ArticleBiochimica et biophysica acta2014
Genetically encoded fluorescent indicator for imaging NAD(+)/NADH ratio changes in different cellular compartments.
Article in Biochimica et biophysica acta, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 64 papers.
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64 citing papers in PubMed, 117 citations in OpenAlex.
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- Regulation of and challenges in targeting NADNature reviews. Molecular cell biology · 2024Review
- Inferring mitochondrial and cytosolic metabolism by coupling isotope tracing and deconvolution.Nature communications · 2023Article
- In vivo investigation of mitochondria in lateral line afferent neurons and hair cells.Hearing research · 2023Review
- Genetically encoded fluorescent sensors for imaging neuronal dynamics in vivo.Journal of neurochemistry · 2023Review
- Ultrasensitive sensors reveal the spatiotemporal landscape of lactate metabolism in physiology and disease.Cell metabolism · 2023Article
- Fluorescence microscopy imaging of mitochondrial metabolism in cancer cells.Frontiers in oncology · 2023Review
- Identification and demonstration of roGFP2 as an environmental sensor for cryogenic correlative light and electron microscopy.Journal of structural biology · 2022Article
- Reduced nicotinamide adenine dinucleotide phosphate in redox balance and diseases: a friend or foe?Acta pharmacologica Sinica · 2022Review
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- Mitochondria as the Target of Hepatotoxicity and Drug-Induced Liver Injury: Molecular Mechanisms and Detection Methods.International journal of molecular sciences · 2022Review
- Monitoring NAD(H) and NADP(H) dynamics during organismal development with genetically encoded fluorescent biosensors.Cell regeneration (London, England) · 2022Review
- Cell cycle induction in human cardiomyocytes is dependent on biosynthetic pathway activation.Redox biology · 2021Article
- Genetically Encoded Fluorescent Redox Indicators for Unveiling Redox Signaling and Oxidative Toxicity.Chemical research in toxicology · 2021Review
4 more citing papers are in PubMed but not listed here.
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Authors and funding
6 authors at 5 institutions in 3 countries.
Funding
Abstract
backgroundThe 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.
methodsWe 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.
resultsThe 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.
conclusionRexYFP 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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