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A transcriptional reporter of intracellular Ca2+ in Drosophila

Abstract

Intracellular Ca2+ is a widely used neuronal activity indicator. Here we describe a transcriptional reporter of intracellular Ca2+ (TRIC) in Drosophila that uses a binary expression system to report Ca2+-dependent interactions between calmodulin and its target peptide. We found that in vitro assays predicted in vivo properties of TRIC and that TRIC signals in sensory systems depend on neuronal activity. TRIC was able to quantitatively monitor neuronal responses that changed slowly, such as those of neuropeptide F–expressing neurons to sexual deprivation and neuroendocrine pars intercerebralis cells to food and arousal. Furthermore, TRIC-induced expression of a neuronal silencer in nutrient-activated cells enhanced stress resistance, providing a proof of principle that TRIC can be used for circuit manipulation. Thus, TRIC facilitates the monitoring and manipulation of neuronal activity, especially those reflecting slow changes in physiological states that are poorly captured by existing methods. TRIC's modular design should enable optimization and adaptation to other organisms.

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Figure 1: Proof of principle of TRIC in cultured cells and transgenic flies.
Figure 2: TRIC signals in the optic lobes depend on visual transduction and visual experience.
Figure 3: Characterization of TRIC in olfactory PNs.
Figure 4: Stoichiometric tuning of TRIC and its application in NPF neurons.
Figure 5: Monitoring PI cell activity with TRIC.
Figure 6: Improved signal-to-noise ratio using mutant TRIC components allows quantitative analysis and manipulation of PI cells.

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Acknowledgements

We thank D. Luginbuhl (Stanford University) for generating transgenic flies, R. Alfa, J. Cao, X. Dong, Y. Fisher, D.M. Gohl, M. Lin, S. Park, C. Ran, K. Shen, M. Silies, X. Wei, Z. Yang and C. Zhou for advice and technical support, H.A. Dierick (Baylor College of Medicine), G. Dietzl (Stanford University), T. Lee (Janelia Farm), A. Rajan (Harvard University), G.M. Rubin (Janelia Farm), J.W. Wang (University of California, San Diego), M. Zeidler (University of Sheffield) and Bloomington Stock Center for fly strains, Addgene for plasmids, and L. DeNardo Wilke, C.J. Guenthner, T.J. Mosca and X. Wang for critiques on the manuscript. X.J.G. is supported by an Enlight Foundation Interdisciplinary Fellowship. L.L. receives funding from the Howard Hughes Medical Institute. This study was also supported by US National Institutes of Health grants R01-DC005982 (L.L.), R01-EY022638 (T.R.C.) and R01-DC013070 (C.J.P.), and a grant from Whitehall Foundation (C.J.P.).

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Authors and Affiliations

Authors

Contributions

X.J.G. designed, performed and analyzed the experiments, aided by J.L. during revision. L.L. and T.R.C. supervised the project. O.R. and C.J.P. provided the unpublished nsyb-QF2 line. X.J.G., L.L. and T.R.C. wrote the manuscript, with inputs from the other authors.

Corresponding authors

Correspondence to Xiaojing J Gao, Thomas R Clandinin or Liqun Luo.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Initial testing of TRIC in cultured cells and flies.

(a) CaM/M13-mediated TRIC in S2 cells (ActP-M13::GAL4DBDo, ActP-p65AD::CaM). The expression of UAS-GFP in S2 cells depends on the presence of ActP-dtrpA1 (right), yet weaker and sparser than split GAL4 (Fig. 1c). (b) CaM/skMLCKN5A-mediated TRIC in S2 cells. The expression of the reporter in the presence of dTrpA1 (right) is comparable to split GAL4 (Fig. 1c), but the background is strong in the absence of dTrpA1 (left). (c) CaM/M13-mediated TRIC in transgenic flies (nsyb-M13::GAL4DBDo, QUAS-p65AD::CaM, nsyb-QF2, tubP-QS, UAS-mCD8::RFP, n ≥ 3). The TRIC signal is present only in ORNs projecting to several glomeruli (c, “control”, compare to Fig. 1d). We hypothesized that this signal reflects ORN activity induced by the food-derived olfactory input. Indeed, the signal was diminished when the flies were separated from food (and thus food-related odors; c, “no food”). Two-tailed unpaired t-test for the quantification (P = 0.0038). (d) CaM/MKII-mediated TRIC signal in the ventral nerve cord of transgenic flies (same genotype as Fig. 1d).

