Indexed on: 14 Feb '06Published on: 14 Feb '06Published in: Methods in enzymology
Cell-cell adhesion can occur in a calcium-dependent or calcium-independent manner, depending on the type of receptor involved. Establishment of cell contacts by either type of cell-cell adhesion (calcium-dependent or calcium-independent) has been shown to activate Rho GTPases in different cells. In this chapter, we describe the method used to assess the activation of Rho GTPases by cadherins, the prototype calcium-dependent adhesion receptor in epithelial cells. We cover the optimal cell culture conditions and controls to ensure that the activation of the GTPases is specifically triggered by the formation of cadherin-dependent cell-cell contacts. Controls described herein determine the specificity of activation of Rho proteins with respect to cadherin adhesion and exclude the contribution of other adhesive receptors, calcium-signaling, cell spreading, and migration. Although we focus on cadherin receptors and normal human keratinocytes as our model system, the methods described can be easily adapted to other adhesion receptors and different cell types.