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

Abstract

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.

Data availability

Raw and processed microscopy data, related analysis scripts (ImageJ and MATLAB), raw size-exclusion chromatography files, and mass spectrometry data have been deposited in a freely accessible dataset on Dryad (Dataset DOI: https://doi.org/10.5061/dryad.gb5mkkwt3). Figure source data and MATLAB code are also included in this study as supplemental or source data files. Plasmids generated in this study are available upon request from the corresponding author.

The following data sets were generated

Article and author information

Author details

  1. Michael J Rale

    Department of Molecular Biology, 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:" 0000-0003-1426-6611
  2. Brianna Romer

    Department of Molecular Biology, 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:" 0000-0003-1772-4243
  3. Brian P Mahon

    Department of Molecular Biology, 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:" 0000-0002-5571-8058
  4. Sophie M Travis

    Department of Molecular Biology, 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:" 0000-0002-1728-1705
  5. 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

Howard Hughes Medical Institute (Gilliam Graduate Student Fellowship)

  • Michael J Rale

National Science Foundation (Graduate Research Fellowship)

  • Michael J Rale

National Institutes of Health (New Innovator Award,1DP2GM123493)

  • Sabine Petry

Pew Charitable Trusts (Pew Scholars Program in the Biomedical Sciences,00027340)

  • Sabine Petry

David and Lucile Packard Foundation (2014-40376)

  • Sabine Petry

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

Reviewing Editor

  1. Suzanne R Pfeffer, Stanford University, United States

Ethics

Animal experimentation: Experimental use of Xenopus laevis frogs was done in strict accordance with our approved Institutional Animal Care and Use Committee (IACUC) protocol # 1941-06 (Princeton University).

Version history

  1. Preprint posted: April 11, 2022 (view preprint)
  2. Received: May 13, 2022
  3. Accepted: December 7, 2022
  4. Accepted Manuscript published: December 14, 2022 (version 1)
  5. Version of Record published: January 20, 2023 (version 2)

Copyright

© 2022, Rale 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. Michael J Rale
  2. Brianna Romer
  3. Brian P Mahon
  4. Sophie M Travis
  5. Sabine Petry
(2022)
The conserved centrosomin motif, γTuNA, forms a dimer that directly activates microtubule nucleation by the γ-tubulin ring complex (γTuRC)
eLife 11:e80053.
https://doi.org/10.7554/eLife.80053

Share this article

https://doi.org/10.7554/eLife.80053

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