Dmitry S Bilan1, Mikhail E Matlashov1, Andrey Yu Gorokhovatsky2, Carsten Schultz3, Grigori Enikolopov4, Vsevolod V Belousov5. 1. Moscow Institute of Physics and Technology, 141700 Moscow Region, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 117997 Moscow, Russia. 2. Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 117997 Moscow, Russia. 3. European Molecular Biology Laboratory, 69117 Heidelberg, Germany. 4. Moscow Institute of Physics and Technology, 141700 Moscow Region, Russia; Cold Spring Harbor Laboratory, 11724 Cold Spring Harbor, NY, USA. 5. Moscow Institute of Physics and Technology, 141700 Moscow Region, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 117997 Moscow, Russia. Electronic address: vsevolod.belousov@gmail.com.
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.
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.
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