In Vivo Determination Of Organellar PH Using A Universal ... - PLOS

In vivo monitoring of pHi changes in individual cells during different stages of yeast colony development

Yeast colonies pass through distinct developmental phases characterized by changes in external pH and ammonia production [6], [7], [8]. The wavelength-based measurements from discrete compartments enabled the non-invasive intracellular quantification of actual physiological pH changes in cells taken from colonies at different stages of growth as well as cells exposed to volatile ammonia (NH3). Three different target proteins were used for this purpose: The first one, Ato1p, is a transmembrane protein with a cytosolic C-terminus whose expression is triggered by NH3 at the beginning of the alkali phase and maintained during the 2nd acidic phase. The other targets are transmembrane protein Jen1p, also with a cytosolic C-terminus, and cytoplasmic protein Met17p. The expression of both Jen1p and Met17p begins at the 1st acidic phase, long before ammonia production, and is maintained throughout the alkali and 2nd acidic phases of colony development (Figure 5). Ato1p-mCherry, Ato1p-pHluorin and Jen1p-mCherry constructs allowed us to monitor cytosolic pH in the periphery of the plasma membrane; while the Met17p-pHluorin construct provided information on the pH from internal regions of the cytoplasm.

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Figure 5. Confocal wavelength-based pH quantifications.

Developmental phases of yeast giant colonies growing on GMA plates with BKP pH indicator (A) and extracellular pH changes in the colony surroundings (B). Timings of ATO1, MET17 and JEN1 gene expression (black, violet and yellow bars) and the ammonia-producing alkali phase (purple bar) are indicated above. Emission wavelength box plots from peripheral ROIs within Ato1p-pHluorin (C) and Ato1p-mCherry cells (D) suspended in N-buffer of pH 6 under near-native conditions. CFM scans were performed during 1st acidic, alkali and 2nd acidic phases. 2nd acidic cells treated with 100 mM NH3 served as an alkalinization positive control. Each sample subset was selected to represent cell variability within the colony and contained a total of 30 subcellular ROIs from 3 independent biological repeats. Box plots indicate median, quartiles, extremes and individual values within each subset. Titration-based pH box plots of Ato1p-pHluorin (E) Ato1p-mCherry (F) Met17p-pHluorin (G) and Jen1p-mCherry (H) extrapolated from wavelength data. The effective range of the extrapolations was strictly defined by the pKa and quantitation limits of the titrations. Extrapolations from wavelength populations displaying out-of-range interquartile or medians (yellow box plots) must therefore be treated as non-quantitative and are included for comparison.

https://doi.org/10.1371/journal.pone.0033229.g005

Cells from Ato1p-mCherry, Ato1p-pHluorin, Jen1p-mCherry and Met17p-pHluorin yeast colonies occurring in different developmental phases were harvested and suspended in pH 6 N-buffer for immediate CFM analysis (Figure 5, A). Additional suspensions of 2nd-acidic cells were treated with high-dose NH3 (100 mM) for 9 minutes before measurements and analyzed as alkalinization positive controls. The timing of the phase transitions was monitored with control GMA plates containing pH indicator bromocresol purple (BKP) (pKa 6.3). Fluorescent signals from peripheral ROIs within both mCherry- and pHluorin-tagged strains underwent large changes in emission wavelength during the alkali/acid transition (Figure 5, C and D). One-way ANOVA and Tukey's post-hoc tests (α 0.05) of our Ato1p and Jen1p peripheral ROI data confirmed very significant differences (P<0.001) between alkali and acidic populations, while NH3 controls were comparable to alkali populations. Median, interquartiles and extreme values from the box plots do not represent measurement error, but the variability among the measurements from the 30 individual subcellular ROIs which comprise each sample cell population. Wavelength-based pHi extrapolations from Ato1p-mCherry and Jen1p-mCherry peripheral ROIs during the alkali phase or NH3 treatment (Figure 5, F and H) implied a substantial cytosolic alkalinization near the plasma membrane while those from 2nd-acidic Ato1p-pHluorin peripheral ROIs (Figure 5, E) indicated usual pHi in yeast cells under acidic conditions [6], [8], [14], [15], [16]; evidencing a correlation between peripheral cytosolic pH and the pH surrounding the colonies (Figure 5, B). In comparison, Met17p-pHluorin cells exhibited relatively smaller changes: extrapolations from Met17p-pHluorin cytosolic ROIs suggested more stable pHi conditions in deeper cytosolic regions throughout all stages of colony development (Figures 5, G and 6). In all experiments, samples exposed to high-dose NH3 exhibited significantly lower wavelength variability than untreated alkali populations due to the treatment's homogenizing effect on all cells. The physiological relevance of this finding is discussed below.

Comparative spectrofluorometric pH extrapolations with Ato1p-pHluorin cells and based on pHluorin's dual intensity ratios showed much lower pH fluctuations in the peripheral cytoplasm (Figure S5, A–B). Alkalinization was only noticeable after artificial NH3 treatment. These results were in opposition to the CFM wavelength-based measurements with identical cells, but strongly resembled the Met17p-pHluorin deep cytosolic results.