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

Journal name:
Nature Cell Biology
Volume:
12,
Pages:
902–908
Year published:
DOI:
doi:10.1038/ncb2094
Received
Accepted
Published online

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

Figures

  1. Marked transformation of plasma membrane sheets induced by incubation with brain cytosol.
    Figure 1: Marked transformation of plasma membrane sheets induced by incubation with brain cytosol.

    (a) Plasma membrane sheets labelled with PM–GFP seem homogeneous without cytosol treatment but become punctate after 15 min incubation with cytosol (Cyto), ATP and GTPγS (see also Supplementary Information, Movie 1). (b) Membrane puncta that accumulate on plasma membrane sheets endogenously labelled with PM–GFP or exogenously labelled with the fluorescent lipid marker BTR colocalize with clathrin (Alexa 555-labelled clathrin heavy chain) or transferrin receptor-pHluorin (TfR) puncta. (c) Time sequence of transferrin receptor-pHluorin clustering (see Supplementary Information, Movie 2). (d) 3D-STORM reconstruction of the transformed plasma membrane. Membrane sheets labelled with PM–GFP were incubated with cytosol, ATP and GTPγS for 15 min, fixed and labelled with anti-GFP, followed by secondary antibody (see Supplementary Information, Movie 3). The colour of individual points in the image encodes distance from the substrate. (e) Circular membrane profiles in en face thin sections demonstrating the tubular nature of the invaginations. (f) Thin section cut perpendicular to the substrate showing longitudinal view of the deep tubular structures that are connected with clathrin-coated pits via constricted necks. Inset is a magnified image of a coated pit at the tip of a tubular structure. (g) Electron microscopy of the platinum replica of quick-frozen deep-etched membrane sheet illustrating deep tubular extensions capped with multiple clathrin-coated pits. Samples in eg were incubated with cytosol, ATP and GTPγS for 15 min. Scale bars, 5 μm (a); 2 μm (b); 5 μm (c); 2 μm (d); 1 μm (e); 1 μm (f); 100 nm (f, inset; g).

  2. Localization and axial segregation of dynamin and FBP17 along the tubular invaginations.
    Figure 2: Localization and axial segregation of dynamin and FBP17 along the tubular invaginations.

    (a) Alexa488 dynamin and Alexa 488 FBP17 were both recruited to the membrane invaginations as indicated by fluorescence lipid puncta (BTR). (b) Stacked 3D-STORM images of 59 membrane invaginations labelled with FBP17 (immunofluorescence with Cy3–Cy5-labelled secondary antibodies) and aligned according to the position of the coat region (Alexa 405-Cy5-labelled clathrin heavy chain), and of 96 membrane invaginations aligned according to the position of the constriction site where dynamin (Alexa 405–Cy5-labelled dynamin) was localized. (c) The distribution of clathrin and FBP17 along the length of tubules as a function of the depth of invaginations. The x-z 3D-STORM images of 207 membrane invaginations labelled with FBP17 (immunofluorescence with Cy3/Cy5-labelled secondary antibodies) and clathrin (Alexa 405–Cy5 labelled clathrin heavy chain) were used to generate this scatter plot. Clathrin localization points follow the diagonal line as expected, whereas FBP17 points fill the space underneath the clathrin structure regardless of the depth of the invagination. (d) Immunogold localization of FBP17 and dynamin (polyclonal antibody DG1) on cross-sections of the tubular invaginations. The top section captured a plane closer to the tip of the tubules. Inset shows a side view. All samples in Fig. 2 were prepared by incubating membrane sheets with cytosol, ATP and GTPγS for 15 min then fixed. Scale bars, 2 μm (a); 200 nm (bd);

  3. Tubular invaginations require spatially coordinated action of clathrin, actin and FBP17.
    Figure 3: Tubular invaginations require spatially coordinated action of clathrin, actin and FBP17.

    (a) 3D-STORM images of clathrin (Alexa 405-Cy5-labelled clathrin heavy chain) at different time points after incubation with cytosol, ATP plus GTPγS, and in the absence or presence of latrunculin B (Latru). Two-colour 3D imaging was performed together with PM–GFP (not shown here). (b) High-magnification view of 3D-STORM images of a single clathrin-coated pit. In the right panels, the horizontal and vertical projections of the 3D image of one clathrin structure shows the half-spherical shape of the pit, whereas 50-nm thick cross-sections through the geometric centre of the pit reveals that inside is hollow (in both x-y and y-z views) and there is an opening at the bottom (only in y-z view), as expected for clathrin-coated pits. In both a and b, the colour of individual points in the images encodes distance from the mean height of the basal membrane sheet. (c) Thin-section electron microscopy confirms the presence of clathrin-coated pits, but a lack of tubulation at the early point (5 min) of incubation with cytosol, ATP plus GTPγS, or at later time point (15 min) in the presence of latrunculin B, anti-FBP17 antibodies, or GTP instead of GTPγS. In the absence of cytosol or in the presence of anti-clathrin antibodies, clathrin-coated pits are rare. (d) Fluorescence images of plasma membrane sheets (labelled with PM–GFP) after incubations as indicated. Scale bars, 1 μm (a); 100 nm (b); 500 nm (c); 5 μm (d).

  4. Occurrence of membrane fission on replacement of GTP[gamma]S with GTP.
    Figure 4: Occurrence of membrane fission on replacement of GTPγS with GTP.

    (a) Appearance of free vesicles (labelled with PM–GFP) in the medium in the presence of GTP, but not in the continued presence of GTPγS or latrunculin B. Each image is the maximum intensity projection of 50 frames by confocal microscopy (see also Supplementary Information, Movie 4). (b) Quantification of the number of free vesicles formed under various incubation conditions. The number for each condition was obtained by counting the mean number of vesicles per frame. Data represent means ± s.e.m. (n values are indicated at the bottom). (c) Immunocapture of vesicles (PM–GFP positive) on beads coated with anti-GFP antibodies and colocalization with a membrane marker (BTR; see Supplementary Information, Movie 5). (d) Electron microscopy of bead-bound vesicles and distribution of their diameters. (e) Electron microscopy of plasma membrane sheets at various times after replacement of GTPγS with GTP. Scale bars, 10 μm (a); 20 μm (c); 200 nm (d); 500 nm (e).

  5. Schematic representation of the membrane invaginations described in this study and of their fission.
    Figure 5: Schematic representation of the membrane invaginations described in this study and of their fission.

    The model illustrates how an endocytic reaction triggered by clathrin may lead to both classical clathrin-coated vesicles derived from the coated bud and non-coated larger vesicles derived from the tubular region.

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, 558563 (1987).
  2. Smythe, E., Pypaert, M., Lucocq, J. & Warren, G. Formation of coated vesicles from coated pits in broken A431 cells. J. Cell Biol. 108, 843853 (1989).
  3. Schmid, S. L. & Smythe, E. Stage-specific assays for coated pit formation and coated vesicle budding in vitro . J. Cell Biol. 114, 869880 (1991).
  4. Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793796 (2006).
  5. Bates, M., Huang, B., Dempsey, G. T. & Zhuang, X. Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes. Science 317, 17491753 (2007).
  6. Huang, B., Wang, W., Bates, M. & Zhuang, X. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810813 (2008).
  7. Hinshaw, J. E. Dynamin and its role in membrane fission. Annu. Rev. Cell Dev. Biol. 16, 483519 (2000).
  8. Farsad, K. et al. Generation of high curvature membranes mediated by direct endophilin bilayer interactions. J. Cell Biol. 155, 193200 (2001).
  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, 4009140099 (2004).
  10. Itoh, T. et al. Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins. Dev. Cell 9, 791804 (2005).
  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, 269279 (2006).
  12. Frost, A. et al. Structural basis of membrane invagination by F-BAR domains. Cell 132, 807817 (2008).
  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, 186190 (1995).
  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, 143154 (1999).
  15. Ho, H.-Y. H. et al. Toca-1 mediates Cdc42-dependent actin nucleation by activating the N-WASP-WIP complex. Cell 118, 203216 (2004).
  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, 28172828 (2008).
  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, 330340 (2010).
  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, 761772 (2007).
  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).
  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, 3339 (1999).
  21. Ferguson, S. et al. Coordinated actions of actin and BAR proteins upstream of dynamin at endocytic clathrin-coated pits. Dev. Cell 17, 811822 (2009).
  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, 20582067 (2005).
  23. Henne, W. M. et al. FCHo proteins are nucleators of clathrin-mediated endocytosis. Science 328, 12811284 (2010).
  24. Sheetz, M. P. & Dai, J. Modulation of membrane dynamics and cell motility by membrane tension. Trends Cell Biol. 6, 8589 (1996).
  25. Zimmerberg, J. & Kozlov, M. M. How proteins produce cellular membrane curvature. Nat. Rev. Mol. Cell Biol. 7, 919 (2006).
  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, 499507 (1983).
  27. Willingham, M. et al. Receptor-mediated endocytosis in cultured fibroblasts: cryptic coated pits and the formation of receptosomes. J. Histochem. Cytochem. 29, 10031013 (1981).
  28. He, W. et al. FcRn-mediated antibody transport across epithelial cells revealed by electron tomography. Nature 455, 542546 (2008).
  29. Veiga, E. et al. Invasive and adherent bacterial pathogens co-opt host clathrin for infection. Cell Host Microbe 2, 340351 (2007).
  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).
  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, 13551367 (1997).
  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, 10471052 (2008).
  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, 44204426 (1984).
  34. Roux, A., Uyhazi, K., Frost, A. & De Camilli, P. GTP-dependent twisting of dynamin implicates constriction and tension in membrane fission. Nature 441, 528531 (2006).
  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, 27662771 (2005).
  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, 593606 (2005).
  37. Heuser, J. The production of 'cell cortices' for light and electron microscopy. Traffic 1, 545552 (2000).
  38. Lin, H., Moore, M., Sanan, D. & Anderson, R. Reconstitution of clathrin-coated pit budding from plasma membranes. J. Cell Biol. 114, 881891 (1991).
  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, 1680616811 (2002).
  40. Heuser, J. Effects of cytoplasmic acidification on clathrin lattice morphology. J. Cell Biol. 108, 401411 (1989).

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Author information

Affiliations

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

    • Min Wu,
    • Andrea Raimondi &
    • Pietro De Camilli
  2. Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06520, USA.

    • Min Wu,
    • Morven Graham,
    • Andrea Raimondi &
    • Pietro De Camilli
  3. Department of Neurobiology, Yale University School of Medicine, New Haven, CT 06520, USA.

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

    • Min Wu,
    • Andrea Raimondi &
    • Pietro De Camilli
  5. Department of Chemistry and Chemical Biology, Harvard University, Cambridge MA 02138, USA.

    • Bo Huang &
    • Xiaowei Zhuang
  6. Department of Physics, Harvard University, Cambridge MA 02138, USA.

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

    • John E. Heuser
  8. Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA.

    • Bo Huang &
    • Xiaowei Zhuang
  9. Current address: Department of Pharmaceutical Chemistry, Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158, USA.

    • Bo Huang

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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The authors declare no competing financial interests.

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