Function of cortical formins in the motility of strongly adherent cells

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The actin cortex is a thin layer of cross-linked actin filaments, nonmuscle myosin II, and associated proteins beneath the plasma membrane of eukaryotic cells. Assembly, contraction and mechanics of this layer is the decisive factor of cell shape and therefore plays a central role in various processes including migration, cell division or tissue morphogenesis. As yet, we could demonstrate that integrity of the cell cortex in Dictyostelium cells is safeguarded by three cortical formins. Conserved functions in cell cortex regulation can now also be ascribed to synergistic activities of mDia1 and mDia3 in mammalian cells. Interestingly, however, analyses of all these formin mutants clearly showed that cell cortex defects in the strongly adhering NIH 3T3 cells affect relative migration rate to a much larger extent as compared to less adhesive cell types. The characterization of the mechanical properties and the mechanisms associated with loss of these cortical formins in the strongly adhering NIH 3T3 fibroblasts is therefore one of the main objectives in this follow-up proposal. In order to fully understand the cellular function and synergy of formins in the establishment and maintenance of the cell cortex, it is furthermore absolutely indispensable to have knowledge about the complete inventory of these actin-assembly factors. Due to the lack of a noticeable cytokinesis defect in mDia1/3-KO cells, it is more than likely that functionality of the cell cortex in animal cells is backed up by at least one additional formin. The most likely candidate is the Rho effector mDia2. Thus, we also aim to generate mDia2-deficient mouse fibroblast using CRISPR/Cas9 technology to assess the function of this remaining formin from the mDia-subfamily in cell migration and cortex regulation. The dissection of formin function in cell cortex mechanics in animal cells is undoubtedly a challenging task, but we anticipate that examination by various complementary approaches including advanced biophysical and comprehensive cell biological analyses as well as high-resolution imaging of knockout cell lines, as specified in the work programme, will have substantial and wide reaching implications on our understanding on cell cortex assembly and cell mechanics.