothelial layers expressed adherens junction proteins, indicating the presence of cell–cell contacts and the existence of polarized morphology of these layers over corneal equivalents. PMID:24715756

  4. Reduction and Redistribution of Gap and Adherens Junction Proteins After Ischemia and Reperfusion

    PubMed Central

    Tansey, Erin E.; Kwaku, Kevin F.; Hammer, Peter E.; Cowan, Douglas B.; Federman, Micheline; Levitsky, Sidney; McCully, James D.

    2007-01-01

    Background Previous studies have demonstrated that alterations in myocardial structure, consistent with tissue and sarcomere disruption as well as myofibril dissociation, occur after myocardial ischemia and reperfusion. In this study we determine the onset of these structural changes and their contribution to electrical conduction. Methods Langendorff perfused rabbit hearts (n = 47) were subjected to 0, 5, 10, 15, 20, 25, and 30 minutes global ischemia, followed by 120 minutes reperfusion. Hemodynamics were recorded and tissue samples were collected for histochemical and immunohistochemical studies. Orthogonal epicardial conduction velocities were measured, with temperature controlled, in a separate group of 10 hearts subjected to 0 or 30 minutes of global ischemia, followed by 120 minutes of reperfusion. Results Histochemical and quantitative light microscopy spatial analysis showed significantly increased longitudinal and transverse interfibrillar separation after 15 minutes or more of ischemia (p < 0.05 versus control). Confocal immunohistochemistry and Western blot analysis demonstrated significant reductions (p < .05 versus control) of the intercellular adherens junction protein, N-cadherin, and the active phosphorylated isoform of the principal gap junction protein, connexin 43 at more than 15 minutes of ischemia. Cellular redistribution of connexin 43 was also evidenced on immunohistochemistry. No change in integrin-β1, an extracellular matrix attachment protein, or in epicardial conduction velocity anisotropy was observed. Conclusions These data indicate that there are significant alterations in the structural integrity of the myocardium as well as gap and adherens junction protein expression with increasing global ischemia time. The changes occur coincident with previously observed significant decreases in postischemic functional recovery, but are not associated with altered expression of matrix binding proteins or electrical anisotropic conduction. PMID

  5. A Cell Motility Screen Reveals Role for MARCKS-Related Protein in Adherens Junction Formation and Tumorigenesis

    PubMed Central

    Finlayson, Alexander E.; Freeman, Kevin W.

    2009-01-01

    Invasion through the extracellular matrix (ECM) is important for wound healing, immunological responses and metastasis. We established an invasion-based cell motility screen using Boyden chambers overlaid with Matrigel to select for pro-invasive genes. By this method we identified antisense to MARCKS related protein (MRP), whose family member MARCKS is a target of miR-21, a microRNA involved in tumor growth, invasion and metastasis in multiple human cancers. We confirmed that targeted knockdown of MRP, in both EpRas mammary epithelial cells and PC3 prostate cancer cells, promoted in vitro cell migration that was blocked by trifluoperazine. Additionally, we observed increased immunofluoresence of E-cadherin, β-catenin and APC at sites of cell-cell contact in EpRas cells with MRP knockdown suggesting formation of adherens junctions. By wound healing assay we observed that reduced MRP supported collective cell migration, a type of cell movement where adherens junctions are maintained. However, destabilized adherens junctions, like those seen in EpRas cells, are frequently important for oncogenic signaling. Consequently, knockdown of MRP in EpRas caused loss of tumorigenesis in vivo, and reduced Wnt3a induced TCF reporter signaling in vitro. Together our data suggest that reducing MRP expression promotes formation of adherens junctions in EpRas cells, allowing collective cell migration, but interferes with oncogenic β-catenin signaling and tumorigenesis. PMID:19924305

  6. Adherens junctional associated protein-1: a novel 1p36 tumor suppressor candidate in gliomas (Review).

    PubMed

    Zeng, Liang; Fee, Brian E; Rivas, Miriam V; Lin, James; Adamson, David Cory

    2014-07-01

    In a broad range of human cancers 1p36 has been a mutational hotspot which strongly suggests that the loss of tumor suppressor activity maps to this genomic region during tumorigenesis. Adherens junctional associated protein-1 (AJAP1; also known as Shrew1) was initially discovered as a novel transmembrane protein of adherent junctions in epithelial cells. Gene profiling showed AJAP1 on 1p36 is frequently lost or epigenetically silenced. AJAP1 may affect cell motility, migration, invasion and proliferation by unclear mechanisms. AJAP1 may be translocated to the nucleus, via its interaction with β-catenin complexes, where it can regulate gene transcription, then possibly have a potent impact on cell cycling and apoptosis. Significantly, loss of AJAP1 expression predicts poor clinical outcome of patients with malignant gliomas such as GBM and it may serve as a promising tumor suppressor-related target. In this review, we summarize and discuss current knowledge that may identify AJAP1 as a tumor suppressor in gliomas.

  7. Photoperiod-Dependent Effects of 4-tert-Octylphenol on Adherens and Gap Junction Proteins in Bank Vole Seminiferous Tubules

    PubMed Central

    Kuras, Paulina; Lydka-Zarzycka, Marta; Bilinska, Barbara

    2013-01-01

    In the present study we evaluated in vivo and in vitro effects of 4-tert-octylphenol (OP) on the expression and distribution of adherens and gap junction proteins, N-cadherin, β-catenin, and connexin 43 (Cx43), in testes of seasonally breeding rodents, bank voles. We found that in bank vole testes expression and distribution of N-cadherin, β-catenin, and Cx43 were photoperiod dependent. Long-term treatment with OP (200 mg/kg b.w.) resulted in the reduction of junction proteins expressions (P < 0.05, P < 0.01) and their delocalization in the testes of males kept in long photoperiod, whereas in short-day animals slight increase of Cx43 (P < 0.05), N-cadherin, and β-catenin (statistically nonsignificant) levels was observed. Effects of OP appeared to be independent of FSH and were maintained during in vitro organ culture, indicating that OP acts directly on adherens and gap junction proteins in the testes. An experiment performed using an antiestrogen ICI 182,780 demonstrated that the biological effects of OP on β-catenin and Cx43 involve an estrogen receptor-mediated response. Taken together, in bank vole organization of adherens and gap junctions and their susceptibility to OP are related to the length of photoperiod. Alterations in cadherin/catenin and Cx43-based junction may partially result from activation of estrogen receptor α and/or β signaling pathway. PMID:23737770

  8. FSGS3/CD2AP is a barbed-end capping protein that stabilizes actin and strengthens adherens junctions

    PubMed Central

    Brieher, William M.

    2013-01-01

    By combining in vitro reconstitution biochemistry with a cross-linking approach, we have identified focal segmental glomerulosclerosis 3/CD2-associated protein (FSGS3/CD2AP) as a novel actin barbed-end capping protein responsible for actin stability at the adherens junction. FSGS3/CD2AP colocalizes with E-cadherin and α-actinin-4 at the apical junction in polarized Madin-Darby canine kidney (MDCK) cells. Knockdown of FSGS3/CD2AP compromised actin stability and decreased actin accumulation at the adherens junction. Using a novel apparatus to apply mechanical stress to cell–cell junctions, we showed that knockdown of FSGS3/CD2AP compromised adhesive strength, resulting in tearing between cells and disruption of barrier function. Our results reveal a novel function of FSGS3/CD2AP and a previously unrecognized role of barbed-end capping in junctional actin dynamics. Our study underscores the complexity of actin regulation at cell–cell contacts that involves actin activators, inhibitors, and stabilizers to control adhesive strength, epithelial behavior, and permeability barrier integrity. PMID:24322428

  9. The F-BAR protein pacsin2 inhibits asymmetric VE-cadherin internalization from tensile adherens junctions

    PubMed Central

    Dorland, Yvonne L.; Malinova, Tsveta S.; van Stalborch, Anne-Marieke D.; Grieve, Adam G.; van Geemen, Daphne; Jansen, Nicolette S.; de Kreuk, Bart-Jan; Nawaz, Kalim; Kole, Jeroen; Geerts, Dirk; Musters, René J. P.; de Rooij, Johan; Hordijk, Peter L.; Huveneers, Stephan

    2016-01-01

    Vascular homoeostasis, development and disease critically depend on the regulation of endothelial cell–cell junctions. Here we uncover a new role for the F-BAR protein pacsin2 in the control of VE-cadherin-based endothelial adhesion. Pacsin2 concentrates at focal adherens junctions (FAJs) that are experiencing unbalanced actomyosin-based pulling. FAJs move in response to differences in local cytoskeletal geometry and pacsin2 is recruited consistently to the trailing end of fast-moving FAJs via a mechanism that requires an intact F-BAR domain. Photoconversion, photobleaching, immunofluorescence and super-resolution microscopy reveal polarized dynamics, and organization of junctional proteins between the front of FAJs and their trailing ends. Interestingly, pacsin2 recruitment inhibits internalization of the VE-cadherin complex from FAJ trailing ends and is important for endothelial monolayer integrity. Together, these findings reveal a novel junction protective mechanism during polarized trafficking of VE-cadherin, which supports barrier maintenance within dynamic endothelial tissue. PMID:27417273

  10. The F-BAR protein pacsin2 inhibits asymmetric VE-cadherin internalization from tensile adherens junctions.

    PubMed

    Dorland, Yvonne L; Malinova, Tsveta S; van Stalborch, Anne-Marieke D; Grieve, Adam G; van Geemen, Daphne; Jansen, Nicolette S; de Kreuk, Bart-Jan; Nawaz, Kalim; Kole, Jeroen; Geerts, Dirk; Musters, René J P; de Rooij, Johan; Hordijk, Peter L; Huveneers, Stephan

    2016-01-01

    Vascular homoeostasis, development and disease critically depend on the regulation of endothelial cell-cell junctions. Here we uncover a new role for the F-BAR protein pacsin2 in the control of VE-cadherin-based endothelial adhesion. Pacsin2 concentrates at focal adherens junctions (FAJs) that are experiencing unbalanced actomyosin-based pulling. FAJs move in response to differences in local cytoskeletal geometry and pacsin2 is recruited consistently to the trailing end of fast-moving FAJs via a mechanism that requires an intact F-BAR domain. Photoconversion, photobleaching, immunofluorescence and super-resolution microscopy reveal polarized dynamics, and organization of junctional proteins between the front of FAJs and their trailing ends. Interestingly, pacsin2 recruitment inhibits internalization of the VE-cadherin complex from FAJ trailing ends and is important for endothelial monolayer integrity. Together, these findings reveal a novel junction protective mechanism during polarized trafficking of VE-cadherin, which supports barrier maintenance within dynamic endothelial tissue. PMID:27417273

  11. The F-BAR protein pacsin2 inhibits asymmetric VE-cadherin internalization from tensile adherens junctions.

    PubMed

    Dorland, Yvonne L; Malinova, Tsveta S; van Stalborch, Anne-Marieke D; Grieve, Adam G; van Geemen, Daphne; Jansen, Nicolette S; de Kreuk, Bart-Jan; Nawaz, Kalim; Kole, Jeroen; Geerts, Dirk; Musters, René J P; de Rooij, Johan; Hordijk, Peter L; Huveneers, Stephan

    2016-01-01

    Vascular homoeostasis, development and disease critically depend on the regulation of endothelial cell-cell junctions. Here we uncover a new role for the F-BAR protein pacsin2 in the control of VE-cadherin-based endothelial adhesion. Pacsin2 concentrates at focal adherens junctions (FAJs) that are experiencing unbalanced actomyosin-based pulling. FAJs move in response to differences in local cytoskeletal geometry and pacsin2 is recruited consistently to the trailing end of fast-moving FAJs via a mechanism that requires an intact F-BAR domain. Photoconversion, photobleaching, immunofluorescence and super-resolution microscopy reveal polarized dynamics, and organization of junctional proteins between the front of FAJs and their trailing ends. Interestingly, pacsin2 recruitment inhibits internalization of the VE-cadherin complex from FAJ trailing ends and is important for endothelial monolayer integrity. Together, these findings reveal a novel junction protective mechanism during polarized trafficking of VE-cadherin, which supports barrier maintenance within dynamic endothelial tissue.

  12. Brain barriers: Crosstalk between complex tight junctions and adherens junctions

    PubMed Central

    Tietz, Silvia

    2015-01-01

    Unique intercellular junctional complexes between the central nervous system (CNS) microvascular endothelial cells and the choroid plexus epithelial cells form the endothelial blood–brain barrier (BBB) and the epithelial blood–cerebrospinal fluid barrier (BCSFB), respectively. These barriers inhibit paracellular diffusion, thereby protecting the CNS from fluctuations in the blood. Studies of brain barrier integrity during development, normal physiology, and disease have focused on BBB and BCSFB tight junctions but not the corresponding endothelial and epithelial adherens junctions. The crosstalk between adherens junctions and tight junctions in maintaining barrier integrity is an understudied area that may represent a promising target for influencing brain barrier function. PMID:26008742

  13. Dynamic features of adherens junctions during Drosophila embryonic epithelial morphogenesis revealed by a Dalpha-catenin-GFP fusion protein.

    PubMed

    Oda, H; Tsukita, S

    1999-04-01

    Cell-cell adherens junctions (AJs), comprised of the cadherin-catenin adhesion system, contribute to cell shape changes and cell movements in epithelial morphogenesis. However, little is known about the dynamic features of AJs in cells of the developing embryo. In this study, we constructed Dalpha-catenin fused with a green fluorescent protein (Dalpha-catenin-GFP), and found that it targeted apically located AJ-based contacts but not other lateral contacts in epithelial cells of living Drosophila embryos. Using time-lapse fluorescence microscopy, we examined the dynamic performance of AJs containing Dalpha-catenin-GFP in epithelial morphogenetic movements. In the ventral ectoderm of stage 11 embryos, concentration and deconcentration of Dalpha-catenin-GFP occurred concomitantly with changes in length of AJ contacts. In the lateral ectoderm of embryos at the same stage, dynamic behaviour of AJs was concerted with division and delamination of sensory organ precursor (SOP) cells. Moreover, changes in patterns of AJ networks during tracheal extension could be followed. Finally, we utilized Dalpha-catenin-GFP to precisely observe the defects in tracheal fusion in shotgun mutants. Thus, the Dalpha-catenin-GFP fusion protein is a helpful tool to simultaneously observe morphogenetic movements and AJ dynamics at high spatio-temporal resolution.

  14. 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

  15. Alpha-catenin-Dependent Recruitment of the Centrosomal Protein CAP350 to Adherens Junctions Allows Epithelial Cells to Acquire a Columnar Shape

    PubMed Central

    Zurbano, Angel; Formstecher, Etienne; Martinez-Morales, Juan R.; Bornens, Michel; Rios, Rosa M.

    2015-01-01

    Epithelial morphogenesis involves a dramatic reorganisation of the microtubule cytoskeleton. How this complex process is controlled at the molecular level is still largely unknown. Here, we report that the centrosomal microtubule (MT)-binding protein CAP350 localises at adherens junctions in epithelial cells. By two-hybrid screening, we identified a direct interaction of CAP350 with the adhesion protein α-catenin that was further confirmed by co-immunoprecipitation experiments. Block of epithelial cadherin (E-cadherin)-mediated cell-cell adhesion or α-catenin depletion prevented CAP350 localisation at cell-cell junctions. Knocking down junction-located CAP350 inhibited the establishment of an apico-basal array of microtubules and impaired the acquisition of columnar shape in Madin-Darby canine kidney II (MDCKII) cells grown as polarised epithelia. Furthermore, MDCKII cystogenesis was also defective in junctional CAP350-depleted cells. CAP350-depleted MDCKII cysts were smaller and contained either multiple lumens or no lumen. Membrane polarity was not affected, but cortical microtubule bundles did not properly form. Our results indicate that CAP350 may act as an adaptor between adherens junctions and microtubules, thus regulating epithelial differentiation and contributing to the definition of cell architecture. We also uncover a central role of α-catenin in global cytoskeleton remodelling, in which it acts not only on actin but also on MT reorganisation during epithelial morphogenesis. PMID:25764135

  16. Dynamics of adherens junctions in epithelial establishment, maintenance, and remodeling

    PubMed Central

    Baum, Buzz

    2011-01-01

    The epithelial cadherin (E-cadherin)–catenin complex binds to cytoskeletal components and regulatory and signaling molecules to form a mature adherens junction (AJ). This dynamic structure physically connects neighboring epithelial cells, couples intercellular adhesive contacts to the cytoskeleton, and helps define each cell’s apical–basal axis. Together these activities coordinate the form, polarity, and function of all cells in an epithelium. Several molecules regulate AJ formation and integrity, including Rho family GTPases and Par polarity proteins. However, only recently, with the development of live-cell imaging, has the extent to which E-cadherin is actively turned over at junctions begun to be appreciated. This turnover contributes to junction formation and to the maintenance of epithelial integrity during tissue homeostasis and remodeling. PMID:21422226

  17. Folliculin, the product of the Birt-Hogg-Dube tumor suppressor gene, interacts with the adherens junction protein p0071 to regulate cell-cell adhesion.

    PubMed

    Medvetz, Doug A; Khabibullin, Damir; Hariharan, Venkatesh; Ongusaha, Pat P; Goncharova, Elena A; Schlechter, Tanja; Darling, Thomas N; Hofmann, Ilse; Krymskaya, Vera P; Liao, James K; Huang, Hayden; Henske, Elizabeth P

    2012-01-01

    Birt-Hogg-Dube (BHD) is a tumor suppressor gene syndrome associated with fibrofolliculomas, cystic lung disease, and chromophobe renal cell carcinoma. In seeking to elucidate the pathogenesis of BHD, we discovered a physical interaction between folliculin (FLCN), the protein product of the BHD gene, and p0071, an armadillo repeat containing protein that localizes to the cytoplasm and to adherens junctions. Adherens junctions are one of the three cell-cell junctions that are essential to the establishment and maintenance of the cellular architecture of all epithelial tissues. Surprisingly, we found that downregulation of FLCN leads to increased cell-cell adhesion in functional cell-based assays and disruption of cell polarity in a three-dimensional lumen-forming assay, both of which are phenocopied by downregulation of p0071. These data indicate that the FLCN-p0071 protein complex is a negative regulator of cell-cell adhesion. We also found that FLCN positively regulates RhoA activity and Rho-associated kinase activity, consistent with the only known function of p0071. Finally, to examine the role of Flcn loss on cell-cell adhesion in vivo, we utilized keratin-14 cre-recombinase (K14-cre) to inactivate Flcn in the mouse epidermis. The K14-Cre-Bhd(flox/flox) mice have striking delays in eyelid opening, wavy fur, hair loss, and epidermal hyperplasia with increased levels of mammalian target of rapamycin complex 1 (mTORC1) activity. These data support a model in which dysregulation of the FLCN-p0071 interaction leads to alterations in cell adhesion, cell polarity, and RhoA signaling, with broad implications for the role of cell-cell adhesion molecules in the pathogenesis of human disease, including emphysema and renal cell carcinoma. PMID:23139756

  18. 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

  19. Flow mechanotransduction regulates traction forces, intercellular forces, and adherens junctions

    PubMed Central

    Ting, Lucas H.; Jahn, Jessica R.; Jung, Joon I.; Shuman, Benjamin R.; Feghhi, Shirin; Han, Sangyoon J.; Rodriguez, Marita L.

    2012-01-01

    Endothelial cells respond to fluid shear stress through mechanotransduction responses that affect their cytoskeleton and cell-cell contacts. Here, endothelial cells were grown as monolayers on arrays of microposts and exposed to laminar or disturbed flow to examine the relationship among traction forces, intercellular forces, and cell-cell junctions. Cells under laminar flow had traction forces that were higher than those under static conditions, whereas cells under disturbed flow had lower traction forces. The response in adhesion junction assembly matched closely with changes in traction forces since adherens junctions were larger in size for laminar flow and smaller for disturbed flow. Treating the cells with calyculin-A to increase myosin phosphorylation and traction forces caused an increase in adherens junction size, whereas Y-27362 cause a decrease in their size. Since tugging forces across cell-cell junctions can promote junctional assembly, we developed a novel approach to measure intercellular forces and found that these forces were higher for laminar flow than for static or disturbed flow. The size of adherens junctions and tight junctions matched closely with intercellular forces for these flow conditions. These results indicate that laminar flow can increase cytoskeletal tension while disturbed flow decreases cytoskeletal tension. Consequently, we found that changes in cytoskeletal tension in response to shear flow conditions can affect intercellular tension, which in turn regulates the assembly of cell-cell junctions. PMID:22447948

  20. The Drosophila genes crumbs and stardust are involved in the biogenesis of adherens junctions.

    PubMed

    Grawe, F; Wodarz, A; Lee, B; Knust, E; Skaer, H

    1996-03-01

    Morphogenetic movements of epithelia during development underlie the normal elaboration of the final body plan. The tissue integrity critical for these movements is conferred by anchorage of the cytoskeleton by adherens junctions, initially spot and later belt-like, zonular structures, which encircle the apical side of the cell. Loss-of-function mutations in the Drosophila genes crumbs and stardust lead to the loss of cell polarity in most ectodermally derived epithelia, followed in some, such as the epidermis, by extensive apoptosis. Here we show that both mutants fail to establish proper zonulae adherentes in the epidermis. Our results suggest that the two genes are involved in different aspects of this process. Further, they are compatible with the hypothesis that crumbs delimits the apical border, where the zonula adherens usually forms and where Crumbs protein is normally most abundant. In contrast, stardust seems to be required at an earlier stage for the assembly of the spot adherence junctions. In both mutants, the defect observed at the ultrastructural level are preceded by a misdistribution of Armadillo and DE-cadherin, the homologues of beta-catenin and E-cadherin, respectively, which are two constituents of the vertebrate adherens junctions. Strikingly, expansion of the apical membrane domain in epidermal cells by overexpression of crumbs also abolishes the formation of adherens junctions and results in the disruption of tissue integrity, but without loss of membrane polarity. This result supports the view that membrane polarity is independent of the formation of adherens junctions in epidermal cells.

  1. CCL2 disrupts the adherens junction: implications for neuroinflammation.

    PubMed

    Roberts, Toni K; Eugenin, Eliseo A; Lopez, Lillie; Romero, Ignacio A; Weksler, Babette B; Couraud, Pierre-Olivier; Berman, Joan W

    2012-08-01

    Alterations to blood-brain barrier (BBB) adhesion molecules and junctional integrity during neuroinflammation can promote central nervous system (CNS) pathology. The chemokine CCL2 is elevated during CNS inflammation and is associated with endothelial dysfunction. The effects of CCL2 on endothelial adherens junctions (AJs) have not been defined. We demonstrate that CCL2 transiently induces Src-dependent disruption of human brain microvascular endothelial AJ. β-Catenin is phosphorylated and traffics from the AJ to PECAM-1 (platelet endothelial cell adhesion molecule-1), where it is sequestered at the membrane. PECAM-1 is also tyrosine-phosphorylated, an event associated with recruitment of the phosphatase SHP-2 (Src homology 2 domain-containing protein phosphatase) to PECAM-1, β-catenin release from PECAM-1, and reassociation of β-catenin with the AJ. Surface localization of PECAM-1 is increased in response to CCL2. This may enable the endothelium to sustain CCL2-induced alterations in AJ and facilitate recruitment of leukocytes into the CNS. Our novel findings provide a mechanism for CCL2-mediated disruption of endothelial junctions that may contribute to BBB dysfunction and increased leukocyte recruitment in neuroinflammatory diseases. PMID:22641100

  2. Calcium-induced assembly of adherens junctions in keratinocytes

    PubMed Central

    1987-01-01

    Extracellular calcium concentration has been shown to control the stratification of cultured keratinocytes, presumably by regulation of formation of desmosomes. Previous studies have shown that keratinocytes cultured in medium containing 0.1 mM Ca++ form loose colonies without desmosomes. If the Ca++ is raised to 1 mM, desmosomes are assembled and the distribution of keratin filaments is altered. We have examined the disposition of vinculin and actin in keratinocytes under similar conditions. Using immunofluorescence microscopy we show that raising [Ca++] in the medium dramatically alters the distribution of vinculin and actin and results in the formation of adherens-type junctions within 15 min after switching to high calcium medium. Borders of cells at the edge of colonies, which are not proximal to other cells, are not affected, while cells in the interior of the colony form junctions around their periphery. Attachment plaques in keratinocytes grown in low calcium medium are located at the ventral plane of the cell, but junctions formed after switching to high calcium are not, as demonstrated by interference reflection microscopy. In cells colabeled with antibodies against vinculin and desmoplakin, vinculin-containing adherens junctions were visible before desmosomal junctions when cells were switched to high calcium. Although newly formed vinculin- containing structures in high calcium cells, like desmosomes, colocalize with phase-dense structures, superimposition of video fluorescence images using digitized fluorescence microscopy indicates that adherens junctions and desmosomes are discrete structures. Adherens junctions, like desmosomes, may play an essential role in controlling stratification of keratinocytes. PMID:2442175

  3. The adherens junction-associated protein 1 is a negative transcriptional regulator of MAGEA2, which potentiates temozolomide-induced apoptosis in GBM.

    PubMed

    Zeng, Liang; Kang, Chunsheng; Di, Chunhui; Fee, Brian E; Rivas, Miriam; Lin, James; Adamson, David Cory

    2014-04-01

    Previous studies identified the frequent loss of adherens junction-associated protein 1 (AJAP1) expression in glioblastoma (GBM) and its correlation with worse survival. AJAP1 may suppress glioma cell migration, which plays an important role in tumor progression in malignant gliomas such as GBM. However, the role of AJAP1 in cell cycle arrest or apoptosis and resistance to chemotherapy remains unclear. Based on microarray screening results, quantitative PCR and luciferase plasmid reporter constructs were used to evaluate the possible regulatory role of AJAP1 on MAGEA2 expression and function. Cell death assays, TUNEL and other markers of apoptosis were utilized to detect cell apoptosis. Restoration of AJAP1 expression in glioma cells was analyzed after temozolomide exposure. AJAP1 suppressed the expression of MAGEA2 and inhibited the transcriptional activity of MAGEA2 in glioma cells. As AJAP1 expression decreased MAGEA2 protein expression apoptosis increased moderately. Consistent with increased cell death, the induced loss of MAGEA2 expression correlated with increased caspase 3/7 activity, BCL2/BAX ratio and TUNEL signal. AJAP1 expression enhanced cell death in the presence of temozolomide. This study suggests AJAP1 may also function as a pro-apoptotic factor and potentiate cell death by temozolomide in glioma cells. This effect may be partially explained by AJAP1-mediated gene regulation of MAGEA2. PMID:24481586

  4. The mechanotransduction machinery at work at adherens junctions

    PubMed Central

    Ladoux, B.; Nelson, W. J.; Yan, J.; Mège, R.M.

    2015-01-01

    The shaping of a multicellular body, and the maintenance and repair of adult tissues require fine-tuning of cell adhesion responses and the transmission of mechanical load between the cell, its neighbors and the underlying extracellular matrix. A growing field of research is focused on how single cells sense mechanical properties of their micro-environment (extracellular matrix, other cells), and on how mechanotranduction pathways affect cell shape, migration, survival as well as differentiation. Within multicellular assemblies, the mechanical load imposed by the physical properties of the environment is transmitted to neighboring cells. Force imbalance at cell-cell contacts induces essential morphogenetic processes such as cell-cell junction remodeling, cell polarization and migration, cell extrusion and cell intercalation. However, how cells respond and adapt to the mechanical properties of neighboring cells, transmit forces, and transform mechanical signals into chemical signals remain open questions. A defining feature of compact tissues is adhesion between cells at the specialized Adherens Junction (AJ) involving the cadherin super-family of Ca2+-dependent cell-cell adhesion proteins (e.g., E-cadherin in epithelia). Cadherins bind to the cytoplasmic protein β-catenin, which in turn binds to the filamentous (F)-actin binding adaptor protein α-catenin, which can also recruit vinculin, making the mechanical connection between cell-cell adhesion proteins and the contractile actomyosin cytoskeleton. The cadherin-catenin adhesion complex is a key component of the AJ, and contributes to cell assembly stability and dynamic cell movements. It has also emerged as the main route of propagation of forces within epithelial and non-epithelial tissues. Here, we discuss recent molecular studies that point toward force-dependent conformational changes in α-catenin that regulate protein interactions in the cadherin-catenin adhesion complex, and show that α-catenin is the

  5. The mechanotransduction machinery at work at adherens junctions.

    PubMed

    Ladoux, B; Nelson, W J; Yan, J; Mège, R M

    2015-10-01

    The shaping of a multicellular body, and the maintenance and repair of adult tissues require fine-tuning of cell adhesion responses and the transmission of mechanical load between the cell, its neighbors and the underlying extracellular matrix. A growing field of research is focused on how single cells sense mechanical properties of their micro-environment (extracellular matrix, other cells), and on how mechanotransduction pathways affect cell shape, migration, survival as well as differentiation. Within multicellular assemblies, the mechanical load imposed by the physical properties of the environment is transmitted to neighboring cells. Force imbalance at cell-cell contacts induces essential morphogenetic processes such as cell-cell junction remodeling, cell polarization and migration, cell extrusion and cell intercalation. However, how cells respond and adapt to the mechanical properties of neighboring cells, transmit forces, and transform mechanical signals into chemical signals remain open questions. A defining feature of compact tissues is adhesion between cells at the specialized adherens junction (AJ) involving the cadherin super-family of Ca(2+)-dependent cell-cell adhesion proteins (e.g., E-cadherin in epithelia). Cadherins bind to the cytoplasmic protein β-catenin, which in turn binds to the filamentous (F)-actin binding adaptor protein α-catenin, which can also recruit vinculin, making the mechanical connection between cell-cell adhesion proteins and the contractile actomyosin cytoskeleton. The cadherin-catenin adhesion complex is a key component of the AJ, and contributes to cell assembly stability and dynamic cell movements. It has also emerged as the main route of propagation of forces within epithelial and non-epithelial tissues. Here, we discuss recent molecular studies that point toward force-dependent conformational changes in α-catenin that regulate protein interactions in the cadherin-catenin adhesion complex, and show that α-catenin is the

  6. The adherens junctions control susceptibility to Staphylococcus aureus α-toxin.

    PubMed

    Popov, Lauren M; Marceau, Caleb D; Starkl, Philipp M; Lumb, Jennifer H; Shah, Jimit; Guerrera, Diego; Cooper, Rachel L; Merakou, Christina; Bouley, Donna M; Meng, Wenxiang; Kiyonari, Hiroshi; Takeichi, Masatoshi; Galli, Stephen J; Bagnoli, Fabio; Citi, Sandra; Carette, Jan E; Amieva, Manuel R

    2015-11-17

    Staphylococcus aureus is both a transient skin colonizer and a formidable human pathogen, ranking among the leading causes of skin and soft tissue infections as well as severe pneumonia. The secreted bacterial α-toxin is essential for S. aureus virulence in these epithelial diseases. To discover host cellular factors required for α-toxin cytotoxicity, we conducted a genetic screen using mutagenized haploid human cells. Our screen identified a cytoplasmic member of the adherens junctions, plekstrin-homology domain containing protein 7 (PLEKHA7), as the second most significantly enriched gene after the known α-toxin receptor, a disintegrin and metalloprotease 10 (ADAM10). Here we report a new, unexpected role for PLEKHA7 and several components of cellular adherens junctions in controlling susceptibility to S. aureus α-toxin. We find that despite being injured by α-toxin pore formation, PLEKHA7 knockout cells recover after intoxication. By infecting PLEKHA7(-/-) mice with methicillin-resistant S. aureus USA300 LAC strain, we demonstrate that this junctional protein controls disease severity in both skin infection and lethal S. aureus pneumonia. Our results suggest that adherens junctions actively control cellular responses to a potent pore-forming bacterial toxin and identify PLEKHA7 as a potential nonessential host target to reduce S. aureus virulence during epithelial infections.

  7. The adherens junctions control susceptibility to Staphylococcus aureus α-toxin

    PubMed Central

    Popov, Lauren M.; Marceau, Caleb D.; Starkl, Philipp M.; Lumb, Jennifer H.; Shah, Jimit; Guerrera, Diego; Cooper, Rachel L.; Merakou, Christina; Bouley, Donna M.; Meng, Wenxiang; Kiyonari, Hiroshi; Takeichi, Masatoshi; Galli, Stephen J.; Bagnoli, Fabio; Citi, Sandra; Carette, Jan E.; Amieva, Manuel R.

    2015-01-01

    Staphylococcus aureus is both a transient skin colonizer and a formidable human pathogen, ranking among the leading causes of skin and soft tissue infections as well as severe pneumonia. The secreted bacterial α-toxin is essential for S. aureus virulence in these epithelial diseases. To discover host cellular factors required for α-toxin cytotoxicity, we conducted a genetic screen using mutagenized haploid human cells. Our screen identified a cytoplasmic member of the adherens junctions, plekstrin-homology domain containing protein 7 (PLEKHA7), as the second most significantly enriched gene after the known α-toxin receptor, a disintegrin and metalloprotease 10 (ADAM10). Here we report a new, unexpected role for PLEKHA7 and several components of cellular adherens junctions in controlling susceptibility to S. aureus α-toxin. We find that despite being injured by α-toxin pore formation, PLEKHA7 knockout cells recover after intoxication. By infecting PLEKHA7−/− mice with methicillin-resistant S. aureus USA300 LAC strain, we demonstrate that this junctional protein controls disease severity in both skin infection and lethal S. aureus pneumonia. Our results suggest that adherens junctions actively control cellular responses to a potent pore-forming bacterial toxin and identify PLEKHA7 as a potential nonessential host target to reduce S. aureus virulence during epithelial infections. PMID:26489655

  8. Cadherin controls nectin recruitment into adherens junctions by remodeling the actin cytoskeleton

    PubMed Central

    Troyanovsky, Regina B.; Indra, Indrajyoti; Chen, Chi-Shuo; Hong, Soonjin; Troyanovsky, Sergey M.

    2015-01-01

    ABSTRACT The mechanism that coordinates activities of different adhesion receptors is poorly understood. We investigated this mechanism by focusing on the nectin-2 and E-cadherin adherens junction receptors. We found that, cadherin was not required for the basic process of nectin junction formation because nectin-2 formed junctions in cadherin-deficient A431D cells. Formation of nectin-2 junctions in these cells, however, became regulated by cadherin as soon as E-cadherin was re-expressed. E-cadherin recruited nectin-2 into adherens junctions, where both proteins formed distinct but tightly associated clusters. Live-cell imaging showed that the appearance of E-cadherin clusters often preceded that of nectin-2 clusters at sites of junction assembly. Inactivation of E-cadherin clustering by different strategies concomitantly suppressed the formation of nectin clusters. Furthermore, cadherin significantly increased the stability of nectin clusters, thereby making them resistant to the BC-12 antibody, which targets the nectin-2 adhesion interface. By testing different E-cadherin–α-catenin chimeras, we showed that the recruitment of nectin into chimera junctions is mediated by the actin-binding domain of α-catenin. Our data suggests that E-cadherin regulates assembly of nectin junctions through α-catenin-induced remodeling of the actin cytoskeleton around the cadherin clusters. PMID:25395582

  9. Plakophilin 3 mediates Rap1-dependent desmosome assembly and adherens junction maturation

    PubMed Central

    Todorovic´, Viktor; Koetsier, Jennifer L.; Godsel, Lisa M.; Green, Kathleen J.

    2014-01-01

    The pathways driving desmosome and adherens junction assembly are temporally and spatially coordinated, but how they are functionally coupled is poorly understood. Here we show that the Armadillo protein plakophilin 3 (Pkp3) mediates both desmosome assembly and E-cadherin maturation through Rap1 GTPase, thus functioning in a manner distinct from the closely related plakophilin 2 (Pkp2). Whereas Pkp2 and Pkp3 share the ability to mediate the initial phase of desmoplakin (DP) accumulation at sites of cell–cell contact, they play distinct roles in later steps: Pkp3 is required for assembly of a cytoplasmic population of DP-enriched junction precursors, whereas Pkp2 is required for transfer of the precursors to the membrane. Moreover, Pkp3 forms a complex with Rap1 GTPase, promoting its activation and facilitating desmosome assembly. We show further that Pkp3 deficiency causes disruption of an E-cadherin/Rap1 complex required for adherens junction sealing. These findings reveal Pkp3 as a coordinator of desmosome and adherens junction assembly and maturation through its functional association with Rap1. PMID:25208567

  10. An Autoinhibited Structure of α-Catenin and Its Implications for Vinculin Recruitment to Adherens Junctions*

    PubMed Central

    Ishiyama, Noboru; Tanaka, Nobutoshi; Abe, Kentaro; Yang, Yoo Jeong; Abbas, Yazan M.; Umitsu, Masataka; Nagar, Bhushan; Bueler, Stephanie A.; Rubinstein, John L.; Takeichi, Masatoshi; Ikura, Mitsuhiko

    2013-01-01

    α-Catenin is an actin- and vinculin-binding protein that regulates cell-cell adhesion by interacting with cadherin adhesion receptors through β-catenin, but the mechanisms by which it anchors the cadherin-catenin complex to the actin cytoskeleton at adherens junctions remain unclear. Here we determined crystal structures of αE-catenin in the autoinhibited state and the actin-binding domain of αN-catenin. Together with the small-angle x-ray scattering analysis of full-length αN-catenin, we deduced an elongated multidomain assembly of monomeric α-catenin that structurally and functionally couples the vinculin- and actin-binding mechanisms. Cellular and biochemical studies of αE- and αN-catenins show that αE-catenin recruits vinculin to adherens junctions more effectively than αN-catenin, partly because of its higher affinity for actin filaments. We propose a molecular switch mechanism involving multistate conformational changes of α-catenin. This would be driven by actomyosin-generated tension to dynamically regulate the vinculin-assisted linkage between adherens junctions and the actin cytoskeleton. PMID:23589308

  11. Viscoelastic response of fibroblasts to tension transmitted through adherens junctions.

    PubMed Central

    Ragsdale, G K; Phelps, J; Luby-Phelps, K

    1997-01-01

    Cytoplasmic deformation was monitored by observing the displacements of 200-nm green fluorescent beads microinjected into the cytoplasm of Swiss 3T3 fibroblasts. We noted a novel protrusion of nonruffling cell margins that was accompanied by axial flow of beads and cytoplasmic vesicles as far as 50 microm behind the protruding plasma membrane. Fluorescent analog cytochemistry and immunofluorescence localization of F-actin, alpha-actinin, N-cadherin, and beta-catenin showed that the protruding margins of deforming cells were mechanically coupled to neighboring cells by adherens junctions. Observations suggested that protrusion resulted from passive linear deformation in response to tensile stress exerted by centripetal contraction of the neighboring cell. The time dependence of cytoplasmic strain calculated from the displacements of beads and vesicles was fit quantitatively by a Kelvin-Voight model for a viscoelastic solid with a mean limiting strain of 0.58 and a mean strain rate of 4.3 x 10(-3) s(-1). In rare instances, the deforming cell and its neighbor spontaneously became uncoupled, and recoil of the protruding margin was observed. The time dependence of strain during recoil also fit a Kelvin-Voight model with similar parameters, suggesting that the kinetics of deformation primarily reflect the mechanical properties of the deformed cell rather than the contractile properties of its neighbor. The existence of mechanical coupling between adjacent fibroblasts through adherens junctions and the viscoelastic responses of cells to tension transmitted directly from cell to cell are factors that must be taken into account to fully understand the role of fibroblasts in such biological processes as wound closure and extracellular matrix remodeling during tissue development. Images FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 PMID:9370474

  12. Interlocked positive and negative feedback network motifs regulate β-catenin activity in the adherens junction pathway

    PubMed Central

    Klinke, David J.; Horvath, Nicholas; Cuppett, Vanessa; Wu, Yueting; Deng, Wentao; Kanj, Rania

    2015-01-01

    The integrity of epithelial tissue architecture is maintained through adherens junctions that are created through extracellular homotypic protein–protein interactions between cadherin molecules. Cadherins also provide an intracellular scaffold for the formation of a multiprotein complex that contains signaling proteins, including β-catenin. Environmental factors and controlled tissue reorganization disrupt adherens junctions by cleaving the extracellular binding domain and initiating a series of transcriptional events that aim to restore tissue homeostasis. However, it remains unclear how alterations in cell adhesion coordinate transcriptional events, including those mediated by β-catenin in this pathway. Here were used quantitative single-cell and population-level in vitro assays to quantify the endogenous pathway dynamics after the proteolytic disruption of the adherens junctions. Using prior knowledge of isolated elements of the overall network, we interpreted these data using in silico model-based inference to identify the topology of the regulatory network. Collectively the data suggest that the regulatory network contains interlocked network motifs consisting of a positive feedback loop, which is used to restore the integrity of adherens junctions, and a negative feedback loop, which is used to limit β-catenin–induced gene expression. PMID:26224311

  13. Myosin-dependent remodeling of adherens junctions protects junctions from Snail-dependent disassembly

    PubMed Central

    Weng, Mo

    2016-01-01

    Although Snail is essential for disassembly of adherens junctions during epithelial–mesenchymal transitions (EMTs), loss of adherens junctions in Drosophila melanogaster gastrula is delayed until mesoderm is internalized, despite the early expression of Snail in that primordium. By combining live imaging and quantitative image analysis, we track the behavior of E-cadherin–rich junction clusters, demonstrating that in the early stages of gastrulation most subapical clusters in mesoderm not only persist, but move apically and enhance in density and total intensity. All three phenomena depend on myosin II and are temporally correlated with the pulses of actomyosin accumulation that drive initial cell shape changes during gastrulation. When contractile myosin is absent, the normal Snail expression in mesoderm, or ectopic Snail expression in ectoderm, is sufficient to drive early disassembly of junctions. In both cases, junctional disassembly can be blocked by simultaneous induction of myosin contractility. Our findings provide in vivo evidence for mechanosensitivity of cell–cell junctions and imply that myosin-mediated tension can prevent Snail-driven EMT. PMID:26754645

  14. Carcinoembryonic antigen promotes colorectal cancer progression by targeting adherens junction complexes

    SciTech Connect

    Bajenova, Olga; Chaika, Nina; Tolkunova, Elena; Davydov-Sinitsyn, Alexander; Gapon, Svetlana; Thomas, Peter; O’Brien, Stephen

    2014-06-10

    Oncomarkers play important roles in the detection and management of human malignancies. Carcinoembryonic antigen (CEA, CEACAM5) and epithelial cadherin (E-cadherin) are considered as independent tumor markers in monitoring metastatic colorectal cancer. They are both expressed by cancer cells and can be detected in the blood serum. We investigated the effect of CEA production by MIP101 colorectal carcinoma cell lines on E-cadherin adherens junction (AJ) protein complexes. No direct interaction between E-cadherin and CEA was detected; however, the functional relationships between E-cadherin and its AJ partners: α-, β- and p120 catenins were impaired. We discovered a novel interaction between CEA and beta-catenin protein in the CEA producing cells. It is shown in the current study that CEA overexpression alters the splicing of p120 catenin and triggers the release of soluble E-cadherin. The influence of CEA production by colorectal cancer cells on the function of E-cadherin junction complexes may explain the link between the elevated levels of CEA and the increase in soluble E-cadherin during the progression of colorectal cancer. - Highlights: • Elevated level of CEA increases the release of soluble E-cadherin during the progression of colorectal cancer. • CEA over-expression alters the binding preferences between E-cadherin and its partners: α-, β- and p120 catenins in adherens junction complexes. • CEA produced by colorectal cancer cells interacts with beta-catenin protein. • CEA over-expression triggers the increase in nuclear beta-catenin. • CEA over-expression alters the splicing of p120 catenin protein.

  15. The Extracellular Architecture of Adherens Junctions Revealed by Crystal Structures of Type I Cadherins

    SciTech Connect

    O Harrison; X Jin; S Hong; F Bahna; G Ahlsen; J Brasch; Y Wu; J Vendome; K Felsovalyi; et al.

    2011-12-31

    Adherens junctions, which play a central role in intercellular adhesion, comprise clusters of type I classical cadherins that bind via extracellular domains extended from opposing cell surfaces. We show that a molecular layer seen in crystal structures of E- and N-cadherin ectodomains reported here and in a previous C-cadherin structure corresponds to the extracellular architecture of adherens junctions. In all three ectodomain crystals, cadherins dimerize through a trans adhesive interface and are connected by a second, cis, interface. Assemblies formed by E-cadherin ectodomains coated on liposomes also appear to adopt this structure. Fluorescent imaging of junctions formed from wild-type and mutant E-cadherins in cultured cells confirm conclusions derived from structural evidence. Mutations that interfere with the trans interface ablate adhesion, whereas cis interface mutations disrupt stable junction formation. Our observations are consistent with a model for junction assembly involving strong trans and weak cis interactions localized in the ectodomain.

  16. Role for Traf4 in Polarizing Adherens Junctions as a Prerequisite for Efficient Cell Shape Changes ▿

    PubMed Central

    Mathew, Sam J.; Rembold, Martina; Leptin, Maria

    2011-01-01

    Apical constriction of epithelial cells is a widely used morphogenetic mechanism. In the Drosophila embryo, the apical constrictions that internalize the mesoderm are controlled by the transcription factor Twist and require intact adherens junctions and a contractile acto-myosin network. We find that adherens junctions in constricting mesodermal cells undergo extensive remodeling. A Twist target gene encoding a member of the tumor necrosis factor (TNF) receptor-associated factor (TRAF) family, Traf4, is involved in this process. While TRAFs are best known for their functions in inflammatory responses, Traf4 appears to have a different role, and its mechanism of action is poorly understood. We show that Traf4 is required for efficient apical constriction during ventral furrow formation and for proper localization of Armadillo to the apical position in constricting cells. Traf4 and Armadillo interact with each other physically and functionally. Traf4 acts in a TNF receptor- and Jun N-terminal protein kinase (JNK)-independent manner to fine-tune the assembly of adherens junctions in the invaginating mesodermal cells. PMID:21986496

  17. Defects in the adherens junction complex (E-cadherin/ β-catenin) in inflammatory bowel disease.

    PubMed

    Mehta, Shameer; Nijhuis, Anke; Kumagai, Tomoko; Lindsay, James; Silver, Andrew

    2015-06-01

    The epithelial monolayer of the intestine is a selective barrier permitting nutrient and electrolyte absorption yet acting to protect the underlying tissue compartments and cellular components from attack and infiltration by antigens, bacteria and bacterial products present in the lumen. Disruption of this barrier has been associated with inflammatory bowel disease (IBD). The adherens junction (AJ), together with tight junctions (TJ) and desmosomes, form an apical junction complex that controls epithelial cell-to-cell adherence and barrier function as well as regulation of the actin cytoskeleton, intracellular signalling pathways and transcriptional regulation. Numerous studies and reviews highlight the responses of TJs to physiological and pathological stimuli. By comparison, the response of AJ proteins, and the subsequent consequences for barrier function, when exposed to the IBD inflammatory milieu, is less well studied. In this review, we will highlight the roles and responses of the AJ proteins in IBD and provide suggestions for future studies. We will also consider recently proposed therapeutic strategies to preserve or restore epithelial barrier functions to prevent and treat IBD. PMID:25238996

  18. 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

  19. Curcumin prevents cisplatin-induced decrease in the tight and adherens junctions: relation to oxidative stress.

    PubMed

    Trujillo, Joyce; Molina-Jijón, Eduardo; Medina-Campos, Omar Noel; Rodríguez-Muñoz, Rafael; Reyes, José Luis; Loredo, María L; Barrera-Oviedo, Diana; Pinzón, Enrique; Rodríguez-Rangel, Daniela Saraí; Pedraza-Chaverri, José

    2016-01-01

    Curcumin is a polyphenol and cisplatin is an antineoplastic agent that induces nephrotoxicity associated with oxidative stress, apoptosis, fibrosis and decrease in renal tight junction (TJ) proteins. The potential effect of curcumin against alterations in TJ structure and function has not been evaluated in cisplatin-induced nephrotoxicity. The present study explored whether curcumin is able to prevent the cisplatin-induced fibrosis and decreased expression of the TJ and adherens junction (AJ) proteins occludin, claudin-2 and E-cadherin in cisplatin-induced nephrotoxicity. Curcumin (200 mg kg(-1)) was administered in three doses, and rats were sacrificed 72 h after cisplatin administration. Curcumin was able to scavenge, in a concentration-dependent way, superoxide anion, hydroxyl radical, peroxyl radical, singlet oxygen, peroxynitrite anion, hypochlorous acid and hydrogen peroxide. Cisplatin-induced renal damage was associated with alterations in plasma creatinine, expression of neutrophil gelatinase-associated lipocalin and of kidney injury molecule-1, histological damage, increase in apoptosis, fibrosis (evaluated by transforming growth factor β1, collagen I and IV and α-smooth muscle actin expressions), increase in oxidative/nitrosative stress (evaluated by Hsp70/72 expression, protein tyrosine nitration, superoxide anion production in isolated glomeruli and proximal tubules, and protein levels of NADPH oxidase subunits p47(phox) and gp91(phox), protein kinase C β2, and Nrf2) as well as by decreased expression of occludin, claudin-2, β-catenin and E-cadherin. Curcumin treatment prevented all the above-described alterations. The protective effect of curcumin against cisplatin-induced fibrosis and decreased proteins of the TJ and AJ was associated with the prevention of glomerular and proximal tubular superoxide anion production induced by NADPH oxidase activity.

  20. Neogenin recruitment of the WAVE regulatory complex maintains adherens junction stability and tension

    PubMed Central

    Lee, Natalie K.; Fok, Ka Wai; White, Amanda; Wilson, Nicole H.; O'Leary, Conor J.; Cox, Hayley L.; Michael, Magdalene; Yap, Alpha S.; Cooper, Helen M.

    2016-01-01

    To maintain tissue integrity during epithelial morphogenesis, adherens junctions (AJs) must resist the mechanical stresses exerted by dynamic tissue movements. Junctional stability is dependent on actomyosin contractility within the actin ring. Here we describe a novel function for the axon guidance receptor, Neogenin, as a key component of the actin nucleation machinery governing junctional stability. Loss of Neogenin perturbs AJs and attenuates junctional tension. Neogenin promotes actin nucleation at AJs by recruiting the Wave regulatory complex (WRC) and Arp2/3. A direct interaction between the Neogenin WIRS domain and the WRC is crucial for the spatially restricted recruitment of the WRC to the junction. Thus, we provide the first example of a functional WIRS–WRC interaction in epithelia. We further show that Neogenin regulates cadherin recycling at the AJ. In summary, we identify Neogenin as a pivotal component of the AJ, where it influences both cadherin dynamics and junctional tension. PMID:27029596

  1. Cadherin flexibility provides a key difference between desmosomes and adherens junctions.

    PubMed

    Tariq, Humera; Bella, Jordi; Jowitt, Thomas A; Holmes, David F; Rouhi, Mansour; Nie, Zhuxiang; Baldock, Clair; Garrod, David; Tabernero, Lydia

    2015-04-28

    Desmosomes and adherens junctions are intercellular adhesive structures essential for the development and integrity of vertebrate tissue, including the epidermis and heart. Their cell adhesion molecules are cadherins: type 1 cadherins in adherens junctions and desmosomal cadherins in desmosomes. A fundamental difference is that desmosomes have a highly ordered structure in their extracellular region and exhibit calcium-independent hyperadhesion, whereas adherens junctions appear to lack such ordered arrays, and their adhesion is always calcium-dependent. We present here the structure of the entire ectodomain of desmosomal cadherin desmoglein 2 (Dsg2), using a combination of small-angle X-ray scattering, electron microscopy, and solution-based biophysical techniques. This structure reveals that the ectodomain of Dsg2 is flexible even in the calcium-bound state and, on average, is shorter than the type 1 cadherin crystal structures. The Dsg2 structure has an excellent fit with the electron tomography reconstructions of human desmosomes. This fit suggests an arrangement in which desmosomal cadherins form trans interactions but are too far apart to interact in cis, in agreement with previously reported observations. Cadherin flexibility may be key to explaining the plasticity of desmosomes that maintain tissue integrity in their hyperadhesive form, but can adopt a weaker, calcium-dependent adhesion during wound healing and early development. PMID:25855637

  2. Cadherin flexibility provides a key difference between desmosomes and adherens junctions.

    PubMed

    Tariq, Humera; Bella, Jordi; Jowitt, Thomas A; Holmes, David F; Rouhi, Mansour; Nie, Zhuxiang; Baldock, Clair; Garrod, David; Tabernero, Lydia

    2015-04-28

    Desmosomes and adherens junctions are intercellular adhesive structures essential for the development and integrity of vertebrate tissue, including the epidermis and heart. Their cell adhesion molecules are cadherins: type 1 cadherins in adherens junctions and desmosomal cadherins in desmosomes. A fundamental difference is that desmosomes have a highly ordered structure in their extracellular region and exhibit calcium-independent hyperadhesion, whereas adherens junctions appear to lack such ordered arrays, and their adhesion is always calcium-dependent. We present here the structure of the entire ectodomain of desmosomal cadherin desmoglein 2 (Dsg2), using a combination of small-angle X-ray scattering, electron microscopy, and solution-based biophysical techniques. This structure reveals that the ectodomain of Dsg2 is flexible even in the calcium-bound state and, on average, is shorter than the type 1 cadherin crystal structures. The Dsg2 structure has an excellent fit with the electron tomography reconstructions of human desmosomes. This fit suggests an arrangement in which desmosomal cadherins form trans interactions but are too far apart to interact in cis, in agreement with previously reported observations. Cadherin flexibility may be key to explaining the plasticity of desmosomes that maintain tissue integrity in their hyperadhesive form, but can adopt a weaker, calcium-dependent adhesion during wound healing and early development.

  3. Mapping of adherens junction components using microscopic resonance energy transfer imaging.

    PubMed

    Kam, Z; Volberg, T; Geiger, B

    1995-03-01

    Quantitative microscopic imaging of resonance energy transfer (RET) was applied for immunological high resolution proximity mapping of several cytoskeletal components of cell adhesions. To conduct this analysis, a microscopic system was developed, consisting of a highly stable field illuminator, computer-controlled filter wheels for rapid multiple-color imaging and a sensitive, high resolution CCD camera, enabling quantitative data recording and processing. Using this system, we have investigated the spatial inter-relationships and organization of four adhesion-associated proteins, namely vinculin, talin, alpha-actinin and actin. Cultured chick lens cells were double labeled for each of the junctional molecules, using fluorescein- and rhodamine-conjugated antibodies or phalloidin. RET images were acquired with fluorescein excitation and rhodamine emission filter setting, corrected for fluorescein and rhodamine fluorescence, and normalized to the fluorescein image. The results pointed to high local densities of vinculin, talin and F-actin in focal adhesions, manifested by mean RET values of 15%, 12% and 10%, respectively. On the other hand, relatively low values (less than 1%) were observed following double immunofluorescence labeling of the same cells for alpha-actinin. Double indirect labeling for pairs of these four proteins (using fluorophore-conjugated antibodies or phalloidin) resulted in RET values of 5% or lower, except for the pair alpha-actinin and actin, which yielded significantly higher values (13-15%). These results suggest that despite their overlapping staining patterns, at the level of resolution of the light microscope, the plaque proteins vinculin and talin are not homogeneously interspersed at the molecular level but form segregated clusters. alpha-Actinin, on the other hand, does not appear to form such clusters but, rather, closely interacts with actin. We discuss here the conceptual and applicative aspects of RET measurements and the

  4. Mammary epithelial cell phagocytosis downstream of TGF-β3 is characterized by adherens junction reorganization

    PubMed Central

    Fornetti, J; Flanders, K C; Henson, P M; Tan, A-C; Borges, V F; Schedin, P

    2016-01-01

    After weaning, during mammary gland involution, milk-producing mammary epithelial cells undergo apoptosis. Effective clearance of these dying cells is essential, as persistent apoptotic cells have a negative impact on gland homeostasis, future lactation and cancer susceptibility. In mice, apoptotic cells are cleared by the neighboring epithelium, yet little is known about how mammary epithelial cells become phagocytic or whether this function is conserved between species. Here we use a rat model of weaning-induced involution and involuting breast tissue from women, to demonstrate apoptotic cells within luminal epithelial cells and epithelial expression of the scavenger mannose receptor, suggesting conservation of phagocytosis by epithelial cells. In the rat, epithelial transforming growth factor-β (TGF-β) signaling is increased during involution, a pathway known to promote phagocytic capability. To test whether TGF-β enhances the phagocytic ability of mammary epithelial cells, non-transformed murine mammary epithelial EpH4 cells were cultured to achieve tight junction impermeability, such as occurs during lactation. TGF-β3 treatment promoted loss of tight junction impermeability, reorganization and cleavage of the adherens junction protein E-cadherin (E-cad), and phagocytosis. Phagocytosis correlated with junction disruption, suggesting junction reorganization is necessary for phagocytosis by epithelial cells. Supporting this hypothesis, epithelial cell E-cad reorganization and cleavage were observed in rat and human involuting mammary glands. Further, in the rat, E-cad cleavage correlated with increased γ-secretase activity and β-catenin nuclear localization. In vitro, pharmacologic inhibitors of γ-secretase or β-catenin reduced the effect of TGF-β3 on phagocytosis to near baseline levels. However, β-catenin signaling through LiCl treatment did not enhance phagocytic capacity, suggesting a model in which both reorganization of cell junctions and

  5. Reduced expression of adherens and gap junction proteins can have a fundamental role in the development of heart failure following cardiac hypertrophy in rats.

    PubMed

    dos Santos, Daniele O; Blefari, Valdecir; Prado, Fernanda P; Silva, Carlos A; Fazan, Rubens; Salgado, Helio C; Ramos, Simone G; Prado, Cibele M

    2016-02-01

    Hypertension causes cardiac hypertrophy, cardiac dysfunction and heart failure (HF). The mechanisms implicated in the transition from compensated to decompensated cardiac hypertrophy are not fully understood. This study was aimed to investigate whether alterations in the expression of intercalated disk proteins could contribute to the transition of compensated cardiac hypertrophy to dilated heart development that culminates in HF. Male rats were submitted to abdominal aortic constriction and at 90 days post surgery (dps), three groups were observed: sham-operated animals (controls), animals with hypertrophic hearts (HH) and animals with hypertrophic + dilated hearts (HD). Blood pressure was evaluated. The hearts were collected and Western blot and immunofluorescence were performed to desmoglein-2, desmocollin-2, N-cadherin, plakoglobin, Bcatenin, and connexin-43. Cardiac systolic function was evaluated using the Vevo 2100 ultrasound system. Data were considered significant when p b 0.05. Seventy percent of the animals presented with HH and 30% were HD at 90 dps. The blood pressure increased in both groups. The amount of desmoglein-2 and desmocollin-2 expression was increased in both groups and no difference was observed in either group. The expression of N-cadherin, plakoglobin and B-catenin increased in the HHgroup and decreased in the HDgroup; and connexin-43 decreased only in theHDgroup. Therewas no difference between the ejection fraction and fractional shortening at 30 and 60 dps; however, they were decreased in the HD group at 90 dps. We found that while some proteins have increased expression accompanied by the increase in the cell volume associated with preserved systolic cardiac function in theHHgroup, these same proteins had decreased expression evenwithout significant reduction in the cell volume associated with decreased systolic cardiac function in HD group. The increased expression of desmoglein-2 and desmocollin-2 in both the HH and HD groups could

  6. Differential regulation of the Hippo pathway by adherens junctions and apical–basal cell polarity modules

    PubMed Central

    Yang, Chih-Chao; Graves, Hillary K.; Moya, Ivan M.; Tao, Chunyao; Hamaratoglu, Fisun; Gladden, Andrew B.; Halder, Georg

    2015-01-01

    Adherens junctions (AJs) and cell polarity complexes are key players in the establishment and maintenance of apical–basal cell polarity. Loss of AJs or basolateral polarity components promotes tumor formation and metastasis. Recent studies in vertebrate models show that loss of AJs or loss of the basolateral component Scribble (Scrib) cause deregulation of the Hippo tumor suppressor pathway and hyperactivation of its downstream effectors Yes-associated protein (YAP) and Transcriptional coactivator with PDZ-binding motif (TAZ). However, whether AJs and Scrib act through the same or independent mechanisms to regulate Hippo pathway activity is not known. Here, we dissect how disruption of AJs or loss of basolateral components affect the activity of the Drosophila YAP homolog Yorkie (Yki) during imaginal disc development. Surprisingly, disruption of AJs and loss of basolateral proteins produced very different effects on Yki activity. Yki activity was cell-autonomously decreased but non–cell-autonomously elevated in tissues where the AJ components E-cadherin (E-cad) or α-catenin (α-cat) were knocked down. In contrast, scrib knockdown caused a predominantly cell-autonomous activation of Yki. Moreover, disruption of AJs or basolateral proteins had different effects on cell polarity and tissue size. Simultaneous knockdown of α-cat and scrib induced both cell-autonomous and non–cell-autonomous Yki activity. In mammalian cells, knockdown of E-cad or α-cat caused nuclear accumulation and activation of YAP without overt effects on Scrib localization and vice versa. Therefore, our results indicate the existence of multiple, genetically separable inputs from AJs and cell polarity complexes into Yki/YAP regulation. PMID:25624491

  7. Differential regulation of the Hippo pathway by adherens junctions and apical-basal cell polarity modules.

    PubMed

    Yang, Chih-Chao; Graves, Hillary K; Moya, Ivan M; Tao, Chunyao; Hamaratoglu, Fisun; Gladden, Andrew B; Halder, Georg

    2015-02-10

    Adherens junctions (AJs) and cell polarity complexes are key players in the establishment and maintenance of apical-basal cell polarity. Loss of AJs or basolateral polarity components promotes tumor formation and metastasis. Recent studies in vertebrate models show that loss of AJs or loss of the basolateral component Scribble (Scrib) cause deregulation of the Hippo tumor suppressor pathway and hyperactivation of its downstream effectors Yes-associated protein (YAP) and Transcriptional coactivator with PDZ-binding motif (TAZ). However, whether AJs and Scrib act through the same or independent mechanisms to regulate Hippo pathway activity is not known. Here, we dissect how disruption of AJs or loss of basolateral components affect the activity of the Drosophila YAP homolog Yorkie (Yki) during imaginal disc development. Surprisingly, disruption of AJs and loss of basolateral proteins produced very different effects on Yki activity. Yki activity was cell-autonomously decreased but non-cell-autonomously elevated in tissues where the AJ components E-cadherin (E-cad) or α-catenin (α-cat) were knocked down. In contrast, scrib knockdown caused a predominantly cell-autonomous activation of Yki. Moreover, disruption of AJs or basolateral proteins had different effects on cell polarity and tissue size. Simultaneous knockdown of α-cat and scrib induced both cell-autonomous and non-cell-autonomous Yki activity. In mammalian cells, knockdown of E-cad or α-cat caused nuclear accumulation and activation of YAP without overt effects on Scrib localization and vice versa. Therefore, our results indicate the existence of multiple, genetically separable inputs from AJs and cell polarity complexes into Yki/YAP regulation.

  8. HIF2α signaling inhibits adherens junctional disruption in acute lung injury

    PubMed Central

    Gong, Haixia; Rehman, Jalees; Tang, Haiyang; Wary, Kishore; Mittal, Manish; Chatturvedi, Pallavi; Zhao, Youyang; Komorova, Yulia A.; Vogel, Stephen M.; Malik, Asrar B.

    2015-01-01

    Vascular endothelial barrier dysfunction underlies diseases such as acute respiratory distress syndrome (ARDS), characterized by edema and inflammatory cell infiltration. The transcription factor HIF2α is highly expressed in vascular endothelial cells (ECs) and may regulate endothelial barrier function. Here, we analyzed promoter sequences of genes encoding proteins that regulate adherens junction (AJ) integrity and determined that vascular endothelial protein tyrosine phosphatase (VE-PTP) is a HIF2α target. HIF2α-induced VE-PTP expression enhanced dephosphorylation of VE-cadherin, which reduced VE-cadherin endocytosis and thereby augmented AJ integrity and endothelial barrier function. Mice harboring an EC-specific deletion of Hif2a exhibited decreased VE-PTP expression and increased VE-cadherin phosphorylation, resulting in defective AJs. Mice lacking HIF2α in ECs had increased lung vascular permeability and water content, both of which were further exacerbated by endotoxin-mediated injury. Treatment of these mice with Fg4497, a prolyl hydroxylase domain 2 (PHD2) inhibitor, activated HIF2α-mediated transcription in a hypoxia-independent manner. HIF2α activation increased VE-PTP expression, decreased VE-cadherin phosphorylation, promoted AJ integrity, and prevented the loss of endothelial barrier function. These findings demonstrate that HIF2α enhances endothelial barrier integrity, in part through VE-PTP expression and the resultant VE-cadherin dephosphorylation-mediated assembly of AJs. Moreover, activation of HIF2α/VE-PTP signaling via PHD2 inhibition has the potential to prevent the formation of leaky vessels and edema in inflammatory diseases such as ARDS. PMID:25574837

  9. A-CAM: a 135-kD receptor of intercellular adherens junctions. II. Antibody-mediated modulation of junction formation

    PubMed Central

    1986-01-01

    Intercellular adherens junctions between cultured lens epithelial cells are highly Ca2+-dependent and are readily dissociated upon chelation of extracellular Ca2+ ions. Addition of Ca2+ to EGTA-treated cells results in the recovery of cell-cell junctions including the reorganization of adherens junction-specific cell adhesion molecule (A-CAM), vinculin, and actin (Volk, T., and B. Geiger, 1986, J. Cell Biol., 103:000-000). Incubation of cells during the recovery phase with Fab' fragments of anti-A-CAM specifically inhibited the re-formation of cell-cell adherens junctions. This inhibition was accompanied by remarkable changes in microfilament organization manifested by an apparent deterioration of stress fibers and the appearance of fragmented actin bundles throughout the cytoplasm. Incubation of EGTA-dissociated cells with intact divalent anti-A-CAM antibodies in normal medium had no apparent inhibitory effect on junction formation and did not affect the assembly of actin microfilament bundles. Moreover, adherens junctions formed in the presence of the divalent antibodies became essentially Ca2+-independent, suggesting that cell-cell adhesion between them was primarily mediated by the antibodies. These studies suggest that A-CAM participates in intercellular adhesion in adherens-type junctions and point to its involvement in microfilament bundle assembly. PMID:3095334

  10. Endothelial Cell Permeability and Adherens Junction Disruption Induced by Junín Virus Infection

    PubMed Central

    Lander, Heather M.; Grant, Ashley M.; Albrecht, Thomas; Hill, Terence; Peters, Clarence J.

    2014-01-01

    Junín virus (JUNV) is endemic to the fertile Pampas of Argentina, maintained in nature by the rodent host Calomys musculinus, and the causative agent of Argentine hemorrhagic fever (AHF), which is characterized by vascular dysfunction and fluid distribution abnormalities. Clinical as well as experimental studies implicate involvement of the endothelium in the pathogenesis of AHF, although little is known of its role. JUNV has been shown to result in productive infection of endothelial cells (ECs) in vitro with no visible cytopathic effects. In this study, we show that direct JUNV infection of primary human ECs results in increased vascular permeability as measured by electric cell substrate impedance sensing and transwell permeability assays. We also show that EC adherens junctions are disrupted during virus infection, which may provide insight into the role of the endothelium in the pathogenesis of AHF and possibly, other viral hemorrhagic fevers. PMID:24710609

  11. The vav oncogene antagonises EGFR signalling and regulates adherens junction dynamics during Drosophila eye development.

    PubMed

    Martín-Bermudo, Maria-Dolores; Bardet, Pierre-Luc; Bellaïche, Yohanns; Malartre, Marianne

    2015-04-15

    Organ shaping and patterning depends on the coordinated regulation of multiple processes. The Drosophila compound eye provides an excellent model to study the coordination of cell fate and cell positioning during morphogenesis. Here, we find that loss of vav oncogene function during eye development is associated with a disorganised retina characterised by the presence of additional cells of all types. We demonstrate that these defects result from two distinct roles of Vav. First, and in contrast to its well-established role as a positive effector of the EGF receptor (EGFR), we show that readouts of the EGFR pathway are upregulated in vav mutant larval eye disc and pupal retina, indicating that Vav antagonises EGFR signalling during eye development. Accordingly, decreasing EGFR signalling in vav mutant eyes restores retinal organisation and rescues most vav mutant phenotypes. Second, using live imaging in the pupal retina, we observe that vav mutant cells do not form stable adherens junctions, causing various defects, such as recruitment of extra primary pigment cells. In agreement with this role in junction dynamics, we observe that these phenotypes can be exacerbated by lowering DE-Cadherin or Cindr levels. Taken together, our findings establish that Vav acts at multiple times during eye development to prevent excessive cell recruitment by limiting EGFR signalling and by regulating junction dynamics to ensure the correct patterning and morphogenesis of the Drosophila eye.

  12. Multicomponent Analysis of Junctional Movements Regulated by Myosin II Isoforms at the Epithelial Zonula Adherens

    PubMed Central

    Smutny, Michael; Wu, Selwin K.; Gomez, Guillermo A.; Mangold, Sabine; Yap, Alpha S.; Hamilton, Nicholas A.

    2011-01-01

    The zonula adherens (ZA) of epithelial cells is a site of cell-cell adhesion where cellular forces are exerted and resisted. Increasing evidence indicates that E-cadherin adhesion molecules at the ZA serve to sense force applied on the junctions and coordinate cytoskeletal responses to those forces. Efforts to understand the role that cadherins play in mechanotransduction have been limited by the lack of assays to measure the impact of forces on the ZA. In this study we used 4D imaging of GFP-tagged E-cadherin to analyse the movement of the ZA. Junctions in confluent epithelial monolayers displayed prominent movements oriented orthogonal (perpendicular) to the ZA itself. Two components were identified in these movements: a relatively slow unidirectional (translational) component that could be readily fitted by least-squares regression analysis, upon which were superimposed more rapid oscillatory movements. Myosin IIB was a dominant factor responsible for driving the unilateral translational movements. In contrast, frequency spectrum analysis revealed that depletion of Myosin IIA increased the power of the oscillatory movements. This implies that Myosin IIA may serve to dampen oscillatory movements of the ZA. This extends our recent analysis of Myosin II at the ZA to demonstrate that Myosin IIA and Myosin IIB make distinct contributions to junctional movement at the ZA. PMID:21799860

  13. VEGFR2 pY949 signalling regulates adherens junction integrity and metastatic spread.

    PubMed

    Li, Xiujuan; Padhan, Narendra; Sjöström, Elisabet O; Roche, Francis P; Testini, Chiara; Honkura, Naoki; Sáinz-Jaspeado, Miguel; Gordon, Emma; Bentley, Katie; Philippides, Andrew; Tolmachev, Vladimir; Dejana, Elisabetta; Stan, Radu V; Vestweber, Dietmar; Ballmer-Hofer, Kurt; Betsholtz, Christer; Pietras, Kristian; Jansson, Leif; Claesson-Welsh, Lena

    2016-01-01

    The specific role of VEGFA-induced permeability and vascular leakage in physiology and pathology has remained unclear. Here we show that VEGFA-induced vascular leakage depends on signalling initiated via the VEGFR2 phosphosite Y949, regulating dynamic c-Src and VE-cadherin phosphorylation. Abolished Y949 signalling in the mouse mutant Vegfr2(Y949F/Y949F) leads to VEGFA-resistant endothelial adherens junctions and a block in molecular extravasation. Vessels in Vegfr2(Y949F/Y949F) mice remain sensitive to inflammatory cytokines, and vascular morphology, blood pressure and flow parameters are normal. Tumour-bearing Vegfr2(Y949F/Y949F) mice display reduced vascular leakage and oedema, improved response to chemotherapy and, importantly, reduced metastatic spread. The inflammatory infiltration in the tumour micro-environment is unaffected. Blocking VEGFA-induced disassembly of endothelial junctions, thereby suppressing tumour oedema and metastatic spread, may be preferable to full vascular suppression in the treatment of certain cancer forms. PMID:27005951

  14. VEGFR2 pY949 signalling regulates adherens junction integrity and metastatic spread

    PubMed Central

    Li, Xiujuan; Padhan, Narendra; Sjöström, Elisabet O.; Roche, Francis P.; Testini, Chiara; Honkura, Naoki; Sáinz-Jaspeado, Miguel; Gordon, Emma; Bentley, Katie; Philippides, Andrew; Tolmachev, Vladimir; Dejana, Elisabetta; Stan, Radu V.; Vestweber, Dietmar; Ballmer-Hofer, Kurt; Betsholtz, Christer; Pietras, Kristian; Jansson, Leif; Claesson-Welsh, Lena

    2016-01-01

    The specific role of VEGFA-induced permeability and vascular leakage in physiology and pathology has remained unclear. Here we show that VEGFA-induced vascular leakage depends on signalling initiated via the VEGFR2 phosphosite Y949, regulating dynamic c-Src and VE-cadherin phosphorylation. Abolished Y949 signalling in the mouse mutant Vegfr2Y949F/Y949F leads to VEGFA-resistant endothelial adherens junctions and a block in molecular extravasation. Vessels in Vegfr2Y949F/Y949F mice remain sensitive to inflammatory cytokines, and vascular morphology, blood pressure and flow parameters are normal. Tumour-bearing Vegfr2Y949F/Y949F mice display reduced vascular leakage and oedema, improved response to chemotherapy and, importantly, reduced metastatic spread. The inflammatory infiltration in the tumour micro-environment is unaffected. Blocking VEGFA-induced disassembly of endothelial junctions, thereby suppressing tumour oedema and metastatic spread, may be preferable to full vascular suppression in the treatment of certain cancer forms. PMID:27005951

  15. Partial recovery of the damaged rat blood-brain barrier is mediated by adherens junction complexes, extracellular matrix remodeling and macrophage infiltration following focal astrocyte loss

    PubMed Central

    Willis, Colin L.; Camire, Ryan B.; Brule, Stephanie A.; Ray, David E.

    2013-01-01

    Blood-brain barrier (BBB) dysfunction is a feature of many neurodegenerative disorders. The mechanisms and interactions between astrocytes, extracellular matrix and vascular endothelial cells in regulating the mature BBB are poorly understood. We have previously shown that transitory GFAP-astrocyte loss, induced by systemic administration of 3-chloropropanediol, leads to reversible disruption of tight junction complexes and BBB integrity to a range of markers. However, early restoration of BBB integrity to dextran (10-70 kDa) and fibrinogen was seen in the absence of paracellular tight junction proteins claudin-5 and occludin. In the present study we show that in the GFAP-astrocyte lesioned rat inferior colliculus, paracellular expression of adherens junction proteins (VE-cadherin and β-catenin) was maintained in vascular endothelial cells that lacked paracellular claudin-5 expression and which showed reversible post-translational occludin modification. Claudin-1 expression paralleled the loss and recovery of claudin-5, while claudin -3 or -12 immunoreactivity was not detected. In addition, the extracellular matrix, as visualized by laminin and fibronectin, underwent extensive reversible remodeling and perivascular CD169 macrophages become abundant throughout the lesioned inferior colliculus. At a time that GFAP-astrocytes repopulated the lesion area and tight junction proteins were returned to paracellular domains, the extracellular matrix and leukocyte profiles normalized and resembled profiles seen in control tissue. This study supports the hypothesis that a combination of paracellular adherens junctional proteins, remodeled basement membrane and the presence perivascular leukocytes provide a temporary barrier to limit extravasation of macromolecules and potentially neurotoxic substances into the brain parenchyma until tight junction proteins are restored to paracellular domains. PMID:23845748

  16. Changes in membrane-microfilament interaction in intercellular adherens junctions upon removal of extracellular Ca2+ ions

    PubMed Central

    1986-01-01

    EGTA-induced depletion of Ca2+ ions from the culture medium of Madin- Darby bovine kidney epithelial cells results in rapid splitting of adherens-type junctions and the detachment of the vinculin- and actin- containing filament bundle from the cytoplasmic faces of the plasma membrane of the zonula adhaerens. This process was monitored by phase- contrast microscopy, combined with electron microscopy and immunofluorescent localization of the two proteins. It is shown that shortly after extracellular free Ca2+ concentration is lowered to the micromolar range, the actin-containing, junction-associated belt of microfilaments, together with the vinculin-rich junctional plaque material, is irreversibly detached as one structural unit from the plasma membrane, contracts, and is displaced towards the perinuclear cytoplasm where it gradually disintegrates. Other actin- and vinculin- containing structures present in the same cells, notably the focal contacts at the substratum, are not similarly affected by the Ca2+ depletion and retain both the adhesion to the external surface and the association with the plaque and microfilament components. Electron microscopic examination has shown that the membrane domain of the zonulae adhaerentes, unlike that of desmosomes, is not endocytosed after Ca2+ removal and that the displaced actin- and vinculin- containing plaque and filament belt are not associated with a particular membrane. It is further shown that upon restoration of normal Ca2+ levels in the culture medium, new intercellular contacts are established gradually by accretion of both vinculin and actin into new belt-like plaque- and microfilament-containing structures. PMID:3084500

  17. Rho1 regulates adherens junction remodeling by promoting recycling endosome formation through activation of myosin II

    PubMed Central

    Yashiro, Hanako; Loza, Andrew J.; Skeath, James B.; Longmore, Gregory D.

