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 gamma-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.

中文翻译：

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酿酒酵母异位微管阵列形成所需的微管相关蛋白和马达

由于运动和微管相关蛋白的协同作用，有时是多余的作用，有丝分裂纺锤体对摄动具有弹性。在这里，我们利用酿酒酵母细胞核中的诱导性异位微管成核位点研究形成双极阵列的三个必要步骤：γ-微管蛋白复合物的募集，微管的成核和伸长以及微管之间的相对组织。这种新颖的工具，Spc110嵌合体，揭示了微管相关蛋白Stu2，Bim1和Bik1以及马达Vik1和Kip3以前未报道的作用。我们报告Stu2和Bim1是成核所必需的，并且Bik1和Kip3促进异位处的成核。Stu2，Bim1和Kip3加入它们的同系物XMAP215，EB1和驱动蛋白8作为微管成核的启动子，而Bik1通过其在SPB定位中的作用间接促进MT成核。此外，我们发现Stu2在体内的成核活性与其在体外的聚合酶活性相关。。最后，我们提供了第一个证据，证明Vik1是Kar3 / Vik1驱动蛋白14的一个亚基，可促进微管负端集中在异位部位。