Experimental scheme of complementation assay. We previously created CENP-H conditional knockout cell line #5-5 in which CENP-H expression is inactivated when tetracycline (tet) is added to culture medium. Various deletion derivatives of the CENP-H cDNA are cloned downstream of the CMV promoter (CMV^pro) in the pEGFP vector. These constructs integrate at random sites in the genome of #5-5 CENP-H conditional knockout cells. Cells express both full-length CENP-H cDNA and mutant CENP-H. When tetracycline is added to culture medium, expression of full-length CENP-H is turned off, but mutant CENP-H is still expressed. If cells survive after addition of TET, the mutant CENP-H complements CENP-H function. We also investigated subcellular localization of mutant CENP-Hs. puro^r, puromycin resistance gene.

CENP-H interacts with Hec1. (A) Two-hybrid analysis of interactions between human CENP-H (hCENP-H) and Hec1 (hHec1) (left) and chicken CENP-H (chCENP-H) and Hec1 (chHec1) (right). As a positive control, the interaction between T antigen (AD/T antigen fusion) and p53 (BD/p53 fusion) is shown. AD indicates the GAL4 transcription activation domain, and BD indicates the DNA binding domain of GAL4. Growth was tested on SD minimal plates (−Ade/−His/−Leu/−Trp). (B) Identification of the region of chicken CENP-H required for interaction with chicken full-length Hec1. As bait, constructs of BD fused with CENP-H deletion derivatives were used. As a prey construct, AD fused with chicken Hec1 was used. Growth was tested on SD minimal plates (−Ade/−His/−Leu/−Trp).

Coimmunoprecipitation analysis to investigate the interaction of CENP-H derivatives with Nuf2 complex. Cell lines expressing several CENP-H deletion derivatives were created. Mitotic extracts of these cell lines were immunoprecipitated with anti-Flag antibody. Immunoprecipitates were separated by SDS-PAGE and analyzed by Western blotting with anti-Nuf2 and Hec1 antibodies. One-eighth of each anti-Flag immunoprecipitate was separated by SDS-PAGE and detected with CENP-H as described in the legend of Fig. 4B. IgG, immunoglobulin G.

Graphic representation of iFRAP data. Quantification of iFRAP kinetics is shown. Red lines indicate loss of fluorescence in unbleached areas. Blue lines indicate relative fluorescence intensities of whole-cell regions. In iFRAP analysis of Hec1-GFP during G₂, fluorescence intensities of whole cells and the unbleached region had equalized by ∼150 s. In contrast, the fluorescence intensity in unbleached regions for both Hec1-GFP during mitosis and CENP-H-GFP during both interphase and mitosis changed little.

Identification of the minimal region required for CENP-H function. A schematic representation of CENP-H-GFP constructs and summary of the complementation and localization analysis are shown. To examine localization of CENP-H derivatives, an anti-CENP-C antibody was used to stain centromeres. Typical images for immunofluorescence are shown. Antibody signals were detected with Cy3-conjugated secondary antibodies (red), and GFP signals appear green. DNA was counterstained with DAPI (blue).

CENP-H interacts with the Nuf2 complex in DT40 cells. (A) Localization of Hec1-GFP (green) and CENP-H-Flag (red) at various stages of the cell cycle in DT40 cells that express Hec1-GFP and CENP-H-Flag simultaneously. Nuclei and chromosomes are counterstained with DAPI (blue). As shown in the merged images, colocalization of Hec1-GFP with CENP-H-Flag begins during G₂ and is maintained during mitosis. (B) Coimmunoprecipitation analysis to investigate the interaction of CENP-H with Nuf2 and Hec1. CHF3 cells in which expression of CENP-H is replaced with CENP-H-Flag were prepared, and mitotic extracts of CHF3 cells were immunoprecipitated with anti-Flag antibody. Immunoprecipitates were separated by SDS-PAGE and analyzed by Western blotting with anti-Hec1 or anti-Nuf2 antibody. As a control, #5-5 CENP-H conditional knockout cells, which do not express Flag-tagged CENP-H, were used. CHF3-HG5 cells, which express both CENP-H-Flag and Hec1-GFP, were also prepared, and mitotic extracts of CHF3-HG5 cells were immunoprecipitated with anti-GFP antibody. Immunoprecipitateswere separated by SDS-PAGE and analyzed by Western blotting with anti-Flag antibody. One-eighth of anti-Flag immunoprecipitate with anti-Flag was loaded onto gels for CENP-H detection. (C) Coimmunoprecipitation analysis to investigate the interaction of CENP-H with Mad2, Spc24, and Spc25. CHF-MDG1 cells that express both CENP-H-Flag and Mad2-GFP, CHF-SP24G cells that express both CENP-H-Flag and Spc24-GFP, and CHFSP25G11 cells that express both CENP-H-Flag and Spc25-GFP were created. Mitotic extracts of these three cell lines were immunoprecipitated with anti-Flag antibody. Immunoprecipitates were separated by SDS-PAGE and analyzed by Western blotting with anti-Flag and anti-GFP antibodies. IgG, immunoglobulin G.

Images of photobleaching experiments. Turnover of Hec1-GFP and CENP-H-GFP was analyzed. With the exception of a small region of the centromere (small red circle), the entire cell (big red circle) expressing a GFP fusion protein was photobleached with a laser, and imageswere obtained by fluorescence time lapse microscopy. Prebleaching and recovery images are shown. In iFRAP for Hec1-GFP, unbleached centromere signals (arrow) decrease and fluorescence intensities of cytoplasm (arrowhead) and bleached centromeres increase during G₂. The fluorescence intensity of unbleached regions for both Hec1-GFP during mitosis and CENP-H-GFP during both interphase and mitosis remains almost unchanged. The average intensity is plotted and shown in Fig. 7.

Model of the relation between inner and outer kinetochore structures. The inner kinetochore structure is divided into several subcomplexes that may interact with each other. The CENP-H complex is one such subcomplex. The Nuf2 complex is targeted to the inner kinetochore during G₂ and forms a stable association with the CENP-H complex during mitosis. The Nuf2 complex also interacts with the Mad2 complex, which is a component of the outer kinetochore and is associated transiently with centromeres. In this model, we propose that the Nuf2 complex functions as a connector between inner and outer kinetochores.