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Sample records for adherens junction proteins

  1. 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

  2. Targeting of Transmembrane Protein Shrew-1 to Adherens Junctions Is Controlled by Cytoplasmic Sorting Motifs

    PubMed Central

    Jakob, Viktor; Schreiner, Alexander; Tikkanen, Ritva

    2006-01-01

    We recently identified transmembrane protein shrew-1 and showed that it is able to target to adherens junctions in polarized epithelial cells. This suggested shrew-1 possesses specific basolateral sorting motifs, which we analyzed by mutational analysis. Systematic mutation of amino acids in putative sorting signals in the cytoplasmic domain of shrew-1 revealed three tyrosines and a dileucine motif necessary for basolateral sorting. Substitution of these amino acids leads to apical localization of shrew-1. By applying tannic acid to either the apical or basolateral part of polarized epithelial cells, thereby blocking vesicle fusion with the plasma membrane, we obtained evidence that the apically localized mutants were primarily targeted to the basolateral membrane and were then redistributed to the apical domain. Further support for a postendocytic sorting mechanism of shrew-1 was obtained by demonstrating that μ1B, a subunit of the epithelial cell-specific adaptor complex AP-1B, interacts with shrew-1. In conclusion, our data provide evidence for a scenario where shrew-1 is primarily delivered to the basolateral membrane by a so far unknown mechanism. Once there, adaptor protein complex AP-1B is involved in retaining shrew-1 at the basolateral membrane by postendocytic sorting mechanisms. PMID:16707570

  3. Planar cell polarity protein Celsr1 regulates endothelial adherens junctions and directed cell rearrangements during valve morphogenesis.

    PubMed

    Tatin, Florence; Taddei, Andrea; Weston, Anne; Fuchs, Elaine; Devenport, Danelle; Tissir, Fadel; Makinen, Taija

    2013-07-15

    Planar cell polarity (PCP) signaling controls tissue morphogenesis by coordinating collective cell behaviors. We show a critical role for the core PCP proteins Celsr1 and Vangl2 in the complex morphogenetic process of intraluminal valve formation in lymphatic vessels. We found that valve-forming endothelial cells undergo elongation, reorientation, and collective migration into the vessel lumen as they initiate valve leaflet formation. During this process, Celsr1 and Vangl2 are recruited from endothelial filopodia to discrete membrane domains at cell-cell contacts. Celsr1- or Vangl2-deficient mice show valve aplasia due to failure of endothelial cells to undergo rearrangements and adopt perpendicular orientation at valve initiation sites. Mechanistically, we show that Celsr1 regulates dynamic cell movements by inhibiting stabilization of VE-cadherin and maturation of adherens junctions. These findings reveal a role for PCP signaling in regulating adherens junctions and directed cell rearrangements during vascular development. PMID:23792146

  4. 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

  5. Protein tyrosine phosphatase κ and SHP-1 are involved in the regulation of cell-cell contacts at adherens junctions in the exocrine pancreas

    PubMed Central

    Schnekenburger, J; Mayerle, J; Krüger, B; Buchwalow, I; Weiss, F U; Albrecht, E; Samoilova, V E; Domschke, W; Lerch, M M

    2005-01-01

    Background: We have previously shown that cell contacts between pancreatic acinar cells dissociate early in pancreatitis and that this is a prerequisite for the development of pancreatic oedema. Here we studied the underlying mechanism. Methods: Employing experimental caerulein induced pancreatitis in vivo and isolated pancreatic acini ex vivo, in conjunction with protein chemistry, morphology, and electron microscopy, we determined whether cell contact regulation in the pancreas requires or involves: (1) changes in cadherin-catenin protein expression, (2) tyrosine phosphorylation of adhesion proteins, or (3) alterations in the actin cytoskeleton. Results: During initial cell-cell contact dissociation at adherens junctions, expression of adhesion proteins remained stable. At time points of dissociated adherens junctions, the cadherin-catenin complex was found to be tyrosine phosphorylated and internalised. The receptor type protein tyrosine phosphatase (PTP)κ was constitutively associated with the cadherin-catenin complex at intact cell contacts whereas following the dissociation of adherens junctions, the internalised components of the cadherin-catenin complex were tyrosine phosphorylated and associated with the cytosolic PTP SHP-1. In isolated acini, inhibition of endogenous protein tyrosine phosphatases alone was sufficient to induce dissociation of adherens junctions analogous to that found with supramaximal caerulein stimulation. Dissociation of actin microfilaments had no effect on adherens junction integrity. Conclusions: These data identify tyrosine phosphorylation as the key regulator for cell contacts at adherens junctions and suggest a definitive role for the protein tyrosine phosphatases PTPκ and SHP-1 in the regulation, maintenance, and restitution of cell adhesions in a complex epithelial organ such as the pancreas. PMID:15987791

  6. 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

  7. 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

  8. E-cadherin Surface Levels in Epithelial Growth Factor-stimulated Cells Depend on Adherens Junction Protein Shrew-1

    PubMed Central

    Gross, Julia Christina; Schreiner, Alexander; Engels, Knut

    2009-01-01

    Gain- and loss-of-function studies indicate that the adherens junction protein shrew-1 acts as a novel modulator of E-cadherin internalization induced by epithelial growth factor (EGF) or E-cadherin function-blocking antibody during epithelial cell dynamics. Knocking down shrew-1 in MCF-7 carcinoma cells preserves E-cadherin surface levels upon EGF stimulation. Overexpression of shrew-1 leads to preformation of an E-cadherin/EGF receptor (EGFR) HER2/src-kinase/shrew-1 signaling complex and accelerated E-cadherin internalization. Shrew-1 is not sufficient to stimulate E-cadherin internalization, but facilitates the actions of EGFR and thus may promote malignant progression in breast cancer cells with constitutive EGFR stimulation by reducing surface E-cadherin expression. PMID:19515834

  9. 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

  10. Protein 4.1R links E-cadherin/beta-catenin complex to the cytoskeleton through its direct interaction with beta-catenin and modulates adherens junction integrity.

    PubMed

    Yang, Shaomin; Guo, Xinhua; Debnath, Gargi; Mohandas, Narla; An, Xiuli

    2009-07-01

    Protein 4.1R (4.1R) is the prototypical member of the protein 4.1 superfamily comprising of the protein 4.1 family (4.1R, 4.1B, 4.1G and 4.1N) and ERM family (ezrin, radixin and meosin). These proteins in general serve as adaptors between the membrane and the cytoskeleton. Here we show that 4.1R expressed in the gastric epithelial cells associates with adherens junction protein beta-catenin. Biochemical examination of 4.1R-deficient stomach epithelia revealed a selective reduction of beta-catenin which is accompanied by a weaker linkage of E-cadherin to the cytoskeleton. In addition, organization of actin cytoskeleton was altered in 4.1R-deficient cells. Moreover, histological examination revealed that cell-cell contacts are impaired and gastric glands are disorganized in 4.1R null stomach epithelia. These results demonstrate an important and previously unidentified role of 4.1R in linking the cadherin/catenin complex to the cytoskeleton through its direct interaction with beta-catenin and in regulating the integrity of adherens junction. PMID:19376086

  11. 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

  12. 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

  13. 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

  14. Hold on tightly, let go lightly: myosin functions at adherens junctions

    PubMed Central

    Sandquist, Joshua C.; Bement, William M.

    2016-01-01

    Adherens junctions, the sites of cadherin-dependent cell–cell adhesion, are also important for dynamic tension sensing, force transduction and signalling. Different myosin motors contribute to adherens junction assembly and versatility in distinct ways. PMID:20596044

  15. 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

  16. 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

  17. Adherens and Tight Junctions: Structure, Function and Connections to the Actin Cytoskeleton

    PubMed Central

    Hartsock, Andrea; Nelson, W. James

    2009-01-01

    Summary Adherens juctions and Tight junctions comprise two modes of cell-cell adhesion that provide different functions. Both junctional complexes are proposed to associate with the actin cytoskeleton, and formation and maturation of cell-cell contacts involves reorganization of the actin cytoskeleton. Adherens junctions initiate cell-cell contacts, and mediate the maturation and maintenance of the contact. Adherens junctions consist of the transmembrane protein E-cadherin, and intracellular components, p120-catenin, β-catenin and α-catenin. Tight junctions regulate the paracellular pathway for the movement of ions and solutes in-between cells. Tight junctions consist of the transmembrane proteins occludin and claudin, and the cytoplasmic scaffolding proteins ZO-1,-2, and -3. This review discusses the binding interactions of the most studied proteins that occur within each of these two junctional complexes and possible modes of regulation of these interactions, and the different mechanisms that connect and regulate interactions with the actin cytoskeleton. PMID:17854762

  18. Microtubules CLASP to Adherens Junctions in epidermal progenitor cells.

    PubMed

    Shahbazi, Marta N; Perez-Moreno, Mirna

    2014-01-01

    Cadherin-mediated cell adhesion at Adherens Junctions (AJs) and its dynamic connections with the microtubule (MT) cytoskeleton are important regulators of cellular architecture. However, the functional relevance of these interactions and the molecular players involved in different cellular contexts and cellular compartments are still not completely understood. Here, we comment on our recent findings showing that the MT plus-end binding protein CLASP2 interacts with the AJ component p120-catenin (p120) specifically in progenitor epidermal cells. Absence of either protein leads to alterations in MT dynamics and AJ functionality. These findings represent a novel mechanism of MT targeting to AJs that may be relevant for the maintenance of proper epidermal progenitor cell homeostasis. We also discuss the potential implication of other MT binding proteins previously associated to AJs in the wider context of epithelial tissues. We hypothesize the existence of adaptation mechanisms that regulate the formation and stability of AJs in different cellular contexts to allow the dynamic behavior of these complexes during tissue homeostasis and remodeling. PMID:24522006

  19. Microtubules CLASP to Adherens Junctions in epidermal progenitor cells

    PubMed Central

    Shahbazi, Marta N; Perez-Moreno, Mirna

    2014-01-01

    Cadherin-mediated cell adhesion at Adherens Junctions (AJs) and its dynamic connections with the microtubule (MT) cytoskeleton are important regulators of cellular architecture. However, the functional relevance of these interactions and the molecular players involved in different cellular contexts and cellular compartments are still not completely understood. Here, we comment on our recent findings showing that the MT plus-end binding protein CLASP2 interacts with the AJ component p120-catenin (p120) specifically in progenitor epidermal cells. Absence of either protein leads to alterations in MT dynamics and AJ functionality. These findings represent a novel mechanism of MT targeting to AJs that may be relevant for the maintenance of proper epidermal progenitor cell homeostasis. We also discuss the potential implication of other MT binding proteins previously associated to AJs in the wider context of epithelial tissues. We hypothesize the existence of adaptation mechanisms that regulate the formation and stability of AJs in different cellular contexts to allow the dynamic behavior of these complexes during tissue homeostasis and remodeling. PMID:24522006

  20. The adherens junction is lost during normal pregnancy but not during ovarian hyperstimulated pregnancy.

    PubMed

    Dowland, Samson N; Madawala, Romanthi J; Lindsay, Laura A; Murphy, Christopher R

    2016-03-01

    During early pregnancy in the rat, the luminal uterine epithelial cells (UECs) must transform to a receptive state to permit blastocyst attachment and implantation. The implantation process involves penetration of the epithelial barrier, so it is expected that the transformation of UECs includes alterations in the lateral junctional complex. Previous studies have demonstrated a deepening of the tight junction (zonula occludens) and a reduction in the number of desmosomes (macula adherens) in UECs at the time of implantation. However, the adherens junction (zonula adherens), which is primarily responsible for cell-cell adhesion, has been little studied during early pregnancy. This study investigated the adherens junction in rat UECs during the early stages of normal pregnancy and ovarian hyperstimulated (OH) pregnancy using transmission electron microscopy. The adherens junction is present in UECs at the time of fertilisation, but is lost at the time of blastocyst implantation during normal pregnancy. Interestingly, at the time of implantation after OH, adherens junctions are retained and may impede blastocyst penetration of the epithelium. The adherens junction anchors the actin-based terminal web, which is known to be disrupted in UECs during early pregnancy. However, artificial disruption of the terminal web, using cytochalasin D, did not cause removal of the adherens junction in UECs. This study revealed that adherens junction disassembly occurs during early pregnancy, but that this process does not occur during OH pregnancy. Such disassembly does not appear to depend on the disruption of the terminal web. PMID:26738975

  1. 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

  2. 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. PMID:26489655

  3. 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

  4. JNK phosphorylates β-catenin and regulates adherens junctions

    PubMed Central

    Lee, Meng-Horng; Koria, Piyush; Qu, Jun; Andreadis, Stelios T.

    2009-01-01

    The c-Jun amino-terminal kinase (JNK) is an important player in inflammation, proliferation, and apoptosis. More recently, JNK was found to regulate cell migration by phosphorylating paxillin. Here, we report a novel role of JNK in cell adhesion. Specifically, we provide evidence that JNK binds to E-cadherin/β-catenin complex and phosphorylates β-catenin at serine 37 and threonine 41, the sites also phosphorylated by GSK-3β. Inhibition of JNK kinase activity using dominant-negative constructs reduces phosphorylation of β-catenin and promotes localization of E-cadherin/β-catenin complex to cell-cell contact sites. Conversely, activation of JNK induces β-catenin phosphorylation and disruption of cell contacts, which are prevented by JNK siRNA. We propose that JNK binds to β-catenin and regulates formation of adherens junctions, ultimately controlling cell-to-cell adhesion.—Lee, M.-H., Koria, P., Qu, J., Andreadis, S. T. JNK phosphorylates β-catenin and regulates adherens junctions. PMID:19667122

  5. Loss of RhoA in neural progenitor cells causes the disruption of adherens junctions and hyperproliferation

    PubMed Central

    Katayama, Kei-ichi; Melendez, Jaime; Baumann, Jessica M.; Leslie, Jennifer R.; Chauhan, Bharesh K.; Nemkul, Niza; Lang, Richard A.; Kuan, Chia-Yi; Zheng, Yi; Yoshida, Yutaka

    2011-01-01

    The organization of neural progenitors in the developing mammalian neuroepithelium is marked by cadherin-based adherens junctions. Whereas RhoA, a founding member of the small Rho GTPase family, has been shown to play important roles in epithelial adherens junctions, its physiological roles in neural development remain uncertain due to the lack of specific loss-of-function studies. Here, we show that RhoA protein accumulates at adherens junctions in the developing mouse brain and colocalizes to the cadherin–catenin complex. Conditional deletion of RhoA in midbrain and forebrain neural progenitors using Wnt1-Cre and Foxg1-Cre mice, respectively, disrupts apical adherens junctions and causes massive dysplasia of the brain. Furthermore, RhoA-deficient neural progenitor cells exhibit accelerated proliferation, reduction of cell- cycle exit, and increased expression of downstream target genes of the hedgehog pathway. Consequently, both lines of conditional RhoA-deficient embryos exhibit expansion of neural progenitor cells and exencephaly-like protrusions. These results demonstrate a critical role of RhoA in the maintenance of apical adherens junctions and the regulation of neural progenitor proliferation in the developing mammalian brain. PMID:21502507

  6. RhoA mediates cyclooxygenase-2 signaling to disrupt the formation of adherens junctions and increase cell motility.

    PubMed

    Chang, Yu-Wen E; Marlin, Jerry W; Chance, Terry W; Jakobi, Rolf

    2006-12-15

    Cyclooxygenase-2 (COX-2) represents an important target for treatment and prevention of colorectal cancer. Although COX-2 signaling is implicated in promoting tumor cell growth and invasion, the molecular mechanisms that mediate these processes are largely unknown. In this study, we show that the RhoA pathway mediates COX-2 signaling to disrupt the formation of adherens junctions and increase cell motility. Disruption of adherens junctions promotes tumor cell invasion and metastasis and is often associated with tumor progression. We detected high levels of RhoA activity in HCA-7 colon carcinoma cells that constitutively express COX-2. Inhibition of COX-2 significantly reduced the levels of RhoA activity in HCA-7 cells, suggesting that constitutive expression of COX-2 stimulates RhoA activity. Interestingly, inhibition of COX-2 or silencing of COX-2 expression with small interfering RNA (siRNA) stimulated the formation of adherens junctions, concomitant with increased protein levels of E-cadherin and alpha-catenin. Furthermore, inhibition of RhoA or silencing of RhoA expression with siRNA increased the levels of E-cadherin and alpha-catenin. Inhibition of Rho kinases (ROCK), the RhoA effector proteins, also increased levels of E-cadherin and alpha-catenin and stimulated formation of adherens junctions. The motility of HCA-7 cells was significantly decreased when COX-2 or RhoA was inhibited. Therefore, our data reveal a novel molecular mechanism that links COX-2 signaling to disrupt the formation of adherens junctions; COX-2 stimulates the RhoA/ROCK pathway, which reduces levels of E-cadherin and alpha-catenin leading to disruption of adherens junction formation and increased motility. Understanding of COX-2 downstream signaling pathways that promote tumor progression is crucial for the development of novel therapeutic strategies. PMID:17178865

  7. 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

  8. PLEKHA7 Recruits PDZD11 to Adherens Junctions to Stabilize Nectins.

    PubMed

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

    2016-05-20

    PLEKHA7 is a junctional protein implicated in stabilization of the cadherin protein complex, hypertension, cardiac contractility, glaucoma, microRNA processing, and susceptibility to bacterial toxins. To gain insight into the molecular basis for the functions of PLEKHA7, we looked for new PLEKHA7 interactors. Here, we report the identification of PDZ domain-containing protein 11 (PDZD11) as a new interactor of PLEKHA7 by yeast two-hybrid screening and by mass spectrometry analysis of PLEKHA7 immunoprecipitates. We show that PDZD11 (17 kDa) is expressed in epithelial and endothelial cells, where it forms a complex with PLEKHA7, as determined by co-immunoprecipitation analysis. The N-terminal Trp-Trp (WW) domain of PLEKHA7 interacts directly with the N-terminal 44 amino acids of PDZD11, as shown by GST-pulldown assays. Immunofluorescence analysis shows that PDZD11 is localized at adherens junctions in a PLEKHA7-dependent manner, because its junctional localization is abolished by knock-out of PLEKHA7, and is rescued by re-expression of exogenous PLEKHA7. The junctional recruitment of nectin-1 and nectin-3 and their protein levels are decreased via proteasome-mediated degradation in epithelial cells where either PDZD11 or PLEKHA7 have been knocked-out. PDZD11 forms a complex with nectin-1 and nectin-3, and its PDZ domain interacts directly with the PDZ-binding motif of nectin-1. PDZD11 is required for the efficient assembly of apical junctions of epithelial cells at early time points in the calcium-switch model. These results show that the PLEKHA7-PDZD11 complex stabilizes nectins to promote efficient early junction assembly and uncover a new molecular mechanism through which PLEKHA7 recruits PDZ-binding membrane proteins to epithelial adherens junctions. PMID:27044745

  9. 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

  10. Oxygen deprivation inhibits basal keratinocyte proliferation in a model of human skin and induces regio-specific changes in the distribution of epidermal adherens junction proteins, aquaporin-3, and glycogen

    PubMed Central

    Straseski, Joely A.; Gibson, Angela L.; Thomas-Virnig, Christina L.; Allen-Hoffmann, B. Lynn

    2009-01-01

    It is generally accepted that hypoxia and recovery from oxygen deprivation contribute to the breakdown and ulceration of human skin. The effects of these stresses on proliferation, differentiation and expression of cell-cell adhesion molecules were investigated for the first time in an organotypic model of human skin. Fully stratified tissues were exposed to a time course of oxygen deprivation and subsequent reoxygenation. Regional changes in keratinocyte morphology, glycogen stores and cellular junctions were observed, with more differentiated layers of the epidermis exhibiting the first evidence of oxygen deprivation. Cellular swelling within the granular layer was concurrent with aquaporin-3 depletion. The keratinocyte adherens junction proteins E-cadherin and β-catenin were dramatically decreased in a regio-specific manner throughout the epidermis following oxygen deprivation. In contrast, P-cadherin and the desmosomal proteins desmoplakin and desmoglein-1 were refractory to oxygen deprivation. Relative to normoxic controls, hypoxic tissues exhibited increased mRNA levels of the transcriptional repressor Slug however mRNA levels of the related transcriptional factor Snail were unaffected. All cellular and molecular changes were reversible upon reoxygenation. These results demonstrate that oxygen deprivation and reoxygenation exert differential effects on epidermal adhesion proteins and suggest a novel role for cadherins, β-catenin, and Slug in hypoxia-induced junctional changes occurring in stratified squamous epithelium. PMID:19614926

  11. 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.

  12. HIV-associated disruption of tight and adherens junctions of oral epithelial cells facilitates HSV-1 infection and spread.

    PubMed

    Sufiawati, Irna; Tugizov, Sharof M

    2014-01-01

    Herpes simplex virus (HSV) types 1 and 2 are the most common opportunistic infections in HIV/AIDS. In these immunocompromised individuals, HSV-1 reactivates and replicates in oral epithelium, leading to oral disorders such as ulcers, gingivitis, and necrotic lesions. Although the increased risk of HSV infection may be mediated in part by HIV-induced immune dysfunction, direct or indirect interactions of HIV and HSV at the molecular level may also play a role. In this report we show that prolonged interaction of the HIV proteins tat and gp120 and cell-free HIV virions with polarized oral epithelial cells leads to disruption of tight and adherens junctions of epithelial cells through the mitogen-activated protein kinase signaling pathway. HIV-induced disruption of oral epithelial junctions facilitates HSV-1 paracellular spread between the epithelial cells. Furthermore, HIV-associated disruption of adherens junctions exposes sequestered nectin-1, an adhesion protein and critical receptor for HSV envelope glycoprotein D (gD). Exposure of nectin-1 facilitates binding of HSV-1 gD, which substantially increases HSV-1 infection of epithelial cells with disrupted junctions over that of cells with intact junctions. Exposed nectin-1 from disrupted adherens junctions also increases the cell-to-cell spread of HSV-1 from infected to uninfected oral epithelial cells. Antibodies to nectin-1 and HSV-1 gD substantially reduce HSV-1 infection and cell-to-cell spread, indicating that HIV-promoted HSV infection and spread are mediated by the interaction of HSV gD with HIV-exposed nectin-1. Our data suggest that HIV-associated disruption of oral epithelial junctions may potentiate HSV-1 infection and its paracellular and cell-to-cell spread within the oral mucosal epithelium. This could be one of the possible mechanisms of rapid development of HSV-associated oral lesions in HIV-infected individuals. PMID:24586397

  13. HIV-Associated Disruption of Tight and Adherens Junctions of Oral Epithelial Cells Facilitates HSV-1 Infection and Spread

    PubMed Central

    Sufiawati, Irna; Tugizov, Sharof M.

    2014-01-01

    Herpes simplex virus (HSV) types 1 and 2 are the most common opportunistic infections in HIV/AIDS. In these immunocompromised individuals, HSV-1 reactivates and replicates in oral epithelium, leading to oral disorders such as ulcers, gingivitis, and necrotic lesions. Although the increased risk of HSV infection may be mediated in part by HIV-induced immune dysfunction, direct or indirect interactions of HIV and HSV at the molecular level may also play a role. In this report we show that prolonged interaction of the HIV proteins tat and gp120 and cell-free HIV virions with polarized oral epithelial cells leads to disruption of tight and adherens junctions of epithelial cells through the mitogen-activated protein kinase signaling pathway. HIV-induced disruption of oral epithelial junctions facilitates HSV-1 paracellular spread between the epithelial cells. Furthermore, HIV-associated disruption of adherens junctions exposes sequestered nectin-1, an adhesion protein and critical receptor for HSV envelope glycoprotein D (gD). Exposure of nectin-1 facilitates binding of HSV-1 gD, which substantially increases HSV-1 infection of epithelial cells with disrupted junctions over that of cells with intact junctions. Exposed nectin-1 from disrupted adherens junctions also increases the cell-to-cell spread of HSV-1 from infected to uninfected oral epithelial cells. Antibodies to nectin-1 and HSV-1 gD substantially reduce HSV-1 infection and cell-to-cell spread, indicating that HIV-promoted HSV infection and spread are mediated by the interaction of HSV gD with HIV-exposed nectin-1. Our data suggest that HIV-associated disruption of oral epithelial junctions may potentiate HSV-1 infection and its paracellular and cell-to-cell spread within the oral mucosal epithelium. This could be one of the possible mechanisms of rapid development of HSV-associated oral lesions in HIV-infected individuals. PMID:24586397

  14. Adherens Junction and E-Cadherin complex regulation by epithelial polarity.

    PubMed

    Coopman, Peter; Djiane, Alexandre

    2016-09-01

    E-Cadherin-based Adherens Junctions (AJs) are a defining feature of all epithelial sheets. Through the homophilic association of E-Cadherin molecules expressed on neighboring cells, they ensure intercellular adhesion amongst epithelial cells, and regulate many key aspects of epithelial biology. While their adhesive role requires these structures to remain stable, AJs are also extremely plastic. This plasticity allows for the adaptation of the cell to its changing environment: changes in neighbors after cell division, cell death, or cell movement, and changes in cell shape during differentiation. In this review we focus on the recent advances highlighting the critical role of the apico-basal polarity machinery, and in particular of the Par3/Bazooka scaffold, in the regulation and remodeling of AJs. We propose that by regulating key phosphorylation events on the core E-Cadherin complex components, Par3 and epithelial polarity promote meta-stable protein complexes governing the correct formation, localization, and functioning of AJ. PMID:27151512

  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. RPTPα controls epithelial adherens junctions, linking E-cadherin engagement to c-Src-mediated phosphorylation of cortactin.

    PubMed

    Truffi, Marta; Dubreuil, Véronique; Liang, Xuan; Vacaresse, Nathalie; Nigon, Fabienne; Han, Siew Ping; Yap, Alpha S; Gomez, Guillermo A; Sap, Jan

    2014-06-01

    Epithelial junctions are fundamental determinants of tissue organization, subject to regulation by tyrosine phosphorylation. Homophilic binding of E-cadherin activates tyrosine kinases, such as Src, that control junctional integrity. Protein tyrosine phosphatases (PTPs) also contribute to cadherin-based adhesion and signaling, but little is known about their specific identity or functions at epithelial junctions. Here, we report that the receptor PTP RPTPα (human gene name PTPRA) is recruited to epithelial adherens junctions at the time of cell-cell contact, where it is in molecular proximity to E-cadherin. RPTPα is required for appropriate cadherin-dependent adhesion and for cyst architecture in three-dimensional culture. Loss of RPTPα impairs adherens junction integrity, as manifested by defective E-cadherin accumulation and peri-junctional F-actin density. These effects correlate with a role for RPTPα in cellular (c)-Src activation at sites of E-cadherin engagement. Mechanistically, RPTPα is required for appropriate tyrosine phosphorylation of cortactin, a major Src substrate and a cytoskeletal actin organizer. Expression of a phosphomimetic cortactin mutant in RPTPα-depleted cells partially rescues F-actin and E-cadherin accumulation at intercellular contacts. These findings indicate that RPTPα controls cadherin-mediated signaling by linking homophilic E-cadherin engagement to cortactin tyrosine phosphorylation through c-Src. PMID:24652832

  17. 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

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

    PubMed

    Shahbazi, Marta N; Megias, Diego; Epifano, Carolina; Akhmanova, Anna; Gundersen, Gregg G; Fuchs, Elaine; Perez-Moreno, Mirna

    2013-12-23

    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. PMID:26467482

  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. 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

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

    PubMed

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

    2016-02-01

    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

  4. 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

  5. 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. PMID:25624491

  6. 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

  7. Junctional adhesion molecules (JAMs) are differentially expressed in fibroblasts and co-localize with ZO-1 to adherens-like junctions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Junctional Adhesion Molecules (JAMs) are components and regulators of the well-characterized epithelial and endothelial tight junction. Since the molecular components of native fibroblast adherens-like junctions remain poorly described we determined JAM expression profiles in fibroblasts. We found J...

  8. Regulation of Adherens Junctions in Trabecular Meshwork Cells by Rac GTPase and their influence on Intraocular Pressure

    PubMed Central

    Pattabiraman, Padmanabhan P; Epstein, David L; Rao, Ponugoti Vasantha

    2013-01-01

    Intercellular adherens junctions and cell-extracellular matrix interactions are presumed to influence aqueous humor (AH) drainage via the conventional route, however, their direct role in modulation of intraocular pressure (IOP) is not well understood. Here, we investigated the role of Rac GTPase signaling in basal and growth factor-induced formation of adherens junctions in human trabecular meshwork (HTM) cells as compared to human umbilical vascular endothelial cells, and evaluated the effects of inhibition of Rac GTPase activity on IOP in rabbits. Expression of a constitutively active Rac1 GTPase or treatment with platelet derived growth factor (PDGF), a known activator of Rac GTPase, induced formation of β-catenin-based adherens junctions, actin cytoskeletal reorganization and membrane ruffle in HTM cells. In contrast, treatment of HTM cells with inhibitors of Rac GTPase caused cell-cell separation, a decrease in adherens junctions, and reorganization of actin stress fibers to the cell cortical regions and focal adhesion to the cell leading edges. Both, constitutively active Rac1 and PDGF stimulated generation of Reactive Oxygen Species (ROS) in HTM cells, and ROS were found to increase adherens junction formation and transendothelial electrical resistance (TEER) in HTM cells. Topical application of Rac GTPase inhibitors (EHT1864 and NSC23766), however, only marginally influenced IOP in rabbit eyes. Taken together, these data reveal that while Rac GTPase signaling plays a significant role in regulation of adherens junctions, ROS production and TEER in cells of the AH outflow pathway, Rac inhibitors showed only a marginal influence on IOP in live rabbits. PMID:24932460

  9. Regulation of Adherens Junctions in Trabecular Meshwork Cells by Rac GTPase and their influence on Intraocular Pressure.

    PubMed

    Pattabiraman, Padmanabhan P; Epstein, David L; Rao, Ponugoti Vasantha

    2013-06-01

    Intercellular adherens junctions and cell-extracellular matrix interactions are presumed to influence aqueous humor (AH) drainage via the conventional route, however, their direct role in modulation of intraocular pressure (IOP) is not well understood. Here, we investigated the role of Rac GTPase signaling in basal and growth factor-induced formation of adherens junctions in human trabecular meshwork (HTM) cells as compared to human umbilical vascular endothelial cells, and evaluated the effects of inhibition of Rac GTPase activity on IOP in rabbits. Expression of a constitutively active Rac1 GTPase or treatment with platelet derived growth factor (PDGF), a known activator of Rac GTPase, induced formation of β-catenin-based adherens junctions, actin cytoskeletal reorganization and membrane ruffle in HTM cells. In contrast, treatment of HTM cells with inhibitors of Rac GTPase caused cell-cell separation, a decrease in adherens junctions, and reorganization of actin stress fibers to the cell cortical regions and focal adhesion to the cell leading edges. Both, constitutively active Rac1 and PDGF stimulated generation of Reactive Oxygen Species (ROS) in HTM cells, and ROS were found to increase adherens junction formation and transendothelial electrical resistance (TEER) in HTM cells. Topical application of Rac GTPase inhibitors (EHT1864 and NSC23766), however, only marginally influenced IOP in rabbit eyes. Taken together, these data reveal that while Rac GTPase signaling plays a significant role in regulation of adherens junctions, ROS production and TEER in cells of the AH outflow pathway, Rac inhibitors showed only a marginal influence on IOP in live rabbits. PMID:24932460

  10. Drosophila MAGI interacts with RASSF8 to regulate E-Cadherin-based adherens junctions in the developing eye.

    PubMed

    Zaessinger, Sophie; Zhou, Yanxiang; Bray, Sarah J; Tapon, Nicolas; Djiane, Alexandre

    2015-03-15

    Morphogenesis is crucial during development to generate organs and tissues of the correct size and shape. During Drosophila late eye development, interommatidial cells (IOCs) rearrange to generate the highly organized pupal lattice, in which hexagonal ommatidial units pack tightly. This process involves the fine regulation of adherens junctions (AJs) and of adhesive E-Cadherin (E-Cad) complexes. Localized accumulation of Bazooka (Baz), the Drosophila PAR3 homolog, has emerged as a critical step to specify where new E-Cad complexes should be deposited during junction remodeling. However, the mechanisms controlling the correct localization of Baz are still only partly understood. We show here that Drosophila Magi, the sole fly homolog of the mammalian MAGI scaffolds, is an upstream regulator of E-Cad-based AJs during cell rearrangements, and that Magi mutant IOCs fail to reach their correct position. We uncover a direct physical interaction between Magi and the Ras association domain protein RASSF8 through a WW domain-PPxY motif binding, and show that apical Magi recruits the RASSF8-ASPP complex during AJ remodeling in IOCs. We further show that this Magi complex is required for the cortical recruitment of Baz and of the E-Cad-associated proteins α- and β-catenin. We propose that, by controlling the proper localization of Baz to remodeling junctions, Magi and the RASSF8-ASPP complex promote the recruitment or stabilization of E-Cad complexes at junction sites. PMID:25725070

  11. Adherens junctions determine the apical position of the midbody during follicular epithelial cell division.

    PubMed

    Morais-de-Sá, Eurico; Sunkel, Claudio

    2013-08-01

    Cytokinesis is asymmetric along the apical-basal axis of epithelial cells, positioning the midbody near the apical domain. However, little is known about the mechanism and purpose of this asymmetry. We use live imaging of Drosophila follicle cell division to show that asymmetric cytokinesis does not result from intrinsic polarization of the main contractile ring components. We show that adherens junctions (AJs) maintain close contact with the apical side of the contractile ring during cytokinesis. Asymmetric distribution of AJ components within follicle cells and in the otherwise unpolarized S2 cells is sufficient to recruit the midbody, revealing that asymmetric cytokinesis is determined by apical AJs in the epithelia. We further show that ectopic midbody localization induces epithelial invaginations, shifting the position of the apical interface between daughter cells relative to the AB axis of the tissue. Thus, apical midbody localization is essential to maintain epithelial tissue architecture during proliferation. PMID:23774295

  12. Disruption of Adherens Junctions Liberates Nectin-1 To Serve as Receptor for Herpes Simplex Virus and Pseudorabies Virus Entry

    PubMed Central

    Yoon, Miri; Spear, Patricia G.

    2002-01-01

    Nectin-1, a cell adhesion molecule belonging to the immunoglobulin superfamily, can bind to virion glycoprotein D (gD) to mediate entry of herpes simplex viruses (HSV) and pseudorabies virus (PRV). Nectin-1 colocalizes with E-cadherin at adherens junctions in epithelial cells. The disruption of cell junctions can result in the redistribution of nectin-1. To determine whether disruption of junctions by calcium depletion influenced the susceptibility of epithelial cells to viral entry, Madin-Darby canine kidney cells expressing endogenous nectin-1 or transfected human nectin-1 were tested for the ability to bind soluble forms of viral gD and to be infected by HSV and PRV, before and after calcium depletion. Confocal microscopy revealed that binding of HSV and PRV gD was localized to adherens junctions in cells maintained in normal medium but was distributed, along with nectin-1, over the entire cell surface after calcium depletion. Both the binding of gD and the fraction of cells that could be infected by HSV-1 and PRV were enhanced by calcium depletion. Taken together, these results provide evidence that nectin-1 confined to adherens junctions in epithelial cells is not very accessible to virus, whereas dissociation of cell junctions releases nectin-1 to serve more efficiently as an entry receptor. PMID:12072519

  13. 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

  14. IQGAP1 mediates the disruption of adherens junctions to promote Escherichia coli K1 invasion of brain endothelial cells

    PubMed Central

    Krishnan, Subramanian; Fernandez, G. Esteban; Sacks, David B.; Prasadarao, Nemani V.

    2012-01-01

    The transcellular entry of E. coli K1 through human brain microvascular endothelial cells (HBMEC) is responsible for tight junction disruption, leading to brain edema in neonatal meningitis. Previous studies demonstrated that outer membrane protein A (OmpA) of E. coli K1 interacts with its receptor, Ecgp96 to induce PKC-α phosphorylation, adherens junction (AJ) disassembly (by dislodging β-catenin from VE-cadherin), and remodeling of actin in HBMEC. We report here that IQGAP1 mediates β-catenin dissociation from AJs to promote actin polymerization required for E. coli K1 invasion of HBMEC. Overexpression of C-terminal truncated IQGAP1 (IQΔC) that cannot bind β-catenin prevents both AJ disruption and E. coli K1 entry. Of note, phospho-PKC-α interacts with the C-terminal portion of Ecgp96 as well as with VE-cadherin after IQGAP1 mediated AJ disassembly. HBMEC overexpressing either C-terminal truncated Ecgp96 (Ecgp96Δ200) or IQΔC upon infection with E. coli showed no interaction of phospho-PKC-α with Ecgp96. These data indicate that the binding of OmpA to Ecgp96 induces PKC-α phosphorylation and association of phospho-PKC-α with Ecgp96, and then signals IQGAP1 to detach β-catenin from AJs. Subsequently, IQGAP1/β-catenin bound actin translocates to the site of E. coli K1 attachment to promote invasion. PMID:22519731

  15. 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

  16. 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

  17. Segmentation and Tracking of Adherens Junctions in 3D for the Analysis of Epithelial Tissue Morphogenesis

    PubMed Central

    Cilla, Rodrigo; Mechery, Vinodh; Hernandez de Madrid, Beatriz; Del Signore, Steven; Dotu, Ivan; Hatini, Victor

    2015-01-01

    Epithelial morphogenesis generates the shape of tissues, organs and embryos and is fundamental for their proper function. It is a dynamic process that occurs at multiple spatial scales from macromolecular dynamics, to cell deformations, mitosis and apoptosis, to coordinated cell rearrangements that lead to global changes of tissue shape. Using time lapse imaging, it is possible to observe these events at a system level. However, to investigate morphogenetic events it is necessary to develop computational tools to extract quantitative information from the time lapse data. Toward this goal, we developed an image-based computational pipeline to preprocess, segment and track epithelial cells in 4D confocal microscopy data. The computational pipeline we developed, for the first time, detects the adherens junctions of epithelial cells in 3D, without the need to first detect cell nuclei. We accentuate and detect cell outlines in a series of steps, symbolically describe the cells and their connectivity, and employ this information to track the cells. We validated the performance of the pipeline for its ability to detect vertices and cell-cell contacts, track cells, and identify mitosis and apoptosis in surface epithelia of Drosophila imaginal discs. We demonstrate the utility of the pipeline to extract key quantitative features of cell behavior with which to elucidate the dynamics and biomechanical control of epithelial tissue morphogenesis. We have made our methods and data available as an open-source multiplatform software tool called TTT (http://github.com/morganrcu/TTT) PMID:25884654

  18. Targeted Recycling of the Lateral Border Recycling Compartment Precedes Adherens Junction Dissociation during Transendothelial Migration.

