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Title: | A super-resolution map of the vertebrate kinetochore |
Authors: | Ribeiro, SA Vagnarelli, P Dong, Y Hori, T McEwen, BF Fukagawa, T Flors, C Earnshaw, WC |
Keywords: | CENP-A;Centromere;Super-resolution imaging;Chromosome;Boustrophedon |
Issue Date: | 2010 |
Publisher: | National Academy of Sciences |
Citation: | Proceedings of the National Academy of Sciences, 107(23), 10484 - 10489, 2010 |
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. |
Description: | This article is freely available online through the PNAS open access option. Copyright @ 2010 National Academy of Sciences. |
URI: | http://www.pnas.org/content/107/23/10484 http://bura.brunel.ac.uk/handle/2438/8940 |
DOI: | http://dx.doi.org/10.1073/pnas.1002325107 |
ISSN: | 1091-6490 |
Appears in Collections: | Biological Sciences Dept of Life Sciences Research Papers |
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