Assessment of Mitochondrial Membrane Potential in Intact and Detergent-Permeabilized Trypanosoma brucei Insect and...

Mitochondrial membrane potential (ΔΨ_m) is a critical component of the protonmotive force that drives ATP synthesis and supports essential mitochondrial functions, including metabolite transport, ion homeostasis, and protein import. In the parasitic protist Trypanosoma brucei, ΔΨ_m regulation is uniquely adapted across life cycle stages to meet changing metabolic demands. In the insect-stage procyclic form (PF), ΔΨ_m is generated by a canonical electron transport system (ETS), while in the bloodstream form (BF), where complexes III and IV are absent, ΔΨ_m is maintained by the reverse operation of ATP synthase, consuming ATP to pump protons. In T. brucei evansi, which lacks mitochondrial DNA, the ATP synthase is unable to translocate protons, and ΔΨ_m is sustained solely by electrogenic ADP/ATP exchange through the mitochondrial carrier. This chapter presents three complementary fluorescence-based methods to evaluate ΔΨ_m in T. brucei and T. b. evansi cells, highlighting their applicability to both intact and permeabilized parasites. We detail the use of two ΔΨ_m-sensitive dyes-TMRE, a cell-permeable dye suited for live-cell assays, and Safranine O, used in permeabilized preparations-and describe protocols for flow cytometry and fluorescence spectroscopy, respectively. These approaches allow robust, qualitative and semi-quantitative analysis of ΔΨ_m under different metabolic and experimental conditions. We address specific challenges associated with using fluorescent dyes to measure ΔΨ_m including issues of dye concentration, cellular permeability and potential artifacts that can affect interpretation of ΔΨ_m measurements.