DEVELOPMENT OF THE FUN-1 FAMILY OF FLUORESCENT-PROBES FOR VACUOLE LABELING AND VIABILITY TESTING OF YEASTS

A new family of fluorescent probes has been developed for assessing th e viability and metabolic activity of yeasts, This class of halogenate d unsymmetric cyanine dyes is exemplified by the FUN-1 ro-4-(2,3-dihyd ro-3-methyl-(benzo-1,3-thiazol-2-yl -methylidene)-1-phenylquinolinium iodide] stain, a membrane-permeant nucleic acid-binding dye that has b een found to give rise to cylindrical intravacuolar structures (CIVS) in Saccharomyces cerevisiae. Biochemical processing of the dye by acti ve yeasts yielded CIVS that were markedly red shifted in fluorescence emission and therefore spectrally distinct from the nucleic acid-bound form of the dye, The formation of CIVS occurred under both aerobic an d anaerobic conditions and was highly temperature dependent. Treatment of yeasts with the nonmetabolizable glucose analog 2-deoxy-D-glucose reduced cellular ATP levels similar to 6-fold and completely inhibited CIVS formation, Under aerobic conditions, the formation of CIVS was a brogated by the cytochrome oxidase inhibitors azide and cyanide; howev er, the H+ transport uncoupler carbonyl cyanide m-chlorophenylhydrazon e inhibited CIVS formation under both aerobic and anaerobic conditions . Depletion of cellular thiols, including glutathione, with millimolar concentrations of N-ethylmaleimide, iodoacetamide, or allyl alcohol c ompletely inhibited CIVS production, Marked reduction in the formation of CIVS by ethacrynic acid and sulfobromophthalein, inhibitors of glu tathione S-transferase, suggested that dye processing can involve enzy me-mediated formation of glutathione conjugates, The conversion of FUN -1 by S. cerevisiae was studied quantitatively by using several techni ques, including fluorometry, flow cytometry, and wide-field and confoc al laser scanning fluorescence microscopy.