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Published online 27 January 2003. doi:10.1083/jcb.200207148
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© The Rockefeller University Press, 0021-9525/2003/2/387 $5.00
The Journal of Cell Biology, Volume 160, Number 3, 387-398

Article

 A biomimetic motility assay provides insight into the mechanism of actin-based motility

Sebastian Wiesner1, Emmanuele Helfer1, Dominique Didry1, Guylaine Ducouret2, Françoise Lafuma2, Marie-France Carlier1 and Dominique Pantaloni1

1 Dynamique du cytosquelette, Laboratoire d'Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette, France
2 Physicochimie des Polymères et des Milieux Dispersés, UMR 7615, 75231 Paris cedex 05, France

Address correspondence to Marie-France Carlier, CNRS, LEBS, Bât. 34, 1 Avenue de la Terrasse, F-91198 Gif-sur-Yvette, France. Tel.: 33-16-982-3465. Fax: 33-16-982-3129. E-mail: carlier{at}lebs.cnrs-gif.fr

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 µm) nor by increasing the viscosity of the medium by 105-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 Alexa®488-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.

Key Words: actin; cell motility; N-WASP; Arp2/3 complex; biomimetics


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