Dissection of the actin assembly machinery at the lamellipodium tip

arp2 /3催化的肌动蛋白丝组装与多种细胞运动过程有关。arp2 /3复合物被成核促进因子（npf）激活，例如WASP和WAVE家族蛋白，它们驱动Rho- gtpase Cdc42和Rad下游的肌动蛋白重组。一个由无处不在的WAVE2和组成相关因子（WAVE复合物）组成的大分子单元在Rac1激活时转移到细胞外周，对于arp2 /3复合物激活和诱导板足突出至关重要。然而，这是如何发生的分子细节，例如关于质膜脂在这一过程中的潜在作用或关于板足中单个波复合物单位的寿命是难以捉摸的。在本提案中，我们着手通过(1)开发WAVE复合物介导的肌动蛋白丝组装的体内重建分析，以及(2)通过FRAP（光漂白后荧光恢复）实验评估板足突出过程中WAVE-和arp2 /3复合物的周转率来解决这些问题。这些方法不仅有助于绘制更全面的波复合物激活需求图，而且还有助于开发和评估新的分析方法，以揭示肌动蛋白聚合复合物的分子特征。

Arp2/3-catalysed actin filament assembly is implicated in various cellular motility processes. The Arp2/3-complex is activated by nucleation promoting factors (NPFs), for instance WASP and WAVE family proteins, which drive actin reorganizations downstream of the Rho- GTPases Cdc42 and Rad. A macromolecular unit comprising ubiquitous WAVE2 and constitutively associated factors (WAVE-complex) is translocated to the cell periphery upon Rac1 activation and essential for Arp2/3-complex activation and induction of lamellipodia protrusion. However, the molecular details of how this occurs e.g. concerning the potential role of plasma membrane lipids in this process or regarding the lifetime of individual WAVE-complex units in lamellipodia are elusive. In this proposal, we set out to address these questions by (1) developing an in vivo reconstitution assay of WAVE-complex mediated actin filament assembly, and by (2) assessing the turnover of both WAVE- and Arp2/3-complexes during lamellipodia protrusion e.g. by FRAP (fluorescence recovery after photobleaching) experiments. These approaches will help not only to draw a more comprehensive picture of the requirements for WAVE-complex activation, but also to develop and evaluate new assays for unravelling the molecular features of actin polymerisation complexes in vivo.