AbstractpHluorin is a pH-sensitive variant of green fluorescent protein for measuring intracellular pH (pH in ) in living cells. We constructed a new pHluorin plasmid with the dominant selection marker KanMX. This plasmid allows pH measurements in cells without auxotrophic mutations and/or grown in chemically indefinite media. We observed differing values of pH in for three prototrophic wild-types. The new construct was also used to determine the pH in in strains differing in the activity of the plasma membrane Pma1 H + -ATPase and the influence of glucose on pH in . We describe in detail pHluorin measurements performed in a microplate reader, which require much less hands-on time and much lower cell culture volumes compared to standard cuvettes measurements. We also utilized pHluorin in a new method of measuring the buffering capacity of yeast cell cytosol in vivo, shown to be ca. 52 mM/pH for wild-type yeast and moderately decreased in mutants with affected potassium transport.
Fluorescence spectral properties of calcofluor (a popular stain used to visualize cell walls of bacteria, yeast and fungi) has been studied. The analysis of calcofluor fluorescence emission spectra measured in a wide range of solvents (including media containing chitin), and in yeast cell suspensions has revealed that the solvatochromic properties of calcofluor ensue essentially from the by solvent-solute hydrogen bonding, or from the hydrogen bonding to cell wall polysaccharides with an eventual contribution of calcofluor aggregation at the cell surface. Preliminary data suggest that calcofluor emission spectra can be employed as a practical marker of variations in the quality of yeast cell wall.
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