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First published online 26 June 2003
doi: 10.1242/jcs.00645

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Dynamic behavior of Nuf2-Hec1 complex that localizes to the centrosome and centromere and is essential for mitotic progression in vertebrate cells

Tetsuya Hori1, Tokuko Haraguchi2, Yasushi Hiraoka2, Hiroshi Kimura3 and Tatsuo Fukagawa1,*

1 PRESTO, The Japan Science and Technology Corporation, National Institute of Genetics and The Graduate University for Advanced Studies, Mishima, Shizuoka 411-8540, Japan
2 CREST Research Project, The Japan Science and Technology Corporation, Kansai Advanced Research Center, Communications Research Laboratory, 588-2 Iwaoka, Nishi-ku, Kobe 651-2492, Japan
3 Department of Functional Genomics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510, Japan



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  		Fig. 1. Nuf2 associates tightly with chicken Hec1 throughout the cell cycle. (A) Immunoprecipitation with pre-immune IgG or an anti-GFP antibody from extracts of Nuf39-15 cells in which Nuf2 was replaced with Nuf2-GFP and from Hec25-1/5 cells in which Hec1 was replaced with Hec1-GFP. Total extract (input) and the immune complexes (anti-GFP) were analyzed by immunoblotting with anti-Hec1 or anti-Nuf2 antibodies. (B) Localization of Nuf2-GFP (green) and Hec1-HcRed (red) at progressive stages of the cell cycle in DT40 cells that expressed Nuf2-GFP and Hec1-HcRed simultaneously. Nuclei and chromosomes are counterstained with DAPI (blue). The scale bars correspond to 10 µm. As shown in the merged images, Nuf2-GFP signals are colocalized with Hec1-HcRed signals throughout the cell cycle.

 


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  		Fig. 2. Dynamic localization of the Nuf2-Hec1 complex during the cell cycle. (A) Localization of Nuf2-GFP at progressive stages of the cell cycle in DT40 cells. Cells were fixed and stained with anti-CENP-C antibody (red). Green signals are specific for Nuf2-GFP. Nuclei and chromosomes are counterstained with DAPI (blue). As shown in the merged images, Nuf2-GFP signals begin to colocalize with CENP-C signals in G2 and are colocalized with CENP-C throughout mitosis. (B) Cells that express Nuf2-GFP (green) were fixed and stained with anti-{gamma}-tubulin (red). Nuclei and chromosomes are counterstained with DAPI (blue). (C) Selected images of chromosomes (Hoechst; top row) and Nuf2-GFP (middle row) in living cells. In the superimposed images (lower row), chromosomes and Nuf2-GFP are displayed in red and green, respectively.

 


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  		Fig. 3. Generation of {Delta}Nuf2 and {Delta}Hec1 clones carrying transgenes under the control of a tet-repressible promoter. (A) Restriction maps of the Nuf2 and Hec1 loci, gene disruption constructs, and targeted loci. Black boxes indicate the positions of exons. Several restriction enzyme sites are shown. The position of the probe used for Southern hybridization is indicated. (B) Restriction analysis of targeted integration of the Nuf2 and Hec1 disruption constructs. In Nuf2 disruption, genomic DNAs from wild-type DT40 cells (Wt), a clone after first round targeting (+/–, 1st), a clone after first round targeting and random integration of the Nuf2 transgene (+/–Nuf2+, 1st +cDNA), and a clone after second round targeting (–/–Nuf2+, 2nd +cDNA) were analyzed by Southern hybridization with the probe indicated in (A). In Hec1 disruption, genomic DNAs from wild-type DT40 cells (Wt), a clone after first round targeting (+/+/–, 1st), a clone after second round targeting (+/–/–, 2nd), a clone after second round targeting and random integration of the Hec1 transgene (+/–/–Hec1+, 2nd+cDNA), and a clone after third round targeting (–/–/–Hec1+, 3rd+cDNA) were analyzed by Southern hybridization with the probe indicated in A. (C) Western blot analysis of whole cell extracts of Nuf23-63 and Hec25-1 cells with anti-Nuf2 and anti-Hec1 antibodies at the indicated times following addition of tet. Equal amounts of extracts were separated by SDS-PAGE and analyzed by western blotting. Anti-{alpha}-tubulin antibody was used as a loading control.

 


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  		Fig. 4. Both Nuf2 and Hec1 are essential for normal progression of the cell cycle. (A) Representative growth curves for the indicated cell cultures. Tet was added at time 0 to the Nuf2- or Hec1-deficient cell tet (+) cultures, and the number of cells not stained with trypan blue was counted. Each experiment was performed twice, and each time point was examined in duplicate. (B) Cell-cycle distribution of Nuf23-63 and Hec25-1 in cells following inhibition of transgene expression due to addition of tet at time 0. Cells were stained with FITC-anti-BrdU (y-axis, log scale) to detect BrdU incorporation (DNA replication), and with propidium iodide to detect total DNA (x axis, linear scale). The lower-left box represents G1-phase cells, the upper box represents S-phase cells, and the lower-right box represents G2/M-phase cells. The numbers given in the boxes specify the percentage of gated events.

