Abstract
SummaryThe multisubunit γ-tubulin complex (γ-TuC) is critical for microtubule nucleation in eukaryotic cells [1, 2], but it remains unclear how the γ-TuC becomes active specifically at microtubule-organizing centers (MTOCs) and not more broadly throughout the cytoplasm [3, 4]. In the fission yeast Schizosaccharomyces pombe, the proteins Mto1 and Mto2 form the Mto1/2 complex, which interacts with the γ-TuC and recruits it to several different types of cytoplasmic MTOC sites [5–10]. Here, we show that the Mto1/2 complex activates γ-TuC-dependent microtubule nucleation independently of localizing the γ-TuC. This was achieved through the construction of a “minimal” version of Mto1/2, Mto1/2[bonsai], that does not localize to any MTOC sites. By direct imaging of individual Mto1/2[bonsai] complexes nucleating single microtubules in vivo, we further determine the number and stoichiometry of Mto1, Mto2, and γ-TuC subunits Alp4 (GCP2) and Alp6 (GCP3) within active nucleation complexes. These results are consistent with active nucleation complexes containing ∼13 copies each of Mto1 and Mto2 per active complex and likely equimolar amounts of γ-tubulin. Additional experiments suggest that Mto1/2 multimers act to multimerize the fission yeast γ-tubulin small complex and that multimerization of Mto2 in particular may underlie assembly of active microtubule nucleation complexes.
Highlights
During anaphase elongation of the intranuclear mitotic spindle, Mto1/2 recruits the g-tubulin complex (g-TuC) to the cytoplasmic face of the spindle pole body (SPB) to support astral MT nucleation, and during late mitosis, Mto1/2 recruits the g-TuC to an equatorial microtubule-organizing centers (MTOCs) associated with the cytokinetic actomyosin ring, to generate a postanaphase MT array that stabilizes the actomyosin ring [12]
We reasoned that if we could construct a mutant version of Mto1/2 that failed to localize to conventional MTOC sites but promoted MT nucleation, this would provide strong support for the second model
Our results suggest that multimerization of g-TuSCs by Mto1/2 may generate the same type of supramolecular architecture as is found in conventional g-tubulin ring complex (g-TuRC), but via an alternative mode of assembly (Figure S3I)
Summary
The multisubunit g-tubulin complex (g-TuC) is critical for microtubule nucleation in eukaryotic cells [1, 2], but it remains unclear how the g-TuC becomes active at microtubule-organizing centers (MTOCs) and not more broadly throughout the cytoplasm [3, 4]. We show that the Mto1/2 complex activates g-TuC-dependent microtubule nucleation independently of localizing the g-TuC This was achieved through the construction of a ‘‘minimal’’ version of Mto1/2, Mto1/2[bonsai], that does not localize to any MTOC sites. By direct imaging of individual Mto1/2[bonsai] complexes nucleating single microtubules in vivo, we further determine the number and stoichiometry of Mto, Mto, and g-TuC subunits Alp (GCP2) and Alp (GCP3) within active nucleation complexes. These results are consistent with active nucleation complexes containing w13 copies each of Mto and Mto per active complex and likely equimolar amounts of g-tubulin. Additional experiments suggest that Mto1/2 multimers act to multimerize the fission yeast g-tubulin small complex and that multimerization of Mto in particular may underlie assembly of active microtubule nucleation complexes
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