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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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Influence of the surface density of N-WASP on actin-based motility of N-WASP–coated beads in the motility medium. (A) Efficient movement requires a minimum surface density of N-WASP. Beads (2 μm in diameter) were coated with different densities of N-WASP, corresponding to the indicated average distance between the adsorbed molecules. The percent of beads that moved with comet tails in the standard motility medium was recorded by phase contrast microscopy after a 1-h incubation. Average values were determined for sets of 100 beads. (B) The density of actin filaments in the actin tail correlates with the surface density of N-WASP. The assay contained 5.5 μM ADF. (B, open circles) The optical density of the actin filaments in the tail attached to beads of 2 μm diameter was measured, as the mean average gray value, in a 2 × 2-μm square placed at a distance of 2 μm from the center of the bead, with two opposite sides of the square arranged parallel to the tail axis (open circles). The optical density of the background is subtracted. (B, closed circles) Evaluation of the amount of actin in tails by sedimentation followed by SDS-PAGE. (C) The steady-state velocity of beads was reached in 10–20 min after addition of beads to the motility medium. Mean velocities of beads (2 μm diameter; ds = 0.064, 0.13, and 0.016, top to bottom curves) were determined as described, and average mean velocities were calculated from sets of 6–12 beads per coating density. Conditions are the same as in B. (D) The inhibition of movement by an excess of gelsolin is more prominent at a low surface density of N-WASP. The number of motile beads coated with N-WASP at surface densities of 0.008 (open circles) and 0.1 (closed circles) was scored in the motility medium containing the indicated concentrations of gelsolin. A mixture of fluorescent high-density beads and nonfluorescent low-density beads was analyzed in the same sample. After a 45-min incubation in the motility medium, aliquots of the assays were deposited onto slides. Five phase contrast and fluorescence images per assay were immediately taken, and average values for tail formation were determined from overlay images for sets of 20–40 beads per coating type.
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fig2: Influence of the surface density of N-WASP on actin-based motility of N-WASP–coated beads in the motility medium. (A) Efficient movement requires a minimum surface density of N-WASP. Beads (2 μm in diameter) were coated with different densities of N-WASP, corresponding to the indicated average distance between the adsorbed molecules. The percent of beads that moved with comet tails in the standard motility medium was recorded by phase contrast microscopy after a 1-h incubation. Average values were determined for sets of 100 beads. (B) The density of actin filaments in the actin tail correlates with the surface density of N-WASP. The assay contained 5.5 μM ADF. (B, open circles) The optical density of the actin filaments in the tail attached to beads of 2 μm diameter was measured, as the mean average gray value, in a 2 × 2-μm square placed at a distance of 2 μm from the center of the bead, with two opposite sides of the square arranged parallel to the tail axis (open circles). The optical density of the background is subtracted. (B, closed circles) Evaluation of the amount of actin in tails by sedimentation followed by SDS-PAGE. (C) The steady-state velocity of beads was reached in 10–20 min after addition of beads to the motility medium. Mean velocities of beads (2 μm diameter; ds = 0.064, 0.13, and 0.016, top to bottom curves) were determined as described, and average mean velocities were calculated from sets of 6–12 beads per coating density. Conditions are the same as in B. (D) The inhibition of movement by an excess of gelsolin is more prominent at a low surface density of N-WASP. The number of motile beads coated with N-WASP at surface densities of 0.008 (open circles) and 0.1 (closed circles) was scored in the motility medium containing the indicated concentrations of gelsolin. A mixture of fluorescent high-density beads and nonfluorescent low-density beads was analyzed in the same sample. After a 45-min incubation in the motility medium, aliquots of the assays were deposited onto slides. Five phase contrast and fluorescence images per assay were immediately taken, and average values for tail formation were determined from overlay images for sets of 20–40 beads per coating type.

Mentions: The proportion of motile beads (2 μm diameter) forming actin tails in the reconstituted motility medium increased with the surface density of N-WASP above a threshold value corresponding to an average distance of 20 nm between the N-WASP molecules, and reached 100% at an average distance of 8 nm between the immobilized N-WASP molecules (Fig. 2 A). The amount of F-actin in the tails increased with the surface density of N-WASP (Fig. 2 B). The velocity of beads also increased with the surface density of N-WASP and reached a plateau in the range 0.032–0.13 N-WASP/nm2, corresponding to average distances of 5.6–2.8 nm between the N-WASP molecules. At all surface densities of N-WASP, the steady-state velocity of beads was reached after an ∼10-min incubation in the medium and remained constant for >1 h (Fig. 2 C).


