N-WASP involvement in dorsal ruffle formation in mouse embryonic fibroblasts.
The Wiskott-Aldrich syndrome protein (WASP) 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 immortalized 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 small interfering RNA (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 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.
Legg
John A
JA
School of Biosciences, The University of Birmingham, Birmingham, Edgbaston, West Midlands, B15 2TT, United Kingdom.
Bompard
Guillaume
G
Dawson
John
J
Morris
Hannah L
HL
Andrew
Natalie
N
Cooper
Lisa
L
Johnston
Simon A
SA
Tramountanis
Giorgos
G
Machesky
Laura M
LM
D008841:Actin Cytoskeleton / Q000648:ultrastructure; D051376:Actin-Related Protein 2-3 Complex / Q000502:physiology*; D000818:Animals; D002227:Carbazoles / Q000494:pharmacology; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D051379:Mice; D020409:Molecular Motor Proteins / Q000235:genetics / Q000502:physiology*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011412:Propanolamines / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology; D051300:Wiskott-Aldrich Syndrome Protein Family / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*
D051376:Actin-Related Protein 2-3 Complex; D002227:Carbazoles; D020409:Molecular Motor Proteins; D010982:Platelet-Derived Growth Factor; D011412:Propanolamines; D034741:RNA, Small Interfering; C495774:Wasf1 protein, mouse; C492303:Wasf2 protein, mouse; D051300:Wiskott-Aldrich Syndrome Protein Family; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; C489942:wiskostatin