Requirement of a Vasodilator-stimulated Phosphoprotein Family Member for Cell Adhesion, the Formation of Filopodia, and Chemotaxis in Dictyostelium

Abstract

We have examined the function of a member of the vasodilator-stimulated phosphoprotein family of proteins (DdVASP) in Dictyostelium. Ddvasp null cells lack filopodia, whereas targeting DdVASP to the plasma membrane with a myristoyl tag results in a significant increase in filopodia. The proline-rich domain-Ena/ VASP homology 2 structure is required for both actin polymerization activity and filopodia formation. Ddvasp null cells exhibit a chemotaxis defect, which appears to be due to a defect in the ability of the cells to properly adhere to the substratum and to suppress lateral pseudopod extension. We demonstrate that during chemotaxis, the anterior ϳ50% of the cell lifts from the substratum and remains elevated for up to 1 min. These defects lead to a significant decrease in chemotaxis efficiency. DdVASP localizes to the leading edge in migrating cells and to the tips of filopodia. In addition, Ddvasp null cells have a defect in particle adhesion but internalize particles normally. Our results provide new insights into the function of DdVASP in controlling the actin cytoskeleton during chemotaxis and filopodia formation. Vasodilator-stimulated phosphoprotein (VASP)1 was originally identified as a protein phosphorylated in response to an elevation of intracellular cAMP or cGMP levels by cAMP-and cGMP-dependent protein kinase in human platelets (1). VASP is a member of the Ena/VASP family of proteins that share a domain structure composed of three conserved domains: an N-terminal Ena/VASP homology 1 (EVH1) domain; a central proline-rich, Src homology 3-binding domain (PRD); and a Cterminal EVH2 domain. The EVH1 domain, which is also present in Wiskott-Aldrich syndrome protein (WASP) and N-WASP, binds to proteins containing the consensus sequence FPPPP, such as Listeria monocytogenes surface protein ActA, and interacts with the PRD of WASP (2-4). The PRD mediates VASP's interaction with the Src homology 3 domain on Src and F-actin-binding protein profilin (5-8). The EVH2 domain, which is unique to the Ena/VASP family, mediates VASP's tetramerization, F-actin binding, and actin bundling (9).Involvement of VASP in the regulation of actin assembly and cell motility was suggested by the results of studies on actinbased movement of Listeria. Listeria movement was abolished in VASP-depleted platelet extracts and could be restored by the addition of recombinant VASP (10). VASP and EVL (Ena/ VASP-like protein) induce polymerization of G-actin into Factin bundles in in vitro assays in the presence of low salt and stabilize F-actin in a phosphorylation-dependent manner (10). As expected from the ability of VASP to induce actin polymerization, in vitro reconstitution of Listeria movement using purified proteins demonstrated that the speed of bacterial movement is greatly enhanced if VASP is added to the reconstitution mixture (11). These findings suggested that VASP enhances actin polymerization at a site on the Listeria surface to generate propelling power. The mechanism of membrane protrusion through localized actin poly...

Filopodia: Complex models for simple rods

“…In agreement with this, Ena/VASP proteins were shown to promote the growth of filopodia in different cell types (Han et al, 2002, Lebrand et al, 2004, Mejillano et al, 2004. Although not essential for filopodium formation in mammals per se (Dent et al, 2007, Mattila and, the recent elimination of all three Ena/VASP proteins resulted in a markedly reduced formation of filopodia in neuronal cells, and is consistent with the critical function of the single VASP member in Dictyostelium cells (Figure 2) (Dent et al, 2007, Han et al, 2002, Schirenbeck et al, 2006, arguing for a conserved function of Ena/VASP proteins in filopodia formation. Despite these findings, the precise function of these proteins in promoting actin assembly has remained controversial (Trichet et al, 2008).…”

Migfilin Interacts with Vasodilator-stimulated Phosphoprotein (VASP) and Regulates VASP Localization to Cell-Matrix Adhesions and Migration

“…37 and 38). In Dictyostelium, DdVASP promotes cell adhesion, filopodia formation, and directional cell movement (39). In the nervous system, members of the Ena/VASP protein family are required for proper neuronal migration and axon guidance (40 -48).…”

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

“…In Dictyostelium discoideum cells VASP localizes to the leading edge and the tips of filopodia and was found to play an important role in the formation of these structures (16,24). Mouse mDia1 FH2 interacts with full-length murine VASP, and this interaction is enhanced by the presence of the FH1 domain (24).…”