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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 present at kinetochore after CENP-A depletion by RNAi. (A) Immunoblotting of CENP-A in HeLa cell extracts after 68 h. Anti-PSTAIR was used as loading control. The level of CENP-A protein in cell extracts was greatly reduced after incubation with siRNA (right), whereas no reduction occurred in control, no RNAi cells (left). (B) HeLa cells transfected by siRNA for CENP-A (top three rows) or by control buffer only (bottom row) were fixed at 68 h and stained by Hoechst 33342 (blue), anti-hMis12 (green), and anti–CENP-A (red) antibodies. Centromere signals of CENP-A, but not of hMis12, disappeared by RNAi. (C) Costaining by anti–CENP-C and anti–CENP-A antibodies. Centromere signals of CENP-C disappeared by CENP-A knockdown. (D) Costaining by anti-hMis6 and anti-hMis12 antibodies. Centromere signals of hMis6 are greatly diminished by CENP-A RNAi. Control, no RNAi cell is shown at bottom. (E) The frequency of misaligned chromosome appearance in CENP-A RNAi and no RNAi, control mitotic cells. Red columns indicate metaphase cells containing aligned chromosomes, and blue columns represent cells not containing aligned chromosomes. (F) Immunostaining by kinetochore marker (hMis12) and tubulin in CENP-A–knockdown mitotic cells. Cells were fixed at 68 h after siRNA transfection. Misaligned chromosomes were frequently observed in CENP-A–knockdown mitotic cells. Mitotic spindles in such cells were greatly expanded compared with control nontreated cells. (G) Micronuclei (arrows) or abnormal donut-shaped nuclei (middle row) seen in CENP-A–reduced interphase cells. Bars, 10 μm.
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fig5: hMis12 is present at kinetochore after CENP-A depletion by RNAi. (A) Immunoblotting of CENP-A in HeLa cell extracts after 68 h. Anti-PSTAIR was used as loading control. The level of CENP-A protein in cell extracts was greatly reduced after incubation with siRNA (right), whereas no reduction occurred in control, no RNAi cells (left). (B) HeLa cells transfected by siRNA for CENP-A (top three rows) or by control buffer only (bottom row) were fixed at 68 h and stained by Hoechst 33342 (blue), anti-hMis12 (green), and anti–CENP-A (red) antibodies. Centromere signals of CENP-A, but not of hMis12, disappeared by RNAi. (C) Costaining by anti–CENP-C and anti–CENP-A antibodies. Centromere signals of CENP-C disappeared by CENP-A knockdown. (D) Costaining by anti-hMis6 and anti-hMis12 antibodies. Centromere signals of hMis6 are greatly diminished by CENP-A RNAi. Control, no RNAi cell is shown at bottom. (E) The frequency of misaligned chromosome appearance in CENP-A RNAi and no RNAi, control mitotic cells. Red columns indicate metaphase cells containing aligned chromosomes, and blue columns represent cells not containing aligned chromosomes. (F) Immunostaining by kinetochore marker (hMis12) and tubulin in CENP-A–knockdown mitotic cells. Cells were fixed at 68 h after siRNA transfection. Misaligned chromosomes were frequently observed in CENP-A–knockdown mitotic cells. Mitotic spindles in such cells were greatly expanded compared with control nontreated cells. (G) Micronuclei (arrows) or abnormal donut-shaped nuclei (middle row) seen in CENP-A–reduced interphase cells. Bars, 10 μm.

Mentions: The RNAi experiment was done for CENP-A in comparison with the result of hMis12 RNAi. Immunoblot patterns obtained using antibodies against CENP-A showed that the level of cellular CENP-A greatly decreased 68 h after siRNA transfection (Fig. 5 A). Anti–CENP-A immunostaining of HeLa cells treated with the CENP-A siRNA exhibited no signal at this time point (Fig. 5 B). At earlier time points (24 and 48 h), most cells still revealed centromere signals of CENP-A (unpublished data). The detailed observation of CENP-A–knockdown cells described below was hence done for 68-h samples (Fig. 5, B–G).


