Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators

Colette T. Dooley, Timothy Dore, George T. Hanson, W. Coyt Jackson, S. James Remington, Roger Y. Tsien

    Research output: Contribution to journalArticle

    Abstract

    Changes in the redox equilibrium of cells influence a host of cell functions. Alterations in the redox equilibrium are precipitated by changing either the glutathione/glutathione-disulfide ratio (GSH/GSSG) and/or the reduced/oxidized thioredoxin ratio. Redox-sensitive green fluorescent proteins (GFP) allow real time visualization of the oxidation state of the indicator. Ratios of fluorescence from excitation at 400 and 490 nm indicate the extent of oxidation and thus the redox potential while canceling out the amount of indicator and the absolute optical sensitivity. Because the indicator is genetically encoded, it can be targeted to specific proteins or organelles of interest and expressed in a wide variety of cells and organisms. We evaluated roGFP1 (GFP with mutations C48S, S147C, and Q204C) and roGFP2 (the same plus S65T) with physiologically or toxicologically relevant oxidants both in vitro and in living mammalian cells. Furthermore, we investigated the response of the redox probes under physiological redox changes during superoxide bursts in macrophage cells, hyperoxic and hypoxic conditions, and in responses to H 2O2-stimulating agents, e.g. epidermal growth factor and lysophosphatidic acid.

    Original languageEnglish (US)
    Pages (from-to)22284-22293
    Number of pages10
    JournalJournal of Biological Chemistry
    Volume279
    Issue number21
    DOIs
    StatePublished - May 21 2004

    Fingerprint

    Green Fluorescent Proteins
    Oxidation-Reduction
    Cells
    Imaging techniques
    Glutathione Disulfide
    Oxidation
    Thioredoxins
    Macrophages
    Epidermal Growth Factor
    Oxidants
    Superoxides
    Organelles
    Glutathione
    Visualization
    Fluorescence
    Mutation
    Proteins

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology

    Cite this

    Dooley, C. T., Dore, T., Hanson, G. T., Jackson, W. C., Remington, S. J., & Tsien, R. Y. (2004). Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators. Journal of Biological Chemistry, 279(21), 22284-22293. https://doi.org/10.1074/jbc.M312847200

    Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators. / Dooley, Colette T.; Dore, Timothy; Hanson, George T.; Jackson, W. Coyt; Remington, S. James; Tsien, Roger Y.

    In: Journal of Biological Chemistry, Vol. 279, No. 21, 21.05.2004, p. 22284-22293.

    Research output: Contribution to journalArticle

    Dooley, CT, Dore, T, Hanson, GT, Jackson, WC, Remington, SJ & Tsien, RY 2004, 'Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators', Journal of Biological Chemistry, vol. 279, no. 21, pp. 22284-22293. https://doi.org/10.1074/jbc.M312847200
    Dooley, Colette T. ; Dore, Timothy ; Hanson, George T. ; Jackson, W. Coyt ; Remington, S. James ; Tsien, Roger Y. / Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 21. pp. 22284-22293.
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