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. 2001 Sep 15;29(18):3796-803.
doi: 10.1093/nar/29.18.3796.

Creation and characterization of temperature-sensitive CENP-C mutants in vertebrate cells

T Fukagawa  1 , V Regnier, T Ikemura
Affiliations

Creation and characterization of temperature-sensitive CENP-C mutants in vertebrate cells

T Fukagawa et al. Nucleic Acids Res. .

Abstract

CENP-C is an evolutionarily conserved centromere protein that is thought to be an important component in kinetochore assembly in vertebrate cells. However, the functional role of CENP-C in cell cycle progression remains unclear. To further understand CENP-C function, we developed a method incorporating the hyper-recombinogenic chicken B lymphocyte cell line DT40 to create several temperature-sensitive CENP-C mutants in DT40 cells. We found that, under restrictive conditions, one temperature-sensitive mutant, ts4-11, displayed metaphase delay and chromosome missegregation but proceeded through the cell cycle until arrest at G(1) phase. Furthermore, ts4-11 cells were transfected with a human HeLa cell cDNA library maintained in a retroviral vector, and genes that suppressed the temperature-sensitive phenotype were identified. One of these suppressor genes encodes SUMO-1, which is a ubiquitin-like protein. This finding suggests that SUMO-1 may be involved in centromere function in vertebrate cells. The novel strategy reported here will be useful and applicable to a wide range of proteins that have general cell-autonomous function in vertebrate cells.

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Figures

Figure 1
Figure 1
Creation of temperature-sensitive CENP-C mutants. (A) Experimental strategy for isolation of temperature-sensitive mutant clones. (B) Positions of mutations in temperature-sensitive CENP-C mutants. Cells were transfected with 10 different constructs as described in the Materials and Methods. The four mutations shown here displayed temperature sensitivity. (C) Sequencing analysis of an RT–PCR product from the temperature-sensitive CENP-C mutant ts4-11.
Figure 2
Figure 2
A temperature-sensitive CENP-C mutant displays growth arrest at 43°C. Representative growth curves for the indicated cell cultures. Cell viability was determined by trypan blue exclusion. Each experiment was performed twice, and each time point was examined in duplicate. When ts4-11 cells were incubated at 43°C, cells ceased proliferating by 12 h, and no viable cells were observed at 60 h. Wild-type DT40 cells proliferated normally at 43°C.
Figure 3
Figure 3
Localization of mutant CENP-C to the centromere is not changed under restrictive conditions. (A) Immunofluorescence analysis of ts4-11 cells stained with both anti-CENP-C and anti-CENP-H antibodies after 24 h at 43°C. The CENP-H signal is red, and the CENP-C signal is green. DNA was counterstained with DAPI (blue). (B) Immunofluorescence analysis of ts4-11 cells stained with both anti-CENP-A and anti-CENP-C antibodies after 24 h at 43°C. CENP-A signal is red, and CENP-C signal is green. DNA was counterstained with DAPI (blue). (C) Western blot analysis with anti-CENP-C antibody of ts4-11 cell extracts at the indicated times following incubation at 43°C. We also analyzed wild-type DT40 cells.
Figure 3
Figure 3
Localization of mutant CENP-C to the centromere is not changed under restrictive conditions. (A) Immunofluorescence analysis of ts4-11 cells stained with both anti-CENP-C and anti-CENP-H antibodies after 24 h at 43°C. The CENP-H signal is red, and the CENP-C signal is green. DNA was counterstained with DAPI (blue). (B) Immunofluorescence analysis of ts4-11 cells stained with both anti-CENP-A and anti-CENP-C antibodies after 24 h at 43°C. CENP-A signal is red, and CENP-C signal is green. DNA was counterstained with DAPI (blue). (C) Western blot analysis with anti-CENP-C antibody of ts4-11 cell extracts at the indicated times following incubation at 43°C. We also analyzed wild-type DT40 cells.
Figure 4
Figure 4
A temperature-sensitive CENP-C mutant displays chromosome missegregation under restrictive conditions. (A) To examine chromosome loss, we used FISH analysis with chromosome-specific painting probes. We used probes specific for chicken chromosomes 1 and 2. Because DT40 has three copies of chromosome 2, we observed five painted chromosomes in ts4-11 cells at the permissive temperature (left panel). ts4-11 cells showing loss of chromosomes (right panel) were detected after incubation of cells at 43°C. (B) Distribution of the number of painted chromosomes per cell. ts4-11 cells were cultured at 43°C for 24 h. Wild-type DT40 cells were cultured at 43°C as a control. The numbers of painted chromosomes 1 and 2 were scored in approximately 200 metaphase cells. After colcemid treatment, we observed that ∼50% of cells were in metaphase in control cultures and ∼25% of cells were in metaphase after 24 h at 43°C.
Figure 5
Figure 5
Cell cycle progression of a temperature-sensitive CENP-C mutant. (A) Cell-cycle distribution of ts4-11 cells following culture at 43°C. Cells were stained with FITC-anti-BrdU (y-axis, log scale) to detect BrdU incorporation 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 indicate the percentage of gated events. (B) Cell cycle progression of synchronized cultures of DT40 (left) or ts4-11 (right) cells. Both cell lines were cultured at 43°C. Cells were synchronized at mitosis with nocodazole. After release of the block, we measured DNA content by FACS at 2-h intervals.
Figure 5
Figure 5
Cell cycle progression of a temperature-sensitive CENP-C mutant. (A) Cell-cycle distribution of ts4-11 cells following culture at 43°C. Cells were stained with FITC-anti-BrdU (y-axis, log scale) to detect BrdU incorporation 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 indicate the percentage of gated events. (B) Cell cycle progression of synchronized cultures of DT40 (left) or ts4-11 (right) cells. Both cell lines were cultured at 43°C. Cells were synchronized at mitosis with nocodazole. After release of the block, we measured DNA content by FACS at 2-h intervals.
Figure 6
Figure 6
Identification of genes that suppress the temperature-sensitive CENP-C mutant phenotype. (A) Experimental strategy for isolation of genes that suppress the temperature-sensitive CENP-C mutant phenotype. (B) The temperature-sensitive phenotype was suppressed by introduction of the human SUMO-1 but not human CENP-A or chicken CENP-H gene.
Figure 6
Figure 6
Identification of genes that suppress the temperature-sensitive CENP-C mutant phenotype. (A) Experimental strategy for isolation of genes that suppress the temperature-sensitive CENP-C mutant phenotype. (B) The temperature-sensitive phenotype was suppressed by introduction of the human SUMO-1 but not human CENP-A or chicken CENP-H gene.
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