    PubMed

    Gonzalez, Annette M; Cyrus, Bita F; Muller, William A

    2016-05-01

    Leukocyte transendothelial migration (TEM) requires two major events: local dissociation of adherens junctions manifested as gaps in vascular endothelial (VE)-cadherin staining at the site of TEM and targeted trafficking of the lateral border recycling compartment (LBRC) to the site of TEM. However, the association between LBRC recycling and VE-cadherin gaps remains unknown. We found that when targeting of the LBRC is selectively inhibited using established methods, such as a function blocking anti-platelet endothelial cell adhesion molecule 1 antibody, depolymerizing microtubules, or microinjection of an antibody that inhibits kinesin, VE-cadherin gaps do not form around the blocked leukocyte. This is the first time that the LBRC has been implicated in this process. We obtained similar results for neutrophils and monocytes and in studies using live cell imaging microscopy conducted under fluid shear conditions. Depolymerizing microtubules did not affect the ability of leukocytes to induce tyrosine phosphorylation of VE-cadherin. A VE-cadherin double mutant (Y658F, Y731F) expressed in endothelial cells acted as a dominant negative and inhibited VE-cadherin gap formation and TEM, yet targeting of the LBRC still occurred. These data suggest that targeting of the LBRC to the site of TEM precedes VE-cadherin clearance. Recruitment of the LBRC may play a role in clearing VE-cadherin from the site of TEM. PMID:26968345

  19. Evidence of a common mechanism of disassembly of adherens junctions through Gα13 targeting of VE-cadherin

    PubMed Central

    Gong, Haixia; Gao, Xiaopei; Feng, Shaoting; Siddiqui, M. Rizwan; Garcia, Alexander; Bonini, Marcelo G.; Komarova, Yulia; Vogel, Stephen M.; Mehta, Dolly

    2014-01-01

    The heterotrimeric G protein Gα13 transduces signals from G protein–coupled receptors (GPCRs) to induce cell spreading, differentiation, migration, and cell polarity. Here, we describe a novel GPCR-independent function of Gα13 in regulating the stability of endothelial cell adherens junctions (AJs). We observed that the oxidant H2O2, which is released in response to multiple proinflammatory mediators, induced the interaction of Gα13 with VE-cadherin. Gα13 binding to VE-cadherin in turn induced Src activation and VE-cadherin phosphorylation at Tyr 658, the p120-catenin binding site thought to be responsible for VE-cadherin internalization. Inhibition of Gα13–VE-cadherin interaction using an interfering peptide derived from the Gα13 binding motif on VE-cadherin abrogated the disruption of AJs in response to inflammatory mediators. These studies identify a unique role of Gα13 binding to VE-cadherin in mediating VE-cadherin internalization and endothelial barrier disruption and inflammation. PMID:24590762

  20. 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

  1. 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

  2. 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. PMID:27220534

  3. A Novel Role of E-Cadherin-Based Adherens Junctions in Neoplastic Cell Dissemination

    PubMed Central

    Gloushankova, Natalya A.

    2015-01-01

    Using confocal microscopy, we analyzed the behavior of IAR-6-1, IAR1170, and IAR1162 transformed epithelial cells seeded onto the confluent monolayer of normal IAR-2 epithelial cells. Live-cell imaging of neoplastic cells stably expressing EGFP and of normal epithelial cells stably expressing mKate2 showed that transformed cells retaining expression of E-cadherin were able to migrate over the IAR-2 epithelial monolayer and invade the monolayer. Transformed IAR cells invaded the IAR-2 monolayer at the boundaries between normal cells. Studying interactions of IAR-6-1 transformed cells stably expressing GFP-E-cadherin with the IAR-2 epithelial monolayer, we found that IAR-6-1 cells established E-cadherin-based adhesions with normal epithelial cells: dot-like dynamic E-cadherin-based adhesions in protrusions and large adherens junctions at the cell sides and rear. A comparative study of a panel of transformed IAR cells that differ by their ability to form E-cadherin-based AJs, either through loss of E-cadherin expression or through expression of a dominant negative E-cadherin mutant, demonstrated that E-cadherin-based AJs are key mediators of the interactions between neoplastic and normal epithelial cells. IAR-6-1DNE cells expressing a dominant-negative mutant form of E-cadherin with the mutation in the first extracellular domain practically lost the ability to adhere to IAR-2 cells and invade the IAR-2 epithelial monolayer. The ability of cancer cells to form E-cadherin-based AJs with the surrounding normal epithelial cells may play an important role in driving cancer cell dissemination in the body. PMID:26207916

  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. 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

  6. The tyrosine phosphatase SHP2 regulates recovery of endothelial adherens junctions through control of β-catenin phosphorylation

    PubMed Central

    Timmerman, Ilse; Hoogenboezem, Mark; Bennett, Anton M.; Geerts, Dirk; Hordijk, Peter L.; van Buul, Jaap D.

    2012-01-01

    Impaired endothelial barrier function results in a persistent increase in endothelial permeability and vascular leakage. Repair of a dysfunctional endothelial barrier requires controlled restoration of adherens junctions, comprising vascular endothelial (VE)-cadherin and associated β-, γ-, α-, and p120-catenins. Little is known about the mechanisms by which recovery of VE-cadherin–mediated cell–cell junctions is regulated. Using the inflammatory mediator thrombin, we demonstrate an important role for the Src homology 2-domain containing tyrosine phosphatase (SHP2) in mediating recovery of the VE-cadherin–controlled endothelial barrier. Using SHP2 substrate-trapping mutants and an in vitro phosphatase activity assay, we validate β-catenin as a bona fide SHP2 substrate. SHP2 silencing and SHP2 inhibition both result in delayed recovery of endothelial barrier function after thrombin stimulation. Moreover, on thrombin challenge, we find prolonged elevation in tyrosine phosphorylation levels of VE-cadherin–associated β-catenin in SHP2-depleted cells. No disassembly of the VE-cadherin complex is observed throughout the thrombin response. Using fluorescence recovery after photobleaching, we show that loss of SHP2 reduces the mobility of VE-cadherin at recovered cell–cell junctions. In conclusion, our data show that the SHP2 phosphatase plays an important role in the recovery of disrupted endothelial cell–cell junctions by dephosphorylating VE-cadherin–associated β-catenin and promoting the mobility of VE-cadherin at the plasma membrane. PMID:22956765

  7. 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

  8. Pivotal Role of MUC1 Glycosylation by Cigarette Smoke in Modulating Disruption of Airway Adherens Junctions In Vitro

    PubMed Central

    Zhang, Lili; Gallup, Marianne; Zlock, Lorna; Chen, Yu Ting Feeling; Finkbeiner, Walter E.; McNamara, Nancy A.

    2014-01-01

    Cigarette smoke increases the risk of lung cancer by 20-fold and accounts for 87% of lung cancer deaths. In the normal airway, heavily O-glycosylated mucin-1 (MUC1) and adherens junctions (AJs) establish a structural barrier that protects the airway from infectious, inflammatory and noxious stimuli. Smoke disrupts cell-cell adhesion via its damaging effects on the AJ protein, epithelial cadherin (E-cad). Loss of E-cad is a major hallmark of epithelial-mesenchymal transition (EMT) and has been reported in lung cancer where it is associated with invasion, metastasis and poor prognosis. Using organotypic cultures of primary human bronchial epithelial (HBE) cells treated with smoke-concentrated medium (Smk), we have demonstrated that E-cad loss is regulated through the aberrant interaction of its AJ binding partner, p120-catenin (p120ctn), and the C-terminus of MUC1 (MUC1-C). Here, we reported that even before MUC1-C became bound to p120ctn, smoke promoted the generation of a novel 400kDa glycoform of MUC1’s N-terminus (MUC1-N) differing from the 230kDa and 150kDa glycoforms in untreated control cells. The subsequent smoke-induced, time-dependent shedding of glycosylated MUC1-N exposed MUC1-C as a putative receptor for interactions with EGFR, Src and p120ctn. Smoke-induced MUC1-C glycosylation modulated MUC1-C tyrosine phosphorylation (TyrP) that was essential for MUC1-C/p120ctn interaction through dose-dependent bridging of Src/MUC1-C/galectin-3/EGFR signalosomes. Chemical deglycosylation of MUC1 using a mixture of N-glycosylation inhibitor tunicamycin and O-glycosylation inhibitor benzyl-α-GalNAc disrupted the Src/MUC1-C/galectin-3/EGFR complexes and thereby abolished smoke-induced MUC1-C-TyrP and MUC1-C/p120ctn interaction. Similarly, inhibition of smoke-induced MUC1-N glycosylation using adenoviral shRNA directed against N-acetyl-galactosaminyl transferase-6 (GALNT6, an enzyme that controls the initiating step of O-glycosylation) successfully suppressed MUC1-C

  9. Microtubule plus-end and minus-end capture at adherens junctions is involved in the assembly of apico-basal arrays in polarised epithelial cells.

    PubMed

    Bellett, Gemma; Carter, Jane M; Keynton, Jennifer; Goldspink, Deborah; James, Colin; Moss, David K; Mogensen, Mette M

    2009-10-01

    Apico-basal polarisation of epithelial cells involves a dramatic reorganisation of the microtubule cytoskeleton. The classic radial array of microtubules focused on a centrally located centrosome typical of many animal cells is lost or greatly reduced and a non-centrosomal apico-basal array develops. The molecules and mechanisms responsible for the assembly and positioning of these non-centrosomal microtubules have not been fully elucidated. Using a Nocodazole induced regrowth assay in invitro culture (MDCK) and in situ epithelial (cochlear Kolliker's) cell models we establish that the apico-basal array originates from the centrosome and that the non-centrosomal microtubule minus-end anchoring sites do not contribute significantly to their nucleation. Confocal and electron microscopy revealed that an extended radial array assembles with microtubule plus-ends targeting cadheren sites at adherens junctions and EB1 and CLIP-170 co-localising with beta-catenin and dynein clusters at the junction sites. The extended radial array is likely to be a vital intermediate step in the assembly process with cortical anchored dynein providing the mechanical force required for microtubule release, translocation and capture. Ultrastructural analyses of the apico-basal arrays in fully polarised MDCK and Kolliker's cells revealed microtubule minus-end association with the most apical adherens junction (Zonula adherens). We propose that a release and capture model involving both microtubule plus- and minus-end capture at adherens junctions is responsible for the generation of non-centrosomal apico-basal arrays in most centrosome containing polarised epithelial cells. PMID:19479825

  10. 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

  11. 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.

  12. FoxM1 regulates re-annealing of endothelial adherens junctions through transcriptional control of β-catenin expression

    PubMed Central

    Mirza, Muhammad K.; Sun, Ying; Zhao, Yidan D.; S.K. Potula, Hari-Hara; Frey, Randall S.; Vogel, Steven M.; Malik, Asrar B.

    2010-01-01

    Repair of the injured vascular intima requires a series of coordinated events that mediate both endothelial regeneration and reannealing of adherens junctions (AJs) to form a restrictive endothelial barrier. The forkhead transcription factor FoxM1 is essential for endothelial proliferation after vascular injury. However, little is known about mechanisms by which FoxM1 regulates endothelial barrier reannealing. Here, using a mouse model with endothelial cell (EC)-restricted disruption of FoxM1 (FoxM1 CKO) and primary cultures of ECs with small interfering RNA (siRNA)-mediated knockdown of FoxM1, we demonstrate a novel requisite role of FoxM1 in mediating endothelial AJ barrier repair through the transcriptional control of β-catenin. In the FoxM1 CKO lung vasculature, we observed persistent microvessel leakage characterized by impaired reannealing of endothelial AJs after endothelial injury. We also showed that FoxM1 directly regulated β-catenin transcription and that reexpression of β-catenin rescued the defective AJ barrier–reannealing phenotype of FoxM1-deficient ECs. Knockdown of β-catenin mimicked the phenotype of defective barrier recovery seen in FoxM1-deficient ECs. These data demonstrate that FoxM1 is required for reannealing of endothelial AJs in order to form a restrictive endothelial barrier through transcriptional control of β-catenin expression. Therefore, means of activating FoxM1-mediated endothelial repair represent a new therapeutic strategy for the treatment of inflammatory vascular diseases associated with persistent vascular barrier leakiness such as acute lung injury. PMID:20660612

  13. Tight Junction Proteins in Human Schwann Cell Autotypic Junctions

    PubMed Central

    Alanne, Maria H.; Pummi, Kati; Heape, Anthony M.; Grènman, Reidar; Peltonen, Juha; Peltonen, Sirkku

    2009-01-01

    Tight junctions (TJs) form physical barriers in various tissues and regulate paracellular transport of ions, water, and molecules. Myelinating Schwann cells form highly organized structures, including compact myelin, nodes of Ranvier, paranodal regions, Schmidt-Lanterman incisures, periaxonal cytoplasmic collars, and mesaxons. Autotypic TJs are formed in non-compacted myelin compartments between adjacent membrane lamellae of the same Schwann cell. Using indirect immunofluorescence and RT-PCR, we analyzed the expression of adherens junction (E-cadherin) and TJ [claudins, zonula occludens (ZO)-1, occludin] components in human peripheral nerve endoneurium, showing clear differences with published rodent profiles. Adult nerve paranodal regions contained E-cadherin, claudin-1, claudin-2, and ZO-1. Schmidt-Lanterman incisures contained E-cadherin, claudin-1, claudin-2, claudin-3, claudin-5, ZO-1, and occludin. Mesaxons contained E-cadherin, claudin-1, claudin-2, claudin-3, ZO-1, and occludin. None of the proteins studied were associated with nodal inter-Schwann cell junctions. Fetal nerve expression of claudin-1, claudin-3, ZO-1, and occludin was predominantly punctate, with a mesaxonal labeling pattern, but paranodal (ZO-1, claudin-3) and Schmidt-Lanterman incisure (claudins-1 and -3) expression profiles typical of compact myelin were visible by gestational week 37. The clear differences observed between human and published rodent nerve profiles emphasize the importance of human studies when translating the results of animal models to human diseases. (J Histochem Cytochem 57:523–529, 2009) PMID:19153196

  14. The Wnt pathway destabilizes adherens junctions and promotes cell migration via β-catenin and its target gene cyclin D1.

    PubMed

    Vlad-Fiegen, Annica; Langerak, Anette; Eberth, Sonja; Müller, Oliver

    2012-01-01

    The Wnt pathway regulates cell proliferation, mobility and differentiation. Among the many Wnt target genes is CCND1 which codes for cyclin D1. Cyclin D1, in complex with cdk4 and cdk6, regulates G1/S phase transition during cell cycle. Independently of CDK, cyclin D1 also regulates the migration of macrophages. Here we analyzed the effects of cyclin D1 on the migration of cancer cell lines using the transwell migration and scratch assays. We also tested the effect of cyclin D1 and β-catenin on E-cadherin-mediated cell-cell contacts. Our results show that the Wnt pathway promotes cellular migration via its target gene cyclin D1. Moreover we show that cyclin D1 influences the actin cytoskeleton and destabilizes adherens junctions. PMID:23650577

  15. The Wnt pathway destabilizes adherens junctions and promotes cell migration via β-catenin and its target gene cyclin D1

    PubMed Central

    Vlad-Fiegen, Annica; Langerak, Anette; Eberth, Sonja; Müller, Oliver

    2012-01-01

    The Wnt pathway regulates cell proliferation, mobility and differentiation. Among the many Wnt target genes is CCND1 which codes for cyclin D1. Cyclin D1, in complex with cdk4 and cdk6, regulates G1/S phase transition during cell cycle. Independently of CDK, cyclin D1 also regulates the migration of macrophages. Here we analyzed the effects of cyclin D1 on the migration of cancer cell lines using the transwell migration and scratch assays. We also tested the effect of cyclin D1 and β-catenin on E-cadherin-mediated cell–cell contacts. Our results show that the Wnt pathway promotes cellular migration via its target gene cyclin D1. Moreover we show that cyclin D1 influences the actin cytoskeleton and destabilizes adherens junctions. PMID:23650577

  16. Rab14 and Its Exchange Factor FAM116 Link Endocytic Recycling and Adherens Junction Stability in Migrating Cells

    PubMed Central

    Linford, Andrea; Yoshimura, Shin-ichiro; Bastos, Ricardo Nunes; Langemeyer, Lars; Gerondopoulos, Andreas; Rigden, Daniel J.; Barr, Francis A.

    2012-01-01

    Summary Rab GTPases define the vesicle trafficking pathways underpinning cell polarization and migration. Here, we find that Rab4, Rab11, and Rab14 and the candidate Rab GDP-GTP exchange factors (GEFs) FAM116A and AVL9 are required for cell migration. Rab14 and its GEF FAM116A localize to and act on an intermediate compartment of the transferrin-recycling pathway prior to Rab11 and after Rab5 and Rab4. This Rab14 intermediate recycling compartment has specific functions in migrating cells discrete from early and recycling endosomes. Rab14-depleted cells show increased N-cadherin levels at junctional complexes and cannot resolve cell-cell junctions. This is due to decreased shedding of cell-surface N-cadherin by the ADAM family protease ADAM10/Kuzbanian. In FAM116A- and Rab14-depleted cells, ADAM10 accumulates in a transferrin-positive endocytic compartment, and the cell-surface level of ADAM10 is correspondingly reduced. FAM116 and Rab14 therefore define an endocytic recycling pathway needed for ADAM protease trafficking and regulation of cell-cell junctions. PMID:22595670

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

    PubMed Central

    Bazzoun, Dana; Lelièvre, Sophie; Talhouk, Rabih

    2013-01-01

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

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

    PubMed Central

    Baranwal, Somesh

    2015-01-01

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

  19. Caveolin-1 regulates expression of junction-associated proteins in brain microvascular endothelial cells

    PubMed Central

    Song, Li; Ge, Shujun; Pachter, Joel S.

    2007-01-01

    Recent evidence from this laboratory indicated that reduced expression of caveolin-1 accompanied the diminished expression of tight junction (TJ)–associated proteins occludin and zonula occludens-1 (ZO-1) following stimulation of brain microvascular endothelial cells (BMECs) with the chemokine CCL2 (formerly called MCP-1). Because attenuated caveolin-1 levels have also been correlated with heightened permeability of other endothelia, the objective of this study was to test the hypothesis that reduced caveolin-1 expression is causally linked to the action of CCL2 on BMEC junctional protein expression and barrier integrity. This was achieved using adenovirus to nondestructively deliver caveolin-1 siRNA (Ad-siCav-1) to BMEC monolayers, which model the blood-brain barrier (BBB). Treatment with siRNA reduced the caveolin-1 protein level as well as occludin and ZO-1. Additionally, occludin exhibited dissociation from the cytoskeletal framework. These changes were attended by comparable alterations in adherens junction (AJ)–associated proteins, VE-cadherin and β-catenin, increased BMEC paracellular permeability, and facilitated the ability of CCL2 to stimulate monocytic transendothelial migration. Furthermore, treating BMECs with cavtratin, a synthetic cell-permeable peptide encoding the caveolin-1 scaffolding domain, antagonized effects of both Ad-siCav-1 and CCL2. These results collectively highlight caveolin-1 loss as a critical step in CCL2-induced modulation of BMEC junctional protein expression and integrity, and possibly serve a crucial role in regulating inflammation at the BBB. PMID:17023578

  20. 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

  1. 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

  2. Exercise regulation of intestinal tight junction proteins.

    PubMed

    Zuhl, Micah; Schneider, Suzanne; Lanphere, Katherine; Conn, Carole; Dokladny, Karol; Moseley, Pope

    2014-06-01

    Gastrointestinal distress, such as diarrhoea, cramping, vomiting, nausea and gastric pain are common among athletes during training and competition. The mechanisms that cause these symptoms are not fully understood. The stress of heat and oxidative damage during exercise causes disruption to intestinal epithelial cell tight junction proteins resulting in increased permeability to luminal endotoxins. The endotoxin moves into the blood stream leading to a systemic immune response. Tight junction integrity is altered by the phosphoylation state of the proteins occludin and claudins, and may be regulated by the type of exercise performed. Prolonged exercise and high-intensity exercise lead to an increase in key phosphorylation enzymes that ultimately cause tight junction dysfunction, but the mechanisms are different. The purpose of this review is to (1) explain the function and physiology of tight junction regulation, (2) discuss the effects of prolonged and high-intensity exercise on tight junction permeability leading to gastrointestinal distress and (3) review agents that may increase or decrease tight junction integrity during exercise. PMID:23134759

  3. Evidence for differential changes of junctional complex proteins in murine neurocysticerosis dependent upon CNS vasculature

    PubMed Central

    Alvarez, Jorge I.; Teale, Judy M.

    2009-01-01

    The delicate balance required to maintain homeostasis of the central nervous system (CNS) is controlled by the blood brain barrier (BBB). Upon injury, the BBB is disrupted compromising the CNS. BBB disruption has been represented as a uniform event. However, our group has shown in a murine model of neurocysticercosis (NCC) that BBB disruption varies depending upon the anatomical site/vascular bed analyzed. In this study further understanding of the mechanisms of BBB disruption were explored in blood vessels located in leptomeninges (pial vessels) and brain parenchyma (parenchymal vessels) by examining the expression of junctional complex proteins in murine brain infected with Mesocestoides corti. Both pial and parenchymal vessels from mock infected animals showed significant colocalization of junctional proteins and displayed an organized architecture. Upon infection, the patterned organization was disrupted and in some cases, particular tight junction and adherens junction proteins were undetectable or appeared to be undergoing proteolysis. The extent and timing of these changes differed between both types of vessels (pial vessel disruption within days versus weeks for parenchymal vessels). To approach potential mechanisms, the expression and activity of matrix metalloproteinase-9 (MMP-9) was evaluated by in situ zymography. The results indicated an increase in MMP-9 activity at sites of BBB disruption exhibiting leukocyte infiltration. Moreover, the timing of MMP activity in pial and parenchymal vessels correlated with the timing of permeability disruption. Thus, breakdown of the BBB is a mutable process despite the similar structure of the junctional complex between pial and parenchymal vessels and involvement of MMP activity. PMID:17686468

  4. 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

  5. A complex of ZO-1 and the BAR-domain protein TOCA-1 regulates actin assembly at the tight junction

    PubMed Central

    Van Itallie, Christina M.; Tietgens, Amber Jean; Krystofiak, Evan; Kachar, Bechara; Anderson, James M.

    2015-01-01

    Assembly and sealing of the tight junction barrier are critically dependent on the perijunctional actin cytoskeleton, yet little is known about physical and functional links between barrier-forming proteins and actin. Here we identify a novel functional complex of the junction scaffolding protein ZO-1 and the F-BAR–domain protein TOCA-1. Using MDCK epithelial cells, we show that an alternative splice of TOCA-1 adds a PDZ-binding motif, which binds ZO-1, targeting TOCA-1 to barrier contacts. This isoform of TOCA-1 recruits the actin nucleation–promoting factor N-WASP to tight junctions. CRISPR-Cas9–mediated knockout of TOCA-1 results in increased paracellular flux and delayed recovery in a calcium switch assay. Knockout of TOCA-1 does not alter FRAP kinetics of GFP ZO-1 or occludin, but longer term (12 h) time-lapse microscopy reveals strikingly decreased tight junction membrane contact dynamics in knockout cells compared with controls. Reexpression of TOCA-1 with, but not without, the PDZ-binding motif rescues both altered flux and membrane contact dynamics. Ultrastructural analysis shows actin accumulation at the adherens junction in TOCA-1–knockout cells but unaltered freeze-fracture fibril morphology. Identification of the ZO-1/TOCA-1 complex provides novel insights into the underappreciated dependence of the barrier on the dynamic nature of cell-to-cell contacts and perijunctional actin. PMID:26063734

  6. PLEKHA7: Cytoskeletal adaptor protein at center stage in junctional organization and signaling.

    PubMed

    Shah, Jimit; Guerrera, Diego; Vasileva, Ekaterina; Sluysmans, Sophie; Bertels, Eva; Citi, Sandra

    2016-06-01

    PLEKHA7 is a recently characterized component of the cytoplasmic region of epithelial adherens junctions (AJ). It comprises two WW domains, a pleckstrin-homology domain, and proline-rich and coiled-coil domains. PLEKHA7 interacts with cytoplasmic components of the AJ (p120-catenin, paracingulin, afadin), stabilizes the E-cadherin complex by linking it to the minus ends of noncentrosomal microtubules, and stabilizes junctional nectins through the newly identified interactor PDZD11. Similarly to afadin, and unlike E-cadherin and p120-catenin, the localization of PLEKHA7 at AJ is strictly zonular (in the zonula adhaerens subdomain of AJ), and does not extend along the basolateral contacts. Genome-wide association studies and experiments on animal and cellular models show that although PLEKHA7 is not required for organism viability, it is implicated in cardiovascular physiology, hypertension, primary angle closure glaucoma, susceptibility to staphylococcal α-toxin, and epithelial morphogenesis and growth. Thus, PLEKHA7 is a cytoskeletal adaptor protein important for AJ organization, and at the center of junction-associated signaling pathways which fine-tune important pathophysiological processes. PMID:27072621

  7. 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. PMID:27291930

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

    PubMed

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

    2016-04-25

    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

  9. Chronic hypoxia down‐regulates tight junction protein ZO‐2 expression in children with cyanotic congenital heart defect

    PubMed Central

    Jenkins, Emma L.; Caputo, Massimo; Angelini, Gianni D.

    2015-01-01

    Abstract Aims Tight junction protein zonula occludens protein 2 (ZO‐2) is a member of the membrane‐associated guanylate kinases protein family known to be expressed at tight junctions of epithelial and endothelial cells and at adherens junctions (AJs) in cardiomyocytes. Little is known about ZO‐2 expression and function in the human heart. Here, we examined the hypothesis that chronic hypoxia down‐regulates ZO‐2 expression in human myocardium and cultured rat cardiomyocytes. Methods and results Patients with a diagnosis of cyanotic (n = 10) or acyanotic (n = 10) Tetralogy of Fallot undergoing surgical repair were used to examine ZO‐2 messenger RNA and protein expression by real time‐PCR, immunohistochemistry, and western blotting. A model of cultured rat cardiomyocytes was used to measure ZO‐2 and AJ proteins levels in response to hypoxia and to investigate ZO‐2 cellular localization. We showed that ZO‐2 is expressed in myocardial tissue in acyanotic and cyanotic children with congenital heart defects. ZO‐2 was specifically down‐regulated in cyanotic myocardium at both the messenger RNA and protein levels when compared with acyanotic patients. This specific down‐regulation can be mimicked in cultured rat cardiomyocytes by treating them with hypoxic conditions confirming that ZO‐2 gene down‐regulation is specifically due to cyanosis. Furthermore, in addition to its cytoplasmic expression, ZO‐2 showed nuclear expression in cultured rat cardiomyocytes suggesting potential role in transcription regulation. Conclusions Hypoxia down‐regulates ZO‐2 expression in both cyanotic patient's myocardium and cultured rat cardiomyocytes. This down‐regulation suggest an involvement of ZO‐2 in cardiac remodelling of AJs in cyanotic children and may explain the greater susceptibility of cyanotic patients to corrective heart surgery.

  10. Evolution and cell physiology. 4. Why invent yet another protein complex to build junctions in epithelial cells?

    PubMed

    Le Bivic, André

    2013-12-15

    The formation of the first epithelium was an essential step for animal evolution, since it has allowed coordination of the behavior of a cell layer and creation of a selective barrier between the internal medium and the outside world. The possibility of coupling the cells in a single layer has allowed morphogenetic events, such as tube formation, or gastrulation, to form more complex animal morphologies. The invention of sealed junctions between cells has allowed, on the other hand, creation of an asymmetry of nutrients or salts between the apical and the basal side of the epithelial layer. Creation of an internal medium has led to homeostasis, allowing the evolution of more complex physiological functions and the emergence of sophisticated animal shapes. During evolution, the origins of the first animals coincided with the invention of several protein complexes, including true cadherins and the polarity protein complexes. How these complexes regulate formation of the apicolateral border and the adherens junctions is still not fully understood. This review focuses on the role of these apical polarity complexes and, in particular, the Crumbs complex, which is essential for proper organization of epithelial layers from Drosophila to humans. PMID:24025867

  11. Cyclic stretch disrupts apical junctional complexes in Caco-2 cell monolayers by a JNK-2-, c-Src-, and MLCK-dependent mechanism

    PubMed Central

    Samak, Geetha; Gangwar, Ruchika; Crosby, Lynn M.; Desai, Leena P.; Wilhelm, Kristina; Waters, Christopher M.

    2014-01-01

    The intestinal epithelium is subjected to various types of mechanical stress. In this study, we investigated the impact of cyclic stretch on tight junction and adherens junction integrity in Caco-2 cell monolayers. Stretch for 2 h resulted in a dramatic modulation of tight junction protein distribution from a linear organization into wavy structure. Continuation of cyclic stretch for 6 h led to redistribution of tight junction proteins from the intercellular junctions into the intracellular compartment. Disruption of tight junctions was associated with redistribution of adherens junction proteins, E-cadherin and β-catenin, and dissociation of the actin cytoskeleton at the actomyosin belt. Stretch activates JNK2, c-Src, and myosin light-chain kinase (MLCK). Inhibition of JNK, Src kinase or MLCK activity and knockdown of JNK2 or c-Src attenuated stretch-induced disruption of tight junctions, adherens junctions, and actin cytoskeleton. Paracellular permeability measured by a novel method demonstrated that cyclic stretch increases paracellular permeability by a JNK, Src kinase, and MLCK-dependent mechanism. Stretch increased tyrosine phosphorylation of occludin, ZO-1, E-cadherin, and β-catenin. Inhibition of JNK or Src kinase attenuated stretch-induced occludin phosphorylation. Immunofluorescence localization indicated that phospho-MLC colocalizes with the vesicle-like actin structure at the actomyosin belt in stretched cells. On the other hand, phospho-c-Src colocalizes with the actin at the apical region of cells. This study demonstrates that cyclic stretch disrupts tight junctions and adherens junctions by a JNK2, c-Src, and MLCK-dependent mechanism. PMID:24722904

  12. The cytoskeletal mechanisms of cell–cell junction formation in endothelial cells

    PubMed Central

    Hoelzle, Matthew K.; Svitkina, Tatyana

    2012-01-01

    The actin cytoskeleton and associated proteins play a vital role in cell–cell adhesion. However, the procedure by which cells establish adherens junctions remains unclear. We investigated the dynamics of cell–cell junction formation and the corresponding architecture of the underlying cytoskeleton in cultured human umbilical vein endothelial cells. We show that the initial interaction between cells is mediated by protruding lamellipodia. On their retraction, cells maintain contact through thin bridges formed by filopodia-like protrusions connected by VE-cadherin–rich junctions. Bridges share multiple features with conventional filopodia, such as an internal actin bundle associated with fascin along the length and vasodilator-stimulated phosphoprotein at the tip. It is striking that, unlike conventional filopodia, transformation of actin organization from the lamellipodial network to filopodial bundle during bridge formation occurs in a proximal-to-distal direction and is accompanied by recruitment of fascin in the same direction. Subsequently, bridge bundles recruit nonmuscle myosin II and mature into stress fibers. Myosin II activity is important for bridge formation and accumulation of VE-cadherin in nascent adherens junctions. Our data reveal a mechanism of cell–cell junction formation in endothelial cells using lamellipodia as the initial protrusive contact, subsequently transforming into filopodia-like bridges connected through adherens junctions. Moreover, a novel lamellipodia-to-filopodia transition is used in this context. PMID:22090347

  13. 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

  14. Minireview: Steroid/Nuclear Receptor-Regulated Dynamics of Occluding and Anchoring Junctions

    PubMed Central

    Kapadia, Bhumika J.

    2014-01-01

    A diverse set of physiological signals control intercellular interactions by regulating the structure and function of occluding junctions (tight junctions) and anchoring junctions (adherens junctions and desmosomes). These plasma membrane junctions are comprised of multiprotein complexes of transmembrane and cytoplasmic peripheral plasma membrane proteins. Evidence from many hormone-responsive tissues has shown that expression, modification, molecular interactions, stability, and localization of junctional complex-associated proteins can be targeted by nuclear hormone receptors and their ligands through transcriptional and nontranscriptional mechanisms. The focus of this minireview is to discuss molecular, cellular, and physiological studies that directly link nuclear receptor- and ligand-triggered signaling pathways to the regulation of occluding and anchoring junction dynamics. PMID:25203673

  15. 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

  16. Differences in expression of junctional adhesion molecule-A and beta-catenin in multiple sclerosis brain tissue: increasing evidence for the role of tight junction pathology.

    PubMed

    Padden, Maureen; Leech, Susie; Craig, Beverly; Kirk, John; Brankin, Brenda; McQuaid, Stephen

    2007-02-01

    Previously we have employed antibodies to the tight junction (TJ)-associated proteins ZO-1 and occludin to describe endothelial tight junction abnormalities, in lesional and normal appearing white matter, in primary and secondary progressive multiple sclerosis (MS). This work is extended here by use of antibodies to the independent TJ-specific proteins and junctional adhesion molecule A & B (JAM-A, JAM-B). We have also assessed the expression in MS of beta-catenin, a protein specific to the TJ-associated adherens junction. Immunocytochemistry and semiquantitative confocal microscopy for JAM-A and beta-catenin was performed on snap-frozen sections from MS cases (n=11) and controls (n=6). Data on 1,443 blood vessels was acquired from active lesions (n=13), inactive lesions (n=13), NAWM (n=20) and control white matter (n=13). In MS abnormal JAM-A expression was found in active (46%) and inactive lesions (21%), comparable to previous data using ZO-1. However, a lower level of TJ abnormality was found in MS NAWM using JAM-A (3%) compared to ZO-1 (13%). JAM-B was strongly expressed on a small number of large blood vessels in control and MS tissues but at too low a level for quantitative analysis. By comparison with the high levels of abnormality observed with the TJ proteins, the adherens junction protein beta-catenin was normally expressed in all MS and control tissue categories. These results confirm, by use of the independent marker JAM-A, that TJ abnormalities are most frequent in active white matter lesions. Altered expression of JAM-A, in addition to affecting junctional tightness may also both reflect and affect leukocyte trafficking, with implications for immune status within the diseased CNS. Conversely, the adherens junction component of the TJ, as indicated by beta-catenin expression is normally expressed in all MS and control tissue categories. PMID:17024496

  17. STIM Proteins and the Endoplasmic Reticulum-Plasma Membrane Junctions

    PubMed Central

    Carrasco, Silvia; Meyer, Tobias

    2013-01-01

    Eukaryotic organelles can interact with each other through stable junctions where the two membranes are kept in close apposition. The junction that connects the endoplasmic reticulum to the plasma membrane (ER-PM junction) is unique in providing a direct communication link between the ER and the PM. In a recently discovered signaling process, STIM (stromal-interacting molecule) proteins sense a drop in ER Ca2+ levels and directly activate Orai PM Ca2+ channels across the junction space. In an inverse process, a voltage-gated PM Ca2+ channel can directly open ER ryanodine-receptor Ca2+ channels in striated-muscle cells. Although ER-PM junctions were first described 50 years ago, their broad importance in Ca2+ signaling, as well as in the regulation of cholesterol and phosphatidylinositol lipid transfer, has only recently been realized. Here, we discuss research from different fields to provide a broad perspective on the structures and unique roles of ER-PM junctions in controlling signaling and metabolic processes. PMID:21548779

  18. 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.

  19. The tight junction: a multifunctional complex.

    PubMed

    Schneeberger, Eveline E; Lynch, Robert D

    2004-06-01

    Multicellular organisms are separated from the external environment by a layer of epithelial cells whose integrity is maintained by intercellular junctional complexes composed of tight junctions, adherens junctions, and desmosomes, whereas gap junctions provide for intercellular communication. The aim of this review is to present an updated overview of recent developments in the area of tight junction biology. In a relatively short time, our knowledge of the tight junction has evolved from a relatively simple view of it being a permeability barrier in the paracellular space and a fence in the plane of the plasma membrane to one of it acting as a multicomponent, multifunctional complex that is involved in regulating numerous and diverse cell functions. A group of integral membrane proteins-occludin, claudins, and junction adhesion molecules-interact with an increasingly complex array of tight junction plaque proteins not only to regulate paracellular solute and water flux but also to integrate such diverse processes as gene transcription, tumor suppression, cell proliferation, and cell polarity. PMID:15151915

  20. 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. PMID:26365579

  1. Pasiflora proteins are novel core components of the septate junction.

    PubMed

    Deligiannaki, Myrto; Casper, Abbie L; Jung, Christophe; Gaul, Ulrike

    2015-09-01

    Epithelial sheets play essential roles as selective barriers insulating the body from the environment and establishing distinct chemical compartments within it. In invertebrate epithelia, septate junctions (SJs) consist of large multi-protein complexes that localize at the apicolateral membrane and mediate barrier function. Here, we report the identification of two novel SJ components, Pasiflora1 and Pasiflora2, through a genome-wide glial RNAi screen in Drosophila. Pasiflora mutants show permeable blood-brain and tracheal barriers, overelongated tracheal tubes and mislocalization of SJ proteins. Consistent with the observed phenotypes, the genes are co-expressed in embryonic epithelia and glia and are required cell-autonomously to exert their function. Pasiflora1 and Pasiflora2 belong to a previously uncharacterized family of tetraspan membrane proteins conserved across the protostome-deuterostome divide. Both proteins localize at SJs and their apicolateral membrane accumulation depends on other complex components. In fluorescence recovery after photobleaching experiments we demonstrate that pasiflora proteins are core SJ components as they are required for complex formation and exhibit restricted mobility within the membrane of wild-type epithelial cells, but rapid diffusion in cells with disrupted SJs. Taken together, our results show that Pasiflora1 and Pasiflora2 are novel integral components of the SJ and implicate a new family of tetraspan proteins in the function of these ancient and crucial cell junctions. PMID:26329602

  2. Pasiflora proteins are novel core components of the septate junction

    PubMed Central

    Deligiannaki, Myrto; Casper, Abbie L.; Jung, Christophe; Gaul, Ulrike

    2015-01-01

    Epithelial sheets play essential roles as selective barriers insulating the body from the environment and establishing distinct chemical compartments within it. In invertebrate epithelia, septate junctions (SJs) consist of large multi-protein complexes that localize at the apicolateral membrane and mediate barrier function. Here, we report the identification of two novel SJ components, Pasiflora1 and Pasiflora2, through a genome-wide glial RNAi screen in Drosophila. Pasiflora mutants show permeable blood-brain and tracheal barriers, overelongated tracheal tubes and mislocalization of SJ proteins. Consistent with the observed phenotypes, the genes are co-expressed in embryonic epithelia and glia and are required cell-autonomously to exert their function. Pasiflora1 and Pasiflora2 belong to a previously uncharacterized family of tetraspan membrane proteins conserved across the protostome-deuterostome divide. Both proteins localize at SJs and their apicolateral membrane accumulation depends on other complex components. In fluorescence recovery after photobleaching experiments we demonstrate that pasiflora proteins are core SJ components as they are required for complex formation and exhibit restricted mobility within the membrane of wild-type epithelial cells, but rapid diffusion in cells with disrupted SJs. Taken together, our results show that Pasiflora1 and Pasiflora2 are novel integral components of the SJ and implicate a new family of tetraspan proteins in the function of these ancient and crucial cell junctions. PMID:26329602

  3. 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. PMID:27120348

  4. 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

  5. Contribution of Annexin 2 to the Architecture of Mature Endothelial Adherens Junctions▿ †

    PubMed Central

    Heyraud, Stéphanie; Jaquinod, Michel; Durmort, Claire; Dambroise, Emilie; Concord, Evelyne; Schaal, Jean Patrick; Huber, Philippe; Gulino-Debrac, Danielle

    2008-01-01

    The vascular endothelial cadherin (VE-cad)-based complex is involved in the maintenance of vascular endothelium integrity. Using immunoprecipitation experiments, we have demonstrated that, in confluent human umbilical vein endothelial cells, the VE-cad-based complex interacts with annexin 2 and that annexin 2 translocates from the cytoplasm to the cell-cell contact sites as cell confluence is established. Annexin 2, located in cholesterol rafts, binds to both the actin cytoskeleton and the VE-cad-based complex so the complex is docked to cholesterol rafts. These multiple connections prevent the lateral diffusion of the VE-cad-based complex, thus strengthening adherens junctions in the ultimate steps of maturation. Moreover, we observed that the down-regulation of annexin 2 by small interfering RNA induces a delocalization of VE-cad from adherens junctions and consequently a destabilization of these junctions. Furthermore, our data indicate that the decoupling of the annexin 2/p11 complex from the VE-cad-based junction, triggered by vascular endothelial growth factor treatment, facilitates the switch from a quiescent to an immature state. PMID:18160703

  6. Transmissible gastroenteritis virus and porcine epidemic diarrhoea virus infection induces dramatic changes in the tight junctions and microfilaments of polarized IPEC-J2 cells.

