title: Genetically encoded fluorescent indicator for imaging NAD(+)/NADH ratio changes in different cellular compartments.
abstract: BACKGROUND
  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.
  
  
  CONCLUSIONS
  RexYFP is suitable for detecting the NAD(H) redox state in different cellular compartments.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Moscow Institute of Physics and Technology, 141700 Moscow Region, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 117997 Moscow, Russia.
authors.forename: Dmitry S
authors.initials: DS
authors.lastname: Bilan
authors.affiliation: Moscow Institute of Physics and Technology, 141700 Moscow Region, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 117997 Moscow, Russia.
authors.forename: Mikhail E
authors.initials: ME
authors.lastname: Matlashov
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 117997 Moscow, Russia.
authors.forename: Andrey Yu
authors.initials: AY
authors.lastname: Gorokhovatsky
authors.affiliation: European Molecular Biology Laboratory, 69117 Heidelberg, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Schultz
authors.affiliation: Moscow Institute of Physics and Technology, 141700 Moscow Region, Russia; Cold Spring Harbor Laboratory, 11724 Cold Spring Harbor, NY, USA.
authors.forename: Grigori
authors.initials: G
authors.lastname: Enikolopov
authors.affiliation: Moscow Institute of Physics and Technology, 141700 Moscow Region, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 117997 Moscow, Russia. Electronic address: vsevolod.belousov@gmail.com.
authors.forename: Vsevolod V
authors.initials: VV
authors.lastname: Belousov
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D003593:Cytoplasm / Q000737:chemistry; D005456:Fluorescent Dyes; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008164:Luminescent Proteins / Q000235:genetics*; D008928:Mitochondria / Q000737:chemistry; D009243:NAD / Q000032:analysis*; D010084:Oxidation-Reduction
chemical_list: D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D009243:NAD
keywords: Fluorescent probe; NAD(+)/NADH ratio; Redox sensor