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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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Localization of outer plate proteins in CENP-S– or CENP-X–deficient cells. (A) Immunofluorescence analysis in wild-type (WT) DT40 or CENP-S– or CENP-X–deficient cells with anti-Ndc80 and -Mis12 antibodies. Signal intensities at each kinetochore were measured in these cells. Error bars represent SD. (B) Immunofluorescence analysis with anti-Ndc80, -KNL1, or -Dsn1 antibodies in HeLa cells treated with control or CENP-X siRNAs. The numbers in the micrographs are relative signal intensities of kinetochore signals. (C) A model for the assembly of kinetochore proteins at mitosis. The CENP-S–CENP-X complex is essential for the stable assembly of outer kinetochore proteins. In addition, the CENP-S–CENP-X complex generates a discrete CCAN structure to prevent the kinetochore from overstretching (left). In CENP-S– and CENP-X–deficient cells, the amount of KNL1 and Ndc80 at kinetochores is reduced, and CCAN structure is less tight, which causes kinetochore to be overstretched (right). Bars, 10 µm.
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fig5: Localization of outer plate proteins in CENP-S– or CENP-X–deficient cells. (A) Immunofluorescence analysis in wild-type (WT) DT40 or CENP-S– or CENP-X–deficient cells with anti-Ndc80 and -Mis12 antibodies. Signal intensities at each kinetochore were measured in these cells. Error bars represent SD. (B) Immunofluorescence analysis with anti-Ndc80, -KNL1, or -Dsn1 antibodies in HeLa cells treated with control or CENP-X siRNAs. The numbers in the micrographs are relative signal intensities of kinetochore signals. (C) A model for the assembly of kinetochore proteins at mitosis. The CENP-S–CENP-X complex is essential for the stable assembly of outer kinetochore proteins. In addition, the CENP-S–CENP-X complex generates a discrete CCAN structure to prevent the kinetochore from overstretching (left). In CENP-S– and CENP-X–deficient cells, the amount of KNL1 and Ndc80 at kinetochores is reduced, and CCAN structure is less tight, which causes kinetochore to be overstretched (right). Bars, 10 µm.

Mentions: The EM analysis suggests that there are defects in the proper formation of a functional kinetochore outer plate in CENP-S–deficient cells. Therefore, we next analyzed the localization of outer kinetochore proteins in CENP-S– or CENP-X–deficient cells. The KMN network is located at the outer plate (DeLuca et al., 2005) and has a central role in forming kinetochore–microtubule attachments (Cheeseman et al., 2006). Although the KMN network subunits KNL1, Mis12, and Ndc80 all localized to kinetochores in CENP-S– and CENP-X–deficient DT40 cells, we found that the signal intensities of Ndc80 (∼80% relative to control; Fig. 5 A) and KNL1 (not depicted) were reduced in CENP-S– and CENP-X–deficient cells relative to controls. In contrast, we did not detect significant differences of Mis12 signals between the control and mutant cells.


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)

Localization of outer plate proteins in CENP-S– or CENP-X–deficient cells. (A) Immunofluorescence analysis in wild-type (WT) DT40 or CENP-S– or CENP-X–deficient cells with anti-Ndc80 and -Mis12 antibodies. Signal intensities at each kinetochore were measured in these cells. Error bars represent SD. (B) Immunofluorescence analysis with anti-Ndc80, -KNL1, or -Dsn1 antibodies in HeLa cells treated with control or CENP-X siRNAs. The numbers in the micrographs are relative signal intensities of kinetochore signals. (C) A model for the assembly of kinetochore proteins at mitosis. The CENP-S–CENP-X complex is essential for the stable assembly of outer kinetochore proteins. In addition, the CENP-S–CENP-X complex generates a discrete CCAN structure to prevent the kinetochore from overstretching (left). In CENP-S– and CENP-X–deficient cells, the amount of KNL1 and Ndc80 at kinetochores is reduced, and CCAN structure is less tight, which causes kinetochore to be overstretched (right). Bars, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig5: Localization of outer plate proteins in CENP-S– or CENP-X–deficient cells. (A) Immunofluorescence analysis in wild-type (WT) DT40 or CENP-S– or CENP-X–deficient cells with anti-Ndc80 and -Mis12 antibodies. Signal intensities at each kinetochore were measured in these cells. Error bars represent SD. (B) Immunofluorescence analysis with anti-Ndc80, -KNL1, or -Dsn1 antibodies in HeLa cells treated with control or CENP-X siRNAs. The numbers in the micrographs are relative signal intensities of kinetochore signals. (C) A model for the assembly of kinetochore proteins at mitosis. The CENP-S–CENP-X complex is essential for the stable assembly of outer kinetochore proteins. In addition, the CENP-S–CENP-X complex generates a discrete CCAN structure to prevent the kinetochore from overstretching (left). In CENP-S– and CENP-X–deficient cells, the amount of KNL1 and Ndc80 at kinetochores is reduced, and CCAN structure is less tight, which causes kinetochore to be overstretched (right). Bars, 10 µm.
Mentions: The EM analysis suggests that there are defects in the proper formation of a functional kinetochore outer plate in CENP-S–deficient cells. Therefore, we next analyzed the localization of outer kinetochore proteins in CENP-S– or CENP-X–deficient cells. The KMN network is located at the outer plate (DeLuca et al., 2005) and has a central role in forming kinetochore–microtubule attachments (Cheeseman et al., 2006). Although the KMN network subunits KNL1, Mis12, and Ndc80 all localized to kinetochores in CENP-S– and CENP-X–deficient DT40 cells, we found that the signal intensities of Ndc80 (∼80% relative to control; Fig. 5 A) and KNL1 (not depicted) were reduced in CENP-S– and CENP-X–deficient cells relative to controls. In contrast, we did not detect significant differences of Mis12 signals between the control and mutant cells.

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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