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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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Both CENP-S– and CENP-X–deficient DT40 cells are viable but show defects in mitotic progression. (A, left) Dynamics of chromosomes in wild-type (WT) or CENP-S–deficient cells visualized by time-lapse observation of living cells. Selected images of chromosomes from prophase to anaphase in these cells are shown. (right) Quantification of the time for progression from prophase to anaphase in wild-type and CENP-S–deficient cells as determined by time-lapse microscopy of living cells. (B) Chromosome morphology and α-tubulin staining (green) in human HeLa cells after siRNA-based knockdown for CENP-X. Human anticentromere antibodies (ACA) were used to detect the position of centromeres (red), and DNA (blue) was stained with Hoechst. Bars, 10 µm.
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fig2: Both CENP-S– and CENP-X–deficient DT40 cells are viable but show defects in mitotic progression. (A, left) Dynamics of chromosomes in wild-type (WT) or CENP-S–deficient cells visualized by time-lapse observation of living cells. Selected images of chromosomes from prophase to anaphase in these cells are shown. (right) Quantification of the time for progression from prophase to anaphase in wild-type and CENP-S–deficient cells as determined by time-lapse microscopy of living cells. (B) Chromosome morphology and α-tubulin staining (green) in human HeLa cells after siRNA-based knockdown for CENP-X. Human anticentromere antibodies (ACA) were used to detect the position of centromeres (red), and DNA (blue) was stained with Hoechst. Bars, 10 µm.

Mentions: To examine the presence of any errors in mitosis, we visualized the behavior of individual CENP-S–deficient cells expressing histone H2B-RFP. Time-lapse imaging of wild-type and CENP-S–deficient cells (Fig. 2 A) indicated the presence of some mitotic defects in the absence of CENP-S. Although most CENP-S–deficient cells progressed through mitosis, these cells took a mean of 25.1 ± 6.4 min (±SD; n = 42) to progress from prophase to anaphase, which is significantly longer than the time observed in wild-type cells (19.3 ± 4.3 min; n = 36; P = 0.000012). In addition, we observed that >30% of CENP-S–deficient cells displayed anaphase bridging of chromosomes (Fig. 2 A, CENP-S KO 1–3). These results indicate that CENP-S is important for proper mitotic progression and chromosome segregation in DT40 cells even though it is dispensable for viability in a population.


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)

Both CENP-S– and CENP-X–deficient DT40 cells are viable but show defects in mitotic progression. (A, left) Dynamics of chromosomes in wild-type (WT) or CENP-S–deficient cells visualized by time-lapse observation of living cells. Selected images of chromosomes from prophase to anaphase in these cells are shown. (right) Quantification of the time for progression from prophase to anaphase in wild-type and CENP-S–deficient cells as determined by time-lapse microscopy of living cells. (B) Chromosome morphology and α-tubulin staining (green) in human HeLa cells after siRNA-based knockdown for CENP-X. Human anticentromere antibodies (ACA) were used to detect the position of centromeres (red), and DNA (blue) was stained with Hoechst. Bars, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2717651&req=5

fig2: Both CENP-S– and CENP-X–deficient DT40 cells are viable but show defects in mitotic progression. (A, left) Dynamics of chromosomes in wild-type (WT) or CENP-S–deficient cells visualized by time-lapse observation of living cells. Selected images of chromosomes from prophase to anaphase in these cells are shown. (right) Quantification of the time for progression from prophase to anaphase in wild-type and CENP-S–deficient cells as determined by time-lapse microscopy of living cells. (B) Chromosome morphology and α-tubulin staining (green) in human HeLa cells after siRNA-based knockdown for CENP-X. Human anticentromere antibodies (ACA) were used to detect the position of centromeres (red), and DNA (blue) was stained with Hoechst. Bars, 10 µm.
Mentions: To examine the presence of any errors in mitosis, we visualized the behavior of individual CENP-S–deficient cells expressing histone H2B-RFP. Time-lapse imaging of wild-type and CENP-S–deficient cells (Fig. 2 A) indicated the presence of some mitotic defects in the absence of CENP-S. Although most CENP-S–deficient cells progressed through mitosis, these cells took a mean of 25.1 ± 6.4 min (±SD; n = 42) to progress from prophase to anaphase, which is significantly longer than the time observed in wild-type cells (19.3 ± 4.3 min; n = 36; P = 0.000012). In addition, we observed that >30% of CENP-S–deficient cells displayed anaphase bridging of chromosomes (Fig. 2 A, CENP-S KO 1–3). These results indicate that CENP-S is important for proper mitotic progression and chromosome segregation in DT40 cells even though it is dispensable for viability in a population.

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