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

Wiesner S, Helfer E, Didry D, Ducouret G, Lafuma F, Carlier MF, Pantaloni D - J. Cell Biol. (2003)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Dynamique du cytosquelette, Laboratoire d'Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette, France.

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.

Show MeSH

Related in: MedlinePlus

The velocity of beads is independent of their size. (A) Beads of different diameters (0.2–3 μm, relative sizes are drawn at the bottom of the panel) were coated with N-WASP (ds = 0.016). Movement was recorded in the standard motility medium. (B) Trajectories of beads of different diameters (1, 3 μm; 2, 1 μm; 3, 0.5 μm) moving in the same field in the standard motility medium were recorded over a period of 60 min and traced in white on the last frame image (see also Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200207148/DC1). Bar, 15 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172664&req=5

fig3: The velocity of beads is independent of their size. (A) Beads of different diameters (0.2–3 μm, relative sizes are drawn at the bottom of the panel) were coated with N-WASP (ds = 0.016). Movement was recorded in the standard motility medium. (B) Trajectories of beads of different diameters (1, 3 μm; 2, 1 μm; 3, 0.5 μm) moving in the same field in the standard motility medium were recorded over a period of 60 min and traced in white on the last frame image (see also Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200207148/DC1). Bar, 15 μm.

Mentions: To understand the dependence of bead velocity on external load, movement of beads of different diameters in the range of 0.2–3 μm was monitored. At a given surface density of N-WASP, all beads moved at identical rates (Fig. 3 A). Typical trajectories of beads of different sizes in the same field are shown in Fig. 3 B (see also Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200207148/DC1). This result suggests that the velocity is not affected by the load due to the increase in size.


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

Wiesner S, Helfer E, Didry D, Ducouret G, Lafuma F, Carlier MF, Pantaloni D - J. Cell Biol. (2003)

The velocity of beads is independent of their size. (A) Beads of different diameters (0.2–3 μm, relative sizes are drawn at the bottom of the panel) were coated with N-WASP (ds = 0.016). Movement was recorded in the standard motility medium. (B) Trajectories of beads of different diameters (1, 3 μm; 2, 1 μm; 3, 0.5 μm) moving in the same field in the standard motility medium were recorded over a period of 60 min and traced in white on the last frame image (see also Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200207148/DC1). Bar, 15 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172664&req=5

fig3: The velocity of beads is independent of their size. (A) Beads of different diameters (0.2–3 μm, relative sizes are drawn at the bottom of the panel) were coated with N-WASP (ds = 0.016). Movement was recorded in the standard motility medium. (B) Trajectories of beads of different diameters (1, 3 μm; 2, 1 μm; 3, 0.5 μm) moving in the same field in the standard motility medium were recorded over a period of 60 min and traced in white on the last frame image (see also Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200207148/DC1). Bar, 15 μm.
Mentions: To understand the dependence of bead velocity on external load, movement of beads of different diameters in the range of 0.2–3 μm was monitored. At a given surface density of N-WASP, all beads moved at identical rates (Fig. 3 A). Typical trajectories of beads of different sizes in the same field are shown in Fig. 3 B (see also Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200207148/DC1). This result suggests that the velocity is not affected by the load due to the increase in size.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Dynamique du cytosquelette, Laboratoire d'Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette, France.

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.

Show MeSH
Related in: MedlinePlus