Transient light-induced intracellular oxidation revealed by redox biosensor

Vladimir L. Kolossov, Jessica N. Beaudoin, William P. Hanafin, Stephen J. DiLiberto, Paul J A Kenis, H Rex Gaskins

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)517-521
Number of pages5
JournalBiochemical and Biophysical Research Communications
Volume439
Issue number4
DOIs
StatePublished - Oct 4 2013

Fingerprint

Biosensing Techniques
Biosensors
Oxidation-Reduction
Cells
Glutaredoxins
Light
Oxidation
Image acquisition
Green Fluorescent Proteins
Sulfhydryl Compounds
Disulfides
Glutathione
Fusion reactions
Imaging techniques
Wavelength
Cytosol
Cues
Proteins

Keywords

  • Glutathione
  • Green fluorescent protein (GFP)
  • Light-induced oxidation
  • Live cell imaging
  • Redox-sensitive probe

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology
  • Molecular Biology

Cite this

Transient light-induced intracellular oxidation revealed by redox biosensor. / Kolossov, Vladimir L.; Beaudoin, Jessica N.; Hanafin, William P.; DiLiberto, Stephen J.; Kenis, Paul J A; Gaskins, H Rex.

In: Biochemical and Biophysical Research Communications, Vol. 439, No. 4, 04.10.2013, p. 517-521.

Research output: Contribution to journalArticle

Kolossov, Vladimir L. ; Beaudoin, Jessica N. ; Hanafin, William P. ; DiLiberto, Stephen J. ; Kenis, Paul J A ; Gaskins, H Rex. / Transient light-induced intracellular oxidation revealed by redox biosensor. In: Biochemical and Biophysical Research Communications. 2013 ; Vol. 439, No. 4. pp. 517-521.
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