XMAP215 and γ-tubulin additively promote microtubule nucleation in purified solutions.
XMAP215 and γ-tubulin additively promote microtubule nucleation in purified solutions.
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DOI：
10.1091/mbc.e20-02-0160
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发表时间：
2020-09-15
影响因子：
3.3
通讯作者：
Davis TN
中科院分区：
文献类型：
--
作者：
King BR;Moritz M;Kim H;Agard DA;Asbury CL;Davis TN
细胞中的微管成核受几种已知分子的时空调控，包括模板γ-微管蛋白和聚合酶XMAP215。XMAP215在成核中的作用仍有争议，特别是它是作为一种聚合酶独立作用还是与γ-微管蛋白依赖作用。我们首先确认XMAP215是一种经典定义的成核剂，可以减少微管蛋白组装中的成核滞后。其次，利用缺失结构，我们探索了XMAP215促进微管成核所需的结构域。我们表明，它在纯化溶液中成核微管的能力与其延长现有微管的能力相关，而不依赖于肿瘤过表达基因（TOG）结构域的数量。最后，我们发现XMAP215和γ-微管蛋白以加性而非协同的方式促进α - β-微管蛋白的组装。因此，它们在微管成核过程中的作用模式是不同的。这些发现表明在成核过程中至少有两个独立的过程，一个是由γ-微管蛋白促进的，另一个是由XMAP215促进的。我们提出XMAP215加速了亚基加入到现有的成核中间体中，无论是自发形成的还是由γ-微管蛋白的低聚物形成的。
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 tumor overexpressed gene (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.