A genetically-encoded cysteine biosensor to monitor cysteine dynamics across life domains

Abstract Cysteine is a central metabolite in cellular redox regulation and iron-sulfur cluster assembly. Despite its critical role, monitoring cysteine dynamics in living systems has remained a challenge due to the lack of tools that avoid cysteine oxidation and/or do not destroy the cell in the process. Here, we report the development of Cystector (from Cys teine De tector ), a genetically encoded, ratiometric green fluorescent biosensor for cysteine that exhibits an exceptional selectivity, minimal pH sensitivity in the physiological range, and a dynamic range of up to 4500%. Furthermore, the sensor retains functionality in the presence of physiological glutathione concentrations. We demonstrate the live-cell functionality of Cystector by monitoring intracellular and extracellular cysteine dynamics in different organisms. In E. coli , we show how cystine reduction in Escherichia coli is dependent on glutathione and glutaredoxins, and that the reduced cysteine is then exported into the extracellular environment. In yeast, we demonstrate how energy metabolism and oxidative stress determine cysteine homeostasis. In mammalian cells, we show how Cystector effectively monitors cysteine depletion in response to treatments such as H 2 O 2 , erastin2, and glutamate. Finally, we demonstrate, via a mitochondrially targeted variant, that Cystector can be used to monitor subcellular cysteine dynamics. These results together establish Cystector as a robust tool to unravel cysteine metabolism and transport in live cells across life domains.