Investigating Mitochondrial Redox Potential with Redox-sensitive Green Fluorescent Protein Indicators*

By George T. Hanson, R. Aggeler, D. Oglesbee, M. Cannon, Roderick A. Capaldi, Roger Tsien, S. J. Remington. Journal of Biological Chemistry, 2004

955 citations recorded by the source corpus.

Current methods for determining ambient redox potential in cells are labor-intensive and generally require destruction of tissue. This precludes single cell or real time studies of changes in redox poise that result from metabolic processes or environmental influences. By substitution of surface-exposed residues on the Aequorea victoria green fluorescent protein (GFP) with cysteines in appropriate positions to form disulfide bonds, reduction-oxidation-sensitive GFPs (roGFPs) have been created. roGFPs have two fluorescence excitation maxima at about 400 and 490 nm and display rapid and reversible ratiometric changes in fluorescence in response to changes in ambient redox potential in vitro and in vivo. Crystal structure analyses of reduced and oxidized crystals of roGFP2 at 2.0- and 1.9-Å resolution, respectively, reveal in the oxidized state a highly strained disulfide and localized main chain structural changes that presumably account for the state-dependent spectral changes. roGFP1 has been targeted to the mitochondria in HeLa cells. Fluorometric measurements on these cells using a fluorescence microscope or in cell suspension using a fluorometer reveal that the roGFP1 probe is in dynamic equilibrium with the mitochondrial redox status and responds to membrane-permeable reductants and oxidants. The roGFP1 probe reports that the matrix space in HeLa cell mitochondria is highly reducing, with a midpoint potential near -360 mV (assuming mitochondrial pH ∼8.0 at 37 °C). In other work (C. T. Dooley, T. M. Dore, G. Hanson, W. C. Jackson, S. J. Remington, and R. Y. Tsien, submitted for publication), it is shown that the cytosol of HeLa cells is also unusually reducing but somewhat less so than the mitochondrial matrix.

Subfields: Biophysics, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/e77940dbca86de6a59d6dc77db5acde5ff7b6129

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Citations in the corpus

Mitochondria-Ros Crosstalk in the Control of Cell Death and Aging (2011, neutral): “proteins (roGFPs) [103, 104].”
Glutathione (2011, neutral): “These include glutathione-specifi c redox-sensitive protein variants of the yellow (rxYFP) or green (roGFP) fl uorescent protein (Hanson et al., 2004; Maulucci et al., 2009).”
Imaging Dynamic Redox Changes in Mammalian Cells with Green Fluorescent Protein Indicators* (2004, methodology): “These values, E0’ = -294 and –287 mV for roGFP1 and 2 respectively, are somewhat more negative than the redox potentials reported by Hanson et al.(9) based on DTT as standard, -288 and –272 mV, though the ordering of roGFP1 as more reducing than roGFP2 is preserved.”
Genetically encoded fluorescent indicator for intracellular hydrogen peroxide (2006, methodology): “Experiments that demonstrate temporal H 2 O 2 increase upon growth factor stimulation allowed comparison of HyPer with two other probes—H 2 DCFDA, the widely used dye for detection of intracellular ROS production, and roGFPs, recently developed redox-sensitive mutants of GF”
Energy Substrate Modulates Mitochondrial Structure and Oxidative Capacity in Cancer Cells (2004, background): “The ratiometric GFP pH (18) and rosGFP1 (20) probes allow”
Fluorescent proteins and their applications in imaging living cells and tissues. (2010, neutral): “value (147, 193, 216, 280, 286, 362), concentrations of chloride (190, 464) and metal ions (31, 246), and redox potential (94, 146).”
Redox compartmentalization in eukaryotic cells. (2008, methodology): “The approaches have been complemented by techniques to measure redox potential in living cells, largely based upon partitioning of lipophilic cationic fluorescent chemicals into mitochondria dependent upon the high mitochondrial membrane potential (Δψ) [6] and development of redox-sensitive forms of”
Endocytosis of Nanomedicines (2010, background): “Examples include rab5 in early endosomes [13], caveolin-1 in caveolae [14], human Golgiresident enzyme N-acetylgalactosaminyltranferase-2 in Golgi [15], lysosomal association protein 1 (Lamp1) in lysosomes [16], signal sequence of calreticulin and endoplasmic reticulum (ER) retention signal KDEL in ”
Superoxide Flashes in Single Mitochondria (2008, positive): “Unlike GFP-based redox biosensors ( Hanson et al., 2004; Ostergaard et al., 2001), the fluorescence emission of cpYFP is unaltered when the redox potential varies between � 319 mV and � 7.5 mV (produced by differential mixing of reduced and oxidized DTT, Figure 1F).”
Unravelling the Biological Roles of Reactive Oxygen Species (2011, background): “These proteins incorporate redox-sensitive cysteines into the b barrel of GFP (Hanson et al., 2004) or integrate GFP and related derivatives into redox-regulated protein motifs, such as the hydrogen peroxide sensor from OxyR (Belousov et al., 2006) or the yeast peroxiredoxin Orp1 (Gutscher et al., 2”