DISCRIMINATION OF RESPIRATORY DYSFUNCTION IN YEAST MUTANTS BY CONFOCAL MICROSCOPY, IMAGE, AND FLOW-CYTOMETRY

Living yeast cells can be selectively stained with the lipophilic cati onic cyanine dye DiOC(6)(3) in a mitochondrial membrane potential-depe ndent manner. Our study extends the use of now cytometric analysis and sorting to DiOC(6)(3)-stained yeast cells. Experimental conditions we re developed that prevented the toxic side effect of the probe and gav e a quantitative correlation between fluorescence and mitochondrial me mbrane potential, without any staining of other membranes. The localiz ation of the fluorochrome was checked by confocal microscopy and image cytometry. The mitochondrial membrane alterations were also tested th rough cardiolipin staining with nonyl acridine orange. Differences in light scattering and in fluorescence were detected in mutants (rho(-), rho degrees, mit(-), or pet(-)) and wild-type (rho(+)mit(+)) populati ons of yeast. The dye uptake of respiratory-deficient yeast strains wa s significantly reduced as compared to that of the wild-type. Applicat ion of an uncoupler (mClCCP), which collapsed the mitochondrial membra ne potential (Delta Psi(m)), led to a drastic reduction of the dye upt ake, It was observed that a decrease in Delta Psi(m) was usually corre lated with a decrease in cardiolipin stainability by nonyl acridine or ange (NAO). Quantitative flow cytometry is a fast and reproducible tec hnique for rapid screening of yeast strains that might be suspected of respiratory dysfunction and/or mitochondrial structural changes. We g ive evidence that it is an adequate method to characterize and isolate respiratory mutants through sorting procedure, with selective enrichm ent of the population studied in respiring or nonrespiring yeast cells . Confocal microscopy and image cytometry corroborate the now cytometr y results. (C) 1996 Wiley-Liss, Inc.