High-throughput assessment of mitochondrial membrane potential in situ using fluorescence resonance energy transfer

James A. Dykens, Beth Fleck, Soumitra Ghosh, Michelle Lewis, Gonul Velicelebi, Manus Ward

    Research output: Contribution to journalArticlepeer-review

    14 Scopus citations

    Abstract

    Mitochondrial dysfunction causes dozens of debilitating diseases, and is implicated in the etiology of type 2 diabetes, Parkinson's and Alzheimer's diseases, among others. However, development of mitochondrially targeted therapeutic agents has been impeded by the lack of high-throughput screening techniques that are capable of distinguishing in intact cells the mitochondrial membrane potential (Δψp) from the plasma membrane potential, (Δψp). We report here a fluorescence resonance energy transfer (FRET) assay that specifically monitors Δψp that is not confounded by background signal arising from potentiometric dye responding to Δψp. The technique relies on energy transfer between nonyl acridine orange (NAO), which stains diphosphatidyl glycerol (cardiolipin) that is indigenous to the inner mitochondrial membrane, and tetramethylrhodamine methyl ester (TMR), a potentiometric dye that is sequestered by mitochondria as a Nernstian function of Δψm and concentration. FRET occurs only when both dyes co-localize to the mitochondria, and results in quenching of NAO emission by TMR in proportion to Δψm. Validation studies using compounds with well-characterized mitochondrial effects, including oligomycin, CCCP+, bongkrekie acid, cyclosporin A, nigericin, ADP, and ruthenium red, demonstrate that the FRET-based Δψm assay responds in accord with the known pharmacology. Validation studies assessing the suitability of the technique for high-throughput compound screening indicate that the assay provides a sensitive and robust assessment not only of mitochondrial integrity in situ, but also, when used in conjunction with agents such as cyclosporin A, an indicator of permeability transition.

    Original languageEnglish (US)
    Pages (from-to)461-473
    Number of pages13
    JournalMitochondrion
    Volume1
    Issue number5
    DOIs
    StatePublished - Jan 1 2002

    Keywords

    • Alzheimer's disease
    • Apoptosis
    • Excitotoxicity
    • High-throughput screening
    • Neurodegenerative diseases
    • Parkinson's disease
    • Reperfusion injury
    • Type 2 diabetes

    ASJC Scopus subject areas

    • Molecular Medicine
    • Molecular Biology
    • Cell Biology

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