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

SCAR/WAVE复合体将上游rho家族GTPase信号通路与人类、苍蝇和变形虫等不同生物中保守的ARP2/3复合体的激活联系起来。SCAR/ wave诱导的和arp2 /3复合物介导的肌动蛋白在质膜下成核对于肌动蛋白在凸出的板足中组装以驱动细胞迁移至关重要。本质上无活性的SCAR/WAVE复合物是由SCAR/WAVE、Abi、Nap、Pir和一个小多肽Hspc300组成的稳定的异对映体，最初有报道称活性Rac与Pir结合时解离，从而释放SCAR/WAVE的c端VCA结构域，激活Arp2/3复合物。然而，这种激活机制受到了最近的数据和挑战，并且该复合体及其亚基在体内的精确调控仍不清楚。此外，五聚体复合物的单个亚基的消除或消耗经常导致SCAR/WAVE复合物剩余多肽的减少，这意味着该复合物在很大程度上起着分子实体的作用。然而，Abi也被发现与N-WASP，媒质形成蛋白和Eps8/SOS1相关，这表明单个SCAR/ wave复合物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复合物亚基，无论是单独的还是通过基因破坏在盘基骨柱细胞中的组合。初步分析显示，不同突变体之间的细胞形态2和F-actin组织存在显著差异，证实了单个亚基除了在SCAR/ wave介导的arp2 /3复合物激活的线性信号级联中起作用外，还有其他作用的观点。该项目的目的是鉴定和表征这些功能，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曲目。

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.