Dissection of formin functions in cell edge protrusion and migration

• Applicant: Professor Dr. Klemens Rottner
• Subject Area: Cell Biology
• Term: from 2018 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 409168965

Migratory processes and any type of shape change in cells critically depend on dynamic reorganization of filaments of the actin cytoskeleton. For this, many cell types employ web-like, flat and fingerlike, pointed structures termed lamellipodia and filopodia, respectively. Formation of these structures is directly and indirectly driven by a large number of signalling molecules and regulators of the actin cytoskeleton, but the intricate interplay of all these factors is incompletely understood. Direct regulation of actin filaments is accomplished by cytosolic molecules binding either actin monomers or filaments, or even catalysing the transition of monomer to filament, called nucleation, as prominently exemplified by the formin family of proteins. Mammals express 15 formin family members, the precise individual functions of which are still understudied. Some members of this group of proteins, such as mDia and FMNL are known to affect the formation of cellular protrusions such as lamellipodia and filopodia, but how precisely they do this, how many of the 15 formin family members can share this function, as well as their precise relative contributions to these processes have largely remained unknown. In particular, the formation of filopodia at the protrusive front of migrating cells, which have been ascribed sensory functions, are proposed to be regulated by formin activity, but which members of the formin family or if any at all are essential for the formation of these structures in mammalian cells could not be established to date. The project proposed here will clarify these questions, starting from cell lines already established in the lab using CRISPR/Cas9, for instance lines lacking FMNL2 and FMNL3 individually and in combination, as well as extending those to novel cell lines lacking formin family members constituting promising members concerning involvement in filopodia formation, such as mDia2, mDia3 and DAAM. All these formin members will be genetically targeted and analysed for their contribution to filopodia formation individually and in combination with previously characterised lines. To do these, growth and signalling conditions of respective cell lines will be optimized allowing robust and easily quantifiable filopodia formation in a computer-aided fashion. Moreover, establishment of commonalities of defined molecular mechanisms of filopodia formation in at least two complementary parent cell lines of distinct origin will guarantee to select for general mechanisms and pathways, relevant for a broad range of cell types and/or developmental stages. Finally, established cell lines will also be employed for identification of hitherto unknown formins potentially involved in the generation of so called mother filaments for Arp2/3 complex-dependent branching of filaments in lamellipodial actin networks. Clarification of all these issues will significantly refine our understanding or cellular actin reorganization and motility processes.

• DFG Programme: Research Grants