Department of Molecular and Cell Biology, University of California-Berkeley, Berkeley, CA 94720, USA.
Cytoskeleton (Hoboken). 2010 Mar;67(3):193-206. doi: 10.1002/cm.20437.
Activators of the Arp2/3 complex, termed nucleation-promoting factors (NPFs), are required for the proper spatial and temporal control of actin assembly in cells. Mammalian cells express several NPFs, each of which functions in a distinct cellular process, including WASP and N-WASP in phagocytosis and endocytosis, WAVE and JMY in cell migration, and WHAMM in ER-to-Golgi transport. Although another NPF called WASH was recently identified, the cellular localization and function of this protein were unclear. Here we demonstrated that human WASH alone potently activated the Arp2/3 complex in vitro and in cells, suggesting that the protein is not autoinhibited like N-WASP, but is likely regulated by interacting proteins. In cells, WASH was associated with Rab5-positive early endosomes and Rab11-positive recycling endosomes that were enriched for actin filaments. Silencing of WASH or Arp2/3 complex expression by RNAi, or disruption of actin function by drug treatments, caused enlargement and elongation of endosomes. Intriguingly, WASH silencing, as well as actin disruption, delayed EGF transport to LAMP1-positive late endosomes. These observations indicate that actin polymerization by WASH influences the shape and maturation of endosomes, and highlight a previously unrecognized role for WASH and the Arp2/3 complex in the degradative steps of endocytic trafficking.
Arp2/3 复合物的激活剂，称为成核促进因子 (NPF)，对于细胞中肌动蛋白组装的适当空间和时间控制是必需的。哺乳动物细胞表达几种 NPF，每种 NPF 都在不同的细胞过程中发挥作用，包括吞噬作用和内吞作用中的 WASP 和 N-WASP、细胞迁移中的 WAVE 和 JMY 以及内质网到高尔基体运输中的 WHAMM。尽管最近发现了另一种称为 WASH 的 NPF，但该蛋白的细胞定位和功能尚不清楚。在这里，我们证明了人类 WASH 本身可以在体外和细胞中强烈激活 Arp2/3 复合物，这表明该蛋白不像 N-WASP 那样自动抑制，但可能受相互作用蛋白的调节。在细胞中，WASH 与 Rab5 阳性早期内体和 Rab11 阳性再循环内体相关，这些内体富含肌动蛋白丝。通过 RNAi 沉默 WASH 或 Arp2/3 复合物的表达，或通过药物处理破坏肌动蛋白功能，导致内体增大和伸长。有趣的是，WASH 沉默以及肌动蛋白破坏会延迟 EGF 向 LAMP1 阳性晚期内体的运输。这些观察结果表明，WASH 引发的肌动蛋白聚合影响内体的形状和成熟，并强调了 WASH 和 Arp2/3 复合物在胞吞作用降解步骤中的先前未被认识到的作用。