    PubMed

    Zhao, Shanshan; Gao, Junkai; Zhu, Liqi; Yang, Qian

    2014-11-01

    Viral infection converts the normal constitution of a cell to optimise viral entry, replication, and virion production. These conversions contain alterations or disruptions of the tight and adherens junctions between cells as part of their pathogenesis, and reorganise cellular microfilaments that initiate, sustain and spread the viral infections and so on. Using porcine epidemic diarrhoea virus (PEDV), transmissible gastroenteritis virus (TGEV) and a model of normal intestinal epithelial cells (IPEC-J2), we researched the interaction between tight and adherens junctions and microfilaments of IPEC-J2 cells with these viruses. In our work, the results showed that IPEC-J2 cells were susceptible to TGEV and PEDV infection. And TGEV could impair the barrier integrity of IPEC-J2 cells at early stages of infection through down-regulating some proteins of tight and adherens junctions, while PEDV cloud cause a slight of damage in the integrity of epithelial barrier. In addition, they also could affect the microfilaments remodelling of IPEC-J2 cells, and the drug-interfered microfilaments could inhibit viral replication and release. Furthermore, PEDV+TGEV co-infection was more aggravating to damage of tight junctions and remodelling of microfilaments than their single infection. Finally, the PEDV and TGEV infection affected the MAPK pathway, and inhibition of MAPK pathway regulated the changes of tight junctions and microfilaments of cells. These studies provide a new insight from the perspective of the epithelial barrier and microfilaments into the pathogenesis of PEDV and TGEV. PMID:25173696

  7. 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

  8. 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

  9. PATJ, a Tight Junction-Associated PDZ Protein, Is a Novel Degradation Target of High-Risk Human Papillomavirus E6 and the Alternatively Spliced Isoform 18 E6*▿

    PubMed Central

    Storrs, Carina H.; Silverstein, Saul J.

    2007-01-01

    The E6 protein from high-risk human papillomavirus types interacts with and degrades several PDZ domain-containing proteins that localize to adherens junctions or tight junctions in polarized epithelial cells. We have identified the tight junction-associated multi-PDZ protein PATJ (PALS1-associated TJ protein) as a novel binding partner and degradation target of high-risk types 16 and 18 E6. PATJ functions in the assembly of the evolutionarily conserved CRB-PALS1-PATJ and Par6-aPKC-Par3 complexes and is critical for the formation of tight junctions in polarized cells. The ability of type 18 E6 full-length to bind to, and the subsequent degradation of, PATJ is dependent on its C-terminal PDZ binding motif. We demonstrate that the spliced 18 E6* protein, which lacks a C-terminal PDZ binding motif, associates with and degrades PATJ independently of full-length 18 E6. Thus, PATJ is the first binding partner that is degraded in response to both isoforms of 18 E6. The ability of E6 to utilize a non-E6AP ubiquitin ligase for the degradation of several PDZ binding partners has been suggested. We also demonstrate that 18 E6-mediated degradation of PATJ is not inhibited in cells where E6AP is silenced by shRNA. This suggests that the E6-E6AP complex is not required for the degradation of this protein target. PMID:17287269

  10. Calcium-Ask1-MKK7-JNK2-c-Src Signaling Cascade Mediates Disruption of Intestinal Epithelial Tight Junctions by Dextran Sulfate Sodium

    PubMed Central

    Samak, Geetha; Chaudhry, Kamaljit K.; Gangwar, Ruchika; Narayanan, Damodaran; Jaggar, Jonathan H.; Rao, RadhaKrishna

    2015-01-01

    Disruption of intestinal epithelial tight junctions is an important event in the pathogenesis of ulcerative colitis. Dextran sodium sulfate (DSS) induces colitis in mice with the symptoms similar to ulcerative colitis. However, the mechanism of DSS-induced colitis is unknown. We investigated the mechanism of DSS-induced disruption of intestinal epithelial tight junctions and barrier dysfunction in Caco-2 cell monolayers in vitro and mouse colon in vivo. DSS treatment resulted in disruption of tight junctions, adherens junctions and actin cytoskeleton leading to barrier dysfunction in Caco-2 cell monolayers. DSS induced a rapid activation of c-jun N-terminal kinase (JNK), and the inhibition or knockdown of JNK2 attenuated DSS-induced tight junction disruption and barrier dysfunction. In mice, DSS administration for 4 days caused redistribution of tight junction and adherens junction proteins from the epithelial junctions, which was blocked by JNK inhibitor. In Caco-2 cell monolayers, DSS increased intracellular Ca2+ concentration, and depletion of intracellular Ca2+ by BAPTA or thapsigargin attenuated DSS-induced JNK activation, tight junction disruption and barrier dysfunction. Knockdown of Ask1 or MKK7 blocked DSS-induced tight junction disruption and barrier dysfunction. DSS activated c-Src by a Ca2+ and JNK-dependent mechanism. Inhibition of Src kinase activity or knockdown of c-Src blocked DSS-induced tight junction disruption and barrier dysfunction. DSS increased Tyr-phosphorylation of occludin, ZO-1, E-cadherin and β-catenin. SP600125 abrogated DSS-induced Tyr-phosphorylation of junctional proteins. Recombinant JNK2 induced threonine phosphorylation and auto phosphorylation of c-Src. This study demonstrates that Ca2+-Ask1-MKK7-JNK2-cSrc signaling cascade mediates DSS-induced tight junction disruption and barrier dysfunction. PMID:25377781

  11. Emerging Multifunctional Roles of Claudin Tight Junction Proteins in Bone

    PubMed Central

    Alshbool, Fatima. Z.

    2014-01-01

    The imbalance between bone formation and resorption during bone remodeling has been documented to be a major factor in the pathogenesis of osteoporosis. Recent evidence suggests a significant role for the tight junction proteins, Claudins (Cldns), in the regulation of bone remodeling processes. In terms of function, whereas Cldns act “canonically” as key determinants of paracellular permeability, there is considerable recent evidence to suggest that Cldns also participate in cell signaling, ie, a “noncanonical function”. To this end, Cldns have been shown to regulate cell proliferation, differentiation, and gene expression in a variety of cell types. The present review will discuss Cldns' structure, their expression profile, regulation of expression, and their canonical and non- canonical functions in general with special emphasis on bone cells. In order to shed light on the noncanonical functions of Cldns in bone, we will highlight the role of Cldn-18 in regulating bone resorption and osteoclast differentiation. Collectively, we hope to provide a framework for guiding future research on understanding how Cldns modulate osteoblast and osteoclast function and overall bone homeostasis. Such studies should provide valuable insights into the pathogenesis of osteoporosis, and may highlight Cldns as novel targets for the diagnosis and therapeutic management of osteoporosis. PMID:24758302

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

    PubMed Central

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

    2015-01-01

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

  13. The cell-cell junctions of mammalian testes: I. The adhering junctions of the seminiferous epithelium represent special differentiation structures.

    PubMed

    Domke, Lisa M; Rickelt, Steffen; Dörflinger, Yvette; Kuhn, Caecilia; Winter-Simanowski, Stefanie; Zimbelmann, Ralf; Rosin-Arbesfeld, Rina; Heid, Hans; Franke, Werner W

    2014-09-01

    The seminiferous tubules and the excurrent ducts of the mammalian testis are physiologically separated from the mesenchymal tissues and the blood and lymph system by a special structural barrier to paracellular translocations of molecules and particles: the "blood-testis barrier", formed by junctions connecting Sertoli cells with each other and with spermatogonial cells. In combined biochemical as well as light and electron microscopical studies we systematically determine the molecules located in the adhering junctions of adult mammalian (human, bovine, porcine, murine, i.e., rat and mouse) testis. We show that the seminiferous epithelium does not contain desmosomes, or "desmosome-like" junctions, nor any of the desmosome-specific marker molecules and that the adhering junctions of tubules and ductules are fundamentally different. While the ductules contain classical epithelial cell layers with E-cadherin-based adherens junctions (AJs) and typical desmosomes, the Sertoli cells of the tubules lack desmosomes and "desmosome-like" junctions but are connected by morphologically different forms of AJs. These junctions are based on N-cadherin anchored in cytoplasmic plaques, which in some subforms appear thick and dense but in other subforms contain only scarce and loosely arranged plaque structures formed by α- and β-catenin, proteins p120, p0071 and plakoglobin, together with a member of the striatin family and also, in rodents, the proteins ZO-1 and myozap. These N-cadherin-based AJs also include two novel types of junctions: the "areae adhaerentes", i.e., variously-sized, often very large cell-cell contacts and small sieve-plate-like AJs perforated by cytoplasm-to-cytoplasm channels of 5-7 nm internal diameter ("cribelliform junctions"). We emphasize the unique character of this epithelium that totally lacks major epithelial marker molecules and structures such as keratin filaments and desmosomal elements as well as EpCAM- and PERP-containing junctions. We also

  14. Multiple Protein Interactions Involving Proposed Extracellular Loop Domains of the Tight Junction Protein Occludin

    PubMed Central

    Nusrat, Asma; Brown, G. Thomas; Tom, Jeffrey; Drake, Alex; Bui, Tam T.T.; Quan, Cliff; Mrsny, Randall J.

    2005-01-01

    Occludin is a tetraspan integral membrane protein in epithelial and endothelial tight junction (TJ) structures that is projected to have two extracellular loops. We have used peptides emulating central regions of human occludin's first and second loops, termed O-A:101–121 and O-B:210–228, respectively, to examine potential molecular interactions between these two regions of occludin and other TJ proteins. A superficial biophysical assessment of A:101–121 and O-B:210–228 showed them to have dissimilar solution conformation characteristics. Although O-A:101–121 failed to strongly interact with protein components of the human epithelial intestinal cell line T84, O-B:210–228 selectively associated with occludin, claudin-one and the junctional adhesion molecule (JAM)-A. Further, the presence of O-B:210–228, but not O-A:101–121, impeded the recovery of functional TJ structures. A scrambled peptide sequences of O-B:210–228 failed to influence TJ assembly. These studies demonstrate distinct properties for these two extracellular segments of the occludin protein and provide an improved understanding of how specific domains of occludin may interact with proteins present at TJ structures. PMID:15659655

  15. Kinetic Measurements Reveal Enhanced Protein-Protein Interactions at Intercellular Junctions

    PubMed Central

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

    2016-01-01

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

  16. Kinetic Measurements Reveal Enhanced Protein-Protein Interactions at Intercellular Junctions.

    PubMed

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

    2016-01-01

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

  17. 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

  18. 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

  19. 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.

  20. Lateral interactions among membrane proteins. Implications for the organization of gap junctions.

    PubMed Central

    Abney, J R; Braun, J; Owicki, J C

    1987-01-01

    We have studied the relationship between interprotein forces and the lateral distribution of proteins in disordered mouse liver gap junctions. Data on protein positions are obtained from freeze-fracture electron micrographs. Short-ranged correlations in observed positions are characteristic of interacting particles in a fluid state. An analysis derived from statistical mechanics allows the determination of the magnitude and functional form of interprotein forces. We find that jap junction proteins are mutually repulsive, in a manner consistent with electrostatics and excluded volume. This dictates that long-ranged protein aggregation into jap junction plaques cannot arise solely from interparticle interactions. An alternative is the balance of lateral pressures between the junction and the surrounding glycocalyx. This idea is quantified into a model. Junctional pressure arises from protein-protein interactions and is computed from a pressure equation based on the force and a radial distribution function describing order. The pressure from the glycocalyx is assumed to arise from mixing, electrostatic, and elastic interactions of sugar residues, and is described with terms from Flory-Krigbaum and McMillan-Mayer theories. The results of this modeling are in reasonable agreement with available experimental data. Images FIGURE 1 PMID:3651562

  1. 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

  2. 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

  3. Tight junctions in Hailey-Hailey and Darier's diseases

    PubMed Central

    Raiko, Laura; Leinonen, Pekka; Hägg, Päivi M.; Peltonen, Juha; Oikarinen, Aarne; Peltonen, Sirkku

    2009-01-01

    Hailey-Hailey disease (HHD) and Darier's disease (DD) are caused by mutations in Ca2+-ATPases with the end result of desmosomal disruption and suprabasal acantholysis. Tight junctions (TJ) are located in the granular cell layer in normal skin and contribute to the epidermal barrier. Aberrations in the epidermal differentiation, such as in psoriasis, have been shown to lead to changes in the expression of TJ components. Our aim was to elucidate the expression and dynamics of the TJ proteins during the disruption of desmosomes in HHD and DD lesions. Indirect immunofluorescence and avidin-biotin labeling for TJ, desmosomal and adherens junction proteins, and subsequent analyses with the confocal laser scanning microscope were carried out on 14 HHD and 14 DD skin samples. Transepidermal water loss (TEWL) was measured in normal and lesional epidermis of nine HHD and eight DD patients to evaluate the function of the epidermal barrier in HHD and DD skin. The localization of TJ proteins claudin-1, claudin-4, ZO-1, and occludin in perilesional HHD and DD epidermis was similar to that previously described in normal skin. In HHD lesions the tissue distribution of ZO-1 expanded to the acantholytic spinous cells. In agreement with previous findings, desmoplakin was localized intracellularly. In contrast claudin-1 and ZO-1 persisted in the cell-cell contact sites of acantholytic cells. TEWL was increased in the lesional skin. The current results suggest that TJ components follow different dynamics in acantholysis of HHD and DD compared to desmosomal and adherens junction proteins. PMID:25386233

  4. 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. PMID:27250245

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

    PubMed Central

    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. PMID:27250245

  6. 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

  7. p120-catenin-dependent junctional recruitment of Shroom3 is required for apical constriction during lens pit morphogenesis

    PubMed Central

    Lang, Richard A.; Herman, Ken; Reynolds, Albert B.; Hildebrand, Jeffrey D.; Plageman, Timothy F.

    2014-01-01

    Apical constriction (AC) is a widely utilized mechanism of cell shape change whereby epithelial cells transform from a cylindrical to conical shape, which can facilitate morphogenetic movements during embryonic development. Invertebrate epithelial cells undergoing AC depend on the contraction of apical cortex-spanning actomyosin filaments that generate force on the apical junctions and pull them toward the middle of the cell, effectively reducing the apical circumference. A current challenge is to determine whether these mechanisms are conserved in vertebrates and to identify the molecules responsible for linking apical junctions with the AC machinery. Utilizing the developing mouse eye as a model, we have uncovered evidence that lens placode AC may be partially dependent on apically positioned myosin-containing filaments associated with the zonula adherens. In addition we found that, among several junctional components, p120-catenin genetically interacts with Shroom3, a protein required for AC during embryonic morphogenesis. Further analysis revealed that, similar to Shroom3, p120-catenin is required for AC of lens cells. Finally, we determined that p120-catenin functions by recruiting Shroom3 to adherens junctions. Together, these data identify a novel role for p120-catenin during AC and further define the mechanisms required for vertebrate AC. PMID:25038041

  8. 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

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

    PubMed Central

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

    2014-01-01

    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. PMID:24385580

  10. 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

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

    PubMed

    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

  12. Protein kinase C isoforms at the neuromuscular junction: localization and specific roles in neurotransmission and development

    PubMed Central

    Lanuza, Maria A; Santafe, Manel M; Garcia, Neus; Besalduch, Núria; Tomàs, Marta; Obis, Teresa; Priego, Mercedes; Nelson, Phillip G; Tomàs, Josep

    2014-01-01

    The protein kinase C family (PKC) regulates a variety of neural functions including neurotransmitter release. The selective activation of a wide range of PKC isoforms in different cells and domains is likely to contribute to the functional diversity of PKC phosphorylating activity. In this review, we describe the isoform localization, phosphorylation function, regulation and signalling of the PKC family at the neuromuscular junction. Data show the involvement of the PKC family in several important functions at the neuromuscular junction and in particular in the maturation of the synapse and the modulation of neurotransmission in the adult. PMID:24102585

  13. 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

  14. HMG box proteins bind to four-way DNA junctions in their open conformation.

    PubMed

    P-ohler, J R; Norman, D G; Bramham, J; Bianchi, M E; Lilley, D M

    1998-02-01

    The HMG box is an 80 amino acid domain found in a variety of eukaryotic chromosomal proteins and transcription factors. Binding to DNA is associated with recognition of structural distortion or manipulation of DNA structure. All the HMG box domains bind to four-way DNA junctions, which must therefore present some feature that is common to the binding targets of this wide variety of proteins. Since the four-way junction can itself adopt a variety of structures depending upon conditions, it is important to determine in which form it exists in complexes with HMG boxes. We find that a single HMG box domain is bound exclusively to the open square form of the junction and that conditions that stabilize the stacked X structure significantly lower affinity for the HMG box. We suggest that the HMG domain binds to one arm of the junction in the minor groove at the point of strand exchange and we present a model for the structure of the complex. PMID:9451006

  15. Sertoli cell junctional proteins as early targets for different classes of reproductive toxicants.

    PubMed

    Fiorini, Céline; Tilloy-Ellul, Anne; Chevalier, Stephan; Charuel, Claude; Pointis, Georges

    2004-05-01

    In the testis, Sertoli cells establish intercellular junctions that are essential for spermatogenesis. The SerW3 Sertoli cell line displays some features of native Sertoli cells. Western blot and immunofluorescence analyses showed that SerW3 Sertoli cells expressed typical components of tight (occludin and zonula occludens-1), anchoring (N-cadherin) and gap (connexin 43) junctions. Testicular toxicants (DDT, pentachlorophenol, dieldrin, dinitrobenzene, cadmium chloride, cisplatin, gossypol, bisphenol A and tert-octylphenol) affected intercellular junctions by either reducing the amount or inducing aberrant intracellular localization of these membranous proteins. Phosphodiesterase inhibitors (isobutyl methylxantine, rolipram, zaprinast, zardaverine) did not alter junctional-complex component levels but caused a rapid and reversible redistribution of these proteins to the cytoplasmic compartment. The present study showed that occludin, ZO-1, N-cadherin and specifically Cx43 could be early targets for testicular toxicants. The SerW3 cell line therefore appears as a useful in vitro model to evaluate molecules with potential anti-reproductive effects. PMID:15082077

  16. Expression of occludin, tight-junction-associated protein, in human digestive tract.

    PubMed Central

    Kimura, Y.; Shiozaki, H.; Hirao, M.; Maeno, Y.; Doki, Y.; Inoue, M.; Monden, T.; Ando-Akatsuka, Y.; Furuse, M.; Tsukita, S.; Monden, M.

    1997-01-01

    The tight junction seals cells together at a subapical location and functionally separates the plasma membrane into an apical and a basolateral domain. This junction is one of the most characteristic structural markers of the polarized epithelial cell. Recently, occludin has been identified as an integral transmembrane protein localizing at the tight junction and directly associated with ZO-1, an undercoat-constitutive cytoplasmic protein. We have investigated occludin expression in conjunction with ZO-1 in normal epithelia and cancers of human digestive tract by immunostaining with a new antibody raised against human occludin. In the normal simple columnar epithelium, occludin was expressed together with ZO-1 as a single line at the apical cell border. However, in the esophagus, which has a stratified squamous epithelium, no occludin expression could be detected, but ZO-1 was expressed in the spinous layer. As for cancers, both occludin and ZO-1 showed the same expression in differentiated adenocarcinoma cells as in normal epithelium, but in poorly differentiated adenocarcinomas, the expression of these two proteins was reduced. There was significant correlation between tumor differentiation and expression of these proteins. These results suggest that occludin, together with ZO-1, is involved in the formation of gland-like structures. In addition, occludin expression can serve as a histopathological indicator for differentiation in gastrointestinal adenocarcinomas. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:9212730

  17. Phylogenetic and bioinformatic analysis of gap junction-related proteins, innexins, pannexins and connexins.

    PubMed

    Fushiki, Daisuke; Hamada, Yasuo; Yoshimura, Ryoichi; Endo, Yasuhisa

    2010-04-01

    All multi-cellular animals, including hydra, insects and vertebrates, develop gap junctions, which communicate directly with neighboring cells. Gap junctions consist of protein families called connexins in vertebrates and innexins in invertebrates. Connexins and innexins have no homology in their amino acid sequence, but both are thought to have some similar characteristics, such as a tetra-membrane-spanning structure, formation of a channel by hexamer, and transmission of small molecules (e.g. ions) to neighboring cells. Pannexins were recently identified as a homolog of innexins in vertebrate genomes. Although pannexins are thought to share the function of intercellular communication with connexins and innexins, there is little information about the relationship among these three protein families of gap junctions. We phylgenetically and bioinformatically examined these protein families and other tetra-membrane-spanning proteins using a database and three analytical softwares. The clades formed by pannexin families do not belong to the species classification but do to paralogs of each member of pannexins. Amino acid sequences of pannexins are closely related to those of innexins but less to those of connexins. These data suggest that innexins and pannexins have a common origin, but the relationship between innexins/pannexins and connexins is as slight as that of other tetra-membrane-spanning members. PMID:20460741

  18. The impact of intrinsic ageing on the protein composition of the dermal-epidermal junction.

    PubMed

    Langton, Abigail K; Halai, Poonam; Griffiths, Christopher E M; Sherratt, Michael J; Watson, Rachel E B

    2016-06-01

    The dermal-epidermal junction of human skin exhibits age-related remodelling, resulting in a flattened appearance and reduced surface area. Despite this, a paucity of information is available regarding which protein components change with advancing age. Here we report a significant reduction in the protein distribution of collagen IV (P<0.0001), collagen VII (P<0.001), collagen XVII (P<0.01), integrin β4 (P<0.001) and laminin-332 (P<0.0001) in intrinsically aged skin. The functional implication of this altered protein composition appears to be loss of structural integrity and may, in part, explain the increased fragility of aged skin. PMID:27013376

  19. Protein Kinase Cδ-mediated Phosphorylation of Connexin43 Gap Junction Channels Causes Movement within Gap Junctions followed by Vesicle Internalization and Protein Degradation*

    PubMed Central

    Cone, Angela C.; Cavin, Gabriel; Ambrosi, Cinzia; Hakozaki, Hiroyuki; Wu-Zhang, Alyssa X.; Kunkel, Maya T.; Newton, Alexandra C.; Sosinsky, Gina E.

    2014-01-01

    Phosphorylation of gap junction proteins, connexins, plays a role in global signaling events involving kinases. Connexin43 (Cx43), a ubiquitous and important connexin, has several phosphorylation sites for specific kinases. We appended an imaging reporter tag for the activity of the δ isoform of protein kinase C (PKCδ) to the carboxyl terminus of Cx43. The FRET signal of this reporter is inversely related to the phosphorylation of serine 368 of Cx43. By activating PKC with the phorbol ester phorbol 12,13-dibutyrate (PDBu) or a natural stimulant, UTP, time lapse live cell imaging movies indicated phosphorylated Ser-368 Cx43 separated into discrete domains within gap junctions and was internalized in small vesicles, after which it was degraded by lysosomes and proteasomes. Mutation of Ser-368 to an Ala eliminated the response to PDBu and changes in phosphorylation of the reporter. A phosphatase inhibitor, calyculin A, does not change this pattern, indicating PKC phosphorylation causes degradation of Cx43 without dephosphorylation, which is in accordance with current hypotheses that cells control their intercellular communication by a fast and constant turnover of connexins, using phosphorylation as part of this mechanism. PMID:24500718

  20. 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

  1. 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

  2. Abnormal Junctions and Permeability of Myelin in PMP22-Deficient Nerves

    PubMed Central

    Guo, Jiasong; Wang, Leiming; Zhang, Yang; Wu, Jiawen; Arpag, Sezgi; Hu, Bo; Imhof, Beat A.; Tian, Xinxia; Carter, Bruce D.; Suter, Ueli; Li, Jun

    2014-01-01

    Objective The peripheral myelin protein-22 (PMP22) gene is associated with the most common types of inherited neuropathies, including hereditary neuropathy with liability to pressure palsies (HNPP) caused by PMP22 deficiency. However, the function of PMP22 has yet to be defined. Our previous study has shown that PMP22 deficiency causes an impaired propagation of nerve action potentials in the absence of demyelination. In the present study, we tested an alternative mechanism relating to myelin permeability. Methods Utilizing Pmp22+/− mice as a model of HNPP, we evaluated myelin junctions and their permeability using morphological, electrophysiological, and biochemical approaches. Results We show disruption of multiple types of cell junction complexes in peripheral nerve, resulting in increased permeability of myelin and impaired action potential propagation. We further demonstrate that PMP22 interacts with immunoglobulin domain–containing proteins known to regulate tight/adherens junctions and/or transmembrane adhesions, including junctional adhesion molecule-C (JAM-C) and myelin-associated glycoprotein (MAG). Deletion of Jam-c or Mag in mice recapitulates pathology in HNPP. Interpretation Our study reveals a novel mechanism by which PMP22 deficiency affects nerve conduction not through removal of myelin, but through disruption of myelin junctions. PMID:24339129

  3. Effects of formaldehyde inhalation on the junctional proteins of nasal respiratory mucosa of rats.

    PubMed

    Arican, R Yavuz; Sahin, Zeliha; Ustunel, Ismail; Sarikcioglu, Levent; Ozdem, Sadi; Oguz, Nurettin

    2009-07-01

    Exposure to formaldehyde, which is an organic compound, disturbs the integrity of nasal mucosa. In this study, we aimed to clarify the protein changes in the junctional complex of nasal mucosa of Wistar rats exposed to formaldehyde inhalation. The study was performed in 20 female Wistar rats. Rats were divided into two groups randomly. Control rats were allowed free access to standard rat chaw and tap water (n:10). Experimental group was exposed to formaldehyde vapor at 15ppm, 6h/day, 5 days/week for 12 weeks (n:10). Histological evaluation of the experimental model was determined by hematoxylin-eosin (HE) and periodic acid Schiff (PAS) stainings of paraffin-embedded nasal mucosa tissues and by electron microscopy. The effects of formaldehyde inhalation on the distribution of occludin, E-cadherin, and gamma-catenin were assessed by immunohistochemistry. The nasal mucosa of the experimental group was correlated with hypertrophy in goblet cell, degeneration in basal lamina, stratification of epithelium, and proliferation. Thickness of basal lamina and also local degenerative regions, vacuole increase in cytoplasmic areas, irregular forms of kinocilium and loss of sharpness in the kinocilium membrane were the findings at the ultrastructural level. The expressions of E-cadherin, occludin, gamma-catenin proteins in intercellular junctional complexes of rat nasal mucosa were also decreased in experimental group compared to control group. The findings of the present study indicated that formaldehyde vapor inhalation in the concentrations and duration of exposure used in the present experiment significantly decreased the density of structural proteins of the junctional complex in the nasoepithelium. It was suggested that, the formaldehyde inhalation could cause complete impairment of intercellular junctional complexes and disturb the tissue integrity in nasal mucosa at higher concentrations. PMID:18996001

  4. 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

  5. Age-related differential gene and protein expression in postnatal cartilage canal and osteochondral junction chondrocytes.

    PubMed

    Duesterdieck-Zellmer, Katja; Semevolos, Stacy; Kinsley, Marc; Riddick, Tara

    2015-01-01

    Wnt/β-catenin, Indian hedgehog (Ihh)/Parathyroid-related peptide (PTHrP) and retinoid signaling pathways regulate cartilage differentiation, growth, and function during development and play a key role in endochondral ossification. The objective of this study was to elucidate the gene and protein expression of signaling molecules of these regulatory pathways in chondrocytes surrounding cartilage canals and the osteochondral junction during neonatal and pre-adolescent development. This study revealed cell-specific and age-related differences in gene and protein expression of signaling molecules of these regulatory pathways. A trend for higher gene expression of PTHrP along the cartilage canals and Ihh along the osteochondral junction suggests the presence of paracrine feedback in articular-epiphyseal cartilage. Differential expression of canonical (β-catenin, Wnt-4, Lrp4, Lrp6) and noncanonical Wnt signaling (Wnt-5b, Wnt-11) and their inhibitors (Dkk1, Axin1, sFRP3, sFRP5, Wif-1) surrounding the cartilage canals and osteochondral junction provides evidence of the complex interactions occurring during endochondral ossification. PMID:25479004

  6. Depletion of E-Cadherin Disrupts Establishment but Not Maintenance of Cell Junctions in Madin-Darby Canine Kidney Epithelial Cells

    PubMed Central

    Capaldo, Christopher T.

    2007-01-01

    E-cadherin forms calcium-dependent homophilic intercellular adhesions between epithelial cells. These contacts regulate multiple aspects of cell behavior, including the organization of intercellular tight junctions (TJs). To distinguish between the roles of E-cadherin in formation versus maintenance of junctions, Madin-Darby canine kidney (MDCK) cells were depleted of E-cadherin by RNA interference. Surprisingly, reducing E-cadherin expression had little effect on the protein levels or localization of adherens junction (AJ) or TJ markers. The cells underwent morphological changes, as the normally flat apical surface swelled into a dome. However, apical–basal polarity was not compromised, transmembrane resistance was normal, and zonula occludin protein 1 dynamics at the TJs were unchanged. Additionally, an E-cadherin/Cadherin-6 double knockdown also failed to disrupt established TJs, although β-catenin was lost from the cell cortex. Nevertheless, cells depleted of E-cadherin failed to properly reestablish cell polarity after junction disassembly. Recovery of cell–cell adhesion, transepithelial resistance, and the localization of TJ and AJ markers were all delayed. In contrast, depletion of α-catenin caused long-term disruption of junctions. These results indicate that E-cadherin and Cadherin-6 function as a scaffold for the construction of polarized structures, and they become largely dispensable in mature junctions, whereas α-catenin is essential for the maintenance of functional junctions. PMID:17093058

  7. The tight junction protein CAR regulates cardiac conduction and cell–cell communication

    PubMed Central

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

    2008-01-01

    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. PMID:18794341

  8. Immunohistological study of tight junction protein expression in mal de Meleda.

    PubMed

    Kacem, Monia; Agili, Faouzia; Tounsi, Haifa; Zribi, Hela; Zaraa, Ines; Mokni, Mourad; Boubaker, Samir

    2016-01-01

    Mal de Meleda (MdM, MIM: 248300) is a rare autosomal recessive skin disorder characterized by diffuse palmoplantar keratoderma and transgressive keratosis with onset in early infancy. The gene responsible for MdM, ARS, encodes for Secreted Lys6/Plaur domain-containing protein 1 which is essential for epidermal homeostasis. Tight junctions have been proposed to have two mutually exclusive functions: a fence function which prevents the mixing of membrane proteins between the apical and basolateral membranes; and a gate function which controls the paracellular passage of ions and solutes between cells. In this study we report immunohistochemical investigations of tight junction proteins claudin-1 and occludin in MdM Tunisian families. Nine skin biopsies from patients with MdM were analyzed. The control group was formed by skin biopsies belonging to healthy individuals. Immunohistochemical study was performed on fixed sections from biopsies of four microns with the following polyclonal antibodies: anti-claudin-1 and anti-occludin. In control skin, claudin-1 exhibited membrane expression throughout the epidermis with increasing and upward intensity, whereas occludin was detected in the cell membrane of keratinocytes of the stratum granulosum. In MdM skin, claudin-1 was expressed throughout the thickness of the spinous layers with membrane staining, and occludin had cytoplasmic staining in the granular layer. The immunohistochemical expression of TJ proteins in MdM patients harbors premature expression of occludin and decreased expression of claudin-1, highlighting further evidence for disorders in epidermal homeostasis. PMID:26986447

  9. A role for the retinoblastoma protein as a regulator of mouse osteoblast cell adhesion: implications for osteogenesis and osteosarcoma formation.

    PubMed

    Sosa-García, Bernadette; Gunduz, Volkan; Vázquez-Rivera, Viviana; Cress, W Douglas; Wright, Gabriela; Bian, Haikuo; Hinds, Philip W; Santiago-Cardona, Pedro G

    2010-01-01

    The retinoblastoma protein (pRb) is a cell cycle regulator inactivated in most human cancers. Loss of pRb function results from mutations in the gene coding for pRb or for any of its upstream regulators. Although pRb is predominantly known as a cell cycle repressor, our data point to additional pRb functions in cell adhesion. Our data show that pRb regulates the expression of a wide repertoire of cell adhesion genes and regulates the assembly of the adherens junctions required for cell adhesion. We conducted our studies in osteoblasts, which depend on both pRb and on cell-to-cell contacts for their differentiation and function. We generated knockout mice in which the RB gene was excised specifically in osteoblasts using the cre-lox P system and found that osteoblasts from pRb knockout mice did not assemble adherens junction at their membranes. pRb depletion in wild type osteoblasts using RNAi also disrupted adherens junctions. Microarrays comparing pRb-expressing and pRb-deficient osteoblasts showed that pRb controls the expression of a number of cell adhesion genes, including cadherins. Furthermore, pRb knockout mice showed bone abnormalities consistent with osteoblast adhesion defects. We also found that pRb controls the function of merlin, a well-known regulator of adherens junction assembly, by repressing Rac1 and its effector Pak1. Using qRT-PCR, immunoblots, co-immunoprecipitation assays, and immunofluorescent labeling, we observed that pRb loss resulted in Rac1 and Pak1 overexpression concomitant with merlin inactivation by Pak1, merlin detachment from the membrane, and adherens junction loss. Our data support a pRb function in cell adhesion while elucidating the mechanism for this function. Our work suggests that in some tumor types pRb inactivation results in both a loss of cell cycle control that promotes initial tumor growth as well as in a loss of cell-to-cell contacts, which contributes to later stages of metastasis. PMID:21085651

  10. Connexin 33: a rodent-specific member of the gap junction protein family?

    PubMed

    Fischer, Petra; Brehm, Ralph; Konrad, Lutz; Hartmann, Sonja; Kliesch, Sabine; Bohle, Rainer M; Bergmann, Martin

    2005-01-01

    Gap junctional intercellular communication between Sertoli cells and between Sertoli cells and spermatogonia is considered to play a key role in the regulation of both proliferation and differentiation of germ cells. A member of the gap junction protein family, Connexin 33 (cx33), probably has an inhibitory effect on the formation of gap junctions and so far it is the only cx that has been exclusively found in rat and mouse testes. Thus, this connexin seems to be a special member of the cx family. Using immunohistochemistry, Western blot analysis, polymerase chain reaction, and reverse transcription (RT)-PCR (tissue homogenate and microdissected cells), we studied the possible occurrence of cx33 at the protein, the DNA, and the RNA level in human testis. Whereas immunohistochemistry using the only commercially available anti-cx33 antibody showed similar labeling to the rat within the seminiferous epithelium, we could not find any further evidence for the existence of cx33 using Western blot analysis, PCR, and RT-PCR in human testis. Based on the demonstration of the staining pattern of mitochondria in human germ cells and on preabsorption studies, we could demonstrate anti-cx33 antibody cross-reacting with mitochondrial ferritin, a protein localized in the mitochondria of human testicular spermatids. Therefore, we were not able to abide by the suspicion that cx33 is present in human testis. Additionally, it was not possible to demonstrate cx33 via PCR and immunohistochemistry in the testis of different mammals (dog, cattle, pig, horse, and marmoset monkey) with normal spermatogenesis. These data indicate that cx33 seems to be the first rodent-specific testicular cx. PMID:15611570

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

    PubMed Central

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

    2015-01-01

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

  12. 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

  13. Ezrin: a regulator of actin microfilaments in cell junctions of the rat testis.

    PubMed

    Gungor-Ordueri, N Ece; Celik-Ozenci, Ciler; Cheng, C Yan

    2015-01-01

    Ezrin, radixin, moesin and merlin (ERM) proteins are highly homologous actin-binding proteins that share extensive sequence similarity with each other. These proteins tether integral membrane proteins and their cytoplasmic peripheral proteins (e.g., adaptors, nonreceptor protein kinases and phosphatases) to the microfilaments of actin-based cytoskeleton. Thus, these proteins are crucial to confer integrity of the apical membrane domain and its associated junctional complex, namely the tight junction and the adherens junction. Since ectoplasmic specialization (ES) is an F-actin-rich testis-specific anchoring junction-a highly dynamic ultrastructure in the seminiferous epithelium due to continuous transport of germ cells, in particular spermatids, across the epithelium during the epithelial cycle-it is conceivable that ERM proteins are playing an active role in these events. Although these proteins were first reported almost 25 years and have since been extensively studied in multiple epithelia/endothelia, few reports are found in the literature to examine their role in the actin filament bundles at the ES. Studies have shown that ezrin is also a constituent protein of the actin-based tunneling nanotubes (TNT) also known as intercellular bridges, which are transient cytoplasmic tubular ultrastructures that transport signals, molecules and even organelles between adjacent and distant cells in an epithelium to coordinate cell events that occur across an epithelium. Herein, we critically evaluate recent data on ERM in light of recent findings in the field in particular ezrin regarding its role in actin dynamics at the ES in the testis, illustrating additional studies are warranted to examine its physiological significance in spermatogenesis. PMID:25652626

  14. Tight junction proteins in gallbladder epithelium: different expression in acute acalculous and calculous cholecystitis.

    PubMed

    Laurila, Jouko J; Karttunen, Tuomo; Koivukangas, Vesa; Laurila, Päivi A; Syrjälä, Hannu; Saarnio, Juha; Soini, Ylermi; Ala-Kokko, Tero I

    2007-06-01

    There is a paucity of information of tight junction (TJ) proteins in gallbladder epithelium, and disturbances in the structure of these proteins may play a role in the pathogenesis of acute acalculous cholecystitis (AAC) and acute calculous cholecystitis (ACC). Using immunohistochemistry, we investigated the expression of TJ proteins claudin-1, -2, -3, and -4, occludin, zonula occludens (ZO-1), and E-cadherin in 9 normal gallbladders, 30 gallbladders with AAC, and 21 gallbladders with ACC. The number of positive epithelial and endothelial cells and the intensity of the immunoreaction were determined. Membrane-bound and cytoplasmic immunoreactivities were separately assessed. We found that TJ proteins were uniformly expressed in normal gallbladder epithelium, with the exception of claudin-2, which was present in less than half of the cells. In AAC, expression of cytoplasmic occludin and claudin-1 were decreased, as compared with normal gallbladder. In ACC, expression of claudin-2 was increased, and expression of claudin-1, -3, and -4, occludin, and ZO-1 were decreased, as compared with normal gallbladder or AAC. We conclude that there are significant differences in expression of TJ proteins in AAC and ACC, supporting the idea that AAC represents a manifestation of systemic inflammatory disease, whereas ACC is a local inflammatory and often infectious disease. PMID:17283368

  15. 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

  16. 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

  17. Reinforcing endothelial junctions prevents microvessel permeability increase and tumor cell adhesion in microvessels in vivo

    NASA Astrophysics Data System (ADS)

    Fu, Bingmei M.; Yang, Jinlin; Cai, Bin; Fan, Jie; Zhang, Lin; Zeng, Min

    2015-10-01

    Tumor cell adhesion to the microvessel wall is a critical step during tumor metastasis. Vascular endothelial growth factor (VEGF), a secretion of tumor cells, can increase microvessel permeability and tumor cell adhesion in the microvessel. To test the hypothesis that inhibiting permeability increase can reduce tumor cell adhesion, we used in vivo fluorescence microscopy to measure both microvessel permeability and adhesion rates of human mammary carcinoma MDA-MB-231 cells in post-capillary venules of rat mesentery under the treatment of VEGF and a cAMP analog, 8-bromo-cAMP, which can decrease microvessel permeability. By immunostaining adherens junction proteins between endothelial cells forming the microvessel wall, we further investigated the structural mechanism by which cAMP abolishes VEGF-induced increase in microvessel permeability and tumor cell adhesion. Our results demonstrate that 1) Pretreatment of microvessels with cAMP can abolish VEGF-enhanced microvessel permeability and tumor cell adhesion; 2) Tumor cells prefer to adhere to the endothelial cell junctions instead of cell bodies; 3) VEGF increases microvessel permeability and tumor cell adhesion by compromising endothelial junctions while cAMP abolishes these effects of VEGF by reinforcing the junctions. These results suggest that strengthening the microvessel wall integrity can be a potential approach to inhibiting hematogenous tumor metastasis.