    2014-01-01

    Once adherens junctions (AJs) are formed between polarized epithelial cells they must be maintained because AJs are constantly remodeled in dynamic epithelia. AJ maintenance involves endocytosis and subsequent recycling of E-cadherin to a precise location along the basolateral membrane. In the Drosophila pupal eye epithelium, Rho1 GTPase regulates AJ remodeling through Drosophila E-cadherin (DE-cadherin) endocytosis by limiting Cdc42/Par6/aPKC complex activity. We demonstrate that Rho1 also influences AJ remodeling by regulating the formation of DE-cadherin–containing, Rab11-positive recycling endosomes in Drosophila postmitotic pupal eye epithelia. This effect of Rho1 is mediated through Rok-dependent, but not MLCK-dependent, stimulation of myosin II activity yet independent of its effects upon actin remodeling. Both Rho1 and pMLC localize on endosomal vesicles, suggesting that Rho1 might regulate the formation of recycling endosomes through localized myosin II activation. This work identifies spatially distinct functions for Rho1 in the regulation of DE-cadherin–containing vesicular trafficking during AJ remodeling in live epithelia. PMID:25079692

  18. Rab35 regulates cadherin-mediated adherens junction formation and myoblast fusion

    PubMed Central

    Charrasse, Sophie; Comunale, Franck; De Rossi, Sylvain; Echard, Arnaud; Gauthier-Rouvière, Cécile

    2013-01-01

    Cadherins are homophilic cell–cell adhesion molecules implicated in many fundamental processes, such as morphogenesis, cell growth, and differentiation. They accumulate at cell–cell contact sites and assemble into large macromolecular complexes named adherens junctions (AJs). Cadherin targeting and function are regulated by various cellular processes, many players of which remain to be uncovered. Here we identify the small GTPase Rab35 as a new regulator of cadherin trafficking and stabilization at cell–cell contacts in C2C12 myoblasts and HeLa cells. We find that Rab35 accumulates at cell–cell contacts in a cadherin-dependent manner. Knockdown of Rab35 or expression of a dominant-negative form of Rab35 impaired N- and M-cadherin recruitment to cell–cell contacts, their stabilization at the plasma membrane, and association with p120 catenin and led to their accumulation in transferrin-, clathrin-, and AP-2–positive intracellular vesicles. We also find that Rab35 function is required for PIP5KIγ accumulation at cell–cell contacts and phosphatidyl inositol 4,5-bisphosphate production, which is involved in cadherin stabilization at contact sites. Finally, we show that Rab35 regulates myoblast fusion, a major cellular process under the control of cadherin-dependent signaling. Taken together, these results reveal that Rab35 regulates cadherin-dependent AJ formation and myoblast fusion. PMID:23197472

  19. Visualization of adherent cell monolayers by cryo-electron microscopy: A snapshot of endothelial adherens junctions.

    PubMed

    Le Bihan, Olivier; Decossas, Marion; Gontier, Etienne; Gerbod-Giannone, Marie-Christine; Lambert, Olivier

    2015-12-01

    Cryo-electron microscopy (cryo-EM) allows the visualization of the cell architecture in its native state. We developed a robust solution to adapt cryo-electron microscopy of vitreous sections (CEMOVIS) to a monolayer of adherent cells using a functionalized polyacrylamide hydrogel growing substrate. We applied this method to reconstitute an endothelial cell monolayer to visualize the morphology of adherens junctions (AJs) which regulate permeability and integrity of the vascular barrier. The fine morphology and ultrastructure of AJs from cultured primary human coronary artery endothelial cells (HCAECs) were analyzed in their native state by using CEMOVIS. Doxycycline and sphingosine-1-phosphate (S1P) are known as efficient regulators of endothelial permeability. Doxycycline and S1P treatments both led to a drastic morphological switch from very uneven to standardized 14-17 nm wide AJs over several microns indicative of a better membrane tethering. Repetitive structures were occasionally noticed within the AJ cleft reflecting a local improved structural organization of VE-cadherin molecules. The ultrastructural stabilization of AJs observed upon treatment likely indicates a better adhesion and thus provides structural clues on the mechanism by which these treatments improve the endothelial barrier function. This method was also successfully extended to a thick epithelial barrier model. We expect our strategy to extend the reliable application of CEMOVIS to virtually any adherent cultured cell systems.

  20. Negatively charged silver nanoparticles cause retinal vascular permeability by activating plasma contact system and disrupting adherens junction.

    PubMed

    Long, Yan-Min; Zhao, Xing-Chen; Clermont, Allen C; Zhou, Qun-Fang; Liu, Qian; Feener, Edward P; Yan, Bing; Jiang, Gui-Bin

    2016-01-01

    Silver nanoparticles (AgNPs) have been extensively used as antibacterial component in numerous healthcare, biomedical and consumer products. Therefore, their adverse effects to biological systems have become a major concern. AgNPs have been shown to be absorbed into circulation and redistributed into various organs. It is thus of great importance to understand how these nanoparticles affect vascular permeability and uncover the underlying molecular mechanisms. A negatively charged mecaptoundeonic acid-capped silver nanoparticle (MUA@AgNP) was investigated in this work. Ex vivo experiments in mouse plasma revealed that MUA@AgNPs caused plasma prekallikrein cleavage, while positively charged or neutral AgNPs, as well as Ag ions had no effect. In vitro tests revealed that MUA@AgNPs activated the plasma kallikrein-kinin system (KKS) by triggering Hageman factor autoactivation. By using specific inhibitors aprotinin and HOE 140, we demonstrated that KKS activation caused the release of bradykinin, which activated B2 receptors and induced the shedding of adherens junction protein, VE-cadherin. These biological perturbations eventually resulted in endothelial paracellular permeability in mouse retina after intravitreal injection of MUA@AgNPs. The findings from this work provided key insights for toxicity modulation and biomedical applications of AgNPs. PMID:26399585

  1. SPECC1L deficiency results in increased adherens junction stability and reduced cranial neural crest cell delamination

    PubMed Central

    Wilson, Nathan R.; Olm-Shipman, Adam J.; Acevedo, Diana S.; Palaniyandi, Kanagaraj; Hall, Everett G.; Kosa, Edina; Stumpff, Kelly M.; Smith, Guerin J.; Pitstick, Lenore; Liao, Eric C.; Bjork, Bryan C.; Czirok, Andras; Saadi, Irfan

    2016-01-01

    Cranial neural crest cells (CNCCs) delaminate from embryonic neural folds and migrate to pharyngeal arches, which give rise to most mid-facial structures. CNCC dysfunction plays a prominent role in the etiology of orofacial clefts, a frequent birth malformation. Heterozygous mutations in SPECC1L have been identified in patients with atypical and syndromic clefts. Here, we report that in SPECC1L-knockdown cultured cells, staining of canonical adherens junction (AJ) components, β-catenin and E-cadherin, was increased, and electron micrographs revealed an apico-basal diffusion of AJs. To understand the role of SPECC1L in craniofacial morphogenesis, we generated a mouse model of Specc1l deficiency. Homozygous mutants were embryonic lethal and showed impaired neural tube closure and CNCC delamination. Staining of AJ proteins was increased in the mutant neural folds. This AJ defect is consistent with impaired CNCC delamination, which requires AJ dissolution. Further, PI3K-AKT signaling was reduced and apoptosis was increased in Specc1l mutants. In vitro, moderate inhibition of PI3K-AKT signaling in wildtype cells was sufficient to cause AJ alterations. Importantly, AJ changes induced by SPECC1L-knockdown were rescued by activating the PI3K-AKT pathway. Together, these data indicate SPECC1L as a novel modulator of PI3K-AKT signaling and AJ biology, required for neural tube closure and CNCC delamination. PMID:26787558

  2. Negatively charged silver nanoparticles cause retinal vascular permeability by activating plasma contact system and disrupting adherens junction.

    PubMed

    Long, Yan-Min; Zhao, Xing-Chen; Clermont, Allen C; Zhou, Qun-Fang; Liu, Qian; Feener, Edward P; Yan, Bing; Jiang, Gui-Bin

    2016-01-01

    Silver nanoparticles (AgNPs) have been extensively used as antibacterial component in numerous healthcare, biomedical and consumer products. Therefore, their adverse effects to biological systems have become a major concern. AgNPs have been shown to be absorbed into circulation and redistributed into various organs. It is thus of great importance to understand how these nanoparticles affect vascular permeability and uncover the underlying molecular mechanisms. A negatively charged mecaptoundeonic acid-capped silver nanoparticle (MUA@AgNP) was investigated in this work. Ex vivo experiments in mouse plasma revealed that MUA@AgNPs caused plasma prekallikrein cleavage, while positively charged or neutral AgNPs, as well as Ag ions had no effect. In vitro tests revealed that MUA@AgNPs activated the plasma kallikrein-kinin system (KKS) by triggering Hageman factor autoactivation. By using specific inhibitors aprotinin and HOE 140, we demonstrated that KKS activation caused the release of bradykinin, which activated B2 receptors and induced the shedding of adherens junction protein, VE-cadherin. These biological perturbations eventually resulted in endothelial paracellular permeability in mouse retina after intravitreal injection of MUA@AgNPs. The findings from this work provided key insights for toxicity modulation and biomedical applications of AgNPs.

  3. Negative pressure induces p120-catenin-dependent adherens junction disassembly in keratinocytes during wound healing.

    PubMed

    Huang, Ching-Hui; Hsu, Chih-Chin; Chen, Carl Pai-Chu; Chow, Shu-Er; Wang, Jong-Shyan; Shyu, Yu-Chiau; Lu, Mu-Jie

    2016-09-01

    A negative-pressure of 125mmHg (NP) has been widely used to treat chronic wounds in modern medicine. Keratinocytes under NP treatment have shown accelerated cell movement and decreased E-cadherin expression. However, the molecular mechanism of E-cadherin regulation under NP remains incompletely understood. Therefore, we investigated the E-cadherin regulation in keratinocytes (HaCaT cells) under NP. HaCaT cells were treated at ambient pressure (AP) and NP for 12h. Cell movement was measured by traditional and electric wound healing assays at the 2 different pressures. Mutants with overexpression of p120-catenin (p120(ctn)) were used to observe the effect of NP on p120(ctn) and E-cadherin expression during wound healing. Cell fractionation and immunoblotting data showed that NP increased Y228-phosphorylated p120(ctn) level and resulted in the translocation of p120(ctn) from the plasma membrane to cytoplasm. Immunofluorescence images revealed that NP decreased the co-localization of p120(ctn) and E-cadherin on the plasma membrane. Knockdown of p120(ctn) reduced E-cadherin expression and accelerated cell movement under AP. Overexpression of the Y228-phosphorylation-mimic p120(ctn) decreased E-cadherin membrane expression under both AP and NP. Phosphorylation-deficient mutants conferred restored adherens junctions (AJs) under NP. The Src inhibitor blocked the phosphorylation of p120(ctn) and impeded cell migration under NP. In conclusion, Src-dependent phosphorylation of p120(ctn) can respond rapidly to NP and contribute to E-cadherin downregulation. The NP-induced disassembly of the AJ further accelerates wound healing.

  4. VASP, zyxin and TES are tension-dependent members of Focal Adherens Junctions independent of the α-catenin-vinculin module

    PubMed Central

    Oldenburg, Joppe; van der Krogt, Gerard; Twiss, Floor; Bongaarts, Annika; Habani, Yasmin; Slotman, Johan A.; Houtsmuller, Adriaan; Huveneers, Stephan; de Rooij, Johan

    2015-01-01

    Mechanical forces are integrated at cadherin-based adhesion complexes to regulate morphology and strength of cell-cell junctions and organization of associated F-actin. A central mechanosensor at the cadherin complex is α-catenin, whose stretching recruits vinculin to regulate adhesion strength. The identity of the F-actin regulating signals that are also activated by mechanical forces at cadherin-based junctions has remained elusive. Here we identify the actin-regulators VASP, zyxin and TES as members of punctate, tensile cadherin-based junctions called Focal Adherens Junctions (FAJ) and show that they display mechanosensitive recruitment similar to that of vinculin. However, this recruitment is not altered by destroying or over-activating the α-catenin/vinculin module. Structured Illumination Microscopy (SIM) indicates that these tension sensitive proteins concentrate at locations within FAJs that are distinct from the core cadherin complex proteins. Furthermore, localization studies using mutated versions of VASP and zyxin indicate that these two proteins require binding to each other in order to localize to the FAJs. We conclude that there are multiple force sensitive modules present at the FAJ that are activated at distinct locations along the cadherin-F-actin axis and regulate specific aspects of junction dynamics. PMID:26611125

  5. Polymorphonuclear leukocyte adhesion triggers the disorganization of endothelial cell-to-cell adherens junctions

    PubMed Central

    1996-01-01

    Polymorphonuclear leukocytes (PMN) infiltration into tissues is frequently accompanied by increase in vascular permeability. This suggests that PMN adhesion and transmigration could trigger modifications in the architecture of endothelial cell-to-cell junctions. In the present paper, using indirect immunofluorescence, we found that PMN adhesion to tumor necrosis factor-activated endothelial cells (EC) induced the disappearance from endothelial cell-to-cell contacts of adherens junction (AJ) components: vascular endothelial (VE)-cadherin, alpha-catenin, beta-catenin, and plakoglobin. Immunoprecipitation and Western blot analysis of the VE- cadherin/catenin complex showed that the amount of beta-catenin and plakoglobin was markedly reduced from the complex and from total cell extracts. In contrast, VE-cadherin and alpha-catenin were only partially affected. Disorganization of endothelial AJ by PMN was not accompanied by EC retraction or injury and was specific for VE- cadherin/catenin complex, since platelet/endothelial cell adhesion molecule 1 (PECAM-1) distribution at cellular contacts was unchanged. PMN adhesion to EC seems to be a prerequisite for VE-cadherin/catenin complex disorganization. This phenomenon could be fully inhibited by blocking PMN adhesion with an anti-integrin beta 2 mAb, while it could be reproduced by any condition that induced increase of PMN adhesion, such as addition of PMA or an anti-beta 2-activating mAb. The effect on endothelial AJ was specific for PMN since adherent activated lymphocytes did not induce similar changes. High concentrations of protease inhibitors and oxygen metabolite scavengers were unable to prevent AJ disorganization mediated by PMN. PMN adhesion to EC was accompanied by increase in EC permeability in vitro. This effect was dependent on PMN adhesion, was not mediated by proteases and oxygen- reactive metabolites, and could be reproduced by EC treatment with EGTA. Finally, immunohistochemical analysis showed that VE

  6. Formation of adherens and communicating junctions coordinate the differentiation of the shedding-layer and beta-epidermal generation in regenerating lizard epidermis.

    PubMed

    Alibardi, Lorenzo

    2014-06-01

    In the lizard epidermis, the formation of a stratified alpha- and beta-layer, separated by a shedding complex for molting, suggests that keratinocytes communicate in a coordinated manner after they leave the basal layers during the shedding cycle. I have therefore studied the localization of cell junctional proteins such as beta-catenin and connexins 43 and 26 during scale regeneration in lizard using immunocytochemistry. Beta-catenin is also detected in nuclei of basal cells destined to give rise to the Oberhäutchen and beta-cells suggesting activation of the Wnt-pathway during beta-cell differentiation. The observations show that cells of the entire shedding layer (clear and Oberhäutchen) and beta-layer are connected by beta-catenin (adherens junctions) and connexins (communicating junctions) during their differentiation. This likely cell coupling determines the formation of a distinct shedding and beta-layer within the regenerating epidermis. The observed pattern of cell junctional stratification suggests that after departing from the basal layer Oberhäutchen and beta-cells form a continuous communicating compartment that coordinates the contemporaneous differentiation along the entire scale. While the beta-layer matures the junctions are lost while other cell junctions are formed in the following mesos- and alpha-cell layers. This process determines the formation of layers with different texture (harder or softer) and the precise localization of the shedding layer within lizard epidermis.

  7. Genetic deletion of afadin causes hydrocephalus by destruction of adherens junctions in radial glial and ependymal cells in the midbrain.

    PubMed

    Yamamoto, Hideaki; Maruo, Tomohiko; Majima, Takashi; Ishizaki, Hiroyoshi; Tanaka-Okamoto, Miki; Miyoshi, Jun; Mandai, Kenji; Takai, Yoshimi

    2013-01-01

    Adherens junctions (AJs) play a role in mechanically connecting adjacent cells to maintain tissue structure, particularly in epithelial cells. The major cell-cell adhesion molecules at AJs are cadherins and nectins. Afadin binds to both nectins and α-catenin and recruits the cadherin-β-catenin complex to the nectin-based cell-cell adhesion site to form AJs. To explore the role of afadin in radial glial and ependymal cells in the brain, we generated mice carrying a nestin-Cre-mediated conditional knockout (cKO) of the afadin gene. Newborn afadin-cKO mice developed hydrocephalus and died neonatally. The afadin-cKO brain displayed enlarged lateral ventricles and cerebral aqueduct, resulting from stenosis of the caudal end of the cerebral aqueduct and obliteration of the ventral part of the third ventricle. Afadin deficiency further caused the loss of ependymal cells from the ventricular and aqueductal surfaces. During development, radial glial cells, which terminally differentiate into ependymal cells, scattered from the ventricular zone and were replaced by neurons that eventually covered the ventricular and aqueductal surfaces of the afadin-cKO midbrain. Moreover, the denuded ependymal cells were only occasionally observed in the third ventricle and the cerebral aqueduct of the afadin-cKO midbrain. Afadin was co-localized with nectin-1 and N-cadherin at AJs of radial glial and ependymal cells in the control midbrain, but these proteins were not concentrated at AJs in the afadin-cKO midbrain. Thus, the defects in the afadin-cKO midbrain most likely resulted from the destruction of AJs, because AJs in the midbrain were already established before afadin was genetically deleted. These results indicate that afadin is essential for the maintenance of AJs in radial glial and ependymal cells in the midbrain and is required for normal morphogenesis of the cerebral aqueduct and ventral third ventricle in the midbrain.

  8. A novel amphioxus cadherin that localizes to epithelial adherens junctions has an unusual domain organization with implications for chordate phylogeny.

    PubMed

    Oda, Hiroki; Wada, Hiroshi; Tagawa, Kunifumi; Akiyama-Oda, Yasuko; Satoh, Nori; Humphreys, Tom; Zhang, Shicui; Tsukita, Shoichiro

    2002-01-01

    Although data are available from only vertebrates, urochordates, and three nonchordate animals, there are definite differences in the structures of classic cadherins between vertebrates plus urochordates and nonchordates. In this study we examined structural diversity of classic cadherins among bilaterian animals by obtaining new data from an amphioxus (Cephalochordata, Chordata), an acorn worm (Hemichordata), a sea star (Echinodermata), and an oyster (Mollusca). The structures of newly identified nonchordate cadherins are grouped together with those of the known sea urchin and Drosophila cadherins, whereas the structure of an amphioxus (Branchiostoma belcheri) cadherin, designated BbC, is differently categorized from those of other known chordate cadherins. BbC is identified as a cadherin by its cytoplasmic domain whose sequence is highly related to the cytoplasmic sequences of all known classic cadherins, but it lacks all of the five repeats constituting the extracellular homophilic-binding domain of other chordate cadherins. The ectodomains of BbC match the ectodomains found in nonchordate cadherins but not present in other chordate cadherins. We show that the BbC functions as a cell-cell adhesion molecule when expressed in Drosophila S2 cells and localizes to adherens junctions in the ectodermal epithelia in amphioxus embryos. We argue that BbC is the amphioxus homologue of the classic cadherins involved in the formation of epithelial adherens junctions. The structural relationships of the cadherin molecules allow us to propose a possibility that cephalochordates might be basal to the sister-groups vertebrates and urochordates. PMID:12492143

  9. A novel amphioxus cadherin that localizes to epithelial adherens junctions has an unusual domain organization with implications for chordate phylogeny.

    PubMed

    Oda, Hiroki; Wada, Hiroshi; Tagawa, Kunifumi; Akiyama-Oda, Yasuko; Satoh, Nori; Humphreys, Tom; Zhang, Shicui; Tsukita, Shoichiro

    2002-01-01

    Although data are available from only vertebrates, urochordates, and three nonchordate animals, there are definite differences in the structures of classic cadherins between vertebrates plus urochordates and nonchordates. In this study we examined structural diversity of classic cadherins among bilaterian animals by obtaining new data from an amphioxus (Cephalochordata, Chordata), an acorn worm (Hemichordata), a sea star (Echinodermata), and an oyster (Mollusca). The structures of newly identified nonchordate cadherins are grouped together with those of the known sea urchin and Drosophila cadherins, whereas the structure of an amphioxus (Branchiostoma belcheri) cadherin, designated BbC, is differently categorized from those of other known chordate cadherins. BbC is identified as a cadherin by its cytoplasmic domain whose sequence is highly related to the cytoplasmic sequences of all known classic cadherins, but it lacks all of the five repeats constituting the extracellular homophilic-binding domain of other chordate cadherins. The ectodomains of BbC match the ectodomains found in nonchordate cadherins but not present in other chordate cadherins. We show that the BbC functions as a cell-cell adhesion molecule when expressed in Drosophila S2 cells and localizes to adherens junctions in the ectodermal epithelia in amphioxus embryos. We argue that BbC is the amphioxus homologue of the classic cadherins involved in the formation of epithelial adherens junctions. The structural relationships of the cadherin molecules allow us to propose a possibility that cephalochordates might be basal to the sister-groups vertebrates and urochordates.

  10. Striatins as plaque molecules of zonulae adhaerentes in simple epithelia, of tessellate junctions in stratified epithelia, of cardiac composite junctions and of various size classes of lateral adherens junctions in cultures of epithelia- and carcinoma-derived cells.

    PubMed

    Franke, Werner W; Rickelt, Steffen; Zimbelmann, Ralf; Dörflinger, Yvette; Kuhn, Caecilia; Frey, Norbert; Heid, Hans; Rosin-Arbesfeld, Rina

    2015-03-01

    Proteins of the striatin family (striatins 1-4; sizes ranging from 90 to 110 kDa on SDS-polyacrylamide gel electrophoresis) are highly homologous in their amino acid sequences but can differ in their cell-type-specific gene expression patterns and biological functions. In various cell types, we have found one, two or three polypeptides of this evolutionarily old and nearly ubiquitous family of proteins known to serve as scaffold proteins for diverse protein complexes. Light and electron microscopic immunolocalization methods have revealed striatins in mammalian cell-cell adherens junctions (AJs). In simple epithelia, we have localized striatins as constitutive components of the plaques of the subapical zonulae adhaerentes of cells, including intestinal, glandular, ductal and urothelial cells and hepatocytes. Striatins colocalize with E-cadherin or E-N-cadherin heterodimers and with the plaque proteins α- and β-catenin, p120 and p0071. In some epithelia and carcinomas and in cultured cells derived therefrom, striatins are also seen in lateral AJs. In stratified epithelia and in corresponding squamous cell carcinomas, striatins can be found in plaques of some forms of tessellate junctions. Moreover, striatins are major plaque proteins of composite junctions (CJs; areae compositae) in the intercalated disks connecting cardiomyocytes, colocalizing with other CJ molecules, including plectin and ankyrin-G. We discuss the "multimodulator" scaffold roles of striatins in the initiation and regulation of the formation of various complex particles and structures. We propose that striatins are included in the diagnostic candidate list of proteins that, in the CJs of human hearts, can occur in mutated forms in the pathogeneses of hereditary cardiomyopathies, as seen in some types of genetically determined heart damage in boxer dogs. PMID:25501894

  11. N-cadherin-based adherens junction regulates the maintenance, proliferation, and differentiation of neural progenitor cells during development

    PubMed Central

    Miyamoto, Yasunori; Sakane, Fumi; Hashimoto, Kei

    2015-01-01

    This review addresses our current understanding of the regulatory mechanism by which N-cadherin, a classical cadherin, affects neural progenitor cells (NPCs) during development. N-cadherin is responsible for the integrity of adherens junctions (AJs), which develop in the sub-apical region of NPCs in the neural tube and brain cortex. The apical domain, which contains the sub-apical region, is involved in the switching from symmetric proliferative division to asymmetric neurogenic division of NPCs. In addition, N-cadherin-based AJ is deeply involved in the apico-basal polarity of NPCs and the regulation of Wnt-β-catenin, hedgehog (Hh), and Notch signaling. In this review, we discuss the roles of N-cadherin in the maintenance, proliferation, and differentiation of NPCs through components of AJ, β-catenin and αE-catenin. PMID:25869655

  12. N-cadherin-based adherens junction regulates the maintenance, proliferation, and differentiation of neural progenitor cells during development.

    PubMed

    Miyamoto, Yasunori; Sakane, Fumi; Hashimoto, Kei

    2015-01-01

    This review addresses our current understanding of the regulatory mechanism by which N-cadherin, a classical cadherin, affects neural progenitor cells (NPCs) during development. N-cadherin is responsible for the integrity of adherens junctions (AJs), which develop in the sub-apical region of NPCs in the neural tube and brain cortex. The apical domain, which contains the sub-apical region, is involved in the switching from symmetric proliferative division to asymmetric neurogenic division of NPCs. In addition, N-cadherin-based AJ is deeply involved in the apico-basal polarity of NPCs and the regulation of Wnt-β-catenin, hedgehog (Hh), and Notch signaling. In this review, we discuss the roles of N-cadherin in the maintenance, proliferation, and differentiation of NPCs through components of AJ, β-catenin and αE-catenin. PMID:25869655

  13. Disruption of CDH2/N-cadherin-based adherens junctions leads to apoptosis of ependymal cells and denudation of brain ventricular walls.

    PubMed

    Oliver, Cristian; González, César A; Alvial, Genaro; Flores, Carlos A; Rodríguez, Esteban M; Bátiz, Luis Federico

    2013-09-01

    Disruption/denudation of the ependymal lining has been associated with the pathogenesis of various human CNS disorders, including hydrocephalus, spina bifida aperta, and periventricular heterotopia. It has been traditionally considered that ependymal denudation is a consequence of mechanical forces such as ventricular enlargement. New evidence indicates that ependymal disruption can precede ventricular dilation, but the cellular and molecular mechanisms involved in the onset of ependymal denudation are unknown. Here, we present a novel model to study ependymal cell pathophysiology and demonstrate that selective disruption of N-cadherin-based adherens junctions is sufficient to provoke progressive ependymal denudation. Blocking N-cadherin function using specific peptides that interfere with the histidine-alanine-valine extracellular homophilic interaction domain caused early pathologic changes characterized by disruption of zonula adherens and abnormal intracellular accumulation of N-cadherin. These changes then triggered massive apoptosis of ependymal cells and denudation of brain ventricular walls. Because no typical extrinsic mechanical factors such as elevated pressure or stretching forces are involved in this model, the critical role of N-cadherin-based adherens junctions in ependymal survival/physiology is highlighted. Furthermore, the results suggest that abnormal adherens junctions between ependymal cells should be considered as key components of the pathogenesis of CNS disorders associated with ependymal denudation.

  14. Processus and recessus adhaerentes: giant adherens cell junction systems connect and attract human mesenchymal stem cells.

    PubMed

    Wuchter, Patrick; Boda-Heggemann, Judit; Straub, Beate K; Grund, Christine; Kuhn, Caecilia; Krause, Ulf; Seckinger, Anja; Peitsch, Wiebke K; Spring, Herbert; Ho, Anthony D; Franke, Werner W

    2007-06-01

    Substrate-adherent cultured cells derived from human bone marrow or umbilical cord blood ("mesenchymal stem cells") are of special interest for regenerative medicine. We report that such cells, which can display considerable heterogeneity with respect to their cytoskeletal protein complement, are often interconnected by special tentacle-like cell processes contacting one or several other cells. These processus adhaerentes, studded with many (usually small) puncta adhaerentia and varying greatly in length (up to more than 400 microm long), either contact each other in the intercellular space ("ET touches") or insert in a tight-fitting manner into deep plasma membrane invaginations (recessus adhaerentes), thus forming a novel kind of long (up to 50 microm) continuous cuff-like junction (manubria adhaerentia). The cell processes contain an actin microfilament core that is stabilized with ezrin, alpha-actinin, and myosin and accompanied by microtubules, and their adhering junctions are characterized by a molecular complement comprising the transmembrane glycoproteins N-cadherin and cadherin-11, in combination with the cytoplasmic plaque proteins alpha- and beta-catenin, together with p120(ctn), plakoglobin, and afadin. The processes are also highly dynamic and rapidly foreshorten as cell colonies approach a denser state of cell packing. These structures are obviously able to establish cell-cell connections, even over long distances, and can form deep-rooted and tight cell-cell adhesions. The possible relationship to similar cell processes in the embryonic primary mesenchyme and their potential in cell sorting and tissue formation processes in the body are discussed. PMID:17372769

  15. Nectin-4 mutations causing ectodermal dysplasia with syndactyly perturb the rac1 pathway and the kinetics of adherens junction formation.

    PubMed

    Fortugno, Paola; Josselin, Emmanuelle; Tsiakas, Konstantinos; Agolini, Emanuele; Cestra, Gianluca; Teson, Massimo; Santer, René; Castiglia, Daniele; Novelli, Giuseppe; Dallapiccola, Bruno; Kurth, Ingo; Lopez, Marc; Zambruno, Giovanna; Brancati, Francesco

    2014-08-01

    Defective nectin-1 and -4 have been implicated in ectodermal dysplasia (ED) syndromes with variably associated features including orofacial and limb defects. In particular, nectin-1 mutations cause cleft lip/palate ED (CLPED1; OMIM#225060), whereas defective nectin-4 is associated with ED-syndactyly syndrome (EDSS1; OMIM#613573). Although the broad phenotypic overlap suggests a common mode of action of nectin-1 and -4, little is known about the pathogenic mechanisms involved. We report the identification of, to our knowledge, a previously undescribed nectin-4 homozygous p.Val242Met missense mutation in a patient with EDSS1. We used patient skin biopsy and primary keratinocytes, as well as nectin-4 ectopic expression in epithelial cell lines, to characterize functional consequences of p.Val242Met and p.Thr185Met mutations, the latter previously identified in compound heterozygosity with a truncating mutation. We show that nectin-4-altered expression perturbs nectin-1 clustering at keratinocyte contact sites and delays, but does not impede cell-cell aggregation and cadherin recruitment at adherens junctions (AJs). Moreover, trans-interaction of nectin-1 and -4 induces the activation of Rac1, a member of the Rho family of small GTPases, and regulates E-cadherin-mediated cell-cell adhesion. These data outline a synergistic action of nectin-1 and -4 in the early steps of AJ formation and implicate this interaction in modulating the Rac1 signaling pathway.

  16. Lateral assembly of N-cadherin drives tissue integrity by stabilizing adherens junctions

    PubMed Central

    Garg, S.; Fischer, S. C.; Schuman, E. M.; Stelzer, E. H. K.

    2015-01-01

    Cadherin interactions ensure the correct registry and anchorage of cells during tissue formation. Along the plasma membrane, cadherins form inter-junctional lattices via cis- and trans-dimerization. While structural studies have provided models for cadherin interactions, the molecular nature of cadherin binding in vivo remains unexplored. We undertook a multi-disciplinary approach combining live cell imaging of three-dimensional cell assemblies (spheroids) with a computational model to study the dynamics of N-cadherin interactions. Using a loss-of-function strategy, we demonstrate that each N-cadherin interface plays a distinct role in spheroid formation. We found that cis-dimerization is not a prerequisite for trans-interactions, but rather modulates trans-interfaces to ensure tissue stability. Using a model of N-cadherin junction dynamics, we show that the absence of cis-interactions results in low junction stability and loss of tissue integrity. By quantifying the binding and unbinding dynamics of the N-cadherin binding interfaces, we determined that mutating either interface results in a 10-fold increase in the dissociation constant. These findings provide new quantitative information on the steps driving cadherin intercellular adhesion and demonstrate the role of cis-interactions in junction stability. PMID:25589573

  17. Structural alteration of tight and adherens junctions in villous and crypt epithelium of the small and large intestine of conventional nursing piglets infected with porcine epidemic diarrhea virus.

    PubMed

    Jung, Kwonil; Eyerly, Bryan; Annamalai, Thavamathi; Lu, Zhongyan; Saif, Linda J

    2015-06-12

    Integrity of the intestinal epithelium is critical for proper functioning of the barrier that regulates absorption of water and restricts uptake of luminal bacteria. It is maintained mainly by tight junctions (TJs) and adherens junctions (AJs). We conducted immunofluorescence (IF) staining for in situ identification of zonula occludin (ZO)-1 proteins for TJ and E-Cadherin proteins for AJ in the small and large intestinal villous and crypt epithelium of nursing pigs infected with porcine epidemic diarrhea virus (PEDV). Twenty 9-day-old piglets [PEDV-infected (n=9) and Mock (n=11)] from PEDV seronegative sows, were orally inoculated [8.9 log₁₀ genomic equivalents/pig] with PEDV PC21A strain or mock. At post-inoculation days (PIDs) 1-5, infected pigs showed severe watery diarrhea and/or vomiting and severe atrophic enteritis. By immunohistochemistry, PEDV antigens were evident in enterocytes lining the villous epithelium. At PIDs 1-5, PEDV-infected pigs exhibited mildly to extensively disorganized, irregular distribution and reduced expression of ZO-1 or E-Cadherin in villous, but not crypt epithelial cells of the jejunum and ileum, but not in the large intestine, when compared to the negative controls. The structural destruction and disorganization of TJ and AJ were extensive in PEDV-infected pigs at PIDs 1-3, but then appeared to reversibly recover at PID 5, as evident by increased numbers of ZO-1-positive epithelial cells and markedly improved appearance of E-Cadherin-positive villous epithelium. Our results suggest a possible involvement of structurally impaired TJ and AJ in the pathogenesis of PEDV, potentially leading to secondary bacterial infections.

  18. Id-1 promotes TGF-{beta}1-induced cell motility through HSP27 activation and disassembly of adherens junction in prostate epithelial cells

    SciTech Connect

    Di Kaijun; Wong, Y.C. Wang Xianghong

    2007-11-15

    Id-1 (inhibitor of differentiation or DNA binding-1) has been positively associated with cell proliferation, cell cycle progression, and invasiveness during tumorigenesis. In addition, Id-1 has been shown to modulate cellular sensitivity to TGF-{beta}1 (transforming growth factor {beta}1). Here we demonstrate a novel role of Id-1 in promoting TGF-{beta}1-induced cell motility in a non-malignant prostate epithelial cell line, NPTX. We found that Id-1 promoted F-actin stress fiber formation in response to TGF-{beta}1, which was associated with increased cell-substrate adhesion and cell migration in NPTX cells. In addition, this positive effect of Id-1 on TGF-{beta}1-induced cell motility was mediated through activation of MEK-ERK signaling pathway and subsequent phosphorylation of HSP27 (heat shock protein 27). Furthermore, Id-1 disrupted the adherens junction complex in TGF-{beta}1-treated cells through down-regulation of E-cadherin, redistribution of {beta}-catenin, along with up-regulation of N-cadherin. These lines of evidence reveal a novel tumorigenic role of Id-1 through reorganization of actin cytoskeleton and disassembly of cell-cell adhesion in response to TGF-{beta}1 in human prostate epithelial cells, and suggest that intracellular Id-1 levels might be a determining factor for switching TGF-{beta}1 from a growth inhibitor to a tumor promoter during prostate carcinogenesis.

  19. Type Iγ phosphatidylinositol phosphate kinase modulates adherens junction and E-cadherin trafficking via a direct interaction with μ1B adaptin

    PubMed Central

    Ling, Kun; Bairstow, Shawn F.; Carbonara, Chateen; Turbin, Dmitry A.; Huntsman, David G.; Anderson, Richard A.

    2007-01-01

    Assembly of E-cadherin–based adherens junctions (AJ) is obligatory for establishment of polarized epithelia and plays a key role in repressing the invasiveness of many carcinomas. Here we show that type Iγ phosphatidylinositol phosphate kinase (PIPKIγ) directly binds to E-cadherin and modulates E-cadherin trafficking. PIPKIγ also interacts with the μ subunits of clathrin adaptor protein (AP) complexes and acts as a signalling scaffold that links AP complexes to E-cadherin. Depletion of PIPKIγ or disruption of PIPKIγ binding to either E-cadherin or AP complexes results in defects in E-cadherin transport and blocks AJ assembly. An E-cadherin germline mutation that loses PIPKIγ binding and shows disrupted basolateral membrane targeting no longer forms AJs and leads to hereditary gastric cancers. These combined results reveal a novel mechanism where PIPKIγ serves as both a scaffold, which links E-cadherin to AP complexes and the trafficking machinery, and a regulator of trafficking events via the spatial generation of phosphatidylinositol-4,5-bisphosphate. PMID:17261850

  20. Transient receptor potential channel 1 maintains adherens junction plasticity by suppressing sphingosine kinase 1 expression to induce endothelial hyperpermeability.

    PubMed

    Tauseef, Mohammad; Farazuddin, Mohammad; Sukriti, Sukriti; Rajput, Charu; Meyer, James Otto; Ramasamy, Suresh Kumar; Mehta, Dolly

    2016-01-01

    Stability of endothelial cell (EC) adherens junctions (AJs) is central for prevention of tissue edema, the hallmark of chronic inflammatory diseases including acute respiratory distress syndrome. Here, we demonstrate a previously unsuspected role of sphingosine kinase 1 (SPHK1) in the mechanism by which transient receptor potential channel 1 (Trpc1)-mediated Ca(2+) entry destabilizes AJs. Trpc1(-/-) monolayers showed a 2.2-fold increase in vascular endothelial (VE)-cadherin cell-surface expression above wild-type (WT) monolayers. Thrombin increased endothelial permeability (evident by a 5-fold increase in interendothelial gap area and 60% decrease in transendothelial electrical resistance) in WT but not Trpc1(-/-) ECs. Trpc1(-/-) mice resisted the hyperpermeability effects of the edemagenic agonists used and exhibited 60% less endotoxin-induced mortality. Because sphingosine-1-phosphate (S1P) strengthens AJs, we determined if TRPC1 functioned by inhibiting SPHK1 activity, which generates S1P. Intriguingly, Trpc1(-/-) ECs or ECs transducing a TRPC1-inactive mutant showed a 1.5-fold increase in basal SPHK1 expression compared with WT ECs, resulting in a 2-fold higher S1P level. SPHK1 inhibitor SK1-I decreased basal transendothelial electrical resistance more in WT ECs (48 and 72% reduction at 20 and 50 μM, respectively) than in Trpc1(-/-) ECs. However, SK1-I pretreatment rescued thrombin-induced EC permeability in Trpc1(-/-) ECs. Thus, TRPC1 suppression of basal SPHK1 activity enables EC-barrier destabilization by edemagenic agonists. PMID:26316271

  1. Microtubule-dependent apical restriction of recycling endosomes sustains adherens junctions during morphogenesis of the Drosophila tracheal system.

    PubMed

    Le Droguen, Pierre-Marie; Claret, Sandra; Guichet, Antoine; Brodu, Véronique

    2015-01-15

    Epithelial remodelling is an essential mechanism for organogenesis, during which cells change shape and position while maintaining contact with each other. Adherens junctions (AJs) mediate stable intercellular cohesion but must be actively reorganised to allow morphogenesis. Vesicle trafficking and the microtubule (MT) cytoskeleton contribute to regulating AJs but their interrelationship remains elusive. We carried out a detailed analysis of the role of MTs in cell remodelling during formation of the tracheal system in the Drosophila embryo. Induction of MT depolymerisation specifically in tracheal cells shows that MTs are essential during a specific time frame of tracheal cell elongation while the branch extends. In the absence of MTs, one tracheal cell per branch overelongates, ultimately leading to branch break. Three-dimensional quantifications revealed that MTs are crucial to sustain E-Cadherin (Shotgun) and Par-3 (Bazooka) levels at AJs. Maintaining E-Cadherin/Par-3 levels at the apical domain requires de novo synthesis rather than internalisation and recycling from and to the apical plasma membrane. However, apical targeting of E-Cadherin and Par-3 requires functional recycling endosomes, suggesting an intermediate role for this compartment in targeting de novo synthesized E-Cadherin to the plasma membrane. We demonstrate that apical enrichment of recycling endosomes is dependent on the MT motor Dynein and essential for the function of this vesicular compartment. In addition, we establish that E-Cadherin dynamics and MT requirement differ in remodelling tracheal cells versus planar epithelial cells. Altogether, our results uncover an MT-Dynein-dependent apical restriction of recycling endosomes that controls adhesion by sustaining Par-3 and E-Cadherin levels at AJs during morphogenesis.

  2. Symplekin, a novel type of tight junction plaque protein

    PubMed Central

    1996-01-01

    Using a monoclonal antibody we have identified and cDNA-cloned a novel type of protein localized, by light and electron microscopy, to the plaque associated with the cytoplasmic face of the tight junction- containing zone (zonula occludens) of polar epithelial cells and of Sertoli cells of testis, but absent from the junctions of vascular endothelia. The approximately 3.7-kb mRNA encodes a polypeptide of 1142 amino acids (calculated molecular weight 126.5 kD, pI 6.25), for which the name "symplekin" (from Greek sigma upsilon mu pi lambda epsilon kappa epsilon iota, nu, to tie together, to weave, to be intertwined) is proposed. However, both the mRNA and the protein can also be detected in a wide range of cell types that do not form tight junctions or are even completely devoid of any stable cell contacts. Careful analyses have revealed that the protein occurs in all these diverse cells in the nucleoplasm, and only in those cells forming tight junctions is it recruited, partly but specifically, to the plaque structure of the zonula occludens. We discuss symplekin as a representative of a group of dual residence proteins which occur and probably function in the nucleus as well as in the plaques exclusive for either tight junctions, adherens junctions, or desmosomes. PMID:8769423

  3. Sertoli-germ cell adherens junction dynamics in the testis are regulated by RhoB GTPase via the ROCK/LIMK signaling pathway.

    PubMed

    Lui, Wing-yee; Lee, Will M; Cheng, C Yan

    2003-06-01

    During spermatogenesis, cell-cell actin-based adherens junctions (AJs), such as ectoplasmic specializations (ESs), between Sertoli and germ cells undergo extensive restructuring in the seminiferous epithelium to facilitate germ cell movement across the epithelium. Although the mechanism(s) that regulates AJ dynamics in the testis is virtually unknown, Rho GTPases have been implicated in the regulation of these events in other epithelia. Studies have shown that the in vitro assembly of the Sertoli-germ cell AJs but not of the Sertoli cell tight junctions (TJs) is associated with a transient but significant induction of RhoB. Immunohistochemistry has shown that the localization of RhoB in the seminiferous epithelium is stage specific, being lowest in stages VII-VIII prior to spermiation, and displays cell-specific association during the epithelial cycle. Throughout the cycle, RhoB was localized near the site of basal and apical ESs but was restricted to the periphery of the nuclei in elongating (but not elongated) spermatids, spermatocytes, and Sertoli cells. However, RhoB was not detected near the site of apical ESs at stages VII-VIII. Furthermore, disruption of AJs in Sertoli-germ cell cocultures either by hypotonic treatment or by treatment with 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364) also induced RhoB expression. When adult rats were treated with AF-2364 to perturb Sertoli-germ cell AJs in vivo, a approximately 4-fold induction in RhoB in the testis, but not in kidney and brain, was detected within 1 h, at least approximately 1-4 days before germ cell loss from the epithelium could be detected by histological analysis. The signaling pathway(s) by which AF-2364 perturbed the Sertoli-germ cell AJs apparently began with an initial activation of integrin, which in turn activated RhoB, ROCK1, (Rho-associated protein kinase 1, also called ROKbeta), LIMK1 (LIM kinase 1, also called lin-11 isl-1 mec3 kinase 1), and cofilin but not p140mDia and profilin

  4. MicroRNAs regulate tight junction proteins and modulate epithelial/endothelial barrier functions.

    PubMed

    Cichon, Christoph; Sabharwal, Harshana; Rüter, Christian; Schmidt, M Alexander

    2014-01-01

    Tightly controlled epithelial and endothelial barriers are a prerequisite for life as these barriers separate multicellular organisms from their environment and serve as first lines of defense. Barriers between neighboring epithelial cells are formed by multiple intercellular junctions including the 'apical junctional complex-AJC' with tight junctions (TJ), adherens junctions (AJ), and desmosomes. TJ consist of tetraspan transmembrane proteins like occludin, various claudins that directly control paracellular permeability, and the 'Junctional Adhesion Molecules' (JAMs). For establishing tight barriers TJ are essential but at the same time have to allow also selective permeability. For this, TJ need to be tightly regulated and controlled. This is organized by a variety of adaptor molecules, i.e., protein kinases, phosphatases and GTPases, which in turn are regulated and fine-tuned involving microRNAs (miRNAs). In this review we summarize available data on the role and targeting of miRNAs in the maintenance of epithelial and/or endothelial barriers.

  5. Junction Protein Shrew-1 Influences Cell Invasion and Interacts with Invasion-promoting Protein CD147

    PubMed Central

    Schreiner, Alexander; Ruonala, Mika; Jakob, Viktor; Suthaus, Jan; Boles, Eckhard; Wouters, Fred

    2007-01-01

    Shrew-1 was previously isolated from an endometriotic cell line in our search for invasion-associated genes. It proved to be a membrane protein that targets to the basolateral membrane of polarized epithelial cells, interacting with E-cadherin–catenin complexes of adherens junctions. Paradoxically, the existence of adherens junctions is incompatible with invasion. To investigate whether shrew-1 can indeed influence cellular invasion, we overexpressed it in HT1080 fibrosarcoma cells. This resulted in enhanced invasiveness, accompanied by an increased matrix metalloprotease (MMP)-9 level in the supernatant, raising the question about the role of shrew-1 in this process. Logic suggested we looked for an interaction with CD147, a known promoter of invasiveness and MMP activity. Indeed, genetics-based, biochemical, and microscopy experiments revealed shrew-1– and CD147-containing complexes in invasive endometriotic cells and an interaction in epithelial cells, which was stronger in MCF7 tumor cells, but weaker in Madin-Darby canine kidney cells. In contrast to the effect mediated by overexpression, small interfering RNA-mediated down-regulation of either shrew-1 or CD147 in HeLa cells decreased invasiveness without affecting the proliferation behavior of HeLa cells, but the knockdown cells displayed decreased motility. Altogether, our results imply that shrew-1 has a function in the regulation of cellular invasion, which may involve its interaction with CD147. PMID:17267690

  6. MicroRNAs regulate tight junction proteins and modulate epithelial/endothelial barrier functions

    PubMed Central

    Cichon, Christoph; Sabharwal, Harshana; Rüter, Christian; Schmidt, M Alexander

    2014-01-01

    Tightly controlled epithelial and endothelial barriers are a prerequisite for life as these barriers separate multicellular organisms from their environment and serve as first lines of defense. Barriers between neighboring epithelial cells are formed by multiple intercellular junctions including the ‘apical junctional complex—AJC’ with tight junctions (TJ), adherens junctions (AJ), and desmosomes. TJ consist of tetraspan transmembrane proteins like occludin, various claudins that directly control paracellular permeability, and the ‘Junctional Adhesion Molecules’ (JAMs). For establishing tight barriers TJ are essential but at the same time have to allow also selective permeability. For this, TJ need to be tightly regulated and controlled. This is organized by a variety of adaptor molecules, i.e., protein kinases, phosphatases and GTPases, which in turn are regulated and fine-tuned involving microRNAs (miRNAs). In this review we summarize available data on the role and targeting of miRNAs in the maintenance of epithelial and/or endothelial barriers. PMID:25610754

  7. Expression of TM4SF10, a Claudin/EMP/PMP22 family cell junction protein, during mouse kidney development and podocyte differentiation.

    PubMed

    Bruggeman, Leslie A; Martinka, Scott; Simske, Jeffrey S

    2007-02-01

    Cell junctions in the nephron are highly specialized to perform specific and distinct filtration and reabsorption functions. The mature kidney forms complex cell junctions including slit diaphragms that prevent the passage of serum proteins into the filtrate, and tubule cell junctions that regulate specific paracellular ion reuptake. We have investigated the expression of TM4SF10 (Trans-Membrane tetra(4)-Span Family 10) in mouse kidneys. TM4SF10 is the vertebrate orthologue of Caenorhabditis elegans VAB-9, a tetraspan adherens junction protein in the PMP22/EMP/Claudin family of proteins. We found that TM4SF10 localizes at the basal-most region of podocyte precursors before the capillary loop stage, at some tubule precursors, and at the ureteric bud junction with S-shaped bodies. Overall expression of TM4SF10 peaked at postnatal day 4 and was virtually absent in adult kidneys. The very limited expression of TM4SF10 protein that persisted into adulthood was restricted to a few tubule segments but remained localized to the basal region of lateral membranes. In undifferentiated cultured podocytes, TM4SF10 localized to the perinuclear region and translocated to the cell membrane after Cadherin appearance at cell-cell contacts. TM4SF10 colocalized with ZO1 and p120ctn in undifferentiated confluent podocytes and also colocalized with the tips of actin filaments at cell contacts. Upon differentiation of cultured podocytes, TM4SF10 protein disappeared from cell contacts and expression ceased. These results suggest that TM4SF10 functions during differentiation of podocytes and may participate in the maturation of cell junctions from simple adherens junctions to elaborate slit diaphragms. TM4SF10 may define a new class of Claudin-like proteins that function during junctional development.

  8. Vinculin potentiates E-cadherin mechanosensing and is recruited to actin-anchored sites within adherens junctions in a myosin II–dependent manner

    PubMed Central

    le Duc, Quint; Shi, Quanming; Blonk, Iris; Sonnenberg, Arnoud

    2010-01-01

    Cell surface receptors integrate chemical and mechanical cues to regulate a wide range of biological processes. Integrin complexes are the mechanotransducers between the extracellular matrix and the actomyosin cytoskeleton. By analogy, cadherin complexes may function as mechanosensors at cell–cell junctions, but this capacity of cadherins has not been directly demonstrated. Furthermore, the molecular composition of the link between E-cadherin and actin, which is needed to sustain such a function, is unresolved. In this study, we describe nanomechanical measurements demonstrating that E-cadherin complexes are functional mechanosensors that transmit force between F-actin and E-cadherin. Imaging experiments reveal that intercellular forces coincide with vinculin accumulation at actin-anchored cadherin adhesions, and nanomechanical measurements show that vinculin potentiates the E-cadherin mechanosensory response. These investigations directly demonstrate the mechanosensory capacity of the E-cadherin complex and identify a novel function for vinculin at cell–cell junctions. These findings have implications for barrier function, morphogenesis, cell migration, and invasion and may extend to all soft tissues in which classical cadherins regulate cell–cell adhesion. PMID:20584916

  9. Crucial Role of Rapgef2 and Rapgef6, a Family of Guanine Nucleotide Exchange Factors for Rap1 Small GTPase, in Formation of Apical Surface Adherens Junctions and Neural Progenitor Development in the Mouse Cerebral Cortex123

    PubMed Central

    Maeta, Kazuhiro; Edamatsu, Hironori; Nishihara, Kaori; Ikutomo, Junji; Bilasy, Shymaa E.

    2016-01-01

    Abstract Cerebral neocortex development in mammals requires highly orchestrated events involving proliferation, differentiation, and migration of neural progenitors and neurons. Rapgef2 and Rapgef6 constitute a unique family of guanine nucleotide exchange factors for Rap1 small GTPase, which is known to play crucial roles in migration of postmitotic neurons. We previously reported that conditional knockout of Rapgef2 in dorsal telencephalon (Rapgef2-cKO) resulted in the formation of an ectopic cortical mass (ECM) resembling that of subcortical band heterotopia. Here we show that double knockout of Rapgef6 in Rapgef2-cKO mice (Rapgef2/6-dKO) results in marked enlargement of the ECM. While Rapgef2-cKO affects late-born neurons only, Rapgef2/6-dKO affects both early-born and late-born neurons. The Rapgef2-cKO cortex at embryonic day (E) 15.5, and the Rapgef2/6-dKO cortex at E13.5 and E15.5 show disruption of the adherens junctions (AJs) on the apical surface, detachment of radial glial cells (RGCs) from the apical surface and disorganization of the radial glial fiber system, which are accompanied by aberrant distribution of RGCs and intermediate progenitors, normally located in the ventricular zone and the subventricular zone, respectively, over the entire cerebral cortex. Moreover, intrauterine transduction of Cre recombinase into the Rapgef2flox/flox brains also results in the apical surface AJ disruption and the RGC detachment from the apical surface, both of which are effectively suppressed by cotransduction of the constitutively active Rap1 mutant Rap1G12V. These results demonstrate a cell-autonomous role of the Rapgef2/6-Rap1 pathway in maintaining the apical surface AJ structures, which is necessary for the proper development of neural progenitor cells. PMID:27390776

  10. Meprin A impairs epithelial barrier function, enhances monocyte migration, and cleaves the tight junction protein occludin

    PubMed Central

    Bao, Jialing; Yura, Renee E.; Matters, Gail L.; Bradley, S. Gaylen; Shi, Pan; Tian, Fang

    2013-01-01

    Meprin metalloproteases are highly expressed at the luminal interface of the intestine and kidney and in certain leukocytes. Meprins cleave a variety of substrates in vitro, including extracellular matrix proteins, adherens junction proteins, and cytokines, and have been implicated in a number of inflammatory diseases. The linkage between results in vitro and pathogenesis, however, has not been elucidated. The present study aimed to determine whether meprins are determinative factors in disrupting the barrier function of the epithelium. Active meprin A or meprin B applied to Madin-Darby canine kidney (MDCK) cell monolayers increased permeability to fluorescein isothiocyanate-dextran and disrupted immunostaining of the tight junction protein occludin but not claudin-4. Meprin A, but not meprin B, cleaved occludin in MDCK monolayers. Experiments with recombinant occludin demonstrated that meprin A cleaves the protein between Gly100 and Ser101 on the first extracellular loop. In vivo experiments demonstrated that meprin A infused into the mouse bladder increased the epithelium permeability to sodium fluorescein. Furthermore, monocytes from meprin knockout mice on a C57BL/6 background were less able to migrate through an MDCK monolayer than monocytes from their wild-type counterparts. These results demonstrate the capability of meprin A to disrupt epithelial barriers and implicate occludin as one of the important targets of meprin A that may modulate inflammation. PMID:23804454

  11. 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.

  12. Connexins, E-cadherin, Claudin-7 and β-catenin transiently form junctional nexuses during the post-natal mammary gland development.

    PubMed

    Dianati, Elham; Poiraud, Jérémy; Weber-Ouellette, Anne; Plante, Isabelle

    2016-08-01

    Gap junctions are intercellular channels made of connexins (Cxs) that allow direct communication between adjacent cells. Modulation of Cxs has been associated with abnormal development and function of the mammary gland and breast cancer. However, the mechanisms underlying their expression during normal mammary gland are not yet known. Cxs interact with components of tight and adherens junctions. Thus, we hypothesized that the expression levels of Cxs vary during mammary gland development and are regulated through stage-dependent interactions with members of the tight and adherens junctions. Our specific objectives were to: 1) determine the expression of Cxs and tight and adherens junction proteins throughout development and 2) characterize Cxs interactions with components of tight and adherens junctions. Murine mammary glands were sampled at various developmental stages (pre-pubescent to post-weaning). RT-qPCR and western-blot analyses demonstrated differential expression patterns for all gap (Cx43, Cx32, Cx26, Cx30), tight (Claudin-1, -3, -4, -7) and adherens (β-catenin, E- and P-cadherins) junctions throughout development. Interestingly, co-immunoprecipitation demonstrated interactions between these different types of junctions. Cx30 interacted with Cx26 just at the late pregnancy stage. While Cx43 showed a persistent interaction with β-catenin from virginity to post-weaning, its interactions with E-cadherin and Claudin-7 were transient. Cx32 interacted with Cx26, E-cadherin and β-catenin during lactation. Immunofluorescence results confirmed the existence of a junctional nexus that remodeled during mammary gland development. Together, our results confirm that the expression levels of Cxs vary concomitantly and that Cxs form junctional nexuses with tight and adherens junctions, suggesting the existence of common regulatory pathways.

  13. Remodeling the zonula adherens in response to tension and the role of afadin in this response

    PubMed Central

    Acharya, Bipul R.; Peyret, Grégoire; Fardin, Marc-Antoine; Mège, René-Marc; Ladoux, Benoit; Yap, Alpha S.; Fanning, Alan S.

    2016-01-01

    Morphogenesis requires dynamic coordination between cell–cell adhesion and the cytoskeleton to allow cells to change shape and move without losing tissue integrity. We used genetic tools and superresolution microscopy in a simple model epithelial cell line to define how the molecular architecture of cell–cell zonula adherens (ZA) is modified in response to elevated contractility, and how these cells maintain tissue integrity. We previously found that depleting zonula occludens 1 (ZO-1) family proteins in MDCK cells induces a highly organized contractile actomyosin array at the ZA. We find that ZO knockdown elevates contractility via a Shroom3/Rho-associated, coiled-coil containing protein kinase (ROCK) pathway. Our data suggest that each bicellular border is an independent contractile unit, with actin cables anchored end-on to cadherin complexes at tricellular junctions. Cells respond to elevated contractility by increasing junctional afadin. Although ZO/afadin knockdown did not prevent contractile array assembly, it dramatically altered cell shape and barrier function in response to elevated contractility. We propose that afadin acts as a robust protein scaffold that maintains ZA architecture at tricellular junctions. PMID:27114502

  14. The transmembrane protein occludin of epithelial tight junctions is a functional target for serine peptidases from faecal pellets of Dermatophagoides pteronyssinus.

    PubMed

    Wan, H; Winton, H L; Soeller, C; Taylor, G W; Gruenert, D C; Thompson, P J; Cannell, M B; Stewart, G A; Garrod, D R; Robinson, C

    2001-02-01

    There have been only a few studies of how allergens cross the airway epithelium to cause allergic sensitization. House dust mite fecal pellets (HDMFP) contain several proteolytic enzymes. Group 1 allergens are cysteine peptidases, whilst those of groups 3, 6 and 9 have catalytic sites indicative of enzymes that mechanistically behave as serine peptidases. We have previously shown that the group 1 allergen Der p 1 leads to cleavage of tight junctions (TJs), allowing allergen delivery to antigen presenting cells. In this study we determined whether HDMFP serine peptidases similarly compromise the airway epithelium by attacking TJs, desmosomes and adherens junctions. Experiments were performed in monolayers of MDCK, Calu-3 or 16HBE14o-epithelial cells. Cell junction morphology was examined by 2-photon molecular excitation microscopy and digital image analysis. Barrier function was measured as mannitol permeability. Cleavage of cell adhesion proteins was studied by immunoblotting and mass spectrometry. HDMFP serine peptidases led to a progressive cleavage of TJs and increased epithelial permeability. Desmosomal puncta became more concentrated. Cleavage of TJs involved proteolysis of the TJ proteins, occludin and ZO-1. This was associated with activation of intracellular proteolysis of ZO-1. In contrast to occludin, E-cadherin of adherens junctions was cleaved less extensively. Although Calu-3 and 16HBE14o-cells expressed tethered ligand receptors for serine peptidases, these were not responsible for transducing the changes in TJs. HDMFP serine peptidases cause cleavage of TJs. This study identifies a second general class of HDM peptidase capable of increasing epithelial permeability and thereby creating conditions that would favour transepithelial delivery of allergens.

  15. The stardust family protein MPP7 forms a tripartite complex with LIN7 and DLG1 that regulates the stability and localization of DLG1 to cell junctions.

    PubMed

    Bohl, Joanna; Brimer, Nicole; Lyons, Charles; Vande Pol, Scott B

    2007-03-30

    MPP7, a previously uncharacterized member of the p55 Stardust family of membrane-associated guanylate kinase (MAGUK) proteins, was found in a tripartite complex with DLG1 and LIN7A or LIN7C. MPP7 dimerizes with all three LIN7 family members (LIN7A, -B, and -C) through interaction of the single L27 domain of LIN7 with the carboxyl-terminal L27 domain of MPP7, thereby stabilizing both proteins. The dimer of MPP7 with LIN7A or LIN7C associates with DLG1 through an interaction requiring the amino-terminal L27 domain of MPP7. The amino-terminal L27 domain of MPP7 is not sufficient for interaction with DLG1 but interacts efficiently only if MPP7 is in a complex with LIN7A or -C. Thus the specificity of interaction of DLG1 with the LIN7-MPP7 complex is determined by L27 interactions with both MPP7 and LIN7. The tripartite complex forms in a ratio of 1:1:1 and localizes to epithelial adherens junctions in a manner dependent upon MPP7. Expression of MPP7 stabilizes DLG1 in an insoluble compartment. Expression of MPP7 deleted of the PDZ or Src homology 3 domain redistributes MPP7, DLG1, and LIN7 out of adherens junctions and into the soluble cytoplasmic fraction without changing the localization of E-cadherin. Thus, the stability and localization of DLG1 to cell-cell junctions are complex functions determined by the expression and association of particular Stardust family members together with particular LIN7 family members.

  16. Differences between liver gap junction protein and lens MIP 26 from rat: implications for tissue specificity of gap junctions.

    PubMed

    Nicholson, B J; Takemoto, L J; Hunkapiller, M W; Hood, L E; Revel, J P

    1983-03-01

    Liver gap junctions and gap-junction-like structures from eye lenses are each comprised of a single major protein (Mr 28,000 and 26,000, respectively). These proteins display different two-dimensional peptide fingerprints, distinct amino acid compositions, nonhomologous N-terminal amino acid sequences and different sensitivities to proteases when part of the intact junction. However, the junctional protein of each tissue is well conserved between species, as demonstrated previously for lens and now for liver in several mammalian species. The possiblity of tissue-specific gap junction proteins is discussed in the light of data suggesting that rat heart gap junctions are comprised of yet a third protein. PMID:6299583

  17. Protein kinase C activation disrupts epithelial apical junctions via ROCK-II dependent stimulation of actomyosin contractility

    PubMed Central

    Ivanov, Andrei I; Samarin, Stanislav N; Bachar, Moshe; Parkos, Charles A; Nusrat, Asma

    2009-01-01

    Background Disruption of epithelial cell-cell adhesions represents an early and important stage in tumor metastasis. This process can be modeled in vitro by exposing cells to chemical tumor promoters, phorbol esters and octylindolactam-V (OI-V), known to activate protein kinase C (PKC). However, molecular events mediating PKC-dependent disruption of epithelial cell-cell contact remain poorly understood. In the present study we investigate mechanisms by which PKC activation induces disassembly of tight junctions (TJs) and adherens junctions (AJs) in a model pancreatic epithelium. Results Exposure of HPAF-II human pancreatic adenocarcinoma cell monolayers to either OI-V or 12-O-tetradecanoylphorbol-13-acetate caused rapid disruption and internalization of AJs and TJs. Activity of classical PKC isoenzymes was responsible for the loss of cell-cell contacts which was accompanied by cell rounding, phosphorylation and relocalization of the F-actin motor nonmuscle myosin (NM) II. The OI-V-induced disruption of AJs and TJs was prevented by either pharmacological inhibition of NM II with blebbistatin or by siRNA-mediated downregulation of NM IIA. Furthermore, AJ/TJ disassembly was attenuated by inhibition of Rho-associated kinase (ROCK) II, but was insensitive to blockage of MLCK, calmodulin, ERK1/2, caspases and RhoA GTPase. Conclusion Our data suggest that stimulation of PKC disrupts epithelial apical junctions via ROCK-II dependent activation of NM II, which increases contractility of perijunctional actin filaments. This mechanism is likely to be important for cancer cell dissociation and tumor metastasis. PMID:19422706

  18. Reciprocal influence of connexins and apical junction proteins on their expressions and functions

    PubMed Central

    Derangeon, Mickaël; Spray, David C.; Bourmeyster, Nicolas; Sarrouilhe, Denis; Hervé, Jean-Claude

    2009-01-01

    Membranes of adjacent cells form intercellular junctional complexes to mechanically anchor neighbour cells (anchoring junctions), to seal the paracellular space and to prevent diffusion of integral proteins within the plasma membrane (tight junctions) and to allow cell-to-cell diffusion of small ions and molecules (gap junctions). These different types of specialised plasma membrane microdomains, sharing common adaptor molecules, particularly zonula occludens proteins, frequently present intermingled relationships where the different proteins co-assemble into macromolecular complexes and their expressions are co-ordinately regulated. Proteins forming gap junction channels (connexins, particularly) and proteins fulfilling cell attachment or forming tight junction strands mutually influence expression and functions of one another. PMID:19046940

  19. Switch in Gap Junction Protein Expression is Associated with Selective Changes in Junctional Permeability During Keratinocyte Differentiation

    NASA Astrophysics Data System (ADS)

    Brissette, Janice L.; Kumar, Nalin M.; Gilula, Norton B.; Hall, James E.; Dotto, G. Paolo

    1994-07-01

    Gap junctional communication provides a mechanism for regulating multicellular activities by allowing the exchange of small diffusible molecules between neighboring cells. The diversity of gap junction proteins may exist to form channels that have different permeability properties. We report here that induction of terminal differentiation in mouse primary keratinocytes by calcium results in a specific switch in gap junction protein expression. Expression of α_1 (connexin 43) and β_2 (connexin 26) gap junction proteins is down-modulated, whereas that of β_3 (connexin 31) and β_4 (connexin 31.1) proteins is induced. Although both proliferating and differentiating keratinocytes are electrically coupled, there are significant changes in the permeability properties of the junctions to small molecules. In parallel with the changes in gap junction protein expression during differentiation, the intercellular transfer of the small dyes neurobiotin, carboxyfluorescein, and Lucifer yellow is significantly reduced, whereas that of small metabolites, such as nucleotides and amino acids, proceeds unimpeded. Thus, a switch in gap junction protein expression in differentiating keratinocytes is accompanied by selective changes in junctional permeability that may play an important role in the coordinate control of the differentiation process.

  20. Switch in gap junction protein expression is associated with selective changes in junctional permeability during keratinocyte differentiation.

    PubMed Central

    Brissette, J L; Kumar, N M; Gilula, N B; Hall, J E; Dotto, G P

    1994-01-01

    Gap junctional communication provides a mechanism for regulating multicellular activities by allowing the exchange of small diffusible molecules between neighboring cells. The diversity of gap junction proteins may exist to form channels that have different permeability properties. We report here that induction of terminal differentiation in mouse primary keratinocytes by calcium results in a specific switch in gap junction protein expression. Expression of alpha 1 (connexin 43) and beta 2 (connexin 26) gap junction proteins is down-modulated, whereas that of beta 3 (connexin 31) and beta 4 (connexin 31.1) proteins is induced. Although both proliferating and differentiating keratinocytes are electrically coupled, there are significant changes in the permeability properties of the junctions to small molecules. In parallel with the changes in gap junction protein expression during differentiation, the intercellular transfer of the small dyes neurobiotin, carboxyfluorescein, and Lucifer yellow is significantly reduced, whereas that of small metabolites, such as nucleotides and amino acids, proceeds unimpeded. Thus, a switch in gap junction protein expression in differentiating keratinocytes is accompanied by selective changes in junctional permeability that may play an important role in the coordinate control of the differentiation process. Images PMID:8022804

  1. Alteration in synaptic junction proteins following traumatic brain injury.

    PubMed

    Merlo, Lucia; Cimino, Francesco; Angileri, Filippo Flavio; La Torre, Domenico; Conti, Alfredo; Cardali, Salvatore Massimiliano; Saija, Antonella; Germanò, Antonino

    2014-08-15

    Extensive research and scientific efforts have been focused on the elucidation of the pathobiology of cellular and axonal damage following traumatic brain injury (TBI). Conversely, few studies have specifically addressed the issue of synaptic dysfunction. Synaptic junction proteins may be involved in post-TBI alterations, leading to synaptic loss or disrupted plasticity. A Synapse Protein Database on synapse ontology identified 109 domains implicated in synaptic activities and over 5000 proteins, but few of these demonstrated to play a role in the synaptic dysfunction after TBI. These proteins are involved in neuroplasticity and neuromodulation and, most importantly, may be used as novel neuronal markers of TBI for specific intervention.

  2. Restoration of desmosomal junction protein expression and inhibition of H3K9-specific histone demethylase activity by cytostatic proline-rich polypeptide-1 leads to suppression of tumorigenic potential in human chondrosarcoma cells

    PubMed Central

    GALOIAN, KARINA; QURESHI, AMIR; WIDEROFF, GINA; TEMPLE, H.T.

    2015-01-01

    Disruption of cell-cell junctions and the concomitant loss of polarity, downregulation of tumor-suppressive adherens junctions and desmosomes represent hallmark phenotypes for several different cancer cells. Moreover, a variety of evidence supports the argument that these two common phenotypes of cancer cells directly contribute to tumorigenesis. In this study, we aimed to determine the status of intercellular junction proteins expression in JJ012 human malignant chondrosarcoma cells and investigate the effect of the antitumorigenic cytokine, proline-rich polypeptide-1 (PRP-1) on their expression. The cell junction pathway array data indicated downregulation of desmosomal proteins, such as desmoglein (1,428-fold), desmoplakin (620-fold) and plakoglobin (442-fold). The tight junction proteins claudin 11 and E-cadherin were also downregulated (399- and 52-fold, respectively). Among the upregulated proteins were the characteristic for tumors gap junction β-5 protein (connexin 31.1) and the pro-inflammatory pathway protein intercellular adhesion molecule (upregulated 129- and 43-fold, respectively). We demonstrated that PRP-1 restored the expression of the abovementioned downregulated in chondrosarcoma desmosomal proteins. PRP-1 inhibited H3K9-specific histone demethylase activity in chondrosarcoma cells in a dose-dependent manner (0.5 µg/ml PRP, 63%; 1 µg/ml PRP, 74%; and 10 µg/ml PRP, 91% inhibition). Members of the H3K9 family were shown to transcriptionally repress tumor suppressor genes and contribute to cancer progression. Our experimental data indicated that PRP-1 restores tumor suppressor desmosomal protein expression in JJ012 human chondrosarcoma cells and inhibits H3K9 demethylase activity, contributing to the suppression of tumorigenic potential in chondrosarcoma cells. PMID:25469290

  3. Hedgehog signaling regulates E-cadherin expression for the maintenance of the actin cytoskeleton and tight junctions.

    PubMed

    Xiao, Chang; Ogle, Sally A; Schumacher, Michael A; Schilling, Neal; Tokhunts, Robert A; Orr-Asman, Melissa A; Miller, Marian L; Robbins, David J; Hollande, Frederic; Zavros, Yana

    2010-12-01

    In the stomach, strictly regulated cell adherens junctions are crucial in determining epithelial cell differentiation. Sonic Hedgehog (Shh) regulates epithelial cell differentiation in the adult stomach. We sought to identify whether Shh plays a role in regulating adherens junction protein E-cadherin as a mechanism for epithelial cell differentiation. Mouse nontumorigenic gastric epithelial (IMGE-5) cells treated with Hedgehog signaling inhibitor cyclopamine and anti-Shh 5E1 antibody or transduced with short hairpin RNA against Skinny Hedgehog (IMGE-5(Ski)) were cultured. A mouse model expressing a parietal cell-specific deletion of Shh (HKCre/Shh(KO)) was used to identify further changes in adherens and tight junctions. Inhibition of Hedgehog signaling in IMGE-5 cells caused loss of E-cadherin expression accompanied by disruption of F-actin cortical expression and relocalization of zonula occludens-1 (ZO-1). Loss of E-cadherin was also associated with increased proliferation in IMGE-5(Ski) cells and increased expression of the mucous neck cell lineage marker MUC6. Compared with membrane-expressed E-cadherin and ZO-1 protein in controls, dissociation of E-cadherin/β-catenin and ZO-1/occludin protein complexes was observed in HKCre/Shh(KO) mice. In conclusion, we demonstrate that Hedgehog signaling regulates E-cadherin expression that is required for the maintenance of F-actin cortical expression and stability of tight junction protein ZO-1.

  4. CK2-dependent phosphorylation of occludin regulates the interaction with ZO-proteins and tight junction integrity

    PubMed Central

    2013-01-01

    Background Casein kinase 2 (CK2) is a ubiquitously expressed Ser/Thr kinase with multiple functions in the regulation of cell proliferation and transformation. In targeting adherens and tight junctions (TJs), CK2 modulates the strength and dynamics of epithelial cell-cell contacts. Occludin previously was identified as a substrate of CK2, however the functional consequences of CK2-dependent occludin phosphorylation on TJ function were unknown. Results Here, we present evidence that phosphorylation of a Thr400-XXX-Thr404-XXX-Ser408 motif in the C-terminal cytoplasmic tail of human occludin regulates assembly/disassembly and barrier properties of TJs. In contrast to wildtype and T400A/T404A/S408A-mutated occludin, a phospho-mimetic Occ-T400E/T404E/S408E construct was impaired in binding to ZO-2. Interestingly, pre-phosphorylation of a GST-Occ C-terminal domain fusion protein attenuated binding to ZO-2, whereas, binding to ZO-1 was not affected. Moreover, Occ-T400E/T404E/S408E showed delayed reassembly into TJs in Ca2+-switch experiments. Stable expression of Occ-T400E/T404E/S408E in MDCK C11 cells augments barrier properties in enhancing paracellular resistance in two-path impedance spectroscopy, whereas expression of wildtype and Occ-T400A/T404A/S408A did not affect transepithelial resistance. Conclusions These results suggest an important role of CK2 in epithelial tight junction regulation. The occludin sequence motif at amino acids 400–408 apparently represents a hotspot for Ser/Thr-kinase phosphorylation and depending on the residue(s) which are phosphorylated it differentially modulates the functional properties of the TJ. PMID:23758859

  5. Bacterial lipopolysaccharide increases tyrosine phosphorylation of zonula adherens proteins and opens the paracellular pathway in lung microvascular endothelia through TLR4, TRAF6, and src family kinase activation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective: LPS is a key mediator in vascular leak syndromes associated with Gram-negative bacterial infections and opens the pulmonary vascular endothelial paracellular pathway through protein tyrosine kinase (PTK) activation. We asked which PTKs and signaling molecules mediate LPS-induced endothel...

  6. Loss of αT-catenin alters the hybrid adhering junctions in the heart and leads to dilated cardiomyopathy and ventricular arrhythmia following acute ischemia.

    PubMed

    Li, Jifen; Goossens, Steven; van Hengel, Jolanda; Gao, Erhe; Cheng, Lan; Tyberghein, Koen; Shang, Xiying; De Rycke, Riet; van Roy, Frans; Radice, Glenn L

    2012-02-15

    It is generally accepted that the intercalated disc (ICD) required for mechano-electrical coupling in the heart consists of three distinct junctional complexes: adherens junctions, desmosomes and gap junctions. However, recent morphological and molecular data indicate a mixing of adherens junctional and desmosomal components, resulting in a 'hybrid adhering junction' or 'area composita'. The α-catenin family member αT-catenin, part of the N-cadherin-catenin adhesion complex in the heart, is the only α-catenin that interacts with the desmosomal protein plakophilin-2 (PKP2). Thus, it has been postulated that αT-catenin might serve as a molecular integrator of the two adhesion complexes in the area composita. To investigate the role of αT-catenin in the heart, gene targeting technology was used to delete the Ctnna3 gene, encoding αT-catenin, in the mouse. The αT-catenin-null mice are viable and fertile; however, the animals exhibit progressive cardiomyopathy. Adherens junctional and desmosomal proteins were unaffected by loss of αT-catenin, with the exception of the desmosomal protein PKP2. Immunogold labeling at the ICD demonstrated in the αT-catenin-null heart a preferential reduction of PKP2 at the area composita compared with the desmosome. Furthermore, gap junction protein Cx43 was reduced at the ICD, including its colocalization with N-cadherin. Gap junction remodeling in αT-catenin-knockout hearts was associated with an increased incidence of ventricular arrhythmias after acute ischemia. This novel animal model demonstrates for the first time how perturbation in αT-catenin can affect both PKP2 and Cx43 and thereby highlights the importance of understanding the crosstalk between the junctional proteins of the ICD and its implications for arrhythmogenic cardiomyopathy.

  7. Glutamine supplementation attenuates ethanol-induced disruption of apical junctional complexes in colonic epithelium and ameliorates gut barrier dysfunction and fatty liver in mice.

    PubMed

    Chaudhry, Kamaljit K; Shukla, Pradeep K; Mir, Hina; Manda, Bhargavi; Gangwar, Ruchika; Yadav, Nikki; McMullen, Megan; Nagy, Laura E; Rao, RadhaKrishna

    2016-01-01

    Previous in vitro studies showed that glutamine (Gln) prevents acetaldehyde-induced disruption of tight junctions and adherens junctions in Caco-2 cell monolayers and human colonic mucosa. In the present study, we evaluated the effect of Gln supplementation on ethanol-induced gut barrier dysfunction and liver injury in mice in vivo. Ethanol feeding caused a significant increase in inulin permeability in distal colon. Elevated permeability was associated with a redistribution of tight junction and adherens junction proteins and depletion of detergent-insoluble fractions of these proteins, suggesting that ethanol disrupts apical junctional complexes in colonic epithelium and increases paracellular permeability. Ethanol-induced increase in colonic mucosal permeability and disruption of junctional complexes were most severe in mice fed Gln-free diet. Gln supplementation attenuated ethanol-induced mucosal permeability and disruption of tight junctions and adherens junctions in a dose-dependent manner, indicating the potential role of Gln in nutritional intervention to alcoholic tissue injury. Gln supplementation dose-dependently elevated reduced-protein thiols in colon without affecting the level of oxidized-protein thiols. Ethanol feeding depleted reduced protein thiols and elevated oxidized protein thiols. Ethanol-induced protein thiol oxidation was most severe in mice fed with Gln-free diet and absent in mice fed with Gln-supplemented diet, suggesting that antioxidant effect is one of the likely mechanisms involved in Gln-mediated amelioration of ethanol-induced gut barrier dysfunction. Ethanol feeding elevated plasma transaminase and liver triglyceride, which was accompanied by histopathologic lesions in the liver; ethanol-induced liver damage was attenuated by Gln supplementation. These results indicate that Gln supplementation ameliorates alcohol-induced gut and liver injury.

  8. Expression of Tight Junction Proteins and Cadherin 17 in the Small Intestine of Young Goats Offered a Reduced N and/or Ca Diet.

    PubMed

    Elfers, Kristin; Marr, Isabell; Wilkens, Mirja R; Breves, Gerhard; Langeheine, Marion; Brehm, Ralph; Muscher-Banse, Alexandra S

    2016-01-01

    Diets fed to ruminants should contain nitrogen (N) as low as possible to reduce feed costs and environmental pollution. Though possessing effective N-recycling mechanisms to maintain the N supply for rumen microbial protein synthesis and hence protein supply for the host, an N reduction caused substantial changes in calcium (Ca) and phosphate homeostasis in young goats including decreased intestinal transepithelial Ca absorption as reported for monogastric species. In contrast to the transcellular component of transepithelial Ca transport, the paracellular route has not been investigated in young goats. Therefore, the aim of the present study was to characterise the effects of dietary N and/or Ca reduction on paracellular transport mechanisms in young goats. Electrophysiological properties of intestinal epithelia were investigated by Ussing chamber experiments. The expression of tight junction (TJ) and adherens junction (AJ) proteins in intestinal epithelia were examined on mRNA level by qPCR and on protein level by western blot analysis. Dietary N reduction led to a segment specific increase in tissue conductances in the proximal jejunum which might be linked to concomitantly decreased expression of cadherin 17 mRNA. Expression of occludin (OCLN) and zonula occludens protein 1 was increased in mid jejunal epithelia of N reduced fed goats on mRNA and partly on protein level. Reduced dietary Ca supply resulted in a segment specific increase in claudin 2 and claudin 12 expression and decreased the expression of OCLN which might have been mediated at least in part by calcitriol. These data show that dietary N as well as Ca reduction affected expression of TJ and AJ proteins in a segment specific manner in young goats and may thus be involved in modulation of paracellular Ca permeability.

  9. Expression of Tight Junction Proteins and Cadherin 17 in the Small Intestine of Young Goats Offered a Reduced N and/or Ca Diet

    PubMed Central

    Wilkens, Mirja R.; Breves, Gerhard; Langeheine, Marion; Brehm, Ralph; Muscher-Banse, Alexandra S.

