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  • 1
    ISSN: 0749-503X
    Keywords: carbocyanine fluorescent probes ; membrane potential ; yeast ; cell wall ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Membrane-potential-dependent accumulation of diS-C3(3) in intact yeast cells in suspension is accompanied by a red shift of the maximum of its fluorescence emission spectrum, λmax, caused by a readily reversible probe binding to cell constituents. Membrane depolarization by external KCl (with or without valinomycin) or by ionophores causes a fast and reproducible blue shift. As the potential-reporting parameter, the λmax shift is less affected by probe binding to cuvette walls and possible photobleaching than, for example, fluorescence intensity. The magnitude of the potential-dependent red λmax shift depends on relative cell-to-probe concentration ratio, a maximum shift (572→582 nm) being found in very thick suspensions and in cell lysates. The potential therefore has to be assessed at reasonably low cell (≤5×106 cells/ml) and probe (10-7 M) concentrations at which a clearly defined relationship exists between the λmax shift and the potential-dependent accumulation of the dye in the cells. The redistribution of the probe between the medium and yeast protoplasts takes about 5 min, but in intact cells it takes 10-30 min because the cell wall acts as a barrier, hampering probe penetration into the cells. The barrier properties of the cell wall correlate with its thickness: cells grown in 0·2% glucose (cell wall thickness 0·175±0·015 μm, n=30) are stained much faster and the λmax is more red-shifted than in cells grown in 2% glucose (cell wall thickness 0·260±0·043 μm, n=44). At a suitable cell and probe concentration and under standard conditions, the λmax shift of diS-C3(3) fluorescence provides reliable information on even fast changes in membrane potential in Saccharomyces cerevisiae. © 1998 John Wiley & Sons, Ltd.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
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