Article Figures & Data
Figures
- FIG. 1.
Subcellular localization of S. pombe PKA regulatory and catalytic subunits. (A) Immunoblot analysis of GFP fusion protein expression in cell lysates prepared from SP870 (Control), YMSM105 (cgs1-GFP), and YMSM101 (pka1-GFP) strains cultured overnight to log phase in YEAU medium. (B) Fluorescence photomicrographs of SP870 (Control), cgs1-GFP (Cgs1-GFP), and pka1-GFP (Pka1-GFP) S. pombe strains cultured in YEAU medium to mid-log phase. (C) Fluorescence photomicrographs of cgs1-GFP (Cgs1-GFP) and pka1-GFP (Pka1-GFP) cells were cultured in glucose-limited YEAU medium (YEAU-Glycerol) to mid-log phase.
- FIG. 2.
cAMP is required for nuclear localization of Cgs1-GFP and Pka1-GFP. (A) Fluorescence photomicrographs of cyr1Δ cgs1-GFP (left panels) and cyr1Δ pka1-GFP (right panels) cells cultured in YEAU (YEAU-glucose) or YEAU-glycerol to mid-log phase. (B) Fluorescence photomicrographs of cyr1Δ cgs1-GFP and cyr1Δ pka1-GFP cells cultured in YEAU with 100 mM cAMP for 1.5 h and YEAU with 5 mM cAMP for 0.5 h, respectively. (C) Fluorescence photomicrographs of pka1Δ cgs1-GFP cells cultured in YEAU to mid-log phase.
- FIG. 3.
Cgs1p contributes to regulation of subcellular localization of Pka1p. Fluorescence photomicrographs of cgs1Δ pka1-GFP (A) and cyr1Δ cgs1Δ pka1-GFP (B) strains cultured in YEAU (YEAU-Glucose) (left panels) or YEAU-Glycerol (right panels) to mid-log phase are shown.
- FIG. 4.
KCl-induced nuclear-cytoplasmic redistribution of Cgs1-GFP and Pka1-GFP. (A) Fluorescence photomicrographs of cgs1-GFP (Cgs1-GFP) and pka1-GFP (Pka1-GFP) cells cultured to mid-log phase in YEAU containing 1.2 M KCl (YEAU+KCl). cgs1-GFP (B) and pka1-GFP (C) strains were cultured in YEAU to 2 × 106 cells/ml and then diluted with YEAU containing 3 M KCl for a final concentration of 1.2 M KCl. The cultures were then examined by fluorescence microscopy at the indicated time points. (D) Fluorescence photomicrograph of cgs1Δ pka1-GFP cells cultured to mid-log phase in YEAU containing 1.2 M KCl.
- FIG. 5.
Sorbitol-induced nuclear-cytoplasmic redistribution of Cgs1p. (A) Fluorescence photomicrographs of cgs1-GFP (Cgs1-GFP) and pka1-GFP (Pka1-GFP) cells cultured to mid-log phase in YEAU containing 2 M sorbitol. (B) The cgs1-GFP strain was cultured in YEAU to 2 × 106 cells/ml and then diluted with an equal volume of YEAU containing 4 M sorbitol for a final concentration of 2 M sorbitol. The culture was then examined by fluorescence microscopy at the indicated time points.
- FIG. 6.
Nuclear-cytoplasmic redistribution of Cgs1 and Pka1 is induced during stationary phase. SP870 wild-type (A to C), cgs1-GFP (A), pka1-GFP (B), and cgs1Δ pka1-GFP (C) S. pombe strains were cultured in YEAU and examined by fluorescence microscopy at late log phase (6 × 107 cells/ml) and at 3-h intervals upon reaching stationary-phase density (about 108 cells/ml). (A) Cells imaged with 3-s digital camera exposures. (B and C) Cells imaged with 4-s camera exposures.
- FIG. 7.
Subcellular localization of Cgs1-GFP and Pka1-GFP during S. pombe sexual differentiation. Fluorescence photomicrographs of cgs1-GFP (left panels) and pka1-GFP (right panels) nitrogen-starved cells (Ai), zygotes (Aii to Aiv), and zygotic asci (Bi to Biii). (Ai) Cells were cultured in EMM, washed three times with EMM-nitrogen, resuspended in EMM-nitrogen at 2 × 106 cells/ml to induce nitrogen starvation, and incubated for 18 h at 28°C prior to photomicroscopy. (Aii to Aiv and B) Same conditions as for panel Ai, except the cells were resuspended at 2 × 107 cells/ml to induce mating (Aii to Aiv) and sporulation (B).
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