CLATHPOL · Dynamic study of shape and force generation by clathrin polymerization onto lipid membranes in vitro

The “CLATHPOL” project focuses upon a fundamental aspect of Clathrin Coat Vesicle formation. The critical biochemical (clathrin/adaptor interactions, membrane composition) and physical parameters (deformation forces, role of pre-induced curvature) controlling the shape and polymerization rate of clathrin coats will be determined and the forces applied on the membrane by clathrin polymerization will be measured. The “CLATHPOL” project will investigate the complex interactions driving clathrin coat assembly by developing improved bio-mimetic model systems to capture the complexity of biological membranes. The kinetics of clathrin polymerization and the dynamics of membrane shape generation will be studied by fast videomicroscopy using a membrane sheet assay to track coat formation on membranes exposed to various mixtures of clathrin and adaptor proteins. Membrane deformations will also be visualized using fluorescently labeled proteins. In the second stage, the forces on the membrane caused by clathrin polymerization will be measured using Giant Unilamellar Vesicles (GUVs). The membrane tension of the GUVs will be modulated by micropipette aspiration while membrane rigidity will be varied by changing lipid composition. Finally, the influence of membrane curvature on the dynamics and structure/geometry of the clathrin lattice will be determined using nano-patterned substrates with controllable and well-defined surface curvature. Membrane binding and lattice formation will be followed by Total internal reflection fluorescence (TIRF) microscopy and high speed atomic force microscopy.