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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.

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Evaluation of the branch spacing in actin tails: dependence on gelsolin. (A) Typical images of tail morphologies for high-density beads (diameter 2 μm) at the indicated gelsolin concentrations. Note the presence of “fishbone” actin tails at low gelsolin, as previously reported (Pantaloni et al., 2000), and the decrease in tail length upon increasing gelsolin. (B) Quantitation of actin (rhodamine labeled) and Arp2/3 (Alexa®488 labeled) in the actin tails at different concentrations of gelsolin. The gallery shows typical images of high-density beads (diameter 2 μm) at indicated gelsolin concentrations. Alexa® fluorescence is saturated on the beads at 100 and 200 nM gelsolin. Note the decrease in the intensities ratio (IR/IA) as the gelsolin concentration increases. (C) Branch spacing is a decreasing function of the filament capping rate. Average IR/IA ratios were determined for sets of high-density (ds = 0.064, pink squares) and low-density beads (ds = 0.016, blue squares) at different gelsolin concentrations. For both bead types, branch spacing decreases sharply upon increasing the gelsolin concentration. (D) Bead velocity shows a bell-shaped dependence on filament capping. Average velocities are shown for high-density (ds = 0.064, open squares/pink curve) and low-density beads (ds = 0.016, closed squares/blue curve) at varying gelsolin concentrations.
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fig7: Evaluation of the branch spacing in actin tails: dependence on gelsolin. (A) Typical images of tail morphologies for high-density beads (diameter 2 μm) at the indicated gelsolin concentrations. Note the presence of “fishbone” actin tails at low gelsolin, as previously reported (Pantaloni et al., 2000), and the decrease in tail length upon increasing gelsolin. (B) Quantitation of actin (rhodamine labeled) and Arp2/3 (Alexa®488 labeled) in the actin tails at different concentrations of gelsolin. The gallery shows typical images of high-density beads (diameter 2 μm) at indicated gelsolin concentrations. Alexa® fluorescence is saturated on the beads at 100 and 200 nM gelsolin. Note the decrease in the intensities ratio (IR/IA) as the gelsolin concentration increases. (C) Branch spacing is a decreasing function of the filament capping rate. Average IR/IA ratios were determined for sets of high-density (ds = 0.064, pink squares) and low-density beads (ds = 0.016, blue squares) at different gelsolin concentrations. For both bead types, branch spacing decreases sharply upon increasing the gelsolin concentration. (D) Bead velocity shows a bell-shaped dependence on filament capping. Average velocities are shown for high-density (ds = 0.064, open squares/pink curve) and low-density beads (ds = 0.016, closed squares/blue curve) at varying gelsolin concentrations.

Mentions: Computational simulations have indicated that branch spacing (the reciprocal of the frequency of filament branching) provides a means to discriminate between different models (Carlsson, 2001). Bead velocity and average branch spacing were measured at different concentrations of gelsolin in the range of 20–200 nM, using rhodamine-labeled actin and Alexa®488-labeled Arp2/3. The change in actin tail morphology upon increasing gelsolin is visible in phase contrast microscopy (Fig. 7 A). The density of rhodamine-labeled actin in the tail decreased upon increasing gelsolin, reflecting the role of gelsolin in regulating the life time of growing filaments (Pantaloni et al., 2000). On the other hand, the fluorescence ratio of rhodamine–actin to Alexa®–Arp2/3, which is indicative of the branch spacing, decreased upon increasing the capping rate (Fig. 7, B and C). A fourfold higher density of branching was associated with a 10-fold increase in gelsolin concentration. This result suggests that due to the very high density of activated Arp2/3 at the surface of the bead, the competition between Arp2/3 and capping protein for barbed end branching is less effective at the bead surface. The result is consistent with models assuming that barbed end branching occurs at the surface of the bead (Carlsson, 2001). At two different densities of N-WASP, bead velocity displayed a bell-shaped dependence on gelsolin concentration (Fig. 7 D), as previously observed with Listeria or Shigella (Loisel et al., 1999). This result too is consistent with computational simulations, which predict that velocity should decrease at high capping rates if uncapping does not occur, whereas a constant velocity should be observed if uncapping occurs (Carlsson, 2001).


