Imaging cytosolic NADH-NAD(+) redox state with a genetically encoded fluorescent biosensor - PubMed (original) (raw)

Imaging cytosolic NADH-NAD(+) redox state with a genetically encoded fluorescent biosensor

Yin Pun Hung et al. Cell Metab. 2011.

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.

Copyright © 2011 Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. Characterization of purified P0, a Rex-cpFP chimera

(A) Schematic showing the sensor design, with a cpFP (PDB: 3evp) interposed between the two T-Rex subunits (blue and orange), and a change of fluorescence upon binding of NADH (black). (B) Excitation and emission spectra in the control condition (solid black), after addition of 100 μM NAD+ (dash purple), or 100 μM NAD+ and 0.2 μM NADH (solid green), normalized to the peak intensity in the control condition. For excitation spectra, emission was measured at 510 ± 5 nm; for emission spectra, excitation was at 400 ± 2.5 nm. (C) Green to red fluorescence ratios at the indicated [NAD+] at pH 7.2 plotted against [NADH]. (D) Fluorescence ratios at the indicated [NAD+] and pH plotted against R′. (E) Fluorescence ratios at the indicated [NAD+] and pH plotted against [NADH] × [H+]/[NAD+]. Fluorescence ratios (mean ± SEM, n = 3) were normalized to the control condition in the absence of pyridine nucleotides at pH 7.2 at 25°C.

Figure 2

Figure 2. Characterization of purified Peredox

(A) Green to red fluorescence ratios at the indicated pH, plotted against R′ or R (above the plot), with 80 μM NAD+ at 25°C. (B) Fluorescence ratios at the indicated pH, temperature, and ATP:ADP ratios (low, 0.3; high, 3.6; with total adenine nucleotides of 4.6 mM), plotted against R′ or R (above the plot), with 80 μM NAD+. Fluorescence ratios (mean ± SEM, n = 3) were normalized to the control condition in the absence of pyridine nucleotides at pH 7.2. (C) Kinetics of fluorescence signal upon addition of pyruvate and LDH to Peredox pre-equilibrated with saturating NADH at 25°C or 35°C, normalized to initial and final values.

Figure 3

Figure 3. Imaging cytosolic NADH-NAD+ redox state in various extracellular lactate:pyruvate ratios

(A) Left: Confocal green and red fluorescence images of two cultured mouse neuroblastoma Neuro-2a cells expressing Peredox supplied with 10 mM lactate, 10 mM lactate and 0.2 mM pyruvate, or 10 mM pyruvate. Scale bar 20 μm. Middle: Pseudocolored pixel by pixel green to red ratio images. The slight edge effect seen is likely due to the optical z-shift of 1.5 μm between the green and red confocal images. Right: Widefield differential interference contrast (DIC), nuclear staining, and the overlay image. (B) Time course of fluorescence ratios of four Neuro-2a cells perfused with the indicated lactate:pyruvate ratios. (C) Steady state fluorescence responses of Neuro-2a cells plotted against extracellular lactate:pyruvate ratios, with lactate of 10 mM or 20 mM (mean ± SEM, n = 12–15 cells from two independent experiments). (D) Steady state fluorescence responses in (D) plotted against the predicted R′, by assuming the LDH reaction was at equilibrium and a pH of 7.4. Data of purified Peredox proteins were with 80 μM NAD+ and 4.6 mM total adenine nucleotides at 35°C. Line fitted with a logistic function using a Hill coefficient of 1.8.

Figure 4

Figure 4. Cultured mouse neuroblastoma Neuro-2a cells supplied with glucose show a more reduced cytosolic NADH-NAD+ redox state

(A) Steady state fluorescence responses of Neuro-2a cells plotted against concentrations of extracellular glucose (mean ± SEM, n = 19 cells from two independent experiments). (B) Steady state fluorescence responses of Neuro-2a cells plotted against total concentrations of extracellular lactate and pyruvate, with a constant lactate:pyruvate ratio of 10, and glucose of 10 mM or 0 mM (mean ± SEM, n = 7 cells from three independent experiments). For the alternate y axis, the predicted NAD+:NADH ratio was calculated from purified protein measurements. p < 0.001 (paired _t_-test) for all conditions in 10 mM vs. 0 mM glucose.

Figure 5

Figure 5. Primary cultured mouse cortical astrocytes and neurons differ in their cytosolic NADH-NAD+ redox states

Time course of fluorescence ratios of primary mouse cortical astrocytes or neurons expressing Peredox-NLS and perfused with solutions as indicated (mean ± SEM, n = 4–5 cells from four independent experiments). For the alternate y axis, the predicted NAD+:NADH ratio was calculated from purified protein measurements. p < 0.01 (paired _t_-test) for astrocytes vs. neurons prior to the 10 mM lactate condition.

Figure 6

Figure 6. In stably-expressed mammary epithelial MCF-10A cells, Peredox reports cytosolic NADH decrease upon PI3K pathway inhibition

(A) Heat maps of normalized fluorescence ratios of MCF-10A cells stably expressing Peredox-NLS plotted against time. After 38 minutes of baseline, cells were treated with 1 μM NVP-BEZ235 (left) or DMSO (right). As a control, 20 mM lactate, 20 mM lactate and 1 mM pyruvate, and 20 mM pyruvate and 0.4 mM iodoacetate were applied at red, blue, and black arrows, respectively. For each group, ~700 cells from six fields in two experiments were collected. While the dynamic range was less than the usual 2.5-fold, this was likely due to inadequate control of extracellular lactate and pyruvate concentrations, as these cells were imaged in the 24-well plate formats and solutions were changed without rinsing, as opposed to imaging in a chamber under continuous perfusion of fresh solutions. Fluorescence ratios binned in increments of 0.012. (B) Histograms of normalized fluorescence ratios after one-hour treatment with DMSO (black) or NVP-BEZ235 (blue), binned in increments of 0.027. (C) Histograms of normalized fluorescence ratios from the indicated groups after calibration with 20 mM lactate (L), 20 mM lactate and 1 mM pyruvate (L+P), and 20 mM pyruvate and 0.4 mM iodoacetate (P+I).

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