title: Separating NADH and NADPH fluorescence in live cells and tissues using FLIM.
abstract: NAD is a key determinant of cellular energy metabolism. In contrast, its phosphorylated form, NADP, plays a central role in biosynthetic pathways and antioxidant defence. The reduced forms of both pyridine nucleotides are fluorescent in living cells but they cannot be distinguished, as they are spectrally identical. Here, using genetic and pharmacological approaches to perturb NAD(P)H metabolism, we find that fluorescence lifetime imaging (FLIM) differentiates quantitatively between the two cofactors. Systematic manipulations to change the balance between oxidative and glycolytic metabolism suggest that these states do not directly impact NAD(P)H fluorescence decay rates. The lifetime changes observed in cancers thus likely reflect shifts in the NADPH/NADH balance. Using a mathematical model, we use these experimental data to quantify the relative levels of NADH and NADPH in different cell types of a complex tissue, the mammalian cochlea. This reveals NADPH-enriched populations of cells, raising questions about their distinct metabolic roles.
authors.affiliation: 1] Centre for Mathematics and Physics in the Life Sciences and Experimental Biology, University College London, London WC1E 6BT, UK [2] Research Department of Cell & Developmental Biology, University College London, London WC1E 6BT, UK [3] Department of Physics and Astronomy, University College London, London WC1E 6BT, UK.
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Blacker
authors.affiliation: 1] Research Department of Cell & Developmental Biology, University College London, London WC1E 6BT, UK [2] UCL Ear Institute, University College London, London WC1X 8EE, UK.
authors.forename: Zoe F
authors.initials: ZF
authors.lastname: Mann
authors.affiliation: 1] Research Department of Cell & Developmental Biology, University College London, London WC1E 6BT, UK [2] UCL Ear Institute, University College London, London WC1X 8EE, UK.
authors.forename: Jonathan E
authors.initials: JE
authors.lastname: Gale
authors.affiliation: Department of Molecular Biology, University of Bergen, N-5008 Bergen, Norway.
authors.forename: Mathias
authors.initials: M
authors.lastname: Ziegler
authors.affiliation: Department of Physics and Astronomy, University College London, London WC1E 6BT, UK.
authors.forename: Angus J
authors.initials: AJ
authors.lastname: Bain
authors.affiliation: 1] Research Department of Cell & Developmental Biology, University College London, London WC1E 6BT, UK [2] Department of Biomedical Sciences, University of Padua and CNR Neuroscience Institute, Padua 35121, Italy [3].
authors.forename: Gyorgy
authors.initials: G
authors.lastname: Szabadkai
authors.affiliation: 1] Research Department of Cell & Developmental Biology, University College London, London WC1E 6BT, UK [2].
authors.forename: Michael R
authors.initials: MR
authors.lastname: Duchen
mesh_terms: D000818:Animals; D003051:Cochlea / Q000737:chemistry*; D004734:Energy Metabolism; D005453:Fluorescence; D006019:Glycolysis; D057809:HEK293 Cells; D006801:Humans; D009243:NAD / Q000032:analysis* / Q000378:metabolism; D009249:NADP / Q000032:analysis* / Q000378:metabolism; D061848:Optical Imaging / Q000379:methods*; D010084:Oxidation-Reduction; D051381:Rats
chemical_list: D009243:NAD; D009249:NADP