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First published online 2 July 2003
doi: 10.1242/jcs.00635

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Cell cycle behavior of human HP1 subtypes: distinct molecular domains of HP1 are required for their centromeric localization during interphase and metaphase

Tomohiro Hayakawa1,2, Tokuko Haraguchi1,2, Hiroshi Masumoto3 and Yasushi Hiraoka1,2,*

1 CREST Research Project, Kansai Advanced Research Center, Communications Research Laboratory, 588-2 Iwaoka, Iwaoka-cho, Nishi-ku, Kobe 651-2492, Japan
2 Department of Biology, Graduate School of Science, Osaka University,1-1 Machikaneyama, Osaka 560-0043, Japan
3 Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan



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  		Fig. 7. Human HP1 molecular domains. (A) Amino acid sequence of human HP1 subtypes {alpha}, ß, and {gamma}. Asterisks indicate amino acids conserved among the three subtypes and dots indicate amino acids conserved among two of the subtypes. The red-shading is the chromo domain (CD), and the blue shading is the chromo shadow domain (CSD). These domains are connected by the IVR. The domain that mediates centromere localization during metaphase is indicated by a red outline. (B) In interphase, the CD motif (red) is required for localization to centromeric heterochromatin and other heterochromatin, and the CSD motif (blue) is required for localization to PML nuclear bodies. However, during metaphase, centromeric localization of HP1 requires the region containing a portion of the IVR and the CSD motif.

 


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  		Fig. 1. Intracellular localization of human HP1 subtypes. (A) Localization of human HP1 subtypes in interphase nuclei. HeLa cells transiently expressing YFP-HP1{alpha} (a-e), YFP-HP1ß (f-j), or YFP-HP1{gamma} (k-o) were fixed, and centromeres and PML nuclear bodies were viewed by indirect immunofluorescent staining. (b,g,l) centromeres, (c,h,m) YFP-HP1 and (d,i,n) PML nuclear bodies in the same cells. Merged images in a, f and k, of HP1 (green) and of centromeres (red) and in e, j and o for HP1 (green) and the PML nuclear bodies (red). Bar, 10 µm. (B) Metaphase localization of HP1 subtypes. HeLa cells transiently expressing YFP-HP1{alpha} (a-c), YFP-HP1ß (d-f), or YFP-HP1{gamma} (g-i) were fixed and stained with DAPI (a, d and g). (c, f, and i) Merged images of HP1 (green) and DAPI (red). Bar, 10 µm. (C) HeLa cells transiently expressing YFP-HP1{alpha} were arrested at mitosis by treatment with 650 nM nocodazole for 8 hours. Centromeres were stained with human antisera that recognizes CENP-A, CENP-B, and CENP-C proteins (Masumoto et al., 1989Go). (a) DAPI, (b) YFP-HP1{alpha} and (c) centromeres. (d) A merged image of DAPI (blue), YFP-HP1{alpha} (green) and centromeres (red). (e) An enlarged image of the boxed area in d showing YFP-HP1{alpha} (green) and centromeres (red). Scale bar, 10 µm.

 


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  		Fig. 2. Localization of HP1 subtypes in metaphase chromosome spreads. HP1{alpha} (a,b), HP1ß (c,d) and HP1{gamma} (e,f) are shown in green on metaphase chromosomes (red). Metaphase chromosome spreads were prepared from cells collected with (a,c,e) or without (b,d,f) enrichment of mitotic cells (see Materials and Methods). Mitotic cells were about 22-24% of the total cell population in enriched preparation and about 6-9% in the non-enriched prerapation. Scale bar, 10 µm.

 


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  		Fig. 3. Domains of HP1{alpha} required for its localization. HeLa cells transiently expressing HP1{alpha} fragments fused with YFP and the CENP-B N-terminal region fused with CFP. (A) Diagram of YFP-HP1{alpha} truncation constructs and their intracellular localization: +, localized; –, not localized; +/–, ambiguous; and ND, not determined. Quantitative data were obtained from three independent experiments. (B) YFP-HP1{alpha} fragment localization during metaphase: YFP-HP1{alpha} fragment (left column), CFP-CENPB-N-terminal fragment (middle column), and merged images (right column). (C) Localization of HP1{alpha} fragments in the interphase nucleus. (D) Localization of HP1{alpha} and PML nuclear bodies. Numbers on the left of each row refer to the amino acid numbers of the fragments. Scale bar, 10 µm.

 


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  		Fig. 4. Domains of HP1ß required for its localization. HeLa cells transiently expressing HP1ß fragments fused with YFP and the CENP-B N-terminal region fused with CFP. (A) Diagram of YFP-HP1ß truncation constructs and their intracellular localization: +, localized; –, not localized; +/–, ambiguous; ND, not determined. Quantitative data were obtained from three independent experiments. (B) YFP-HP1ß fragment localization during metaphase: YFP-HP1ß fragment (left column), CFP-CENPB-N-terminal fragment (middle column), and merged images (right column). (C) Localization of HP1ß fragments in the interphase nucleus. (D) Localization of HP1ß and PML nuclear bodies. Numbers on the left of each row refer to the amino acid numbers of the fragments. Scale bar, 10 µm.

 


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  		Fig. 5. Domains of HP1{gamma} required for its localization. HeLa cells transiently expressing HP1{gamma} fragments fused with YFP and the CENP-B N-terminal region fused with CFP. (A) Diagram of YFP- HP1{gamma} truncation constructs and their intracellular localization: +, localized; –, not localized; +/–, ambiguous; and ND, not determined. Quantitative data were obtained from three independent experiments. (B) YFP- HP1{gamma} fragment localization during metaphase: YFP- HP1{gamma} fragment (left column), CFP-CENPB-N-terminal fragment (middle column), and merged images (right column). (C) Localization of HP1{gamma} fragments in the interphase nucleus. (D) Localization of HP1{gamma} and PML nuclear bodies. Scale bar, 10 µm.

 


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  		Fig. 6. Simultaneous observation of HP1{alpha} C-terminal fragment and HP1ß N-terminal fragment. Living HeLa cells transiently expressing a DsRed2-fused HP1{alpha} fragment that localized to the mitotic centromere, the N-terminal fragment of HP1ß fused with YFP, and the N-terminal fragment of CENP-B fused with CFP. From left to right, merged images of the HP1{alpha} fragment (green) and CENP-B (magenta), the HP1{alpha} fragment, CENP-B, the HP1ß fragment, and merged images of the HP1ß fragment (green) and CENP-B (magenta). Numbers on the left indicate time in hours and minutes. Scale bar, 10 µm.

 

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© The Company of Biologists Ltd 2003