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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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Fluorescent labeling of Arp2/3 complex. Arp2/3 complex was labeled with Alexa®488-C5-maleimide as described in the Materials and methods. (A) SDS-PAGE of Alexa®488-labeled Arp2/3 (right, CB staining; left, UV illumination) reveals the incorporation of the label in the p40 subunit (80%) and in Arp3 (20%). M, fluorescent markers. (B) UV-visible spectrum of the labeled Arp2/3 complex. (C) The filament branching activity of Arp2/3 complex is not affected by Alexa®488 labeling. Actin (2.5 μM, 10% pyrenyl labeled) was polymerized in the presence of 0.5 μM VCA, in the absence (a) or presence of 30 nM Arp2/3 before addition (b) and after a 1-h incubation with Alexa®488-C5-maleimide (c).
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fig5: Fluorescent labeling of Arp2/3 complex. Arp2/3 complex was labeled with Alexa®488-C5-maleimide as described in the Materials and methods. (A) SDS-PAGE of Alexa®488-labeled Arp2/3 (right, CB staining; left, UV illumination) reveals the incorporation of the label in the p40 subunit (80%) and in Arp3 (20%). M, fluorescent markers. (B) UV-visible spectrum of the labeled Arp2/3 complex. (C) The filament branching activity of Arp2/3 complex is not affected by Alexa®488 labeling. Actin (2.5 μM, 10% pyrenyl labeled) was polymerized in the presence of 0.5 μM VCA, in the absence (a) or presence of 30 nM Arp2/3 before addition (b) and after a 1-h incubation with Alexa®488-C5-maleimide (c).

Mentions: The relationship between velocity and frequency of filament branching during propulsion was examined using rhodamine-labeled actin and Alexa®488-labeled Arp2/3 complex in the motility medium. Several labeling procedures of Arp2/3 complex using a variety of fluorophores were initially tried. The most satisfactory results were obtained using Alexa®488-C5-maleimide (see Materials and methods). Routinely, 3–4 mol of Alexa®488 fluorophore were covalently incorporated per mole of Arp2/3 complex without any loss in filament branching activity. 80% of the label was bound to the ARCp41 subunit, which contains five cysteines (Fig. 5).


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)

Fluorescent labeling of Arp2/3 complex. Arp2/3 complex was labeled with Alexa®488-C5-maleimide as described in the Materials and methods. (A) SDS-PAGE of Alexa®488-labeled Arp2/3 (right, CB staining; left, UV illumination) reveals the incorporation of the label in the p40 subunit (80%) and in Arp3 (20%). M, fluorescent markers. (B) UV-visible spectrum of the labeled Arp2/3 complex. (C) The filament branching activity of Arp2/3 complex is not affected by Alexa®488 labeling. Actin (2.5 μM, 10% pyrenyl labeled) was polymerized in the presence of 0.5 μM VCA, in the absence (a) or presence of 30 nM Arp2/3 before addition (b) and after a 1-h incubation with Alexa®488-C5-maleimide (c).
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Fluorescent labeling of Arp2/3 complex. Arp2/3 complex was labeled with Alexa®488-C5-maleimide as described in the Materials and methods. (A) SDS-PAGE of Alexa®488-labeled Arp2/3 (right, CB staining; left, UV illumination) reveals the incorporation of the label in the p40 subunit (80%) and in Arp3 (20%). M, fluorescent markers. (B) UV-visible spectrum of the labeled Arp2/3 complex. (C) The filament branching activity of Arp2/3 complex is not affected by Alexa®488 labeling. Actin (2.5 μM, 10% pyrenyl labeled) was polymerized in the presence of 0.5 μM VCA, in the absence (a) or presence of 30 nM Arp2/3 before addition (b) and after a 1-h incubation with Alexa®488-C5-maleimide (c).
Mentions: The relationship between velocity and frequency of filament branching during propulsion was examined using rhodamine-labeled actin and Alexa®488-labeled Arp2/3 complex in the motility medium. Several labeling procedures of Arp2/3 complex using a variety of fluorophores were initially tried. The most satisfactory results were obtained using Alexa®488-C5-maleimide (see Materials and methods). Routinely, 3–4 mol of Alexa®488 fluorophore were covalently incorporated per mole of Arp2/3 complex without any loss in filament branching activity. 80% of the label was bound to the ARCp41 subunit, which contains five cysteines (Fig. 5).

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