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 present at kinetochore after CENP-A depletion by RNAi. (A) Immunoblotting of CENP-A in HeLa cell extracts after 68 h. Anti-PSTAIR was used as loading control. The level of CENP-A protein in cell extracts was greatly reduced after incubation with siRNA (right), whereas no reduction occurred in control, no RNAi cells (left). (B) HeLa cells transfected by siRNA for CENP-A (top three rows) or by control buffer only (bottom row) were fixed at 68 h and stained by Hoechst 33342 (blue), anti-hMis12 (green), and anti–CENP-A (red) antibodies. Centromere signals of CENP-A, but not of hMis12, disappeared by RNAi. (C) Costaining by anti–CENP-C and anti–CENP-A antibodies. Centromere signals of CENP-C disappeared by CENP-A knockdown. (D) Costaining by anti-hMis6 and anti-hMis12 antibodies. Centromere signals of hMis6 are greatly diminished by CENP-A RNAi. Control, no RNAi cell is shown at bottom. (E) The frequency of misaligned chromosome appearance in CENP-A RNAi and no RNAi, control mitotic cells. Red columns indicate metaphase cells containing aligned chromosomes, and blue columns represent cells not containing aligned chromosomes. (F) Immunostaining by kinetochore marker (hMis12) and tubulin in CENP-A–knockdown mitotic cells. Cells were fixed at 68 h after siRNA transfection. Misaligned chromosomes were frequently observed in CENP-A–knockdown mitotic cells. Mitotic spindles in such cells were greatly expanded compared with control nontreated cells. (G) Micronuclei (arrows) or abnormal donut-shaped nuclei (middle row) seen in CENP-A–reduced interphase cells. Bars, 10 μm.
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Related In: Results  -  Collection

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fig5: hMis12 is present at kinetochore after CENP-A depletion by RNAi. (A) Immunoblotting of CENP-A in HeLa cell extracts after 68 h. Anti-PSTAIR was used as loading control. The level of CENP-A protein in cell extracts was greatly reduced after incubation with siRNA (right), whereas no reduction occurred in control, no RNAi cells (left). (B) HeLa cells transfected by siRNA for CENP-A (top three rows) or by control buffer only (bottom row) were fixed at 68 h and stained by Hoechst 33342 (blue), anti-hMis12 (green), and anti–CENP-A (red) antibodies. Centromere signals of CENP-A, but not of hMis12, disappeared by RNAi. (C) Costaining by anti–CENP-C and anti–CENP-A antibodies. Centromere signals of CENP-C disappeared by CENP-A knockdown. (D) Costaining by anti-hMis6 and anti-hMis12 antibodies. Centromere signals of hMis6 are greatly diminished by CENP-A RNAi. Control, no RNAi cell is shown at bottom. (E) The frequency of misaligned chromosome appearance in CENP-A RNAi and no RNAi, control mitotic cells. Red columns indicate metaphase cells containing aligned chromosomes, and blue columns represent cells not containing aligned chromosomes. (F) Immunostaining by kinetochore marker (hMis12) and tubulin in CENP-A–knockdown mitotic cells. Cells were fixed at 68 h after siRNA transfection. Misaligned chromosomes were frequently observed in CENP-A–knockdown mitotic cells. Mitotic spindles in such cells were greatly expanded compared with control nontreated cells. (G) Micronuclei (arrows) or abnormal donut-shaped nuclei (middle row) seen in CENP-A–reduced interphase cells. Bars, 10 μm.
Mentions: The RNAi experiment was done for CENP-A in comparison with the result of hMis12 RNAi. Immunoblot patterns obtained using antibodies against CENP-A showed that the level of cellular CENP-A greatly decreased 68 h after siRNA transfection (Fig. 5 A). Anti–CENP-A immunostaining of HeLa cells treated with the CENP-A siRNA exhibited no signal at this time point (Fig. 5 B). At earlier time points (24 and 48 h), most cells still revealed centromere signals of CENP-A (unpublished data). The detailed observation of CENP-A–knockdown cells described below was hence done for 68-h samples (Fig. 5, B–G).

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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