An impressive variety of motile and morphogenetic processes are driven by site-directed polarized asssembly of actin filaments. In the past ten years, breathtaking advances coming from cell biology, cell biophysics, and biochemistry have brought insight into the molecular bases for production of force and movement by site-directed actin polymerization. Yet, we do not know, with the detail sufficient to understand how force is produced, by which molecular mechanisms the filaments are nucleated or created by branching. We do not know by which elementary steps insertional polymerization of barbed ends of filaments against the membrane is performed by different protein machineries, nor how these machineries work in a coordinated fashion. Here we propose a multiscale and interdisciplinary approach of the mechanisms used by the major actin nucleators to organize the motile response of actin. The elementary reactions involved in the processive walk of formin at the growing barbed ends of filaments and the role of ATP hydrolysis in force production will be analyzed by a combination of biochemical solution studies and physical methods using functionalized GUVs and optical tweezers. The multifunctionality of WH2 domains involved in actin sequestration, filament nucleation severing and processive elongation will be similarly examined in an interdisciplinary perspective from structural biology at atomic resolution to physics at the mesoscopic scale. Biochemical and structural methods and single molecule measurements (TIRFM) will shed light into the elementary steps and structural mechanism of filament branching. Biomimetic assays with functionalized GUVs associated with biophysical methods like FRAP or fluorescence correlation spectroscopy will elucidate how different filament initiating machineries segregate in the membrane as a consequence of their interactions with growing filaments and function in a coordinated fashion during actin-based motility.
Fields of science
Call for proposal
See other projects for this call
Funding SchemeERC-AG - ERC Advanced Grant