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Updated: Mar 9, 2026

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins
Published on: March 3, 2016
Molecular basis of outer kinetochore assembly on CENP-T
Pim J Huis In 't Veld1, Sadasivam Jeganathan1, Arsen Petrovic1
1Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
Abstract:
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.
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