Brain Actin regulation by Microtubule-Associated Proteins: unveiling the Neurocytoskeleton dynamics. – MAPping-BRAIN-ACTIN

The MAPping-BRAIN-ACTIN project aims to reconstitute a pure neurocytoskeleton by integrating brain actin and microtubules to explore their dynamic interplay. While microtubule-associated proteins (MAPs) are primarily known for regulating microtubules, emerging evidence points to their involvement in actin organization as well. Brain actin, pivotal for neuronal shape, plasticity, and function, remains largely underexplored, with most insights drawn from muscle actin. This project will focus on characterizing the distinct properties of brain actin and understanding its regulation by neuronal MAPs. The final goal is to reconstitute an integrated neurocytoskeleton by incorporating both brain actin and microtubules to uncover its molecular organization.

Building on robust unpublished data showing that brain and muscle actin cannot co-assemble, the coordinator hypothesizes that brain-specific actin isoforms possess distinct properties that not only shape cytoskeletal architecture but also directly influence their interactions with microtubules. The first objective is to characterize brain actin using cutting-edge cell-free approaches, combining spectrofluorometry, Total Internal Reflection Fluorescence microscopy, and cryo-electron microscopy to study its polymerization kinetics, structure, and interactions with actin-binding proteins. The next step will be to reconstitute brain actin networks that mimic the branched and linear actin arrays found in neurons and evaluate their homeostasis, defined as the equilibrium between these branched and linear structures, in the presence of neuronal MAPs. Finally, microtubules will be integrated into specialized brain actin networks, together with neuronal MAPs, to reconstitute an integrated neurocytoskeleton. This strategy will be complemented by in-cell experiments using high-resolution nanoscopy, combining expansion and advanced light microscopy techniques, to localize key MAPs on cytoskeletal networks within various neuronal compartments, including axons, dendrites and synapses.

Overall, this project introduces a novel and powerful approach to understanding the molecular principles governing brain actin organization and its interactions with microtubules. By reconstituting highly-organized brain actin networks and integrating brain microtubules, it will enable the first-ever reconstitution of a pure neuronal cytoskeleton. While the effects of neuronal MAPs on microtubule assembly and stability are well-studied, their interactions with brain actin remain largely unexplored, leaving the underlying mechanisms poorly understood. Deciphering the function of MAPs as actin regulators is a critical step in unraveling the coordination between microtubules and actin. Moreover, it will provide new insights into cytoskeletal abnormalities observed in various brain pathologies where these MAPs are deregulated.

The coordinator of the MAPping-BRAIN-ACTIN program will apply his strong expertise in actin cytoskeleton to drive this project and establish an independent research direction focused on brain actin. This work will complement the ongoing research in his team, which is primarily centered on microtubule biology. He has already brought together team members to collaborate on this project, ensuring a strong foundation for its successful execution. The ANR JCJC funding will be critical in enabling him to develop a highly innovative and independent research axis. Moreover, the Grenoble Institute of Neurosciences (GIN) offers an optimal environment, with advanced imaging facilities and a dynamic local cytoskeletal research community, ensuring the success of the project.