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First published online September 29, 2004
doi: 10.1242/10.1242/jcs.01395

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Characterisation of Cdc25B localisation and nuclear export during the cell cycle and in response to stress

Department of Cell and Molecular Biology, Karolinska Institutet, S-171 77 Stockholm, Sweden

View larger version (65K): [in a new window]   Fig. 1. Characterisation of a Cdc25B peptide antibody. (A) The guinea pig anti-Cdc25B antibody B1:5 recognises YFP-Cdc25B, but not YFP-Cdc25A. Cell lysates from HeLa cells transfected with YFP-Cdc25A (lane A) and YFP-Cdc25B (lane B) were subjected to immunoblotting using B1:5 (anti-Cdc25B) and anti-GFP antibodies as indicated in the figure. (B) The B1:5 antibody recognises Cdc25B in whole cell lysates. HeLa cells were transfected with siRNA against Cdc25A (lane A) or Cdc25B (lane B). Cell lysates were harvested 48 hours after transfection and analysed by immunoblotting using the B1:5 antibody. (C) Assessment of antibody specificity by RNA interference. HeLa cells were microinjected with siRNA against Cdc25B (upper panel) or lamin A/C (lower panel) together with a pCFP-Golgi plasmid to mark injected cells. Thirty-five hours later, cells were fixed and stained with B1:5 and cyclin B1 antibodies. The B1:5 antibody stained all cells expressing cyclin B1. In cells microinjected with Cdc25B siRNA, but not with lamin A/C siRNA, this staining is diminished. Cells expressing cyclin B1 that also are microinjected with siRNA are indicated by the letter a.

View larger version (47K): [in a new window]   Fig. 2. Subcellular localisation of Cdc25B. (A) Representative images of Cdc25B-staining using the B1:5 antibody. Cells were co-stained with cyclin B1 antibodies and Hoechst 33342 was used to stain nuclei. (B) Nuclear and cytoplasmic Cdc25B levels increase as cyclin B1 accumulates. The fluorescence signal from a deconvoluted image was quantified, the average background from cells not expressing Cdc25B was subtracted and the quantified B1:5 signal (y-axis) was plotted against the quantified cyclin B1 signal (x-axis). Each point in the graph corresponds to one cell. (C) Cdc25B immunofluorescence of cells at different stages of mitosis.

View larger version (65K): [in a new window]   Fig. 3. Cdc25B nuclear export is mediated by a region between amino acids 54 and 67. (A) Schematic presentation of the N-terminus of Cdc25B. (B) Localisation patterns of EYFP fusion proteins containing parts of the Cdc25B N-terminus as indicated, in the presence or absence of leptomycin B (LMB). hTERT immortalized human fibroblasts were microinjected with constructs expressing parts of the Cdc25B N-terminus fused to YFP. During the last 4 hours before fixation half of the cells were incubated with leptomycin B. (C) Nuclear export of GST Cdc25B 1-67. Purified GST-Cdc25B was microinjected together with Texas Red-conjugated dextran into the nucleus of hTERT immortalized human fibroblasts. One hour later, cells were fixed and stained with anti-GST antibodies (green).

View larger version (36K): [in a new window]   Fig. 4. Mutation of leucine 62 to alanine impairs nuclear export of Cdc25B. (A) The amino acids 54 to 67 of Cdc25B, which constitutes NES2. The three leucines are indicated in bold and the residue subjected to site-directed mutagenesis is indicated above. (B) Localisation of YFP-Cdc25B3 or YFP-Cdc25B3(L62A) 30 hours after transfection in G0 hTERT immortalised human fibroblasts. (C) Localisation patterns of YFP-Cdc25B3, YFP-Cdc25B3 L62A, YFP-Cdc25B3 S323G and YFP-Cdc25B3 NLS- in G2 HeLa cells. Cells were synchronised by a double thymidine-block, transfected immediately after release and fixed after 8 hours. Cells with interphase nuclei were scored for nuclear, both nuclear and cytoplasmic, or cytoplasmic YFP fluorescence.

