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.

Dynamic life of a microtubule

“…These data confirm that CDK5RAP2 directly interacts with γ-TuRC 26 and suggest that in our assays, it can activate the majority of γ-TuRCs to which it binds. The percentage of CDK5RAP2-positive dots colocalizing with γ-TuRCs was rather low in these experiments (~13% with tubulin, ~41% without tubulin), likely because CDK5RAP2 was present in excess, or due to the autoinhibitory regulation of CDK5RAP2 that is controlled by its phosphorylation state 33,34 . Compared to the samples with tubulin alone or with mCherry-EB3, mCherry-CDK5RAP2 had no significant effect on microtubule growth rate or catastrophe frequency, but strongly increased the frequency of microtubule re-nucleation after depolymerization, indicating that CDK5RAP2 can maintain γ-TuRC in an active state (Fig.…”

“…However, we were successful in examining the effect of CDK5RAP2. This protein is well-known for its ability to bind γ-TuRC through the domain called γ-TuNA (γ-TuRC nucleation activator), but there was some conflicting evidence about its ability to activate γ-TuRC in vitro 19,26,27,34,51 . Two studies showed a stimulatory effect of nanomolar γ-TuNA concentrations on microtubule nucleation by γ-TuRC purified from mammalian cells using its affinity for γ-TuNA 26,27 , whereas, more recently, two other groups found no to little effect of even micromolar γ-TuNA concentrations on Xenopus γ-TuRC when purified using a γ-tubulin antibody 19,51 .…”

“…Two studies showed a stimulatory effect of nanomolar γ-TuNA concentrations on microtubule nucleation by γ-TuRC purified from mammalian cells using its affinity for γ-TuNA 26,27 , whereas, more recently, two other groups found no to little effect of even micromolar γ-TuNA concentrations on Xenopus γ-TuRC when purified using a γ-tubulin antibody 19,51 . A recent study attributed these differences to the presence of bulkier N-terminal tags on γ-TuNA and purification methods involving different affinity tags 34 .…”

“…The centrosomal scaffold protein CDK5RAP2 (CDK5 regulatory subunit-associated protein 2), also known as CEP215, is a human microcephaly associated pericentriolar material (PCM) protein that is involved in the recruitment of γ-TuRC to centrosomes and Golgi membranes (Bond et al, 2005;Fong et al, 2008;. Its D. melanogaster homolog, centrosomin, or yeast Spc110/Mto1/Mto2, or C. elegans SPD5, all have been shown to stimulate microtubule nucleation from γ-TuRC Woodruff et al, 2017;Tovey et al, 2021;Brilot et al, 2021;Rale et al, 2022). They all consist of a conserved centrosomin motif 1 (CM1) domain at the N-terminus, which binds to γ-TuRC, and another conserved motif, CM2, at the C-terminus, which localizes the protein to the centrosome or the Golgi complex by binding to its interacting partners like pericentrin or AKAP9 (also know as AKAP450) (Fig.…”

“…6) Wang et al, 2010;Wu and Akhmanova, 2017b;Watanabe et al, 2020;Tovey et al, 2021). A minimal domain of CDK5RAP2/centrosomin containing just the CM1 region, referred as γ-TuRC nucleation activator (γ-TuNA), was sufficient to stimulate microtubule nucleation from γ-TuRC, both in vivo and in vitro Tovey et al, 2021;Rale et al, 2022). Recently, structural work has shown that a γ-TuNA dimer forms a coiled-coil and binds to the MZT2/GCP2-NHD module at position 13 and docks on the outer surface of γ-TuRC, similar to its yeast counterpart (Brilot et al, 2021;.…”