EVALUATION OF RHODAMINE-123 AS A PROBE FOR MONITORING MITOCHONDRIAL-FUNCTION IN TRYPANOSOMA-BRUCEI SPP

Rhodamine 123, a membrane potential-specific dye, has been evaluated a s a probe to monitor the function of the mitochondrion in long slender bloodstream and procyclic trypomastigotes of several Trypanosoma bruc ei spp. By epifluorescence microscopy, mitochondrial development has b een followed in long slender bloodstream and procyclic organisms stain ed with rhodamine 123. To photograph stained long slender bloodstream forms, it was necessary to develop a method to completely immobilize v iable organisms. In both parasite forms, as the cell cycle progressed, the mitochondrion developed from a thread-like structure to a highly branched organelle. A dramatic reorganization occurred preceding cytok inesis to produce two progeny thread-like structures which were partit ioned into newly formed daughter cells. The organelle within the long slender trypomastigote was found to stain optimally at 0.3 mug/ml of r hodamine 123, while the procyclic for-m required 3.0 mug/ml. The resul ts suggest that the plasma membrane potential is higher in the long sl ender parasite than in the procyclic form. The effects of inhibitors t hat disrupt mitochondrial function were examined in long slender and p rocyclic parasites, and some of these agents were shown to affect rhod amine 123 accumulation and retention. In long slender trypomastigotes the trypanosome alternative oxidase does not appear to be coupled to p roton pumping, whereas in procyclic organisms the effects of inhibitor s indicate that this oxidase may be coupled to a pathway that is branc hed preceding an antimycin A1-sensitive site.