  18. 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

  19. 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

  20. Maternal treatment with glucocorticoids modulates gap junction protein expression in the ovine fetal brain.

    PubMed

    Sadowska, G B; Stonestreet, B S

    2014-09-01

    Gap junctions facilitate intercellular communication and are important in brain development. Connexins (Cx) comprise a transmembrane protein family that forms gap junctions. Cx-32 is expressed in oligodendrocytes and neurons, Cx-36 in neurons, and Cx-43 in astrocytes. Although single antenatal steroid courses are recommended for fetal lung maturation, multiple courses can be given to women at recurrent risk for premature delivery. We examined the effects of single and multiple glucocorticoid courses on Cx-32, Cx-36, and Cx-43 protein expressions in the fetal cerebral cortex, cerebellum, and spinal cord, and differences in Cx expression among brain regions under basal conditions. In the single-course groups, the ewes received dexamethasone (6 mg) or placebo as four intramuscular injections every 12h over 48 h. In the multiple-course groups, the ewes received the same treatment, once a week for 5 weeks starting at 76-78 days of gestation. Cx were measured by Western immunoblot on brain samples from 105 to 108-day gestation fetuses. A single dexamethasone course was associated with increases (P<0.05) in cerebral cortical and spinal cord Cx-36 and Cx-43 and multiple courses with increases in cerebellar and spinal cord Cx-36, and cerebral cortical and cerebellar Cx-43. Cx-32 did not change. Cx-32 was higher in the cerebellum than cerebral cortex and spinal cord, Cx-36 higher in the spinal cord than cerebellum, and Cx-43 higher in the cerebellum and spinal cord than cerebral cortex during basal conditions. In conclusion, maternal glucocorticoid therapy increases specific Cx, responses to different maternal courses vary among Cx and brain regions, and Cx expression differs among brain regions under basal conditions. Maternal treatment with glucocorticoids differentially modulates Cx in the fetal brain. PMID:24929069

  1. 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

  2. HPV oncoprotein E6 is a structure-dependent DNA-binding protein that recognizes four-way junctions.

    PubMed

    Ristriani, T; Masson, M; Nominé, Y; Laurent, C; Lefevre, J F; Weiss, E; Travé, G

    2000-03-10

    E6 is an oncoprotein implicated in cervical cancers, produced by "high-risk" human papillomaviruses. E6 is thought to promote tumorigenesis by stimulating cellular degradation of the tumour suppressor p53, but it might display other activities. Sequence similarity was recently detected between E6 and endonuclease VII, a protein of phage T4 that recognizes and cleaves four-way DNA junctions. Here, we purified recombinant E6 proteins and demonstrated that high-risk E6 s bind selectively to four-way junctions in a structure-dependent manner. Several residues in the C-terminal zinc-binding domain, the region of E6 similar to endonuclease VII, are necessary for the junction-binding activity. E6 binds to the junction as a monomer. Comparative electrophoresis shows that E6-bound junctions migrate in an extended square conformation. Magnesium inhibits the electrophoretic migration of the complexes but does not seem to influence their formation at equilibrium. This work is the first demonstration of specific binding of purified active E6 to a well-characterized DNA ligand, and suggests new modes of action of E6 in oncogenesis. PMID:10698626

  3. Macroglobulin complement-related encodes a protein required for septate junction organization and paracellular barrier function in Drosophila

    PubMed Central

    Hall, Sonia; Bone, Courtney; Oshima, Kenzi; Zhang, Liang; McGraw, Molly; Lucas, Bethany; Fehon, Richard G.; Ward, Robert E.

    2014-01-01

    Polarized epithelia play crucial roles as barriers to the outside environment and enable the formation of specialized compartments for organs to carry out essential functions. Barrier functions are mediated by cellular junctions that line the lateral plasma membrane between cells, principally tight junctions in vertebrates and septate junctions (SJs) in invertebrates. Over the last two decades, more than 20 genes have been identified that function in SJ biogenesis in Drosophila, including those that encode core structural components of the junction such as Neurexin IV, Coracle and several claudins, as well as proteins that facilitate the trafficking of SJ proteins during their assembly. Here we demonstrate that Macroglobulin complement-related (Mcr), a gene previously implicated in innate immunity, plays an essential role during embryonic development in SJ organization and function. We show that Mcr colocalizes with other SJ proteins in mature ectodermally derived epithelial cells, that it shows interdependence with other SJ proteins for SJ localization, and that Mcr mutant epithelia fail to form an effective paracellular barrier. Tissue-specific RNA interference further demonstrates that Mcr is required cell-autonomously for SJ organization. Finally, we show a unique interdependence between Mcr and Nrg for SJ localization that provides new insights into the organization of the SJ. Together, these studies demonstrate that Mcr is a core component of epithelial SJs and also highlight an interesting relationship between innate immunity and epithelial barrier functions. PMID:24496625

  4. 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.

  5. Protein-anchoring Strategy for Delivering Acetylcholinesterase to the Neuromuscular Junction

    PubMed Central

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

    2012-01-01

    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. PMID:22371845

  6. TRAF4 Is a Novel Phosphoinositide-Binding Protein Modulating Tight Junctions and Favoring Cell Migration

    PubMed Central

    Rousseau, Adrien; McEwen, Alastair G.; Poussin-Courmontagne, Pierre; Rognan, Didier; Nominé, Yves; Rio, Marie-Christine; Tomasetto, Catherine; Alpy, Fabien

    2013-01-01

    Tumor necrosis factor (TNF) receptor-associated factor 4 (TRAF4) is frequently overexpressed in carcinomas, suggesting a specific role in cancer. Although TRAF4 protein is predominantly found at tight junctions (TJs) in normal mammary epithelial cells (MECs), it accumulates in the cytoplasm of malignant MECs. How TRAF4 is recruited and functions at TJs is unclear. Here we show that TRAF4 possesses a novel phosphoinositide (PIP)-binding domain crucial for its recruitment to TJs. Of interest, this property is shared by the other members of the TRAF protein family. Indeed, the TRAF domain of all TRAF proteins (TRAF1 to TRAF6) is a bona fide PIP-binding domain. Molecular and structural analyses revealed that the TRAF domain of TRAF4 exists as a trimer that binds up to three lipids using basic residues exposed at its surface. Cellular studies indicated that TRAF4 acts as a negative regulator of TJ and increases cell migration. These functions are dependent from its ability to interact with PIPs. Our results suggest that TRAF4 overexpression might contribute to breast cancer progression by destabilizing TJs and favoring cell migration. PMID:24311986

  7. 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

  8. 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

  9. 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

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

    PubMed Central

    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

  11. Low levels of cadmium chloride alter the immunoprecipitation of corneal cadherin-complex proteins.

    PubMed

    Weidner, W J; Waddell, D S; Sillman, A J

    2000-12-01

    The effect of cadmium chloride on the immunoprecipitation of cadherin and the associated adherens junctional proteins, alpha- and beta-catenin, was examined in isolated bullfrog (Rana catesbeiana) corneas utilizing Western blot and enhanced chemoluminescent techniques. Application of either 1.0 microM or 75.0 microM CdCl2 to the corneal endothelium for 2 h markedly decreased the immunoprecipitation of cadherins as compared to paired control corneas. Immunoprecipitation of alpha-catenin was increased in response to both doses of CdCl2, while the immunoprecipitation of beta-catenin was little changed by either cadmium dose. There is accumulating evidence that cadmium may increase epithelial paracellular permeability by interfering with cadherin complex activity at intercellular junctions. The present study suggests that inorganic cadmium in low micromolar concentrations may decrease the integrity of the corneal endothelium, at least in part through a similar mechanism involving disruption of junctional cadherin complex function. PMID:11201663

  12. 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. PMID:22791339

  13. Junctional Adhesion Molecule-A Is Required for Hematogenous Dissemination of Reovirus

    PubMed Central

    Antar, Annukka A. R.; Konopka, Jennifer L.; Campbell, Jacquelyn A.; Henry, Rachel A.; Perdigoto, Ana L.; Carter, Bruce D.; Pozzi, Ambra; Abel, Ty W.; Dermody, Terence S.

    2009-01-01

    SUMMARY Diverse families of viruses bind immunoglobulin superfamily (IgSF) proteins located in tight junctions (TJs) and adherens junctions of epithelium and endothelium. However, little is known about the roles of these receptors in the pathogenesis of viral disease. Junctional adhesion molecule-A (JAM-A) is an IgSF protein that localizes to TJs and serves as a receptor for mammalian reovirus. We inoculated wild-type (wt) and isogenic JAM-A−/− mice perorally with reovirus and found that JAM-A is dispensable for viral replication in the intestine but required for systemic dissemination. Reovirus replication in the brain and tropism for discrete neural regions are equivalent in wt and JAM-A−/− mice following intracranial inoculation, suggesting a function for JAM-A in reovirus spread to extra-intestinal sites. JAM-A promotes reovirus infection of endothelial cells, providing a conduit for the virus into the bloodstream. These findings indicate that a broadly expressed IgSF viral receptor specifically mediates hematogenous dissemination in the host. PMID:19154988

  14. IQGAP1 controls tight junction formation through differential regulation of claudin recruitment

    PubMed Central

    Tanos, Barbara E.; Perez Bay, Andres E.; Salvarezza, Susana; Vivanco, Igor; Mellinghoff, Ingo; Osman, Mahasin; Sacks, David B.; Rodriguez-Boulan, Enrique

    2015-01-01

    ABSTRACT IQGAP1 is a scaffolding protein previously implicated in adherens junction formation. However, its role in the establishment or maintenance of tight junctions (TJs) has not been explored. We hypothesized that IQGAP1 could regulate TJ formation by modulating the expression and/or localization of junctional proteins, and we systematically tested this hypothesis in the model Madin-Darby canine kidney (MDCK) cell line. We find that IQGAP1 silencing enhances a transient increase in transepithelial electrical resistance (TER) observed during the early stages of TJ formation (Cereijido et al., 1978). Quantitative microscopy and biochemical experiments suggest that this effect of IQGAP1 on TJ assembly is accounted for by reduced expression and TJ recruitment of claudin 2, and increased TJ recruitment of claudin 4. Furthermore, we show that IQGAP1 also regulates TJ formation through its interactor CDC42, because IQGAP1 knockdown increases the activity of the CDC42 effector JNK and dominant-negative CDC42 prevents the increase in TER caused by IQGAP1 silencing. Hence, we provide evidence that IQGAP1 modulates TJ formation by a twofold mechanism: (1) controlling the expression and recruitment of claudin 2 and recruitment of claudin 4 to the TJ, and (2) transient inhibition of the CDC42–JNK pathway. PMID:25588839

  15. Expression of gap junctional connexin proteins in ovine fetal ovaries: Effects of maternal diet

    PubMed Central

    Grazul-Bilska, A.T.; Vonnahme, K.A.; Bilski, J.J.; Borowczyk, E.; Soni, D.; Mikkelson, B.; Johnson, M.L.; Reynolds, L.P.; Redmer, D.A.; Caton, J.S.

    2011-01-01

    Gap junctions have been implicated in the regulation of cellular metabolism and the coordination of cellular functions during growth and differentiation of organs and tissues, and gap junctions play a major role in direct cell-cell communication. Gap junctional channels and connexin (Cx) proteins have been detected in adult ovaries in several species. Furthermore, it has been demonstrated that several environmental factors including maternal diet may affect fetal organ growth and function. To determine if maternal diet impacts expression of connexin (Cx) 26, 32, 37 and 43 in fetal ovaries, sheep were fed a maintenance (M) diet with adequate (A) selenium (Se) or high (H) Se levels from 21 days before breeding to day 132 of pregnancy. From day 50 to 132 of pregnancy (tissue collection day), a portion of the ewes from ASe and HSe groups was fed restricted (R; 60% of M) diet. Sections of fetal ovaries were immunostained for the presence of connexins followed by image analysis. All four connexins were detected, but the distribution pattern differed. Cx26 was immunolocalized in the oocytes from primordial, primary, secondary and antral follicles, in granulosa and theca layers of secondary and antral follicles, stroma and blood vessels; Cx32 was in oocytes, granulosa and theca cells in a portion of antral follicles; Cx37 was on the borders between oocyte and granulosa/cumulus cells of primordial to antral follicles, and in endothelium; and Cx43 was on cellular borders in granulosa and theca layers, and between oocyte and granulosa/cumulus cells of primordial to antral follicles. Maternal diet affected Cx26 and Cx43 expression; Cx26 in granulosa layer of antral follicles was decreased (P<0.01) by HSe in M and R diets, and Cx43 in granulosa layer of primary and granulosa and theca of antral follicles was increased (P<0.05) by M diet with HSe. Thus, connexins may be differentially involved in regulation of fetal ovarian function in sheep. These data emphasize the importance

  16. 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

  17. 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. PMID:23965379

  18. Identification and dynamic regulation of tight junction protein expression in human neural stem cells.

    PubMed

    Watters, Andrea K; Rom, Slava; Hill, Jeremy D; Dematatis, Marie K; Zhou, Yu; Merkel, Steven F; Andrews, Allison M; Cena, Jonathan; Potula, Raghava; Skuba, Andrew; Son, Young-Jin; Persidsky, Yuri; Ramirez, Servio H

    2015-06-15

    Recent reports indicate that neural stem cells (NSCs) exist in a cluster-like formation in close proximity to cerebral microvessels. Similar appearing clusters can be seen ex vivo in NSC cultures termed neurospheres. It is known that this neurosphere configuration is important for preserving stemness and a proliferative state. How NSCs form neurospheres or neuroclusters remains largely undetermined. In this study, we show that primary human NSCs express the tight junction proteins (TJPs): zonula occludens-1 (ZO-1), occludin, claudin-1, -3, -5, and -12. The relative mRNA expression was measured by quantitative polymerase chain reaction, and protein expression was confirmed by flow cytometry and immunofluorescence microscopy. Our results show that downregulation of TJPs occurs as neuronal differentiation is induced, suggesting that control of TJPs may be tied to the neuronal differentiation program. Importantly, upon specific knockdown of the accessory TJP, ZO-1, undifferentiated NSCs showed decreased levels of key stem cell markers. Taken together, our results indicate that TJPs possibly aid in maintaining the intercellular configuration of NSCs and that reduction in TJP expression consequently affects the stemness status. PMID:25892136

  19. Crooked, coiled and crimpled are three Ly6-like proteins required for proper localization of septate junction components.

    PubMed

    Nilton, Anna; Oshima, Kenzi; Zare, Fariba; Byri, Sunitha; Nannmark, Ulf; Nyberg, Kevin G; Fehon, Richard G; Uv, Anne E

    2010-07-01

    Cellular junction formation is an elaborate process that is dependent on the regulated synthesis, assembly and membrane targeting of constituting components. Here, we report on three Drosophila Ly6-like proteins essential for septate junction (SJ) formation. SJs provide a paracellular diffusion barrier and appear molecularly and structurally similar to vertebrate paranodal septate junctions. We show that Crooked (Crok), a small GPI-anchored Ly6-like protein, is required for septa formation and barrier functions. In embryos that lack Crok, SJ components are produced but fail to accumulate at the plasma membrane. Crok is detected in intracellular puncta and acts tissue-autonomously, which suggests that it resides in intracellular vesicles to assist the cell surface localization of SJ components. In addition, we demonstrate that two related Ly6 proteins, Coiled (Cold) and Crimpled (Crim), are required for SJ formation and function in a tissue-autonomous manner, and that Cold also localizes to intracellular vesicles. Specifically, Crok and Cold are required for correct membrane trafficking of Neurexin IV, a central SJ component. The non-redundant requirement for Crok, Cold, Crim and Boudin (Bou; another Ly6 protein that was recently shown to be involved in SJ formation) suggests that members of this conserved family of proteins cooperate in the assembly of SJ components, possibly by promoting core SJ complex formation in intracellular compartments associated with membrane trafficking. PMID:20570942

  20. 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

  1. Tight junction proteins expression and modulation in immune cells and multiple sclerosis

    PubMed Central

    Mandel, Ilana; Paperna, Tamar; Glass-Marmor, Lea; Volkowich, Anat; Badarny, Samih; Schwartz, Ilya; Vardi, Pnina; Koren, Ilana; Miller, Ariel

    2012-01-01

    Abstract The tight junction proteins (TJPs) are major determinants of endothelial cells comprising physiological vascular barriers such as the blood–brain barrier, but little is known about their expression and role in immune cells. In this study we assessed TJP expression in human leukocyte subsets, their induction by immune activation and modulation associated with autoimmune disease states and therapies. A consistent expression of TJP complexes was detected in peripheral blood leukocytes (PBLs), predominantly in B and T lymphocytes and monocytes, whereas the in vitro application of various immune cell activators led to an increase of claudin 1 levels, yet not of claudin 5. Claudins 1 and 5 levels were elevated in PBLs of multiple sclerosis (MS) patients in relapse, relative to patients in remission, healthy controls and patients with other neurological disorders. Interestingly, claudin 1 protein levels were elevated also in PBLs of patients with type 1 diabetes (T1D). Following glucocorticoid treatment of MS patients in relapse, RNA levels of JAM3 and CLDN5 and claudin 5 protein levels in PBLs decreased. Furthermore, a correlation between CLDN5 pre-treatment levels and clinical response phenotype to interferon-β therapy was detected. Our findings indicate that higher levels of leukocyte claudins are associated with immune activation and specifically, increased levels of claudin 5 are associated with MS disease activity. This study highlights a potential role of leukocyte TJPs in physiological states, and autoimmunity and suggests they should be further evaluated as biomarkers for aberrant immune activity and response to therapy in immune-mediated diseases such as MS. PMID:21762372

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

    PubMed

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

    2015-10-23

    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 Tyr(595-596) 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

  3. 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. PMID:26348651

  4. Distribution of the gap junction protein connexin 35 in the central nervous system of developing zebrafish larvae

    PubMed Central

    Jabeen, Shaista; Thirumalai, Vatsala

    2013-01-01

    Gap junctions are membrane specializations that allow the passage of ions and small molecules from one cell to another. In vertebrates, connexins are the protein subunits that assemble to form gap junctional plaques. Connexin-35 (Cx35) is the fish ortholog of mammalian Cx36, which is enriched in the retina and the brain and has been shown to form neuronal gap junctions. As a first step toward understanding the role of neuronal gap junctions in central nervous system (CNS) development, we describe here the distribution of Cx35 in the CNS during zebrafish development. Cx35 expression is first seen at 1 day post fertilization (dpf) along cell boundaries throughout the nervous system. At 2 dpf, Cx35 immunoreactivity appears in commissures and fiber tracts throughout the CNS and along the edges of the tectal neuropil. In the rhombencephalon, the Mauthner neurons and fiber tracts show strong Cx35 immunoreactivity. As the larva develops, the commissures and fiber tracts continue to be immunoreactive for Cx35. In addition, the area of the tectal neuropil stained increases vastly and tectal commissures are visible. Furthermore, at 4–5 dpf, Cx35 is seen in the habenulae, cerebellum and in radial glia lining the rhombencephalic ventricle. This pattern of Cx35 immunoreactivity is stable at least until 15 dpf. To test whether the Cx35 immunoreactivity seen corresponds to functional gap junctional coupling, we documented the number of dye-coupled neurons in the hindbrain. We found several dye-coupled neurons within the reticulospinal network indicating functional gap junctional connectivity in the developing zebrafish brain. PMID:23717264

  5. 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

  6. Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization, in vitro.

    PubMed Central

    Fasano, A; Fiorentini, C; Donelli, G; Uzzau, S; Kaper, J B; Margaretten, K; Ding, X; Guandalini, S; Comstock, L; Goldblum, S E

    1995-01-01

    The intracellular signaling involved in the mechanism of action of zonula occludens toxin (ZOT) was studied using several in vitro and ex vivo models. ZOT showed a selective effect among various cell lines tested, suggesting that it may interact with a specific receptor, whose surface expression on various cells differs. When tested in IEC6 cell monolayers, ZOT-containing supernatants induced a redistribution of the F-actin cytoskeleton. Similar results were obtained with rabbit ileal mucosa, where the reorganization of F-actin paralleled the increase in tissue permeability. In endothelial cells, the cytoskeletal rearrangement involved a decrease of the soluble G-actin pool (-27%) and a reciprocal increase in the filamentous F-actin pool (+22%). This actin polymerization was time- and dose-dependent, and was reversible. Pretreatment with a specific protein kinase C inhibitor, CGP41251, completely abolished the ZOT effects on both tissue permeability and actin polymerization. In IEC6 cells ZOT induced a peak increment of the PKC-alpha isoform after 3 min incubation. Taken together, these results suggest that ZOT activates a complex intracellular cascade of events that regulate tight junction permeability, probably mimicking the effect of physiologic modulator(s) of epithelial barrier function. Images PMID:7635964

  7. Coxsackievirus and Adenovirus Receptor, a Tight Junction Protein, in Peri-Implantation Mouse Embryos.

    PubMed

    Oh, Yeong Seok; Nah, Won Heum; Choi, Bomi; Kim, Seok Hyun; Gye, Myung Chan

    2016-07-01

    To understand the role of Coxsackievirus and adenovirus receptor (CAR), a tight junction (TJ) protein, in peri-implantation embryos, developmental expression of CAR and its role in paracellular permeability were examined in mouse embryos. Splice variants for transmembrane CAR, Car1, Car2, and Car3 mRNA, were expressed from 2-cell, morula, and blastocyst stages onward, respectively, whereas mRNA for soluble CAR was expressed in MII oocytes and 4-cell stage onward. On Western blot, ∼46 kDa CAR proteins were detected in blastocysts. During the 4-cell embryos to morula stage, CAR was gradually concentrated at the contacts between blastomeres. In blastocysts, CAR was expressed at the cell contacts within the inner cell mass as well as in the trophectoderm (TE) where CAR was found together with ZO1 at the apical contacts, suggesting that CAR builds up apical TJs in TE and mediates cell adhesion in TE and inner cell mass. In blastocysts, CAR-blocking antibodies under Ca(2+) switching increased the dextran permeability and decreased the volume of blastocoel and H19 and Cdx2 mRNA, suggesting the pivotal role of CAR in the blastocyst development and paracellular permeability barrier in TE. CAR was expressed in TE of implanting embryos as well as endometrial epithelium, suggesting the involvement of CAR in the interaction between implanting embryos and endometrium. At 5-6 days postcoitum, CAR was expressed together with ZO1 in the primitive endoderm, visceral endoderm, and epiblasts facing the pro-amniotic cavity, suggesting that CAR TJs contribute to the separation of epiblast from the blastocoel and development of the pro-amniotic cavity within epiblasts. PMID:27226313

  8. 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

  9. 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

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. Characterization of the interaction between protein 4.1R and ZO-2. A possible link between the tight junction and the actin cytoskeleton.

    PubMed

    Mattagajasingh, S N; Huang, S C; Hartenstein, J S; Benz, E J

    2000-09-29

    Multiple isoforms of the red cell protein 4.1R are expressed in nonerythroid cells, including novel 135-kDa isoforms. Using a yeast two-hybrid system, immunocolocalization, immunoprecipitation, and in vitro binding studies, we found that two 4.1R isoforms of 135 and 150 kDa specifically interact with the protein ZO-2 (zonula occludens-2). 4.1R is colocalized with ZO-2 and occludin at Madin-Darby canine kidney (MDCK) cell tight junctions. Both isoforms of 4.1R coprecipitated with proteins that organize tight junctions such as ZO-2, ZO-1, and occludin. Western blot analysis also revealed the presence of actin and alpha-spectrin in these immunoprecipitates. Association of 4.1R isoforms with these tight junction and cytoskeletal proteins was found to be specific for the tight junction and was not seen in nonconfluent MDCK cells. The amino acid residues that sustain the interaction between 4.1R and ZO-2 reside within the amino acids encoded by exons 19-21 of 4.1R and residues 1054-1118 of ZO-2. Exogenously expressed 4.1R containing the spectrin/actin- and ZO-2-binding domains was recruited to tight junctions in confluent MDCK cells. Taken together, our results suggest that 4.1R might play an important role in organization and function of the tight junction by establishing a link between the tight junction and the actin cytoskeleton. PMID:10874042

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

    PubMed Central

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

    2012-01-01

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

  18. Burn-induced gut barrier injury is attenuated by phosphodiesterase inhibition: effects on tight junction structural proteins.

    PubMed

    Costantini, Todd W; Loomis, William H; Putnam, James G; Drusinsky, Dana; Deree, Jessica; Choi, Sunghyuk; Wolf, Paul; Baird, Andrew; Eliceiri, Brian; Bansal, Vishal; Coimbra, Raul

    2009-04-01

    Loss of intestinal barrier function after burn injury allows movement of intraluminal contents across the mucosa, which can lead to the development of distant organ injury and multiple organ failure. Tight junction function is highly regulated by membrane-associated proteins including occludin and zonula occludens protein 1 (ZO-1), which can be modulated by systemic inflammation. We hypothesized that (1) burn injury leads to gut barrier injury, and (2) phosphodiesterase inhibition will attenuate these burn-induced changes. Male balb/c mice undergoing a 30% steam burn were randomized to resuscitation with normal saline or normal saline + pentoxifylline (PTX; 12.5 mg/kg). Intestinal injury was assessed by histological diagnosis and TNF-alpha levels using enzyme-linked immunosorbent assay. Intestinal permeability was assessed by measuring the plasma concentration of fluorescein isothiocyanate-dextran after intraluminal injection in the distal ileum. Occludin and ZO-1 levels were analyzed by immunoblotting and immunohistochemistry. Thirty percent total body surface area (TBSA) burn results in a significant increase in intestinal permeability. Treatment with PTX after burn attenuates intestinal permeability to sham levels. Burn injury resulted in a marked decrease in the levels of tight junction proteins occludin and ZO-1 at 6 and 24 h. The use of PTX after burn significantly decreases the breakdown of occludin and ZO-1. Pentoxifylline also attenuates the burn-induced increase in plasma and intestinal TNF-alpha. Confocal microscopy demonstrates that PTX attenuates the burn-induced reorganization of occludin and ZO-1 away from the tight junction. Pentoxifylline attenuates burn-induced intestinal permeability and decreases the breakdown and reorganization of intestinal occludin and ZO-1. Therefore, phosphodiesterase inhibition may be a useful adjunct strategy in the attenuation of burn-induced gut barrier injury. PMID:18791495

  19. Internalization of a GFP-tetanus toxin C-terminal fragment fusion protein at mature mouse neuromuscular junctions.

    PubMed

    Roux, Sylvie; Colasante, Cesare; Saint Cloment, Cécile; Barbier, Julien; Curie, Thomas; Girard, Emmanuelle; Molgó, Jordi; Brûlet, Philippe

    2005-09-01

    The distribution, dynamics, internalization, and retrograde axonal traffic of a fusion protein composed of green fluorescent protein (GFP) and the atoxic C-terminal fragment of tetanus toxin (TTC) were studied after its in vivo injection. Confocal microscopy and immunogold electron microscopy revealed that the fusion protein (GFP-TTC) rapidly clustered in motor nerve terminals of the neuromuscular junction. Clathrin-coated pits, and axolemma infoldings located between active zones appeared to be involved in the internalization of the fusion protein. Biochemical analysis of detergent-extracted neuromuscular preparations showed that the GFP-TTC fusion protein was associated with lipid microdomains. We suggest that GFP-TTC clustering in these lipid microdomains favors the recruitment of other proteins involved in its endocytosis and internalization in motor nerve terminals. During its retrograde trafficking, GFP-TTC accumulated in different axonal compartments than those used by cholera toxin B-subunit suggesting that these two proteins are transported by different pathways and cargos. PMID:16023367

  20. Internalization of a GFP-tetanus toxin C-terminal fragment fusion protein at mature mouse neuromuscular junctions.

    PubMed

    Roux, Sylvie; Colasante, Cesare; Saint Cloment, Cécile; Barbier, Julien; Curie, Thomas; Girard, Emmanuelle; Molgó, Jordi; Brûlet, Philippe

    2005-12-01

    The distribution, dynamics, internalization, and retrograde axonal traffic of a fusion protein composed of green fluorescent protein (GFP)and the atoxic C-terminal fragment of tetanus toxin (TTC) were studied after its in vivo injection. Confocal microscopy and immuno-gold electron microscopy revealed that the fusion protein (GFP-TTC) rapidly clustered in motor nerve terminals of the neuromuscular junction. Clathrin-coated pits, and axolemma infoldings located between active zones appeared to be involved in the internalization of the fusion protein. Biochemical analysis of detergent-extracted neuromuscular preparations showed that the GFP-TTC fusion protein was associated with lipid microdomains. We suggest that GFP-TTC clustering in these lipid microdomains favors the recruitment of other proteins involved in its endocytosis and internalization in motor nerve terminals. During its retrograde trafficking, GFP-TTC accumulated indifferent axonal compartments than those used by cholera toxin B-subunit suggesting that these two proteins are transported by different pathways and cargos. PMID:16456925

  1. 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

  2. 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

  3. Zoledronic acid suppresses metastasis of esophageal squamous cell carcinoma cells through upregulating the tight junction protein occludin.

    PubMed

    Lin, Canfeng; Xin, Shubo; Qin, Xin; Li, Haijun; Lin, Lianxing; You, Yanjie

    2016-08-01

    We have previously demonstrated the radio-sensitizing effect of zoledronic acid (ZOL), a third generation bisphosphonate, on human esophageal squamous cell carcinoma (ESCC) cells. Here we show that ZOL suppresses metastatic progression of ESCC cells mainly through up-regulating the tight junction protein occludin. Exposure to ZOL at lower concentrations dramatically reduced migration and invasion of ESCC cells. In addition, ZOL treatment decreased the expression of mesenchymal markers, vimentin and N-cadherin, while increased the expression of the tight junction protein occludin. Moreover, ectopic expression of Slug, a well-known transcriptional repressor of occludin, partially but significantly abrogated the effect of ZOL on occludin expression and subsequently rescued the malignant metastatic phenotype, suggesting that Slug is one of the mediators underlying the anti-metastatic effect of ZOL. The present study is the first to report the significance of ZOL on ESCC metastasis. These data are promising for the future application of this drug regimen in patients with ESCC. PMID:26204820

  4. Paradigms lost-an emerging role for over-expression of tight junction adhesion proteins in cancer pathogenesis.

    PubMed

    Leech, Astrid O; Cruz, Rodrigo G B; Hill, Arnold D K; Hopkins, Ann M

    2015-08-01

    Tight junctions (TJ) are multi-protein complexes located at the apicalmost tip of the lateral membrane in polarised epithelial and endothelial cells. Their principal function is in mediating intercellular adhesion and polarity. Accordingly, it has long been a paradigm that loss of TJ proteins and consequent deficits in cell-cell adhesion are required for tumour cell dissemination in the early stages of the invasive/metastatic cascade. However it is becoming increasingly apparent that TJ proteins play important roles in not just adhesion but also intracellular signalling events, activation of which can contribute to, or even drive, tumour progression and metastasis. In this review, we shall therefore highlight cases wherein the gain of TJ proteins has been associated with signals promoting tumour progression. We will also discuss the potential of overexpressed TJ proteins to act as therapeutic targets in cancer treatment. The overall purpose of this review is not to disprove the fact that loss of TJ-based adhesion contributes to the progression of several cancers, but rather to introduce the growing body of evidence that gain of TJ proteins may have adhesion-independent consequences for promoting progression in other cancers. PMID:26366401

  5. Expression and modulation of connexin 30.2, a novel gap junction protein in the mouse retina.

    PubMed

    Müller, Luis Pérez de Sevilla; Dedek, Karin; Janssen-Bienhold, Ulrike; Meyer, Arndt; Kreuzberg, Maria M; Lorenz, Susanne; Willecke, Klaus; Weiler, Reto

    2010-07-01

    Mammalian retinae express multiple connexins that mediate the metabolic and electrical coupling of various cell types. In retinal neurons, only connexin 36, connexin 45, connexin 50, and connexin 57 have been described so far. Here, we present an analysis of a novel retinal connexin, connexin 30.2 (Cx30.2), and its regulation in the mouse retina. To analyze the expression of Cx30.2, we used a transgenic mouse line in which the coding region of Cx30.2 was replaced by lacZ reporter DNA. We detected the lacZ signal in the nuclei of neurons located in the inner nuclear layer and the ganglion cell layer (GCL). In this study, we focused on the GCL and characterized the morphology of the Cx30.2-expressing cells. Using immunocytochemistry and intracellular dye injections, we found six different types of Cx30.2-expressing ganglion cells: one type of ON-OFF, three types of OFF, and two types of ON ganglion cells; among the latter was the RG A1 type. We show that RG A1 cells were heterologously coupled to numerous displaced amacrine cells. Our results suggest that these gap junction channels may be heterotypic, involving Cx30.2 and a connexin yet unidentified in the mouse retina. Gap junction coupling can be modulated by protein kinases, a process that plays a major role in retinal adaptation. Therefore, we studied the protein kinase-induced modulation of coupling between RG A1 and displaced amacrine cells. Our data provide evidence that coupling of RG A1 cells to displaced amacrine cells is mediated by Cx30.2 and that the extent of this coupling is modulated by protein kinase C. PMID:20537217

  6. Chlorogenic Acid Decreases Intestinal Permeability and Increases Expression of Intestinal Tight Junction Proteins in Weaned Rats Challenged with LPS

    PubMed Central

    Ruan, Zheng; Liu, Shiqiang; Zhou, Yan; Mi, Shumei; Liu, Gang; Wu, Xin; Yao, Kang; Assaad, Houssein; Deng, Zeyuan; Hou, Yongqing; Wu, Guoyao; Yin, Yulong

    2014-01-01

    Chlorogenic acid, a natural phenolic acid present in fruits and plants, provides beneficial effects for human health. The objectives of this study were to investigate whether chlorogenic acid (CHA) could improve the intestinal barrier integrity for weaned rats with lipopolysaccharide (LPS) challenge. Thirty-two weaned male Sprague Dawley rats (21±1 d of age; 62.26±2.73 g) were selected and randomly allotted to four treatments, including weaned rat control, LPS-challenged and chlorogenic acid (CHA) supplemented group (orally 20 mg/kg and 50 mg/kg body). Dietary supplementation with CHA decreased (P<0.05) the concentrations of urea and albumin in the serum, compared to the LPS-challenged group. The levels of IFN-γ and TNF-α were lower (P<0.05) in the jejunal and colon of weaned rats receiving CHA supplementation, in comparison with the control group. CHA supplementation increased (P<0.05) villus height and the ratio of villus height to crypt depth in the jejunal and ileal mucosae under condictions of LPS challenge. CHA supplementation decreased (P<0.05) intestinal permeability, which was indicated by the ratio of lactulose to mannitol and serum DAO activity, when compared to weaned rats with LPS challenge. Immunohistochemical analysis of tight junction proteins revealed that ZO-1 and occludin protein abundances in the jejunum and colon were increased (P<0.05) by CHA supplementation. Additionally, results of immunoblot analysis revealed that the amount of occludin in the colon was also increased (P<0.05) in CHA-supplemented rats. In conclusion, CHA decreases intestinal permeability and increases intestinal expression of tight junction proteins in weaned rats challenged with LPS. PMID:24887396

  7. 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

  8. Cingulin, a specific protein component of tight junctions, is expressed in normal and neoplastic human epithelial tissues.

    PubMed Central

    Citi, S.; Amorosi, A.; Franconi, F.; Giotti, A.; Zampi, G.

    1991-01-01

    Cingulin is a 140-kd protein localized on the cytoplasmic face of avian tight junctions. The expression of cingulin in human normal and neoplastic colonic tissue has been investigated with an antiserum against chicken cingulin. Human cingulin shares its apparent molecular mass and localization with avian cingulin. In normal colonic epithelium, villous adenomas, and differentiated adenocarcinomas, cingulin staining is observed in the junctional region of the polarized cells lining the surface, the crypts, and the glandular lumina. In poorly differentiated adenocarcinomas, labeling also is observed at the interface between cancer tissue and stroma, or in clumps of malignant cells, forming a pattern that highlights the presence of small, compressed lumina. The cingulin content of four adenocarcinomas, estimated by immunoblotting and densitometry, was higher than that of the normal tissue (150% to 230%). Cingulin was detected in a metastasis from a colon adenocarcinoma but not in nonepithelial tissues and neoplasias, suggesting that cingulin may be a useful marker in the characterization of colonic and probably other epithelial neoplasias. Images Figure 1 Figure 2 Figure 3 PMID:2012170

  9. 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

  10. The gap junctional protein INX-14 functions in oocyte precursors to promote C. elegans sperm guidance

    PubMed Central

    Edmonds, Johnathan W.; McKinney, Shauna L.; Prasain, Jeevan K.; Miller, Michael A.

    2011-01-01

    Innexins are the subunits of invertebrate gap junctions. Here we show that the innexin INX-14 promotes sperm guidance to the fertilization site in the C. elegans hermaphrodite reproductive tract. inx-14 loss causes cell nonautonomous defects in sperm migration velocity and directional velocity. Results from genetic and immunocytochemical analyses provide strong evidence that INX-14 acts in transcriptionally active oocyte precursors in the distal gonad, not in transcriptionally inactive oocytes that synthesize prostaglandin sperm-attracting cues. Somatic gonadal sheath cell interaction is necessary for INX-14 function, likely via INX-8 and INX-9 expressed in sheath cells. However, electron microscopy has not identified gap junctions in oocyte precursors, suggesting that INX-14 acts in a channel-independent manner or INX-14 channels are difficult to document. INX-14 promotes prostaglandin signaling to sperm at a step after F-series prostaglandin synthesis in oocytes. Taken together, our results support the model that INX-14 functions in a somatic gonad/germ cell signaling mechanism essential for sperm function. We propose that this mechanism regulates the transcription of a factor(s) that modulates prostaglandin metabolism, transport, or activity in the reproductive tract. PMID:21889935

  11. 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

  12. Leukocyte Diapedesis In Vivo Induces Transient Loss of Tight Junction Protein at the Blood–Retina Barrier

    PubMed Central

    Xu, Heping; Dawson, Rosemary; Crane, Isabel J.; Liversidge, Janet

    2008-01-01

    Purpose Although much is now understood about the molecular structure of tight junctions (TJs), little is known about the regulation of their function during neural inflammatory disease processes in vivo. The mechanisms by which leukocytes transmigrate the blood-retina barrier (BRB) without affecting endothelial permeability are controversial. Methods Confocal immunofluorescence microscopy of ex vivo retinal wholemounts was used to study BRB integrity during leukocyte adhesion and migration during experimental autoimmune uveoretinitis (EAU). Western blot analysis was used to measure levels of TJ proteins in EAU retina and RPE and in normal retina or RPE cultured with cytokines or chemokines. Results No evidence for discontinuity or other weakness of the endothelial or epithelial barrier at tricellular corners was observed, and maximum disruption of TJ protein expression was focused in retinal venules correlating with sites of leukocyte extravasation. Areas of maximum TJ protein loss in vivo also correlated with redistribution or loss of ensheathing astrocyte processes on venules but not adjacent capillaries or arterioles. Exposure of normal choroidal and retinal explants ex vivo to cytokines and chemokines alone did not downregulate total occludin-1 or claudin-3 TJ protein expression. Conclusions The data presented herein support an active role for leukocytes in TJ disruption and blood-retina barrier (BRB) breakdown during retinal inflammation and further implicate venule microenvironment as a key factor in leukocyte recruitment to retinal tissue in vivo. PMID:15980240

  13. 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. PMID:19679145

  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

    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.

  16. 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

  17. 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

  18. Effect of dexmedetomidine on rats with renal ischemia-reperfusion injury and the expression of tight junction protein in kidney.

