Inner centromere formation requires hMis14, a trident kinetochore protein that specifically recruits HP1 to human chromosomes - PubMed (original) (raw)
Inner centromere formation requires hMis14, a trident kinetochore protein that specifically recruits HP1 to human chromosomes
Tomomi Kiyomitsu et al. J Cell Biol. 2010.
Abstract
Centromeric DNA forms two structures on the mitotic chromosome: the kinetochore, which interacts with kinetochore microtubules, and the inner centromere, which connects sister kinetochores. The assembly of the inner centromere is poorly understood. In this study, we show that the human Mis14 (hMis14; also called hNsl1 and DC8) subunit of the heterotetrameric hMis12 complex is involved in inner centromere architecture through a direct interaction with HP1 (heterochromatin protein 1), mediated via a PXVXL motif and a chromoshadow domain. We present evidence that the mitotic function of hMis14 and HP1 requires their functional association at interphase. Alterations in the hMis14 interaction with HP1 disrupt the inner centromere, characterized by the absence of hSgo1 (Shugoshin-like 1) and aurora B. The assembly of HP1 in the inner centromere and the localization of hMis14 at the kinetochore are mutually dependent in human chromosomes. hMis14, which contains a tripartite-binding domain for HP1 and two other kinetochore proteins, hMis13 and blinkin, is a cornerstone for the assembly of the inner centromere and kinetochore.
Figures
Figure 1.
Direct interaction between hMis14 and HP1 and construction of the hMis14 m2 mutant. (A) Human 293 cells were transfected with a plasmid that expressed the Flag-tagged CD or CSD of HP1-α. Extracts were prepared 30 h after transfection using the cytoskeleton buffer and subjected to immunoprecipitation. Input and immunoprecipitates (IP) were immunoblotted using the antibodies indicated. Empty vector was transfected as a control. H, hinge. (B) Y2H experiments between the four subunits of the hMis12 complex and the CSD of HP1-α, -β, or -γ. (C) Y2H analysis between hMis14 and an HP1-α CSD mutant. (D) Amino acid sequences of Mis14 family members. Conserved amino acids are boxed in dark purple, and similar amino acids are boxed in light purple. HP1-binding PXVXL motifs are shown in red. Y2H analysis was performed between deletions or substitutions of hMis14 and HP1-α CSD. (E) Immunoprecipitation of GFP-tagged WT and mutant constructs (m2E) of hMis14. Immunoblot was performed using the antibodies indicated. The band position of HP1-α is indicated by the arrowheads. The band indicated by the asterisks is contaminating IgG. (F) Y2H analysis between various deletions and substitutions of hMis14 and hMis13, HP1-α CSD, and the blinkin C-terminal fragment, blinkin-C (aa 1981–2316). (G) A cartoon depicting interactions detected between the hMis12 complex, blinkin, and HP1.
Figure 2.
Interaction between hMis14 and HP1 in interphase. (A and B) Immunoprecipitation (IP) of GFP-tagged hMis14 (A) and HP1-α (B). Cultures of HeLa cells that stably expressed GFP-tagged hMis14 or HP1-α were used: AS culture is predominantly interphase, and nocodazole-arrested (Noc) culture is mitotically arrested. (C) Interphase HeLa cells that stably expressed GFP-hMis14 were stained with antibodies against HP1-α (top). Metaphase spread chromosomes of HeLa cells stably expressing GFP–HP1-α were stained with anti-hMis14 antibodies (bottom). Cross section images were deconvolved and stacked. Insets show a higher magnification view of the boxed areas. Bars, 10 µm.
Figure 3.
hMis14-dependent enrichment of GFP–HP1-α at the inner centromere. (A) Immunoblot of hMis14 after RNAi treatment. The loading control was tubulin (TUB). Bands indicated by the asterisk are proteins that cross react with anti-hMis14 antibodies (top). HeLa cells were fixed and stained with anti-hMis14 antibody after hMis14 or control RNAi (bottom). (B) Interphase HeLa cells stably expressing GFP–HP1-α were stained with anti–CENP-B antibodies after hMis14 RNAi or control RNAi. Higher magnification views of the boxed areas are shown to the right. (C) HeLa cells stably expressing GFP–HP1-α were cultured for 46 h after hMis14 RNAi or control RNAi and for an additional 2 h after the addition of nocodazole and MG132. Spread chromosomes were stained with anti-hSgo1 and anti–CENP-B antibodies. Cross section images were deconvolved and stacked. Nondeconvolved images are shown for hSgo1. (D) Spread chromosomes were stained with anti-hSgo1 antibodies after hMis14, HP1-α, and hSgo1 RNAi. Representative spreads are shown (n = 100). Insets show a higher magnification view of a representative chromosome. Bars, 10 µm.
