Mechanisms of FHOD1 regulated stress fiber formation

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Regulation of the actin cytoskeleton is critical for many processes such as cell migration and cell division. Downstream of Rho GTPases multiple members of the formin protein family have been identified as critical regulators of actin nucleation in vitro and in vivo. In particular, the formins mDia1 and FHOD1 are thought to regulate actin based stress fibers and cell contractility. mDia1 itself is not predominantly bound to contractile structures, such as stress fibers, but has been implicated in their regulation. In contrast, FHOD1 is strongly associated with stress fibers themselves and our preliminary results also identify this actin nucleator at focal adhesions, which form anchor points for stress fibers. At the current stage of research, many biochemical properties of these formins have been elucidated, which draw a fairly precise picture, how they work as individual molecular machines. However, their interplay with the cellular machinery is still poorly understood. In particular, in is not clear, how the recruitment and spatio-temporal activation dynamics of these formins controls localized actin polymerization to build dynamic cellular structures, such as stress fibers. Rho GTPases have been shown to play crucial role in the activation mechanism of formins. Interestingly, although for both mDia1 and FHOD1, the biochemical activation mechanism – via opening of a head-to-tail intramolecular inhibition - is similar, specificity of regulation downstream of Rho GTPases in the cellular context is still poorly understood – especially for the less studied formin FHOD1. Here, we propose to use a multi-facetted approach including RNA interference, fluorescence activity biosensors and TIRF speckle microscopy to study how formin actin nucleators control cell contractility downstream of Rho signaling pathways. First, we will study how cell contractility is regulated by formins and the upstream GEF-H1/RhoA/ROCK pathway. Second, we will assess the dependence of mDia1 and FHOD1 localization dynamics on the Rho GTPases RhoA, Rac1 and Cdc42. Third, we will correlate formin dynamics with spatio-temporal Rho GTPase activity patterns. Finally, we will use a light inducible technique to manipulate the subcellular distribution of Rho GTPase activity and study, how such external manipulations affect formin function.