Cytoplasmic microtubular system implicated in de novo formation of a Rabl-like orientation of chromosomes in fission yeast
Bunshiro Goto1,2,
Koei Okazaki1 and
Osami Niwa1,2,*
1 Kazusa DNA Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan 2 Chiba University Graduate School of Science, 1-33 Yayoi-cho, Inage-ku, Chiba, Chiba 263, Japan
Fig. 1. Increase of cell length during RTG. Number of cells with indicated length after the transfer to the rich medium (A) and those kept in the conjugation medium (B). Percentages of cells containing a spindle are shown in (C).
Fig. 2. Emergence of the Sad1-containing bodies in the zygotes of strain BG993. (A-C) Chr, DAPI; Sad1, anti-Sad1 antibody; Cut12, GFP-tagged Cut12; Merged: cyan, DAPI; red, Sad1; green, Cut12; overlapped region of Sad1 and Cut12, yellow. Bar, 2.5 µm. (A) 0 hours, (B,C) 4 hours after transferring to the rich medium. (D) Number of cells containing indicated number of the Sad1-bodies per nucleus at the indicated time after transfer.
Fig. 3. Presence of Kms1 and Spo15 in the Sad1-containing bodies. (A,C) 0 hours, (B,D) 4 hours after the nutritional transfer. In merged images: chromosomes, cyan; Sad1, red; Kms1 and Spo15, green.
Fig. 4. Association of the Sad1-bodies with cytoplasmic microtubules. Wild-type zygotes (BG991) were stained with DAPI (Chr), anti-Sad1 antibody (Sad1) and TAT1 (MT, microtubule). Merged: blue, DAPI; green, MT; and red, Sad1. (A,B) 3 hours, (C) 4 hours after the transfer. A spindle formed during RTG is shown in C. Bar, 2.5 µm.
Fig. 5. Positioning of telomeres and centromeres during RTG in wild-type zygotes of BG991. Chr, DAPI; Tel, FISH with cos212; Cen, FISH with pRS140; Sad1, anti-Sad1 antibody; Merged: blue, DAPI; green, cos212 (in A,B) or pRS140 (in C-F); red, Sad1. (A) 0 hours, (B) 4 hours (C,D) 0 hours, (E,F) 3 hours after transfer. Bars, 2.5 µm.
Fig. 6. Accumulation of centromeres near the telomere cluster. Wild-type zygotes (BG991) were prepared only for FISH. (A-C) Chr, DAPI; Cen, Cy3-labeled pRS104; Tel, Cy5-labeled cos212; Merged: cyan, DAPI; red, Cen; and green, Tel. (A) 0 hours, (B,C) 4 hours after transfer. Bar, 2.5 µm. (D) The frequency of the telomere clusters that were associated with at least one centromere.
Fig. 7. Accumulation of a centromere-proximal sequence near the telomere cluster. Zygotes of BG991 were prepared only for FISH. (A-D) Chr, DAPI; cos1228, Cy3-labelled cos1228; Tel, Cy5-labelled cos212; Merged: blue, DAPI; green, cos1228; red, cos212. Bar, 2.5 µm. Images were taken 0.5 hours (A,B) and 3 hours (C,D) after transfer. (E) Number of cells with indicated type of relative positioning of centromeres against the telomere cluster.
Fig. 8. Clustering of centromeres in the cdc25 mutant zygotes during RTG. Number of cells with indicated number of centromere signals are plotted against the time of incubation. When centromere signals were in contact, they were regarded as one signal. The single centromere cluster was always observed at the SPB. 0 hours is the time point when the incubation temperature was shifted to 35°C.
Fig. 9. Effect of a microtubule-destabilizing agent on the centromere clustering. Zygotes of the cdc25 mutant (BG992) were transferred into the rich medium and 2 hours later the incubation temperature was raised to 35°C (time 0). Cells were stained with DAPI (Chr), Cy3-labelled pRS140 (Cen) and anti-Sad1 antibody (Sad1). Merged images: white, DAPI; green, centromeres; red, Sad1. (A) 0 hours, (B-D) 3 hours after transfer. YE (B) or DMSO (C) was added as a control. (D) Cells were treated with 100 µg/ml of thiabendazole (TBZ). (E) Number of cells with clustered or scattered centromeres. Scattered centromeres are present in at least two sites. (F) In a separate experiment, TBZ was added 3 hours after the temperature shift and centromere clustering was observed 1 hour later. (E,F) Red, presumed SPB; green, centromeres. *This number includes two nuclei with clustered centromeres but detached from the SPB.
Fig. 10. Spindle dynamics and chromosome segregation in the first mitosis during RTG. Live observation of spindle elongation in RTG (B). (A) Spindles in ordinary mitoses of diploid cells were measured. (C,D) Typical images of early-to-late anaphase of the first mitosis. Chr, DAPI; cos1228, Cy3-labelled cos1228; MT, TAT1/Cy5; Cen, Cy5-labelled pRS140; Merged: white, DAPI; red, cos1228; green, TAT1 (C) and pRS140 (D).
