Mitochondria and Metabolism Center, Department of Anesthesiology and Pain Medicine, University of Washington, Seattle, WA 98109, USA.
These authors contributed equally.
Cell Rep Methods. 2021 Nov 22;1(7). doi: 10.1016/j.crmeth.2021.100116. Epub 2021 Nov 15.
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 . 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.
氧化型与还原型烟酰胺腺嘌呤二核苷酸（NAD/NADH）的比值可调节细胞内氧化还原平衡和抗氧化能力。细胞内 NAD 存在区室化，线粒体 NAD/NADH 比值与细胞功能密切相关。本研究通过改良的遗传生物传感器（SoNar），在线粒体和细胞质中监测活细胞内 NAD/NADH 比值。靶向线粒体（mt-SoNar）或细胞质（ct-SoNar）的 SoNar 的荧光信号与生理 NAD/NADH 比值呈线性相关。细胞质与线粒体 NAD/NADH 比值对急性代谢扰动的反应迅速但不同，表明存在不同的 NAD 池。通过苹果酸-天冬氨酸穿梭，亚细胞 NAD 氧化还原平衡通过通讯恢复到稳态。NAD 前体水平影响线粒体和细胞质 NAD/NADH 比值，在病理生理条件下受到明显调控。靶向区室的生物传感器和实时成像可评估活细胞内亚细胞 NAD/NADH 氧化还原信号，为 NAD 氧化还原在细胞生物学和疾病发展中的机制研究提供可能。
