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Unravelling the Mechanosensitivity of Actin Bundles in Filopodia

Periodic Reporting for period 2 - BUNDLEFORCE (Unravelling the Mechanosensitivity of Actin Bundles in Filopodia)

Reporting period: 2017-09-01 to 2019-02-28

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 the filopodia mechanosensitive are not known. In particular how the actin network underlying the structure is regulated in response to mechanical cues is not known. To tackle this challenge we propose unique biophysical in vitro and in vivo experiments of increasing complexity.
The actin filaments are crosslinked together in parallel by fascin proteins to create bundles. Their elongation is regulated at their barbed-end by formins, homodimeric proteins that speed up elongation and remain processively attached to filament barbed ends.
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.
Work Package1:
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 form bundle thanks to the action of fascin, the change in actin filament conformation directly impact on formin processivity, already visible . So far, those results have been observed and characterized for the minimal bundle size (ie 2 filaments). Further, investigation and technical development are currently on-going to assess the fasscin/formin crosstalk on higher order structures.

Work package 2:
- we have now 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. Therefore, we reveal in vitro that formin processivity in cells is probably much shorter than previously anticipated.
- To investigate the mechanical response of actin bundles, the optical tweezers setup has been built and is now functional. Some software development has just started to integrate optical tweezers manipulation, fluorescence images recording and microfluidics control. This setup will allow more physiological situations to be tested and force measurement bundle under compression force will be a major breakthrough. All technical aspect of the experimental procedure have been identified and there should be no major obstacle to conduct experiments.

Work Package 3:
- we now have designed different formin constructs that integrate tension sensor modules, express them in Hela cells in order to probe the mechanical pulling state of mDia2 formins at the tip of filopodia.
- preliminary experiments are currently being analyzed.
In general, our experimental systems are unique and allow unprecedented molecular details on the actin assembly dynamics.
We now have established that formin processivity is extremely sensitive to mechanical pulling forces but also to formin ability to accommodate filament rotation as elongation proceeds.