Supplementary Figure 2 Reporter choices and TRIC signal-to-baseline ratio.

(a) Visual deprivation does not significantly alter TRIC signal using mCD8::RFP as the reporter (nsyb-MKII::GAL4DBDo, QUAS-p65AD::CaM, nsyb-QF2, tubP-QS, UAS-mCD8::RFP, n ≥ 5). (b) nsyb::GFP is less stable than CD8::RFP in ORNs (n ≥ 6). Shown are the axonal signals in the antennal lobes one day after removing ORN cell bodies with antennectomy and preventing the further synthesis of reporter proteins from the ORN cell bodies. For either reporter, the signals were normalized to the mock surgery control. (c, d) TRIC signal is reduced by visual deprivation after replacing mCD8::RFP with nsyb::GFP (c, n ≥ 10) or hsFLP, UAS-FRT-stop-FRT-mCD8::GFP (d, n ≥ 5) as the reporter. For d, flies were raised at 18 °C in the dark until eclosion, heat shocked at 37 °C for short periods to remove the stop sequences, and then kept at room temperature for three days in light or dark. Two-tailed unpaired t-test for a (P = 0.1426), c (P < 0.0001), and d (P < 0.0001). Two-way ANOVA for b (interaction P = 0.0244).

Supplementary Figure 3 Characterization of TRIC in PNs

(a) Antenna removal mildly reduces UAS-nsyb::GFP directly driven by GH146-GAL4 (n ≥ 6, compared to Fig. 3b). (b) Unilateral antennectomy causes no difference in TRIC signals in the ipsi- vs. contra-lateral PNs (n = 9). (c) Antenna removal reduces signal in PN-specific TRIC using a luciferase reporter (same genotype as Fig. 3c) but not UAS-luciferase directly driven by GH146-GAL4 (n ≥ 5). The two genotypes were tested in separate batches, and normalized to a UAS-luciferase/+ control. (d) Artificial activation of PNs increases TRIC signal, using the luciferase reporter (n ≥ 3). The LexA system was used to express dTrpA1 in PNs, and the flies were subject to repetitive heat-shocks. nsyb-MKII::GAL4DBDo, QUAS-p65AD::CaM, GH146-QF, UAS-luciferase for the left half of c and d; GH146-GAL4, UAS-luciferase for the right half of c. Two-tailed unpaired t-test for a (P = 0.0152), d (P = 0.025); two-tailed paired t-test for b (P = 0.0683); two-way ANOVA for c (interaction P < 0.0001).

Supplementary Figure 4 Comparing TRIC to CaLexA.

(a) Signal from LexA-based TRIC centered around the antennal lobes (nsyb-MKII::nlsLexADBDo, QUAS-p65AD::CaM, GH146-QF, lexAop2-mCD8::GFP, representative of 7 brains). (b) Signal from CaLexA centered around the antennal lobes, with multiple copies of reporters (UAS-CaLexA, GH146-GAL4, lexAop-mCD2::GFP, lexAop-mCD8::GFP::2A::mCD8::GFP, representative of 7 brains). (c) Signal from pan-neuronal expression of CaLexA (LexAop2-CD8::GFP, UAS-CaLexA, nsyb-GAL4, representative of 7 samples) is substantially weaker and sparser than that of TRIC (Fig. 1d), despite being imaged at a higher gain.

Supplementary Figure 5 TRIC signal in neuromodulatory circuits.

