Immobilized dimers of N-cadherin-Fc chimera mimic cadherin-mediated cell contact formation: contribution of both outside-in and inside-out signals.

Cell adhesion receptors of the cadherin family are involved in various developmental processes, affecting cell adhesion and migration, and also cell proliferation and differentiation. In order to dissect the molecular mechanisms of cadherin-based cell-cell adhesion and subsequent signal transduction to the cytoskeleton and/or cytoplasm leading to adapted cell responses, we developed an approach allowing us to mimic and control cadherin activation. We produced a dimeric N-cadherin-Fc chimera (Ncad-Fc) which retains structural and functional properties of cadherins, including glycosylation, Ca(2+)-dependent trypsin sensitivity and the ability to mediate Ca(2+)-dependent self-aggregation of covered microbeads. Beads covered with either Ncad-Fc or anti-N-cadherin antibodies specifically bound to N-cadherin expressing cells. Both types of beads induced the recruitment of N-cadherin, beta-catenin, alpha-catenin and p120, by lateral mobilization of preexisting cell membrane complexes. Furthermore, cadherin clustering elicited by Ncad-Fc beads triggered local accumulations of tyrosine phosphorylated proteins, a recruitment and redistribution of actin filaments, as well as local membrane remodeling. These results support a model where the adhesion of cadherin ectodomains is followed by clustering of cadherin/catenin complexes allowing signal transduction affecting both cytoskeletal reorganization and cytoplasmic signal mobilization (outside-in signaling). Interestingly, bead-cell binding was altered by agents promoting microfilament and microtubule depolymerization or tyrosine phosphorylation, indicating a possible regulation of the adhesive properties of the extracellular domain of N-cadherin by intracellular factors (inside-out signaling).