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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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hMis6 RNAi leads to accumulation of abnormal mitotic cells. (A) Immunoblotting of hMis6 in HeLa cell extracts after 48 h. Anti-PSTAIR was used as a loading control. The level of hMis6 was greatly reduced after incubation with siRNA (right). No RNAi shown at left. (B) HeLa cells transfected by siRNA for hMis6 (top and middle rows) or by control buffer only (bottom row) were fixed at 48 h and stained by Hoechst 33342, anti-hMis6, and anti-hMis12 antibodies. Centromere signals of hMis6, but not of hMis12, disappeared by RNAi. Misaligned chromosomes were seen by Hoechst staining. (C) Costaining by anti–CENP-A and anti–CENP-C antibodies. Centromere signals of CENP-C remained by hMis6 RNAi. (D) Costaining by anti-hMis12 and anti–CENP-H antibodies. Centromere signals of CENP-H disappeared by hMis6 RNAi. (E) Accumulation of mitotic cells after hMis6 RNAi (red column) in hMis6-depleted cells. Interphase (blue) was abundant in cells not treated by RNAi. (F) Mitotically arrested cells became abundant when HeLa cells were hMis6 depleted. Cells were fixed and stained by Hoechst 33342 and anti-hMis6 antibodies. (G) Immunostaining of kinetochore marker (CENP-C) and tubulin in hMis6-knockdown cells. Cells were fixed 48 h after siRNA transfection. Cells with misaligned chromosomes and expanded bipolar or tripolar spindles (arrows indicate poles) were observed. Bars, 10 μm.
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fig6: hMis6 RNAi leads to accumulation of abnormal mitotic cells. (A) Immunoblotting of hMis6 in HeLa cell extracts after 48 h. Anti-PSTAIR was used as a loading control. The level of hMis6 was greatly reduced after incubation with siRNA (right). No RNAi shown at left. (B) HeLa cells transfected by siRNA for hMis6 (top and middle rows) or by control buffer only (bottom row) were fixed at 48 h and stained by Hoechst 33342, anti-hMis6, and anti-hMis12 antibodies. Centromere signals of hMis6, but not of hMis12, disappeared by RNAi. Misaligned chromosomes were seen by Hoechst staining. (C) Costaining by anti–CENP-A and anti–CENP-C antibodies. Centromere signals of CENP-C remained by hMis6 RNAi. (D) Costaining by anti-hMis12 and anti–CENP-H antibodies. Centromere signals of CENP-H disappeared by hMis6 RNAi. (E) Accumulation of mitotic cells after hMis6 RNAi (red column) in hMis6-depleted cells. Interphase (blue) was abundant in cells not treated by RNAi. (F) Mitotically arrested cells became abundant when HeLa cells were hMis6 depleted. Cells were fixed and stained by Hoechst 33342 and anti-hMis6 antibodies. (G) Immunostaining of kinetochore marker (CENP-C) and tubulin in hMis6-knockdown cells. Cells were fixed 48 h after siRNA transfection. Cells with misaligned chromosomes and expanded bipolar or tripolar spindles (arrows indicate poles) were observed. Bars, 10 μm.

Mentions: We next analyzed the RNAi phenotype of hMis6 in HeLa cells. This is the first functional analysis of mammalian Mis6, though chicken CENP-I gene disruption analysis was recently reported (Nishihashi et al., 2002). Immunoblot against hMis6 showed that hMis6 protein greatly reduced 48 h after siRNA transfection (Fig. 6 A). Consistently, no kinetochore localization of hMis6 was detected in the transfected cells by immunostaining at this time point (Fig. 6 B). The signal distributed throughout the cell was due to the cross-reaction of anti-hMis6 antibodies, so they did not disappear after RNAi. The localization of other kinetochore proteins was determined by immunostaining after hMis6 RNAi transfection (48 h). As shown in Fig. 6 (B and C), punctate localization of hMis12, CENP-A, and CENP-C was clearly visible after hMis6 depletion, indicating that kinetochore localization of these proteins was unaffected by the reduction of hMis6. Identical results were obtained also in interphase cells. Kinetochore localization of CENP-H, however, was not detected at all in the hMis6 RNAi cells (Fig. 6 D). This CENP-H localization result was a positive control for the RNAi experiment.


