title: Imaging cytosolic NADH-NAD(+) redox state with a genetically encoded fluorescent biosensor.
abstract: NADH is a key metabolic cofactor whose sensitive and specific detection in the cytosol of live cells has been difficult. We constructed a fluorescent biosensor of the cytosolic NADH-NAD(+) redox state by combining a circularly permuted GFP T-Sapphire with a bacterial NADH-binding protein, Rex. Although the initial construct reported [NADH] × [H(+)] / [NAD(+)], its pH sensitivity was eliminated by mutagenesis. The engineered biosensor Peredox reports cytosolic NADH:NAD(+) ratios and can be calibrated with exogenous lactate and pyruvate. We demonstrated its utility in several cultured and primary cell types. We found that glycolysis opposed the lactate dehydrogenase equilibrium to produce a reduced cytosolic NADH-NAD(+) redox state. We also observed different redox states in primary mouse astrocytes and neurons, consistent with hypothesized metabolic differences. Furthermore, using high-content image analysis, we monitored NADH responses to PI3K pathway inhibition in hundreds of live cells. As an NADH reporter, Peredox should enable better understanding of bioenergetics.
authors: Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA.
authors: Yin Pun
authors: YP
authors: Hung
authors: John G
authors: JG
authors: Albeck
authors: Mathew
authors: M
authors: Tantama
authors: Gary
authors: G
authors: Yellen
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015374:Biosensing Techniques; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism*; D006019:Glycolysis; D006863:Hydrogen-Ion Concentration; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009243:NAD / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D007770:L-Lactate Dehydrogenase; D008164:Luminescent Proteins; D009243:NAD; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011993:Recombinant Fusion Proteins