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Published on: November 6, 2018
N-WASP involvement in dorsal ruffle formation in mouse embryonic fibroblasts
John A Legg1, Guillaume Bompard, John Dawson
1School of Biosciences, The University of Birmingham, Birmingham, Edgbaston, West Midlands, B15 2TT, United Kingdom.
Abstract:
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.
Insights
N-WASP and Arp2/3 complex are crucial for dorsal ruffle formation in mouse cells, while Scar1 and Scar2 are not essential. Inhibition or absence of N-WASP disrupts ruffle stability and size.
Area of Science:
- Cell biology
- Cytoskeletal dynamics
- Actin polymerization
Background:
- The Wiskott-Aldrich syndrome protein (WASP) family is known to activate the Arp2/3 complex, a key regulator of actin filament formation.
- Dorsal ruffles are dynamic cell structures involved in processes like cell migration and phagocytosis.
- Understanding the molecular machinery behind dorsal ruffle formation is crucial for comprehending cell motility and related biological functions.
Purpose of the Study:
- To investigate the specific roles of Scar1, Scar2, N-WASP, and the Arp2/3 complex in the formation of dorsal ruffles in mouse embryonic fibroblasts (MEFs).
- To determine whether Scar1 and Scar2 are essential for dorsal ruffle assembly.
- To elucidate the necessity of N-WASP and Arp2/3 complex in generating stable and properly sized dorsal ruffles.
Main Methods:
- Stimulation of dorsal ruffle assembly in primary and immortalized MEFs using platelet-derived growth factor.
- Genetic manipulation including the use of Scar1 null MEFs and siRNA to reduce Scar2 protein levels.
- Pharmacological inhibition of N-WASP using wiskostatin.
- siRNA-mediated knockdown of N-WASP and Arp2.
- Expression of an N-WASP truncation mutant lacking Arp2/3 binding capability.
- Analysis of dorsal ruffles in N-WASP knockout (N-WASP(-/-)) fibroblasts.
Main Results:
- Loss of Scar1 did not impair dorsal ruffle formation in MEFs.
- Reduction of Scar2 levels using siRNA did not affect dorsal ruffle production.
- Wiskostatin treatment potently suppressed dorsal ruffle formation in a dose-dependent manner.
- N-WASP and Arp2 siRNA significantly decreased dorsal ruffle formation.
- Expression of an N-WASP mutant unable to bind Arp2/3 blocked ruffle formation.
- N-WASP(-/-) fibroblasts exhibited aberrant, unstable dorsal ruffles depleted of Arp2/3 and diminished in size.
Conclusions:
- N-WASP and the Arp2/3 complex are essential components of a multiprotein assembly required for the generation of dorsal ruffles.
- Scar1 and Scar2 are dispensable for the formation of dorsal ruffles.
- N-WASP plays a critical role in stabilizing dorsal ruffles and recruiting the Arp2/3 complex for proper actin polymerization.
