title: The architecture of CCAN proteins creates a structural integrity to resist spindle forces and achieve proper Intrakinetochore stretch.
abstract: Constitutive centromere-associated network (CCAN) proteins, particularly CENP-C, CENP-T, and the CENP-H/-I complex, mechanically link CENP-A-containing centromeric chromatin within the inner kinetochore to outer kinetochore proteins, such as the Ndc80 complex, that bind kinetochore microtubules. Accuracy of chromosome segregation depends critically upon Aurora B phosphorylation of Ndc80/Hec1. To determine how CCAN protein architecture mechanically constrains intrakinetochore stretch between CENP-A and Ndc80/Hec1 for proper Ndc80/Hec1 phosphorylation, we used super-resolution fluorescence microscopy and selective protein depletion. We found that at bi-oriented chromosomes in late prometaphase cells, CENP-T is stretched ∼16 nm to the inner end of Ndc80/Hec1, much less than expected for full-length CENP-T. Depletion of various CCAN linker proteins induced hyper-intrakinetochore stretch (an additional 20-60 nm) with corresponding significant decreases in Aurora B phosphorylation of Ndc80/Hec1. Thus, proper intrakinetochore stretch is required for normal kinetochore function and depends critically on all the CCAN mechanical linkers to the Ndc80 complex.
authors.affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address: suzukia@email.unc.edu.
authors.forename: Aussie
authors.initials: A
authors.lastname: Suzuki
authors.affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
authors.forename: Benjamin L
authors.initials: BL
authors.lastname: Badger
authors.affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
authors.forename: Xiaohu
authors.initials: X
authors.lastname: Wan
authors.affiliation: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: Jennifer G
authors.initials: JG
authors.lastname: DeLuca
authors.affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address: tsalmon@email.unc.edu.
authors.forename: Edward D
authors.initials: ED
authors.lastname: Salmon
mesh_terms: D064107:Aurora Kinase B / Q000378:metabolism; D001324:Autoantigens / Q000378:metabolism*; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D008941:Spindle Apparatus / Q000378:metabolism*
chemical_list: D001324:Autoantigens; C000619700:CENPA protein, human; C534001:CENPT protein, human; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone; D003598:Cytoskeletal Proteins; C120529:NDC80 protein, human; D009687:Nuclear Proteins; D064107:Aurora Kinase B