Factors underlying membrane potential-dependent and -independent fluorescence responses of potentiometric dyes in stressed cells: diS-C-3(3) in yeast

The redistribution fluorescent dye diS-C-3(3) responds to yeast plasma memb rane depolarisation or hyperpolarisation by Delta psi -dependent outflow fr om or uptake into the cells, reflected in changes in the fluorescence maxim um lambda (max) and fluorescence intensity. Upon membrane permeabilisation the dye redistributes between the cell and the medium in a purely concentra tion-dependent manner, which gives rise to Delta psi -independent fluoresce nce responses that may mimic Delta psi -dependent blue or red shift in lamb da (max). These lambda (max) shifts after cell permeabilisation depend on p robe and ion concentrations inside and outside the cells at the moment of p ermeabilisation and reflect (a) permeabilisation-induced Delta psi collapse , (b) changing probe binding capacity of cell constituents (inverse to the ambient ionic strength) and (c) hampering of probe equilibration by the poo rly permeable cell wall. At low external ion concentrations, cell permeabil isation causes ion outflow and probe influx (hyperpolarisation-like red shi ft in lambda (max)) caused by an increase in the probe-binding capacity of the cell interior and, in the case of heat shock, protein denaturation unma sking additional probe-binding sites. At high external ion levels minimisin g net ion efflux and at high intracellular probe concentrations at the mome nt of permeabilisation, the Delta psi collapse causes a blue lambda (max) s hift mimicking an apparent depolarisation. (C) 2001 Elsevier Science B.V. A ll rights reserved.