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      Vinculin-dependent Cadherin mechanosensing regulates efficient epithelial barrier formation

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          Summary

          Proper regulation of the formation and stabilization of epithelial cell–cell adhesion is crucial in embryonic morphogenesis and tissue repair processes. Defects in this process lead to organ malformation and defective epithelial barrier function. A combination of chemical and mechanical cues is used by cells to drive this process. We have investigated the role of the actomyosin cytoskeleton and its connection to cell–cell junction complexes in the formation of an epithelial barrier in MDCK cells. We find that the E-cadherin complex is sufficient to mediate a functional link between cell–cell contacts and the actomyosin cytoskeleton. This link involves the actin binding capacity of α-catenin and the recruitment of the mechanosensitive protein Vinculin to tensile, punctate cell–cell junctions that connect to radial F-actin bundles, which we name Focal Adherens Junctions (FAJ). When cell–cell adhesions mature, these FAJs disappear and linear junctions are formed that do not contain Vinculin. The rapid phase of barrier establishment (as measured by Trans Epithelial Electrical Resistance (TER)) correlates with the presence of FAJs. Moreover, the rate of barrier establishment is delayed when actomyosin contraction is blocked or when Vinculin recruitment to the Cadherin complex is prevented. Enhanced presence of Vinculin increases the rate of barrier formation. We conclude that E-cadherin-based FAJs connect forming cell–cell adhesions to the contractile actomyosin cytoskeleton. These specialized junctions are sites of Cadherin mechanosensing, which, through the recruitment of Vinculin, is a driving force in epithelial barrier formation.

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          Local force and geometry sensing regulate cell functions.

          The shapes of eukaryotic cells and ultimately the organisms that they form are defined by cycles of mechanosensing, mechanotransduction and mechanoresponse. Local sensing of force or geometry is transduced into biochemical signals that result in cell responses even for complex mechanical parameters such as substrate rigidity and cell-level form. These responses regulate cell growth, differentiation, shape changes and cell death. Recent tissue scaffolds that have been engineered at the micro- and nanoscale level now enable better dissection of the mechanosensing, transduction and response mechanisms.
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            alpha-Catenin as a tension transducer that induces adherens junction development.

            Adherens junctions (AJs), which are organized by adhesion proteins and the underlying actin cytoskeleton, probably sense pulling forces from adjacent cells and modulate opposing forces to maintain tissue integrity, but the regulatory mechanism remains unknown at the molecular level. Although the possibility that alpha-catenin acts as a direct linker between the membrane and the actin cytoskeleton for AJ formation and function has been minimized, here we show that alpha-catenin recruits vinculin, another main actin-binding protein of AJs, through force-dependent changes in alpha-catenin conformation. We identified regions in the alpha-catenin molecule that are required for its force-dependent binding of vinculin by introducing mutant alpha-catenin into cells and using in vitro binding assays. Fluorescence recovery after photobleaching analysis for alpha-catenin mobility and the existence of an antibody recognizing alpha-catenin in a force-dependent manner further supported the notion that alpha-catenin is a tension transducer that translates mechanical stimuli into a chemical response, resulting in AJ development.
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              Direct Binding of Three Tight Junction-Associated Maguks, Zo-1, Zo-2, and Zo-3, with the Cooh Termini of Claudins

              ZO-1, ZO-2, and ZO-3, which contain three PDZ domains (PDZ1 to -3), are concentrated at tight junctions (TJs) in epithelial cells. TJ strands are mainly composed of two distinct types of four-transmembrane proteins, occludin, and claudins, between which occludin was reported to directly bind to ZO-1/ZO-2/ZO-3. However, in occludin-deficient intestinal epithelial cells, ZO-1/ZO-2/ZO-3 were still recruited to TJs. We then examined the possible interactions between ZO-1/ZO-2/ZO-3 and claudins. ZO-1, ZO-2, and ZO-3 bound to the COOH-terminal YV sequence of claudin-1 to -8 through their PDZ1 domains in vitro. Then, claudin-1 or -2 was transfected into L fibroblasts, which express ZO-1 but not ZO-2 or ZO-3. Claudin-1 and -2 were concentrated at cell–cell borders in an elaborate network pattern, to which endogenous ZO-1 was recruited. When ZO-2 or ZO-3 were further transfected, both were recruited to the claudin-based networks together with endogenous ZO-1. Detailed analyses showed that ZO-2 and ZO-3 are recruited to the claudin-based networks through PDZ2 (ZO-2 or ZO-3)/PDZ2 (endogenous ZO-1) and PDZ1 (ZO-2 or ZO-3)/COOH-terminal YV (claudins) interactions. In good agreement, PDZ1 and PDZ2 domains of ZO-1/ZO-2/ZO-3 were also recruited to claudin-based TJs, when introduced into cultured epithelial cells. The possible molecular architecture of TJ plaque structures is discussed.
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                Author and article information

                Journal
                Biol Open
                Biol Open
                biolopen
                bio
                Biology Open
                The Company of Biologists (Bidder Building, 140 Cowley Road, Cambridge, CB4 0DL, UK )
                2046-6390
                15 November 2012
                11 September 2012
                : 1
                : 11
                : 1128-1140
                Affiliations
                [1 ]Hubrecht Institute for Developmental Biology and Stem Cell Research and University Medical Centre Utrecht , PO Box 85164, 3508 AD Utrecht, The Netherlands
                [2 ]Department for Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign , Champaign, IL 61801, USA
                [3 ]Department of Chemistry, University of Illinois at Urbana-Champaign , Champaign, IL 61801, USA
                [4 ]Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign , Champaign, IL 61801, USA
                [5 ]Department of Molecular Cancer Research, Centre of Biomedical Genetics and Cancer Genomics Centre, University Medical Centre Utrecht , 3584 CG Utrecht, The Netherlands
                [6 ]Present address: Sanquin Research, Department of Molecular Cell Biology and University of Amsterdam, Swammerdam Institute for Life Sciences, 1098 XH Amsterdam, The Netherlands
                Author notes
                []Author for correspondence ( j.derooij@ 123456hubrecht.eu )
                Article
                BIO20122428
                10.1242/bio.20122428
                3507192
                23213393
                aa052c37-c42e-4fd0-b543-a8573e31b0ef
                © 2012. Published by The Company of Biologists Ltd

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Share Alike License ( http://creativecommons.org/licenses/by-nc-sa/3.0/).

                History
                : 2 July 2012
                : 13 August 2012
                Categories
                Research Article

                Life sciences
                actomyosin,cell–cell adhesion,mechanotransduction,vinculin,junction formation,e-cadherin

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