Branching microtubule (MT) nucleation is mediated by the augmin complex and γ-tubulin ring complex (γ-TuRC). However, how these two complexes work together to promote this process remains elusive. Here, using purified components from native and recombinant sources, we demonstrate that human augmin and γ-TuRC are sufficient to reconstitute the minimal MT branching machinery, in which NEDD1 bridges between augmin holo complex and GCP3/MZT1 subcomplex of γ-TuRC. The single-molecule experiment suggests that oligomerization of augmin may activate the branching machinery. We provide direct biochemical evidence that CDK1- and PLK1-dependent phosphorylation are crucial for NEDD1 binding to augmin, for their synergistic MT-binding activities, and hence for branching MT nucleation. In addition, we unveil that NEDD1 possesses an unanticipated intrinsic affinity for MTs via its WD40 domain, which also plays a pivotal role in the branching process. In summary, our study provides a comprehensive understanding of the underlying mechanisms of branching MT nucleation in human cells.

中文翻译：

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人体微管分支机器的重建和机械解剖


分支微管 (MT) 成核由 Augmin 复合物和 γ-微管蛋白环复合物 (γ-TuRC) 介导。然而，这两种复合物如何共同促进这一过程仍然难以捉摸。在这里，使用来自天然和重组来源的纯化成分，我们证明人augmin和γ-TuRC足以重建最小的MT分支机器，其中NEDD1在augmin全息复合物和γ-TuRC的GCP3/MZT1亚复合物之间桥接。单分子实验表明，augmin 的寡聚化可能会激活分支机制。我们提供了直接的生化证据，表明 CDK1 和 PLK1 依赖性磷酸化对于 NEDD1 与 augmin 的结合、它们的协同 MT 结合活性以及因此对于分支 MT 成核至关重要。此外，我们还发现 NEDD1 通过其 WD40 结构域对 MT 具有意想不到的内在亲和力，这在分支过程中也发挥着关键作用。总之，我们的研究提供了对人类细胞中分支 MT 成核的潜在机制的全面了解。