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

TitleThe conserved centrosomin motif, γTuNA, forms a dimer that directly activates microtubule nucleation by the γ-tubulin ring complex (γTuRC).
Publication TypeJournal Article
Year of Publication2022
AuthorsRale, MJ, Romer, B, Mahon, BP, Travis, SM, Petry, S
JournalElife
Volume11
Date Published2022 Dec 14
ISSN2050-084X
KeywordsAnimals, Centrosome, Microtubule-Associated Proteins, Microtubule-Organizing Center, Microtubules, Tubulin, Xenopus laevis
Abstract

<p>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 γTuRCs in vitro (Thawani et al., , 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 γTuRC activator. We further validate this effect in 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.</p>

DOI10.7554/eLife.80053
Alternate JournalElife
PubMed ID36515268
PubMed Central IDPMC9859039
Grant ListNew Innovator Award,1DP2GM123493 / NH / NIH HHS / United States
Gilliam Graduate Student Fellowship / HHMI / Howard Hughes Medical Institute / United States
New Innovator Award / NH / NIH HHS / United States
1DP2GM123493 / NH / NIH HHS / United States