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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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hMad2 localization after RNAi against hMis12, CENP-A, and hMis6. (A) hMad2 localization in wild-type HeLa cells. Punctate signals observed in prometaphase disappeared after metaphase. (B and C) hMad2 localization after hMis12 (B) or CENP-A RNAi (C). Dots were observed in most of the misaligned chromosomes (arrows), but invisible on the lagging chromosomes (arrowheads). (D) hMad2 localization after hMis6 RNAi. Dots were visible on the condensed chromosomes in many cases. See text for details. (E) Hoechst 33342 staining of the cells after nocodazole treatment. Nocodazole treatment caused mitotic arrest after hMis12 (85 h) or CENP-A (68 h) RNAi. Bars, 10 μm.
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fig7: hMad2 localization after RNAi against hMis12, CENP-A, and hMis6. (A) hMad2 localization in wild-type HeLa cells. Punctate signals observed in prometaphase disappeared after metaphase. (B and C) hMad2 localization after hMis12 (B) or CENP-A RNAi (C). Dots were observed in most of the misaligned chromosomes (arrows), but invisible on the lagging chromosomes (arrowheads). (D) hMad2 localization after hMis6 RNAi. Dots were visible on the condensed chromosomes in many cases. See text for details. (E) Hoechst 33342 staining of the cells after nocodazole treatment. Nocodazole treatment caused mitotic arrest after hMis12 (85 h) or CENP-A (68 h) RNAi. Bars, 10 μm.

Mentions: The spindle checkpoint monitors the proper kinetochore–spindle attachment and controls the trigger of metaphase–anaphase transition (for review see Zhou et al., 2002). The localization of hMad2, a conserved spindle checkpoint protein, can be a marker of checkpoint activation, as it is localized at unattached kinetochores and transmits signals that prevent metaphase–anaphase transition (for review see Shah and Cleveland, 2000). In control HeLa cells without siRNA addition, dozens of intense hMad2 punctate signals were observed at kinetochore regions by immunofluorescence during prophase and prometaphase, whereas several or no dots were in metaphase (Fig. 7 A; Chen et al., 1996; Li and Benezra, 1996). No punctate signals were visible on chromosomes after anaphase. We then observed hMad2 localization after RNAi against kinetochore proteins. The localization pattern after the RNAi of hMis12 or CENP-A was similar to control cells: intense kinetochore dots in prophase and prometaphase and several dots on the metaphase-like chromosomes (Fig. 7, B and C). The punctate signals were observed in 73% of the cells with orphan chromosomes (arrows; n = 25), whereas they were not observed after anaphase, even on the lagging chromatids (arrowheads). In the case of hMis6 RNAi, 32% of the mitotic cells had dozens of punctate signals, indicating the prolonged hMad2 localization at kinetochores (Fig. 7 D). It is suggested that the mitotic delay was caused by spindle checkpoint activation. The reason why the remaining 68% of the prometaphase-like hMis6-knockdown cells had no punctate hMad2 signals is unclear.


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)

hMad2 localization after RNAi against hMis12, CENP-A, and hMis6. (A) hMad2 localization in wild-type HeLa cells. Punctate signals observed in prometaphase disappeared after metaphase. (B and C) hMad2 localization after hMis12 (B) or CENP-A RNAi (C). Dots were observed in most of the misaligned chromosomes (arrows), but invisible on the lagging chromosomes (arrowheads). (D) hMad2 localization after hMis6 RNAi. Dots were visible on the condensed chromosomes in many cases. See text for details. (E) Hoechst 33342 staining of the cells after nocodazole treatment. Nocodazole treatment caused mitotic arrest after hMis12 (85 h) or CENP-A (68 h) RNAi. Bars, 10 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172742&req=5

fig7: hMad2 localization after RNAi against hMis12, CENP-A, and hMis6. (A) hMad2 localization in wild-type HeLa cells. Punctate signals observed in prometaphase disappeared after metaphase. (B and C) hMad2 localization after hMis12 (B) or CENP-A RNAi (C). Dots were observed in most of the misaligned chromosomes (arrows), but invisible on the lagging chromosomes (arrowheads). (D) hMad2 localization after hMis6 RNAi. Dots were visible on the condensed chromosomes in many cases. See text for details. (E) Hoechst 33342 staining of the cells after nocodazole treatment. Nocodazole treatment caused mitotic arrest after hMis12 (85 h) or CENP-A (68 h) RNAi. Bars, 10 μm.
Mentions: The spindle checkpoint monitors the proper kinetochore–spindle attachment and controls the trigger of metaphase–anaphase transition (for review see Zhou et al., 2002). The localization of hMad2, a conserved spindle checkpoint protein, can be a marker of checkpoint activation, as it is localized at unattached kinetochores and transmits signals that prevent metaphase–anaphase transition (for review see Shah and Cleveland, 2000). In control HeLa cells without siRNA addition, dozens of intense hMad2 punctate signals were observed at kinetochore regions by immunofluorescence during prophase and prometaphase, whereas several or no dots were in metaphase (Fig. 7 A; Chen et al., 1996; Li and Benezra, 1996). No punctate signals were visible on chromosomes after anaphase. We then observed hMad2 localization after RNAi against kinetochore proteins. The localization pattern after the RNAi of hMis12 or CENP-A was similar to control cells: intense kinetochore dots in prophase and prometaphase and several dots on the metaphase-like chromosomes (Fig. 7, B and C). The punctate signals were observed in 73% of the cells with orphan chromosomes (arrows; n = 25), whereas they were not observed after anaphase, even on the lagging chromatids (arrowheads). In the case of hMis6 RNAi, 32% of the mitotic cells had dozens of punctate signals, indicating the prolonged hMad2 localization at kinetochores (Fig. 7 D). It is suggested that the mitotic delay was caused by spindle checkpoint activation. The reason why the remaining 68% of the prometaphase-like hMis6-knockdown cells had no punctate hMad2 signals is unclear.

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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