Dynamic imaging of cellular pH and redox homeostasis with a genetically encoded dual-functional biosensor, pHaROS, in yeast

Abstract

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 <u>p</u><u>H</u><u>a</u>nd <u>r</u>ed<u>o</u>x-<u>s</u>ensitive fluorescent protein (pHaROS), for simultaneous real-time detection of changes in redox potential and pH in living cells. pHaROS consists of the <i>Arabidopsis</i> flavin mononucleotide-binding fluorescent protein iLOV and an mKATE variant, mBeRFP. Using pHaROS in <i>Saccharomyces cerevisiae</i> 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 (<i>Saccharomyces cerevisiae</i>) 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.