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Endophilin marks and controls a clathrin-independent endocytic pathway

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


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).

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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


  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


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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.

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  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.

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