Myosins are fascinating proteins with unique biochemical and physical properties. The multiple roles that they play in the dynamics of intracellular membranes are only beginning to emerge. Recent findings from the research team have highlighted unexpected roles in membrane deformation and in membrane fission played by two myosins (myosin 1b and myosin II) functioning at the interface between the Golgi, TGN (Trans-Golgi Network) and endosomes. Building on these results, we propose to establish a comprehensive model describing how several myosins work in concert with F-actin and with microtubule-based motors for sustaining transport events and membrane dynamics in a region of the cell at the crossroads of complex trafficking pathways.Towards this general objective, our main goals are:Goal 1: to understand the role of myosin 1b in membrane deformationGoal 2: to understand the role of nonmuscle myosin II in membrane fissionGoal 3: to characterize the actin structures required for myosin functionsGoal 4: to identify and to characterize other myosins functioning at the Golgi/TGN/endosome interface and to investigate their functional coordinationGoal 5: to understand how myosins are functionally coordinated with microtubule-based motors.The function of myosins will be studied both at the cellular and physical level using two main original methodological approaches available to the research team: minimal in vitro assays (giant liposomes and membrane nanotubes) and normalized cell systems (micropatterns).This proposal represents a new development in the activity of the research team composed of cell biologists, experimental and theoretical physicists. Success of this proposal will rely on the strong experience of cross-disciplinary approaches that allowed the research team in the past to elucidate several physical mechanisms underlying transport processes and membrane dynamics.