The State Key Laboratory Breeding Base of Basic Science of Stomatology and Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, China.
Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University, Wuhan, China.
J Cell Biol. 2022 Jul 4;221(7). doi: 10.1083/jcb.202109053. Epub 2022 May 23.
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.
分支微管（MT）的成核是由增敏复合物和γ-微管环复合物（γ-TuRC）介导的。然而，这两个复合物如何协同作用来促进这个过程仍然难以捉摸。在这里，我们使用来自天然和重组来源的纯化成分，证明人类增敏复合物和γ-TuRC 足以重建最小的 MT 分支机器，其中 NEDD1 在增敏全复合物和 γ-TuRC 的 GCP3/MZT1 亚复合物之间形成桥接。单分子实验表明，增敏复合物的寡聚化可能激活分支机器。我们提供了直接的生化证据，证明 CDK1 和 PLK1 依赖性磷酸化对于 NEDD1 与增敏复合物的结合、它们的协同 MT 结合活性以及因此对于分支 MT 成核是至关重要的。此外，我们揭示了 NEDD1 通过其 WD40 结构域具有对 MT 的意想不到的内在亲和力，这在分支过程中也起着关键作用。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理解。
