iATPSnFR2: A high-dynamic-range fluorescent sensor for monitoring intracellular ATP, 2024, Jonathan S. Marvin et al

Significance
Adenosine triphosphate (ATP) is a key metabolite necessary for cellular life. Here, we develop a next-generation genetically encoded ratiometric fluorescent ATP sensor that allows subcellular tracking of ATP levels in living cells. The large dynamic range makes it possible to follow the dynamics of this metabolite across cells and subcellular regions under different metabolic stressors. We expect that iATPSnFR2, combined with proper controls for assessing changes in pH, will provide researchers with exciting opportunities to study ATP dynamics with high temporal and spatial resolution.

Abstract
We developed a significantly improved genetically encoded quantitative adenosine triphosphate (ATP) sensor to provide real-time dynamics of ATP levels in subcellular compartments. iATPSnFR2 is a variant of iATPSnFR1, a previously developed sensor that has circularly permuted superfolder green fluorescent protein (GFP) inserted between the ATP-binding helices of the ε-subunit of a bacterial F0-F1 ATPase.

Optimizing the linkers joining the two domains resulted in a ~fivefold to sixfold improvement in the dynamic range compared to the previous-generation sensor, with excellent discrimination against other analytes, and affinity variants varying from 4 µM to 500 µM. A chimeric version of this sensor fused to either the HaloTag protein or a suitable spectrally separated fluorescent protein provides an optional ratiometric readout allowing comparisons of ATP across cellular regions. Subcellular targeting the sensor to nerve terminals reveals previously uncharacterized single-synapse metabolic signatures, while targeting to the mitochondrial matrix allowed direct quantitative probing of oxidative phosphorylation dynamics.

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Primarily, measuring pH changes and O2 consumption are just proxies for ATP production, and not a direct measurement. Other metabolic events or perturbations that affect pH and O2 use will also be interpreted as ATP consumption. Importantly, the Seahorse system is limited to cell culture analysis analysis and is not compatible with vivo applications or measurement of ATP consumption with other manipulations, such as electrical stimulation. This technology also affords no spatial resolution either at the single cell or subcellular length scale.

Furthermore, once cells have been treated with mitochondrial poisons, they are no longer viable and cannot be assayed again.

Finally, the time course of measurement using such devices requires integration of signals over time and is not compatible with subsecond resolution. We expect that iATPSnFR2 will provide researchers with opportunities to study ATP dynamics with temporal and spatial resolution that has, until now, been unobtainable.

 

I love when this sort of step forward happens in basic science, allowing us to see what is happening in cells at a basic level. I bet some assumptions get overturned!

For us, I bet there's many uses. One occurs to me: This may be a way to test the purinergic signalling theory (atp is a 'purine', hence purinergic). Are some cells squirting out ATP as a signal? Does that lead to a shortage in the cell?

One of the really basic ways vasodilation happens is via ATP release. It's a really simple thing: when red blood cells get squished and jostled inside a narrow blood vessel, they release ATP and that signals to the blood vessel to dilate.

If we have endothelial problems that mean the blood vessel is poor at dilating, the atp release could be quite relentless. Extracellular atp is considered to be a credible danger signal, a cell would only release its most valuable energy under conditions of bad stress.

(alternative theory: red bood cells with stiff walls aren't getting squished enough to rleease atp, and that's why the blood cells don't dilate; to prefer this theory you need to posit a membrane problem upstream of the vasodilation problem; perhaps our peroxisomes are doing a terrible job making the kind of phospholipids that lead to bendy cell membranes).