RGB-Color Intensiometric Indicators to Visualize Spatiotemporal Dynamics of ATP in Single Cells

Abstract

Adenosine triphosphate (ATP) provides energy for the regulation of multiple cellular processes in living organisms. Capturing the spatiotemporal dynamics of ATP in single cells is fundamental to our understanding of the mechanisms underlying cellular energy metabolism. However, it has remained challenging to visualize the dynamics of ATP in and between distinct intracellular organelles and its interplay with other signaling molecules. Using single fluorescent proteins, multicolor ATP indicators were developed, enabling the simultaneous visualization of subcellular ATP dynamics in the cytoplasm and mitochondria of cells derived from mammals, plants, and worms. Furthermore, in combination with additional fluorescent indicators, the dynamic interplay of ATP, cAMP, and Ca²⁺ could be visualized in activated brown adipocyte. This set of indicator tools will facilitate future research into energy metabolism.

Keywords: ATP; brown adipocytes; fluorescent probes; live cell imaging; protein engineering.

Figure 1.. Characterization of MaLions in vitro.

(a-c) Normalized excitation (a), emission (b), and absorption spectra (c) of MaLionB (blue), MaLionG (green), and MaLionR (red) in the absence (dashed line) or presence of 10 mM ATP (solid line). (d) Normalized fluorescence of MaLions at different ATP concentrations. Dissociation constants (K_d), and Hill coefficients were calculated as 1.1 mM and 2.0 (MaLionG), 0.34 mM and 1.6 (MaLionR), and 0.46 mM and 1.4 (MaLionB), respectively. The limits of the detection of MaLionG, R, and B were estimated to be 0.50, 0.02, and 0.11 mM. (e) Specificity of MaLions. The fluorescence change (F/F₀) in response to the other nucleotides (10 mM) was normalized to that in response to ATP (10 mM). Error bars indicate mean ± standard deviation.

Figure 2.. Validation of MaLions in HeLa cells.

(a) Fluorescence images of HeLa cells expressing MaLions before (top) and after inhibition (NaF, 10 mM) (bottom). Scale bars, 20 μm. (b) Timelapse experiments of MaLion and negMaLion fluorescence following NaF treatment. Average normalized fluorescence intensity (FI) in single cells was calculated per dish (gray lines). Thick color lines represent the average of three independent experiments. (c) Bar graph of (b). The average normalized FI at the time-point where the biggest change occurred was calculated from cells expressing each negMaLions or MaLions (9–11 cells). *P<0.05, Student’s t-test. (d) Triple-color imaging of MaLionB, G and R in HeLa cells. Scale bar, 10 μm. Error bars indicate mean ± standard deviation.

Figure 3.. Dual-color imaging of subcellular ATP.

(a,b) Monitoring of ATP dynamics in the cytoplasm and mitochondria of a single HeLa cell. Oligomycin (20 μg/ml) was added at 3 min. The light green and red lines represent the normalized FI change of MaLionG and mitoMaLionR in each cell, respectively. Thick color lines are representative of three independent experiments. Scale bar, 20 μm. (c,d) Experiments described in (a,b) were repeated in brown adipocytes. Scale bar, 20 μm. (e) Images of Arabidopsis protoplast expressing MaLionG and mitoMaLionR in three different channels. Scale bar, 5 μm. (f) Monitoring of ATP dynamics in Arabidopsis protoplast following light stimulation-induced photosynthesis. During light stimulation using a 458-nm laser, the recording was paused for 1 min. (g) Images of cytoplasmic and mitochondrial ATP in the pharyngeal muscle of C. elegans. Scale bar, 25 μm. (h) Anesthesia of C. elegans was applied at 3 min using 1-phenoxy-2-propanol (0.5 %). Light green and red lines represent the normalized FI change of MaLionG and mitoMaLionR, respectively, in each C. elegans. Thick color lines are representative of three independent experiments.

Figure 4.. Triple-color imaging of a single brown adipocyte.

(a,b) Visualization of the dynamics of Ca²⁺ (turn-on type, B-GECO), cAMP (turn-off type, Flamindo2), and mitochondrial ATP (mitoMaLionR) in brown adipocytes. Timelapse images in three different channels and normalized FI changes in a representative cell are shown in (a) and (b). Isoproterenol (1 μM) was added at 5 min. (c) Light green (Flamindo2) and red (mitoMaLionR) lines represent normalized FI changes in individual cells. Thick lines represent the average of three independent experiments. (d,e,f) Experiments in (a,b,c) repeated using phenylephrine stimulation (10 μM). Scale bar, 10 μm.

Scheme 1.

Design of single fluorescence protein-based ATP indicators. MaLionG, MaLionR, and MaLionB (PDB ID: 3DPW, 2E5Y, and 2E5U).