title: A super-resolution map of the vertebrate kinetochore.
abstract: A longstanding question in centromere biology has been the organization of CENP-A-containing chromatin and its implications for kinetochore assembly. Here, we have combined genetic manipulations with deconvolution and super-resolution fluorescence microscopy for a detailed structural analysis of chicken kinetochores. Using fluorescence microscopy with subdiffraction spatial resolution and single molecule sensitivity to map protein localization in kinetochore chromatin unfolded by exposure to a low salt buffer, we observed robust amounts of H3K9me3, but only low levels of H3K4me2, between CENP-A subdomains in unfolded interphase prekinetochores. Constitutive centromere-associated network proteins CENP-C and CENP-H localize within CENP-A-rich subdomains (presumably on H3-containing nucleosomes) whereas CENP-T localizes in interspersed H3-rich blocks. Although interphase prekinetochores are relatively more resistant to unfolding than sur-rounding pericentromeric heterochromatin, mitotic kinetochores are significantly more stable, reflecting mitotic kinetochore maturation. Loss of CENP-H, CENP-N, or CENP-W had little or no effect on the unfolding of mitotic kinetochores. However, loss of CENP-C caused mitotic kinetochores to unfold to the same extent as their interphase counterparts. Based on our results we propose a new model for inner centromeric chromatin architecture in which chromatin is folded as a layered boustrophedon, with planar sinusoids containing interspersed CENP-A-rich and H3-rich subdomains oriented toward the outer kinetochore. In mitosis, a CENP-C-dependent mechanism crosslinks CENP-A blocks of different layers together, conferring extra stability to the kinetochore.
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, UK.
authors.forename: Susana Abreu
authors.initials: SA
authors.lastname: Ribeiro
authors.forename: Paola
authors.initials: P
authors.lastname: Vagnarelli
authors.forename: Yimin
authors.initials: Y
authors.lastname: Dong
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Hori
authors.forename: Bruce F
authors.initials: BF
authors.lastname: McEwen
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Fukagawa
authors.forename: Cristina
authors.initials: C
authors.lastname: Flors
authors.forename: William C
authors.initials: WC
authors.lastname: Earnshaw
mesh_terms: D000818:Animals; D002460:Cell Line; D002645:Chickens / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism; D018386:Kinetochores; D008938:Mitosis; D008954:Models, Biological; D017510:Protein Folding
chemical_list: D002868:Chromosomal Proteins, Non-Histone