Development of a genetically encoded supersulfide-dependent translocation reporter

Supersulfides are emerging sulfur-containing signaling molecules involved in redox regulation, mitochondrial function, and protein S-sulfhydration. However, their dynamic behavior in living mammalian systems remains poorly understood because existing analytical methods require destructive sample preparation or lack sufficient intracellular applicability. Here, we developed a genetically encoded supersulfide-dependent translocation reporter (SuTR) for mammalian cells and in vivo imaging. Although the previously reported probe psGFP failed to respond to supersulfides in mammalian cells, fusion of psGFP with the sulfide-responsive transcriptional repressor (SqrR) generated SuTR, a novel reporter that exhibited supersulfide-dependent translocation from the nucleus to the cytoplasm. Na2S2 and Na2S induced dose-dependent cytosolic translocation of SuTR, whereas Na2S showed no effect. Fluorescence recovery after photobleaching (FRAP) analysis revealed accelerated fluorescence recovery shortly after supersulfide stimulation, and overexpression of the endogenous supersulfide-producing enzyme Cysteinyl-tRNA Synthetase 2 (CARS2) similarly altered reporter dynamics. Mutational analyses demonstrated that reporter responsiveness depends on the DNA-binding activity of SqrR. Furthermore, SuTR successfully detected supersulfide induction in mouse liver in vivo following Na2S administration. These findings establish SuTR as a genetically encoded reporter for monitoring supersulfide dynamics in mammalian cells and tissues. HighlightsO_LIWe developed SuTR, a genetically encoded supersulfide-dependent translocation reporter. C_LIO_LISupersulfides induce nuclear-to-cytoplasmic translocation of SuTR C_LIO_LIFRAP enables rapid detection of endogenous and exogenous supersulfide responses C_LIO_LISuTR activity depends on the DNA-binding function of SqrR C_LIO_LISuTR enables visualization of supersulfide dynamics in mouse liver in vivo C_LI