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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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Metaphase chromosome alignment is impaired by the RNAi method for hMis12. (A) Immunoblotting of hMis12 in HeLa cell extracts after 85 h. Anti-PSTAIR was used as loading control. The level of hMis12 protein in cell extracts was greatly reduced after incubation with siRNA (lane 2). (B) HeLa cells transfected by siRNA for hMis12 (top) or by control buffer only (bottom) were fixed at 85 h and stained by Hoechst 33342 (blue), anti-hMis12 (green), and anti-tubulin antibodies (red). Centromere signals disappeared by hMis12 RNAi. (C) RNAi for CENP-E induced accumulation of mitotic cells. (D) Micrograph taken by phase contrast. Arrows indicate mitotic cells, whereas cells indicated by arrowheads have an abnormal shape. These abnormally shaped cells were positively detected by the TUNEL assay (not depicted). (E) Immunostaining of cyclin B1 (green). DNA was stained by Hoechst 33342 (red). Frequencies (%) of each population are also shown in the inset table. WT, no RNAi. (F, I, and J) Immunostaining of kinetochores (CENP-C) and tubulin. Cells were fixed 85 h after siRNA transfection. (F) Misaligned chromosomes (arrow) observed in metaphase of hMis12-knockdown cells. (G) Quantitative results indicated that the frequencies of misaligned chromosomes are frequent in metaphase cells after hMis12 RNAi depletion. (H) Quantitative results show that spindles in such hMis12-knockdown cells (red columns) were expanded compared with control nontreated cells. (I, top) Lagging chromosomes (indicated by arrowheads) seen in anaphase cells. (I, bottom) Micronuclei (indicated by arrows) are observed in telophase cells. (J) Micronuclei are frequently seen in interphase cells. (K and L) Kinetochore localization of CENP-A (K) and hMis6 (L) in hMis12-depleted cells. Cells treated with hMis12 siRNA were stained by anti–CENP-A or hMis6 antibodies. Bars, 10 μm.
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fig4: Metaphase chromosome alignment is impaired by the RNAi method for hMis12. (A) Immunoblotting of hMis12 in HeLa cell extracts after 85 h. Anti-PSTAIR was used as loading control. The level of hMis12 protein in cell extracts was greatly reduced after incubation with siRNA (lane 2). (B) HeLa cells transfected by siRNA for hMis12 (top) or by control buffer only (bottom) were fixed at 85 h and stained by Hoechst 33342 (blue), anti-hMis12 (green), and anti-tubulin antibodies (red). Centromere signals disappeared by hMis12 RNAi. (C) RNAi for CENP-E induced accumulation of mitotic cells. (D) Micrograph taken by phase contrast. Arrows indicate mitotic cells, whereas cells indicated by arrowheads have an abnormal shape. These abnormally shaped cells were positively detected by the TUNEL assay (not depicted). (E) Immunostaining of cyclin B1 (green). DNA was stained by Hoechst 33342 (red). Frequencies (%) of each population are also shown in the inset table. WT, no RNAi. (F, I, and J) Immunostaining of kinetochores (CENP-C) and tubulin. Cells were fixed 85 h after siRNA transfection. (F) Misaligned chromosomes (arrow) observed in metaphase of hMis12-knockdown cells. (G) Quantitative results indicated that the frequencies of misaligned chromosomes are frequent in metaphase cells after hMis12 RNAi depletion. (H) Quantitative results show that spindles in such hMis12-knockdown cells (red columns) were expanded compared with control nontreated cells. (I, top) Lagging chromosomes (indicated by arrowheads) seen in anaphase cells. (I, bottom) Micronuclei (indicated by arrows) are observed in telophase cells. (J) Micronuclei are frequently seen in interphase cells. (K and L) Kinetochore localization of CENP-A (K) and hMis6 (L) in hMis12-depleted cells. Cells treated with hMis12 siRNA were stained by anti–CENP-A or hMis6 antibodies. Bars, 10 μm.

Mentions: To determine the cellular role of hMis12, the RNAi method was employed using the small interfering RNA (siRNA) sequences (see Materials and methods). The amount of hMis12 in HeLa cells was found to be greatly decreased (<20%) after 85 h, whereas the control of no RNA showed the normal level of hMis12 (Fig. 4 A; unpublished data). The control CDK signals (PSTAIR) were not altered in cells treated with siRNA. Fig. 4 B shows immunostained micrographs of HeLa cells after the hMis12 RNAi. Cells transfected with hMis12 siRNA exhibited no signals at the time point (85 h). At earlier time points (48 and 68 h), however, most of the cells still showed centromere and nuclear signals of hMis12 (unpublished data). Detailed observation of hMis12-knockdown cells described below was hence done for the 85-h cells. The siRNA against CENP-E (an outer kinetochore protein) was transfected into HeLa cells as a positive control of the RNAi experiment. Consistent with the previous study (Harborth et al., 2001), mitotic cells with condensed chromosomes were highly accumulated (>80% of total cells; DNA stained in Fig. 4 C).


