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 transport1, 2, 3. 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)4, 5, 6, revealed F-BAR (FBP17) domain coated tubules nucleated by clathrin-coated buds when fission was blocked by GTPγS. Triggering fission by replacing 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.
At a glance
- Assembly of clathrin-coated pits onto purified plasma membranes. Science 236, 558–563 (1987). , , &
- Formation of coated vesicles from coated pits in broken A431 cells. J. Cell Biol. 108, 843–853 (1989). , , &
- Stage-specific assays for coated pit formation and coated vesicle budding in vitro . J. Cell Biol. 114, 869–880 (1991). &
- Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–796 (2006). , &
- Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes. Science 317, 1749–1753 (2007). , , &
- Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810–813 (2008). , , &
- Dynamin and its role in membrane fission. Annu. Rev. Cell Dev. Biol. 16, 483–519 (2000).
- Generation of high curvature membranes mediated by direct endophilin bilayer interactions. J. Cell Biol. 155, 193–200 (2001). et al.
- A novel dynamin-associating molecule, formin-binding protein 17, induces tubular membrane invaginations and participates in endocytosis. J. Biol. Chem. 279, 40091–40099 (2004). et al.
- Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins. Dev. Cell 9, 791–804 (2005). et al.
- Coordination between the actin cytoskeleton and membrane deformation by a novel membrane tubulation domain of PCH proteins is involved in endocytosis. J. Cell Biol. 172, 269–279 (2006). et al.
- Structural basis of membrane invagination by F-BAR domains. Cell 132, 807–817 (2008). et al.
- Tubular membrane invaginations coated by dynamin rings are induced by GTP-γS in nerve terminals. Nature 374, 186–190 (1995). , , &
- Endophilin/SH3p4 is required for the transition from early to late stages in clathrin-mediated synaptic vesicle endocytosis. Neuron 24, 143–154 (1999). et al.
- Toca-1 mediates Cdc42-dependent actin nucleation by activating the N-WASP-WIP complex. Cell 118, 203–216 (2004). et al.
- EFC/F-BAR proteins and the N-WASP-WIP complex induce membrane curvature-dependent actin polymerization. EMBO J. 27, 2817–2828 (2008). , &
- Protein complexes containing CYFIP/Sra/PIR121 coordinate Arf1 and Rac1 signalling during clathrin-AP-1-coated carrier biogenesis at the TGN. Nat. Cell Biol. 12, 330–340 (2010). et al.
- Curved EFC/F-BAR-domain dimers are joined end to end into a filament for membrane invagination in endocytosis. Cell 129, 761–772 (2007). et al.
- Requirements for F-BAR proteins TOCA-1 and TOCA-2 in actin dynamics and membrane trafficking during Caenorhabditis elegans oocyte growth and embryonic epidermal morphogenesis. PLoS Genet. 5, e1000675 (2009). et al.
- Functional partnership between amphiphysin and dynamin in clathrin-mediated endocytosis. Nat. Cell Biol. 1, 33–39 (1999). , , &
- Coordinated actions of actin and BAR proteins upstream of dynamin at endocytic clathrin-coated pits. Dev. Cell 17, 811–822 (2009). et al.
- SNX9 regulates dynamin assembly and is required for efficient clathrin-mediated endocytosis. Mol. Biol. Cell 16, 2058–2067 (2005). , , &
- FCHo proteins are nucleators of clathrin-mediated endocytosis. Science 328, 1281–1284 (2010). et al.
- Modulation of membrane dynamics and cell motility by membrane tension. Trends Cell Biol. 6, 85–89 (1996). &
- How proteins produce cellular membrane curvature. Nat. Rev. Mol. Cell Biol. 7, 9–19 (2006). &
- Reversible blockage of membrane retrieval and endocytosis in the garland cell of the temperature-sensitive mutant of Drosophila melanogaster, shibirets. J. Cell Biol. 97, 499–507 (1983). &
- Receptor-mediated endocytosis in cultured fibroblasts: cryptic coated pits and the formation of receptosomes. J. Histochem. Cytochem. 29, 1003–1013 (1981). et al.
- FcRn-mediated antibody transport across epithelial cells revealed by electron tomography. Nature 455, 542–546 (2008). et al.
- Invasive and adherent bacterial pathogens co-opt host clathrin for infection. Cell Host Microbe 2, 340–351 (2007). et al.
- Vesicular stomatitis virus enters cells through vesicles incompletely coated with clathrin that depend upon actin for internalization. PLoS Pathog. 5, e1000394 (2009). , , , &
- Amphiphysin II (SH3P9; BIN1), a member of the amphiphysin/Rvs family, is concentrated in the cortical cytomatrix of axon initial sSegments and nodes of Ranvier in brain and around T tubules in skeletal muscle. J. Cell Biol. 137, 1355–1367 (1997). et al.
- Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution. Nature Methods 5, 1047–1052 (2008). , , &
- Identification of a protein kinase as an intrinsic component of rat liver coated vesicles. Biochemistry 23, 4420–4426 (1984). , , , &
- GTP-dependent twisting of dynamin implicates constriction and tension in membrane fission. Nature 441, 528–531 (2006). , , &
- The association of epsin with ubiquitinated cargo along the endocytic pathway is negatively regulated by its interaction with clathrin. Proc. Natl Acad. Sci. USA 102, 2766–2771 (2005). &
- Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells. Cell 121, 593–606 (2005). , &
- The production of 'cell cortices' for light and electron microscopy. Traffic 1, 545–552 (2000).
- Reconstitution of clathrin-coated pit budding from plasma membranes. J. Cell Biol. 114, 881–891 (1991). , , &
- Imaging direct, dynamin-dependent recapture of fusing secretory granules on plasma membrane lawns from PC12 cells. Proc. Natl Acad. Sci. USA 99, 16806–16811 (2002). , , , &
- Effects of cytoplasmic acidification on clathrin lattice morphology. J. Cell Biol. 108, 401–411 (1989).
- Supplementary Information (3M)