Analysis of the uptake of the fluorescent marker 2′,7′-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) by hydrogenosomes in Trichomonas vaginalis

Abstract

The Duorescent dye 2′,7′-bis-(2-carboxyethyl)-5(and-6)-carboxyftuorescein (BCECF) has been widely used as an indicator of cytosolic pH. Here we report that BCECF localizes to hydrogenosomes (hydrogengenerating organelles found in several phylogenetically separate groups of anaerobic protists) in Trichomonas vaginalis, where it was observable by Duorescence microscopy. Its cellular location was confirmed by treatment of BCECF-loaded cells with diaminobenzidine and hydrogen peroxide together with UV illumination. This produced an osmiophilic precipitate in the matrix of hydrogenosomes, observable by electron microscopy. Use of a short (7.5 min) loading period, loading on ice, use of concentrations of BCECF (acetoxymethyl ester) down to 10 nM, and inclusion of the anion channel blockers probenicid or sulfmpyrazone, or the K+/H+ ionophore nigericin in the loading buffer all failed to prevent hydrogenosomal 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 electroporation of intact cells. Hydrogenosomal BCECF loading was also obtained with washed cell lysates, without cytoplasm or metabolic substrates. We tested a range of other Duorogenic dyes designed for cytosolic labeling, and found that the calcium indicator fura-2 (acetoxymethyl ester) and the cell viability marker Duorescein diacetate also labeled hydrogenosomes. The results illustrate a novel use for BCECF as a Duorescen marker for hydrogenosomes (the first such marker), but present a warning against the indiscriminate use of Duorogenic ester dyes to measure properties ofthe cytosol in hydrogenosome-containing organisms — the dyes may also be indicating the properties of the hydrogenosome.