The conserved centrosomin motif, γTuNA, forms a dimer that directly activates microtubule nucleation by the γ-tubulin ring complex (γTuRC)
- Princeton University, United States
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.
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Data for "The conserved centrosomin motif, γTuNA, forms a dimer that directly activates microtubule nucleation by the γ-tubulin ring complex."Dryad Digital Repository, doi:10.5061/dryad.gb5mkkwt3.
Article and author information
Author details
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.
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).
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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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
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