Understanding the roles of WASP in Arp2/3 complex activation and branched actin network assembly

项目总结 肌动蛋白相关蛋白(Arp)2/3复合体是一个225 kDa的7亚单位肌动蛋白细丝核蛋白 分支肌动蛋白细丝。聚合分支肌动蛋白网络提供了驱动 无数的细胞过程，包括运动、囊泡运输和内吞作用。为了协调好这些 功能，细胞利用与Arp2/3复合体结合并激活的蛋白质，这种复合体被称为成核促进因子 (NPFS)。最普遍和研究最充分的一类NPF，Wiskott-Aldrich综合征蛋白(WASP)是 具有与肌动蛋白结合的保守的C-末端“VCA”(异丙氨酸同源，中心，酸性)基序的特征 单体(V)和Arp2/3络合物(CA)。在没有WASP的情况下，Arp2/3复合体处于非活性状态 两个肌动蛋白相关蛋白Arp2和Arp3以端到端的方向排列的构象 这里指的是展开状态。激活依赖于移动Arp2和Arp2的大的构象变化 Arp3呈丝状排列，称为短螺距构象。在此之前，我们演示了 黄蜂结合刺激短节距构象的形成，这是主要的激活功能 WASP.然而，WASP结合如何将伸展到短间距构象平衡的确切方式是 不清楚，也是这项提案的重点。我们将使用HIGH从结构-功能的角度来解决这个问题 非活动状态的解析结构和活动状态的假设模型，以确定 在没有WASP的情况下，复合体保持不活跃(目标1)。此外，我们将解决两个基本方面 WASP介导的复合体的调节，这些都是该领域的关键开放问题。首先，虽然最近的数据 表明WASP与复合体上的两个不同的结合部位结合，每个部位的结合如何起作用 在体外或细胞内对肌动蛋白网络复合体和组装的激活作用尚不清楚。我们将解决 目标2中的这个问题，利用最近在Arp2/3上确定的WASP结合位点图 通过交联质谱联用确定了络合物。其次，最近的数据表明，要激活Arp2/3 复杂，WASP必须首先绑定以刺激短节距构象，但随后必须释放以允许 成核继续进行。因为WASP结合了细胞内的膜，所以WASP的释放被认为起到了关键作用 在产生力量的肌动蛋白网络的组装中发挥作用；即，它在 网络到膜，促进推动，但通过成核后释放确保聚合 网络没有如此紧密地捆绑在一起，以至于会导致网络压缩。目前还没有研究表明， WASP与络合物的相互作用被调节，以最佳地平衡其成核能力与其能力 作为肌动蛋白网络和膜之间的纽带，尽管这两种活动都是至关重要的 组装富有成效的肌动蛋白网络。因此，在目标3中，我们将确定WASP之间的交互 和Arp2/3被调整以平衡WASP的成核促进和肌动蛋白网络拴系作用 组装肌动蛋白网络。

PROJECT SUMMARY The actin-related-protein (Arp) 2/3 complex is a 225-kDa seven-subunit actin filament nucleator that nucleates branched actin filaments. Polymerizing branched actin networks provide protrusive forces necessary to drive a myriad of cellular processes, including motility, vesicle trafficking, and endocytosis. To orchestrate these functions, cells utilize proteins that bind to and activate Arp2/3 complex known as nucleation promotion factors (NPFs). The most ubiquitous and well-studied class of NPFs, Wiskott-Aldrich syndrome proteins (WASP), are characterized by a conserved C-terminal “VCA” (verprolin homology, central, acidic) motif that binds to actin monomers (V) and Arp2/3 complex (CA). In the absence of WASP, Arp2/3 complex is held in an inactive conformation in which the two actin-related proteins Arp2 and Arp3 are arranged in an end-to-end orientation referred to here as the splayed state. Activation depends on a large conformational change that moves Arp2 and Arp3 into filament-like arrangement known as the short-pitch conformation. Previously, we demonstrated that WASP binding stimulates formation of the short-pitch conformation and that this is the main activating function of WASP. However, exactly how WASP binding shifts the splayed to short-pitch conformational equilibrium is unclear, and is the focus of this proposal. We will address this from a structure-function perspective using high resolution structures of the inactive state and hypothetical models of the active state to determine how the complex is held inactive in the absence of WASP (Aim 1). In addition, we will address two fundamental aspects of WASP-mediated regulation of the complex that are critical open questions in the field. First, while recent data indicated that WASP binds to two distinct binding sites on the complex, how engagement at each site contributes to activation of the complex and assembly of actin networks in vitro or in cells remains unknown. We will address this question in Aim 2, taking advantage of a recently determined map of the WASP binding sites on Arp2/3 complex determined by crosslinking/mass-spectrometry. Second, recent data show that to activate Arp2/3 complex, WASP must first bind to stimulate the short pitch conformation, but then must be released to allow nucleation to proceed. Because WASP binds membranes in cells, release of WASP is thought to play a critical role in the assembly of force-producing actin networks; i.e., it provides a transient connection between the network to the membrane that facilitates pushing, yet by releasing after nucleation ensures that polymerizing networks are not so tightly bound they cause network compression. No studies have addressed how the interactions of WASP with the complex are tuned to optimally balance its nucleation potency versus its ability to serve as a tether between actin networks and membranes, despite the fact that both of these activities are critical in assembling productive actin networks. Therefore, in Aim 3 we will determine how interactions between WASP and Arp2/3 are tuned to balance the nucleation-promoting versus actin network-tethering roles of WASP in assembling actin networks.