Live-Cell Imaging of Cytosolic NADH–NAD+ Redox State Using a Genetically Encoded Fluorescent Biosensor

  • Yin Pun Hung
  • Gary Yellen
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1071)

Abstract

NADH is an essential redox cofactor in numerous metabolic reactions, and the cytosolic NADH–NAD+ redox state is a key parameter in glycolysis. Conventional NADH measurements rely on chemical determination or autofluorescence imaging, which cannot assess NADH specifically in the cytosol of individual live cells. By combining a bacterial NADH-binding protein and a fluorescent protein variant, we have created a genetically encoded fluorescent biosensor of the cytosolic NADH–NAD+ redox state, named Peredox (Hung et al., Cell Metab 14:545–554, 2011). Here, we elaborate on imaging methods and technical considerations of using Peredox to measure cytosolic NADH:NAD+ ratios in individual live cells.

Key words

NADH Glycolysis Lactate dehydrogenase Sensor calibration Single cell imaging 

Notes

Acknowledgments

We thank Mathew Tantama for careful reading of this manuscript. This work was supported by the Albert J. Ryan fellowship, the Stuart H.Q. and Victoria Quan predoctoral fellowship in neurobiology (both to Y.P.H.), and the U.S. National Institutes of Health (R01 NS055031 to G.Y.).

References

  1. 1.
    Nicholls DG, Ferguson SJ (2002) Bioenergetics, 3rd edn. Academic, LondonGoogle Scholar
  2. 2.
    Avi-Dor Y, Olson JM, Doherty MD, Kaplan NO (1962) Fluorescence of pyridine nucleotides in mitochondria. J Biol Chem 237:2377–2383Google Scholar
  3. 3.
    Rocheleau JV, Head WS, Piston DW (2004) Quantitative NAD(P)H/flavoprotein autofluorescence imaging reveals metabolic mechanisms of pancreatic islet pyruvate response. J Biol Chem 279:31780–31787PubMedCrossRefGoogle Scholar
  4. 4.
    Hung YP, Albeck JG, Tantama M, Yellen G (2011) Imaging cytosolic NADH–NAD+ redox state with a genetically encoded fluorescent biosensor. Cell Metab 14:545–554PubMedCrossRefGoogle Scholar
  5. 5.
    Zapata-Hommer O, Griesbeck O (2003) Efficiently folding and circularly permuted variants of the Sapphire mutant of GFP. BMC Biotechnol 3:5PubMedCrossRefGoogle Scholar
  6. 6.
    Sickmier EA, Brekasis D, Paranawithana S, Bonanno JB, Paget MSB, Burley SK, Kielkopf CL (2005) X-ray structure of a Rex-family repressor/NADH complex insights into the mechanism of redox sensing. Structure 13:43–54PubMedCrossRefGoogle Scholar
  7. 7.
    Wang E, Bauer MC, Rogstam A, Linse S, Logan DT, von Wachenfeldt C (2008) Structure and functional properties of the Bacillus subtilis transcriptional repressor Rex. Mol Microbiol 69:466–478PubMedCrossRefGoogle Scholar
  8. 8.
    McLaughlin KJ, Strain-Damerell CM, Xie K, Brekasis D, Soares AS, Paget MSB, Kielkopf CL (2010) Structural basis for NADH/NAD+ redox sensing by a Rex family repressor. Mol Cell 38:563–575PubMedCrossRefGoogle Scholar
  9. 9.
    Berg J, Hung YP, Yellen G (2009) A genetically encoded fluorescent reporter of ATP:ADP ratio. Nat Methods 6:161–166PubMedCrossRefGoogle Scholar
  10. 10.
    Frommer WB, Davidson MW, Campbell RE (2009) Genetically encoded biosensors based on engineered fluorescent proteins. Chem Soc Rev 38:2833–2841PubMedCrossRefGoogle Scholar
  11. 11.
    Tantama M, Hung YP, Yellen G (2012) Optogenetic reporters: Fluorescent protein-based genetically encoded indicators of signaling and metabolism in the brain. Prog Brain Res 196:235–263PubMedCrossRefGoogle Scholar
  12. 12.
    Bücher T, Brauser B, Conze A, Klein F, Langguth O, Sies H (1972) State of oxidation-reduction and state of binding in the cytosolic NADH-system as disclosed by equilibration with extracellular lactate-pyruvate in hemoglobin-free perfused rat liver. Eur J Biochem 27:301–317PubMedCrossRefGoogle Scholar
  13. 13.
    Tantama M, Hung YP, Yellen G (2011) Imaging intracellular pH in live cells with a genetically encoded red fluorescent protein sensor. J Am Chem Soc 133:10034–10037PubMedCrossRefGoogle Scholar
  14. 14.
    Debnath J, Muthuswamy SK, Brugge JS (2003) Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods 30:256–268PubMedCrossRefGoogle Scholar
  15. 15.
    Katayama H, Yamamoto A, Mizushima N, Yoshimori T, Miyawaki A (2008) GFP-like proteins stably accumulate in lysosomes. Cell Struct Funct 33:1–12PubMedCrossRefGoogle Scholar
  16. 16.
    Passonneau JV, Lowry OH (1993) Enzymatic analysis: a practical guide. Humana, TotowaCrossRefGoogle Scholar
  17. 17.
    Schulz I (1990) Permeabilizing cells: some methods and applications for the study of intracellular processes. Methods Enzymol 192:280–300PubMedCrossRefGoogle Scholar
  18. 18.
    Williamson DH, Lund P, Krebs HA (1967) The redox state of free nicotinamide-adenine dinucleotide in the cytoplasm and mitochondria of rat liver. Biochem J 103:514–527PubMedGoogle Scholar
  19. 19.
    Zhao Y, Jin J, Hu Q, Zhou H-M, Yi J, Yu Z, Xu L, Wang X, Yang Y, Loscalzo J (2011) Genetically encoded fluorescent sensors for intracellular NADH detection. Cell Metab 14:555–566PubMedCrossRefGoogle Scholar
  20. 20.
    Schafer ZT, Grassian AR, Song L, Jiang Z, Gerhart-Hines Z, Irie HY, Gao S, Puigserver P, Brugge JS (2009) Antioxidant and oncogene rescue of metabolic defects caused by loss of matrix attachment. Nature 461:109–113PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Yin Pun Hung
    • 1
  • Gary Yellen
    • 1
  1. 1.Department of NeurobiologyHarvard Medical SchoolCambridgeUSA

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