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The CENP-S complex is essential for the stable assembly of outer kinetochore structure.

Amano M, Suzuki A, Hori T, Backer C, Okawa K, Cheeseman IM, Fukagawa T - J. Cell Biol. (2009)

Bottom Line: However, CENP-S- and CENP-X-deficient cells show a significant reduction in the size of the kinetochore outer plate.In addition, we found that intrakinetochore distance was increased in CENP-S- and CENP-X-deficient cells.These results suggest that the CENP-S complex is essential for the stable assembly of the outer kinetochore.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, National Institute of Genetics, The Graduate University for Advanced Studies, Mishima, Shizuoka 411-8540, Japan.

ABSTRACT
The constitutive centromere-associated network (CCAN) proteins are central to kinetochore assembly. To define the molecular architecture of this critical kinetochore network, we sought to determine the full complement of CCAN components and to define their relationships. This work identified a centromere protein S (CENP-S)-containing subcomplex that includes the new constitutive kinetochore protein CENP-X. Both CENP-S- and CENP-X-deficient chicken DT40 cells are viable but show abnormal mitotic behavior based on live cell analysis. Human HeLa cells depleted for CENP-X also showed mitotic errors. The kinetochore localization of CENP-S and -X is abolished in CENP-T- or CENP-K-deficient cells, but reciprocal experiments using CENP-S-deficient cells did not reveal defects in the localization of CCAN components. However, CENP-S- and CENP-X-deficient cells show a significant reduction in the size of the kinetochore outer plate. In addition, we found that intrakinetochore distance was increased in CENP-S- and CENP-X-deficient cells. These results suggest that the CENP-S complex is essential for the stable assembly of the outer kinetochore.

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The length of the outer plate and the distance of the interkinetochore in CENP-S–deficient cells. (A) Electron micrograph showing a typical image of the kinetochore outer plate in wild-type (1 and 1′) or CENP-S–deficient cells (2 and 2′). (B) Numbers of identifiable outer plates per cell (mean ± SD). Approximately 35 serial sections were made for each cell, and the numbers of outer plates were counted. (C) Distribution of the outer plate length in wild-type or CENP-S–deficient cells. In both cell lines, two peaks with distribution were observed. The two peaks in the distribution of the plate length are likely caused by differences in sample orientation. As the orientation of some cells is not plane of the section, the length of plates varies depending on the angle of the section against a chromosome. (D) Distance between outer plate and electron-dense chromatin region in wild-type or CENP-S–deficient cells. (E) Distances between Ndc80 and Ndc80 or CENP-T and CENP-T of control or CENP-S– or CENP-X–deficient metaphase cells in the absence of microtubules. Arrows indicate sister kinetochore pairs. (D and E) Error bars represent SD. Bars: (A) 200 nm; (E) 5 µm.
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fig4: The length of the outer plate and the distance of the interkinetochore in CENP-S–deficient cells. (A) Electron micrograph showing a typical image of the kinetochore outer plate in wild-type (1 and 1′) or CENP-S–deficient cells (2 and 2′). (B) Numbers of identifiable outer plates per cell (mean ± SD). Approximately 35 serial sections were made for each cell, and the numbers of outer plates were counted. (C) Distribution of the outer plate length in wild-type or CENP-S–deficient cells. In both cell lines, two peaks with distribution were observed. The two peaks in the distribution of the plate length are likely caused by differences in sample orientation. As the orientation of some cells is not plane of the section, the length of plates varies depending on the angle of the section against a chromosome. (D) Distance between outer plate and electron-dense chromatin region in wild-type or CENP-S–deficient cells. (E) Distances between Ndc80 and Ndc80 or CENP-T and CENP-T of control or CENP-S– or CENP-X–deficient metaphase cells in the absence of microtubules. Arrows indicate sister kinetochore pairs. (D and E) Error bars represent SD. Bars: (A) 200 nm; (E) 5 µm.

Mentions: Although we did not observe an apparent reduction in the kinetochore localization of established CCAN proteins in CENP-S– or CENP-X–deficient cells (Fig. 3), it is possible that CENP-S and -X play a role in controlling outer kinetochore assembly. To determine whether CENP-S or -X play a global role in controlling the assembly of non-CCAN proteins, we examined the structural morphology of the kinetochore outer plate by EM. For these experiments, we imaged 30–40 170-nm-thick serial sections for individual mitotic cells treated with nocodazole. In both CENP-S–deficient cells and wild-type DT40 cells, we observed similar numbers of clear electron-dense kinetochore outer plates (Fig. 4, A and B). We also observed a similar number (26.6 ± 8.8) of outer plates in CENP-50 (U)–deficient cells. In contrast, we observed that CENP-H deficiency causes a severe reduction in the number of visible outer plates (unpublished data), as previously shown for CENP-T deficiency (Hori et al., 2008a).


The CENP-S complex is essential for the stable assembly of outer kinetochore structure.

Amano M, Suzuki A, Hori T, Backer C, Okawa K, Cheeseman IM, Fukagawa T - J. Cell Biol. (2009)

The length of the outer plate and the distance of the interkinetochore in CENP-S–deficient cells. (A) Electron micrograph showing a typical image of the kinetochore outer plate in wild-type (1 and 1′) or CENP-S–deficient cells (2 and 2′). (B) Numbers of identifiable outer plates per cell (mean ± SD). Approximately 35 serial sections were made for each cell, and the numbers of outer plates were counted. (C) Distribution of the outer plate length in wild-type or CENP-S–deficient cells. In both cell lines, two peaks with distribution were observed. The two peaks in the distribution of the plate length are likely caused by differences in sample orientation. As the orientation of some cells is not plane of the section, the length of plates varies depending on the angle of the section against a chromosome. (D) Distance between outer plate and electron-dense chromatin region in wild-type or CENP-S–deficient cells. (E) Distances between Ndc80 and Ndc80 or CENP-T and CENP-T of control or CENP-S– or CENP-X–deficient metaphase cells in the absence of microtubules. Arrows indicate sister kinetochore pairs. (D and E) Error bars represent SD. Bars: (A) 200 nm; (E) 5 µm.
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Related In: Results  -  Collection

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fig4: The length of the outer plate and the distance of the interkinetochore in CENP-S–deficient cells. (A) Electron micrograph showing a typical image of the kinetochore outer plate in wild-type (1 and 1′) or CENP-S–deficient cells (2 and 2′). (B) Numbers of identifiable outer plates per cell (mean ± SD). Approximately 35 serial sections were made for each cell, and the numbers of outer plates were counted. (C) Distribution of the outer plate length in wild-type or CENP-S–deficient cells. In both cell lines, two peaks with distribution were observed. The two peaks in the distribution of the plate length are likely caused by differences in sample orientation. As the orientation of some cells is not plane of the section, the length of plates varies depending on the angle of the section against a chromosome. (D) Distance between outer plate and electron-dense chromatin region in wild-type or CENP-S–deficient cells. (E) Distances between Ndc80 and Ndc80 or CENP-T and CENP-T of control or CENP-S– or CENP-X–deficient metaphase cells in the absence of microtubules. Arrows indicate sister kinetochore pairs. (D and E) Error bars represent SD. Bars: (A) 200 nm; (E) 5 µm.
Mentions: Although we did not observe an apparent reduction in the kinetochore localization of established CCAN proteins in CENP-S– or CENP-X–deficient cells (Fig. 3), it is possible that CENP-S and -X play a role in controlling outer kinetochore assembly. To determine whether CENP-S or -X play a global role in controlling the assembly of non-CCAN proteins, we examined the structural morphology of the kinetochore outer plate by EM. For these experiments, we imaged 30–40 170-nm-thick serial sections for individual mitotic cells treated with nocodazole. In both CENP-S–deficient cells and wild-type DT40 cells, we observed similar numbers of clear electron-dense kinetochore outer plates (Fig. 4, A and B). We also observed a similar number (26.6 ± 8.8) of outer plates in CENP-50 (U)–deficient cells. In contrast, we observed that CENP-H deficiency causes a severe reduction in the number of visible outer plates (unpublished data), as previously shown for CENP-T deficiency (Hori et al., 2008a).

Bottom Line: However, CENP-S- and CENP-X-deficient cells show a significant reduction in the size of the kinetochore outer plate.In addition, we found that intrakinetochore distance was increased in CENP-S- and CENP-X-deficient cells.These results suggest that the CENP-S complex is essential for the stable assembly of the outer kinetochore.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, National Institute of Genetics, The Graduate University for Advanced Studies, Mishima, Shizuoka 411-8540, Japan.

ABSTRACT
The constitutive centromere-associated network (CCAN) proteins are central to kinetochore assembly. To define the molecular architecture of this critical kinetochore network, we sought to determine the full complement of CCAN components and to define their relationships. This work identified a centromere protein S (CENP-S)-containing subcomplex that includes the new constitutive kinetochore protein CENP-X. Both CENP-S- and CENP-X-deficient chicken DT40 cells are viable but show abnormal mitotic behavior based on live cell analysis. Human HeLa cells depleted for CENP-X also showed mitotic errors. The kinetochore localization of CENP-S and -X is abolished in CENP-T- or CENP-K-deficient cells, but reciprocal experiments using CENP-S-deficient cells did not reveal defects in the localization of CCAN components. However, CENP-S- and CENP-X-deficient cells show a significant reduction in the size of the kinetochore outer plate. In addition, we found that intrakinetochore distance was increased in CENP-S- and CENP-X-deficient cells. These results suggest that the CENP-S complex is essential for the stable assembly of the outer kinetochore.

Show MeSH
Related in: MedlinePlus