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Coupling between clathrin-dependent endocytic budding and F-BAR-dependent tubulation in a cell-free system

Nature Cell Biology volume 12, pages 902–908 (2010)Cite this article

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A Corrigendum to this article was published on 01 October 2010

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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 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.

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Figure 1: Marked transformation of plasma membrane sheets induced by incubation with brain cytosol.
Figure 2: Localization and axial segregation of dynamin and FBP17 along the tubular invaginations.
Figure 3: Tubular invaginations require spatially coordinated action of clathrin, actin and FBP17.
Figure 4: Occurrence of membrane fission on replacement of GTPγS with GTP.
Figure 5: Schematic representation of the membrane invaginations described in this study and of their fission.

Change history

  • 31 August 2010

    In the version of this letter initially published online, the author affiliations were incorrect. This error has been corrected in both the HTML and PDF versions of the letter.

References

  1. Moore, M., Mahaffey, D., Brodsky, F. & Anderson, R. Assembly of clathrin-coated pits onto purified plasma membranes. Science 236, 558–563 (1987).

    Article  CAS  Google Scholar 

  2. Smythe, E., Pypaert, M., Lucocq, J. & Warren, G. Formation of coated vesicles from coated pits in broken A431 cells. J. Cell Biol. 108, 843–853 (1989).

    Article  CAS  Google Scholar 

  3. Schmid, S. L. & Smythe, E. Stage-specific assays for coated pit formation and coated vesicle budding in vitro. J. Cell Biol. 114, 869–880 (1991).

    Article  CAS  Google Scholar 

  4. Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–796 (2006).

    Article  CAS  Google Scholar 

  5. Bates, M., Huang, B., Dempsey, G. T. & Zhuang, X. Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes. Science 317, 1749–1753 (2007).

    Article  CAS  Google Scholar 

  6. Huang, B., Wang, W., Bates, M. & Zhuang, X. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810–813 (2008).

    Article  CAS  Google Scholar 

  7. Hinshaw, J. E. Dynamin and its role in membrane fission. Annu. Rev. Cell Dev. Biol. 16, 483–519 (2000).

    Article  CAS  Google Scholar 

  8. Farsad, K. et al. Generation of high curvature membranes mediated by direct endophilin bilayer interactions. J. Cell Biol. 155, 193–200 (2001).

    Article  CAS  Google Scholar 

  9. Kamioka, Y. 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).

    Article  CAS  Google Scholar 

  10. Itoh, T. et al. Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins. Dev. Cell 9, 791–804 (2005).

    Article  CAS  Google Scholar 

  11. Tsujita, K. 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).

    Article  CAS  Google Scholar 

  12. Frost, A. et al. Structural basis of membrane invagination by F-BAR domains. Cell 132, 807–817 (2008).

    Article  CAS  Google Scholar 

  13. Takei, K., McPherson, P. S., Schmid, S. L. & Camilli, P. D. Tubular membrane invaginations coated by dynamin rings are induced by GTP-γS in nerve terminals. Nature 374, 186–190 (1995).

    Article  CAS  Google Scholar 

  14. Ringstad, N. et al. Endophilin/SH3p4 is required for the transition from early to late stages in clathrin-mediated synaptic vesicle endocytosis. Neuron 24, 143–154 (1999).

    Article  CAS  Google Scholar 

  15. Ho, H.-Y. H. et al. Toca-1 mediates Cdc42-dependent actin nucleation by activating the N-WASP-WIP complex. Cell 118, 203–216 (2004).

    Article  CAS  Google Scholar 

  16. Takano, K., Toyooka, K. & Suetsugu, S. EFC/F-BAR proteins and the N-WASP-WIP complex induce membrane curvature-dependent actin polymerization. EMBO J. 27, 2817–2828 (2008).

    Article  CAS  Google Scholar 

  17. Anitei, M. et al. 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).

    Article  CAS  Google Scholar 

  18. Shimada, A. 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).

    Article  CAS  Google Scholar 

  19. Giuliani, C. 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).

    Article  Google Scholar 

  20. Takei, K., Slepnev, V. I., Haucke, V. & De Camilli, P. Functional partnership between amphiphysin and dynamin in clathrin-mediated endocytosis. Nat. Cell Biol. 1, 33–39 (1999).

    Article  CAS  Google Scholar 

  21. Ferguson, S. et al. Coordinated actions of actin and BAR proteins upstream of dynamin at endocytic clathrin-coated pits. Dev. Cell 17, 811–822 (2009).

    Article  CAS  Google Scholar 

  22. Soulet, F., Yarar, D., Leonard, M. & Schmid, S. L. SNX9 regulates dynamin assembly and is required for efficient clathrin-mediated endocytosis. Mol. Biol. Cell 16, 2058–2067 (2005).

    Article  CAS  Google Scholar 

  23. Henne, W. M. et al. FCHo proteins are nucleators of clathrin-mediated endocytosis. Science 328, 1281–1284 (2010).

    Article  CAS  Google Scholar 

  24. Sheetz, M. P. & Dai, J. Modulation of membrane dynamics and cell motility by membrane tension. Trends Cell Biol. 6, 85–89 (1996).

