1. Genetically encoded fluorescent sensors for intracellular NADH detection.
- Author
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Zhao Y, Jin J, Hu Q, Zhou HM, Yi J, Yu Z, Xu L, Wang X, Yang Y, and Loscalzo J
- Subjects
- Bacterial Proteins genetics, Cell Line, Cytosol metabolism, Glucose pharmacology, Humans, Hydrogen Peroxide pharmacology, Lactic Acid pharmacology, Luminescent Proteins genetics, Mitochondria drug effects, Mitochondria metabolism, Oxidation-Reduction, Pyruvic Acid pharmacology, Bacterial Proteins metabolism, Luminescent Proteins metabolism, Microscopy, Fluorescence, NAD analysis
- Abstract
We have developed genetically encoded fluorescent sensors for reduced nicotinamide adenine dinucleotide (NADH), which manifest a large change in fluorescence upon NADH binding. We demonstrate the utility of these sensors in mammalian cells by monitoring the dynamic changes in NADH levels in subcellular organelles as affected by NADH transport, glucose metabolism, electron transport chain function, and redox environment, and we demonstrate the temporal separation of changes in mitochondrial and cytosolic NADH levels with perturbation. These results support the view that cytosolic NADH is sensitive to environmental changes, while mitochondria have a strong tendency to maintain physiological NADH homeostasis. These sensors provide a very good alternative to existing techniques that measure endogenous fluorescence of intracellular NAD(P)H and, owing to their superior sensitivity and specificity, allow for the selective monitoring of total cellular and compartmental responses of this essential cofactor., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
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