PT - JOURNAL ARTICLE AU - Pim J. Huis in ’t Veld AU - Sadasivam Jeganathan AU - Arsen Petrovic AU - Juliane John AU - Priyanka Singh AU - Florian Weissmann AU - Tanja Bange AU - Andrea Musacchio TI - Molecular basis of outer kinetochore assembly on CENP-T AID - 10.1101/071613 DP - 2016 Jan 01 TA - bioRxiv PG - 071613 4099 - http://biorxiv.org/content/early/2016/08/25/071613.short 4100 - http://biorxiv.org/content/early/2016/08/25/071613.full AB - Stable kinetochore-microtubule attachment is essential for cell division. It requires recruitment of outer kinetochore microtubule binders by centromere proteins C and T (CENP-C and CENP-T). To study the molecular requirements of kinetochore formation, we reconstituted the binding of the MIS12 and NDC80 outer kinetochore subcomplexes to CENP-C and CENP-T. Whereas CENP-C recruits a single MIS12:NDC80 complex, we show here that CENP-T binds one MIS12:NDC80 and two NDC80 complexes upon phosphorylation by the mitotic CDK1:Cyclin B complex at three distinct CENP-T sites. Visualization of reconstituted complexes by electron microscopy supports this model. Binding of CENP-C and CENP-T to MIS12 is competitive, and therefore CENP-C and CENP-T act in parallel to recruit two MIS12 and up to four NDC80 complexes. Our observations provide a molecular explanation for the stoichiometry of kinetochore components and its cell cycle regulation, and highlight how outer kinetochore modules bridge distances of well over 100 nm.