Figure 4.
Virtually complete loss of FoF1–ATPase leads to a sudden collapse of ΔΨm, lower ADP/ATP ratio, and reduced respiration rate in BSF parasites.A, flow cytometry analysis of TMRE-stained BSF Tb1 3’ RNAi–noninduced cells (−tet) and cells induced for 1 day and 2 days (+tet, 1 day and 2 days) to detect changes in the ΔΨm. The addition of FCCP served as a control for mitochondrial membrane depolarization (+FCCP) (means ± SD, n = 4, Student’s unpaired t-test). B, flow cytometry analysis of TMRE-stained BSF OSCP cDKO cells grown in the presence (+tet) or absence of tetracycline for 1 day and 2 days (−tet, 1 day and 2 days) to detect changes in the ΔΨm. The addition of FCCP served as a control for mitochondrial membrane depolarization (+FCCP) (means ± SD, n = 4, Student’s unpaired t-test). C, mitochondrial membrane polarization detected using safranin O dye in digitonin-permeabilized BSF Tb1 3’ RNAi–noninduced cells (−tet, black line) and cells induced for 2 days (+tet, 2 days, red line) in the presence of ATP. ATP, oligomycin (OLM), carboxyatractyloside (cATR), and SF 6847, an uncoupler, were added where indicated. cATR was added after OLM to test for any further depolarization of the mitochondrial membrane due to inhibition of the AAC, whose electrogenic activity can potentially contribute in the generation of ATP-stimulated ΔΨm. D, mitochondrial membrane polarization detected using safranin O dye in digitonin-permeabilized BSF OSCP cDKO cells grown in the presence (+tet, black line) or absence of tetracycline for 2 days (−tet, 2 days, red line) after the addition of ATP. ATP, oligomycin (OLM), carboxyatractyloside (cATR), and SF 6847, an uncoupler, were added where indicated. cATR was added after OLM to test for any further depolarization of the mitochondrial membrane due to inhibition of the AAC, whose electrogenic activity can potentially contribute in the generation of ATP-stimulated ΔΨm. E, relative ADP/ATP ratio in BSF Tb1 3’ RNAi–noninduced cells (−tet) and cells induced for 1 day and 2 days (+tet, 1 day and 2 days) (means ± SD, n = 6, Student’s unpaired t-test). F, relative ADP/ATP ratio in BSF OSCP cDKO cells cultured in the presence (+tet) or absence of tetracycline for 1 day and 2 days (−tet, 1 day and 2 days) (means ± SD, n = 6, Student’s unpaired t-test). G, oxygen consumption rates of digitonin-permeabilized BSF Tb1 3’ RNAi–noninduced cells (−tet, black line) and cells induced for 2 days (+tet, 2 days, red line) in the presence of glycerol-3-phosphate. Respiration was arrested by the addition of SHAM where indicated. H, oxygen consumption rates of digitonin-permeabilized BSF OSCP cDKO cells grown in the presence (+tet, black line) or absence of tetracycline for 2 days (−tet, 2 days, red line) after addition of glycerol-3-phosphate. Respiration was arrested by addition of SHAM where indicated. ΔΨm, mitochondrial membrane potential; AAC, ADP/ATP carrier; BSF, bloodstream form; cDKO, conditional double knock-out; FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; OSCP, oligomycin sensitivity-conferring protein; SHAM, salicylhydroxamic acid; TMRE, tetramethylrhodamine ethyl ester.