N-WASP involvement in dorsal ruffle formation in mouse embryonic fibroblasts
N-WASP involvement in dorsal ruffle formation in mouse embryonic fibroblasts
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DOI：
10.1091/mbc.e06-06-0569
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发表时间：
2007-02-01
影响因子：
3.3
通讯作者：
Machesky, Laura M.
中科院分区：
文献类型：
--
作者：
Legg, John A.;Bompard, Guillaume;Machesky, Laura M.
Wiskott-Aldrich综合征蛋白（WASP）家族激活Arp 2/3复合物，导致新肌动蛋白丝的形成。在这里，我们研究了Scar 1，Scar 2，N-WASP，Arp 2/3复合物在小鼠胚胎成纤维细胞（MEFs）背褶形成的参与。使用血小板源性生长因子刺激圆形背褶大会在原E13和永生E9 Scar 1（+/+）和Scar 1空MEFs，我们建立Scar 1的损失不会损害背褶的形成。通过小干扰RNA（siRNA）降低Scar 2蛋白水平也不影响背部皱褶的产生。相比之下，wiskostatin，一种化学抑制剂的N-WASP，有力地抑制背部皱褶形成的剂量依赖性方式。此外，N-WASP和Arp 2 siRNA处理显著减少MEFs中背褶的形成。此外，不能结合Arp 2/3复合物的N-WASP截短突变体的表达阻断了这些结构的形成。最后，N-WASP（-/-）成纤维细胞样细胞产生异常的背部皱褶。这些皱褶高度不稳定，Arp 2/3复合物严重耗尽，尺寸减小。我们假设N-WASP和Arp 2/3复合物是一个多蛋白组装的一部分，对产生背部皱褶很重要，Scar 1和Scar 2在这个过程中起着重要作用。
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.