Tariq Ammarah, Green Lucy, Jeynes J Charles G, Soeller Christian, Wakefield James G
Living Systems Institute, University of Exeter, Exeter, United Kingdom.
Elife. 2020 Jan 14;9:e49769. doi: 10.7554/eLife.49769.
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-bulin ing omplex (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 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.
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，它能精确地排列和分离复制后的染色体。纺锤体形成的动力学主要由细胞内微管成核剂γ-微管蛋白复合体（γ-TuRC）的正确定位所决定。一种保守的微管相关蛋白复合体Augmin，将γ-TuRC招募到预先存在的纺锤体微管上，增加微管数量，这是一种被称为“分支”微管成核的重要细胞现象。在这里，我们使用一种新颖的一步亲和技术，从胚胎中纯化出近乎均一的内源性绿色荧光蛋白标记的Augmin和γ-TuRC。我们证明，在体外，虽然单独的Augmin不影响微管蛋白的聚合动力学，但它以细胞周期依赖的方式刺激γ-TuRC依赖的微管成核。我们还使用光学显微镜组装并观察了微管-Augmin-γ-TuRC-微管连接。因此，我们的工作最终重建了分支微管成核。它还提供了一种强大的合成方法，用以研究从组成部分中产生的细胞现象，如有丝分裂纺锤体的形成。