    PubMed

    Liu, Yun-En; Tong, Chang-Ci; Zhang, Yu-Biao; Jin, Hong-Xu; Gao, Yan; Hou, Ming-Xiao

    2015-01-01

    To explore the protective effect of dexmedetomidine (Dex) on rats with renal ischemia-reperfusion injury and the influence of Dex on the expression of tight junction protein in kidney. Grouped 40 SPF male rats into 4 groups, sham operation group (group S), ischemia-reperfusion group (group I/R), pretreatment with Dex group (group Pre/Dex), post-treatment with Dex group (group Post/Dex), randomly, 10 rats each group. Rats in group S were anaesthetized and set up with removal of right kidney; rats in group I/R were set up with removal of right kidney and left renal artery clamping for 45 min followed by 60 min reperfusion; rats in group Pre/Dex were intravenous injected with Dex (1 μg/kg) for 30 min after indwelling catheter via femoral vein puncture; rats in group Post/Dex were intravenous injected with Dex (1 μg/kg) for 30 min after left renal reperfusion. The kidneys in each group were made out pathologic slices after 6 h I/R, stained with HE; blood samples were taken with separation plasma, creatinine (Scr) and urea nitrogen (BUN) were detected by automatic biochemical analyzer; IL-1β and TNF-α were detected by Enzyme-linked Immunosorbent Assay (ELISA); the expression level of tight junction protein ZO-1 and protein occludin in kidney were detected by Western-blot. The results of HE staining showed that, comparing to group S, the tissue of kidney in group I/R were damaged heavily with tubules dilatation and inflammation obviously, while lightened in group Pre/Dex and group Post/Dex. The results of detection of renal function and inflammatory factors showed that, comparing to group S, Scr, BUN, IL-1β and TNF-α were all enhanced in group I/R, group Pre/Dex and group Post/Dex, significantly (P < 0.05), while the inflammatory factors in group Pre/Dex and group Post/Dex were lower than in group I/R, significantly (P < 0.05). The results of Western-blot showed that the expression of protein ZO-1 and occludin in group Pre/Dex and group Post/Dex were higher than in

  19. 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

  20. 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

  1. Planar cell polarity protein localization in the secretory ameloblasts of rat incisors.

    PubMed

    Nishikawa, Sumio; Kawamoto, Tadafumi

    2012-05-01

    The localization of the planar cell polarity proteins Vang12, frizzled-3, Vang11, and Celsr1 in the rat incisors was examined using immunocytochemistry. The results showed that Vang12 was localized at two regions of the Tomes' processes of inner enamel-secretory ameloblasts in rat incisors: a proximal and a distal region. In contrast, frizzled-3 was localized at adherens junctions of the proximal and distal areas of inner enamel- and outer enamel-secretory ameloblasts, where N-cadherin and β-catenin were localized. frizzled-3 was also localized in differentiating inner enamel epithelial cells. Vang11 was localized sparsely in differentiating preameloblasts and extensively at the cell boundary of stratum intermedium. Celsr1 was not localized in ameloblasts but localized in odontoblasts extensively. These results suggest the involvement of planar cell polarity proteins in odontogenesis. PMID:22378702

  2. Endothelial Alpha-Parvin Controls Integrity of Developing Vasculature and Is Required for Maintenance of Cell–Cell Junctions

    PubMed Central

    Fraccaroli, Alessia; Pitter, Bettina; Taha, Abdallah Abu; Seebach, Jochen; Huveneers, Stephan; Kirsch, Julian; Casaroli-Marano, Ricardo P.; Zahler, Stefan; Pohl, Ulrich; Gerhardt, Holger; Schnittler, Hans-J.

    2015-01-01

    Rationale: Angiogenesis and vessel integrity depend on the adhesion of endothelial cells (ECs) to the extracellular matrix and to adjacent ECs. The focal adhesion protein α-parvin (α-pv) is essential for vascular development. However, the role of α-pv in ECs in vivo is not known. Objective: To determine the function of α-pv in ECs during vascular development in vivo and the underlying mechanisms. Methods and Results: We deleted the α-pv gene specifically in ECs of mice to study its role in angiogenesis and vascular development. Here, we show that endothelial-specific deletion of α-pv in mice results in late embryonic lethality associated with hemorrhages and reduced vascular density. Postnatal-induced EC-specific deletion of α-pv leads to retinal hypovascularization because of reduced vessel sprouting and excessive vessel regression. In the absence of α-pv, blood vessels display impaired VE-cadherin junction morphology. In vitro, α-pv–deficient ECs show reduced stable adherens junctions, decreased monolayer formation, and impaired motility, associated with reduced formation of integrin-mediated cell–extracellular matrix adhesion structures and an altered actin cytoskeleton. Conclusions: Endothelial α-pv is essential for vessel sprouting and for vessel stability. PMID:25925587

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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. PMID:23710457

  10. The degradation of airway tight junction protein under acidic conditions is probably mediated by transient receptor potential vanilloid 1 receptor

    PubMed Central

    Xu, Rui; Li, Qi; Zhou, Jia; Zhou, Xiang-dong; Perelman, Juliy M.; Kolosov, Victor P.

    2013-01-01

    Acidic airway microenvironment is one of the representative pathophysiological features of chronic inflammatory respiratory diseases. Epithelial barrier function is maintained by TJs (tight junctions), which act as the first physical barrier against the inhaled substances and pathogens of airway. As previous studies described, acid stress caused impaired epithelial barriers and led the hyperpermeability of epithelium. However, the specific mechanism is still unclear. We have showed previously the existence of TRPV (transient receptor potential vanilloid) 1 channel in airway epithelium, as well as its activation by acidic stress in 16HBE cells. In this study, we explored the acidic stress on airway barrier function and TJ proteins in vitro with 16HBE cell lines. Airway epithelial barrier function was determined by measuring by TER (trans-epithelial electrical resistance). TJ-related protein [claudin-1, claudin-3, claudin-4, claudin-5, claudin-7 and ZO-1 (zonula occluden 1)] expression was examined by western blotting of insoluble fractions of cell extraction. The localization of TJ proteins were visualized by immunofluorescent staining. Interestingly, stimulation by pH 6.0 for 8 h slightly increased the epithelial resistance in 16HBE cells insignificantly. However, higher concentration of hydrochloric acid (lower than pH 5.0) did reduce the airway epithelial TER of 16HBE cells. The decline of epithelial barrier function induced by acidic stress exhibited a TRPV1-[Ca2+]i-dependent pathway. Of the TJ proteins, claudin-3 and claudin-4 seemed to be sensitive to acidic stress. The degradation of claudin-3 and claudin-4 induced by acidic stress could be attenuated by the specific TRPV1 blocker or intracellular Ca2+ chelator BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)]. PMID:24073800

  11. Atelectrauma disrupts pulmonary epithelial barrier integrity and alters the distribution of tight junction proteins ZO-1 and claudin 4.

    PubMed

    Jacob, Anne-Marie; Gaver, Donald P

    2012-11-01

    Mechanical ventilation inevitably exposes the delicate tissues of the airways and alveoli to abnormal mechanical stresses that can induce pulmonary edema and exacerbate conditions such as acute respiratory distress syndrome. The goal of our research is to characterize the cellular trauma caused by the transient abnormal fluid mechanical stresses that arise when air is forced into a liquid-occluded airway (i.e., atelectrauma). Using a fluid-filled, parallel-plate flow chamber to model the "airway reopening" process, our in vitro study examined consequent increases in pulmonary epithelial plasma membrane rupture, paracellular permeability, and disruption of the tight junction (TJ) proteins zonula occludens-1 and claudin-4. Computational analysis predicts the normal and tangential surface stresses that develop between the basolateral epithelial membrane and underlying substrate due to the interfacial stresses acting on the apical cell membrane. These simulations demonstrate that decreasing the velocity of reopening causes a significant increase in basolateral surface stresses, particularly in the region between neighboring cells where TJs concentrate. Likewise, pulmonary epithelial wounding, paracellular permeability, and TJ protein disruption were significantly greater following slower reopening. This study thus demonstrates that maintaining a higher velocity of reopening, which reduces the damaging fluid stresses acting on the airway wall, decreases the mechanical stresses on the basolateral cell surface while protecting cells from plasma membrane rupture and promoting barrier integrity. PMID:22898551

  12. 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

  13. Atelectrauma disrupts pulmonary epithelial barrier integrity and alters the distribution of tight junction proteins ZO-1 and claudin 4

    PubMed Central

    Jacob, Anne-Marie

    2012-01-01

    Mechanical ventilation inevitably exposes the delicate tissues of the airways and alveoli to abnormal mechanical stresses that can induce pulmonary edema and exacerbate conditions such as acute respiratory distress syndrome. The goal of our research is to characterize the cellular trauma caused by the transient abnormal fluid mechanical stresses that arise when air is forced into a liquid-occluded airway (i.e., atelectrauma). Using a fluid-filled, parallel-plate flow chamber to model the “airway reopening” process, our in vitro study examined consequent increases in pulmonary epithelial plasma membrane rupture, paracellular permeability, and disruption of the tight junction (TJ) proteins zonula occludens-1 and claudin-4. Computational analysis predicts the normal and tangential surface stresses that develop between the basolateral epithelial membrane and underlying substrate due to the interfacial stresses acting on the apical cell membrane. These simulations demonstrate that decreasing the velocity of reopening causes a significant increase in basolateral surface stresses, particularly in the region between neighboring cells where TJs concentrate. Likewise, pulmonary epithelial wounding, paracellular permeability, and TJ protein disruption were significantly greater following slower reopening. This study thus demonstrates that maintaining a higher velocity of reopening, which reduces the damaging fluid stresses acting on the airway wall, decreases the mechanical stresses on the basolateral cell surface while protecting cells from plasma membrane rupture and promoting barrier integrity. PMID:22898551

  14. [Gap junction and diabetic foot].

    PubMed

    Zou, Xiao-rong; Tao, Jian; Wang, Yun-kai

    2015-11-01

    Gap junctions play a critical role in electrical synchronization and exchange of small molecules between neighboring cells; connexins are a family of structurally related transmembrane proteins that assemble to form vertebrate gap junctions. Hyperglycemia changes the structure gap junction proteins and their expression, resulting in obstruction of neural regeneration, vascular function and wound healing, and also promoting vascular atherosclerosis. These pathogenic factors would cause diabetic foot ulcers. This article reviews the involvement of connexins in pathogenesis of diabetic foot. PMID:26822053

  15. When proteome meets genome: the alpha helix and the beta strand of proteins are eschewed by mRNA splice junctions and may define the minimal indivisible modules of protein architecture

    PubMed Central

    Barik, Sailen

    2008-01-01

    The significance of the intron-exon structure of genes is a mystery. As eukaryotic proteins are made up of modular functional domains, each exon was suspected to encode some form of module; however, the definition of a module remained vague. Comparison of pre-mRNA splice junctions with the three-dimensional architecture of its protein product from different eukaryotes revealed that the junctions were far less likely to occur inside the α-helices and β-strands of proteins than within the more flexible linker regions (‘turns’ and ‘loops’) connecting them. The splice junctions were equally distributed in the different types of linkers and throughout the linker sequence, although a slight preference for the central region of the linker was observed. The avoidance of the α-helix and the β-strand by splice junctions suggests the existence of a selection pressure against their disruption, perhaps underscoring the investment made by nature in building these intricate secondary structures. A corollary is that the helix and the strand are the smallest integral architectural units of a protein and represent the minimal modules in the evolution of protein structure. These results should find use in comparative genomics, designing of cloning strategies, and in the mutual verification of genome sequences with protein structures. PMID:15381847

  16. Rapid disruption of intestinal epithelial tight junction and barrier dysfunction by ionizing radiation in mouse colon in vivo: protection by N-acetyl-l-cysteine.

    PubMed

    Shukla, Pradeep K; Gangwar, Ruchika; Manda, Bhargavi; Meena, Avtar S; Yadav, Nikki; Szabo, Erzsebet; Balogh, Andrea; Lee, Sue Chin; Tigyi, Gabor; Rao, RadhaKrishna

    2016-05-01

    The goals of this study were to evaluate the effects of ionizing radiation on apical junctions in colonic epithelium and mucosal barrier function in mice in vivo. Adult mice were subjected to total body irradiation (4 Gy) with or without N-acetyl-l-cysteine (NAC) feeding for 5 days before irradiation. At 2-24 h postirradiation, the integrity of colonic epithelial tight junctions (TJ), adherens junctions (AJ), and the actin cytoskeleton was assessed by immunofluorescence microscopy and immunoblot analysis of detergent-insoluble fractions for TJ and AJ proteins. The barrier function was evaluated by measuring vascular-to-luminal flux of fluorescein isothiocyanate (FITC)-inulin in vivo and luminal-to-mucosal flux in vitro. Oxidative stress was evaluated by measuring protein thiol oxidation. Confocal microscopy showed that radiation caused redistribution of occludin, zona occludens-1, claudin-3, E-cadherin, and β-catenin, as well as the actin cytoskeleton as early as 2 h postirradiation, and this effect was sustained for at least 24 h. Feeding NAC before irradiation blocked radiation-induced disruption of TJ, AJ, and the actin cytoskeleton. Radiation increased mucosal permeability to inulin in colon, which was blocked by NAC feeding. The level of reduced-protein thiols in colon was depleted by radiation with a concomitant increase in the level of oxidized-protein thiol. NAC feeding blocked the radiation-induced protein thiol oxidation. These data demonstrate that radiation rapidly disrupts TJ, AJ, and the actin cytoskeleton by an oxidative stress-dependent mechanism that can be prevented by NAC feeding. PMID:26822914

  17. Sphingosine 1-Phosphate Induces Myoblast Differentiation through Cx43 Protein Expression: A Role for a Gap Junction-dependent and -independent Function

    PubMed Central

    Squecco, R.; Sassoli, C.; Nuti, F.; Martinesi, M.; Chellini, F.; Nosi, D.; Zecchi-Orlandini, S.; Francini, F.; Formigli, L.

    2006-01-01

    Although sphingosine 1-phosphate (S1P) has been considered a potent regulator of skeletal muscle biology, acting as a physiological anti-mitogenic and prodifferentiating agent, its downstream effectors are poorly known. In the present study, we provide experimental evidence for a novel mechanism by which S1P regulates skeletal muscle differentiation through the regulation of gap junctional protein connexin (Cx) 43. Indeed, the treatment with S1P greatly enhanced Cx43 expression and gap junctional intercellular communication during the early phases of myoblast differentiation, whereas the down-regulation of Cx43 by transfection with short interfering RNA blocked myogenesis elicited by S1P. Moreover, calcium and p38 MAPK-dependent pathways were required for S1P-induced increase in Cx43 expression. Interestingly, enforced expression of mutated Cx43Δ130–136 reduced gap junction communication and totally inhibited S1P-induced expression of the myogenic markers, myogenin, myosin heavy chain, caveolin-3, and myotube formation. Notably, in S1P-stimulated myoblasts, endogenous or wild-type Cx43 protein, but not the mutated form, coimmunoprecipitated and colocalized with F-actin and cortactin in a p38 MAPK-dependent manner. These data, together with the known role of actin remodeling in cell differentiation, strongly support the important contribution of gap junctional communication, Cx43 expression and Cx43/cytoskeleton interaction in skeletal myogenesis elicited by S1P. PMID:16957055

  18. ER-mitochondrial junctions can be bypassed by dominant mutations in the endosomal protein Vps13.

    PubMed

    Lang, Alexander B; John Peter, Arun T; Walter, Peter; Kornmann, Benoît

    2015-09-14

    The endoplasmic reticulum-mitochondria encounter structure (ERMES) complex tethers the endoplasmic reticulum and the mitochondria. It is thought to facilitate interorganelle lipid exchange and influence mitochondrial dynamics and mitochondrial DNA maintenance. Despite this important role, ERMES is not found in metazoans. Here, we identified single amino acid substitutions in Vps13 (vacuolar protein sorting 13), a large universally conserved eukaryotic protein, which suppress all measured phenotypic consequences of ERMES deficiency. Combined loss of VPS13 and ERMES is lethal, indicating that Vps13 and ERMES function in redundant pathways. Vps13 dynamically localizes to vacuole-mitochondria and to vacuole-nucleus contact sites depending on growth conditions, suggesting that ERMES function can be bypassed by the activity of other contact sites, and that contact sites establish a growth condition-regulated organelle network. PMID:26370498

  19. 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.

  20. 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.

  1. Mitotic regulator Mis18β interacts with and specifies the centromeric assembly of molecular chaperone holliday junction recognition protein (HJURP).

    PubMed

    Wang, Jianyu; Liu, Xing; Dou, Zhen; Chen, Liang; Jiang, Hao; Fu, Chuanhai; Fu, Guosheng; Liu, Dan; Zhang, Jiancun; Zhu, Tongge; Fang, Jingwen; Zang, Jianye; Cheng, Jinke; Teng, Maikun; Ding, Xia; Yao, Xuebiao

    2014-03-21

    The centromere is essential for precise and equal segregation of the parental genome into two daughter cells during mitosis. CENP-A is a unique histone H3 variant conserved in eukaryotic centromeres. The assembly of CENP-A to the centromere is mediated by Holliday junction recognition protein (HJURP) in early G1 phase. However, it remains elusive how HJURP governs CENP-A incorporation into the centromere. Here we show that human HJURP directly binds to Mis18β, a component of the Mis18 complex conserved in the eukaryotic kingdom. A minimal region of HJURP for Mis18β binding was mapped to residues 437-460. Depletion of Mis18β by RNA interference dramatically impaired HJURP recruitment to the centromere, indicating the importance of Mis18β in HJURP loading. Interestingly, phosphorylation of HJURP by CDK1 weakens its interaction with Mis18β, consistent with the notion that assembly of CENP-A to the centromere is achieved after mitosis. Taken together, these data define a novel molecular mechanism underlying the temporal regulation of CENP-A incorporation into the centromere by accurate Mis18β-HJURP interaction. PMID:24519934

  2. Mitotic Regulator Mis18β Interacts with and Specifies the Centromeric Assembly of Molecular Chaperone Holliday Junction Recognition Protein (HJURP)*

    PubMed Central

    Wang, Jianyu; Liu, Xing; Dou, Zhen; Chen, Liang; Jiang, Hao; Fu, Chuanhai; Fu, Guosheng; Liu, Dan; Zhang, Jiancun; Zhu, Tongge; Fang, Jingwen; Zang, Jianye; Cheng, Jinke; Teng, Maikun; Ding, Xia; Yao, Xuebiao

    2014-01-01

    The centromere is essential for precise and equal segregation of the parental genome into two daughter cells during mitosis. CENP-A is a unique histone H3 variant conserved in eukaryotic centromeres. The assembly of CENP-A to the centromere is mediated by Holliday junction recognition protein (HJURP) in early G1 phase. However, it remains elusive how HJURP governs CENP-A incorporation into the centromere. Here we show that human HJURP directly binds to Mis18β, a component of the Mis18 complex conserved in the eukaryotic kingdom. A minimal region of HJURP for Mis18β binding was mapped to residues 437–460. Depletion of Mis18β by RNA interference dramatically impaired HJURP recruitment to the centromere, indicating the importance of Mis18β in HJURP loading. Interestingly, phosphorylation of HJURP by CDK1 weakens its interaction with Mis18β, consistent with the notion that assembly of CENP-A to the centromere is achieved after mitosis. Taken together, these data define a novel molecular mechanism underlying the temporal regulation of CENP-A incorporation into the centromere by accurate Mis18β-HJURP interaction. PMID:24519934

  3. 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

  4. 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

  5. Effects of sevoflurane on tight junction protein expression and PKC-α translocation after pulmonary ischemia-reperfusion injury.

    PubMed

    Chai, Jun; Long, Bo; Liu, Xiaomei; Li, Yan; Han, Ning; Zhao, Ping; Chen, Weimin

    2015-01-01

    Pulmonary dysfunction caused by ischemia-reperfusion injury is the leading cause of mortality in lung transplantation. We aimed to investigate the effects of sevoflurane pretreatment on lung permeability, tight junction protein occludin and zona occludens 1 (ZO-1) expression, and translocation of protein kinase C (PKC)-α after ischemia-reperfusion. A lung ischemia-reperfusion injury model was established in 96 male Wistar rats following the modified Eppinger method. The rats were divided into four groups with 24 rats in each group: a control (group C), an ischemia-reperfusion group (IR group), a sevoflurane control group (sev-C group), and a sevoflurane ischemia-reperfusion group (sev-IR group). There were three time points in each group: ischemic occlusion for 45 min, reperfusion for 60 min and reperfusion for 120 min; and there were six rats per time point. For the 120-min reperfusion group, six extra rats underwent bronchoalveolar lavage. Mean arterial pressure (MAP) and pulse oxygen saturation (SpO2) were recorded at each time point. The wet/dry weight ratio and lung permeability index (LPI) were measured. Quantitative RT-PCR and Western blot were used to measure pulmonary occludin and ZO-1, and Western blot was used to measure cytosolic and membranous PKC-α in the lung. Lung permeability was significantly increased after ischemia-reperfusion. Sevoflurane pretreatment promoted pulmonary expression of occludin and ZO-1 after reperfusion and inhibited the translocation of PKC-α. In conclusion, sevoflurane pretreatment alleviated lung permeability by upregulating occludin and ZO-1 after ischemia-reperfusion. Sevoflurane pretreatment inhibited the translocation and activation of PKC-α, which also contributed to the lung-protective effect of sevoflurane. PMID:26045255

  6. Volatile Anesthetics Influence Blood-Brain Barrier Integrity by Modulation of Tight Junction Protein Expression in Traumatic Brain Injury

    PubMed Central

    Schaible, Eva-Verena; Timaru-Kast, Ralph; Hedrich, Jana; Luhmann, Heiko J.; Engelhard, Kristin

    2012-01-01

    Disruption of the blood-brain barrier (BBB) results in cerebral edema formation, which is a major cause for high mortality after traumatic brain injury (TBI). As anesthetic care is mandatory in patients suffering from severe TBI it may be important to elucidate the effect of different anesthetics on cerebral edema formation. Tight junction proteins (TJ) such as zonula occludens-1 (ZO-1) and claudin-5 (cl5) play a central role for BBB stability. First, the influence of the volatile anesthetics sevoflurane and isoflurane on in-vitro BBB integrity was investigated by quantification of the electrical resistance (TEER) in murine brain endothelial monolayers and neurovascular co-cultures of the BBB. Secondly brain edema and TJ expression of ZO-1 and cl5 were measured in-vivo after exposure towards volatile anesthetics in native mice and after controlled cortical impact (CCI). In in-vitro endothelial monocultures, both anesthetics significantly reduced TEER within 24 hours after exposure. In BBB co-cultures mimicking the neurovascular unit (NVU) volatile anesthetics had no impact on TEER. In healthy mice, anesthesia did not influence brain water content and TJ expression, while 24 hours after CCI brain water content increased significantly stronger with isoflurane compared to sevoflurane. In line with the brain edema data, ZO-1 expression was significantly higher in sevoflurane compared to isoflurane exposed CCI animals. Immunohistochemical analyses revealed disruption of ZO-1 at the cerebrovascular level, while cl5 was less affected in the pericontusional area. The study demonstrates that anesthetics influence brain edema formation after experimental TBI. This effect may be attributed to modulation of BBB permeability by differential TJ protein expression. Therefore, selection of anesthetics may influence the barrier function and introduce a strong bias in experimental research on pathophysiology of BBB dysfunction. Future research is required to investigate adverse or

  7. 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

  8. The 90-kDa junctional sarcoplasmic reticulum protein forms an integral part of a supramolecular triad complex in skeletal muscle.

    PubMed

    Froemming, G R; Pette, D; Ohlendieck, K

    1999-08-11

    Although it is well established that voltage-sensing of the alpha(1)-dihydropyridine receptor triggers Ca(2+)-release via the ryanodine receptor during excitation-contraction coupling in skeletal muscle fibers, it remains to be determined which junctional components are responsible for the assembly, maintenance, and stabilization of triads. Here, we analyzed the expression pattern and neighborhood relationship of a novel 90-kDa sarcoplasmic reticulum protein. This protein is highly enriched in the triad fraction and is predominantly expressed in fast-twitching muscle fibers. Chronic low-frequency electro-stimulation induced a drastic decrease in the relative abundance of this protein. Chemical crosslinking showed a potential overlap between the 90-kDa junctional face membrane protein and the ryanodine receptor Ca(2+)-release channel, suggesting tight protein-protein interactions between these two triad components. Hence, Ca(2+)-regulatory muscle proteins have a strong tendency to oligomerize and the triad region of skeletal muscle fibers forms supramolecular membrane complexes involved in the regulation of Ca(2+)-homeostasis and signal transduction. PMID:10441473

  9. Solubilization and characterization of a ouabain-sensitive protein from transverse tubule membrane-junctional sarcoplasmic reticulum complexes (TTM-JSR) in cat cardiac muscle.

    PubMed

    Fujino, S; Satoh, K; Bando, T; Kurokawa, T; Nakai, T; Takashima, K; Fujino, M

    1989-05-15

    A new glycoprotein of 31,500 dalton, which has a high affinity for ouabain, and is independent of (Na+-K+)-ATPase, was solubilized from transverse tubule membrane and junctional sarcoplasmic reticulum complexes (TTM-JSR) of cat cardiac muscle. This protein could be extracted only in small amounts from sarcolemma-plasma membrane (SLM-PL) fragments, suggesting that it indeed originates from the TTM-JSR. PMID:2721638

  10. Proteins other than the Locus of Enterocyte Effacement-encoded proteins may contribute to Escherichia coli O157:H7 adherence to bovine rectoanal junction stratified squamous epithelial cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study, the Type III Secretion System (TTSS) proteins considered critical for Escherichia coli O157 (O157) adherence to the follicle-associated epithelial (FAE) cells at the bovine recto-anal junction (RAJ), did not appear to contribute to O157 adherence to the RAJ squamous epithelial (RSE) ...

  11. Involvement of YAP, TAZ and HSP90 in Contact Guidance and Intercellular Junction Formation in Corneal Epithelial Cells

    PubMed Central

    Morgan, Joshua T.; Tuyen, Binh C.; Rose, Brad W.; Reilly, Christopher M.; Russell, Paul; Murphy, Christopher J.

    2014-01-01

    The extracellular environment possesses a rich milieu of biophysical and biochemical signaling cues that are simultaneously integrated by cells and influence cellular phenotype. Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (WWTR1; TAZ), two important signaling molecules of the Hippo pathway, have been recently implicated as nuclear relays of cytoskeletal changes mediated by substratum rigidity and topography. These proteins intersect with other important intracellular signaling pathways (e.g. Wnt and TGFβ). In the cornea, epithelial cells adhere to the stroma through a 3-dimensional topography-rich basement membrane, with features in the nano-submicron size-scale that are capable of profoundly modulating a wide range of fundamental cell behaviors. The influences of substratum-topography, YAP/TAZ knockdown, and HSP90 inhibition on cell morphology, YAP/TAZ localization, and the expression of TGFβ2 and CTGF, were investigated. The results demonstrate (a) that knockdown of TAZ enhances contact guidance in a YAP dependent manner, (b) that CTGF is predominantly regulated by YAP and not TAZ, and (c) that TGFβ2 is regulated by both YAP and TAZ in these cells. Additionally, inhibition of HSP90 resulted in nuclear localization and subsequent transcriptional-activation of YAP, formation of cell-cell junctions and co-localization of E-cadherin and β-catenin at adherens junctions. Results presented in this study reflect the complexities underlying the molecular relationships between the cytoskeleton, growth factors, heat shock proteins, and co-activators of transcription that impact mechanotransduction. The data reveal the importance of YAP/TAZ on the cell behaviors, and gene and protein expression. PMID:25290150

  12. Nitric Oxide and Airway Epithelial Barrier Function: Regulation of Tight Junction Proteins and Epithelial Permeability

    PubMed Central

    Olson, Nels; Greul, Anne-Katrin; Hristova, Milena; Bove, Peter F.; Kasahara, David I.; van der Vliet, Albert

    2008-01-01

    Acute airway inflammation is associated with enhanced production of nitric oxide (NO•) and altered airway epithelial barrier function, suggesting a role of NO• or its metabolites in epithelial permeability. While high concentrations of S-nitrosothiols disrupted transepithelial resistance (TER) and increased permeability in 16HBE14o- cells, no significant barrier disruption was observed by NONOates, in spite of altered distribution and expression of some TJ proteins. Barrier disruption of mouse tracheal epithelial (MTE) cell monolayers in response to inflammatory cytokines was independent of NOS2, based on similar effects in MTE cells from NOS2-/- mice and a lack of effect of the NOS2-inhibitor 1400W. Cell pre-incubation with LPS protected MTE cells from TER loss and increased permeability by H2O2, which was independent of NOS2. However, NOS2 was found to contribute to epithelial wound repair and TER recovery after mechanical injury. Overall, our results demonstrate that epithelial NOS2 is not responsible for epithelial barrier dysfunction during inflammation, but may contribute to restoration of epithelial integrity. PMID:19100237

  13. The Src Homology 3 Domain Is Required for Junctional Adhesion Molecule Binding to the Third PDZ Domain of the Scaffolding Protein ZO-1

    SciTech Connect

    Nomme, Julian; Fanning, Alan S.; Caffrey, Michael; Lye, Ming F.; Anderson, James M.; Lavie, Arnon

    2012-01-20

    Tight junctions are cell-cell contacts that regulate the paracellular flux of solutes and prevent pathogen entry across cell layers. The assembly and permeability of this barrier are dependent on the zonula occludens (ZO) membrane-associated guanylate kinase (MAGUK) proteins ZO-1, -2, and -3. MAGUK proteins are characterized by a core motif of protein-binding domains that include a PDZ domain, a Src homology 3 (SH3) domain, and a region of homology to guanylate kinase (GUK); the structure of this core motif has never been determined for any MAGUK. To better understand how ZO proteins organize the assembly of protein complexes we have crystallized the entire PDZ3-SH3-GUK core motif of ZO-1. We have also crystallized this core motif in complex with the cytoplasmic tail of the ZO-1 PDZ3 ligand, junctional adhesion molecule A (JAM-A) to determine how the activity of different domains is coordinated. Our study shows a new feature for PDZ class II ligand binding that implicates the two highly conserved Phe{sup -2} and Ser{sup -3} residues of JAM. Our x-ray structures and NMR experiments also show for the first time a role for adjacent domains in the binding of ligands to PDZ domains in the MAGUK proteins family.

  14. 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

  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. 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.

  17. 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

  18. Polyinosinic:polycytidylic acid induces protein kinase D–dependent disassembly of apical junctions and barrier dysfunction in airway epithelial cells

    PubMed Central

    Rezaee, Fariba; Meednu, Nida; Emo, Jason A.; Saatian, Bahman; Chapman, Timothy J.; Naydenov, Nayden G.; De Benedetto, Anna; Beck, Lisa A.; Ivanov, Andrei I.; Georas, Steve N.

    2011-01-01

    Background Disruption of the epithelial barrier might be a risk factor for allergen sensitization and asthma. Viral respiratory tract infections are strongly associated with asthma exacerbation, but the effects of respiratory viruses on airway epithelial barrier function are not well understood. Many viruses generate double-stranded RNA, which can lead to airway inflammation and initiate an antiviral immune response. Objectives We investigated the effects of the synthetic double-stranded RNA polyinosinic:polycytidylic acid (polyI:C) on the structure and function of the airway epithelial barrier in vitro. Methods 16HBE14o- human bronchial epithelial cells and primary airway epithelial cells at an air-liquid interface were grown to confluence on Transwell inserts and exposed to polyI:C. We studied epithelial barrier function by measuring transepithelial electrical resistance and paracellular flux of fluorescent markers and structure of epithelial apical junctions by means of immunofluorescence microscopy. Results PolyI:C induced a profound decrease in transepithelial electrical resistance and increase in paracellular permeability. Immunofluorescence microscopy revealed markedly reduced junctional localization of zonula occludens-1, occludin, E-cadherin, β-catenin, and disorganization of junction-associated actin filaments. PolyI:C induced protein kinase D (PKD) phosphorylation, and a PKD antagonist attenuated polyI:C-induced disassembly of apical junctions and barrier dysfunction. Conclusions PolyI:C has a powerful and previously unsuspected disruptive effect on the airway epithelial barrier. PolyI:C-dependent barrier disruption is mediated by disassembly of epithelial apical junctions, which is dependent on PKD signaling. These findings suggest a new mechanism potentially underlying the associations between viral respiratory tract infections, airway inflammation, and allergen sensitization. PMID:21996340

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. Bimetallic junctions

    NASA Technical Reports Server (NTRS)

    Arcella, F. G.; Lessmann, G. G.; Lindberg, R. A. (Inventor)

    1977-01-01

    The formation of voids through interdiffusion in bimetallic welded structures exposed to high operating temperatures is inhibited by utilizing an alloy of the parent materials in the junction of the parent materials or by preannealing the junction at an ultrahigh temperature. These methods are also used to reduce the concentration gradient of a hardening agent.

  4. Diets high in fermentable protein and fibre alter tight junction protein composition with minor effects on barrier function in piglet colon.

    PubMed

    Richter, Jan F; Pieper, Robert; Zakrzewski, Silke S; Günzel, Dorothee; Schulzke, Joerg D; Van Kessel, Andrew G

    2014-03-28

    Protein fermentation end products may damage the colonic mucosa, which could be counteracted by dietary inclusion of fermentable carbohydrates (fCHO). Although fermentable crude protein (fCP) and fCHO are known to affect microbial ecology, their interactive effects on epithelial barrier function are unknown. In the present study, in a 2 × 2 factorial experiment, thirty-two weaned piglets were fed low-fCP/low-fCHO (14·5 % crude protein (CP)/14·5 % total dietary fibre (TDF)), low-fCP/high-fCHO (14·8 % CP/16·6 % TDF), high-fCP/low-fCHO (19·8 % CP/14·5 % TDF) and high-fCP/high-fCHO (20·1 % CP/18·0 % TDF) diets. After 21-23 d, samples of proximal and distal colonic mucosae were investigated in Ussing chambers with respect to the paracellular and transcytotic passages of macromolecules and epithelial ion transport. The high-fCHO diets were found to reduce the permeability of the distal colon to the transcytotic marker horseradish peroxidase (HRP, 44 kDa; P <0·05) and also reduce the paracellular permeation of N-hydroxysuccinimide-biotin into the submucosa (443 Da; P <0·05), whereas that of HRP was decreased by the high-fCP diets (P <0·01). Short-circuit current (active ion transport), transepithelial resistance (barrier function) and charge selectivity were largely unaffected in both the segments. However, the high-fCP diets were found to suppress the aldosterone-induced epithelial Na channel activity (P <0·01) irrespective of fCHO inclusion. The high-fCP diets generally reduced the expression of colonic claudin-1, claudin-2 and claudin-3 (P <0·01), while that of claudin-4 was increased by the high-fCHO diets (P <0·01). The high-fCHO diets also altered the ratio between occludin forms (P <0·05) and increased the expression of tricellulin in the proximal colon, which was not observed with high-fCP diets. In conclusion, dietary fCHO and fCP exerted few and largely independent effects on functional measurements, but altered tight junction

  5. 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

  6. The effect of hyperglycaemia on permeability and the expression of junctional complex molecules in human retinal and choroidal endothelial cells.

    PubMed

    Saker, S; Stewart, E A; Browning, A C; Allen, C L; Amoaku, W M

    2014-04-01

    Diabetic retinopathy is the leading cause of preventable blindness in the working population and its prevalence continues to increase as the worldwide prevalence of diabetes grows. Diabetic choroidopathy is less well studied and occurs in the late stages of diabetic eye disease. The main cause of visual loss in diabetic eye disease is diabetic macular oedema caused by an increase in microvascular endothelial permeability. Endothelial cell permeability is influenced by multiple factors which have not been fully elucidated, particularly in human models. In addition, the gene and protein expression between retinal and choroidal endothelial cells, even in humans, has been shown to be heterogeneous. The aim of this project was to determine, in vitro, the effect of high glucose (25 mM) on human paracellular permeability in retinal and choroidal endothelial cells. The expression of selected tight junction molecules (Occludin, Claudin-5, JAM-A and JAM-C) and adheren junction (VE-Cadherin) molecules was also compared between retinal and choroidal endothelial cells and with high glucose. High glucose conditions significantly increased the permeability in both retinal and choroidal endothelial cells monolayers although the increase was higher in retinal endothelial cells. Under normal glucose culture conditions microarray analysis determined that occludin and claudin-5 gene expression was higher in retinal endothelial cells than choroidal endothelial cells, and western blotting indicated that claudin-5 protein expression was also higher in retinal endothelial cells whilst JAM-A, and C and VE-Cadherin levels were similar. In retinal endothelial cells exposed to high glucose claudin-5, occludin and JAM-A was found to be reduced, whereas the expression of VE-Cadherin and JAM-C was unchanged when evaluated with western blotting, immunofluorescence and qPCR. None of the proteins were significantly decreased by high glucose in choroidal endothelial cells. The increase in retinal

  7. Recessive Mutations in the Gene Encoding the Tight Junction Protein Occludin Cause Band-like Calcification with Simplified Gyration and Polymicrogyria

    PubMed Central

    O'Driscoll, Mary C.; Daly, Sarah B.; Urquhart, Jill E.; Black, Graeme C.M.; Pilz, Daniela T.; Brockmann, Knut; McEntagart, Meriel; Abdel-Salam, Ghada; Zaki, Maha; Wolf, Nicole I.; Ladda, Roger L.; Sell, Susan; D'Arrigo, Stefano; Squier, Waney; Dobyns, William B.; Livingston, John H.; Crow, Yanick J.

    2010-01-01

    Band-like calcification with simplified gyration and polymicrogyria (BLC-PMG) is a rare autosomal-recessive neurological disorder showing highly characteristic clinical and neuroradiological features. Affected individuals demonstrate early-onset seizures, severe microcephaly, and developmental arrest with bilateral, symmetrical polymicrogyria (PMG) and a band of gray matter calcification on brain imaging; as such, the disorder can be considered as a “pseudo-TORCH” syndrome. By using autozygosity mapping and copy number analysis we identified intragenic deletions and mutations in OCLN in nine patients from six families with BLC-PMG. The OCLN gene encodes occludin, an integral component of tight junctions. Neuropathological analysis of an affected individual showed similarity to the mouse model of occludin deficiency with calcification predominantly associated with blood vessels. Both intracranial calcification and PMG are heterogeneous in etiology. Neuropathological and clinical studies of PMG have suggested that in utero ischemic or vascular insults may contribute to this common cortical abnormality. Tight junctions are functional in cerebral blood vessels early in fetal development and continue to play a vital role in maintenance of the blood-brain barrier during postnatal life. We provide evidence that the tight junction protein occludin (encoded by the OCLN gene) is involved in the pathogenesis of malformations of cortical development. PMID:20727516

  8. A novel component of epidermal cell-matrix and cell-cell contacts: transmembrane protein type XIII collagen.

    PubMed

    Peltonen, S; Hentula, M; Hägg, P; Ylä-Outinen, H; Tuukkanen, J; Lakkakorpi, J; Rehn, M; Pihlajaniemi, T; Peltonen, J

    1999-10-01

    Type XIII collagen is a short chain collagen which has recently been shown to be a transmembrane protein. The purpose of this study was to elucidate the presence and localization of type XIII collagen in normal human skin and cultured keratinocytes. Expression of type XIII collagen was demonstrated in normal human skin and epidermis at the RNA level using reverse transcription followed by polymerase chain reaction and at the protein level using western blotting and indirect immunofluorescence labeling. Immunolabeling of epidermis revealed type XIII collagen both in the cell-cell contact sites and in the dermal-epidermal junction. In cultured keratinocytes type XIII collagen epitopes were detected in focal contacts and in intercellular contacts. The results of this study show very little colocalization of type XIII collagen and desmosomal components at the light microscopic level. Thus, these results suggest that type XIII collagen is unlikely to be a component of desmosomes. Instead, the punctate labeling pattern of type XIII collagen at the cell-cell contact sites and high degree of colocalization with E-cadherin suggests that type XIII collagen is very likely to be closely associated with adherens type junctions, and may, in fact, be a component of these junctions. PMID:10504453

  9. Proteins facilitating Escherichia coli O157 persistence at the bovine recto-anal junction (RAJ) squamous epithelial cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli O157 (O157) persist at the recto-anal junction (RAJ) of gastrointestinal tracts (GIT) of cattle, the primary reservoirs of this human pathogen. We recently reported (Kudva et al., BMC Microbiol. 2012, 12: 103) that the previously identified and extensively documented principal O157...

  10. 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

  11. 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

  12. 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

  13. The response of claudin-like transmembrane septate junction proteins to altered environmental ion levels in the larval mosquito Aedes aegypti.

