A super-resolution map of the vertebrate kinetochore

• DOI: 10.1073/pnas.1002325107
• Publication Date: 2010-05-19T02:02:14Z
• Authors: Susana Abreu Ribeiro; Paola Vagnarelli; Yimin Dong; Tetsuya Hori; Bruce F. McEwen; Tatsuo Fukagawa; Cristina Flors; William C. Earnshaw
• Keywords: 0301 basic medicine; 570; Protein Folding; 0303 health sciences; Chromosomal Proteins, Non-Histone; Centromere; Boustrophedon; boustrophedon; Mitosis; 540; Chromosome; Models, Biological; Cell Line; Super-resolution imaging; 03 medical and health sciences; centromere; /dk/atira/pure/subjectarea/asjc/1000; Animals; chromosome; General; Kinetochores; CENP-A; Chickens; super-resolution imaging

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.