title: Molecular basis of outer kinetochore assembly on CENP-T.
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.
authors.affiliation: Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
authors.forename: Pim J
authors.identifier: 0000-0003-0234-6390
authors.initials: PJ
authors.lastname: Huis In 't Veld
authors.affiliation: Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
authors.forename: Sadasivam
authors.initials: S
authors.lastname: Jeganathan
authors.affiliation: Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
authors.forename: Arsen
authors.initials: A
authors.lastname: Petrovic
authors.affiliation: Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
authors.forename: Priyanka
authors.initials: P
authors.lastname: Singh
authors.affiliation: Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
authors.forename: Juliane
authors.initials: J
authors.lastname: John
authors.affiliation: Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
authors.forename: Veronica
authors.initials: V
authors.lastname: Krenn
authors.affiliation: Research Institute of Molecular Pathology (IMP), Vienna, Austria.|Vienna Biocenter (VBC), Vienna, Austria.
authors.forename: Florian
authors.initials: F
authors.lastname: Weissmann
authors.affiliation: Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
authors.forename: Tanja
authors.initials: T
authors.lastname: Bange
authors.affiliation: Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.|Centre for Medical Biotechnology, University Duisburg-Essen, Essen, Germany.|Faculty of Biology, University Duisburg-Essen, Essen, Germany.
authors.forename: Andrea
authors.identifier: 0000-0003-2362-8784
authors.initials: A
authors.lastname: Musacchio
mesh_terms: D016203:CDC2 Protein Kinase / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D019926:Cyclin B / Q000378:metabolism; D003598:Cytoskeletal Proteins; D018386:Kinetochores / Q000378:metabolism*; D046911:Macromolecular Substances / Q000648:ultrastructure; D008854:Microscopy, Electron; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational
chemical_list: C534001:CENPT protein, human; D002868:Chromosomal Proteins, Non-Histone; D019926:Cyclin B; D003598:Cytoskeletal Proteins; C492664:MIS12 protein, human; D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins; C120529:NDC80 protein, human; D009687:Nuclear Proteins; C060905:centromere protein C; D016203:CDC2 Protein Kinase
keywords: biochemistry; biophysics; centromere; human; kinetochore; mitosis; structural biology