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)

Metaphase chromosome alignment is impaired by the RNAi method for hMis12. (A) Immunoblotting of hMis12 in HeLa cell extracts after 85 h. Anti-PSTAIR was used as loading control. The level of hMis12 protein in cell extracts was greatly reduced after incubation with siRNA (lane 2). (B) HeLa cells transfected by siRNA for hMis12 (top) or by control buffer only (bottom) were fixed at 85 h and stained by Hoechst 33342 (blue), anti-hMis12 (green), and anti-tubulin antibodies (red). Centromere signals disappeared by hMis12 RNAi. (C) RNAi for CENP-E induced accumulation of mitotic cells. (D) Micrograph taken by phase contrast. Arrows indicate mitotic cells, whereas cells indicated by arrowheads have an abnormal shape. These abnormally shaped cells were positively detected by the TUNEL assay (not depicted). (E) Immunostaining of cyclin B1 (green). DNA was stained by Hoechst 33342 (red). Frequencies (%) of each population are also shown in the inset table. WT, no RNAi. (F, I, and J) Immunostaining of kinetochores (CENP-C) and tubulin. Cells were fixed 85 h after siRNA transfection. (F) Misaligned chromosomes (arrow) observed in metaphase of hMis12-knockdown cells. (G) Quantitative results indicated that the frequencies of misaligned chromosomes are frequent in metaphase cells after hMis12 RNAi depletion. (H) Quantitative results show that spindles in such hMis12-knockdown cells (red columns) were expanded compared with control nontreated cells. (I, top) Lagging chromosomes (indicated by arrowheads) seen in anaphase cells. (I, bottom) Micronuclei (indicated by arrows) are observed in telophase cells. (J) Micronuclei are frequently seen in interphase cells. (K and L) Kinetochore localization of CENP-A (K) and hMis6 (L) in hMis12-depleted cells. Cells treated with hMis12 siRNA were stained by anti–CENP-A or hMis6 antibodies. Bars, 10 μm.
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fig4: Metaphase chromosome alignment is impaired by the RNAi method for hMis12. (A) Immunoblotting of hMis12 in HeLa cell extracts after 85 h. Anti-PSTAIR was used as loading control. The level of hMis12 protein in cell extracts was greatly reduced after incubation with siRNA (lane 2). (B) HeLa cells transfected by siRNA for hMis12 (top) or by control buffer only (bottom) were fixed at 85 h and stained by Hoechst 33342 (blue), anti-hMis12 (green), and anti-tubulin antibodies (red). Centromere signals disappeared by hMis12 RNAi. (C) RNAi for CENP-E induced accumulation of mitotic cells. (D) Micrograph taken by phase contrast. Arrows indicate mitotic cells, whereas cells indicated by arrowheads have an abnormal shape. These abnormally shaped cells were positively detected by the TUNEL assay (not depicted). (E) Immunostaining of cyclin B1 (green). DNA was stained by Hoechst 33342 (red). Frequencies (%) of each population are also shown in the inset table. WT, no RNAi. (F, I, and J) Immunostaining of kinetochores (CENP-C) and tubulin. Cells were fixed 85 h after siRNA transfection. (F) Misaligned chromosomes (arrow) observed in metaphase of hMis12-knockdown cells. (G) Quantitative results indicated that the frequencies of misaligned chromosomes are frequent in metaphase cells after hMis12 RNAi depletion. (H) Quantitative results show that spindles in such hMis12-knockdown cells (red columns) were expanded compared with control nontreated cells. (I, top) Lagging chromosomes (indicated by arrowheads) seen in anaphase cells. (I, bottom) Micronuclei (indicated by arrows) are observed in telophase cells. (J) Micronuclei are frequently seen in interphase cells. (K and L) Kinetochore localization of CENP-A (K) and hMis6 (L) in hMis12-depleted cells. Cells treated with hMis12 siRNA were stained by anti–CENP-A or hMis6 antibodies. Bars, 10 μm.
Mentions: To determine the cellular role of hMis12, the RNAi method was employed using the small interfering RNA (siRNA) sequences (see Materials and methods). The amount of hMis12 in HeLa cells was found to be greatly decreased (<20%) after 85 h, whereas the control of no RNA showed the normal level of hMis12 (Fig. 4 A; unpublished data). The control CDK signals (PSTAIR) were not altered in cells treated with siRNA. Fig. 4 B shows immunostained micrographs of HeLa cells after the hMis12 RNAi. Cells transfected with hMis12 siRNA exhibited no signals at the time point (85 h). At earlier time points (48 and 68 h), however, most of the cells still showed centromere and nuclear signals of hMis12 (unpublished data). Detailed observation of hMis12-knockdown cells described below was hence done for the 85-h cells. The siRNA against CENP-E (an outer kinetochore protein) was transfected into HeLa cells as a positive control of the RNAi experiment. Consistent with the previous study (Harborth et al., 2001), mitotic cells with condensed chromosomes were highly accumulated (>80% of total cells; DNA stained in Fig. 4 C).

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.

Show MeSH
Related in: MedlinePlus