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. 2010 Jun 8;107(23):10484-9.
doi: 10.1073/pnas.1002325107. Epub 2010 May 18.

A super-resolution map of the vertebrate kinetochore

Susana Abreu Ribeiro  1 , Paola Vagnarelli, Yimin Dong, Tetsuya Hori, Bruce F McEwen, Tatsuo Fukagawa, Cristina Flors, William C Earnshaw
Affiliations

A super-resolution map of the vertebrate kinetochore

Susana Abreu Ribeiro et al. Proc Natl Acad Sci U S A. .

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Mapping of pericentromeric chromatin. Localization of histone modifications and INCENP in pericentromeric region in SMC2ON (A) and SMC2OFF (B) metaphase cells expressing CENP-H-GFP: a, H3K4me2; b, H3T3ph; c, H3K9me3; and d, INCENP. (Scale bars in all figures: 5 μm, unless otherwise stated.)
Fig. 2.
Fig. 2.
Mitotic kinetochores are more stable than interphase prekinetochores when subjected to unfolding induced by TEEN buffer. (A) Experimental procedure for unfolding centromeric region of mitotic and interphase cells. Examples of interphase (B) and mitotic (C) fibers immunostained for H3K9me3 (Upper) and H3K4me2 (Lower). (D) Quantification of the percentage of the total length of the CENP-A–containing fiber occupied by H3K9me3 (yellow) and H3K4me2 (gray). (E) Distribution of CENP-A fiber lengths measured for interphase (red) and mitotic (black) cells. Gray area indicates fibers larger than 3 μm, used to define a separation between the two groups.
Fig. 3.
Fig. 3.
CENP-C is essential to confer extra stability to mitotic kinetochores. (A and B) Distribution of lengths of unfolded mitotic CENP-A chromatin fibers measured in the presence or absence of (A) CENP-N (orange) and CENP-H (blue), and (B) CENP-W (green) and condensin (pink). (C) Distribution of unfolded CENP-A chromatin fibers from mitotic CENP-CON and CENP-COFF cells. (D) In the absence of CENP-C (CENP-COFF), mitotic centromeres unravel to levels similar to those observed in interphase with CENP-CON.
Fig. 4.
Fig. 4.
Characterization of unfolded prekinetochores using fluorescence microscopy with subdiffraction spatial resolution. (A) Example of a 13.4-μm interphase fiber in which H3K9me3 blocks are clearly observed between CENP-A arrays. (B) Example of a 15.1-μm fiber in which H3K4me2 is also detected between CENP-A arrays, but with a more diffuse distribution. Dronpa-CENP-A is represented as green, H3K9me3 and H3K4me2 labeled with Alexa647 are represented as red. Yellow represents colocalization. Each dot corresponds to the localization of a single molecule switching event. (C) Frequency distribution histogram of stretched fiber widths measured from super-resolution reconstructed images of (C) Dronpa-labeled CENP-A and (C′) Alexa Fluor 647–labeled H3K9me3. The solid lines represent the best fit to bimodal Gaussian distributions centered at 46 and 67 nm for CENP-A (n = 53) and 40 and 57 nm for H3K9me3 (n = 30).
Fig. 5.
Fig. 5.
Localization of CCAN components in interphase CENP-A chromatin fibers. CENP-H (A) and CENP-C (B) in red colocalize with CENP-A (green) both in short (A, B) and more extended (A′, B′) fibers. (C) CENP-T (red) colocalizes with CENP-A (green) in short fibers (C); however, in more extended fibers (C′), CENP-T is interspersed between CENP-A domains.
Fig. 6.
Fig. 6.
Model showing proposed boustrophedon arrangement of kinetochore chromatin. (A) A single continuous chromatin segment is arranged in a sinusoidal wave in a series of layers linked at both ends to heterochromatin. (B) Top and side view as indicated in A. (C) CCAN protein distribution in the kinetochore. (D) The KMN network assembles in mitosis on top of the CCAN and may confer stability to the mitotic kinetochore by crosslinking the CENP-C either directly or indirectly (see text for details).
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