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

We evaluated roGFP1 and roGFP2 with physiologically or toxicologically relevant oxidants both in vitro and in living mammalian cells

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

2004

Scholarcy highlights

  • Cells have elaborate homeostatic mechanisms to regulate of the indicator
  • It has been speculated that Disulfide formation between the cysteine residues promotes the cytoplasm is reducing because many metabolic reactions protonation of the chromophore and increases the excitation evolved before oxygen became abundant in the atmosphere . spectrum peak near 400 nm at the expense of the peak near 490
  • Confirmation of Redox Potential—Preliminary experiments showed that roGFP1 and roGFP2 in the cytosol of mammalian cells were largely reduced, so it was important to characterize the behavior of reduced roGFP1 and roGFP2 in vitro
  • We found distinct redox potentials when using lipoate or BMES as standard solutions; the values for roGFP1 and roGFP2 differed by the identical values
  • These values, EЈ0 ϭ Ϫ294 and Ϫ287 mV for roGFP1 and roGFP2, respectively, are somewhat more negative than the redox potentials reported by Hanson et al based on DTT as standard, Ϫ288 and Ϫ272 mV, the ordering of roGFP1 as more reducing than roGFP2 is preserved
  • Cells were grown in IMDM for 2 days before oxidation experiments
  • Genetic encoding means that the probes can be introduced into any cell or organism that can express recombinant cDNA, that the proteins can be targeted to specific subcellular locations or tissue distributions, and that reagent distribution costs are minimized
  • Using roGFPs, we were unable to demonstrate any response of the redox probes to two growth factor stimuli reported to generate cellular H2O2: epidermal growth factor in NR6 cells or lysophosphatidic acid in HeLa cells

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