Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 294, Issue 43, Pages 15768-15780Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.RA119.007557
Keywords
biosensor; organellar pH homeostasis; Saccharomyces cerevisiae; flavoprotein; redox signaling; fluorescent probe; iLOV; light; oxygen; or voltage sensing (LOV); mBeRFP
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Funding
- National Key Basic Special Funds [2012CB1143001]
- National Natural Science Foundation of China [31430061]
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Intracellular pH and redox states are critical for multiple processes and partly determine cell behavior. Here, we developed a genetically encoded dual-function probe, named p H and redox-sensitive fluorescent protein (pHaROS), for simultaneous real-time detection of changes in redox potential and pH in living cells. pHaROS consists of the Arabidopsis flavin mononucleotide-binding fluorescent protein iLOV and an mKATE variant, mBeRFP. Using pHaROS in Saccharomyces cerevisiae cells, we confirmed that H2O2 raises the overall redox potential of the cell and found that this increase is accompanied by a decrease in cytosolic pH. Furthermore, we observed spatiotemporal pH and redox homeostasis within the nucleus at various stages of the cell cycle in budding yeast (Saccharomyces cerevisiae) during cellular development and responses to oxidative stress. Importantly, we could tailor pHaROS to specific applications, including measurements in different organelles and cell types and the GSH/GSSG ratio, highlighting pHaROS's high flexibility and versatility. In summary, we have developed pHaROS as a dual-function probe that can be used for simultaneously measuring cellular pH and redox potential, representing a very promising tool for determining the cross-talk between intracellular redox- and pH-signaling processes in yeast and mammalian U87 cell.
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