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Human centromere chromatin protein hMis12, essential for equal segregation, is independent of CENP-A loading pathway.

Goshima G, Kiyomitsu T, Yoda K, Yanagida M - J. Cell Biol. (2003)

Bottom Line: RNA interference (RNAi) analysis of HeLa cells shows that the reduced hMis12 results in misaligned metaphase chromosomes, lagging anaphase chromosomes, and interphase micronuclei without mitotic delay, while CENP-A is located at kinetochores.RNAi for hMis6, like that of a kinetochore kinesin CENP-E, induces mitotic arrest.Kinetochore localization of hMis12 is unaffected by CENP-A RNAi, demonstrating an independent pathway of CENP-A in human kinetochores.

View Article: PubMed Central - PubMed

Affiliation: COE Research Project, Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan.

ABSTRACT
Kinetochores are the chromosomal sites for spindle interaction and play a vital role for chromosome segregation. The composition of kinetochore proteins and their cellular roles are, however, poorly understood in higher eukaryotes. We identified a novel kinetochore protein family conserved from yeast to human that is essential for equal chromosome segregation. The human homologue hMis12 of yeast spMis12/scMtw1 retains conserved sequence features and locates at the kinetochore region indistinguishable from CENP-A, a centromeric histone variant. RNA interference (RNAi) analysis of HeLa cells shows that the reduced hMis12 results in misaligned metaphase chromosomes, lagging anaphase chromosomes, and interphase micronuclei without mitotic delay, while CENP-A is located at kinetochores. Further, the metaphase spindle length is abnormally extended. Spindle checkpoint protein hMad2 temporally localizes at kinetochores at early mitotic stages after RNAi. The RNAi deficiency of CENP-A leads to a similar mitotic phenotype, but the kinetochore signals of other kinetochore proteins, hMis6 and CENP-C, are greatly diminished. RNAi for hMis6, like that of a kinetochore kinesin CENP-E, induces mitotic arrest. Kinetochore localization of hMis12 is unaffected by CENP-A RNAi, demonstrating an independent pathway of CENP-A in human kinetochores.

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hMis12 is localized at centromere regions throughout the cell cycle. Paraformaldehyde-fixed HeLa cells were stained by anti-hMis12 and anti–CENP-A antibodies. DNA was stained by Hoechst 33342. The merged images were produced by hMis12 (green), CENP-A (red), and DNA (blue). hMis12 and CENP-A dots show identical localization patterns throughout the cell cycle, though hMis12 also showed dispersed nuclear signals in interphase. Both hMis12 and CENP-A signals in telophase were rather weak, the reason of which remains unclear. I; interphase, Pro; prophase, Prometa; prometaphase, Meta; metaphase, Ana; anaphase, Telo; telophase. Bar, 10 μm.
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fig2: hMis12 is localized at centromere regions throughout the cell cycle. Paraformaldehyde-fixed HeLa cells were stained by anti-hMis12 and anti–CENP-A antibodies. DNA was stained by Hoechst 33342. The merged images were produced by hMis12 (green), CENP-A (red), and DNA (blue). hMis12 and CENP-A dots show identical localization patterns throughout the cell cycle, though hMis12 also showed dispersed nuclear signals in interphase. Both hMis12 and CENP-A signals in telophase were rather weak, the reason of which remains unclear. I; interphase, Pro; prophase, Prometa; prometaphase, Meta; metaphase, Ana; anaphase, Telo; telophase. Bar, 10 μm.

Mentions: To determine whether the signals corresponded to the kinetochores, immunolocalization experiments were done using antibodies against human CENP-A, a variant of histone H3 known to be specifically located at the centromere regions throughout the cell cycle (Palmer et al., 1987; Sullivan et al., 1994; Warburton et al., 1997). As seen in Fig. 2, the punctate signals derived from anti-hMis12 and anti–CENP-A antibodies were identical throughout various mitotic stages. The punctate signals of hMis12 were present on the condensed chromosomes during prophase and prometaphase, congressed at metaphase, and split into two sets during anaphase. This behavior is typical for kinetochore proteins. In interphase, whole chromatin regions were stained by anti-hMis12 antibodies, and, among them, several brighter punctate signals colocalized with CENP-A were visible. Reduction of the cellular hMis12 protein level by the RNAi method described below resulted in the disappearance of both punctate and dispersed nuclear signals, suggesting that both signals were indeed derived from hMis12 protein.


