Novel roles of SCAR/WAVE subunits in the regulation of actin dynamics

SCAR/WAVE复合体将Rho家族GTP酶上游信号与不同生物中保守的Arp2/3复合体激活联系在一起，包括人、蝇和网柄金藻变形虫。SCAR/WAVE诱导和Arp2/3-复合体介导的肌动蛋白在质膜下的成核对于肌动蛋白在突起的片状脂体中组装以驱动细胞迁移是至关重要的。固有失活的SCAR/WAVE复合体是由SCAR/WAVE、Abi、Nap、Pir和一个小的多肽Hspc300组成的稳定的异五聚体，最初的报道是当活性Rac与Pir结合时解离，从而释放SCAR/WAVE的C-末端VCA结构域来激活Arp2/3复合体。然而，这一激活机制受到了最近的数据和挑战，而且在体内对该复合体及其亚单位的精确调控仍不清楚。此外，五聚体复合体的单个亚基的消除或耗尽经常会导致SCAR/WAVE复合体中剩余多肽的减少，这意味着该复合体在很大程度上是一个分子实体。然而，Abi也被发现与N-WASP、MDIA Forins和Eps8/SOS1相关，这表明单个SCAR/WAVE-Complex亚基在调节基于肌动蛋白的过程中具有额外的功能。为了系统地解决这个问题，我们已经开始消除所有的SCAR/WAVE复合体亚单位，无论是单独的还是通过基因破坏的方式在Dictyostelials细胞中联合消除。初步分析表明，不同突变体之间在细胞形态2和F-肌动蛋白组织上存在显著差异，这证实了单个亚基的额外作用的观点，而不是作用于SCAR/WAVE介导的Arp2/3-复合体激活的线性信号级联。该项目的目标是确定和表征这些功能，以揭示归因于这些重要的肌动蛋白组装调节因子的生化和细胞活动的完整谱系。

The SCAR/WAVE complex links upstream Rho-family GTPase signaling to the activation of the conserved ARP2/3 complex in different organisms including man, fly and Dictyostelium amoebae. SCAR/WAVE-induced and ARP2/3-complex-mediated actin nucleation beneath the plasma membrane is crucial for the assembly of actin in protruding lamellipodia to drive cell migration. The intrinsically inactive SCAR/WAVE complex is a stable heteropentamer composed of SCAR/WAVE, Abi, Nap, Pir and a small polypeptide Hspc300, and has initially been reported to dissociate upon binding of active Rac to Pir, thereby releasing the C-terminal VCA domain of SCAR/WAVE to activate the Arp2/3 complex. However, this activation mechanism has been challenged by recent data and, and the precise regulation of this complex and its subunits in vivo remains unclear. In addition, the elimination or depletion of individual subunits of the pentameric complex frequently results in diminished amounts of the remaining polypeptides of the SCAR/WAVE complex, implying that the complex largely acts as a molecular entity. However, Abi was also found to associate with N-WASP, mDia formins and Eps8/SOS1, indicating additional functions of individual SCAR/WAVE-complex subunits in the regulation of actin-based processes. To address this issue systematically, we have begun to eliminate all SCAR/WAVE-complex subunits, either alone or in combination by gene disruption in Dictyostelium cells. Preliminary analyses revealed significant differences in cell morphology 2 and F-actin organization between different mutants, corroborating the view of additional roles for individual subunits, beyond operating in a linear signaling cascade to SCAR/WAVEmediated Arp2/3-complex activation. The objective of this project is to identify and characterize these functions, in order to uncover the complete repertoire of biochemical and cellular activities ascribed to these important regulators of actin assembly.