View larger version (35K): [in a new window]   Fig. 5. Mutation of NES2 reduces the export rate of Cdc25B3. Fluorescence loss in photobleaching (FLIP) of YFP-Cdc25B3, YFP-Cdc25B3(L62A), and YFP-Cdc25B3 together with leptomycin B (LMB) in G1 HeLa cells. Mitotic nocodazole-arrested HeLa cells were microinjected with expression vectors and released from nocodazole block. Six hours later, differential interference contrast (DIC) and YFP fluorescence images were acquired and cells were bleached in the cytoplasm for 45 seconds. The imaging and bleaching cycle was repeated 15 times. (A) Example of a FLIP experiment with YFP-Cdc25B3. Top four panels: Image before and after bleaching the right of the two daughter cells from the microinjected mitotic cell. The bleach area is circled in red. Bottom four panels: The same cells as above, after fixation and immunofluorescence with anti-GFP antibodies (red). (B) Quantification of FLIP. The nuclear fluorescence of the two daughter cells was quantified. After background subtraction, the value of nuclear fluorescence at the start of the experiment was normalised to 1. Measurements of nuclear fluorescence of the bleached cell relative to the unbleached cell are plotted in the graph. Each curve in the graph shows the average of six cells from three independent experiments (bars indicate standard deviation).

View larger version (29K): [in a new window]   Fig. 6. Induction of premature mitosis by overexpressing Cdc25B mutants. Cells were synchronised by a double thymidine-block, transfected with YFP-Cdc25B3, YFP-Cdc25B3(L62A), YFP-Cdc25B3(S323G), YFP-Cdc25B3(NLS-) or YFP alone immidiately after release and fixed after 8 hours. The different constructs were expressed at similar levels as determined by the intensity of YFP fluorescence. Cells expressing the constructs were counted and scored for premature chromatin condensation (% PCC) as judged by Hoechst 33342 staining. Data show the mean of three independent experiments. Images on the right show examples of a typical cell displaying prematurely condensed chromatin (a) and an interphase cell (b).

View larger version (46K): [in a new window]   Fig. 7. YFP-Cdc25B3 translocates to the cytoplasm after treatment with cycloheximide or exposure to UV-C. (A) Cycloheximide induces cytoplasmic translocation of Cdc25B. HeLa cells were microinjected with YFP-Cdc25B3, treated with 100 µg/ml cycloheximide and followed by time-lapse microscopy. The time after cycloheximide addition is indicated (in minutes). (B) Representative images of cells expressing YFP-Cdc25B3, YFP-Cdc25B3(L62A) and YFP-Cdc25B3(S323G) with or without UV-C irradiation. The different constructs were transfected into HeLa cells. Five hours after transfection, cells were irradiated with 50 J/m2 UV-C and fixed 1 hour later. (C) Representative images of cells expressing YFP-Cdc25B3 after UV-C treatment and addition of kinase inhibitors or leptomycin B. YFP-Cdc25B3 was transfected into HeLa cells. Four hours after transfection, cells were treated with SB202190 (SB, 10 µM), PD98059 (MEK, 50 µM), or leptomycin B (LMB, 20 nM) for 1 hour. At that point, cells were exposed to 50 J/m2 UV-C and fixed 1 hour later. (D) The cytoplasmic translocation of YFP-Cdc25B3 after UV-C treatment is abolished by a p38 inhibitor. HeLa cells were transfected with YFP-Cdc25B3 and 3 hours later SB202190 was added. Four hours after transfection cells were exposed to 50 J/m2 UV-C and fixed 1 hour later. Cells were scored as in Fig. 4. The graph shows the mean of 200 cells counted in three independent experiments.

View larger version (62K): [in a new window]   Fig. 8. Endogenous Cdc25B partly translocates to the cytoplasm after exposure to UV-C. One hour before 50 J/m2 UV-C treatment, HeLa cells were incubated with the p38 inhibitor SB202190 (SB), leptomycin B (LMB) or the MEK1 inhibitor PD98059 (MEK) as indicated in images on the left. Cells were fixed 1 hour after UV-C exposure. Representative images of the B1:5 and cyclin B1 immunostaining are shown. Cells that express high levels of cyclin B1 (G2 cells) are indicated with the letter a in the images showing Cdc25B staining (B1:5). The cytoplasmic translocation of Cdc25B is observed in cells treated with UV-C and with UV-C in combination with the MEK1 inhibitor but not in untreated cells, or cells treated with UV-C in combination with leptomycin B or the p38 inhibitor.