Biology Department, University of Massachusetts, Amherst, MA.
Biology Department, University of Massachusetts, Amherst, MA
J Cell Biol. 2019 Sep 2;218(9):2829-2840. doi: 10.1083/jcb.201904114. Epub 2019 Jul 24.
Centrosome-mediated microtubule (MT) nucleation has been well characterized; however, numerous noncentrosomal MT nucleation mechanisms exist. The branching MT nucleation pathway envisages that the γ-tubulin ring complex (γ-TuRC) is recruited to MTs by the augmin complex to initiate nucleation of new MTs. While the pathway is well conserved at a molecular and functional level, branching MT nucleation by core constituents has never been directly observed in animal cells. Here, multicolor TIRF microscopy was applied to visualize and quantitatively define the entire process of branching MT nucleation in dividing cells during anaphase. The steps of a stereotypical branching nucleation event entailed augmin binding to a mother MT and recruitment of γ-TuRC after 15 s, followed by nucleation 16 s later of a daughter MT at a 36° branch angle. Daughters typically remained attached throughout their ∼40-s lifetime unless the mother depolymerized past the branch point. Assembly of branched MT arrays, which did not require TPX2, enhanced localized RhoA activation during cytokinesis.
中心体介导的微管（MT）成核已经得到了很好的描述；然而，存在许多非中心体的 MT 成核机制。分支 MT 成核途径设想γ-微管蛋白环复合物（γ-TuRC）被增敏复合物募集到 MT 上，从而启动新 MT 的成核。虽然该途径在分子和功能水平上得到了很好的保守，但在动物细胞中从未直接观察到核心成分的分支 MT 成核。在这里，多色 TIRF 显微镜被应用于可视化和定量定义有丝分裂后期分裂细胞中分支 MT 成核的整个过程。一个典型的分支成核事件的步骤包括增敏复合物与母 MT 结合，15 秒后招募 γ-TuRC，随后 16 秒后在 36°分支角处生成子 MT。除非母 MT 解聚到分支点之外，否则子 MT 通常在其大约 40 秒的生命周期内保持附着。不需要 TPX2 的分支 MT 阵列的组装增强了胞质分裂过程中局部 RhoA 的激活。
