Barrero Daniel J, Wijeratne Sithara S, Zhao Xiaowei, Cunningham Grace F, Rui Yan, Nelson Christian R, Yasuhiro Arimura, Funabiki Hironori, Asbury Charles L, Yu Zhiheng, Subramanian Radhika, Biggins Sue
Howard Hughes Medical Institute, Division of Basic Sciences, Fred Hutchinson Cancer Center, 1100 Fairview Ave. N., Seattle, WA 98109, USA.
Molecular and Cellular Biology Program, University of Washington, 1705 NE Pacific Street, Seattle, WA 98195, USA.
bioRxiv. 2024 Feb 29:2024.02.28.582571. doi: 10.1101/2024.02.28.582571.
Eukaryotic chromosome segregation requires kinetochores, multi-megadalton protein machines that assemble on the centromeres of chromosomes and mediate attachments to dynamic spindle microtubules. Kinetochores are built from numerous complexes, and understanding how they are arranged is key to understanding how kinetochores perform their multiple functions. However, an integrated understanding of kinetochore architecture has not yet been established. To address this, we purified functional, native kinetochores from and examined them by electron microscopy, cryo-electron tomography and atomic force microscopy. The kinetochores are extremely large, flexible assemblies that exhibit features consistent with prior models. We assigned kinetochore polarity by visualizing their interactions with microtubules and locating the microtubule binder Ndc80c. This work shows that isolated kinetochores are more dynamic and complex than what might be anticipated based on the known structures of recombinant subassemblies, and provides the foundation to study the global architecture and functions of kinetochores at a structural level.
真核生物染色体分离需要动粒，动粒是组装在染色体着丝粒上的多兆道尔顿蛋白质机器，介导与动态纺锤体微管的附着。动粒由众多复合体构成，了解它们的排列方式是理解动粒如何执行其多种功能的关键。然而，尚未建立对动粒结构的综合理解。为解决这一问题，我们从……中纯化了功能性天然动粒，并通过电子显微镜、冷冻电子断层扫描和原子力显微镜对其进行研究。动粒是极其庞大且灵活的组装体，呈现出与先前模型一致的特征。我们通过观察动粒与微管的相互作用并定位微管结合蛋白Ndc80c来确定动粒极性。这项工作表明，分离出的动粒比基于重组亚组装体的已知结构所预期的更加动态和复杂，并为在结构水平上研究动粒的整体结构和功能奠定了基础。
