RESUMO
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 10(8) 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.
Assuntos
Actinas/fisiologia , Moléculas de Adesão Celular/fisiologia , Proteínas Contráteis , Proteínas do Citoesqueleto , Proteínas de Ligação a DNA/fisiologia , Listeria monocytogenes/fisiologia , Fosfoproteínas/fisiologia , Actinas/química , Actinas/ultraestrutura , Animais , Sequência de Bases , Sítios de Ligação , Plaquetas/metabolismo , Encéfalo/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Moléculas de Adesão Celular/genética , Primers do DNA/genética , Proteínas de Ligação a DNA/genética , Listeria monocytogenes/genética , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/fisiologia , Microscopia Eletrônica , Modelos Biológicos , Movimento/fisiologia , Fosfoproteínas/genética , Profilinas , Ligação Proteica , Proteínas/genética , Proteínas/fisiologiaRESUMO
Mammalian enabled (Mena) is a member of a protein family thought to link signal transduction pathways to localized remodeling of the actin cytoskeleton. Mena binds directly to Profilin, an actin-binding protein that modulates actin polymerization. In primary neurons, Mena is concentrated at the tips of growth cone filopodia. Mena-deficient mice are viable; however, axons projecting from interhemispheric cortico-cortical neurons are misrouted in early neonates, and failed decussation of the corpus callosum as well as defects in the hippocampal commissure and the pontocerebellar pathway are evident in the adult. Mena-deficient mice that are heterozygous for a Profilin I deletion die in utero and display defects in neurulation, demonstrating an important functional role for Mena in regulation of the actin cytoskeleton.