    2016-01-01

    Diets fed to ruminants should contain nitrogen (N) as low as possible to reduce feed costs and environmental pollution. Though possessing effective N-recycling mechanisms to maintain the N supply for rumen microbial protein synthesis and hence protein supply for the host, an N reduction caused substantial changes in calcium (Ca) and phosphate homeostasis in young goats including decreased intestinal transepithelial Ca absorption as reported for monogastric species. In contrast to the transcellular component of transepithelial Ca transport, the paracellular route has not been investigated in young goats. Therefore, the aim of the present study was to characterise the effects of dietary N and/or Ca reduction on paracellular transport mechanisms in young goats. Electrophysiological properties of intestinal epithelia were investigated by Ussing chamber experiments. The expression of tight junction (TJ) and adherens junction (AJ) proteins in intestinal epithelia were examined on mRNA level by qPCR and on protein level by western blot analysis. Dietary N reduction led to a segment specific increase in tissue conductances in the proximal jejunum which might be linked to concomitantly decreased expression of cadherin 17 mRNA. Expression of occludin (OCLN) and zonula occludens protein 1 was increased in mid jejunal epithelia of N reduced fed goats on mRNA and partly on protein level. Reduced dietary Ca supply resulted in a segment specific increase in claudin 2 and claudin 12 expression and decreased the expression of OCLN which might have been mediated at least in part by calcitriol. These data show that dietary N as well as Ca reduction affected expression of TJ and AJ proteins in a segment specific manner in young goats and may thus be involved in modulation of paracellular Ca permeability. PMID:27120348

  10. Tight junctions and the regulation of gene expression.

    PubMed

    Balda, Maria S; Matter, Karl

    2009-04-01

    Cell adhesion is a key regulator of cell differentiation. Cell interactions with neighboring cells and the extracellular matrix regulate gene expression, cell proliferation, polarity and apoptosis. Apical cell-cell junctions participate in these processes using different types of proteins, some of them exhibit nuclear and junctional localization and are called NACos for Nuclear Adhesion Complexes. Tight junctions are one type of such cell-cell junctions and several signaling complexes have been identified to associate with them. In general, expression of tight junction components suppresses proliferation to allow differentiation in a coordinated manner with adherens junctions and extracellular matrix adhesion. These tight junction components have been shown to affect several signaling and transcriptional pathways, and changes in the expression of tight junction proteins are associated with several disease conditions, such as cancer. Here, we will review how tight junction proteins participate in the regulation of gene expression and cell proliferation, as well as how they are regulated themselves by different mechanisms involved in gene expression and cell differentiation.

  11. Human Articular Chondrocytes Express Multiple Gap Junction Proteins

    PubMed Central

    Mayan, Maria D.; Carpintero-Fernandez, Paula; Gago-Fuentes, Raquel; Martinez-de-Ilarduya, Oskar; Wang, Hong-Zhang; Valiunas, Virginijus; Brink, Peter; Blanco, Francisco J.

    2014-01-01

    Osteoarthritis (OA) is the most common joint disease and involves progressive degeneration of articular cartilage. The aim of this study was to investigate if chondrocytes from human articular cartilage express gap junction proteins called connexins (Cxs). We show that human chondrocytes in tissue express Cx43, Cx45, Cx32, and Cx46. We also find that primary chondrocytes from adults retain the capacity to form functional voltage-dependent gap junctions. Immunohistochemistry experiments in cartilage from OA patients revealed significantly elevated levels of Cx43 and Cx45 in the superficial zone and down through the next approximately 1000 μm of tissue. These zones corresponded with regions damaged in OA that also had high levels of proliferative cell nuclear antigen. An increased number of Cxs may help explain the increased proliferation of cells in clusters that finally lead to tissue homeostasis loss. Conversely, high levels of Cxs in OA cartilage reflect the increased number of adjacent cells in clusters that are able to interact directly by gap junctions as compared with hemichannels on single cells in normal cartilage. Our data provide strong evidence that OA patients have a loss of the usual ordered distribution of Cxs in the damaged zones and that the reductions in Cx43 levels are accompanied by the loss of correct Cx localization in the nondamaged areas. PMID:23416160

  12. Cell culture model predicts human disease: Altered expression of junction proteins and matrix metalloproteinases in cervical dysplasia

    PubMed Central

    2012-01-01

    Background Cervical cancer is necessarily caused by human papillomaviruses, which encode three oncogenes manifesting their functions by interfering with a number of cellular proteins and pathways: the E5, E6, and E7 proteins. We have earlier found in our microarray studies that the E5 oncogene crucially affects the expression of cellular genes involved in adhesion and motility of epithelial cells. Methods In order to biologically validate our previous experimental findings we performed immunohistochemical staining of a representative set of tissue samples from different grades of high-risk human papillomavirus associated cervical disease as well as normal squamous and columnar cervical epithelium. Three-dimensional collagen raft cultures established from E5-expressing and control epithelial cells were also examined. The expression of p16, matrix metalloproteinase (MMP) -7, MMP-16, cytokeratin (CK) 8/18, laminin, E-cadherin and beta-catenin was studied. Results In agreement with our previous microarray studies, we found intense staining for E-cadherin and beta-catenin in adherens junctions even in high-grade cervical lesions. Staining for MMP-16 was increased in severe disease as well. No significant change in staining for MMP-7 and cytokeratin 8/18 along with the grade of cervical squamous epithelial disease was observed. Conclusions Here we have confirmed, using tissue material from human papillomavirus associated lesions, some of the cellular gene expression modifications that we earlier reported in an experimental system studying specifically the E5 oncogene of papillomaviruses. These findings were partially surprising in the context of cervical carcinogenesis and emphasize that the complexity of carcinogenesis is not yet fully understood. Microarray approaches provide a wide overwiev of gene expression in experimental settings, which may yield biologically valid biomarkers for disease diagnostics, prognosis, and follow-up. PMID:22863036

  13. Gap junction proteins and their role in spinal cord injury

    PubMed Central

    Tonkin, Ryan S.; Mao, Yilin; O’Carroll, Simon J.; Nicholson, Louise F. B.; Green, Colin R.; Gorrie, Catherine A.; Moalem-Taylor, Gila

    2015-01-01

    Gap junctions are specialized intercellular communication channels that are formed by two hexameric connexin hemichannels, one provided by each of the two adjacent cells. Gap junctions and hemichannels play an important role in regulating cellular metabolism, signaling, and functions in both normal and pathological conditions. Following spinal cord injury (SCI), there is damage and disturbance to the neuronal elements of the spinal cord including severing of axon tracts and rapid cell death. The initial mechanical disruption is followed by multiple secondary cascades that cause further tissue loss and dysfunction. Recent studies have implicated connexin proteins as playing a critical role in the secondary phase of SCI by propagating death signals through extensive glial networks. In this review, we bring together past and current studies to outline the distribution, changes and roles of various connexins found in neurons and glial cells, before and in response to SCI. We discuss the contribution of pathologically activated connexin proteins, in particular connexin 43, to functional recovery and neuropathic pain, as well as providing an update on potential connexin specific pharmacological agents to treat SCI. PMID:25610368

  14. Tricellulin is a tight-junction protein necessary for hearing.

    PubMed

    Riazuddin, Saima; Ahmed, Zubair M; Fanning, Alan S; Lagziel, Ayala; Kitajiri, Shin-ichiro; Ramzan, Khushnooda; Khan, Shaheen N; Chattaraj, Parna; Friedman, Penelope L; Anderson, James M; Belyantseva, Inna A; Forge, Andrew; Riazuddin, Sheikh; Friedman, Thomas B

    2006-12-01

    The inner ear has fluid-filled compartments of different ionic compositions, including the endolymphatic and perilymphatic spaces of the organ of Corti; the separation from one another by epithelial barriers is required for normal hearing. TRIC encodes tricellulin, a recently discovered tight-junction (TJ) protein that contributes to the structure and function of tricellular contacts of neighboring cells in many epithelial tissues. We show that, in humans, four different recessive mutations of TRIC cause nonsyndromic deafness (DFNB49), a surprisingly limited phenotype, given the widespread tissue distribution of tricellulin in epithelial cells. In the inner ear, tricellulin is concentrated at the tricellular TJs in cochlear and vestibular epithelia, including the structurally complex and extensive junctions between supporting and hair cells. We also demonstrate that there are multiple alternatively spliced isoforms of TRIC in various tissues and that mutations of TRIC associated with hearing loss remove all or most of a conserved region in the cytosolic domain that binds to the cytosolic scaffolding protein ZO-1. A wild-type isoform of tricellulin, which lacks this conserved region, is unaffected by the mutant alleles and is hypothesized to be sufficient for structural and functional integrity of epithelial barriers outside the inner ear. PMID:17186462

  15. A lens intercellular junction protein, MP26, is a phosphoprotein.

    PubMed

    Johnson, K R; Lampe, P D; Hur, K C; Louis, C F; Johnson, R G

    1986-04-01

    The major protein present in the plasma membrane of the bovine lens fiber cell (MP26), thought to be a component of intercellular junctions, was phosphorylated in an in vivo labeling procedure. After fragments of decapsulated fetal bovine lenses were incubated with [32P]orthophosphate, membranes were isolated and analyzed by SDS PAGE and autoradiography. A number of lens membrane proteins were routinely phosphorylated under these conditions. These proteins included species at Mr 17,000 and 26,000 as well as a series at both 34,000 and 55,000. The label at Mr 26,000 appeared to be associated with MP26, since (a) boiling the membrane sample in SDS led to both an aggregation of MP26 and a loss of label at Mr 26,000, (b) the label at 26,000 was resistant to both urea and nonionic detergents, and (c) two-dimensional gels showed that a phosphorylated Mr 24,000 fragment was derived from MP26 with V8 protease. Studies with proteases also provided for a localization of most label within approximately 20 to 40 residues from the COOH-terminus of MP26. Published work indicates that the phosphorylated portion of MP26 resides on the cytoplasmic side of the membrane, and that this region of MP26 contains a number of serine residues. The same region of MP26 was labeled when isolated lens membranes were reacted with a cAMP-dependent protein kinase prepared from the bovine lens. After the in vivo labeling of lens fragments, phosphoamino acid analysis of MP26 demonstrated primarily labeled serines, with 5-10% threonines and no tyrosines. Treatments that lowered the intracellular calcium levels in the in vivo system led to a selective reduction of MP26 phosphorylation. In addition, forskolin and cAMP stimulated the phosphorylation of MP26 and other proteins in concentrated lens homogenates. These findings are of interest because MP26 appears to serve as a protein of cell-to-cell channels in the lens, perhaps as a lens gap junction protein. PMID:3958048

  16. Fibrinogen Induces Alterations of Endothelial Cell Tight Junction Proteins

    PubMed Central

    PATIBANDLA, PHANI K.; TYAGI, NEETU; DEAN, WILLIAM L.; TYAGI, SURESH C.; ROBERTS, ANDREW M.; LOMINADZE, DAVID

    2009-01-01

    We previously showed that an elevated content of fibrinogen (Fg) increased formation of filamentous actin and enhanced endothelial layer permeability. In the present work we tested the hypothesis that Fg binding to endothelial cells (ECs) alters expression of actin-associated endothelial tight junction proteins (TJP). Rat cardiac microvascular ECs were grown in gold plated chambers of an electrical cell-substrate impedance system, 8-well chambered, or in 12-well plates. Confluent ECs were treated with Fg (2 or 4 mg/ml), Fg (4 mg/ml) with mitogen-activated protein kinase (MEK) kinase inhibitors (PD98059 or U0126), Fg (4 mg/ml) with anti-ICAM-1 antibody or BQ788 (endothelin type B receptor blocker), endothelin-1, endothelin-1 with BQ788, or medium alone for 24 h. Fg induced a dose-dependent decrease in EC junction integrity as determined by transendothelial electrical resistance (TEER). Western blot analysis and RT-PCR data showed that the higher dose of Fg decreased the contents of TJPs, occludin, zona occluden-1 (ZO-1), and zona occluden-2 (ZO-2) in ECs. Fg-induced decreases in contents of the TJPs were blocked by PD98059, U0126, or anti-ICAM-1 antibody. While BQ788 inhibited endothelin-1-induced decrease in TEER, it did not affect Fg-induced decrease in TEER. These data suggest that Fg increases EC layer permeability via the MEK kinase signaling pathway by affecting occludin, ZO-1, and ZO-2, TJPs, which are bound to actin filaments. Therefore, increased binding of Fg to its major EC receptor, ICAM-1, during cardiovascular diseases may increase microvascular permeability by altering the content and possibly subcellular localization of endothelial TJPs. PMID:19507189

  17. A General Method for Insertion of Functional Proteins within Proteins via Combinatorial Selection of Permissive Junctions.

    PubMed

    Peng, Yingjie; Zeng, Wenwen; Ye, Hui; Han, Kyung Ho; Dharmarajan, Venkatasubramanian; Novick, Scott; Wilson, Ian A; Griffin, Patrick R; Friedman, Jeffrey M; Lerner, Richard A

    2015-08-20

    A major goal of modern protein chemistry is to create new proteins with different functions. One approach is to amalgamate secondary and tertiary structures from different proteins. This is difficult for several reasons, not the least of which is the fact that the junctions between secondary and tertiary structures are not degenerate and usually affect the function and folding of the entire complex. Here, we offer a solution to this problem by coupling a large combinatorial library of about 10(7) different N- and C-terminal junctions to a powerful system that selects for function. Using this approach, the entire Leptin and follicle-stimulating hormone (FSH) were inserted into an antibody. Complexes with full retention of function in vivo and in vitro, although rare, were found easily by using an autocrine selection system to search for hormonal activity. Such large diversity systems, when coupled to robust selection systems, should enable construction of novel therapeutic proteins.

  18. Organization of multiprotein complexes at cell–cell junctions

    PubMed Central

    2008-01-01

    The formation of stable cell–cell contacts is required for the generation of barrier-forming sheets of epithelial and endothelial cells. During various physiological processes like tissue development, wound healing or tumorigenesis, cellular junctions are reorganized to allow the release or the incorporation of individual cells. Cell–cell contact formation is regulated by multiprotein complexes which are localized at specific structures along the lateral cell junctions like the tight junctions and adherens junctions and which are targeted to these site through their association with cell adhesion molecules. Recent evidence indicates that several major protein complexes exist which have distinct functions during junction formation. However, this evidence also indicates that their composition is dynamic and subject to changes depending on the state of junction maturation. Thus, cell–cell contact formation and integrity is regulated by a complex network of protein complexes. Imbalancing this network by oncogenic proteins or pathogens results in barrier breakdown and eventually in cancer. Here, I will review the molecular organization of the major multiprotein complexes at junctions of epithelial cells and discuss their function in cell–cell contact formation and maintenance. PMID:18365233

  19. A protein homologous to the Torpedo postsynaptic 58K protein is present at the myotendinous junction

    PubMed Central

    1990-01-01

    The 58K protein is a peripheral membrane protein enriched in the acetylcholine receptor (AChR)-rich postsynaptic membrane of Torpedo electric organ. Because of its coexistence with AChRs in the postsynaptic membrane in both electrocytes and skeletal muscle, it is thought to be involved in the formation and maintenance of AChR clusters. Using an mAb against the 58K protein of Torpedo electric organ, we have identified a single protein band in SDS-PAGE analysis of Xenopus myotomal muscle with an apparent molecular mass of 48 kD. With this antibody, the distribution of this protein was examined in the myotomal muscle fibers with immunofluorescence techniques. We found that the 48K protein is concentrated at the myotendinous junctions (MTJs) of these muscle fibers. The MTJ is also enriched in talin and vinculin. By double labeling muscle fibers with antibodies against talin and the 48K protein, these two proteins were found to colocalize at the membrane invaginations of the MTJ. In cultured myotomal muscle cells, the 48K protein and talin are also colocalized at sites of membrane-myofibril interaction. The 48K protein is, however, not found at focal adhesion sites in nonmuscle cells, which are enriched in talin. These data suggest that the 48K protein is specifically involved in the interaction of myofibrillar actin filaments with the plasma membrane at the MTJ. In addition to the MTJ localization, 48K protein is also present at AChR clusters both in vivo and in vitro. Thus, this protein is shared by both the MTJ and the neuromuscular junction. PMID:2112550

  20. Transitions of protein traffic from cardiac ER to junctional SR

    PubMed Central

    Sleiman, Naama H.; McFarland, Timothy P.; Jones, Larry R.; Cala, Steven E.

    2015-01-01

    The junctional sarcoplasmic reticulum (jSR) is an important and unique ER subdomain in the adult myocyte that concentrates resident proteins to regulate Ca2+ release. To investigate cellular mechanisms for sorting and trafficking proteins to jSR, we overexpressed canine forms of junctin (JCT) or triadin (TRD) in adult rat cardiomyocytes. Protein accumulation over time was visualized by confocal fluorescence microscopy using species-specific antibodies. Newly synthesized JCTdog and TRDdog appeared by 12-24 h as bright fluorescent puncta close to the nuclear surface, decreasing in intensity with increasing radial distance. With increasing time (24-48 h), fluorescent puncta appeared at further radial distances from the nuclear surface, eventually populating jSR similar to steady-state patterns. CSQ2-DsRed, a form of CSQ that polymerizes ectopically in rough ER, prevented anterograde traffic of newly made TRDdog and JCTdog, demonstrating common pathways of intracellular trafficking as well as in situ binding to CSQ2 in juxtanuclear rough ER. Reversal of CSQD-sRed interactions occurred when a form of TRDdog was used in which CSQ2-binding sites are removed (delTRD). With increasing levels of expression, CSQ2-DsRed revealed a novel smooth ER network that surrounds nuclei and connects the nuclear axis. TRDdog was retained in smooth ER by binding to CSQ2-DsRed, but escaped to populate jSR puncta. TRDdog and del TRD were therefore able to elucidate areas of ER-SR transition. High levels of CSQ2-DsRed in the ER led to loss of jSR puncta labeling, suggesting a plasticity of ER-SR transition sites. We propose a model of ER and SR protein traffic along microtubules, with prominent transverse/radial ER trafficking of JCT and TRD along Z-lines to populate jSR, and an abundant longitudinal/axial smooth ER between and encircling myonuclei, from which jSR proteins traffic. PMID:25640161

  1. The Mr 28,000 gap junction proteins from rat heart and liver are different but related.

    PubMed

    Nicholson, B J; Gros, D B; Kent, S B; Hood, L E; Revel, J P

    1985-06-10

    The sequence of the amino-terminal 32 residues of the rat heart Mr 28,000 gap junction protein presented here allows, for the first time, a sequence comparison of gap junctional proteins from different tissues (heart and liver). Comparison of the rat heart gap junction protein sequence and that available from rat liver reveals 43% sequence identity and conservative changes at an additional 25% of the positions. Both proteins exhibit a hydrophobic domain which could represent a transmembrane span of the junction. This result unequivocally demonstrates the existence of at least two forms of the gap junction protein. As yet, no homology is evident between the gap junctional proteins of either heart or liver and main intrinsic protein from rat eye lens. PMID:2987225

  2. AMP-activated protein kinase regulates the assembly of epithelial tight junctions

    PubMed Central

    Zhang, Li; Li, Ji; Young, Lawrence H.; Caplan, Michael J.

    2006-01-01

    AMP activated protein kinase (AMPK), a sensor of cellular energy status in all eukaryotic cells, is activated by LKB1-dependent phosphorylation. Recent studies indicate that activated LKB1 induces polarity in epithelial cells and that this polarization is accompanied by the formation of tight junction structures. We wished to determine whether AMPK also contributes to the assembly of tight junctions in the epithelial cell polarization process. We found that AMPK is activated during calcium-induced tight junction assembly. Activation of AMPK by 5-aminoimidazole-4-carboxamide ribonucleoside facilitates tight junction assembly under conditions of normal extracellular Ca2+ concentrations and initiates tight junction assembly in the absence of Ca2+ as revealed by the relocation of zonula occludens 1, the establishment of transepithelial electrical resistance, and the paracellular flux assay. Expression of a dominant negative AMPK construct inhibits tight junction assembly in MDCK cells, and this defect in tight junction assembly can be partially ameliorated by rapamycin. These results suggest that AMPK plays a role in the regulation of tight junction assembly. PMID:17088526

  3. AMP-activated protein kinase regulates the assembly of epithelial tight junctions.

    PubMed

    Zhang, Li; Li, Ji; Young, Lawrence H; Caplan, Michael J

    2006-11-14

    AMP activated protein kinase (AMPK), a sensor of cellular energy status in all eukaryotic cells, is activated by LKB1-dependent phosphorylation. Recent studies indicate that activated LKB1 induces polarity in epithelial cells and that this polarization is accompanied by the formation of tight junction structures. We wished to determine whether AMPK also contributes to the assembly of tight junctions in the epithelial cell polarization process. We found that AMPK is activated during calcium-induced tight junction assembly. Activation of AMPK by 5-aminoimidazole-4-carboxamide ribonucleoside facilitates tight junction assembly under conditions of normal extracellular Ca2+ concentrations and initiates tight junction assembly in the absence of Ca2+ as revealed by the relocation of zonula occludens 1, the establishment of transepithelial electrical resistance, and the paracellular flux assay. Expression of a dominant negative AMPK construct inhibits tight junction assembly in MDCK cells, and this defect in tight junction assembly can be partially ameliorated by rapamycin. These results suggest that AMPK plays a role in the regulation of tight junction assembly. PMID:17088526

  4. Estrogen Modulates Expression of Tight Junction Proteins in Rat Vagina

    PubMed Central

    Oh, Kyung-Jin; Ahn, Kyuyoun

    2016-01-01

    Background. The objectives of this study were to investigate the localization of tight junctions and the modulation of zonula occludens- (ZO-) 1, occludin and claudin-1 expression by estrogen in castrated female rat vagina. Female Sprague-Dawley rats (230–240 g, n = 45) were divided into three groups and subjected to a sham operation (control group, n = 15), bilateral ovariectomy (Ovx group, n = 15), or bilateral ovariectomy followed by daily subcutaneous injection of 17β-estradiol (50 μg/kg/day, Ovx + Est group, n = 15). The cellular localization and expression of ZO-1, occludin, and claudin-1 were determined in each group by immunohistochemistry and western blot. Results. Expression of ZO-1 was diffuse in all groups, with the highest intensity in the superficial epithelium in the control group. Occludin was localized in the intermediate and basal epithelium. Claudin-1 was most intense in the superficial layer of the vaginal epithelium in the control group. Expression of ZO-1, occludin, and claudin-1 was significantly decreased after ovariectomy and was restored to the level of the control after estrogen replacement. Conclusions. Tight junctions are distinctly localized in rat vagina, and estrogen modulates the expression of tight junctions. Further researches are needed to clarify the functional role of tight junctions in vaginal lubrication. PMID:27127786

  5. A Cul-3-BTB ubiquitylation pathway regulates junctional levels and asymmetry of core planar polarity proteins

    PubMed Central

    Strutt, Helen; Searle, Elizabeth; Thomas-MacArthur, Victoria; Brookfield, Rosalind; Strutt, David

    2013-01-01

    The asymmetric localisation of core planar polarity proteins at apicolateral junctions is required to specify cell polarity in the plane of epithelia. This asymmetric distribution of the core proteins is proposed to require amplification of an initial asymmetry by feedback loops. In addition, generation of asymmetry appears to require the regulation of core protein levels, but the importance of such regulation and the underlying mechanisms is unknown. Here we show that ubiquitylation acts through more than one mechanism to control core protein levels in Drosophila, and that without this regulation cellular asymmetry is compromised. Levels of Dishevelled at junctions are regulated by a Cullin-3-Diablo/Kelch ubiquitin ligase complex, the activity of which is most likely controlled by neddylation. Furthermore, activity of the deubiquitylating enzyme Fat facets is required to maintain Flamingo levels at junctions. Notably, ubiquitylation does not alter the total cellular levels of Dishevelled or Flamingo, but only that of the junctional population. When junctional core protein levels are either increased or decreased by disruption of the ubiquitylation machinery, their asymmetric localisation is reduced and this leads to disruption of planar polarity at the tissue level. Loss of asymmetry by altered core protein levels can be explained by reference to feedback models for amplification of asymmetry. PMID:23487316

  6. Hepatic immunohistochemical localization of the tight junction protein ZO-1 in rat models of cholestasis.

    PubMed Central

    Anderson, J. M.; Glade, J. L.; Stevenson, B. R.; Boyer, J. L.; Mooseker, M. S.

    1989-01-01

    Structural alterations in hepatocyte tight junctions accompanying cholestasis were investigated using immunolocalization of ZO-1, the first known protein component of the tight junction. Disruption in the paracellular barrier function of the tight junction has been proposed to allow reflux of bile into the blood. Cholestasis was induced in 210 to 235 g male Sprague-Dawley rats either by five consecutive daily subcutaneous injections of 17-alpha-ethinyl estradiol (0.5 mg/kg/d in propylene glycol) or ligation of the common bile duct for 72 hours. The structural organization of the tight junction was assessed in each model by indirect immunofluorescent and immunoperoxidase staining for ZO-1 on frozen sections of liver and compared with controls. In control, sham-operated, and estradiol-injected animals, ZO-1 localizes in a uniform continuous manner along the margins of the canaliculi. In contrast, bile duct ligation results in the appearance of numerous discontinuities in ZO-1 staining accompanied by dilation or collapse of the lumenal space. Tissue content of the ZO-1 protein, as determined by quantitative immunoblotting, was unaffected in either cholestatic model compared with controls. These findings indicate that the molecular organization of the tight junction can be assessed from immunostaining patterns of ZO-1 in frozen sections of cholestatic livers. Under these experimental conditions, the organization of the tight junction at the level of the ZO-1 protein is altered by bile duct obstruction but not by ethinyl estradiol. Images Figure 1 Figure 2 PMID:2719075

  7. LRP6 acts as a scaffold protein in cardiac gap junction assembly.

    PubMed

    Li, Jun; Li, Changming; Liang, Dandan; Lv, Fei; Yuan, Tianyou; The, Erlinda; Ma, Xiue; Wu, Yahan; Zhen, Lixiao; Xie, Duanyang; Wang, Shiyi; Liu, Yuan; Huang, Jian; Shi, Jingyi; Liu, Yi; Shi, Dan; Xu, Liang; Lin, Li; Peng, Luying; Cui, Jianmin; Zhu, Weidong; Chen, Yi-Han

    2016-01-01

    Low-density lipoprotein receptor-related protein 6 (LRP6) is a Wnt co-receptor in the canonical Wnt/β-catenin signalling. Here, we report the scaffold function of LRP6 in gap junction formation of cardiomyocytes. Cardiac LRP6 is spatially restricted to intercalated discs and binds to gap junction protein connexin 43 (Cx43). A deficiency in LRP6 disrupts Cx43 gap junction formation and thereby impairs the cell-to-cell coupling, which is independent of Wnt/β-catenin signalling. The defect in Cx43 gap junction resulting from LRP6 reduction is attributable to the defective traffic of de novo Cx43 proteins from the endoplasmic reticulum to the Golgi apparatus, leading to the lysosomal degradation of Cx43 proteins. Accordingly, the hearts of conditional cardiac-specific Lrp6-knockout mice consistently exhibit overt reduction of Cx43 gap junction plaques without any abnormality in Wnt signalling and are predisposed to lethal arrhythmias. These findings uncover a distinct role of LRP6 as a platform for intracellular protein trafficking.

  8. Intraepithelial lymphocytes express junctional molecules in murine small intestine

    SciTech Connect

    Inagaki-Ohara, Kyoko . E-mail: INAGAKI@med.miyazaki-u.ac.jp; Sawaguchi, Akira; Suganuma, Tatsuo; Matsuzaki, Goro; Nawa, Yukifumi

    2005-06-17

    Intestinal intraepithelial lymphocytes (IEL) that reside at basolateral site regulate the proliferation and differentiation of epithelial cells (EC) for providing a first line of host defense in intestine. However, it remains unknown how IEL interact and communicate with EC. Here, we show that IEL express junctional molecules like EC. We identified mRNA expression of the junctional molecules in IEL such as zonula occludens (ZO)-1, occludin and junctional adhesion molecule (JAM) (tight junction), {beta}-catenin and E-cadherin (adherens junction), and connexin26 (gap junction). IEL constitutively expressed occludin and E-cadherin at protein level, while other T cells in the thymus, spleen, liver, mesenteric lymph node, and Peyer's patches did not. {gamma}{delta} IEL showed higher level of these expressions than {alpha}{beta} IEL. The expression of occludin was augmented by anti-CD3 Ab stimulation. These results suggest the possibility of a novel role of IEL concerning epithelial barrier and communication between IEL and EC.

  9. Protein-RNA Dynamics in the Central Junction Control 30S Ribosome Assembly.

    PubMed

    Baker, Kris Ann; Lamichhane, Rajan; Lamichhane, Tek; Rueda, David; Cunningham, Philip R

    2016-09-11

    Interactions between ribosomal proteins (rproteins) and ribosomal RNA (rRNA) facilitate the formation of functional ribosomes. S15 is a central domain primary binding protein that has been shown to trigger a cascade of conformational changes in 16S rRNA, forming the functional structure of the central domain. Previous biochemical and structural studies in vitro have revealed that S15 binds a three-way junction of helices 20, 21, and 22, including nucleotides 652-654 and 752-754. All junction nucleotides except 653 are highly conserved among the Bacteria. To identify functionally important motifs within the junction, we subjected nucleotides 652-654 and 752-754 to saturation mutagenesis and selected and analyzed functional mutants. Only 64 mutants with greater than 10% ribosome function in vivo were isolated. S15 overexpression complemented mutations in the junction loop in each of the partially active mutants, although mutations that produced inactive ribosomes were not complemented by overexpression of S15. Single-molecule Förster or fluorescence resonance energy transfer (smFRET) was used to study the Mg(2+)- and S15-induced conformational dynamics of selected junction mutants. Comparison of the structural dynamics of these mutants with the wild type in the presence and absence of S15 revealed specific sequence and structural motifs in the central junction that are important in ribosome function. PMID:27192112

  10. Protein tyrosine phosphatase σ targets apical junction complex proteins in the intestine and regulates epithelial permeability.

    PubMed

    Murchie, Ryan; Guo, Cong-Hui; Persaud, Avinash; Muise, Aleixo; Rotin, Daniela

    2014-01-14

    Protein tyrosine phosphatase (PTP)σ (PTPRS) was shown previously to be associated with susceptibility to inflammatory bowel disease (IBD). PTPσ(-/-) mice exhibit an IBD-like phenotype in the intestine and show increased susceptibility to acute models of murine colitis. However, the function of PTPσ in the intestine is uncharacterized. Here, we show an intestinal epithelial barrier defect in the PTPσ(-/-) mouse, demonstrated by a decrease in transepithelial resistance and a leaky intestinal epithelium that was determined by in vivo tracer analysis. Increased tyrosine phosphorylation was observed at the plasma membrane of epithelial cells lining the crypts of the small bowel and colon of the PTPσ(-/-) mouse, suggesting the presence of PTPσ substrates in these regions. Using mass spectrometry, we identified several putative PTPσ intestinal substrates that were hyper-tyrosine-phosphorylated in the PTPσ(-/-) mice relative to wild type. Among these were proteins that form or regulate the apical junction complex, including ezrin. We show that ezrin binds to and is dephosphorylated by PTPσ in vitro, suggesting it is a direct PTPσ substrate, and identified ezrin-Y353/Y145 as important sites targeted by PTPσ. Moreover, subcellular localization of the ezrin phosphomimetic Y353E or Y145 mutants were disrupted in colonic Caco-2 cells, similar to ezrin mislocalization in the colon of PTPσ(-/-) mice following induction of colitis. Our results suggest that PTPσ is a positive regulator of intestinal epithelial barrier, which mediates its effects by modulating epithelial cell adhesion through targeting of apical junction complex-associated proteins (including ezrin), a process impaired in IBD.

  11. Epiprofin/Sp6 regulates Wnt-BMP signaling and the establishment of cellular junctions during the bell stage of tooth development.

    PubMed

    Ibarretxe, Gaskon; Aurrekoetxea, Maitane; Crende, Olatz; Badiola, Iker; Jimenez-Rojo, Lucia; Nakamura, Takashi; Yamada, Yoshihiko; Unda, Fernando

    2012-10-01

    Epiprofin/Specificity Protein 6 (Epfn) is a Krüppel-like family (KLF) transcription factor that is critically involved in tooth morphogenesis and dental cell differentiation. However, its mechanism of action is still not fully understood. We have employed both loss-of-function and gain-of-function approaches to address the role of Epfn in the formation of cell junctions in dental cells and in the regulation of junction-associated signal transduction pathways. We have evaluated the expression of junction proteins in bell-stage incisor and molar tooth sections from Epfn(-/-) mice and in dental pulp MDPC-23 cells overexpressing Epfn. In Epfn(-/-) mice, a dramatic reduction occurs in the expression of tight junction and adherens junction proteins and of the adherens-junction-associated β-catenin protein, a major effector of canonical Wnt signaling. Loss of cell junctions and β-catenin in Epfn(-/-) mice is correlated with a clear decrease in bone morphogenetic protein 4 (BMP-4) expression, a decrease in nestin in the tooth mesenchyme, altered cell proliferation, and failure of ameloblast cell differentiation. Overexpression of Epfn in MDPC-23 cells results in an increased cellular accumulation of β-catenin protein, indicative of upregulation of canonical Wnt signaling. Together, these results suggest that Epfn enhances canonical Wnt/β-catenin signaling in the developing dental pulp mesenchyme, a condition that promotes the activity of other downstream signaling pathways, such as BMP, which are fundamental for cellular induction and ameloblast differentiation. These altered signaling events might underlie some of the most prominent dental defects observed in Epfn(-/-) mice, such as the absence of ameloblasts and enamel, and might throw light on developmental malformations of the tooth, including hyperdontia.

  12. Neisseria gonorrhoeae induced disruption of cell junction complexes in epithelial cells of the human genital tract.

    PubMed

    Rodríguez-Tirado, Carolina; Maisey, Kevin; Rodríguez, Felipe E; Reyes-Cerpa, Sebastián; Reyes-López, Felipe E; Imarai, Mónica

    2012-03-01

    Pathogenic microorganisms, such as Neisseria gonorrhoeae, have developed mechanisms to alter epithelial barriers in order to reach subepithelial tissues for host colonization. The aim of this study was to examine the effects of gonococci on cell junction complexes of genital epithelial cells of women. Polarized Ishikawa cells, a cell line derived from endometrial epithelium, were used for experimental infection. Infected cells displayed a spindle-like shape with an irregular distribution, indicating potential alteration of cell-cell contacts. Accordingly, analysis by confocal microscopy and cellular fractionation revealed that gonococci induced redistribution of the adherens junction proteins E-cadherin and its adapter protein β-catenin from the membrane to a cytoplasmic pool, with no significant differences in protein levels. In contrast, gonococcal infection did not induce modification of either expression or distribution of the tight junction proteins Occludin and ZO-1. Similar results were observed for Fallopian tube epithelia. Interestingly, infected Ishikawa cells also showed an altered pattern of actin cytoskeleton, observed in the form of stress fibers across the cytoplasm, which in turn matched a strong alteration on the expression of fibronectin, an adhesive glycoprotein component of extracellular matrix. Interestingly, using western blotting, activation of the ERK pathway was detected after gonococcal infection while p38 pathway was not activated. All effects were pili and Opa independent. Altogether, results indicated that gonococcus, as a mechanism of pathogenesis, induced disruption of junction complexes with early detaching of E-cadherin and β-catenin from the adherens junction complex, followed by a redistribution and reorganization of actin cytoskeleton and fibronectin within the extracellular matrix. PMID:22146107

  13. Septate Junction Proteins Play Essential Roles in Morphogenesis Throughout Embryonic Development in Drosophila

    PubMed Central

    Hall, Sonia; Ward, Robert E.

    2016-01-01

    The septate junction (SJ) is the occluding junction found in the ectodermal epithelia of invertebrate organisms, and is essential to maintain chemically distinct compartments in epithelial organs, to provide the blood–brain barrier in the nervous system, and to provide an important line of defense against invading pathogens. More than 20 genes have been identified to function in the establishment or maintenance of SJs in Drosophila melanogaster. Numerous studies have demonstrated the cell biological function of these proteins in establishing the occluding junction, whereas very few studies have examined further developmental roles for them. Here we examined embryos with mutations in nine different core SJ genes and found that all nine result in defects in embryonic development as early as germ band retraction, with the most penetrant defect observed in head involution. SJ genes are also required for cell shape changes and cell rearrangements that drive the elongation of the salivary gland during midembryogenesis. Interestingly, these developmental events occur at a time prior to the formation of the occluding junction, when SJ proteins localize along the lateral membrane and have not yet coalesced into the region of the SJ. Together, these observations reveal an underappreciated role for a large group of SJ genes in essential developmental events during embryogenesis, and suggest that the function of these proteins in facilitating cell shape changes and rearrangements is independent of their role in the occluding junction. PMID:27261004

  14. Morphological adaptation and protein modulation of myotendinous junction following moderate aerobic training.

    PubMed

    Curzi, Davide; Baldassarri, Valentina; De Matteis, Rita; Salamanna, Francesca; Bolotta, Alessandra; Frizziero, Antonio; Fini, Milena; Marini, Marina; Falcieri, Elisabetta

    2015-04-01

    Myotendinous junction is the muscle-tendon interface through which the contractile force can be transferred from myofibrils to the tendon extracellular matrix. At the ultrastructural level, aerobic training can modify the distal myotendinous junction of rat gastrocnemius, increasing the contact area between tissues. The aim of this work is to investigate the correlation between morphological changes and protein modulation of the myotendinous junction following moderate training. For this reason, talin, vinculin and type IV collagen amount and spatial distribution were investigated by immunohistochemistry and confocal microscopy. The images were then digitally analyzed by evaluating fluorescence intensity. Morphometric analysis revealed a significant increased thickening of muscle basal lamina in the trained group (53.1 ± 0.4 nm) with respect to the control group (43.9 ± 0.3 nm), and morphological observation showed the presence of an electron-dense area in the exercised muscles, close to the myotendinous junction. Protein concentrations appeared significantly increased in the trained group (talin +22.2%; vinculin +22.8% and type IV collagen +11.8%) with respect to the control group. Therefore, our findings suggest that moderate aerobic training induces/causes morphological changes at the myotendinous junction, correlated to the synthesis of structural proteins of the muscular basal lamina and of the cytoskeleton.

  15. West Nile virus infection causes endocytosis of a specific subset of tight junction membrane proteins.

    PubMed

    Xu, Zaikun; Waeckerlin, Regula; Urbanowski, Matt D; van Marle, Guido; Hobman, Tom C

    2012-01-01

    West Nile virus (WNV) is a blood-borne pathogen that causes systemic infections and serious neurological disease in human and animals. The most common route of infection is mosquito bites and therefore, the virus must cross a number of polarized cell layers to gain access to organ tissue and the central nervous system. Resistance to trans-cellular movement of macromolecules between epithelial and endothelial cells is mediated by tight junction complexes. While a number of recent studies have documented that WNV infection negatively impacts the barrier function of tight junctions, the intracellular mechanism by which this occurs is poorly understood. In the present study, we report that endocytosis of a subset of tight junction membrane proteins including claudin-1 and JAM-1 occurs in WNV infected epithelial and endothelial cells. This process, which ultimately results in lysosomal degradation of the proteins, is dependent on the GTPase dynamin and microtubule-based transport. Finally, infection of polarized cells with the related flavivirus, Dengue virus-2, did not result in significant loss of tight junction membrane proteins. These results suggest that neurotropic flaviviruses such as WNV modulate the host cell environment differently than hemorrhagic flaviviruses and thus may have implications for understanding the molecular basis for neuroinvasion.

  16. Participation of plasma membrane proteins in the formation of tight junction by cultured epithelial cells

    PubMed Central

    Griepp, EB; Dolan, WJ; Robbins, ES; Sabatini, DD

    1983-01-01

    Measurements of the transepithelial electrical resistance correlated with freeze-fracture observations have been used to study the process of tight junction formation under various experimental conditions in monolayers of the canine kidney epithelial cell line MDCK. Cells derived from previously confluent cultures and plated immediately after trypsin- EDTA dissociation develop a resistance that reaches its maximum value of several hundred ohms-cm(2) after approximately 24 h and falls to a steady-state value of 80-150 ohms- cm(2) by 48 h. The rise in resistance and the development of tight junctions can be completely and reversibly prevented by the addition of 10 μg/ml cycloheximide at the time of plating, but not when this inhibitor is added more than 10 h after planting. Thus tight junction formation consists of separable synthetic and assembly phases. These two phases can also be dissociated and the requirement for protein synthesis after plating eliminated if, following trypsinization, the cells are maintained in spinner culture for 24 h before plating. The requirement for protein synthesis is restored, however, if cells maintained in spinner culture are treated with trypsin before plating. Actinomycin D prevents development of resistance only in monolayers formed from cells derived from sparse rather than confluent cultures, but new mRNA synthesis is not required if cells obtained from sparse cultures are maintained for 24 h in spinner culture before plating. Once a steady-state resistance has been reached, its maintenance does not require either mRNA or protein synthesis; in fact, inhibition of protein synthesis causes a rise in the resistance over a 30-h period. Following treatments that disrupt the junctions in steady- state monolayers recovery of resistance also does not require protein synthesis. These observations suggest that proteins are involved in tight junction formation. Such proteins, which do not turn over rapidly under steady-state conditions

  17. Interaction of HMG proteins and H1 with hybrid PNA-DNA junctions.

    PubMed

    Totsingan, Filbert; Bell, Anthony J

    2013-11-01

    The objective of this study was to evaluate the effects of inserting peptide nucleic acid (PNA) sequences into the protein-binding surface of an immobilized four-way junction (4WJ). Here we compare the classic immobile DNA junction, J1, with two PNA containing hybrid junctions (4WJ-PNA1 and 4WJ-PNA3 ). The protein interactions of each 4WJ were evaluated using recombinant high mobility group proteins from rat (HMGB1b and HMGB1b/R26A) and human histone H1. In vitro studies show that both HMG and H1 proteins display high binding affinity toward 4WJ's. A 4WJ can access different conformations depending on ionic environment, most simply interpreted by a two-state equilibrium between: (i) an open-x state favored by absence of Mg(2+), low salt, and protein binding, and (ii) a compact stacked-x state favored by Mg(2+). 4WJ-PNA3, like J1, shifts readily from an open to stacked conformation in the presence of Mg(+2), while 4WJ-PNA1 does not. Circular dichroism spectra indicate that HMGB1b recognizes each of the hybrid junctions. H1, however, displays a strong preference for J1 relative to the hybrids. More extensive binding analysis revealed that HMGB1b binds J1 and 4WJ-PNA3 with nearly identical affinity (K(D)s) and 4WJ-PNA1 with two-fold lower affinity. Thus both the sequence/location of the PNA sequence and the protein determine the structural and protein recognition properties of 4WJs.

  18. Toll-like receptor 4 signaling is coupled to src family kinase activation, tyrosine phosphorylation of zonula adherens proteins, and opening of the paracellular pathway in human lung microvascular endothelia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacterial lipopolysaccharide (LPS) is a key mediator in the vascular leak syndromes associated with Gram-negative bacterial infections. LPS opens the paracellular pathway in pulmonary vascular endothelia through protein tyrosine phosphorylation. We now have identified the protein tyrosine kinase (PT...

  19. Effects of intercellular junction protein expression on intracellular ice formation in mouse insulinoma cells.

    PubMed

    Higgins, Adam Z; Karlsson, Jens O M

    2013-11-01

    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

  20. Modulation of gap junction transcript and protein expression during pregnancy in the rat

    PubMed Central

    1990-01-01

    The expression of three different gap junction transcripts, alpha 1 (Cx43), beta 1 (Cx32), and beta 2 (Cx26) was examined in several organs during pregnancy in the rat. In all of the organs that were examined-- uterus, ovary, heart, and liver--there was a strong correlation between levels of gap junction mRNA and gap junction antigens that were detected at different stages of pregnancy. A striking change in alpha 1 transcript levels (a 5.5-fold increase) was detected in the uterine myometrium on the day before parturition. This elevation of the alpha 1 transcript is thought to be associated with the formation of gap junctions that are required for synchronizing the contractility of the myometrial cells during parturition. 2 d before parturition, there was a detectable elevation of beta 2 transcripts and protein in the endometrial epithelium, which was then followed by a dramatic decrease in beta 2 gap junctional protein on the day before parturition. There was also a substantial elevation of alpha 1 transcripts (a 6.7-fold increase) in the stromal regions of the ovary on the day before parturition that was identical to the temporal pattern of alpha 1 expression in the myometrium. In all three instances--the alpha 1 transcripts in the myometrium, beta 2 transcripts in the endometrium, and alpha 1 transcripts in the ovary--the transcript modulation appeared to be cell specific, because the changes in transcript levels of these three gene products occurred independently of the poly(A) + RNA concentrations at the same pregnancy stages in the respective organs. There were no specific changes detected in gap junction transcript levels in the heart and liver during pregnancy. These observations indicate that a cell-specific modulation of gap junction expression occurs in two regions of the uterus and the ovary during pregnancy. Further, it appears that the same gap junction gene in different organs, such as the alpha 1 gene in the uterine myometrium and the heart, can be

  1. Migration of a Holliday junction through a nucleosome directed by the E. coli RuvAB motor protein.

    PubMed

    Grigoriev, M; Hsieh, P

    1998-09-01

    Chromatin plays a critical role in regulating access to DNA by proteins that direct recombination and repair. The E. coli RuvAB protein complex promotes branch migration of the Holliday junction recombination intermediate. The ability of RuvAB to negotiate passage of the junction through nucleosomal DNA is examined. The model system involves the formation of a Holliday junction positioned upstream of a nucleosome. Unassisted, the junction is blocked by a histone octamer. In the presence of RuvAB and ATP, rapid branch migration through the nucleosome is observed. It results in disruption of the histone-DNA interactions leading to the removal of the octamer from the junction intermediate. These results suggest that eukaryotic DNA motor proteins analogous to RuvAB could function during recombination to promote branch migration through chromatin.

  2. Sequence and developmental expression of mRNA coding for a gap junction protein in Xenopus

    PubMed Central

    1988-01-01

    Cloned complementary DNAs representing the complete coding sequence for an embryonic gap junction protein in the frog Xenopus laevis have been isolated and sequenced. The cDNAs hybridize with an RNA of 1.5 kb that is first detected in gastrulating embryos and accumulates throughout gastrulation and neurulation. By the tailbud stage, the highest abundance of the transcript is found in the region containing ventroposterior endoderm and the rudiment of the liver. In the adult, transcripts are present in the lungs, alimentary tract organs, and kidneys, but are not detected in the brain, heart, body wall and skeletal muscles, spleen, or ovary. The gene encoding this embryonic gap junction protein is present in only one or a few copies in the frog genome. In vitro translation of RNA synthesized from the cDNA template produces a 30-kD protein, as predicted by the coding sequence. This product has extensive sequence similarity to mammalian gap junction proteins in its putative transmembrane and extracellular domains, but has diverged substantially in two of its intracellular domains. PMID:2843548

  3. MicroRNA-205 Targets Tight Junction-related Proteins during Urothelial Cellular Differentiation *

    PubMed Central

    Chung, Pei-Jung Katy; Chi, Lang-Ming; Chen, Chien-Lun; Liang, Chih-Lung; Lin, Chung-Tzu; Chang, Yu-Xun; Chen, Chun-Hsien; Chang, Yu-Sun

    2014-01-01

    The mammalian bladder urothelium classified as basal, intermediate, and terminally differentiated umbrella cells offers one of the most effective permeability barrier functions known to exist in nature because of the formation of apical uroplakin plaques and tight junctions. To improve our understanding of urothelial differentiation, we analyzed the microRNA (miRNA) expression profiles of mouse urinary tissues and by TaqMan miRNA analysis of microdissected urothelial layers and in situ miRNA-specific hybridization to determine the dependence of these miRNAs on the differentiation stage. Our in situ hybridization studies revealed that miR-205 was enriched in the undifferentiated basal and intermediate cell layers. We then used a quantitative proteomics approach to identify miR-205 target genes in primary cultured urothelial cells subjected to antagomir-mediated knockdown of specific miRNAs. Twenty-four genes were reproducibly regulated by miR-205; eleven of them were annotated as cell junction- and tight junction-related molecules. Western blot analysis demonstrated that antagomir-induced silencing of miR-205 in primary cultured urothelial cells elevated the expression levels of Tjp1, Cgnl1, and Cdc42. Ectopic expression of miR-205 in MDCK cells inhibited the expression of tight junction proteins and the formation of tight junctions. miR-205- knockdown urothelial cells showed alterations in keratin synthesis and increases of uroplakin Ia and Ib, which are the urothelial differentiation products. These results suggest that miR-205 may contribute a role in regulation of urothelial differentiation by modulating the expression of tight junction-related molecules. PMID:24912853

  4. Rapid electrical stimulation causes alterations in cardiac intercellular junction proteins of cardiomyocytes.

    PubMed

    Nakashima, Tadamitsu; Ohkusa, Tomoko; Okamoto, Yoko; Yoshida, Masaaki; Lee, Jong-Kook; Mizukami, Yoichi; Yano, Masafumi

    2014-05-01

    The intercellular junctions contain two complexes, adhesion junctions (AJ) and connexin (Cx) gap junctions (GJs). GJs provide the pathway for intercellular current flow. AJs mediate normal mechanical coupling and play an important role in the stability of GJs. We investigated the effects of rapid electrical stimulation (RES) on cardiac intercellular junctions, especially β-catenin and Cx43 alterations. We also studied the effects of ANG II receptor blockade on intercellular junction remodeling. Neonatal rats were euthanized by decapitation, and cardiomyocytes were prepared, cultured, and subjected to RES. We used real-time PCR, western blot analysis, and immunohistochemical methods. Conduction properties were examined by an extracellular potential mapping system. Cx43 protein expression in cardiomyocytes was significantly increased after 60 min. β-Catenin expression in the total cell fraction was significantly increased after 30 min. The expression level of β-catenin in the nucleus, which functions as a T cell factor/lymphocyte enhancer binding factor transcriptional activator of Cx43 with its degradation regulated by glycogen synthase kinase-3β, was dramatically increased after 10 min. Conduction velocity was increased significantly by RES for 60 min. Olmesartan prevented most these effects of RES. We showed an increase of phosphorylated glycogen synthase kinase-3β, which is phosphorylated by activated MAPKs and inhibits β-catenin degradation, was attenuated by olmesartan. The changes in β-catenin precede Cx43 GJ remodeling and might play an important role in the formation and stability of GJs. Olmesartan might be a new upstream arrhythmia therapy by modulating intercellular junction remodeling through the β-catenin signaling pathway.

  5. 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

  6. Nonmammalian vertebrate skeletal muscles express two triad junctional foot protein isoforms.

    PubMed Central

    Olivares, E B; Tanksley, S J; Airey, J A; Beck, C F; Ouyang, Y; Deerinck, T J; Ellisman, M H; Sutko, J L

    1991-01-01

    Mammalian skeletal muscles express a single triad junctional foot protein, whereas avian muscles have two isoforms of this protein. We investigated whether either case is representative of muscles from other vertebrate classes. We identified two foot proteins in bullfrog and toadfish muscles on the basis of (a) copurification with [3H]epiryanodine binding; (b) similarity to avian muscle foot proteins in native and subunit molecular weights; (c) recognition by anti-foot protein antibodies. The bullfrog and toadfish proteins exist as homooligomers. The subunits of the bullfrog muscle foot protein isoforms are shown to be unique by peptide mapping. In addition, immunocytochemical localization established that the bullfrog muscle isoforms coexist in the same muscle cells. The isoforms in either bullfrog and chicken muscles have comparable [3H]epiryanodine binding capacities, whereas in toadfish muscle the isoforms differ in their levels of ligand binding. Additionally, chicken thigh and breast muscles differ in the relative amounts of the two isoforms they contain, the amounts being similar in breast muscle and markedly different in thigh muscle. In conclusion, in contrast to mammalian skeletal muscle, two foot protein isoforms are present in amphibian, avian, and piscine skeletal muscles. This may represent a general difference in the architecture and/or a functional specialization of the triad junction in mammalian and nonmammalian vertebrate muscles. Images FIGURE 1 FIGURE 2 FIGURE 4 FIGURE 5 FIGURE 6 FIGURE 7 FIGURE 8 FIGURE 9 PMID:1873458

  7. The Triple-Repeat Protein Anakonda Controls Epithelial Tricellular Junction Formation in Drosophila.

    PubMed

    Byri, Sunitha; Misra, Tvisha; Syed, Zulfeqhar A; Bätz, Tilmann; Shah, Jimit; Boril, Lukas; Glashauser, Jade; Aegerter-Wilmsen, Tinri; Matzat, Till; Moussian, Bernard; Uv, Anne; Luschnig, Stefan

    2015-06-01

    In epithelia, specialized tricellular junctions (TCJs) mediate cell contacts at three-cell vertices. TCJs are fundamental to epithelial biology and disease, but only a few TCJ components are known, and how they assemble at tricellular vertices is not understood. Here we describe a transmembrane protein, Anakonda (Aka), which localizes to TCJs and is essential for the formation of tricellular, but not bicellular, junctions in Drosophila. Loss of Aka causes epithelial barrier defects associated with irregular TCJ structure and geometry, suggesting that Aka organizes cell corners. Aka is necessary and sufficient for accumulation of Gliotactin at TCJs, suggesting that Aka initiates TCJ assembly by recruiting other proteins to tricellular vertices. Aka's extracellular domain has an unusual tripartite repeat structure that may mediate self-assembly, directed by the geometry of tricellular vertices. Conversely, Aka's cytoplasmic tail is dispensable for TCJ localization. Thus, extracellular interactions, rather than TCJ-directed intracellular transport, appear to mediate TCJ assembly.

  8. Molecular cloning of cDNA for rat liver gap junction protein

    PubMed Central

    1986-01-01

    An affinity-purified antibody directed against the 27-kD protein associated with isolated rat liver gap junctions was produced. Light and electron microscopic immunocytochemistry showed that this antigen was localized specifically to the cytoplasmic surfaces of gap junctions. The antibody was used to select cDNA from a rat liver library in the expression vector lambda gt11. The largest cDNA selected contained 1,494 bp and coded for a protein with a calculated molecular mass of 32,007 daltons. Northern blot analysis indicated that brain, kidney, and stomach express an mRNA with similar size and homology to that expressed in liver, but that heart and lens express differently sized, less homologous mRNA. PMID:3013898

  9. Effects of Soybean Agglutinin on Intestinal Barrier Permeability and Tight Junction Protein Expression in Weaned Piglets

    PubMed Central

    Zhao, Yuan; Qin, Guixin; Sun, Zewei; Che, Dongsheng; Bao, Nan; Zhang, Xiaodong

    2011-01-01

    This study was developed to provide further information on the intestinal barrier permeability and the tight junction protein expression in weaned piglets fed with different levels of soybean agglutinin (SBA). Twenty-five weaned crossbred barrows (Duroc × Landrace × Yorkshire) were selected and randomly allotted to five groups, each group with five replicates. The piglets in the control group were not fed with leguminous products. 0.05, 0.1, 0.15 and 0.2% SBA was added to the control diet to form four experimental diets, respectively. After the experimental period of 7 days (for each group), all the piglets were anesthetized with excess procaine and slaughtered. The d-lactic acid in plasma and the Ileal mucosa diamine oxidase (DAO) was analyzed to observe the change in the intestinal permeability. The tight junction proteins occludin and ZO-1 in the jejunum tissue distribution and relative expression were detected by immunohistochemistry and Western Blot. The results illustrated that a high dose of SBA (0.1–0.2%) could increase the intestinal permeability and reduce piglet intestinal epithelial tight junction protein occludin or ZO-1 expression, while low dose of SBA (0.05% of total diet) had no significant affects. The contents of DAO, d-lactic acid, occludin or ZO-1, had a linear relationship with the SBA levels (0–0.2%) in diets. The high dose SBA (0.1–0.2%) could increase the intestinal permeability and reduce piglet intestinal epithelial tight junction protein occludin or ZO-1 expression, while low dose of SBA (0.05% of total diet) had no affects. PMID:22272087

  10. Differential expression of active zone proteins in neuromuscular junctions suggests functional diversification.

    PubMed

    Juranek, Judyta; Mukherjee, Konark; Rickmann, Michael; Martens, Henrik; Calka, Jaroslaw; Südhof, Thomas C; Jahn, Reinhard

    2006-12-01

    Nerve terminals of the central nervous system (CNS) contain specialized release sites for synaptic vesicles, referred to as active zones. They are characterized by electron-dense structures that are tightly associated with the presynaptic plasma membrane and organize vesicle docking and priming sites. Recently, major protein constituents of active zones have been identified, including the proteins Piccolo, Bassoon, RIM, Munc13, ERCs/ELKs/CASTs and liprins. While it is becoming apparent that each of these proteins is essential for synaptic function in the CNS, it is not known to what extent these proteins are involved in synaptic function of the peripheral nervous system. Somatic neuromuscular junctions contain morphologically and functionally defined active zones with similarities to CNS synapses. In contrast, sympathetic neuromuscular varicosities lack active zone-like morphological specializations. Using immunocytochemistry at the light and electron microscopic level we have now performed a systematic investigation of all five major classes of active zone proteins in peripheral neuromuscular junctions. Our results show that somatic neuromuscular endplates contain a full complement of all active zone proteins. In contrast, varicosities of the vas deferens contain a subset of active zone proteins including Bassoon and ELKS2, with the other four components being absent. We conclude that Bassoon and ELKS2 perform independent and specialized functions in synaptic transmission of autonomic synapses.

  11. Proteomic Analysis of Proteins Surrounding Occludin and Claudin-4 Reveals Their Proximity to Signaling and Trafficking Networks

    PubMed Central

    Fredriksson, Karin; Van Itallie, Christina M.; Aponte, Angel; Gucek, Marjan; Tietgens, Amber J.; Anderson, James M.

    2015-01-01

    Tight junctions are complex membrane structures that regulate paracellular movement of material across epithelia and play a role in cell polarity, signaling and cytoskeletal organization. In order to expand knowledge of the tight junction proteome, we used biotin ligase (BioID) fused to occludin and claudin-4 to biotinylate their proximal proteins in cultured MDCK II epithelial cells. We then purified the biotinylated proteins on streptavidin resin and identified them by mass spectrometry. Proteins were ranked by relative abundance of recovery by mass spectrometry, placed in functional categories, and compared not only among the N- and C- termini of occludin and the N-terminus of claudin-4, but also with our published inventory of proteins proximal to the adherens junction protein E-cadherin and the tight junction protein ZO-1. When proteomic results were analyzed, the relative distribution among functional categories was similar between occludin and claudin-4 proximal proteins. Apart from already known tight junction- proteins, occludin and claudin-4 proximal proteins were enriched in signaling and trafficking proteins, especially endocytic trafficking proteins. However there were significant differences in the specific proteins comprising the functional categories near each of the tagging proteins, revealing spatial compartmentalization within the junction complex. Taken together, these results expand the inventory of known and unknown proteins at the tight junction to inform future studies of the organization and physiology of this complex structure. PMID:25789658

  12. Antofine-induced connexin43 gap junction disassembly in rat astrocytes involves protein kinase Cβ.

    PubMed

    Huang, Yu-Fang; Liao, Chih-Kai; Lin, Jau-Chen; Jow, Guey-Mei; Wang, Hwai-Shi; Wu, Jiahn-Chun

    2013-03-01

    Antofine, a phenanthroindolizidine alkaloid derived from Cryptocaryachinensis and Ficusseptica in the Asclepiadaceae milkweed family, is cytotoxic for various cancer cell lines. In this study, we demonstrated that treatment of rat primary astrocytes with antofine induced dose-dependent inhibition of gap junction intercellular communication (GJIC), as assessed by scrape-loading 6-carboxyfluorescein dye transfer. Levels of Cx43 protein were also decreased in a dose- and time-dependent manner following antofine treatment. Double-labeling immunofluorescence microscopy showed that antofine (10ng/ml) induced endocytosis of surface gap junctions into the cytoplasm, where Cx43 was co-localized with the early endosome marker EEA1. Inhibition of lysosomes or proteasomes by co-treatment with antofine and their respective specific inhibitors, NH4Cl or MG132, partially inhibited the antofine-induced decrease in Cx43 protein levels, but did not inhibit the antofine-induced inhibition of GJIC. After 30min of treatment, antofine induced a rapid increase in the intracellular Ca(2+) concentration and activation of protein kinase C (PKC)α/βII, which was maintained for at least 6h. Co-treatment of astrocytes with antofine and the intracellular Ca(2+) chelator BAPTA-AM prevented downregulation of Cx43 and inhibition of GJIC. Moreover, co-treatment with antofine and a specific PKCβ inhibitor prevented endocytosis of gap junctions, downregulation of Cx43, and inhibition of GJIC. Taken together, these findings indicate that antofine induces Cx43 gap junction disassembly by the PKCβ signaling pathway. Inhibition of GJIC by antofine may undermine the neuroprotective effect of astrocytes in CNS. PMID:23403203

  13. Desmosomal molecules in and out of adhering junctions: normal and diseased States of epidermal, cardiac and mesenchymally derived cells.

    PubMed

    Pieperhoff, Sebastian; Barth, Mareike; Rickelt, Steffen; Franke, Werner W

    2010-01-01

    Current cell biology textbooks mention only two kinds of cell-to-cell adhering junctions coated with the cytoplasmic plaques: the desmosomes (maculae adhaerentes), anchoring intermediate-sized filaments (IFs), and the actin microfilament-anchoring adherens junctions (AJs), including both punctate (puncta adhaerentia) and elongate (fasciae adhaerentes) structures. In addition, however, a series of other junction types has been identified and characterized which contain desmosomal molecules but do not fit the definition of desmosomes. Of these special cell-cell junctions containing desmosomal glycoproteins or proteins we review the composite junctions (areae compositae) connecting the cardiomyocytes of mature mammalian hearts and their importance in relation to human arrhythmogenic cardiomyopathies. We also emphasize the various plakophilin-2-positive plaques in AJs (coniunctiones adhaerentes) connecting proliferatively active mesenchymally-derived cells, including interstitial cells of the heart and several soft tissue tumor cell types. Moreover, desmoplakin has also been recognized as a constituent of the plaques of the complexus adhaerentes connecting certain lymphatic endothelial cells. Finally, we emphasize the occurrence of the desmosomal transmembrane glycoprotein, desmoglein Dsg2, out of the context of any junction as dispersed cell surface molecules in certain types of melanoma cells and melanocytes. This broadening of our knowledge on the diversity of AJ structures indicates that it may still be too premature to close the textbook chapters on cell-cell junctions. PMID:20671973

  14. The tight junction protein CAR regulates cardiac conduction and cell-cell communication.

    PubMed

    Lisewski, Ulrike; Shi, Yu; Wrackmeyer, Uta; Fischer, Robert; Chen, Chen; Schirdewan, Alexander; Jüttner, Rene; Rathjen, Fritz; Poller, Wolfgang; Radke, Michael H; Gotthardt, Michael

    2008-09-29

    The Coxsackievirus-adenovirus receptor (CAR) is known for its role in virus uptake and as a protein of the tight junction. It is predominantly expressed in the developing brain and heart and reinduced upon cardiac remodeling in heart disease. So far, the physiological functions of CAR in the adult heart are largely unknown. We have generated a heart-specific inducible CAR knockout (KO) and found impaired electrical conduction between atrium and ventricle that increased with progressive loss of CAR. The underlying mechanism relates to the cross talk of tight and gap junctions with altered expression and localization of connexins that affect communication between CAR KO cardiomyocytes. Our results indicate that CAR is not only relevant for virus uptake and cardiac remodeling but also has a previously unknown function in the propagation of excitation from the atrium to the ventricle that could explain the association of arrhythmia and Coxsackievirus infection of the heart.

  15. Overexpression of Galectin-7 in Mouse Epidermis Leads to Loss of Cell Junctions and Defective Skin Repair

    PubMed Central

    Dang, Tien; Deshayes, Frédérique; Delacour, Delphine; Pichard, Evelyne; Advedissian, Tamara; Sidhu, Sukhvinder S.; Viguier, Mireille; Magnaldo, Thierry; Poirier, Francoise

    2015-01-01

    Background The proteins of the galectin family are implicated in many cellular processes, including cell interactions, polarity, intracellular trafficking, and signal transduction. In human and mouse, galectin-7 is almost exclusively expressed in stratified epithelia, notably in the epidermis. Galectin-7 expression is also altered in several human tumors of epithelial origin. This study aimed at dissecting the consequences of galectin-7 overexpression on epidermis structure and functions in vivo. Methods We established transgenic mice specifically overexpressing galectin-7 in the basal epidermal keratinocytes and analyzed the consequences on untreated skin and after UVB irradiation or mechanical injury. Results The intercellular cohesion of the epidermis is impaired in transgenic animals, with gaps developing between adjacent keratinocytes, associated with loss of adherens junctions. The epidermal architecture is aberrant with perturbations in the multilayered cellular organisation of the tissue, and structural defects in the basement membrane. These transgenic animals displayed a reduced re-epithelialisation potential following superficial wound, due to a defective collective migration of keratinocytes. Finally, a single mild dose of UVB induced an abnormal apoptotic response in the transgenic epidermis. Conclusion These results indicate that an excess of galectin-7 leads to a destabilisation of adherens junctions associated with defects in epidermal repair. As this phenotype shares similarities with that of galectin-7 null mutant mice, we conclude that a critical level of this protein is required for maintaining proper epidermal homeostasis. This study brings new insight into the mode of action of galectins in normal and pathological situations. PMID:25741714

  16. The tight junction protein ZO-2 blocks cell cycle progression and inhibits cyclin D1 expression.

    PubMed

    Gonzalez-Mariscal, Lorenza; Tapia, Rocio; Huerta, Miriam; Lopez-Bayghen, Esther

    2009-05-01

    ZO-2 is an adaptor protein of the tight junction that belongs to the MAGUK protein family. ZO-2 is a dual localization protein that in sparse cultures is present at the cell borders and the nuclei, whereas in confluent cultures it is concentrated at the cell boundaries. Here we have studied whether ZO-2 is able to regulate the expression of cyclin D1 (CD1) and cell proliferation. We have demonstrated that ZO-2 negatively regulates CD1 transcription by interacting with c-Myc at an E box present in CD1 promoter. We have further found that ZO-2 transfection into epithelial MDCK cells triggers a diminished expression of CD1 protein and decreases the rate of cell proliferation in a wound-healing assay.

  17. Nuclear localization of the tight junction protein ZO-2 in epithelial cells.

    PubMed

    Islas, Socorro; Vega, Jesús; Ponce, Lissette; González-Mariscal, Lorenza

    2002-03-10

    The tight junction constitutes the major barrier to solute and water flow through the paracellular space of epithelia and endothelia. It is formed by transmembrane proteins and submembranous molecules such as the MAGUKs ZOs. We have previously found that several MAGUKs, including those of the tight (ZO-1, ZO-2, and ZO-3) and septate junction (tamou and Dlg), contain one or two nuclear sorting signals located at their first PDZ and GK domains. Now we show that these proteins also contain a nuclear export signal and focus our study on the nuclear membrane shuttling of ZO-2. In sparse cultures this molecule concentrates at the nucleus in clusters, where it partially colocalizes with splicing factor SC35. Nuclear staining diminishes as the monolayer acquires confluence through a process sensitive to the nuclear export inhibitor leptomycin B. Nuclear localization can be induced by impairing cell-cell contacts, by mechanical injury. ZO-2 that shuttles from the cell periphery into the nucleus is not newly synthesized but originates from a preexistent pool. The movement of this protein is mediated by the actin cytoskeleton.

  18. Simvastatin Sodium Salt and Fluvastatin Interact with Human Gap Junction Gamma-3 Protein.

    PubMed

    Marsh, Andrew; Casey-Green, Katherine; Probert, Fay; Withall, David; Mitchell, Daniel A; Dilly, Suzanne J; James, Sean; Dimitri, Wade; Ladwa, Sweta R; Taylor, Paul C; Singer, Donald R J

    2016-01-01

    Finding pleiomorphic targets for drugs allows new indications or warnings for treatment to be identified. As test of concept, we applied a new chemical genomics approach to uncover additional targets for the widely prescribed lipid-lowering pro-drug simvastatin. We used mRNA extracted from internal mammary artery from patients undergoing coronary artery surgery to prepare a viral cardiovascular protein library, using T7 bacteriophage. We then studied interactions of clones of the bacteriophage, each expressing a different cardiovascular polypeptide, with surface-bound simvastatin in 96-well plates. To maximise likelihood of identifying meaningful interactions between simvastatin and vascular peptides, we used a validated photo-immobilisation method to apply a series of different chemical linkers to bind simvastatin so as to present multiple orientations of its constituent components to potential targets. Three rounds of biopanning identified consistent interaction with the clone expressing part of the gene GJC3, which maps to Homo sapiens chromosome 7, and codes for gap junction gamma-3 protein, also known as connexin 30.2/31.3 (mouse connexin Cx29). Further analysis indicated the binding site to be for the N-terminal domain putatively 'regulating' connexin hemichannel and gap junction pores. Using immunohistochemistry we found connexin 30.2/31.3 to be present in samples of artery similar to those used to prepare the bacteriophage library. Surface plasmon resonance revealed that a 25 amino acid synthetic peptide representing the discovered N-terminus did not interact with simvastatin lactone, but did bind to the hydrolysed HMG CoA inhibitor, simvastatin acid. This interaction was also seen for fluvastatin. The gap junction blockers carbenoxolone and flufenamic acid also interacted with the same peptide providing insight into potential site of binding. These findings raise key questions about the functional significance of GJC3 transcripts in the vasculature and

  19. Simvastatin Sodium Salt and Fluvastatin Interact with Human Gap Junction Gamma-3 Protein.

    PubMed

    Marsh, Andrew; Casey-Green, Katherine; Probert, Fay; Withall, David; Mitchell, Daniel A; Dilly, Suzanne J; James, Sean; Dimitri, Wade; Ladwa, Sweta R; Taylor, Paul C; Singer, Donald R J

    2016-01-01

    Finding pleiomorphic targets for drugs allows new indications or warnings for treatment to be identified. As test of concept, we applied a new chemical genomics approach to uncover additional targets for the widely prescribed lipid-lowering pro-drug simvastatin. We used mRNA extracted from internal mammary artery from patients undergoing coronary artery surgery to prepare a viral cardiovascular protein library, using T7 bacteriophage. We then studied interactions of clones of the bacteriophage, each expressing a different cardiovascular polypeptide, with surface-bound simvastatin in 96-well plates. To maximise likelihood of identifying meaningful interactions between simvastatin and vascular peptides, we used a validated photo-immobilisation method to apply a series of different chemical linkers to bind simvastatin so as to present multiple orientations of its constituent components to potential targets. Three rounds of biopanning identified consistent interaction with the clone expressing part of the gene GJC3, which maps to Homo sapiens chromosome 7, and codes for gap junction gamma-3 protein, also known as connexin 30.2/31.3 (mouse connexin Cx29). Further analysis indicated the binding site to be for the N-terminal domain putatively 'regulating' connexin hemichannel and gap junction pores. Using immunohistochemistry we found connexin 30.2/31.3 to be present in samples of artery similar to those used to prepare the bacteriophage library. Surface plasmon resonance revealed that a 25 amino acid synthetic peptide representing the discovered N-terminus did not interact with simvastatin lactone, but did bind to the hydrolysed HMG CoA inhibitor, simvastatin acid. This interaction was also seen for fluvastatin. The gap junction blockers carbenoxolone and flufenamic acid also interacted with the same peptide providing insight into potential site of binding. These findings raise key questions about the functional significance of GJC3 transcripts in the vasculature and

  20. Cell–cell junction remodeling in the heart: Possible role in cardiac conduction system function and arrhythmias?

    PubMed Central

    Mezzano, Valeria; Sheikh, Farah

    2012-01-01

    Anchoring Cell–cell junctions (desmosomes, fascia adherens) play crucial roles in maintaining mechanical integrity of cardiac muscle cells and tissue. Genetic mutations and/or loss of critical components in these macromolecular structures are increasingly being associated with arrhythmogenic cardiomyopathies; however, their specific roles have been primarily attributed to effects within the working (ventricular) cardiac muscle. Growing evidence also points to a key role for anchoring Cell–cell junction components in cardiac muscle cells of the cardiac conduction system. This is not only evidenced by the molecular and ultra-structural presence of anchoring cell junctions in specific compartments/structures of the cardiac conduction system (sinoatrial node, atrioventricular node, His-Purkinje system), but also because conduction system-related arrhythmias can be found in humans and mouse models of cardiomyopathies harboring defects and/or mutations in key anchoring Cell–cell junction proteins. These studies emphasize the clinical need to understand the molecular and cellular role(s) for anchoring Cell–cell junctions in cardiac conduction system function and arrhythmias. This review will focus on (i) experimental findings that underline an important role for anchoring Cell–cell junctions in the cardiac conduction system, (ii) insights regarding involvement of these structures in age-related cardiac remodeling of the conduction system, (iii) summarizing available genetic mouse models that can target cardiac conduction system structures and (iv) implications of these findings on future therapies for arrhythmogenic heart diseases. PMID:22227473

  1. 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

    2005-09-01

    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.

  2. PATJ connects and stabilizes apical and lateral components of tight junctions in human intestinal cells.

    PubMed

    Michel, Didier; Arsanto, Jean-Pierre; Massey-Harroche, Dominique; Béclin, Christophe; Wijnholds, Jan; Le Bivic, André

    2005-09-01

    The Crumbs complex that also contains the cortical proteins Stardust and DPATJ (a homologue of PATJ), is crucial for the building of epithelial monolayers in Drosophila. Although loss of function of the Crumbs or Stardust genes prevents the stabilization of a belt of adherens junctions at the apico-lateral border of the cells, no phenotype has been described for the Dpatj gene and its role in epithelial morphogenesis and polarity remains unknown. We have produced downregulated PATJ stable lines of Caco2 to clarify its role in epithelial morphogenesis. In PATJ knockdown cells, Pals1 (a Stardust homologue) is no longer associated with tight junctions whereas Crumbs3 (Crb3) is accumulated into a compartment spatially close to the apical membrane and related to early endosomes. Furthermore, occludin and ZO-3, two proteins of tight junctions are mislocalized on the lateral membrane indicating that PATJ plays a novel role in the building of tight junctions by providing a link between their lateral and apical components. Thus, PATJ stabilizes the Crb3 complex and regulates the spatial concentration of several components at the border between the apical and lateral domains.

  3. armadillo, bazooka, and stardust are critical for early stages in formation of the zonula adherens and maintenance of the polarized blastoderm epithelium in Drosophila.

    PubMed

    Müller, H A; Wieschaus, E

    1996-07-01

    Cellularization of the Drosophila embryo results in the formation of a cell monolayer with many characteristics of a polarized epithelium. We have used antibodies specific to cellular junctions and nascent plasma membranes to study the formation of the zonula adherens (ZA) in relation to the establishment of basolateral membrane polarity. The same approach was then used as a test system to identify X-linked zygotically active genes required for ZA formation. We show that ZA formation begins during cellularization and that the basolateral membrane domain is established at mid-gastrulation. By creating deficiencies for defined regions of the X chromosome, we have identified genes that are required for the formation of the ZA and the generation of basolateral membrane polarity. We show that embryos mutant for both stardust (sdt) and bazooka (baz) fail to form a ZA. In addition to the failure to establish the ZA, the formation of the monolayered epithelium is disrupted after cellularization, resulting in formation of a multilayered cell sheet by mid-gastrulation. SEM analysis of mutant embryos revealed a conversion of cells exhibiting epithelial characteristics into cells exhibiting mesenchymal characteristics. To investigate how mutations that affect an integral component of the ZA itself influence ZA formation, we examined embryos with reduced maternal and zygotic supply of wild-type Arm protein. These embryos, like embryos mutant for both sdt and baz, exhibit an early disruption of ZA formation. These results suggest that early stages in the assembly of the ZA are critical for the stability of the polarized blastoderm epithelium.

  4. Role of extracellular matrix proteins and their receptors in the development of the vertebrate neuromuscular junction.

    PubMed

    Singhal, Neha; Martin, Paul T

    2011-11-01

    The vertebrate neuromuscular junction (NMJ) remains the best-studied model for understanding the mechanisms involved in synaptogenesis, due to its relatively large size, its simplicity of patterning, and its unparalleled experimental accessibility. During neuromuscular development, each skeletal myofiber secretes and deposits around its extracellular surface an assemblage of extracellular matrix (ECM) proteins that ultimately form a basal lamina. This is also the case at the NMJ, where the motor nerve contributes additional factors. Before most of the current molecular components were known, it was clear that the synaptic ECM of adult skeletal muscles was unique in composition and contained factors sufficient to induce the differentiation of both pre- and postsynaptic membranes. Biochemical, genetic, and microscopy studies have confirmed that agrin, laminin (221, 421, and 521), collagen IV (α3-α6), collagen XIII, perlecan, and the ColQ-bound form of acetylcholinesterase are all synaptic ECM proteins with important roles in neuromuscular development. The roles of their many potential receptors and/or binding proteins have been more difficult to assess at the genetic level due to the complexity of membrane interactions with these large proteins, but roles for MuSK-LRP4 in agrin signaling and for integrins, dystroglycan, and voltage-gated calcium channels in laminin-dependent phenotypes have been identified. Synaptic ECM proteins and their receptors are involved in almost all aspects of synaptic development, including synaptic initiation, topography, ultrastructure, maturation, stability, and transmission. PMID:21766463

  5. Effect of High Dietary Tryptophan on Intestinal Morphology and Tight Junction Protein of Weaned Pig

    PubMed Central

    Tossou, Myrlene Carine B.; Bai, Miaomiao; Chen, Shuai; Cai, Yinghua; Duraipandiyan, Veeramuthu; Liu, Hongbin; Adebowale, Tolulope O.; Al-Dhabi, Naif Abdullah; Long, Lina; Tarique, Hussain; Oso, Abimbola O.; Liu, Gang; Yin, Yulong

    2016-01-01

    Tryptophan (Trp) plays an essential role in pig behavior and growth performances. However, little is known about Trp's effects on tight junction barrier and intestinal health in weaned pigs. In the present study, twenty-four (24) weaned pigs were randomly assigned to one of the three treatments with 8 piglets/treatments. The piglets were fed different amounts of L-tryptophan (L-Trp) as follows: 0.0%, 0.15, and 0.75%, respectively, named zero Trp (ZTS), low Trp (LTS), and high Trp (HTS), respectively. No significant differences were observed in average daily gain (ADG), average daily feed intake (ADFI), and gain: feed (G/F) ratio between the groups. After 21 days of the feeding trial, results showed that dietary Trp significantly increased (P < 0.05) crypt depth and significantly decreased (P < 0.05) villus height to crypt depth ratio (VH/CD) in the jejunum of pig fed HTS. In addition, pig fed HTS had higher (P < 0.05) serum diamine oxidase (DAO) and D-lactate. Furthermore, pig fed HTS significantly decreased mRNA expression of tight junction proteins occludin and ZO-1 but not claudin-1 in the jejunum. The number of intraepithelial lymphocytes and goblet cells were not significantly different (P > 0.05) between the groups. Collectively, these data suggest that dietary Trp supplementation at a certain level (0.75%) may negatively affect the small intestinal structure in weaned pig. PMID:27366740

  6. Modulation of tight junction barrier function by outer membrane proteins of enteropathogenic Escherichia coli: role of F-actin and junctional adhesion molecule-1.

