An expanded palette of ATP sensors for subcellular and multiplexed imaging
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Abstract
Genetically encoded fluorescent biosensors that detect changes in ATP levels in live cells have enabled the discovery of novel roles for ATP in cellular processes and signaling. Many of these available ATP biosensors have a limited dynamic range, or have ATP affinities that are not suitable for sensing the physiological concentrations of ATP in mammalian cells. To address these limitations, we developed a FRET-based ATP biosensor with enhanced dynamic range and signal to noise ratio, eATeam. Using eATeam, we uncovered distinct spatiotemporal dynamics of ATP changes upon inhibition of cellular energy production. We also developed dimerization-dependent GFP and RFP-based ATP biosensors with enhanced dynamic ranges compared to the current standard in the field. Using the single-color ATP biosensors, we visualized the complex interplay between AMPK activity, ATP, lactate, and calcium by multiplexed imaging in single cells. This palette of ATP sensors expands the toolbox for interrogating subcellular ATP regulation and metabolic signaling in living cells.
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Thank you for the input! 1. it would be very interesting to see how ATP levels change in these compartments. We have not previously used such a targeting sequence but a ciliary targeting sequence could definitely be fused to this sensor. 2. There are ATP sensors optimized for bacterial use (the Queen series), but use in these other organisms might require some sensor optimization. 3. There are some existing GTP sensors available, such as the GEVAL sensor https://doi.org/10.1038/nmeth.4404
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Thank you for the suggestion! We will include the text in the figure to make it easier to interpret
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The development of these new genetically-encoded ATP biosensors is impressive! The targeting of the sensor to different parts of the cell enables studies of cellular energy expenditure in different regions or compartments of the cell. I have a few questions. 1. Have you considered making a ciliary ATP biosensor? You could fuse a ciliary targeting sequence to your sensor. I think the cilia community would be interested in this tool. 2. How well do you think this tool would work in other organisms, such as nematodes, algae, or ciliates? 3. How difficult would it be to develop a similar sensor for GTP?
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Figure 2.
Great figure! It clearly shows the utility of the ATP sensor for studying energy expenditure locally in the cell.
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Figure 1.
It would be useful to the reader to have a legend describing the different traces directly in the figure, so the reader can understand the experiments even without reading the text. For example, you could have small boxes that are black or cyan with text right besides that says "Control ( DMSO)" or "ATP depletion (2-DG)". Readers will appreciate clear and easy-to-interpret figures!
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