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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 colocalized with CENP-A and CENP-C. (A) Coimmunostaining of hMis12 with CENP-B (top), or with hMis6 (bottom) was done. DNA was stained by Hoechst 33342. The merged images were produced by green-colored hMis12, red-colored hMis6 or CENP-B, and blue-colored DNA. hMis12 signals colocalized with hMis6 dots but not CENP-B rod signals in metaphase, as shown in the enlarged images (insets). Bar, 10 μm. (B) GFP–hMis12 is colocalized with CENP-A at centromeres throughout the cell cycle. HeLa cells transfected with GFP–hMis12 were fixed and stained by anti–CENP-A antibody. The merged images were produced by green-colored GFP–hMis12, red-colored CENP-A, and blue-colored DNA. Bar, 10 μm. (C–E) Kinetochore localization of GFP–hMis12 on the spread chromosomes. GFP–hMis12 was expressed in HeLa cells and metaphase spread chromosomes were prepared. Immunostaining of CENP-A (C), CENP-B (D), and CENP-C (E) was performed. hMis12 was colocalized with CENP-A and CENP-C, components of the inner kinetochore plate. Panel 1, chromosome stained by Hoechst 33342; panel 2, GFP-hMis12; panel 3, CENP-A/B/C; panel 4, merged image (blue for chromosome, green for GFP–hMis12, and red for CENPs). Bar, 1 μm.
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fig3: hMis12 is colocalized with CENP-A and CENP-C. (A) Coimmunostaining of hMis12 with CENP-B (top), or with hMis6 (bottom) was done. DNA was stained by Hoechst 33342. The merged images were produced by green-colored hMis12, red-colored hMis6 or CENP-B, and blue-colored DNA. hMis12 signals colocalized with hMis6 dots but not CENP-B rod signals in metaphase, as shown in the enlarged images (insets). Bar, 10 μm. (B) GFP–hMis12 is colocalized with CENP-A at centromeres throughout the cell cycle. HeLa cells transfected with GFP–hMis12 were fixed and stained by anti–CENP-A antibody. The merged images were produced by green-colored GFP–hMis12, red-colored CENP-A, and blue-colored DNA. Bar, 10 μm. (C–E) Kinetochore localization of GFP–hMis12 on the spread chromosomes. GFP–hMis12 was expressed in HeLa cells and metaphase spread chromosomes were prepared. Immunostaining of CENP-A (C), CENP-B (D), and CENP-C (E) was performed. hMis12 was colocalized with CENP-A and CENP-C, components of the inner kinetochore plate. Panel 1, chromosome stained by Hoechst 33342; panel 2, GFP-hMis12; panel 3, CENP-A/B/C; panel 4, merged image (blue for chromosome, green for GFP–hMis12, and red for CENPs). Bar, 1 μm.

Mentions: CENP-B was shown to be present in the innermost heterochromatic region of the kinetochores of human chromosomes, whereas CENP-A was located outside of CENP-B at a region called the inner plate (Masumoto et al., 1989; Cooke et al., 1990; Warburton et al., 1997). HeLa cells were fixed by paraformaldehyde and immunostained by anti-hMis12 and anti–CENP-B antibodies. The results shown in Fig. 3 A showed that in metaphase, the signals of hMis12 were rounded dots differing from the rod-like signals of CENP-B, which were located further inside (top row). In the enlarged, merged micrographs, CENP-B (red) was distributed over large areas and clearly distinct from hMis12 (green), which overlapped with the ends of rod-like CENP-B signals.


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 colocalized with CENP-A and CENP-C. (A) Coimmunostaining of hMis12 with CENP-B (top), or with hMis6 (bottom) was done. DNA was stained by Hoechst 33342. The merged images were produced by green-colored hMis12, red-colored hMis6 or CENP-B, and blue-colored DNA. hMis12 signals colocalized with hMis6 dots but not CENP-B rod signals in metaphase, as shown in the enlarged images (insets). Bar, 10 μm. (B) GFP–hMis12 is colocalized with CENP-A at centromeres throughout the cell cycle. HeLa cells transfected with GFP–hMis12 were fixed and stained by anti–CENP-A antibody. The merged images were produced by green-colored GFP–hMis12, red-colored CENP-A, and blue-colored DNA. Bar, 10 μm. (C–E) Kinetochore localization of GFP–hMis12 on the spread chromosomes. GFP–hMis12 was expressed in HeLa cells and metaphase spread chromosomes were prepared. Immunostaining of CENP-A (C), CENP-B (D), and CENP-C (E) was performed. hMis12 was colocalized with CENP-A and CENP-C, components of the inner kinetochore plate. Panel 1, chromosome stained by Hoechst 33342; panel 2, GFP-hMis12; panel 3, CENP-A/B/C; panel 4, merged image (blue for chromosome, green for GFP–hMis12, and red for CENPs). Bar, 1 μm.
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fig3: hMis12 is colocalized with CENP-A and CENP-C. (A) Coimmunostaining of hMis12 with CENP-B (top), or with hMis6 (bottom) was done. DNA was stained by Hoechst 33342. The merged images were produced by green-colored hMis12, red-colored hMis6 or CENP-B, and blue-colored DNA. hMis12 signals colocalized with hMis6 dots but not CENP-B rod signals in metaphase, as shown in the enlarged images (insets). Bar, 10 μm. (B) GFP–hMis12 is colocalized with CENP-A at centromeres throughout the cell cycle. HeLa cells transfected with GFP–hMis12 were fixed and stained by anti–CENP-A antibody. The merged images were produced by green-colored GFP–hMis12, red-colored CENP-A, and blue-colored DNA. Bar, 10 μm. (C–E) Kinetochore localization of GFP–hMis12 on the spread chromosomes. GFP–hMis12 was expressed in HeLa cells and metaphase spread chromosomes were prepared. Immunostaining of CENP-A (C), CENP-B (D), and CENP-C (E) was performed. hMis12 was colocalized with CENP-A and CENP-C, components of the inner kinetochore plate. Panel 1, chromosome stained by Hoechst 33342; panel 2, GFP-hMis12; panel 3, CENP-A/B/C; panel 4, merged image (blue for chromosome, green for GFP–hMis12, and red for CENPs). Bar, 1 μm.
Mentions: CENP-B was shown to be present in the innermost heterochromatic region of the kinetochores of human chromosomes, whereas CENP-A was located outside of CENP-B at a region called the inner plate (Masumoto et al., 1989; Cooke et al., 1990; Warburton et al., 1997). HeLa cells were fixed by paraformaldehyde and immunostained by anti-hMis12 and anti–CENP-B antibodies. The results shown in Fig. 3 A showed that in metaphase, the signals of hMis12 were rounded dots differing from the rod-like signals of CENP-B, which were located further inside (top row). In the enlarged, merged micrographs, CENP-B (red) was distributed over large areas and clearly distinct from hMis12 (green), which overlapped with the ends of rod-like CENP-B signals.

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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