J Biol Chem. 2023 Apr 13. pii: S0021-9258(23)01739-8. [Epub ahead of print]
104711
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 towards the spindle pole during early mitosis. The microtubule-binding 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 specifically targeted to study kMT capture, we show that mitotic cells lacking Ndc80 exhibit severe 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 TIR-FM 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 physical interaction between Ndc80 and dynein that we identify in prometaphase extracts might be critical for efficient kMT 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.