Abstract
Cell-free reconstitution of membrane traffic reactions and the morphological characterization of membrane intermediates that accumulate under these conditions have helped elucidate the physical and molecular mechanisms involved in membrane transport1–3. Towards an improved understanding of endocytosis we have reconstituted vesicle budding and fission from isolated plasma membrane sheets and imaged these events. Electron and fluorescence microscopy, including sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) 4–6, revealed F-BAR (FBP17) domain coated tubules nucleated by clathrin-coated buds when fission was blocked (presence of GTPγS). Triggering fission by replacement of GTPγS with GTP led not only to separation of clathrin-coated buds, but also to vesicle formation by fragmentation of the tubules. These results suggest a functional link between FBP17 dependent membrane tubulation and clathrin-dependent budding. They also show that clathrin spatially directs plasma membrane invaginations that lead to the generation of endocytic vesicles larger than those enclosed by the coat.
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