Department of Cell and Developmental Biology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
Department of Cell and Developmental Biology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
J Biol Chem. 2023 Jun;299(6):104711. doi: 10.1016/j.jbc.2023.104711. Epub 2023 Apr 14.
Mitotic kinetochores are initially captured by dynamic microtubules via a "search-and-capture" mechanism. The microtubule motor, dynein, is critical for kinetochore capture as it has been shown to transport microtubule-attached chromosomes toward the spindle pole during prometaphase. The microtubule-binding nuclear division cycle 80 (Ndc80) complex that is recruited to kinetochores in prophase is known to play a central role in forming kinetochore-microtubule (kMT) attachments in metaphase. It is not yet clear, however, how Ndc80 contributes to initial kMT capture during prometaphase. Here, by combining CRISPR/Cas9-mediated knockout and RNAi technology with assays specific to study kMT capture, we show that mitotic cells lacking Ndc80 exhibit substantial defects in this function during prometaphase. Rescue experiments show that Ndc80 mutants deficient in microtubule-binding are unable to execute proper kMT capture. While cells inhibited of dynein alone are predominantly able to make initial kMT attachments, cells co-depleted of Ndc80 and dynein show severe defects in kMT capture. Further, we use an in vitro total internal reflection fluorescence microscopy assay to reconstitute microtubule capture events, which suggest that Ndc80 and dynein coordinate with each other for microtubule plus-end capture and that the phosphorylation status of Ndc80 is critical for productive kMT capture. A novel interaction between Ndc80 and dynein that we identify in prometaphase extracts might be critical for efficient plus-end capture. Thus, our studies, for the first time, identify a distinct event in the formation of initial kMT attachments, which is directly mediated by Ndc80 and in coordination with dynein is required for efficient kMT capture and chromosome alignment.
有丝分裂的动粒最初通过“搜索和捕获”机制被动态微管捕获。微管动力蛋白 dynein 对于动粒捕获至关重要，因为它已经被证明在前期将附着微管的染色体运向纺锤体极。在前期被招募到动粒的微管结合核分裂周期 80（Ndc80）复合物，已知在中期形成动粒-微管（kMT）连接中起核心作用。然而，目前尚不清楚 Ndc80 如何在前期为初始 kMT 捕获做出贡献。在这里，我们通过结合 CRISPR/Cas9 介导的敲除和 RNAi 技术与专门用于研究 kMT 捕获的测定方法，表明缺乏 Ndc80 的有丝分裂细胞在前期表现出该功能的显著缺陷。挽救实验表明，缺乏微管结合的 Ndc80 突变体无法执行适当的 kMT 捕获。虽然单独抑制 dynein 的细胞主要能够进行初始 kMT 连接，但同时耗尽 Ndc80 和 dynein 的细胞在 kMT 捕获方面表现出严重缺陷。此外，我们使用体外全内反射荧光显微镜测定法重新构建微管捕获事件，这表明 Ndc80 和 dynein 相互协调以捕获微管的正极，并且 Ndc80 的磷酸化状态对于有效 kMT 捕获至关重要。我们在前期提取物中鉴定的 Ndc80 和 dynein 之间的新相互作用对于有效的正极捕获可能是关键的。因此，我们的研究首次确定了初始 kMT 连接形成中的一个独特事件，该事件直接由 Ndc80 介导，并与 dynein 协调，对于有效的 kMT 捕获和染色体排列是必需的。
