Genetically encoded fluorescent sensors reveal dynamic regulation of NADPH metabolism.
Rongkun TaoYuzheng ZhaoHuanyu ChuAoxue WangJiahuan ZhuXianjun ChenYejun ZouMei ShiRenmei LiuNi SuJiulin DuHai-Meng ZhouLinyong ZhuXuhong QianHaiyan LiuJoseph LoscalzoYi YangPublished in: Nature methods (2017)
Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is essential for biosynthetic reactions and antioxidant functions; however, detection of NADPH metabolism in living cells remains technically challenging. We develop and characterize ratiometric, pH-resistant, genetically encoded fluorescent indicators for NADPH (iNap sensors) with various affinities and wide dynamic range. iNap sensors enabled quantification of cytosolic and mitochondrial NADPH pools that are controlled by cytosolic NAD+ kinase levels and revealed cellular NADPH dynamics under oxidative stress depending on glucose availability. We found that mammalian cells have a strong tendency to maintain physiological NADPH homeostasis, which is regulated by glucose-6-phosphate dehydrogenase and AMP kinase. Moreover, using the iNap sensors we monitor NADPH fluctuations during the activation of macrophage cells or wound response in vivo. These data demonstrate that the iNap sensors will be valuable tools for monitoring NADPH dynamics in live cells and gaining new insights into cell metabolism.
Keyphrases
- reactive oxygen species
- living cells
- oxidative stress
- induced apoptosis
- fluorescent probe
- quantum dots
- single cell
- single molecule
- cell cycle arrest
- blood glucose
- stem cells
- machine learning
- metabolic syndrome
- signaling pathway
- tyrosine kinase
- sensitive detection
- gene expression
- blood pressure
- endoplasmic reticulum stress
- cell death
- bone marrow
- ischemia reperfusion injury
- cell therapy
- deep learning
- hydrogen peroxide
- anti inflammatory