Pj. Millard et al., DEVELOPMENT OF THE FUN-1 FAMILY OF FLUORESCENT-PROBES FOR VACUOLE LABELING AND VIABILITY TESTING OF YEASTS, Applied and environmental microbiology, 63(7), 1997, pp. 2897-2905
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.