Imaging energy status in live cells with a fluorescent biosensor of the intracellular ATP-to-ADP ratio

Abstract

The ATP:ADP ratio is a critical parameter of cellular energy status that regulates many metabolic activities. Here we report an optimized genetically encoded fluorescent biosensor, PercevalHR, that senses the ATP:ADP ratio. PercevalHR is tuned to the range of intracellular ATP:ADP expected in mammalian cells, and it can be used with one-or two-photon microscopy in live samples. We use PercevalHR to visualize activity-dependent changes in ATP:ADP when neurons are exposed to multiple stimuli, demonstrating that it is a sensitive reporter of physiological changes in energy consumption and production. We also use PercevalHR to visualize intracellular ATP:ADP while simultaneously recording currents from ATP-sensitive potassium (K ATP ) channels in single cells, showing that PercevalHR enables the study of coordinated variation in ATP:ADP and K ATP channel open probability in intact cells. With its ability to monitor changes in cellular energetics within seconds, PercevalHR should be a versatile tool for metabolic research.

Mitochondrial biogenesis is required for axonal growth

“…Next, we estimated the cell ATP/ADP ratio using the genetically encoded fluorescent ratiometric probe PercevalHR, which senses ATP/ADP ratio (Tantama et al, 2013). Control experiments demonstrated either an increase in PercevalHR signal (F 488nm / F 405nm ) for individual axonal endings after reinfusion of glucose to glucose-deprived cells, or a decrease in signal after inhibition of mitochondrial ATP production by oligomycin, confirming the suitability of PercevalHR for tracking intracellular ATP/ADP levels in our model (Fig.…”

The Pancreatic β-Cell: A Bioenergetic Perspective

“…Perceval was subsequently modified ("PercevalHR") to respond to ATP/ADP ratios up to 40:1, and with a 3.5-fold dynamic range (507). It should be noted, however, that even this range might be insufficient to accurately record maximal ATP/ADP ratios.…”

“…It is therefore surprising that such a powerful and quantitative technique as the monitoring of the PCr/Cr ratio has not been more exploited. One potential application, perhaps best performed with a muscle-derived cell line such as C2C12, would be to calibrate the fluorescent response of Perceval or PercevalHR (507) in terms of the absolute ATP/ADP ratios calculated from the PCr/Cr pool as ⌬p is varied, rather than by extrapolation from cell-free determinations (507).…”

Neuronal energy consumption: biophysics, efficiency and evolution

“…Such an approach depends upon electrophysiological measurements of currents and conductances, as well as membrane capacitance. Recently, traditional electrophysiological methods have been augmented by live imaging of molecules that are directly involved in energy metabolism, such as ADP/ATP or NADH-NAD+ [71][72][73][74]. This imaging has enormous potential for estimating energy consumption within spatially extensive neurons and specific structures such as synapses [73,74], providing bounds for cellular-and subcellularlevel bottom-up energy budgets.…”

“…Recently, traditional electrophysiological methods have been augmented by live imaging of molecules that are directly involved in energy metabolism, such as ADP/ATP or NADH-NAD+ [71][72][73][74]. This imaging has enormous potential for estimating energy consumption within spatially extensive neurons and specific structures such as synapses [73,74], providing bounds for cellular-and subcellularlevel bottom-up energy budgets. When combined with details of molecular processes that occur within neurons, such as the structure of second messenger cascades, this approach can yield detailed energy budgets for neurons that quantify the consumption of specific processes.…”