    Article  CAS  Google Scholar 

  25. Zimmerberg, J. & Kozlov, M. M. How proteins produce cellular membrane curvature. Nat. Rev. Mol. Cell Biol. 7, 9–19 (2006).

    Article  CAS  Google Scholar 

  26. Kosaka, T. & Ikeda, K. 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).

    Article  CAS  Google Scholar 

  27. Willingham, M. et al. Receptor-mediated endocytosis in cultured fibroblasts: cryptic coated pits and the formation of receptosomes. J. Histochem. Cytochem. 29, 1003–1013 (1981).

    Article  CAS  Google Scholar 

  28. He, W. et al. FcRn-mediated antibody transport across epithelial cells revealed by electron tomography. Nature 455, 542–546 (2008).

    Article  CAS  Google Scholar 

  29. Veiga, E. et al. Invasive and adherent bacterial pathogens co-opt host clathrin for infection. Cell Host Microbe 2, 340–351 (2007).

    Article  CAS  Google Scholar 

  30. Cureton, D. K., Massol, R. H., Saffarian, S., Kirchhausen, T. L. & Whelan, S. P. J. Vesicular stomatitis virus enters cells through vesicles incompletely coated with clathrin that depend upon actin for internalization. PLoS Pathog. 5, e1000394 (2009).

    Article  Google Scholar 

  31. Butler, M. H. et al. 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).

    Article  CAS  Google Scholar 

  32. Huang, B., Jones, S. A., Brandenburg, B. & Zhuang, X. Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution. Nature Methods 5, 1047–1052 (2008).

    Article  CAS  Google Scholar 

  33. Campbell, C., Squicciarini, J., Shia, M., Pilch, P. F. & Fine, R. E. Identification of a protein kinase as an intrinsic component of rat liver coated vesicles. Biochemistry 23, 4420–4426 (1984).

    Article  CAS  Google Scholar 

  34. Roux, A., Uyhazi, K., Frost, A. & De Camilli, P. GTP-dependent twisting of dynamin implicates constriction and tension in membrane fission. Nature 441, 528–531 (2006).

    Article  CAS  Google Scholar 

  35. Chen, H. & De Camilli, P. 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).

    Article  CAS  Google Scholar 

  36. Merrifield, C. J., Perrais, D. & Zenisek, D. Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells. Cell 121, 593–606 (2005).

    Article  CAS  Google Scholar 

  37. Heuser, J. The production of 'cell cortices' for light and electron microscopy. Traffic 1, 545–552 (2000).

    Article  CAS  Google Scholar 

  38. Lin, H., Moore, M., Sanan, D. & Anderson, R. Reconstitution of clathrin-coated pit budding from plasma membranes. J. Cell Biol. 114, 881–891 (1991).

    Article  CAS  Google Scholar 

  39. Holroyd, P., Lang, T., Wenzel, D., De Camilli, P. & Jahn, R. 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).

    Article  CAS  Google Scholar 

  40. Heuser, J. Effects of cytoplasmic acidification on clathrin lattice morphology. J. Cell Biol. 108, 401–411 (1989).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Aurelien Roux and Yuxin Mao for advice and help; Lijun Liu, Frank Wilson and Christoph Rahner for critical technical assistance. This work was supported in part by the G. Harold and Leila Y. Mathers Charitable Foundation, the W.M. Keck Foundation and the National Institutes of Health grants (NS36251 and DK45735) to P.D.C., and National Institutes of Health grant (GM068518) to X.Z.; P.D.C. and X.Z. are Howard Hughes Medical Institute Investigators.

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Author notes

  1. Bo Huang

    Present address: Current address: Department of Pharmaceutical Chemistry, Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158, USA.,

Authors and Affiliations

  1. Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, 06520, CT, USA

    Min Wu, Andrea Raimondi & Pietro De Camilli

  2. Department of Cell Biology, Yale University School of Medicine, New Haven, 06520, CT, USA

    Min Wu, Morven Graham, Andrea Raimondi & Pietro De Camilli

  3. Department of Neurobiology, Yale University School of Medicine, New Haven, 06520, CT, USA

    Pietro De Camilli

  4. Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, 06520, CT, USA

    Min Wu, Andrea Raimondi & Pietro De Camilli

  5. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, 02138, MA, USA

    Bo Huang & Xiaowei Zhuang

  6. Department of Physics, Harvard University, Cambridge, 02138, MA, USA

    Xiaowei Zhuang

  7. Department of Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, 63110, MO, USA

    John E. Heuser

  8. Howard Hughes Medical Institute, Harvard University, Cambridge, 02138, MA, USA

    Bo Huang & Xiaowei Zhuang

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Contributions

M.W. and P.D.C. designed the experiments and wrote the manuscript; M.W. performed experiments. Experimental work was also contributed by B.H. (STORM), J.E.H. (electron microscopy), A.R. (electron microscopy) and M.G. (electron microscopy). B.H. and X.Z. also contributed to discussion and manuscript preparation.

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Correspondence to Pietro De Camilli.

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Wu, M., Huang, B., Graham, M. et al. Coupling between clathrin-dependent endocytic budding and F-BAR-dependent tubulation in a cell-free system. Nat Cell Biol 12, 902–908 (2010). https://doi.org/10.1038/ncb2094

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