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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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Arp2/3 complex is incorporated in the actin tail after branching of filaments at the bead surface. N-WASP–coated beads (2 μm in diameter; ds = 0.032) were placed in the motility medium containing 2% rhodamine-labeled actin and 100% Alexa®488-labeled Arp2/3. A typical bead and its actin tail are shown in rhodamine fluorescence (A) and Alexa®488 fluorescence (B). The actin/Arp2/3 stoichiometry is constant along the actin tail (C), the figure shows the fluorescences of rhodamine–actin (red curve) and Alexa®488–Arp2/3 (green curve) along an actin tail. Note the sharp peak in the Alexa®488 signal that corresponds to Arp2/3 binding to the bead. Green dotted line is a fourfold expansion of the continuous green curve, emphasizing the identical rhodamine and Alexa®488 decays. Beads in a stationary regime of propulsion in the motility medium containing unlabeled Arp2/3 were supplemented with fluorescent Arp2/3 complex and observed in phase contrast (D) and in Alexa®488 fluorescence (E) at 8, 15, and 22 min after addition of fluorescent Arp2/3. The medium contained 50 nM gelsolin and 1.5 μM ADF, ensuring slow movement (0.4 μm/min) to facilitate measurement of the early steps of Arp2/3 incorporation in the tail.
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fig6: Arp2/3 complex is incorporated in the actin tail after branching of filaments at the bead surface. N-WASP–coated beads (2 μm in diameter; ds = 0.032) were placed in the motility medium containing 2% rhodamine-labeled actin and 100% Alexa®488-labeled Arp2/3. A typical bead and its actin tail are shown in rhodamine fluorescence (A) and Alexa®488 fluorescence (B). The actin/Arp2/3 stoichiometry is constant along the actin tail (C), the figure shows the fluorescences of rhodamine–actin (red curve) and Alexa®488–Arp2/3 (green curve) along an actin tail. Note the sharp peak in the Alexa®488 signal that corresponds to Arp2/3 binding to the bead. Green dotted line is a fourfold expansion of the continuous green curve, emphasizing the identical rhodamine and Alexa®488 decays. Beads in a stationary regime of propulsion in the motility medium containing unlabeled Arp2/3 were supplemented with fluorescent Arp2/3 complex and observed in phase contrast (D) and in Alexa®488 fluorescence (E) at 8, 15, and 22 min after addition of fluorescent Arp2/3. The medium contained 50 nM gelsolin and 1.5 μM ADF, ensuring slow movement (0.4 μm/min) to facilitate measurement of the early steps of Arp2/3 incorporation in the tail.

Mentions: Both rhodamine–actin and Alexa®488–Arp2/3 were localized throughout the actin tails. In addition, Alexa®488 fluorescence was intense on the bead, consistent with the tight binding of Arp2/3 to immobilized N-WASP (Fig. 6, A and B). The distribution of the rhodamine and Alexa®488 fluorescence intensities along the tails shows the same exponential decrease for actin and Arp2/3 from the proximal to the distal regions of the tail, suggesting that actin and Arp2/3 complex are lost at identical rates by pointed end depolymerization (Fig. 6 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)

Arp2/3 complex is incorporated in the actin tail after branching of filaments at the bead surface. N-WASP–coated beads (2 μm in diameter; ds = 0.032) were placed in the motility medium containing 2% rhodamine-labeled actin and 100% Alexa®488-labeled Arp2/3. A typical bead and its actin tail are shown in rhodamine fluorescence (A) and Alexa®488 fluorescence (B). The actin/Arp2/3 stoichiometry is constant along the actin tail (C), the figure shows the fluorescences of rhodamine–actin (red curve) and Alexa®488–Arp2/3 (green curve) along an actin tail. Note the sharp peak in the Alexa®488 signal that corresponds to Arp2/3 binding to the bead. Green dotted line is a fourfold expansion of the continuous green curve, emphasizing the identical rhodamine and Alexa®488 decays. Beads in a stationary regime of propulsion in the motility medium containing unlabeled Arp2/3 were supplemented with fluorescent Arp2/3 complex and observed in phase contrast (D) and in Alexa®488 fluorescence (E) at 8, 15, and 22 min after addition of fluorescent Arp2/3. The medium contained 50 nM gelsolin and 1.5 μM ADF, ensuring slow movement (0.4 μm/min) to facilitate measurement of the early steps of Arp2/3 incorporation in the tail.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Arp2/3 complex is incorporated in the actin tail after branching of filaments at the bead surface. N-WASP–coated beads (2 μm in diameter; ds = 0.032) were placed in the motility medium containing 2% rhodamine-labeled actin and 100% Alexa®488-labeled Arp2/3. A typical bead and its actin tail are shown in rhodamine fluorescence (A) and Alexa®488 fluorescence (B). The actin/Arp2/3 stoichiometry is constant along the actin tail (C), the figure shows the fluorescences of rhodamine–actin (red curve) and Alexa®488–Arp2/3 (green curve) along an actin tail. Note the sharp peak in the Alexa®488 signal that corresponds to Arp2/3 binding to the bead. Green dotted line is a fourfold expansion of the continuous green curve, emphasizing the identical rhodamine and Alexa®488 decays. Beads in a stationary regime of propulsion in the motility medium containing unlabeled Arp2/3 were supplemented with fluorescent Arp2/3 complex and observed in phase contrast (D) and in Alexa®488 fluorescence (E) at 8, 15, and 22 min after addition of fluorescent Arp2/3. The medium contained 50 nM gelsolin and 1.5 μM ADF, ensuring slow movement (0.4 μm/min) to facilitate measurement of the early steps of Arp2/3 incorporation in the tail.
Mentions: Both rhodamine–actin and Alexa®488–Arp2/3 were localized throughout the actin tails. In addition, Alexa®488 fluorescence was intense on the bead, consistent with the tight binding of Arp2/3 to immobilized N-WASP (Fig. 6, A and B). The distribution of the rhodamine and Alexa®488 fluorescence intensities along the tails shows the same exponential decrease for actin and Arp2/3 from the proximal to the distal regions of the tail, suggesting that actin and Arp2/3 complex are lost at identical rates by pointed end depolymerization (Fig. 6 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