Vinculin localizes to tension-bearing cell–cell junctions to help transmit signals from E-cadherin to the actin cytoskeleton in response to mechanical stress.
The authors regret an error in the Materials and methods section of this paper pertaining to the use of a certain antibody.The correct sentence appears below:Alternatively, beads were labeled with monoclonal anti-E-cadherin antibodies (also used as a blocking antibody in these experiments; DECMA-1; Sigma-Aldrich) using an identical protocol.
SummaryProper 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.
The scattering of cultured epithelial cells in response to hepatocyte growth factor (HGF) is a model system that recapitulates key features of metastatic cell behavior in vitro, including disruption of cell-cell adhesions and induction of cell migration. We have developed image analysis tools to automatically track and characterize this process in live cells without the need for fluorescent tagging, in three different physical aspects: increase in cell motility, loss of cell-cell adhesion, and spatial dispersion of cells. These tools were used to screen a library of drugs. We identified several efficient inhibitors of scattering and classified them as selective inhibitors of either motility or loss of cell-cell adhesion, or as non-selective inhibitors. We validated the inhibitors and putative targets from this screen in two unrelated model cell lines using pharmacological treatments and RNA interference (RNAi). We found that non-steroidal anti-inflammatory drugs inhibited cell-cell dissociation, that indirubins inhibited cell motility, and that cyclin-dependent kinase 1 and ribosomal S6 kinase were signaling intermediates in HGF-induced cell scattering. The assay will be suitable for larger-scale screenings of chemical compounds or RNAi libraries.
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