Figure 4.
hMis14 m2 mutants fail to restore GFP–HP1-α at the inner centromere. (A) Schematized experimental procedure of the rescue experiment. See “hMis14 mutants are deficient in the enrichment of HP1-α and hSgo1 at the inner centromere” for details. (B) Spread chromosomes were stained with anti-hSgo1 antibodies. Control HeLa cells that were not transfected with mCherry-hMis14 (bottom) and mCherry-hMis14–transfected cells (top) were observed in the same field. (C) GFP–HP1-α–expressing HeLa cells were treated according to the procedures depicted in A, using plasmids as indicated. Nocodazole and MG132 were added for the last 2 h. Spread chromosomes were stained with anti-hSgo1 antibodies and Hoechst 33342 (magenta). Bars, 10 µm.
Figure 5.
hMis14 m2 mutants fail to restore aurora B at the inner centromere. (A) Schematized experimental procedure of the rescue experiment. Four stable cell lines were generated using the Flip-In system. (B) Immunoblot of hMis14, Flag, and Ponceau staining after treatment with tetracycline and siRNA as indicated. The bands indicated by one or two asterisks represent endogenous hMis14 or exogenous Flag-mCherry (mCh)–tagged hMis14, respectively. (C) The substitute experiment was performed according to the procedures depicted in A using the cell lines indicated. Spread chromosomes were stained with the antibodies indicated. Insets show a higher magnification view of the chromosomes. Bar, 10 µm.
Figure 6.
Failure of the hMis14 m2E mutant to localize at the mitotic kinetochore and interphase centromere. (A) The substitute experiment was performed according to the procedures depicted in Fig. 5 A using the cell lines indicated. Cells were fixed and stained with the antibodies indicated. mCh, mCherry. (B) Intensity of the kinetochore signals of Flag-mCherry hMis14 WT or m2E relative to that of CENP-C. 30 kinetochore signals from three cells were measured for each sample. Error bars represent standard deviation. (C) Chromatin fractionation assay. Nocodazole (Noc)-arrested extracts of cells that expressed Flag-mCherry hMis14 WT or m2E were fractionated. The supernatant (Sup) and pellet (PT) fractions were immunoblotted using the antibodies indicated. (D) Simultaneous imaging of GFP-hMis14 WT and m2E mutant in double thymidine–arrested cells. See “Centromeric localization of hMis14 m2 in interphase is diminished” for details. Cross section images were deconvolved and stacked (right). (E) Chromatin fractionation assay. AS HeLa cell extracts stably expressing GFP-hMis14 WT or m2E were used. Sup and PT stand for the supernatant and pellet fraction, respectively, of the first fractionation. S and P stand for supernatant and pellet fraction, respectively, of the second fractionation. See “Centromeric localization of hMis14 m2 in interphase is diminished” for details. Bars, 10 µm.
Figure 7.
The hMis14 m2E mutant fails to suppress chromosome misalignment. (A) Schematized experimental procedures. See “The hMis14 m2E mutant causes misalignment and abnormal anaphase.” (B) Immunoblot of hMis14, GFP, and tubulin (TUB) in transfected HeLa cells. The bands indicated by an asterisk are contaminating proteins that cross react with anti-hMis14 antibodies. (C) Summary of phenotypes in four different RNAi cells. (D) Time-lapse micrographs of HeLa cells. GFP and GFP-tagged RNAi-resistant hMis14 WT or m2E mutant were expressed in hMis14 RNAi cells. The numbers and arrowheads indicate the time (minutes) and the timing of anaphase onset, respectively. Bar, 10 µm.
Figure 8.
HP1 RNAi prevents the proper formation of the inner centromere and the kinetochore localization of hMis14. (A) Immunoblot of HP1-α and -γ after RNAi. The loading control was tubulin (TUB). (B and C) Time-lapse micrographs of HeLa cells stably expressing histone H2B–GFP after control RNAi (B) and HP1-α+γ RNAi (C). The number indicates the time (minutes). Arrows and arrowheads indicate misaligned and lagging chromosomes, respectively. (D) Mitotic spread chromosomes were stained with anti–CENP-B antibodies and Hoechst 33342 (magenta) after HP1-α+γ and HP1-α RNAi. (E) Spread chromosomes were immunostained with anti–aurora B and anti-hSgo1 antibodies after HP1-α, aurora B, and hSgo1 RNAi. (F) Spread chromosomes were immunostained with anti-hMis14 and anti–CENP-C antibodies after HP1-α+γ RNAi. (E and F) Insets show a higher magnification view of a representative chromosome. (G) The intensity of kinetochore signals of hMis14 relative to that of CENP-C was measured after HP1 RNAi. 40 kinetochore signals from four cells were measured for each sample. Error bars represent standard deviation. (H) A model of the inner centromere formation by hMis14 and HP1. See Discussion. Bars: (B, C, E, and F) 10 µm; (D) 2 µm.
References
- Brasher S.V., Smith B.O., Fogh R.H., Nietlispach D., Thiru A., Nielsen P.R., Broadhurst R.W., Ball L.J., Murzina N.V., Laue E.D. 2000. The structure of mouse HP1 suggests a unique mode of single peptide recognition by the shadow chromo domain dimer. EMBO J. 19:1587–1597 10.1093/emboj/19.7.1587 - DOI - PMC - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Molecular Biology Databases
Research Materials