title: Transient light-induced intracellular oxidation revealed by redox biosensor.
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 30s 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.
authors.affiliation: Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801, USA. Electronic address: viadimer@illinois.edu.
authors.forename: Vladimir L
authors.initials: VL
authors.lastname: Kolossov
authors.forename: Jessica N
authors.initials: JN
authors.lastname: Beaudoin
authors.forename: William P
authors.initials: WP
authors.lastname: Hanafin
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: DiLiberto
authors.forename: Paul J A
authors.initials: PJ
authors.lastname: Kenis
authors.forename: H Rex
authors.initials: HR
authors.lastname: Gaskins
mesh_terms: D000818:Animals; D015374:Biosensing Techniques; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D054477:Glutaredoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008027:Light; D010084:Oxidation-Reduction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism
chemical_list: D004220:Disulfides; C516005:GLRX protein, human; D054477:Glutaredoxins; D011993:Recombinant Fusion Proteins; D013438:Sulfhydryl Compounds; D049452:Green Fluorescent Proteins
keywords: GSH; GSSG; Glutathione; Green fluorescent protein (GFP); Grx1; Light-induced oxidation; Live cell imaging; Redox-sensitive probe; human glutaredoxin 1; oxidized glutathione; redox-sensitive green fluorescent proteins; reduced glutathione; roGFP
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