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Uncoupling of dynamin polymerization and GTPase activity revealed by the conformation-specific nanobody Dynab

  1. Valentina Galli
  2. Rafael Sebastian
  3. Sandrine Moutel
  4. Jason Ecard
  5. Franck Perez
  6. Aurélien Roux Is a corresponding author
  1. University of Geneva, Switzerland
  2. University of Valencia, Spain
  3. Institut Curie, France
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Cite as: eLife 2017;6:e25197 doi: 10.7554/eLife.25197

Abstract

Dynamin is a large GTPase that forms a helical collar at the neck of endocytic pits, and catalyzes membrane fission (1, 2). Dynamin fission reaction is strictly dependent on GTP hydrolysis, but how fission is mediated is still debated (3): GTP energy could be spent in membrane constriction required for fission, or in disassembly of the dynamin polymer to trigger fission. To follow dynamin GTP hydrolysis at endocytic pits, we generated a conformation-specific nanobody called dynab, that binds preferentially to the GTP hydrolytic state of dynamin-1. Dynab allowed us to follow the GTPase activity of dynamin-1 in real-time. We show that in fibroblasts, dynamin GTP hydrolysis occurs as stochastic bursts, which are randomly distributed relatively to the peak of dynamin assembly. Thus, dynamin disassembly is not coupled to GTPase activity, supporting that the GTP energy is primarily spent in constriction.

Article and author information

Author details

  1. Valentina Galli

    Department of Biochemistry, University of Geneva, Geneva, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  2. Rafael Sebastian

    Department of Computer Sciences, University of Valencia, Valencia, Spain
    Competing interests
    The authors declare that no competing interests exist.
  3. Sandrine Moutel

    Institut Curie, Paris, France
    Competing interests
    The authors declare that no competing interests exist.
  4. Jason Ecard

    Institut Curie, Paris, France
    Competing interests
    The authors declare that no competing interests exist.
  5. Franck Perez

    Institut Curie, Paris, France
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon 0000-0002-9129-9401
  6. Aurélien Roux

    Department of Biochemistry, University of Geneva, Geneva, Switzerland
    For correspondence
    aurelien.roux@unige.ch
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon 0000-0002-6088-0711

Funding

Human Frontier Science Program (CDA-0061-08)

  • Aurélien Roux

Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Grant 31003A_130520)

  • Aurélien Roux

H2020 European Research Council (Starting Grant 311536 (2011 call))

  • Aurélien Roux

Seventh Framework Programme (Marie Curie ITN grant #264399)

  • Aurélien Roux

Agence Nationale de la Recherche (ANR-12-BSV2-0003-01)

  • Franck Perez

Centre National de la Recherche Scientifique

  • Franck Perez

Fondation pour la Recherche Médicale (DEQ20120323723)

  • Franck Perez

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

Reviewing Editor

  1. Christien Merrifield, Reviewing Editor, Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, France

Publication history

  1. Received: January 17, 2017
  2. Accepted: October 11, 2017
  3. Accepted Manuscript published: October 12, 2017 (version 1)

Copyright

© 2017, Galli 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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