The conserved centrosomin motif, γTuNA, forms a dimer that directly activates microtubule nucleation by the γ-tubulin ring complex (γTuRC)
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This paper is of interest to cell biologists studying the mechanisms and control of microtubule nucleation. In this work, the authors use a novel protocol for the purification of gamma-TuRCs and for the production of gamma-TuNA that enables them to demonstrate a clear activating effect of gamma-TuNA on microtubule nucleation that depends on the dimerization of gamma-TuNA protein chains.
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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.
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eLife assessment
This paper is of interest to cell biologists studying the mechanisms and control of microtubule nucleation. In this work, the authors use a novel protocol for the purification of gamma-TuRCs and for the production of gamma-TuNA that enables them to demonstrate a clear activating effect of gamma-TuNA on microtubule nucleation that depends on the dimerization of gamma-TuNA protein chains.
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Reviewer #1 (Public Review):
In this paper, the authors use purified Xenopus γ-TuRCs and experiments in cell extract combined with cutting edge imaging techniques to investigate whether binding of the γ-TuNA fragment can activate γ-TuRCs. The authors show that γ-TuNA fragments from both humans and Xenopus are obligate dimers and that dimerization is necessary for γ-TuRC binding. They further show, using direct visualisation of microtubule nucleation from individual purified γ-TuRCs, that γ-TuNA binding increases the nucleation efficiency of γ-TuRCs by ~20 fold, helping to overcome negative regulation by Strathmin.
γ-TuNA, otherwise known as the CM1 domain, CM1 motif or CM1 helix, is well conserved and found within the N-terminal region of proteins across evolution. These proteins bind and recruit γ-TuRCs to MTOCs, such as the …
Reviewer #1 (Public Review):
In this paper, the authors use purified Xenopus γ-TuRCs and experiments in cell extract combined with cutting edge imaging techniques to investigate whether binding of the γ-TuNA fragment can activate γ-TuRCs. The authors show that γ-TuNA fragments from both humans and Xenopus are obligate dimers and that dimerization is necessary for γ-TuRC binding. They further show, using direct visualisation of microtubule nucleation from individual purified γ-TuRCs, that γ-TuNA binding increases the nucleation efficiency of γ-TuRCs by ~20 fold, helping to overcome negative regulation by Strathmin.
γ-TuNA, otherwise known as the CM1 domain, CM1 motif or CM1 helix, is well conserved and found within the N-terminal region of proteins across evolution. These proteins bind and recruit γ-TuRCs to MTOCs, such as the centrosome, meaning that γ-TuRC recruitment and activation could be closely linked. Earlier studies had provided strong evidence that binding of γ-TuNA activated γ-TuRCs, hence the name "γ-TuRC mediated nucleation activator" (Choi et al., 2010), and this claim was supported by similar work a few years later (Muroyama et al. 2016). Moreover, several other studies showed that expressing in cells γ-TuNA, or equivalent protein fragments, led to ectopic microtubule nucleation in the cytoplasm, with some of the studies showing that mutations preventing the binding of these fragments to γ-TuRCs ablated this effect (Choi et al., 2010; Lynch et al., 2014; Hanafusa et al., 2015; Cota et al., 2016; Tovey et al., 2021). Collectively, therefore, it was accepted that binding of these fragments somehow activated γ-TuRCs. More recent data, however, including from the authors themselves, had provided evidence that γ-TuNA binding did not activate γ-TuRCs (Liu et al., 2019; Thawani et al., 2020). A major objective of this paper was therefore to help resolve this controversy. The author's data suggest that the ability of these fragments to activate γ-TuRCs depends upon the type and position of tag attached to the N-terminus of the γ-TuNA fragment, with large tags seemingly turning γ-TuNA into a γ-TuRC inhibitor (although they also note that one of the previous studies, which concluded γ-TuNA was an activator, had also used fragments with large N-terminal tags). The authors also insist that the new results benefit from a much-improved γ-TuRC purification protocol that results in higher yield and purity. This purification approach uses the affinity of the γ-TuNA fragment and so could be adopted by others in the field.
The major strength of this paper is directly showing, using very powerful single molecule imaging and their improved protocols, that γ-TuNA is a γ-TuRC activator, thus resolving the controversy that has existed for the last few years. The weakness is that we still don't learn how γ-TuNA binding activates γ-TuRCs (this has been proposed to be via structural changes but other mechanisms can be considered), and thus there is little conceptual advance from the original Choi et al. 2010 paper, which had already concluded that γ-TuNA binding increased the nucleation efficiency of γ-TuRCs. Moreover, the authors do not include experiments with the other proposed γ-TuRC activator, XMAP215, which they have investigated previously (Thawani et al., 2020), and so we are left wondering whether γ-TuNA and XMAP215 work together or as part of separate activation pathways.
Overall, this paper is timely as it finally resolves the controversy over γ-TuNA and it is admirable that the authors are willing to directly address and correct their previous conclusion. The data is solid and well-presented and the text is clear and has appropriate citations. In my opinion, papers that clarify the literature are just as important as those that make conceptual advances.
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Reviewer #2 (Public Review):
This is the first report that establishes gamma-TuNA as an activator of gamma-TuRC-dependent microtubule-nucleation, using purified components. This is an in-depth study that establishes experimental conditions under which gamma-TuNA can function as an activator (dimerization of gamma-TuNA, appropriately sized N-terminal tag) and clarifies why similar attempts to study gamma-TuNA have failed in the past. I think that the information in this manuscript will be of immense value to the scientific community, as it resolves a long-standing mystery concerning the function of gamma-TuNA. A key question that still remains unanswered is whether the gamma-TuNA-dependent activation mechanism involves a conformational change of the gamma-TuRC, from an asymmetric to a ring-shaped template structure, but this may be …
Reviewer #2 (Public Review):
This is the first report that establishes gamma-TuNA as an activator of gamma-TuRC-dependent microtubule-nucleation, using purified components. This is an in-depth study that establishes experimental conditions under which gamma-TuNA can function as an activator (dimerization of gamma-TuNA, appropriately sized N-terminal tag) and clarifies why similar attempts to study gamma-TuNA have failed in the past. I think that the information in this manuscript will be of immense value to the scientific community, as it resolves a long-standing mystery concerning the function of gamma-TuNA. A key question that still remains unanswered is whether the gamma-TuNA-dependent activation mechanism involves a conformational change of the gamma-TuRC, from an asymmetric to a ring-shaped template structure, but this may be beyond the scope of the present submission.
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Reviewer #3 (Public Review):
Rale et. al. convincingly establish the regulatory role of the γ-TuNA motif in microtubule nucleation and settle the conflicting results in the literature. They show that γ-TuNA binds to and activates γ-TuRC-based microtubule nucleation both in Xenopus extracts and in vitro. The authors use real-time imaging of the nucleating microtubules in vitro to show that γ-TuNA activates microtubule nucleation by ~20 fold. They further go on to show that γ-TuNA exists as a dimer and propose that its dimeric state is important for the activating function.
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