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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.

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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.

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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.).

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Correspondence to Tobias P Dick.

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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

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