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.
-
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).
Reviewing Editor
- Suzanne R Pfeffer, Stanford University, United States
Publication history
- Preprint posted: April 11, 2022 (view preprint)
- Received: May 13, 2022
- Accepted: December 7, 2022
- Accepted Manuscript published: December 14, 2022 (version 1)
- 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.
Metrics
-
- 941
- Page views
-
- 151
- Downloads
-
- 0
- Citations
Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
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
Further reading
-
- Biochemistry and Chemical Biology
- Developmental Biology
In mammals, interactions between the bone marrow (BM) stroma and hematopoietic progenitors contribute to bone-BM homeostasis. Perinatal bone growth and ossification provide a microenvironment for the transition to definitive hematopoiesis; however, mechanisms and interactions orchestrating the development of skeletal and hematopoietic systems remain largely unknown. Here, we establish intracellular O-linked β-N-acetylglucosamine (O-GlcNAc) modification as a posttranslational switch that dictates the differentiation fate and niche function of early BM stromal cells (BMSCs). By modifying and activating RUNX2, O-GlcNAcylation promotes osteogenic differentiation of BMSCs and stromal IL-7 expression to support lymphopoiesis. In contrast, C/EBPβ-dependent marrow adipogenesis and expression of myelopoietic stem cell factor (SCF) is inhibited by O-GlcNAcylation. Ablating O-GlcNAc transferase (OGT) in BMSCs leads to impaired bone formation, increased marrow adiposity, as well as defective B-cell lymphopoiesis and myeloid overproduction in mice. Thus, the balance of osteogenic and adipogenic differentiation of BMSCs is determined by reciprocal O-GlcNAc regulation of transcription factors, which simultaneously shapes the hematopoietic niche.
-
- Biochemistry and Chemical Biology
- Neuroscience
Modification by sialylated glycans can affect protein functions, underlying mechanisms that control animal development and physiology. Sialylation relies on a dedicated pathway involving evolutionarily conserved enzymes, including CMP-sialic acid synthetase (CSAS) and sialyltransferase (SiaT) that mediate the activation of sialic acid and its transfer onto glycan termini, respectively. In Drosophila, CSAS and DSiaT genes function in the nervous system, affecting neural transmission and excitability. We found that these genes function in different cells: the function of CSAS is restricted to glia, while DSiaT functions in neurons. This partition of the sialylation pathway allows for regulation of neural functions via a glia-mediated control of neural sialylation. The sialylation genes were shown to be required for tolerance to heat and oxidative stress and for maintenance of the normal level of voltage-gated sodium channels. Our results uncovered a unique bipartite sialylation pathway that mediates glia-neuron coupling and regulates neural excitability and stress tolerance.
