Molecular mechanisms of filopodia formation. (360G-Wellcome-095829_Z_11_Z)

I have developed a reconstitution system using supported lipid bilayers and Xenopus egg extracts that recapitulates several aspects of filopodia formation in a format highly amenable to microscopy and intervention. The filopodia-like structures are comprised of long, parallel bundled actin filaments that polymerise from the membrane surface. At the membrane there is assembly of actin regulators that mimics the filopodial tip complex. To understand how the tip complex functions I propose to: (1 ) determine the assembly mechanism of the tip complex. I will do this by (a) measuring the recruitment of proteins to the tip complex (b) studying the roles of tip complex members toca, IRSp53, srGAP2 (of the BAR domain superfamily) and Cdc42 (the Rho-type GTPase) in initiation using mutant proteins, immunodepletion and kinetics using fluorescent probes and (c) testing the contribution of Ena/VASP/Evl and diaphanous-related formins using similar techniques; (2) explore the molecular architecture of the tip complex using single molecule microscopy to determine the relationship between the structure and function of the tip complex; (3) compare the kinetics of protein recruitment to filopodia formed during early development to determine whether the molecular mechanism is conserved between different cell types.