RAC1 regulates adherens junctions through endocytosis of E-cadherin

The establishment of cadherin-dependent cell-cell contacts in human epiderm al keratinocytes are known to be regulated by the Rac1 small GTP-binding pr otein, although the mechanisms by which Rac1 participates in the assembly o r disruption of cell-cell adhesion are not well understood. In this study w e utilized green fluorescent protein (GFP)-tagged Rac1 expression vectors t o examine the subcellular distribution of Rac1 and its effects on E-cadheri n-mediated cell-cell adhesion. Microinjection of keratinocytes with constit utively active Rac1 resulted in cell spreading and disruption of cell-cell contacts. The ability of Rac1 to disrupt cell-cell adhesion was dependent o n colony size, with large established colonies being resistant to the effec ts of active Rac1. Disruption of cell-cell contacts in small preconfluent c olonies was achieved through the selective recruitment of E-cadherin-cateni n complexes to the perimeter of multiple large intracellular vesicles, whic h were bounded by GFP-tagged L61Rac1. Similar vesicles were observed in non injected keratinocytes when cell-cell adhesion was disrupted by removal of extracellular calcium or with the use of an E-cadherin blocking antibody. M oreover, formation of these structures in noninjected keratinocytes was dep endent on endogenous Rac1 activity. Expression of GFP-tagged effector mutan ts of Rac1 in keratinocytes demonstrated that reorganization of the actin c ytoskeleton was important for vesicle formation. Characterization of these Rac1-induced vesicles revealed that they were endosomal in nature and tight ly colocalized with the transferrin receptor, a marker for recycling endoso mes. Expression of GFP-L61Rac1 inhibited uptake of transferrin-biotin, sugg esting that the endocytosis of E-cadherin was a clathrin-independent mechan ism. This was supported by the observation that caveolin, but not clathrin, localized around these structures. Furthermore, an inhibitory form of dyna min, known to inhibit internalization of caveolae, inhibited formation of c adherin vesicles. Our data suggest that Rac1 regulates adherens junctions v ia clathrin independent endocytosis of E-cadherin.