Abstract
Cell-free reconstitution of membrane traffic reactions and the morphological characterization of membrane intermediates that accumulate under these conditions have helped to elucidate the physical and molecular mechanisms involved in membrane transport. To gain a better understanding of endocytosis, we have reconstituted vesicle budding and fission from isolated plasma membrane sheets and imaged these events. Electron and fluorescence microscopy, including subdiffraction-limit imaging by stochastic optical reconstruction microscopy (STORM), revealed F-BAR (FBP17) domain coated tubules nucleated by clathrin-coated buds when fission was blocked by GTPgammaS. Triggering fission by replacing GTPgammaS 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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Actins / antagonists & inhibitors
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Acyltransferases / metabolism
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Adenosine Triphosphate / pharmacology
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Animals
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Antibodies / immunology
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Antibodies / pharmacology
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Bridged Bicyclo Compounds, Heterocyclic / pharmacology
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Carrier Proteins / immunology
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Carrier Proteins / metabolism*
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Cattle
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Cell Line
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Cell Membrane / drug effects
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Cell Membrane / physiology
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Cell Membrane / ultrastructure
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Cell Membrane Structures / drug effects
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Cell Membrane Structures / physiology*
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Cell Membrane Structures / ultrastructure
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Cell-Free System / drug effects
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Cell-Free System / physiology
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Clathrin / immunology
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Clathrin / metabolism*
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Coated Pits, Cell-Membrane / drug effects
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Coated Pits, Cell-Membrane / physiology*
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Coated Pits, Cell-Membrane / ultrastructure
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Cytosol / metabolism
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Dynamins / metabolism
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Endocytosis / drug effects
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Endocytosis / physiology*
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Fatty Acid-Binding Proteins
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Fibroblasts
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Guanosine 5'-O-(3-Thiotriphosphate) / pharmacology
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Guanosine Triphosphate / pharmacology
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Humans
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Imaging, Three-Dimensional / methods
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Mice
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Microscopy, Electron, Transmission
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Microscopy, Fluorescence
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Models, Biological
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Potoroidae
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Rats
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Receptors, Transferrin / metabolism
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Thiazolidines / pharmacology
Substances
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Actins
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Antibodies
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Bridged Bicyclo Compounds, Heterocyclic
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Carrier Proteins
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Clathrin
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FNBP1 protein, human
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Fatty Acid-Binding Proteins
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Receptors, Transferrin
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Thiazolidines
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Guanosine 5'-O-(3-Thiotriphosphate)
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Guanosine Triphosphate
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Adenosine Triphosphate
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Acyltransferases
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2-acylglycerophosphate acyltransferase
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Dynamins
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latrunculin B