Genetically encoded fluorescent indicator for imaging NAD+/NADH ratio changes in different cellular compartments

Abstract

Background The ratio of NAD+/NADH is a key indicator that reflects the overall redox state of the cells. Until recently, there were no methods for real time NAD+/NADH monitoring in living cells. Genetically encoded fluorescent probes for NAD+/NADH are fundamentally new approach for studying the NAD+/NADH dynamics. Methods We developed a genetically encoded probe for the nicotinamide adenine dinucleotide, NAD(H), redox state changes by inserting circularly permuted YFP into redox sensor T-REX from Thermus aquaticus. We characterized the sensor in vitro using spectrofluorometry and in cultured mammalian cells using confocal fluorescent microscopy. Results The sensor, named RexYFP, reports changes in the NAD+/NADH ratio in different compartments of living cells. Using RexYFP, we were able to track changes in NAD+/NADH in cytoplasm and mitochondrial matrix of cells under a variety of conditions. The affinity of the probe enables comparison of NAD+/NADH in compartments with low (cytoplasm) and high (mitochondria) NADH concentration. We developed a method of eliminating pH-driven artifacts by normalizing the signal to the signal of the pH sensor with the same chromophore. Conclusion RexYFP is suitable for detecting the NAD(H) redox state in different cellular compartments. General significance RexYFP has several advantages over existing NAD+/NADH sensors such as smallest size and optimal affinity for different compartments. Our results show that normalizing the signal of the sensor to the pH changes is a good strategy for overcoming pH-induced artifacts in imaging.

Genetically encoded fluorescent sensors for redox processes

“…In contrast to the previous biosensors Frex and Peredox, the operation of RexYFP biosensor [80] is based on interactions of domains in the composition of the single T Rex subunit. cpYFP was integrated BILAN et al into the structure of the bacterial protein between the nucleotide and DNA binding domains during the development of this biosensor.…”

“…It was shown experimentally that SypHer and RexYFP have the same pH depen dence in the physiological pH range [80]. Normaliza tion of RexYFP signal to SypHer signal under the same experimental conditions takes into account the effect of pH oscillations on RexYFP signal [80].…”

“…The comparison of the dynamic of Rex YFP signal change in response to various stimuli with the data obtained using pH stable Peredox sensor [78] was also conducted [80]. The Peredox and RexYFP signals were the same in nature and quite comparable in the response amplitude.…”

“…The fluorescence intensity decreases upon the NADH binding; NADPH is the only compound among others that affects the Rex YFP signal. The RexYFP affinity constants for NADH and NADPH are different in the process: 180 nM and 6.2 μM, respectively [80]. The higher affinity of Rex YFP for NADH and the fact that the intracellular con centration of the total NADP pool is lower in compar ison with the NAD pool (for example, approximately 10 fold in the rat liver cells) allows us to conclude that the RexYFP signal depends to a greater extent on the oscillations of the NAD + /NADH ratio.…”

“…The efficiency of RexYFL biosensor in combina tion with SypHer was demonstrated for mammalian cell lines [80]. The comparison of the dynamic of Rex YFP signal change in response to various stimuli with the data obtained using pH stable Peredox sensor [78] was also conducted [80].…”