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 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.

Impact of branching on the elasticity of actin networks

“…The variation of actin density is at most a factor of 1.6 for gelsolin variation and 1.5 for Arp2/3 variation. Such a weak actin density variation with branching has already been observed in similar networks (25). Meanwhile, the gels strongly stiffen as the concentration of branching protein increases (from 1 kPa at 10 nM to 8 kPa at 100 nM), and the Young's modulus levels off at a concentration above 100 nM ( Fig.…”

“…Both these effects would point to a softening when the concentration of gelsolin increases, in contrast to the observed stiffening. Nevertheless, the protein gelsolin has also been shown to reduce the distance between branches in reconstituted systems (25,32). The major mechanical effect of increasing gelsolin is neither the decreasing connectivity nor the decrease of the filaments total length but rather the reduction of the distance between branching points.…”

Forces generated during actin-based propulsion: A direct measurement by micromanipulation

“…Indirect approaches have been developed recently to evaluate the magnitude of the propulsive force. Increasing the viscosity of the medium by several orders of magnitude only slightly slowed down bead movement (20), suggesting that the propulsive force is much larger than 50 pN. This result is in disagreement with the reported observation of a dramatic decrease in Listeria velocity under a 0-50 pN viscous force (21).…”

“…By using a mesoscopic elastic analysis, Gerbal et al (36) find a shallow force-velocity curve and predict that forces of the order of nanonewtons are necessary to slow down Listeria movement significantly. Two independent experiments in which external forces were applied on actin-propelled objects by adjusting the external viscosity led to differing results (20,21). A sharp velocity drop at very low forces (10 pN) followed by a slow decrease is observed for Listeria in extracts, whereas beads in a pure reconstituted protein medium display a very weak decrease in velocity over a similar force range.…”

“…By using this experimental design, a constant force in the Ϫ1.7 to 4.3 nN range is applied to the actin-propelled bead, and the associated velocity is measured, yielding to a force-velocity diagram. The convention for the sign of the force is that pushing forces (opposing free-bead movement) have a positive value to keep the convention that has been used in studies (20,21,30). This force is then the opposite of the external force, ϪF ext , applied to the bead.…”