Recruitment dynamics of ESCRT-III and Vps4 to endosomes and implications for reverse membrane budding

  1. Manuel Alonso Y Adell
  2. Simona M Migliano
  3. Srigokul Upadhyayula
  4. Yury S Bykov
  5. Simon Sprenger
  6. Mehrshad Pakdel
  7. Georg F Vogel
  8. Gloria Jih
  9. Wesley Skillern
  10. Reza Behrouzi
  11. Markus Babst
  12. Oliver Schmidt
  13. Michael W Hess
  14. John AG Briggs
  15. Tomas Kirchhausen  Is a corresponding author
  16. David Teis  Is a corresponding author
  1. Medical University of Innsbruck, Austria
  2. Harvard Medical School, United States
  3. European Molecular Biology Laboratory, Germany
  4. Boston Children's Hospital, United States
  5. University of Utah, United States

Abstract

The ESCRT machinery mediates reverse membrane scission. By quantitative fluorescence lattice light-sheet microscopy, we have shown that ESCRT-III subunits polymerize rapidly on yeast endosomes, together with the recruitment of at least two Vps4 hexamers. During their 3-45 second lifetimes, the ESCRT-III assemblies accumulated 75-200 Snf7 and 15-50 Vps24 molecules. Productive budding events required at least two additional Vps4 hexamers. Membrane budding was associated with continuous, stochastic exchange of Vps4 and ESCRT-III components, rather than steady growth of fixed assemblies, and depended on Vps4 ATPase activity. An all-or-none step led to final release of ESCRT-III and Vps4. Tomographic electron microscopy demonstrated that acute disruption of Vps4 recruitment stalled membrane budding. We propose a model in which multiple Vps4 hexamers (four or more) draw together several ESCRT-III filaments. This process induces cargo crowding and inward membrane buckling, followed by constriction of the nascent bud neck and ultimately ILV generation by vesicle fission.

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Author details

  1. Manuel Alonso Y Adell

    Division of Cell Biology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria
    Competing interests
    The authors declare that no competing interests exist.
  2. Simona M Migliano

    Division of Cell Biology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria
    Competing interests
    The authors declare that no competing interests exist.
  3. Srigokul Upadhyayula

    Department of Cell Biology, Harvard Medical School, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Yury S Bykov

    Structural and Computational Unit, European Molecular Biology Laboratory, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2959-4108
  5. Simon Sprenger

    Division of Cell Biology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria
    Competing interests
    The authors declare that no competing interests exist.
  6. Mehrshad Pakdel

    Division of Cell Biology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria
    Competing interests
    The authors declare that no competing interests exist.
  7. Georg F Vogel

    Division of Cell Biology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria
    Competing interests
    The authors declare that no competing interests exist.
  8. Gloria Jih

    Department of Cell Biology, Harvard Medical School, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Wesley Skillern

    Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Reza Behrouzi

    Department of Cell Biology, Harvard Medical School, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3064-9743
  11. Markus Babst

    Center for Cell and Genome Science, Department of Biology, University of Utah, Salt Lake City, United States
    Competing interests
    The authors declare that no competing interests exist.
  12. Oliver Schmidt

    Division of Cell Biology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria
    Competing interests
    The authors declare that no competing interests exist.
  13. Michael W Hess

    Division of Histology and Embryology, Medical University of Innsbruck, Innsbruck, Austria
    Competing interests
    The authors declare that no competing interests exist.
  14. John AG Briggs

    Structural and Computational Unit, European Molecular Biology Laboratory, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  15. Tomas Kirchhausen

    Department of Cell Biology, Harvard Medical School, Boston, United States
    For correspondence
    kirchhau@crystal.harvard.edu
    Competing interests
    The authors declare that no competing interests exist.
  16. David Teis

    Division of Cell Biology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria
    For correspondence
    david.teis@i-med.ac.at
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8181-0253

Funding

National Institutes of Health (GM075252)

  • Tomas Kirchhausen

Austrian Science Fund (Y444-B12)

  • David Teis

Austrian Science Fund (P30263)

  • David Teis

Austrian Science Fund (W1101-B18)

  • David Teis

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Christopher G Burd, Yale School of Medicine, United States

Publication history

  1. Received: August 30, 2017
  2. Accepted: September 25, 2017
  3. Accepted Manuscript published: October 11, 2017 (version 1)
  4. Version of Record published: November 1, 2017 (version 2)

Copyright

© 2017, Adell et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Manuel Alonso Y Adell
  2. Simona M Migliano
  3. Srigokul Upadhyayula
  4. Yury S Bykov
  5. Simon Sprenger
  6. Mehrshad Pakdel
  7. Georg F Vogel
  8. Gloria Jih
  9. Wesley Skillern
  10. Reza Behrouzi
  11. Markus Babst
  12. Oliver Schmidt
  13. Michael W Hess
  14. John AG Briggs
  15. Tomas Kirchhausen
  16. David Teis
(2017)
Recruitment dynamics of ESCRT-III and Vps4 to endosomes and implications for reverse membrane budding
eLife 6:e31652.
https://doi.org/10.7554/eLife.31652

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