    PubMed

    Puthenedam, Manjula; Williams, Peter H; Lakshmi, B S; Balakrishnan, Arun

    2007-08-01

    Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhea. In this work we investigated the effect of outer membrane proteins (OMP) of EPEC on barrier integrity and the role of actin, junctional adhesion molecule (JAM) and signaling pathways contributing to these changes. Barrier function was assessed by transepithelial electrical resistance (TER). OMP of wild type EPEC, eaeA and maltoporin mutants decreased TER levels of Caco-2 cells. The OMP of espB mutant was deficient in decreasing TER of Caco-2 cells. The proteinase K-digested wild type OMP and EAF mutant OMP did not cause any change in barrier function. Our previous studies have demonstrated that EPEC OMP induced changes in cadherin junctions of Caco-2 cells. Immunofluorescence revealed disruption in actin cytoskeleton by EPEC OMP. However, no change in expression of junctional adhesion molecule-1 was observed. NF-kappaB inhibitor slightly blocked the decrease in TER and protected against actin disruption while ERK1/2 inhibitor had no effect in blocking these changes. In conclusion, our data suggest that the OMP of EPEC alter intestinal barrier function by disrupting actin cytoskeleton and signaling pathways like NF-kappaB may have a role in regulating barrier changes.

  7. Tissue and species conservation of the vertebrate and arthropod forms of the low molecular weight (16-18000) proteins of gap junctions.

    PubMed

    Buultjens, T E; Finbow, M E; Lane, N J; Pitts, J D

    1988-03-01

    Gap junctions have been isolated from four murine tissues, from rat and Xenopus laevis liver, and from Nephrops norvegicus (Norway lobster) hepatopancreas. The preparations of gap junctions from each vertebrate tissue contain a single major protein, Mr 16,000, and those from Nephrops hepatopancreas a protein, Mr 18,000. Immunocytochemical studies using affinity-purified antibodies raised against gap junctions from Nephrops show the junctional origin of the 18k protein. Immunological studies using Western blotting and biochemical studies using tryptic peptide mapping show no significant differences between the 16k junctional proteins of mouse and hence provide no evidence of tissue variation. These studies also suggest that the mouse, rat, and Xenopus 16k proteins and the Nephrops 18k protein share some common structural features.

  8. Requirement of enhanced Survival Motoneuron protein imposed during neuromuscular junction maturation

    PubMed Central

    Kariya, Shingo; Obis, Teresa; Garone, Caterina; Akay, Turgay; Sera, Fusako; Iwata, Shinichi; Homma, Shunichi; Monani, Umrao R.

    2014-01-01

    Spinal muscular atrophy is a common motor neuron disease caused by low survival motoneuron (SMN), a key protein in the proper splicing of genes. Restoring the protein is therefore a promising therapeutic strategy. Implementation of this strategy, however, depends on defining the temporal requirements for SMN. Here, we used controlled knockdown of SMN in transgenic mice to determine the precise postnatal stage requirements for this protein. Reducing SMN in neonatal mice resulted in a classic SMA-like phenotype. Unexpectedly, depletion of SMN in adults had relatively little effect. Insensitivity to low SMN emerged abruptly at postnatal day 17, which coincided with establishment of the fully mature neuromuscular junction (NMJ). Mature animals depleted of SMN eventually exhibited evidence of selective neuromuscular pathology that was made worse by traumatic injury. The ability to regenerate the mature NMJ in aged or injured SMN-depleted mice was grossly impaired, a likely consequence of the inability to meet the surge in demand for motoneuronal SMN that was seen in controls. Our results demonstrate that relative maturity of the NMJ determines the temporal requirement for the SMN protein. These observations suggest that the use of potent but potentially deleterious SMN-enhancing agents could be tapered in human patients once the neuromuscular system matures and reintroduced as needed to enhance SMN for remodeling aged or injured NMJs. PMID:24463453

  9. Specific motifs in the external loops of connexin proteins can determine gap junction formation between chick heart myocytes.

    PubMed Central

    Warner, A; Clements, D K; Parikh, S; Evans, W H; DeHaan, R L

    1995-01-01

    1. Gap junction formation was compared in the absence and presence of small peptides containing extracellular loop sequences of gap junction (connexin) proteins by measuring the time taken for pairs of spontaneously beating embryonic chick heart myoballs to synchronize beat rates. Test peptides were derived from connexin 32. Non-homologous peptides were used as controls. Control pairs took 42 +/- 0.5 min (mean +/- S.E.M.; n = 1088) to synchronize. 2. Connexins 32 and 43, but not 26, were detected in gap junction plaques. The density and distribution of connexin immunolabelling varied between myoballs. 3. Peptides containing conserved motifs from extracellular loops 1 and 2 delayed gap junction formation. The steep portion of the dose-response relation lay between 30 and 300 microM peptide. 4. In loop 1, the conserved motifs QPG and SHVR were identified as being involved in junction formation. In loop 2, the conserved SRPTEK motif was important. The ability of peptides containing the SRPTEK motif to interfere with the formation of gap junctions was enhanced by amino acids from the putative membrane-spanning region. 5. Peptides from loop 1 and loop 2 were equivalently effective; there was no synergism between them. 6. The inclusion of conserved cysteines in test peptides did not make them more effective in the competition assay. Images Figure 1 PMID:8576861

  10. The junctions that don't fit the scheme: special symmetrical cell-cell junctions of their own kind.

    PubMed

    Franke, Werner W; Rickelt, Steffen; Barth, Mareike; Pieperhoff, Sebastian

    2009-10-01

    Immunocytochemical, electron-, and immunoelectron-microscopical studies have revealed that, in addition to the four major "textbook categories" of cell-cell junctions (gap junctions, tight junctions, adherens junctions, and desmosomes), a broad range of other junctions exists, such as the tiny puncta adhaerentia minima, the taproot junctions (manubria adhaerentia), the plakophilin-2-containing adherens junctions of mesenchymal or mesenchymally derived cell types including malignantly transformed cells, the composite junctions (areae compositae) of the mature mammalian myocardium, the cortex adhaerens of the eye lens, the interdesmosomal "sandwich" or "stud" junctions in the subapical layers of stratified epithelia and the tumors derived therefrom, and the complexus adhaerentes of the endothelial and virgultar cells of the lymph node sinus. On the basis of their sizes and shapes, other morphological criteria, and their specific molecular ensembles, these junctions and the genes that encode them cannot be subsumed under one of the major categories mentioned above but represent special structures in their own right, appear to serve special functions, and can give rise to specific pathological disorders. PMID:19680692

  11. Increased beta-catenin protein and somatic APC mutations in sporadic aggressive fibromatoses (desmoid tumors).

    PubMed

    Alman, B A; Li, C; Pajerski, M E; Diaz-Cano, S; Wolfe, H J

    1997-08-01

    Sporadic aggressive fibromatosis (also called desmoid tumor) is a monoclonal proliferation of spindle (fibrocyte-like) cells that is locally invasive but does not metastasize. A similarity to abdominal fibromatoses (desmoids) in familial adenomatous polyposis and a cytogenetic study showing partial deletion of 5q in a subset of aggressive fibromatoses suggests that the adenomatous polyposis coli (APC) gene plays a role in its pathogenesis. APC helps regulate the cellular level of beta-catenin, which is a downstream mediator in Wnt (Wingless) signaling. beta-Catenin has a nuclear function (binds transcription factors) and a cell membrane function (is a component of epithelial cell adherens junctions). Six cases of aggressive fibromatosis of the extremities from patients without familial adenomatous polyposis, or a family history of colon cancer, were studied. Immunohistochemistry, using carboxy and amino terminus antibodies to APC, and DNA sequencing showed that three of the six contained an APC-truncating mutation, whereas normal tissues did not contain a mutation. Western blot and Northern dot blot showed that all six tumors had a higher level of beta-catenin protein than surrounding normal tissues, despite containing similar levels of beta-catenin mRNA. Immunohistochemistry localized beta-catenin throughout the cell in tumor tissues, although it localized more to the periphery in cells from normal tissues. Reverse transcription polymerase chain reaction showed that the tumors expressed N-cadherin but not E-cadherin (a pattern of expression of proteins making up adherens junctions similar to fibrocytes), suggesting that the specific adherens junctions present in epithelial cells are not necessary for beta-catenin function. Increased beta-catenin may cause the growth advantage of cells in this tumor through a nuclear mechanism. The increased protein level, relative to the RNA level, suggests that beta-catenin is degraded at a lower rate compared with normal tissues

  12. Increased beta-catenin protein and somatic APC mutations in sporadic aggressive fibromatoses (desmoid tumors).

    PubMed Central

    Alman, B. A.; Li, C.; Pajerski, M. E.; Diaz-Cano, S.; Wolfe, H. J.

    1997-01-01

    Sporadic aggressive fibromatosis (also called desmoid tumor) is a monoclonal proliferation of spindle (fibrocyte-like) cells that is locally invasive but does not metastasize. A similarity to abdominal fibromatoses (desmoids) in familial adenomatous polyposis and a cytogenetic study showing partial deletion of 5q in a subset of aggressive fibromatoses suggests that the adenomatous polyposis coli (APC) gene plays a role in its pathogenesis. APC helps regulate the cellular level of beta-catenin, which is a downstream mediator in Wnt (Wingless) signaling. beta-Catenin has a nuclear function (binds transcription factors) and a cell membrane function (is a component of epithelial cell adherens junctions). Six cases of aggressive fibromatosis of the extremities from patients without familial adenomatous polyposis, or a family history of colon cancer, were studied. Immunohistochemistry, using carboxy and amino terminus antibodies to APC, and DNA sequencing showed that three of the six contained an APC-truncating mutation, whereas normal tissues did not contain a mutation. Western blot and Northern dot blot showed that all six tumors had a higher level of beta-catenin protein than surrounding normal tissues, despite containing similar levels of beta-catenin mRNA. Immunohistochemistry localized beta-catenin throughout the cell in tumor tissues, although it localized more to the periphery in cells from normal tissues. Reverse transcription polymerase chain reaction showed that the tumors expressed N-cadherin but not E-cadherin (a pattern of expression of proteins making up adherens junctions similar to fibrocytes), suggesting that the specific adherens junctions present in epithelial cells are not necessary for beta-catenin function. Increased beta-catenin may cause the growth advantage of cells in this tumor through a nuclear mechanism. The increased protein level, relative to the RNA level, suggests that beta-catenin is degraded at a lower rate compared with normal tissues

  13. Ischemia-reperfusion impairs blood-brain barrier function and alters tight junction protein expression in the ovine fetus.

    PubMed

    Chen, X; Threlkeld, S W; Cummings, E E; Juan, I; Makeyev, O; Besio, W G; Gaitanis, J; Banks, W A; Sadowska, G B; Stonestreet, B S

    2012-12-13

    The blood-brain barrier is a restrictive interface between the brain parenchyma and the intravascular compartment. Tight junctions contribute to the integrity of the blood-brain barrier. Hypoxic-ischemic damage to the blood-brain barrier could be an important component of fetal brain injury. We hypothesized that increases in blood-brain barrier permeability after ischemia depend upon the duration of reperfusion and that decreases in tight junction proteins are associated with the ischemia-related impairment in blood-brain barrier function in the fetus. Blood-brain barrier function was quantified with the blood-to-brain transfer constant (K(i)) and tight junction proteins by Western immunoblot in fetal sheep at 127 days of gestation without ischemia, and 4, 24, or 48 h after ischemia. The largest increase in K(i) (P<0.05) was 4 h after ischemia. Occludin and claudin-5 expressions decreased at 4 h, but returned toward control levels 24 and 48 h after ischemia. Zonula occludens-1 and -2 decreased after ischemia. Inverse correlations between K(i) and tight junction proteins suggest that the decreases in tight junction proteins contribute to impaired blood-brain barrier function after ischemia. We conclude that impaired blood-brain barrier function is an important component of hypoxic-ischemic brain injury in the fetus, and that increases in quantitatively measured barrier permeability (K(i)) change as a function of the duration of reperfusion after ischemia. The largest increase in permeability occurs 4 h after ischemia and blood-brain barrier function improves early after injury because the blood-brain barrier is less permeable 24 and 48 than 4 h after ischemia. Changes in the tight junction molecular composition are associated with increases in blood-brain barrier permeability after ischemia.

  14. Probiotics modify tight-junction proteins in an animal model of nonalcoholic fatty liver disease

    PubMed Central

    Briskey, David; Heritage, Mandy; Jaskowski, Lesley-Anne; Peake, Jonathan; Gobe, Glenda; Subramaniam, V. Nathan; Crawford, Darrell; Campbell, Catherine; Vitetta, Luis

    2016-01-01

    Background: We have investigated the effects of a multispecies probiotic preparation containing a combination of probiotic bacterial genera that included Bifidobacteria, Lactobacilli and a Streptococcus in a mouse model of high-fat diet or obesity-induced liver steatosis. Methods: Three groups of C57B1/6J mice were fed either a standard chow or a high-fat diet for 20 weeks, while a third group was fed a high-fat diet for 10 weeks and then concomitantly administered probiotics for a further 10 weeks. Serum, liver and large bowel samples were collected for analysis. Results: The expression of the tight-junction proteins ZO-1 and ZO-2 was reduced (p < 0.05) in high-fat diet-fed mice compared to chow-fed mice. Probiotic supplementation helped to maintain tight ZO-1 and ZO-2 expression compared with the high-fat diet group (p < 0.05), but did not restore ZO-1 or ZO-2 expression compared with chow-fed mice. Mice fed a high-fat diet ± probiotics had significant steatosis development compared with chow-fed mice (p < 0.05); steatosis was less severe in the probiotics group compared with the high-fat diet group. Hepatic triglyceride concentration was higher in mice fed a high-fat diet ± probiotics compared with the chow group (p < 0.05), and was lower in the probiotics group compared with the high-fat diet group (p < 0.05). Compared with chow-fed mice, serum glucose, cholesterol concentration and the activity of alanine transaminase were higher (p < 0.05), whereas serum triglyceride concentration was lower (p < 0.05) in mice fed a high-fat diet ± probiotics. Conclusions: Supplementation with a multispecies probiotic formulation helped to maintain tight-junction proteins ZO-1 and ZO-2, and reduced hepatic triglyceride concentration compared with a high-fat diet alone. PMID:27366215

  15. Simvastatin Sodium Salt and Fluvastatin Interact with Human Gap Junction Gamma-3 Protein

    PubMed Central

    Marsh, Andrew; Casey-Green, Katherine; Probert, Fay; Withall, David; Mitchell, Daniel A.; Dilly, Suzanne J.; James, Sean; Dimitri, Wade; Ladwa, Sweta R.; Taylor, Paul C.; Singer, Donald R. J.

    2016-01-01

    Finding pleiomorphic targets for drugs allows new indications or warnings for treatment to be identified. As test of concept, we applied a new chemical genomics approach to uncover additional targets for the widely prescribed lipid-lowering pro-drug simvastatin. We used mRNA extracted from internal mammary artery from patients undergoing coronary artery surgery to prepare a viral cardiovascular protein library, using T7 bacteriophage. We then studied interactions of clones of the bacteriophage, each expressing a different cardiovascular polypeptide, with surface-bound simvastatin in 96-well plates. To maximise likelihood of identifying meaningful interactions between simvastatin and vascular peptides, we used a validated photo-immobilisation method to apply a series of different chemical linkers to bind simvastatin so as to present multiple orientations of its constituent components to potential targets. Three rounds of biopanning identified consistent interaction with the clone expressing part of the gene GJC3, which maps to Homo sapiens chromosome 7, and codes for gap junction gamma-3 protein, also known as connexin 30.2/31.3 (mouse connexin Cx29). Further analysis indicated the binding site to be for the N-terminal domain putatively ‘regulating’ connexin hemichannel and gap junction pores. Using immunohistochemistry we found connexin 30.2/31.3 to be present in samples of artery similar to those used to prepare the bacteriophage library. Surface plasmon resonance revealed that a 25 amino acid synthetic peptide representing the discovered N-terminus did not interact with simvastatin lactone, but did bind to the hydrolysed HMG CoA inhibitor, simvastatin acid. This interaction was also seen for fluvastatin. The gap junction blockers carbenoxolone and flufenamic acid also interacted with the same peptide providing insight into potential site of binding. These findings raise key questions about the functional significance of GJC3 transcripts in the vasculature and

  16. Protein-anchoring strategy for delivering acetylcholinesterase to the neuromuscular junction.

    PubMed

    Ito, Mikako; Suzuki, Yumi; Okada, Takashi; Fukudome, Takayasu; Yoshimura, Toshiro; Masuda, Akio; Takeda, Shin'ichi; Krejci, Eric; Ohno, Kinji

    2012-07-01

    Acetylcholinesterase (AChE) at the neuromuscular junction (NMJ) is anchored to the synaptic basal lamina via a triple helical collagen Q (ColQ). Congenital defects of ColQ cause endplate AChE deficiency and myasthenic syndrome. A single intravenous administration of adeno-associated virus serotype 8 (AAV8)-COLQ to Colq(-/-) mice recovered motor functions, synaptic transmission, as well as the morphology of the NMJ. ColQ-tailed AChE was specifically anchored to NMJ and its amount was restored to 89% of the wild type. We next characterized the molecular basis of this efficient recovery. We first confirmed that ColQ-tailed AChE can be specifically targeted to NMJ by an in vitro overlay assay in Colq(-/-) mice muscle sections. We then injected AAV1-COLQ-IRES-EGFP into the left tibialis anterior and detected AChE in noninjected limbs. Furthermore, the in vivo injection of recombinant ColQ-tailed AChE protein complex into the gluteus maximus muscle of Colq(-/-) mice led to accumulation of AChE in noninjected forelimbs. We demonstrated for the first time in vivo that the ColQ protein contains a tissue-targeting signal that is sufficient for anchoring itself to the NMJ. We propose that the protein-anchoring strategy is potentially applicable to a broad spectrum of diseases affecting extracellular matrix molecules.

  17. LDL-receptor-related protein 4 is crucial for formation of the neuromuscular junction.

    PubMed

    Weatherbee, Scott D; Anderson, Kathryn V; Niswander, Lee A

    2006-12-01

    Low-density lipoprotein receptor-related protein 4 (Lrp4) is a member of a family of structurally related, single-pass transmembrane proteins that carry out a variety of functions in development and physiology, including signal transduction and receptor-mediated endocytosis. Lrp4 is expressed in multiple tissues in the mouse, and is important for the proper development and morphogenesis of limbs, ectodermal organs, lungs and kidneys. We show that Lrp4 is also expressed in the post-synaptic endplate region of muscles and is required to form neuromuscular synapses. Lrp4-mutant mice die at birth with defects in both presynaptic and postsynaptic differentiation, including aberrant motor axon growth and branching, a lack of acetylcholine receptor and postsynaptic protein clustering, and a failure to express postsynaptic genes selectively by myofiber synaptic nuclei. Our data show that Lrp4 is required during the earliest events in postsynaptic neuromuscular junction (NMJ) formation and suggest that it acts in the early, nerveindependent steps of NMJ assembly. The identification of Lrp4 as a crucial factor for NMJ formation may have implications for human neuromuscular diseases such as myasthenia syndromes. PMID:17119023

  18. The role of gap junction proteins in the development of neural network functional topology.

    PubMed

    Anava, S; Saad, Y; Ayali, A

    2013-10-01

    Gap junctions (GJs) provide a common form of intercellular communication in most animal cells and tissues, from Hydra to human, including electrical synaptic signalling. Cell coupling via GJs has an important role in development in general, and in neural network development in particular. However, quantitative studies monitoring GJ proteins throughout nervous system development are few. Direct investigations demonstrating a role for GJ proteins by way of experimental manipulation of their expression are also rare. In the current work we focused on the role of invertebrate GJ proteins (innexins) in the in vitro development of neural network functional topology, using two-dimensional neural culture preparations derived from the frontal ganglion of the desert locust, Schistocerca gregaria. Immunocytochemistry and quantitative real-time PCR revealed a dynamic expression pattern of the innexins during development of the cultured networks. Changes were observed both in the levels and in the localization of expression. Down-regulating the expression of innexins, by using double-strand RNA for the first time in locust neural cultures, induced clear changes in network morphology, as well as inhibition of synaptogenesis, thus suggesting a role for GJs during the development of the functional topology of neuronal networks.

  19. Protein phosphatase 2A activity is required for functional adherent junctions in endothelial cells.

    PubMed

    Kása, Anita; Czikora, István; Verin, Alexander D; Gergely, Pál; Csortos, Csilla

    2013-09-01

    Reversible Ser/Thr phosphorylation of cytoskeletal and adherent junction (AJ) proteins has a critical role in the regulation of endothelial cell (EC) barrier function. We have demonstrated earlier that protein phosphatase 2A (PP2A) activity is important in EC barrier integrity. In the present work, macro- and microvascular EC were examined and we provided further evidence on the significance of PP2A in the maintenance of EC cytoskeleton and barrier function with special focus on the Bα (regulatory) subunit of PP2A. Immunofluorescent staining revealed that the inhibition of PP2A results in changes in the organization of EC cytoskeleton as microtubule dissolution and actin re-arrangement were detected. Depletion of Bα regulatory subunit of PP2A had similar effect on the cytoskeleton structure of the cells. Furthermore, transendothelial electric resistance measurements demonstrated significantly slower barrier recovery of Bα depleted EC after thrombin treatment. AJ proteins, VE-cadherin and β-catenin, were detected along with Bα in pull-down assay. Also, the inhibition of PP2A (by okadaic acid or fostriecin) or depletion of Bα caused β-catenin translocation from the membrane to the cytoplasm in parallel with its phosphorylation on Ser552. In conclusion, our data suggest that the A/Bα/C holoenzyme form of PP2A is essential in EC barrier integrity both in micro- and macrovascular EC. PMID:23721711

  20. Endotoxemia alters tight junction gene and protein expression in the kidney.

    PubMed

    Eadon, Michael T; Hack, Bradley K; Xu, Chang; Ko, Benjamin; Toback, F Gary; Cunningham, Patrick N

    2012-09-15

    Intact tight junctional (TJ) proteins are required for tubular ion transport and waste excretion. Disruption of TJs may contribute to a decreased glomerular filtration rate in acute kidney injury (AKI) via tubular backleak. The effect of LPS-mediated AKI on murine TJs has not been studied extensively. We hypothesized LPS endotoxin administration to mice would disrupt tubular TJ proteins including zonula occludens-1 (ZO-1), occludin, and claudins. ZO-1 and occludin immunofluorescence 24 h post-LPS revealed a marked change in localization from the usual circumferential fencework pattern to one with substantial fragmentation. Renal ZO-1 expression was significantly reduced 24 h after LPS (decrease of 56.1 ± 7.4%, P < 0.001), with subsequent recovery. ZO-1 mRNA expression was increased 24 h post-LPS (4.34 ± 0.87-fold, P = 0.0019), suggesting disruption of ZO-1 protein is not mediated by transcriptional regulation, but rather by degradation or changes in translation. Similarly, claudin-4 protein expression was decreased despite elevated mRNA. LPS administration resulted in dephosphorylation of occludin and fragmented tubular redistribution. Protein expression of claudin-1, and -3 was increased after LPS. ZO-1, occludin, and claudin-1, -3, and -4 gene expression were increased 48 h after LPS, suggesting a renal response to strengthen TJs following injury. Interestingly, reduced mRNA expression was found only for claudin-8. This study provides further support that LPS-induced AKI is associated with structural injury and is not merely due to hemodynamic changes.

  1. AMP-18 protects barrier function of colonic epithelial cells: role of tight junction proteins

    PubMed Central

    Walsh-Reitz, Margaret M.; Huang, Erick F.; Musch, Mark W.; Chang, Eugene B.; Martin, Terence E.; Kartha, Sreedharan; Toback, F. Gary

    2005-01-01

    AMP-18, a novel gastric antrum mucosal protein, and a synthetic peptide of amino acids 77-97, have mitogenic and motogenic properties for epithelial cells. The possibility that AMP-18 is also protective was evaluated in the colonic mucosa of mice and monolayer cultures of human colonic epithelial Caco2/bbe (C2) cells. Administration of AMP peptide to mice with dextran sulfate sodium (DSS)-induced colonic injury delayed the onset of bloody diarrhea, and reduced weight loss. Treatment of C2 cells with AMP peptide protected monolayers against decreases in transepithelial electrical resistance (TER) induced by the oxidant monochloramine, indomethacin, or DSS. A molecular mechanism for these barrier-protective effects was sought by asking if AMP peptide acted on specific tight junction (TJ) proteins. Immunoblots of detergent-insoluble fractions of C2 cells treated with AMP peptide exhibited increased accumulation of specific TJ proteins. Occludin immunoreactivity was also increased in detergent-insoluble fractions obtained from colonic mucosal cells of mice injected with AMP peptide. Laser scanning confocal microscopy (CF) supported the capacity of AMP peptide to enhance accumulation of occludin and ZO-1 in TJ domains of C2 cell monolayers, and together with immunoblot analysis showed that the peptide protected against loss of these TJ proteins following oxidant injury. AMP peptide also protected against a fall in TER during disruption of actin filaments by cytochalasin D, and stabilized perijunctional actin during oxidant injury when assessed by CF. These findings suggest that AMP-18 could protect the intestinal mucosal barrier by acting on specific TJ proteins and stabilizing perijunctional actin. PMID:15961882

  2. Bile salts disrupt human esophageal squamous epithelial barrier function by modulating tight junction proteins.

    PubMed

    Chen, Xin; Oshima, Tadayuki; Shan, Jing; Fukui, Hirokazu; Watari, Jiro; Miwa, Hiroto

    2012-07-15

    Reflux of acid and bile acids contributes to epithelial tissue injury in gastro-esophageal reflux disease. However, the influence of refluxed material on human esophageal stratified epithelial barrier function and tight junction (TJ) proteins has not been fully elucidated. Here, we investigated the influence of acid and bile acids on barrier function and TJ protein distribution using a newly developed air-liquid interface (ALI) in vitro culture model of stratified squamous epithelium based on primary human esophageal epithelial cells (HEECs). Under ALI conditions, HEECs formed distinct epithelial layers on Transwell inserts after 7 days of culture. The epithelial layers formed TJ, and the presence of claudin-1, claudin-4, and occludin were detected by immunofluorescent staining. The NP-40-insoluble fraction of these TJ proteins was significantly higher by day 7 of ALI culture. Exposure of HEECs to pH 2, and taurocholic acid (TCA) and glycocholic acid (GCA) at pH 3, but not pH 4, for 1 h decreased transepithelial electrical resistance (TEER) and increased paracellular permeability. Exposure of cell layers to GCA (pH 3) and TCA (pH 3) for 1 h also markedly reduced the insoluble fractions of claudin-1 and -4. We found that deoxycholic acid (pH 7.4 or 6, 1 h) and pepsin (pH 3, 24 h) significantly decreased TEER and increased permeability. Based on these findings, ALI-cultured HEECs represent a new in vitro model of human esophageal stratified epithelium and are suitable for studying esophageal epithelial barrier functions. Using this model, we demonstrated that acid, bile acids, and pepsin disrupt squamous epithelial barrier function partly by modulating TJ proteins. These results provide new insights into understanding the role of TJ proteins in esophagitis.

  3. c-Jun N-terminal kinase mediates disassembly of apical junctions in model intestinal epithelia.

    PubMed

    Naydenov, Nayden G; Hopkins, Ann M; Ivanov, Andrei I

    2009-07-01

    Dynamic remodeling of intercellular junctions is a critical determinant of epithelial barrier function in both physiological and pathophysiological states. While the disassembly of epithelial tight junctions (TJ) and adherens junctions (AJ) has been well-described in response to pathogens and other external stressors, the role of stress-related signaling in TJ/AJ regulation remains poorly understood. The aim of this study was to define the role of stress-activated c-Jun N-terminal kinase (JNK) in disruption of intercellular junctions in model intestinal epithelia. We show that rapid AJ/TJ disassembly triggered by extracellular calcium depletion of T84 and SK-CO15 cell monolayers was accompanied by activation (phosphorylation) of JNK, and prevented by pharmacological inhibitors of JNK. The opposite process, TJ/AJ reassembly, was accelerated by JNK inhibition and suppressed by the JNK activator anisomycin. JNK1 but not JNK2 was found to colocalize with intercellular junctions, and siRNA-mediated downregulation of JNK1 attenuated the TJ/AJ disruption caused by calcium depletion. JNK inhibition also blocked formation of characteristic contractile F-actin rings in calcium-depleted epithelial cells, suggesting that JNK regulates junctions by remodeling the actin cytoskeleton. In this role JNK acts downstream of the actin-reorganizing Rho-dependent kinase (ROCK), since ROCK inhibition abrogated JNK phosphorylation and TJ/AJ disassembly after calcium depletion. Furthermore, JNK acts upstream of F-actin-membrane linker proteins of the ERM (ezrin-radixin-moesin) family, but in a complex relationship yet to be fully elucidated. Taken together, our findings suggest a novel role for JNK in the signaling pathway that links ROCK and F-actin remodeling during disassembly of epithelial junctions.

  4. Lymphocytes accelerate epithelial tight junction assembly: role of AMP-activated protein kinase (AMPK).

    PubMed

    Tang, Xiao Xiao; Chen, Hao; Yu, Sidney; Zhang, Li; Caplan, Michael J; Chan, Hsiao Chang

    2010-01-01

    The tight junctions (TJs), characteristically located at the apicolateral borders of adjacent epithelial cells, are required for the proper formation of epithelial cell polarity as well as for sustaining the mucosal barrier to the external environment. The observation that lymphocytes are recruited by epithelial cells to the sites of infection [1] suggests that they may play a role in the modulation of epithelial barrier function and thus contribute to host defense. To test the ability of lymphocytes to modulate tight junction assembly in epithelial cells, we set up a lymphocyte-epithelial cell co-culture system, in which Madin-Darby canine kidney (MDCK) cells, a well-established model cell line for studying epithelial TJ assembly [2], were co-cultured with mouse lymphocytes to mimic an infection state. In a typical calcium switch experiment, the TJ assembly in co-culture was found to be accelerated compared to that in MDCK cells alone. This accelaration was found to be mediated by AMP-activated protein kinase (AMPK). AMPK activation was independent of changes in cellular ATP levels but it was found to be activated by the pro-inflammatory cytokine TNF-alpha. Forced suppression of AMPK, either with a chemical inhibitor or by knockdown, abrogated the accelerating effect of lymphocytes on TJ formation. Similar results were also observed in a co-culture with lymphocytes and Calu-3 human airway epithelial cells, suggesting that the activation of AMPK may be a general mechanism underlying lymphocyte-accelerated TJ assembly in different epithelia. These results suggest that signals from lymphocytes, such as cytokines, facilitate TJ assembly in epithelial cells via the activation of AMPK. PMID:20808811

  5. Characterization of the tight junction protein ZO-2 localized at the nucleus of epithelial cells.

    PubMed

    Jaramillo, Blanca Estela; Ponce, Arturo; Moreno, Jacqueline; Betanzos, Abigail; Huerta, Miriam; Lopez-Bayghen, Esther; Gonzalez-Mariscal, Lorenza

    2004-07-01

    ZO-2 is a MAGUK protein that in confluent epithelial sheets localizes at tight junctions (TJ) whereas in sparse cultures accumulates in clusters at the nucleus. Here, we have characterized several nuclear properties of ZO-2. We observe that ZO-2 is present in the nuclear matrix and co-immunoprecipitates with lamin B(1) and actin from the nuclei of sparse cultures. We show that ZO-2 presents several NLS at its amino region, that when deleted, diminish the nuclear import of the ZO-2 amino segment and impair the ability of the region to regulate the transcriptional activity of promoters controlled by AP-1. Several RS repeats are detected in the ZO-2 amino segment, however, their deletion does not preclude the display of a speckled nuclear pattern. ZO-2 displays two putative NES. However, only the second one appears to be functional, as when conjugated to ovalbumin (OV), it is able to translocate this protein from the nucleus to the cytoplasm in a leptomycin B-sensitive way.

  6. Adaptive evolution of tight junction protein claudin-14 in echolocating whales.

    PubMed

    Xu, Huihui; Liu, Yang; He, Guimei; Rossiter, Stephen J; Zhang, Shuyi

    2013-11-10

    Toothed whales and bats have independently evolved specialized ultrasonic hearing for echolocation. Recent findings have suggested that several genes including Prestin, Tmc1, Pjvk and KCNQ4 appear to have undergone molecular adaptations associated with the evolution of this ultrasonic hearing in mammals. Here we studied the hearing gene Cldn14, which encodes the claudin-14 protein and is a member of tight junction proteins that functions in the organ of Corti in the inner ear to maintain a cationic gradient between endolymph and perilymph. Particular mutations in human claudin-14 give rise to non-syndromic deafness, suggesting an essential role in hearing. Our results uncovered two bursts of positive selection, one in the ancestral branch of all toothed whales and a second in the branch leading to the delphinid, phocoenid and ziphiid whales. These two branches are the same as those previously reported to show positive selection in the Prestin gene. Furthermore, as with Prestin, the estimated hearing frequencies of whales significantly correlate with numbers of branch-wise non-synonymous substitutions in Cldn14, but not with synonymous changes. However, in contrast to Prestin, we found no evidence of positive selection in bats. Our findings from Cldn14, and comparisons with Prestin, strongly implicate multiple loci in the acquisition of echolocation in cetaceans, but also highlight possible differences in the evolutionary route to echolocation taken by whales and bats.

  7. Autophagy enhances intestinal epithelial tight junction barrier function by targeting claudin-2 protein degradation.

    PubMed

    Nighot, Prashant K; Hu, Chien-An Andy; Ma, Thomas Y

    2015-03-13

    Autophagy is an intracellular degradation pathway and is considered to be an essential cell survival mechanism. Defects in autophagy are implicated in many pathological processes, including inflammatory bowel disease. Among the innate defense mechanisms of intestinal mucosa, a defective tight junction (TJ) barrier has been postulated as a key pathogenic factor in the causation and progression of inflammatory bowel disease by allowing increased antigenic permeation. The cross-talk between autophagy and the TJ barrier has not yet been described. In this study, we present the novel finding that autophagy enhances TJ barrier function in Caco-2 intestinal epithelial cells. Nutrient starvation-induced autophagy significantly increased transepithelial electrical resistance and reduced the ratio of sodium/chloride paracellular permeability. Nutrient starvation reduced the paracellular permeability of small-sized urea but not larger molecules. The role of autophagy in the modulation of paracellular permeability was confirmed by pharmacological induction as well as pharmacological and genetic inhibition of autophagy. Consistent with the autophagy-induced reduction in paracellular permeability, a marked decrease in the level of the cation-selective, pore-forming TJ protein claudin-2 was observed after cell starvation. Starvation reduced the membrane presence of claudin-2 and increased its cytoplasmic, lysosomal localization. Therefore, our data show that autophagy selectively reduces epithelial TJ permeability of ions and small molecules by lysosomal degradation of the TJ protein claudin-2.

  8. Autophagy Enhances Intestinal Epithelial Tight Junction Barrier Function by Targeting Claudin-2 Protein Degradation*

    PubMed Central

    Nighot, Prashant K.; Hu, Chien-An Andy; Ma, Thomas Y.

    2015-01-01

    Autophagy is an intracellular degradation pathway and is considered to be an essential cell survival mechanism. Defects in autophagy are implicated in many pathological processes, including inflammatory bowel disease. Among the innate defense mechanisms of intestinal mucosa, a defective tight junction (TJ) barrier has been postulated as a key pathogenic factor in the causation and progression of inflammatory bowel disease by allowing increased antigenic permeation. The cross-talk between autophagy and the TJ barrier has not yet been described. In this study, we present the novel finding that autophagy enhances TJ barrier function in Caco-2 intestinal epithelial cells. Nutrient starvation-induced autophagy significantly increased transepithelial electrical resistance and reduced the ratio of sodium/chloride paracellular permeability. Nutrient starvation reduced the paracellular permeability of small-sized urea but not larger molecules. The role of autophagy in the modulation of paracellular permeability was confirmed by pharmacological induction as well as pharmacological and genetic inhibition of autophagy. Consistent with the autophagy-induced reduction in paracellular permeability, a marked decrease in the level of the cation-selective, pore-forming TJ protein claudin-2 was observed after cell starvation. Starvation reduced the membrane presence of claudin-2 and increased its cytoplasmic, lysosomal localization. Therefore, our data show that autophagy selectively reduces epithelial TJ permeability of ions and small molecules by lysosomal degradation of the TJ protein claudin-2. PMID:25616664

  9. EMP-induced alterations of tight junction protein expression and disruption of the blood-brain barrier.

    PubMed

    Ding, Gui-Rong; Qiu, Lian-Bo; Wang, Xiao-Wu; Li, Kang-Chu; Zhou, Yong-Chun; Zhou, Yan; Zhang, Jie; Zhou, Jia-Xing; Li, Yu-Rong; Guo, Guo-Zhen

    2010-07-15

    The blood-brain barrier (BBB) is critical to maintain cerebral homeostasis. In this study, we examined the effects of exposure to electromagnetic pulse (EMP) on the functional integrity of BBB and, on the localization and expression of tight junction (TJ) proteins (occludin and ZO-1) in rats. Animals were sham or whole-body exposed to EMP at 200 kV/m for 400 pulses. The permeability of BBB in rat cerebral cortex was examined by using Evans Blue (EB) and lanthanum nitrate as vascular tracers. The localization and expression of TJ proteins were assessed by western blot and immunofluorescence analysis, respectively. The data indicated that EMP exposure caused: (i) increased permeability of BBB, and (ii) altered localization as well as decreased levels of TJ protein ZO-1. These results suggested that the alteration of ZO-1 may play an important role in the disruption of tight junctions, which may lead to dysfunction of BBB after EMP exposure.

  10. p38 MAPK Regulates Cavitation and Tight Junction Function in the Mouse Blastocyst

    PubMed Central

    Bell, Christine E.; Watson, Andrew J.

    2013-01-01

    Blastocyst formation is essential for implantation and maintenance of pregnancy and is dependent on the expression and coordinated function of a series of proteins involved in establishing and maintaining the trans-trophectoderm ion gradient that enables blastocyst expansion. These consist of Na/K-ATPase, adherens junctions, tight junctions (TJ) and aquaporins (AQP). While their role in supporting blastocyst formation is established, the intracellular signaling pathways that coordinate their function is unclear. The p38 MAPK pathway plays a role in regulating these proteins in other cell types and is required for embryo development at the 8–16 cell stage, but its role has not been investigated in the blastocyst. Hypothesis p38 MAPK regulates blastocyst formation by regulating blastocyst formation gene expression and function. Methods Embryos were cultured from the early blastocyst stage for 12 h or 24 h in the presence of a potent and specific p38 MAPK inhibitor, SB 220025. Blastocyst expansion, hatching, gene family expression and localization, TJ function and apoptosis levels were analyzed. Results Inhibition of the p38 MAPK pathway reduced blastocyst expansion and hatching, increased tight junction permeability, affected TJP1 localization, reduced Aqp3 expression, and induced a significant increase in apoptosis. Conclusion The p38 MAPK pathway coordinates the overall events that regulate blastocyst formation. PMID:23593143

  11. Comparative analysis of theophylline and cholera toxin in rat colon reveals an induction of sealing tight junction proteins.

    PubMed

    Markov, Alexander G; Falchuk, Evgeny L; Kruglova, Natalia M; Rybalchenko, Oksana V; Fromm, Michael; Amasheh, Salah

    2014-11-01

    Claudin tight junction proteins have been identified to primarily determine intestinal epithelial barrier properties. While functional contribution of single claudins has been characterized in detail, information on the interplay with secretory mechanisms in native intestinal epithelium is scarce. Therefore, effects of cholera toxin and theophylline on rat colon were analyzed, including detection of sealing claudins. Tissue specimens were stripped off submucosal tissue layers and mounted in Ussing chambers, and short-circuit current (ISC) and transepithelial resistance (TER) were recorded. In parallel, expression and localization of claudins was analyzed and histological studies were performed employing hematoxylin-eosin staining and light and electron microscopy. Theophylline induced a strong increase of ISC in colon tissue specimens. In parallel, a decrease of TER was observed. In contrast, cholera toxin did not induce a significant increase of ISC, whereas an increase of TER was detected after 120 min. Western blots of membrane fractions revealed an increase of claudin-3 and -4 after incubation with cholera toxin, and theophylline induced an increase of claudin-4. In accordance, confocal laser-scanning microscopy exhibited increased signals of claudin-3 and -4 after incubation with cholera toxin, and increased signals of claudin-4 after incubation with theophylline, within tight junction complexes. Morphological analyses revealed no general changes of tight junction complexes, but intercellular spaces were markedly widened after incubation with cholera toxin and theophylline. We conclude that cholera toxin and theophylline have different effects on sealing tight junction proteins in native colon preparations, which may synergistically contribute to transport functions, in vitro.

  12. Arsenic-induced cutaneous hyperplastic lesions are associated with the dysregulation of Yap, a Hippo signaling-related protein

    SciTech Connect

    Li, Changzhao; Srivastava, Ritesh K.; Elmets, Craig A.; Afaq, Farrukh; Athar, Mohammad

    2013-09-06

    Highlights: •Arsenic activates canonical Hippo signaling pathway and up-regulates αCatenin in the skin. •Arsenic activates transcriptional activity of Yap by its nuclear translocation. •Yap is involved in the disruption of tight/adherens junctions in arsenic-exposed animals. -- Abstract: Arsenic exposure in humans causes a number of toxic manifestations in the skin including cutaneous neoplasm. However, the mechanism of these alterations remains elusive. Here, we provide novel observations that arsenic induced Hippo signaling pathway in the murine skin. This pathway plays crucial roles in determining organ size during the embryonic development and if aberrantly activated in adults, contributes to the pathogenesis of epithelial neoplasm. Arsenic treatment enhanced phosphorylation-dependent activation of LATS1 kinase and other Hippo signaling regulatory proteins Sav1 and MOB1. Phospho-LATS kinase is known to catalyze the inactivation of a transcriptional co-activator, Yap. However, in arsenic-treated epidermis, we did not observed its inactivation. Thus, as expected, unphosphorylated-Yap was translocated to the nucleus in arsenic-treated epidermis. Yap by binding to the transcription factors TEADs induces transcription of its target genes. Consistently, an up-regulation of Yap-dependent target genes Cyr61, Gli2, Ankrd1 and Ctgf was observed in the skin of arsenic-treated mice. Phosphorylated Yap is important in regulating tight and adherens junctions through its binding to αCatenin. We found disruption of these junctions in the arsenic-treated mouse skin despite an increase in αCatenin. These data provide evidence that arsenic-induced canonical Hippo signaling pathway and Yap-mediated disruption of tight and adherens junctions are independently regulated. These effects together may contribute to the carcinogenic effects of arsenic in the skin.

  13. Characterization of the structural and protein recognition properties of hybrid PNA-DNA four-way junctions.

    PubMed

    Iverson, Douglas; Serrano, Crystal; Brahan, Ann Marie; Shams, Arik; Totsingan, Filbert; Bell, Anthony J

    2015-12-01

    The objective of this study is to evaluate the structure and protein recognition properties of hybrid four-way junctions (4WJs) composed of DNA and peptide nucleic acid (PNA) strands. We compare a classic immobile DNA junction, J1, vs. six PNA-DNA junctions, including a number with blunt DNA ends and multiple PNA strands. Circular dichroism (CD) analysis reveals that hybrid 4WJs are composed of helices that possess structures intermediate between A- and B-form DNA, the apparent level of A-form structure correlates with the PNA content. The structure of hybrids that contain one PNA strand is sensitive to Mg(+2). For these constructs, the apparent B-form structure and conformational stability (Tm) increase in high Mg(+2). The blunt-ended junction, b4WJ-PNA3, possesses the highest B-form CD signals and Tm (40.1 °C) values vs. all hybrids and J1. Protein recognition studies are carried out using the recombinant DNA-binding protein, HMGB1b. HMGB1b binds the blunt ended single-PNA hybrids, b4WJ-PNA1 and b4WJ-PNA3, with high affinity. HMGB1b binds the multi-PNA hybrids, 4WJ-PNA1,3 and b4WJ-PNA1,3, but does not form stable protein-nucleic acid complexes. Protein interactions with hybrid 4WJs are influenced by the ratio of A- to B-form helices: hybrids with helices composed of higher levels of B-form structure preferentially associate with HMGB1b.

  14. Tight junction protein expression and barrier properties of immortalized mouse brain microvessel endothelial cells.

    PubMed

    Brown, Rachel C; Morris, Andrew P; O'Neil, Roger G

    2007-01-26

    Understanding the molecular and biochemical mechanisms regulating the blood-brain barrier is aided by in vitro model systems. Many studies have used primary cultures of brain microvessel endothelial cells for this purpose. However, primary cultures limit the generation of material for molecular and biochemical assays since cells grow slowly, are prone to contamination by other neurovascular unit cells, and lose blood-brain barrier characteristics when passaged. To address these issues, immortalized cell lines have been generated. In these studies, we assessed the suitability of the immortalized mouse brain endothelial cell line, bEnd3, as a blood-brain barrier model. RT-PCR and immunofluorescence indicated expression of multiple tight junction proteins. bEnd3 cells formed barriers to radiolabeled sucrose, and responded like primary cultures to disrupting stimuli. Exposing cells to serum-free media on their basolateral side significantly decreased paracellular permeability; astrocyte-conditioned media did not enhance barrier properties. The serum-free media-induced decrease in permeability was correlated with an increase in claudin-5 and zonula occludens-1 immunofluorescence at cell-cell contracts. We conclude that bEnd3 cells are an attractive candidate as a model of the blood-brain barrier due to their rapid growth, maintenance of blood-brain barrier characteristics over repeated passages, formation of functional barriers and amenability to numerous molecular interventions.

  15. Tight junction protein expression and barrier properties of immortalized mouse brain microvessel endothelial cells.

    PubMed

    Brown, Rachel C; Morris, Andrew P; O'Neil, Roger G

    2007-01-26

    Understanding the molecular and biochemical mechanisms regulating the blood-brain barrier is aided by in vitro model systems. Many studies have used primary cultures of brain microvessel endothelial cells for this purpose. However, primary cultures limit the generation of material for molecular and biochemical assays since cells grow slowly, are prone to contamination by other neurovascular unit cells, and lose blood-brain barrier characteristics when passaged. To address these issues, immortalized cell lines have been generated. In these studies, we assessed the suitability of the immortalized mouse brain endothelial cell line, bEnd3, as a blood-brain barrier model. RT-PCR and immunofluorescence indicated expression of multiple tight junction proteins. bEnd3 cells formed barriers to radiolabeled sucrose, and responded like primary cultures to disrupting stimuli. Exposing cells to serum-free media on their basolateral side significantly decreased paracellular permeability; astrocyte-conditioned media did not enhance barrier properties. The serum-free media-induced decrease in permeability was correlated with an increase in claudin-5 and zonula occludens-1 immunofluorescence at cell-cell contracts. We conclude that bEnd3 cells are an attractive candidate as a model of the blood-brain barrier due to their rapid growth, maintenance of blood-brain barrier characteristics over repeated passages, formation of functional barriers and amenability to numerous molecular interventions. PMID:17169347

  16. Three mechanisms control E-cadherin localization to the zonula adherens.

    PubMed

    Woichansky, Innokenty; Beretta, Carlo Antonio; Berns, Nicola; Riechmann, Veit

    2016-01-01

    E-cadherin localization to the zonula adherens is fundamental for epithelial differentiation but the mechanisms controlling localization are unclear. Using the Drosophila follicular epithelium we genetically dissect E-cadherin transport in an in vivo model. We distinguish three mechanisms mediating E-cadherin accumulation at the zonula adherens. Two membrane trafficking pathways deliver newly synthesized E-cadherin to the plasma membrane. One is Rab11 dependent and targets E-cadherin directly to the zonula adherens, while the other transports E-cadherin to the lateral membrane. Lateral E-cadherin reaches the zonula adherens by endocytosis and targeted recycling. We show that this pathway is dependent on RabX1, which provides a functional link between early and recycling endosomes. Moreover, we show that lateral E-cadherin is transported to the zonula adherens by an apically directed flow within the plasma membrane. Differential activation of these pathways could facilitate cell shape changes during morphogenesis, while their misregulation compromises cell adhesion and tissue architecture in differentiated epithelia. PMID:26960923

  17. Contactin-associated protein (Caspr) and contactin form a complex that is targeted to the paranodal junctions during myelination.

    PubMed

    Rios, J C; Melendez-Vasquez, C V; Einheber, S; Lustig, M; Grumet, M; Hemperly, J; Peles, E; Salzer, J L

    2000-11-15

    Specialized paranodal junctions form between the axon and the closely apposed paranodal loops of myelinating glia. They are interposed between sodium channels at the nodes of Ranvier and potassium channels in the juxtaparanodal regions; their precise function and molecular composition have been elusive. We previously reported that Caspr (contactin-associated protein) is a major axonal constituent of these junctions (Einheber et al., 1997). We now report that contactin colocalizes and forms a cis complex with Caspr in the paranodes and juxtamesaxon. These proteins coextract and coprecipitate from neurons, myelinating cultures, and myelin preparations enriched in junctional markers; they fractionate on sucrose gradients as a high-molecular-weight complex, suggesting that other proteins may also be associated with this complex. Neurons express two contactin isoforms that differ in their extent of glycosylation: a lower-molecular-weight phosphatidylinositol phospholipase C (PI-PLC)-resistant form is associated specifically with Caspr in the paranodes, whereas a higher-molecular-weight form of contactin, not associated with Caspr, is present in central nodes of Ranvier. These results suggest that the targeting of contactin to different axonal domains may be determined, in part, via its association with Caspr. Treatment of myelinating cocultures of Schwann cells and neurons with RPTPbeta-Fc, a soluble construct containing the carbonic anhydrase domain of the receptor protein tyrosine phosphatase beta (RPTPbeta), a potential glial receptor for contactin, blocks the localization of the Caspr/contactin complex to the paranodes. These results strongly suggest that a preformed complex of Caspr and contactin is targeted to the paranodal junctions via extracellular interactions with myelinating glia. PMID:11069942

  18. 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

  19. Cdc42-dependent Modulation of Tight Junctions and Membrane Protein Traffic in Polarized Madin-Darby Canine Kidney Cells

    PubMed Central

    Rojas, Raul; Ruiz, Wily G.; Leung, Som-Ming; Jou, Tzuu-Shuh; Apodaca, Gerard

    2001-01-01

    Polarized epithelial cells maintain the asymmetric composition of their apical and basolateral membrane domains by at least two different processes. These include the regulated trafficking of macromolecules from the biosynthetic and endocytic pathway to the appropriate membrane domain and the ability of the tight junction to prevent free mixing of membrane domain-specific proteins and lipids. Cdc42, a Rho family GTPase, is known to govern cellular polarity and membrane traffic in several cell types. We examined whether this protein regulated tight junction function in Madin-Darby canine kidney cells and pathways that direct proteins to the apical and basolateral surface of these cells. We used Madin-Darby canine kidney cells that expressed dominant-active or dominant-negative mutants of Cdc42 under the control of a tetracycline-repressible system. Here we report that expression of dominant-active Cdc42V12 or dominant-negative Cdc42N17 altered tight junction function. Expression of Cdc42V12 slowed endocytic and biosynthetic traffic, and expression of Cdc42N17 slowed apical endocytosis and basolateral to apical transcytosis but stimulated biosynthetic traffic. These results indicate that Cdc42 may modulate multiple cellular pathways required for the maintenance of epithelial cell polarity. PMID:11514615

  20. 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.

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

    PubMed

    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

  2. The single Drosophila ZO-1 protein Polychaetoid regulates embryonic morphogenesis in coordination with Canoe/afadin and Enabled

    PubMed Central

    Choi, Wangsun; Jung, Kuo-Chen; Nelson, Kevin S.; Bhat, Manzoor A.; Beitel, Greg J.; Peifer, Mark; Fanning, Alan S.

    2011-01-01

    Adherens and tight junctions play key roles in assembling epithelia and maintaining barriers. In cell culture zonula occludens (ZO)–family proteins are important for assembly/maturation of both tight and adherens junctions (AJs). Genetic studies suggest that ZO proteins are important during normal development, but interpretation of mouse and fly studies is limited by genetic redundancy and/or a lack of null alleles. We generated null alleles of the single Drosophila ZO protein Polychaetoid (Pyd). Most embryos lacking Pyd die with striking defects in morphogenesis of embryonic epithelia including the epidermis, segmental grooves, and tracheal system. Pyd loss does not dramatically affect AJ protein localization or initial localization of actin and myosin during dorsal closure. However, Pyd loss does affect several cell behaviors that drive dorsal closure. The defects, which include segmental grooves that fail to retract, a disrupted leading edge actin cable, and reduced zippering as leading edges meet, closely resemble defects in canoe zygotic null mutants and in embryos lacking the actin regulator Enabled (Ena), suggesting that these proteins act together. Canoe (Cno) and Pyd are required for proper Ena localization during dorsal closure, and strong genetic interactions suggest that Cno, Pyd, and Ena act together in regulating or anchoring the actin cytoskeleton during dorsal closure. PMID:21508316

  3. Impact of obesity on 7,12-dimethylbenz[a]anthracene-induced altered ovarian connexin gap junction proteins in female mice

    SciTech Connect

    Ganesan, Shanthi Nteeba, Jackson Keating, Aileen F.

    2015-01-01

    The ovarian gap junction proteins alpha 4 (GJA4 or connexin 37; CX37), alpha 1 (GJA1 or connexin 43; CX43) and gamma 1 (GJC1 or connexin 45; CX45) are involved in cell communication and folliculogenesis. 7,12-dimethylbenz[a]anthracene (DMBA) alters Cx37 and Cx43 expression in cultured neonatal rat ovaries. Additionally, obesity has an additive effect on DMBA-induced ovarian cell death and follicle depletion, thus, we investigated in vivo impacts of obesity and DMBA on CX protein levels. Ovaries were collected from lean and obese mice aged 6, 12, 18, or 24 wks. A subset of 18 wk old mice (lean and obese) were dosed with sesame oil or DMBA (1 mg/kg; ip) for 14 days and ovaries collected 3 days thereafter. Cx43 and Cx45 mRNA and protein levels decreased (P < 0.05) after 18 wks while Cx37 mRNA and protein levels decreased (P < 0.05) after 24 wks in obese ovaries. Cx37 mRNA and antral follicle protein staining intensity were reduced (P < 0.05) by obesity while total CX37 protein was reduced (P < 0.05) in DMBA exposed obese ovaries. Cx43 mRNA and total protein levels were decreased (P < 0.05) by DMBA in both lean and obese ovaries while basal protein staining intensity was reduced (P < 0.05) in obese controls. Cx45 mRNA, total protein and protein staining intensity level were decreased (P < 0.05) by obesity. These data support that obesity temporally alters gap junction protein expression and that DMBA-induced ovotoxicity may involve reduced gap junction protein function. - Highlights: • Ovarian gap junction proteins are affected by ovarian aging and obesity. • DMBA exposure negatively impacts gap junction proteins. • Altered gap junction proteins may contribute to infertility.

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

    SciTech Connect

    Tkachuk, Natalia; Tkachuk, Sergey; Patecki, Margret; Kusch, Angelika; Korenbaum, Elena; Haller, Hermann; Dumler, Inna

    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), 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.

  5. A protein interaction map for cell-cell adhesion regulators identifies DUSP23 as a novel phosphatase for β-catenin

    PubMed Central

    Gallegos, Lisa Leon; Ng, Mei Rosa; Sowa, Mathew E.; Selfors, Laura M.; White, Anne; Zervantonakis, Ioannis K.; Singh, Pragya; Dhakal, Sabin; Harper, J. Wade; Brugge, Joan S.

    2016-01-01

    Cell-cell adhesion is central to morphogenesis and maintenance of epithelial cell state. We previously identified 27 candidate cell-cell adhesion regulatory proteins (CCARPs) whose down-regulation disrupts epithelial cell-cell adhesion during collective migration. Using a protein interaction mapping strategy, we found that 18 CCARPs link to core components of adherens junctions or desmosomes. We further mapped linkages between the CCARPs and other known cell-cell adhesion proteins, including hits from recent screens uncovering novel components of E-cadherin adhesions. Mechanistic studies of one novel CCARP which links to multiple cell-cell adhesion proteins, the phosphatase DUSP23, revealed that it promotes dephosphorylation of β-catenin at Tyr 142 and enhances the interaction between α- and β-catenin. DUSP23 knockdown specifically diminished adhesion to E-cadherin without altering adhesion to fibronectin matrix proteins. Furthermore, DUSP23 knockdown produced “zipper-like” cell-cell adhesions, caused defects in transmission of polarization cues, and reduced coordination during collective migration. Thus, this study identifies multiple novel connections between proteins that regulate cell-cell interactions and provides evidence for a previously unrecognized role for DUSP23 in regulating E-cadherin adherens junctions through promoting the dephosphorylation of β-catenin. PMID:27255161

  6. The tight junction protein JAM-A functions as coreceptor for rotavirus entry into MA104 cells.

    PubMed

    Torres-Flores, Jesús M; Silva-Ayala, Daniela; Espinoza, Marco A; López, Susana; Arias, Carlos F

    2015-01-15

    Several molecules have been identified as receptors or coreceptors for rotavirus infection, including glycans, integrins, and hsc70. In this work we report that the tight junction proteins JAM-A, occludin, and ZO-1 play an important role during rotavirus entry into MA104 cells. JAM-A was found to function as coreceptor for rotavirus strains RRV, Wa, and UK, but not for rotavirus YM. Reassortant viruses derived from rotaviruses RRV and YM showed that the virus spike protein VP4 determines the use of JAM-A as coreceptor.

  7. N-glycosylation controls the function of junctional adhesion molecule-A

    PubMed Central

    Scott, David W.; Tolbert, Caitlin E.; Graham, David M.; Wittchen, Erika; Bear, James E.; Burridge, Keith

    2015-01-01

    Junctional adhesion molecule-A (JAM-A) is an adherens and tight junction protein expressed by endothelial and epithelial cells. JAM-A serves many roles and contributes to barrier function and cell migration and motility, and it also acts as a ligand for the leukocyte receptor LFA-1. JAM-A is reported to contain N-glycans, but the extent of this modification and its contribution to the protein’s functions are unknown. We show that human JAM-A contains a single N-glycan at N185 and that this residue is conserved across multiple mammalian species. A glycomutant lacking all N-glycans, N185Q, is able to reach the cell surface but exhibits decreased protein half-life compared with the wild- type protein. N-glycosylation of JAM-A is required for the protein’s ability to reinforce barrier function and contributes to Rap1 activity. We further show that glycosylation of N185 is required for JAM-A–mediated reduction of cell migration. Finally, we show that N-glycosylation of JAM-A regulates leukocyte adhesion and LFA-1 binding. These findings identify N-glycosylation as critical for JAM-A’s many functions. PMID:26224316

  8. Kin of IRRE-like Protein 2 Is a Phosphorylated Glycoprotein That Regulates Basal Insulin Secretion*

    PubMed Central

    Yesildag, Burcak; Bock, Thomas; Herrmanns, Karolin; Wollscheid, Bernd; Stoffel, Markus

    2015-01-01

    Direct interactions among pancreatic β-cells via cell surface proteins inhibit basal and enhance stimulated insulin secretion. Here, we functionally and biochemically characterized Kirrel2, an immunoglobulin superfamily protein with β-cell-specific expression in the pancreas. Our results show that Kirrel2 is a phosphorylated glycoprotein that co-localizes and interacts with the adherens junction proteins E-cadherin and β-catenin in MIN6 cells. We further demonstrate that the phosphosites Tyr595–596 are functionally relevant for the regulation of Kirrel2 stability and localization. Analysis of the extracellular and intracellular domains of Kirrel2 revealed that it is cleaved and shed from MIN6 cells and that the remaining membrane spanning cytoplasmic domain is processed by γ-secretase complex. Kirrel2 knockdown with RNA interference in MIN6 cells and ablation of Kirrel2 from mice with genetic deletion resulted in increased basal insulin secretion from β-cells, with no immediate influence on stimulated insulin secretion, total insulin content, or whole body glucose metabolism. Our results show that in pancreatic β-cells Kirrel2 localizes to adherens junctions, is regulated by multiple post-translational events, including glycosylation, extracellular cleavage, and phosphorylation, and engages in the regulation of basal insulin secretion. PMID:26324709

  9. Ultrastructural studies of the junctional complex in the musculature of the arrow-worm (Sagitta setosa) (Chaetognatha).

    PubMed

    Duvert, M; Gros, D; Salat, C

    1980-01-01

    In the A fibres of the primary musculature of Sagitta, the junctional complex is made up of three kinds of junctions. From the apex to the base they occur in the following order: an apical zonula adherens, a columnar zonula then columnar maculae intermingled with gap junction. Each columnar junction joins two intracellular filament networks in adjacent cells; this cytoskeleton is largely developed around the nucleus of the A fibres and in close relation with the contractile apparatus, especially at the I band level. The B fibres, which never reach the general cavity, lack zonula adherens and columnar zonula. The columnar junction constitutes a new type of junction which seems to belong to the adherens kind. At their level fibrous columns cross the extracellular space, joining the membranes. Each column faces two cytoplasmic densities localized against the cytoplasmic leaflets of the membranes. A cytoskeleton composed of bunldes of cytoplasmic filaments is in close contact with these cytoplasmic densities. The great number of columnar junctions and associated cytoskeleton assure the cohesion of the tissue and the distribution of contractile forces in the absence of connective tissue. The abundance of gap junctions can account for the metabolic and ionic coupling of the fibres. PMID:7189067

  10. Ultrastructural studies of the junctional complex in the musculature of the arrow-worm (Sagitta setosa) (Chaetognatha).

    PubMed

    Duvert, M; Gros, D; Salat, C

    1980-01-01

    In the A fibres of the primary musculature of Sagitta, the junctional complex is made up of three kinds of junctions. From the apex to the base they occur in the following order: an apical zonula adherens, a columnar zonula then columnar maculae intermingled with gap junction. Each columnar junction joins two intracellular filament networks in adjacent cells; this cytoskeleton is largely developed around the nucleus of the A fibres and in close relation with the contractile apparatus, especially at the I band level. The B fibres, which never reach the general cavity, lack zonula adherens and columnar zonula. The columnar junction constitutes a new type of junction which seems to belong to the adherens kind. At their level fibrous columns cross the extracellular space, joining the membranes. Each column faces two cytoplasmic densities localized against the cytoplasmic leaflets of the membranes. A cytoskeleton composed of bunldes of cytoplasmic filaments is in close contact with these cytoplasmic densities. The great number of columnar junctions and associated cytoskeleton assure the cohesion of the tissue and the distribution of contractile forces in the absence of connective tissue. The abundance of gap junctions can account for the metabolic and ionic coupling of the fibres.

  11. Oncostatin M induces upregulation of claudin-2 in rodent hepatocytes coinciding with changes in morphology and function of tight junctions

    SciTech Connect

    Imamura, Masafumi; Kojima, Takashi . E-mail: ktakashi@sapmed.ac.jp; Lan, Mengdong; Son, Seiichi; Murata, Masaki; Osanai, Makoto; Chiba, Hideki; Hirata, Koichi; Sawada, Norimasa

    2007-05-15

    In rodent livers, integral tight junction (TJ) proteins claudin-1, -2, -3, -5 and -14 are detected and play crucial roles in the barrier to keep bile in bile canaculi away from the blood circulation. Claudin-2 shows a lobular gradient increasing from periportal to pericentral hepatocytes, whereas claudin-1 and -3 are expressed in the whole liver lobule. Although claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells, the physiological functions and regulation of claudin-2 in hepatocytes remain unclear. Oncostatin M (OSM) is a multifunctional cytokine implicated in the differentiation of hepatocytes that induces formation of E-cadherin-based adherens junctions in fetal hepatocytes. In this study, we examined whether OSM could induce expression and function of claudin-2 in rodent hepatocytes, immortalized mouse and primary cultured proliferative rat hepatocytes. In the immortalized mouse and primary cultured proliferative rat hepatocytes, treatment with OSM markedly increased mRNA and protein of claudin-2 together with formation of developed networks of TJ strands. The increase of claudin-2 enhanced the paracellular barrier function which depended on molecular size. The increase of claudin-2 expression induced by OSM in rodent hepatocytes was regulated through distinct signaling pathways including PKC. These results suggest that expression of claudin-2 in rodent hepatocytes may play a specific role as controlling the size of paracellular permeability in the barrier to keep bile in bile canaculi.

  12. The role of aquaporin and tight junction proteins in the regulation of water movement in larval zebrafish (Danio rerio).

    PubMed

    Kwong, Raymond W M; Kumai, Yusuke; Perry, Steve F

    2013-01-01

    Teleost fish living in freshwater are challenged by passive water influx; however the molecular mechanisms regulating water influx in fish are not well understood. The potential involvement of aquaporins (AQP) and epithelial tight junction proteins in the regulation of transcellular and paracellular water movement was investigated in larval zebrafish (Danio rerio). We observed that the half-time for saturation of water influx (K(u)) was 4.3±0.9 min, and reached equilibrium at approximately 30 min. These findings suggest a high turnover rate of water between the fish and the environment. Water influx was reduced by the putative AQP inhibitor phloretin (100 or 500 μM). Immunohistochemistry and confocal microscopy revealed that AQP1a1 protein was expressed in cells on the yolk sac epithelium. A substantial number of these AQP1a1-positive cells were identified as ionocytes, either H⁺-ATPase-rich cells or Na⁺/K⁺-ATPase-rich cells. AQP1a1 appeared to be expressed predominantly on the basolateral membranes of ionocytes, suggesting its potential involvement in regulating ionocyte volume and/or water flux into the circulation. Additionally, translational gene knockdown of AQP1a1 protein reduced water influx by approximately 30%, further indicating a role for AQP1a1 in facilitating transcellular water uptake. On the other hand, incubation with the Ca²⁺-chelator EDTA or knockdown of the epithelial tight junction protein claudin-b significantly increased water influx. These findings indicate that the epithelial tight junctions normally act to restrict paracellular water influx. Together, the results of the present study provide direct in vivo evidence that water movement can occur through transcellular routes (via AQP); the paracellular routes may become significant when the paracellular permeability is increased.

  13. Developmental expression and molecular characterization of two gap junction channel proteins expressed during embryogenesis in the grasshopper Schistocerca americana.

    PubMed

    Ganfornina, M D; Sánchez, D; Herrera, M; Bastiani, M J

    1999-01-01

    Gap junctions are membrane channels that directly connect the cytoplasm of neighboring cells, allowing the exchange of ions and small molecules. Two analogous families of proteins, the connexins and innexins, are the channel-forming molecules in vertebrates and invertebrates, respectively. In order to study the role of gap junctions in the embryonic development of the nervous system, we searched for innexins in the grasshopper Schistocerca americana. Here we present the molecular cloning and sequence analysis of two novel innexins, G-Inx(1) and G-Inx(2), expressed during grasshopper embryonic development. The analysis of G-Inx(1) and G-Inx(2) proteins suggests they bear four transmembrane domains, which show strong conservation in members of the innexin family. The study of the phylogenetic relationships between members of the innexin family and the new grasshopper proteins suggests that G-Inx(1) is orthologous to the Drosophila 1(1)-ogre. However, G-Inx(2) seems to be a member of a new group of insect innexins. We used in situ hybridization with the G-Inx(1) and G-Inx(2) cDNA clones, and two polyclonal sera raised against different regions of G-Inx(1) to study the mRNA and protein expression patterns and the subcellular localization of the grasshopper innexins. G-Inx(1) is primarily expressed in the embryonic nervous system, in neural precursors and glial cells. In addition, a restricted stripe of epithelial cells in the developing limb, involved in the guidance of sensory growth cones, expresses G-Inx(1). G-Inx(2) expression is more widespread in the grasshopper embryo, but a restricted expression is found in a subset of neural precursors. The generally different but partially overlapping expression patterns of G-Inx(1) and G-Inx(2) supports the combinatorial character of gap junction formation in invertebrates, an essential property to generate specificity in this form of cell-cell communication.

  14. The apical polarity protein network in Drosophila epithelial cells: regulation of polarity, junctions, morphogenesis, cell growth, and survival.

    PubMed

    Tepass, Ulrich

    2012-01-01

    Epithelial tissue formation and function requires the apical-basal polarization of individual epithelial cells. Apical polarity regulators (APRs) are an evolutionarily conserved group of key factors that govern polarity and several other aspects of epithelial differentiation. APRs compose a diverse set of molecules including a transmembrane protein (Crumbs), a serine/threonine kinase (aPKC), a lipid phosphatase (PTEN), a small GTPase (Cdc42), FERM domain proteins (Moesin, Yurt), and several adaptor or scaffolding proteins (Bazooka/Par3, Par6, Stardust, Patj). These proteins form a dynamic cooperative network that is engaged in negative-feedback regulation with basolateral polarity factors to set up the epithelial apical-basal axis. APRs support the formation of the apical junctional complex and the segregation of the junctional domain from the apical membrane. It is becoming increasingly clear that APRs interact with the cytoskeleton and vesicle trafficking machinery, regulate morphogenesis, and modulate epithelial cell growth and survival. Not surprisingly, APRs have multiple fundamental links to human diseases such as cancer and blindness.

  15. Drosophila Syncrip modulates the expression of mRNAs encoding key synaptic proteins required for morphology at the neuromuscular junction

    PubMed Central

    McDermott, Suzanne M.; Yang, Lu; Halstead, James M.; Hamilton, Russell S.; Meignin, Carine

    2014-01-01

    Localized mRNA translation is thought to play a key role in synaptic plasticity, but the identity of the transcripts and the molecular mechanism underlying their function are still poorly understood. Here, we show that Syncrip, a regulator of localized translation in the Drosophila oocyte and a component of mammalian neuronal mRNA granules, is also expressed in the Drosophila larval neuromuscular junction, where it regulates synaptic growth. We use RNA-immunoprecipitation followed by high-throughput sequencing and qRT-PCR to show that Syncrip associates with a number of mRNAs encoding proteins with key synaptic functions, including msp-300, syd-1, neurexin-1, futsch, highwire, discs large, and α-spectrin. The protein levels of MSP-300, Discs large, and a number of others are significantly affected in syncrip null mutants. Furthermore, syncrip mutants show a reduction in MSP-300 protein levels and defects in muscle nuclear distribution characteristic of msp-300 mutants. Our results highlight a number of potential new players in localized translation during synaptic plasticity in the neuromuscular junction. We propose that Syncrip acts as a modulator of synaptic plasticity by regulating the translation of these key mRNAs encoding synaptic scaffolding proteins and other important components involved in synaptic growth and function. PMID:25171822

  16. Interaction of Ubinuclein-1, a nuclear and adhesion junction protein, with the 14-3-3 epsilon protein in epithelial cells: implication of the PKA pathway.

    PubMed

    Conti, Audrey; Sueur, Charlotte; Lupo, Julien; Brazzolotto, Xavier; Burmeister, Wim P; Manet, Evelyne; Gruffat, Henri; Morand, Patrice; Boyer, Véronique

    2013-03-01

    Ubinuclein-1 is a NACos (Nuclear and Adhesion junction Complex components) protein which shuttles between the nucleus and tight junctions, but its function in the latter is not understood. Here, by co-immunoprecipitation and confocal analysis, we show that Ubinuclein-1 interacts with the 14-3-3ɛ protein both in HT29 colon cells, and AGS gastric cells. This interaction is mediated by an Ubinuclein-1 phosphoserine motif. We show that the arginine residues (R56, R60 and R132) which form the 14-3-3ɛ ligand binding site are responsible for the binding of 14-3-3ɛ to phosphorylated Ubinuclein-1. Furthermore, we demonstrate that in vitro Ubinuclein-1 can be directly phosphorylated by cAMP-dependent protein kinase A. This in vitro phosphorylation allows binding of wildtype 14-3-3ɛ. Moreover, treatment of the cells with inhibitors of the cAMP-dependent protein kinase, KT5720 or H89, modifies the subcellular localization of Ubinuclein-1. Indeed, KT5720 and H89 greatly increase the staining of Ubinuclein-1 at the tight junctions in AGS gastric cells. In the presence of the kinase inhibitor KT5720, the amount of Ubinuclein-1 in the NP40 insoluble fraction is increased, together with actin. Moreover, treatment of the cells with KT5720 or H89 induces the concentration of Ubinuclein-1 at tricellular intersections of MDCK cells. Taken together, our findings demonstrate novel cell signaling trafficking by Ubinuclein-1 via association with 14-3-3ɛ following Ubinuclein-1 phosphorylation by the cAMP-dependent protein kinase-A.

  17. Guidance of subcellular tubulogenesis by actin under the control of a synaptotagmin-like protein and Moesin.

    PubMed

    JayaNandanan, N; Mathew, Renjith; Leptin, Maria

    2014-01-01

    Apical membranes in many polarized epithelial cells show specialized morphological adaptations that fulfil distinct physiological functions. The air-transporting tubules of Drosophila tracheal terminal cells represent an extreme case of membrane specialization. Here we show that Bitesize (Btsz), a synaptotagmin-like protein family member, is needed for luminal membrane morphogenesis. Unlike in multicellular tubes and other epithelia, where it influences apical integrity by affecting adherens junctions, Btsz here acts at a distance from junctions. Localized at the luminal membrane through its tandem C2 domain, it recruits activated Moesin. Both proteins are needed for the integrity of the actin cytoskeleton at the luminal membrane, but not for other pools of F-actin in the cell, nor do actin-dependent processes at the outer membrane, such as filopodial activity or membrane growth depend on Btsz. Btsz and Moesin guide luminal membrane morphogenesis through organizing actin and allowing the incorporation of membrane containing the apical determinant Crumbs.

  18. Angelica archengelica extract induced perturbation of rat skin and tight junctional protein (ZO-1) of HaCaT cells

    PubMed Central

    Kaushal, N.; Naz, S.; Tiwary, AK.

    2011-01-01

    Background and purpose of the study Herbal enhancers compared to the synthetic ones have shown less toxis effects. Coumarins have been shown at concentrations inhibiting phospoliphase C-Y (Phc-Y) are able to enhance tight junction (TJ) permeability due to hyperpoalation of Zonolous Occludense-1 (ZO-1) proteins. The purpose of this study was to evaluate the influence of ethanolic extract of Angelica archengelica (AA-E) which contain coumarin on permeation of repaglinide across rat epidermis and on the tight junction plaque protein ZO-1 in HaCaT cells. Methods Transepidermal water loss (TEWL) from the rat skin treated with different concentrations of AA-E was assessed by Tewameter. Scanning and Transmission Electron Microscopy (TEM) on were performed on AA-E treated rat skin portions. The possibility of AA-E influence on the architecture of tight junctions by adverse effect on the cytoplasmic ZO-1 in HaCaT cells was investigated. Finally, the systemic delivery of repaglinide from the optimized transdermal formulation was investigated in rats. Results The permeation of repaglinide across excised rat epidermis was 7-fold higher in the presence of AA-E (5% w/v) as compared to propylene glycol:ethanol (7:3) mixture. The extract was found to perturb the lipid microconstituents in both excised and viable rat skin, although, the effect was less intense in the later. The enhanced permeation of repaglinide across rat epidermis excised after treatment with AA-E (5% w/v) for different periods was in concordance with the high TEWL values of similarly treated viable rat skin. Further, the observed increase in intercellular space, disordering of lipid structure and corneocyte detachment indicated considerable effect on the ultrastructure of rat epidermis. Treatment of HaCaT cell line with AA-E (0.16% w/v) for 6 hrs influenced ZO-1 as evidenced by reduced immunofluorescence of anti-TJP1 (ZO-1) antibody in Confocal Laser Scanning Microscopy studies (CLSM) studies. The plasma

  19. Molecular cloning, expression analysis, and functional characterization of connexin44.1: a zebrafish lens gap junction protein.

    PubMed

    Cason, N; White, T W; Cheng, S; Goodenough, D A; Valdimarsson, G

    2001-06-01

    The connexin family of genes codes for proteins that oligomerize into a connexon of six subunits to form one half of the gap junction channel. Gap junctions are plasma membrane structures that mediate intercellular communication by joining the cytoplasm of two cells, allowing the passage of small molecules and metabolites, and contributing significantly to the maintenance of tissue homeostasis. The signaling mediated by these junctions appears to be necessary for the correct timing of key developmental events. This communication is especially important in the avascular lens where the intercellular passage of metabolites, second messengers, and ions is necessary to maintain the correct ionic balance in the lens fibre cells, and prevent cataract formation. To characterize the role that the connexin genes play in development, a novel connexin was cloned from zebrafish. A genomic clone was isolated that contained a 1,173 base open reading frame. The nucleotide sequence in this open reading frame shows extensive sequence similarity to mouse connexin50 (Cx50), chicken Cx45.6, sheep Cx49, and human Cx50. The protein encoded by this open reading frame contains 391 amino acids, with a predicted molecular weight of 44.1 kDa and a typical connexin transmembrane topology. By using the LN54 radiation hybrid panel, the Cx44.1 gene was mapped to linkage group 1. Whole-mount in situ hybridization and Northern blot analyses were performed on zebrafish embryos at various developmental stages to characterize the developmental expression of the Cx44.1 message. The ocular lens was the only tissue in which Cx44.1 transcripts were detected. The transcripts were first detected in the lens around 24 hr post fertilization and remained detectable until 120 hr post fertilization. Electrophysiological analysis of Cx44.1 channels revealed gating properties that were virtually identical to the mouse and chicken orthologues of Cx44.1.

  20. Phosphorylation of connexin 32, a hepatocyte gap-junction protein, by cAMP-dependent protein kinase, protein kinase C and Ca2+/calmodulin-dependent protein kinase II.

    PubMed

    Sáez, J C; Nairn, A C; Czernik, A J; Spray, D C; Hertzberg, E L; Greengard, P; Bennett, M V

    1990-09-11

    Phosphorylation of connexin 32, the major liver gap-junction protein, was studied in purified liver gap junctions and in hepatocytes. In isolated gap junctions, connexin 32 was phosphorylated by cAMP-dependent protein kinase (cAMP-PK), by protein kinase C (PKC) and by Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM-PK II). Connexin 26 was not phosphorylated by these three protein kinases. Phosphopeptide mapping of connexin 32 demonstrated that cAMP-PK and PKC primarily phosphorylated a seryl residue in a peptide termed peptide 1. PKC also phosphorylated seryl residues in additional peptides. CA2+/CaM-PK II phosphorylated serine and to a lesser extent, threonine, at sites different from those phosphorylated by the other two protein kinases. A synthetic peptide PSRKGSGFGHRL-amine (residues 228-239 based on the deduced amino acid sequence of rat connexin 32) was phosphorylated by cAMP-PK and by PKC, with kinetic properties being similar to those for other physiological substrates phosphorylated by these enzymes. Ca2+/CaM-PK II did not phosphorylate the peptide. Phosphopeptide mapping and amino acid sequencing of the phosphorylated synthetic peptide indicated that Ser233 of connexin 32 was present in peptide 1 and was phosphorylated by cAMP-PK or by PKC. In hepatocytes labeled with [32P]orthophosphoric acid, treatment with forskolin or 20-deoxy-20-oxophorbol 12,13-dibutyrate (PDBt) resulted in increased 32P-incorporation into connexin 32. Phosphopeptide mapping and phosphoamino acid analysis showed that a seryl residue in peptide 1 was most prominently phosphorylated under basal conditions. Treatment with forskolin or PDBt stimulated the phosphorylation of peptide 1. PDBt treatment also increased the phosphorylation of seryl residues in several other peptides. PDBt did not affect the cAMP-PK activity in hepatocytes. It has previously been shown that phorbol ester reduces dye coupling in several cell types, however in rat hepatocytes, dye coupling was not reduced

  1. Different modes of internalization of proteins associated with adhaerens junctions and desmosomes: experimental separation of lateral contacts induces endocytosis of desmosomal plaque material.

