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Unravelling the Mechanosensitivity of Actin Bundles in Filopodia

Periodic Reporting for period 4 - BUNDLEFORCE (Unravelling the Mechanosensitivity of Actin Bundles in Filopodia)

Reporting period: 2020-09-01 to 2021-08-31

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.

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.
Work Package 1:
WP1 objectives have been reached: Protein engineering and purification to investigate the synergy between formin and fascin activities were successfully completed. Formin and fascin proteins have minimal crosstalks when simultaneously bound on actin filaments. When filaments are crosslinked in small bundles (2 or 3 filaments) thanks to fascin, the change in actin filament conformation directly reduces formin processivity and actin filament elongation rate.

Work Package 2:
Using microfluidics we have been able to study the activity of oformins on fascin-induced actin filament bundles. We show that formin anchoring has a direct impact of actin bundle elongation by formins: length of the tether connecting the formin to the bundle, as well as the ability of formin to reorganize on a fluid lipid bilayer. We thus revealed that formin processivity in cells is probably much shorter than previously anticipated, due to the crosstalk of formin anchoring and filament crosslinking, highly hindering formin activity.
By developing new experimental approaches, using lipid micropatterns in microfluidics chambers, we have shown that higher bundle structures profoundly affect formin activity and that the build up of compressive mechanical load readily provokes the detachment of formin from actin filament barbed ends.
Fascin crosslinked bundles are somehow resistant to cofilin disassembly activity. This is a stake with previous publications, reporting a cooperative effect that results in an increase of bundle disassembly by cofilin proteins. We have investigated the molecular events of bundle disassembly. We show that cofilin domain nucleation is highly impaired on bundles, even more so for bigger bundles. The local departure of fascin crosslinks due to cofilin domain growth and the change of filament twist being transmitted across the bundle from one filament to another, favor the nucleation of new cofilin domains that will overlap with the initial one. This causes an acceleration of cofilin severing. Still this effect is milder than what is observed for single filaments, in the intermediate cofilin concentration regime.

Work Package 3:
- In Hela cells, we have probed the mechanical state of mDia2 formins at the tip of filopodia and in the cytoplasm. We show that mDia2 formins are under compression at the filopodia tip. This level of compression is dependent on cell polarity and the overall profilin-actin availability for polymerization.
- Preliminary work on the activity of myosinX on dynamic filaments revealed that myosin X can crosslink in a filament density dependent manner. Once a bundle, myosin X causes the bundle to curve or bend. This dynamic bending can cause oscillation patterns that need to be further investigated.
Our unique experimental approaches have now established that actin polymerization is both highly dependent on both the mechanical and geometrical environment. In particular, as formins are processively elongating filaments that are helical structures, the torsional rigidity and force transmission/relaxation in actin bundles, through the dynamic crosslinking of the filament by fascin and the anchoring of the filament to lipid surfaces, have a huge impact on the ability of an actin network to grow. Our in vitro results therefore shed light on the actin cytoskeleton dynamics as a complex system where biochemical processes are finely tuned by mechanics and the geometrical organization of its parts.
Actin filament assembly and disassembly by regulatory proteins