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Intracellular pH homeostasis during cell-cycle progression and growth state transition in Schizosaccharomyces pombe

Department of Biology, Queen's University, Kingston, ON, Canada, K7L 3N6
^* Author for correspondence (e-mail: youngpg{at}biology.queensu.ca )

View larger version (49K): [in a new window]   Fig. 1. Phluorin expression in S. pombe. (A) Wild-type cells carrying empty pREP1 or pREP1-phluorin vectors were grown to early logarithmic phase in EMM pH 5.5 and examined. Images were acquired using Nomarski optics or dual channel fluorescence microscopy. Emission from the low excitation channel was arbitrarily given a false colour of blue, whereas emission from the high excitation channel was given a false colour of green. The final colour of the image is the combination of the two channels. (B) FACS analysis of phluorin-expressing cells. (a) Wild-type carrying pREP1-phluorin. (b) cdc2-1w carrying pREP1-phluorin. (c) Wildtype carrying empty pREP1. (d) Wild-type cells grown in rich media to logarithmic phase (2C control). (e) cdc10-129 cells arrested in G1 (1C control). (C) Phluorin standard curve. Phluorin-expressing wild-type cells were permeabilized in buffers of the indicated pH and photographed at the two excitation wavelengths. The means of the calculated ratios of emission intensity (low/high) are plotted for 40 cells at each pH. Error bars indicate standard deviations. False colour images for each pH are shown to the right of the figure. (D) Phluorin-expressing wild-type cells were grown in EMM pH 5.5 to early logarithmic phase and examined. Examples of cells in different phases of the cell cycle along with their respective cell lengths are indicated: (a) short uni-nucleate (late S/early G2); (b) long uni-nucleate (late G2); (c) non-septated binucleate (late M/early G1); (d) septated binucleate (late G1/early S). Note that cells in late G2 to septation are in the constant length phase of the cell cycle (Mitchison, 1957). (E) pHi as a function of emission intensity. Phluorin-expressing wild-type cells were grown to early logarithmic phase in EMM pH 3.5 (), EMM pH 4.5 (), EMM pH 5.5 () or EMM pH 6.5 ([UNK]) and examined. The plotted results are normalized to the most weakly expressing cell in each data set. n ranges from 60-120. (F) pHi as a function of external pH. Phluorin-expressing wild-type cells were grown to early logarithmic phase in EMM of the indicated pH and examined. The plotted results are the average of three independent replicates. In each replicate, 20-40 cells were sampled for every external pH value tested. Error bars indicate s.d. (G) Phluorin-expressing wild-type cells were grown to early logarithmic phase in EMM pH 5.5 and mounted in the same medium in the flow chamber. After a 30-60 minute equilibration time, medium was switched (t=0) to EMM (adjusted to a final pH of 5.5) containing either 15 mM acetic acid () or 200 mM bicarbonate (). Images were acquired at the indicated times. 20 cells were sampled per time point. Error bars indicate s.d.

View larger version (17K): [in a new window]   Fig. 2. Effects of various stresses on pHi homeostasis. pHi was measured in wild-type cells treated as described in Materials and Methods. pHi after heat shock, cold shock, hyper-osmotic shock, and hypo-osmotic shock were measured at t=1 hour, whereas pHi after high density incubation was measured at t=3 hours. pHi measurements after all other stresses were made within 12 minutes of the insult. n ranges from 20 to 40.

View larger version (30K): [in a new window]   Fig. 3. pHi during the cell cycle. Phluorin-expressing wild-type or cdc2-1w cells were grown to early logarithmic phase in EMM pH 5.5 and the pHi and cell length measured for each individual cell. Top: scatter plots of internal pH as a function of cell length. Open diamonds indicate binucleate cells, whereas closed diamonds indicate uni-nucleates. The linear line of best fit is shown in red. Approximate cell-cycle phases are indicated. Bottom: cells were classified into arbitrary 1 µm size classes and the mean internal pH of the created classes calculated. Error bars indicate s.d.

View larger version (41K): [in a new window]   Fig. 4. Effects of nutritional shifts on pHi. Phluorin-expressing wild-type cells were grown to early logarithmic phase in EMM(glutamine) pH 5.5 (left) or EMM(proline) pH 5.5 (right) and mounted in the same media in the flow chamber. After a 30-60 minute equilibration time the perfusion medium was switched (t=0) to EMM(proline) pH 5.5 (left) or EMM(glutamine) pH 5.5 (right). The top panels show the mean internal pH of 20-40 cells plotted as a function of time. Error bars indicate s.d. Lower panels are scatter plots of internal pH as a function of cell length at the indicated times. Open diamonds indicate binucleate cells whereas closed diamonds indicate uni-nucleates.

View larger version (13K): [in a new window]   Fig. 5. pHi homeostasis in wild-type and temperature-sensitive cell-cycle mutants under restrictive conditions. (A) Phluorin-expressing wild-type (), cdc10-129 (), cdc22-M45 (), or cdc25-22 ([UNK]) cells were grown to early logarithmic phase at 25°C in EMM pH 5.5 and then mounted in the same media in the flow chamber. After a 60-minute equilibration time the temperature was shifted (t=0 minutes) to 36°C. A wild-type strain () maintained at 25°C for the entire experiment is included as a control. n=20 for each time point for each individual strain. (B) Phluorin-expressing wild-type (open bars) or nda3-KM311 (solid bars) were grown to early logarithmic phase at 35°C in EMM pH 5.5. The culture was then split in two, half being maintained at 35°C and half being transferred to 20°C for 8 hours before images were acquired. n=20 for each time point for each individual strain.

View larger version (55K): [in a new window]   Fig. 6. Measurement of pHi 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).

View larger version (22K): [in a new window]   Fig. 7. Phluorin-expressing wild-type cells were grown to early logarithmic phase in EMM pH 5.5 and mounted in the same media in the flow chamber. After a 30-60 minute equilibration time the medium was switched (t=0) to EMM(minus nitrogen) pH 5.5. The top panel shows the mean internal pH of 20-40 cells plotted as a function of time. Error bars indicate s.d. Lower panels are scatter plots of internal pH as a function of cell length at the times indicated. Open diamonds indicate binucleate cells, whereas closed diamonds indicate uni-nucleates.

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