Fig. 6. Measurement of pH_i in stationary phase cells. (A) Phluorin-expressing ade6-210 cells were grown to early logarithmic phase in EMM pH 5.5. The culture was subsequently transferred to EMM(minus NH₄Cl plus 1x10^-4 mg/ml adenine). Images were acquired 36 hours later using both the rhodamine (left) and GFP channels (right). (B) Phluorin-NLS-expressing wild-type cells were grown to early log phase in EMM pH 5.5. Images were acquired using Nomarski optics (left) or dual channel fluorescence microscopy (right). (C) Phluorin- and phluorin-NLS-expressing wild-type cells were grown concurrently (i.e. as a mixed population in the same culture) to early log phase in EMM pH 5.5 and analyzed. Emission intensities were calculated for both the nuclear and cytoplasmic compartments. Error bars indicate s.d. n=40 cells/compartment. (D) Phluorin-NLS-expressing wild-type cells were grown to early log phase in EMM pH 5.5 before being transferred to EMM(minus nitrogen). Images were acquired 36 hours after the transfer using Nomarski optics (left) and dual channel fluorescence microscopy (right). (E) Phluorin-NLS-expressing homothallic, or heterothallic cells were grown to early log phase in EMM pH 5.5 before being transferred to EMM(minus nitrogen) of the indicated pH. Images were acquired 36 hours after the transfer. Error bars indicate s.d. 20-40 cells/spores were sampled for every external pH value tested. Logarithmically growing control (); Vegetative cells ([UNK]); Vacuoles (); Ascospores (). (F) Phluorin-NLS-expressing homothallic wild-type cells were grown to early log phase in EMM pH 5.5 and transferred to EMM(minus nitrogen) to induce mating and the formation of asci. Images were acquired 36 hours after the transfer using Nomarski optics (left) and dual channel fluorescence microscopy (right). (G) Wild-type cells expressing either phluorin (open symbols) or phluorin-NLS (solid symbols) were grown to early log phase in EMM pH 5.5 and transferred to EMM(minus glucose) in the absence, or presence, of 1% ethanol at the indicated pH. Images were acquired 36 hours after the transfer. Error bars indicate s.d. 20-40 cells were sampled for every external pH value tested. Logarithmically growing control (); Vegetative cells+ethanol ([UNK]); Vegetative cells-ethanol (; Vacuoles (). (H) Phluorin-NLS-expressing wild-type cells were grown to early log phase in EMM(1% ethanol) and transferred to EMM(minus glucose, 1% ethanol) at a pH of 5.5. Images were acquired 36 hours after the transfer using Nomarski optics (left) and dual channel fluorescence microscopy (right).

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