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First published online 14 April 2009
doi: 10.1242/jcs.046466

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Genetic control of cellular quiescence in S. pombe

¹ Okinawa Institute of Science and Technology (OIST), Initial Research Project, Uruma 904-2234, Okinawa, Japan
² Graduate School of Biological Science, Nara Institute of Science and Technology (NAIST), Ikoma, Nara 630-0192, Japan
³ Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan

View larger version (21K): [in this window] [in a new window]   Fig. 1. Requirement of seven genes for the entry into quiescence after the withdrawal of nitrogen source. (A) The life cycle of S. pombe. The entry into, the maintenance of, and the exit from the quiescence state are depicted. See also supplementary material Movie 1. (B) The DNA content of wild-type (WT) quiescent cells (24 hours in the -N medium) is shown. (C) Seven genes required for the quiescence entry are schematically shown.

View larger version (43K): [in this window] [in a new window]   Fig. 2. Quiescence entry requires stress-responsive kinases, MAPK and MAPKK. (A) DAPI-stained wild-type (WT), wis1-558, wis1-982 and sty1-989 cells cultured after 24 hours in -N medium at 26°C. Mutant cells revealed the extended nuclear chromatin. Scale bars: 1 µm. (B) The time-courses of the cell number increase (upper panel) and cell viability decrease (lower panel) were measured after the -N shift at 26°C. (C) The changes in DNA content over time were measured by FACScan after the -N shift at 26°C. (D) Electron micrographs of sty1-989 and wis1-982 after 24 hours in the -N medium. WT control is shown in Fig. 3E. Scale bars: 1 µm. (E) DAPI-stained WT and wis1-982 after the shift to -N medium. The nuclear extension occurred around 12 hours in wis1-982. (F) Extracts of WT, wis1-982 and sty1-989 cells cultured in the -N medium for 0-24 hours at 26°C were immunoblotted using antibodies against Rum1 (CDK inhibitor), Cdc13 (cyclin), Cig2 (S-phase cyclin), Cdc2 (PSTAIR) and -tubulin (TUB). (G) The deletion mutant rum1 was cultured in the -N medium at 26°C for 24 hours. DAPI-stained cells (scale bar: 1 µm) and their DNA content are shown.

View larger version (52K): [in this window] [in a new window]   Fig. 3. Vacuole dynamics by Ypt5, Vam6 and Vps11 controls entry into quiescence. (A) The time-courses of the cell number increase (upper panel) and the change in cell viability (lower panel) for vps11-319, vam6-532 and ypt5-909 cells after nitrogen starvation for 24 hours at 26°C are shown. (B) DAPI-stained cells and DNA contents of mutants after 24 hours at 26°C in the -N medium are shown. Scale bars: 1 µm. (C) Immunoblot of wild-type (WT), vps11-319, vam6-532 and ypt5-909 cells cultured in the -N medium for 0-24 hours at 26°C using indicated antibodies against Rum1, Cdc13, Cig2, PSTAIR and tubulin. (D) WT and mutant cells cultured in the -N medium after 24 hours were stained with FM4-64 specific for vacuole membranes. (E-H) Thin-section electron micrographs of WT (E), vps11-319 (F), vam6-532 (G) and ypt5-909 (H) cells after 24 hours in the -N medium, fixing with potassium permanganate and observation under a transmission microscope are shown. N, nucleus; V, vacuole; D, putative lipid droplets that are abundant in quiescence but scarce in proliferative cells. Scale bars: 1 µm.

View larger version (64K): [in this window] [in a new window]   Fig. 4. Osmo-environment in quiescence requires actin-interacting Wsp1 and End4. (A) DAPI-stained cells and DNA contents of wsp1-318 and end4-507 cells after 24 hours at 26°C in the -N medium. Scale bars: 1 µm. (B) The time-courses of the changes in cell number (upper panel) and cell viability (lower panel) after the shift to -N for 0-144 hours are shown. See text for an explanation of the adaptation of end4-507 in the -N medium. (C) Rhodamin-conjugated phalloidin was used to stain actin in wild-type (WT), wsp1-318 and end4-507 cells. Scale bars: 1 µm. (D-F) Time-lapse micrographs of wsp1-318 (D), end4-507 (E) and WT (F) cultured in the -N medium for 24 hours (96 hours for the right panel of end4) and replenished with the nutrient-rich YPD medium. Many cells of wsp1 and end4 after 14 hours in the YPD medium were physically disrupted or erupted followed with shrinkage (indicated by the arrows in D), although WT and end4-507 cells pre-cultured in the -N medium for 96 hours produced dividing cells in the replenished YPD medium (mono-polar growth for end4, 96 hours). See supplementary material Movies 5, 6 and 7. (G,H) Electron micrographs of wsp1-318 (G) and end4-507 (H) cells after 24 hours in the -N medium. In H, the mushroom-like structure is observed at the cortex (indicated by the red arrow and shown enlarged on the right). Scale bars: 1 µm. (I) Suppression of the viability loss by 1.2 M sorbitol. After 24 hours in the liquid -N medium, in the presence or absence of 1.2 M sorbitol, cells were plated on YPD with or without sorbitol. Each 300 cells of WT, wsp1-318 and end4-507 were plated at 26°C for 6 days.

View larger version (62K): [in this window] [in a new window]   Fig. 5. Twenty-six genes required for the maintenance of S. pombe quiescence. Seven genes for the quiescence entry are also shown (gray circles). See Table 2 and text for further details.

View larger version (54K): [in this window] [in a new window]   Fig. 6. Fcp1 CTD phosphatase affects a broad range of transcripts in the +N and -N media. (A) Fcp1 chromosomally tagged with GFP was observed in the quiescent -N medium of WT (left) and fcp1-452 mutant (right). Images stained with GFP (top), DAPI (middle) and merged (bottom) are shown. Scale bars: 1 µm. (B) Chromosomally tagged WT Fcp1-GFP and mutant Fcp1-452-GFP were expressed in the quiescent -N medium and detected by immunoblot using antibodies against GFP. The band is indicated by an arrowhead. (C) Transcriptomic patterns of WT and fcp1-452 mutant in the +N or -N medium at 26 and 37°C. Color panel indicates relative increase (red) and decrease (green) to the median (white) of eight different culture conditions for each transcript. Transcripts of proliferating (+N) cultures were obtained by shifting from 26 to 37°C (26°C in control) for 4 hours in the +N medium. Transcripts of quiescent (-N) cultures were obtained by shifting from 26°C (pre-culture for 24 hours in the -N medium) to 37°C (26°C in control) for 24 hours in the -N medium. (D) The number of gene transcripts for each category and overlapped categories.

View larger version (121K): [in this window] [in a new window]   Fig. 7. Thin-section electron microscopy of fcp1-452 mutant cells in the +N and the -N medium. Electron micrographs of fcp1-452 in the +N medium at 26°C (A) and 37°C (B), and in the -N medium at 26°C (C) and 37°C (D). Scale bars: 1 µm.

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