    PubMed Central

    Kartenbeck, J; Schmid, E; Franke, W W; Geiger, B

    1982-01-01

    The distribution and fate of two junctional complexes, zonula adhaerens and desmosomes, after dissociation of cell-cell contacts is described in MDBK cells. Junctions were split between adjacent cells by treatment with EGTA and proteins associated with the plaques of zonulae adhaerentes and desmosomes were localized by immunological methods. Splitting of these junctions is accompanied by the dislocation of desmosomal plaque protein from the cell periphery and its distribution in punctate arrays over the whole cytoplasm. By contrast, vinculin associated with zonulae adhaerentes is still seen at early times (0.5-1 h) in a conspicuous belt-like structure which, however, is displaced from the plasma membrane. Strong vinculin staining is maintained on leading edges of free cell surfaces. Electron microscopy of EGTA-treated cells exposed to colloidal gold particles reveals the disappearance of junctional structures from the cell periphery and the concomitant appearance of a distinct class of gold particle-containing vesicles which are coated by dense plaques. These vesicle plaques react with antibodies to desmosomal plaque proteins and are associated with filaments of the cytokeratin type. In the same cells, extended dense aggregates are seen which are most probably the membrane-detached vinculin-rich material from the zonula adhaerens . The experiments show that, upon release from their junction-mediated connections with adjacent cells, major proteins associated with the cytoplasmic side of the junctions remain, for several hours, clustered within plaques displaced from the cell surface. While plaque material of adhaerens junctions containing vinculin is recovered in large belt-like aggregates, desmosomal plaque protein remains attached to membrane structures and appears on distinct vesicles endocytotically formed from half-desmosomal equivalents.(ABSTRACT TRUNCATED AT 250 WORDS) Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:6821357

  2. Localization and Function of Pals1-associated Tight Junction Protein in Drosophila Is Regulated by Two Distinct Apical Complexes.

    PubMed

    Sen, Arnab; Sun, Rui; Krahn, Michael P

    2015-05-22

    The transmembrane protein Crumbs (Crb) and its intracellular adaptor protein Pals1 (Stardust, Sdt in Drosophila) play a crucial role in the establishment and maintenance of apical-basal polarity in epithelial cells in various organisms. In contrast, the multiple PDZ domain-containing protein Pals1-associated tight junction protein (PATJ), which has been described to form a complex with Crb/Sdt, is not essential for apical basal polarity or for the stability of the Crb/Sdt complex in the Drosophila epidermis. Here we show that, in the embryonic epidermis, Sdt is essential for the correct subcellular localization of PATJ in differentiated epithelial cells but not during cellularization. Consistently, the L27 domain of PATJ is crucial for the correct localization and function of the protein. Our data further indicate that the four PDZ domains of PATJ function, to a large extent, in redundancy, regulating the function of the protein. Interestingly, the PATJ-Sdt heterodimer is not only recruited to the apical cell-cell contacts by binding to Crb but depends on functional Bazooka (Baz). However, biochemical experiments show that PATJ associates with both complexes, the Baz-Sdt and the Crb-Sdt complex, in the mature epithelium of the embryonic epidermis, suggesting a role of these two complexes for the function of PATJ during the development of Drosophila.

  3. Localization and Function of Pals1-associated Tight Junction Protein in Drosophila Is Regulated by Two Distinct Apical Complexes*

    PubMed Central

    Sen, Arnab; Sun, Rui; Krahn, Michael P.

    2015-01-01

    The transmembrane protein Crumbs (Crb) and its intracellular adaptor protein Pals1 (Stardust, Sdt in Drosophila) play a crucial role in the establishment and maintenance of apical-basal polarity in epithelial cells in various organisms. In contrast, the multiple PDZ domain-containing protein Pals1-associated tight junction protein (PATJ), which has been described to form a complex with Crb/Sdt, is not essential for apical basal polarity or for the stability of the Crb/Sdt complex in the Drosophila epidermis. Here we show that, in the embryonic epidermis, Sdt is essential for the correct subcellular localization of PATJ in differentiated epithelial cells but not during cellularization. Consistently, the L27 domain of PATJ is crucial for the correct localization and function of the protein. Our data further indicate that the four PDZ domains of PATJ function, to a large extent, in redundancy, regulating the function of the protein. Interestingly, the PATJ-Sdt heterodimer is not only recruited to the apical cell-cell contacts by binding to Crb but depends on functional Bazooka (Baz). However, biochemical experiments show that PATJ associates with both complexes, the Baz-Sdt and the Crb-Sdt complex, in the mature epithelium of the embryonic epidermis, suggesting a role of these two complexes for the function of PATJ during the development of Drosophila. PMID:25847234

  4. CHOLESTEROL DEPLETION ALTERS DETERGENT-SPECIFIC SOLUBILITY PROFILES OF SELECTED TIGHT JUNCTION PROTEINS AND THE PHOSPHORYLATION OF OCCLUDIN

    PubMed Central

    Lynch, Robert D.; Francis, Stacy A.; McCarthy, Karin M.; Casas, Elizabeth; Thiele, Christoph; Schneeberger, Eveline E.

    2007-01-01

    Differential centrifugation of Triton X-100 or CHAPS lysates from control and cholesterol (CH) depleted MDCK II cells, segregated integral tight junction (TJ) proteins associated with detergent resistant membranes (DRMs) into two groups. Group A proteins (occludin, claudin-2 and -3) were detected in large, intermediate and small aggregates in both detergents, whereas group B proteins (claudin-1, -4 and -7) were observed in small aggregates in TX-100 and in intermediate and small aggregates in CHAPS. Depletion of CH altered the distribution of group A and B proteins among the three size categories in a detergent-specific manner. In lysates produced with octyl glucoside, a detergent that selectively extracts proteins from DRMs, group A proteins were undetectable in large aggregates and CH depletion did not alter the distribution of either group A or B proteins in intermediate or small aggregates. Neither occludin (group A) nor claudin-1 (group B) was in intimate enough contact with CH to be cross-linked to [3H]-photo-cholesterol. However, antibodies to either TJ protein co-immunoprecipitated caveolin-1, a CH-binding protein. Unlike claudins, occludin’s presence in TJs and DRMs did not require palmitoylation. Equilibrium density centrifugation on discontinuous OptiPrep gradients revealed detergent-related differences in the densities of TJ-bearing DRMs. There was little or no change in those densities after CH depletion. Removing CH from the plasma membrane increased tyrosine and threonine phosphorylation of occludin, and transepithelial electrical resistance (TER) within 30 min. After 2 h of CH efflux, phospho-occludin levels and TER fell below control values. We conclude that the association of integral TJ proteins with DRMS, pelleted at low speeds, is partially CH dependent. However, the buoyant density of TJ-associated DRMs is a function of the detergent used and is insensitive to decreases in CH. PMID:17574235

  5. The transmembrane protein Crumbs displays complex dynamics during follicular morphogenesis and is regulated competitively by Moesin and aPKC

    PubMed Central

    Sherrard, Kristin M.; Fehon, Richard G.

    2015-01-01

    The transmembrane protein Crumbs (Crb) functions in apical polarity and epithelial integrity. To better understand its role in epithelial morphogenesis, we examined Crb localization and dynamics in the late follicular epithelium of Drosophila. Crb was unexpectedly dynamic during middle-to-late stages of egg chamber development, being lost from the marginal zone (MZ) in stage 9 before abruptly returning at the end of stage 10b, then undergoing a pulse of endocytosis in stage 12. The reappearance of MZ Crb is necessary to maintain an intact adherens junction and MZ. Although Crb has been proposed to interact through its juxtamembrane domain with Moesin (Moe), a FERM domain protein that regulates the cortical actin cytoskeleton, the functional significance of this interaction is poorly understood. We found that whereas the Crb juxtamembrane domain was not required for adherens junction integrity, it was necessary for MZ localization of Moe, aPKC and F-actin. Furthermore, Moe and aPKC functioned antagonistically, suggesting that Moe limits Crb levels by reducing its interactions with the apical Par network. Additionally, Moe mutant cells lost Crb from the apical membrane and accumulated excess Crb at the MZ, suggesting that Moe regulates Crb distribution at the membrane. Together, these studies reveal reciprocal interactions between Crb, Moe and aPKC during cellular morphogenesis. PMID:25926360

  6. The Zinc Finger Protein Zfr1p Is Localized Specifically to Conjugation Junction and Required for Sexual Development in Tetrahymena thermophila

    PubMed Central

    Xu, Jing; Tian, Huaru; Wang, Wei; Liang, Aihua

    2012-01-01

    Conjugation in Tetrahymena thermophila involves a developmental program consisting of three prezygotic nuclear divisions, pronuclear exchange and fusion, and postzygotic and exconjugant stages. The conjugation junction structure appears during the initiation of conjugation development, and disappears during the exconjugant stage. Many structural and functional proteins are involved in the establishment and maintenance of the junction structure in T. thermophila. In the present study, a zinc finger protein-encoding gene ZFR1 was found to be expressed specifically during conjugation and to localize specifically to the conjugation junction region. Truncated Zfr1p localized at the plasma membrane in ordered arrays and decorated Golgi apparatus located adjacent to basal body. The N-terminal zinc finger and C-terminal hydrophobic domains of Zfr1p were found to be required for its specific conjugation junction localization. Conjugation development of ZFR1 somatic knockout cells was aborted at the pronuclear exchange and fusion conjugation stages. Furthermore, Zfr1p was found to be important for conjugation junction stability during the prezygotic nuclear division stage. Taken together, our data reveal that Zfr1p is required for the stability and integrity of the conjugation junction structure and essential for the sexual life cycle of the Tetrahymena cell. PMID:23251712

  7. Tight junction modulation of the blood brain barrier: CNS delivery of small molecules.

    PubMed

    Greene, Chris; Campbell, Matthew

    2016-01-01

    The blood brain barrier (BBB) represents a major obstacle for targeted drug delivery to the brain for the treatment of central nervous system (CNS) disorders. Significant advances in barrier research over the past decade has led to the discovery of an increasing number of structural and regulatory proteins in tight junctions (TJ) and adherens junctions (AJ). These discoveries are providing the framework for the development of novel TJ modulators which can act specifically and temporarily to alter BBB function and regulate paracellular uptake of molecules. TJ modulators that have shown therapeutic potential in preclinical models include claudin-5 and occludin siRNAs, peptides derived from zonula occludens toxin as well as synthetic peptides targeting the extracellular loops of TJs. Adding to the array of modulating agents are novel mechanisms of BBB regulation such as focused ultrasound (FUS). This review will give a succinct overview of BBB biology and TJ modulation in general. Novel insights into BBB regulation in health and disease will also be summarized.

  8. The tight junction protein ZO-2 associates with Jun, Fos and C/EBP transcription factors in epithelial cells.

    PubMed

    Betanzos, Abigail; Huerta, Miriam; Lopez-Bayghen, Esther; Azuara, Elisa; Amerena, José; González-Mariscal, Lorenza

    2004-01-01

    ZO-2 is a membrane-associated guanylate kinase (MAGUK) protein present at the tight junction (TJ) of epithelial cells. While confluent monolayers have ZO-2 at their cellular borders, sparse cultures conspicuously show ZO-2 at the nuclei. To study the role of nuclear ZO-2, we tested by pull-down assays and gel shift analysis the interaction between ZO-2 GST fusion proteins and different transcription factors. We identified the existence of a specific interaction of ZO-2 with Fos, Jun and C/EBP (CCAAT/enhancer binding protein). To analyze if this association is present "in vivo", we performed immunoprecipitation and immunolocalization experiments, which revealed an interaction of ZO-2 with Jun, Fos and C/EBP not only at the nucleus but also at the TJ region. To test if the association of ZO-2 with AP-1 (activator protein-1) modulates gene transcription, we performed reporter gene assays employing chloramphenicol acetyltransferase (CAT) constructs with promoters under the control of AP-1 sites. We observed that the co-transfected ZO-2 down-regulates CAT expression in a dose-dependent manner. Since ZO-2 is a multidomain protein, we proceeded to determine which region of the molecule is responsible for the modulation of gene expression, and observed that both the amino and the carboxyl domains are capable of inhibiting gene transcription.

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

    PubMed Central

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

    2015-01-01

    ABSTRACT 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. PMID:26126850

  10. Tight junction protein Par6 interacts with an evolutionarily conserved region in the amino terminus of PALS1/stardust.

    PubMed

    Wang, Qian; Hurd, Toby W; Margolis, Ben

    2004-07-16

    Tight junctions are the structures in mammalian epithelial cells that separate the apical and basolateral membranes and may also be important in the establishment of cell polarity. Two evolutionarily conserved multiprotein complexes, Crumbs-PALS1 (Stardust)-PATJ and Cdc42-Par6-Par3-atypical protein kinase C, have been implicated in the assembly of tight junctions and in polarization of Drosophila melanogaster epithelia. These two complexes have been linked physically and functionally by an interaction between PALS1 and Par6. Here we identify an evolutionarily conserved region in the amino terminus of PALS1 as the Par6 binding site and identify valine and aspartic acid residues in this region as essential for interacting with the PDZ domain of Par6. We have also characterized, in more detail, the amino terminus of Drosophila Stardust and demonstrate that the interaction mechanism between Stardust and Drosophila Par6 is evolutionarily conserved. Par6 interferes with PATJ in binding PALS1, and these two interactions do not appear to function synergistically. Taken together, these results define the molecular mechanisms linking two conserved polarity complexes.

  11. Cholera toxin disrupts barrier function by inhibiting exocyst-mediated trafficking of host proteins to intestinal cell junctions

    PubMed Central

    Guichard, Annabel; Moreno, Beatriz Cruz; Aguilar, Berenice; van Sorge, Nina M.; Kuang, Jennifer; Kurkciyan, Adrianne A.; Wang, Zhipeng; Hang, Saiyu; Pineton de Chambrun, Guillaume P.; McCole, Declan F.; Watnick, Paula; Nizet, Victor; Bier, Ethan

    2013-01-01

    Summary Cholera toxin (CT), a virulence factor elaborated by Vibrio cholerae, is sufficient to induce the severe diarrhea characteristic of cholera. The enzymatic moiety of CT (CtxA) increases cAMP synthesis in intestinal epithelial cells, leading to chloride ion (Cl−) efflux through the CFTR Cl− channel. To preserve electroneutrality and osmotic balance, sodium ions and water also flow into the intestinal lumen via a paracellular route. We find that CtxA-driven cAMP increase also inhibits Rab11/exocyst-mediated trafficking of host proteins including E-cadherin and Notch signaling components to cell-cell junctions in Drosophila, human intestinal epithelial cells, and ligated mouse ileal loops, thereby disrupting barrier function. Additionally, CtxA induces junctional damage, weight loss, and dye leakage in the Drosophila gut, contributing to lethality from live V. cholerae infection, all of which can be rescued by Rab11 over-expression. These barrier-disrupting effects of CtxA may act in parallel with Cl− secretion to drive the pathophysiology of cholera. PMID:24034615

  12. Cholera toxin disrupts barrier function by inhibiting exocyst-mediated trafficking of host proteins to intestinal cell junctions.

    PubMed

    Guichard, Annabel; Cruz-Moreno, Beatriz; Cruz-Moreno, Beatriz Cruz; Aguilar, Berenice; van Sorge, Nina M; Kuang, Jennifer; Kurkciyan, Adrianne A; Wang, Zhipeng; Hang, Saiyu; Pineton de Chambrun, Guillaume P; McCole, Declan F; Watnick, Paula; Nizet, Victor; Bier, Ethan

    2013-09-11

    Cholera toxin (CT), a virulence factor elaborated by Vibrio cholerae, is sufficient to induce the severe diarrhea characteristic of cholera. The enzymatic moiety of CT (CtxA) increases cAMP synthesis in intestinal epithelial cells, leading to chloride ion (Cl(-)) efflux through the CFTR Cl(-) channel. To preserve electroneutrality and osmotic balance, sodium ions and water also flow into the intestinal lumen via a paracellular route. We find that CtxA-driven cAMP increase also inhibits Rab11/exocyst-mediated trafficking of host proteins including E-cadherin and Notch signaling components to cell-cell junctions in Drosophila, human intestinal epithelial cells, and ligated mouse ileal loops, thereby disrupting barrier function. Additionally, CtxA induces junctional damage, weight loss, and dye leakage in the Drosophila gut, contributing to lethality from live V. cholerae infection, all of which can be rescued by Rab11 overexpression. These barrier-disrupting effects of CtxA may act in parallel with Cl(-) secretion to drive the pathophysiology of cholera. PMID:24034615

  13. Microfluidic chips with multi-junctions: an advanced tool in recovering proteins from inclusion bodies.

    PubMed

    Yamaguchi, Hiroshi; Miyazaki, Masaya

    2015-01-01

    Active recombinant proteins are used for studying the biological functions of genes and for the development of therapeutic drugs. Overexpression of recombinant proteins in bacteria often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. Protein refolding is an important process for obtaining active recombinant proteins from inclusion bodies. However, the conventional refolding method of dialysis or dilution is time-consuming and recovered active protein yields are often low, and a cumbersome trial-and-error process is required to achieve success. To circumvent these difficulties, we used controllable diffusion through laminar flow in microchannels to regulate the denaturant concentration. This method largely aims at reducing protein aggregation during the refolding procedure. This Commentary introduces the principles of the protein refolding method using microfluidic chips and the advantage of our results as a tool for rapid and efficient recovery of active recombinant proteins from inclusion bodies.

  14. Effects of phenol on barrier function of a human intestinal epithelial cell line correlate with altered tight junction protein localization

    PubMed Central

    McCall, Ingrid C.; Betanzos, Abigail; Weber, Dominique A.; Nava, Porfirio; Miller, Gary W.; Parkos, Charles A.

    2010-01-01

    Phenol contamination of soil and water has raised concerns among people living near phenol-producing factories and hazardous waste sites containing the chemical. Phenol, particularly in high concentrations, is an irritating and corrosive substance, making mucosal membranes targets of toxicity in humans. However, few data on the effects of phenol after oral exposure exist. We used an in vitro model employing human intestinal epithelial cells (SK-CO15) cultured on permeable supports to examine effects of phenol on epithelial barrier function. We hypothesized that phenol disrupts epithelial barrier by altering tight junction (TJ) protein expression. The dose-response effect of phenol on epithelial barrier function was determined using transepithelial electrical resistance (TER) and FITC-dextran permeability measurements. We studied phenol-induced changes in cell morphology and expression of several tight junction proteins by immunofluorescence and Western blot analysis. Effects on cell viability were assessed by MTT, Trypan blue, propidium iodide and TUNEL staining. Exposure to phenol resulted in decreased TER and increased paracellular flux of FITC-dextran in a dose-dependent manner. Delocalization of claudin-1 and ZO-1 from TJs to cytosol correlated with the observed increase in permeability after phenol treatment. Additionally, the decrease in TER correlated with changes in the distribution of a membrane raft marker, suggesting phenol-mediated effects on membrane fluidity. Such observations were independent of effects of phenol on cell viability as enhanced permeability occurred at doses of phenol that did not cause cell death. Overall, these findings suggest that phenol may affect transiently the lipid bilayer of the cell membrane, thus destabilizing TJ-containing microdomains. PMID:19679145

  15. Effects of phenol on barrier function of a human intestinal epithelial cell line correlate with altered tight junction protein localization.

    PubMed

    McCall, Ingrid C; Betanzos, Abigail; Weber, Dominique A; Nava, Porfirio; Miller, Gary W; Parkos, Charles A

    2009-11-15

    Phenol contamination of soil and water has raised concerns among people living near phenol-producing factories and hazardous waste sites containing the chemical. Phenol, particularly in high concentrations, is an irritating and corrosive substance, making mucosal membranes targets of toxicity in humans. However, few data on the effects of phenol after oral exposure exist. We used an in vitro model employing human intestinal epithelial cells (SK-CO15) cultured on permeable supports to examine effects of phenol on epithelial barrier function. We hypothesized that phenol disrupts epithelial barrier by altering tight junction (TJ) protein expression. The dose-response effect of phenol on epithelial barrier function was determined using transepithelial electrical resistance (TER) and FITC-dextran permeability measurements. We studied phenol-induced changes in cell morphology and expression of several tight junction proteins by immunofluorescence and Western blot analysis. Effects on cell viability were assessed by MTT, Trypan blue, propidium iodide and TUNEL staining. Exposure to phenol resulted in decreased TER and increased paracellular flux of FITC-dextran in a dose-dependent manner. Delocalization of claudin-1 and ZO-1 from TJs to cytosol correlated with the observed increase in permeability after phenol treatment. Additionally, the decrease in TER correlated with changes in the distribution of a membrane raft marker, suggesting phenol-mediated effects on membrane fluidity. Such observations were independent of effects of phenol on cell viability as enhanced permeability occurred at doses of phenol that did not cause cell death. Overall, these findings suggest that phenol may affect transiently the lipid bilayer of the cell membrane, thus destabilizing TJ-containing microdomains.

  16. Effects of phenol on barrier function of a human intestinal epithelial cell line correlate with altered tight junction protein localization

    SciTech Connect

    McCall, Ingrid C.; Betanzos, Abigail; Weber, Dominique A.; Nava, Porfirio; Miller, Gary W.; Parkos, Charles A.

    2009-11-15

    Phenol contamination of soil and water has raised concerns among people living near phenol-producing factories and hazardous waste sites containing the chemical. Phenol, particularly in high concentrations, is an irritating and corrosive substance, making mucosal membranes targets of toxicity in humans. However, few data on the effects of phenol after oral exposure exist. We used an in vitro model employing human intestinal epithelial cells (SK-CO15) cultured on permeable supports to examine effects of phenol on epithelial barrier function. We hypothesized that phenol disrupts epithelial barrier by altering tight junction (TJ) protein expression. The dose-response effect of phenol on epithelial barrier function was determined using transepithelial electrical resistance (TER) and FITC-dextran permeability measurements. We studied phenol-induced changes in cell morphology and expression of several tight junction proteins by immunofluorescence and Western blot analysis. Effects on cell viability were assessed by MTT, Trypan blue, propidium iodide and TUNEL staining. Exposure to phenol resulted in decreased TER and increased paracellular flux of FITC-dextran in a dose-dependent manner. Delocalization of claudin-1 and ZO-1 from TJs to cytosol correlated with the observed increase in permeability after phenol treatment. Additionally, the decrease in TER correlated with changes in the distribution of a membrane raft marker, suggesting phenol-mediated effects on membrane fluidity. Such observations were independent of effects of phenol on cell viability as enhanced permeability occurred at doses of phenol that did not cause cell death. Overall, these findings suggest that phenol may affect transiently the lipid bilayer of the cell membrane, thus destabilizing TJ-containing microdomains.

  17. The rotavirus surface protein VP8 modulates the gate and fence function of tight junctions in epithelial cells.

    PubMed

    Nava, Porfirio; López, Susana; Arias, Carlos F; Islas, Socorro; González-Mariscal, Lorenza

    2004-11-01

    Rotaviruses constitute a major cause of diarrhea in young mammals. Rotaviruses utilize different integrins as cell receptors, therefore upon their arrival to the intestinal lumen their integrin receptors will be hidden below the tight junction (TJ), on the basolateral membrane. Here we have studied whether the rotavirus outer capsid proteins are capable of opening the paracellular space sealed by the TJ. From the outermost layer of proteins of the rotavirus, 60 spikes formed of protein VP4 are projected. VP4 is essential for virus-cell interactions and is cleaved by trypsin into peptides VP5 and VP8. Here we found that when these peptides are added to confluent epithelial monolayers (Madin-Darby canine kidney cells), VP8 is capable of diminishing in a dose dependent and reversible manner the transepithelial electrical resistance. VP5 exerted no effect. VP8 can also inhibit the development of newly formed TJs in a Ca-switch assay. Treatment with VP8 augments the paracellular passage of non-ionic tracers, allows the diffusion of a fluorescent lipid probe and the apical surface protein GP135, from the luminal to the lateral membrane, and triggers the movement of the basolateral proteins Na+-K+-ATPase, alphanubeta3 integrin and beta1 integrin subunit, to the apical surface. VP8 generates a freeze-fracture pattern of TJs characterized by the appearance of loose end filaments, that correlates with an altered distribution of several TJ proteins. VP8 given orally to diabetic rats allows the enteral administration of insulin, thus indicating that it can be employed to modulate epithelial permeability. PMID:15494377

  18. Mutational Analysis of Rab3 Function for Controlling Active Zone Protein Composition at the Drosophila Neuromuscular Junction.

    PubMed

    Chen, Shirui; Gendelman, Hannah K; Roche, John P; Alsharif, Peter; Graf, Ethan R

    2015-01-01

    At synapses, the release of neurotransmitter is regulated by molecular machinery that aggregates at specialized presynaptic release sites termed active zones. The complement of active zone proteins at each site is a determinant of release efficacy and can be remodeled to alter synapse function. The small GTPase Rab3 was previously identified as playing a novel role that controls the distribution of active zone proteins to individual release sites at the Drosophila neuromuscular junction. Rab3 has been extensively studied for its role in the synaptic vesicle cycle; however, the mechanism by which Rab3 controls active zone development remains unknown. To explore this mechanism, we conducted a mutational analysis to determine the molecular and structural requirements of Rab3 function at Drosophila synapses. We find that GTP-binding is required for Rab3 to traffick to synapses and distribute active zone components across release sites. Conversely, the hydrolytic activity of Rab3 is unnecessary for this function. Through a structure-function analysis we identify specific residues within the effector-binding switch regions that are required for Rab3 function and determine that membrane attachment is essential. Our findings suggest that Rab3 controls the distribution of active zone components via a vesicle docking mechanism that is consistent with standard Rab protein function.

  19. Mutational Analysis of Rab3 Function for Controlling Active Zone Protein Composition at the Drosophila Neuromuscular Junction

    PubMed Central

    Roche, John P.; Alsharif, Peter; Graf, Ethan R.

    2015-01-01

    At synapses, the release of neurotransmitter is regulated by molecular machinery that aggregates at specialized presynaptic release sites termed active zones. The complement of active zone proteins at each site is a determinant of release efficacy and can be remodeled to alter synapse function. The small GTPase Rab3 was previously identified as playing a novel role that controls the distribution of active zone proteins to individual release sites at the Drosophila neuromuscular junction. Rab3 has been extensively studied for its role in the synaptic vesicle cycle; however, the mechanism by which Rab3 controls active zone development remains unknown. To explore this mechanism, we conducted a mutational analysis to determine the molecular and structural requirements of Rab3 function at Drosophila synapses. We find that GTP-binding is required for Rab3 to traffick to synapses and distribute active zone components across release sites. Conversely, the hydrolytic activity of Rab3 is unnecessary for this function. Through a structure-function analysis we identify specific residues within the effector-binding switch regions that are required for Rab3 function and determine that membrane attachment is essential. Our findings suggest that Rab3 controls the distribution of active zone components via a vesicle docking mechanism that is consistent with standard Rab protein function. PMID:26317909

  20. Effects of elevated circulating cortisol levels on hydromineral status and gill tight junction protein abundance in the stenohaline goldfish.

    PubMed

    Chasiotis, Helen; Kelly, Scott P

    2012-01-15

    A role for cortisol in the regulation of hydromineral balance and gill tight junction (TJ) protein transcript abundance in the stenohaline freshwater goldfish was investigated. Intraperitoneal cortisol implants (50, 100, 200, 400 μg cortisol/g body weight) were used to dose-dependently elevate circulating cortisol levels over a 4 day period. Elevated cortisol did not significantly alter serum osmolality, serum Na(+) or muscle water content, however serum glucose and gill Na(+)-K(+)-ATPase activity were significantly increased and serum Cl(-) levels were significantly reduced when compared to control groups. Transcript levels for glucocorticoid receptor 1 (GR1) and mineralocorticoid receptor (MR) in the gill remained unchanged by cortisol treatment, however glucocorticoid receptor 2 (GR2) mRNA abundance was significantly down-regulated. Conversely, cortisol treatment significantly increased transcript and protein abundance of the TJ protein occludin in goldfish gill tissue, as well as mRNA abundance for claudin e, 7 and 8d. Goldfish tissue expression profiles demonstrated that transcripts encoding for these claudins are particularly abundant in the gill. Overall, results suggest a 'tightened' gill epithelium in response to elevated cortisol levels in goldfish. However, negative autoregulation of gill GR2 transcript suggests a lessened capacity to respond to cortisol and thus a potentially 'dampened' corticosteroid-mediated effect in the gill. Reduced systemic Cl(-) levels also suggest that sustained cortisol elevation in goldfish may have a detrimental effect on other ionoregulatory tissues.

  1. ROCK activity regulates functional tight junction assembly during blastocyst formation in porcine parthenogenetic embryos

    PubMed Central

    Kwon, Jeongwoo

    2016-01-01

    The Rho-associated coiled-coil-containing protein serine/threonine kinases 1 and 2 (ROCK1 and ROCK2) are Rho subfamily GTPase downstream effectors that regulate cell migration, intercellular adhesion, cell polarity, and cell proliferation by stimulating actin cytoskeleton reorganization. Inhibition of ROCK proteins affects specification of the trophectoderm (TE) and inner cell mass (ICM) lineages, compaction, and blastocyst cavitation. However, the molecules involved in blastocyst formation are not known. Here, we examined developmental competence and levels of adherens/tight junction (AJ/TJ) constituent proteins, such as CXADR, OCLN, TJP1, and CDH1, as well as expression of their respective mRNAs, after treating porcine parthenogenetic four-cell embryos with Y-27632, a specific inhibitor of ROCK, at concentrations of 0, 10, 20, 100 µM for 24 h. Following this treatment, the blastocyst development rates were 39.1, 20.7, 10.0, and 0% respectively. In embryos treated with 20 µM treatment, expression levels of CXADR, OCLN, TJP1, and CDH1 mRNA and protein molecules were significantly reduced (P < 0.05). FITC-dextran uptake assay revealed that the treatment caused an increase in TE TJ permeability. Interestingly, the majority of the four-cell and morula embryos treated with 20 µM Y-27643 for 24 h showed defective compaction and cavitation. Taken together, our results indicate that ROCK activity may differentially affect assembly of AJ/TJs as well as regulate expression of genes encoding junctional proteins. PMID:27077008

  2. 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

  3. HPV16 E6 Controls the Gap Junction Protein Cx43 in Cervical Tumour Cells

    PubMed Central

    Sun, Peng; Dong, Li; MacDonald, Alasdair I.; Akbari, Shahrzad; Edward, Michael; Hodgins, Malcolm B.; Johnstone, Scott R.; Graham, Sheila V.

    2015-01-01

    Human papillomavirus type 16 (HPV16) causes a range of cancers including cervical and head and neck cancers. HPV E6 oncoprotein binds the cell polarity regulator hDlg (human homologue of Drosophila Discs Large). Previously we showed in vitro, and now in vivo, that hDlg also binds Connexin 43 (Cx43), a major component of gap junctions that mediate intercellular transfer of small molecules. In HPV16-positive non-tumour cervical epithelial cells (W12G) Cx43 localised to the plasma membrane, while in W12T tumour cells derived from these, it relocated with hDlg into the cytoplasm. We now provide evidence that E6 regulates this cytoplasmic pool of Cx43. E6 siRNA depletion in W12T cells resulted in restoration of Cx43 and hDlg trafficking to the cell membrane. In C33a HPV-negative cervical tumour cells expressing HPV16 or 18 E6, Cx43 was located primarily in the cytoplasm, but mutation of the 18E6 C-terminal hDlg binding motif resulted in redistribution of Cx43 to the membrane. The data indicate for the first time that increased cytoplasmic E6 levels associated with malignant progression alter Cx43 trafficking and recycling to the membrane and the E6/hDlg interaction may be involved. This suggests a novel E6-associated mechanism for changes in Cx43 trafficking in cervical tumour cells. PMID:26445057

  4. Tight junction proteins and oxidative stress in heavy metals-induced nephrotoxicity.

    PubMed

    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.

  5. Nonmechanical Roles of Dystrophin and Associated Proteins in Exercise, Neuromuscular Junctions, and Brains

    PubMed Central

    Nichols, Bailey; Takeda, Shin’ichi; Yokota, Toshifumi

    2015-01-01

    Dystrophin-glycoprotein complex (DGC) is an important structural unit in skeletal muscle that connects the cytoskeleton (f-actin) of a muscle fiber to the extracellular matrix (ECM). Several muscular dystrophies, such as Duchenne muscular dystrophy, Becker muscular dystrophy, congenital muscular dystrophies (dystroglycanopathies), and limb-girdle muscular dystrophies (sarcoglycanopathies), are caused by mutations in the different DGC components. Although many early studies indicated DGC plays a crucial mechanical role in maintaining the structural integrity of skeletal muscle, recent studies identified novel roles of DGC. Beyond a mechanical role, these DGC members play important signaling roles and act as a scaffold for various signaling pathways. For example, neuronal nitric oxide synthase (nNOS), which is localized at the muscle membrane by DGC members (dystrophin and syntrophins), plays an important role in the regulation of the blood flow during exercise. DGC also plays important roles at the neuromuscular junction (NMJ) and in the brain. In this review, we will focus on recently identified roles of DGC particularly in exercise and the brain. PMID:26230713

  6. Muscle-regulated expression and determinants for neuromuscular junctional localization of the mouse RIα regulatory subunit of cAMP- dependent protein kinase

    PubMed Central

    Barradeau, Sébastien; Imaizumi-Scherrer, Tereza; Weiss, Mary C.; Faust, Daniela M.

    2001-01-01

    In skeletal muscle, transcription of the gene encoding the mouse type Iα (RIα) subunit of the cAMP-dependent protein kinase is initiated from the alternative noncoding first exons 1a and 1b. Here, we report that activity of the promoter upstream of exon 1a (Pa) depends on two adjacent E boxes (E1 and E2) in NIH 3T3-transfected fibroblasts as well as in intact muscle. Both basal activity and MyoD transactivation of the Pa promoter require binding of the upstream stimulating factors (USF) to E1. E2 binds either an unknown protein in a USF/E1 complex-dependent manner or MyoD. Both E2-bound proteins seem to function as repressors, but with different strengths, of the USF transactivation potential. Previous work has shown localization of the RIα protein at the neuromuscular junction. Using DNA injection into muscle of plasmids encoding segments of RIα or RIIα fused to green fluorescent protein, we demonstrate that anchoring at the neuromuscular junction is specific to RIα subunits and requires the amino-terminal residues 1–81. Mutagenesis of Phe-54 to Ala in the full-length RIα–green fluorescent protein template abolishes localization, indicating that dimerization of RIα is essential for anchoring. Moreover, two other hydrophobic residues, Val-22 and Ile-27, are crucial for localization of RIα at the neuromuscular junction. These amino acids are involved in the interaction of the Caenorhabditis elegans type Iα homologue RCE with AKAPCE and for in vitro binding of RIα to dual A-kinase anchoring protein 1. We also show enrichment of dual A-kinase anchoring protein 1 at the neuromuscular junction, suggesting that it could be responsible for RIα tethering at this site. PMID:11296260

  7. Specific Deletion of AMP-Activated Protein Kinase (α1AMPK) in Murine Oocytes Alters Junctional Protein Expression and Mitochondrial Physiology

    PubMed Central

    Bertoldo, Michael J.; Guibert, Edith; Faure, Melanie; Ramé, Christelle; Foretz, Marc; Viollet, Benoit; Dupont, Joëlle; Froment, Pascal

    2015-01-01

    Oogenesis and folliculogenesis are dynamic processes that are regulated by endocrine, paracrine and autocrine signals. These signals are exchanged between the oocyte and the somatic cells of the follicle. Here we analyzed the role of AMP-activated protein kinase (AMPK), an important regulator of cellular energy homeostasis, by using transgenic mice deficient in α1AMPK specifically in the oocyte. We found a decrease of 27% in litter size was observed in ZP3-α1AMPK-/- (ZP3-KO) female mice. Following in vitro fertilization, where conditions are stressful for the oocyte and embryo, ZP3-KO oocytes were 68% less likely to pass the 2-cell stage. In vivo and in cumulus-oocyte complexes, several proteins involved in junctional communication, such as connexin37 and N-cadherin were down-regulated in the absence of α1AMPK. While the two signalling pathways (PKA and MAPK) involved in the junctional communication between the cumulus/granulosa cells and the oocyte were stimulated in control oocytes, ZP3-KO oocytes exhibited only low phosphorylation of MAPK or CREB proteins. In addition, MII oocytes deficient in α1AMPK had a 3-fold lower ATP concentration, an increase in abnormal mitochondria, and a decrease in cytochrome C and PGC1α levels, suggesting perturbed energy production by mitochondria. The absence of α1AMPK also induced a reduction in histone deacetylase activity, which was associated with an increase in histone H3 acetylation (K9/K14 residues). Together, the results of the present study suggest that absence of AMPK, modifies oocyte quality through energy processes and oocyte/somatic cell communication. The limited effect observed in vivo could be partly due to a favourable follicle microenvironment where nutrients, growth factors, and adequate cell interaction were present. Whereas in a challenging environment such as that of in vitro culture following IVF, the phenotype is revealed. PMID:25767884

  8. Specific deletion of AMP-activated protein kinase (α1AMPK) in murine oocytes alters junctional protein expression and mitochondrial physiology.

    PubMed

    Bertoldo, Michael J; Guibert, Edith; Faure, Melanie; Ramé, Christelle; Foretz, Marc; Viollet, Benoit; Dupont, Joëlle; Froment, Pascal

    2015-01-01

    Oogenesis and folliculogenesis are dynamic processes that are regulated by endocrine, paracrine and autocrine signals. These signals are exchanged between the oocyte and the somatic cells of the follicle. Here we analyzed the role of AMP-activated protein kinase (AMPK), an important regulator of cellular energy homeostasis, by using transgenic mice deficient in α1AMPK specifically in the oocyte. We found a decrease of 27% in litter size was observed in ZP3-α1AMPK-/- (ZP3-KO) female mice. Following in vitro fertilization, where conditions are stressful for the oocyte and embryo, ZP3-KO oocytes were 68% less likely to pass the 2-cell stage. In vivo and in cumulus-oocyte complexes, several proteins involved in junctional communication, such as connexin37 and N-cadherin were down-regulated in the absence of α1AMPK. While the two signalling pathways (PKA and MAPK) involved in the junctional communication between the cumulus/granulosa cells and the oocyte were stimulated in control oocytes, ZP3-KO oocytes exhibited only low phosphorylation of MAPK or CREB proteins. In addition, MII oocytes deficient in α1AMPK had a 3-fold lower ATP concentration, an increase in abnormal mitochondria, and a decrease in cytochrome C and PGC1α levels, suggesting perturbed energy production by mitochondria. The absence of α1AMPK also induced a reduction in histone deacetylase activity, which was associated with an increase in histone H3 acetylation (K9/K14 residues). Together, the results of the present study suggest that absence of AMPK, modifies oocyte quality through energy processes and oocyte/somatic cell communication. The limited effect observed in vivo could be partly due to a favourable follicle microenvironment where nutrients, growth factors, and adequate cell interaction were present. Whereas in a challenging environment such as that of in vitro culture following IVF, the phenotype is revealed. PMID:25767884

  9. Tight Junction Disruption Induced by Type 3 Secretion System Effectors Injected by Enteropathogenic and Enterohemorrhagic Escherichia coli

    PubMed Central

    Ugalde-Silva, Paul; Gonzalez-Lugo, Octavio; Navarro-Garcia, Fernando

    2016-01-01

    The intestinal epithelium consists of a single cell layer, which is a critical selectively permeable barrier to both absorb nutrients and avoid the entry of potentially harmful entities, including microorganisms. Epithelial cells are held together by the apical junctional complexes, consisting of adherens junctions, and tight junctions (TJs), and by underlying desmosomes. TJs lay in the apical domain of epithelial cells and are mainly composed by transmembrane proteins such as occludin, claudins, JAMs, and tricellulin, that are associated with the cytoplasmic plaque formed by proteins from the MAGUK family, such as ZO-1/2/3, connecting TJ to the actin cytoskeleton, and cingulin and paracingulin connecting TJ to the microtubule network. Extracellular bacteria such as EPEC and EHEC living in the intestinal lumen inject effectors proteins directly from the bacterial cytoplasm to the host cell cytoplasm, where they play a relevant role in the manipulation of the eukaryotic cell functions by modifying or blocking cell signaling pathways. TJ integrity depends on various cell functions such as actin cytoskeleton, microtubule network for vesicular trafficking, membrane integrity, inflammation, and cell survival. EPEC and EHEC effectors target most of these functions. Effectors encoded inside or outside of locus of enterocyte effacement (LEE) disrupt the TJ strands. EPEC and EHEC exploit the TJ dynamics to open this structure, for causing diarrhea. EPEC and EHEC secrete effectors that mimic host proteins to manipulate the signaling pathways, including those related to TJ dynamics. In this review, we focus on the known mechanisms exploited by EPEC and EHEC effectors for causing TJ disruption. PMID:27606286

  10. Tight Junction Disruption Induced by Type 3 Secretion System Effectors Injected by Enteropathogenic and Enterohemorrhagic Escherichia coli.

    PubMed

    Ugalde-Silva, Paul; Gonzalez-Lugo, Octavio; Navarro-Garcia, Fernando

    2016-01-01

    The intestinal epithelium consists of a single cell layer, which is a critical selectively permeable barrier to both absorb nutrients and avoid the entry of potentially harmful entities, including microorganisms. Epithelial cells are held together by the apical junctional complexes, consisting of adherens junctions, and tight junctions (TJs), and by underlying desmosomes. TJs lay in the apical domain of epithelial cells and are mainly composed by transmembrane proteins such as occludin, claudins, JAMs, and tricellulin, that are associated with the cytoplasmic plaque formed by proteins from the MAGUK family, such as ZO-1/2/3, connecting TJ to the actin cytoskeleton, and cingulin and paracingulin connecting TJ to the microtubule network. Extracellular bacteria such as EPEC and EHEC living in the intestinal lumen inject effectors proteins directly from the bacterial cytoplasm to the host cell cytoplasm, where they play a relevant role in the manipulation of the eukaryotic cell functions by modifying or blocking cell signaling pathways. TJ integrity depends on various cell functions such as actin cytoskeleton, microtubule network for vesicular trafficking, membrane integrity, inflammation, and cell survival. EPEC and EHEC effectors target most of these functions. Effectors encoded inside or outside of locus of enterocyte effacement (LEE) disrupt the TJ strands. EPEC and EHEC exploit the TJ dynamics to open this structure, for causing diarrhea. EPEC and EHEC secrete effectors that mimic host proteins to manipulate the signaling pathways, including those related to TJ dynamics. In this review, we focus on the known mechanisms exploited by EPEC and EHEC effectors for causing TJ disruption. PMID:27606286

  11. Tight Junction Disruption Induced by Type 3 Secretion System Effectors Injected by Enteropathogenic and Enterohemorrhagic Escherichia coli

    PubMed Central

    Ugalde-Silva, Paul; Gonzalez-Lugo, Octavio; Navarro-Garcia, Fernando

    2016-01-01

    The intestinal epithelium consists of a single cell layer, which is a critical selectively permeable barrier to both absorb nutrients and avoid the entry of potentially harmful entities, including microorganisms. Epithelial cells are held together by the apical junctional complexes, consisting of adherens junctions, and tight junctions (TJs), and by underlying desmosomes. TJs lay in the apical domain of epithelial cells and are mainly composed by transmembrane proteins such as occludin, claudins, JAMs, and tricellulin, that are associated with the cytoplasmic plaque formed by proteins from the MAGUK family, such as ZO-1/2/3, connecting TJ to the actin cytoskeleton, and cingulin and paracingulin connecting TJ to the microtubule network. Extracellular bacteria such as EPEC and EHEC living in the intestinal lumen inject effectors proteins directly from the bacterial cytoplasm to the host cell cytoplasm, where they play a relevant role in the manipulation of the eukaryotic cell functions by modifying or blocking cell signaling pathways. TJ integrity depends on various cell functions such as actin cytoskeleton, microtubule network for vesicular trafficking, membrane integrity, inflammation, and cell survival. EPEC and EHEC effectors target most of these functions. Effectors encoded inside or outside of locus of enterocyte effacement (LEE) disrupt the TJ strands. EPEC and EHEC exploit the TJ dynamics to open this structure, for causing diarrhea. EPEC and EHEC secrete effectors that mimic host proteins to manipulate the signaling pathways, including those related to TJ dynamics. In this review, we focus on the known mechanisms exploited by EPEC and EHEC effectors for causing TJ disruption.

  12. Impact of obesity on 7,12-dimethylbenz[a]anthracene-induced altered ovarian connexin gap junction proteins in female mice

    PubMed Central

    Ganesan, Shanthi; Nteeba, Jackson; Keating, Aileen F.

    2014-01-01

    The ovarian gap junction proteins alpha 4 (GJA4 or connexin 37; CX37), alpha 1 (GJA1 or connexin 43; CX43) and gamma 1 (GJC1 or connexin 45; CX45) are involved in cell communication and folliculogenesis. 7,12-dimethylbenz[a]anthracene (DMBA) alters Cx37 and Cx43 expression in cultured neonatal rat ovaries. Additionally, obesity has an additive effect on DMBA-induced ovarian cell death and follicle depletion, thus, we investigated in vivo impacts of obesity and DMBA on CX protein levels. Ovaries were collected from lean and obese mice aged 6, 12, 18, or 24 wks. A subset of 18 wk old mice (lean and obese) were dosed with sesame oil or DMBA (1mg/kg; ip) for 14 days and ovaries collected 3 days thereafter. Cx43 and Cx45 mRNA and protein levels decreased (P < 0.05) after 18 wks while Cx37 mRNA and protein levels decreased (P < 0.05) after 24 wks in obese ovaries. Cx37 mRNA and antral follicle protein staining intensity were reduced (P < 0.05) by obesity while total CX37 protein was reduced (P < 0.05) in DMBA exposed obese ovaries. Cx43 mRNA and total protein levels were decreased (P < 0.05) by DMBA in both lean and obese ovaries while basal protein staining intensity was reduced (P < 0.05) in obese controls. Cx45 mRNA, total protein and protein staining intensity level were decreased (P < 0.05) by obesity. These data support that obesity temporally alters gap junction protein expression and that DMBA-induced ovotoxicity may involve reduced gap junction protein function. PMID:25447408

  13. Protein Kinase Cβ Phosphorylates Occludin Regulating Tight Junction Trafficking in Vascular Endothelial Growth Factor–Induced Permeability In Vivo

    PubMed Central

    Murakami, Tomoaki; Frey, Tiffany; Lin, Chengmao; Antonetti, David A.

    2012-01-01

    Vascular endothelial growth factor (VEGF)–induced breakdown of the blood-retinal barrier requires protein kinase C (PKC)β activation. However, the molecular mechanisms related to this process remain poorly understood. In this study, the role of occludin phosphorylation and ubiquitination downstream of PKCβ activation in tight junction (TJ) trafficking and endothelial permeability was investigated. Treatment of bovine retinal endothelial cells and intravitreal injection of PKCβ inhibitors as well as expression of dominant-negative kinase was used to determine the contribution of PKCβ to endothelial permeability and occludin phosphorylation at Ser490 detected with a site-specific antibody. In vitro kinase assay was used to demonstrate direct occludin phosphorylation by PKCβ. Ubiquitination was measured by immunoblotting after occludin immunoprecipitation. Confocal microscopy revealed organization of TJ proteins. The results reveal that inhibition of VEGF-induced PKCβ activation blocks occludin Ser490 phosphorylation, ubiquitination, and TJ trafficking in retinal vascular endothelial cells both in vitro and in vivo and prevents VEGF-stimulated vascular permeability. Occludin Ser490 is a direct target of PKCβ, and mutating Ser490 to Ala (S490A) blocks permeability downstream of PKCβ. Therefore, PKCβ activation phosphorylates occludin on Ser490, leading to ubiquitination required for VEGF-induced permeability. These data demonstrate a novel mechanism for PKCβ targeted inhibitors in regulating vascular permeability. PMID:22438576

  14. Polycystin-2 activity is controlled by transcriptional coactivator with PDZ binding motif and PALS1-associated tight junction protein.

    PubMed

    Duning, Kerstin; Rosenbusch, Deike; Schlüter, Marc A; Tian, Yuemin; Kunzelmann, Karl; Meyer, Nina; Schulze, Ulf; Markoff, Arseni; Pavenstädt, Hermann; Weide, Thomas

    2010-10-29

    Autosomal dominant polycystic kidney disease (ADPKD) is the most frequent monogenic cause of kidney failure, characterized by the development of renal cysts. ADPKD is caused by mutations of the polycystin-1 (PC1) or polycystin-2 (PC2) genes. PC2 encodes a Ca(2+)-permeable cation channel, and its dysfunction has been implicated in cyst development. The transcriptional coactivator with PDZ binding motif (TAZ) is required for the integrity of renal cilia. Its absence results in the development of renal cysts in a knock-out mouse model. TAZ directly interacts with PC2, and it has been suggested that another yet unidentified PDZ domain protein may be involved in the TAZ/PC2 interaction. Here we describe a novel interaction of TAZ with the multi-PDZ-containing PALS1-associated tight junction protein (PATJ). TAZ interacts with both the N-terminal PDZ domains 1-3 and the C-terminal PDZ domains 8-10 of PATJ, suggesting two distinct TAZ binding domains. We also show that the C terminus of PC2 strongly interacts with PDZ domains 8-10 and to a weaker extent with PDZ domains 1-3 of PATJ. Finally, we demonstrate that both TAZ and PATJ impair PC2 channel activity when co-expressed with PC2 in oocytes of Xenopus laevis. These results implicate TAZ and PATJ as novel regulatory elements of the PC2 channel and might thus be involved in ADPKD pathology.

  15. Nanotube junctions

    DOEpatents

    Crespi, Vincent Henry; Cohen, Marvin Lou; Louie, Steven Gwon; Zettl, Alexander Karlwalte

    2004-12-28

    The present invention comprises a new nanoscale metal-semiconductor, semiconductor-semiconductor, or metal-metal junction, designed by introducing topological or chemical defects in the atomic structure of the nanotube. Nanotubes comprising adjacent sections having differing electrical properties are described. These nanotubes can be constructed from combinations of carbon, boron, nitrogen and other elements. The nanotube can be designed having different indices on either side of a junction point in a continuous tube so that the electrical properties on either side of the junction vary in a useful fashion. For example, the inventive nanotube may be electrically conducting on one side of a junction and semiconducting on the other side. An example of a semiconductor-metal junction is a Schottky barrier. Alternatively, the nanotube may exhibit different semiconductor properties on either side of the junction. Nanotubes containing heterojunctions, Schottky barriers, and metal-metal junctions are useful for microcircuitry.

  16. Nanotube junctions

    DOEpatents

    Crespi, Vincent Henry; Cohen, Marvin Lou; Louie, Steven Gwon Sheng; Zettl, Alexander Karlwalter

    2003-01-01

    The present invention comprises a new nanoscale metal-semiconductor, semiconductor-semiconductor, or metal-metal junction, designed by introducing topological or chemical defects in the atomic structure of the nanotube. Nanotubes comprising adjacent sections having differing electrical properties are described. These nanotubes can be constructed from combinations of carbon, boron, nitrogen and other elements. The nanotube can be designed having different indices on either side of a junction point in a continuous tube so that the electrical properties on either side of the junction vary in a useful fashion. For example, the inventive nanotube may be electrically conducting on one side of a junction and semiconducting on the other side. An example of a semiconductor-metal junction is a Schottky barrier. Alternatively, the nanotube may exhibit different semiconductor properties on either side of the junction. Nanotubes containing heterojunctions, Schottky barriers, and metal-metal junctions are useful for microcircuitry.

  17. ADAM10 Is Involved in Cell Junction Assembly in Early Porcine Embryo Development.

    PubMed

    Kwon, Jeongwoo; Jeong, Sung-min; Choi, Inchul; Kim, Nam-Hyung

    2016-01-01

    ADAM10 (A Disintegrin and Metalloprotease domain-containing protein 10) is a cell surface protein with a unique structure possessing both potential adhesion and protease domains. However, the role of ADAM10 in preimplantation stage embryos is not clear. In this study, we examined the expression patterns and functional roles of ADAM10 in porcine parthenotes during preimplantation development. The transcription level of ADAM10 dramatically increased from the morula stage onward. Immunostaining revealed that ADAM10 was present in both the nucleus and cytoplasm in early cleavage stage embryos, and localized to the apical region of the outer cells in morula and blastocyst embryos. Knockdown (KD) of ADAM10 using double strand RNA did not alter preimplantation embryo development until morula stage, but resulted in significantly reduced development to blastocyst stage. Moreover, the KD blastocyst showed a decrease in gene expression of adherens and tight junction (AJ/TJ), and an increase in trophectoderm TJ permeability by disrupting TJ assembly. Treatment with an ADAM10 specific chemical inhibitor, GI254023X, at the morula stage also inhibited blastocyst development and led to disruption of TJ assembly. An in situ proximity ligation assay demonstrated direct interaction of ADAM10 with coxsackie virus and adenovirus receptor (CXADR), supporting the involvement of ADAM10 in TJ assembly. In conclusion, our findings strongly suggest that ADADM10 is important for blastocyst formation rather than compaction, particularly for TJ assembly and stabilization in preimplantation porcine parthenogenetic development. PMID:27043020

  18. ADAM10 Is Involved in Cell Junction Assembly in Early Porcine Embryo Development

    PubMed Central

    Kwon, Jeongwoo; Jeong, Sung-min; Choi, Inchul; Kim, Nam-Hyung

    2016-01-01

    ADAM10 (A Disintegrin and Metalloprotease domain-containing protein 10) is a cell surface protein with a unique structure possessing both potential adhesion and protease domains. However, the role of ADAM10 in preimplantation stage embryos is not clear. In this study, we examined the expression patterns and functional roles of ADAM10 in porcine parthenotes during preimplantation development. The transcription level of ADAM10 dramatically increased from the morula stage onward. Immunostaining revealed that ADAM10 was present in both the nucleus and cytoplasm in early cleavage stage embryos, and localized to the apical region of the outer cells in morula and blastocyst embryos. Knockdown (KD) of ADAM10 using double strand RNA did not alter preimplantation embryo development until morula stage, but resulted in significantly reduced development to blastocyst stage. Moreover, the KD blastocyst showed a decrease in gene expression of adherens and tight junction (AJ/TJ), and an increase in trophectoderm TJ permeability by disrupting TJ assembly. Treatment with an ADAM10 specific chemical inhibitor, GI254023X, at the morula stage also inhibited blastocyst development and led to disruption of TJ assembly. An in situ proximity ligation assay demonstrated direct interaction of ADAM10 with coxsackie virus and adenovirus receptor (CXADR), supporting the involvement of ADAM10 in TJ assembly. In conclusion, our findings strongly suggest that ADADM10 is important for blastocyst formation rather than compaction, particularly for TJ assembly and stabilization in preimplantation porcine parthenogenetic development. PMID:27043020

  19. JUNCTIONAL COMPLEX AND FOCAL ADHESION REARRANGEMENT MEDIATES PULMONARY ENDOTHELIAL BARRIER ENHANCEMENT BY FTY720 S-PHOSPHONATE

    PubMed Central

    Wang, Lichun; Bittman, Robert; Garcia, Joe G.N.; Dudek, Steven M.

    2015-01-01

    Rationale Modulation of pulmonary vascular barrier function is an important clinical goal given the devastating effects of vascular leak in acute lung injury (ALI). We previously demonstrated that FTY720 S-phosphonate (Tys), an analog of sphingosine 1-phosphate (S1P) and FTY720, has more potent pulmonary barrier protective effects than these agents in vitro and in mouse models of ALI. Tys preserves expression of the barrier-promoting S1P1 receptor (S1PR1), whereas S1P and FTY720 induce its ubiquitination and degradation. Here we further characterize the novel barrier promoting effects of Tys in cultured human pulmonary endothelial cells (EC). Methods/Results : In human lung EC, Tys significantly increased peripheral redistribution of adherens junction proteins VE-cadherin and β-catenin and tight junction protein ZO-1. Inhibition of VE-cadherin with blocking antibody significantly attenuated Tys-induced transendothelial resistance (TER) elevation, while ZO-1 siRNA partially inhibited this elevation. Tys significantly increased focal adhesion formation and phosphorylation of focal adhesion kinase (FAK). Pharmacologic inhibition of FAK significantly attenuated Tys-induced TER elevation. Tys significantly increased phosphorylation and peripheral redistribution of the actin-binding protein, cortactin, while cortactin siRNA partially attenuated Tys-induced TER elevation. Although Tys significantly increased phosphorylation of Akt and GSK3β, neither PI3 kinase nor GSK3β inhibition altered Tys-induced TER elevation. Tys significantly increased Rac1 activity, while inhibition of Rac1 activity significantly attenuated Tys-induced VE-cadherin redistribution and TER elevation. Conclusion Junctional complex, focal adhesion rearrangement and Rac1 activation play critical roles in Tys-mediated barrier protection in pulmonary EC. These results provide mechanistic insights into the effects of this potential ALI therapy. PMID:25862132

  20. Connexin 35: a gap-junctional protein expressed preferentially in the skate retina.

    PubMed

    O'Brien, J; al-Ubaidi, M R; Ripps, H

    1996-02-01

    We have used low stringency hybridization to clone a novel connexin from a skate retinal cDNA library. A rat connexin 32 clone was used to isolate a single partial clone that was subsequently used to isolate seven more overlapping clones of the same cDNA. Two clones containing the entire open reading frame have a consensus sequence of 1456 bp and predict a protein of 302 amino acids length and molecular mass of 35,044 daltons, referred to as connexin 35 or Cx35. Southern blot analysis suggests that the cloned sequence lies in a single gene with one intron. Polymerase chain reaction amplification from genomic DNA and partial sequencing of this intron showed that it was approximately 950 bp in length, and located within the coding region 71 bp after the translation start site. Hydropathy analysis of the predicted protein and alignments with previously cloned connexins indicate that Cx35 has a long cytoplasmic loop and a relatively short carboxyl terminal tail. Multiple sequence alignments show that Cx35 has similarities to both alpha and beta groups of connexins and suggests that its origins may be near the divergence point for the two groups. Consensus sequences consistent with sites for phosphorylation by protein kinase C and by cAMP - or cGMP -dependent protein kinase were identified. Two transcripts were detected in Northern blot analysis: a 1.95-kb primary transcript and a 4.6-kb minor transcript. In RNA samples from 10 tissues, transcripts were detected only in the retina.

  1. FERM protein EPB41L5 is a novel member of the mammalian CRB-MPP5 polarity complex.

    PubMed

    Gosens, Ilse; Sessa, Alessandro; den Hollander, Anneke I; Letteboer, Stef J F; Belloni, Valentina; Arends, Maarten L; Le Bivic, André; Cremers, Frans P M; Broccoli, Vania; Roepman, Ronald

    2007-11-15

    Cell polarity is induced and maintained by separation of the apical and basolateral domains through specialized cell-cell junctions. The Crumbs protein and its binding partners are involved in formation and stabilization of adherens junctions. In this study, we describe a novel component of the mammalian Crumbs complex, the FERM domain protein EPB41L5, which associates with the intracellular domains of all three Crumbs homologs through its FERM domain. Surprisingly, the same FERM domain is involved in binding to the HOOK domain of MPP5/PALS1, a previously identified interactor of Crumbs. Co-expression and co-localization studies suggested that in several epithelial derived tissues Epb4.1l5 interacts with at least one Crumbs homolog, and with Mpp5. Although at early embryonic stages Epb4.1l5 is found at the basolateral membrane compartment, in adult tissues it co-localizes at the apical domain with Crumbs proteins and Mpp5. Overexpression of Epb4.1l5 in polarized MDCK cells affects tightness of cell junctions and results in disorganization of the tight junction markers ZO-1 and PATJ. Our results emphasize the importance of a conserved Crumbs-MPP5-EPB41L5 polarity complex in mammals. PMID:17920587

  2. Effects of Soybean Agglutinin on Mechanical Barrier Function and Tight Junction Protein Expression in Intestinal Epithelial Cells from Piglets

    PubMed Central

    Pan, Li; Qin, Guixin; Zhao, Yuan; Wang, Jun; Liu, Feifei; Che, Dongsheng

    2013-01-01

    In this study, we sought to investigate the role of soybean agglutinin (SBA) in mediating membrane permeability and the mechanical barrier function of intestinal epithelial cells. The IPEC-J2 cells were cultured and treated with 0, 0.5, 1.0, 1.5, 2.0, 2.5, or 3.0 mg/mL SBA. Transepithelial electrical resistance (TEER) and alkaline phosphatase (AP) activity were measured to evaluate membrane permeability. The results showed a significant decrease in TEER values (p < 0.05) in a time- and dose-dependent manner, and a pronounced increase in AP activity (p < 0.05). Cell growth and cell morphology were used to evaluate the cell viability. A significant cell growth inhibition (p < 0.05) and alteration of morphology were observed when the concentration of SBA was increased. The results of western blotting showed that the expression levels of occludin and claudin-3 were decreased by 31% and 64% compared to those of the control, respectively (p < 0.05). In addition, immunofluorescence labeling indicated an obvious decrease in staining of these targets and changes in their localizations. In conclusion, SBA increased the membrane permeability, inhibited the cell viability and reduced the levels of tight junction proteins (occludin and claudin-3), leading to a decrease in mechanical barrier function in intestinal epithelial cells. PMID:24189218

  3. Electroacupuncture Treatment Improves Neurological Function Associated with Regulation of Tight Junction Proteins in Rats with Cerebral Ischemia Reperfusion Injury

    PubMed Central

    Zhang, Ya-min; Xu, Hong; Sun, Hua; Chen, Su-hui; Wang, Fu-ming

    2014-01-01

    Strategies to develop effective neuroprotective therapy to reduce brain damage and related behavioral deficits in stroke patients are of great significance. Electroacupuncture (EA), which derives from traditional Chinese medicine, may be effective as a complementary and alternative method for promoting recovery of neurological function and quality of life. Adult Sprague-Dawley rats were randomly divided into 3 groups: (1) sham, (2) middle cerebral artery occlusion (MCAO) model groups of 2 h MCAO followed by 1, 3, 5, or 7 d of reperfusion, and (3) EA groups of 2 h MCAO followed by 1, 3, 5, or 7 d of reperfusion. EA groups received EA therapy by needling at GV20 and left ST36. The results show that EA therapy improved the neurological function and reduced infarct volume, confirmed by modified neurological severity scores and TTC staining. Real-time PCR, immunohistochemistry, and western blot assay verified that EA upregulated the expression of tight junction (TJ) claudin-5, occludin, and zonula occluding-1 from 1 to 7 d after reperfusion. Our findings suggest that EA reduces brain damage and related behavioral deficits via upregulation of the TJ proteins. PMID:25009574

  4. MOSFET-BJT hybrid mode of the gated lateral bipolar junction transistor for C-reactive protein detection.

    PubMed

    Yuan, Heng; Kwon, Hyurk-Choon; Yeom, Se-Hyuk; Kwon, Dae-Hyuk; Kang, Shin-Won

    2011-10-15

    In this study, we propose a novel biosensor based on a gated lateral bipolar junction transistor (BJT) for biomaterial detection. The gated lateral BJT can function as both a BJT and a metal-oxide-semiconductor field-effect transistor (MOSFET) with both the emitter and source, and the collector and drain, coupled. C-reactive protein (CRP), which is an important disease marker in clinical examinations, can be detected using the proposed device. In the MOSFET-BJT hybrid mode, the sensitivity, selectivity, and reproducibility of the gated lateral BJT for biosensors were evaluated in this study. According to the results, in the MOSFET-BJT hybrid mode, the gated lateral BJT shows good selectivity and reproducibility. Changes in the emitter (source) current of the device for CRP antigen detection were approximately 0.65, 0.72, and 0.80 μA/decade at base currents of -50, -30, and -10 μA, respectively. The proposed device has significant application in the detection of certain biomaterials that require a dilution process using a common biosensor, such as a MOSFET-based biosensor. PMID:21835604

  5. The Tight Junction Associated Signalling Proteins ZO-1 and ZONAB Regulate Retinal Pigment Epithelium Homeostasis in Mice

    PubMed Central

    Bainbridge, James W. B.; Balaggan, Kamaljit S.; Mowat, Freya; West, Emma L.; Munro, Peter M. G.; Thrasher, Adrian J.; Matter, Karl; Balda, Maria S.; Ali, Robin R.

    2010-01-01

    Cell-cell adhesion regulates the development and function of epithelia by providing mechanical support and by guiding cell proliferation and differentiation. The tight junction (TJ) protein zonula occludens (ZO)-1 regulates cell proliferation and gene expression by inhibiting the activity of the Y-box transcription factor ZONAB in cultured epithelial cells. We investigated the role of this TJ-associated signalling pathway in the retinal pigment epithelium (RPE) in vivo by lentivirally-mediated overexpression of ZONAB, and knockdown of its cellular inhibitor ZO-1. Both overexpression of ZONAB or knockdown of ZO-1 resulted in increased RPE proliferation, and induced ultrastructural changes of an epithelial-mesenchymal transition (EMT)-like phenotype. Electron microscopy analysis revealed that transduced RPE monolayers were disorganised with increased pyknosis and monolayer breaks, correlating with increased expression of several EMT markers. Moreover, fluorescein angiography analysis demonstrated that the increased proliferation and EMT-like phenotype induced by overexpression of ZONAB or downregulation of ZO-1 resulted in RPE dysfunction. These findings demonstrate that ZO-1 and ZONAB are critical for differentiation and homeostasis of the RPE monolayer and may be involved in RPE disorders such as proliferative vitroretinopathy and atrophic age-related macular degeneration. PMID:21209887

  6. Distinct Changes in Synaptic Protein Composition at Neuromuscular Junctions of Extraocular Muscles versus Limb Muscles of ALS Donors

    PubMed Central

    Liu, Jing-Xia; Brännström, Thomas; Andersen, Peter M.; Pedrosa-Domellöf, Fatima

    2013-01-01

    The pathophysiology of amyotrophic lateral sclerosis (ALS) is very complex and still rather elusive but in recent years evidence of early involvement of the neuromuscular junctions (NMJs) has accumulated. We have recently reported that the human extraocular muscles (EOMs) are far less affected than limb muscles at the end-stage of ALS from the same donor. The present study aimed to compare the differences in synaptic protein composition at NMJ and in nerve fibers between EOM and limb muscles from ALS donors and controls. Neurofilament light subunit and synaptophysin decreased significantly at NMJs and in nerve fibers in limb muscles with ALS whereas they were maintained in ALS EOMs. S100B was significantly decreased at NMJs and in nerve fibers in both EOMs and limb muscles of ALS donors, but other markers confirmed the presence of terminal Schwann cells in these NMJs. p75 neurotrophin receptor was present in nerve fibers but absent at NMJs in ALS limb muscles. The EOMs were able to maintain the integrity of their NMJs to a very large extent until the end-stage of ALS, in contrast to the limb muscles. Changes in Ca2+ homeostasis, reflected by altered S100B distribution, might be involved in the breakdown of nerve-muscle contact at NMJs in ALS. PMID:23468993

  7. An Atypical PKC Directly Associates and Colocalizes at the Epithelial Tight Junction with ASIP, a Mammalian Homologue of Caenorhabditis elegans Polarity Protein PAR-3

    PubMed Central

    Izumi, Yasushi; Hirose, Tomonori; Tamai, Yoko; Hirai, Syu-ichi; Nagashima, Yoji; Fujimoto, Toyoshi; Tabuse, Yo; Kemphues, Kenneth J.; Ohno, Shigeo

    1998-01-01

    Cell polarity is fundamental to differentiation and function of most cells. Studies in mammalian epithelial cells have revealed that the establishment and maintenance of cell polarity depends upon cell adhesion, signaling networks, the cytoskeleton, and protein transport. Atypical protein kinase C (PKC) isotypes PKCζ and PKCλ have been implicated in signaling through lipid metabolites including phosphatidylinositol 3-phosphates, but their physiological role remains elusive. In the present study we report the identification of a protein, ASIP (atypical PKC isotype–specific interacting protein), that binds to aPKCs, and show that it colocalizes with PKCλ to the cell junctional complex in cultured epithelial MDCKII cells and rat intestinal epithelia. In addition, immunoelectron microscopy revealed that ASIP localizes to tight junctions in intestinal epithelial cells. Furthermore, ASIP shows significant sequence similarity to Caenorhabditis elegans PAR-3. PAR-3 protein is localized to the anterior periphery of the one-cell embryo, and is required for the establishment of cell polarity in early embryos. ASIP and PAR-3 share three PDZ domains, and can both bind to aPKCs. Taken together, our results suggest a role for a protein complex containing ASIP and aPKC in the establishment and/or maintenance of epithelial cell polarity. The evolutionary conservation of the protein complex and its asymmetric distribution in polarized cells from worm embryo to mammalian-differentiated cells may mean that the complex functions generally in the organization of cellular asymmetry. PMID:9763423

  8. Ultrafast endocytosis at Caenorhabditis elegans neuromuscular junctions

    PubMed Central

    Watanabe, Shigeki; Liu, Qiang; Davis, M Wayne; Hollopeter, Gunther; Thomas, Nikita; Jorgensen, Nels B; Jorgensen, Erik M

    2013-01-01

    Synaptic vesicles can be released at extremely high rates, which places an extraordinary demand on the recycling machinery. Previous ultrastructural studies of vesicle recycling were conducted in dissected preparations using an intense stimulation to maximize the probability of release. Here, a single light stimulus was applied to motor neurons in intact Caenorhabditis elegans nematodes expressing channelrhodopsin, and the animals rapidly frozen. We found that docked vesicles fuse along a broad active zone in response to a single stimulus, and are replenished with a time constant of about 2 s. Endocytosis occurs within 50 ms adjacent to the dense projection and after 1 s adjacent to adherens junctions. These studies suggest that synaptic vesicle endocytosis may occur on a millisecond time scale following a single physiological stimulus in the intact nervous system and is unlikely to conform to current models of endocytosis. DOI: http://dx.doi.org/10.7554/eLife.00723.001 PMID:24015355

  9. Effects of oral glutamine supplementation on exercise-induced gastrointestinal permeability and tight junction protein expression.

    PubMed

    Zuhl, Micah N; Lanphere, Kathryn R; Kravitz, Len; Mermier, Christine M; Schneider, Suzanne; Dokladny, Karol; Moseley, Pope L

    2014-01-15

    The objectives of this study are threefold: 1) to assess whether 7 days of oral glutamine (GLN) supplementation reduces exercise-induced intestinal permeability; 2) whether supplementation prevents the proinflammatory response; and 3) whether these changes are associated with upregulation of the heat shock response. On separate occasions, eight human subjects participated in baseline testing and in GLN and placebo (PLA) supplementation trials, followed by a 60-min treadmill run. Intestinal permeability was higher in the PLA trial compared with baseline and GLN trials (0.0604 ± 0.047 vs. 0.0218 ± 0.008 and 0.0272 ± 0.007, respectively; P < 0.05). IκBα expression in peripheral blood mononuclear cells was higher 240 min after exercise in the GLN trial compared with the PLA trial (1.411 ± 0.523 vs. 0.9839 ± 0.343, respectively; P < 0.05). In vitro using the intestinal epithelial cell line Caco-2, we measured effects of GLN supplementation (0, 4, and 6 mM) on heat-induced (37° or 41.8°C) heat shock protein 70 (HSP70), heat shock factor-1 (HSF-1), and occludin expression. HSF-1 and HSP70 levels increased in 6 mM supplementation at 41°C compared with 0 mM at 41°C (1.785 ± 0.495 vs. 0.6681 ± 0.290, and 1.973 ± 0.325 vs. 1.133 ± 0.129, respectively; P < 0.05). Occludin levels increased after 4 mM supplementation at 41°C and 6 mM at 41°C compared with 0 mM at 41°C (1.236 ± 0.219 and 1.849 ± 0.564 vs. 0.7434 ± 0.027, respectively; P < 0.001). GLN supplementation prevented exercise-induced permeability, possibly through HSF-1 activation. PMID:24285149

  10. Josephson junction

    DOEpatents

    Wendt, Joel R.; Plut, Thomas A.; Martens, Jon S.

    1995-01-01

    A novel method for fabricating nanometer geometry electronic devices is described. Such Josephson junctions can be accurately and reproducibly manufactured employing photolithographic and direct write electron beam lithography techniques in combination with aqueous etchants. In particular, a method is described for manufacturing planar Josephson junctions from high temperature superconducting material.

  11. Josephson junction

    DOEpatents

    Wendt, J.R.; Plut, T.A.; Martens, J.S.

    1995-05-02

    A novel method for fabricating nanometer geometry electronic devices is described. Such Josephson junctions can be accurately and reproducibly manufactured employing photolithographic and direct write electron beam lithography techniques in combination with aqueous etchants. In particular, a method is described for manufacturing planar Josephson junctions from high temperature superconducting material. 10 figs.

  12. Dietary calcium concentration and cereals differentially affect mineral balance and tight junction proteins expression in jejunum of weaned pigs.

    PubMed

    Metzler-Zebeli, Barbara U; Mann, Evelyne; Ertl, Reinhard; Schmitz-Esser, Stephan; Wagner, Martin; Klein, Dieter; Ritzmann, Mathias; Zebeli, Qendrim

    2015-04-14

    Ca plays an essential role in bone development; however, little is known about its effect on intestinal gene expression in juvenile animals. In the present study, thirty-two weaned pigs (9·5 (SEM 0·11) kg) were assigned to four diets that differed in Ca concentration (adequate v. high) and cereal composition (wheat-barley v. maize) to assess the jejunal and colonic gene expression of nutrient transporters, tight junction proteins, cytokines and pathogen-associated molecular patterns, nutrient digestibility, Ca balance and serum acute-phase response. To estimate the impact of mucosal bacteria on colonic gene expression, Spearman's correlations between colonic gene expression and bacterial abundance were computed. Faecal Ca excretion indicated that more Ca was available along the intestinal tract of the pigs fed high Ca diets as compared to the pigs fed adequate Ca diets (P> 0.05). High Ca diets decreased jejunal zonula occludens 1 (ZO1) and occludin (OCLN) expression, up-regulated jejunal expression of toll-like receptor 2 (TLR2) and down-regulated colonic GLUT2 expression as compared to the adequate Ca diets (P< 0.05). Dietary cereal composition up-regulated jejunal TLR2 expression and interacted (P= 0.021) with dietary Ca on colonic IL1B expression; high Ca concentration up-regulated IL1B expression with wheat-barley diets and down-regulated it with maize diets. Spearman's correlations (r> 0·35; P< 0·05) indicated an association between operational taxonomic units assigned to the phyla Bacteroidetes, Firmicutes and Proteobacteria and bacterial metabolites and mucosal gene expression in the colon. The present results indicate that high Ca diets have the potential to modify the jejunal and colonic mucosal gene expression response which, in turn, interacts with the composition of the basal diet and mucosa-associated bacteria in weaned pigs. PMID:25761471

  13. Role of gap junctions and protein kinase A during the development of oocyte maturational competence in Ayu (Plecoglossus altivelis)

    USGS Publications Warehouse

    Yamamoto, Y.; Yoshizaki, G.; Takeuchi, T.; Soyano, K.; Patino, R.

    2008-01-01

    Meiotic resumption in teleost oocytes is induced by a maturation-inducing hormone (MIH). The sensitivity of oocytes to MIH, also known as oocyte maturational competence (OMC), is induced by LH via mechanisms that are not fully understood. A previous study of Ayu (Plecoglossus altivelis) showed the presence of functional heterologous gap junctions (GJs) between oocytes and their surrounding granulosa cells. The objectives of this study were to determine the role of ovarian GJs and of protein kinase A (PKA) during the acquisition of OMC. We examined the effects of the specific GJ inhibitor carbenoxolone (CBX) and 18??-glycyrrhetinic acid (??-GA) on the LH-(hCG)-dependent acquisition of OMC and on MIH-(17,20??-dihydroxy-4-pregnen-3-one)-dependent meiotic resumption; measured the cAMP content of ovarian follicles during the hCG-dependent acquisition of OMC; and determined the effects of PK activators and inhibitors on hCG-dependent OMC. Production of follicular cAMP increased during the hCG-dependent acquisition of OMC. Both GJ inhibitors and the PKA inhibitor H8-dihydrochloride, but not the PKC inhibitor GF109203X, suppressed the hCG-dependent acquisition of OMC in a dose-dependent manner. The PKA activator forskolin induced OMC with a similar potency to hCG. Unlike previous observations with teleosts where disruption of heterologous GJ either blocks or stimulates meiotic resumption, treatment with GJ inhibitors did not affect MIH-dependent meiotic resumption in maturationally competent follicles of Ayu. These observations suggest that ovarian GJs are essential for LH-dependent acquisition of OMC but not for MIH-dependent meiotic resumption, and that the stimulation of OMC by LH is mediated by cAMP-dependent PKA. They are also consistent with the view that a precise balance between GJ-mediated signals (positive or negative) and oocyte maturational readiness is required for hormonally regulated meiotic resumption. ?? 2007 Elsevier Inc. All rights reserved.

  14. Novel role of zonula occludens-1: A tight junction protein closely associated with the odontoblast differentiation of human dental pulp cells.

    PubMed

    Xu, Jue; Shao, Meiying; Pan, Hongying; Wang, Huning; Cheng, Li; Yang, Hui; Hu, Tao

    2016-07-01

    Zonula occludens-1 (ZO-1), a tight junction protein, contributes to the maintenance of the polarity of odontoblasts and junctional complex formation in odontoblast layer during tooth development. However, expression and possible role of ZO-1 in human dental pulp cells (hDPCs) during repair process remains unknown. Here, we investigated the expression of ZO-1 in hDPCs and the relationship with odontoblast differentiation. We found the processes of two adjacent cells were fused and formed junction-like structure using scanning electron microscopy. Fluorescence immunoassay and Western blot confirmed ZO-1 expression in hDPCs. Especially, ZO-1 was high expressed at the cell-cell junction sites. More interestingly, ZO-1 accumulated at the leading edge of lamellipodia in migrating cells when a scratch assay was performed. Furthermore, ZO-1 gradual increased during odontoblast differentiation and ZO-1 silencing greatly inhibited the differentiation. ZO-1 binds directly to actin filaments and RhoA/ROCK signaling mainly regulates cell cytoskeleton, thus RhoA/ROCK might play a role in regulating ZO-1. Lysophosphatidic acid (LPA) and Y-27632 were used to activate and inhibit RhoA/ROCK signaling, respectively, with or without mineralizing medium. In normal cultured hDPCs, RhoA activation increased ZO-1 expression and especially in intercellular contacts, whereas ROCK inhibition attenuated the effects induced by LPA. However, expression of ZO-1 was upregulated by Y-27632 but not significantly affected by LPA after odontoblast differentiation. Hence, ZO-1 highly expresses in cell-cell junctions and is related to odontoblast differentiation, which may contribute to dental pulp repair or even the formation of an odontoblast layer. RhoA/ROCK signaling is involved in the regulation of ZO-1. PMID:27109589

  15. ZO-3, a Novel Member of the MAGUK Protein Family Found at the Tight Junction, Interacts with ZO-1 and Occludin

    PubMed Central

    Haskins, Julie; Gu, Lijie; Wittchen, Erika S.; Hibbard, Jennifer; Stevenson, Bruce R.

