The Ndc80 complex is essential for the initial kinetochore-microtubule capture during early mitosis

Abstract

ABSTRACTMitotic 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. In metaphase, the kinetochore localized, microtubule-binding complex, Ndc80, plays a central role in stabilizing kinetochore-microtubule (kMT) attachments. It is not yet clear, however, if Ndc80, which is recruited to kinetochores very early during mitosis contributes to initial kMT capture. 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 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 and proper chromosome alignment.