 


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  		Fig. 5. Nuf2- and Hec1-deficient cells show prometaphase arrest associated with aberrant chromosomes and spindles that lead to chromosome missegregation. (A) Chromosome morphology and {alpha}-tubulin staining (green) of Nuf23-63 and Hec25-1 cells in the absence or presence of tet. DNA was counterstained with DAPI (blue). In the absence of tet, cells show the normal staining pattern for {alpha}-tubulin (upper panels in both cell lines). In the presence of tet, chromosomes are not aligned at the metaphase plate. Arrows indicate misaligned chromosomes at the metaphase plate. Apoptotic cells were observed in both mutants 48 hours after addition of tet. We also detected cells with monopolar and multipolar spindles. (B) Quantitation of aberrant Nuf23-63 and Hec25-1 cells after inhibition of transgene expression following addition of tet at time 0. We scored the number of interphase cells, normal metaphase cells, aberrant metaphase cells, anaphase cells and apoptotic cells. Apoptotic cells were detected by TUNEL assay. We scored approximately 3000 cells for each time point. (C) To examine chromosome loss, we used FISH analysis with chromosome-specific painting probes. We used painting probes for chicken chromosomes 1 and 2. Because DT40 cells have three copies of chromosome 2, five painted chromosomes were observed in normal cells. Nuf23-63 and Hec25-1 cells were cultured after addition of tet. At the indicated times cells were treated with colcemid for 1.5 hours, and the number of painted chromosomes per cell was determined. The number of painted chromosomes was scored in approximately 1000 metaphase cells.

 


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  		Fig. 6. Chromosome and microtubule dynamics in Nuf2-deficient cells. (A) Selected images of chromosomes (upper) and microtubules (middle) in Nuf23-63 cells (–tet) from prophase to telophase. In the merged images (lower), chromosomes and microtubules are displayed in red and green, respectively. The numbers at the top of each image represent the time in minutes. (B) Observation of a single cell beginning 18 hours after addition of tet. Selected frames are shown. Once the cell entered mitosis, it arrested at prometaphase for approximately 400 minutes. The cell died during the mitotic stage between 380 and 460 minutes.

 


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  		Fig. 7. Localization of centromere- and checkpoint-related proteins in Nuf2- and Hec1-deficient cells. (A) Localization analysis of Hec1-GFP in Nuf2-deficient cells and Nuf2-GFP in Hec1-deficient cells. In control cells (–tet), Hec1-GFP (green) and Nuf2-GFP (green) signals are localized at the centrosome (G1/S phase) and centromere (mitosis). After addition of tet, both signals are reduced. DNA is counterstained with DAPI (blue). The panel on the right shows a western blot analysis with a mixture of anti-Nuf2 and anti-Hec1 antibodies of Nuf2- and Hec1-deficient cells. The levels of Hec1 in Nuf2-deficient cells and of Nuf2 in Hec1-deficient cells are reduced. (B) Immunofluorescence analysis with antibodies against CENP-A, -C and -H of prometaphase chromosomes in Nuf2- and Hec1-deficient cells after addition of tet. (C) Localization of checkpoint proteins in Nuf2- and Hec1-deficient cells. Both control cells (-tet) have strong Mad2-DsRed signals. After addition of tet, the Mad2-DsRed signals are diffuse in both mutants. Both mutants were also stained with anti-BubR1 (red) in the presence or absence of tet. BubR1 signals are present on the centromeres of cells in mitotic arrest caused by Nuf2 or Hec1 depletion. (D) Immunofluorescence analysis with antibody against {gamma}-tubulin of Nuf2- and Hec1-deficient cells.

 


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  		Fig. 8. Nuf2 and Hec1 localization in CENP-H- and CENP-I-deficient cells. (A) Localization of Nuf2-GFP and Hec1-GFP in #5-5 ({Delta}/CENP-H, CENP-H transgene) cells grown in the absence (–tet) or presence (+tet) of tet for 48 hours. Hec1-GFP (green) or Nuf2-GFP (green) signals are reduced after addition of tet (+tet). (B) Localization analysis of Nuf2-GFP and Hec1-GFP in M6-90 ({Delta}/CENP-I, CENP-I transgene) cells grown in the absence (–tet) or presence (+tet) of tet for 48 hours. Hec1-GFP (green) or Nuf2-GFP (green) signals are reduced after addition of tet (+tet).

 


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  		Fig. 9. Turnover of Nuf2-GFP and Hec1-GFP at centrosomes and centromeres. (A) The centrosomes and centromeres (white boxes) were targeted for laser photobleaching and then followed by fluorescence time-lapse microscopy. Pre-bleach, post-bleach, and recovery images are shown. See Movies 4-7 (http://jcs.biologists.org/supplemental). (B) Graphs illustrating the ratio of relative fluorescence recovery. For centrosome analysis, we used Origin software (OriginLab Corp.) to determine recovery half-times.

 


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  		Fig. 10. Dynamic behavior of the Nuf2-Hec1 complex during the cell cycle. Illustration of the dynamic behavior of the Nuf2-Hec1 complex during the cell cycle. Association of the Nuf2-Hec1 complex with centrosomes is dynamic during G1 and S phases. The complex moves through the nuclear membrane to centromeres during G2 and mitosis. Association of the complex with centromeres is stable. In the centromere, the complex provides a place for the Mad2 complex to attach to the kinetochore.

 

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© The Company of Biologists Ltd 2003