    1998-01-01

    A 130-kD protein that coimmunoprecipitates with the tight junction protein ZO-1 was bulk purified from Madin-Darby canine kidney (MDCK) cells and subjected to partial endopeptidase digestion and amino acid sequencing. A resulting 19–amino acid sequence provided the basis for screening canine cDNA libraries. Five overlapping clones contained a single open reading frame of 2,694 bp coding for a protein of 898 amino acids with a predicted molecular mass of 98,414 daltons. Sequence analysis showed that this protein contains three PSD-95/SAP90, discs-large, ZO-1 (PDZ) domains, a src homology (SH3) domain, and a region similar to guanylate kinase, making it homologous to ZO-1, ZO-2, the discs large tumor suppressor gene product of Drosophila, and other members of the MAGUK family of proteins. Like ZO-1 and ZO-2, the novel protein contains a COOH-terminal acidic domain and a basic region between the first and second PDZ domains. Unlike ZO-1 and ZO-2, this protein displays a proline-rich region between PDZ2 and PDZ3 and apparently contains no alternatively spliced domain. MDCK cells stably transfected with an epitope-tagged construct expressed the exogenous polypeptide at an apparent molecular mass of ∼130 kD. Moreover, this protein colocalized with ZO-1 at tight junctions by immunofluorescence and immunoelectron microscopy. In vitro affinity analyses demonstrated that recombinant 130-kD protein directly interacts with ZO-1 and the cytoplasmic domain of occludin, but not with ZO-2. We propose that this protein be named ZO-3. PMID:9531559

  16. 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

  17. 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.

  18. Interaction of Ddc1 and RPA with single-stranded/double-stranded DNA junctions in yeast whole cell extracts: Proteolytic degradation of the large subunit of replication protein A in ddc1Δ strains.

    PubMed

    Sukhanova, Maria V; D'Herin, Claudine; Boiteux, Serge; Lavrik, Olga I

    2014-10-01

    To characterize proteins that interact with single-stranded/double-stranded (ss/ds) DNA junctions in whole cell free extracts of Saccharomyces cerevisiae, we used [(32)P]-labeled photoreactive partial DNA duplexes containing a 3'-ss/ds-junction (3'-junction) or a 5'-ss/ds-junction (5'-junction). Identification of labeled proteins was achieved by MALDI-TOF mass spectrometry peptide mass fingerprinting and genetic analysis. In wild-type extract, one of the components of the Ddc1-Rad17-Mec3 complex, Ddc1, was found to be preferentially photocrosslinked at a 3'-junction. On the other hand, RPAp70, the large subunit of the replication protein A (RPA), was the predominant crosslinking product at a 5'-junction. Interestingly, ddc1Δ extracts did not display photocrosslinking of RPAp70 at a 5'-junction. The results show that RPAp70 crosslinked to DNA with a 5'-junction is subject to limited proteolysis in ddc1Δ extracts, whereas it is stable in WT, rad17Δ, mec3Δ and mec1Δ extracts. The degradation of the RPAp70-DNA adduct in ddc1Δ extract is strongly reduced in the presence of the proteasome inhibitor MG 132. We also addressed the question of the stability of free RPA, using anti-RPA antibodies. The results show that RPAp70 is also subject to proteolysis without photocrosslinking to DNA upon incubation in ddc1Δ extract. The data point to a novel property of Ddc1, modulating the turnover of DNA binding proteins such as RPAp70 by the proteasome.

  19. Poly-L-arginine-Induced internalization of tight junction proteins increases the paracellular permeability of the Caco-2 cell monolayer to hydrophilic macromolecules.

    PubMed

    Yamaki, Tsutomu; Ohtake, Kazuo; Ichikawa, Keiko; Uchida, Masaki; Uchida, Hiroyuki; Oshima, Shinji; Ohshima, Shinji; Juni, Kazuhiko; Kobayashi, Jun; Morimoto, Yasunori; Natsume, Hideshi

    2013-01-01

    We investigated whether poly-L-arginine (PLA) enhances the paracellular permeability of the Caco-2 monolayer to hydrophilic macromolecules and clarified the disposition of tight junction (TJ) proteins. The transepithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)-dextran (FD-4) permeation were determined after treatment with PLA. TJ proteins were visualized using immunofluorescence microscopy after PLA exposure and depletion, and their expression levels were determined. The barrier function of TJs was also evaluated by measuring the alterations in the TEER and in the localization of TJ proteins. PLA induced an increase in hydrophilic macromolecule, FD-4, permeation through Caco-2 cell monolayers and a decrease in the TEER in a concentration-dependent manner, without any significant impact on the cell viability. This increased paracellular permeability induced by PLA was found to be internalized of claudin-4, ZO-1, tricellulin and mainly occludin from cell-cell junction to the subcellular space. ZO-1 appeared to play an important role in the reconstitution of TJ strand structures following PLA depletion. These results indicate that the PLA led to the internalization of TJ proteins to the subcellular space, subsequently increasing the permeability of the Caco-2 cell monolayer to FD-4 via a paracellular route.

  20. EspF Interacts with Nucleation-Promoting Factors To Recruit Junctional Proteins into Pedestals for Pedestal Maturation and Disruption of Paracellular Permeability ▿

    PubMed Central

    Peralta-Ramírez, Janneth; Hernandez, J. Manuel; Manning-Cela, Rebeca; Luna-Muñoz, José; Garcia-Tovar, Carlos; Nougayréde, Jean-Philippe; Oswald, Eric; Navarro-Garcia, Fernando

    2008-01-01

    Many pathogenic bacteria subvert normal host cell processes by delivering effector proteins which mimic eukaryotic functions directly into target cells. EspF is a multifunctional protein injected into host cells by attaching and effacing pathogens, but its mechanism of action is not understood completely. In silico analyses of EspF revealed two key motifs: proline-rich domains and PDZ domain binding motifs. Such functional domains may allow EspF to act as an actin nucleation-promoting factor by mimicking host proteins. In agreement with these predictions, we found that EspF from rabbit enteropathogenic Escherichia coli (E22) participates in the regulation of actin polymerization by binding to a complex of proteins at the tight junctions (TJ). EspF bound to actin and profilin throughout the course of infection. However, after 2 h of infection, EspF also bound to the neural Wiskott-Aldrich syndrome protein and to the Arp2/3, zonula occludens-1 (ZO-1), and ZO-2 proteins. Moreover, EspF caused occludin, claudin, ZO-1, and ZO-2 redistribution and loss of transepithelial electrical resistance, suggesting that actin sequestration by EspF may cause local actin depolymerization leading to EspF-induced TJ disruption. Furthermore, EspF caused recruitment of these TJ proteins into the pedestals. An E22 strain lacking EspF did not cause TJ disruption and pedestals were smaller than those induced by the wild-type strain. Additionally, the pedestals were located mainly in the TJ. The overexpression of EspF caused bigger pedestals located along the length of the cells. Thus, actin sequestration by EspF allows the recruitment of junctional proteins into the pedestals, leading to the maturation of actin pedestals and the disruption of paracellular permeability. PMID:18559425

  1. Modulation of adrenal gap junction expression.

    PubMed

    Murray, S A; Shah, U S

    1998-01-01

    To increase our knowledge of the role of peptide hormone stimulation in gap junction protein expression and adrenal cortical cell function, primary rat adrenal cortical cells were treated with adrenocorticotropin, and gap junction proteins were measured. Immunocytochemistry and western blot analysis were used to detect and characterize gap junction type and distribution. The gap junction protein, connexin 43 (alpha 1), was detected. Analysis of six connexin protein types did not reveal gap junction species other than alpha 1. Cells of the inner adrenal cortical zones, zonae fasciculata and reticularis, were demonstrated to have the highest number of gap junctions per cell in the adrenal gland. Adrenal cell cultures enriched for the two inner cortical adrenal zones were established and demonstrated also to express alpha 1 gap junction protein. Adrenocorticotropin (40 mUnits/ml) and dibutyryl cyclic adenosine monophosphate (1 mM) treatments increased alpha 1 gap junction protein levels and decreased cell proliferation rates in the cell cultures. The results are consistent with the hypothesis that gap junction expression can be regulated by adrenocorticotropin acting through the second messenger cyclic adenosine monophosphate. It can be suggested that gap junction expression in the adrenal gland may be under hormonal influence, and that gap junctions serve as passage for movement of molecules involved in control of cell proliferation. PMID:9694574

  2. CAR regulates epithelial cell junction stability through control of E-cadherin trafficking

    PubMed Central

    Morton, Penny E.; Hicks, Alexander; Nastos, Theodoros; Santis, George; Parsons, Maddy

    2013-01-01

    CAR (Coxsackie and Adenovirus Receptor) is the primary docking receptor for typeB coxsackie viruses and subgroup C adenoviruses. CAR is a member of the JAM family of adhesion receptors and is located to both tight and adherens junctions between epithelial cells where it can assemble adhesive contacts through homodimerisation in trans. However, the role of CAR in controlling epithelial junction dynamics remains poorly understood. Here we demonstrate that levels of CAR in human epithelial cells play a key role in determining epithelial cell adhesion through control of E-cadherin stability at cell-cell junctions. Mechanistically, we show that CAR is phosphorylated within the C-terminus by PKCδ and that this in turn controls Src-dependent endocytosis of E-cadherin at cell junctions. This data demonstrates a novel role for CAR in regulating epithelial homeostasis. PMID:24096322

  3. The septate junction protein caspr is required for structural support and retention of KCNQ4 at calyceal synapses of vestibular hair cells.

    PubMed

    Sousa, Aurea D; Andrade, Leonardo R; Salles, Felipe T; Pillai, Anilkumar M; Buttermore, Elizabeth D; Bhat, Manzoor A; Kachar, Bechara

    2009-03-11

    The afferent innervation contacting the type I hair cells of the vestibular sensory epithelia form distinct calyceal synapses. The apposed presynaptic and postsynaptic 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 postsynaptic membranes of these calyceal synapses. Immunolabeling and ultrastructural analyses of Caspr knock-out 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 postsynaptic 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

  4. The nuclear and adherent junction complex component protein ubinuclein negatively regulates the productive cycle of Epstein-Barr virus in epithelial cells.

    PubMed

    Gruffat, Henri; Lupo, Julien; Morand, Patrice; Boyer, Véronique; Manet, Evelyne

    2011-01-01

    The Epstein-Barr Virus (EBV) productive cycle is initiated by the expression of the viral trans-activator EB1 (also called Zebra, Zta, or BZLF1), which belongs to the basic leucine zipper transcription factor family. We have previously identified the cellular NACos (nuclear and adherent junction complex components) protein ubinuclein (Ubn-1) as a partner for EB1, but the function of this complex has never been studied. Here, we have evaluated the consequences of this interaction on the EBV productive cycle and find that Ubn-1 overexpression represses the EBV productive cycle whereas Ubn-1 downregulation by short hairpin RNA (shRNA) increases virus production. By a chromatin immunoprecipitation (ChIP) assay, we show that Ubn-1 blocks EB1-DNA interaction. We also show that in epithelial cells, relocalization and sequestration of Ubn-1 to the tight junctions of nondividing cells allow increased activation of the productive cycle. We propose a model in which Ubn-1 is a modulator of the EBV productive cycle: in proliferating epithelial cells, Ubn-1 is nuclear and inhibits activation of the productive cycle, whereas in differentiated cells, Ubn-1 is sequestrated to tight junctions, thereby allowing EB1 to fully function in the nucleus.

  5. The Nuclear and Adherent Junction Complex Component Protein Ubinuclein Negatively Regulates the Productive Cycle of Epstein-Barr Virus in Epithelial Cells▿

    PubMed Central

    Gruffat, Henri; Lupo, Julien; Morand, Patrice; Boyer, Véronique; Manet, Evelyne

    2011-01-01

    The Epstein-Barr Virus (EBV) productive cycle is initiated by the expression of the viral trans-activator EB1 (also called Zebra, Zta, or BZLF1), which belongs to the basic leucine zipper transcription factor family. We have previously identified the cellular NACos (nuclear and adherent junction complex components) protein ubinuclein (Ubn-1) as a partner for EB1, but the function of this complex has never been studied. Here, we have evaluated the consequences of this interaction on the EBV productive cycle and find that Ubn-1 overexpression represses the EBV productive cycle whereas Ubn-1 downregulation by short hairpin RNA (shRNA) increases virus production. By a chromatin immunoprecipitation (ChIP) assay, we show that Ubn-1 blocks EB1-DNA interaction. We also show that in epithelial cells, relocalization and sequestration of Ubn-1 to the tight junctions of nondividing cells allow increased activation of the productive cycle. We propose a model in which Ubn-1 is a modulator of the EBV productive cycle: in proliferating epithelial cells, Ubn-1 is nuclear and inhibits activation of the productive cycle, whereas in differentiated cells, Ubn-1 is sequestrated to tight junctions, thereby allowing EB1 to fully function in the nucleus. PMID:21084479

  6. Perineuronal satellite cells in mouse spinal ganglia express the gap junction protein connexin43 throughout life with decline in old age.

    PubMed

    Procacci, Patrizia; Magnaghi, Valerio; Pannese, Ennio

    2008-03-28

    Satellite glial cells that envelope the bodies of sensory neurons in spinal ganglia are connected to each other by gap junctions and exhibit dye coupling. These junctions may endow perineuronal satellite cells with the coordination necessary for the efficient performance of functions such as buffering of K(+) in the perineuronal microenvironment, provision of metabolic support to ganglionic neurons, and neuroprotection. Our knowledge of gap junctions has increased considerably in recent years, but little information is available on the connexins that form these junctions in spinal ganglia. In the present study we set out to determine whether the perineuronal satellite cells of mouse spinal ganglia express the connexins that are mainly present in neuroglial cells (Cx32 and Cx43). In young (3 months) mice, PCR showed the presence of both Cx32 and Cx43 transcripts. By immunocytochemistry, we localized Cx32 to axon-ensheathing Schwann cells, but not to other parts of the ganglion. We found Cx43 positivity in the perineuronal satellite cells, which were identified by their immunoreactivity to S100 protein and to glutamine synthetase. PCR showed Cx43 transcripts also in the spinal ganglia of adult (8 months) and old (24 months) animals. Cx43 immunostaining was present in satellite cells surrounding all nerve cell bodies, irrespective of size. The mean number of Cx43-immunoreactive puncta was significantly lower in the perineuronal satellite cells of aged mice compared to young and adult animals. This latter finding is consistent with observations in non-nervous tissues, and the hypothesis that a prominent decrease in Cx43 is a marker of senescence. PMID:18355632

  7. The actin-binding protein EPS8 binds VE-cadherin and modulates YAP localization and signaling

    PubMed Central

    Disanza, Andrea; Bravi, Luca; Barrios-Rodiles, Miriam; Corada, Monica; Frittoli, Emanuela; Savorani, Cecilia; Lampugnani, Maria Grazia; Boggetti, Barbara; Niessen, Carien; Wrana, Jeff L.

    2015-01-01

    Vascular endothelial (VE)–cadherin transfers intracellular signals contributing to vascular hemostasis. Signaling through VE-cadherin requires association and activity of different intracellular partners. Yes-associated protein (YAP)/TAZ transcriptional cofactors are important regulators of cell growth and organ size. We show that EPS8, a signaling adapter regulating actin dynamics, is a novel partner of VE-cadherin and is able to modulate YAP activity. By biochemical and imaging approaches, we demonstrate that EPS8 associates with the VE-cadherin complex of remodeling junctions promoting YAP translocation to the nucleus and transcriptional activation. Conversely, in stabilized junctions, 14–3-3–YAP associates with the VE–cadherin complex, whereas Eps8 is excluded. Junctional association of YAP inhibits nuclear translocation and inactivates its transcriptional activity both in vitro and in vivo in Eps8-null mice. The absence of Eps8 also increases vascular permeability in vivo, but did not induce other major vascular defects. Collectively, we identified novel components of the adherens junction complex, and we introduce a novel molecular mechanism through which the VE-cadherin complex controls YAP transcriptional activity. PMID:26668327

  8. FAP20 is an inner junction protein of doublet microtubules essential for both the planar asymmetrical waveform and stability of flagella in Chlamydomonas

    PubMed Central

    Yanagisawa, Haru-aki; Mathis, Garrison; Oda, Toshiyuki; Hirono, Masafumi; Richey, Elizabeth A.; Ishikawa, Hiroaki; Marshall, Wallace F.; Kikkawa, Masahide; Qin, Hongmin

    2014-01-01

    The axoneme—the conserved core of eukaryotic cilia and flagella—contains highly specialized doublet microtubules (DMTs). A long-standing question is what protein(s) compose the junctions between two tubules in DMT. Here we identify a highly conserved flagellar-associated protein (FAP), FAP20, as an inner junction (IJ) component. The flagella of Chlamydomonas FAP20 mutants have normal length but beat with an abnormal symmetrical three-dimensional pattern. In addition, the mutant axonemes are liable to disintegrate during beating, implying that interdoublet connections may be weakened. Conventional electron microscopy shows that the mutant axonemes lack the IJ, and cryo–electron tomography combined with a structural labeling method reveals that the labeled FAP20 localizes at the IJ. The mutant axonemes also lack doublet-specific beak structures, which are localized in the proximal portion of the axoneme and may be involved in planar asymmetric flagellar bending. FAP20 itself, however, may not be a beak component, because uniform localization of FAP20 along the entire length of all nine DMTs is inconsistent with the beak's localization. FAP20 is the first confirmed component of the IJ. Our data also suggest that the IJ is important for both stabilizing the axoneme and scaffolding intra–B-tubular substructures required for a planar asymmetrical waveform. PMID:24574454

  9. FAP20 is an inner junction protein of doublet microtubules essential for both the planar asymmetrical waveform and stability of flagella in Chlamydomonas.

    PubMed

    Yanagisawa, Haru-aki; Mathis, Garrison; Oda, Toshiyuki; Hirono, Masafumi; Richey, Elizabeth A; Ishikawa, Hiroaki; Marshall, Wallace F; Kikkawa, Masahide; Qin, Hongmin

    2014-05-01

    The axoneme-the conserved core of eukaryotic cilia and flagella-contains highly specialized doublet microtubules (DMTs). A long-standing question is what protein(s) compose the junctions between two tubules in DMT. Here we identify a highly conserved flagellar-associated protein (FAP), FAP20, as an inner junction (IJ) component. The flagella of Chlamydomonas FAP20 mutants have normal length but beat with an abnormal symmetrical three-dimensional pattern. In addition, the mutant axonemes are liable to disintegrate during beating, implying that interdoublet connections may be weakened. Conventional electron microscopy shows that the mutant axonemes lack the IJ, and cryo-electron tomography combined with a structural labeling method reveals that the labeled FAP20 localizes at the IJ. The mutant axonemes also lack doublet-specific beak structures, which are localized in the proximal portion of the axoneme and may be involved in planar asymmetric flagellar bending. FAP20 itself, however, may not be a beak component, because uniform localization of FAP20 along the entire length of all nine DMTs is inconsistent with the beak's localization. FAP20 is the first confirmed component of the IJ. Our data also suggest that the IJ is important for both stabilizing the axoneme and scaffolding intra-B-tubular substructures required for a planar asymmetrical waveform.

  10. Histidine Prevents Cu-Induced Oxidative Stress and the Associated Decreases in mRNA from Encoding Tight Junction Proteins in the Intestine of Grass Carp (Ctenopharyngodon idella)

    PubMed Central

    Feng, Lin; Jiang, Jun; Kuang, Sheng-Yao; Wu, Pei; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu; Liu, Yang

    2016-01-01

    Copper (Cu) is a common heavy metal pollutant in aquatic environments that originates from natural as well as anthropogenic sources. The present study investigated whether Cu causes oxidative damage and induces changes in the expression of genes that encode tight junction (TJ) proteins, cytokines and antioxidant-related genes in the intestine of the grass carp (Ctenopharyngodon idella). We demonstrated that Cu decreases the survival rate of fish and increases oxidative damage as measured by increases in malondialdehyde and protein carbonyl contents. Cu exposure significantly decreased the expression of genes that encode the tight junction proteins, namely, claudin (CLDN)-c, -3 and -15 as well as occludin and zonula occludens-1, in the intestine of fish. In addition, Cu exposure increases the mRNA levels of the pro-inflammatory cytokines, specifically, IL-8, TNF-α and its related signalling factor (nuclear factor kappa B, NF-κB), which was partly correlated to the decreased mRNA levels of NF-κB inhibitor protein (IκB). These changes were associated with Cu-induced oxidative stress detected by corresponding decreases in glutathione (GSH) content, as well as decreases in the copper, zinc-superoxide dismutase (SOD1) and glutathione peroxidase (GPx) activities and mRNA levels, which were associated with the down-regulated antioxidant signalling factor NF-E2-related factor-2 (Nrf2) mRNA levels, and the Kelch-like-ECH-associated protein1 (Keap1) mRNA levels in the intestine of fish. Histidine supplementation in diets (3.7 up to 12.2 g/kg) blocked Cu-induced changes. These results indicated that Cu-induced decreases in intestinal TJ proteins and cytokine mRNA levels might be partially mediated by oxidative stress and are prevented by histidine supplementation in fish diet. PMID:27280406

  11. Regulation of tight junction proteins occludin and claudin 5 in the primate ovary during the ovulatory cycle and after inhibition of vascular endothelial growth factor.

    PubMed

    Rodewald, M; Herr, D; Fraser, H M; Hack, G; Kreienberg, R; Wulff, C

    2007-11-01

    Ovarian follicular and corpus luteum development, including angiogenesis, are characterized by cell-cell rearrangements that may require dynamic changes in cell-cell adhesion. The present study investigates the expression of tight junction proteins occludin and claudin 5 during follicular and luteal development in the primate ovary and after inhibition of vascular endothelial growth factor (VEGF) by VEGF trap treatment. Occludin was localized to the plasma membrane of granulosa cells. During follicular development occludin staining decreased significantly (P < 0.05) and disappeared completely by the ovulatory stage. After inhibition of VEGF, occludin staining was significantly (P < 0.05) higher in the granulosa of secondary and tertiary follicles compared with controls. Claudin 5 was exclusively localized to the theca vasculature. A significant (P < 0.05) increase in staining was detected from the pre-antral to the antral and ovulatory stage. However, dual staining with CD31 revealed that within the theca endothelium the amount of claudin 5 remained constant during follicular development. Treatment with VEGF trap throughout the follicular phase revealed a lack of claudin 5 staining in the theca interna but no difference was observed in the remaining theca externa vasculature. In the corpus luteum, claudin 5 was also localized in the vasculature. Treatment with VEGF trap in the mid-luteal phase resulted in a significant increase in staining (P < 0.05). These results led us to hypothesize that tight junctions are involved in regulation of follicular growth, antrum transition and follicular angiogenesis which is compromised by VEGF inhibition. VEGF may influence luteal vascular permeability by regulation of the endothelial specific tight junction protein claudin 5.

  12. Testicular cell junction: a novel target for male contraception.

    PubMed

    Lee, Nikki P Y; Wong, Elissa W P; Mruk, Dolores D; Cheng, C Yan

    2009-01-01

    Even though various contraceptive methods are widely available, the number of unwanted pregnancies is still on the rise in developing countries, pressurizing the already resource limited nations. One of the major underlying reasons is the lack of effective, low cost, and safe contraceptives for couples. During the past decade, some studies were performed using animal models to decipher if the Sertoli-germ cell junction in the testis is a target for male fertility regulation. Some of these study models were based on the use of hormones and/or chemicals to disrupt the hypothalamic-pituitary-testicular axis (e.g., androgen-based implants or pills) and others utilized a panel of chemical entities or synthetic peptides to perturb spermatogenesis either reversibly or non-reversibly. Among them, adjudin, a potential male contraceptive, is one of the compounds exerting its action on the unique adherens junctions, known as ectoplasmic specializations, in the testis. Since the testis is equipped with inter-connected cell junctions, an initial targeting of one junction type may affect the others and these accumulative effects could lead to spermatogenic arrest. This review attempts to cover an innovative theme on how male infertility can be achieved by inducing junction instability and defects in the testis, opening a new window of research for male contraceptive development. While it will still take much time and effort of intensive investigation before a product can reach the consumable market, these findings have provided hope for better family planning involving men.

  13. Models for the binary complex of bacteriophage T4 gp59 helicase loading protein: gp32 single-stranded DNA-BINDING protein and ternary complex with pseudo-Y junction DNA.

    PubMed

    Hinerman, Jennifer M; Dignam, J David; Mueser, Timothy C

    2012-05-25

    Bacteriophage T4 gp59 helicase assembly protein (gp59) is required for loading of gp41 replicative helicase onto DNA protected by gp32 single-stranded DNA-binding protein. The gp59 protein recognizes branched DNA structures found at replication and recombination sites. Binding of gp32 protein (full-length and deletion constructs) to gp59 protein measured by isothermal titration calorimetry demonstrates that the gp32 protein C-terminal A-domain is essential for protein-protein interaction in the absence of DNA. Sedimentation velocity experiments with gp59 protein and gp32ΔB protein (an N-terminal B-domain deletion) show that these proteins are monomers but form a 1:1 complex with a dissociation constant comparable with that determined by isothermal titration calorimetry. Small angle x-ray scattering (SAXS) studies indicate that the gp59 protein is a prolate monomer, consistent with the crystal structure and hydrodynamic properties determined from sedimentation velocity experiments. SAXS experiments also demonstrate that gp32ΔB protein is a prolate monomer with an elongated A-domain protruding from the core. Fitting structures of gp59 protein and the gp32 core into the SAXS-derived molecular envelope supports a model for the gp59 protein-gp32ΔB protein complex. Our earlier work demonstrated that gp59 protein attracts full-length gp32 protein to pseudo-Y junctions. A model of the gp59 protein-DNA complex, modified to accommodate new SAXS data for the binary complex together with mutational analysis of gp59 protein, is presented in the accompanying article (Dolezal, D., Jones, C. E., Lai, X., Brister, J. R., Mueser, T. C., Nossal, N. G., and Hinton, D. M. (2012) J. Biol. Chem. 287, 18596-18607).

  14. Models for the Binary Complex of Bacteriophage T4 Gp59 Helicase Loading Protein. GP32 Single-Stranded DNA-Binding Protein and Ternary Complex with Pseudo-Y Junction DNA

    SciTech Connect

    Hinerman, Jennifer M.; Dignam, J. David; Mueser, Timothy C.

    2012-04-05

    The bacteriophage T4 gp59 helicase assembly protein (gp59) is required for loading of gp41 replicative helicase onto DNA protected by gp32 single-stranded DNA-binding protein. The gp59 protein recognizes branched DNA structures found at replication and recombination sites. Binding of gp32 protein (full-length and deletion constructs) to gp59 protein measured by isothermal titration calorimetry demonstrates that the gp32 protein C-terminal A-domain is essential for protein-protein interaction in the absence of DNA. Sedimentation velocity experiments with gp59 protein and gp32ΔB protein (an N-terminal B-domain deletion) show that these proteins are monomers but form a 1:1 complex with a dissociation constant comparable with that determined by isothermal titration calorimetry. Small angle x-ray scattering (SAXS) studies indicate that the gp59 protein is a prolate monomer, consistent with the crystal structure and hydrodynamic properties determined from sedimentation velocity experiments. SAXS experiments also demonstrate that gp32ΔB protein is a prolate monomer with an elongated A-domain protruding from the core. Moreover, fitting structures of gp59 protein and the gp32 core into the SAXS-derived molecular envelope supports a model for the gp59 protein-gp32ΔB protein complex. Our earlier work demonstrated that gp59 protein attracts full-length gp32 protein to pseudo-Y junctions. A model of the gp59 protein-DNA complex, modified to accommodate new SAXS data for the binary complex together with mutational analysis of gp59 protein, is presented in the accompanying article (Dolezal, D., Jones, C. E., Lai, X., Brister, J. R., Mueser, T. C., Nossal, N. G., and Hinton, D. M. (2012) J. Biol. Chem. 287, 18596–18607).

  15. aPKC Inhibition by Par3 CR3 Flanking Regions Controls Substrate Access and Underpins Apical-Junctional Polarization.

    PubMed

    Soriano, Erika V; Ivanova, Marina E; Fletcher, Georgina; Riou, Philippe; Knowles, Philip P; Barnouin, Karin; Purkiss, Andrew; Kostelecky, Brenda; Saiu, Peter; Linch, Mark; Elbediwy, Ahmed; Kjær, Svend; O'Reilly, Nicola; Snijders, Ambrosius P; Parker, Peter J; Thompson, Barry J; McDonald, Neil Q

    2016-08-22

    Atypical protein kinase C (aPKC) is a key apical-basal polarity determinant and Par complex component. It is recruited by Par3/Baz (Bazooka in Drosophila) into epithelial apical domains through high-affinity interaction. Paradoxically, aPKC also phosphorylates Par3/Baz, provoking its relocalization to adherens junctions (AJs). We show that Par3 conserved region 3 (CR3) forms a tight inhibitory complex with a primed aPKC kinase domain, blocking substrate access. A CR3 motif flanking its PKC consensus site disrupts the aPKC kinase N lobe, separating P-loop/αB/αC contacts. A second CR3 motif provides a high-affinity anchor. Mutation of either motif switches CR3 to an efficient in vitro substrate by exposing its phospho-acceptor site. In vivo, mutation of either CR3 motif alters Par3/Baz localization from apical to AJs. Our results reveal how Par3/Baz CR3 can antagonize aPKC in stable apical Par complexes and suggests that modulation of CR3 inhibitory arms or opposing aPKC pockets would perturb the interaction, promoting Par3/Baz phosphorylation. PMID:27554858

  16. Functional expression of Ca²⁺ dependent mammalian transmembrane gap junction protein Cx43 in slime mold Dictyostelium discoideum.

    PubMed

    Kaufmann, Stefan; Weiss, Ingrid M; Eckstein, Volker; Tanaka, Motomu

    2012-03-01

    In this paper, we expressed murine gap junction protein Cx43 in Dictyostelium discoideum by introducing the specific vector pDXA. In the first step, the successful expression of Cx43 and Cx43-eGFP was verified by (a) Western blot (anti-Cx43, anti-GFP), (b) fluorescence microscopy (eGFP-Cx43 co-expression, Cx43 immunostaining), and (c) flow cytometry analysis (eGFP-Cx43 co-expression). Although the fluorescence signals from cells expressing Cx43-eGFP detected by fluorescence microscopy seem relatively low, analysis by flow cytometry demonstrated that more than 60% of cells expressed Cx43-eGFP. In order to evaluate the function of expressed Cx43 in D. discoideum, we examined the hemi-channel function of Cx43. In this series of experiments, the passive uptake of carboxyfluorescein was monitored using flow cytometric analysis. A significant number of the transfected cells showed a prominent dye uptake in the absence of Ca(2+). The dye uptake by transfected cells in the presence of Ca(2+) was even lower than the non-specific dye uptake by non-transformed Ax3 orf+ cells, confirming that Cx43 expressed in D. discoideum retains its Ca(2+)-dependent, specific gating function. The expression of gap junction proteins expressed in slime molds opens a possibility to the biological significance of intercellular communications in development and maintenance of multicellular organisms.

  17. Functional expression of Ca²⁺ dependent mammalian transmembrane gap junction protein Cx43 in slime mold Dictyostelium discoideum.

    PubMed

    Kaufmann, Stefan; Weiss, Ingrid M; Eckstein, Volker; Tanaka, Motomu

    2012-03-01

    In this paper, we expressed murine gap junction protein Cx43 in Dictyostelium discoideum by introducing the specific vector pDXA. In the first step, the successful expression of Cx43 and Cx43-eGFP was verified by (a) Western blot (anti-Cx43, anti-GFP), (b) fluorescence microscopy (eGFP-Cx43 co-expression, Cx43 immunostaining), and (c) flow cytometry analysis (eGFP-Cx43 co-expression). Although the fluorescence signals from cells expressing Cx43-eGFP detected by fluorescence microscopy seem relatively low, analysis by flow cytometry demonstrated that more than 60% of cells expressed Cx43-eGFP. In order to evaluate the function of expressed Cx43 in D. discoideum, we examined the hemi-channel function of Cx43. In this series of experiments, the passive uptake of carboxyfluorescein was monitored using flow cytometric analysis. A significant number of the transfected cells showed a prominent dye uptake in the absence of Ca(2+). The dye uptake by transfected cells in the presence of Ca(2+) was even lower than the non-specific dye uptake by non-transformed Ax3 orf+ cells, confirming that Cx43 expressed in D. discoideum retains its Ca(2+)-dependent, specific gating function. The expression of gap junction proteins expressed in slime molds opens a possibility to the biological significance of intercellular communications in development and maintenance of multicellular organisms. PMID:22330805

  18. The cytoskeletal protein α-catenin unfurls upon binding to vinculin.

    PubMed

    Rangarajan, Erumbi S; Izard, Tina

    2012-05-25

    Adherens junctions (AJs) are essential for cell-cell contacts, morphogenesis, and the development of all higher eukaryotes. AJs are formed by calcium-dependent homotypic interactions of the ectodomains of single membrane-pass cadherin family receptors. These homotypic interactions in turn promote binding of the intracellular cytoplasmic tail domains of cadherin receptors with β-catenin, a multifunctional protein that plays roles in both transcription and AJs. The cadherin receptor-β-catenin complex binds to the cytoskeletal protein α-catenin, which is essential for both the formation and the stabilization of these junctions. Precisely how α-catenin contributes to the formation and stabilization of AJs is hotly debated, although the latter is thought to involve its interactions with the cytoskeletal protein vinculin. Here we report the crystal structure of the vinculin binding domain (VBD) of α-catenin in complex with the vinculin head domain (Vh1). This structure reveals that α-catenin is in a unique unfurled mode allowing dimer formation when bound to vinculin. Finally, binding studies suggest that vinculin must be in an activated state to bind to α-catenin and that this interaction is stabilized by the formation of a ternary α-catenin-vinculin-F-actin complex, which can be formed via the F-actin binding domain of either protein. We propose a feed-forward model whereby α-catenin-vinculin interactions promote their binding to the actin cytoskeleton to stabilize AJs. PMID:22493458

  19. The expression of gingival epithelial junctions in response to subgingival biofilms.

    PubMed

    Belibasakis, Georgios N; Kast, Jeannette I; Thurnheer, Thomas; Akdis, Cezmi A; Bostanci, Nagihan

    2015-01-01

    Periodontitis is an infectious inflammatory disease that destroys the tooth-supporting tissues. It is caused by the formation of subgingival biofilms on the surface of the tooth. Characteristic bacteria associated with subgingival biofilms are the Gram-negative anaerobes Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola, collectively known as the "red complex" species. Inter-epithelial junctions ensure the barrier integrity of the gingival epithelium. This may however be disrupted by the biofilm challenge. The aim of this in vitro study was to investigate the effect of subgingival biofilms on the expression of inter-epithelial junctions by gingival epithelia, and evaluate the relative role of the red complex. Multi-layered human gingival epithelial cultures were challenged with a 10-species in vitro subgingival biofilm model, or its variant without the red complex, for 3 h and 24 h. A low-density array microfluidic card platform was then used for analyzing the expression of 62 genes encoding for tight junctions, gap junctions, adherens junctions, and desmosomes. Although there was a limited effect of the biofilms on the expression of tight, adherens and gap junctions, the expression of a number of desmosomal components was affected. In particular, Desmoglein-1 displayed a limited and transient up-regulation in response to the biofilm. In contrast, Desmocollin-2, Desmoplakin and Plakoglobin were down-regulated equally by both biofilm variants, after 24 h. In conclusion, this subgingival biofilm model may down-regulate selected desmosomal junctions in the gingival epithelium, irrespective of the presence of the "red complex." In turn, this could compromise the structural integrity of the gingival tissue, favoring bacterial invasion and chronic infection.

  20. The expression of gingival epithelial junctions in response to subgingival biofilms

    PubMed Central

    Belibasakis, Georgios N; Kast, Jeannette I; Thurnheer, Thomas; Akdis, Cezmi A; Bostanci, Nagihan

    2015-01-01

    Periodontitis is an infectious inflammatory disease that destroys the tooth-supporting tissues. It is caused by the formation of subgingival biofilms on the surface of the tooth. Characteristic bacteria associated with subgingival biofilms are the Gram-negative anaerobes Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola, collectively known as the “red complex” species. Inter-epithelial junctions ensure the barrier integrity of the gingival epithelium. This may however be disrupted by the biofilm challenge. The aim of this in vitro study was to investigate the effect of subgingival biofilms on the expression of inter-epithelial junctions by gingival epithelia, and evaluate the relative role of the red complex. Multi-layered human gingival epithelial cultures were challenged with a 10-species in vitro subgingival biofilm model, or its variant without the red complex, for 3 h and 24 h. A low-density array microfluidic card platform was then used for analyzing the expression of 62 genes encoding for tight junctions, gap junctions, adherens junctions, and desmosomes. Although there was a limited effect of the biofilms on the expression of tight, adherens and gap junctions, the expression of a number of desmosomal components was affected. In particular, Desmoglein-1 displayed a limited and transient up-regulation in response to the biofilm. In contrast, Desmocollin-2, Desmoplakin and Plakoglobin were down-regulated equally by both biofilm variants, after 24 h. In conclusion, this subgingival biofilm model may down-regulate selected desmosomal junctions in the gingival epithelium, irrespective of the presence of the “red complex.” In turn, this could compromise the structural integrity of the gingival tissue, favoring bacterial invasion and chronic infection. PMID:26305580

  1. Quantitative proteomic analysis of milk fat globule membrane (MFGM) proteins in human and bovine colostrum and mature milk samples through iTRAQ labeling.

    PubMed

    Yang, Mei; Cong, Min; Peng, Xiuming; Wu, Junrui; Wu, Rina; Liu, Biao; Ye, Wenhui; Yue, Xiqing

    2016-05-18

    Milk fat globule membrane (MFGM) proteins have many functions. To explore the different proteomics of human and bovine MFGM, MFGM proteins were separated from human and bovine colostrum and mature milk, and analyzed by the iTRAQ proteomic approach. A total of 411 proteins were recognized and quantified. Among these, 232 kinds of differentially expressed proteins were identified. These differentially expressed proteins were analyzed based on multivariate analysis, gene ontology (GO) annotation and KEGG pathway. Biological processes involved were response to stimulus, localization, establishment of localization, and the immune system process. Cellular components engaged were the extracellular space, extracellular region parts, cell fractions, and vesicles. Molecular functions touched upon were protein binding, nucleotide binding, and enzyme inhibitor activity. The KEGG pathway analysis showed several pathways, including regulation of the actin cytoskeleton, focal adhesion, neurotrophin signaling pathway, leukocyte transendothelial migration, tight junction, complement and coagulation cascades, vascular endothelial growth factor signaling pathway, and adherens junction. These results enhance our understanding of different proteomes of human and bovine MFGM across different lactation phases, which could provide important information and potential directions for the infant milk powder and functional food industries. PMID:27159491

  2. Stimulus complexity dependent memory impairment and changes in motor performance after deletion of the neuronal gap junction protein connexin36 in mice.

    PubMed

    Frisch, C; De Souza-Silva, M A; Söhl, G; Güldenagel, M; Willecke, K; Huston, J P; Dere, E

    2005-02-10

    Gap junction channels, composed of connexin (Cx) proteins, are conduits for intercellular communication and metabolic exchange in the central nervous system. Connexin36 (Cx36) is expressed in distinct subpopulations of neurons throughout the mammalian brain. Deletion of the Cx36 gene in the mouse affected power and frequency of gamma and sharp wave-ripple oscillations, putative correlates of memory engram inscription. Here, we present a behavioral analysis of Cx36-deficient mice. Activity patterns, exploratory- and anxiety-related responses were largely unaffected by elimination of Cx36, while sensorimotor capacities and learning and memory processes were impaired. Repeated testing on the rotarod suggested that the Cx36-deficient mice showed slower motor-coordination learning. After a retention interval of 24 h the Cx36-deficient mice showed habituation to an open-field, but failed to habituate to a more complex spatial environment (Y-maze). A more pronounced memory impairment was found when Cx36 knockout mice had to remember recently explored objects. Cx36-deficient mice were unable to recognize objects after short delays of 15 and 45 min. These data suggest that lack of Cx36 induces memory impairments that vary in dependence of the complexity of the stimuli presented. Our results suggest that neuronal gap junctions incorporating Cx36 play a role in learning and memory.

  3. Exon Junction Complexes Show a Distributional Bias toward Alternatively Spliced mRNAs and against mRNAs Coding for Ribosomal Proteins.

    PubMed

    Hauer, Christian; Sieber, Jana; Schwarzl, Thomas; Hollerer, Ina; Curk, Tomaz; Alleaume, Anne-Marie; Hentze, Matthias W; Kulozik, Andreas E

    2016-08-01

    The exon junction complex (EJC) connects spliced mRNAs to posttranscriptional processes including RNA localization, transport, and regulated degradation. Here, we provide a comprehensive analysis of bona fide EJC binding sites across the transcriptome including all four RNA binding EJC components eIF4A3, BTZ, UPF3B, and RNPS1. Integration of these data sets permits definition of high-confidence EJC deposition sites as well as assessment of whether EJC heterogeneity drives alternative nonsense-mediated mRNA decay pathways. Notably, BTZ (MLN51 or CASC3) emerges as the EJC subunit that is almost exclusively bound to sites 20-24 nucleotides upstream of exon-exon junctions, hence defining EJC positions. By contrast, eIF4A3, UPF3B, and RNPS1 display additional RNA binding sites suggesting accompanying non-EJC functions. Finally, our data show that EJCs are largely distributed across spliced RNAs in an orthodox fashion, with two notable exceptions: an EJC deposition bias in favor of alternatively spliced transcripts and against the mRNAs that encode ribosomal proteins. PMID:27475226

  4. Localization of the tight junction protein gene TJP1 to human chromosome 15q13, distal to the Prader-Willi/Angelman region, and to mouse chromosome 7

    SciTech Connect

    Mohandas, T.K.; Chen, X.N.; Korenberg, J.R.

    1995-12-10

    The gene encoding the tight junction (zonula occludens) protein, TJP1, was mapped to human chromosome 15q13 by fluorescence in situ hybridization (FISH) using a cDNA probe. The Jackson Laboratory backcross DNA panel derived from the cross (C57BL/6JEi X SPRET/Ei) F1 females X SPRET/Ei males was used to map the mouse Tjp1 to chromosome 7 near position 30 on the Chromosome Committee Map, a region with conserved homology to human chromosome 15q13. FISH studies on metaphases from patients with the Prader-Willi (PWS) or the Angelman syndrome (AS) showed that TJP1 maps close but distal to the PWS/AS chromosome region. 13 refs., 2 figs.

  5. The Chlamydia trachomatis Protease CPAF Contains a Cryptic PDZ-Like Domain with Similarity to Human Cell Polarity and Tight Junction PDZ-Containing Proteins

    PubMed Central

    Mou, Rui; Valdivia, Raphael H.; McCafferty, Dewey G.

    2016-01-01

    The need for more effective anti-chlamydial therapeutics has sparked research efforts geared toward further understanding chlamydial pathogenesis mechanisms. Recent studies have implicated the secreted chlamydial serine protease, chlamydial protease-like activity factor (CPAF) as potentially important for chlamydial pathogenesis. By mechanisms that remain to be elucidated, CPAF is directed to a discrete group of substrates, which are subsequently cleaved or degraded. While inspecting the previously solved CPAF crystal structure, we discovered that CPAF contains a cryptic N-terminal PSD95 Dlg ZO-1 (PDZ) domain spanning residues 106–212 (CPAF106-212). This PDZ domain is unique in that it bears minimal sequence similarity to canonical PDZ-forming sequences and displays little sequence and structural similarity to known chlamydial PDZ domains. We show that the CPAF106-212 sequence is homologous to PDZ domains of human tight junction proteins. PMID:26829550

  6. The levels of epithelial anchor proteins β-catenin and zona occludens-1 are altered by E7 of human papillomaviruses 5 and 8.

    PubMed

    Heuser, Sandra; Hufbauer, Martin; Marx, Benjamin; Tok, Ali; Majewski, Slawomir; Pfister, Herbert; Akgül, Baki

    2016-02-01

    Infection with viruses of the genus Betapapillomavirus, β-human papillomaviruses (β-HPV), is implicated in the development of non-melanoma skin cancer. This was first evidenced for HPV5 and HPV8 in patients with the skin disease epidermodysplasia verruciformis (EV). The relocalization of the junctional bridging proteins β-catenin and zona occludens-1 (ZO-1) from the adherens and tight junctions are common processes of the epithelial-mesenchymal transition (EMT) associated with tumour invasion. Here, we report that β-catenin and ZO-1 are strongly upregulated by the E7 oncoproteins of HPV5 and HPV8 in keratinocytes grown in organotypic skin cultures. Although the membrane-tethered form of β-catenin was elevated, no signs of β-catenin activity within the canonical Wnt signalling pathway could be detected. The upregulation of β-catenin and ZO-1 could also be confirmed in the skin of HPV8 transgenic mice as well as in cutaneous squamous cell carcinomas of EV patients. These data provide the first evidence that β-catenin and ZO-1 are direct targets of E7 of the oncogenic β-HPV types 5 and 8. The ability to deregulate these epithelial junction proteins may contribute to the oncogenic potential of these viruses in human skin. PMID:26645068

  7. The levels of epithelial anchor proteins β-catenin and zona occludens-1 are altered by E7 of human papillomaviruses 5 and 8.

    PubMed

    Heuser, Sandra; Hufbauer, Martin; Marx, Benjamin; Tok, Ali; Majewski, Slawomir; Pfister, Herbert; Akgül, Baki

    2016-02-01

    Infection with viruses of the genus Betapapillomavirus, β-human papillomaviruses (β-HPV), is implicated in the development of non-melanoma skin cancer. This was first evidenced for HPV5 and HPV8 in patients with the skin disease epidermodysplasia verruciformis (EV). The relocalization of the junctional bridging proteins β-catenin and zona occludens-1 (ZO-1) from the adherens and tight junctions are common processes of the epithelial-mesenchymal transition (EMT) associated with tumour invasion. Here, we report that β-catenin and ZO-1 are strongly upregulated by the E7 oncoproteins of HPV5 and HPV8 in keratinocytes grown in organotypic skin cultures. Although the membrane-tethered form of β-catenin was elevated, no signs of β-catenin activity within the canonical Wnt signalling pathway could be detected. The upregulation of β-catenin and ZO-1 could also be confirmed in the skin of HPV8 transgenic mice as well as in cutaneous squamous cell carcinomas of EV patients. These data provide the first evidence that β-catenin and ZO-1 are direct targets of E7 of the oncogenic β-HPV types 5 and 8. The ability to deregulate these epithelial junction proteins may contribute to the oncogenic potential of these viruses in human skin.

  8. Deficiency of angulin-2/ILDR1, a tricellular tight junction-associated membrane protein, causes deafness with cochlear hair cell degeneration in mice.

    PubMed

    Higashi, Tomohito; Katsuno, Tatsuya; Kitajiri, Shin-Ichiro; Furuse, Mikio

    2015-01-01

    Tricellular tight junctions seal the extracellular spaces of tricellular contacts, where the vertices of three epithelial cells meet, and are required for the establishment of a strong barrier function of the epithelial cellular sheet. Angulins and tricellulin are known as specific protein components of tricellular tight junctions, where angulins recruit tricellulin. Mutations in the genes encoding angulin-2/ILDR1 and tricellulin have been reported to cause human hereditary deafness DFNB42 and DFNB49, respectively. To investigate the pathogenesis of DFNB42, we analyzed mice with a targeted disruption of Ildr1, which encodes angulin-2/ILDR1. Ildr1 null mice exhibited profound deafness. Hair cells in the cochlea of Ildr1 null mice develop normally, but begin to degenerate by two weeks after birth. Tricellulin localization at tricellular contacts of the organ of Corti in the cochlea was retained in Ildr1 null mice, but its distribution along the depth of tricellular contacts was affected. Interestingly, compensatory tricellular contact localization of angulin-1/LSR was observed in the organ of Corti in Ildr1 null mice although it was hardly detected in the organ of Corti in wild-type mice. The onset of hair cell degeneration in Ildr1 null mice was earlier than that in the reported Tric mutant mice, which mimic one of the tricellulin mutations in DFNB49 deafness. These results indicate that the angulin-2/ILDR1 deficiency causes the postnatal degenerative loss of hair cells in the cochlea, leading to human deafness DFNB42. Our data also suggest that angulin family proteins have distinct functions in addition to their common roles of tricellulin recruitment and that the function of angulin-2/ILDR1 for hearing cannot be substituted by angulin-1/LSR.

  9. Epidermal growth factor stimulates the disruption of gap junctional communication and connexin43 phosphorylation independent of 12-0-tetradecanoylphorbol 13-acetate-sensitive protein kinase C: the possible involvement of mitogen-activated protein kinase.

    PubMed

    Kanemitsu, M Y; Lau, A F

    1993-08-01

    We previously reported that epidermal growth factor (EGF) induced the disruption of gap junctional communication (gjc) and serine phosphorylation of connexin43 (Cx43) in T51B rat liver epithelial cells. However, the cascade of events linking EGF receptor activation to these particular responses have not been fully characterized. Furthermore, the serine kinase(s) acting directly on Cx43 remain unidentified. In the current study, we demonstrate that downmodulation of 12-0-tetradecanoylphorbol 13-acetate (TPA)-sensitive protein kinase C (PKC) activity does not affect EGF's ability to reduce junctional permeability or phosphorylate Cx43 in T51B cells. EGF in the presence or absence of chronic TPA treatment stimulated marked increases in Cx43 phosphorylation on numerous sites as determined by two-dimensional tryptic phosphopeptide mapping. Computer-assisted sequence analysis of Cx43 identified several protein kinase phosphorylation consensus sites including two sites for mitogen-activated protein (MAP) kinase. EGF stimulated activation of MAP kinase in a time- and dose-dependent manner where the kinetics of kinase activity corroborated its possible involvement in mediating EGF's effects. Moreover, purified MAP kinase directly phosphorylated Cx43 on serine residues in vitro. Two-dimensional tryptic and chymotryptic phosphopeptide mapping demonstrated that the in vitro phosphopeptides represented a specific subset of the in vivo phosphopeptides produced in response to EGF after chronic TPA treatment. Therefore, EGF-induced disruption of gjc and phosphorylation of Cx43 may be mediated in part by MAP kinase in vivo.

  10. Ajuba LIM proteins are Snail/Slug corepressors required for neural crest development in Xenopus

    PubMed Central

    Langer, Ellen M.; Feng, Yunfeng; Zhaoyuan, Hou; Rauscher, Frank J.; Kroll, Kristen L.; Longmore, Gregory D.

    2008-01-01

    Snail family transcriptional repressors regulate epithelial mesenchymal transitions during physiological and pathological processes. A conserved SNAG repression domain present in all vertebrate Snail proteins is necessary for repressor complex assembly. Here, we identify the Ajuba family of LIM proteins as functional corepressors of the Snail family via an interaction with the SNAG domain. Ajuba LIM proteins interact with Snail in the nucleus on endogenous E-cadherin promoters and contribute to Snail-dependent repression of E-cadherin. Using Xenopus neural crest as a model of in vivo Snail- or Slug-induced EMT, we demonstrate that Ajuba LIM proteins contribute to neural crest development as Snail/Slug corepressors and are required for in vivo Snail/Slug function. Because Ajuba LIM proteins are also components of adherens junction and contribute to their assembly or stability, their functional interaction with Snail proteins in the nucleus suggests that Ajuba LIM proteins are important regulators of epithelia dynamics communicating surface events with nuclear responses. PMID:18331720

  11. Dynamic behavior of Arabidopsis eIF4A-III, putative core protein of exon junction complex: fast relocation to nucleolus and splicing speckles under hypoxia.

    PubMed

    Koroleva, O A; Calder, G; Pendle, A F; Kim, S H; Lewandowska, D; Simpson, C G; Jones, I M; Brown, J W S; Shaw, P J

    2009-05-01

    Here, we identify the Arabidopsis thaliana ortholog of the mammalian DEAD box helicase, eIF4A-III, the putative anchor protein of exon junction complex (EJC) on mRNA. Arabidopsis eIF4A-III interacts with an ortholog of the core EJC component, ALY/Ref, and colocalizes with other EJC components, such as Mago, Y14, and RNPS1, suggesting a similar function in EJC assembly to animal eIF4A-III. A green fluorescent protein (GFP)-eIF4A-III fusion protein showed localization to several subnuclear domains: to the nucleoplasm during normal growth and to the nucleolus and splicing speckles in response to hypoxia. Treatment with the respiratory inhibitor sodium azide produced an identical response to the hypoxia stress. Treatment with the proteasome inhibitor MG132 led to accumulation of GFP-eIF4A-III mainly in the nucleolus, suggesting that transition of eIF4A-III between subnuclear domains and/or accumulation in nuclear speckles is controlled by proteolysis-labile factors. As revealed by fluorescence recovery after photobleaching analysis, the nucleoplasmic fraction was highly mobile, while the speckles were the least mobile fractions, and the nucleolar fraction had an intermediate mobility. Sequestration of eIF4A-III into nuclear pools with different mobility is likely to reflect the transcriptional and mRNA processing state of the cell. PMID:19435936

  12. Curcumin protects against the intestinal ischemia-reperfusion injury: involvement of the tight junction protein ZO-1 and TNF-α related mechanism

    PubMed Central

    Tian, Shuying; Guo, Ruixue; Kong, Yu; Wei, Xinliang; Wang, Weiwei; Shi, Xiaomeng; Jiang, Hongyu

    2016-01-01

    Present study aimed to investigate the eff ect of curcumin-pretreatment on intestinal I/R injury and on intestinal mucosa barrier. Thirty Wistar rats were randomly divided into: sham, I/R, and curcumin groups (n=10). Animals in curcumin group were pretreated with curcumin by gastric gavage (200 mg/kg) for 2 days before I/R. Small intestine tissues were prepared for Haematoxylin & Eosin (H&E) staining. Serum diamine oxidase (DAO) and tumor necrosis factor (TNF)-α levels were measured. Expression of intestinal TNF-α and tight junction protein (ZO-1) proteins was detected by Western blot and/or immunohistochemistry. Serum DAO level and serum and intestinal TNF-α leves were signifi cantly increased after I/R, and the values were markedly reduced by curcumin pretreatment although still higher than that of sham group (p<0.05 or p<0.001). H&E staining showed the significant injury to intestinal mucosa following I/R, and curcumin pretreatment signifi cantly improved the histological structure of intestinal mucosa. I/R insult also induced significantly down-regulated expression of ZO-1, and the eff ect was dramatically attenuated by curcumin-pretreatment. Curcumin may protect the intestine from I/R injury through restoration of the epithelial structure, promotion of the recovery of intestinal permeability, as well as enhancement of ZO-1 protein expression, and this eff ect may be partly attributed to the TNF-α related pathway. PMID:26937210

  13. Dynamic behavior of Arabidopsis eIF4A-III, putative core protein of exon junction complex: fast relocation to nucleolus and splicing speckles under hypoxia.

    PubMed

    Koroleva, O A; Calder, G; Pendle, A F; Kim, S H; Lewandowska, D; Simpson, C G; Jones, I M; Brown, J W S; Shaw, P J

    2009-05-01

    Here, we identify the Arabidopsis thaliana ortholog of the mammalian DEAD box helicase, eIF4A-III, the putative anchor protein of exon junction complex (EJC) on mRNA. Arabidopsis eIF4A-III interacts with an ortholog of the core EJC component, ALY/Ref, and colocalizes with other EJC components, such as Mago, Y14, and RNPS1, suggesting a similar function in EJC assembly to animal eIF4A-III. A green fluorescent protein (GFP)-eIF4A-III fusion protein showed localization to several subnuclear domains: to the nucleoplasm during normal growth and to the nucleolus and splicing speckles in response to hypoxia. Treatment with the respiratory inhibitor sodium azide produced an identical response to the hypoxia stress. Treatment with the proteasome inhibitor MG132 led to accumulation of GFP-eIF4A-III mainly in the nucleolus, suggesting that transition of eIF4A-III between subnuclear domains and/or accumulation in nuclear speckles is controlled by proteolysis-labile factors. As revealed by fluorescence recovery after photobleaching analysis, the nucleoplasmic fraction was highly mobile, while the speckles were the least mobile fractions, and the nucleolar fraction had an intermediate mobility. Sequestration of eIF4A-III into nuclear pools with different mobility is likely to reflect the transcriptional and mRNA processing state of the cell.

  14. Molecular cloning of complementary DNA to Newcastle disease virus, and nucleotide sequence analysis of the junction between the genes encoding the haemagglutinin-neuraminidase and the large protein.

    PubMed

    Chambers, P; Millar, N S; Bingham, R W; Emmerson, P T

    1986-03-01

    Complementary DNA clones to 90% of the Newcastle disease virus (NDV) genome have been produced and mapped. These clones cover the entire HN, F and M genes, most if not all of the L gene and parts of the NP and P genes. The map of overlapping clones gives the gene order 3'-NP-P-M-F-HN-L-5' for NDV, identical to the gene order of Sendai virus, on the assumption that the NP gene of NDV is at the 3' end of the genome as previously suggested by inactivation of NDV transcription by u.v. light. The nucleotide sequence of 453 bases covering the junction between the HN and L genes has been determined. There is nucleotide sequence homology to the consensus polyadenylation and mRNA start sites of Sendai virus and vesicular stomatitis virus. The deduced amino acid sequence of the C terminus of the HN protein of NDV shows homology to the C-terminal amino acid sequences of the HN proteins of simian virus 5 and Sendai virus. An explanation for the presence of HN0, the precursor to HN in some strains of NDV, is suggested by the presence of a long non-coding region at the 3' terminus of the mRNA encoding the HN protein of NDV that could, by mutation, allow synthesis of a larger polypeptide.

  15. Shear-induced reorganization of renal proximal tubule cell actin cytoskeleton and apical junctional complexes.

    PubMed

    Duan, Yi; Gotoh, Nanami; Yan, Qingshang; Du, Zhaopeng; Weinstein, Alan M; Wang, Tong; Weinbaum, Sheldon

    2008-08-12

    In this study, we demonstrate that fluid shear stress (FSS)-induced actin cytoskeletal reorganization and junctional formation in renal epithelial cells are nearly completely opposite the corresponding changes in vascular endothelial cells (ECs) [Thi MM et al. (2004) Proc Natl Acad Sci USA 101:16483-16488]. Mouse proximal tubule cells (PTCs) were subjected to 5 h of FSS (1 dyn/cm(2)) to investigate the dynamic responses of the cytoskeletal distribution of filamentous actin (F-actin), ZO-1, E-cadherin, vinculin, and paxillin to FSS. Immunofluorescence analysis revealed that FSS caused basal stress fiber disruption, more densely distributed peripheral actin bands (DPABs), and the formation of both tight junctions (TJs) and adherens junctions (AJs). A dramatic reinforcement of vinculin staining was found at the cell borders as well as the cell interior. These responses were abrogated by the actin-disrupting drug, cytochalasin D. To interpret these results, we propose a "junctional buttressing" model for PTCs in which FSS enables the DPABs, TJs, and AJs to become more tightly connected. In contrast, in the "bumper-car" model for ECs, all junctional connections were severely disrupted by FSS. This "junctional buttressing" model explains why a FSS of only 1/10 of that used in the EC study can cause a similarly dramatic, cytoskeletal response in these tall, cuboidal epithelial cells; and why junctional buttressing between adjacent cells may benefit renal epithelium in maximizing flow-activated, brush border-dependent, transcellular salt and water reabsorption. PMID:18685100

  16. Intestinal immune function, antioxidant status and tight junction proteins mRNA expression in young grass carp (Ctenopharyngodon idella) fed riboflavin deficient diet.

    PubMed

    Chen, Liang; Feng, Lin; Jiang, Wei-Dan; Jiang, Jun; Wu, Pei; Zhao, Juan; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu; Liu, Yang

    2015-11-01

    This study investigated the effects of riboflavin on intestinal immunity, tight junctions and antioxidant status of young grass carp (Ctenopharyngodon idella). Fish were fed diets containing graded levels of riboflavin (0.63-10.04 mg/kg diet) for 8 weeks. The study indicated that riboflavin deficiency decreased lysozyme, acid phosphatase, copper/zinc superoxide dismutase, glutathione reductase and glutathione peroxidase activities, and contents of complement component 3 and reduced glutathione in the intestine of fish (P < 0.05). Meanwhile, riboflavin deficiency increased reactive oxygen species, malondialdehyde and protein carbonyl contents and catalase activity (P < 0.05) in the intestine of fish. Furthermore, real-time polymerase chain reaction analysis was used to investigate mRNA expression patterns and found that the mRNA levels of interleukin 10 and transforming growth factor β1, Occludin, zonula occludens 1, Claudin-b and Claudin-c, inhibitor protein κBα, target of rapamycin, ribosomal S6 protein kinase 1 and NF-E2-related factor 2, copper/zinc superoxide dismutase, glutathione peroxidase and glutathione reductase were decreased (P < 0.05) in the intestine of fish fed riboflavin-deficient diet. Conversely, the mRNA levels of tumor necrosis factor α, interleukin 1β, interleukin 8, nuclear factor kappa B p65, Ikappa B kinase β, Ikappa B kinase γ, Kelch-like-ECH-associated protein 1b, p38 mitogen-activated protein kinase, myosin light chain kinase and Claudin-12 were increased (P < 0.05) in the intestine of fish fed riboflavin-deficient diet. In conclusion, riboflavin deficiency decreased immunity and structural integrity of fish intestine. The optimum riboflavin level for intestinal acid phosphatase activity of young grass carp was estimated to be 6.65 mg/kg diet.

  17. Sonic hedgehog, TBX18, and TSHZ3 proteins involved in pyeloureteral motility development are overexpressed in ureteropelvic junction obstruction

    PubMed Central

    Yilmaz, Omer; Nese, Nalan; Dalgic, Mustafa; Kesici, Gonca P.; Genc, Abdulkadir; Taneli, Can; Gunsar, Cuneyt; Sencan, Aydın; Cayirli, Hasan; Isisag, Aydın

    2016-01-01

    Objectives: To compare pathological samples obtained from cases that underwent surgery for ureteropelvic junction (UPJ) obstruction with samples obtained during autopsies of subjects. Methods: Retrospectively, 42 patients who had undergone surgery due to UPJ obstruction (group 1) were included in the study. Histopathological and immunohistochemical features for sonic hedgehog (SHH), TBX18, and TSHZ3 of UPJ were evaluated and findings were compared with 20 autopsy cases (group 2). Results: In group 1, the scores were statistically significantly higher in terms of cytoplasmic SHH, nuclear TBX18, cytoplasmic and nuclear TSHZ3 staining. Statistically, no correlation was found between age and the staining scores belonging to these 3 antibodies in group 1 and group 2. Intense inflammation was found to be related with nuclear staining for TBX18. Conclusion: Gene product expressions of SHH, TBX18 and TSHZ3 are statistically higher in patients with UPJ obstruction, when compared with control group. The explanation may be the reactivation of the processes, which had shown their effects in the embryological period, due to the chronic inflammation and long-term micro-trauma created by the disease. PMID:27381532

  18. Multiplexed multi-scale imaging: novel roles for the scaffold protein IQGAP1 in epithelial cell development (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Schweikhard, Volker

    2016-02-01

    The precise sub-cellular spatial localization of multi-protein complexes is increasingly recognized as a key mechanism governing the organization of mammalian cells. Consequently, there is a need for novel microscopy techniques capable of investigating such sub-cellular architectures in comprehensive detail. Here, we applied a novel multiplexed STORM super-resolution microscopy technique, in combination with high-throughput immunofluorescence microscopy and live-cell imaging, to investigate the roles of the scaffold protein IQGAP1 in epithelial cells. IQGAP1 is known to orchestrate a wide range of biological processes, including intracellular signaling, cytoskeletal regulation, cell-cell adhesion, and protein trafficking, by forming distinct complexes with a number of known interaction partners, and recruiting these complexes to specific subcellular locations. Our results demonstrate that, in addition to supporting epithelial adherens junctions by associating with specialized cortical actin structures, IQGAP1 plays a second role in which it controls the confinement of a unique, previously undocumented class of membranous compartments to the basal actin cortex. These largely immotile yet highly dynamic structures appear transiently as cells merge into clusters and establish of apical-basolateral (epithelial) polarity, and are identified as an intermediate compartment in the endocytic recycling pathways for cell junction complexes and cell surface receptors. Although these two functions of IQGAP1 occur in parallel and largely independently of each other, they both support the maturation and maintenance of polarized epithelial cell architectures.

  19. MgcRacGAP restricts active RhoA at the cytokinetic furrow and both RhoA and Rac1 at cell–cell junctions in epithelial cells

    PubMed Central

    Breznau, Elaina B.; Semack, Ansley C.; Higashi, Tomohito; Miller, Ann L.

    2015-01-01

    Localized activation of Rho GTPases is essential for multiple cellular functions, including cytokinesis and formation and maintenance of cell–cell junctions. Although MgcRacGAP (Mgc) is required for spatially confined RhoA-GTP at the equatorial cortex of dividing cells, both the target specificity of Mgc's GAP activity and the involvement of phosphorylation of Mgc at Ser-386 are controversial. In addition, Mgc's function at cell–cell junctions remains unclear. Here, using gastrula-stage Xenopus laevis embryos as a model system, we examine Mgc's role in regulating localized RhoA-GTP and Rac1-GTP in the intact vertebrate epithelium. We show that Mgc's GAP activity spatially restricts accumulation of both RhoA-GTP and Rac1-GTP in epithelial cells—RhoA at the cleavage furrow and RhoA and Rac1 at cell–cell junctions. Phosphorylation at Ser-386 does not switch the specificity of Mgc's GAP activity and is not required for successful cytokinesis. Furthermore, Mgc regulates adherens junction but not tight junction structure, and the ability to regulate adherens junctions is dependent on GAP activity and signaling via the RhoA pathway. Together these results indicate that Mgc's GAP activity down-regulates the active populations of RhoA and Rac1 at localized regions of epithelial cells and is necessary for successful cytokinesis and cell–cell junction structure. PMID:25947135

  20. Mutation Analysis of Gap Junction Protein Beta 1 and Genotype–Phenotype Correlation in X-linked Charcot–Marie–Tooth Disease in Chinese Patients

    PubMed Central

    Sun, Bo; Chen, Zhao-Hui; Ling, Li; Li, Yi-Fan; Liu, Li-Zhi; Yang, Fei; Huang, Xu-Sheng

    2016-01-01

    Background: Among patients with Charcot–Marie–Tooth disease (CMT), the X-linked variant (CMTX) caused by gap junction protein beta 1 (GJB1) gene mutation is the second most frequent type, accounting for approximately 90% of all CMTX. More than 400 mutations have been identified in the GJB1 gene that encodes connexin 32 (CX32). CX32 is thought to form gap junctions that promote the diffusion pathway between cells. GJB1 mutations interfere with the formation of the functional channel and impair the maintenance of peripheral myelin, and novel mutations are continually discovered. Methods: We included 79 unrelated patients clinically diagnosed with CMT at the Department of Neurology of the Chinese People's Liberation Army General Hospital from December 20, 2012, to December 31, 2015. Clinical examination, nerve conduction studies, and molecular and bioinformatics analyses were performed to identify patients with CMTX1. Results: Nine GJB1 mutations (c.283G>A, c.77C>T, c.643C>T, c.515C>T, c.191G>A, c.610C>T, c.490C>T, c.491G>A, and c.44G>A) were discovered in nine patients. Median motor nerve conduction velocities of all nine patients were < 38 m/s, resembling CMT Type 1. Three novel mutations, c.643C>T, c.191G>A, and c.610C>T, were revealed and bioinformatics analyses indicated high pathogenicity. Conclusions: The three novel missense mutations within the GJB1 gene broaden the mutational diversity of CMT1X. Molecular analysis of family members and bioinformatics analyses of the afflicted patients confirmed the pathogenicity of these mutations. PMID:27098783

  1. Age-related deficits in skeletal muscle recovery following disuse are associated with neuromuscular junction instability and ER stress, not impaired protein synthesis

    PubMed Central

    Baehr, Leslie M.; West, Daniel W.D.; Marcotte, George; Marshall, Andrea G.; De Sousa, Luis Gustavo; Baar, Keith; Bodine, Sue C.

    2016-01-01

    Age-related loss of muscle mass and strength can be accelerated by impaired recovery of muscle mass following a transient atrophic stimulus. The aim of this study was to identify the mechanisms underlying the attenuated recovery of muscle mass and strength in old rats following disuse-induced atrophy. Adult (9 month) and old (29 month) male F344BN rats underwent hindlimb unloading (HU) followed by reloading. HU induced significant atrophy of the hindlimb muscles in both adult (17-38%) and old (8-29%) rats, but only the adult rats exhibited full recovery of muscle mass and strength upon reloading. Upon reloading, total RNA and protein synthesis increased to a similar extent in adult and old muscles. At baseline and upon reloading, however, proteasome-mediated degradation was suppressed leading to an accumulation of ubiquitin-tagged proteins and p62. Further, ER stress, as measured by CHOP expression, was elevated at baseline and upon reloading in old rats. Analysis of mRNA expression revealed increases in HDAC4, Runx1, myogenin, Gadd45a, and the AChRs in old rats, suggesting neuromuscular junction instability/denervation. Collectively, our data suggests that with aging, impaired neuromuscular transmission and deficits in the proteostasis network contribute to defects in muscle fiber remodeling and functional recovery of muscle mass and strength. PMID:26826670

  2. Exogenous GDF9 but not Activin A, BMP15 or TGFβ alters tight junction protein transcript abundance in zebrafish ovarian follicles.

    PubMed

    Clelland, Eric S; Kelly, Scott P

    2011-04-01

    The tight junction (TJ) complex plays an important role in regulating paracellular permeability and provides mechanical stability in vertebrate epithelia and endothelia. In zebrafish ovarian follicles, TJ complexes in the follicular envelope degenerate as the follicles develop towards maturation. In the current study, transcript abundance of claudins (cldn d, g, h, 1, and 12) and occludins (ocln, and ocln b) were assessed in mid-vitellogenic follicles in response to treatment with exogenous growth factors that are reported to be involved in zebrafish follicle development (i.e. Activin A, BMP15, GDF9 and TGFβ). Exogenous GDF9 reduced the transcript abundance of cldn g, ocln and ocln b in mid-vitellogenic follicles, whereas Activin A, BMP15, and TGFβ had no effect. Subsequent studies with GDF9 revealed that this factor did not alter TJ protein transcript abundance in pre-vitellogenic follicles but did increase the abundance of ocln b in fully grown (maturing) follicles. GDF9 was also seen to increase the abundance of StAR mRNA in all but primary stage follicles. These data suggest a role for GDF9 in the regulation of TJ integrity in zebrafish ovarian follicles, perhaps in the facilitation of ovulation, and support a previously postulated role for GDF9 in zebrafish ovarian follicle development. In addition, data also support the idea that endocrine factors play an important role in the regulation of TJ proteins during ovarian follicle development.

  3. Age-related deficits in skeletal muscle recovery following disuse are associated with neuromuscular junction instability and ER stress, not impaired protein synthesis.

    PubMed

    Baehr, Leslie M; West, Daniel W D; Marcotte, George; Marshall, Andrea G; De Sousa, Luis Gustavo; Baar, Keith; Bodine, Sue C

    2016-01-01

    Age-related loss of muscle mass and strength can be accelerated by impaired recovery of muscle mass following a transient atrophic stimulus. The aim of this study was to identify the mechanisms underlying the attenuated recovery of muscle mass and strength in old rats following disuse-induced atrophy. Adult (9 month) and old (29 month) male F344BN rats underwent hindlimb unloading (HU) followed by reloading. HU induced significant atrophy of the hindlimb muscles in both adult (17-38%) and old (8-29%) rats, but only the adult rats exhibited full recovery of muscle mass and strength upon reloading. Upon reloading, total RNA and protein synthesis increased to a similar extent in adult and old muscles. At baseline and upon reloading, however, proteasome-mediated degradation was suppressed leading to an accumulation of ubiquitin-tagged proteins and p62. Further, ER stress, as measured by CHOP expression, was elevated at baseline and upon reloading in old rats. Analysis of mRNA expression revealed increases in HDAC4, Runx1, myogenin, Gadd45a, and the AChRs in old rats, suggesting neuromuscular junction instability/denervation. Collectively, our data suggests that with aging, impaired neuromuscular transmission and deficits in the proteostasis network contribute to defects in muscle fiber remodeling and functional recovery of muscle mass and strength.

  4. The roles of tricellular tight junction protein lipolysis-stimulated lipoprotein receptor in malignancy of human endometrial cancer cells

    PubMed Central

    Shimada, Hiroshi; Satohisa, Seiro; Kohno, Takayuki; Takahashi, Syunta; Hatakeyama, Tsubasa; Konno, Takumi; Tsujiwaki, Mitsuhiro; Saito, Tsuyoshi; Kojima, Takashi

    2016-01-01

    Lipolysis-stimulated lipoprotein receptor (LSR) has been identified as a novel molecular constituent of tricellular contacts that have a barrier function for the cellular sheet. LSR recruits tricellulin (TRIC), which is the first molecular component of tricellular tight junctions. Knockdown of LSR increases cell motility and invasion of certain cancer cells. However, the behavior and the roles of LSR in endometrial cancer remain unknown. In the present study, we investigated the behavior and roles of LSR in normal and endometrial cancer cells in vivo and in vitro. In endometriosis and endometrial cancer, LSR was observed not only in the subapical region but also throughout the lateral region as well as in normal endometrial epithelial cells in the secretory phase, and LSR in the cancer was reduced in correlation with the malignancy. Knockdown of LSR by the siRNA in cells of the endometrial cancer cell line Sawano, induced cell migration, invasion and proliferation, while TRIC relocalized from the tricellular region to the bicellular region at the membrane. In Sawano cells and normal HEEs, a decrease of LSR induced by leptin and an increase of LSR induced by adiponectin and the drugs for type 2 diabetes metformin and berberine were observed via distinct signaling pathways including JAK2/STAT. In Sawano cells, metformin and berberine prevented cell migration and invasion induced by downregulation of LSR by the siRNA and leptin treatment. The dissection of the mechanism in the downregulation of endometrial LSR during obesity is important in developing new diagnostic and therapy for endometrial cancer. PMID:27036040

  5. Mutations in contactin-1, a neural adhesion and neuromuscular junction protein, cause a familial form of lethal congenital myopathy.

    PubMed

    Compton, Alison G; Albrecht, Douglas E; Seto, Jane T; Cooper, Sandra T; Ilkovski, Biljana; Jones, Kristi J; Challis, Daniel; Mowat, David; Ranscht, Barbara; Bahlo, Melanie; Froehner, Stanley C; North, Kathryn N

    2008-12-01

    We have previously reported a group of patients with congenital onset weakness associated with a deficiency of members of the syntrophin-alpha-dystrobrevin subcomplex and have demonstrated that loss of syntrophin and dystrobrevin from the sarcolemma of skeletal muscle can also be associated with denervation. Here, we have further studied four individuals from a consanguineous Egyptian family with a lethal congenital myopathy inherited in an autosomal-recessive fashion and characterized by a secondary loss of beta2-syntrophin and alpha-dystrobrevin from the muscle sarcolemma, central nervous system involvement, and fetal akinesia. We performed homozygosity mapping and candidate gene analysis and identified a mutation that segregates with disease within CNTN1, the gene encoding for the neural immunoglobulin family adhesion molecule, contactin-1. Contactin-1 transcripts were markedly decreased on gene-expression arrays of muscle from affected family members compared to controls. We demonstrate that contactin-1 is expressed at the neuromuscular junction (NMJ) in mice and man in addition to the previously documented expression in the central and peripheral nervous system. In patients with secondary dystroglycanopathies, we show that contactin-1 is abnormally localized to the sarcolemma instead of exclusively at the NMJ. The cntn1 null mouse presents with ataxia, progressive muscle weakness, and postnatal lethality, similar to the affected members in this family. We propose that loss of contactin-1 from the NMJ impairs communication or adhesion between nerve and muscle resulting in the severe myopathic phenotype. This disorder is part of the continuum in the clinical spectrum of congenital myopathies and congenital myasthenic syndromes.

  6. Export of a Toxoplasma gondii Rhoptry Neck Protein Complex at the Host Cell Membrane to Form the Moving Junction during Invasion

    PubMed Central

    Poncet, Joël; Dubremetz, Jean-François; Lebrun, Maryse

    2009-01-01

    One of the most conserved features of the invasion process in Apicomplexa parasites is the formation of a moving junction (MJ) between the apex of the parasite and the host cell membrane that moves along the parasite and serves as support to propel it inside the host cell. The MJ was, up to a recent period, completely unknown at the molecular level. Recently, proteins originated from two distinct post-Golgi specialised secretory organelles, the micronemes (for AMA1) and the neck of the rhoptries (for RON2/RON4/RON5 proteins), have been shown to form a complex. AMA1 and RON4 in particular, have been localised to the MJ during invasion. Using biochemical approaches, we have identified RON8 as an additional member of the complex. We also demonstrated that all RON proteins are present at the MJ during invasion. Using metabolic labelling and immunoprecipitation, we showed that RON2 and AMA1 were able to interact in the absence of the other members. We also discovered that all MJ proteins are subjected to proteolytic maturation during trafficking to their respective organelles and that they could associate as non-mature forms in vitro. Finally, whereas AMA1 has previously been shown to be inserted into the parasite membrane upon secretion, we demonstrated, using differential permeabilization and loading of RON-specific antibodies into the host cell, that the RON complex is targeted to the host cell membrane, where RON4/5/8 remain associated with the cytoplasmic face. Globally, these results point toward a model of MJ organization where the parasite would be secreting and inserting interacting components on either side of the MJ, both at the host and at its own plasma membranes. PMID:19247437

  7. Exposure to vehicle emissions results in altered blood brain barrier permeability and expression of matrix metalloproteinases and tight junction proteins in mice

    PubMed Central

    2013-01-01

    Background Traffic-generated air pollution-exposure is associated with adverse effects in the central nervous system (CNS) in both human exposures and animal models, including neuroinflammation and neurodegeneration. While alterations in the blood brain barrier (BBB) have been implicated as a potential mechanism of air pollution-induced CNS pathologies, pathways involved have not been elucidated. Objectives To determine whether inhalation exposure to mixed vehicle exhaust (MVE) mediates alterations in BBB permeability, activation of matrix metalloproteinases (MMP) -2 and −9, and altered tight junction (TJ) protein expression. Methods Apolipoprotein (Apo) E−/− and C57Bl6 mice were exposed to either MVE (100 μg/m3 PM) or filtered air (FA) for 6 hr/day for 30 days and resulting BBB permeability, expression of ROS, TJ proteins, markers of neuroinflammation, and MMP activity were assessed. Serum from study mice was applied to an in vitro BBB co-culture model and resulting alterations in transport and permeability were quantified. Results MVE-exposed Apo E−/− mice showed increased BBB permeability, elevated ROS and increased MMP-2 and −9 activity, compared to FA controls. Additionally, cerebral vessels from MVE-exposed mice expressed decreased levels of TJ proteins, occludin and claudin-5, and increased levels of inducible nitric oxide synthase (iNOS) and interleukin (IL)-1β in the parenchyma. Serum from MVE-exposed animals also resulted in increased in vitro BBB permeability and altered P-glycoprotein transport activity. Conclusions These data indicate that inhalation exposure to traffic-generated air pollutants promotes increased MMP activity and degradation of TJ proteins in the cerebral vasculature, resulting in altered BBB permeability and expression of neuroinflammatory markers. PMID:24344990

  8. Impact of 7,12-dimethylbenz[a]anthracene exposure on connexin gap junction proteins in cultured rat ovaries

    SciTech Connect

    Ganesan, Shanthi Keating, Aileen F.

