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.
-
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.
Metrics
-
- 1,700
- views
-
- 241
- downloads
-
- 14
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
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)
Further reading
-
- Biochemistry and Chemical Biology
The conformational ensemble and function of intrinsically disordered proteins (IDPs) are sensitive to their solution environment. The inherent malleability of disordered proteins, combined with the exposure of their residues, accounts for this sensitivity. One context in which IDPs play important roles that are concomitant with massive changes to the intracellular environment is during desiccation (extreme drying). The ability of organisms to survive desiccation has long been linked to the accumulation of high levels of cosolutes such as trehalose or sucrose as well as the enrichment of IDPs, such as late embryogenesis abundant (LEA) proteins or cytoplasmic abundant heat-soluble (CAHS) proteins. Despite knowing that IDPs play important roles and are co-enriched alongside endogenous, species-specific cosolutes during desiccation, little is known mechanistically about how IDP-cosolute interactions influence desiccation tolerance. Here, we test the notion that the protective function of desiccation-related IDPs is enhanced through conformational changes induced by endogenous cosolutes. We find that desiccation-related IDPs derived from four different organisms spanning two LEA protein families and the CAHS protein family synergize best with endogenous cosolutes during drying to promote desiccation protection. Yet the structural parameters of protective IDPs do not correlate with synergy for either CAHS or LEA proteins. We further demonstrate that for CAHS, but not LEA proteins, synergy is related to self-assembly and the formation of a gel. Our results suggest that functional synergy between IDPs and endogenous cosolutes is a convergent desiccation protection strategy seen among different IDP families and organisms, yet the mechanisms underlying this synergy differ between IDP families.
-
- Biochemistry and Chemical Biology
- Stem Cells and Regenerative Medicine
Human induced pluripotent stem cells (hiPSCs) have great potential to be used as alternatives to embryonic stem cells (hESCs) in regenerative medicine and disease modelling. In this study, we characterise the proteomes of multiple hiPSC and hESC lines derived from independent donors and find that while they express a near-identical set of proteins, they show consistent quantitative differences in the abundance of a subset of proteins. hiPSCs have increased total protein content, while maintaining a comparable cell cycle profile to hESCs, with increased abundance of cytoplasmic and mitochondrial proteins required to sustain high growth rates, including nutrient transporters and metabolic proteins. Prominent changes detected in proteins involved in mitochondrial metabolism correlated with enhanced mitochondrial potential, shown using high-resolution respirometry. hiPSCs also produced higher levels of secreted proteins, including growth factors and proteins involved in the inhibition of the immune system. The data indicate that reprogramming of fibroblasts to hiPSCs produces important differences in cytoplasmic and mitochondrial proteins compared to hESCs, with consequences affecting growth and metabolism. This study improves our understanding of the molecular differences between hiPSCs and hESCs, with implications for potential risks and benefits for their use in future disease modelling and therapeutic applications.