Imaging Dynamic Redox Changes in Mammalian Cells with Green Fluorescent Protein Indicators

TLDR

Redox equilibrium shifts influence many cellular functions, and redox‑sensitive GFPs provide genetically encoded, real‑time visualization of oxidation states through fluorescence ratios that normalize for probe amount. The authors evaluated roGFP1 and roGFP2 variants in vitro and in live mammalian cells, testing their responses to physiological oxidants, superoxide bursts, oxygen fluctuations, and H₂O₂‑stimulating agents such as epidermal growth factor and lysophosphatidic acid.

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(2)O(2)-stimulating agents, e.g. epidermal growth factor and lysophosphatidic acid.