Bilan, Dmitry S., Matlashov, Mikhail E., Gorokhovatsky, Andrey Yu, Schultz, Carsten, Enikolopov, Grigori, Belousov, Vsevolod V. (January 2014) Genetically encoded fluorescent indicator for imaging NAD+/NADH ratio changes in different cellular compartments. Biochimica et Biophysica Acta (BBA) - General Subjects, 1840 (3). pp. 951-957. ISSN 0304-4165
Abstract
AbstractBackground 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. Methods 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. Results 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. Conclusion RexYFP is suitable for detecting the NAD(H) redox state in different cellular compartments. General significance 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.
| Item Type: | Paper |
|---|---|
| Uncontrolled Keywords: | NAD+/NADH ratio fluorescent probe redox sensor |
| Subjects: | Investigative techniques and equipment Investigative techniques and equipment > imaging organs, tissues, organelles, cell types and functions > sub-cellular tissues: types and functions |
| CSHL Authors: | |
| Communities: | CSHL Cancer Center Program > Signal Transduction CSHL Cancer Center Shared Resources > Microscopy Service CSHL labs > Enikopolov lab |
| Depositing User: | Matt Covey |
| Date: | January 2014 |
| Date Deposited: | 09 Dec 2013 16:54 |
| Last Modified: | 16 Jul 2021 16:18 |
| PMCID: | PMC4398388 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/28891 |
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