title: A biomimetic motility assay provides insight into the mechanism of actin-based motility.
abstract: Abiomimetic motility assay is used to analyze the mechanism of force production by site-directed polymerization of actin. Polystyrene microspheres, functionalized in a controlled fashion by the N-WASP protein, the ubiquitous activator of Arp2/3 complex, undergo actin-based propulsion in a medium that consists of five pure proteins. We have analyzed the dependence of velocity on N-WASP surface density, on the concentration of capping protein, and on external force. Movement was not slowed down by increasing the diameter of the beads (0.2 to 3 microm) nor by increasing the viscosity of the medium by 10(5)-fold. This important result shows that forces due to actin polymerization are balanced by internal forces due to transient attachment of filament ends at the surface. These forces are greater than the viscous drag. Using Alexa488-labeled Arp2/3, we show that Arp2/3 is incorporated in the actin tail like G-actin by barbed end branching of filaments at the bead surface, not by side branching, and that filaments are more densely branched upon increasing gelsolin concentration. These data support models in which the rates of filament branching and capping control velocity, and autocatalytic branching of filament ends, rather than filament nucleation, occurs at the particle surface.
authors.affiliation: Dynamique du cytosquelette, Laboratoire d'Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette, France.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Wiesner
authors.forename: Emmanuele
authors.initials: E
authors.lastname: Helfer
authors.forename: Dominique
authors.initials: D
authors.lastname: Didry
authors.forename: Guylaine
authors.initials: G
authors.lastname: Ducouret
authors.forename: Françoise
authors.initials: F
authors.lastname: Lafuma
authors.forename: Marie-France
authors.initials: MF
authors.lastname: Carlier
authors.forename: Dominique
authors.initials: D
authors.lastname: Pantaloni
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D051377:Actin-Related Protein 2; D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002465:Cell Movement / Q000502:physiology*; D003598:Cytoskeletal Proteins / Q000378:metabolism; D005057:Eukaryotic Cells / Q000378:metabolism*; D018260:Gelsolin / Q000378:metabolism; D006801:Humans; D008863:Microspheres; D008954:Models, Biological; D015394:Molecular Structure; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011554:Pseudopodia / Q000378:metabolism*; D013314:Stress, Mechanical; D014783:Viscosity; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal
chemical_list: C495926:ACTR2 protein, human; D051377:Actin-Related Protein 2; D003598:Cytoskeletal Proteins; D018260:Gelsolin; D009419:Nerve Tissue Proteins; C495771:WASL protein, human; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal