Article | Published:

Endophilin marks and controls a clathrin-independent endocytic pathway

Nature volume 517, pages 460465 (22 January 2015) | Download Citation

Abstract

Endocytosis is required for internalization of micronutrients and turnover of membrane components. Endophilin has been assigned as a component of clathrin-mediated endocytosis. Here we show in mammalian cells that endophilin marks and controls a fast-acting tubulovesicular endocytic pathway that is independent of AP2 and clathrin, activated upon ligand binding to cargo receptors, inhibited by inhibitors of dynamin, Rac, phosphatidylinositol-3-OH kinase, PAK1 and actin polymerization, and activated upon Cdc42 inhibition. This pathway is prominent at the leading edges of cells where phosphatidylinositol-3,4-bisphosphate—produced by the dephosphorylation of phosphatidylinositol-3,4,5-triphosphate by SHIP1 and SHIP2—recruits lamellipodin, which in turn engages endophilin. This pathway mediates the ligand-triggered uptake of several G-protein-coupled receptors such as α2a- and β1-adrenergic, dopaminergic D3 and D4 receptors and muscarinic acetylcholine receptor 4, the receptor tyrosine kinases EGFR, HGFR, VEGFR, PDGFR, NGFR and IGF1R, as well as interleukin-2 receptor. We call this new endocytic route fast endophilin-mediated endocytosis (FEME).

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    , & The SH3p4/Sh3p8/SH3p13 protein family: binding partners for synaptojanin and dynamin via a Grb2-like Src homology 3 domain. Proc. Natl Acad. Sci. USA 94, 8569–8574 (1997)

  2. 2.

    et al. Essential role of endophilin A in synaptic vesicle budding at the Drosophila neuromuscular junction. EMBO J. 21, 1661–1672 (2002)

  3. 3.

    et al. Endophilin mutations block clathrin-mediated endocytosis but not neurotransmitter release. Cell 109, 101–112 (2002)

  4. 4.

    et al. Endophilin is required for synaptic vesicle endocytosis by localizing synaptojanin. Neuron 40, 749–762 (2003)

  5. 5.

    et al. Recruitment of endophilin to clathrin-coated pit necks is required for efficient vesicle uncoating after fission. Neuron 72, 587–601 (2011)

  6. 6.

    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)

  7. 7.

    et al. Fission and uncoating of synaptic clathrin-coated vesicles are perturbed by disruption of interactions with the SH3 domain of endophilin. Neuron 27, 301–312 (2000)

  8. 8.

    , & Selective perturbation of the BAR domain of endophilin impairs synaptic vesicle endocytosis. Synapse 64, 556–560 (2010)

  9. 9.

    et al. An endophilin-dynamin complex promotes budding of clathrin-coated vesicles during synaptic vesicle recycling. J. Cell Sci. 124, 133–143 (2011)

  10. 10.

    , , , & Two synaptojanin 1 isoforms are recruited to clathrin-coated pits at different stages. Proc. Natl Acad. Sci. USA 103, 19332–19337 (2006)

  11. 11.

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

  12. 12.

    , & A high precision survey of the molecular dynamics of mammalian clathrin-mediated endocytosis. PLoS Biol. 9, e1000604 (2011)

  13. 13.

    et al. Cooperative recruitment of dynamin and BIN/amphiphysin/Rvs (BAR) domain-containing proteins leads to GTP-dependent membrane scission. J. Biol. Chem. 288, 6651–6661 (2013)

  14. 14.

    , , & Clathrin-dependent and clathrin-independent retrieval of synaptic vesicles in retinal bipolar cells. Neuron 46, 869–878 (2005)

  15. 15.

    et al. Endophilin drives the fast mode of vesicle retrieval in a ribbon synapse. J. Neurosci. 31, 8512–8519 (2011)

  16. 16.

    et al. Identification of the endophilins (SH3p4/p8/p13) as novel binding partners for the beta1-adrenergic receptor. Proc. Natl Acad. Sci. USA 96, 12559–12564 (1999)

  17. 17.

    et al. The endophilin-CIN85-Cbl complex mediates ligand-dependent downregulation of c-Met. Nature 416, 187–190 (2002)

  18. 18.

    , , , & Cbl-CIN85-endophilin complex mediates ligand-induced downregulation of EGF receptors. Nature 416, 183–187 (2002)

  19. 19.

    et al. Essential role of phosphoinositide metabolism in synaptic vesicle recycling. Cell 99, 179–188 (1999)

  20. 20.

    & Caveolae as plasma membrane sensors, protectors and organizers. Nature Rev. Mol. Cell Biol. 14, 98–112 (2013)

  21. 21.

    , & Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells. Nature Cell Biol. 8, 46–54 (2006)

  22. 22.

    et al. The GTPase-activating protein GRAF1 regulates the CLIC/GEEC endocytic pathway. Curr. Biol. 18, 1802–1808 (2008)

  23. 23.

    et al. Clathrin-mediated internalization is essential for sustained EGFR signaling but dispensable for degradation. Dev. Cell 15, 209–219 (2008)

  24. 24.

    et al. Interleukin 2 receptors and detergent-resistant membrane domains define a clathrin-independent endocytic pathway. Mol. Cell 7, 661–671 (2001)

  25. 25.

    , & Cortactin and dynamin are required for the clathrin-independent endocytosis of gammac cytokine receptor. J. Cell Biol. 168, 155–163 (2005)

  26. 26.

    , & Clathrin-independent endocytosis: a cargo-centric view. Exp. Cell Res. 319, 2759–2769 (2013)

  27. 27.

    , , , & Clathrin-independent endocytosis used by the IL-2 receptor is regulated by Rac1, Pak1 and Pak2. EMBO Rep. 9, 356–362 (2008)

  28. 28.

    & SnapShot: Class I PI3K isoform signaling. Cell 154, 940 (2013)

  29. 29.

    et al. Polarization of chemoattractant receptor signaling during neutrophil chemotaxis. Science 287, 1037–1040 (2000)

  30. 30.

    , & The functions and regulation of the PTEN tumour suppressor. Nature Rev. Mol. Cell Biol. 13, 283–296 (2012)

  31. 31.

    , & How does SHIP1/2 balance PtdIns(3,4)P2 and does it signal independently of its phosphatase activity? Bioessays 35, 733–743 (2013)

  32. 32.

    et al. The control of phosphatidylinositol 3,4-bisphosphate concentrations by activation of the Src homology 2 domain containing inositol polyphosphate 5-phosphatase 2, SHIP2. Biochem. J. 407, 255–266 (2007)

  33. 33.

    et al. Evidence that inositol polyphosphate 4-phosphatase type II is a tumor suppressor that inhibits PI3K signaling. Cancer Cell 16, 115–125 (2009)

  34. 34.

    et al. Spatiotemporal control of endocytosis by phosphatidylinositol-3,4-bisphosphate. Nature 499, 233–237 (2013)

  35. 35.

    , & Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) specifically induces membrane penetration and deformation by Bin/amphiphysin/Rvs (BAR) domains. J. Biol. Chem. 287, 34078–34090 (2012)

  36. 36.

    et al. Lamellipodin, an Ena/VASP ligand, is implicated in the regulation of lamellipodial dynamics. Dev. Cell 7, 571–583 (2004)

  37. 37.

    et al. Endophilin, Lamellipodin, and Mena cooperate to regulate F-actin-dependent EGF-receptor endocytosis. EMBO J. 32, 2722–2734 (2013)

  38. 38.

    et al. Clathrin/AP-2 mediate synaptic vesicle reformation from endosome-like vacuoles but are not essential for membrane retrieval at central synapses. Neuron 82, 981–988 (2014)

  39. 39.

    et al. Ultrafast endocytosis at mouse hippocampal synapses. Nature 504, 242–247 (2013)

  40. 40.

    & Molecular mechanism and physiological functions of clathrin-mediated endocytosis. Nature Rev. Mol. Cell Biol. 12, 517–533 (2011)

  41. 41.

    et al. Membrane fission is promoted by insertion of amphipathic helices and is restricted by crescent BAR domains. Cell 149, 124–136 (2012)

  42. 42.

    et al. Shiga toxin induces tubular membrane invaginations for its uptake into cells. Nature 450, 670–675 (2007)

  43. 43.

    et al. GM1 structure determines SV40-induced membrane invagination and infection. Nature Cell Biol. 12, 11–18 (2010)

  44. 44.

    et al. Endophilin-A2 functions in membrane scission in clathrin-independent endocytosis. Nature (2014)

  45. 45.

    et al. Improving the photostability of bright monomeric orange and red fluorescent proteins. Nature Methods 5, 545–551 (2008)

  46. 46.

    , & Potent rescue of human immunodeficiency virus type 1 late domain mutants by ALIX/AIP1 depends on its CHMP4 binding site. J. Virol. 81, 6614–6622 (2007)

  47. 47.

    et al. Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on membranes. Science 291, 1051–1055 (2001)

  48. 48.

    et al. GTPase activity of dynamin and resulting conformation change are essential for endocytosis. Nature 410, 231–235 (2001)

  49. 49.

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

  50. 50.

    , , & Clathrin-mediated endocytosis in AP-2-depleted cells. J. Cell Biol. 162, 909–918 (2003)

  51. 51.

    , & Rapid endocytosis of interleukin 2 receptors when clathrin-coated pit endocytosis is inhibited. J. Cell Sci. 107, 3461–3468 (1994)

  52. 52.

    et al. Secramine inhibits Cdc42-dependent functions in cells and Cdc42 activation in vitro. Nature Chem. Biol. 2, 39–46 (2006)

  53. 53.

    et al. Dynasore, a cell-permeable inhibitor of dynamin. Dev. Cell 10, 839–850 (2006)

  54. 54.

    et al. Clathrin-independent carriers form a high capacity endocytic sorting system at the leading edge of migrating cells. J. Cell Biol. 190, 675–691 (2010)

  55. 55.

    et al. Inhibition of dynamin mediated endocytosis by the dynoles–synthesis and functional activity of a family of indoles. J. Med. Chem. 52, 3762–3773 (2009)

  56. 56.

    et al. The dynamin inhibitors MiTMAB and OcTMAB induce cytokinesis failure and inhibit cell proliferation in human cancer cells. Mol. Cancer Ther. 9, 1995–2006 (2010)

  57. 57.

    , , & Amantadine and dansylcadaverine inhibit vesicular stomatitis virus uptake and receptor-mediated endocytosis of alpha 2-macroglobulin. Proc. Natl Acad. Sci. USA 79, 2291–2295 (1982)

  58. 58.

    , & Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation. J. Cell Biol. 123, 1107–1117 (1993)

  59. 59.

    , , & Phenylarsine oxide inhibition of endocytosis: effects on asialofetuin internalization. Am. J. Physiol. 257, C182–C184 (1989)

  60. 60.

    & Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation. J. Cell Biol. 108, 389–400 (1989)

  61. 61.

    , , & Depletion of intracellular potassium arrests coated pit formation and receptor-mediated endocytosis in fibroblasts. Cell 33, 273–285 (1983)

  62. 62.

    et al. Quantification of PtdIns(3,4,5)P3 dynamics in EGF-stimulated carcinoma cells: a comparison of PH-domain-mediated methods with immunological methods. Biochem. J. 411, 441–448 (2008)

Download references

Acknowledgements

We thank S. Y. Peak-Chew for mass spectrometry, M. Edwards and K. McGourty for technical help and P. De Camilli, T. Kirchhausen, G. B. Hammond, P. Robinson, M. Robinson, M. McNiven, B. Nichols, A. Benmerah, M. Krause and Genentech for the gift of reagents, and the members of the McMahon and Boucrot laboratories for comments. The research was funded by the Medical Research Council UK (grant number U105178805) (H.T.M., L.A.-S., G.H., Y.V. and E.B. in part) and a Royal Society grant (research grant number RG120481) (E.B.). A.P.A.F is supported by the Fundação para a Ciência e Tecnologia, L.A.-S. is a EMBO Long Term fellow and is supported by Marie Curie Actions, and E.B. is a Biotechnology and Biological Sciences Research Council (BBSRC) David Phillips Research Fellow.

Author information

Affiliations

  1. MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK

    • Emmanuel Boucrot
    • , Leonardo Almeida-Souza
    • , Yvonne Vallis
    • , Gillian Howard
    •  & Harvey T. McMahon
  2. Institute of Structural and Molecular Biology, University College London & Birkbeck College, London WC1E 6BT, UK

    • Emmanuel Boucrot
    • , Antonio P. A. Ferreira
    •  & Sylvain Debard
  3. Department of Biology, Ecole Normale Supérieure de Cachan, 94235 Cachan, France

    • Sylvain Debard
  4. Institut Pasteur, Unité de Pathogenie Moleculaire Microbienne, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France

    • Laetitia Bertot
    •  & Nathalie Sauvonnet

Authors

  1. Search for Emmanuel Boucrot in:

  2. Search for Antonio P. A. Ferreira in:

  3. Search for Leonardo Almeida-Souza in:

  4. Search for Sylvain Debard in:

  5. Search for Yvonne Vallis in:

  6. Search for Gillian Howard in:

  7. Search for Laetitia Bertot in:

  8. Search for Nathalie Sauvonnet in:

  9. Search for Harvey T. McMahon in:

Contributions

H.T.M. and E.B. designed the research and supervised the project. H.T.M. and A.P.A.F. performed pull-down experiments. L.A.-S., A.P.A.F. and S.D. did the signalling experiments. L.A.-S. did the super-resolution imaging. A.P.A.F. did the cell migration experiments. S.D. performed the plasma membrane isolations; Y.V. performed the PC12 cell experiments. G.H. prepared and acquired the electron-microscopy data. L.B. and N.S. provided critical reagents. E.B. performed and analysed all the other experiments. E.B. and H.T.M. wrote the manuscript with input from all the other authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Emmanuel Boucrot or Harvey T. McMahon.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains a Supplementary Discussion and Supplementary References.

Videos

  1. 1.

    Spinning-disk confocal microscopy of a BSC1 cell stably expressing σ2-EGFP (AP2, green) and transiently expressing low levels of endophilin A2-RFP (red) and imaged at 0.5 Hz.

    The cell was imaged at 37 °C in normal imaging medium (5 % serum). Note the numerous endophilin puncta devoid of AP2 at the leading edge of the cell. The video is playing at 10 frames/sec.

  2. 2.

    Spinning-disk confocal microscopy of a confluent BSC1 cell transiently expressing low levels of EGFP-LCa (clathrin, green) and endophilin A2-RFP (red) and imaged at 2 Hz.

    The cell was imaged at 37 °C in normal imaging medium (5 % serum). Note the numerous endophilin puncta devoid of clathrin. The video is playing at 10 frames/sec.

  3. 3.

    Spinning-disk confocal microscopy (focal plane ~1 μm above the bottom surface) of a BSC1 cell transiently expressing low levels of endophilin A2-RFP (red) and imaged at 0.5 Hz.

    The cell was imaged at 37 °C in normal imaging medium (5 % serum). Additional 10 μM isoproterenol was added at the time frame 5. Note the numerous endophilin-coated tubules and vesicles budding from the periphery of the cell and accumulating toward the perinuclear area. The video is playing at 10 frames/sec.

  4. 4.

    Spinning-disk confocal microscopy (focal plane ~1 μm above the bottom surface) of a BSC1 cell transiently expressing low levels of endophilin A2-RFP (red) and imaged at 0.5 Hz.

    The cell was imaged at 37 °C in serum-free imaging medium (changed right before imaging). Additional 2 ng/mL was added at the time frame 0. Note the numerous endophilin-coated tubules and vesicles budding from the periphery of the cell and moving toward the perinuclear area. The video is playing at 10 frames/sec.

  5. 5.

    Spinning-disk confocal microscopy (focal plane ~1 μm above the bottom surface) of a BSC1 cell transiently expressing endophilin A2-RFP (red) and Cdc42-T17N dominant negative mutant and imaged at 0.5 Hz.

    The cell was imaged at 37 °C in normal imaging medium (5 % serum). Note the recruitment of endophilin all around the edge of the cell and the numerous endophilin-coated tubules and vesicles budding from the periphery of the cell even though the cell was not stimulated with additional growth factor or β1-AR agonist. The video is playing at 10 frames/sec.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature14067

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.