A green lifetime biosensor for calcium that remains bright over its full dynamic range

Reviewer #1 (Public review):

Summary:

van der Linden et al. report on the development of a new green-fluorescent sensor for calcium, following a novel rational design strategy based on the modification of the cyan-emissive sensor mTq2-CaFLITS. Through a mutational strategy similar to the one used to convert EGFP into EYFP, coupled with optimization of strategic amino acids located in proximity of the chromophore, they identify a novel sensor, G-CaFLITS. Through a careful characterization of the photophysical properties in vitro and the expression level in cell cultures, the authors demonstrate that G-CaFLITS combines a large lifetime response with a good brightness in both the bound and unbound states. This relative independence of the brightness on calcium binding, compared with existing sensors that often feature at least one very dim form, is an interesting feature of this new type of sensors, which allows for a more robust usage in fluorescence lifetime imaging. Furthermore, the authors evaluate the performance of G-CaFLITS in different subcellular compartments and under two-photon excitation in Drosophila. While the data appears robust and the characterization thorough, the interpretation of the results in some cases appears less solid, and alternative explanations cannot be excluded.

Strengths:

- The approach is innovative and extends the excellent photophysical properties of the mTq2-based to more red-shifted variants. While the spectral shift might appear relatively minor, as the authors correctly point out, it has interesting practical implications, such as the possibility to perform FLIM imaging of calcium using widely available laser wavelengths, or to reduce background autofluorescence, which can be a significant problem in FLIM.
- The screening was simple and rationally guided, demonstrating that, at least for this class of sensors, a careful choice of screening positions is an excellent strategy to obtain variants with large FLIM responses without the need of high-throughput screening.
- The description of the methodologies is very complete and accurate, greatly facilitating the reproduction of the results by others, or the adoption of similar methods. This is particularly true for the description of the experimental conditions for optimal screening of sensor variants in lysed bacterial cultures.
- The photophysical characterization is very thorough and complete, and the vast amount of data reported in the supporting information is a valuable reference for other researchers willing to attempt a similar sensor development strategy. Particularly well done is the characterization of the brightness in cells, and the comparison on multiple parameters with existing sensors.
- Overall, G-CaFLITS displays excellent properties for a FLIM sensor: very large lifetime change, bright emission in both forms and independence from pH in the physiological range.

Weaknesses:

- The paper demonstrates the application of G-CaFLITS in various cellular sub-compartments without providing direct evidence that the sensor's response is not affected by the targeting. Showing at least that the lifetime values in the saturated state are similar in all compartments would improve the robustness of the claims.
- In some cases, the interpretation of the results is not fully convincing, leaving alternative hypotheses as a possibility. This is particularly the case for the claim of the origin of the strongly reduced brightness of G-CaFLITS in Drosophila. The explanation of the intensity changes of G-CaFLITS also shows some inconsistency with the basic photophysical characterization.
- While the claims generally appear robust, in some cases they are conveyed with a lack of precision. Several sentences in the introduction and discussion could be improved in this regard. Furthermore, the use of the signal-to-noise ratio as a means of comparison between sensors appears to be imprecise, since it is dependent on experimental conditions.

Reviewer #2 (Public review):

Summary:

Van der Linden et al. describe the addition of the T203Y mutation to their previously described fluorescence lifetime calcium sensor Tq-Ca-FLITS to shift the fluorescence to green emission. This mutation was previously described to similarly red-shift the emission of green and cyan FPs. Tq-Ca-FLITS_T203Y behaves as a green calcium sensor with opposite polarity compared with the original (lifetime goes down upon calcium binding instead of up). They then screen a library of variants at two linker positions and identify a variant with slightly improved lifetime contrast (Tq-Ca-FLITS_T203Y_V27A_N271D, named G-Ca-FLITS). The authors then characterize the performance of G-Ca-FLITS relative to Tq-Ca-FLITS in purified protein samples, in cultured cells, and in the brains of fruit flies.

Strengths:

This work is interesting as it extends their prior work generating a calcium indicator scaffold for fluorescent protein-based lifetime sensors with large contrast at a single wavelength, which is already being adopted by the community for production of other FLIM biosensors. This work effectively extends that from cyan to green fluorescence. While the cyan and green sensors are not spectrally distinct enough (~20-30nm shift) to easily multiplex together, it at least shifts the spectra to wavelengths that are more commonly available on commercial microscopes.

The observations of organellar calcium concentrations were interesting and could potentially lead to new biological insight if followed up.

Weaknesses:

The new G-Ca-FLITS sensor doesn't appear to be significantly improved in performance over the original Tq-Ca-FLITS, no specific benefits are demonstrated.

Although it was admirable to attempt in vivo demonstration in Drosophila with these sensors, depolarizing the whole brain with high potassium is not a terribly interesting or physiological stimulus and doesn't really highlight any advantages of their sensors; G-Ca-FLITS appears to be quite dim in the flies.

Reviewer #3 (Public review):

Summary:

The authours present a variant of a previously described fluorescence lifetime sensor for calcium. Much of the manuscript describes the process of developing appropriate assays for screening sensor variants, and thorough characterization of those variants (inherent fluorescence characteristics, response to calcium and pH, comparisons to other calcium sensors). The final two figures show how the sensor performs in cultured cells and in vivo drosophila brains.

Strengths:

The work is presented clearly and the conclusion (this is a new calcium sensor that could be useful in some circumstances) is supported by the data.

Weaknesses:

There are probably few circumstances where this sensor would facilitate experiments (calcium measurements) that other sensors would prove insufficient.