(a-d) Expressing TRIC with specific GAL4s in different neuromodulatory circuits (nsyb-MKII::nlsLexADBDo, UAS-p65AD::CaM, X-GAL4, UAS-mCD8::RFP, LexAop2-mCD8::GFP, representative of ≥ 5 samples). ple-, trh-, tdc-, and dimm-GAL4s target dopaminergic, serotonergic, tyraminergic/octopaminergic, and peptidergic neurons respectively. (e) TRIC in the PI cells with RU486-inducible expression of DBD (same genotype as Fig. 4c, representative of ≥ 11 samples). In this figure, the GAL4 expression pattern is visualized in the first column, and TRIC signal in the second column.

Supplementary Figure 6 Characterizing TRIC in PI cells.

a) TRIC signal is not significantly affected by starvation when 75 mg QA per vial was used to induce TRIC expressions (n ≥ 4, compared to Fig. 5c), probably due to signal saturation. (b) Starvation still reduces TRIC signal in upd2 mutant (n ≥ 14). (c) TRIC signal in PI neurons increases with the duration of food exposure (n ≥ 7). We first induced TRIC with QA for 24 hours, and then exposed the flies to food for 0, 12, or 24 hours. (d) The decay of TRIC signal. The flies were first exposed to food to induce TRIC signal in PI cells, and then kept on wet Kimwipe for the specified durations, the half life was determined to be 0.55 day, after fitting with an exponential curve. (e) MKIIK11A improves the fold of TRIC signal induction in response to 10% yeast (n ≥ 9, compared to Fig. 5d). (f) MKIIK11A improves the fold of TRIC signal induction by 10 mg/mL OA (n ≥ 10, compared to Fig. 5e). (g) Left, TRIC signal in response to mianserin titration in the presence of OA (n ≥ 8), to determine an intermediate mianserin dose. Right, differential TRIC signals in response to saturating OA concentrations in the presence of mianserin (n ≥ 9, compared to Fig. 6e). (h) TRIC signals in the brain at Day 0 under the “control” and “experiment” conditions as in Fig. 6h (representative of 10 samples). The only notable difference of expression is in the PI cells (dashed circles). Both a and b are the same genotype as Fig. 1d, except that mCD8::RFP is replaced with mCD8::GFP. The rest of the figures are of the genotype nsyb-MKIIK11A::GAL4DBDo, QUAS-p65AD::CaM, nsyb-QF2, tubP-QS, UAS-mCD8::RFP. Two-tailed unpaired t-test for a (P = 0.1591), b (P = 0.0291), c (P = 0.0040, 0.0002), d (P = 0.0171, 0.0040), e (P = 0.0072), f (P < 0.0001, < 0.0001), and g (P = 0.0009), with Holm-Bonferroni correction for multiple comparisons.

Supplementary Figure 7 Simulating the effects of alanine mutation and endogenous competition on TRIC signal.

(a) The K11A mutation reduces TRIC signal (left) without affecting the shape of the response curve (right). (b) Competition from endogenous CaM and its target peptides reduces TRIC signal (left) without affecting the shape of the response curve (right). (c) The K11A mutation reduces TRIC signal (left) and lowers the sensitivity of the response curve (right) in the presence of 5 × endogenous competition. All the variables and parameters for this figure are identical to that of Fig. 4b, unless stated otherwise. (d) Correlation between in silico and in vitro alanine scan over the non-charged residues. The reporter expression in S2 cells were visually assigned to four ranks according to intensity, and correlated with the simulated ΔΔG using Spearman’s rank. Robetta calculates ΔΔG by subtracting the CaM-MKII binding energy (ΔG) from the binding energy between CaM and the alanine mutant of MKII.

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Gao, X., Riabinina, O., Li, J. et al. A transcriptional reporter of intracellular Ca2+ in Drosophila. Nat Neurosci 18, 917–925 (2015). https://doi.org/10.1038/nn.4016

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