Vinculin is essential for sustaining normal levels of endogenous forces at cell-cell contacts.

Transmission of cell-generated (i.e., endogenous) tension at cell-cell contacts is crucial for tissue shape changes during morphogenesis and adult tissue repair in tissues such as epithelia. E-cadherin-based adhesions at cell-cell contacts are the primary means by which endogenous tension is transmitted between cells. The E-cadherin-ß-catenin-α-catenin complex mechanically couples to the actin cytoskeleton (and thereby the cell's contractile machinery) both directly and indirectly. However, the key adhesion constituents required for substantial endogenous force transmission at these adhesions in cell-cell contacts are unclear. Due to the role of α-catenin as a mechanotransducer that recruits vinculin at cell-cell contacts, we expected α-catenin to be essential for sustaining normal levels of force transmission. Instead, using the traction force imbalance method to determine the inter-cellular force at a single cell-cell contact between cell pairs, we found that it is vinculin that is essential for sustaining normal levels of endogenous force transmission, with absence of vinculin decreasing the inter-cellular tension by over 50%. Our results constrain the potential mechanical pathways of force transmission at cell-cell contacts and suggest that vinculin can transmit forces at E-cadherin adhesions independent of α-catenin, possibly through ß-catenin. Furthermore, we tested the ability of lateral cell-cell contacts to withstand external stretch and found that both vinculin and α-catenin are essential to maintain cell-cell contact stability under external forces.

α-Catenin-dependent vinculin recruitment to adherens junctions is antagonistic to focal adhesions.

Vinculin is a protein found in both focal adhesions (FAs) and adherens junctions (AJs) which regulates actin connectivity to these structures. Many studies have demonstrated that mechanical perturbations of cells result in enhanced recruitment of vinculin to FAs and/or AJs. Likewise, many other studies have shown "cross-talk" between FAs and AJs. Vinculin itself has been suggested to be a probable regulator of this adhesion cross-talk. In this study we used MDCK as a model system of epithelia, developing cell lines in which vinculin recruitment was reduced or enhanced at AJs. Careful analysis of these cells revealed that perturbing vinculin recruitment to AJs resulted in a reduction of detectable FAs. Interestingly the cross-talk between these two structures was not due to a limited pool of vinculin, as increasing expression of vinculin did not rescue FA formation. Instead, we demonstrate that vinculin translocation between AJs and FAs is necessary for actin cytoskeleton rearrangements that occur during cell migration, which is necessary for large, well-formed FAs. Last, we show using a wound assay that collective cell migration is similarly hindered when vinculin recruitment is reduced or enhanced at AJs, highlighting that vinculin translocation between each compartment is necessary for efficient collective migration.

Epithelial Cell-Like Elasticity Modulates Actin-Dependent E-Cadherin Adhesion Organization.

E-cadherin adhesions are essential for cell-to-cell cohesion and mechanical coupling between epithelial cells and reside in a microenvironment that comprises the adjoining epithelial cells. While E-cadherin has been shown to be a mechanosensor, it is unknown if E-cadherin adhesions can differentially sense stiffness within the range of that of epithelial cells. A survey of literature shows that epithelial cells' Young's moduli of elasticity lie predominantly in the sub-kPa to few-kPa range, with cancer cells often being softer than noncancerous ones. Here, we devised oriented E-cadherin-coated soft silicone substrates with sub-kPa or few-kPa elasticity but with similar viscous moduli and found that E-cadherin adhesions differentially organize depending on the magnitude of epithelial cell-like elasticity. Our results show that the actin cytoskeleton organizes E-cadherin adhesions in two ways─by supporting irregularly shaped adhesions at localized regions of high actin density and linear shaped adhesions at the end of linear actin bundles. Linearly shaped E-cadherin adhesions associated with radially oriented actin─but not irregularly shaped E-cadherin adhesions associated with circumferential actin foci─were much more numerous on 2.4 kPa E-cadherin substrates compared to 0.3 kPa E-cadherin substrates. However, the total amount of E-cadherin in both types of adhesions taken together was similar on the 0.3 and 2.4 kPa E-cadherin substrates across many cells. Our results show how the distribution of E-cadherin adhesions, supported by actin density and architecture, is modulated by epithelial cell-like elasticity and have significant implications for disease states like carcinomas characterized by altered epithelial cell elasticity.