TY - JOUR
T1 - Genetically encoded fluorescent indicator for imaging NAD+/NADH ratio changes in different cellular compartments
AU - Bilan, Dmitry S.
AU - Matlashov, Mikhail E.
AU - Gorokhovatsky, Andrey Yu
AU - Schultz, Carsten
AU - Enikolopov, Grigori
AU - Belousov, Vsevolod V.
N1 - Funding Information:
This work was supported by the Russian Foundation for Basic Research ( 13-04-40333-H ), a joint EMBL-RFBR grant ( 12-04-92427 ), Measures to Attract Leading Scientists to Russian Educational Institutions program ( 11.G34.31.0071 ), National Institute of Aging (grant R01AG040209 ) and the Molecular and Cell Biology program of Russian Academy of Sciences .
PY - 2014/3
Y1 - 2014/3
N2 - 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.
AB - 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.
KW - Fluorescent probe
KW - NAD/NADH ratio
KW - Redox sensor
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U2 - 10.1016/j.bbagen.2013.11.018
DO - 10.1016/j.bbagen.2013.11.018
M3 - Article
C2 - 24286672
AN - SCOPUS:84890450161
SN - 0304-4165
VL - 1840
SP - 951
EP - 957
JO - Biochimica et Biophysica Acta - General Subjects
JF - Biochimica et Biophysica Acta - General Subjects
IS - 3
ER -