The “Bundleforce” project focuses on the physical and chemical characterization of the dynamics of actin bundles as encountered in filopodia.
Eukaryotic cells constantly convert signals between biochemical energy and mechanical work to timely accomplish many key functions such as migration, division or development. Filopodia are essential finger- like structures that emerge at the cell front to orient the cell in response to its chemical and mechanical environment. Yet, the molecular interactions that make this type of actin network mechanosensitive are not known. To tackle this challenge we propose unique biophysical in vitro and in vivo experiments of increasing complexity.
Dynamic actin filaments are crosslinked together in parallel by fascin proteins to create bundles. Formins are proteins that speed up actin filament elongation and remain processively attached to filament barbed ends. How formins control the growth of actin bundles in filopodia is not understood:
We aim to:
1) Elucidate how formin and fascin functions are regulated by mechanics at the single filament level. We will investigate how formin partners and competitors present in filopodia affect formin processivity; how fascin affinity for the side of filaments is modified by filament tension and formin presence at the barbed- end.
2) Reconstitute filopodia-like actin bundles in vitro to understand how actin bundle size and fate are regulated down to the molecular scale. Using a unique experimental setup that combines microfluidics and optical tweezers, we will uncover for the first time actin bundles mechanosensitive capabilities, both in tension and compression. Conversely, fascin-crosslinked bundle disassembly by proteins from the ADF/cofilin family is not well understood. We propose to address bundle severing by Cofilin-A, taking advantage of our reconstituted system.
3) Decipher in vivo the mechanics of actin bundles in filopodia, using formin with integrated fluorescent tension sensor modules.
This framework spanning from in vitro single filament assays to in vivo meso-scale actin networks will bring unprecedented insights into the role of actin bundles in filopodia mechanosensitivity.
Conclusion:
Over the course of the ERC funded project, we have deciphered many important aspects of actin bundle growth and disassembly:
1. mDia1 formins are highly sensitive to pulling forces, and their unbinding from the barbed ends of filaments can follow two independent routes, one being more sensitive to force.
2. mDia1 formin processivity is dependent on the occupancy of its FH1 domains by profilin-actin complexes. Profilin-loaded FH1 forms a so-called 'ring complex' by interacting with the filament barbed end, preventing the FH2 domain unbinding.
3. When elongating fascin-crosslinked bundles, mDia1 & mDia2 formin functions are altered. In particular, their processivity is highly reduced when filament barbed ends are in the vicinity of fascin molecules in charge of zippering filament together.
4. mDia1 formins are also highly sensitive to their ability to freely rotate, relative to the filament long axis. If they cannot relax their accumulating torque, they will unbind from barbed ends.
5. When exposed to the disassembly factor, ADF/Cofilin, fascin-crosslinked bundles are more resistant to disassembly than single filaments.
6. Cofilin domain nucleation on filaments that are bundled by fascin is reduced up to 20-fold compared to single filaments. This effect is both fascin concentration and bundle size dependent.
7. Cofilin domain growth causes fascin to locally depart from the filament side, thus neighboring filaments to be locally unbundled from each other.
8. We report a similar mechanism on alpha-actinin bundles
10. In cells, mDia2 formins at the tip of filopodia are under compression, due to plasma membrane tension. This compression is more pronounced for filopodia at the migration edge than at the retracting back.
