α-catenin cytomechanics--role in cadherin-dependent adhesion and mechanotransduction.

The findings presented here demonstrate the role of α-catenin in cadherin-based adhesion and mechanotransduction in different mechanical contexts. Bead-twisting measurements in conjunction with imaging, and the use of different cell lines and α-catenin mutants reveal that the acute local mechanical manipulation of cadherin bonds triggers vinculin and actin recruitment to cadherin adhesions in an actin- and α-catenin-dependent manner. The modest effect of α-catenin on the two-dimensional binding affinities of cell surface cadherins further suggests that force-activated adhesion strengthening is due to enhanced cadherin-cytoskeletal interactions rather than to α-catenin-dependent affinity modulation. Complementary investigations of cadherin-based rigidity sensing also suggest that, although α-catenin alters traction force generation, it is not the sole regulator of cell contractility on compliant cadherin-coated substrata.

Vinculin-dependent Cadherin mechanosensing regulates efficient epithelial barrier formation.

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.

Cadherin-dependent mechanotransduction depends on ligand identity but not affinity.

This study investigates the relationship between classical cadherin binding affinities and mechanotransduction through cadherin-mediated adhesions. The mechanical properties of cadherin-dependent intercellular junctions are generally attributed to differences in the binding affinities of classical cadherin subtypes that contribute to cohesive energies between cells. However, cell mechanics and mechanotransduction may also regulate intercellular contacts. We used micropipette measurements to quantify the two-dimensional affinities of cadherins at the cell surface, and two complementary mechanical measurements to assess ligand-dependent mechanotransduction through cadherin adhesions. At the cell surface, the classical cadherins investigated in this study form both homophilic and heterophilic bonds with two-dimensional affinities that differ by less than threefold. In contrast, mechanotransduction through cadherin adhesions is strongly ligand dependent such that homophilic, but not heterophilic ligation mediates mechanotransduction, independent of the cadherin binding affinity. These findings suggest that ligand-selective mechanotransduction may supersede differences in cadherin binding affinities in regulating intercellular contacts.

Cadherin point mutations alter cell sorting and modulate GTPase signaling.

This study investigated the impact of cadherin binding differences on both cell sorting and GTPase activation. The use of N-terminal domain point mutants of Xenopus C-cadherin enabled us to quantify binding differences and determine their effects on cadherin-dependent functions without any potential complications arising as a result of differences in cytodomain interactions. Dynamic cell-cell binding measurements carried out with the micropipette manipulation technique quantified the impact of these mutations on the two-dimensional binding affinities and dissociation rates of cadherins in the native context of the cell membrane. Pairwise binding affinities were compared with in vitro cell-sorting specificity and ligation-dependent GTPase signaling. Two-dimensional affinity differences greater than five-fold correlated with cadherin-dependent in vitro cell segregation, but smaller differences failed to induce cell sorting. Comparison of the binding affinities with GTPase signaling amplitudes further demonstrated that differential binding also proportionally modulates intracellular signaling. These results show that differential cadherin affinities have broader functional consequences than merely controlling cell-cell cohesion.