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

SCAR/WAVE复合物将上游Rho家族GT3信号传导与不同生物体（包括人、蝇和网骨藻）中保守的ARP 2/3复合物的激活联系起来。SCAR/WAVE诱导和ARP 2/3复合物介导的质膜下肌动蛋白成核对于肌动蛋白在突出的板状伪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和小多肽Hspc 300组成的稳定的异五聚体，并且最初已报道在活性Rac与Pir结合时解离，从而释放SCAR/WAVE的C末端VCA结构域以激活Arp 2/3复合物。然而，这种激活机制受到了最近数据的挑战，并且这种复合物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复合物的剩余多肽的量减少，这意味着复合物主要作为分子实体起作用。然而，阿比特龙也被发现与N-WASP，mDia formins和Eps 8/SOS 1，表明个别SCAR/WAVE复合物亚基在肌动蛋白为基础的过程中的调节的额外功能。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，无论是单独或组合在Dictyosteoblasts细胞中的基因破坏。初步分析显示，不同突变体之间的细胞形态2和F-肌动蛋白组织存在显着差异，证实了单个亚基的额外作用的观点，超出了SCAR/WAVE介导的Arp 2/3复合物激活的线性信号级联。本项目的目的是确定和表征这些功能，以揭示归因于这些重要的调节肌动蛋白组装的生化和细胞活动的完整剧目。

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.