Estimation of the electric plasma membrane potential difference in yeast with fluorescent dyes: comparative study of methods

Abstract

Different methods to estimate the plasma membrane potential difference (PMP) of yeast cells with fluorescent monitors were compared. The validity of the methods was tested by the fluorescence difference with or without glucose, and its decrease by the addition of 10 mM KCl. Low CaCl₂ concentrations avoid binding of the dye to the cell surface, and low CCCP concentrations avoid its accumulation by mitochondria. Lower concentrations of Ba²+ produce a similar effect as Ca²+, without producing the fluorescence changes derived from its transport. Fluorescence changes without considering binding of the dyes to the cells and accumulation by mitochondria are overshadowed by their distribution between this organelle and the cytoplasm. Other factors, such as yeast starvation, dye used, parameters of the fluorescence changes, as well as buffers and incubation times were analyzed. An additional approach to measure the actual or relative values of PMP, determining the accumulation of the dye, is presented.

Effects of Chitosan onCandida albicans: Conditions for Its Antifungal Activity

“…For this purpose, the effects of the polymer were studied by estimating the membrane potential by the fluorescence changes of DiSC 3 (3). This method can be used in this yeast, as shown by the fluorescence increase observed when adding glucose to starved cells, as well as by the decrease produced by the addition of K + on this parameter [17]. It was found (Figure 3) that the addition of chitosan in fact produced an increase of the fluorescence, in agreement with what was suspected, that is, that the efflux of K + is electrogenic and hyperpolarizes the plasma membrane.…”

“…The plasma membrane potential difference was estimated as described before [17] following the fluorescence changes of DiSC 3 (3). Starved cells (12.5 mg·mL −1 ) were added to the spectrofluorometer cell containing 10 mM MES-TEA buffer, 20  μ M CCCP, 10  μ M BaCl 2 , and 200 nM cyanine, in a final volume of 2.0 mL.…”

High Osmolarity Environments Activate the Mitochondrial Alternative Oxidase in Debaryomyces Hansenii

“…Mitochondrial membrane potentials were estimated in situ in a cell suspension (1 mg.mL -1 ) by monitoring fluorescence changes of 3,3’-dihexiloxacarbocianine (1 nM, DiOC 6 ), (λ ex 482 nm, λ em 524 nm) [11] in a Hitachi F4500 fluorescence spectrophotometer. The incubation media was the same as in oxygen consumption experiments.…”

Proton-associated sucrose transport of mammalian solute carrier family 45: an analysis in Saccharomyces cerevisiae

“…To assess whether transport is coupled to protons as is the case for plant sucrose transporters as well as for two other members of the SLC45 family, the mammalian SLC45A1 [20] and the insect Slc45-1 [4], we checked transport in the presence of the specific protonophore carbonyl cyanide mchlorophenylhydrazone (CCCP) which has been shown to cause depolarization of the yeast plasma membrane [33,34]. Figure 2(A) shows a significant reduction in sucrose uptake by SLC45A2, SLC45A3 and SLC45A4 when CCCP was added, indicating proton-coupled sucrose transport.…”