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© The Rockefeller University Press, 0021-9525/1999//1245 $5.00
The Journal of Cell Biology, Volume 144, Number 6, , 1999 1245-1258

Role of Proteins of the Ena/VASP Family in Actin-based Motility of Listeria monocytogenes

Valérie Laurent^*, Thomas P. Loisel^*, Birgit Harbeck, Ann Wehman, Lothar Gröbe^||, Brigitte M. Jockusch, Jürgen Wehland^||, Frank B. Gertler, and Marie-France Carlier^*

^* Dynamique du Cytosquelette, Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, 91198 Gif-sur-Yvette, France; Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; Cell Biology, Zoological Institute, Technical University, 38092 Braunschweig, Germany; and ^|| National Research Center for Biotechnology, 38124 Braunschweig, Germany

Intracellular propulsion of Listeria monocytogenes is the best understood form of motility dependent on actin polymerization. We have used in vitro motility assays of Listeria in platelet and brain extracts to elucidate the function of the focal adhesion proteins of the Ena (Drosophila Enabled)/VASP (vasodilator-stimulated phosphoprotein) family in actin-based motility. Immunodepletion of VASP from platelet extracts and of Evl (Ena/VASP-like protein) from brain extracts of Mena knockout (–/–) mice combined with add-back of recombinant (bacterial or eukaryotic) VASP and Evl show that VASP, Mena, and Evl play interchangeable roles and are required to transform actin polymerization into active movement and propulsive force. The EVH1 (Ena/VASP homology 1) domain of VASP is in slow association–dissociation equilibrium high-affinity binding to the zyxin-homologous, proline-rich region of ActA. VASP also interacts with F-actin via its COOH-terminal EVH2 domain. Hence VASP/ Ena/Evl link the bacterium to the actin tail, which is required for movement. The affinity of VASP for F-actin is controlled by phosphorylation of serine 157 by cAMP-dependent protein kinase. Phospho-VASP binds with high affinity (0.5 x 10⁸ M^–1); dephospho-VASP binds 40-fold less tightly. We propose a molecular ratchet model for insertional polymerization of actin, within which frequent attachment–detachment of VASP to F-actin allows its sliding along the growing filament.

Key Words: actin polymerization • Listeria monocytogenes • Ena/VASP/Mena • motility • platelet

Abbreviations used in this paper: Ena, Drosophila Enabled protein; VASP, vasodilator-stimulated phosphoprotein.

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