Dissection of formin pathways coordinating polarity with cell migration downstream of Ras signalling

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Diaphanous-related formins constitute a conserved family of ubiquitous multidomain proteins that catalyze the de novo nucleation of linear actin filaments and act as downstream effectors of Rho GTPase signaling pathways to control numerous cellular functions including the establishment of cell shape or cell migration. Our ongoing analysis of the 10 Dictyostelium formins unexpectedly revealed that the elimination of the regulated formin dDia1 strongly accelerated cell motility, whereas migration of dDia1-overexpressing cells was significantly suppressed. As assessed by imaging of an active dDia1 variant, the formin accumulated in a thin layer around the plasma membrane and was most strongly enriched in vesicular structures at the cell cortex. The aim of this project is to characterize these highly dynamic structures by epifluorescence and correlative electron microscopy and find out how exactly dDia1 brings about it unexpected negative effect on cell migration. In vivo formins seem to strictly depend on the recruitment of profilin-actin complexes by the FH1-domain for further assembly by the FH2 2 domain. Therefore, we also intend to thoroughly study actin polymerization by Dictyostelium ForC, because it is the only known formin lacking the FH1-region. As ForC is also devoid of other canonical G-actin binding sites, the second project aims to identify and characterize possible mechanisms by which this formin accomplishes to recruit actin subunits for subsequent filament assembly. We expect that both projects will greatly add to our knowledge of formin-mediated actin assembly.