University of Birmingham The small GTPases Rho and Rac are required for the establishment of cadherin-dependent cell-cell contacts

Cadherins are calcium-dependent cell–cell adhesion molecules that require the interaction of the cytoplasmic tail with the actin cytoskeleton for adhesive activity. Because of the functional relationship between cadherin receptors and actin filament organization, we investigated whether members of the Rho family of small GTPases are necessary for cadherin adhesion. In fibroblasts, the Rho family members Rho and Rac regulate actin polymerization to produce stress fibers and lamellipodia, respectively. In epithelial cells, we demonstrate that Rho and Rac are required for the establishment of cadherin-mediated cell–cell adhesion and the actin reorganization necessary to stabilize the receptors at sites of intercellular junctions. Blocking endogenous Rho or Rac selectively removed cadherin complexes from junctions induced for up to 3 h, while desmosomes were not perturbed. In addition, withdrawal of cadherins from intercellular junctions temporally precedes the removal of CD44 and integrins, other microfilament-associated receptors. Our data showed that the concerted action of Rho and Rac modulate the establishment of cadherin adhesion: a constitutively active form of Rac was not sufficient to stabilize cadherindependent cell–cell contacts when endogenous Rho was inhibited. Upon induction of calcium-dependent intercellular adhesion, there was a rapid accumulation of actin at sites of cell–cell contacts, which was prevented by blocking cadherin function, Rho or Rac activity. However, if cadherin complexes are clustered by specific antibodies attached to beads, actin recruitment to the receptors was perturbed by inhibiting Rac but not Rho. Our results provide new insights into the role of the small GTPases in the cadherin-dependent cell– cell contact formation and the remodelling of actin filaments in epithelial cells. A dhesion of cells to their neighbors is an important process for the differentiation of multicellular organisms. During embryonic development and adult life, the cadherin superfamily of cell–cell adhesion receptors has been implicated in many processes such as cell segregation, proliferation, differentiation, and invasive behavior (for review see Takeichi, 1991, 1993). In epithelial tissues, cadherins are required for the assembly of cells into multiple layers and the establishment and maintenance of the epithelial phenotype (Rodriguez-Boulan and Nelson, 1989; Wheelock and Jensen, 1992; Hodivala and Watt, 1994; Lewis et al., 1994). Functional cadherin receptors can influence the reorganization of the microfilament, microtubule, and intermediate cytoskeleton, the distribution of cytoplasmic and transmembrane proteins in a polarized fashion, and the formation of gap junctions and other adhesive structures (desmosomes and tight junctions; Green et al., 1987; Magee et al., 1987; Gumbiner et al., 1988; Mege et al., 1988; McNeill et al., 1990; Jongen et al., 1991; Zamansky et al., 1991; Lewis et al., 1994; Amagai et al., 1995). Cadherins are transmembrane molecules that bind to the same type of cadherin in adjacent cells (homophilic binding; Takeichi, 1991). Their adhesive function requires calcium ions and the association of the cytoplasmic tail with the actin cytoskeleton, which is mediated by the interaction with intracellular proteins called catenins ( a -catenin and the armadillo proteins b -catenin, plakoglobin, and p120; for review see Cowin and Burke, 1996). In addition to their role in cadherin-mediated adhesion, the armadillo proteins also bind to other cytoplasmic and nuclear partners and participate in signal transduction events (Rubinfeld et al., 1993, 1996; Su et al., 1993; Behrens et al., 1996; for review see Gumbiner, 1995). Binding to different partners (including the cadherin cytoplasmic tail) occurs via repeats of 42 amino acids, a new protein–protein interaction motif ( armadillo repeats; Peifer et al., 1994 a ). It is believed that the association of cadherin–catenin complexes N.A. Hotchin’s present address is School of Biochemistry, University of Birmingham, Birmingham, B15 2TT, U.K. Please address all correspondence to Vania M.M. Braga, MRC-Laboratory for Molecular Cell Biology, University College London, Gower Street, WCIE 6BT, London, U.K. Tel. : (44) 171-380-7814; Fax: (44) 171380-7805; E-mail: v.braga@ucl.ac. uk