ANALYSIS OF THE UPTAKE OF THE FLUORESCENT MARKER '-BIS-(2-CARBOXYETHYL)-5(AND-6)-CARBOXYFLUORESCEIN (BCECF) BY HYDROGENOSOMES IN TRICHOMONAS-VAGINALIS

The fluorescent dye '-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) has been widely used as an indicator of cytosolic pH. Here we report that BCECF localizes to hydrogenosomes (hydrogen-generating or ganelles found in several phylogenetically separate groups of anaerobi c protists) in Trichomonas vaginalis, where it was observable by fluor escence microscopy. Its cellular location was confirmed by treatment o f BCECF-loaded cells with diaminobenzidine and hydrogen peroxide toget her with UV illumination. This produced an osmiophilic precipitate in the matrix of hydrogenosomes, observable by electron microscopy. Use o f a short (7.5 min) loading period, loading on ice, use of concentrati ons of BCECF (acetoxymethyl ester) down to 10nM, and inclusion of the anion channel blockers probenicid or sulfinpyrazone, or the K+/H+ iono phore nigericin in the loading buffer all failed to prevent hydrogenos omal accumulation of BCECF. This uptake was best observed when intact cells were loaded with the ester form of BCECF, but could also be seen using free BCECF following either incubation with ruptured cells or e lectroporation of intact cells. Hydrogenosomal BCECF loading was also obtained with washed cell lysates, without cytoplasm or metabolic subs trates. We tested a range of other fluorogenic dyes designed for cytos olic labeling, and found that the calcium indicator fura-2 (acetoxymet hyl ester) and the cell viability marker fluorescein diacetate also la beled hydrogenosomes. The results illustrate a novel use for BCECF as a fluorescent marker for hydrogenosomes (the first such marker), but p resent a warning against the indiscriminate use of fluorogenic ester d yes to measure properties of the cytosol in hydrogenosome-containing o rganisms - the dyes may also be indicating the properties of the hydro genosome.