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Imaging changes in the cytosolic ATP-to-ADP ratio - PubMed

Imaging changes in the cytosolic ATP-to-ADP ratio

Mathew Tantama et al. Methods Enzymol. 2014.

Abstract

Adenosine triphosphate (ATP) is a central metabolite that plays fundamental roles as an energy transfer molecule, a phosphate donor, and a signaling molecule inside the cells. The phosphoryl group transfer potential of ATP provides a thermodynamic driving force for many metabolic reactions, and phosphorylation of both small metabolites and large proteins can serve as a regulatory modification. In the process of phosphoryl transfer from ATP, the diphosphate ADP is produced, and as a result, the ATP-to-ADP ratio is an important physiological control parameter. The ATP-to-ADP ratio is directly proportional to cellular energy charge and phosphorylation potential. Furthermore, several ATP-dependent enzymes and signaling proteins are regulated by ADP, and their activation profiles are a function of the ATP-to-ADP ratio. Finally, regeneration of ATP from ADP can serve as an important readout of energy metabolism and mitochondrial function. We, therefore, developed a genetically encoded fluorescent biosensor tuned to sense ATP-to-ADP ratios in the physiological range of healthy mammalian cells. Here, we present a protocol for using this biosensor to visualize energy status using live-cell fluorescence microscopy.

Keywords: ADP; ATP; Energy; Fluorescence; Genetically encoded; Imaging; Metabolism; Microscopy; Ratiometric; Sensor.

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Figures

Figure 1
Figure 1

Design of PercevalHR. Left, the MgATP-binding protein GlnK (PDB 2J9C) is a homotrimer. Monomers are colored (purple, green, blue), and MgATP spheres are shown in red. Right, the yellow fluorescent protein Venus (PDB 1MYW). Center, a diagram of PercevalHR, a single gene product in which GlnK has been fused to a circularly-permuted version of Venus. When MgATP (red) binds, the conformational change in GlnK is transduced to Venus, altering its fluorescence.

Figure 2
Figure 2

Ligand-dependent PercevalHR fluorescence. ADP (blue) bound to the GlnK domain (grey) shifts the Venus (green) chromophore charge state equilibrium (box), favoring the neutral protonated A-state. MgATP (red) binding favors the anionic deprotonated B-state. Ligand binding affects the shape of the fluorescence excitation spectrum (middle). The shape of the fluorescence emission spectrum is not significantly different between states (bottom).

Figure 3
Figure 3

Ratiometric image analysis of a cell expressing PercevalHR. Pixelated cartoons of fluorescence images of a cell (left). Images from Channels 1 (top, green) and 2 (bottom, blue) show uneven distribution of the fluorescence intensity. This could be due to cell structure (e.g., nucleus) or a true difference in ATP-to-ADP ratio. The fluorescence intensity images alone cannot distinguish the two possibilities. Pixel-by-pixel division of the Channel 1 image by the Channel 2 image results in the Ratio image (right, red). The ratio image is normalized for biosensor concentration, revealing a homogeneous signal and ATP-to-ADP ratio throughout the cell.

Figure 4
Figure 4

PercevalHR expression in the cytosol of Neuro2A cells (left) and in a sparse co-culture of neurons and astrocytes (right).

Figure 5
Figure 5

Metabolic poisoning and performance of the pH calibration in HEK293 cells co-expressing PercevalHR and pHRed. (A) Green fluorescence overlay on a DIC image of cells. (B) Experimental time course. Left y-axis, green data: PercevalHR fluorescence ratio. Right y-axis, red data: pHRed fluorescence ratio. Note that pH varies inversely with pHRed ratio. Top bars indicate applications of NH4Cl at (1), (3) 5 mM and (2), (4) 15 mM for approximately 4 minutes. Arrow indicates application of 1 mM iodoacetic acid (iaa). Cells were imaged in ACSF containing 20 mM glucose. Dead time for perfusion was ∼ 2 minutes. (C) PercevalHR ratio versus pHRed ratio pH calibration curves from NH4Cl pre-pulses in the (2) ATP-loaded state (orange, circles) and (4) ADP-loaded state (blue, diamonds). (D) PercevalHR occupancy shows that iodoacetic acid caused a slow reduction in ATP-to-ADP ratio as expected. pH correction using calibration data from the (2) ATP-loaded state (orange, circles) versus (4) ADP-loaded state (blue, diamonds) are not significantly different.

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