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

摘要 为了协调复杂的过程，如运动和内吞作用，细胞依赖于控制 肌动蛋白细胞骨架的动力学和结构。肌动蛋白微丝核蛋白是重要的肌动蛋白调节因子 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。Arp2/3复合体是一种七亚单位肌动蛋白细丝成核机，专门生成 分支肌动蛋白细丝。Arp2/3复合体调节的分支不仅对过程很重要 正常细胞功能所必需的，如内吞作用和片状脂血症突出，但也在致病 宿主细胞被细菌感染或肿瘤细胞转移等过程。大多数对Arp2/3的研究 复杂的调控集中在WASP蛋白上，WASP蛋白是一种介导分支肌动蛋白传播的激活剂 网络。这些研究揭示了WASP介导的激活的基本原理；WASP与Arp2/3结合 复合体，招募肌动蛋白单体，并刺激激活的构象变化。然而， WASP介导的激活的分子方面对于理解WASP的生物学功能最重要 复杂的情况仍是未知的。例如，目前尚不清楚为什么WASP蛋白需要肌动蛋白细丝来激活 Arp2/3复合体，这是一种允许Arp2/3复合体作为分支肌动蛋白细丝的特殊功能 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招募到Arp2/3 触发成核的复合体，尽管这一要求调节了分支细丝的密度 末端为Arp2/3-组装的肌动蛋白网络。除了WASP，现在还有其他几种调节蛋白 已知能激活Arp2/3复合体。我们最近发现Wish/DIP/SPIN90(WDS)蛋白是 与WASP的不同之处在于，它们可以激活Arp2/3复合体，而不需要预先形成的细丝来诱导它 有核的线状肌动蛋白细丝。这些线状细丝用作启动WASP介导的分枝的种子 从而启动肌动蛋白网络组装。尽管印心可能是最重要的 控制分支网络组装的步骤，WDS蛋白如何激活复合体来创建种子 细丝是未知的。此外，目前还不清楚WDS蛋白的活性是如何与 其他激活剂，如WASP，或WDS蛋白本身是如何受到调控的。在这里，我们提出了一种组合 生化、生物物理、结构、计算和细胞生物学方法来研究这些 问题，长期目标是了解这些调控机制如何允许Arp2/3 Complex 控制复杂细胞过程中的肌动蛋白动力学。

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.