    2014-01-15

    7,12-Dimethylbenz[a]anthracene (DMBA) destroys ovarian follicles in a concentration-dependent manner. The impact of DMBA on connexin (CX) proteins that mediate communication between follicular cell types along with pro-apoptotic factors p53 and Bax were investigated. Postnatal day (PND) 4 Fisher 344 rat ovaries were cultured for 4 days in vehicle medium (1% DMSO) followed by a single exposure to vehicle control (1% DMSO) or DMBA (12.5 nM or 75 nM) and cultured for 4 or 8 days. RT-PCR was performed to quantify Cx37, Cx43, p53 and Bax mRNA level. Western blotting and immunofluorescence staining were performed to determine CX37 or CX43 level and/or localization. Cx37 mRNA and protein increased (P < 0.05) at 4 days of 12.5 nM DMBA exposure. Relative to vehicle control-treated ovaries, mRNA encoding Cx43 decreased (P < 0.05) but CX43 protein increased (P < 0.05) at 4 days by both DMBA exposures. mRNA expression of pro-apoptotic p53 was decreased (P < 0.05) but no changes in Bax expression were observed after 4 days of DMBA exposures. In contrast, after 8 days, DMBA decreased Cx37 and Cx43 mRNA and protein but increased both p53 and Bax mRNA levels. CX43 protein was located between granulosa cells, while CX37 was located at the oocyte cell surface of all follicle stages. These findings support that DMBA exposure impacts ovarian Cx37 and Cx43 mRNA and protein prior to both observed changes in pro-apoptotic p53 and Bax and follicle loss. It is possible that such interference in follicular cell communication is detrimental to follicle viability, and may play a role in DMBA-induced follicular atresia. - Highlights: • DMBA increases Cx37 and Cx43 expression prior to follicle loss. • During follicle loss both Cx37 and Cx43 expressions are reduced. • CX43 protein is absent in follicle remnants lacking an oocyte.

  9. Hypertonicity-induced transmitter release at Drosophila neuromuscular junctions is partly mediated by integrins and cAMP/protein kinase A

    NASA Technical Reports Server (NTRS)

    Suzuki, Kazuhiro; Grinnell, Alan D.; Kidokoro, Yoshiaki

    2002-01-01

    The frequency of quantal transmitter release increases upon application of hypertonic solutions. This effect bypasses the Ca(2+) triggering step, but requires the presence of key molecules involved in vesicle fusion, and hence could be a useful tool for dissecting the molecular process of vesicle fusion. We have examined the hypertonicity response at neuromuscular junctions of Drosophila embryos in Ca(2+)-free saline. Relative to wild-type, the response induced by puff application of hypertonic solution was enhanced in a mutant, dunce, in which the cAMP level is elevated, or in wild-type embryos treated with forskolin, an activator of adenylyl cyclase, while protein kinase A (PKA) inhibitors decreased it. The response was also smaller in a mutant, DC0, which lacks the major subunit of PKA. Thus the cAMP/PKA cascade is involved in the hypertonicity response. Peptides containing the sequence Arg-Gly-Asp (RGD), which inhibit binding of integrins to natural ligands, reduced the response, whereas a peptide containing the non-binding sequence Arg-Gly-Glu (RGE) did not. A reduced response persisted in a mutant, myospheroid, which expresses no integrins, and the response in DC0 was unaffected by RGD peptides. These data indicate that there are at lease two components in the hypertonicity response: one that is integrin mediated and involves the cAMP/PKA cascade, and another that is not integrin mediated and does not involve the cAMP/PKA cascade.

  10. Amphiregulin co-operates with bone morphogenetic protein 15 to increase bovine oocyte developmental competence: effects on gap junction-mediated metabolite supply.

    PubMed

    Sugimura, Satoshi; Ritter, Lesley J; Sutton-McDowall, Melanie L; Mottershead, David G; Thompson, Jeremy G; Gilchrist, Robert B

    2014-06-01

    This study assessed the participation of amphiregulin (AREG) and bone morphogenetic protein 15 (BMP15) during maturation of bovine cumulus-oocyte complexes (COCs) on cumulus cell function and their impact on subsequent embryo development. AREG treatment of COCs enhanced blastocyst formation and quality only when in the presence of BMP15. Expression of hyaluronan synthase 2 was enhanced by follicle-stimulating hormone (FSH) but not by AREG, which was reflected in the level of cumulus expansion. Although both FSH and AREG stimulated glycolysis, AREG-treated COCs had higher glucose consumption, lactate production and ratio of lactate production to glucose uptake. Autofluorescence levels in oocytes, indicative of NAD(P)H and FAD(++), were increased with combined AREG and BMP15 treatment of COCs. In contrast, these treatments did not alter autofluorescence levels when cumulus cells were removed from oocytes, even in the presence of other COCs, suggesting that oocyte-cumulus gap-junctional communication (GJC) is required. FSH contributed to maintaining GJC for an extended period of time. Remarkably, BMP15 was equally effective at maintaining GJC even in the presence of AREG. Hence, AREG stimulation of COC glycolysis and BMP15 preservation of GJC may facilitate efficient transfer of metabolites from cumulus cells to the oocyte thereby enhancing oocyte developmental competence. These results have implications for improving in vitro oocyte maturation systems.

  11. Dexamethasone potentiates in vitro blood-brain barrier recovery after primary blast injury by glucocorticoid receptor-mediated upregulation of ZO-1 tight junction protein.

    PubMed

    Hue, Christopher D; Cho, Frances S; Cao, Siqi; Dale Bass, Cameron R; Meaney, David F; Morrison, Barclay

    2015-07-01

    Owing to the frequent incidence of blast-induced traumatic brain injury (bTBI) in recent military conflicts, there is an urgent need to develop effective therapies for bTBI-related pathologies. Blood-brain barrier (BBB) breakdown has been reported to occur after primary blast exposure, making restoration of BBB function and integrity a promising therapeutic target. We tested the hypothesis that treatment with dexamethasone (DEX) after primary blast injury potentiates recovery of an in vitro BBB model consisting of mouse brain endothelial cells (bEnd.3). DEX treatment resulted in complete recovery of transendothelial electrical resistance and hydraulic conductivity 1 day after injury, compared with 3 days for vehicle-treated injured cultures. Administration of RU486 (mifepristone) inhibited effects of DEX, confirming that barrier restoration was mediated by glucocorticoid receptor signaling. Potentiated recovery with DEX treatment was accompanied by stronger zonula occludens (ZO)-1 tight junction immunostaining and expression, suggesting that increased ZO-1 expression was a structural correlate to BBB recovery after blast. Interestingly, augmented ZO-1 protein expression was associated with specific upregulation of the α(+) isoform but not the α(-) isoform. This is the first study to provide a mechanistic basis for potentiated functional recovery of an in vitro BBB model because of glucocorticoid treatment after primary blast injury.

  12. MicroRNA-19b Downregulates Gap Junction Protein Alpha1 and Synergizes with MicroRNA-1 in Viral Myocarditis

    PubMed Central

    Lin, Junyi; Xue, Aimin; Li, Liliang; Li, Beixu; Li, Yuhua; Shen, Yiwen; Sun, Ning; Chen, Ruizhen; Xu, Hongfei; Zhao, Ziqin

    2016-01-01

    Viral myocarditis (VMC) is a life-threatening disease that leads to heart failure or cardiac arrhythmia. A large number of researches have revealed that mircroRNAs (miRNAs) participate in the pathological processes of VMC. We previously reported that miR-1 repressed the expression of gap junction protein α1 (GJA1) in VMC. In this study, miR-19b was found to be significantly upregulated using the microarray analysis in a mouse model of VMC, and overexpression of miR-19b led to irregular beating pattern in human cardiomyocytes derived from the induced pluripotent stem cells (hiPSCs-CMs). The upregulation of miR-19b was associated with decreased GJA1 in vivo. Furthermore, a miR-19b inhibitor increased, while its mimics suppressed the expression of GJA1 in HL-1 cells. When GJA1 was overexpressed, the miR-19b mimics-mediated irregular beating was reversed in hiPSCs-CMs. In addition, the effect of miR-19b on GJA1 was enhanced by miR-1 in a dose-dependent manner. These data suggest miR-19b contributes to irregular beating through regulation of GJA1 by cooperating with miR-1. Based on the present and our previous studies, it could be indicated that miR-19b and miR-1 might be critically involved in cardiac arrhythmia associated with VMC. PMID:27213338

  13. Neuropeptide Y, substance P, and human bone morphogenetic protein 2 stimulate human osteoblast osteogenic activity by enhancing gap junction intercellular communication

    PubMed Central

    Ma, W.H.; Liu, Y.J.; Wang, W.; Zhang, Y.Z.

    2015-01-01

    Bone homeostasis seems to be controlled by delicate and subtle “cross talk” between the nervous system and “osteo-neuromediators” that control bone remodeling. The purpose of this study was to evaluate the effect of interactions between neuropeptides and human bone morphogenetic protein 2 (hBMP2) on human osteoblasts. We also investigated the effects of neuropeptides and hBMP2 on gap junction intercellular communication (GJIC). Osteoblasts were treated with neuropeptide Y (NPY), substance P (SP), or hBMP2 at three concentrations. At various intervals after treatment, cell viability was measured by the MTT assay. In addition, cellular alkaline phosphatase (ALP) activity and osteocalcin were determined by colorimetric assay and radioimmunoassay, respectively. The effects of NPY, SP and hBMP on GJIC were determined by laser scanning confocal microscopy. The viability of cells treated with neuropeptides and hBMP2 increased significantly in a time-dependent manner, but was inversely associated with the concentration of the treatments. ALP activity and osteocalcin were both reduced in osteoblasts exposed to the combination of neuropeptides and hBMP2. The GJIC of osteoblasts was significantly increased by the neuropeptides and hBMP2. These results suggest that osteoblast activity is increased by neuropeptides and hBMP2 through increased GJIC. Identification of the GJIC-mediated signal transduction capable of modulating the cellular activities of bone cells represents a novel approach to studying the biology of skeletal innervation. PMID:25714881

  14. Expression and role of gap junction protein connexin43 in immune challenge-induced extracellular ATP release in Japanese flounder (Paralichthys olivaceus).

    PubMed

    Li, Shuo; Peng, Weijiao; Chen, Xiaoli; Geng, Xuyun; Zhan, Wenbin; Sun, Jinsheng

    2016-08-01

    Connexin43 (Cx43) is the best characterized gap junction protein that allows the direct exchange of signaling molecules during cell-to-cell communications. The immunological functions and ATP permeable properties of Cx43 have been insensitively examined in mammals. The similar biological significance of Cx43 in lower vertebrates, however, is not yet understood. In the present study we identified and characterized a Cx43 ortholog (termed PoCx43) from Japanese flounder (Paralichthys olivaceus) and investigated its role in immune challenge-induced extracellular ATP release. PoCx43 mRNA transcripts are widely distributed in all tested normal tissues and cells with predominant expression in the brain, and are significantly up-regulated by LPS, poly(I:C) and zymosan challenges and Edwardsiella tarda infections as well, suggesting that PoCx43 expression was modulated by the inflammatory stresses. In addition, cyclic AMP (cAMP), an essential second messenger, also plays an important role in regulating PoCx43 gene expression, by which the PoCx43-mediated gap junctional communication may be regulated. Furthermore, overexpression of PoCx43 in Japanese flounder FG-9307 cells significantly potentiates the LPS- and poly(I:C)-induced extracellular ATP release and this enhanced ATP release was attenuated by pre-incubation with Cx43 inhibitor carbenoxolone. In a complementary experiment, down-regulation of PoCx43 endogenous expression in FG-9307 cells with small interfering RNA also significantly reduced the PAMP-induced extracellular ATP release, suggesting that PoCx43 is an important ATP release conduit under the immune challenge conditions. Finally, we showed that extracellular ATP stimulation led to an increased PoCx43 expression which probably provides a feedback mechanism in regulating PoCx43 expression at the transcriptional level. These findings suggest that PoCx43 is an inducible immune response gene and an important conduit for immune challenge-induced extracellular ATP

  15. Expression and role of gap junction protein connexin43 in immune challenge-induced extracellular ATP release in Japanese flounder (Paralichthys olivaceus).

    PubMed

    Li, Shuo; Peng, Weijiao; Chen, Xiaoli; Geng, Xuyun; Zhan, Wenbin; Sun, Jinsheng

    2016-08-01

    Connexin43 (Cx43) is the best characterized gap junction protein that allows the direct exchange of signaling molecules during cell-to-cell communications. The immunological functions and ATP permeable properties of Cx43 have been insensitively examined in mammals. The similar biological significance of Cx43 in lower vertebrates, however, is not yet understood. In the present study we identified and characterized a Cx43 ortholog (termed PoCx43) from Japanese flounder (Paralichthys olivaceus) and investigated its role in immune challenge-induced extracellular ATP release. PoCx43 mRNA transcripts are widely distributed in all tested normal tissues and cells with predominant expression in the brain, and are significantly up-regulated by LPS, poly(I:C) and zymosan challenges and Edwardsiella tarda infections as well, suggesting that PoCx43 expression was modulated by the inflammatory stresses. In addition, cyclic AMP (cAMP), an essential second messenger, also plays an important role in regulating PoCx43 gene expression, by which the PoCx43-mediated gap junctional communication may be regulated. Furthermore, overexpression of PoCx43 in Japanese flounder FG-9307 cells significantly potentiates the LPS- and poly(I:C)-induced extracellular ATP release and this enhanced ATP release was attenuated by pre-incubation with Cx43 inhibitor carbenoxolone. In a complementary experiment, down-regulation of PoCx43 endogenous expression in FG-9307 cells with small interfering RNA also significantly reduced the PAMP-induced extracellular ATP release, suggesting that PoCx43 is an important ATP release conduit under the immune challenge conditions. Finally, we showed that extracellular ATP stimulation led to an increased PoCx43 expression which probably provides a feedback mechanism in regulating PoCx43 expression at the transcriptional level. These findings suggest that PoCx43 is an inducible immune response gene and an important conduit for immune challenge-induced extracellular ATP

  16. Moderate hypoxia followed by reoxygenation results in blood-brain barrier breakdown via oxidative stress-dependent tight-junction protein disruption.

    PubMed

    Zehendner, Christoph M; Librizzi, Laura; Hedrich, Jana; Bauer, Nina M; Angamo, Eskedar A; de Curtis, Marco; Luhmann, Heiko J

    2013-01-01

    Re-canalization of cerebral vessels in ischemic stroke is pivotal to rescue dysfunctional brain areas that are exposed to moderate hypoxia within the penumbra from irreversible cell death. Goal of the present study was to evaluate the effect of moderate hypoxia followed by reoxygenation (MHR) on the evolution of reactive oxygen species (ROS) and blood-brain barrier (BBB) integrity in brain endothelial cells (BEC). BBB integrity was assessed in BEC in vitro and in microvessels of the guinea pig whole brain in situ preparation. Probes were exposed to MHR (2 hours 67-70 mmHg O2, 3 hours reoxygenation, BEC) or towards occlusion of the arteria cerebri media (MCAO) with or without subsequent reperfusion in the whole brain preparation. In vitro BBB integrity was evaluated using trans-endothelial electrical resistance (TEER) and transwell permeability assays. ROS in BEC were evaluated using 2',7'-dichlorodihydrofluorescein diacetate (DCF), MitoSox and immunostaining for nitrotyrosine. Tight-junction protein (TJ) integrity in BEC, stainings for nitrotyrosine and FITC-albumin extravasation in the guinea pig brain preparation were assessed by confocal microscopy. Diphenyleneiodonium (DPI) was used to investigate NADPH oxidase dependent ROS evolution and its effect on BBB parameters in BEC. MHR impaired TJ proteins zonula occludens 1 (ZO-1) and claudin 5 (Cl5), decreased TEER, and significantly increased cytosolic ROS in BEC. These events were blocked by the NADPH oxidase inhibitor DPI. MCAO with or without subsequent reoxygenation resulted in extravasation of FITC-albumin and ROS generation in the penumbra region of the guinea pig brain preparation and confirmed BBB damage. BEC integrity may be impaired through ROS in MHR on the level of TJ and the BBB is also functionally impaired in moderate hypoxic conditions followed by reperfusion in a complex guinea pig brain preparation. These findings suggest that the BBB is susceptible towards MHR and that ROS play a key role in this

  17. Reduction of brain barrier tight junctional proteins by lead exposure: role of activation of nonreceptor tyrosine kinase Src via chaperon GRP78.

    PubMed

    Song, Han; Zheng, Gang; Shen, Xue-Feng; Liu, Xin-Qin; Luo, Wen-Jing; Chen, Jing-Yuan

    2014-04-01

    Lead (Pb) has long been recognized as a neurodevelopmental toxin. Developing blood-brain barrier (BBB) is known to be a target of Pb neurotoxicity; however, the underlying mechanisms are still unclear. Recent evidence suggests that intracellular nonreceptor protein tyrosine kinase Src regulates tight junctional proteins (TJPs). This study was designed to investigate whether Pb acted on the Src-mediated cascade event leading to an altered TJP expression at BBB. Rats aged 20-22 days were exposed to Pb in drinking water (0, 100, 200, and 300 ppm Pb) for eight weeks. Electron microscopic and Western blot analyses revealed a severe leakage of BBB and significantly decreased expressions of TJP occludin and ZO-1. When cultured brain endothelial RBE4 cells were exposed to 10μM Pb for 24 h, expressions of phosphor-Src and an upstream regulator GRP78 were significantly increased by 6.42-fold and 8.29-fold (p < 0.01), respectively. Inactivation of Src pathway by a Src-specific inhibitor reversed Pb-induced downregulation of occludin, but not ZO-1; small interfering RNA knockdown of GRP78 attenuated Pb-induced Src phosphorylation and occludin reduction. Furthermore, Pb exposure caused redistribution of GRP78 from endoplasmic reticulum to cytosol and toward cell member. However, the data from immunoneutralization studies did not show the involvement of cell-surface GRP78 in regulating Src phosphorylation upon Pb exposure, suggesting that the cytosolic GRP78, rather than cell-surface GRP78, was responsible to Pb-induced Src activation and ensuing occludin reduction. Taken together, this study provides the evidence of a novel linkage of GRP78, Src activation to downregulation of occludin, and BBB disruption during Pb exposure.

  18. Junctional Adhesion Molecule A Promotes Epithelial Tight Junction Assembly to Augment Lung Barrier Function

    PubMed Central

    Mitchell, Leslie A.; Ward, Christina; Kwon, Mike; Mitchell, Patrick O.; Quintero, David A.; Nusrat, Asma; Parkos, Charles A.; Koval, Michael

    2016-01-01

    Epithelial barrier function is maintained by tight junction proteins that control paracellular fluid flux. Among these proteins is junctional adhesion molecule A (JAM-A), an Ig fold transmembrane protein. To assess JAM-A function in the lung, we depleted JAM-A in primary alveolar epithelial cells using shRNA. In cultured cells, loss of JAM-A caused an approximately 30% decrease in transepithelial resistance, decreased expression of the tight junction scaffold protein zonula occludens 1, and disrupted junctional localization of the structural transmembrane protein claudin-18. Consistent with findings in other organs, loss of JAM-A decreased β1 integrin expression and impaired filamentous actin formation. Using a model of mild systemic endoxotemia induced by i.p. injection of lipopolysaccharide, we report that JAM-A−/− mice showed increased susceptibility to pulmonary edema. On injury, the enhanced susceptibility of JAM-A−/− mice to edema correlated with increased, transient disruption of claudin-18, zonula occludens 1, and zonula occludens 2 localization to lung tight junctions in situ along with a delay in up-regulation of claudin-4. In contrast, wild-type mice showed no change in lung tight junction morphologic features in response to mild systemic endotoxemia. These findings support a key role of JAM-A in promoting tight junction homeostasis and lung barrier function by coordinating interactions among claudins, the tight junction scaffold, and the cytoskeleton. PMID:25438062

  19. Expression of zonula occludens-1 (ZO-1) and the transcription factor ZO-1-associated nucleic acid-binding protein (ZONAB)-MsY3 in glial cells and colocalization at oligodendrocyte and astrocyte gap junctions in mouse brain.

    PubMed

    Penes, Mihai C; Li, Xinbo; Nagy, James I

    2005-07-01

    The PDZ domain-containing protein zonula occludens-1 (ZO-1) interacts with several members of the connexin (Cx) family of gap junction-forming proteins and has been localized to gap junctions, including those containing Cx47 in oligodendrocytes. We now provide evidence for ZO-1 expression in astrocytes in vivo and association with astrocytic connexins by confocal immunofluorescence demonstration of ZO-1 colocalization with astrocytic Cx30 and Cx43, and by ZO-1 coimmunoprecipitation with Cx30 and Cx43. Evidence for direct interaction of Cx30 with ZO-1 was obtained by pull-down assays that indicated binding of Cx30 to the second of the three PDZ domains in ZO-1. Further, we investigated mouse Y-box transcription factor MsY3, the canine ortholog of which has been termed ZO-1-associated nucleic acid-binding protein (ZONAB) and previously reported to interact with ZO-1. By immunofluorescence using specific antimouse ZONAB antibody, ZONAB was found to be associated with oligodendrocytes throughout mouse brain and spinal cord, and to be colocalized with oligodendrocytic Cx47 and Cx32 as well as with astrocytic Cx43. Our results extend the CNS cell types that express the multifunctional protein ZO-1, demonstrate an additional connexin (Cx30) that directly interacts with ZO-1, and show for the first time the association of a transcription factor (ZONAB) with ZO-1 localized to oligodendrocyte and astrocyte gap junctions. Given previous observations that ZONAB and ZO-1 in combination regulate gene expression, our results suggest roles of glial gap junction-mediated anchoring of signalling molecules in a wide variety of glial homeostatic processes. PMID:16045494

  20. H19 Long Noncoding RNA Regulates Intestinal Epithelial Barrier Function via MicroRNA 675 by Interacting with RNA-Binding Protein HuR.

    PubMed

    Zou, Tongtong; Jaladanki, Suraj K; Liu, Lan; Xiao, Lan; Chung, Hee Kyoung; Wang, Jun-Yao; Xu, Yan; Gorospe, Myriam; Wang, Jian-Ying

    2016-05-01

    The disruption of the intestinal epithelial barrier function occurs commonly in various pathologies, but the exact mechanisms responsible are unclear. The H19 long noncoding RNA (lncRNA) regulates the expression of different genes and has been implicated in human genetic disorders and cancer. Here, we report that H19 plays an important role in controlling the intestinal epithelial barrier function by serving as a precursor for microRNA 675 (miR-675). H19 overexpression increased the cellular abundance of miR-675, which in turn destabilized and repressed the translation of mRNAs encoding tight junction protein ZO-1 and adherens junction E-cadherin, resulting in the dysfunction of the epithelial barrier. Increasing the level of the RNA-binding protein HuR in cells overexpressing H19 prevented the stimulation of miR-675 processing from H19, promoted ZO-1 and E-cadherin expression, and restored the epithelial barrier function to a nearly normal level. In contrast, the targeted deletion of HuR in intestinal epithelial cells enhanced miR-675 production in the mucosa and delayed the recovery of the gut barrier function after exposure to mesenteric ischemia/reperfusion. These results indicate that H19 interacts with HuR and regulates the intestinal epithelial barrier function via the H19-encoded miR-675 by altering ZO-1 and E-cadherin expression posttranscriptionally.

  1. H19 Long Noncoding RNA Regulates Intestinal Epithelial Barrier Function via MicroRNA 675 by Interacting with RNA-Binding Protein HuR

    PubMed Central

    Zou, Tongtong; Jaladanki, Suraj K.; Liu, Lan; Xiao, Lan; Chung, Hee Kyoung; Wang, Jun-Yao; Xu, Yan; Gorospe, Myriam

    2016-01-01

    The disruption of the intestinal epithelial barrier function occurs commonly in various pathologies, but the exact mechanisms responsible are unclear. The H19 long noncoding RNA (lncRNA) regulates the expression of different genes and has been implicated in human genetic disorders and cancer. Here, we report that H19 plays an important role in controlling the intestinal epithelial barrier function by serving as a precursor for microRNA 675 (miR-675). H19 overexpression increased the cellular abundance of miR-675, which in turn destabilized and repressed the translation of mRNAs encoding tight junction protein ZO-1 and adherens junction E-cadherin, resulting in the dysfunction of the epithelial barrier. Increasing the level of the RNA-binding protein HuR in cells overexpressing H19 prevented the stimulation of miR-675 processing from H19, promoted ZO-1 and E-cadherin expression, and restored the epithelial barrier function to a nearly normal level. In contrast, the targeted deletion of HuR in intestinal epithelial cells enhanced miR-675 production in the mucosa and delayed the recovery of the gut barrier function after exposure to mesenteric ischemia/reperfusion. These results indicate that H19 interacts with HuR and regulates the intestinal epithelial barrier function via the H19-encoded miR-675 by altering ZO-1 and E-cadherin expression posttranscriptionally. PMID:26884465

  2. Current trends in salivary gland tight junctions.

    PubMed

    Baker, Olga J

    2016-01-01

    Tight junctions form a continuous intercellular barrier between epithelial cells that is required to separate tissue spaces and regulate selective movement of solutes across the epithelium. They are composed of strands containing integral membrane proteins (e.g., claudins, occludin and tricellulin, junctional adhesion molecules and the coxsackie adenovirus receptor). These proteins are anchored to the cytoskeleton via scaffolding proteins such as ZO-1 and ZO-2. In salivary glands, tight junctions are involved in polarized saliva secretion and barrier maintenance between the extracellular environment and the glandular lumen. This review seeks to provide an overview of what is currently known, as well as the major questions and future research directions, regarding tight junction expression, organization and function within salivary glands. PMID:27583188

  3. Angiopoietin 2 regulates the transformation and integrity of lymphatic endothelial cell junctions

    PubMed Central

    Zheng, Wei; Nurmi, Harri; Appak, Sila; Sabine, Amélie; Bovay, Esther; Korhonen, Emilia A.; Orsenigo, Fabrizio; Lohela, Marja; D’Amico, Gabriela; Holopainen, Tanja; Leow, Ching Ching; Dejana, Elisabetta; Petrova, Tatiana V.; Augustin, Hellmut G.; Alitalo, Kari

    2014-01-01

    Primitive lymphatic vessels are remodeled into functionally specialized initial and collecting lymphatics during development. Lymphatic endothelial cell (LEC) junctions in initial lymphatics transform from a zipper-like to a button-like pattern during collecting vessel development, but what regulates this process is largely unknown. Angiopoietin 2 (Ang2) deficiency leads to abnormal lymphatic vessels. Here we found that an ANG2-blocking antibody inhibited embryonic lymphangiogenesis, whereas endothelium-specific ANG2 overexpression induced lymphatic hyperplasia. ANG2 inhibition blocked VE-cadherin phosphorylation at tyrosine residue 685 and the concomitant formation of button-like junctions in initial lymphatics. The defective junctions were associated with impaired lymph uptake. In collecting lymphatics, adherens junctions were disrupted, and the vessels leaked upon ANG2 blockade or gene deletion. ANG2 inhibition also suppressed the onset of lymphatic valve formation and subsequent valve maturation. These data identify ANG2 as the first essential regulator of the functionally important interendothelial cell–cell junctions that form during lymphatic development. PMID:25030698

  4. Angiopoietin 2 regulates the transformation and integrity of lymphatic endothelial cell junctions.

    PubMed

    Zheng, Wei; Nurmi, Harri; Appak, Sila; Sabine, Amélie; Bovay, Esther; Korhonen, Emilia A; Orsenigo, Fabrizio; Lohela, Marja; D'Amico, Gabriela; Holopainen, Tanja; Leow, Ching Ching; Dejana, Elisabetta; Petrova, Tatiana V; Augustin, Hellmut G; Alitalo, Kari

    2014-07-15

    Primitive lymphatic vessels are remodeled into functionally specialized initial and collecting lymphatics during development. Lymphatic endothelial cell (LEC) junctions in initial lymphatics transform from a zipper-like to a button-like pattern during collecting vessel development, but what regulates this process is largely unknown. Angiopoietin 2 (Ang2) deficiency leads to abnormal lymphatic vessels. Here we found that an ANG2-blocking antibody inhibited embryonic lymphangiogenesis, whereas endothelium-specific ANG2 overexpression induced lymphatic hyperplasia. ANG2 inhibition blocked VE-cadherin phosphorylation at tyrosine residue 685 and the concomitant formation of button-like junctions in initial lymphatics. The defective junctions were associated with impaired lymph uptake. In collecting lymphatics, adherens junctions were disrupted, and the vessels leaked upon ANG2 blockade or gene deletion. ANG2 inhibition also suppressed the onset of lymphatic valve formation and subsequent valve maturation. These data identify ANG2 as the first essential regulator of the functionally important interendothelial cell-cell junctions that form during lymphatic development. PMID:25030698

  5. Angiopoietin 2 regulates the transformation and integrity of lymphatic endothelial cell junctions.

    PubMed

    Zheng, Wei; Nurmi, Harri; Appak, Sila; Sabine, Amélie; Bovay, Esther; Korhonen, Emilia A; Orsenigo, Fabrizio; Lohela, Marja; D'Amico, Gabriela; Holopainen, Tanja; Leow, Ching Ching; Dejana, Elisabetta; Petrova, Tatiana V; Augustin, Hellmut G; Alitalo, Kari

    2014-07-15

    Primitive lymphatic vessels are remodeled into functionally specialized initial and collecting lymphatics during development. Lymphatic endothelial cell (LEC) junctions in initial lymphatics transform from a zipper-like to a button-like pattern during collecting vessel development, but what regulates this process is largely unknown. Angiopoietin 2 (Ang2) deficiency leads to abnormal lymphatic vessels. Here we found that an ANG2-blocking antibody inhibited embryonic lymphangiogenesis, whereas endothelium-specific ANG2 overexpression induced lymphatic hyperplasia. ANG2 inhibition blocked VE-cadherin phosphorylation at tyrosine residue 685 and the concomitant formation of button-like junctions in initial lymphatics. The defective junctions were associated with impaired lymph uptake. In collecting lymphatics, adherens junctions were disrupted, and the vessels leaked upon ANG2 blockade or gene deletion. ANG2 inhibition also suppressed the onset of lymphatic valve formation and subsequent valve maturation. These data identify ANG2 as the first essential regulator of the functionally important interendothelial cell-cell junctions that form during lymphatic development.

  6. Odontogenic Ameloblast-associated Protein (ODAM) Mediates Junctional Epithelium Attachment to Teeth via Integrin-ODAM-Rho Guanine Nucleotide Exchange Factor 5 (ARHGEF5)-RhoA Signaling.

    PubMed

    Lee, Hye-Kyung; Ji, Suk; Park, Su-Jin; Choung, Han-Wool; Choi, Youngnim; Lee, Hyo-Jung; Park, Shin-Young; Park, Joo-Cheol

    2015-06-01

    Adhesion of the junctional epithelium (JE) to the tooth surface is crucial for maintaining periodontal health. Although odontogenic ameloblast-associated protein (ODAM) is expressed in the JE, its molecular functions remain unknown. We investigated ODAM function during JE development and regeneration and its functional significance in the initiation and progression of periodontitis and peri-implantitis. ODAM was expressed in the normal JE of healthy teeth but absent in the pathologic pocket epithelium of diseased periodontium. In periodontitis and peri-implantitis, ODAM was extruded from the JE following onset with JE attachment loss and detected in gingival crevicular fluid. ODAM induced RhoA activity and the expression of downstream factors, including ROCK (Rho-associated kinase), by interacting with Rho guanine nucleotide exchange factor 5 (ARHGEF5). ODAM-mediated RhoA signaling resulted in actin filament rearrangement. Reduced ODAM and RhoA expression in integrin β3- and β6-knockout mice revealed that cytoskeleton reorganization in the JE occurred via integrin-ODAM-ARHGEF5-RhoA signaling. Fibronectin and laminin activated RhoA signaling via the integrin-ODAM pathway. Finally, ODAM was re-expressed with RhoA in regenerating JE after gingivectomy in vivo. These results suggest that ODAM expression in the JE reflects a healthy periodontium and that JE adhesion to the tooth surface is regulated via fibronectin/laminin-integrin-ODAM-ARHGEF5-RhoA signaling. We also propose that ODAM could be used as a biomarker of periodontitis and peri-implantitis.

  7. Polychlorinated biphenyls impair blood-brain barrier integrity via disruption of tight junction proteins in cerebrum, cerebellum and hippocampus of female Wistar rats: neuropotential role of quercetin.

    PubMed

    Selvakumar, K; Prabha, R Lakshmi; Saranya, K; Bavithra, S; Krishnamoorthy, G; Arunakaran, J

    2013-07-01

    Polychlorinated biphenyls (PCBs) comprise a ubiquitous class of toxic substances associated with carcinogenic and tumor-promoting effects as well as neurotoxic properties. Reactive oxygen species, which is produced from PCBs, alters blood-brain barrier (BBB) integrity, which is paralleled by cytoskeletal rearrangements and redistribution and disappearance of tight junction proteins (TJPs) like claudin-5 and occludin. Quercetin, a potent antioxidant present in onion and other vegetables, appears to protect brain cells against oxidative stress, a tissue-damaging process associated with Alzheimer's and other neurodegenerative disorders. The aim of this study is to analyze the role of quercetin on oxidative stress markers and transcription of transmembrane and cytoplasmic accessory TJPs on cerebrum, cerebellum and hippocampus of female rats exposed to PCBs. Rats were divided into the following four groups. Group I: received only vehicle (corn oil) intraperitoneally (i.p.); group II: received Aroclor 1254 at a dose of 2 mg/kg body weight (bwt)/day (i.p); group III: received Aroclor 1254 (i.p.) and simultaneously quercetin 50 mg/kg bwt/day through gavage and group IV: received quercetin alone gavage. From the experiment, the levels of hydrogen peroxide, lipid peroxidation and thiobarbituric acid reactive substances were observed to increase significantly in cerebrum, cerebellum and hippocampus as 50%, 25% and 20%, respectively, after exposure to PCB, and the messenger RNA expression of TJP in rats exposed to PCBs is decreased and is retrieved to the normal level simultaneously in quercetin-treated rats. Hence, quercetin can be used as a preventive medicine to PCBs exposure and prevents neurodegenerative disorders.

  8. ATP Induces Disruption of Tight Junction Proteins via IL-1 Beta-Dependent MMP-9 Activation of Human Blood-Brain Barrier In Vitro

    PubMed Central

    Zhao, Kai

    2016-01-01

    Disruption of blood-brain barrier (BBB) follows brain trauma or central nervous system (CNS) stress. However, the mechanisms leading to this process or the underlying neural plasticity are not clearly known. We hypothesized that ATP/P2X7R signaling regulates the integrity of BBB. Activation of P2X7 receptor (P2X7R) by ATP induces the release of interleukin-1β (IL-1β), which in turn enhances the activity of matrix metalloproteinase-9 (MMP-9). Degradation of tight junction proteins (TJPs) such as ZO-1 and occludin occurs, which finally contributes to disruption of BBB. A contact coculture system using human astrocytes and hCMEC/D3, an immortalized human brain endothelial cell line, was used to mimic BBB in vitro. Permeability was used to evaluate changes in the integrity of TJPs. ELISA, Western blot, and immunofluorescent staining procedures were used. Our data demonstrated that exposure to the photoreactive ATP analog, 3′-O-(4-benzoyl)benzoyl adenosine 5′-triphosphate (BzATP), induced a significant decrease in ZO-1 and occludin expression. Meanwhile, the decrease of ZO-1 and occludin was significantly attenuated by P2X7R inhibitors, as well as IL-1R and MMP antagonists. Further, the induction of IL-1β and MMP-9 was closely linked to ATP/P2X7R-associated BBB leakage. In conclusion, our study explored the mechanism of ATP/P2X7R signaling in the disruption of BBB following brain trauma/stress injury, especially focusing on the relationship with IL-1β and MMP-9.

  9. MicroRNA-30a increases tight junction protein expression to suppress the epithelial-mesenchymal transition and metastasis by targeting Slug in breast cancer

    PubMed Central

    Chang, Chia-Wei; Yu, Jyh-Cherng; Hsieh, Yi-Hsien; Yao, Chung-Chin; Chao, Jui-I; Chen, Po-Ming; Hsieh, Hsiao-Yen; Hsiung, Chia-Ni; Chu, Hou-Wei; Shen, Chen-Yang; Cheng, Chun-Wen

    2016-01-01

    The epithelial-to-mesenchymal (EMT) transition is a prerequisite for conferring metastatic potential during tumor progression. microRNA-30a (miR-30a) expression was significantly lower in aggressive breast cancer cell lines compared with non-invasive breast cancer and non-malignant mammary epithelial cell lines. In contrast, miR-30a overexpression reversed the mesenchymal appearance of cancer cells to result in a cobblestone-like epithelial phenotype. We identified Slug, one of the master regulators of EMT, as a target of miR-30a using in silico prediction. Reporter assays indicated that miR-30a could bind to the 3′-untranslted region of Slug mRNA. Furthermore, we linked miR-30a to increased expression of claudins, a family of tight junction transmembrane proteins. An interaction between Slug and E-box in the claudin promoter sequences was reduced upon miR-30a overexpression, further leading to reduction of filopodia formation and decreased invasiveness/metastasis capabilities of breast cancer cells. Consistently, delivery of miR-30a in xenografted mice decreased tumor invasion and migration. In patients with breast cancer, a significantly elevated risk of the miR-30alow/CLDN2low/FSCNhigh genotype was observed, linking to a phenotypic manifestation of larger tumor size, lymph node metastasis, and advanced tumor stage among patients. In conclusion, the miR-30a/Slug axis inhibits mesenchymal tumor development by interfering with metastatic cancer cell programming and may be a potential target for therapy in breast cancer. PMID:26918943

  10. The cytoskeletal adaptor protein band 4.1B is required for the maintenance of paranodal axoglial septate junctions in myelinated axons.

    PubMed

    Buttermore, Elizabeth D; Dupree, Jeffrey L; Cheng, JrGang; An, Xiuli; Tessarollo, Lino; Bhat, Manzoor A

    2011-06-01

    Precise targeting and maintenance of axonal domains in myelinated axons is essential for saltatory conduction. Caspr and Caspr2, which localize at paranodal and juxtaparanodal domains, contain binding sites for the cytoskeletal adaptor protein 4.1B. The exact role of 4.1B in the organization and maintenance of axonal domains is still not clear. Here, we report the generation and characterization of 4.1B-null mice. We show that loss of 4.1B in the PNS results in mislocalization of Caspr at paranodes and destabilization of paranodal axoglial septate junctions (AGSJs) as early as postnatal day 30. In the CNS, Caspr localization is progressively disrupted and ultrastructural analysis showed paranodal regions that were completely devoid of AGSJs, with axolemma separated from the myelin loops, and loops coming off the axolemma. Most importantly, our phenotypic analysis of previously generated 4.1B mutants, used in the study by Horresh et al. (2010), showed that Caspr localization was not affected in the PNS, even after 1 year; and 4.1R was neither expressed, nor enriched at the paranodes. Furthermore, ultrastructural analysis of these 4.1B mutants showed destabilization of CNS AGSJs at ∼ 1 year. We also discovered that the 4.1B locus is differentially expressed in the PNS and CNS, and generates multiple splice isoforms in the PNS, suggesting 4.1B may function differently in the PNS versus CNS. Together, our studies provide direct evidence that 4.1B plays a pivotal role in interactions between the paranodal AGSJs and axonal cytoskeleton, and that 4.1B is critically required for long-term maintenance of axonal domains in myelinated axons. PMID:21632923

  11. The Cytoskeletal Adaptor Protein Band 4.1B is Required for the Maintenance of Paranodal Axo-Glial Septate Junctions in Myelinated Axons

    PubMed Central

    Buttermore, Elizabeth D.; Dupree, Jeffrey L.; Cheng, JrGang; An, Xiuli; Tessarollo, Lino; Bhat, Manzoor A.

    2011-01-01

    Precise targeting and maintenance of axonal domains in myelinated axons is essential for saltatory conduction. Caspr and Caspr2, which localize at paranodal and juxtaparanodal domains, contain binding sites for the cytoskeletal adaptor protein 4.1B. The exact role of 4.1B in the organization and maintenance of axonal domains is still not clear. Here we report the generation and characterization of 4.1B null mice. We show that loss of 4.1B in the PNS results in mislocalization of Caspr at paranodes and destabilization of paranodal axo-glial septate junctions (AGSJs) as early as postnatal day 30. In the CNS, Caspr localization is progressively disrupted and ultrastructural analysis showed paranodal regions that were completely devoid of AGSJs, with axolemma separated from the myelin loops, and loops coming off the axolemma. Most importantly, our phenotypic analysis of previously generated 4.1B mutants, used in Horresh et al. (2010), showed that Caspr localization was not affected in the PNS, even after one year; and 4.1R was neither expressed, nor enriched at the paranodes. Furthermore, ultrastructural analysis of these 4.1B mutants showed destabilization of CNS AGSJs at about one year. We also discovered that the 4.1B locus is differentially expressed in the PNS and CNS, and generates multiple splice isoforms in the PNS, suggesting 4.1B may function differently in the PNS versus CNS. Together, our studies provide direct evidence that 4.1B plays a pivotal role in interactions between the paranodal AGSJs and axonal cytoskeleton, and that 4.1B is critically required for long-term maintenance of axonal domains in myelinated axons. PMID:21632923

  12. Downregulation of gap junctions in cancer cells.

    PubMed

    Leithe, Edward; Sirnes, Solveig; Omori, Yasufumi; Rivedal, Edgar

    2006-12-01

    Gap junctions are intercellular plasma membrane domains enriched in channels that allow direct exchange of ions and small molecules between adjacent cells. Gap junction channels are composed of a family of transmembrane proteins called connexin. Connexins play important roles in the regulation of cell growth and differentiation. Cancer cells usually have downregulated levels of gap junctions, and several lines of evidence suggest that loss of gap junctional intercellular communication is an important step in carcinogenesis. In support of this hypothesis are studies showing that reexpression of connexins in cancer cells causes normalization of cell growth control and reduced tumor growth. To gain a more detailed understanding of the role of connexins as tumor suppressors, a clearer picture of the mechanisms involved in loss of gap junctions in cancer cells is needed. Furthermore, defining the mechanisms involved in downregulation of connexins in carcinogenesis will be an important step toward utilizing the potential of connexins as targets in cancer prevention and therapy. Various mechanisms are involved in the loss of gap junctions in cancer cells, ranging from loss of connexin gene transcription to aberrant trafficking of connexin proteins. This review will discuss our current knowledge on the molecular mechanisms involved in the downregulation of gap junctions in cancer cells. PMID:17425504

  13. Regulation of Embryonic Cell Adhesion by the Prion Protein

    PubMed Central

    Schrock, Yvonne; Geiss, Corinna; Luncz, Lydia; Thomanetz, Venus; Stuermer, Claudia A. O

    2009-01-01

    Prion proteins (PrPs) are key players in fatal neurodegenerative disorders, yet their physiological functions remain unclear, as PrP knockout mice develop rather normally. We report a strong PrP loss-of-function phenotype in zebrafish embryos, characterized by the loss of embryonic cell adhesion and arrested gastrulation. Zebrafish and mouse PrP mRNAs can partially rescue this knockdown phenotype, indicating conserved PrP functions. Using zebrafish, mouse, and Drosophila cells, we show that PrP: (1) mediates Ca+2-independent homophilic cell adhesion and signaling; and (2) modulates Ca+2-dependent cell adhesion by regulating the delivery of E-cadherin to the plasma membrane. In vivo time-lapse analyses reveal that the arrested gastrulation in PrP knockdown embryos is due to deficient morphogenetic cell movements, which rely on E-cadherin–based adhesion. Cell-transplantation experiments indicate that the regulation of embryonic cell adhesion by PrP is cell-autonomous. Moreover, we find that the local accumulation of PrP at cell contact sites is concomitant with the activation of Src-related kinases, the recruitment of reggie/flotillin microdomains, and the reorganization of the actin cytoskeleton, consistent with a role of PrP in the modulation of cell adhesion via signaling. Altogether, our data uncover evolutionarily conserved roles of PrP in cell communication, which ultimately impinge on the stability of adherens cell junctions during embryonic development. PMID:19278297

  14. 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.

  15. Claudins and the Modulation of Tight Junction Permeability

    PubMed Central

    Günzel, Dorothee

    2013-01-01

    Claudins are tight junction membrane proteins that are expressed in epithelia and endothelia and form paracellular barriers and pores that determine tight junction permeability. This review summarizes our current knowledge of this large protein family and discusses recent advances in our understanding of their structure and physiological functions. PMID:23589827

  16. Slit diaphragm junctional complex and regulation of the cytoskeleton.

    PubMed

    Garg, Puneet; Verma, Rakesh; Holzman, Lawrence B

    2007-01-01

    In recent years, identification of proteins found at the slit diaphragm has greatly improved our understanding of the molecular mechanisms responsible for forming and maintaining the glomerular filtration barrier. This review examines the function of proteins identified at the podocyte intercellular junction in coordinating podocyte intercellular junction dynamics or cytoskeletal dynamics.

  17. 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. PMID:26748968

  18. VEGF and Angiopoietin-1 exert opposing effects on cell junctions by regulating the Rho GEF Syx

    PubMed Central

    Ngok, Siu P.; Geyer, Rory; Liu, Miaoliang; Kourtidis, Antonis; Agrawal, Sudesh; Wu, Chuanshen; Seerapu, Himabindu Reddy; Lewis-Tuffin, Laura J.; Moodie, Karen L.; Huveldt, Deborah; Marx, Ruth; Baraban, Jay M.; Storz, Peter

    2012-01-01

    Vascular endothelial growth factor (VEGF) and Ang1 (Angiopoietin-1) have opposing effects on vascular permeability, but the molecular basis of these effects is not fully known. We report in this paper that VEGF and Ang1 regulate endothelial cell (EC) junctions by determining the localization of the RhoA-specific guanine nucleotide exchange factor Syx. Syx was recruited to junctions by members of the Crumbs polarity complex and promoted junction integrity by activating Diaphanous. VEGF caused translocation of Syx from cell junctions, promoting junction disassembly, whereas Ang1 maintained Syx at the junctions, inducing junction stabilization. The VEGF-induced translocation of Syx from EC junctions was caused by PKD1 (protein kinase D1)-mediated phosphorylation of Syx at Ser806, which reduced Syx association to its junctional anchors. In support of the pivotal role of Syx in regulating EC junctions, syx−/− mice had defective junctions, resulting in vascular leakiness, edema, and impaired heart function. PMID:23253477

  19. Polarity Protein Complex Scribble/Lgl/Dlg and Epithelial Cell Barriers

    PubMed Central

    Su, Wen-Hui; Mruk, Dolores D.; Wong, Elissa W.P.; Lui, Wing-Yee; Cheng, C. Yan

    2014-01-01

    The Scribble polarity complex or module is one of the three polarity modules that regulate cell polarity in multiple epithelia including blood-tissue barriers. This protein complex is composed of Scribble, Lethal giant larvae (Lgl) and Discs large (Dlg), which are well conserved across species from fruitflies and worms to mammals. Originally identified in Drosophila and C. elegans where the Scribble complex was found to work with the Par-based and Crumbs-based polarity modules to regulate apicobasal polarity and asymmetry in cells and tissues during embryogenesis, their mammalian homologs have all been identified in recent years. Components of the Scribble complex are known to regulate multiple cellular functions besides cell polarity, which include cell proliferation, assembly and maintenance of adherens junction (AJ) and tight junction (TJ), and they are also tumor suppressors. Herein, we provide an update on the Scribble polarity complex and how this protein complex modulates cell adhesion with some emphasis on its role in Sertoli cell blood-testis barrier (BTB) function. It should be noted that this is a rapidly developing field, in particular the role of this protein module in blood-tissue barriers, and this short chapter attempts to provide the information necessary for investigators studying reproductive biology and blood-tissue barriers to design future studies. We also include results of recent studies from flies and worms since this information will be helpful in planning experiments for future functional studies in the testis to understand how Scribble-based proteins regulate BTB dynamics and spermatogenesis. PMID:23397623

  20. Quantitative Proteomics Identifies Vasopressin-Responsive Nuclear Proteins in Collecting Duct Cells

    PubMed Central

    Schenk, Laura K.; Bolger, Steven J.; Luginbuhl, Kelli; Gonzales, Patricia A.; Rinschen, Markus M.; Yu, Ming-Jiun; Hoffert, Jason D.; Pisitkun, Trairak

    2012-01-01

    Vasopressin controls transport in the renal collecting duct, in part, by regulating transcription. This complex process, which can involve translocation and/or modification of transcriptional regulators, is not completely understood. Here, we applied a method for large-scale profiling of nuclear proteins to quantify vasopressin-induced changes in the nuclear proteome of cortical collecting duct (mpkCCD) cells. Using stable isotope labeling and tandem mass spectrometry, we quantified 3987 nuclear proteins and identified significant changes in the abundance of 65, including previously established targets of vasopressin signaling in the collecting duct. Vasopressin-induced changes in the abundance of the transcription factors JunB, Elf3, Gatad2b, and Hmbox1; transcriptional co-regulators Ctnnb1 (β-catenin) and Crebbp; subunits of the Mediator complex; E3 ubiquitin ligase Nedd4; nuclear transport regulator RanGap1; and several proteins associated with tight junctions and adherens junctions. Bioinformatic analysis showed that many of the quantified transcription factors have putative binding sites in the 5′-flanking regions of genes coding for the channel proteins Aqp2, Aqp3, Scnn1b (ENaCβ), and Scnn1g (ENaCγ), which are known targets of vasopressin. Immunoblotting demonstrated that the increase in β-catenin in nuclear fractions was accompanied by an even larger increase in its phosphorylated form (pSer552). The findings provide a new online database resource for nuclear proteomics (http://helixweb.nih.gov/ESBL/Database/mNPD/) and generate new hypotheses regarding vasopressin-mediated transcriptional regulation in the collecting duct. PMID:22440904

  1. Quantitative proteomics identifies vasopressin-responsive nuclear proteins in collecting duct cells.

    PubMed

    Schenk, Laura K; Bolger, Steven J; Luginbuhl, Kelli; Gonzales, Patricia A; Rinschen, Markus M; Yu, Ming-Jiun; Hoffert, Jason D; Pisitkun, Trairak; Knepper, Mark A

    2012-06-01

    Vasopressin controls transport in the renal collecting duct, in part, by regulating transcription. This complex process, which can involve translocation and/or modification of transcriptional regulators, is not completely understood. Here, we applied a method for large-scale profiling of nuclear proteins to quantify vasopressin-induced changes in the nuclear proteome of cortical collecting duct (mpkCCD) cells. Using stable isotope labeling and tandem mass spectrometry, we quantified 3987 nuclear proteins and identified significant changes in the abundance of 65, including previously established targets of vasopressin signaling in the collecting duct. Vasopressin-induced changes in the abundance of the transcription factors JunB, Elf3, Gatad2b, and Hmbox1; transcriptional co-regulators Ctnnb1 (β-catenin) and Crebbp; subunits of the Mediator complex; E3 ubiquitin ligase Nedd4; nuclear transport regulator RanGap1; and several proteins associated with tight junctions and adherens junctions. Bioinformatic analysis showed that many of the quantified transcription factors have putative binding sites in the 5'-flanking regions of genes coding for the channel proteins Aqp2, Aqp3, Scnn1b (ENaCβ), and Scnn1g (ENaCγ), which are known targets of vasopressin. Immunoblotting demonstrated that the increase in β-catenin in nuclear fractions was accompanied by an even larger increase in its phosphorylated form (pSer552). The findings provide a new online database resource for nuclear proteomics (http://helixweb.nih.gov/ESBL/Database/mNPD/) and generate new hypotheses regarding vasopressin-mediated transcriptional regulation in the collecting duct. PMID:22440904

  2. The Nectin-4/Afadin Protein Complex and Intercellular Membrane Pores Contribute to Rapid Spread of Measles Virus in Primary Human Airway Epithelia

    PubMed Central

    Singh, Brajesh K.; Hornick, Andrew L.; Krishnamurthy, Sateesh; Locke, Anna C.; Mendoza, Crystal A.; Mateo, Mathieu; Miller-Hunt, Catherine L.; Cattaneo, Roberto

    2015-01-01

    ABSTRACT The discovery that measles virus (MV) uses the adherens junction protein nectin-4 as its epithelial receptor provides a new vantage point from which to characterize its rapid spread in the airway epithelium. We show here that in well-differentiated primary cultures of airway epithelial cells from human donors (HAE), MV infectious centers form rapidly and become larger than those of other respiratory pathogens: human respiratory syncytial virus, parainfluenza virus 5, and Sendai virus. While visible syncytia do not form after MV infection of HAE, the cytoplasm of an infected cell suddenly flows into an adjacent cell, as visualized through wild-type MV-expressed cytoplasmic green fluorescent protein (GFP). High-resolution video microscopy documents that GFP flows through openings that form on the lateral surfaces between columnar epithelial cells. To assess the relevance of the protein afadin, which connects nectin-4 to the actin cytoskeleton, we knocked down its mRNA. This resulted in more-limited infectious-center formation. We also generated a nectin-4 mutant without the afadin-binding site in its cytoplasmic tail. This mutant was less effective than wild-type human nectin-4 at promoting MV infection in primary cultures of porcine airway epithelia. Thus, in airway epithelial cells, MV spread requires the nectin-4/afadin complex and is based on cytoplasm transfer between columnar cells. Since the viral membrane fusion apparatus may open the passages that allow cytoplasm transfer, we refer to them as intercellular membrane pores. Virus-induced intercellular pores may contribute to extremely efficient measles contagion by promoting the rapid spread of the virus through the upper respiratory epithelium. IMPORTANCE Measles virus (MV), while targeted for eradication, still causes about 120,000 deaths per year worldwide. The recent reemergence of measles in insufficiently vaccinated populations in Europe and North America reminds us that measles is extremely

  3. Mutant B-Raf(V600E) Promotes Melanoma Paracellular Transmigration by Inducing Thrombin-mediated Endothelial Junction Breakdown.

    PubMed

    Zhang, Pu; Feng, Shan; Liu, Gentao; Wang, Heyong; Zhu, Huifeng; Ren, Qiao; Bai, Huiyuan; Fu, Changliang; Dong, Cheng

    2016-01-29

    Tumor invasiveness depends on the ability of tumor cells to breach endothelial barriers. In this study, we investigated the mechanism by which the adhesion of melanoma cells to endothelium regulates adherens junction integrity and modulates tumor transendothelial migration (TEM) by initiating thrombin generation. We found that the B-Raf(V600E) mutation in metastatic melanoma cells up-regulated tissue factor (TF) expression on cell membranes and promoted thrombin production. Co-culture of endothelial monolayers with metastatic melanoma cells mediated the opening of inter-endothelial spaces near melanoma cell contact sites in the presence of platelet-free plasma (PFP). By using small interfering RNA (siRNA), we demonstrated that B-Raf(V600E) and TF silencing attenuated the focal disassembly of adherens junction induced by tumor contact. Vascular endothelial-cadherin (VE-cadherin) disassembly was dependent on phosphorylation of p120-catenin on Ser-879 and VE-cadherin on Tyr-658, Tyr-685, and Tyr-731, which can be prevented by treatment with the thrombin inhibitor, hirudin, or by silencing the thrombin receptor, protease-activated receptor-1, in endothelial cells. We also provided strong evidence that tumor-derived thrombin enhanced melanoma TEM by inducing ubiquitination-coupled VE-cadherin internalization, focal adhesion formation, and actin assembly in endothelium. Confocal microscopic analysis of tumor TEM revealed that junctions transiently opened and resealed as tumor cells accomplished TEM. In addition, in the presence of PFP, tumor cells preferentially transmigrated via paracellular routes. PFP supported melanoma transmigration under shear conditions via a B-Raf(V600E)-thrombin-dependent mechanism. We concluded that the activation of thrombin generation by cancer cells in plasma is an important process regulating melanoma extravasation by disrupting endothelial junction integrity. PMID:26504080

  4. Spatial and temporal relationships between cadherins and PECAM-1 in cell-cell junctions of human endothelial cells

    PubMed Central

    1994-01-01

    The integrity of the endothelial layer, which lines the entire cavity of the vascular system, depends on tight adhesion of the cells to the underlying basement membrane as well as to each other. It has been previously shown that such interactions occur via membrane receptors that determine the specificity, topology, and mechanical properties of the surface adhesion. Cell-cell junctions between endothelial cells, in culture and in situ, involve both Ca(2+)-dependent and -independent mechanisms that are mediated by distinct adhesion molecules. Ca(2+)- dependent cell-cell adhesion occurs mostly via members of the cadherin family, which locally anchor the microfilament system to the plasma membrane, in adherens junctions. Ca(2+)-independent adhesions were reported to mainly involve members of the Ig superfamily. In this study, we performed three-dimensional microscopic analysis of the relative subcellular distributions of these two endothelial intercellular adhesion systems. We show that cadherins are located at adjacent (usually more apical), yet clearly distinct domains of the lateral plasma membrane, compared to PECAM-1. Moreover, cadherins were first organized in adherens junctions within 2 h after seeding of endothelial cells, forming multiple lateral patches which developed into an extensive belt-like structure over a period of 24 h. PECAM-1 became associated with surface adhesions significantly later and became progressively associated with the cadherin-containing adhesions. Cadherins and PECAM-1 also differed in their detergent extractability, reflecting differences in their mode of association with the cytoskeleton. Moreover, the two adhesion systems could be differentially modulated since short treatment with the Ca2+ chelator EGTA, disrupted the cadherin junctions leaving PECAM-1 apparently intact. These results confirm that endothelial cells possess distinct intercellular contact mechanisms that differ in their spatial and temporal organization as well as

  5. Receptor protein tyrosine phosphatases are novel components of a polycystin complex

    PubMed Central

    Boucher, Catherine A.; Ward, Heather H.; Case, Ruth L.; Thurston, Katie S.; Li, Xiaohong; Needham, Andrew; Romero, Elsa; Hyink, Deborah; Qamar, Seema; Roitbak, Tamara; Powell, Samantha; Ward, Christopher; Wilson, Patricia D.; Wandinger-Ness, Angela; Sandford, Richard N.

    2011-01-01

    Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutation of PKD1 and PKD2 that encode polycystin-1 and polycystin-2. Polycystin-1 is tyrosine phosphorylated and modulates multiple signaling pathways including AP-1, but the identity of the phosphatases regulating polycystin-1 are previously uncharacterized. Here we identify members of the LAR protein tyrosine phosphatase (RPTP) superfamily as members of the polycystin-1complex mediated through extra- and intracellular interactions. The first extracellular PKD1 domain of polycystin-1 interacts with the first Ig domain of RPTPσ, while the polycystin-1 C-terminus of polycystin-1 interacts with the regulatory D2 phosphatase domain of RPTPγ. Additional homo- and heterotypic interactions between RPTPs recruit RPTPδ The multimeric polycystin protein complex is found localised in cilia. RPTPσ and RPTPδ are also part of a polycystin-1/E-cadherin complex known to be important for early events in adherens junction stabilisation. The interaction between polycystin-1 and RPTPγ is disrupted in ADPKD cells, while RPTPσ and RPTPδ remain closely associated with E-cadherin, largely in an intracellular location. The polycystin-1 C-terminus is an in vitro substrate of RPTPγ, which dephosphorylates the c-Src phosphorylated Y4237 residue and activates AP1-mediated transcription. The data identify RPTPs as novel interacting partners of the polycystins both in cilia and at adhesion complexes and demonstrate RPTPγ phosphatase activity is central to the molecular mechanisms governing polycystin-dependent signaling. PMID:21126580

  6. Deficiency of dietary niacin impaired gill immunity and antioxidant capacity, and changes its tight junction proteins via regulating NF-κB, TOR, Nrf2 and MLCK signaling pathways in young grass carp (Ctenopharyngodon idella).

    PubMed

    Li, Shun-Quan; Feng, Lin; Jiang, Wei-Dan; Liu, Yang; Jiang, Jun; Wu, Pei; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu

    2016-08-01

    To investigate the effects of dietary niacin on gill immunity, tight junction proteins, antioxidant system and related signaling molecules mRNA expression, young grass carp (Ctenopharyngodon idella) were fed six diets containing graded levels of niacin (3.95-55.01 mg/kg diet) for 8 weeks. The study indicated that niacin deficiency decreased lysozyme and acid phosphatase activities, and complement 3 content, and caused oxidative damage that might be partly due to the decreased copper, zinc superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase and glutathione-S-transferase activities and reduced glutathione content in fish gills (P < 0.05). Moreover, the relative mRNA levels of antimicrobial peptides (liver expressed antimicrobial peptide 2 and Hepcidin), anti-inflammatory cytokines (interleukin 10 and transforming growth factor β1), tight junction proteins (Occludin, zonula occludens 1, Claudin-15 and -3), signaling molecules (inhibitor of κBα (IκBα), target of rapamycin (TOR), ribosomal protein S6 kinase 1 (S6K1) and NF-E2-related factor 2 (Nrf2)) and antioxidant enzymes were significantly decreased (P < 0.05) in niacin-deficient diet group. Conversely, the mRNA levels of pro-inflammatory cytokines (tumor necrosis factor α, interleukin 8, interferon γ2, and interleukin 1β), signaling molecules (nuclear factor kappa B p65, IκB kinase α, IκB kinase β, IκB kinase γ, Kelch-like-ECH-associated protein 1b, myosin light chain kinase and p38 mitogen-activated protein kinase (p38 MAPK) were significantly increased (P < 0.05) in fish gills fed niacin-deficient diet. Interestingly, the varying niacin levels of 3.95-55.01 mg/kg diet had no effect on the mRNA level of Kelch-like-ECH-associated protein 1a, Claudin-c and -12 in fish gills (P > 0.05). In conclusion, niacin deficiency decreased gill immunity, impaired gill antioxidant system, as well as regulated mRNA expression of gill tight junction proteins and related signaling

  7. Mammalian tight junctions in the regulation of epithelial differentiation and proliferation.

    PubMed

    Matter, Karl; Aijaz, Saima; Tsapara, Anna; Balda, Maria S

    2005-10-01

    Tight junctions are important for the permeability properties of epithelial and endothelial barriers as they restrict diffusion along the paracellular space. Recent observations have revealed that tight junctions also function in the regulation of epithelial proliferation and differentiation. They harbour evolutionarily conserved protein complexes that regulate polarisation and junction assembly. Tight junctions also recruit signalling proteins that participate in the regulation of cell proliferation and differentiation. These signalling proteins include components that affect established signalling cascades and dual localisation proteins that can associate with junctions as well as travel to the nucleus where they regulate gene expression.

  8. Thiamin deficiency induces impaired fish gill immune responses, tight junction protein expression and antioxidant capacity: Roles of the NF-κB, TOR, p38 MAPK and Nrf2 signaling molecules.

    PubMed

    Wen, Ling-Mei; Feng, Lin; Jiang, Wei-Dan; Liu, Yang; Wu, Pei; Zhao, Juan; Jiang, Jun; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu

    2016-04-01

    In this study, we investigate the effects of dietary thiamin deficiency on immune responses, tight junctions, antioxidant capacity and related signaling molecules in the gills of young grass carp (Ctenopharyngodon idella). Fish were fed diets that contained 0.12-2.04 mg thiamin kg(-1) for 8 weeks. We found that dietary thiamin deficiency resulted in reduced complement 3 content, lysozyme and acid phosphatase activities, mRNA levels of hepcidin, liver-expressed antimicrobial peptides 2, transforming growth factor (TGF)-β1, interleukin (IL)-10, inhibitor protein-κBα (IκBα), ribosomal S6 protein kinase 1 and target of rapamycin (TOR) and increased expression of interferon-γ2, tumor necrosis factor-α, TGF-β2, IL-1β, IL-8, IκB kinases (IKKβ and IKKγ) and nuclear factor-κB p65 (NF-κB p65). Our findings showed that thiamin deficiency reduced the immune status of fish gills. Furthermore, thiamin deficiency resulted in reduced mRNA transcript levels of claudin b, claudin 3, claudin 12, zonula occludens 1 (ZO-1) and occludin and increased mRNA transcript levels of claudin 15a, myosin light-chain kinase (MLCK) and p38 mitogen-activated protein kinase (p38 MAPK) in fish gill tissues. These data suggested that thiamin deficiency disrupted tight junction-mediated fish gill barrier function. Additionally, reactive oxygen species, malondialdehyde and protein carbonyl levels and both the activities and expression levels of Cu/Zn superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferases and glutathione reductase, as well as NF-E2-related factor 2 gene expression in fish gills, were lower in fish fed a thiamin-deficient diet. By contrast, thiamin deficiency increased levels of Kelch-like-ECH-associated protein 1a (Keap1a) and Keap1b mRNA transcript expression in fish gills. Taken together, our findings indicated that thiamin deficiency impaired fish gill health by effects on the expression of genes encoding cytokines, tight junction proteins

  9. Role of occludin, a tight junction protein, in blastocoel formation, and in the paracellular permeability and differentiation of trophectoderm in preimplantation mouse embryos.

    PubMed

    Kim, Jinmee; Gye, Myung Chan; Kim, Moon Kyoo

    2004-04-30

    Tight junctions (TJ) are critical for blastocoel formation in mammalian embryos. The present study aimed to examine the role of tight junctions in the differentiation of the trophectoderm (TE), and in the pluripotency of blastomeres, as well as in the formation and integrity of the blastocoel. We examined the effect of occludin antibody on blastocoel formation, blastocyst permeability, and expression of H19 and Oct-4, markers of TE differentiation and blastomere pluripotency, respectively. Eight-cell mouse embryos and morulae were cultured in the presence or absence of occludin antibody for 31 h. Occludin antibody inhibited blastocoel formation and increased permeability of the TE of nascent and expanding blastocysts to FITC-dextran (4 kDa), a permeability tracer. At the same time Oct-4 expression increased while expression of H19 became barely detectable. These observations indicate that occludin is involved in establishing the blastocoel, as well as in maintaining its impermeability, and that the development of tight junction is critical for TE formation in mouse embryos.

  10. Chronic type 2 diabetes reduces the integrity of the blood-brain barrier by reducing tight junction proteins in the hippocampus

    PubMed Central

    YOO, Dae Young; YIM, Hee Sun; JUNG, Hyo Young; NAM, Sung Min; KIM, Jong Whi; CHOI, Jung Hoon; SEONG, Je Kyung; YOON, Yeo Sung; KIM, Dae Won; HWANG, In Koo

    2016-01-01

    In the present study, we investigated the effects of type 2 diabetes-induced hyperglycemia on the integrity of the blood–brain barrier and tight junction markers in the rat hippocampus. Forty-week-old diabetic (Zucker diabetic fatty, ZDF) rats and littermate control (Zucker lean control, ZLC) rats were used in this study. We evaluated the integrity of the blood–brain barrier by measuring sodium fluorescein extravasation and blood vessel ultrastructure. In addition, tight junction markers, such as zona occludens-1, occludin and claudin-5, were quantified by western blot analysis. ZDF rats showed significantly increased sodium fluorescein leakage in the hippocampus. Tight junction markers, such as occludin and claudin-5, were significantly decreased in the hippocampi of ZDF rats compared to those of ZLC rats. In addition, ZDF rats showed ultrastructural changes with phagocytic findings in the blood vessels. These results suggest that chronic untreated diabetes impairs the permeability of the hippocampal blood–brain barrier by down-regulating occludin and claudin-5, indicating that chronic untreated diabetes may cause hippocampus-dependent dysfunction. PMID:26876499

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

    SciTech Connect

    Pereira, Flavia E. . E-mail: flavia.pereira@umontana.edu; Coffin, J. Douglas . E-mail: douglas.coffin@umontana.edu; Beall, Howard D. . E-mail: howard.beall@umontana.edu

    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 that 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.

  12. Three-junction solar cell

    DOEpatents

    Ludowise, Michael J.

    1986-01-01

    A photovoltaic solar cell is formed in a monolithic semiconductor. The cell contains three junctions. In sequence from the light-entering face, the junctions have a high, a medium, and a low energy gap. The lower junctions are connected in series by one or more metallic members connecting the top of the lower junction through apertures to the bottom of the middle junction. The upper junction is connected in voltage opposition to the lower and middle junctions by second metallic electrodes deposited in holes 60 through the upper junction. The second electrodes are connected to an external terminal.

  13. The Effects of Glucagon-like Peptide-2 on the Tight Junction and Barrier Function in IPEC-J2 Cells through Phosphatidylinositol 3-kinase–Protein Kinase B–Mammalian Target of Rapamycin Signaling Pathway

    PubMed Central

    Yu, Changsong; Jia, Gang; Deng, Qiuhong; Zhao, Hua; Chen, Xiaoling; Liu, Guangmang; Wang, Kangning

    2016-01-01

    Glucagon-like peptide-2 (GLP-2) is important for intestinal barrier function and regulation of tight junction (TJ) proteins, but the intracellular mechanisms of action remain undefined. The purpose of this research was to determine the protective effect of GLP-2 mediated TJ and transepithelial electrical resistance (TER) in lipopolysaccharide (LPS) stressed IPEC-J2 cells and to test the hypothesis that GLP-2 regulate TJ and TER through the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) signaling pathway in IPEC-J2 cells. Wortmannin and LY294002 are specific inhibitors of PI3K. The results showed that 100 μg/mL LPS stress decreased TER and TJ proteins occludin, claudin-1 and zonula occludens protein 1 (ZO-1) mRNA, proteins expressions (p<0.01) respectively. GLP-2 (100 nmol/L) promote TER and TJ proteins occludin, claudin-1, and zo-1 mRNA, proteins expressions in LPS stressed and normal IPEC-J2 cells (p<0.01) respectively. In normal cells, both wortmannin and LY294002, PI3K inhibitors, prevented the mRNA and protein expressions of Akt and mTOR increase induced by GLP-2 (p<0.01) following with the significant decreasing of occludin, claudin-1, ZO-1 mRNA and proteins expressions and TER (p<0.01). In conclusion, these results indicated that GLP-2 can promote TJ’s expression and TER in LPS stressed and normal IPEC-J2 cells and GLP-2 could regulate TJ and TER through the PI3K/Akt/mTOR pathway. PMID:26954146

  14. Crumbs Affects Protein Dynamics In Anterior Regions Of The Developing Drosophila Embryo

    PubMed Central

    Firmino, João; Tinevez, Jean-Yves; Knust, Elisabeth

    2013-01-01

    Maintenance of apico-basal polarity is essential for epithelial integrity and requires particular reinforcement during tissue morphogenesis, when cells are reorganised, undergo shape changes and remodel their junctions. It is well established that epithelial integrity during morphogenetic processes depends on the dynamic exchange of adherens junction components, but our knowledge on the dynamics of other proteins and their dynamics during these processes is still limited. The early Drosophila embryo is an ideal system to study membrane dynamics during morphogenesis. Here, morphogenetic activities differ along the anterior-posterior axis, with the extending germband showing a high degree of epithelial remodelling. We developed a Fluorescence Recovery After Photobleaching (FRAP) assay with a higher temporal resolution, which allowed the distinction between a fast and a slow component of recovery of membrane proteins during the germband extension stage. We show for the first time that the recovery kinetics of a general membrane marker, SpiderGFP, differs in the anterior and posterior parts of the embryo, which correlates well with the different morphogenetic activities of the respective embryonic regions. Interestingly, absence of crumbs, a polarity regulator essential for epithelial integrity in the Drosophila embryo, decreases the fast component of SpiderGFP and of the apical marker Stranded at Second-Venus specifically in the anterior region. We suggest that the defects in kinetics observed in crumbs mutant embryos are the first signs of tissue instability in this region, explaining the earlier breakdown of the head epidermis in comparison to that of the trunk, and that diffusion in the plasma membrane is affected by the absence of Crumbs. PMID:23555600

  15. Self-Organizing Actomyosin Patterns on the Cell Cortex at Epithelial Cell-Cell Junctions

    PubMed Central

    Moore, Thomas; Wu, Selwin K.; Michael, Magdalene; Yap, Alpha S.; Gomez, Guillermo A.; Neufeld, Zoltan

    2014-01-01

    The behavior of actomyosin critically determines morphologically distinct patterns of contractility found at the interface between adherent cells. One such pattern is found at the apical region (zonula adherens) of cell-cell junctions in epithelia, where clusters of the adhesion molecule E-cadherin concentrate in a static pattern. Meanwhile, E-cadherin clusters throughout lateral cell-cell contacts display dynamic movements in the plane of the junctions. To gain insight into the principles that determine the nature and organization of these dynamic structures, we analyze this behavior by modeling the 2D actomyosin cell cortex as an active fluid medium. The numerical simulations show that the stability of the actin filaments influences the spatial structure and dynamics of the system. We find that in addition to static Turing-type patterns, persistent dynamic behavior occurs in a wide range of parameters. In the 2D model, mechanical stress-dependent actin breakdown is shown to produce a continuously changing network of actin bridges, whereas with a constant breakdown rate, more isolated clusters of actomyosin tend to form. The model qualitatively reproduces the dynamic and stable patterns experimentally observed at the junctions between epithelial cells. PMID:25468344

  16. Self-organizing actomyosin patterns on the cell cortex at epithelial cell-cell junctions.

    PubMed

    Moore, Thomas; Wu, Selwin K; Michael, Magdalene; Yap, Alpha S; Gomez, Guillermo A; Neufeld, Zoltan

    2014-12-01

    The behavior of actomyosin critically determines morphologically distinct patterns of contractility found at the interface between adherent cells. One such pattern is found at the apical region (zonula adherens) of cell-cell junctions in epithelia, where clusters of the adhesion molecule E-cadherin concentrate in a static pattern. Meanwhile, E-cadherin clusters throughout lateral cell-cell contacts display dynamic movements in the plane of the junctions. To gain insight into the principles that determine the nature and organization of these dynamic structures, we analyze this behavior by modeling the 2D actomyosin cell cortex as an active fluid medium. The numerical simulations show that the stability of the actin filaments influences the spatial structure and dynamics of the system. We find that in addition to static Turing-type patterns, persistent dynamic behavior occurs in a wide range of parameters. In the 2D model, mechanical stress-dependent actin breakdown is shown to produce a continuously changing network of actin bridges, whereas with a constant breakdown rate, more isolated clusters of actomyosin tend to form. The model qualitatively reproduces the dynamic and stable patterns experimentally observed at the junctions between epithelial cells. PMID:25468344

  17. Aberrant expression and function of gap junctions during carcinogenesis.

    PubMed Central

    Yamasaki, H

    1991-01-01

    Gap junctional intercellular communication plays a key role in the maintenance of homeostasis in multicellular organisms. Reflecting deranged homeostasis in cancer cells, most transformed or cancerous cells show aberrant gap junctional intercellular communication; they have decreased junctional communication between each other and/or with surrounding normal cells. Studies with in vitro cell transformation and animal carcinogenesis models suggest an involvement of blocked intercellular communication in later stages of carcinogenesis. Analysis of expression of gap junction proteins (connexins) and corresponding mRNA indicates that a number of regulation sites are involved in aberrant function of gap junctions during carcinogenesis. Suppression of transformed phenotypes is often seen when transformed cells are physically in contact with their normal counterparts. Some studies suggest that gap junctional intercellular communication is involved in such tumor suppression. PMID:1663449

  18. Partial nucleotide and amino acid sequences of the envelope and the envelope/nonstructural protein-1 gene junction of four dengue-2 virus strains isolated during the 1981 Cuban epidemic.

    PubMed

    Guzman, M G; Deubel, V; Pelegrino, J L; Rosario, D; Marrero, M; Sariol, C; Kouri, G

    1995-03-01

    In 1981, an epidemic of dengue hemorrhagic fever (DHF) caused by dengue-2 virus occurred in Cuba. This was the first DHF epidemic reported in the Western Hemisphere. In this study, we have analyzed four dengue-2 Cuban strains for two short genomic fragments: one on the envelope (E) glycoprotein and one at the E/nonstructural protein-1 (NS1) gene junction. The E segment of these 1981 Cuban isolates were more closely related to older dengue-2 virus strains such as New Guinea C 1944, Thailand 1964, Sri Lanka 1968, and Burma 1976 than to more recent isolates of this virus from Jamaica and Vietnam. More than 9% of the divergence with strains isolated from Jamaica and Vietnam was observed at the E/NS1 gene junction. One nucleotide change was observed between the first strain isolated during the epidemic and the rest of the Cuban strains. This mutation induced a nonconserved amino acid change from phenylalanine to leucine at position 43 that was not observed in any of the other strains with which it was compared.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27357130

  20. 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

    2016-01-01

    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. PMID:27357130

  1. 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

    2016-01-01

    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.

  2. Equivalent Josephson junctions

    NASA Astrophysics Data System (ADS)

    Boyadjiev, T. L.; Semerdjieva, E. G.; Shukrinov, Yu. M.

    2008-01-01

    The magnetic field dependences of critical current are numerically constructed for a long Josephson junction with a shunt-or resistor-type microscopic inhomogeneities and compared to the critical curve of a junction with exponentially varying width. The numerical results show that it is adequate to replace the distributed inhomogeneity of a long Josephson junction by an inhomogeneity localized at one of its ends, which has certain technological advantages. It is also shown that the critical curves of junctions with exponentially varying width and inhomogeneities localized at the ends are unaffected by the mixed fluxon-antifluxon distributions of the magnetic flow. This fact may explain the improvement of the spectra of microwave radiation noted in the literature.

