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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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Effects of methylcellulose on solution viscosity, actin polymerization, and bead movement. (A) Methylcellulose bundles actin filaments. Polymerization of 4.7 μM actin (10% pyrenyl labeled) was monitored simultaneously by fluorescence (λexc = 366 nm; λem = 407 nm) and by light scattering at 90° angle (λ = 350 nm) using a Safas spectrofluorimeter in the absence (a and a') and in the presence of methylcellulose M-7140 (15 cP at 2%) (b and b') or M-0512 (4,000 cP at 2%) (c and c'). (B) LTM measurements. The frequency dependence of the viscoelastic modulus G(f) was obtained from the spectral densities, <Δr2(f)>, of beads in Brownian motion in different solutions (from bottom to top: water [1], 1% M-7140 [2], 1% M-0262 [3], 1% M-0512 [4], and 2% M-0512 [5]). (C) The force–velocity relationship for actin-based propulsion in increasingly viscous assays is almost flat up to a viscous force of 50 pN. Viscous forces were derived from the Stokes equation using the velocities and viscosities tabulated in Tables I and II, respectively, for increasing concentrations of methylcellulose M-0512. To account for the effect of methylcellulose not related to viscosity, velocities were normalized by dividing by the velocity measured in the presence of the same mass amount of “short” nonviscous methylcellulose. (D) Methylcellulose increases the density of the actin tails in a manner unrelated to the increase in viscous drag. Phase contrast images of N-WASP–coated beads (diameter 2 μm) moving in media containing (a) no methylcellulose, (b) 1% methylcellulose M-7140, and (c) 1% methylcellulose M-0512.
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fig4: Effects of methylcellulose on solution viscosity, actin polymerization, and bead movement. (A) Methylcellulose bundles actin filaments. Polymerization of 4.7 μM actin (10% pyrenyl labeled) was monitored simultaneously by fluorescence (λexc = 366 nm; λem = 407 nm) and by light scattering at 90° angle (λ = 350 nm) using a Safas spectrofluorimeter in the absence (a and a') and in the presence of methylcellulose M-7140 (15 cP at 2%) (b and b') or M-0512 (4,000 cP at 2%) (c and c'). (B) LTM measurements. The frequency dependence of the viscoelastic modulus G(f) was obtained from the spectral densities, <Δr2(f)>, of beads in Brownian motion in different solutions (from bottom to top: water [1], 1% M-7140 [2], 1% M-0262 [3], 1% M-0512 [4], and 2% M-0512 [5]). (C) The force–velocity relationship for actin-based propulsion in increasingly viscous assays is almost flat up to a viscous force of 50 pN. Viscous forces were derived from the Stokes equation using the velocities and viscosities tabulated in Tables I and II, respectively, for increasing concentrations of methylcellulose M-0512. To account for the effect of methylcellulose not related to viscosity, velocities were normalized by dividing by the velocity measured in the presence of the same mass amount of “short” nonviscous methylcellulose. (D) Methylcellulose increases the density of the actin tails in a manner unrelated to the increase in viscous drag. Phase contrast images of N-WASP–coated beads (diameter 2 μm) moving in media containing (a) no methylcellulose, (b) 1% methylcellulose M-7140, and (c) 1% methylcellulose M-0512.

Mentions: The critical concentrations for actin assembly at either the barbed or the pointed end were not affected by up to 2% of any kind of methylcellulose (unpublished data). The kinetics of pyrenyl–actin fluorescence change upon spontaneous actin assembly were not greatly affected by up to 2% methylcellulose of any kind, however spontaneous nucleation was favored by methylcellulose. In addition, the increase in light scattering or turbidity at 350 nm was much larger in the presence of all species of methylcellulose, reflecting bundle formation (Fig. 4 A), in agreement with previous observations (Takiguchi, 1991). Bundling of filaments increased with the concentration but not with the length of the methylcellulose polymer, i.e., it was independent of the viscosity of the solution. Interestingly, at the same mass concentration, methylglucose (monomer of methylcellulose) failed to bundle actin filaments.


