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

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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.