Structural Dynamics of Endocytosis by High-Speed Atomic Force Microscopy

Clathrin-mediated endocytosis is one of the major endocytosis pathways. Formation of clathrin coated vesicles (CCV) begins from relatively flat plasma membrane regions. Clathrin molecules bind to the CCV budding sites and assemble into a lattice covering growing vesicle. Initial flat clathrin lattice displays a hexagonal honeycomb architecture whereas the final CCV displays a mixture of hexagons and pentagons. The relation between clathrin lattice conversion and curvature generation in that process remains unknown. No dynamic structural analysis of the detailed mechanisms during CCV budding was achieved so far. High-speed atomic force microscopy (HS-AFM) permits observation of structural dynamics in biological processes because it allows performing imaging with nanometer spatial and subsecond temporal resolution. To study the CCV transformation by HS-AFM, we use a cell-free endocytosis assay based on plasma membrane patches. Combination of HS-AFM together with cell-free assay should permit direct, real-time observation of clathrin lattice rearrangement during CCV budding.