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)

Evaluation of the branch spacing in actin tails: dependence on gelsolin. (A) Typical images of tail morphologies for high-density beads (diameter 2 μm) at the indicated gelsolin concentrations. Note the presence of “fishbone” actin tails at low gelsolin, as previously reported (Pantaloni et al., 2000), and the decrease in tail length upon increasing gelsolin. (B) Quantitation of actin (rhodamine labeled) and Arp2/3 (Alexa®488 labeled) in the actin tails at different concentrations of gelsolin. The gallery shows typical images of high-density beads (diameter 2 μm) at indicated gelsolin concentrations. Alexa® fluorescence is saturated on the beads at 100 and 200 nM gelsolin. Note the decrease in the intensities ratio (IR/IA) as the gelsolin concentration increases. (C) Branch spacing is a decreasing function of the filament capping rate. Average IR/IA ratios were determined for sets of high-density (ds = 0.064, pink squares) and low-density beads (ds = 0.016, blue squares) at different gelsolin concentrations. For both bead types, branch spacing decreases sharply upon increasing the gelsolin concentration. (D) Bead velocity shows a bell-shaped dependence on filament capping. Average velocities are shown for high-density (ds = 0.064, open squares/pink curve) and low-density beads (ds = 0.016, closed squares/blue curve) at varying gelsolin concentrations.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172664&req=5

fig7: Evaluation of the branch spacing in actin tails: dependence on gelsolin. (A) Typical images of tail morphologies for high-density beads (diameter 2 μm) at the indicated gelsolin concentrations. Note the presence of “fishbone” actin tails at low gelsolin, as previously reported (Pantaloni et al., 2000), and the decrease in tail length upon increasing gelsolin. (B) Quantitation of actin (rhodamine labeled) and Arp2/3 (Alexa®488 labeled) in the actin tails at different concentrations of gelsolin. The gallery shows typical images of high-density beads (diameter 2 μm) at indicated gelsolin concentrations. Alexa® fluorescence is saturated on the beads at 100 and 200 nM gelsolin. Note the decrease in the intensities ratio (IR/IA) as the gelsolin concentration increases. (C) Branch spacing is a decreasing function of the filament capping rate. Average IR/IA ratios were determined for sets of high-density (ds = 0.064, pink squares) and low-density beads (ds = 0.016, blue squares) at different gelsolin concentrations. For both bead types, branch spacing decreases sharply upon increasing the gelsolin concentration. (D) Bead velocity shows a bell-shaped dependence on filament capping. Average velocities are shown for high-density (ds = 0.064, open squares/pink curve) and low-density beads (ds = 0.016, closed squares/blue curve) at varying gelsolin concentrations.
Mentions: Computational simulations have indicated that branch spacing (the reciprocal of the frequency of filament branching) provides a means to discriminate between different models (Carlsson, 2001). Bead velocity and average branch spacing were measured at different concentrations of gelsolin in the range of 20–200 nM, using rhodamine-labeled actin and Alexa®488-labeled Arp2/3. The change in actin tail morphology upon increasing gelsolin is visible in phase contrast microscopy (Fig. 7 A). The density of rhodamine-labeled actin in the tail decreased upon increasing gelsolin, reflecting the role of gelsolin in regulating the life time of growing filaments (Pantaloni et al., 2000). On the other hand, the fluorescence ratio of rhodamine–actin to Alexa®–Arp2/3, which is indicative of the branch spacing, decreased upon increasing the capping rate (Fig. 7, B and C). A fourfold higher density of branching was associated with a 10-fold increase in gelsolin concentration. This result suggests that due to the very high density of activated Arp2/3 at the surface of the bead, the competition between Arp2/3 and capping protein for barbed end branching is less effective at the bead surface. The result is consistent with models assuming that barbed end branching occurs at the surface of the bead (Carlsson, 2001). At two different densities of N-WASP, bead velocity displayed a bell-shaped dependence on gelsolin concentration (Fig. 7 D), as previously observed with Listeria or Shigella (Loisel et al., 1999). This result too is consistent with computational simulations, which predict that velocity should decrease at high capping rates if uncapping does not occur, whereas a constant velocity should be observed if uncapping occurs (Carlsson, 2001).

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