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)

Influence of the surface density of N-WASP on actin-based motility of N-WASP–coated beads in the motility medium. (A) Efficient movement requires a minimum surface density of N-WASP. Beads (2 μm in diameter) were coated with different densities of N-WASP, corresponding to the indicated average distance between the adsorbed molecules. The percent of beads that moved with comet tails in the standard motility medium was recorded by phase contrast microscopy after a 1-h incubation. Average values were determined for sets of 100 beads. (B) The density of actin filaments in the actin tail correlates with the surface density of N-WASP. The assay contained 5.5 μM ADF. (B, open circles) The optical density of the actin filaments in the tail attached to beads of 2 μm diameter was measured, as the mean average gray value, in a 2 × 2-μm square placed at a distance of 2 μm from the center of the bead, with two opposite sides of the square arranged parallel to the tail axis (open circles). The optical density of the background is subtracted. (B, closed circles) Evaluation of the amount of actin in tails by sedimentation followed by SDS-PAGE. (C) The steady-state velocity of beads was reached in 10–20 min after addition of beads to the motility medium. Mean velocities of beads (2 μm diameter; ds = 0.064, 0.13, and 0.016, top to bottom curves) were determined as described, and average mean velocities were calculated from sets of 6–12 beads per coating density. Conditions are the same as in B. (D) The inhibition of movement by an excess of gelsolin is more prominent at a low surface density of N-WASP. The number of motile beads coated with N-WASP at surface densities of 0.008 (open circles) and 0.1 (closed circles) was scored in the motility medium containing the indicated concentrations of gelsolin. A mixture of fluorescent high-density beads and nonfluorescent low-density beads was analyzed in the same sample. After a 45-min incubation in the motility medium, aliquots of the assays were deposited onto slides. Five phase contrast and fluorescence images per assay were immediately taken, and average values for tail formation were determined from overlay images for sets of 20–40 beads per coating type.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Influence of the surface density of N-WASP on actin-based motility of N-WASP–coated beads in the motility medium. (A) Efficient movement requires a minimum surface density of N-WASP. Beads (2 μm in diameter) were coated with different densities of N-WASP, corresponding to the indicated average distance between the adsorbed molecules. The percent of beads that moved with comet tails in the standard motility medium was recorded by phase contrast microscopy after a 1-h incubation. Average values were determined for sets of 100 beads. (B) The density of actin filaments in the actin tail correlates with the surface density of N-WASP. The assay contained 5.5 μM ADF. (B, open circles) The optical density of the actin filaments in the tail attached to beads of 2 μm diameter was measured, as the mean average gray value, in a 2 × 2-μm square placed at a distance of 2 μm from the center of the bead, with two opposite sides of the square arranged parallel to the tail axis (open circles). The optical density of the background is subtracted. (B, closed circles) Evaluation of the amount of actin in tails by sedimentation followed by SDS-PAGE. (C) The steady-state velocity of beads was reached in 10–20 min after addition of beads to the motility medium. Mean velocities of beads (2 μm diameter; ds = 0.064, 0.13, and 0.016, top to bottom curves) were determined as described, and average mean velocities were calculated from sets of 6–12 beads per coating density. Conditions are the same as in B. (D) The inhibition of movement by an excess of gelsolin is more prominent at a low surface density of N-WASP. The number of motile beads coated with N-WASP at surface densities of 0.008 (open circles) and 0.1 (closed circles) was scored in the motility medium containing the indicated concentrations of gelsolin. A mixture of fluorescent high-density beads and nonfluorescent low-density beads was analyzed in the same sample. After a 45-min incubation in the motility medium, aliquots of the assays were deposited onto slides. Five phase contrast and fluorescence images per assay were immediately taken, and average values for tail formation were determined from overlay images for sets of 20–40 beads per coating type.
Mentions: The proportion of motile beads (2 μm diameter) forming actin tails in the reconstituted motility medium increased with the surface density of N-WASP above a threshold value corresponding to an average distance of 20 nm between the N-WASP molecules, and reached 100% at an average distance of 8 nm between the immobilized N-WASP molecules (Fig. 2 A). The amount of F-actin in the tails increased with the surface density of N-WASP (Fig. 2 B). The velocity of beads also increased with the surface density of N-WASP and reached a plateau in the range 0.032–0.13 N-WASP/nm2, corresponding to average distances of 5.6–2.8 nm between the N-WASP molecules. At all surface densities of N-WASP, the steady-state velocity of beads was reached after an ∼10-min incubation in the medium and remained constant for >1 h (Fig. 2 C).

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