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.

细胞中的微管成核受到几种已知分子的时空调节，包括模板 γ-微管蛋白和聚合酶 XMAP215。XMAP215 在成核中的作用尚存在争议，特别是它是作为聚合酶独立发挥作用还是依赖于 γ-微管蛋白发挥作用。我们首先确认 XMAP215 是一种经典定义的成核剂，可减少批量微管蛋白组装中的成核滞后。其次，使用缺失构建体，我们探讨了 XMAP215 促进微管成核的结构域要求。我们表明，其在纯化溶液中使微管成核的能力与其延长现有微管的能力相关，并且不依赖于 TOG 结构域的数量。最后，我们证明 XMAP215 和 γ-微管蛋白以相加而非协同的方式促进 αβ-微管蛋白组装。因此，它们在微管成核过程中的作用模式是不同的。这些发现表明成核过程中至少有两个独立的过程，一个由 γ-微管蛋白促进，另一个由 XMAP215 促进。我们建议 XMAP215 加速将亚基添加到自发形成或由 γ-微管蛋白寡聚物形成的现有成核中间体中。