Genetically encoded fluorescent indicator for imaging NAD(+)/NADH ratio changes in different cellular compartments.
Genetically encoded fluorescent indicator for imaging NAD(+)/NADH ratio changes in different cellular compartments.
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DOI：
10.1016/j.bbagen.2013.11.018
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发表时间：
2014-03
影响因子：
3
通讯作者：
Belousov, Vsevolod V.
中科院分区：
文献类型：
--
作者：
Bilan, Dmitry S.;Matlashov, Mikhail E.;Gorokhovatsky, Andrey Yu.;Schultz, Carsten;Enikolopov, Grigori;Belousov, Vsevolod V.
NAD +/NADH的比率是反映细胞整体氧化还原状态的关键指标。直到最近，还没有用于活细胞中的真实的时间NAD +/NADH监测的方法。基因编码的NAD +/NADH荧光探针是研究NAD +/NADH动力学的新方法。我们通过将环状排列的YFP插入到来自水生栖热菌的氧化还原传感器T-REX中，开发了用于烟酰胺腺嘌呤二核苷酸（NAD（H））氧化还原状态变化的遗传编码探针。我们的特点是传感器在体外使用荧光分光光度法和在培养的哺乳动物细胞使用共聚焦荧光显微镜。该传感器名为RexYFP，报告活细胞不同隔室中NAD +/NADH比率的变化。使用RexYFP，我们能够在各种条件下跟踪细胞的细胞质和线粒体基质中NAD +/NADH的变化。探针的亲和力使得能够比较具有低（细胞质）和高（线粒体）NADH浓度的区室中的NAD +/NADH。我们开发了一种通过将信号归一化为具有相同发色团的pH传感器的信号来消除pH驱动伪影的方法。RexYFP适用于检测不同细胞区室中的NAD（H）氧化还原状态。RexYFP与现有的NAD +/NADH传感器相比具有几个优点，例如最小的尺寸和对不同隔室的最佳亲和力。我们的研究结果表明，归一化的pH值变化的传感器的信号是一个很好的策略，克服pH值引起的伪影成像。
The ratio of NAD+/NADH is a key indicator that reflects the overall redox state of the cells. Until recently, there were no methods for real time NAD+/NADH monitoring in living cells. Genetically encoded fluorescent probes for NAD+/NADH are fundamentally new approach for studying the NAD+/NADH dynamics. We developed a genetically encoded probe for the nicotinamide adenine dinucleotide, NAD(H), redox state changes by inserting circularly permuted YFP into redox sensor T-REX from Thermus aquaticus. We characterized the sensor in vitro using spectrofluorometry and in cultured mammalian cells using confocal fluorescent microscopy. The sensor, named RexYFP, reports changes in the NAD+/NADH ratio in different compartments of living cells. Using RexYFP, we were able to track changes in NAD+/NADH in cytoplasm and mitochondrial matrix of cells under a variety of conditions. The affinity of the probe enables comparison of NAD+/NADH in compartments with low (cytoplasm) and high (mitochondria) NADH concentration. We developed a method of eliminating pH-driven artifacts by normalizing the signal to the signal of the pH sensor with the same chromophore. RexYFP is suitable for detecting the NAD(H) redox state in different cellular compartments. RexYFP has several advantages over existing NAD+/NADH sensors such as smallest size and optimal affinity for different compartments. Our results show that normalizing the signal of the sensor to the pH changes is a good strategy for overcoming pH-induced artifacts in imaging.
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影响因子：
3.6
作者：
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通讯作者：
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作者：
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作者：
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通讯作者：
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