期刊
NATURE METHODS
卷 14, 期 7, 页码 720-+出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nmeth.4306
关键词
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资金
- National Natural Science Foundation of China [31225008, 91313301, 31470833, 31370755, 91649123, 31671484]
- 973 Program [2013CB531200]
- Shanghai Science and Technology Commission [14XD1401400, 16430723100, 15YF1402600]
- Specialized Research Fund for the Doctoral Program of Higher Education [20100074110010]
- Lift Engineering for Young Talent of China Association for Science and Technology
- Shanghai Young Topnotch Talent
- State Key Laboratory of Bioreactor Engineering
- 111 Project [607023]
- Fundamental Research Funds for the Central Universities
- US National Institutes of Health [HL061795, HL007690, GM107618]
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 mitochondria( 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.
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