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First published online 26 March 2008
doi: 10.1242/dev.020115

Development 135, 1647-1657 (2008)
Published by The Company of Biologists 2008
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Live imaging of the Dictyostelium cell cycle reveals widespread S phase during development, a G2 bias in spore differentiation and a premitotic checkpoint

Tetsuya Muramoto and Jonathan R. Chubb*

Division of Cell and Developmental Biology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.


Figure 1
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  		Fig. 1. Dynamic localisation of GFP-PCNA in Dictyostelium cells. (A) Localisation of GFP-PCNA during and after mitosis. Timing is in minutes relative to mitosis. Arrows indicate appearance of a nuclear peripheral GFP-PCNA spot. (B) Nuclear GFP-PCNA peaks after mitosis and decays in G2. Bars reflect s.d. (C) Comparison of BrdU incorporation (red) with GFP-PCNA localisation (green); DAPI (blue). Two examples are shown for small foci and one-spot nuclei. Arrow indicates a peripheral BrdU spot colocalised to the GFP-PCNA spot, as observed in a number of cells (see table).

 

Figure 2
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  		Fig. 2. Dictyostelium heterochromatin is late replicating. (A) Localisation of RFP-PCNA and heterochromatin (HcpB) and nucleolar (eIF6) markers. Right-hand panels show merged images. (B) Localisation of PCNA and HcpB after mitosis. The HcpB spot is assembled then recruits nucleoplasmic PCNA. Arrows indicate the times at which HcpB and PCNA spots become visible.

 

Figure 3
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  		Fig. 3. Visualising the whole cell cycle of Dictyostelium. Cells expressing GFP-PCNA were imaged for complete cell cycles during asynchronous growth. Time (hours) from the first mitosis is indicated.

 

Figure 4
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  		Fig. 4. Timing and variability of cell cycle phases. Cell cycle analysis was performed on whole cycles of Dictyostelium cells expressing GFP-PCNA, in asynchronous culture. (A) Distribution of cell cycle timing for early S, late S and G2. (B) Frequency distribution of G2 variation between sister cells, representing the time cells remain in G2 after their sister has divided. Includes exponential fit. (C,D) Frequency distributions of S variation between sisters, representing the time cells remain in early (C) or late (D) S phase after their sister has finished dividing. Random pair comparisons are shown on the right.

 

Figure 5
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  		Fig. 5. Visualising mitosis and DNA replication in multicellular development. Mitotic cells were identified using RFP-H2B as a marker for chromatin. These cells were followed for up to 2 hours in the slug stage of development. GFP-PCNA was tracked to identify S-phase cells. The movie is of the prespore region of the slug. The first five frames include both RFP-H2B and GFP-PCNA. The RFP-H2B panels show a cell division in the central cell (arrows). The continuation of the movie (5:00 onwards), showing GFP-PCNA alone, allows one daughter cell (arrows) to be followed into S phase.

 

Figure 6
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  		Fig. 6. Developmental regulation of the cell cycle. (A) Live-cell measurements of lengths of M, early and late S phases in growing (growth), 3-hour developed (3 hrs) and slug cells. Bars indicate s.d. (B) Developmental variation in the proportion of the population in late S phase. Snapshots were collected of cells at different stages in development, using disaggregated cells for multicellular stages. Bars represent s.d. (C) Reconstructed image of an intact slug showing S-phase distribution. S-phase cells are abundant in the middle and posterior of slugs (prespore fate). Few S-phase cells are observed in the anterior of slugs (prestalk fate). (D) Effects of nutrition on cycle phases. Cells were cultured in standard imaging media (25% HL5/75% LF), bacterial and glucose-free media (25% HL5/75% LF without glucose).

 

Figure 7
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  		Fig. 7. Mitosis precedes S phase after spore germination. (A) Percentage of BrdU-positive cells after spore induction with DMSO; labelled with 100 µM BrdU for 30 minutes. Bars reflect s.d.; three replicates. (B) Onset of mitosis after spore germination. Percentage of cells dividing in 2-hour periods. First divisions occur in the 18-20 hour window. Two replicates. (C) Imaging-relative timing of division and S phase after spore germination, using GFP-PCNA as an S-phase marker. Stills from a movie of a germinated spore undergoing mitosis (arrows) are shown. Time after induction of germination (hours) is indicated.

 

Figure 8
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  		Fig. 8. Identification of a DNA damage checkpoint in Dictyostelium. (A) Cells were treated with bleomycin for 3 hours as indicated. The number of cell divisions occurring per hour was scored in mock-treated cells and those treated with 5 mU/ml or 20 mU/ml bleomycin. (B) After removal of bleomycin, cells escape the checkpoint and divide with increased synchronicity, as shown pictorially in these stills from a movie. Arrows indicate dividing cells.

 

Figure 9
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  		Fig. 9. Regulation of the DNA damage response. (A) Treatment of Dictyostelium cells with 30 mM caffeine and 20 mU/ml bleomycin causes a `mitotic catastrophe' phenotype. Two examples (i,ii) are shown. Cells enter an S-like state without division. (B) Response of Ku and DNA-PKcs mutant cells to bleomycin. At 20 mU/ml bleomycin, the Ku and DNA-PKcs mutants are impaired in the recommencement of cell division after checkpoint arrest.

 

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