Periodic Reporting for period 4 - MicMactin (Dissecting active matter: Microscopic origins of macroscopic actomyosin activity) Reporting period: 2020-12-01 to 2022-05-31 Summary of the context and overall objectives of the project Biological motion and forces originate from mechanically active proteins operating at the nanometer scale. These individual active elements interact through the surrounding cellular medium, collectively generating structures spanning tens of micrometers whose mechanical properties are perfectly tuned to their fundamentally out-of-equilibrium biological function. While both individual proteins and the resulting cellular behaviors are well characterized, understanding the relationship between these two scales remains a major challenge in both physics and cell biology. We bridge this gap through multiscale models of the emergence of active material properties in the experimentally well-characterized actin cytoskeleton. We thus investigate unexplored, strongly interacting nonequilibrium regimes. We develop a complete framework for cytoskeletal activity by separately studying all three fundamental processes driving it out of equilibrium: actin filament assembly and disassembly, force exertion by branched actin networks, and the action of molecular motors. We then recombine these approaches into a unified understanding of complex cell motility processes. To tackle the cytoskeleton’s disordered geometry and many-body interactions, we design new nonequilibrium self-consistent methods in statistical mechanics and elasticity theory. Our findings are being validated through simulations and close experimental collaborations. Our work breaks new ground in both biology and physics. In the context of biology, it establishes a new framework to understand how the cell controls its architecture and mechanics through biochemical regulation. On the physics side, it sets up new paradigms for the emergence of original out-of-equilibrium collective behaviors in an experimentally well-characterized system, addressing the foundations of existing macroscopic “active matter” approaches. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Task 1 – fibrous self-organization: We predicted the microstructure of irreversibly growing bundled networks (Nature Communications, bioRxiv publications). We are now focusing on the emergence and role of prestresses in the linear and nonlinear viscoelasticity of these networks. We have also started uncovering nonequilibrium steady-states with implications for the general principles of self-organization in biological fibers (Nature Physics publication). Task 2 – growing branched networks: We have shed light on force generation by growing actin filaments in the presence of actin growth regulators in collaboration with experimentalists (eLife publication). We have set up a simulation framework and a theoretical model that describe branched actin network elasticity as caused by filament entanglements and agree with experiments (Science Advances and Nature Physics publications, manuscript in preparation). We have advanced our fundamental understanding of the viscoelastic behaviour of networks in collaboration with experimentalists (Macromolecules publication). Task 3 – force generation and transmission: We have shown that contractile stresses are both amplified and rectified by fiber networks (publication in PNAS), and are currently establishing these principles as universal in nonlinear elastic networks (publications in Soft Matter). In collaboration with experimentalists, we have established a new inference method allowing the validation of our results in experiments, which will allow monitoring the time evolution of contractility among others (second publication in PNAS). Finally, we have elucidated the fundamentals of force generation in disordered actin networks in the absence of filament nonlinear response (eLife publication).Task 4 – putting it all together: We have uncovered new principles underlying the collective stability of active systems in several different contexts, which provides fundamental insights required to study the collective dynamics of the cell (PNAS, Nat. Commun. and Phys. Rev. Lett.). Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Task 1 – fibrous self-organization: We have demonstrated a new mechanism setting the morphology of actin networks based on kinetic trapping, not equilibrium statistics (which cannot account for experimental results). We plan on further developing the model to describe nonequilibrium steady-states and end-to-end filament coalescence as required to describe actin and intermediate filament vimentin in vivo. At the scale of the single protein filament, we have shown that ill-fitting particles beget fiber formation. We looking into whether this process can happen at equilibrium, and into its broader physical basis.Task 2 – growing branched networks: We have elucidated force-generation mechanism in growing actin filaments in the presence of formin. We have collected results on the entanglement topology of growing actin trees and are putting together simulations on their linear and nonlinear elasticity. We are now moving forward to use these results to obtain a fuller understanding of the elasticity of branched actin networks.Task 3 – force generation and transmission: We have demonstrated new mechanisms for contractile force generation in actin networks in the absence of preexisting filament and motor organization, shedding light on disordered, contractile structures in vivo. We are now working to establish a systematic comparison between these mechanisms and generate specific, testable predictions for active force generation in cells.Task 4 – putting it all together: We have demonstrated unknown stabilization mechanisms in active nematic, polar and chiral active systems. We have started collaborating with experimentalists and will integrate our results on the first three tasks to describe the response of the morphology, mechanics and dynamics of whole cells to osmotic shocks. Model of a network of flexible filaments tensed by molecular motors.