Real-time imaging of the intracellular glutathione redox potential

Abstract

Dynamic analysis of redox-based processes in living cells is now restricted by the lack of appropriate redox biosensors. Conventional redox-sensitive GFPs (roGFPs) are limited by undefined specificity and slow response to changes in redox potential. In this study we demonstrate that the fusion of human glutaredoxin-1 (Grx1) to roGFP2 facilitates specific real-time equilibration between the sensor protein and the glutathione redox couple. The Grx1-roGFP2 fusion protein allowed dynamic live imaging of the glutathione redox potential (EGSH) in different cellular compartments with high sensitivity and temporal resolution. The biosensor detected nanomolar changes in oxidized glutathione (GSSG) against a backdrop of millimolar reduced glutathione (GSH) on a scale of seconds to minutes. It facilitated the observation of redox changes associated with growth factor availability, cell density, mitochondrial depolarization, respiratory burst activity and immune receptor stimulation.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Grx1 dynamically catalyzes rapid redox equilibration between glutathione and roGFP2.
Figure 2: Grx1-roGFP2 fusion protein allows live imaging of rapid intracellular redox changes.
Figure 3: Redox response of HeLa cells to growth-factor starvation and changes in cell density.
Figure 4: Grx1-roGFP2 visualizes redox changes associated with TRAIL-induced apoptosis.
Figure 5: Grx1-roGFP2 visualizes physiological redox changes associated with immune-cell activation.

References

  1. 1

    Dröge, W. Free radicals in the physiological control of cell function. Physiol. Rev. 82, 47–95 (2002).

    Article  Google Scholar 

  2. 2

    Menon, S.G. & Goswami, P.C. A redox cycle within the cell cycle: ring in the old with the new. Oncogene 26, 1101–1109 (2007).

    CAS  Article  Google Scholar 

  3. 3

    Balaban, R.S., Nemoto, S. & Finkel, T. Mitochondria, oxidants, and aging. Cell 120, 483–495 (2005).

    CAS  Article  Google Scholar 

  4. 4

    Filomeni, G. & Ciriolo, M.R. Redox control of apoptosis: an update. Antioxid. Redox Signal. 8, 2187–2192 (2006).

    CAS  Article  Google Scholar 

  5. 5

    Veal, E.A., Day, A.M. & Morgan, B.A. Hydrogen peroxide sensing and signaling. Mol. Cell 26, 1–14 (2007).

    CAS  Article  Google Scholar 

  6. 6

    Ramsey, M.R. & Sharpless, N.E. ROS as a tumour suppressor? Nat. Cell Biol. 8, 1213–1215 (2006).

    CAS  Article  Google Scholar 

  7. 7

    Colavitti, R. & Finkel, T. Reactive oxygen species as mediators of cellular senescence. IUBMB Life 57, 277–281 (2005).

    CAS  Article  Google Scholar 

  8. 8

    Lambeth, J.D. NOX enzymes and the biology of reactive oxygen. Nat. Rev. Immunol. 4, 181–189 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Houstis, N., Rosen, E.D. & Lander, E.S. Reactive oxygen species have a causal role in multiple forms of insulin resistance. Nature 440, 944–948 (2006).

    CAS  Article  Google Scholar 

  10. 10

    Jones, D.P. Redefining oxidative stress. Antioxid. Redox Signal. 8, 1865–1879 (2006).

    CAS  Article  Google Scholar 

  11. 11

    Trotter, E.W. & Grant, C.M. Overlapping roles of the cytoplasmic and mitochondrial redox regulatory systems in the yeast Saccharomyces cerevisiae. Eukaryot. Cell 4, 392–400 (2005).

    CAS  Article  Google Scholar 

  12. 12

    Hansen, J.M., Go, Y.M. & Jones, D.P. Nuclear and mitochondrial compartmentation of oxidative stress and redox signaling. Annu. Rev. Pharmacol. Toxicol. 46, 215–234 (2006).

    CAS  Article  Google Scholar 

  13. 13

    Pani, G. et al. Cell compartmentalization in redox signaling. IUBMB Life 52, 7–16 (2001).

    CAS  Article  Google Scholar 

  14. 14

    Rota, C., Chignell, C.F. & Mason, R.P. Evidence for free radical formation during the oxidation of 2′-7′-dichlorofluorescin to the fluorescent dye 2′-7′-dichlorofluorescein by horseradish peroxidase: possible implications for oxidative stress measurements. Free Radic. Biol. Med. 27, 873–881 (1999).

    CAS  Article  Google Scholar 

  15. 15

    Belousov, V.V. et al. Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat. Methods 3, 281–286 (2006).

    CAS  Article  Google Scholar 

  16. 16

    Ostergaard, H., Henriksen, A., Hansen, F.G. & Winther, J.R. Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein. EMBO J. 20, 5853–5862 (2001).

    CAS  Article  Google Scholar 

  17. 17

    Hanson, G.T. et al. Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators. J. Biol. Chem. 279, 13044–13053 (2004).

    CAS  Article  Google Scholar 

  18. 18

    Dooley, C.T. et al. Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators. J. Biol. Chem. 279, 22284–22293 (2004).

