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Biochim Biophys Acta. 2014 Mar;1840(3):951-7. doi: 10.1016/j.bbagen.2013.11.018. Epub 2013 Nov 25.

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

Author information

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.

KEYWORDS:

Fluorescent probe; NAD(+)/NADH ratio; Redox sensor

PMID:
24286672
PMCID:
PMC4398388
DOI:
10.1016/j.bbagen.2013.11.018
[Indexed for MEDLINE]
Free PMC Article

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