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.

In epithelial tissues, cells are linked to their neighbors through specialized cell-cell adhesion proteins. E-cadherin is one of the most important membrane proteins for the establishment of intimate cell-cell contacts, but the molecular mechanism by which it is recruited to contact sites is largely unknown. We report here that the cytoplasmic domain of E-cadherin interacts with C3G, a guanine nucleotide exchange factor for Rap1. In epithelial cell cultures, ligation of the extracellular domain of E-cadherin enhances Rap1 activity, which in turn is necessary for the proper targeting of E-cadherin molecules to maturing cell-cell contacts. Furthermore, our data suggest that Cdc42 functions downstream of Rap1 in this process. We conclude that Rap1 plays a vital role in the establishment of E-cadherin-based cell-cell adhesion.

Cadherins are cell–cell adhesion receptors whose adhesive function requires their association with the actin cytoskeleton via proteins called catenins. The small guanosine triphosphatases (GTPases), Rho and Rac, are intracellular proteins that regulate the formation of distinct actin structures in different cell types. In keratinocytes and in other epithelial cells, Rho and Rac activities are required for E-cadherin function. Here we show that the regulation of cadherin adhesiveness by the small GTPases is influenced by the maturation status of the junction and the cellular context. E-cadherin localization was disrupted in mature keratinocyte junctions after inhibition of Rho and Rac. However, an incubation of 2 h was required after GTPase inhibition, when compared with newly established E-cadherin contacts (30 min). Regarding other cadherin receptors, P-cadherin was effectively removed from mature keratinocytes junctions by blocking Rho or Rac. In contrast, VE-cadherin localization at endothelial junctions was independent of Rho/Rac activity. We demontrate that the insensitivity of VE-cadherin to inhibition of Rho and Rac was not due to the maturation status of endothelial junction, but rather the cellular background: when transfected into CHO cells, the localization of VE-cadherin was perturbed by inhibition of Rho proteins. Our results suggest that the same stimuli may have different activity in regulating the paracellular activity in endothelial and epithelial cells. In addition, we uncovered possible roles for the small GTPases during the establishment of E-cadherin–dependent contacts. In keratinocytes, Rac activation by itself cannot promote accumulation of actin at the cell periphery in the absence of cadherin-dependent contacts. Moreover, neither Rho nor Rac activation was sufficient to redistribute cadherin molecules to cell borders, indicating that redistribution results mostly from the homophilic binding of the receptors. Our results point out the complexity of the regulation of cadherin-mediated adhesion by the small GTPases, Rho and Rac.

The ability of epithelial cells to polarize requires cell-cell adhesion mediated by cadherin receptors. During cell-cell contact, the mechanism via which a flat, spread cell shape is changed into a tall, cuboidal epithelial morphology is not known. We found that cadherin-dependent adhesion modulates actin dynamics by triggering changes in actin organization both locally at junctions and within the rest of the cell. Upon induction of cell-cell contacts, two spatial actin populations are distinguishable: junctional actin and peripheral thin bundles. With time, the relative position of these two populations changes and becomes indistinguishable to form a cortical actin ring that is characteristic of mature, fully polarized epithelial cells. Junctional actin and thin actin bundles differ in their actin dynamics and mechanism of formation, and interestingly, have distinct roles during epithelial polarization. Whereas junctional actin stabilizes clustered cadherin receptors at cell-cell contacts, contraction of peripheral actin bundle is essential for an increase in the maximum height at the lateral domain during polarization (cuboidal morphology). Thus, both junctional actin and thin bundles are necessary, and cooperate with each other to generate a polarized epithelial morphology.