Fluorescent probing of membrane potential in walled cells: diS-C3(3) assay in Saccharomyces cerevisiae

Yeast. 1998 Sep 30;14(13):1189-97. doi: 10.1002/(sici)1097-0061(19980930)14:13<1189::aid-yea320>3.3.co;2-b.

Abstract

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, lambda max, 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 lambda max 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 lambda max 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 (< or = 5 x 10(6) cells/ml) and probe (10(-7)M) concentrations at which a clearly defined relationship exists between the lambda max 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 micron, n = 30) are stained much faster and the lambda max is more red-shifted than in cells grown in 2% glucose (cell wall thickness 0.260 +/- 0.043 micron, n = 44). At a suitable cell and probe concentration and under standard conditions, the lambda max shift of diS-C3(3) fluorescence provides reliable information on even fast changes in membrane potential in Saccharomyces cerevisiae.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carbocyanines / chemistry
  • Carbonyl Cyanide m-Chlorophenyl Hydrazone / pharmacology
  • Cell Wall / physiology
  • Cell Wall / ultrastructure
  • Fluorescent Dyes / chemistry
  • Glucose / metabolism
  • Membrane Potentials / physiology
  • Microscopy, Electron
  • Microscopy, Fluorescence
  • Potassium / pharmacology
  • Protoplasts / chemistry
  • Protoplasts / physiology
  • Saccharomyces cerevisiae / physiology*
  • Saccharomyces cerevisiae / ultrastructure
  • Valinomycin / pharmacology

Substances

  • 3,3'-dipropylthiacarbocyanine
  • Carbocyanines
  • Fluorescent Dyes
  • Valinomycin
  • Carbonyl Cyanide m-Chlorophenyl Hydrazone
  • Glucose
  • Potassium