Monitoring thioredoxin redox with a genetically encoded red fluorescent biosensor

Thioredoxin (Trx) is one of the two major thiol antioxidants playing essential roles in redox homeostasis and signaling. Despite the importance, there is a lack of method in monitoring Trx redox dynamics in live cells, hindering a better understanding of physiological and pathological roles of the Trx redox system. In this work, we developed the first genetically encoded fluorescent biosensor for Trx redox by engineering a redox relay between the active-site cysteines of human Trx1 and rxRFP1—a redox-sensitive red fluorescent protein. We utilized the resultant biosensor—TrxRFP1—to selectively monitor perturbations of Trx redox in various mammalian cell lines. We subcellularly localized TrxRFP1 to image compartmentalized Trx redox changes. We further combined TrxRFP1 with a green fluorescent Grx1-roGFP2 biosensor to simultaneously monitor Trx and glutathione redox dynamics in live cells in response to chemical and physiologically relevant stimuli.