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MBC in Press, published online ahead of print December 20, 2006
Mol. Biol. Cell 10.1091/mbc.E06-06-0569

A more recent version of this article appeared on February 1, 2007
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Submitted on June 30, 2006
Revised on November 22, 2006
Accepted on November 30, 2006

N-WASP Involvement in Dorsal Ruffle Formation in Mouse Embryonic Fibroblasts

John A. Legg,* Guillaume Bompard,{dagger} John Dawson,* Hannah L. Morris,* Natalie Andrew,* Lisa Cooper,{ddagger} Simon A. Johnston,* Giorgos Tramountanis,* and Laura M. Machesky*

Schools of *Biosciences and {ddagger}Medicine, The University of Birmingham, Birmingham, Edgbaston, West Midlands, B15 2TT, United Kingdom; {dagger}Centre de Recherches de Biochimie Macromoléculaire, Centre National de la Recherche Scientifique FRE2593, 34293 Montpellier, Cedex 5, France

Monitoring Editor: David Drubin

The Wiskott-Aldrich Syndrome protein family activates the Arp2/3 complex leading to the formation of new actin filaments. Here, we study the involvement of Scar1, Scar2, N-WASP and Arp2/3 complex in dorsal ruffle formation in mouse embryonic fibroblasts (MEFs). Using platelet derived growth factor to stimulate circular dorsal ruffle assembly in primary E13 and immortalised E9 Scar1+/+ and Scar1 null MEFs, we establish that Scar1 loss does not impair the formation of dorsal ruffles. Reduction of Scar2 protein levels via siRNA also did not affect dorsal ruffle production. In contrast, wiskostatin, a chemical inhibitor of N-WASP, potently suppressed dorsal ruffle formation in a dose-dependent manner. Furthermore, N-WASP and Arp2 siRNA treatment significantly decreased the formation of dorsal ruffles in MEFs. In addition, the expression of an N-WASP truncation mutant that cannot bind Arp2/3 complex blocked the formation of these structures. Finally, N-WASP-/- fibroblast-like cells (FLCs) generated aberrant dorsal ruffles. These ruffles were highly unstable, severely depleted of Arp2/3 complex and diminished in size. We hypothesize that N-WASP and Arp2/3 complex are part of a multiprotein assembly important for the generation of dorsal ruffles and that Scar1 and Scar2 are dispensable for this process.

Address correspondence to: Laura M. Machesky (l.m.machesky{at}bham.ac.uk)




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