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 transports microtubule attached chromosomes towards the spindle pole during early mitosis. In metaphase, the kinetochore microtubule binding complex, Ndc80, plays a central role in stabilizing kinetochore microtubule (kMT) attachments. It is not yet clear, however, if Ndc80 that is recruited to kinetochores early during mitosis, contributes to initial kMT capture. Here, by combining CRISPR mediated knockout and RNAi technology with assays specific for kMT capture, we show that mitotic cells lacking Ndc80 exhibit severe defects in kMT capture 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 able to make initial kMT attachments, cells codepleted of Ndc80 and dynein show severe defects in kMT capture. Further, we demonstrate a novel physical interaction between Ndc80 and dynein during prometaphase. Thus, our studies, for the first time, identify a distinct event in the formation of initial kMT attachments, which is directly mediated by Ndc80 followed by a coordinated function with dynein, both of which are required for efficient kMT capture.
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