    PubMed

    Jonusaite, Sima; Kelly, Scott P; Donini, Andrew

    2016-07-01

    Septate junctions (SJs) occlude the paracellular pathway and function as paracellular diffusion barriers within invertebrate epithelia. However, integral components of SJs and their contribution to barrier properties have received considerably less attention than those of vertebrate occluding junctions. In arthropods, SJ proteins have only been identified in Drosophila and among these are three integral claudin-like proteins, Megatrachea (Mega), Sinuous (Sinu) and Kune-kune (Kune), as well as a receptor-like transmembrane SJ protein known as Neurexin IV (Nrx IV). In this study, mega, sinu, kune and nrx IV are identified and characterized in aquatic larvae of the mosquito Aedes aegypti and a role for these proteins in ionoregulatory homeostasis is considered. Transcripts encoding Mega, Sinu, Kune and Nrx IV were found in iono/osmoregulatory tissues such as the midgut, Malpighian tubules, hindgut and anal papillae, but abundance was greater in the hindgut and anal papillae. Using immunohistochemical and western blot analysis it was found that Kune localized to the regions of intercellular contact between epithelial cells of the rectum and posterior midgut and in the apical membrane domain of the syncytial epithelium of anal papillae. To investigate a potential role for integral SJ proteins in larval A. aegypti iono/osmoregulation, abundance was examined in animals reared in freshwater or brackish water (30 % seawater). In iono/osmoregulatory epithelia, larvae exhibited tissue-specific alterations in mega mRNA and Kune protein abundance, but not sinu or nrx IV mRNA. These studies provide a first look at the potential contribution of integral SJ components to iono/osmoregulatory homeostasis in an aquatic invertebrate. PMID:27004691

  14. 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

  15. Testicular Cell Junction: A Novel Target for Male Contraception

    PubMed Central

    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. PMID:19275601

  16. 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).

  17. 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

  18. Enhancement of Blood-Brain Barrier Permeability and Reduction of Tight Junction Protein Expression Are Modulated by Chemokines/Cytokines Induced by Rabies Virus Infection

    PubMed Central

    Chai, Qingqing; He, Wen Q.; Zhou, Ming; Lu, Huijun

    2014-01-01

    ABSTRACT Infection with laboratory-attenuated rabies virus (RABV) enhances blood-brain barrier (BBB) permeability, which has been demonstrated to be an important factor for host survival, since it allows immune effectors to enter the central nervous system (CNS) and clear RABV. To probe the mechanism by which RABV infection enhances BBB permeability, the expression of tight junction (TJ) proteins in the CNS was investigated following intracranial inoculation with laboratory-attenuated or wild-type (wt) RABV. BBB permeability was significantly enhanced in mice infected with laboratory-attenuated, but not wt, RABV. The expression levels of TJ proteins (claudin-5, occludin, and zonula occludens-1) were decreased in mice infected with laboratory-attenuated, but not wt, RABV, suggesting that enhancement of BBB permeability is associated with the reduction of TJ protein expression in RABV infection. RABV neither infects the brain microvascular endothelial cells (BMECs) nor modulates the expression of TJ proteins in BMECs. However, brain extracts prepared from mice infected with laboratory-attenuated, but not wt, RABV reduced TJ protein expression in BMECs. It was found that brain extracts from mice infected with laboratory-attenuated RABV contained significantly higher levels of inflammatory chemokines/cytokines than those from mice infected with wt RABV. Pathway analysis indicates that gamma interferon (IFN-γ) is located in the center of the cytokine network in the RABV-infected mouse brain, and neutralization of IFN-γ reduced both the disruption of BBB permeability in vivo and the downregulation of TJ protein expression in vitro. These findings indicate that the enhancement of BBB permeability and the reduction of TJ protein expression are due not to RABV infection per se but to virus-induced inflammatory chemokines/cytokines. IMPORTANCE Previous studies have shown that infection with only laboratory-attenuated, not wild-type, rabies virus (RABV) enhances blood

  19. Gap Junction Proteins in the Blood-Brain Barrier Control Nutrient-Dependent Reactivation of Drosophila Neural Stem Cells

    PubMed Central

    Spéder, Pauline; Brand, Andrea H.

    2014-01-01

    Summary Neural stem cells in the adult brain exist primarily in a quiescent state but are reactivated in response to changing physiological conditions. How do stem cells sense and respond to metabolic changes? In the Drosophila CNS, quiescent neural stem cells are reactivated synchronously in response to a nutritional stimulus. Feeding triggers insulin production by blood-brain barrier glial cells, activating the insulin/insulin-like growth factor pathway in underlying neural stem cells and stimulating their growth and proliferation. Here we show that gap junctions in the blood-brain barrier glia mediate the influence of metabolic changes on stem cell behavior, enabling glia to respond to nutritional signals and reactivate quiescent stem cells. We propose that gap junctions in the blood-brain barrier are required to translate metabolic signals into synchronized calcium pulses and insulin secretion. PMID:25065772

  20. 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

  1. Nitric oxide attenuates hydrogen peroxide-induced barrier disruption and protein tyrosine phosphorylation in monolayers of intestinal epithelial cell.

    PubMed

    Katsube, Takanori; Tsuji, Hideo; Onoda, Makoto

    2007-06-01

    The intestinal epithelium provides a barrier to the transport of harmful luminal molecules into the systemic circulation. A dysfunctional epithelial barrier is closely associated with the pathogenesis of a variety of intestinal and systemic disorders. We investigated here the effects of nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) on the barrier function of a human intestinal epithelial cell line, Caco-2. When treated with H(2)O(2), Caco-2 cell monolayers grown on permeable supports exhibited several remarkable features of barrier dysfunction as follows: a decrease in transepithelial electrical resistance, an increase in paracellular permeability to dextran, and a disruption of the intercellular junctional localization of the scaffolding protein ZO-1. In addition, an induction of tyrosine phosphorylation of numerous cellular proteins including ZO-1, E-cadherin, and beta-catenin, components of tight and adherens junctions, was observed. On the other hand, combined treatment of Caco-2 monolayers with H(2)O(2) and an NO donor (NOC5 or NOC12) relieved the damage to the barrier function and suppressed the protein tyrosine phosphorylation induced by H(2)O(2) alone. These results suggest that NO protects the barrier function of intestinal epithelia from oxidative stress by modulating some intracellular signaling pathways of protein tyrosine phosphorylation in epithelial cells. PMID:17451824

  2. Direct Exposure to Ethanol Disrupts Junctional Cell-Cell Contact and Hippo-YAP Signaling in HL-1 Murine Atrial Cardiomyocytes

    PubMed Central

    Noritake, Kanako; Aki, Toshihiko; Funakoshi, Takeshi; Unuma, Kana; Uemura, Koichi

    2015-01-01

    Direct exposure of cardiomyocytes to ethanol causes cardiac damage such as cardiac arrythmias and apoptotic cell death. Cardiomyocytes are connected to each other through intercalated disks (ID), which are composed of a gap junction (GJ), adherens junction, and desmosome. Changes in the content as well as the subcellular localization of connexin43 (Cx43), the main component of the cardiac GJ, are reportedly involved in cardiac arrythmias and subsequent damage. Recently, the hippo-YAP signaling pathway, which links cellular physical status to cell proliferation, differentiation, and apoptosis, has been implicated in cardiac homeostasis under physiological as well as pathological conditions. This study was conducted to explore the possible involvement of junctional intercellular communication, mechanotransduction through cytoskeletal organization, and the hippo-YAP pathway in cardiac damage caused by direct exposure to ethanol. HL-1 murine atrial cardiac cells were used since these cells retain cardiac phenotypes through ID formation and subsequent synchronous contraction. Cells were exposed to 0.5–2% ethanol; significant apoptotic cell death was observed after exposure to 2% ethanol for 48 hours. A decrease in Cx43 levels was already observed after 3 hours exposure to 2% ethanol, suggesting a rapid degradation of this protein. Upon exposure to ethanol, Cx43 translocated into lysosomes. Cellular cytoskeletal organization was also dysregulated by ethanol, as demonstrated by the disruption of myofibrils and intermediate filaments. Coinciding with the loss of cell-cell adherence, decreased phosphorylation of YAP, a hippo pathway effector, was also observed in ethanol-treated cells. Taken together, the results provide evidence that cells exposed directly to ethanol show 1) impaired cell-cell adherence/communication, 2) decreased cellular mechanotransduction by the cytoskeleton, and 3) a suppressed hippo-YAP pathway. Suppression of hippo-YAP pathway signaling should be

  3. 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

  4. 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

  5. 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

  6. 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.

  7. Drosophila coracle, a Member of the Protein 4.1 Superfamily, Has Essential Structural Functions in the Septate Junctions and Developmental Functions in Embryonic and Adult Epithelial Cells

    PubMed Central

    Lamb, Rebecca S.; Ward, Robert E.; Schweizer, Liang; Fehon, Richard G.

    1998-01-01

    Although extensively studied biochemically, members of the Protein 4.1 superfamily have not been as well characterized genetically. Studies of coracle, a Drosophila Protein 4.1 homologue, provide an opportunity to examine the genetic functions of this gene family. coracle was originally identified as a dominant suppressor of EgfrElp, a hypermorphic form of the Drosophila Epidermal growth factor receptor gene. In this article, we present a phenotypic analysis of coracle, one of the first for a member of the Protein 4.1 superfamily. Screens for new coracle alleles confirm the null coracle phenotype of embryonic lethality and failure in dorsal closure, and they identify additional defects in the embryonic epidermis and salivary glands. Hypomorphic coracle alleles reveal functions in many imaginal tissues. Analysis of coracle mutant cells indicates that Coracle is a necessary structural component of the septate junction required for the maintenance of the transepithelial barrier but is not necessary for apical–basal polarity, epithelial integrity, or cytoskeletal integrity. In addition, coracle phenotypes suggest a specific role in cell signaling events. Finally, complementation analysis provides information regarding the functional organization of Coracle and possibly other Protein 4.1 superfamily members. These studies provide insights into a range of in vivo functions for coracle in developing embryos and adults. PMID:9843584

  8. Long noncoding RNA SPRY4-IT1 regulates intestinal epithelial barrier function by modulating the expression levels of tight junction proteins

    PubMed Central

    Xiao, Lan; Rao, Jaladanki N.; Cao, Shan; Liu, Lan; Chung, Hee Kyoung; Zhang, Yun; Zhang, Jennifer; Liu, Yulan; Gorospe, Myriam; Wang, Jian-Ying

    2016-01-01

    Epithelial cells line the intestinal mucosa and form an important barrier to a wide array of noxious substances in the lumen. Disruption of the barrier integrity occurs commonly in various pathologies. Long noncoding RNAs (lncRNAs) control diverse biological processes, but little is known about the role of lncRNAs in regulation of the gut permeability. Here we show that the lncRNA SPRY4-IT1 regulates the intestinal epithelial barrier function by altering expression of tight junction (TJ) proteins. SPRY4-IT1 silencing led to dysfunction of the epithelial barrier in cultured cells by decreasing the stability of mRNAs encoding TJ proteins claudin-1, claudin-3, occludin, and JAM-1 and repressing their translation. In contrast, increasing the levels of SPRY4-IT1 in the intestinal mucosa protected the gut barrier in mice exposed to septic stress by increasing the abundance of TJ proteins. SPRY4-IT1 directly interacted with TJ mRNAs, and this process was enhanced through the association with the RNA-binding protein HuR. Of interest, the intestinal mucosa from patients with increased gut permeability exhibited a decrease in the levels of SPRY4-IT1. These findings highlight a novel role for SPRY4-IT1 in controlling the intestinal epithelial barrier and define a mechanism by which SPRY4-IT1 modulates TJ expression by altering the stability and translation of TJ mRNAs. PMID:26680741

  9. Bacterial protein AvrA stabilizes intestinal epithelial tight junctions via blockage of the C-Jun N-terminal kinase pathway.

    PubMed

    Zhang, Yongguo; Wu, Shaoping; Ma, Jun; Xia, Yinglin; Ai, Xun; Sun, Jun

    2015-01-01

    The Salmonella type III secretory system secretes virulence proteins, called effectors. Effectors are responsible for the alteration of tight junctions (TJ) and epithelial functions in intestinal infection and inflammation. In a previous study, we have demonstrated that a bacterial effector AvrA plays a role in stabilizing TJs and balancing the opposing action of other bacterial effectors. However, the molecular mechanisms by which AvrA-modulates TJ protein expression remain unknown. AvrA possesses acetyltransferase activity toward specific mitogen-activated protein kinase kinases (MAPKKs) and potently inhibits the c-Jun N-terminal kinase (JNK) pathway in inflammation. Inhibition of the JNK pathway is known to inhibit the TJ protein disassemble. Therefore, we hypothesize that AvrA stabilizes intestinal epithelial TJs via c-Jun and JNK pathway blockage. Using both in vitro and in vivo models, we showed that AvrA targets the c-Jun and JNK pathway that in turn stabilizes TJ protein ZO-1. Inhibition of JNK abolished the effect of AvrA on ZO-1. We further determined that AvrA suppressed the transcription factor activator protein-1, which was regulated by activated JNK. Moreover, we identified the functional domain of AvrA that directly regulated TJs using a series of AvrA mutants. The role of AvrA represents a highly refined bacterial strategy that helps the bacteria survive in the host and dampens the inflammatory response of the host. Our findings have uncovered a novel role of the bacterial protein AvrA in suppressing the inflammatory response of the host through JNK-regulated blockage of epithelial cell barrier function. PMID:25838979

  10. 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.

  11. 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

  12. Deficiency of Angulin-2/ILDR1, a Tricellular Tight Junction-Associated Membrane Protein, Causes Deafness with Cochlear Hair Cell Degeneration in Mice

    PubMed Central

    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. PMID:25822906

  13. 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. PMID:25822906

  14. Tight Junctions Go Viral!

    PubMed Central

    Torres-Flores, Jesús M.; Arias, Carlos F.

    2015-01-01

    Tight junctions (TJs) are highly specialized membrane domains involved in many important cellular processes such as the regulation of the passage of ions and macromolecules across the paracellular space and the establishment of cell polarity in epithelial cells. Over the past few years there has been increasing evidence that different components of the TJs can be hijacked by viruses in order to complete their infectious cycle. Viruses from at least nine different families of DNA and RNA viruses have been reported to use TJ proteins in their benefit. For example, TJ proteins such as JAM-A or some members of the claudin family of proteins are used by members of the Reoviridae family and hepatitis C virus as receptors or co-receptors during their entry into their host cells. Reovirus, in addition, takes advantage of the TJ protein Junction Adhesion Molecule-A (JAM-A) to achieve its hematogenous dissemination. Some other viruses are capable of regulating the expression or the localization of TJ proteins to induce cell transformation or to improve the efficiency of their exit process. This review encompasses the importance of TJs for viral entry, replication, dissemination, and egress, and makes a clear statement of the importance of studying these proteins to gain a better understanding of the replication strategies used by viruses that infect epithelial and/or endothelial cells. PMID:26404354

  15. 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

  16. Curcumin protects against the intestinal ischemia-reperfusion injury: involvement of the tight junction protein ZO-1 and TNF-α related mechanism.

    PubMed

    Tian, Shuying; Guo, Ruixue; Wei, Sichen; Kong, Yu; Wei, Xinliang; Wang, Weiwei; Shi, Xiaomeng; Jiang, Hongyu

    2016-03-01

    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

  17. Necrotizing Enterocolitis in a mouse model leads to widespread renal inflammation, acute kidney injury and disruption of renal tight junction proteins

    PubMed Central

    Garg, Parvesh M; Tatum, Rodney; Ravisankar, Srikanth; Shekhawat, Prem S; Chen, Yan-Hua

    2015-01-01

    BACKGROUND Necrotizing enterocolitis (NEC) is a devastating condition affecting premature infants and leads to high mortality and chronic morbidity. Severe form of NEC is associated with acute renal failure, fluid imbalance, hyponatremia and acidosis. We investigated the effect of NEC on tight junction (TJ) proteins in kidneys using a NEC mouse model to investigate the basis for the observed renal dysfunction. METHODS NEC was induced in C57BL/6 mice by formula feeding and subjecting them to periods of hypoxia and cold stress. NEC was confirmed by gross and histological examination. We studied various markers of inflammation in kidneys and investigated changes in expression of several TJ proteins and AQP2 using immunofluorecent staining and Western blotting. RESULTS We found markedly increased expression of NFκB, TGFβ and ERK1/2 along with claudin-1, -2, -3, -4, -8 and AQP-2 in NEC kidneys. The membrane localization of claudin-2 was altered in the NEC kidneys and its immunostaining signal at TJ was disrupted. CONCLUSION NEC led to a severe inflammatory response not only in the gut but also the kidneys. NEC increased expression of several TJ proteins and caused disruption of claudin-2 in renal tubules. These observed changes can help explain some of the clinical findings observed in NEC. PMID:26270572

  18. 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

  19. 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

  20. Cytoskeletal modulation and tyrosine phosphorylation of tight junction proteins are associated with mainstream cigarette smoke-induced permeability of airway epithelium.

    PubMed

    Olivera, Dorian; Knall, Cindy; Boggs, Susan; Seagrave, JeanClare

    2010-03-01

    Cigarette smoke increases the permeability of the lung epithelium. Consequences of increased permeability include increased access of toxins and pathogens from the air spaces to the interstitium and even the blood stream, and leakage of fluids into the air spaces. The mechanisms for permeability alterations have not been elucidated for airway epithelia. By analogy with other types of epithelia, we hypothesized that changes in the phosphorylation status and function of tight junction (TJ) or cytoskeletal proteins might mediate the smoke-induced permeability changes. We investigated the effects of exposure to mainstream cigarette smoke (MS) on cultures of Calu-3 cells, an airway epithelial cell line. Specifically, MS exposure caused increases in phosphorylation of the myosin-binding subunit (MBS) of myosin phosphatase and myosin light chain (MLC), proteins involved in the regulation of actin polymerization. These results implicate activation of Rho kinase (ROCK), consistent with previously reported data indicating that inhibition of ROCK activation suppressed MS-induced increases in permeability. MS exposure also increased polymerized (filamentous) actin (f-actin) content and caused redistribution of the TJ proteins from the normal apical circumferential band to a more basal location. The translocation of the TJ proteins was spatially associated with local increases in both f-actin and macromolecular permeability. Finally, MS exposure increased tyrosine phosphorylation of occludin but not ZO-1 and decreased association between the two TJ proteins. These results indicate that MS exposure causes alterations in cytoskeletal and TJ structure and function, resulting in increased macromolecular permeability that may contribute to the adverse health effects of MS. PMID:19376691

  1. Disc large 1 expression is altered by human papillomavirus E6/E7 proteins in organotypic cultures of human keratinocytes.

    PubMed

    Valdano, M Bugnon; Cavatorta, A L; Morale, M G; Marziali, F; de Souza Lino, V; Steenbergen, R D M; Boccardo, E; Gardiol, D

    2016-02-01

    Loss of cell polarity is a fundamental process in cell transformation. Among polarity proteins, we focused on human disc large (DLG1), which is localized mainly at adherens junctions and contributes to the control of cell proliferation. We previously demonstrated that its expression is altered in HPV-associated cervical neoplastic lesions, but the mechanisms beyond this remain unknown. In this study, we analysed the contribution of HPV proteins to the changes in DLG1 expression in the squamous epithelium. We observed tissue and intracellular misdistribution of DLG1 when high-risk HPV-18 E7 or E6/E7 proteins were expressed in organotypic raft cultures. The viral oncoproteins induce the loss of DLG1 from the cell borders and an increase in the level of DLG1 protein, reflecting the pattern observed in cervical lesions. These findings were corroborated in cultures bearing the entire HPV-18 genome. Interestingly, changes in tissue distribution and abundance of DLG1 were also detected in organotypic cultures expressing the low-risk HPV-11 E7 or E6/E7 proteins, suggesting a conserved function among different HPV types. However, for low-risk HPVs, the subcellular localization of DLG1 at cell-to-cell contacts was predominantly maintained. This report offers new evidence, we believe, of the involvement of HPV proteins in DLG1 expression pattern and our data support previous observations regarding DLG1 expression in cervical lesions. PMID:26653181

  2. Lipopolysaccharide-Induced Middle Ear Inflammation Disrupts the cochlear Intra-Strial Fluid–Blood Barrier through Down-Regulation of Tight Junction Proteins

    PubMed Central

    Zhang, Jinhui; Chen, Songlin; Hou, Zhiqiang; Cai, Jing; Dong, Mingmin; Shi, Xiaorui

    2015-01-01

    blood barrier, essential for cochlear homeostasis. The barrier is degraded as the expression of tight junction-associated proteins such as zona occludens 1, occludin, and vascular endothelial cadherin are down-regulated. PMID:25815897

  3. 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

  4. Functional expression of NF1 tumor suppressor protein: association with keratin intermediate filaments during the early development of human epidermis

    PubMed Central

    Malminen, Maria; Peltonen, Sirkku; Koivunen, Jussi; Peltonen, Juha

    2002-01-01

    Background NF1 refers to type 1 neurofibromatosis syndrome, which has been linked with mutations of the large NF1 gene. NF1 tumor suppressor protein, neurofibromin, has been shown to regulate ras: the NF1 protein contains a GTPase activating protein (GAP) related domain which functions as p21rasGAP. Our studies have previously demonstrated that the NF1 protein forms a high affinity association with cytokeratin 14 during the formation of desmosomes and hemidesmosomes in cultured keratinocytes. Methods The expression of NF1 protein was studied in developing human epidermis using western transfer analysis, indirect immunofluorescence, confocal laser scanning microscopy, immunoelectron microscopy, and in situ hybridization. Results The expression of NF1 protein was noted to be highly elevated in the periderm at 8 weeks estimated gestational age (EGA) and in the basal cells at 8–14 weeks EGA. During this period, NF1 protein was associated with cytokeratin filaments terminating to desmosomes and hemidesmosomes. NF1 protein did not display colocalization with α-tubulin or actin of the cytoskeleton, or with adherens junction proteins. Conclusions These results depict an early fetal period when the NF1 tumor suppressor is abundantly expressed in epidermis and associated with cytokeratin filaments. This period is characterized by the initiation of differentiation of the basal cells, maturation of the basement membrane zone as well as accentuated formation of selected cellular junctions. NF1 tumor suppressor may function in the regulation of epidermal histogenesis via controlling the organization of the keratin cytoskeleton during the assembly of desmosomes and hemidesmosomes. PMID:12199909

  5. 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

  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. 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

  8. Functional Requirement for a Highly Conserved Charged Residue at Position 75 in the Gap Junction Protein Connexin 32*

    PubMed Central

    Abrams, Charles K.; Islam, Mahee; Mahmoud, Rola; Kwon, Taekyung; Bargiello, Thaddeus A.; Freidin, Mona M.

    2013-01-01

    Charcot Marie Tooth disease (CMT) is a group of inherited disorders characterized clinically by exclusively or predominantly peripheral nerve dysfunction. CMT1X, the most common form of X-linked CMT is caused by mutations in connexin 32 (Cx32). In this work, we used dual whole cell patch clamp recording to examine the functional effects of mutations at the Arg75 position. This residue is highly conserved among members of the connexin family, and disease-causing mutations have been identified at this (or the corresponding) position in Cx26, Cx43, and Cx46. Thus, a better understanding of the effects of mutations of this position in Cx32 may have relevance to pathogenesis of a number of different human diseases. All three mutants associated with CMT1X (R75P, R75Q, and R75W) showed very low levels of coupling similar to those of the cells transfected with vector alone. Heterotypic pairing with Cx32 WT showed that the absence of coupling for these mutants in the homotypic configuration could be explained by shifts in their hemichannel Gj-Vj relations. Examination of the expression levels and gating characteristics of seven additional mutants (R75A, R75D, R75E, R75H, R75K, R75L, and R75V) at this position suggest that the positive charge at position 75 in Cx32 is required for normal channel function but not for gap junction assembly. Our studies also suggest that disease treatment strategies for CMT1X, which correct trafficking abnormalities in Cx32, may be ineffective for the group of mutations also conferring changes in gating properties of Cx32 channels. PMID:23209285

  9. Impact of 7,12-dimethylbenz[a]anthracene exposure on connexin gap junction proteins in cultured rat ovaries

    PubMed Central

    Ganesan, Shanthi; Keating, Aileen F.

    2014-01-01

    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. PMID:24269759

  10. Diosmectite-zinc oxide composite improves intestinal barrier function, modulates expression of pro-inflammatory cytokines and tight junction protein in early weaned pigs.

    PubMed

    Hu, Caihong; Song, Juan; Li, Yali; Luan, Zhaoshuang; Zhu, Kang

    2013-08-01

    The study evaluated whether feeding diosmectite-ZnO composite (DS-ZnO) at 500 mg Zn/kg to early weaned pigs would alleviate the weaning-related intestinal disorders as a substitute for high concentration of ZnO (2250 mg Zn/kg). The pigs weaned at an age of 21 ± 1 d were allotted to four treatments groups as follows: (1) control; (2) DS-ZnO, 500 mg Zn/kg diet; (3) ZnO, 2250 mg Zn/kg diet; and (4) mixture of 2·0 g DS/kg and 500 mg Zn/kg from ZnO (equal amount of DS and ZnO in the DS-ZnO treatment group). The results showed that, compared with the control on days 7 and 14 post-weaning, addition of DS-ZnO at 500 mg Zn/kg improved (P<0·05) daily gain and feed intake, decreased (P<0·05) post-weaning scour scores, increased (P<0·05) jejunal villus height and the ratio of villus height and crypt depth, decreased (P<0·05) jejunal paracellular permeability of fluorescein isothiocyanate dextran 4 kDa and up-regulated (P<0·05) tight junction protein expression of occludin, claudin-1 and zonula occludens-1 in jejunal mucosa. The mRNA levels of TNF-α, IL-6 and interferon-γ (IFN-γ) on day 7 post-weaning were also decreased (P<0·05). The piglets fed DS-ZnO at 500 mg Zn/kg did not differ in the above parameters from those fed ZnO at 2250 mg Zn/kg, while they had better performance than those fed the mixture of DS and ZnO. Supplementation with DS-ZnO at 500 mg Zn/kg was effective in alleviating diarrhoea, barrier dysfunction and inflammatory cytokine expression and up-regulating tight junction protein expression. PMID:23308387

  11. 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.

  12. ANKS1B Interacts with the Cerebral Cavernous Malformation Protein-1 and Controls Endothelial Permeability but Not Sprouting Angiogenesis

    PubMed Central

    Moll, Iris; Yang, Wan-Jen; Wüstehube-Lausch, Joycelyn; Fischer, Andreas

    2015-01-01

    Cerebral cavernous malformations are fragile blood vessel conglomerates in the central nervous system that are caused by mutations in the CCM1/KRIT1, CCM2 or CCM3 genes. The gene products form a protein complex at adherens junctions and loss of either CCM protein disrupts endothelial cell quiescence leading to increased permeability and excessive angiogenesis. We performed a yeast 2-hybrid screen to identify novel proteins directly interacting with KRIT1. The ankyrin repeat and sterile alpha motif domain-containing protein 1B (ANKS1B) was identified as a novel binding partner of KRIT1. Silencing of ANKS1B or the related gene ANKS1A in primary human endothelial cells had no significant effects on cellular proliferation, migration and sprouting angiogenesis. However, silencing of ANKS1B expression disturbed endothelial cell barrier functions leading to increased permeability. Forced ANKS1B expression reduced permeability. This was independent of Rho kinase activity and the presence of KRIT1. Taken together, ANKS1B was identified as a novel KRIT1-interacting protein that selectively controls endothelial permeability but not angiogenesis. PMID:26698571

  13. Identification and characterization of espin, an actin-binding protein localized to the F-actin-rich junctional plaques of Sertoli cell ectoplasmic specializations.

    PubMed

    Bartles, J R; Wierda, A; Zheng, L

    1996-06-01

    Ectoplasmic specializations are membrane-cytoskeletal assemblages found in Sertoli cells at sites of attachment to elongate spermatids or neighboring Sertoli cells. They are characterized in part by the presence of a unique junctional plaque which contains a narrow layer of parallel actin bundles sandwiched between the Sertoli cell plasma membrane and an affiliated cistern of endoplasmic reticulum. Using a monoclonal antibody, we have identified 'espin,' a novel actin-binding protein localized to ectoplasmic specializations. By immunogold electron microscopy, espin was localized to the parallel actin bundles of ectoplasmic specializations at sites where Sertoli cells contacted the heads of elongate spermatids. The protein was also detected at the sites of ectoplasmic specializations between neighboring Sertoli cells. Espin exhibits an apparent molecular mass of approximately 110 kDa in SDS gels. It is encoded by an approximately 2.9 kb mRNA, which was found to be specific to testis among the 11 rat organs and tissues examined. On the basis of cDNA sequence, espin is predicted to be an 836 amino acid protein which contains 8 ankyrin-like repeats in its N-terminal third, a potential P-loop, two proline-rich peptides and two peptides which contain clusters of multiple glutamates bracketed by arginines, lysines and glutamines in a pattern reminiscent of the repetitive motif found in the protein trichohyalin. The ankyrin-like repeats and a 66 amino acid peptide in the C terminus show significant sequence similarity to proteins encoded by the forked gene of Drosophila. A fusion protein containing the C-terminal 378 amino acids of espin was found to bind with high affinity (Kd = approximately 10 nM) to F-actin in vitro with a stoichiometry of approximately 1 espin per 6 actin monomers. When expressed by transfected NRK fibroblasts, the same C-terminal fragment of espin was observed to decorate actin fibers or cables. On the basis of its structure, localization and

  14. Hailey-Hailey disease and tight junctions: Claudins 1 and 4 are regulated by ATP2C1 gene encoding Ca(2+) /Mn(2+) ATPase SPCA1 in cultured keratinocytes.

    PubMed

    Raiko, Laura; Siljamäki, Elina; Mahoney, Mỹ G; Putaala, Heli; Suominen, Erkki; Peltonen, Juha; Peltonen, Sirkku

    2012-08-01

    Mutations in the ATP2C1 gene encoding Ca(2+) /Mn(2+) ATPase SPCA1 cause Hailey-Hailey disease (HHD, OMIM 16960). HHD is characterized by epidermal acantholysis. We attempted to model HHD using normal keratinocytes, in which the SPCA1 mRNA was down-regulated with the small inhibitory RNA (siRNA) method. SiRNA inhibition significantly down-regulated the SPCA1 mRNA, as demonstrated by qPCR, and decreased the SPCA1 protein beyond detectable level, as shown by Western analysis. The expression of selected desmosomal, adherens and tight junction (TJ) proteins was then studied in the SPCA1-deficient and control keratinocytes cultured in low (0.06 mm) or high (1.2 mm) calcium concentration. The mRNA and protein levels of most TJ components were up-regulated in non-treated control keratinocyte cultures upon switch from low to high calcium concentration. In contrast, SPCA1-deficient keratinocytes displayed high levels of TJ proteins claudins 1 and 4 even in low calcium. ZO-1 did not, however, follow similar expression patterns. Protein levels of occludin, beta-catenin, E-cadherin, desmoplakin, desmogleins 1-3, desmocollin 2/desmocollin 3 and plakoglobin did not show marked changes in SPCA1-deficient keratinocytes. Indirect immunofluorescence labelling revealed delayed translocation of desmoplakin and desmoglein 3 in desmosomes and increased intracellular pools of TJ and desmosomal components in SPCA1-inhibited keratinocytes. The results show that SPCA1 regulates the levels of claudins 1 and 4, but does not affect desmosomal protein levels, indicating that TJ proteins are differently regulated. The results also suggest a potential role for claudins in HHD. PMID:22639968

  15. Hailey-Hailey disease and tight junctions: Claudins 1 and 4 are regulated by ATP2C1 gene encoding Ca2+/Mn2+ ATPase SPCA1 in cultured keratinocytes

    PubMed Central

    Raiko, Laura; Siljamäki, Elina; Mahoney, Mỹ G.; Putaala, Heli; Suominen, Erkki; Peltonen, Juha; Peltonen, Sirkku

    2013-01-01

    Mutations in the ATP2C1 gene encoding Ca2+/Mn2+ ATPase SPCA1 cause Hailey-Hailey disease (HHD, OMIM 16960). HHD is characterized by epidermal acantholysis. We attempted to model HHD using normal keratinocytes in which the SPCA1 mRNA was down-regulated with the small inhibitory RNA (siRNA) method. SiRNA inhibition significantly down-regulated the SPCA1 mRNA, as demonstrated by qPCR, and decreased the SPCA1 protein beyond detectable level, as shown by western analysis. The expression of selected desmosomal, adherens and tight junction (TJ) proteins was then studied in the SPCA1-deficient and control keratinocytes cultured in low (0.06 mM) or high (1.2 mM) calcium concentration. The mRNA and protein levels of most TJ components were up-regulated in non-treated control keratinocyte cultures upon switch from low to high calcium concentration. In contrast, SPCA1-deficient keratinocytes displayed high levels of TJ proteins claudin 1 and 4 even in low calcium. ZO-1 did not however follow similar expression patterns. Protein levels of occludin, betacatenin, E-cadherin, desmoplakin, desmogleins 1-3, desmocollin 2/3 and plakoglobin did not show marked changes in SPCA1 deficient keratinocytes. Indirect immunofluorescence labeling revealed delayed translocation of desmoplakin and desmoglein 3 in desmosomes, and increased intracellular pools of TJ and desmosomal components in SPCA1 inhibited keratinocytes. The results show that SPCA1 regulates the levels of claudins 1 and 4, but does not affect desmosomal protein levels, indicating that TJ proteins are differently regulated. The results also suggest a potential role for claudins in HHD. PMID:22639968

  16. 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. PMID:24557840

  17. Expression of tight junction proteins and transporters for xenobiotic metabolism at the blood-CSF barrier during development in the nonhuman primate (P. hamadryas).

    PubMed

    Ek, C Joakim; D'Angelo, Barbara; Lehner, Christine; Nathanielsz, Peter; Li, Cun; Mallard, Carina

    2015-08-15

    The choroid plexus (CP) is rich in barrier mechanisms including transporters and enzymes which can influence drug disposition between blood and brain. We have limited knowledge of their state in fetus. We have studied barrier mechanisms along with metabolism and transporters influencing xenobiotics, using RNAseq and protein analysis, in the CP during the second-half of gestation in a nonhuman primate (Papio hamadryas). There were no differences in the expression of the tight-junctions at the CP suggesting a well-formed fetal blood-CSF barrier during this period of gestation. Further, the fetal CP express many enzymes for phase I-III metabolisms as well as transporters suggesting that it can greatly influence drug disposition and has a significant machinery to deactivate reactive molecules with only minor gestational changes. In summary, the study suggests that from, at least, midgestation, the CP in the nonhuman primate is restrictive and express most known genes associated with barrier function and transport. PMID:26092209

  18. 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.

  19. 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

  20. MicroRNA-19b Downregulates Gap Junction Protein Alpha1 and Synergizes with MicroRNA-1 in Viral Myocarditis.

    PubMed

    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

  1. 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

  2. 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. PMID:24431214

  3. Segmental Differences in Radiation-Induced Alterations of Tight Junction-Related Proteins in Non-Human Primate Jejunum, Ileum and Colon.

    PubMed

    Garg, Sarita; Zheng, Junying; Wang, Junru; Authier, Simon; Pouliot, Mylene; Hauer-Jensen, Martin

    2016-01-01

    Dysfunction of the intestinal epithelial barrier and leakage of luminal antigens and bacteria across the barrier have been linked to various human diseases. Intestinal permeability is regulated by intercellular structures, termed "tight junction" (Tj), which are disrupted after total-body irradiation (TBI). In this study, we investigated radiation-induced alterations in Tj-related proteins in the jejunum, ileum and colon of a non-human primate (NHP) model. NHPs were total-body irradiated with 6.7 and 7.4 Gy and intestines were procured at day 4, 7 and 12. Radiation exposure was found to induce significant increases in claudin-10 mRNA early (day 4) in all three gut segments and claudin-4 mRNA levels were repressed through day 12. TNF-alpha was highly induced in the jejunum and colon at early time points, but little induction was found in the ileum. Claudin-1 was induced only in the colon on day 4 postirradiation. Unlike the colon and jejunum, the ileum levels of claudin-7 were significantly downregulated through day 12 postirradiation. Western blot analysis revealed increased levels of claudin-2 on day 4 and of JAM-1 on day 7 postirradiation in all three gut segments. E-cadherin was downregulated on day 4 postirradiation in all segments, but remained reduced in the jejunum only until day 12. Taken together, these data suggest that exposure to radiation causes segment-specific alterations in the expression of Tj-related proteins. Interruption of Tjs may be a key factor contributing to injury to the intestinal mucosal barrier and increased intestinal permeability. PMID:26720804

  4. Moderate Hypoxia Followed by Reoxygenation Results in Blood-Brain Barrier Breakdown via Oxidative Stress-Dependent Tight-Junction Protein Disruption

    PubMed Central

    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

  5. 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

  6. The inhibitory effects of boldine, glaucine, and probucol on TPA-induced down regulation of gap junction function. Relationships to intracellular peroxides, protein kinase C translocation, and connexin 43 phosphorylation.

    PubMed

    Hu, J; Speisky, H; Cotgreave, I A

    1995-11-01

    The naturally occurring antioxidant boldine and its di-methoxy analogue glucine, as well as the drug antioxidant probucol, all inhibit TPA-induced downregulation of gap junctional intercellular communication in WB-F344 rat liver epithelial cells in dose-dependent manners. The compounds were essentially 100% inhibitory to the effect of TPA (10 nM) at 50 microM each. Analysis of the mechanism of the antitumor promotive action of these agents in vitro revealed that boldine and probucol (both at 10 microM) totally inhibited the TPA-induced accumulation of intracellular oxidants. Additionally, boldine, glaucine, and probucol, each at 50 microM, inhibited TPA-induced translocation of protein kinase C (PKC) to the particulate fraction of the cells, with concomitant inhibition of TPA-induced hyperphosphorylation of gap junctional connexin 43 (cx43) and TPA-induced internalisation of cx43 protein from the plasma membrane of the cells. None of the compounds inhibited the binding of (3H)-PDBu to TPA-specific binding sites in the cells. The results indicate that antioxidant molecules, irrespective of structure, possess common antitumor promotive potential in this model of gap junctional intercellular communication. The data also indicate that the compounds may interfere with the promotive function of TPA, at least in part, by the destruction of oxidants within the cells. Xanthine oxidase was excluded as a major source of such intracellular oxidants because allopurinol (50 microM) did not significantly affect either the accumulation of oxidants in the cells or the downregulation of gap junctional communication in response to TPA. Taken together, these data also suggest that TPA-induced oxidants play a role in the translocation of PKC to cellular membranes and it is at this level where the antioxidants may interfere in TPA-induced downregulation of gap junctional function. PMID:7503766

  7. G alpha12 interaction with alphaSNAP induces VE-cadherin localization at endothelial junctions and regulates barrier function.

    PubMed

    Andreeva, Alexandra V; Kutuzov, Mikhail A; Vaiskunaite, Rita; Profirovic, Jasmina; Meigs, Thomas E; Predescu, Sanda; Malik, Asrar B; Voyno-Yasenetskaya, Tatyana

    2005-08-26

    The involvement of heterotrimeric G proteins in the regulation of adherens junction function is unclear. We identified alphaSNAP as an interactive partner of G alpha12 using yeast two-hybrid screening. Glutathione S-transferase pull-down assays showed the selective interaction of alphaSNAP with G alpha12 in COS-7 as well as in human umbilical vein endothelial cells. Using domain swapping experiments, we demonstrated that the N-terminal region of G alpha12 (1-37 amino acids) was necessary and sufficient for its interaction with alphaSNAP. G alpha13 with its N-terminal extension replaced by that of G alpha12 acquired the ability to bind to alphaSNAP, whereas G alpha12 with its N terminus replaced by that of G alpha13 lost this ability. Using four point mutants of alphaSNAP, which alter its ability to bind to the SNARE complex, we determined that the convex rather than the concave surface of alphaSNAP was involved in its interaction with G alpha12. Co-transfection of human umbilical vein endothelial cells with G alpha12 and alphaSNAP stabilized VE-cadherin at the plasma membrane, whereas down-regulation of alphaSNAP with siRNA resulted in the loss of VE-cadherin from the cell surface and, when used in conjunction with G alpha12 overexpression, decreased endothelial barrier function. Our results demonstrate a direct link between the alpha subunit of G12 and alphaSNAP, an essential component of the membrane fusion machinery, and implicate a role for this interaction in regulating the membrane localization of VE-cadherin and endothelial barrier function. PMID:15980433

  8. Tight junction protein claudin-6 inhibits growth and induces the apoptosis of cervical carcinoma cells in vitro and in vivo.

