Lateral Interactions Affect Cadherin Binding Kinetics and Function

Abstract

Adhesion protein interactions between cell membranes (2D) are indispensable for the formation and maintenance of tissues in multicellular organisms. However, binding and organization of membrane proteins within these interfaces is not well understood at the molecular level. To address this question, we use cell-cell adhesion proteins Cadherins as our model. Cadherins form adhesive interactions by binding to identical proteins on opposite cells, but are also hypothesized to form clusters on a single membrane. The protein-protein bonds proposed to stabilize these “lateral” interactions were not detected in any solution binding studies (3D), and their existence as well as their relevance for establishing cell-cell junctions and transducing signals are not clearly known. We investigated the relevance of these lateral interactions using quantitative micropipette measurements of cadherin-mediated cell-cell binding kinetics. We found that classical E-cadherin exhibits kinetics that exhibit two distinct kinetic processes. This ‘biphasic’ kinetics is not consistent with kinetic models based on solution binding data, and suggested that one of the kinetic steps might be due to clustering of the confined proteins. In support of this interpretation, mutating the proposed lateral interaction sites eliminated the kinetic step that we attributed to clustering. We also demonstrated the functional significance of this putative lateral clustering step, by quantifying the leakiness of cell-cell junctions to macromolecules. Cell junctions formed by the cadherin mutants were leakier than WT E-cadherin. Wound healing assays also showed that cells expressing the mutants migrated faster than cells with WT protein. Together, these data reveal that cadherins in “2D” environments undergo additional interactions than in solution, and these alter the assembly of intercellular junctions. These findings might also be relevant to similar class of adhesion proteins, like nectins and CAMs.