Periodic Reporting for period 3 - BUNDLEFORCE (Unravelling the Mechanosensitivity of Actin Bundles in Filopodia)
Reporting period: 2019-03-01 to 2020-08-31
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 the actin network underlying the filopodia 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 homodimeric proteins that speed up actin filament elongation and remain processively attached to filament barbed ends. Formins are key proteins that control the growth of actin bundles in filopodia, but how their activity is regulated once filaments are tightly connected into bundles 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 filopodium-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.
3) Decipher in vivo the mechanics of actin bundles in filopodia, using fascins and formins with integrated fluorescent tension sensors.2028This framework spanning from in vitro single filament to in vivo meso-scale actin networks will bring unprecedented insights into the role of actin bundles in filopodia mechanosensitivity.
WP1 objectives have been reached: Protein engineering and purification to start investigating synergy between formin and fascin activities were successfully completed to investigate aims using microfluidics/microscopy assays. 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:
We have established that formin anchoring has a direct impact of actin bundle elongation by formins: The length of the tether connecting the formin to the bundle, as well as the ability of formin to reorganize on a fluid lipid bilayer has a direct impact on formin elongation rate and processivity. This is a major finding, as in cells formins are part of proteins complexes that modulate their ability to reorient themselves as actin filaments elongate. 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 acrtivity.
By developing new experimental approaches, using lipid microppatterns in microfluidics chambers, we have shown that higher bundle structures profoundly affect formin activity and that the build up of compressive mechanical load readily provoke the detachment of formin from actin filament barbed ends.
Work Package 3:
- we are currently designing different formin constructs integrating tension sensor modules, and express them in Hela cells, in order to probe the mechanical pulling state of mDia2 formins at the tip of filopodia.
- preliminary experiments reveal that mDia2 activity at the tip of filopodia are performed mostly under compression.