1. Cell Biology
  2. Physics of Living Systems
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The transition state and regulation of γ-TuRC-mediated microtubule nucleation revealed by single molecule microscopy

  1. Akanksha Thawani
  2. Michael J Rale
  3. Nicolas Coudray
  4. Gira Bhabha
  5. Howard A Stone
  6. Joshua W Shaevitz
  7. Sabine Petry  Is a corresponding author
  1. Princeton University, United States
  2. New York University School of Medicine, United States
  3. Skirball Institute of Biomolecular Medicine, United States
Research Article
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Cite this article as: eLife 2020;9:e54253 doi: 10.7554/eLife.54253

Abstract

Determining how microtubules (MTs) are nucleated is essential for understanding how the cytoskeleton assembles. While the MT nucleator, γ-tubulin ring complex (γ-TuRC) has been identified, precisely how γ-TuRC nucleates a MT remains poorly understood. Here we developed a single molecule assay to directly visualize nucleation of a MT from purified Xenopus laevis γ-TuRC. We reveal a high γ-/αβ-tubulin affinity, which facilitates assembly of a MT from γ-TuRC. Whereas spontaneous nucleation requires assembly of 8 αβ-tubulins, nucleation from γ-TuRC occurs efficiently with a cooperativity of 4 αβ-tubulin dimers. This is distinct from pre-assembled MT seeds, where a single dimer is sufficient to initiate growth. A computational model predicts our kinetic measurements and reveals the rate-limiting transition where laterally-associated αβ-tubulins drive γ-TuRC into a closed conformation. Putative activation domain of CDK5RAP2, NME7 and TPX2 do not enhance γ-TuRC-mediated nucleation, while XMAP215 drastically increases the nucleation efficiency by strengthening the longitudinal γ-/αβ-tubulin interaction.

Article and author information

Author details

  1. Akanksha Thawani

    Department of Chemical and Biological Engineering, Princeton University, Princeton, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4168-128X
  2. Michael J Rale

    Department of Molecular Biology, Princeton University, Princeton, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Nicolas Coudray

    Department of Cell Biology and Applied Bioinformatics Laboratory, New York University School of Medicine, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Gira Bhabha

    New York University School of Medicine, Skirball Institute of Biomolecular Medicine, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Howard A Stone

    Department of Mechanical and Aerospace Engineering, Princeton University, Princeton, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9670-0639
  6. Joshua W Shaevitz

    Lewis-Sigler Institute of Integrative Genomics, Princeton University, Princeton, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8809-4723
  7. Sabine Petry

    Department of Molecular Biology, Princeton University, Princeton, United States
    For correspondence
    spetry@Princeton.EDU
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8537-9763

Funding

American Heart Association (17PRE33660328)

  • Akanksha Thawani

Princeton University (Charlotte Elizabeth Procter Honorific Fellowship)

  • Akanksha Thawani

Howard Hughes Medical Institute (Gilliam fellowship)

  • Michael J Rale

National Science Foundation (Graduate Student Fellowship)

  • Michael J Rale

National Institute of General Medical Sciences (R00GM112982)

  • Gira Bhabha

National Institute of General Medical Sciences (1DP2GM123493)

  • Sabine Petry

Pew Charitable Trusts (00027340)

  • Sabine Petry

David and Lucile Packard Foundation (2014-40376)

  • Sabine Petry

National Science Foundation (PHY-1734030)

  • Joshua W Shaevitz

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

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved Institutional Animal Care and Use Committee (IACUC) protocol # 1941-16 of Princeton University.

Reviewing Editor

  1. Jens Lüders, Institute for Research in Biomedicine, Spain

Publication history

  1. Received: December 7, 2019
  2. Accepted: June 15, 2020
  3. Accepted Manuscript published: June 15, 2020 (version 1)

Copyright

© 2020, Thawani 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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