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

Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA.
Cell metabolism (Impact Factor: 17.57). 10/2011; 14(4):545-54. DOI: 10.1016/j.cmet.2011.08.012
Source: PubMed


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.

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    • "Neg shRNA, SPG7, and PPIF KD 293T cells were transiently transfected with NADH-NAD + redox sensor peredox m-cherry (Hung et al., 2011). Confocal images were acquired at 405 and 561 nm excitation every 3 s using Carl Zeiss 710 Meta NLO. "
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    • "These Frex sensors (Zhao et al., 2011) specifically report NADH levels over a large dynamic range; however, they do not adapt an optimal tertiary structure in some cells, and their fluorescence is pH sensitive. Peredox sensors (Hung et al., 2011) are much more pH resistant and partially reflect the more physiologically relevant NAD + /NADH ratio; however , they have a limited dynamic range, and their affinity appears too high to be useful under physiological conditions. Importantly, neither Frex nor Peredox sensors show obvious fluorescence response to NAD "
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