Classical cadherin adhesion molecules are fundamental determinants of cell-cell recognition that function in cooperation with the actin cytoskeleton. Productive cadherin-based cell recognition is characterized by a distinct morphological process of contact zone extension, where limited initial points of adhesion are progressively expanded into broad zones of contact. We recently demonstrated that E-cadherin ligation recruits the Arp2/3 actin nucleator complex to the plasma membrane in regions where cell contacts are undergoing protrusion and extension. This suggested that Arp2/3 might generate the protrusive forces necessary for cell surfaces to extend upon one another during contact assembly. We tested this hypothesis in mammalian cells by exogenously expressing the CA region of N-WASP. This fragment, which potently inhibits Arp2/3-mediated actin assembly in vitro, also effectively reduced actin assembly at cadherin adhesive contacts. Blocking Arp2/3 activity by this strategy profoundly reduced the ability of cells to extend cadherin adhesive contacts but did not affect cell adhesiveness. These findings demonstrate that Arp2/3 activity is necessary for cells to efficiently extend and assemble cadherin-based adhesive contacts.

Functional interactions between classical cadherins and the actin cytoskeleton involve diverse actin activities, including filament nucleation, cross-linking, and bundling. In this report, we explored the capacity of Ena/VASP proteins to regulate the actin cytoskeleton at cadherin-adhesive contacts. We extended the observation that Ena/vasodilator-stimulated phosphoprotein (VASP) proteins localize at cell-cell contacts to demonstrate that E-cadherin homophilic ligation is sufficient to recruit Mena to adhesion sites. Ena/VASP activity was necessary both for F-actin accumulation and assembly at cell-cell contacts. Moreover, we identified two distinct pools of Mena within individual homophilic adhesions that cells made when they adhered to cadherin-coated substrata. These Mena pools localized with Arp2/3-driven cellular protrusions as well as at the tips of cadherin-based actin bundles. Importantly, Ena/VASP activity was necessary for both modes of actin activity to be expressed. Moreover, selective depletion of Ena/VASP proteins from the tips of cadherinbased bundles perturbed the bundles without affecting the protrusive F-actin pool. We propose that Ena/VASP proteins may serve as higher order regulators of the cytoskeleton at cadherin contacts through their ability to modulate distinct modes of actin organization at those contacts. INTRODUCTIONCadherins are a family of calcium-dependent cell-cell adhesion molecules that regulate the actin cytoskeleton. On productive ligation of their adhesive ectodomains, classical cadherins activate cellular responses necessary for a range of morphogenetic processes, including cell sorting and tissue cohesion (Tepass et al., 2000), cell-upon-cell locomotion (Brieher and Gumbiner, 1994), and the coordination of cell migration in dorsal closure and wound repair (Danjo and Gipson, 1998). These morphogenetic responses likely reflect complex functional and biochemical interactions between the cadherin, its associated catenins, cell signaling pathways, and the cytoskeleton. Dynamic activity of the actin cytoskeleton, in particular, must be coordinated with surface adhesion to provide local protrusive force and mechanical stability at cell-cell contacts (Takeichi, 1991;Gumbiner, 1996;Yap et al., 1997). The challenge, then, is to define key molecular regulators responsible for these functional interrelationships.The capacity for cadherins to regulate actin was first suggested by the extensive alterations in actin filament distribution that occur when epithelial cells adhere to one another (Yonemura et al., 1995;Adams et al., 1996). It subsequently became evident that multiple forms of actin activity occur at cadherin contacts, including de novo nucleation and crosslinking, that must presumably be stringently coordinated with the dynamic state of adhesive contacts (Yonemura et al., 1995;Adams et al., 1996;Vasioukhin et al., 2000; WatermanStorer et al., 2000;Kobielak et al., 2004;Verma et al., 2004). Indeed, a growing corpus of actin-regulatory proteins have been identified at cadherin-b...

SummaryCinderella no longer: a a a a-catenin steps out of cadherin's shadow