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)

hMis6 RNAi leads to accumulation of abnormal mitotic cells. (A) Immunoblotting of hMis6 in HeLa cell extracts after 48 h. Anti-PSTAIR was used as a loading control. The level of hMis6 was greatly reduced after incubation with siRNA (right). No RNAi shown at left. (B) HeLa cells transfected by siRNA for hMis6 (top and middle rows) or by control buffer only (bottom row) were fixed at 48 h and stained by Hoechst 33342, anti-hMis6, and anti-hMis12 antibodies. Centromere signals of hMis6, but not of hMis12, disappeared by RNAi. Misaligned chromosomes were seen by Hoechst staining. (C) Costaining by anti–CENP-A and anti–CENP-C antibodies. Centromere signals of CENP-C remained by hMis6 RNAi. (D) Costaining by anti-hMis12 and anti–CENP-H antibodies. Centromere signals of CENP-H disappeared by hMis6 RNAi. (E) Accumulation of mitotic cells after hMis6 RNAi (red column) in hMis6-depleted cells. Interphase (blue) was abundant in cells not treated by RNAi. (F) Mitotically arrested cells became abundant when HeLa cells were hMis6 depleted. Cells were fixed and stained by Hoechst 33342 and anti-hMis6 antibodies. (G) Immunostaining of kinetochore marker (CENP-C) and tubulin in hMis6-knockdown cells. Cells were fixed 48 h after siRNA transfection. Cells with misaligned chromosomes and expanded bipolar or tripolar spindles (arrows indicate poles) were observed. Bars, 10 μm.
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fig6: hMis6 RNAi leads to accumulation of abnormal mitotic cells. (A) Immunoblotting of hMis6 in HeLa cell extracts after 48 h. Anti-PSTAIR was used as a loading control. The level of hMis6 was greatly reduced after incubation with siRNA (right). No RNAi shown at left. (B) HeLa cells transfected by siRNA for hMis6 (top and middle rows) or by control buffer only (bottom row) were fixed at 48 h and stained by Hoechst 33342, anti-hMis6, and anti-hMis12 antibodies. Centromere signals of hMis6, but not of hMis12, disappeared by RNAi. Misaligned chromosomes were seen by Hoechst staining. (C) Costaining by anti–CENP-A and anti–CENP-C antibodies. Centromere signals of CENP-C remained by hMis6 RNAi. (D) Costaining by anti-hMis12 and anti–CENP-H antibodies. Centromere signals of CENP-H disappeared by hMis6 RNAi. (E) Accumulation of mitotic cells after hMis6 RNAi (red column) in hMis6-depleted cells. Interphase (blue) was abundant in cells not treated by RNAi. (F) Mitotically arrested cells became abundant when HeLa cells were hMis6 depleted. Cells were fixed and stained by Hoechst 33342 and anti-hMis6 antibodies. (G) Immunostaining of kinetochore marker (CENP-C) and tubulin in hMis6-knockdown cells. Cells were fixed 48 h after siRNA transfection. Cells with misaligned chromosomes and expanded bipolar or tripolar spindles (arrows indicate poles) were observed. Bars, 10 μm.
Mentions: We next analyzed the RNAi phenotype of hMis6 in HeLa cells. This is the first functional analysis of mammalian Mis6, though chicken CENP-I gene disruption analysis was recently reported (Nishihashi et al., 2002). Immunoblot against hMis6 showed that hMis6 protein greatly reduced 48 h after siRNA transfection (Fig. 6 A). Consistently, no kinetochore localization of hMis6 was detected in the transfected cells by immunostaining at this time point (Fig. 6 B). The signal distributed throughout the cell was due to the cross-reaction of anti-hMis6 antibodies, so they did not disappear after RNAi. The localization of other kinetochore proteins was determined by immunostaining after hMis6 RNAi transfection (48 h). As shown in Fig. 6 (B and C), punctate localization of hMis12, CENP-A, and CENP-C was clearly visible after hMis6 depletion, indicating that kinetochore localization of these proteins was unaffected by the reduction of hMis6. Identical results were obtained also in interphase cells. Kinetochore localization of CENP-H, however, was not detected at all in the hMis6 RNAi cells (Fig. 6 D). This CENP-H localization result was a positive control for the RNAi experiment.

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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