Cadherin-dependent epithelial cell-cell adhesion is thought to be regulated by Rho family small GTPases and PI 3-kinase, but the mechanisms involved are poorly understood. Using time-lapse microscopy and quantitative image analysis, we show that cell-cell contact in MDCK epithelial cells coincides with a spatio-temporal reorganization of plasma membrane Rac1 and lamellipodia from noncontacting to contacting surfaces. Within contacts, Rac1 and lamellipodia transiently concentrate at newest sites, but decrease at older, stabilized sites. Significantly, Rac1 mutants alter kinetics of cell-cell adhesion and strengthening, but not the eventual generation of cell-cell contacts. Products of PI 3-kinase activity also accumulate dynamically at contacts, but are not essential for either initiation or development of cell-cell adhesion. These results define a role for Rac1 in regulating the rates of initiation and strengthening of cell-cell adhesion.

Background: ARHGAP21 is an important Rho-GAP for Cdc42 involved in vesicle trafficking and focal adhesion kinase activity. Results: ARHGAP21 participates in cell-cell adhesion formation and cellular migration, interacts and modulates ␣-tubulin acetylation, and is essential for epithelial-mesenchymal transition. Conclusion: ARHGAP21 is a novel ␣-tubulin partner coordinating cell-cell adhesion, migration, and epithelial-mesenchymal transition. Significance: ARHGAP21 might be involved in cancer metastasis.

Cadherins mediate cell-cell adhesion by linking cell junctions to actin networks. Although several actin regulatory systems have been implicated in cell-cell adhesion, it remains unclear how such systems drive cadherin-actin network formation and how they are regulated to coincide with initiation of adhesion. Previous work implicated VASP in assembly of cell-cell junctions in keratinocytes and the VASP-binding protein zyxin colocalizes with VASP at cellcell junctions. Here we examine how domains in zyxin and its relative LPP contribute to cell-cell junction assembly. Using a quantitative assay for cell-cell adhesion, we demonstrate that zyxin and LPP function to increase the rate of early cell-cell junction assembly through the VASP-binding ActA repeat region. We also identify the LIM region of zyxin and LPP to be a regulatory domain that blocks function of these proteins. Deletion of the LIM domains drives adhesion and increases VASP level in detergent insoluble cadherinactin. Dominant-negative zyxin/LPP mutants reduce the rate of adhesion, lower VASP levels in detergent-insoluble cadherin-actin networks, and allow for the accumulation of capping protein at cellcell contacts. These data implicate the LIM domains of zyxin and LPP in regulating cell-cell junction assembly through VASP.Cadherin-mediated cell-cell adhesion integrates individual cells into tissues (1). Strong cell-cell adhesion is required to maintain cell morphology and tissue homeostasis (2). As individual cadherin-cadherin adhesion interactions are weak (3), strong adhesion is thought to require incorporation of cadherin into actin networks (4, 5). The cytoplasmic domain of cadherin binds either ␤-catenin or plakoglobin (6), which in turn binds ␣-catenin (7, 8). ␣-Catenin links cadherin to actin filaments either directly (8) or by binding the actin-binding proteins vinculin (9) or ␣-actinin (10). Cadherin anchors into detergent-insoluble actin networks concomitant with incorporation of ␣-catenin into cadherin complexes (4, 5). However, it remains unclear how binding of cadherin to actin filaments is regulated to coincide with cell-cell adhesion. Although cadherin has been demonstrated to induce dramatic changes in actin organization (11-13) and several actin regulatory programs have been generally implicated in cell-cell adhesion (13, 14), how actin rearrangements and cadherin engagement are coordinated remains poorly understood.Understanding actin assembly mechanisms has been greatly improved by studies of the intracellular pathogen, Listeria monocytogenes (15). Listeria ActA protein hijacks host actin polymerization machinery, directing assembly of actin comet tails that propel the bacterium through the cytosol (16,17). In addition to actin nucleation by the Arp2/3 complex, normal motility requires the activity of VASP family members (18) that are recruited via an array of proline-rich motifs termed ActA repeats (19). It has been proposed that VASP competes with capping protein (CapZ) for available barbed ends of actin filaments, protecting the...

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...