Mechanotransduction at cell-cell contacts

For nearly 40 years, it was debated as to whether the differences in cadherin- dependent cell-cell adhesion energies could solely predict cell sorting. Although adhesion energies do influence cell sorting in vitro, recent studies suggested that that differences in cell adhesion, determined mainly by cadherin binding affinities and expression levels, were not sufficient to predict cell sorting. This result suggested that other factors contribute. Besides their adhesive function, cadherins are also signaling proteins. Naturally, the next question is whether differences in downstream signaling associated with differences in cadherin affinity might also contribute to cell segregation. Chapter 2 of this dissertation specifically therefore investigates the relationship between cadherin affinities and signaling by small Rho GTPases, which are cytoskeletal regulatory proteins that influence actin polymerization and myosin dependent contractility. Recent findings also suggested that adhesion energies and cell mechanics together influence cell sorting, but the link between the two parameters had not been established. Chapter 3 builds on recent findings that cadherin complexes are also force transducers, and addresses whether differences in cadherin affinity also differentially alter cell mechanics. Studies discovered a significant difference between force transduction triggered by homophilic versus heterophilic cadherin bonds that could differentially influence cell mechanics. I further investigated the mechanism of force transduction at cadherin junctions, as described in Chapter 4 of this thesis. Chapter 4 specifically focused on the role of α-catenin—a key component in cadherin complexes—as a critical force transducer at cell-cell adhesion

A mechanically active heterotypic E-cadherin/N-cadherin adhesion enables fibroblasts to drive cancer cell invasion

Cancer-associated fibroblasts (CAFs) promote tumour invasion and metastasis. We show that CAFs exert a physical force on cancer cells that enables their collective invasion. Force transmission is mediated by a heterophilic adhesion involving N-cadherin at the CAF membrane and E-cadherin at the cancer cell membrane. This adhesion is mechanically active; when subjected to force it triggers β-catenin recruitment and adhesion reinforcement dependent on α-catenin/vinculin interaction. Impairment of E-cadherin/N-cadherin adhesion abrogates the ability of CAFs to guide collective cell migration and blocks cancer cell invasion. N-cadherin also mediates repolarization of the CAFs away from the cancer cells. In parallel, nectins and afadin are recruited to the cancer cell/CAF interface and CAF repolarization is afadin dependent. Heterotypic junctions between CAFs and cancer cells are observed in patient-derived material. Together, our findings show that a mechanically active heterophilic adhesion between CAFs and cancer cells enables cooperative tumour invasion

alpha-Catenin cytomechanics: role in cadherin-dependent adhesion and mechanotransduction

The findings presented here demonstrate the role of alpha-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 alpha-catenin mutants reveal that the acute local mechanical manipulation of cadherin bonds triggers vinculin and actin recruitment to cadherin adhesions in an actin-and alpha-catenin-dependent manner. The modest effect of alpha-catenin on the two-dimensional binding affinities of cell surface cadherins further suggests that forceactivated adhesion strengthening is due to enhanced cadherincytoskeletal interactions rather than to alpha-catenin-dependent affinity modulation. Complementary investigations of cadherin-based rigidity sensing also suggest that, although alpha-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

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

Deletion of the cytoplasmic domain of N-cadherin reduces, but does not eliminate, traction force-transmission

Collective migration of epithelial cells is an integral part of embryonic development, wound healing, tissue renewal and carcinoma invasion. While previous studies have focused on cell-extracellular matrix adhesion as a site of migration-driving, traction force-transmission, cadherin mediated cell-cell adhesion is also capable of force-transmission. Using a soft elastomer coated with purified N-cadherin as a substrate and a Hepatocyte Growth Factor-treated, transformed MDCK epithelial cell line as a model system, we quantified traction transmitted by N-cadherin-mediated contacts. On a substrate coated with purified extracellular domain of N-cadherin, cell surface N-cadherin proteins arranged into puncta. N-cadherin mutants (either the cytoplasmic deletion or actin-binding domain chimera), however, failed to assemble into puncta, suggesting the assembly of focal adhesion like puncta requires the cytoplasmic domain of N-cadherin. Furthermore, the cytoplasmic domain deleted N-cadherin expressing cells exerted lower traction stress than the full-length or the actin binding domain chimeric N-cadherin. Our data demonstrate that N-cadherin junctions exert significant traction stress that requires the cytoplasmic domain of N-cadherin, but the loss of the cytoplasmic domain does not completely eliminate traction force transmission