Molecular Mechanism for Orienting Membrane and Actin Dynamics to Nascent Cell-Cell Contacts in Epithelial Cells

Abstract

The small GTPase Rac1 has been implicated in regulation of cell migration and cell-cell adhesion in epithelial cells. Little is known, however, about the spatial and temporal coordination of Rac1 activity required to balance these competing processes. We fractionated endogenous Rac1-containing protein complexes from membranes of Madin-Darby canine kidney cells and identified three major complexes comprising a Rac1⅐PAK (p21-activated kinase) complex, and 11 S and 16 S Rac1 complexes. Significantly, Rac1 shifts from the 11 S to a 16 S particle during initiation of cell-cell adhesion. This shift may reflect a diffusion trapping mechanism by which these Rac1 complexes are localized to cadherin-mediated cell-cell contacts through an interaction with annexin II.The actin cytoskeleton is highly regulated and organized. Although recent work has provided great insight into how actin dynamics are regulated in cells, mechanisms linking spatial organization with specific functions remain poorly understood. Migratory cells assemble actin-rich lamellae and filopodia at the leading edge, a distinct subdomain of the plasma membrane, ensuring directional movement. In fibroblasts, the small GTPases Rac1 and Cdc42 regulate formation of lamellae and filopodia, respectively (1). In epithelial cells, membrane dynamics observed at developing cell-cell contacts are reminiscent of those observed at the leading edge (2-4). Levels of active Cdc42 and Rac1 increase following initiation of cadherinmediated adhesion (5-7). Rac1 accumulation within cell-cell contacts is juxtaposed with high levels of PI 1 3-kinase products and greatest lamellae activity and occurs concomitantly with depletion of Rac1 from non-contacting membranes (2). However, molecular mechanisms that specify Rac1 localization and retention at cell-cell contacts are not understood.To investigate how Rho family members become localized to cell-cell contacts, we isolated protein complexes containing endogenous Rac1 from membranes of MDCK cells undergoing cell-cell contact formation. We demonstrate that Rac1 shifts from an 11 S to a 16 S particle in response to cadherin-based adhesion. This shift requires PI 3-kinase activity and occurs concomitantly with recruitment and retention of Rac1 at nascent cell contacts. We also show that annexin II interacts with Rac1 complexes and that its localization to cell-cell contacts also requires PI-3 kinase, implicating annexin II as the determinant of specific Rac1 localization.Annexins are peripheral membrane proteins that bind anionic phospholipids, such as PI 3-kinase products, in a Ca 2ϩ -dependent manner (8). Annexin II has been identified in preparations of lamellae (9), implicated in regulation of migration (10), and observed in actin comet tails of rocketing macropinocytic vesicles where it plays a critical, but unidentified, role (11). Annexin II also binds CD44/H-CAM, resulting in lamellipodia formation in a Rac1-dependent manner (12). EXPERIMENTAL PROCEDURESCell Culture-MDCK cells were cultured in Dulbecco's modified Eagle's med...

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