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Genetically encoded biosensors for evaluating NAD(+)/NADH ratio in cytosolic and mitochondrial compartments

Study · Cell reports methods · 2021 · DOI 10.1016/j.crmeth.2021.100116 · PMID 34901920

Plain-language summary

Paraphrased from the published abstract below — not a verdict on whether anything works.

This study (species not specified; conducted in cultured live cells) reports the development of compartment-targeted genetically encoded biosensors, mt-SoNar and ct-SoNar, to monitor the NAD+/NADH ratio in mitochondrial and cytosolic compartments respectively. The abstract reports the fluorescence signal of SoNar responded linearly to physiological NAD+/NADH ratios in situ, that cytosolic and mitochondrial NAD+/NADH ratios responded rapidly but differently to acute metabolic perturbations, indicating distinct NAD pools, and that subcellular NAD redox balance was restored via the malate-aspartate shuttle. Mitochondrial and cytosolic NAD+/NADH ratios were reported to be influenced by NAD+ precursor levels and distinctly regulated under pathophysiological conditions.

Abstract

The ratio of oxidized to reduced NAD (NAD+/NADH) sets intracellular redox balance and antioxidant capacity. Intracellular NAD is compartmentalized and the mitochondrial NAD+/NADH ratio is intricately linked to cellular function. Here, we report the monitoring of the NAD+/NADH ratio in mitochondrial and cytosolic compartments in live cells by using a modified genetic biosensor (SoNar). The fluorescence signal of SoNar targeted to mitochondria (mt-SoNar) or cytosol (ct-SoNar) responded linearly to physiological NAD+/NADH ratios in situ. NAD+/NADH ratios in cytosol versus mitochondria responded rapidly, but differently, to acute metabolic perturbations, indicating distinct NAD pools. Subcellular NAD redox balance regained homeostasis via communications through malate-aspartate shuttle. Mitochondrial and cytosolic NAD+/NADH ratios are influenced by NAD+ precursor levels and are distinctly regulated under pathophysiological conditions. Compartment-targeted biosensors and real-time imaging allow assessment of subcellular NAD+/NADH redox signaling in live cells, enabling future mechanistic research of NAD redox in cell biology and disease development.

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