  3. The Escherichia coli O157:H7 cattle immuno-proteome includes outer membrane protein A (OmpA), a modulator of adherence to bovine recto-anal junction squamous epithelial (RSE) cells

    PubMed Central

    Kudva, Indira T.; Krastins, Bryan; Torres, Alfredo G.; Griffin, Robert W.; Sheng, Haiqing; Sarracino, David A.; Hovde, Carolyn J.; Calderwood, Stephen B.; John, Manohar

    2015-01-01

    SUMMARY Building on previous studies, we defined the repertoire of proteins comprising the immuno-proteome of E. coli O157:H7 (O157) cultured in DMEM supplemented with norepinephrine (NE; O157 immuno-proteome), a β-adrenergic hormone that regulates E. coli O157 gene expression in the gastrointestinal tract, using a variation of a novel proteomics-based platform proteome mining tool for antigen discovery, called Proteomics-based Expression Library Screening (PELS; Kudva et al., 2006). The E. coli O157 immuno-proteome (O157-IP) comprised 91 proteins, and included those identified previously using PELS, and also proteins comprising DMEM- and bovine rumen fluid- proteomes. Outer membrane protein A (OmpA), a common component of the above proteomes, and reportedly a contributor to E. coli O157 adherence to cultured Hep-2 epithelial cells, was interestingly found to be a modulator rather than a contributor to E. coli O157 adherence to bovine recto-anal junction squamous epithelial (RSE) cells. Our results point to a role for yet to be identified members of the O157-IP in E. coli O157 adherence to RSE-cells, and additionally implicate a possible role for the OmpA regulator, TdcA, in the expression of such adhesins. Our observations have implications for development of efficacious vaccines for preventing E. coli O157 colonization of the bovine gastrointestinal tract. PMID:25643951

  4. Effects of Maternal Treatment with Corticosteroids on Tight Junction Protein Expression in the Cerebral Cortex of the Ovine Fetus with and without Exposure to In-Utero Brain Ischemia

    PubMed Central

    Malaeb, Shadi N; Sadowska, Grazyna B; Stonestreet, Barbara S

    2007-01-01

    Maternal treatment with corticosteroids reduces blood-brain barrier permeability in premature ovine fetuses and the incidence of intraventricular hemorrhage in premature infants. We tested the hypothesis that maternally administered corticosteroids increase the expression of tight junction (TJ) proteins in the cerebral cortex of ovine fetuses with and without exposure to in-utero brain ischemia. Fetuses at 80% of gestation were studied 18 h after the last of four 4−6 mg dexamethasone or placebo injections were given over 48 h to ewes. Groups were placebo/control, dexamethasone/control, placebo/ischemic, and dexamethasone/ischemic. Ischemia consisted of 30 min of fetal carotid artery occlusion and 72 h of reperfusion. Cerebral cortex was snap frozen. Western immunoblot was used to measure the protein expression of occludin, claudin-1, claudin-5, zonula occludens (ZO)-1, and ZO-2, and a TJ accessory protein annexin II. Occludin and annexin II protein expression were 48% and 58% higher (P<0.05) in the dexamethasone/ischemic than placebo/control group, respectively. Claudin-5 protein expression was 69% and 73% higher (P<0.05) in the placebo/ischemic and dexamethasone/ischemic than placebo/control group. Claudin-1 expression did not differ among groups. ZO-1 protein expression was 25%, 40%, and 55% lower in the dexamethasone/control, placebo/ischemic and dexamethasone/ischemic than placebo/control group, respectively. ZO-2 expression was 45% and 70% lower (P<0.01) in the placebo/ischemic and dexamethasone/ischemic than placebo/control group. We conclude that maternal corticosteroid treatment differentially regulates the expression of component proteins of TJs in the cerebral cortex of fetuses exposed to brain ischemia. The functional significance of this differential regulation warrants further investigation. PMID:17583681

  5. Presence of functional gap junctions in human embryonic stem cells.

    PubMed

    Wong, Raymond C B; Pébay, Alice; Nguyen, Linh T V; Koh, Karen L L; Pera, Martin F

    2004-01-01

    Gap junctions are intercellular channels that allow both chemical and electrical signaling between two adjacent cells. Gap junction intercellular communication has been implicated in the regulation of various cellular processes, including cell migration, cell proliferation, cell differentiation, and cell apoptosis. This study aimed to determine the presence and functionality of gap junctions in human embryonic stem cells (hESCs). Using reverse transcription--polymerase chain reaction and immunocytochemistry, we demonstrate that human ES cells express two gap junction proteins, connexin 43 and connexin 45. Western blot analysis revealed the presence of three phosphorylated forms (nonphosphorylated [NP], P1, and P2) of connexin 43, NP being prominent. Moreover, scrape loading/dye transfer assay indicates that human ES cells are coupled through functional gap junctions that are inhibited by protein kinase C activation and extracellular signal-regulated kinase inhibition.

  6. Characterization of Skin Aging-Associated Secreted Proteins (SAASP) Produced by Dermal Fibroblasts Isolated from Intrinsically Aged Human Skin.

    PubMed

    Waldera Lupa, Daniel M; Kalfalah, Faiza; Safferling, Kai; Boukamp, Petra; Poschmann, Gereon; Volpi, Elena; Götz-Rösch, Christine; Bernerd, Francoise; Haag, Laura; Huebenthal, Ulrike; Fritsche, Ellen; Boege, Fritz; Grabe, Niels; Tigges, Julia; Stühler, Kai; Krutmann, Jean

    2015-08-01

    Most molecular hallmarks of cellular senescence have been identified in studies of cells aged in vitro by driving them into replicative or stress-induced senescence. Comparatively, less is known about the characteristic features of cells that have aged in vivo. Here we provide a systematic molecular analysis of normal human dermal fibroblasts (NHDFs) that were isolated from intrinsically aged human skin of young versus middle aged versus old donors. Intrinsically aged NHDFs in culture exhibited more frequently nuclear foci positive for p53 binding protein 1 and promyelocytic leukemia protein reminiscent of 'DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS)'. Formation of such foci was neither accompanied by increased DNA double strand breaks, nor decreased cell viability, nor telomere shortening. However, it was associated with the development of a secretory phenotype, indicating incipient cell senescence. By quantitative analysis of the entire secretome present in conditioned cell culture supernatant, combined with a multiplex cytokine determination, we identified 998 proteins secreted by intrinsically aged NHDFs in culture. Seventy of these proteins exhibited an age-dependent secretion pattern and were accordingly denoted 'skin aging-associated secreted proteins (SAASP)'. Systematic comparison of SAASP with the classical senescence-associated secretory phenotype (SASP) revealed that matrix degradation as well as proinflammatory processes are common aspects of both conditions. However, secretion of 27 proteins involved in the biological processes of 'metabolism' and 'adherens junction interactions' was unique for NHDFs isolated from intrinsically aged skin. In conclusion, fibroblasts isolated from intrinsically aged skin exhibit some, but not all, molecular hallmarks of cellular senescence. Most importantly, they secrete a unique pattern of proteins that is distinct from the canonical SASP and might reflect specific processes of skin aging

  7. Characterization of Skin Aging-Associated Secreted Proteins (SAASP) Produced by Dermal Fibroblasts Isolated from Intrinsically Aged Human Skin.

    PubMed

    Waldera Lupa, Daniel M; Kalfalah, Faiza; Safferling, Kai; Boukamp, Petra; Poschmann, Gereon; Volpi, Elena; Götz-Rösch, Christine; Bernerd, Francoise; Haag, Laura; Huebenthal, Ulrike; Fritsche, Ellen; Boege, Fritz; Grabe, Niels; Tigges, Julia; Stühler, Kai; Krutmann, Jean

    2015-08-01

    Most molecular hallmarks of cellular senescence have been identified in studies of cells aged in vitro by driving them into replicative or stress-induced senescence. Comparatively, less is known about the characteristic features of cells that have aged in vivo. Here we provide a systematic molecular analysis of normal human dermal fibroblasts (NHDFs) that were isolated from intrinsically aged human skin of young versus middle aged versus old donors. Intrinsically aged NHDFs in culture exhibited more frequently nuclear foci positive for p53 binding protein 1 and promyelocytic leukemia protein reminiscent of 'DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS)'. Formation of such foci was neither accompanied by increased DNA double strand breaks, nor decreased cell viability, nor telomere shortening. However, it was associated with the development of a secretory phenotype, indicating incipient cell senescence. By quantitative analysis of the entire secretome present in conditioned cell culture supernatant, combined with a multiplex cytokine determination, we identified 998 proteins secreted by intrinsically aged NHDFs in culture. Seventy of these proteins exhibited an age-dependent secretion pattern and were accordingly denoted 'skin aging-associated secreted proteins (SAASP)'. Systematic comparison of SAASP with the classical senescence-associated secretory phenotype (SASP) revealed that matrix degradation as well as proinflammatory processes are common aspects of both conditions. However, secretion of 27 proteins involved in the biological processes of 'metabolism' and 'adherens junction interactions' was unique for NHDFs isolated from intrinsically aged skin. In conclusion, fibroblasts isolated from intrinsically aged skin exhibit some, but not all, molecular hallmarks of cellular senescence. Most importantly, they secrete a unique pattern of proteins that is distinct from the canonical SASP and might reflect specific processes of skin aging.

  8. Lactobacillus sakei OK67 ameliorates high-fat diet-induced blood glucose intolerance and obesity in mice by inhibiting gut microbiota lipopolysaccharide production and inducing colon tight junction protein expression.

    PubMed

    Lim, Su-Min; Jeong, Jin-Ju; Woo, Kyung Hee; Han, Myung Joo; Kim, Dong-Hyun

    2016-04-01

    A high-fat diet (HFD) induces obesity and the associated increases in blood glucose and inflammation through changes in gut microbiota, endotoxemia, and increased gut permeability. To counteract this, researchers have suggested that the use of probiotics that suppress production of proinflammatory lipopolysaccharide (LPS). Here, we tested whether Lactobacillus sakei OK67, which inhibits gut microbiota LPS production selected from among the lactic acid bacteria isolated from kimchi, exerted antihypoglycemic or anti-inflammatory effects in HFD-fed mice. Mice were randomly divided into 2 groups and fed an HFD or a low-fat diet for 4 weeks. These groups were further subdivided; 1 subgroup was treated with L sakei OK67 and fed the experimental diet for 4.5 weeks, whereas the other subgroup was fed the experimental diet alone. L sakei OK67 treatment lowered HFD-elevated LPS levels in blood and colonic fluid and significantly decreased HFD-elevated fasting blood glucose levels and the area under the curve in an oral glucose tolerance test. L sakei OK67 treatment inhibited HFD-induced body and epididymal fat weight gains, suppressed HFD-induced tumor necrosis factor-α and interleukin-1β expression and nuclear factor-κB activation in the colon, and significantly increased HFD-suppressed interleukin-10 and tight junction protein expression in the colon. Oral administration of L sakei OK67 significantly downregulated HFD-induced expression of peroxisome proliferator-activated receptor γ, fatty acid synthase, and tumor necrosis factor-α in adipose tissue. In addition, L sakei OK67 treatment strongly inhibited nuclear factor-κB activation in LPS-stimulated peritoneal macrophages. We report that L sakei OK67 ameliorates HFD-induced hyperglycemia and obesity by reducing inflammation and increasing the expression of colon tight junction proteins in mice. PMID:27001279

  9. The impaired intestinal mucosal immune system by valine deficiency for young grass carp (Ctenopharyngodon idella) is associated with decreasing immune status and regulating tight junction proteins transcript abundance in the intestine.

    PubMed

    Luo, Jian-Bo; Feng, Lin; Jiang, Wei-Dan; Liu, Yang; Wu, Pei; Jiang, Jun; Kuang, Sheng-Yao; Tang, Ling; Zhang, Yong-An; Zhou, Xiao-Qiu

    2014-09-01

    This study investigated the effects of dietary valine on the growth, intestinal immune response, tight junction proteins transcript abundance and gene expression of immune-related signaling molecules in the intestine of young grass carp (Ctenopharyngodon idella). Six iso-nitrogenous diets containing graded levels of valine (4.3-19.1 g kg(-)(1) diet) were fed to the fish for 8 weeks. The results showed that percentage weight gain (PWG), feed intake and feed efficiency of fish were the lowest in fish fed the valine-deficient diet (P < 0.05). In addition, valine deficiency decreased lysozyme, acid phosphatase activities and complement 3 content in the intestine (P < 0.05), down-regulated mRNA levels of interleukin 10, transforming growth factor β1, IκBα and target of rapamycin (TOR) (P < 0.05), and up-regulated tumor necrosis factor α, interleukin 8 and nuclear factor κB P65 (NF-κB P65) gene expression (P < 0.05). Additionally, valine deficiency significantly decreased transcript of Occludin, Claudin b, Claudin c, Claudin 3, and ZO-1 (P < 0.05), and improved Claudin 15 expression in the fish intestine (P < 0.05). However, valine did not have a significant effect on expression of Claudin 12 in the intestine of grass carp (P > 0.05). In conclusion, valine deficiency decreased fish growth and intestinal immune status, as well as regulated gene expression of tight junction proteins, NF-κB P65, IκBα and TOR in the fish intestine. Based on the quadratic regression analysis of lysozyme activity or PWG, the dietary valine requirement of young grass carp (268-679 g) were established to be 14.47 g kg(-1) diet (4.82 g 100 g(-1) CP) or 14.00 g kg(-1) diet (4.77 g 100 g(-1) CP), respectively.

  10. Single molecule imaging of green fluorescent proteins in living cells: E-cadherin forms oligomers on the free cell surface.

    PubMed Central

    Iino, R; Koyama, I; Kusumi, A

    2001-01-01

    Single green fluorescent protein (GFP) molecules were successfully imaged for the first time in living cells. GFP linked to the cytoplasmic carboxyl terminus of E-cadherin (E-cad-GFP) was expressed in mouse fibroblast L cells, and observed using an objective-type total internal reflection fluorescence microscope. Based on the fluorescence intensity of individual fluorescent spots, the majority of E-cad-GFP molecules on the free cell surface were found to be oligomers of various sizes, many of them greater than dimers, suggesting that oligomerization of E-cadherin takes place before its assembly at cell-cell adhesion sites. The translational diffusion coefficient of E-cad-GFP is reduced by a factor of 10 to 40 upon oligomerization. Because such large decreases in translational mobility cannot be explained solely by increases in radius upon oligomerization, an oligomerization-induced trapping model is proposed in which, when oligomers are formed, they are trapped in place due to greatly enhanced tethering and corralling effects of the membrane skeleton on oligomers (compared with monomers). The presence of many oligomers greater than dimers on the free surface suggests that these greater oligomers are the basic building blocks for the two-dimensional cell adhesion structures (adherens junctions). PMID:11371443

  11. Papain Degrades Tight Junction Proteins of Human Keratinocytes In Vitro and Sensitizes C57BL/6 Mice via the Skin Independent of its Enzymatic Activity or TLR4 Activation.

    PubMed

    Stremnitzer, Caroline; Manzano-Szalai, Krisztina; Willensdorfer, Anna; Starkl, Philipp; Pieper, Mario; König, Peter; Mildner, Michael; Tschachler, Erwin; Reichart, Ursula; Jensen-Jarolim, Erika

    2015-07-01

    Papain is commonly used in food, pharmaceutical, textile, and cosmetic industries and is known to induce occupational allergic asthma. We have previously shown that the papain-like cysteine protease Dermatophagoides pteronyssinus 1 from house dust mite exhibits percutaneous sensitization potential. We aimed here to investigate the potential of papain itself in epicutaneous sensitization. The effects of papain on tight junction (TJ) proteins were tested in vitro in human primary keratinocytes. Using C57BL/6 wild-type and Toll-like receptor 4 (TLR4)-deficient mice, we analyzed the sensitization potential of papain, its effects on the skin barrier, and immune cell recruitment. Our results show that papain affects the skin barrier by increasing transepidermal water loss, degrading TJ proteins and inducing vasodilation. When topically applied, papain exhibited a high epicutaneous inflammatory potential by recruiting neutrophils, mast cells, and CD3-positive cells and by induction of a TH2-biased antibody response. However, its high potency for specific sensitization via the skin was TLR4 independent and, in spite of its capacity to degrade epidermal TJ proteins, does not rely on its enzymatic function. From our data, we conclude that papain has all features to act as a strong allergen via the skin.

  12. Papain Degrades Tight Junction Proteins of Human Keratinocytes In Vitro and Sensitizes C57BL/6 Mice via the Skin Independent of its Enzymatic Activity or TLR4 Activation

    PubMed Central

    Willensdorfer, Anna; Starkl, Philipp; Pieper, Mario; König, Peter; Mildner, Michael; Tschachler, Erwin; Reichart, Ursula; Jensen-Jarolim, Erika

    2015-01-01

    Papain is commonly used in food, pharmaceutical, textile, and cosmetic industries and is known to induce occupational allergic asthma. We have previously shown that the papain-like cysteine protease Dermatophagoides pteronyssinus 1 from house dust mite exhibits percutaneous sensitization potential. We aimed here to investigate the potential of papain itself in epicutaneous sensitization. The effects of papain on tight junction (TJ) proteins were tested in vitro in human primary keratinocytes. Using C57BL/6 wild-type and Toll-like receptor 4 (TLR4)-deficient mice, we analyzed the sensitization potential of papain, its effects on the skin barrier, and immune cell recruitment. Our results show that papain affects the skin barrier by increasing transepidermal water loss, degrading TJ proteins and inducing vasodilation. When topically applied, papain exhibited a high epicutaneous inflammatory potential by recruiting neutrophils, mast cells, and CD3-positive cells and by induction of a TH2-biased antibody response. However, its high potency for specific sensitization via the skin was TLR4 independent and, in spite of its capacity to degrade epidermal TJ proteins, does not rely on its enzymatic function. From our data, we conclude that papain has all features to act as a strong allergen via the skin. PMID:25705851

  13. Dietary phenylalanine-improved intestinal barrier health in young grass carp (Ctenopharyngodon idella) is associated with increased immune status and regulated gene expression of cytokines, tight junction proteins, antioxidant enzymes and related signalling molecules.

    PubMed

    Feng, Lin; Li, Wen; Liu, Yang; Jiang, Wei-Dan; Kuang, Sheng-Yao; Jiang, Jun; Tang, Ling; Wu, Pei; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu

    2015-08-01

    The present work evaluated the effects of dietary phenylalanine (Phe) on the intestinal immune response, tight junction proteins transcript abundance, and the gene expression of immune- and antioxidant-related signalling molecules in the intestine. In addition, the dietary Phe (and Phe + Tyr) requirement of young grass carp (Ctenopharyngodon idella) was also estimated. Fish were fed fish meal-casein-gelatin based diets (302.3 g crude protein kg(-1)) containing 3.4 (basal diet), 6.1, 9.1, 11.5, 14.0 and 16.8 g Phe kg(-1) with a fixed amount of 10.7 g tyrosine kg(-1) for 8 weeks. The results showed that Phe deficiency or excess Phe reduced the lysozyme and acid phosphatase activities and complement C 3 content in the intestine (P < 0.05). Moreover, zonula occludens-1 (ZO-1), occludin and claudin c mRNA levels were highest in the fish fed the diet containing 11.5 g Phe kg(-1) (P < 0.05). However, claudin 12 and claudin b mRNA levels were not significantly affected by dietary Phe (P > 0.05). Gene expression of interleukin-10 (IL-10), transforming growth factor-β1 (TGF-β1), target of rapamycin (TOR) and inhibitor of nuclear factor κBα (IκBα) in proximal intestine (PI), mid intestine (MI) and distal intestine (DI) increased as dietary Phe increased up to 6.1, 9.1, 11.5 and 14.0 g kg(-1), respectively (P < 0.05). However, interleukin-8 (IL-8), tumour necrosis factor-α (TNF-α) and nuclear factor-κB p65 (NF-κB p65) mRNA levels showed opposite tendencies. In addition, the mRNA level of superoxide dismutase (SOD) was significantly lower in the intestinal tissue of the group fed a diet with Phe levels of 16.8 g kg(-1) than in those of other groups (P < 0.05). The expression of NF-E2-related factor 2 (Nrf2) gene was increased as dietary Phe increased up to 9.1 g kg(-1) (P < 0.05). In conclusion, Phe improved intestinal immune status, and regulated gene expression of cytokines, tight junction proteins, antioxidant enzymes, NF-κB p65, IκBα, TOR, and Nrf2 in the fish

  14. Changes in barrier health status of the gill for grass carp (Ctenopharyngodon idella) during valine deficiency: Regulation of tight junction protein transcript, antioxidant status and apoptosis-related gene expression.

    PubMed

    Feng, Lin; Luo, Jian-Bo; Jiang, Wei-Dan; Liu, Yang; Wu, Pei; Jiang, Jun; Kuang, Sheng-Yao; Tang, Ling; Zhang, Yong-An; Zhou, Xiao-Qiu

    2015-08-01

    This study investigated the effects of dietary valine on tight junction protein transcription, antioxidant status and apoptosis on grass carp gills (Ctenopharyngodon idella). Fish were fed six different experimental diets containing graded levels of valine (4.3, 8.0, 10.6, 13.1, 16.7, 19.1 g/kg). The results indicated that valine deficiency decreased Claudin b, Claudin 3, Occludin and ZO-1 transcription and increased Claudin 15 expression in the fish gill (P < 0.05). These effects were partly due to the down-regulation of interleukin 10 (IL-10), transforming growth factor β1 (TGF-β1) and IκB α and the up-regulation of relative mRNA expression of interleukin 1β (IL-1β), interleukin 8 (IL-8), tumor necrosis factor-α (TNF-α) and nuclear factor κB P65 (NF-κB P65) (P < 0.05). However, valine deficiency and valine supplementation did not have a significant effect on Claudin c and Claudin 12 expression in grass carp gills (P > 0.05). Valine deficiency also disrupted antioxidant status in the gill by decreasing anti-superoxide radicals and hydroxyl radical capacity, glutathione contents and the activities and mRNA levels of Cu/Zn superoxide dismutase (SOD1), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-S-transferase (GST) (P < 0.05). These results may be ascribed to the down-regulation of NF-E2-related factor 2 (Nrf2), target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1) and the up-regulation of Kelch-like-ECH-associated protein 1 (Keap1) (P < 0.05). Additionally, valine deficiency induced DNA fragmentation via the up-regulation of Caspase 3, Caspase 8 and Caspase 9 expressions (P < 0.05). These results may be ascribed to the improvement in ROS levels in the fish gill (P < 0.05). Taken together, the results showed that valine deficiency impaired the structural integrity of fish gill by disrupted fish antioxidant defenses and regulating the expression of tight junction protein, cytokines, antioxidant

  15. Four-junction superconducting circuit

    PubMed Central

    Qiu, Yueyin; Xiong, Wei; He, Xiao-Ling; Li, Tie-Fu; You, J. Q.

    2016-01-01

    We develop a theory for the quantum circuit consisting of a superconducting loop interrupted by four Josephson junctions and pierced by a magnetic flux (either static or time-dependent). In addition to the similarity with the typical three-junction flux qubit in the double-well regime, we demonstrate the difference of the four-junction circuit from its three-junction analogue, including its advantages over the latter. Moreover, the four-junction circuit in the single-well regime is also investigated. Our theory provides a tool to explore the physical properties of this four-junction superconducting circuit. PMID:27356619

  16. Four-junction superconducting circuit.

    PubMed

    Qiu, Yueyin; Xiong, Wei; He, Xiao-Ling; Li, Tie-Fu; You, J Q

    2016-01-01

    We develop a theory for the quantum circuit consisting of a superconducting loop interrupted by four Josephson junctions and pierced by a magnetic flux (either static or time-dependent). In addition to the similarity with the typical three-junction flux qubit in the double-well regime, we demonstrate the difference of the four-junction circuit from its three-junction analogue, including its advantages over the latter. Moreover, the four-junction circuit in the single-well regime is also investigated. Our theory provides a tool to explore the physical properties of this four-junction superconducting circuit. PMID:27356619

  17. Four-junction superconducting circuit.

    PubMed

    Qiu, Yueyin; Xiong, Wei; He, Xiao-Ling; Li, Tie-Fu; You, J Q

    2016-06-30

    We develop a theory for the quantum circuit consisting of a superconducting loop interrupted by four Josephson junctions and pierced by a magnetic flux (either static or time-dependent). In addition to the similarity with the typical three-junction flux qubit in the double-well regime, we demonstrate the difference of the four-junction circuit from its three-junction analogue, including its advantages over the latter. Moreover, the four-junction circuit in the single-well regime is also investigated. Our theory provides a tool to explore the physical properties of this four-junction superconducting circuit.

  18. Differential roles of proteins involved in migration of Holliday junctions on recombination and tolerance to DNA damaging agents in Rhizobium etli.

    PubMed

    Martínez-Salazar, Jaime M; Zuñiga-Castillo, Jacobo; Romero, David

    2009-03-01

    The recombination genes involved in Holliday junction migration (ruvB, recG, radA) and heteroduplex editing (mutS) were studied in the alpha-proteobacterium Rhizobium etli. The genes were interrupted with a loxPSp interposon and R. etli mutants, either single or in combination, were constructed by marker exchange. Our results show that these systems play a differential role in sensitivity to DNA damaging agents and recombination in R. etli. RuvB appears to be the main system for tolerance toward agents instigating single- or double-strand breaks (such as UV light, methyl methanesulphonate and nalidixic acid) while the RecG and RadA systems play minor roles in tolerance to these agents. Using five different recombination assays, we have found that a ruvB null mutant showed a notable reduction in recombination proficiency, while a radA mutant was only weakly affected. A null mutation in recG had the opposite effect, enhancing recombination in most of our assays. This effect was more clearly seen in an assay that measured recombination between divergent sequences (i.e. homeologous), but is unaffected by inactivation of mutS. These data indicate that RecG in R. etli limits intra- and intergenomic plasticity.

  19. Magnetic Tunnel Junctions

    NASA Astrophysics Data System (ADS)

    Reiss, Günter; Schmalhorst, Jan; Thomas, Andre; Hütten, Andreas; Yuasa, Shinji

    In magnetoelectronic devices large opportunities are opened by the spin dependent tunneling resistance, where a strong dependence of the tunneling current on the relative orientation of the magnetization of the electrodes is found. Within a short time, the amplitude of the resistance change of the junctions increased dramatically. We will cover Al-O and MgO based junctions and present highly spin-polarized electrode materials such as Heusler alloys. Furthermore, we will give a short overview on applications such as read heads in hard disk drives, storage cells in MRAMs, field programmable logic circuits and biochips. Finally, we will discuss the currently growing field of current induced magnetization switching.

  20. Dehydroepiandrosterone Sulfate Stimulates Expression of Blood-Testis-Barrier Proteins Claudin-3 and -5 and Tight Junction Formation via a Gnα11-Coupled Receptor in Sertoli Cells

    PubMed Central

    Papadopoulos, Dimitrios; Dietze, Raimund; Shihan, Mazen; Kirch, Ulrike; Scheiner-Bobis, Georgios

    2016-01-01

    Dehydroepiandrosterone sulfate (DHEAS) is a circulating sulfated steroid considered to be a pro-androgen in mammalian physiology. Here we show that at a physiological concentration (1 μM), DHEAS induces the phosphorylation of the kinase Erk1/2 and of the transcription factors CREB and ATF-1 in the murine Sertoli cell line TM4. This signaling cascade stimulates the expression of the tight junction (TJ) proteins claudin-3 and claudin-5. As a consequence of the increased expression, tight junction connections between neighboring Sertoli cells are augmented, as demonstrated by measurements of transepithelial resistance. Phosphorylation of Erk1/2, CREB, or ATF-1 is not affected by the presence of the steroid sulfatase inhibitor STX64. Erk1/2 phosphorylation was not observed when dehydroepiandrosterone (DHEA) was used instead of DHEAS. Abrogation of androgen receptor (AR) expression by siRNA did not affect DHEAS-stimulated Erk1/2 phosphorylation, nor did it change DHEAS-induced stimulation of claudin-3 and claudin-5 expression. All of the above indicate that desulfation and conversion of DHEAS into a different steroid hormone is not required to trigger the DHEAS-induced signaling cascade. All activating effects of DHEAS, however, are abolished when the expression of the G-protein Gnα11 is suppressed by siRNA, including claudin-3 and -5 expression and TJ formation between neighboring Sertoli cells as indicated by reduced transepithelial resistance. Taken together, these results are consistent with the effects of DHEAS being mediated through a membrane-bound G-protein-coupled receptor interacting with Gnα11 in a signaling pathway that resembles the non-classical signaling pathways of steroid hormones. Considering the fact that DHEAS is produced in reproductive organs, these findings also suggest that DHEAS, by acting as an autonomous steroid hormone and influencing the formation and dynamics of the TJ at the blood-testis barrier, might play a crucial role for the

  1. Role of autophagy in the regulation of epithelial cell junctions.

    PubMed

    Nighot, Prashant; Ma, Thomas

    2016-01-01

    Autophagy is a cell survival mechanism by which bulk cytoplasmic material, including soluble macromolecules and organelles, is targeted for lysosomal degradation. The role of autophagy in diverse cellular processes such as metabolic stress, neurodegeneration, cancer, aging, immunity, and inflammatory diseases is being increasingly recognized. Epithelial cell junctions play an integral role in the cell homeostasis via physical binding, regulating paracellular pathways, integrating extracellular cues into intracellular signaling, and cell-cell communication. Recent data indicates that cell junction composition is very dynamic. The junctional protein complexes are actively regulated in response to various intra- and extra-cellular clues by intracellular trafficking and degradation pathways. This review discusses the recent and emerging information on how autophagy regulates various epithelial cell junctions. The knowledge of autophagy regulation of epithelial junctions will provide further rationale for targeting autophagy in a wide variety of human disease conditions. PMID:27583189

  2. Refinement of the localization of the gene for human intra-acrosomal protein SP-10 (ACRV1) to the junction of bands q23 [yields] q24 of chromosome 11 by nonisotopic in situ hybridization

    SciTech Connect

    Golden, W.L.; Von Kap-Herr, C.; Kurth, B.; Kurth, B.; Wright, R.M.; Flickinger, C.J.; Herr, J.C. ); Eddy, R.; Shows, T. )

    1993-11-01

    The human sperm antigen SP-10 is a testis-specific protein associated with the matrix of the acrosomal vesicle in developing spermatids and the acrosomal matrix and membranes of ejaculated sperm. A previous study, utilizing somatic cell hybrids, localized the gene for SP-10 to chromosome 11 and assigned the gene symbol ACRV1 (acrosomal vesicle protein-1). Although previous analysis of several somatic cell hybrids containing portions of chromosome 11 indicated that ACRV1 was in the p12 [yields] q13 region, the present fluorescence in situ hybridization studies using cDNA, ribo, and genomic versions of probes for SP-10 coupled to analysis of an expanded series of somatic cell hybrids demonstrated the refined localization of ACRV1 to the junction of bands q23 and q24 of chromosome 11. A comparison of the three types of probes used for the in situ study demonstrated that while the genomic probe hybridized most efficiently, the riboprobe hybridized to the same location and was superior to the cDNA probe in mapping this single-copy gene. This report emphasizes the utility of riboprobes for chromosome localization of single-copy genes. 12 refs., 2 figs.

  3. Differential protein expression and oncogenic gene network link tyrosine kinase ephrin B4 receptor to aggressive gastric and gastroesophageal junction cancers.

    PubMed

    Liersch-Löhn, Britta; Slavova, Nadia; Buhr, Heinz J; Bennani-Baiti, Idriss M

    2016-03-01

    Transmembrane tyrosine-kinase Ephrin receptors promote tumor progression and/or metastasis of several malignancies including leukemia, follicular lymphoma, glioma, malignant pleural mesothelioma, papillary thyroid carcinoma, sarcomas and ovarian, breast, bladder and non-small cell lung cancers. They also drive intestinal stem cell proliferation and positioning, control intestinal tissue boundaries and are involved in liver, pancreatic and colorectal cancers, indicating involvement in additional digestive system malignancies. We investigated the role of Ephrin-B4 receptor (EPHB4), and its ligand EFNB2, in gastric and gastroesophageal junction cancers in patient cohorts through computational, mathematical, molecular and immunohistochemical analyses. We show that EPHB4 is upregulated in preneoplastic gastroesophageal lesions and its expression further increased in gastroesophageal cancers in several independent cohorts. The closely related EPHB6 receptor, which also binds EFNB2, was downregulated in all tested cohorts, consistent with its tumor-suppressive properties in other cancers. EFNB2 expression is induced in esophageal cells by acidity, suggesting that gastroesophageal reflux disease (GERD) may constitute an early triggering event in activating EFNB2-EPHB4 signaling. Association of EPHB4 to both Barrett's esophagus and to advanced tumor stages, and its overexpression at the tumor invasion front and vascular endothelial cells intimate the notion that EPHB4 may be associated with multiple steps of gastroesophageal tumorigenesis. Analysis of oncogenomic signatures uncovered the first EPHB4-associated gene network (false discovery rate: 7 × 10(-90) ) composed of a five-transcription factor interconnected gene network that drives proliferation, angiogenesis and invasiveness. The EPHB4 oncogenomic network provides a molecular basis for its role in tumor progression and points to EPHB4 as a potential tumor aggressiveness biomarker and drug target in gastroesophageal

  4. Terbium-based time-gated Förster resonance energy transfer imaging for evaluating protein-protein interactions on cell membranes.

    PubMed

    Lindén, Stina; Singh, Manish Kumar; Wegner, K David; Regairaz, Marie; Dautry, François; Treussart, François; Hildebrandt, Niko

    2015-03-21

    Fluorescence imaging of cells and subcellular compartments is an essential tool to investigate biological processes and to evaluate the development and progression of diseases. In particular, protein-protein interactions can be monitored by Förster resonance energy transfer (FRET) between two proximal fluorophores that are attached to specific recognition biomolecules such as antibodies. We investigated the membrane expression of E- and N-cadherins in three different cell lines used as model systems to study epithelial to mesenchymal transition (EMT) and a possible detection of circulating tumour cells (CTCs). EMT is a key process in cancer metastasis, during which epithelial markers (such as E-cadherin) are down-regulated in the primary tumour whereas mesenchymal markers (such as N-cadherin) are up-regulated, leading to enhanced cell motility, intravasation, and appearance of CTCs. Various FRET donor-acceptor pairs and protein recognition strategies were utilized, in which Lumi4-Tb terbium complexes (Tb) and different organic dyes were conjugated to several distinct E- and N-cadherin-specific antibodies. Pulsed excitation of Tb at low repetition rates (100 Hz) and time-gated (TG) imaging of both the Tb-donor and the dye-acceptor photoluminescence (PL) allowed efficient detection of the EMT markers as well as FRET in the case of sufficient donor-acceptor proximity. Efficient FRET was observed only between two E-cadherin-specific antibodies and further experiments indicated that these antibodies recognized the same E-cadherin molecule, suggesting a limited accessibility of cadherins when they are clustered at adherens junctions. The investigated Tb-to-dye FRET systems provided reduced photobleaching compared to the AlexaFluor 488-568 donor-acceptor pair. Our results demonstrate the applicability and advantages of Tb-based TG FRET for efficient and stable imaging of antibody-antibody interactions on different cell lines. They also reveal the limitations of

  5. Zebrafish Prion Protein PrP2 Controls Collective Migration Process during Lateral Line Sensory System Development

    PubMed Central

    Huc-Brandt, Sylvaine; Hieu, Nelson; Imberdis, Thibaut; Cubedo, Nicolas; Silhol, Michelle; Leighton, Patricia L. A.; Domaschke, Thomas; Allison, W. Ted; Perrier, Véronique; Rossel, Mireille

    2014-01-01

    Prion protein is involved in severe neurodegenerative disorders but its physiological role is still in debate due to an absence of major developmental defects in knockout mice. Previous reports in zebrafish indicate that the two prion genes, PrP1 and PrP2, are both involved in several steps of embryonic development thus providing a unique route to discover prion protein function. Here we investigate the role of PrP2 during development of a mechano-sensory system, the posterior lateral line, using morpholino knockdown and PrP2 targeted inactivation. We confirm the efficiency of the translation blocking morpholino at the protein level. Development of the posterior lateral line is altered in PrP2 morphants, including nerve axonal outgrowth and primordium migration defects. Reduced neuromast deposition was observed in PrP2 morphants as well as in PrP2−/− mutants. Rosette formation defects were observed in PrP2 morphants, strongly suggesting an abnormal primordium organization and reflecting loss of cell cohesion during migration of the primordium. In addition, the adherens junction proteins, E-cadherin and ß-catenin, were mis-localized after reduction of PrP2 expression and thus contribute to the primordium disorganization. Consequently, hair cell differentiation and number were affected and this resulted in reduced functional neuromasts. At later developmental stages, myelination of the posterior lateral line nerve was altered. Altogether, our study reports an essential role of PrP2 in collective migration process of the primordium and in neuromast formation, further implicating a role for prion protein in cell adhesion. PMID:25436888

  6. The selective binding and transmigration of monocytes through the junctional complexes of human endothelium

    PubMed Central

    1988-01-01

    Human monocytes show a high affinity for vascular endothelium both in vitro and in vivo. To explore monocyte-endothelial interaction in greater detail, we have developed a new in vitro model for growth of human endothelial cells (EC). Human umbilical vein EC (HUVEC) cultured upon collagen gels form confluent monolayers of EC that bind silver at their intercellular border similar to cells in situ. Intercellular junctional structures, both adherens and tight junctions, were identified. In contrast, HUVEC grown on plastic surfaces did not stain with silver. The silver-staining characteristic of EC-collagen monolayers was reversible and related to their in vitro maturation and senescence. Silver staining of EC borders provided a grid by which the location of monocyte binding to the luminal surface of individual EC could be assessed. Using this technique, we found that monocytes preferentially bound to the margins of EC, in approximation to the silver-staining junctions. These results suggest that EC determinants recognized by monocytes occur in a unique topographical distribution on the apical face of EC. After binding, monocytes migrated through the EC monolayers at high basal rates. The lack of penetration of collagen gels in the absence of an EC monolayer suggested the generation of EC- specific chemotactic signal(s). Monocytes were observed to pass between EC without evidence of disruption of the monolayer. Silver stain remained present during all phases of migration, and under transmission electron microscopy, junctional complexes were found proximal to monocytes that had just completed their passage through the monolayer. After orientation to the basal surface of the EC monolayer, monocytes migrated randomly into the underlying collagen gel. Monocyte adherence, penetration, migration, and long term survival can be studied under these conditions. PMID:3183575

  7. Victory Junction Gang Camp

    ERIC Educational Resources Information Center

    Shell, Ryan

    2007-01-01

    This article describes the Victory Junction Gang Camp, a not-for-profit, NASCAR-themed camp for children with chronic medical conditions that serves 24 different disease groups. The mission of the camp is to give children life-changing camping experiences that are exciting, fun, and empowering in a safe and medically sound environment. While doing…

  8. Expression of claudins, occludin, junction adhesion molecule A and zona occludens 1 in canine organs.

    PubMed

    Ahn, Changhwan; Shin, Da-Hye; Lee, Dongoh; Kang, Su-Myung; Seok, Ju-Hyung; Kang, Hee Young; Jeung, Eui-Bae

    2016-10-01

    Tight junctions are the outermost structures of intercellular junctions and are classified as transmembrane proteins. These factors form selective permeability barriers between cells, act as paracellular transporters and regulate structural and functional polarity of cells. Although tight junctions have been previously studied, comparison of the transcriptional‑translational levels of these molecules in canine organs remains to be investigated. In the present study, organ‑specific expression of the tight junction proteins, claudin, occludin, junction adhesion molecule A and zona occludens 1 was examined in the canine duodenum, lung, liver and kidney. Results of immunohistochemistry analysis demonstrated that the tight junctions were localized in intestinal villi and glands of the duodenum, bronchiolar epithelia and alveolar walls of the lung, endometrium and myometrium of the hepatocytes, and the distal tubules and glomeruli of the kidney. These results suggest that tight junctions are differently expressed in organs, and therefore may be involved in organ‑specific functions to maintain physiological homeostasis. PMID:27600198

  9. Expression of claudins, occludin, junction adhesion molecule A and zona occludens 1 in canine organs

    PubMed Central

    Ahn, Changhwan; Shin, Da-Hye; Lee, Dongoh; Kang, Su-Myung; Seok, Ju-Hyung; Kang, Hee Young; Jeung, Eui-Bae

    2016-01-01

    Tight junctions are the outermost structures of intercellular junctions and are classified as transmembrane proteins. These factors form selective permeability barriers between cells, act as paracellular transporters and regulate structural and functional polarity of cells. Although tight junctions have been previously studied, comparison of the transcriptional-translational levels of these molecules in canine organs remains to be investigated. In the present study, organ-specific expression of the tight junction proteins, claudin, occludin, junction adhesion molecule A and zona occludens 1 was examined in the canine duodenum, lung, liver and kidney. Results of immunohistochemistry analysis demonstrated that the tight junctions were localized in intestinal villi and glands of the duodenum, bronchiolar epithelia and alveolar walls of the lung, endometrium and myometrium of the hepatocytes, and the distal tubules and glomeruli of the kidney. These results suggest that tight junctions are differently expressed in organs, and therefore may be involved in organ-specific functions to maintain physiological homeostasis. PMID:27600198

  10. Mechanical tugging force regulates the size of cell–cell junctions

    PubMed Central

    Liu, Zhijun; Tan, John L.; Cohen, Daniel M.; Yang, Michael T.; Sniadecki, Nathan J.; Ruiz, Sami Alom; Nelson, Celeste M.; Chen, Christopher S.

    2010-01-01

    Actomyosin contractility affects cellular organization within tissues in part through the generation of mechanical forces at sites of cell–matrix and cell–cell contact. While increased mechanical loading at cell–matrix adhesions results in focal adhesion growth, whether forces drive changes in the size of cell–cell adhesions remains an open question. To investigate the responsiveness of adherens junctions (AJ) to force, we adapted a system of microfabricated force sensors to quantitatively report cell–cell tugging force and AJ size. We observed that AJ size was modulated by endothelial cell–cell tugging forces: AJs and tugging force grew or decayed with myosin activation or inhibition, respectively. Myosin-dependent regulation of AJs operated in concert with a Rac1, and this coordinated regulation was illustrated by showing that the effects of vascular permeability agents (S1P, thrombin) on junctional stability were reversed by changing the extent to which these agents coupled to the Rac and myosin-dependent pathways. Furthermore, direct application of mechanical tugging force, rather than myosin activity per se, was sufficient to trigger AJ growth. These findings demonstrate that the dynamic coordination of mechanical forces and cell–cell adhesive interactions likely is critical to the maintenance of multicellular integrity and highlight the need for new approaches to study tugging forces. PMID:20463286

  11. Electrostatically driven spatial patterns at supported lipid membrane junctions

    NASA Astrophysics Data System (ADS)

    Parthasarathy, Raghuveer

    2005-03-01

    We have recently shown that mobile, membrane-bound proteins sandwiched at simple, cell-free junctions between lipid bilayers can organize themselves into micron-scale spatial patterns. This pattern formation is mechanical in origin, a consequence of the coupling of the lateral mobility of the proteins and inter-membrane adhesion forces. We find that these mechanically driven protein patterns can electrostatically generate patterns of charged membrane lipids. Measuring the magnitude of the electrostatic interaction as a function of lipid composition and ionic strength, and quantitatively analyzing the interplay between thermodynamics and electrostatics via a Poisson-Boltzmann approach, we are able to determine the charge densities and surface potentials of the components of our junctions -- properties that are difficult or impossible to measure by other means. Surprisingly, the electrostatic potential of the proteins is a minor factor in the lipid reorganization; the protein size and its modulation of the junction topography play the dominant role in driving the electrostatic patterns.

  12. Epithelial junctions and Rho family GTPases: the zonular signalosome

    PubMed Central

    Citi, Sandra; Guerrera, Diego; Spadaro, Domenica; Shah, Jimit

    2014-01-01

    The establishment and maintenance of epithelial cell-cell junctions is crucially important to regulate adhesion, apico-basal polarity and motility of epithelial cells, and ultimately controls the architecture and physiology of epithelial organs. Junctions are supported, shaped and regulated by cytoskeletal filaments, whose dynamic organization and contractility are finely tuned by GTPases of the Rho family, primarily RhoA, Rac1 and Cdc42. Recent research has identified new molecular mechanisms underlying the cross-talk between these GTPases and epithelial junctions. Here we briefly summarize the current knowledge about the organization, molecular evolution and cytoskeletal anchoring of cell-cell junctions, and we comment on the most recent advances in the characterization of the interactions between Rho GTPases and junctional proteins, and their consequences with regards to junction assembly and regulation of cell behavior in vertebrate model systems. The concept of “zonular signalosome” is proposed, which highlights the close functional relationship between proteins of zonular junctions (zonulae occludentes and adhaerentes) and the control of cytoskeletal organization and signaling through Rho GTPases, transcription factors, and their effectors. PMID:25483301

  13. Epithelial junctions and Rho family GTPases: the zonular signalosome.

    PubMed

    Citi, Sandra; Guerrera, Diego; Spadaro, Domenica; Shah, Jimit

    2014-01-01

    The establishment and maintenance of epithelial cell-cell junctions is crucially important to regulate adhesion, apico-basal polarity and motility of epithelial cells, and ultimately controls the architecture and physiology of epithelial organs. Junctions are supported, shaped and regulated by cytoskeletal filaments, whose dynamic organization and contractility are finely tuned by GTPases of the Rho family, primarily RhoA, Rac1 and Cdc42. Recent research has identified new molecular mechanisms underlying the cross-talk between these GTPases and epithelial junctions. Here we briefly summarize the current knowledge about the organization, molecular evolution and cytoskeletal anchoring of cell-cell junctions, and we comment on the most recent advances in the characterization of the interactions between Rho GTPases and junctional proteins, and their consequences with regards to junction assembly and regulation of cell behavior in vertebrate model systems. The concept of "zonular signalosome" is proposed, which highlights the close functional relationship between proteins of zonular junctions (zonulae occludentes and adhaerentes) and the control of cytoskeletal organization and signaling through Rho GTPases, transcription factors, and their effectors.

  14. Isolation and purification of gap junction channels.

    PubMed

    Stauffer, K A; Kumar, N M; Gilula, N B; Unwin, N

    1991-10-01

    This paper reports methods we have developed to solubilize gap junction channels, or connexons, from isolated gap junctions and to purify them in milligram quantities. Two sources of material are used: rat liver gap junctions and gap junctions produced by infecting insect cells with a baculovirus containing the cDNA for human liver beta 1 protein (connexin 32). Complete solubilization is obtained with long chain detergents (lauryl dimethyl amineoxide, dodecyl maltoside) and requires high ionic strength and high pH as well as reducing conditions. The purification involves chromatography on hydroxylapatite and gel filtration on Superose 6. A homogeneous product is indicated by a single band on a silver-stained gel and a homogeneous population of doughnut-shaped particles under the electron microscope. These particles have hexameric symmetry. The purified connexons have a tendency to form aggregates: filaments and sheets. The filaments grow by end-to-end association of connexons and are nonpolar, suggesting that the connexons are paired as in the cell-to-cell channel. The sheets grow by lateral association of the filaments.

  15. Holliday Junction Resolvases

    PubMed Central

    Wyatt, Haley D.M.; West, Stephen C.

    2014-01-01

    Four-way DNA intermediates, called Holliday junctions (HJs), can form during meiotic and mitotic recombination, and their removal is crucial for chromosome segregation. A group of ubiquitous and highly specialized structure-selective endonucleases catalyze the cleavage of HJs into two disconnected DNA duplexes in a reaction called HJ resolution. These enzymes, called HJ resolvases, have been identified in bacteria and their bacteriophages, archaea, and eukaryotes. In this review, we discuss fundamental aspects of the HJ structure and their interaction with junction-resolving enzymes. This is followed by a brief discussion of the eubacterial RuvABC enzymes, which provide the paradigm for HJ resolvases in other organisms. Finally, we review the biochemical and structural properties of some well-characterized resolvases from archaea, bacteriophage, and eukaryotes. PMID:25183833

  16. Wireless Josephson Junction Arrays

    NASA Astrophysics Data System (ADS)

    Adams, Laura

    2015-03-01

    We report low temperature, microwave transmission measurements on a wireless two- dimensional network of Josephson junction arrays composed of superconductor-insulator -superconductor tunnel junctions. Unlike their biased counterparts, by removing all electrical contacts to the arrays and superfluous microwave components and interconnects in the transmission line, we observe new collective behavior in the transmission spectra. In particular we will show emergent behavior that systematically responds to changes in microwave power at fixed temperature. Likewise we will show the dynamic and collective response of the arrays while tuning the temperature at fixed microwave power. We discuss these spectra in terms of the Berezinskii-Kosterlitz-Thouless phase transition and Shapiro steps. We gratefully acknowledge the support Prof. Steven Anlage at the University of Maryland and Prof. Allen Goldman at the University of Minnesota. Physics and School of Engineering and Applied Sciences.

  17. Gene knockout using transcription activator-like effector nucleases (TALENs) reveals that human NDUFA9 protein is essential for stabilizing the junction between membrane and matrix arms of complex I.

    PubMed

    Stroud, David A; Formosa, Luke E; Wijeyeratne, Xiaonan W; Nguyen, Thanh N; Ryan, Michael T

    2013-01-18

    Transcription activator-like effector nucleases (TALENs) represent a promising approach for targeted knock-out of genes in cultured human cells. We used TALEN-technology to knock out the nuclear gene encoding NDUFA9, a subunit of mitochondrial respiratory chain complex I in HEK293T cells. Screening for the knock-out revealed a mixture of NDUFA9 cell clones that harbored partial deletions of the mitochondrial N-terminal targeting signal but were still capable of import. A cell line lacking functional copies of both NDUFA9 alleles resulted in a loss of NDUFA9 protein expression, impaired assembly of complex I, and cells incapable of growth in galactose medium. Cells lacking NDUFA9 contained a complex I subcomplex consisting of membrane arm subunits but not marker subunits of the matrix arm. Re-expression of NDUFA9 restored the defects in complex I assembly. We conclude that NDUFA9 is involved in stabilizing the junction between membrane and matrix arms of complex I, a late assembly step critical for complex I biogenesis and activity.

  18. Anchored PKA as a gatekeeper for gap junctions

    PubMed Central

    Pidoux, Guillaume; Taskén, Kjetil

    2015-01-01

    Anchored protein kinase A (PKA) bound to A Kinase Anchoring Protein (AKAP) mediates effects of localized increases in cAMP in defined subcellular microdomains and retains the specificity in cAMP-PKA signaling to distinct extracellular stimuli. Gap junctions are pores between adjacent cells constituted by connexin proteins that provide means of communication and transfer of small molecules. While the PKA signaling is known to promote human trophoblast cell fusion, the gap junction communication through connexin 43 (Cx43) is a prerequisite for this process. We recently demonstrated that trophoblast fusion is regulated by ezrin, a known AKAP, which binds to Cx43 and delivers PKA in the vicinity gap junctions. We found that disruption of the ezrin-Cx43 interaction abolished PKA-dependent phosphorylation of Cx43 as well as gap junction communication and subsequently cell fusion. We propose that the PKA-ezrin-Cx43 macromolecular complex regulating gap junction communication constitutes a general mechanism to control opening of Cx43 gap junctions by phosphorylation in response to cAMP signaling in various cell types. PMID:26478781

  19. Thermoelectricity in molecular junctions.

    PubMed

    Reddy, Pramod; Jang, Sung-Yeon; Segalman, Rachel A; Majumdar, Arun

    2007-03-16

    By trapping molecules between two gold electrodes with a temperature difference across them, the junction Seebeck coefficients of 1,4-benzenedithiol (BDT), 4,4'-dibenzenedithiol, and 4,4''-tribenzenedithiol in contact with gold were measured at room temperature to be +8.7 +/- 2.1 microvolts per kelvin (muV/K), +12.9 +/- 2.2 muV/K, and +14.2 +/- 3.2 muV/K, respectively (where the error is the full width half maximum of the statistical distributions). The positive sign unambiguously indicates p-type (hole) conduction in these heterojunctions, whereas the Au Fermi level position for Au-BDT-Au junctions was identified to be 1.2 eV above the highest occupied molecular orbital level of BDT. The ability to study thermoelectricity in molecular junctions provides the opportunity to address these fundamental unanswered questions about their electronic structure and to begin exploring molecular thermoelectric energy conversion. PMID:17303718

  20. Fractional order junctions

    NASA Astrophysics Data System (ADS)

    Machado, J. Tenreiro

    2015-01-01

    Gottfried Leibniz generalized the derivation and integration, extending the operators from integer up to real, or even complex, orders. It is presently recognized that the resulting models capture long term memory effects difficult to describe by classical tools. Leon Chua generalized the set of lumped electrical elements that provide the building blocks in mathematical models. His proposal of the memristor and of higher order elements broadened the scope of variables and relationships embedded in the development of models. This paper follows the two directions and proposes a new logical step, by generalizing the concept of junction. Classical junctions interconnect system elements using simple algebraic restrictions. Nevertheless, this simplistic approach may be misleading in the presence of unexpected dynamical phenomena and requires including additional "parasitic" elements. The novel γ -junction includes, as special cases, the standard series and parallel connections and allows a new degree of freedom when building models. The proposal motivates the search for experimental and real world manifestations of the abstract conjectures.

  1. Regulatory and junctional proteins of the blood-testis barrier in human Sertoli cells are modified by monobutyl phthalate (MBP) and bisphenol A (BPA) exposure.

    PubMed

    de Freitas, André Teves Aquino Gonçalves; Ribeiro, Mariana Antunes; Pinho, Cristiane Figueiredo; Peixoto, André Rebelo; Domeniconi, Raquel Fantin; Scarano, Wellerson R

    2016-08-01

    The blood-testis barrier (BTB) is responsible for providing a protected environment and coordinating the spermatogenesis. Endocrine disruptors (EDs) might lead to infertility, interfering in the BTB structure and modulation. This study aimed to correlate the actions of two EDs, monobutyl phthalate (MBP) and bisphenol A (BPA) in different periods of exposure, in a low toxicity dose to the human Sertoli cells (HSeC) and its effects on the proteins of the BTB and regulatory proteins involved in its modulation. HSeC cells were exposed to MBP (10μM) and BPA (20μM) for 6 and 48h. Western Blot assay indicated that MBP was able to reduce the expression of occludin, ZO-1, N-cadherin and Androgen Receptor (AR), while BPA leads to a reduction of occludin, ZO-1, β-catenin and AR. TGF-β2 and F-actin were not modified. Phalloidin and Hematoxylin and Eosin assay revealed phenotically disruption in Sertoli cells adhesion, without changes in F-actin expression or localization. Our data suggested both EDs present potential for disrupting the structure and maintenance of the human BTB by AR dependent pathway.

  2. Oregano Essential Oil Improves Intestinal Morphology and Expression of Tight Junction Proteins Associated with Modulation of Selected Intestinal Bacteria and Immune Status in a Pig Model.

    PubMed

    Zou, Yi; Xiang, Quanhang; Wang, Jun; Peng, Jian; Wei, Hongkui

    2016-01-01

    Oregano essential oil (OEO) has long been used to improve the health of animals, particularly the health of intestine, which is generally attributed to its antimicrobial and anti-inflammatory effects. However, how OEO acts in the intestine of pig is still unclear. This study was aimed at elucidating how OEO promotes the intestinal barrier integrity in a pig model. Pigs were fed a control diet alone or one supplemented with 25 mg/kg of OEO for 4 weeks. The OEO-treated pigs showed decreased (P < 0.05) endotoxin level in serum and increased (P < 0.05) villus height and expression of occludin and zonula occludens-1 (ZO-1) in the jejunum. These results demonstrated that the integrity of intestinal barrier was improved by OEO treatment. The OEO-treated pigs had a lower (P < 0.05) population of Escherichia coli in the jejunum, ileum, and colon than the control. This is in accordance with the greater inactivation (P < 0.05) of inflammation, which was reflected by the mitogen-activated protein kinase (MAPK), protein kinase B (Akt), and nuclear factor κB (NF-κB) signaling pathways and expression of inflammatory cytokines in the jejunum. Our results show that OEO promotes intestinal barrier integrity, probably through modulating intestinal bacteria and immune status in pigs. PMID:27314026

  3. Oregano Essential Oil Improves Intestinal Morphology and Expression of Tight Junction Proteins Associated with Modulation of Selected Intestinal Bacteria and Immune Status in a Pig Model

    PubMed Central

    Zou, Yi; Xiang, Quanhang; Wang, Jun; Peng, Jian; Wei, Hongkui

    2016-01-01

    Oregano essential oil (OEO) has long been used to improve the health of animals, particularly the health of intestine, which is generally attributed to its antimicrobial and anti-inflammatory effects. However, how OEO acts in the intestine of pig is still unclear. This study was aimed at elucidating how OEO promotes the intestinal barrier integrity in a pig model. Pigs were fed a control diet alone or one supplemented with 25 mg/kg of OEO for 4 weeks. The OEO-treated pigs showed decreased (P < 0.05) endotoxin level in serum and increased (P < 0.05) villus height and expression of occludin and zonula occludens-1 (ZO-1) in the jejunum. These results demonstrated that the integrity of intestinal barrier was improved by OEO treatment. The OEO-treated pigs had a lower (P < 0.05) population of Escherichia coli in the jejunum, ileum, and colon than the control. This is in accordance with the greater inactivation (P < 0.05) of inflammation, which was reflected by the mitogen-activated protein kinase (MAPK), protein kinase B (Akt), and nuclear factor κB (NF-κB) signaling pathways and expression of inflammatory cytokines in the jejunum. Our results show that OEO promotes intestinal barrier integrity, probably through modulating intestinal bacteria and immune status in pigs. PMID:27314026

  4. Pado, a fluorescent protein with proton channel activity can optically monitor membrane potential, intracellular pH, and map gap junctions

    PubMed Central

    Kang, Bok Eum; Baker, Bradley J.

    2016-01-01

    An in silico search strategy was developed to identify potential voltage-sensing domains (VSD) for the development of genetically encoded voltage indicators (GEVIs). Using a conserved charge distribution in the S2 α-helix, a single in silico search yielded most voltage-sensing proteins including voltage-gated potassium channels, voltage-gated calcium channels, voltage-gated sodium channels, voltage-gated proton channels, and voltage-sensing phosphatases from organisms ranging from mammals to bacteria and plants. A GEVI utilizing the VSD from a voltage-gated proton channel identified from that search was able to optically report changes in membrane potential. In addition this sensor was capable of manipulating the internal pH while simultaneously reporting that change optically since it maintains the voltage-gated proton channel activity of the VSD. Biophysical characterization of this GEVI, Pado, demonstrated that the voltage-dependent signal was distinct from the pH-dependent signal and was dependent on the movement of the S4 α-helix. Further investigation into the mechanism of the voltage-dependent optical signal revealed that inhibiting the dimerization of the fluorescent protein greatly reduced the optical signal. Dimerization of the FP thereby enabled the movement of the S4 α-helix to mediate a fluorescent response. PMID:27040905

  5. Signatures of topological Josephson junctions

    NASA Astrophysics Data System (ADS)

    Peng, Yang; Pientka, Falko; Berg, Erez; Oreg, Yuval; von Oppen, Felix

    2016-08-01

    Quasiparticle poisoning and diabatic transitions may significantly narrow the window for the experimental observation of the 4 π -periodic dc Josephson effect predicted for topological Josephson junctions. Here, we show that switching-current measurements provide accessible and robust signatures for topological superconductivity which persist in the presence of quasiparticle poisoning processes. Such measurements provide access to the phase-dependent subgap spectrum and Josephson currents of the topological junction when incorporating it into an asymmetric SQUID together with a conventional Josephson junction with large critical current. We also argue that pump-probe experiments with multiple current pulses can be used to measure the quasiparticle poisoning rates of the topological junction. The proposed signatures are particularly robust, even in the presence of Zeeman fields and spin-orbit coupling, when focusing on short Josephson junctions. Finally, we also consider microwave excitations of short topological Josephson junctions which may complement switching-current measurements.

  6. GRAF1a is a brain-specific protein that promotes lipid droplet clustering and growth, and is enriched at lipid droplet junctions

    PubMed Central

    Lucken-Ardjomande Häsler, Safa; Vallis, Yvonne; Jolin, Helen E.; McKenzie, Andrew N.; McMahon, Harvey T.

    2014-01-01

    ABSTRACT Lipid droplets are found in all cell types. Normally present at low levels in the brain, they accumulate in tumours and are associated with neurodegenerative diseases. However, little is known about the mechanisms controlling their homeostasis in the brain. We found that GRAF1a, the longest GRAF1 isoform (GRAF1 is also known as ARHGAP26), was enriched in the brains of neonates. Endogenous GRAF1a was found on lipid droplets in oleic-acid-fed primary glial cells. Exclusive localization required a GRAF1a-specific hydrophobic segment and two membrane-binding regions, a BAR and a PH domain. Overexpression of GRAF1a promoted lipid droplet clustering, inhibited droplet mobility and severely perturbed lipolysis following the chase of cells overloaded with fatty acids. Under these conditions, GRAF1a concentrated at the interface between lipid droplets. Although GRAF1-knockout mice did not show any gross abnormal phenotype, the total lipid droplet volume that accumulated in GRAF1−/− primary glia upon incubation with fatty acids was reduced compared to GRAF1+/+ cells. These results provide additional insights into the mechanisms contributing to lipid droplet growth in non-adipocyte cells, and suggest that proteins with membrane sculpting BAR domains play a role in droplet homeostasis. PMID:25189622

  7. Distinct roles of secreted HtrA proteases from gram-negative pathogens in cleaving the junctional protein and tumor suppressor E-cadherin.

    PubMed

    Hoy, Benjamin; Geppert, Tim; Boehm, Manja; Reisen, Felix; Plattner, Patrick; Gadermaier, Gabriele; Sewald, Norbert; Ferreira, Fatima; Briza, Peter; Schneider, Gisbert; Backert, Steffen; Wessler, Silja

    2012-03-23

    The periplasmic chaperone and serine protease HtrA is important for bacterial stress responses and protein quality control. Recently, we discovered that HtrA from Helicobacter pylori is secreted and cleaves E-cadherin to disrupt the epithelial barrier, but it remained unknown whether this maybe a general virulence mechanism. Here, we show that important other pathogens including enteropathogenic Escherichia coli, Shigella flexneri, and Campylobacter jejuni, but not Neisseria gonorrhoeae, cleaved E-cadherin on host cells. HtrA deletion in C. jejuni led to severe defects in E-cadherin cleavage, loss of cell adherence, paracellular transmigration, and basolateral invasion. Computational modeling of HtrAs revealed a conserved pocket in the active center exhibiting pronounced proteolytic activity. Differential E-cadherin cleavage was determined by an alanine-to-glutamine exchange in the active center of neisserial HtrA. These data suggest that HtrA-mediated E-cadherin cleavage is a prevalent pathogenic mechanism of multiple gram-negative bacteria representing an attractive novel target for therapeutic intervention to combat bacterial infections. PMID:22337879

  8. Protein kinase C-mediated changes in synaptic efficacy at the neuromuscular junction in vitro: the role of postsynaptic acetylcholine receptors.

    PubMed

    Lanuza, M A; Li, M X; Jia, M; Kim, S; Davenport, R; Dunlap, V; Nelson, P G

    2000-09-15

    Activation of a mouse in vitro neuromuscular synapse produces a reduction in synaptic efficacy which is greater for nonactivated than for activated inputs to the myotubes. This has been shown to require thrombin and thrombin receptor activation and to involve a protein kinase C (PKC)-mediated step. We show in the present work that phorbol ester activation of PKC produces physiological loss of synapses in a time- and dose-related manner. We observe, using quantitative imaging methods, a parallel loss of acetylcholine receptors (AChR) from synaptically functional neurite-associated receptor aggregates in nerve-muscle cocultures. Biochemical measurements of total AChR show that PKC activation reduces both AChR stability (increases receptor loss) and receptor insertion into the surface membrane. Taken together, the data suggest that PKC activation decreases the stability of AChR aggregates in the muscle surface membrane. We conclude that PKC plays a crucial role in activity-dependent synapse reduction and does so, at least in part, by altering AChR stability. PMID:10972958

  9. An induced junction photovoltaic cell

    NASA Technical Reports Server (NTRS)

    Call, R. L.

    1974-01-01

    Silicon solar cells operating with induced junctions rather than diffused junctions have been fabricated and tested. Induced junctions were created by forming an inversion layer near the surface of the silicon by supplying a sheet of positive charge above the surface. Measurements of the response of the inversion layer cell to light of different wavelengths indicated it to be more sensitive to the shorter wavelengths of the sun's spectrum than conventional cells. The greater sensitivity occurs because of the shallow junction and the strong electric field at the surface.

  10. GUARD RING SEMICONDUCTOR JUNCTION

    DOEpatents

    Goulding, F.S.; Hansen, W.L.

    1963-12-01

    A semiconductor diode having a very low noise characteristic when used under reverse bias is described. Surface leakage currents, which in conventional diodes greatly contribute to noise, are prevented from mixing with the desired signal currents. A p-n junction is formed with a thin layer of heavily doped semiconductor material disposed on a lightly doped, physically thick base material. An annular groove cuts through the thin layer and into the base for a short distance, dividing the thin layer into a peripheral guard ring that encircles the central region. Noise signal currents are shunted through the guard ring, leaving the central region free from such currents. (AEC)

  11. Cementoenamel junction: An insight.

    PubMed

    Vandana, Kharidi Laxman; Haneet, Ryana Kour

    2014-09-01

    The location and nature of cemento-enamel junction (CEJ) are more than descriptive terms used simply to describe some aspects of tooth morphology; however, CEJ gains a lot of clinical significance due to various measurements dependent on it. It may be necessary to determine the location and pathological changes occurring at CEJ to make a diagnosis and treat diseases pertaining to epithelial attachment and gingival margin. However, the information related to CEJ is not discussed commonly. Hence, the present review paper provides an insight on CEJ in both primary and permanent dentition. PMID:25425813

  12. Cementoenamel junction: An insight

    PubMed Central

    Vandana, Kharidi Laxman; Haneet, Ryana Kour

    2014-01-01

    The location and nature of cemento-enamel junction (CEJ) are more than descriptive terms used simply to describe some aspects of tooth morphology; however, CEJ gains a lot of clinical significance due to various measurements dependent on it. It may be necessary to determine the location and pathological changes occurring at CEJ to make a diagnosis and treat diseases pertaining to epithelial attachment and gingival margin. However, the information related to CEJ is not discussed commonly. Hence, the present review paper provides an insight on CEJ in both primary and permanent dentition. PMID:25425813

  13. Aberrant expression of E-cadherin and β-catenin proteins in placenta of bovine embryos derived from somatic cell nuclear transfer.

    PubMed

    Kohan-Ghadr, H R; Smith, L C; Arnold, D R; Murphy, B D; Lefebvre, R C

    2012-01-01

    Abnormal placental development is common in the bovine somatic cell nuclear transfer (SCNT)-derived fetus. In the present study, we characterised the expression of E-cadherin and β-catenin, structural proteins of adherens junctions, in SCNT gestations as a model for impaired placentation. Cotyledonary tissues were separated from pregnant uteri of SCNT (n = 6) and control pregnancies (n = 8) obtained by artificial insemination. Samples were analysed by western blot, quantitative RT-PCR (qRT-PCR) and immunohistochemistry. Bovine trophectoderm cell lines derived from SCNT and control embryos were analysed to compare with the in utero condition. Although no differences in E-cadherin or β-catenin mRNA abundance were observed in fetal tissues between the two groups, proteins encoded by these genes were markedly under-expressed in SCNT trophoblast cells. Immunohistochemistry revealed a different pattern of E-cadherin and total β-catenin localisation in SCNT placentas compared with controls. No difference was observed in subcellular localisation of dephosphorylated active-β-catenin protein in SCNT tissues compared with controls. However, qRT-PCR confirmed that the wingless (WNT)/β-catenin signalling pathway target genes CCND1, CLDN1 and MSX1 were downregulated in SCNT placentas. No differences were detected between two groups of bovine trophectoderm cell lines. Our results suggest that impaired expression of E-cadherin and β-catenin proteins, along with defective β-catenin signalling during embryo attachment, specifically during placentation, is a molecular mechanism explaining insufficient placentation in the bovine SCNT-derived fetus.

  14. Vascular Endothelial Tight Junctions and Barrier Function Are Disrupted by 15(S)-Hydroxyeicosatetraenoic Acid Partly via Protein Kinase Cϵ-mediated Zona Occludens-1 Phosphorylation at Threonine 770/772*

    PubMed Central

    Chattopadhyay, Rima; Dyukova, Elena; Singh, Nikhlesh K.; Ohba, Motoi; Mobley, James A.; Rao, Gadiparthi N.

    2014-01-01

    Disruption of tight junctions (TJs) perturbs endothelial barrier function and promotes inflammation. Previously, we have shown that 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), the major 15-lipoxygenase 1 (15-LO1) metabolite of arachidonic acid, by stimulating zona occludens (ZO)-2 tyrosine phosphorylation and its dissociation from claudins 1/5, induces endothelial TJ disruption and its barrier dysfunction. Here, we have studied the role of serine/threonine phosphorylation of TJ proteins in 15(S)-HETE-induced endothelial TJ disruption and its barrier dysfunction. We found that 15(S)-HETE enhances ZO-1 phosphorylation at Thr-770/772 residues via PKCϵ-mediated MEK1-ERK1/2 activation, causing ZO-1 dissociation from occludin, disrupting endothelial TJs and its barrier function, and promoting monocyte transmigration; these effects were reversed by T770A/T772A mutations. In the arteries of WT mice ex vivo, 15(S)-HETE also induced ZO-1 phosphorylation and endothelial TJ disruption in a PKCϵ and MEK1-ERK1/2-dependent manner. In line with these observations, in WT mice high fat diet feeding induced 12/15-lipoxygenase (12/15-LO) expression in the endothelium and caused disruption of its TJs and barrier function. However, in 12/15-LO−/− mice, high fat diet feeding did not cause disruption of endothelial TJs and barrier function. These observations suggest that the 12/15-LO-12/15(S)-HETE axis, in addition to tyrosine phosphorylation of ZO-2, also stimulates threonine phosphorylation of ZO-1 in the mediation of endothelial TJ disruption and its barrier dysfunction. PMID:24338688

  15. Barley malt increases hindgut and portal butyric acid, modulates gene expression of gut tight junction proteins and Toll-like receptors in rats fed high-fat diets, but high advanced glycation end-products partially attenuate the effects.

    PubMed

    Zhong, Yadong; Teixeira, Cristina; Marungruang, Nittaya; Sae-Lim, Watina; Tareke, Eden; Andersson, Roger; Fåk, Frida; Nyman, Margareta

    2015-09-01

    Barley malt, a product of controlled germination, has been shown to produce high levels of butyric acid in the cecum and portal serum of rats and may therefore have anti-inflammatory effects. The aim of the study was to investigate how four barley malts, caramelized and colored malts, 50-malt and 350-malt, differing in functional characteristics concerning beta-glucan content and color, affect short-chain fatty acids (SCFA), barrier function and inflammation in the hindgut of rats fed high-fat diets. Male Wistar rats were given malt-supplemented high-fat diets for four weeks. Low and high-fat diets containing microcrystalline cellulose were incorporated as controls. All diets contained 70 g kg(-1) dietary fiber. The malt-fed groups were found to have had induced higher amounts of butyric and propionic acids in the hindgut and portal serum compared with controls, while cecal succinic acid only increased to a small extent. Fat increased the mRNA expression of tight junction proteins and Toll-like receptors (TLR) in the small intestine and distal colon of the rats, as well as the concentration of some amino acids in the portal plasma, but malt seemed to counteract these adverse effects to some extent. However, the high content of advanced glycation end-products (AGE) in caramelized malt tended to prohibit the positive effects on occludin in the small intestine and plasma amino acids seen with the other malt products. In conclusion, malting seems to be an interesting process for producing foods with positive health effects, but part of these effects may be destroyed if the malt contains a high content of AGE. PMID:26227569

  16. Mapping of four mouse genes encoding eye lens-specific structural, gap junction, and integral membrane proteins: Cryba1 (crystallin{beta}A3/A1), Crybb2 (crystallin{beta}B2), Gja8 (MP70), and Lim2 (MP19)

    SciTech Connect

    Kerscher, S.; Boyd, Y.; Lyon, M.F.

    1995-09-20

    Four genes encoding eye lens-specific proteins, potential candidate genes for congenital cataract (CC) mutations, were mapped in the mouse genome using a panel of somatic cell hybrids and DNAs from the EUCIB (European Collaborative Interspecific Backcross). Two of them are lens fiber cell structural proteins: the Cryba1 locus encoding crystallin{beta}A3/A1 maps to chromosome 11, 2.5 {+-} 2.5 cM distal to D11Mit31, and the Crybb2 locus encoding crystallin{beta}B2 maps to chromosome 5, 9.1 {+-} 4.3 cM distal to D5Mit88. The other two genes encode lens-specific gap junction and integral membrane proteins, respectively: the Gja8 locus encoding gap junction membrane channel protein {alpha}8, also called connexin50 or MP70, maps to chromosome 3, 11.9 {+-} 5.0 cM distal to D3Mit22, and the Lim2 locus encoding lens intrinsic membrane protein 2, also call MP19, maps to chromosome 7, 2.5 {+-} 2.5 cM proximal to Ngfg. All four map positions, when compared with the corresponding positions in human, lie within known regions of conserved synteny between mouse and human chromosomes. 43 refs., 2 figs., 1 tab.

  17. 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. PMID:26883891

  18. Herlitz junctional epidermolysis bullosa.

    PubMed

    Laimer, Martin; Lanschuetzer, Christoph M; Diem, Anja; Bauer, Johann W

    2010-01-01

    Junctional epidermolysis bullosa type Herlitz (JEB-H) is the autosomal recessively inherited, more severe variant of "lucidolytic" JEB. Characterized by generalized, extensive mucocutaneous blistering at birth and early lethality, this devastating condition is most often caused by homozygous null mutations in the genes LAMA3, LAMB3, or LAMC2, each encoding for 1 of the 3 chains of the heterotrimer laminin-332. The JEB-H subtype usually presents as a severe and clinically diverse variant of the EB group of mechanobullous genodermatoses. This article outlines the epidemiology, presentation, and diagnosis of JEB-H. Morbidity and mortality are high, necessitating optimized protocols for early (including prenatal) diagnosis and palliative care. Gene therapy remains the most promising perspective. PMID:19945616

  19. Ion bipolar junction transistors

    PubMed Central

    Tybrandt, Klas; Larsson, Karin C.; Richter-Dahlfors, Agneta; Berggren, Magnus

    2010-01-01

    Dynamic control of chemical microenvironments is essential for continued development in numerous fields of life sciences. Such control could be achieved with active chemical circuits for delivery of ions and biomolecules. As the basis for such circuitry, we report a solid-state ion bipolar junction transistor (IBJT) based on conducting polymers and thin films of anion- and cation-selective membranes. The IBJT is the ionic analogue to the conventional semiconductor BJT and is manufactured using standard microfabrication techniques. Transistor characteristics along with a model describing the principle of operation, in which an anionic base current amplifies a cationic collector current, are presented. By employing the IBJT as a bioelectronic circuit element for delivery of the neurotransmitter acetylcholine, its efficacy in modulating neuronal cell signaling is demonstrated. PMID:20479274

  20. The Drosophila Claudin Kune-kune is required for septate junction organization and tracheal tube size control.

    PubMed

    Nelson, Kevin S; Furuse, Mikio; Beitel, Greg J

    2010-07-01

    The vertebrate tight junction is a critical claudin-based cell-cell junction that functions to prevent free paracellular diffusion between epithelial cells. In Drosophila, this barrier is provided by the septate junction, which, despite being ultrastructurally distinct from the vertebrate tight junction, also contains the claudin-family proteins Megatrachea and Sinuous. Here we identify a third Drosophila claudin, Kune-kune, that localizes to septate junctions and is required for junction organization and paracellular barrier function, but not for apical-basal polarity. In the tracheal system, septate junctions have a barrier-independent function that promotes lumenal secretion of Vermiform and Serpentine, extracellular matrix modifier proteins that are required to restrict tube length. As with Sinuous and Megatrachea, loss of Kune-kune prevents this secretion and results in overly elongated tubes. Embryos lacking all three characterized claudins have tracheal phenotypes similar to any single mutant, indicating that these claudins act in the same pathway controlling tracheal tube length. However, we find that there are distinct requirements for these claudins in epithelial septate junction formation. Megatrachea is predominantly required for correct localization of septate junction components, while Sinuous is predominantly required for maintaining normal levels of septate junction proteins. Kune-kune is required for both localization and levels. Double- and triple-mutant combinations of Sinuous and Megatrachea with Kune-kune resemble the Kune-kune single mutant, suggesting that Kune-kune has a more central role in septate junction formation than either Sinuous or Megatrachea.

  1. Cellular Redistribution of Protein Tyrosine Phosphatases LAR and PTPσ by Inducible Proteolytic Processing

    PubMed Central

    Aicher, Babette; Lerch, Markus M.; Müller, Thomas; Schilling, James; Ullrich, Axel

    1997-01-01

    Most receptor-like protein tyrosine phosphatases (PTPases) display a high degree of homology with cell adhesion molecules in their extracellular domains. We studied the functional significance of processing for the receptor-like PTPases LAR and PTPσ. PTPσ biosynthesis and intracellular processing resembled that of the related PTPase LAR and was expressed on the cell surface as a two-subunit complex. Both LAR and PTPσ underwent further proteolytical processing upon treatment of cells with either calcium ionophore A23187 or phorbol ester TPA. Induction of LAR processing by TPA in 293 cells did require overexpression of PKCα. Induced proteolysis resulted in shedding of the extracellular domains of both PTPases. This was in agreement with the identification of a specific PTPσ cleavage site between amino acids Pro821 and Ile822. Confocal microscopy studies identified adherens junctions and desmosomes as the preferential subcellular localization for both PTPases matching that of plakoglobin. Consistent with this observation, we found direct association of plakoglobin and β-catenin with the intracellular domain of LAR in vitro. Taken together, these data suggested an involvement of LAR and PTPσ in the regulation of cell contacts in concert with cell adhesion molecules of the cadherin/catenin family. After processing and shedding of the extracellular domain, the catalytically active intracellular portions of both PTPases were internalized and redistributed away from the sites of cell–cell contact, suggesting a mechanism that regulates the activity and target specificity of these PTPases. Calcium withdrawal, which led to cell contact disruption, also resulted in internalization but was not associated with prior proteolytic cleavage and shedding of the extracellular domain. We conclude that the subcellular localization of LAR and PTPσ is regulated by at least two independent mechanisms, one of which requires the presence of their extracellular domains and one of

  2. 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. PMID:16093281

  3. The Onecut Transcription Factor HNF-6 Regulates in Motor Neuro