Assessment of the Peroxisomal Redox State in Living Cells Using NADPH- and NAD+/NADH-Specific Fluorescent Protein Sensors.

Costa, Cláudio F; Li, Hongli; Hussein, Mohamed AF; Yang, Yi; Lismont, Celien; Fransen, Marc

doi:10.1007/978-1-0716-3048-8_13

Author:

Costa, Cláudio F

Li, Hongli ; Hussein, Mohamed AF ; Yang, Yi ; Lismont, Celien ; Fransen, Marc

Keywords:

Fluorescent biosensors, Live-cell imaging, NAD(H), NADP(H), Peroxisomes, Redox state, SoNar, iNAP, NAD, NADP, Oxidation-Reduction, Luminescent Proteins, C14/18/088#54689605, G091819N#54969808, 1213620N#55264054, 0399 Other Chemical Sciences, 0601 Biochemistry and Cell Biology, Developmental Biology, 3101 Biochemistry and cell biology, 3404 Medicinal and biomolecular chemistry

Abstract:

The pyridine nucleotides NAD(H) and NADP(H) are key molecules in cellular metabolism, and measuring their levels and oxidation states with spatiotemporal precision is of great value in biomedical research. Traditional methods to assess the redox state of these metabolites are intrusive and prohibit live-cell quantifications. This obstacle was surpassed by the development of genetically encoded fluorescent biosensors enabling dynamic measurements with subcellular resolution in living cells. Here, we provide step-by-step protocols to monitor the intraperoxisomal NADPH levels and NAD+/NADH redox state in cellulo by using targeted variants of iNAP1 and SoNar, respectively.