Eukaryotic cell division requires the mitotic spindle, a microtubule (MT)-based structure which accurately aligns and segregates duplicated chromosomes. The dynamics of spindle formation are determined primarily by correctly localising the MT nucleator, γ-Tubulin Ring Complex (γ-TuRC), within the cell. A conserved MT-associated protein complex, Augmin, recruits γ-TuRC to pre-existing spindle MTs, amplifying their number, in an essential cellular phenomenon termed ‘branching’ MT nucleation. Here, we purify endogenous, GFP-tagged Augmin and γ-TuRC from Drosophila embryos to near homogeneity using a novel one-step affinity technique. We demonstrate that, in vitro, while Augmin alone does not affect Tubulin polymerisation dynamics, it stimulates γ-TuRC-dependent MT nucleation in a cell cycle-dependent manner. We also assemble and visualise the MT-Augmin-γ-TuRC-MT junction using light microscopy. Our work therefore conclusively reconstitutes branching MT nucleation. It also provides a powerful synthetic approach with which to investigate the emergence of cellular phenomena, such as mitotic spindle formation, from component parts.

中文翻译：

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分支微管成核的体外重建

真核细胞分裂需要有丝分裂纺锤体，这是一种基于微管（MT）的结构，可以准确地对齐和分离重复的染色体。纺锤体形成的动力学是通过正确地本地化的MT成核主要是确定的，γ-涂布林ř荷兰国际集团Ç omplex（γ-TURC），在细胞内。保守的MT相关蛋白复合物Augmin将γ-TuRC募集到预先存在的纺锤MT中，从而放大了它们的数量，这是一种称为“分支” MT成核的基本细胞现象。在这里，我们从果蝇中纯化内源性，带有GFP标签的Augmin和γ-TuRC胚胎使用一种新的一步亲和力技术接近同质性。我们证明，在体外，虽然仅Augmin不会影响微管蛋白聚合动力学，但它会以细胞周期依赖的方式刺激γ-TuRC依赖的MT成核。我们还使用光学显微镜组装并可视化MT-Augmin-γ-TuRC-MT连接。因此，我们的工作最终重建了分支MT形核。它还提供了一种强大的综合方法，可用来研究零件中细胞现象的出现，例如有丝分裂纺锤体的形成。