Contribution of Ena/VASP Proteins to Intracellular Motility ofListeriaRequires Phosphorylation and Proline-rich Core but Not F-Actin Binding or Multimerization

Abstract

The Listeria model system has been essential for the identification and characterization of key regulators of the actin cytoskeleton such as the Arp2/3 complex and Ena/vasodilator-stimulated phosphoprotein (VASP) proteins. Although the role of Ena/VASP proteins in Listeria motility has been extensively studied, little is known about the contributions of their domains and phosphorylation state to bacterial motility. To address these issues, we have generated a panel of Ena/ VASP mutants and, upon expression in Ena/VASP-deficient cells, evaluated their contribution to Ena/VASP function in Listeria motility. The proline-rich region, the putative G-actin binding site, and the Ser/Thr phosphorylation of Ena/VASP proteins are all required for efficient Listeria motility. Surprisingly, the interaction of Ena/VASP proteins with F-actin and their potential ability to form multimers are both dispensable for their involvement in this process. Our data suggest that Ena/VASP proteins contribute to Listeria motility by regulating both the nucleation and elongation of actin filaments at the bacterial surface.

The bundling activity of vasodilator-stimulated phosphoprotein is required for filopodium formation

“…Cells of D. discoideum AX2 WT strain and of derived transformants were cultivated and transformed by electroporation as described (41). MV D7 cells and MV D7 cells stably expressing GFP-tagged human VASP variants were described previously (42). DdVASP was cloned into a pEGFP-C vector (CLONTECH) and expressed in mouse embryonic fibroblasts immortalized by using a temperature-sensitive variant of the large T antigen and growing at 32°C as described previously (43).…”

The arsenal of virulence factors deployed byListeria monocytogenesto promote its cell infection cycle

“…170,172 VASP plays a dual role in Listeria actin tail formation: on one hand, it recruits profilin, an actin monomer-binding protein, that promotes actin polymerization; and on the other hand, it reduces the frequency of actin filament branching, favoring the assembly of long parallel filaments. 173,174 The interaction of ActA with VASP plays a key role in the infection because ActA mutants that do not interact with VASP show a defect in intercellular spread and are avirulent in the mouse model of infection. 172 A p60 mutant (see invasion section) was first shown to have intracellular motility and cell-to-cell spread levels drastically reduced in fibroblast and epithelial cell lines, since it failed to promote the formation of actin tails.…”

Ena/VASP proteins mediate repulsion from ephrin ligands

“…The fact that the level of repulsion was further decreased in MV D7 cells suggests that Mena acts partially redundantly with VASP, Journal of Cell Science 120 (2) similarly to its role in regulation of fibroblast and Listeria motility (Geese et al, 2002;Laurent et al, 1999;. Fibroblast cell lines are not thought to express the other vertebrate Ena/VASP protein, Evl (Lambrechts et al, 2000), and so Evl is unlikely to contribute to repulsion in fibroblast-derived cell types.…”

“…The Ena/VASP family consists of VASP (Ddx4; Mouse Genome Informatics), Mena (Enah; MGI) and Evl in vertebrates (Krause et al, 2003), each of which can function interchangeably, at least with respect to their roles in the regulation of fibroblast and Listeria motility (Geese et al, 2002;Laurent et al, 1999;Loureiro et al, 2002). In addition to these roles, this family has been implicated downstream of several repulsive guidance cue receptors, including Robo, Dlar and Sema6D (Bashaw et al, 2000;Toyofuku et al, 2004;Wills et al, 1999), but as yet there is no evidence suggesting a link downstream of Eph receptors.…”