The conserved centrosomin motif, γTuNA, forms a dimer that directly activates microtubule nucleation by the γ-tubulin ring complex (γTuRC)
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 restricte...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2022-12-01
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| Series: | eLife |
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| Online Access: | https://elifesciences.org/articles/80053 |
| _version_ | 1797946591658639360 |
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| author | Michael J Rale Brianna Romer Brian P Mahon Sophie M Travis Sabine Petry |
| author_facet | Michael J Rale Brianna Romer Brian P Mahon Sophie M Travis Sabine Petry |
| author_sort | Michael J Rale |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-10T21:13:27Z |
| format | Article |
| id | doaj.art-b5b710de454f4e849750ca57d8746531 |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-10T21:13:27Z |
| publishDate | 2022-12-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-b5b710de454f4e849750ca57d87465312023-01-20T15:42:38ZengeLife Sciences Publications LtdeLife2050-084X2022-12-011110.7554/eLife.80053The conserved centrosomin motif, γTuNA, forms a dimer that directly activates microtubule nucleation by the γ-tubulin ring complex (γTuRC)Michael J Rale0https://orcid.org/0000-0003-1426-6611Brianna Romer1https://orcid.org/0000-0003-1772-4243Brian P Mahon2https://orcid.org/0000-0002-5571-8058Sophie M Travis3https://orcid.org/0000-0002-1728-1705Sabine Petry4https://orcid.org/0000-0002-8537-9763Department of Molecular Biology, Princeton University, Princeton, United StatesDepartment of Molecular Biology, Princeton University, Princeton, United StatesDepartment of Molecular Biology, Princeton University, Princeton, United StatesDepartment of Molecular Biology, Princeton University, Princeton, United StatesDepartment of Molecular Biology, Princeton University, Princeton, United StatesTo 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.https://elifesciences.org/articles/80053microtubulesgamma-tubulin ring complexsingle molecule TIRFmicrotubule nucleationcentrosomeCdk5rap2 |
| spellingShingle | Michael J Rale Brianna Romer Brian P Mahon Sophie M Travis Sabine Petry The conserved centrosomin motif, γTuNA, forms a dimer that directly activates microtubule nucleation by the γ-tubulin ring complex (γTuRC) eLife microtubules gamma-tubulin ring complex single molecule TIRF microtubule nucleation centrosome Cdk5rap2 |
| title | The conserved centrosomin motif, γTuNA, forms a dimer that directly activates microtubule nucleation by the γ-tubulin ring complex (γTuRC) |
| title_full | The conserved centrosomin motif, γTuNA, forms a dimer that directly activates microtubule nucleation by the γ-tubulin ring complex (γTuRC) |
| title_fullStr | The conserved centrosomin motif, γTuNA, forms a dimer that directly activates microtubule nucleation by the γ-tubulin ring complex (γTuRC) |
| title_full_unstemmed | The conserved centrosomin motif, γTuNA, forms a dimer that directly activates microtubule nucleation by the γ-tubulin ring complex (γTuRC) |
| title_short | The conserved centrosomin motif, γTuNA, forms a dimer that directly activates microtubule nucleation by the γ-tubulin ring complex (γTuRC) |
| title_sort | conserved centrosomin motif γtuna forms a dimer that directly activates microtubule nucleation by the γ tubulin ring complex γturc |
| topic | microtubules gamma-tubulin ring complex single molecule TIRF microtubule nucleation centrosome Cdk5rap2 |
| url | https://elifesciences.org/articles/80053 |
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