Transient light-induced intracellular oxidation revealed by redox biosensor |
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Authors: | Vladimir L Kolossov Jessica N Beaudoin William P Hanafin Stephen J DiLiberto Paul JA Kenis H Rex Gaskins |
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Institution: | 1. Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801, USA;2. Department of Animal Sciences, University of Illinois at Urbana-Champaign, 1207 W. Gregory Drive, Urbana, IL 61801, USA;3. Department of Chemical & Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 S. Mathews Avenue, Urbana, IL 61801, USA;4. Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 S. Lincoln Avenue, Urbana, IL 61801, USA;5. Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, 905 S. Goodwin Avenue, Urbana, IL 61801, USA |
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Abstract: | We have implemented a ratiometric, genetically encoded redox-sensitive green fluorescent protein fused to human glutaredoxin (Grx1-roGFP2) to monitor real time intracellular glutathione redox potentials of mammalian cells. This probe enabled detection of media-dependent oxidation of the cytosol triggered by short wavelength excitation. The transient nature of light-induced oxidation was revealed by time-lapse live cell imaging when time intervals of less than 30 s were implemented. In contrast, transient ROS generation was not observed with the parental roGFP2 probe without Grx1, which exhibits slower thiol-disulfide exchange. These data demonstrate that the enhanced sensitivity of the Grx1-roGFP2 fusion protein enables the detection of short-lived ROS in living cells. The superior sensitivity of Grx1-roGFP2, however, also enhances responsiveness to environmental cues introducing a greater likelihood of false positive results during image acquisition. |
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Keywords: | GSH reduced glutathione GSSG oxidized glutathione roGFP redox-sensitive green fluorescent proteins Grx1 human glutaredoxin 1 |
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