Control of actin filament networks by Arp2/3 complex and its regulators

总结 为了协调复杂的过程，如运动和内吞作用，细胞依赖于调节蛋白质， 肌动蛋白细胞骨架的动力学和结构。肌动蛋白丝成核剂是重要的肌动蛋白调节剂 因为它们允许细胞控制新肌动蛋白网络的启动，并决定这些网络的结构， 网络. Arp 2/3复合物是一个七亚基肌动蛋白丝成核机器，特异性地产生 分支肌动蛋白丝。Arp 2/3复合物调控的分支不仅对过程很重要， 正常细胞功能所需，如内吞作用和片状伪足突出，但也在致病性 如宿主细胞被细菌感染或肿瘤细胞转移的过程。大多数关于Arp 2/3的研究 复杂的调节集中在WASP蛋白，一种介导分支肌动蛋白增殖的激活剂 网络.这些研究揭示了WASP介导的活化的基本原理：WASP结合Arp 2/3 复合物，招募肌动蛋白单体，并刺激激活构象变化。但 WASP介导的活化的分子方面对于理解WASP的生物学功能是最重要的。 复杂性仍然未知。例如，目前还不清楚为什么WASP蛋白需要肌动蛋白丝来激活 Arp 2/3复合物，一种允许Arp 2/3复合物特异性地作为分支肌动蛋白丝发挥功能的要求 成核剂此外，还不知道为什么WASP（而不是其他激活剂）必须将肌动蛋白单体募集到Arp 2/3 复杂的触发成核，尽管事实上，这一要求调节分支丝的密度 末端为Arp 2/3组装的肌动蛋白网络。除了WASP之外，现在还有几种其他调节蛋白被 已知激活Arp 2/3复合物。我们最近发现WISH/DIP/SPIN 90（WDS）蛋白是 与WASP不同的是，它们可以激活Arp 2/3复合物，而不需要预先形成的细丝来诱导它， 成核的线状肌动蛋白丝。这些线状细丝作为种子启动WASP介导的分支 从而启动肌动蛋白网络组装。尽管入会可能是最重要的 控制分支网络组装的一步，WDS蛋白如何激活复合物以产生种子 灯丝是未知的。此外，还不了解WDS蛋白的活性如何与 其他激活剂如WASP，或WDS蛋白本身是如何调节的。在这里，我们提出了一个组合， 生物化学，生物物理，结构，计算和细胞生物学方法来研究这些 长期目标是了解这些调控机制如何使Arp 2/3复合物 在复杂的细胞过程中控制肌动蛋白动力学。

Summary To orchestrate complex processes like motility and endocytosis, cells rely on regulatory proteins that control the dynamics and architectures of the actin cytoskeleton. Actin filament nucleators are important actin regulators because they allow cells to control the initiation of new actin networks and dictate the architectures of these networks. Arp2/3 complex is a seven subunit actin filament nucleating machine that specifically generates branched actin filaments. Regulated branching by Arp2/3 complex is important not only for processes required for normal cellular function, such as endocytosis and protrusion of lamellipodia, but also in pathogenic processes like host cell infection by bacteria or metastasis of tumor cells. The majority of studies on Arp2/3 complex regulation have focused on WASP proteins, an activator that mediates propagation of branched actin networks. These studies revealed the basic tenets of WASP mediated activation; that WASP binds to Arp2/3 complex, recruits actin monomers, and stimulates an activating conformational change. However, the molecular aspects of WASP mediated-activation most important for understanding the biological function of the complex are still unknown. For instance, it is unknown why WASP proteins require actin filaments to activate Arp2/3 complex, a requirement that allows Arp2/3 complex to function specifically as a branched actin filament nucleator. Further, it is not known why WASP (but not other activators) must recruit actin monomers to Arp2/3 complex to trigger nucleation, despite the fact that this requirement regulates the density of branched filament ends in Arp2/3-assembled actin networks. In addition to WASP, several other regulatory proteins are now known to activate Arp2/3 complex. We recently discovered that the WISH/DIP/SPIN90 (WDS) proteins are distinct from WASP in that they can activate Arp2/3 complex without preformed filaments to induce it to nucleate linear actin filaments. These linear filaments serve as seeds to kickstart WASP-mediated branching thereby initiating actin network assembly. Despite the fact that initiation is perhaps the most important step in controlling branched network assembly, how WDS proteins activate the complex to create seed filaments is unknown. Furthermore, it is not understood how the activity of WDS proteins is coordinated with other activators like WASP, or how WDS proteins themselves are regulated. Here we propose a combination of biochemical, biophysical, structural, computational, and cell biological approaches to investigate these questions, with a long-term goal of understanding how these regulatory mechanisms allow Arp2/3 complex to control actin dynamics in complex cellular processes.