1. Cell Biology
  2. Physics of Living Systems

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
https://doi.org/10.7554/eLife.54253
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  1. Akanksha Thawani
  2. Michael J Rale
  3. Nicolas Coudray
  4. Gira Bhabha
  5. Howard A Stone
  6. Joshua W Shaevitz
  7. Sabine Petry
(2020)
The transition state and regulation of γ-TuRC-mediated microtubule nucleation revealed by single molecule microscopy
eLife 9:e54253.
https://doi.org/10.7554/eLife.54253
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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.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 2, 3, 4, 6, 7 and related supplements.

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.

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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  1. Akanksha Thawani
  2. Michael J Rale
  3. Nicolas Coudray
  4. Gira Bhabha
  5. Howard A Stone
  6. Joshua W Shaevitz
  7. Sabine Petry
(2020)
The transition state and regulation of γ-TuRC-mediated microtubule nucleation revealed by single molecule microscopy
eLife 9:e54253.
https://doi.org/10.7554/eLife.54253

Share this article

https://doi.org/10.7554/eLife.54253

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    The conserved centrosomin motif, γTuNA, forms a dimer that directly activates microtubule nucleation by the γ-tubulin ring complex (γTuRC)

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    To establish the microtubule cytoskeleton, the cell must tightly regulate when and where microtubules are nucleated. This regulation involves controlling the initial nucleation template, the γ-tubulin ring complex (γTuRC). Although γTuRC is present throughout the cytoplasm, its activity is restricted to specific sites including the centrosome and Golgi. The well-conserved γ-tubulin nucleation activator (γTuNA) domain has been reported to increase the number of microtubules (MTs) generated by γTuRCs. However, previously we and others observed that γTuNA had a minimal effect on the activity of antibody-purified Xenopus γTuRCs in vitro (Thawani et al., eLife, 2020; Liu et al., 2020). Here, we instead report, based on improved versions of γTuRC, γTuNA, and our TIRF assay, the first real-time observation that γTuNA directly increases γTuRC activity in vitro, which is thus a bona fide γTuRC activator. We further validate this effect in Xenopus egg extract. Via mutation analysis, we find that γTuNA is an obligate dimer. Moreover, efficient dimerization as well as γTuNA’s L70, F75, and L77 residues are required for binding to and activation of γTuRC. Finally, we find that γTuNA’s activating effect opposes inhibitory regulation by stathmin. In sum, our improved assays prove that direct γTuNA binding strongly activates γTuRCs, explaining previously observed effects of γTuNA expression in cells and illuminating how γTuRC-mediated microtubule nucleation is regulated.

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