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)

Effects of methylcellulose on solution viscosity, actin polymerization, and bead movement. (A) Methylcellulose bundles actin filaments. Polymerization of 4.7 μM actin (10% pyrenyl labeled) was monitored simultaneously by fluorescence (λexc = 366 nm; λem = 407 nm) and by light scattering at 90° angle (λ = 350 nm) using a Safas spectrofluorimeter in the absence (a and a') and in the presence of methylcellulose M-7140 (15 cP at 2%) (b and b') or M-0512 (4,000 cP at 2%) (c and c'). (B) LTM measurements. The frequency dependence of the viscoelastic modulus G(f) was obtained from the spectral densities, <Δr2(f)>, of beads in Brownian motion in different solutions (from bottom to top: water [1], 1% M-7140 [2], 1% M-0262 [3], 1% M-0512 [4], and 2% M-0512 [5]). (C) The force–velocity relationship for actin-based propulsion in increasingly viscous assays is almost flat up to a viscous force of 50 pN. Viscous forces were derived from the Stokes equation using the velocities and viscosities tabulated in Tables I and II, respectively, for increasing concentrations of methylcellulose M-0512. To account for the effect of methylcellulose not related to viscosity, velocities were normalized by dividing by the velocity measured in the presence of the same mass amount of “short” nonviscous methylcellulose. (D) Methylcellulose increases the density of the actin tails in a manner unrelated to the increase in viscous drag. Phase contrast images of N-WASP–coated beads (diameter 2 μm) moving in media containing (a) no methylcellulose, (b) 1% methylcellulose M-7140, and (c) 1% methylcellulose M-0512.
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Related In: Results  -  Collection

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fig4: Effects of methylcellulose on solution viscosity, actin polymerization, and bead movement. (A) Methylcellulose bundles actin filaments. Polymerization of 4.7 μM actin (10% pyrenyl labeled) was monitored simultaneously by fluorescence (λexc = 366 nm; λem = 407 nm) and by light scattering at 90° angle (λ = 350 nm) using a Safas spectrofluorimeter in the absence (a and a') and in the presence of methylcellulose M-7140 (15 cP at 2%) (b and b') or M-0512 (4,000 cP at 2%) (c and c'). (B) LTM measurements. The frequency dependence of the viscoelastic modulus G(f) was obtained from the spectral densities, <Δr2(f)>, of beads in Brownian motion in different solutions (from bottom to top: water [1], 1% M-7140 [2], 1% M-0262 [3], 1% M-0512 [4], and 2% M-0512 [5]). (C) The force–velocity relationship for actin-based propulsion in increasingly viscous assays is almost flat up to a viscous force of 50 pN. Viscous forces were derived from the Stokes equation using the velocities and viscosities tabulated in Tables I and II, respectively, for increasing concentrations of methylcellulose M-0512. To account for the effect of methylcellulose not related to viscosity, velocities were normalized by dividing by the velocity measured in the presence of the same mass amount of “short” nonviscous methylcellulose. (D) Methylcellulose increases the density of the actin tails in a manner unrelated to the increase in viscous drag. Phase contrast images of N-WASP–coated beads (diameter 2 μm) moving in media containing (a) no methylcellulose, (b) 1% methylcellulose M-7140, and (c) 1% methylcellulose M-0512.
Mentions: The critical concentrations for actin assembly at either the barbed or the pointed end were not affected by up to 2% of any kind of methylcellulose (unpublished data). The kinetics of pyrenyl–actin fluorescence change upon spontaneous actin assembly were not greatly affected by up to 2% methylcellulose of any kind, however spontaneous nucleation was favored by methylcellulose. In addition, the increase in light scattering or turbidity at 350 nm was much larger in the presence of all species of methylcellulose, reflecting bundle formation (Fig. 4 A), in agreement with previous observations (Takiguchi, 1991). Bundling of filaments increased with the concentration but not with the length of the methylcellulose polymer, i.e., it was independent of the viscosity of the solution. Interestingly, at the same mass concentration, methylglucose (monomer of methylcellulose) failed to bundle actin filaments.

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