4.8 Article

Imaging Cytosolic NADH-NAD+ Redox State with a Genetically Encoded Fluorescent Biosensor

Journal

CELL METABOLISM
Volume 14, Issue 4, Pages 545-554

Publisher

CELL PRESS
DOI: 10.1016/j.cmet.2011.08.012

Keywords

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Funding

  1. US National Institutes of Health [R01 NS055031]
  2. NIH [F32 NS066613]
  3. U.S. Army Breast Cancer Research [W81XWH-08-1-0609]
  4. Albert J. Ryan fellowship
  5. Victoria Quan

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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] x [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.

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