    PubMed

    Zhang, Xiaowei; Ruan, Yang; Li, Yanru; Lin, Dongjing; Quan, Chengshi

    2015-05-01

    Claudin-6, a member of claudin family integral membrane proteins, has recently been reported to be a tumor suppressor for breast cancer. However, whether it plays a role in other types of cancer remains unclear. In the present study, we showed that the expression of claudin-6 is down-regulated in cervical carcinoma tissues as revealed by immunohistochemistry. Through over-expressing claudin-6 in HeLa and C33A cervical carcinoma cells, we found that claudin-6 is localized at plasma membrane and it increases transepithelial electrical resistance of the cells. Gain of claudin-6 expression suppresses cell proliferation, colony formation in vitro, and tumor growth in vivo. The effects are accompanied and potentially caused by promotion of tumor cell apoptosis. Taken together, these results suggest that claudin-6 may function as a tumor suppressor and loss of claudin-6 contributes to enhanced tumorigenic properties of cervical carcinoma cells. PMID:25822939

  9. 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

  10. Low Level Pro-inflammatory Cytokines Decrease Connexin36 Gap Junction Coupling in Mouse and Human Islets through Nitric Oxide-mediated Protein Kinase Cδ.

    PubMed

    Farnsworth, Nikki L; Walter, Rachelle L; Hemmati, Alireza; Westacott, Matthew J; Benninger, Richard K P

    2016-02-12

    Pro-inflammatory cytokines contribute to the decline in islet function during the development of diabetes. Cytokines can disrupt insulin secretion and calcium dynamics; however, the mechanisms underlying this are poorly understood. Connexin36 gap junctions coordinate glucose-induced calcium oscillations and pulsatile insulin secretion across the islet. Loss of gap junction coupling disrupts these dynamics, similar to that observed during the development of diabetes. This study investigates the mechanisms by which pro-inflammatory cytokines mediate gap junction coupling. Specifically, as cytokine-induced NO can activate PKCδ, we aimed to understand the role of PKCδ in modulating cytokine-induced changes in gap junction coupling. Isolated mouse and human islets were treated with varying levels of a cytokine mixture containing TNF-α, IL-1β, and IFN-γ. Islet dysfunction was measured by insulin secretion, calcium dynamics, and gap junction coupling. Modulators of PKCδ and NO were applied to determine their respective roles in modulating gap junction coupling. High levels of cytokines caused cell death and decreased insulin secretion. Low levels of cytokine treatment disrupted calcium dynamics and decreased gap junction coupling, in the absence of disruptions to insulin secretion. Decreases in gap junction coupling were dependent on NO-regulated PKCδ, and altered membrane organization of connexin36. This study defines several mechanisms underlying the disruption to gap junction coupling under conditions associated with the development of diabetes. These mechanisms will allow for greater understanding of islet dysfunction and suggest ways to ameliorate this dysfunction during the development of diabetes. PMID:26668311

  11. Neuromuscular junction disorders.

    PubMed

    Verschuuren, Jan; Strijbos, Ellen; Vincent, Angela

    2016-01-01

    Diseases of the neuromuscular junction comprise a wide range of disorders. Antibodies, genetic mutations, specific drugs or toxins interfere with the number or function of one of the essential proteins that control signaling between the presynaptic nerve ending and the postsynaptic muscle membrane. Acquired autoimmune disorders of the neuromuscular junction are the most common and are described here. In myasthenia gravis, antibodies to acetylcholine receptors or to proteins involved in receptor clustering, particularly muscle-specific kinase, cause direct loss of acetylcholine receptors or interfere with the agrin-induced acetylcholine receptor clustering necessary for efficient neurotransmission. In the Lambert-Eaton myasthenic syndrome (LEMS), loss of the presynaptic voltage-gated calcium channels results in reduced release of the acetylcholine transmitter. The conditions are generally recognizable clinically and the diagnosis confirmed by serologic testing and electromyography. Screening for thymomas in myasthenia or small cell cancer in LEMS is important. Fortunately, a wide range of symptomatic treatments, immunosuppressive drugs, or other immunomodulating therapies is available. Future research is directed to understanding the pathogenesis, discovering new antigens, and trying to develop disease-specific treatments. PMID:27112691

  12. The Counteradhesive Proteins, Thrombospondin 1 and SPARC/Osteonectin, Open the Tyrosine Phosphorylation-Responsive Paracellular Pathway in Pulmonary Vascular Endothelia

    PubMed Central

    Liu, Anguo; Mosher, Deane F.; Murphy-Ullrich, Joanne E.

    2009-01-01

    The counteradhesive proteins are a group of genetically and structurally distinct multidomain proteins that have been grouped together for their ability to inhibit cell-substrate interactions. Three counteradhesive proteins that influence endothelial cell behavior include thrombospondin (TSP)1, SPARC (Secreted Protein Acidic and Rich in Cysteine), also known as osteonectin, and tenascin. More recently, these proteins have been shown to not only regulate cell-matrix interactions but cell-cell interactions as well. TSP1 increases tyrosine phosphorylation of components of the cell-cell adherens junctions or zonula adherens (ZA) and opens the paracellular pathway in human lung microvascular endothelia. The EGF-like repeats of TSP1 activate the epidermal growth factor receptor (EGFR) and ErbB2 and these two receptor protein tyrosine kinase (PTK)s participate in ZA protein tyrosine phosphorylation and barrier disruption in response to the TSP1 stimulus. For the barrier response to TSP1, EGFR/ErbB2 activation is necessary but insufficient. Protein tyrosine phosphatase (PTP)µ counter-regulates phosphorylation of selected tyrosine residues within the cytoplasmic domain of EGFR. Although tenascin, like TSP1, also contains EGF-like repeats and is known to activate EGFR, whether it too opens the paracellular pathway is unknown. In addition to TSP1, tenascin, and the other TSP family members, there are numerous other proteins that also contain EGF-like repeats and participate in hemostasis, wound healing, and tissue remodeling. EGFR not only responds to direct binding of EGF motif-containing ligands but can be transactivated by a wide range of diverse stimuli. In fact, several established mediators of increased vascular permeability and/or lung injury, including thrombin, tumor necrosis factor-α, platelet-activating factor, bradykinin, angiopoietin, and H2O2, each transactivate EGFR. It is conceivable that EGFR serves a pivotal signaling role in a final common pathway for the

  13. The protein 4.1, ezrin, radixin, moesin (FERM) domain of Drosophila Coracle, a cytoplasmic component of the septate junction, provides functions essential for embryonic development and imaginal cell proliferation.

    PubMed Central

    Ward, R E; Schweizer, L; Lamb, R S; Fehon, R G

    2001-01-01

    Coracle is a member of the Protein 4.1 superfamily of proteins, whose members include Protein 4.1, the Neurofibromatosis 2 tumor suppressor Merlin, Expanded, the ERM proteins, protein tyrosine phosphatases, and unconventional myosins. Recent evidence suggests that members of this family participate in cell signaling events, including those that regulate cell proliferation and the cytoskeleton. Previously, we demonstrated that Coracle protein is localized to the septate junction in epithelial cells and is required for septate junction integrity. Loss of coracle function leads to defects in embryonic development, including failure in dorsal closure, and to proliferation defects. In addition, we determined that the N-terminal 383 amino acids define an essential functional domain possessing membrane-organizing properties. Here we investigate the full range of functions provided by this highly conserved domain and find that it is sufficient to rescue all embryonic defects associated with loss of coracle function. In addition, this domain is sufficient to rescue the reduced cell proliferation defect in imaginal discs, although it is incapable of rescuing null mutants to the adult stage. This result suggests the presence of a second functional domain within Coracle, a notion supported by molecular characterization of a series of coracle alleles. PMID:11560899

  14. The protein 4.1, ezrin, radixin, moesin (FERM) domain of Drosophila Coracle, a cytoplasmic component of the septate junction, provides functions essential for embryonic development and imaginal cell proliferation.

    PubMed

    Ward, R E; Schweizer, L; Lamb, R S; Fehon, R G

    2001-09-01

    Coracle is a member of the Protein 4.1 superfamily of proteins, whose members include Protein 4.1, the Neurofibromatosis 2 tumor suppressor Merlin, Expanded, the ERM proteins, protein tyrosine phosphatases, and unconventional myosins. Recent evidence suggests that members of this family participate in cell signaling events, including those that regulate cell proliferation and the cytoskeleton. Previously, we demonstrated that Coracle protein is localized to the septate junction in epithelial cells and is required for septate junction integrity. Loss of coracle function leads to defects in embryonic development, including failure in dorsal closure, and to proliferation defects. In addition, we determined that the N-terminal 383 amino acids define an essential functional domain possessing membrane-organizing properties. Here we investigate the full range of functions provided by this highly conserved domain and find that it is sufficient to rescue all embryonic defects associated with loss of coracle function. In addition, this domain is sufficient to rescue the reduced cell proliferation defect in imaginal discs, although it is incapable of rescuing null mutants to the adult stage. This result suggests the presence of a second functional domain within Coracle, a notion supported by molecular characterization of a series of coracle alleles. PMID:11560899

  15. 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

  16. 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

  17. MicroRNA-30a increases tight junction protein expression to suppress the epithelial-mesenchymal transition and metastasis by targeting Slug in breast cancer.

    PubMed

    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

    2016-03-29

    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

  18. 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

  19. 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

  20. 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. PMID:25911094

  1. Odontogenic Ameloblast-associated Protein (ODAM) Mediates Junctional Epithelium Attachment to Teeth via Integrin-ODAM-Rho Guanine Nucleotide Exchange Factor 5 (ARHGEF5)-RhoA Signaling*

    PubMed Central

    Lee, Hye-Kyung; Ji, Suk; Park, Su-Jin; Choung, Han-Wool; Choi, Youngnim; Lee, Hyo-Jung; Park, Shin-Young; Park, Joo-Cheol

    2015-01-01

    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. PMID:25911094

  2. Hyperbaric oxygen preconditioning ameliorates blood-brain barrier damage induced by hypoxia through modulation of tight junction proteins in an in vitro model

    PubMed Central

    Hao, Lei; Guo, Xiuming; Zou, Can; Zhou, Huchuan; Tian, Hong; Zhang, Yubo; Song, Chuan; Liu, Lei

    2016-01-01

    Aim To explore the effects of hyperbaric oxygen preconditioning (HBOP) on the permeability of blood-brain barrier (BBB) and expression of tight junction proteins under hypoxic conditions in vitro. Methods A BBB in vitro model was constructed using the hCMEC/D3 cell line and used when its trans-endothelial electrical resistance (TEER) reached 80-120 Ω · cm2 (tested by Millicell-Electrical Resistance System). The cells were randomly divided into the control group cultured under normal conditions, the group cultured under hypoxic conditions (2%O2) for 24 h (hypoxia group), and the group first subjected to HBOP for 2 h and then to hypoxia (HBOP group). Occludin and ZO-1 expression were analyzed by immunofluorescence assay. Results Normal hCMEC/D3 was spindle-shaped and tightly integrated. TEER was significantly reduced in the hypoxia (P = 0.001) and HBOP group (P = 0.014) compared to control group, with a greater decrease in the hypoxia group. Occludin membranous expression was significantly decreased in the hypoxia group (P = 0.001) compared to the control group, but there was no change in the HBOP group. ZO-1 membranous expression was significantly decreased (P = 0.002) and cytoplasmic expression was significantly increased (P = 0.001) in the hypoxia group compared to the control group, although overall expression levels did not change. In the HBOP group, there was no significant change in ZO-1 expression compared to the control group. Conclusion Hyperbaric oxygen preconditioning protected the integrity of BBB in an in vitro model through modulation of occludin and ZO-1 expression under hypoxic conditions. PMID:26935614

  3. Cardiotonic steroid ouabain stimulates expression of blood-testis barrier proteins claudin-1 and -11 and formation of tight junctions in Sertoli cells.

    PubMed

    Dietze, Raimund; Shihan, Mazen; Stammler, Angelika; Konrad, Lutz; Scheiner-Bobis, Georgios

    2015-04-15

    The interaction of ouabain with the sodium pump induces signalling cascades resembling those triggered by hormone/receptor interactions. In the rat Sertoli cell line 93RS2, ouabain at low concentrations stimulates the c-Src/c-Raf/Erk1/2 signalling cascade via its interaction with the α4 isoform of the sodium pump expressed in these cells, leading to the activation of the transcription factor CREB. As a result of this signalling sequence, ouabain stimulates expression of claudin-1 and claudin-11, which are also controlled by a CRE promoter. Both of these proteins are known to be essential constituents of tight junctions (TJ) between Sertoli cells, and as a result of the ouabain-induced signalling TJ formation between neighbouring Sertoli cells is significantly enhanced by the steroid. Thus, ouabain-treated cell monolayers display higher transepithelial resistance and reduced free diffusion of FITC-coupled dextran in tracer diffusion assays. Taking into consideration that the formation of TJ is indispensable for the maintenance of the blood-testis barrier (BTB) and therefore for male fertility, the actions of ouabain described here and the fact that this and other related cardiotonic steroids (CTS) are produced endogenously suggest a direct influence of ouabain/sodium pump interactions on the maintenance of the BTB and thereby an effect on male fertility. Since claudin-1 and claudin-11 are also present in other blood-tissue barriers, one can speculate that ouabain and perhaps other CTS influence the dynamics of these barriers as well. PMID:25666991

  4. 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

  5. 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

  6. The Escherichia coli O157:H7 cattle immunoproteome includes outer membrane protein A (OmpA), a modulator of adherence to bovine rectoanal junction squamous epithelial (RSE) cells.

    PubMed

    Kudva, Indira T; Krastins, Bryan; Torres, Alfredo G; Griffin, Robert W; Sheng, Haiqing; Sarracino, David A; Hovde, Carolyn J; Calderwood, Stephen B; John, Manohar

    2015-06-01

    Building on previous studies, we defined the repertoire of proteins comprising the immunoproteome (IP) of Escherichia coli O157:H7 (O157) cultured in DMEM supplemented with norepinephrine (O157 IP), 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 IP (O157-IP) comprised 91 proteins, and included those identified previously using proteomics-based expression library screening, 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 rectoanal junction squamous epithelial cells. Our results point to a role for yet to be identified members of the O157-IP in E. coli O157 adherence to rectoanal junction squamous epithelial cells, and additionally implicate a possible role for the outer membrane protein A regulator, TdcA, in the expression of such adhesins. Our observations have implications for the development of efficacious vaccines for preventing E. coli O157 colonization of the bovine gastrointestinal tract. PMID:25643951

  7. Interaction of fibrinogen and muramidase-released protein promotes the development of Streptococcus suis meningitis.

    PubMed

    Wang, Junping; Kong, Decong; Zhang, Shengwei; Jiang, Hua; Zheng, Yuling; Zang, Yating; Hao, Huaijie; Jiang, Yongqiang

    2015-01-01

    Muramidase-released protein (MRP) is as an important virulence marker of Streptococcus suis (S. suis) serotype 2. Our previous works have shown that MRP can bind human fibrinogen (hFg); however, the function of this interaction in S. suis meningitis is not known. In this study, we found that the deletion of mrp significantly impairs the hFg-mediated adherence and traversal ability of S. suis across human cerebral microvascular endothelial cells (hCMEC/D3). Measurement of the permeability to Lucifer yellow in vitro and Evans blue extravasation in vivo show that the MRP-hFg interaction significantly increases the permeability of the blood-brain barrier (BBB). In the mouse meningitis model, wild type S. suis caused higher bacterial loads in the brain and more severe histopathological signs of meningitis than the mrp mutant at day 3 post-infection. Western blot analysis and immunofluorescence observations reveal that the MRP-hFg interaction can destroy the cell adherens junction protein p120-catenin of hCMEC/D3. These results indicate that the MRP-hFg interaction is important in the development of S. suis meningitis. PMID:26441928

  8. Dietary fat and bile juice, but not obesity, are responsible for the increase in small intestinal permeability induced through the suppression of tight junction protein expression in LETO and OLETF rats

    PubMed Central

    2010-01-01

    Background An increase in the intestinal permeability is considered to be associated with the inflammatory tone and development in the obesity and diabetes, however, the pathogenesis of the increase in the intestinal permeability is poorly understood. The present study was performed to determine the influence of obesity itself as well as dietary fat on the increase in intestinal permeability. Methods An obese rat strain, Otsuka Long Evans Tokushima Fatty (OLETF), and the lean counter strain, Long Evans Tokushima Otsuka (LETO), were fed standard or high fat diets for 16 weeks. Glucose tolerance, intestinal permeability, intestinal tight junction (TJ) proteins expression, plasma bile acids concentration were evaluated. In addition, the effects of rat bile juice and dietary fat, possible mediators of the increase in the intestinal permeability in the obesity, on TJ permeability were explored in human intestinal Caco-2 cells. Results The OLETF rats showed higher glucose intolerance than did the LETO rats, which became more marked with the prolonged feeding of the high fat diet. Intestinal permeability in the OLETF rats evaluated by the urinary excretion of intestinal permeability markers (Cr-EDTA and phenolsulfonphthalein) was comparable to that in the LETO rats. Feeding the high fat diet increased intestinal permeability in both the OLETF and LETO rats, and the increases correlated with decreases in TJ proteins (claudin-1, claudin-3, occludin and junctional adhesion molecule-1) expression in the small, but not in the large intestine (cecum or colon). The plasma bile acids concentration was higher in rats fed the high fat diet. Exposure to bile juice and the fat emulsion increased TJ permeability with concomitant reductions in TJ protein expression (claudin-1, claudin-3, and junctional adhesion molecule-1) in the Caco-2 cell monolayers. Conclusion Excessive dietary fat and/or increased levels of luminal bile juice, but not genetic obesity, are responsible for the

  9. Jian carp (Cyprinus carpio var. Jian) intestinal immune responses, antioxidant status and tight junction protein mRNA expression are modulated via Nrf2 and PKC in response to dietary arginine deficiency.

    PubMed

    Wang, Biao; Feng, Lin; Chen, Gang-Fu; Jiang, Wei-Dan; Liu, Yang; Kuang, Sheng-Yao; Jiang, Jun; Tang, Ling; Wu, Pei; Tang, Wu-Neng; Zhang, Yong-An; Zhao, Juan; Zhou, Xiao-Qiu

    2016-04-01

    This study investigated the effect of dietary arginine on the immune response, antioxidant status and tight junction mRNA expression in the intestine of juvenile Jian carp (Cyprinus carpio var. Jian). A total of 1200 juvenile Jian carp with an average initial weight of 6.33 ± 0.03 g were fed graded levels of arginine (9.8-24.5 g kg(-1) diet) for nine weeks. The study showed that arginine deficiency up-regulated interleukin 1, interleukin 8 and transforming growth factor-β and down-regulated tumour necrosis factor α gene expression (P < 0.05). Additionally, arginine deficiency increased malondialdehyde (MDA), protein carbonyl (PC) and glutathione contents and decreased the activities of copper/zinc superoxide dismutase (SOD1), glutathione peroxidase (GPx), catalase (CAT) and glutathione reductase (GR) and glutathione-S-transferase (GST) (P < 0.05). Meanwhile, arginine deficiency significantly increased claudin 7, occludin, protein kinase C, NF-E2-related factor 2 and Kelch-like-ECH- associated protein 1 mRNA expression and decreased SOD1, CAT and GR mRNA expression (P < 0.05). All of these results indicated that arginine deficiency impaired intestinal immune function via the regulation of mRNA expression of cytokines, tight junction proteins, antioxidant enzymes, Nrf2/Keap1 and PKC in fish intestine. PMID:26518504

  10. Solitons in Josephson junctions

    NASA Astrophysics Data System (ADS)

    Ustinov, A. V.

    1998-11-01

    Magnetic flux quanta in Josephson junctions, often called fluxons, in many cases behave as solitons. A review of recent experiments and modelling of fluxon dynamics in Josephson circuits is presented. Classic quasi-one-dimensional junctions, stacked junctions (Josephson superlattices), and discrete Josephson transmission lines (JTLs) are discussed. Applications of fluxon devices as high-frequency oscillators and digital circuits are also addressed.

  11. 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

  12. 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

  13. 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

  14. 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

  15. Nonstructural protein 3 of the hepatitis C virus encodes a serine-type proteinase required for cleavage at the NS3/4 and NS4/5 junctions.

    PubMed Central

    Bartenschlager, R; Ahlborn-Laake, L; Mous, J; Jacobsen, H

    1993-01-01

    We have studied processing of the nonstructural (NS) polyprotein of the hepatitis C virus. A series of cDNAs corresponding to predicted NS2/3/4 or NS3/4 regions were constructed, and processing of the polyproteins was studied in an in vitro transcription-translation system. We report that a catalytically active serine-type proteinase is encoded by the NS3 region. Substitution of the serine residue of the putative catalytic triad (H, D, and S) by alanine blocked cleavage at the NS3/4 junction, while processing between NS2 and NS3 was not affected. Thus, cleavage at the NS2/3 junction is mediated either by cellular enzymes or by an NS-2 inherent proteinase activity. Deletion analysis of an NS3/4 cDNA construct mapped the amino terminus of the enzymatically active proteinase between amino acids 1049 and 1065 of the polyprotein. As internal deletions of variable segments of the presumed helicase domain prevented processing at the NS314 junction, a continuous NS3 region appears to be required for processing at this site. To analyze hepatitis C virus polyprotein cleavage in vivo, recombinant vaccinia viruses expressing NS2/3/4 or NS3/4/5 proteins were generated. In agreement with the in vitro data, cleavage between NS2 and NS3 was independent of a catalytically active NS3 proteinase, whereas substitution of the active-site serine residue by the amino acid alanine completely blocked processing at the NS3/4 and NS4/5 junctions. These results demonstrate that NS3 encodes the viral proteinase essential for generating the amino termini of NS4 and NS5. Images PMID:8389908

  16. Lactational exposure of phthalate causes long-term disruption in testicular architecture by altering tight junctional and apoptotic protein expression in Sertoli cells of first filial generation pubertal Wistar rats.

    PubMed

    Sekaran, S; Balaganapathy, P; Parsanathan, R; Elangovan, S; Gunashekar, J; Bhat, F A; Jagadeesan, A

    2015-06-01

    Di(2-ethylhexyl) phthalate (DEHP) is a ubiquitous environmental contaminant and a well-known endocrine disruptor (ED) that interferes with the reproductive function in both humans and animals. This study aimed to find out the impact of lactational exposure of DEHP in testes of first filial generation (F1) progeny male rat postnatal day (PND)-60. Lactating dams were orally treated with DEHP (0, 1, 10 and 100 mg/kg body weight/day, respectively) from the PND-1 to PND-21. Rats were killed at PND 60. Testes were removed and used for histological analysis and for isolation of Sertoli cells (SCs). The histoarchitecture of DEHP-treated rats showed disturbed testicular structure. DEHP-treated rats also showed increased oxidative stress by decreasing antioxidant levels in the SCs; it disrupted SC tight junctional proteins occludin, claudin, junctional adhesion molecule, zona occludens protein-1 (ZO-1), zona occludens protein-2 (ZO-2), and afadin-6 (AF-6), increased apoptosis by altering the apoptotic genes Bax, cytochrome c, caspase-8, -9, -3 and antiapoptotic gene Bcl-2. It is concluded that early postnatal exposure to DEHP disturbs histoarchitecture of testis and SC function in pubertal Wistar rats. PMID:25352649

  17. 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.

  18. 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

  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. 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

  1. Chronic type 2 diabetes reduces the integrity of the blood-brain barrier by reducing tight junction proteins in the hippocampus.

    PubMed

    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

    2016-07-01

    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

  2. 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

  3. 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

  4. 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

  5. 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

  6. ROCK2 primes the endothelium for vascular hyperpermeability responses by raising baseline junctional tension.

    PubMed

    Beckers, Cora M L; Knezevic, Nebojsa; Valent, Erik T; Tauseef, Mohammad; Krishnan, Ramaswamy; Rajendran, Kavitha; Hardin, C Corey; Aman, Jurjan; van Bezu, Jan; Sweetnam, Paul; van Hinsbergh, Victor W M; Mehta, Dolly; van Nieuw Amerongen, Geerten P

    2015-07-01

    Rho kinase mediates the effects of inflammatory permeability factors by increasing actomyosin-generated traction forces on endothelial adherens junctions, resulting in disassembly of intercellular junctions and increased vascular leakage. In vitro, this is accompanied by the Rho kinase-driven formation of prominent radial F-actin fibers, but the in vivo relevance of those F-actin fibers has been debated, suggesting other Rho kinase-mediated events to occur in vascular leak. Here, we delineated the contributions of the highly homologous isoforms of Rho kinase (ROCK1 and ROCK2) to vascular hyperpermeability responses. We show that ROCK2, rather than ROCK1 is the critical Rho kinase for regulation of thrombin receptor-mediated vascular permeability. Novel traction force mapping in endothelial monolayers, however, shows that ROCK2 is not required for the thrombin-induced force enhancements. Rather, ROCK2 is pivotal to baseline junctional tension as a novel mechanism by which Rho kinase primes the endothelium for hyperpermeability responses, independent from subsequent ROCK1-mediated contractile stress-fiber formation during the late phase of the permeability response. PMID:25869521

  7. 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

  8. Tight junction, selective permeability, and related diseases.

    PubMed

    Krug, Susanne M; Schulzke, Jörg D; Fromm, Michael

    2014-12-01

    The tight junction forms a barrier against unlimited paracellular passage but some of the tight junction proteins just do the opposite, they form extracellular channels zigzagging between lateral membranes of neighboring cells. All of these channel-forming proteins and even some of the barrier formers exhibit selectivity, which means that they prefer certain substances over others. All channel formers exhibit at least one of the three types of selectivity: for cations (claudin-2, -10b, -15), for anions (claudin-10a, -17) or for water (claudin-2). Also some, but not all, barrier-forming claudins are charge-selective (claudin-4, -8, -14). Moreover, occludin and tricellulin turned out to be relevant for barrier formation against macromolecule passage. Tight junction proteins are dysregulated or can be genetically defective in numerous diseases, which may lead to three effects: (i) impaired paracellular transport e.g. causing magnesium loss in the kidney, (ii) increased paracellular transport of solutes and water e.g. causing leak-flux diarrhea in the intestine, and (iii) increased permeability to large molecules e.g. unwanted intestinal pathogen uptake fueling inflammatory processes. This review gives an overview on the properties of tight junction proteins featuring selective permeability, and in this context explains how these proteins induce or aggravate diseases. PMID:25220018

  9. 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.

  10. Molecular organization of tricellular tight junctions.

    PubMed

    Furuse, Mikio; Izumi, Yasushi; Oda, Yukako; Higashi, Tomohito; Iwamoto, Noriko

    2014-01-01

    When the apicolateral border of epithelial cells is compared with a polygon, its sides correspond to the apical junctional complex, where cell adhesion molecules assemble from the plasma membranes of two adjacent cells. On the other hand, its vertices correspond to tricellular contacts, where the corners of three cells meet. Vertebrate tricellular contacts have specialized structures of tight junctions, termed tricellular tight junctions (tTJs). tTJs were identified by electron microscopic observations more than 40 years ago, but have been largely forgotten in epithelial cell biology since then. The identification of tricellulin and angulin family proteins as tTJ-associated membrane proteins has enabled us to study tTJs in terms of not only the paracellular barrier function but also unknown characteristics of epithelial cell corners via molecular biological approaches. PMID:25097825

  11. 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

  12. 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

  13. 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. PMID:25014314

  14. 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

  15. Doped semiconductor nanocrystal junctions

    NASA Astrophysics Data System (ADS)

    Borowik, Ł.; Nguyen-Tran, T.; Roca i Cabarrocas, P.; Mélin, T.

    2013-11-01

    Semiconductor junctions are the basis of electronic and photovoltaic devices. Here, we investigate junctions formed from highly doped (ND≈1020-1021cm-3) silicon nanocrystals (NCs) in the 2-50 nm size range, using Kelvin probe force microscopy experiments with single charge sensitivity. We show that the charge transfer from doped NCs towards a two-dimensional layer experimentally follows a simple phenomenological law, corresponding to formation of an interface dipole linearly increasing with the NC diameter. This feature leads to analytically predictable junction properties down to quantum size regimes: NC depletion width independent of the NC size and varying as ND-1/3, and depleted charge linearly increasing with the NC diameter and varying as ND1/3. We thus establish a "nanocrystal counterpart" of conventional semiconductor planar junctions, here however valid in regimes of strong electrostatic and quantum confinements.

  16. 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

  17. Dynamic Behavior of Arabidopsis eIF4A-III, Putative Core Protein of Exon Junction Complex: Fast Relocation to Nucleolus and Splicing Speckles under Hypoxia[W

    PubMed Central

    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.

    2009-01-01

    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

  18. Quantum junction solar cells.

    PubMed

    Tang, Jiang; Liu, Huan; Zhitomirsky, David; Hoogland, Sjoerd; Wang, Xihua; Furukawa, Melissa; Levina, Larissa; Sargent, Edward H

    2012-09-12

    Colloidal quantum dot solids combine convenient solution-processing with quantum size effect tuning, offering avenues to high-efficiency multijunction cells based on a single materials synthesis and processing platform. The highest-performing colloidal quantum dot rectifying devices reported to date have relied on a junction between a quantum-tuned absorber and a bulk material (e.g., TiO(2)); however, quantum tuning of the absorber then requires complete redesign of the bulk acceptor, compromising the benefits of facile quantum tuning. Here we report rectifying junctions constructed entirely using inherently band-aligned quantum-tuned materials. Realizing these quantum junction diodes relied upon the creation of an n-type quantum dot solid having a clean bandgap. We combine stable, chemically compatible, high-performance n-type and p-type materials to create the first quantum junction solar cells. We present a family of photovoltaic devices having widely tuned bandgaps of 0.6-1.6 eV that excel where conventional quantum-to-bulk devices fail to perform. Devices having optimal single-junction bandgaps exhibit certified AM1.5 solar power conversion efficiencies of 5.4%. Control over doping in quantum solids, and the successful integration of these materials to form stable quantum junctions, offers a powerful new degree of freedom to colloidal quantum dot optoelectronics. PMID:22881834

  19. 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

  20. Oxidative Stress, Lens Gap Junctions, and Cataracts

    PubMed Central

    Beyer, Eric C.

    2009-01-01

    Abstract The eye lens is constantly subjected to oxidative stress from radiation and other sources. The lens has several mechanisms to protect its components from oxidative stress and to maintain its redox state, including enzymatic pathways and high concentrations of ascorbate and reduced glutathione. With aging, accumulation of oxidized lens components and decreased efficiency of repair mechanisms can contribute to the development of lens opacities or cataracts. Maintenance of transparency and homeostasis of the avascular lens depend on an extensive network of gap junctions. Communication through gap junction channels allows intercellular passage of molecules (up to 1 kDa) including antioxidants. Lens gap junctions and their constituent proteins, connexins (Cx43, Cx46, and Cx50), are also subject to the effects of oxidative stress. These observations suggest that oxidative stress-induced damage to connexins (and consequent altered intercellular communication) may contribute to cataract formation. Antioxid. Redox Signal. 11, 339–353. PMID:18831679

  1. IQ Domain GTPase-Activating Protein 1 is Involved in Shear Stress-Induced Progenitor-Derived Endothelial Cell Alignment

    PubMed Central

    Rami, Lila; Auguste, Patrick; Thebaud, Noélie B.; Bareille, Reine; Daculsi, Richard; Ripoche, Jean; Bordenave, Laurence

    2013-01-01

    Shear stress is one of mechanical constraints which are exerted by blood flow on endothelial cells (ECs). To adapt to shear stress, ECs align in the direction of flow through adherens junction (AJ) remodeling. However, mechanisms regulating ECs alignment under shear stress are poorly understood. The scaffold protein IQ domain GTPase activating protein 1 (IQGAP1) is a scaffold protein which couples cell signaling to the actin and microtubule cytoskeletons and is involved in cell migration and adhesion. IQGAP1 also plays a role in AJ organization in epithelial cells. In this study, we investigated the potential IQGAP1 involvement in the endothelial cells alignment under shear stress. Progenitor-derived endothelial cells (PDECs), transfected (or not) with IQGAP1 small interfering RNA, were exposed to a laminar shear stress (1.2 N/m2) and AJ proteins (VE-cadherin and β-catenin) and IQGAP1 were labeled by immunofluorescence. We show that IQGAP1 is essential for ECs alignment under shear stress. We studied the role of IQGAP1 in AJs remodeling of PDECs exposed to shear stress by studying cell localization and IQGAP1 interactions with VE-cadherin and β-catenin by immunofluorescence and Proximity Ligation Assays. In static conditions, IQGAP1 interacts with VE-cadherin but not with β-catenin at the cell membrane. Under shear stress, IQGAP1 lost its interaction from VE-cadherin to β-catenin. This “switch” was concomitant with the loss of β-catenin/VE-cadherin interaction at the cell membrane. This work shows that IQGAP1 is essential to ECs alignment under shear stress and that AJ remodeling represents one of the mechanisms involved. These results provide a new approach to understand ECs alignment under to shear stress. PMID:24278215

  2. β-asarone and levodopa co-administration increase striatal dopamine level in 6-hydroxydopamine induced rats by modulating P-glycoprotein and tight junction proteins at the blood-brain barrier and promoting levodopa into the brain.

    PubMed

    Huang, Liping; Deng, Minzhen; He, Yuping; Lu, Shiyao; Ma, Ruanxin; Fang, Yongqi

    2016-06-01

    Levodopa (L-dopa) is widely considered as one of the most effective drug constituents in the treatment of Parkinson's disease (PD), but the blood-brain barrier (BBB) permeability of L-dopa is <5%, which causes low efficacy. Neuroprotective effects of β-asarone on 6-hydroxydopamine (6-OHDA)-induced PD rats were demonstrated by our previous studies. Co-administration of β-asarone and L-dopa has not been explored until being investigated on PD rats in this study. PD rats were divided into four groups: untreated, L-dopa-treated, β-asarone-treated and co-administered-treated groups. All of the treatments were administered to the rats twice per day for 30 days. The L-dopa, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), S100β and neuron-specific enolase (NSE) levels were subsequently determined. The P-glycoprotein (P-gp), zonula occludens-1 (ZO-1), claudin-5, occludin and actin expression was also assessed in cortex. Changes in BBB ultrastructure were observed using transmission electron microscopy. Our results showed that the co-administered treatment increased levels of L-dopa, DA, DOPAC and HVA in striatum, and S100β in plasma, but down-regulated NSE, P-gp, ZO-1, occludin, actin and claudin-5 in cortex. Crevices were observed between capillary endothelial cells at intercellular tight junction of the striatum in co-administered-treated group, while the endothelial cells in untreated group were tightly jointing each other. In addition, the correlations of L-dopa or DA and P-gp or tight junction proteins respectively were significantly negative in co-administered- and β-asarone-treated groups. These findings suggest that co-administered treatment may enhance the L-dopa BBB permeability and attenuate brain injury, which may be beneficial to PD treatment. PMID:26991136

  3. 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

    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

  4. 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

  5. Carbon nanotube intramolecular junctions

    NASA Astrophysics Data System (ADS)

    Yao, Zhen; Postma, Henk W. Ch.; Balents, Leon; Dekker, Cees

    1999-11-01

    The ultimate device miniaturization would be to use individual molecules as functional devices. Single-wall carbon nanotubes (SWNTs) are promising candidates for achieving this: depending on their diameter and chirality, they are either one-dimensional metals or semiconductors. Single-electron transistors employing metallic nanotubes and field-effect transistors employing semiconducting nanotubes have been demonstrated. Intramolecular devices have also been proposed which should display a range of other device functions. For example, by introducing a pentagon and a heptagon into the hexagonal carbon lattice, two tube segments with different atomic and electronic structures can be seamlessly fused together to create intramolecular metal-metal, metal-semiconductor, or semiconductor-semiconductor junctions. Here we report electrical transport measurements on SWNTs with intramolecular junctions. We find that a metal-semiconductor junction behaves like a rectifying diode with nonlinear transport characteristics that are strongly asymmetric with respect to bias polarity. In the case of a metal-metal junction, the conductance appears to be strongly suppressed and it displays a power-law dependence on temperatures and applied voltage, consistent with tunnelling between the ends of two Luttinger liquids. Our results emphasize the need to consider screening and electron interactions when designing and modelling molecular devices. Realization of carbon-based molecular electronics will require future efforts in the controlled production of these intramolecular nanotube junctions.

  6. 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

  7. Four-junction superconducting circuit

    NASA Astrophysics Data System (ADS)

    Qiu, Yueyin; Xiong, Wei; He, Xiao-Ling; Li, Tie-Fu; You, J. Q.

    2016-06-01

    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.

  8. 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

  9. 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

  10. T-Junction Benchmark

    SciTech Connect

    2010-01-01

    Part 1: Two different volume renderings of fluid temperatures in a turbulent T-junction mixing problem at Reynolds number Re=40,000. Part 2: Volume rendering of fluid temperatures in a turbulent T-junction mixing problem at Reynolds number Re=40,000, simulated using Nek5000 at three different resolutions. Part 3: Temperature distribution for a turbulent T-junction mixing problem at Reynolds number Re=40,000, simulated using Nek5000 with 89056 spectral elements of order N=9 (65 million grid points). Credits: Science: Aleks Obabko and Paul Fisher, Argonne National Laboratory
 Visualization: Hank Childs, Lawrence Berkeley National Laboratory

 This research used resources of the Argonne Leadership Computing Facility at Argonne National Laboratory, which is supported by the Office of Science of the U.S. Department of Energy under contract DE-AC02-06CH11357

  11. 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

  12. Insights into the C-terminal Peptide Binding Specificity of the PDZ Domain of Neuronal Nitric-oxide Synthase: CHARACTERIZATION OF THE INTERACTION WITH THE TIGHT JUNCTION PROTEIN CLAUDIN-3.

    PubMed

    Merino-Gracia, Javier; Costas-Insua, Carlos; Canales, María Ángeles; Rodríguez-Crespo, Ignacio

    2016-05-27

    Neuronal nitric-oxide synthase, unlike its endothelial and inducible counterparts, displays a PDZ (PSD-95/Dlg/ZO-1) domain located at its N terminus involved in subcellular targeting. The C termini of various cellular proteins insert within the binding groove of this PDZ domain and determine the subcellular distribution of neuronal NOS (nNOS). The molecular mechanisms underlying these interactions are poorly understood because the PDZ domain of nNOS can apparently exhibit class I, class II, and class III binding specificity. In addition, it has been recently suggested that the PDZ domain of nNOS binds with very low affinity to the C termini of target proteins, and a necessary simultaneous lateral interaction must take place for binding to occur. We describe herein that the PDZ domain of nNOS can behave as a bona fide class III PDZ domain and bind to C-terminal sequences with acidic residues at the P-2 position with low micromolar binding constants. Binding to C-terminal sequences with a hydrophobic residue at the P-2 position plus an acidic residue at the P-3 position (class II) can also occur, although interactions involving residues extending up to the P-7 position mediate this type of binding. This promiscuous behavior also extends to its association to class I sequences, which must display a Glu residue at P-3 and a Thr residue at P-2 By means of site-directed mutagenesis and NMR spectroscopy, we have been able to identify the residues involved in each specific type of binding and rationalize the mechanisms used to recognize binding partners. Finally, we have analyzed the high affinity association of the PDZ domain of nNOS to claudin-3 and claudin-14, two tight junction tetraspan membrane proteins that are essential components of the paracellular barrier. PMID:27030110

  13. Aβ(1-42) oligomer-induced leakage in an in vitro blood-brain barrier model is associated with up-regulation of RAGE and metalloproteinases, and down-regulation of tight junction scaffold proteins.

