Requirement of a Vasodilator-stimulated Phosphoprotein Family Member for Cell Adhesion, the Formation of Filopodia, and Chemotaxis in Dictyostelium* 210

We have examined the function of a member of the vasodilator-stimulated phosphoprotein family of proteins (DdVASP) inDictyostelium. 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 ∼50% 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.

[1]  P. Cossart,et al.  Inducible recruitment of Cdc42 or WASP to a cell-surface receptor triggers actin polymerization and filopodium formation , 1999, Current Biology.

[2]  R. Firtel,et al.  Paka, a Putative Pak Family Member, Is Required for Cytokinesis and the Regulation of the Cytoskeleton in Dictyostelium discoideum Cells during Chemotaxis , 1999, The Journal of cell biology.

[3]  U. Walter,et al.  Vasodilator-stimulated protein phosphorylation in platelets is mediated by cAMP- and cGMP-dependent protein kinases. , 1987, European journal of biochemistry.

[4]  James E Bear,et al.  Negative Regulation of Fibroblast Motility by Ena/VASP Proteins , 2000, Cell.

[5]  C. Goodman,et al.  Repulsive Axon Guidance Abelson and Enabled Play Opposing Roles Downstream of the Roundabout Receptor , 2000, Cell.

[6]  C. Nobes,et al.  Rho GTPases: molecular switches that control the organization and dynamics of the actin cytoskeleton. , 2000, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[7]  J. Cooper,et al.  Cell motility: Bare bones of the cytoskeleton , 1999, Nature.

[8]  R. Firtel,et al.  The molecular genetics of chemotaxis: sensing and responding to chemoattractant gradients , 2000, BioEssays : news and reviews in molecular, cellular and developmental biology.

[9]  J. Condeelis,et al.  Relationship of pseudopod extension to chemotactic hormone‐induced actin polymerization in amoeboid cells , 1988, Journal of cellular biochemistry.

[10]  J. Murray,et al.  Three-dimensional dynamics of pseudopod formation and the regulation of turning during the motility cycle of Dictyostelium. , 1994, Cell motility and the cytoskeleton.

[11]  D R Soll,et al.  Three-dimensional reconstruction and motion analysis of living, crawling cells. , 2006, Scanning.

[12]  D. Soll,et al.  Motion Analysis of Living Cells , 1997 .

[13]  Gary G. Borisy,et al.  Antagonism between Ena/VASP Proteins and Actin Filament Capping Regulates Fibroblast Motility , 2002, Cell.

[14]  A. Aszódi,et al.  The vasodilator‐stimulated phosphoprotein (VASP) is involved in cGMP‐ and cAMP‐mediated inhibition of agonist‐induced platelet aggregation, but is dispensable for smooth muscle function , 1999, The EMBO journal.

[15]  K. Rottner,et al.  VASP dynamics during lamellipodia protrusion , 1999, Nature Cell Biology.

[16]  J. Spudich,et al.  Structure and function of the cytoskeleton of a Dictyostelium myosin- defective mutant , 1990, The Journal of cell biology.

[17]  P. Devreotes,et al.  Two cAMP receptors activate common signaling pathways in Dictyostelium. , 1994, Molecular biology of the cell.

[18]  F. Alt,et al.  Cdc42 is required for PIP2-induced actin polymerization and early development but not for cell viability , 2000, Current Biology.

[19]  G. Gerisch,et al.  Chemoattractant-controlled accumulation of coronin at the leading edge of Dictyostelium cells monitored using a green fluorescent protein–coronin fusion protein , 1995, Current Biology.

[20]  D. Purich,et al.  Profilin interacts with the Gly-Pro-Pro-Pro-Pro-Pro sequences of vasodilator-stimulated phosphoprotein (VASP): implications for actin-based Listeria motility. , 1997, Biochemistry.

[21]  H. Schwarz,et al.  Mechanism of phagocytosis in dictyostelium discoideum: phagocytosis is mediated by different recognition sites as disclosed by mutants with altered phagocytotic properties , 1980, The Journal of cell biology.

[22]  U. Walter,et al.  The EVH2 Domain of the Vasodilator-stimulated Phosphoprotein Mediates Tetramerization, F-actin Binding, and Actin Bundle Formation* , 1999, The Journal of Biological Chemistry.

[23]  J. Wehland,et al.  The bacterial actin nucleator protein ActA of Listeria monocytogenes contains multiple binding sites for host microfilament proteins , 1995, Current Biology.

[24]  Richard A. Firtel,et al.  Role of Phosphatidylinositol 3′ Kinase and a Downstream Pleckstrin Homology Domain–Containing Protein in Controlling Chemotaxis inDictyostelium , 2001, The Journal of cell biology.

[25]  M. Resh,et al.  Amino-terminal basic residues of Src mediate membrane binding through electrostatic interaction with acidic phospholipids. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[26]  Marie-France Carlier,et al.  Role of Proteins of the Ena/VASP Family in Actin-based Motility of Listeria monocytogenes , 1999, The Journal of cell biology.

[27]  J. Wehland,et al.  A novel proline‐rich motif present in ActA of Listeria monocytogenes and cytoskeletal proteins is the ligand for the EVH1 domain, a protein module present in the Ena/VASP family , 1997, The EMBO journal.

[28]  D. Soll,et al.  T cell syncytia induced by HIV release. T cell chemoattractants: demonstration with a newly developed single cell chemotaxis chamber. , 1998, Journal of cell science.

[29]  Lorene M Lanier,et al.  Mena Is Required for Neurulation and Commissure Formation , 1999, Neuron.

[30]  M. Titus,et al.  A role for myosin VII in dynamic cell adhesion , 2001, Current Biology.

[31]  P. Chavrier,et al.  A WASp–VASP complex regulates actin polymerization at the plasma membrane , 2001, The EMBO journal.

[32]  D. Knecht,et al.  Use of a fusion protein between GFP and an actin-binding domain to visualize transient filamentous-actin structures , 1998, Current Biology.

[33]  U. Walter,et al.  Identification, purification, and characterization of a zyxin-related protein that binds the focal adhesion and microfilament protein VASP (vasodilator-stimulated phosphoprotein). , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[34]  R. Firtel,et al.  Role of Rac in controlling the actin cytoskeleton and chemotaxis in motile cells. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[35]  J. Vandekerckhove,et al.  The mammalian profilin isoforms display complementary affinities for PIP2 and proline‐rich sequences , 1997, The EMBO journal.

[36]  U. Walter,et al.  Identification of profilin and src homology 3 domains as binding partners for Drosophila enabled. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[37]  L. Machesky.  Rocket-based motility: a universal mechanism? , 1999, Nature Cell Biology.