SUPRAMOLECULAR ASSEMBLIES |
Arp2/3 controls the motile behavior of N-WASP-functionalized GUVs and modulates N-WASP surface distribution by mediating transient links with actin filaments
Vincent Delatour 1, Emmanuèle Helfer 1, Dominique Didry 1, Kim Hô Diêp Lê 1, Jean-François Gaucher 2, Marie-France Carlier 1 and Guillaume Romet-Lemonne 1*
1 CNRS
2 Université Paris Diderot
* To whom correspondence should be addressed. E-mail: romet{at}lebs.cnrs-gif.fr.
Submitted on August 9, 2007
Revised on October 9, 2007
Accepted on 12 February 2008
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Abstract |
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Spatially controlled assembly of actin in branched filaments generates cell protrusions or the propulsion of intracellular vesicles and pathogens. The propulsive movement of giant unilamellar vesicles (GUVs) functionalized by N-WASP (full-length or truncated) is reconstituted in a biochemically controlled medium, and analyzed using phase contrast and fluorescence microscopy to elucidate the links between membrane components and the actin cytoskeleton that determine motile behavior. Actin-based propulsion displays a continuous regime or a periodic saltatory regime. The transition between the two regimes is controlled by the concentration of Arp2/3 complex, which branches filaments by interacting with N-WASP at the liposome surface. Saltatory motion is linked to cycles in the distribution of N-WASP at the membrane between a homogeneous and a segregated state. Comparison of the changes in distribution of N-WASP, Arp2/3 and actin during propulsion demonstrates that actin filaments bind to N-WASP, and that these bonds are transitory. This interaction, mediated by Arp2/3, drives N-WASP segregation. VC-fragments of N-WASP, that interact more weakly than N-WASP with the Arp2/3 complex, segregate less than N-WASP at the rear of the GUVs. GUV propulsion is inhibited by the presence of VCA-actin covalent complex, showing that the release of actin from the nuleator is required for movement. The balance between segregation and free diffusion determines whether continuous movement can be sustained. Computed surface distributions of N-WASP, derived from a theoretical description of this segregation-diffusion mechanism, account satisfactorily for the measured density profiles of N-WASP, Arp2/3 complex, and actin.
Key Words:
Arp2/3 filament branching, N-WASP functionalized liposome, actin, light microscopy, reconstituted motility, systems biology