    PubMed

    Wan, Wenbin; Cao, Lan; Liu, Lumei; Zhang, Chunyan; Kalionis, Bill; Tai, Xiantao; Li, Yaming; Xia, Shijin

    2015-07-01

    Accumulating evidence indicates that abnormal deposition of amyloid-β (Aβ) peptide in the brain is responsible for endothelial cell damage and consequently leads to blood-brain barrier (BBB) leakage. However, the mechanisms underlying BBB disruption are not well described. We employed an monolayer BBB model comprising bEnd.3 cell and found that BBB leakage was induced by treatment with Aβ(1-42), and the levels of tight junction (TJ) scaffold proteins (ZO-1, Claudin-5, and Occludin) were decreased. Through comparisons of the effects of the different components of Aβ(1-42), including monomer (Aβ(1-42)-Mono), oligomer (Aβ(1-42)-Oligo), and fibril (Aβ(1-42)-Fibril), our data confirmed that Aβ(1-42)-Oligo is likely to be the most important damage factor that results in TJ damage and BBB leakage in Alzheimer's disease. We found that the incubation of bEnd.3 cells with Aβ(1-42) significantly up-regulated the level of receptor for advanced glycation end-products (RAGE). Co-incubation of a polyclonal antibody to RAGE and Aβ(1-42)-Oligo in bEnd.3 cells blocked RAGE suppression of Aβ(1-42)-Oligo-induced alterations in TJ scaffold proteins and reversed Aβ(1-42)-Oligo-induced up-regulation of RAGE, matrix metalloproteinase (MMP)-2, and MMP-9. Furthermore, we found that these effects induced by Aβ(1-42)-Oligo treatment were effectively suppressed by knockdown of RAGE using small interfering RNA (siRNA) transfection. We also found that GM 6001, a broad-spectrum MMP inhibitor, partially reversed the Aβ(1-42)-Oligo-induced inhibitor effects in bEnd.3 cells. Thus, these results suggested that RAGE played an important role in Aβ-induced BBB leakage and alterations of TJ scaffold proteins, through a mechanism that involved up-regulation of MMP-2 and MMP-9. PMID:25866188

  14. 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…

  15. Josephson junction mixing.

    NASA Technical Reports Server (NTRS)

    Thompson, E. D.

    1973-01-01

    A theory is presented which, though too simple to explain quantitative details in the Josephson junction mixing response, is sufficient for explaining qualitatively the results observed. Crucial to the theory presented, and that which differentiates it from earlier ones, is the inclusion of harmonic voltages across the ideal Josephson element.

  16. Squeezable electron tunneling junctions

    NASA Astrophysics Data System (ADS)

    Moreland, J.; Alexander, S.; Cox, M.; Sonnenfeld, R.; Hansma, P. K.

    1983-09-01

    We report a versatile new technique for constructing electron tunneling junctions with mechanically-adjusted artificial barriers. I-V curves are presented for tunneling between Ag electrodes with vacuum, gas, liquid or solid in the barrier. An energy gap is apparent in the measured I-V curve when tunneling occurs between superconducting Pb electrodes.

  17. Doped semiconductor nanocrystal junctions

    SciTech Connect

    Borowik, Ł.; Mélin, T.; Nguyen-Tran, T.; Roca i Cabarrocas, P.

    2013-11-28

    Semiconductor junctions are the basis of electronic and photovoltaic devices. Here, we investigate junctions formed from highly doped (N{sub D}≈10{sup 20}−10{sup 21}cm{sup −3}) silicon nanocrystals (NCs) in the 2–50 nm size range, using Kelvin probe force microscopy experiments with single charge sensitivity. We show that the charge transfer from doped NCs towards a two-dimensional layer experimentally follows a simple phenomenological law, corresponding to formation of an interface dipole linearly increasing with the NC diameter. This feature leads to analytically predictable junction properties down to quantum size regimes: NC depletion width independent of the NC size and varying as N{sub D}{sup −1/3}, and depleted charge linearly increasing with the NC diameter and varying as N{sub D}{sup 1/3}. We thus establish a “nanocrystal counterpart” of conventional semiconductor planar junctions, here however valid in regimes of strong electrostatic and quantum confinements.

  18. 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

  19. Study of Gap Junctions in Human Embryonic Stem Cells.

    PubMed

    Pébay, Alice; Wong, Raymond C B

    2016-01-01

    Gap junctional intercellular communication (GJIC) has been described in different cell types including stem cells and has been involved in different biological events. GJIC is required for mouse embryonic stem cell maintenance and proliferation and various studies suggest that functional GJIC is a common characteristic of human embryonic stem cells (hESC) maintained in different culture conditions. This chapter introduces methods to study gap junctions in hESC, from expression of gap junction proteins to functional study of GJIC in hESC proliferation, apoptosis, colony growth, and pluripotency. PMID:24859928

  20. Preparation of Gap Junctions in Membrane Microdomains for Immunoprecipitation and Mass Spectrometry Interactome Analysis.

    PubMed

    Fowler, Stephanie; Akins, Mark; Bennett, Steffany A L

    2016-01-01

    Protein interaction networks at gap junction plaques are increasingly implicated in a variety of intracellular signaling cascades. Identifying protein interactions of integral membrane proteins is a valuable tool for determining channel function. However, several technical challenges exist. Subcellular fractionation of the bait protein matrix is usually required to identify less abundant proteins in complex homogenates. Sufficient solvation of the lipid environment without perturbation of the protein interactome must also be achieved. The present chapter describes the flotation of light and heavy liver tissue membrane microdomains to facilitate the identification and analysis of endogenous gap junction proteins and includes technical notes for translation to other integral membrane proteins, tissues, or cell culture models. These procedures are valuable tools for the enrichment of gap junction membrane compartments and for the identification of gap junction signaling interactomes. PMID:27207290

  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. PMID:26922907

  2. 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

  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

    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

    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. 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

  6. 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.

  7. 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

  8. Hydrogen supplemented air inhalation reduces changes of prooxidant enzyme and gap junction protein levels after transient global cerebral ischemia in the rat hippocampus.

    PubMed

    Hugyecz, Marietta; Mracskó, Eva; Hertelendy, Péter; Farkas, Eszter; Domoki, Ferenc; Bari, Ferenc

    2011-08-01

    Transient global cerebral ischemia (TGCI) occurs during acute severe hypotension depriving the brain of oxygen and glucose for a short period of time. During reperfusion, several mechanisms can induce secondary neuronal damage, including the increased production of reactive oxygen species (ROS). Hydrogen gas-enriched air inhalation is a neuroprotective approach with proven antioxidant potential, which has not yet been examined in TGCI. Accordingly, we set out to describe the effect of inhalation of 2.1% hydrogen supplemented room air (H(2)-RA) in comparison with a well studied neuroprotective agent, rosiglitazone (RSG) in a TGCI rat model. Male Wistar rats were exposed to TGCI (n=26) or sham operation (n=26), while a third group served as intact control (naive, n=5). The operated groups were further divided into non-treated, H(2)-RA, RSG (6 mg/kg i.v.) and vehicle treated animals. Tissue samples from the hippocampus and frontal cortex were taken 3 days following surgery. Western blot analysis was applied to determine the expressions of cyclooxygenase-2 (COX-2), neuronal and endothelial nitric oxide synthase (nNOS and eNOS, respectively), manganese superoxide dismutase (MnSOD) and glial connexin proteins: connexin 30 and connexin 43. The expressions of COX-2, and connexin proteins were upregulated, while nNOS was downregulated 3 days after TGCI. Both RSG and H(2)-RA prevented the changes of enzyme and connexin levels. Considering the lack of harmful side effects, inhalation of H(2)-RA can be a promising approach to reduce neuronal damage after TGCI. PMID:21718970

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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

  14. Pallidal gap junctions-triggers of synchrony in Parkinson's disease?

    PubMed Central

    Schwab, Bettina C; Heida, Tjitske; Zhao, Yan; van Gils, Stephan A; van Wezel, Richard J A

    2014-01-01

    Although increased synchrony of the neural activity in the basal ganglia may underlie the motor deficiencies exhibited in Parkinson's disease (PD), how this synchrony arises, propagates through the basal ganglia, and changes under dopamine replacement remains unknown. Gap junctions could play a major role in modifying this synchrony, because they show functional plasticity under the influence of dopamine and after neural injury. In this study, confocal imaging was used to detect connexin-36, the major neural gap junction protein, in postmortem tissues of PD patients and control subjects in the putamen, subthalamic nucleus (STN), and external and internal globus pallidus (GPe and GPi, respectively). Moreover, we quantified how gap junctions affect synchrony in an existing computational model of the basal ganglia. We detected connexin-36 in the human putamen, GPe, and GPi, but not in the STN. Furthermore, we found that the number of connexin-36 spots in PD tissues increased by 50% in the putamen, 43% in the GPe, and 109% in the GPi compared with controls. In the computational model, gap junctions in the GPe and GPi strongly influenced synchrony. The basal ganglia became especially susceptible to synchronize with input from the cortex when gap junctions were numerous and high in conductance. In conclusion, connexin-36 expression in the human GPe and GPi suggests that gap junctional coupling exists within these nuclei. In PD, neural injury and dopamine depletion could increase this coupling. Therefore, we propose that gap junctions act as a powerful modulator of synchrony in the basal ganglia. PMID:25124148

  15. Pallidal gap junctions-triggers of synchrony in Parkinson's disease?

    PubMed

    Schwab, Bettina C; Heida, Tjitske; Zhao, Yan; van Gils, Stephan A; van Wezel, Richard J A

    2014-10-01

    Although increased synchrony of the neural activity in the basal ganglia may underlie the motor deficiencies exhibited in Parkinson's disease (PD), how this synchrony arises, propagates through the basal ganglia, and changes under dopamine replacement remains unknown. Gap junctions could play a major role in modifying this synchrony, because they show functional plasticity under the influence of dopamine and after neural injury. In this study, confocal imaging was used to detect connexin-36, the major neural gap junction protein, in postmortem tissues of PD patients and control subjects in the putamen, subthalamic nucleus (STN), and external and internal globus pallidus (GPe and GPi, respectively). Moreover, we quantified how gap junctions affect synchrony in an existing computational model of the basal ganglia. We detected connexin-36 in the human putamen, GPe, and GPi, but not in the STN. Furthermore, we found that the number of connexin-36 spots in PD tissues increased by 50% in the putamen, 43% in the GPe, and 109% in the GPi compared with controls. In the computational model, gap junctions in the GPe and GPi strongly influenced synchrony. The basal ganglia became especially susceptible to synchronize with input from the cortex when gap junctions were numerous and high in conductance. In conclusion, connexin-36 expression in the human GPe and GPi suggests that gap junctional coupling exists within these nuclei. In PD, neural injury and dopamine depletion could increase this coupling. Therefore, we propose that gap junctions act as a powerful modulator of synchrony in the basal ganglia. PMID:25124148

  16. Tricellular Tight Junctions in the Inner Ear

    PubMed Central

    2016-01-01

    Tight junctions (TJs) are structures that seal the space between the epithelial cell sheets. In the inner ear, the barrier function of TJs is indispensable for the separation of the endolymphatic and perilymphatic spaces, which is essential for the generation and maintenance of the endocochlear potential (EP). TJs are formed by the intercellular binding of membrane proteins, known as claudins, and mutations in these proteins cause deafness in humans and mice. Within the epithelial cell sheet, however, a bound structure is present at the site where the corners of three cells meet (tricellular tight junctions (tTJs)), and the maintenance of the barrier function at this location cannot be explained by the claudins alone. Tricellulin and the angulin family of proteins (angulin-1/LSR, angulin-2/ILDR1, and angulin-3/ILDR2) have been identified as tTJ-associated proteins. Tricellulin and ILDR1 are localized at the tTJ and alterations in these proteins have been reported to be involved in deafness. In this review, we will present the current state of knowledge for tTJs. PMID:27195292

  17. Tricellular Tight Junctions in the Inner Ear.

    PubMed

    Kitajiri, Shin-Ichiro; Katsuno, Tatsuya

    2016-01-01

    Tight junctions (TJs) are structures that seal the space between the epithelial cell sheets. In the inner ear, the barrier function of TJs is indispensable for the separation of the endolymphatic and perilymphatic spaces, which is essential for the generation and maintenance of the endocochlear potential (EP). TJs are formed by the intercellular binding of membrane proteins, known as claudins, and mutations in these proteins cause deafness in humans and mice. Within the epithelial cell sheet, however, a bound structure is present at the site where the corners of three cells meet (tricellular tight junctions (tTJs)), and the maintenance of the barrier function at this location cannot be explained by the claudins alone. Tricellulin and the angulin family of proteins (angulin-1/LSR, angulin-2/ILDR1, and angulin-3/ILDR2) have been identified as tTJ-associated proteins. Tricellulin and ILDR1 are localized at the tTJ and alterations in these proteins have been reported to be involved in deafness. In this review, we will present the current state of knowledge for tTJs. PMID:27195292

  18. 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.

  19. Claudin heterogeneity and control of lung tight junctions.

    PubMed

    Koval, Michael

    2013-01-01

    Lung epithelial cells interconnected by tight junctions provide a barrier to the free diffusion of solutes into airspaces. Transmembrane tight junction proteins known as claudins are essential for epithelial barrier function. Claudins are regulated through interactions with each other that are coordinated with other transmembrane tight junction proteins and cytosolic scaffold proteins. Of the 14 claudins expressed by the alveolar epithelium, claudin-3, claudin-4, and claudin-18 are the most prominent; each confers unique properties to alveolar barrier function. In particular, a protective role for claudin-4 in preventing lung injury has emerged. By contrast, lung diseases that affect claudin expression and impair barrier function, including alcoholic lung syndrome and sepsis, prime the lung for pulmonary edema. Thus, approaches to restore and/or augment lung claudin expression provide potential targets for promoting healthy barrier function. PMID:23072447

  20. 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

  1. 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.

  2. Josephson junction simulation of neurons

    NASA Astrophysics Data System (ADS)

    Crotty, Patrick; Schult, Dan; Segall, Ken

    2010-07-01

    With the goal of understanding the intricate behavior and dynamics of collections of neurons, we present superconducting circuits containing Josephson junctions that model biologically realistic neurons. These “Josephson junction neurons” reproduce many characteristic behaviors of biological neurons such as action potentials, refractory periods, and firing thresholds. They can be coupled together in ways that mimic electrical and chemical synapses. Using existing fabrication technologies, large interconnected networks of Josephson junction neurons would operate fully in parallel. They would be orders of magnitude faster than both traditional computer simulations and biological neural networks. Josephson junction neurons provide a new tool for exploring long-term large-scale dynamics for networks of neurons.

  3. 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)

  4. 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.

  5. Junctional actin assembly is mediated by Formin-like 2 downstream of Rac1

    PubMed Central

    Grikscheit, Katharina; Frank, Tanja; Wang, Ying

    2015-01-01

    Epithelial integrity is vitally important, and its deregulation causes early stage cancer. De novo formation of an adherens junction (AJ) between single epithelial cells requires coordinated, spatial actin dynamics, but the mechanisms steering nascent actin polymerization for cell–cell adhesion initiation are not well understood. Here we investigated real-time actin assembly during daughter cell–cell adhesion formation in human breast epithelial cells in 3D environments. We identify formin-like 2 (FMNL2) as being specifically required for actin assembly and turnover at newly formed cell–cell contacts as well as for human epithelial lumen formation. FMNL2 associates with components of the AJ complex involving Rac1 activity and the FMNL2 C terminus. Optogenetic control of Rac1 in living cells rapidly drove FMNL2 to epithelial cell–cell contact zones. Furthermore, Rac1-induced actin assembly and subsequent AJ formation critically depends on FMNL2. These data uncover FMNL2 as a driver for human epithelial AJ formation downstream of Rac1. PMID:25963818

  6. Junctional actin assembly is mediated by Formin-like 2 downstream of Rac1.

    PubMed

    Grikscheit, Katharina; Frank, Tanja; Wang, Ying; Grosse, Robert

    2015-05-11

    Epithelial integrity is vitally important, and its deregulation causes early stage cancer. De novo formation of an adherens junction (AJ) between single epithelial cells requires coordinated, spatial actin dynamics, but the mechanisms steering nascent actin polymerization for cell-cell adhesion initiation are not well understood. Here we investigated real-time actin assembly during daughter cell-cell adhesion formation in human breast epithelial cells in 3D environments. We identify formin-like 2 (FMNL2) as being specifically required for actin assembly and turnover at newly formed cell-cell contacts as well as for human epithelial lumen formation. FMNL2 associates with components of the AJ complex involving Rac1 activity and the FMNL2 C terminus. Optogenetic control of Rac1 in living cells rapidly drove FMNL2 to epithelial cell-cell contact zones. Furthermore, Rac1-induced actin assembly and subsequent AJ formation critically depends on FMNL2. These data uncover FMNL2 as a driver for human epithelial AJ formation downstream of Rac1. PMID:25963818

  7. Dynamic gap junctional communication: a delimiting model for tissue responses.

    PubMed Central

    Christ, G J; Brink, P R; Ramanan, S V

    1994-01-01

    Gap junctions are aqueous intercellular channels formed by a diverse class of membrane-spanning proteins, known as connexins. These aqueous pores provide partial cytoplasmic continuity between cells in most tissues, and are freely permeable to a host of physiologically relevant second messenger molecules/ionic species (e.g., Ca2+, IP3, cAMP, cGMP). Despite the fact that these second messenger molecules/ionic species have been shown to alter junctional patency, there is no clear basis for understanding how dynamic and transient changes in the intracellular concentration of second messenger molecules might modulate the extent of intercellular communication among coupled cells. Thus, we have modified the tissue monolayer model of Ramanan and Brink (1990) to account for both the up-regulatory and down-regulatory effects on junctions by second messenger molecules that diffuse through gap junctions. We have chosen the vascular wall as our morphological correlate because of its anisotropy and large investment of gap junctions. The model allows us to illustrate the putative behavior of gap junctions under a variety of physiologically relevant conditions. The modeling studies demonstrated that transient alterations in intracellular second messenger concentrations are capable of producing 50-125% changes in the number of cells recruited into a functional syncytial unit, after activation of a single cell. Moreover, the model conditions required to demonstrate such physiologically relevant changes in intercellular diffusion among coupled cells are commonly observed in intact tissues and cultured cells. Images FIGURE 2 PMID:7811948

  8. 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

  9. Ion bipolar junction transistors.

    PubMed

    Tybrandt, Klas; Larsson, Karin C; Richter-Dahlfors, Agneta; Berggren, Magnus

    2010-06-01

    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

  10. 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

  11. Disordered graphene Josephson junctions

    NASA Astrophysics Data System (ADS)

    Muñoz, W. A.; Covaci, L.; Peeters, F. M.

    2015-02-01

    A tight-binding approach based on the Chebyshev-Bogoliubov-de Gennes method is used to describe disordered single-layer graphene Josephson junctions. Scattering by vacancies, ripples, or charged impurities is included. We compute the Josephson current and investigate the nature of multiple Andreev reflections, which induce bound states appearing as peaks in the density of states for energies below the superconducting gap. In the presence of single-atom vacancies, we observe a strong suppression of the supercurrent, which is a consequence of strong intervalley scattering. Although lattice deformations should not induce intervalley scattering, we find that the supercurrent is still suppressed, which is due to the presence of pseudomagnetic barriers. For charged impurities, we consider two cases depending on whether the average doping is zero, i.e., existence of electron-hole puddles, or finite. In both cases, short-range impurities strongly affect the supercurrent, similar to the vacancies scenario.

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

    PubMed

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

    2016-01-01

    Internalization of gap junction plaques results in the formation of annular gap junction vesicles. The factors that regulate the coordinated internalization of the gap junction plaques to form annular gap junction vesicles, and the subsequent events involved in annular gap junction processing have only relatively recently been investigated in detail. However it is becoming clear that while annular gap junction vesicles have been demonstrated to be degraded by autophagosomal and endo-lysosomal pathways, they undergo a number of additional processing events. Here, we characterize the morphology of the annular gap junction vesicle and review the current knowledge of the processes involved in their formation, fission, fusion, and degradation. In addition, we address the possibility for connexin protein recycling back to the plasma membrane to contribute to gap junction formation and intercellular communication. Information on gap junction plaque removal from the plasma membrane and the subsequent processing of annular gap junction vesicles is critical to our understanding of cell-cell communication as it relates to events regulating development, cell homeostasis, unstable proliferation of cancer cells, wound healing, changes in the ischemic heart, and many other physiological and pathological cellular phenomena. PMID:27230503

  13. The Onecut Transcription Factor HNF-6 Regulates in Motor Neurons the Formation of the Neuromuscular Junctions

    PubMed Central

    Audouard, Emilie; Schakman, Olivier; René, Frédérique; Huettl, Rosa-Eva; Huber, Andrea B.; Loeffler, Jean-Philippe; Gailly, Philippe; Clotman, Frédéric

    2012-01-01

    The neuromuscular junctions are the specialized synapses whereby spinal motor neurons control the contraction of skeletal muscles. The formation of the neuromuscular junctions is controlled by a complex interplay of multiple mechanisms coordinately activated in motor nerve terminals and in their target myotubes. However, the transcriptional regulators that control in motor neurons the genetic programs involved in neuromuscular junction development remain unknown. Here, we provide evidence that the Onecut transcription factor HNF-6 regulates in motor neurons the formation of the neuromuscular junctions. Indeed, adult Hnf6 mutant mice exhibit hindlimb muscle weakness and abnormal locomotion. This results from defects of hindlimb neuromuscular junctions characterized by an abnormal morphology and defective localization of the synaptic vesicle protein synaptophysin at the motor nerve terminals. These defects are consequences of altered and delayed formation of the neuromuscular junctions in newborn mutant animals. Furthermore, we show that the expression level of numerous regulators of neuromuscular junction formation, namely agrin, neuregulin-2 and TGF-ß receptor II, is downregulated in the spinal motor neurons of Hnf6 mutant newborn animals. Finally, altered formation of neuromuscular junction-like structures in a co-culture model of wildtype myotubes with mutant embryonic spinal cord slices is rescued by recombinant agrin and neuregulin, indicating that depletion in these factors contributes to defective neuromuscular junction development in the absence of HNF-6. Thus, HNF-6 controls in spinal motor neurons a genetic program that coordinates the formation of hindlimb neuromuscular junctions. PMID:23227180

  14. Breaking into the epithelial apical-junctional complex--news from pathogen hackers.

    PubMed

    Vogelmann, Roger; Amieva, Manuel R; Falkow, Stanley; Nelson, W James

    2004-02-01

    The epithelial apical-junctional complex is a key regulator of cellular functions. In addition, it is an important target for microbial pathogens that manipulate the cell to survive, proliferate and sometimes persist within a host. Out of a myriad of potential molecular targets, some bacterial and viral pathogens have selected a subset of protein targets at the apical-junctional complex of epithelial cells. Studying how microbes use these targets also teaches us about the inherent physiological properties of host molecules in the context of normal junctional structure and function. Thus, we have learned that three recently uncovered components of the apical-junctional complex of the Ig superfamily--junctional adhesion molecule, Nectin and the coxsackievirus and adenovirus receptor--are important regulators of junction structure and function and represent critical targets of microbial virulence gene products. PMID:15037310

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

    PubMed

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

    2016-02-01

    The intercellular junctions restrict the free passage of hydrophilic compounds through the paracellular clefts. Reversible opening of the tight junctions of biological barriers is investigated as one of the ways to increase drug delivery to the systemic circulation or the central nervous system. Six peptides, ADT-6, HAV-6, C-CPE, 7-mer (FDFWITP, PN-78), AT-1002, and PN-159, acting on different integral membrane and linker junctional proteins were tested on Caco-2 intestinal epithelial cell line and a coculture model of the blood-brain barrier. All peptides tested in nontoxic concentrations showed a reversible tight junctions modulating effect and were effective to open the paracellular pathway for the marker molecules fluorescein and albumin. The change in the structure of cell-cell junctions was verified by immunostaining for occludin, claudin-4,-5, ZO-1, β-catenin, and E-cadherin. Expression levels of occludin and claudins were measured in both models. We could demonstrate a selectivity of C-CPE, ADT-6, and HAV-6 peptides for epithelial cells and 7-mer and AT-1002 peptides for brain endothelial cells. PN-159 was the most effective modulator of junctional permeability in both models possibly acting via claudin-1 and -5. Our results indicate that these peptides can be effectively and selectively used as potential pharmaceutical excipients to improve drug delivery across biological barriers. PMID:26869428

  16. Thermopower measurements in molecular junctions.

    PubMed

    Rincón-García, Laura; Evangeli, Charalambos; Rubio-Bollinger, Gabino; Agraït, Nicolás

    2016-08-01

    The measurement of thermopower in molecular junctions offers complementary information to conductance measurements and is becoming essential for the understanding of transport processes at the nanoscale. In this review, we discuss the recent advances in the study of the thermoelectric properties of molecular junctions. After presenting the theoretical background for thermoelectricity at the nanoscale, we review the experimental techniques for measuring the thermopower in these systems and discuss the main results. Finally, we consider the challenges in the application of molecular junctions in viable thermoelectric devices. PMID:27277330

  17. Electronic properties of nanotube junctions

    NASA Astrophysics Data System (ADS)

    Lambin, Ph.; Meunier, V.

    1998-08-01

    The possibility of realizing junctions between two different nanotubes has recently attracted a great interest, even though much remains to be done for putting this idea in concrete form. Pentagon-heptagon pair defects in the otherwise perfect graphitic network make such connections possible, with virtually infinite varieties. In this paper, the literature devoted to nanotube junctions is briefly reviewed. A special emphasize is put on the electronic properties of C nanotube junctions, together with an indication on how their current-voltage characteristics may look like.

  18. Josephson junction Q-spoiler

    DOEpatents

    Clarke, J.; Hilbert, C.; Hahn, E.L.; Sleator, T.

    1986-03-25

    An automatic Q-spoiler comprising at least one Josephson tunnel junction connected in an LC circuit for flow of resonant current therethrough. When in use in a system for detecting the magnetic resonance of a gyromagnetic particle system, a high energy pulse of high frequency energy irradiating the particle system will cause the critical current through the Josephson tunnel junctions to be exceeded, causing the tunnel junctions to act as resistors and thereby damp the ringing of the high-Q detection circuit after the pulse. When the current has damped to below the critical current, the Josephson tunnel junctions revert to their zero-resistance state, restoring the Q of the detection circuit and enabling the low energy magnetic resonance signals to be detected.

  19. Josephson junction Q-spoiler

    DOEpatents

    Clarke, John; Hilbert, Claude; Hahn, Erwin L.; Sleator, Tycho

    1988-01-01

    An automatic Q-spoiler comprising at least one Josephson tunnel junction connected in an LC circuit for flow of resonant current therethrough. When in use in a system for detecting the magnetic resonance of a gyromagnetic particle system, a high energy pulse of high frequency energy irradiating the particle system will cause the critical current through the Josephson tunnel junctions to be exceeded, causing the tunnel junctions to act as resistors and thereby damp the ringing of the high-Q detection circuit after the pulse. When the current has damped to below the critical current, the Josephson tunnel junctions revert to their zero-resistance state, restoring the Q of the detection circuit and enabling the low energy magnetic resonance signals to be detected.

  20. Thermal conductance of superlattice junctions

    SciTech Connect

    Lu, Simon; McGaughey, Alan J. H.

    2015-05-15

    We use molecular dynamics simulations and the lattice-based scattering boundary method to compute the thermal conductance of finite-length Lennard-Jones superlattice junctions confined by bulk crystalline leads. The superlattice junction thermal conductance depends on the properties of the leads. For junctions with a superlattice period of four atomic monolayers at temperatures between 5 and 20 K, those with mass-mismatched leads have a greater thermal conductance than those with mass-matched leads. We attribute this lead effect to interference between and the ballistic transport of emergent junction vibrational modes. The lead effect diminishes when the temperature is increased, when the superlattice period is increased, and when interfacial disorder is introduced, but is reversed in the harmonic limit.

  1. Electronic thermometry in tunable tunnel junction

    DOEpatents

    Maksymovych, Petro

    2016-03-15

    A tunable tunnel junction thermometry circuit includes a variable width tunnel junction between a test object and a probe. The junction width is varied and a change in thermovoltage across the junction with respect to the change in distance across the junction is determined. Also, a change in biased current with respect to a change in distance across the junction is determined. A temperature gradient across the junction is determined based on a mathematical relationship between the temperature gradient, the change in thermovoltage with respect to distance and the change in biased current with respect to distance. Thermovoltage may be measured by nullifying a thermoelectric tunneling current with an applied voltage supply level. A piezoelectric actuator may modulate the probe, and thus the junction width, to vary thermovoltage and biased current across the junction. Lock-in amplifiers measure the derivatives of the thermovoltage and biased current modulated by varying junction width.

  2. Neuromuscular junctional disorders.

    PubMed

    Girija, A S; Ashraf, V V

    2008-07-01

    Neuromuscular junctional disorders (NMJ) in children are distinct entity. They may be acquired or hereditary. They pose problem in diagnosis because of the higher occurrence of sero negative Myasthenia Gravis (MG) cases in children. The identity of MusK antibody positivity in a good percentage of sero negative cases further adds to problems in diagnosis. The Congenital Myasthenic Syndrome (CMS) which are rare disorders of hereditary neuromuscular transmission (NMT) has to be differentiated because immunotherapy has no benefit in this group. Molecular genetic studies of these diseases helps to identify specific type of CMS which is important as other drugs like Fluoxetine, Quinidine are found to be effective in some. In infancy, all can manifest as floppy infant syndrome. The important key to diagnosis is by detailed electrophysiological studies including repetitive nerve stimulation at slow and high rates and its response to anticholinesterases and estimation of Acetyl choline receptor antibodies. Other causes of neuromuscular transmission defects viz. snake venom poisoning and that due to drugs are discussed. PMID:18716738

  3. Confocal Annular Josephson Tunnel Junctions

    NASA Astrophysics Data System (ADS)

    Monaco, Roberto

    2016-04-01

    The physics of Josephson tunnel junctions drastically depends on their geometrical configurations and here we show that also tiny geometrical details play a determinant role. More specifically, we develop the theory of short and long annular Josephson tunnel junctions delimited by two confocal ellipses. The behavior of a circular annular Josephson tunnel junction is then seen to be simply a special case of the above result. For junctions having a normalized perimeter less than one, the threshold curves in the presence of an in-plane magnetic field of arbitrary orientations are derived and computed even in the case with trapped Josephson vortices. For longer junctions, a numerical analysis is carried out after the derivation of the appropriate motion equation for the Josephson phase. We found that the system is modeled by a modified and perturbed sine-Gordon equation with a space-dependent effective Josephson penetration length inversely proportional to the local junction width. Both the fluxon statics and dynamics are deeply affected by the non-uniform annulus width. Static zero-field multiple-fluxon solutions exist even in the presence of a large bias current. The tangential velocity of a traveling fluxon is not determined by the balance between the driving and drag forces due to the dissipative losses. Furthermore, the fluxon motion is characterized by a strong radial inward acceleration which causes electromagnetic radiation concentrated at the ellipse equatorial points.

  4. Confocal Annular Josephson Tunnel Junctions

    NASA Astrophysics Data System (ADS)

    Monaco, Roberto

    2016-09-01

    The physics of Josephson tunnel junctions drastically depends on their geometrical configurations and here we show that also tiny geometrical details play a determinant role. More specifically, we develop the theory of short and long annular Josephson tunnel junctions delimited by two confocal ellipses. The behavior of a circular annular Josephson tunnel junction is then seen to be simply a special case of the above result. For junctions having a normalized perimeter less than one, the threshold curves in the presence of an in-plane magnetic field of arbitrary orientations are derived and computed even in the case with trapped Josephson vortices. For longer junctions, a numerical analysis is carried out after the derivation of the appropriate motion equation for the Josephson phase. We found that the system is modeled by a modified and perturbed sine-Gordon equation with a space-dependent effective Josephson penetration length inversely proportional to the local junction width. Both the fluxon statics and dynamics are deeply affected by the non-uniform annulus width. Static zero-field multiple-fluxon solutions exist even in the presence of a large bias current. The tangential velocity of a traveling fluxon is not determined by the balance between the driving and drag forces due to the dissipative losses. Furthermore, the fluxon motion is characterized by a strong radial inward acceleration which causes electromagnetic radiation concentrated at the ellipse equatorial points.

  5. GAP junctional communication in brain secondary organizers.

    PubMed

    Bosone, Camilla; Andreu, Abraham; Echevarria, Diego

    2016-06-01

    Gap junctions (GJs) are integral membrane proteins that enable the direct cytoplasmic exchange of ions and low molecular weight metabolites between adjacent cells. They are formed by the apposition of two connexons belonging to adjacent cells. Each connexon is formed by six proteins, named connexins (Cxs). Current evidence suggests that gap junctions play an important part in ensuring normal embryo development. Mutations in connexin genes have been linked to a variety of human diseases, although the precise role and the cell biological mechanisms of their action remain almost unknown. Among the big family of Cxs, several are expressed in nervous tissue but just a few are expressed in the anterior neural tube of vertebrates. Many efforts have been made to elucidate the molecular bases of Cxs cell biology and how they influence the morphogenetic signal activity produced by brain signaling centers. These centers, orchestrated by transcription factors and morphogenes determine the axial patterning of the mammalian brain during its specification and regionalization. The present review revisits the findings of GJ composed by Cx43 and Cx36 in neural tube patterning and discuss Cx43 putative enrollment in the control of Fgf8 signal activity coming from the well known secondary organizer, the isthmic organizer. PMID:27273333

  6. Octagonal Defects at Carbon Nanotube Junctions

    PubMed Central

    Jaskólski, W.; Pelc, M.; Chico, Leonor; Ayuela, A.

    2013-01-01

    We investigate knee-shaped junctions of semiconductor zigzag carbon nanotubes. Two dissimilar octagons appear at such junctions; one of them can reconstruct into a pair of pentagons. The junction with two octagons presents two degenerate localized states at Fermi energy (EF). The reconstructed junction has only one state near EF, indicating that these localized states are related to the octagonal defects. The inclusion of Coulomb interaction splits the localized states in the junction with two octagons, yielding an antiferromagnetic system. PMID:24089604

  7. Wnt Signaling in Neuromuscular Junction Development

    PubMed Central

    Koles, Kate

    2012-01-01

    Wnt proteins are best known for their profound roles in cell patterning, because they are required for the embryonic development of all animal species studied to date. Besides regulating cell fate, Wnt proteins are gaining increasing recognition for their roles in nervous system development and function. New studies indicate that multiple positive and negative Wnt signaling pathways take place simultaneously during the formation of vertebrate and invertebrate neuromuscular junctions. Although some Wnts are essential for the formation of NMJs, others appear to play a more modulatory role as part of multiple signaling pathways. Here we review the most recent findings regarding the function of Wnts at the NMJ from both vertebrate and invertebrate model systems. PMID:22510459

  8. Chlorpromazine reduces the intercellular communication via gap junctions in mammalian cells

    SciTech Connect

    Orellana, Juan A.; Palacios-Prado, Nicolas; Saez, Juan C. . E-mail: jsaez@bio.puc.cl

    2006-06-15

    In the work presented herein, we evaluated the effect of chlorpromazine (CPZ) on gap junctions expressed by two mammalian cell types; Gn-11 cells (cell line derived from mouse LHRH neurons) and rat cortical astrocytes maintained in culture. We also attempted to elucidate possible mechanisms of action of CPZ effects on gap junctions. CPZ, in concentrations comparable with doses used to treat human diseases, was found to reduce the intercellular communication via gap junctions as evaluated with measurements of dye coupling (Lucifer yellow). In both cell types, maximal inhibition of functional gap junctions was reached within about 1 h of treatment with CPZ, an recovery was almost complete at about 5 h after CPZ wash out. In both cell types, CPZ treatment increased the phosphorylation state of connexin43 (Cx43), a gap junction protein subunit. Moreover, CPZ reduced the reactivity of Cx43 (immunofluorescence) at cell interfaces and concomitantly increased its reactivity in intracellular vesicles, suggesting an increased retrieval from and/or reduced insertion into the plasma membrane. CPZ also caused cellular retraction reducing cell-cell contacts in a reversible manner. The reduction in contact area might destabilize existing gap junctions and abrogate formation of new ones. Moreover, the CPZ-induced reduction in gap junctional communication may depend on the connexins (Cxs) forming the junctions. If Cx43 were the only connexin expressed, MAPK-dependent phosphorylation of this connexin would induce closure of gap junction channels.

  9. Epithelial tight junctions in intestinal inflammation.

    PubMed

    Schulzke, Joerg D; Ploeger, Svenja; Amasheh, Maren; Fromm, Anja; Zeissig, Sebastian; Troeger, Hanno; Richter, Jan; Bojarski, Christian; Schumann, Michael; Fromm, Michael

    2009-05-01

    The epithelium in inflamed intestinal segments of patients with Crohn's disease is characterized by a reduction of tight junction strands, strand breaks, and alterations of tight junction protein content and composition. In ulcerative colitis, epithelial leaks appear early due to micro-erosions resulting from upregulated epithelial apoptosis and in addition to a prominent increase of claudin-2. Th1-cytokine effects by interferon-gamma in combination with TNFalpha are important for epithelial damage in Crohn's disease, while interleukin-13 (IL-13) is the key effector cytokine in ulcerative colitis stimulating apoptosis and upregulation of claudin-2 expression. Focal lesions caused by apoptotic epithelial cells contribute to barrier disturbance in IBD by their own conductivity and by confluence toward apoptotic foci or erosions. Another type of intestinal barrier defect can arise from alpha-hemolysin harboring E. coli strains among the physiological flora, which can gain pathologic relevance in combination with proinflammatory cytokines under inflammatory conditions. On the other hand, intestinal barrier impairment can also result from transcellular antigen translocation via an initial endocytotic uptake into early endosomes, and this is intensified by proinflammatory cytokines as interferon-gamma and may thus play a relevant role in the onset of IBD. Taken together, barrier defects contribute to diarrhea by a leak flux mechanism (e.g., in IBD) and can cause mucosal inflammation by luminal antigen uptake. Immune regulation of epithelial functions by cytokines may cause barrier dysfunction not only by tight junction impairments but also by apoptotic leaks, transcytotic mechanisms, and mucosal gross lesions. PMID:19538319

  10. Transport in Carbon Nanotube Junctions

    NASA Astrophysics Data System (ADS)

    Khoo, K. H.; Chelikowsky, James R.

    2008-03-01

    There is growing interest in the use of carbon nanotube thin films as transparent electrical conductors and thin-film transistors owing to their high optical transmittance, low sheet resistivity, and ease of fabrication. [1,2] A major contribution to the sheet resistivity originates at nanotube junctions, as electrical contact is typically poor between adjacent nanotubes. It is thus important to characterize carbon nanotube junctions in order to understand the conduction properties of nanotube thin films. To this end, we have performed ab initio density functional theory calculations to investigate the structural, electronic and transport properties of carbon nanotube junctions as a function of nanotube chirality and contact geometry [1] Z. Wu et al., Science 305, 1273 (2004) [2] E. S. Snow, J. P. Novak, P. M. Campbell, and D. Park, Appl. Phys. Lett. 82, 2145 (2003).

  11. Tight Junction Pore and Leak Pathways: A Dynamic Duo

    PubMed Central

    Raleigh, David R.; Yu, Dan; Turner, Jerrold R.

    2015-01-01

    Tissue barriers that restrict passage of liquids, ions, and larger solutes are essential for the development of multicellular organisms. In simple organisms this allows distinct cell types to interface with the external environment. In more complex species, the diversity of cell types capable of forming barriers increases dramatically. Although the plasma membranes of these barrier-forming cells prevent flux of most hydrophilic solutes, the paracellular, or shunt, pathway between cells must also be sealed. This function is accomplished in vertebrates by the zonula occludens, or tight junction. The tight junction barrier is not absolute but is selectively permeable and is able to discriminate between solutes on the basis of size and charge. Many tight junction components have been identified over the past 20 years, and recent progress has provided new insights into the proteins and interactions that regulate structure and function. This review presents these data in a historical context and proposes an integrated model in which dynamic regulation of tight junction protein interactions determines barrier function. PMID:20936941

  12. Functionally Active Gap Junctions between Connexin 43-Positive Mesenchymal Stem Cells and Glioma Cells.

    PubMed

    Gabashvili, A N; Baklaushev, V P; Grinenko, N F; Levinskii, A B; Mel'nikov, P A; Cherepanov, S A; Chekhonin, V P

    2015-05-01

    The formation of functional gap junctions between mesenchymal stem cells and cells of low-grade rat glioma C6