Bilan 2014 Biochim Biophys Acta
| Bilan DS, Matlashov ME, Gorokhovatsky AY, Schultz C, Enikolopov G, Belousov VV (2014) Genetically encoded fluorescent indicator for imaging NAD+/NADH ratio changes in different cellular compartments. Biochim Biophys Acta 1840:951-7. |
Bilan DS, Matlashov ME, Gorokhovatsky AY, Schultz C, Enikolopov G, Belousov VV (2014) Biochim Biophys Acta
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. • Keywords: Fluorescent probe, NAD(+)/NADH ratio, redox sensor • Bioblast editor: Doerrier C
Labels: MiParea: Instruments;methods
Tissue;cell: HEK, HeLa
