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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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N-WASP–coated beads stimulate actin polymerization via local activation of Arp2/3 complex. (A) Characterization of the surface density of N-WASP. Beads of 2 μm diameter (3 × 107 beads) were incubated in 50 μl of buffer C containing the indicated amount of N-WASP (in pmol). The amount of immobilized N-WASP was derived from SDS-PAGE and immunoblots as described in the Materials and methods. Data are represented in terms of a binding isotherm (open circles) or of the average distance between the immobilized N-WASP molecules, calculated as the square root of the reciprocal of the surface density (closed circles). (B) Typical fluorescence polymerization curves of actin (2.5 μM, 10% pyrenyl labeled) in the presence of 25 nM Arp2/3 complex and either soluble N-WASP (thin lines) or immobilized N-WASP (2-μm-diameter beads, thick lines). The number on each curve represents the concentration of N-WASP (in nM), in the case of soluble N-WASP. In the case of immobilized N-WASP, this number represents the concentration that would be obtained if all immobilized molecules were free in solution. (C) Maximum rates of polymerization were measured at half-time of polymerization in a series of assays as shown in B; N-WASP was free in solution (closed triangles) or immobilized on beads (open triangles). The rates are plotted versus the concentration of N-WASP in the assay (as the number of pmol of N-WASP per ml of solution). (D) Actin (2.5 μM) was polymerized in the presence of Arp2/3 complex (25 nM) and 8 × 106/ml N-WASP–coated beads (2 μm diameter). Beads were observed in the microscope after addition of 2.5 μM rhodamine-phalloidine and a 500-fold dilution in microscopy buffer. Bar, 5 μm.
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fig1: N-WASP–coated beads stimulate actin polymerization via local activation of Arp2/3 complex. (A) Characterization of the surface density of N-WASP. Beads of 2 μm diameter (3 × 107 beads) were incubated in 50 μl of buffer C containing the indicated amount of N-WASP (in pmol). The amount of immobilized N-WASP was derived from SDS-PAGE and immunoblots as described in the Materials and methods. Data are represented in terms of a binding isotherm (open circles) or of the average distance between the immobilized N-WASP molecules, calculated as the square root of the reciprocal of the surface density (closed circles). (B) Typical fluorescence polymerization curves of actin (2.5 μM, 10% pyrenyl labeled) in the presence of 25 nM Arp2/3 complex and either soluble N-WASP (thin lines) or immobilized N-WASP (2-μm-diameter beads, thick lines). The number on each curve represents the concentration of N-WASP (in nM), in the case of soluble N-WASP. In the case of immobilized N-WASP, this number represents the concentration that would be obtained if all immobilized molecules were free in solution. (C) Maximum rates of polymerization were measured at half-time of polymerization in a series of assays as shown in B; N-WASP was free in solution (closed triangles) or immobilized on beads (open triangles). The rates are plotted versus the concentration of N-WASP in the assay (as the number of pmol of N-WASP per ml of solution). (D) Actin (2.5 μM) was polymerized in the presence of Arp2/3 complex (25 nM) and 8 × 106/ml N-WASP–coated beads (2 μm diameter). Beads were observed in the microscope after addition of 2.5 μM rhodamine-phalloidine and a 500-fold dilution in microscopy buffer. Bar, 5 μm.

Mentions: The adsorption of N-WASP to carboxylated polystyrene beads, as determined by SDS-PAGE and quantitative immunoblot analysis, was linear up to a saturating surface density of 20 N-WASP molecules per 100 nm2 (ds = 0.2). This corresponds to an average distance of 2 nm between the immobilized N-WASP molecules, consistent with a close packing arrangement (Fig. 1 A). The ability of immobilized N-WASP to activate Arp2/3 complex and stimulate filament branching was assayed in polymerization assays and in motility assays.


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)

N-WASP–coated beads stimulate actin polymerization via local activation of Arp2/3 complex. (A) Characterization of the surface density of N-WASP. Beads of 2 μm diameter (3 × 107 beads) were incubated in 50 μl of buffer C containing the indicated amount of N-WASP (in pmol). The amount of immobilized N-WASP was derived from SDS-PAGE and immunoblots as described in the Materials and methods. Data are represented in terms of a binding isotherm (open circles) or of the average distance between the immobilized N-WASP molecules, calculated as the square root of the reciprocal of the surface density (closed circles). (B) Typical fluorescence polymerization curves of actin (2.5 μM, 10% pyrenyl labeled) in the presence of 25 nM Arp2/3 complex and either soluble N-WASP (thin lines) or immobilized N-WASP (2-μm-diameter beads, thick lines). The number on each curve represents the concentration of N-WASP (in nM), in the case of soluble N-WASP. In the case of immobilized N-WASP, this number represents the concentration that would be obtained if all immobilized molecules were free in solution. (C) Maximum rates of polymerization were measured at half-time of polymerization in a series of assays as shown in B; N-WASP was free in solution (closed triangles) or immobilized on beads (open triangles). The rates are plotted versus the concentration of N-WASP in the assay (as the number of pmol of N-WASP per ml of solution). (D) Actin (2.5 μM) was polymerized in the presence of Arp2/3 complex (25 nM) and 8 × 106/ml N-WASP–coated beads (2 μm diameter). Beads were observed in the microscope after addition of 2.5 μM rhodamine-phalloidine and a 500-fold dilution in microscopy buffer. Bar, 5 μm.
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Related In: Results  -  Collection

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fig1: N-WASP–coated beads stimulate actin polymerization via local activation of Arp2/3 complex. (A) Characterization of the surface density of N-WASP. Beads of 2 μm diameter (3 × 107 beads) were incubated in 50 μl of buffer C containing the indicated amount of N-WASP (in pmol). The amount of immobilized N-WASP was derived from SDS-PAGE and immunoblots as described in the Materials and methods. Data are represented in terms of a binding isotherm (open circles) or of the average distance between the immobilized N-WASP molecules, calculated as the square root of the reciprocal of the surface density (closed circles). (B) Typical fluorescence polymerization curves of actin (2.5 μM, 10% pyrenyl labeled) in the presence of 25 nM Arp2/3 complex and either soluble N-WASP (thin lines) or immobilized N-WASP (2-μm-diameter beads, thick lines). The number on each curve represents the concentration of N-WASP (in nM), in the case of soluble N-WASP. In the case of immobilized N-WASP, this number represents the concentration that would be obtained if all immobilized molecules were free in solution. (C) Maximum rates of polymerization were measured at half-time of polymerization in a series of assays as shown in B; N-WASP was free in solution (closed triangles) or immobilized on beads (open triangles). The rates are plotted versus the concentration of N-WASP in the assay (as the number of pmol of N-WASP per ml of solution). (D) Actin (2.5 μM) was polymerized in the presence of Arp2/3 complex (25 nM) and 8 × 106/ml N-WASP–coated beads (2 μm diameter). Beads were observed in the microscope after addition of 2.5 μM rhodamine-phalloidine and a 500-fold dilution in microscopy buffer. Bar, 5 μm.
Mentions: The adsorption of N-WASP to carboxylated polystyrene beads, as determined by SDS-PAGE and quantitative immunoblot analysis, was linear up to a saturating surface density of 20 N-WASP molecules per 100 nm2 (ds = 0.2). This corresponds to an average distance of 2 nm between the immobilized N-WASP molecules, consistent with a close packing arrangement (Fig. 1 A). The ability of immobilized N-WASP to activate Arp2/3 complex and stimulate filament branching was assayed in polymerization assays and in motility assays.

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