Ena/VASP Proteins Can Regulate Distinct Modes of Actin Organization at Cadherin-adhesive Contacts

Abstract

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...

G-protein-coupled signals control cortical actin assembly by controlling cadherin expression in the earlyXenopusembryo

“…Development 134 (14) It is already known that cadherin-containing punctae form at sites of initial contact of epithelial cells in culture Kobielak et al, 2004;Kovacs et al, 2002b;Scott et al, 2006;Vasioukhin et al, 2000;Verma et al, 2004), and that these associate with actin filaments and actin assembly components such as Arp2/3 (Kovacs et al, 2002b;Verma et al, 2004), formin 1 (Kobielak et al, 2004) and Ena/Vasp (Scott et al, 2006). Here we show, in a developing system in vivo, that such punctae and their associated actin filament assemblies form the skeleton of the early embryo.…”

“…E-cadherin engagement in cultured cells causes activation of Rac1 and Cdc42, both of which are involved in actin polymerization and organization (Betson et al, 2002;Kim et al, 2000;Kovacs et al, 2002a;Nakagawa et al, 2001;Noren et al, 2001). Actin nucleation proteins such as members of the Arp2/3 complex and formin 1 are associated with nascent cadherin-mediated adhesive contacts (Kobielak et al, 2004;Kovacs et al, 2002b;Verma et al, 2004), as are nucleationpromoting factors such as Ena/Vasp and cortactin Scott et al, 2006;Vasioukhin et al, 2000). However, the detailed mechanism by which cortical actin assembles at the cadherin complex is still unknown Drees et al, 2005;Perez-Moreno et al, 2003;Yamada et al, 2005).…”

Cortactin: Coordinating adhesion and the actin cytoskeleton at cellular protrusions

“…This is observed in cell culture [Adams et al, 1998] and also in Xenopus embryos, where oriented cell protrusion distinguishes morphogenetic cell-upon-cell locomotion during gastrulation [Keller, 2002]. Indeed, when cells are allowed to make cadherin-based adhesive contacts with substrata coated with cadherin ligands, they spread in two dimensions, as they do on matrix proteins, with cell protrusion at leading margins being the initiating event in this process [Kovacs et al, 2002b;Gavard et al, 2004;Scott et al, 2006]. As well, cell-cell contacts are sites for actin assembly [Vasioukhin et al, 2000] which is also evident at the protrusive leading margins of cells spreading on cadherin ligands [Kovacs et al, 2002b].…”

“…This implies that both these adhesion systems can coopt Arp2/3 depending on the cell's functional context. Moreover, the subcellular localization of Arp2/3 at both these forms of adhesive contacts is strikingly dynamic and stringently controlled, being apparently confined to the leading margins, despite the fact that adhesive ligand-receptor interactions occur at many other places, even within the same adhesive contacts [Scott et al, 2006]. Does such a shared module have a definable conserved function at both integrin and cadherin adhesions?…”

α-Actinin links LPP, but not zyxin, to cadherin-based junctions

“…VASP family members are actin regulatory proteins that localize to cell-cell junctions and have been implicated in cell-cell adhesion [8]. They are recruited to specific cellular sites through interactions with FPPPPP motifs [9].…”