Microtubule nucleation is spatiotemporally regulated in cells by several known molecules, including the template γ-tubulin and the polymerase XMAP215. The role of XMAP215 in nucleation is under debate, specifically whether it acts independently as a polymerase or acts dependently with γ-tubulin. We first confirm XMAP215 as a classically defined nucleator that reduces the nucleation lag seen in bulk tubulin assembly. Secondly, using deletion constructs, we probe the domain requirements for XMAP215 to promote microtubule nucleation. We show that its ability to nucleate microtubules in purified solutions correlates with its ability to elongate existing microtubules and does not depend on the number of TOG domains. Finally, we show that XMAP215 and γ-tubulin promote αβ-tubulin assembly in an additive, not synergistic, manner. Thus, their modes of action during microtubule nucleation are distinct. These findings suggest there are at least two independent processes in nucleation, one promoted by γ-tubulin and one promoted by XMAP215. We propose that XMAP215 accelerates the addition of subunits to existing nucleation intermediates formed either spontaneously or by oligomers of γ-tubulin.

The mitotic spindle is resilient to perturbation due to the concerted, and sometimes redundant, action of motors and microtubule-associated proteins. Here we utilize an inducible ectopic microtubule nucleation site in the nucleus of Saccharomyces cerevisiae to study three necessary steps in the formation of a bipolar array: the recruitment of the γ-tubulin complex, nucleation and elongation of microtubules, and the organization of microtubules relative to each other. This novel tool, an Spc110 chimera, reveals previously unreported roles of the microtubule-associated proteins Stu2, Bim1, and Bik1, and the motors Vik1 and Kip3. We report that Stu2 and Bim1 are required for nucleation and that Bik1 and Kip3 promote nucleation at the ectopic site. Stu2, Bim1, and Kip3 join their homologs XMAP215, EB1 and kinesin-8 as promoters of microtubule nucleation, while Bik1 promotes MT nucleation indirectly via its role in SPB positioning. Further, we find that the nucleation activity of Stu2 in vivo correlates with its polymerase activity in vitro. Finally, we provide the first evidence that Vik1, a subunit of Kar3/Vik1 kinesin-14, promotes microtubule minus end focusing at the ectopic site.

Microtubule nucleation is spatiotemporally regulated in cells by several molecules, including the template -tubulin and the polymerase XMAP215. Recently, XMAP215 and the -tubulin ring complex were reported to function synergistically, and this synergy was hypothesized to be due to direct binding between XMAP215 and -tubulin. Here, we address this hypothesis by 1) probing domain requirements for XMAP215 to promote microtubule nucleation and 2) testing whether XMAP215 functions synergistically with -tubulin in the absence of the other ring complex proteins. We confirm that -tubulin and XMAP215 are classically defined nucleators that reduce the nucleation lag seen in bulk tubulin assembly. Then, using deletion constructs, we show that XMAP215's ability to nucleate microtubules in purified solutions correlates with its ability to elongate existing microtubules and does not depend on the number of TOG domains. Finally, we show that XMAP215 and -tubulin promote -tubulin assembly in an additive, not synergistic, manner. Thus, their modes of action during microtubule nucleation are distinct, and the synergy reported between XMAP215 and the -tubulin ring complex is not due to -tubulin alone.We thank Albion Baucom and Koji Yonekura for help with reconstructions of the tubulin arrays. We thank Per Widlund for XMAP215 expression plasmids and helpful discussion.

The mitotic spindle is resilient to perturbation due to the concerted, and sometimes redundant, action of motors and microtubule-associated proteins. Here we utilize an inducible ectopic microtubule nucleation site in the nucleus of Saccharomyces cerevisiae to study three necessary steps in the formation of a bipolar array: the recruitment of the γ-tubulin complex, nucleation and elongation of microtubules, and the organization of microtubules relative to each other. This novel tool, an Spc110 chimera, reveals previously unreported roles of the microtubule-associated proteins Stu2, Bim1, and Bik1, and the motors Vik1 and Kip3. We report that Stu2 and Bim1 are required for nucleation and that Bik1 and Kip3 promote nucleation at the ectopic site. Stu2, Bim1, and Kip3 join their homologs XMAP215, EB1 and kinesin-8 as promoters of microtubule nucleation, while Bik1 promotes MT nucleation indirectly via its role in SPB positioning. Further, we find that the nucleation activity of Stu2 in vivo correlates with its polymerase activity in vitro. Finally, we provide the first evidence that Vik1, a subunit of Kar3/Vik1 kinesin-14, promotes microtubule minus end focusing at the ectopic site.