Dissection of the actin assembly machinery at the lamellipodium tip

Arp2/3催化的肌动蛋白微丝组装参与多种细胞运动过程。Arp2/3-复合体被核化促进因子(NPF)激活，例如WASP和WAVE家族蛋白，它们驱动Rho-GTP酶CDc42和Rad下游的肌动蛋白重组。由普遍存在的WAVE2和结构性相关因子(波-复合体)组成的大分子单位在rac1激活时被转移到细胞外周，对于Arp2/3-复合体的激活和诱导片状脂体突出是必不可少的。然而，这是如何发生的，例如关于质膜脂在这一过程中的潜在作用，或者关于片状脂蛋白中单个波-复合体单位的寿命的分子细节是难以捉摸的。在这项建议中，我们着手于通过(1)建立波复合体介导的肌动蛋白细丝组装的体内重建实验，以及(2)通过FRAP(光漂白后荧光恢复)实验来评估波状和Arp2/3-复合体在片状脂虫突起过程中的周转。这些方法不仅有助于更全面地描绘波复合体激活的要求，而且还将开发和评估新的分析方法，以在体内解开肌动蛋白聚合复合体的分子特征。

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.