Estimation of the electric plasma membrane potential difference in yeast with fluorescent dyes: comparative study of methods.
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.
Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Ciudad Universitaria, 04510 Mexico, D. F., México. apd@ifc.unam.mx
Antonio
A
Peña
Norma Silvia
NS
Sánchez
Martha
M
Calahorra
D001693:Biological Transport, Active / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D002122:Calcium Chloride / Q000378:metabolism; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000378:metabolism; D002462:Cell Membrane / Q000502:physiology*; D019069:Cell Respiration / Q000502:physiology; D005456:Fluorescent Dyes; D008564:Membrane Potentials / Q000502:physiology*; D018613:Microscopy, Confocal; D011188:Potassium / Q000378:metabolism; D011189:Potassium Chloride / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000502:physiology*
D005456:Fluorescent Dyes; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D011189:Potassium Chloride; D002122:Calcium Chloride; D011188:Potassium; D002118:Calcium