Real-time imaging of the intracellular glutathione redox potential.
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 (E(GSH)) 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.
Gutscher
Marcus
M
Redox Regulation Research Group, German Cancer Research Center (DKFZ/A160), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.
Pauleau
Anne-Laure
AL
Marty
Laurent
L
Brach
Thorsten
T
Wabnitz
Guido H
GH
Samstag
Yvonne
Y
Meyer
Andreas J
AJ
Dick
Tobias P
TP
D017209:Apoptosis; D015374:Biosensing Techniques; D001709:Biotechnology / Q000379:methods*; D004220:Disulfides / Q000737:chemistry; D054477:Glutaredoxins / Q000737:chemistry; D005978:Glutathione / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012680:Sensitivity and Specificity; D013879:Thioredoxins / Q000737:chemistry
D004220:Disulfides; D054477:Glutaredoxins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D013879:Thioredoxins; D005978:Glutathione; D010100:Oxygen