Human centromere chromatin protein hMis12, essential for equal segregation, is independent of CENP-A loading pathway.

Goshima G, Kiyomitsu T, Yoda K, Yanagida M - J. Cell Biol. (2003)

hMis12 is localized at centromere regions throughout the cell cycle. Paraformaldehyde-fixed HeLa cells were stained by anti-hMis12 and anti–CENP-A antibodies. DNA was stained by Hoechst 33342. The merged images were produced by hMis12 (green), CENP-A (red), and DNA (blue). hMis12 and CENP-A dots show identical localization patterns throughout the cell cycle, though hMis12 also showed dispersed nuclear signals in interphase. Both hMis12 and CENP-A signals in telophase were rather weak, the reason of which remains unclear. I; interphase, Pro; prophase, Prometa; prometaphase, Meta; metaphase, Ana; anaphase, Telo; telophase. Bar, 10 μm.
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Related In: Results  -  Collection

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fig2: hMis12 is localized at centromere regions throughout the cell cycle. Paraformaldehyde-fixed HeLa cells were stained by anti-hMis12 and anti–CENP-A antibodies. DNA was stained by Hoechst 33342. The merged images were produced by hMis12 (green), CENP-A (red), and DNA (blue). hMis12 and CENP-A dots show identical localization patterns throughout the cell cycle, though hMis12 also showed dispersed nuclear signals in interphase. Both hMis12 and CENP-A signals in telophase were rather weak, the reason of which remains unclear. I; interphase, Pro; prophase, Prometa; prometaphase, Meta; metaphase, Ana; anaphase, Telo; telophase. Bar, 10 μm.
Mentions: To determine whether the signals corresponded to the kinetochores, immunolocalization experiments were done using antibodies against human CENP-A, a variant of histone H3 known to be specifically located at the centromere regions throughout the cell cycle (Palmer et al., 1987; Sullivan et al., 1994; Warburton et al., 1997). As seen in Fig. 2, the punctate signals derived from anti-hMis12 and anti–CENP-A antibodies were identical throughout various mitotic stages. The punctate signals of hMis12 were present on the condensed chromosomes during prophase and prometaphase, congressed at metaphase, and split into two sets during anaphase. This behavior is typical for kinetochore proteins. In interphase, whole chromatin regions were stained by anti-hMis12 antibodies, and, among them, several brighter punctate signals colocalized with CENP-A were visible. Reduction of the cellular hMis12 protein level by the RNAi method described below resulted in the disappearance of both punctate and dispersed nuclear signals, suggesting that both signals were indeed derived from hMis12 protein.

Bottom Line: RNA interference (RNAi) analysis of HeLa cells shows that the reduced hMis12 results in misaligned metaphase chromosomes, lagging anaphase chromosomes, and interphase micronuclei without mitotic delay, while CENP-A is located at kinetochores.RNAi for hMis6, like that of a kinetochore kinesin CENP-E, induces mitotic arrest.Kinetochore localization of hMis12 is unaffected by CENP-A RNAi, demonstrating an independent pathway of CENP-A in human kinetochores.

View Article: PubMed Central - PubMed

Affiliation: COE Research Project, Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan.

ABSTRACT
Kinetochores are the chromosomal sites for spindle interaction and play a vital role for chromosome segregation. The composition of kinetochore proteins and their cellular roles are, however, poorly understood in higher eukaryotes. We identified a novel kinetochore protein family conserved from yeast to human that is essential for equal chromosome segregation. The human homologue hMis12 of yeast spMis12/scMtw1 retains conserved sequence features and locates at the kinetochore region indistinguishable from CENP-A, a centromeric histone variant. RNA interference (RNAi) analysis of HeLa cells shows that the reduced hMis12 results in misaligned metaphase chromosomes, lagging anaphase chromosomes, and interphase micronuclei without mitotic delay, while CENP-A is located at kinetochores. Further, the metaphase spindle length is abnormally extended. Spindle checkpoint protein hMad2 temporally localizes at kinetochores at early mitotic stages after RNAi. The RNAi deficiency of CENP-A leads to a similar mitotic phenotype, but the kinetochore signals of other kinetochore proteins, hMis6 and CENP-C, are greatly diminished. RNAi for hMis6, like that of a kinetochore kinesin CENP-E, induces mitotic arrest. Kinetochore localization of hMis12 is unaffected by CENP-A RNAi, demonstrating an independent pathway of CENP-A in human kinetochores.

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