    CAS  Article  Google Scholar 

  19. 19

    Gravina, S.A. & Mieyal, J.J. Thioltransferase is a specific glutathionyl mixed disulfide oxidoreductase. Biochemistry 32, 3368–3376 (1993).

    CAS  Article  Google Scholar 

  20. 20

    Antunes, F. & Cadenas, E. Estimation of H2O2 gradients across biomembranes. FEBS Lett. 475, 121–126 (2000).

    CAS  Article  Google Scholar 

  21. 21

    Winterbourn, C.C. & Metodiewa, D. Reactivity of biologically important thiol compounds with superoxide and hydrogen peroxide. Free Radic. Biol. Med. 27, 322–328 (1999).

    CAS  Article  Google Scholar 

  22. 22

    Satoh, T., Sakai, N., Enokido, Y., Uchiyama, Y. & Hatanaka, H. Survival factor-insensitive generation of reactive oxygen species induced by serum deprivation in neuronal cells. Brain Res. 733, 9–14 (1996).

    CAS  Article  Google Scholar 

  23. 23

    Pani, G. et al. A redox signaling mechanism for density-dependent inhibition of cell growth. J. Biol. Chem. 275, 38891–38899 (2000).

    CAS  Article  Google Scholar 

  24. 24

    Franco, R. & Cidlowski, J.A. SLCO/OATP-like transport of glutathione in FasL-induced apoptosis: glutathione efflux is coupled to an organic anion exchange and is necessary for the progression of the execution phase of apoptosis. J. Biol. Chem. 281, 29542–29557 (2006).

    CAS  Article  Google Scholar 

  25. 25

    Danial, N.N. & Korsmeyer, S.J. Cell death: critical control points. Cell 116, 205–219 (2004).

    CAS  Article  Google Scholar 

  26. 26

    Ferri, K.F. & Kroemer, G. Mitochondria–the suicide organelles. Bioessays 23, 111–115 (2001).

    CAS  Article  Google Scholar 

  27. 27

    Zorov, D.B., Filburn, C.R., Klotz, L.O., Zweier, J.L. & Sollott, S.J. Reactive oxygen species (ROS)-induced ROS release: a new phenomenon accompanying induction of the mitochondrial permeability transition in cardiac myocytes. J. Exp. Med. 192, 1001–1014 (2000).

    CAS  Article  Google Scholar 

  28. 28

    Green, S.P. & Phillips, W.A. Activation of the macrophage respiratory burst by phorbol myristate acetate: evidence for both tyrosine-kinase-dependent and -independent pathways. Biochim. Biophys. Acta 1222, 241–248 (1994).

    CAS  Article  Google Scholar 

  29. 29

    Jackson, S.H., Devadas, S., Kwon, J., Pinto, L.A. & Williams, M.S. T cells express a phagocyte-type NADPH oxidase that is activated after T cell receptor stimulation. Nat. Immunol. 5, 818–827 (2004).

    CAS  Article  Google Scholar 

  30. 30

    Gulow, K. et al. HIV-1 trans-activator of transcription substitutes for oxidative signaling in activation-induced T cell death. J. Immunol. 174, 5249–5260 (2005).

    Article  Google Scholar 

  31. 31

    Ghezzi, P. Regulation of protein function by glutathionylation. Free Radic. Res. 39, 573–580 (2005).

    CAS  Article  Google Scholar 

  32. 32

    Meyer, A.J. et al. Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the redox potential of the cellular glutathione redox buffer. Plant J. 52, 973–986 (2007).

    CAS  Article  Google Scholar 

  33. 33

    Peltoniemi, M.J., Karala, A.R., Jurvansuu, J.K., Kinnula, V.L. & Ruddock, L.W. Insights into deglutathionylation reactions: different intermediates in the glutaredoxin and protein disulphide isomerase catalysed reactions are defined by the gamma-linkage present in glutathione J. Biol. Chem. 281, 33107–33114 (2006).

    CAS  Article  Google Scholar 

  34. 34

    Fernandes, A.P. & Holmgren, A. Glutaredoxins: glutathione-dependent redox enzymes with functions far beyond a simple thioredoxin backup system. Antioxid. Redox Signal. 6, 63–74 (2004).

    CAS  Article  Google Scholar 

  35. 35

    Björnberg, O., Østergaard, H. & Winther, J.R. Mechanistic insight provided by glutaredoxin within a fusion to redox-sensitive yellow fluorescent protein. Biochemistry 45, 2362–2371 (2006).

    Article  Google Scholar 

Download references

Acknowledgements

We thank J. Remington (University of Oregon) for providing the roGFP2 construct; M. Winkler, S. Hoffmann and E. Sollner for technical assistance; H. Walczak (German Cancer Research Center) for TRAIL protein; U. Engel and C. Ackermann (Nikon Imaging Center at the University of Heidelberg) for providing microscope access and assistance. Supported by the European Commission (Marie Curie Excellence Grant 2761 'Redox signaling' to T.P.D.).

Author information

Affiliations

Authors

Corresponding author

Correspondence to Tobias P Dick.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10, Supplementary Methods (PDF 366 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Gutscher, M., Pauleau, AL., Marty, L. et al. Real-time imaging of the intracellular glutathione redox potential. Nat Methods 5, 553–559 (2008). https://doi.org/10.1038/nmeth.1212

Download citation

Further reading

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing