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A transcriptional reporter of intracellular Ca2+ in Drosophila

Nature Neuroscience volume 18, pages 917–925 (2015)Cite this article

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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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References

  1. Tsien, R.Y. Fluorescence measurement and photochemical manipulation of cytosolic free calcium. Trends Neurosci. 11, 419–424 (1988).

    CAS  PubMed  Google Scholar 

  2. Looger, L.L. & Griesbeck, O. Genetically encoded neural activity indicators. Curr. Opin. Neurobiol. 22, 18–23 (2012).

    CAS  PubMed  Google Scholar 

  3. Marella, S., Mann, K. & Scott, K. Dopaminergic modulation of sucrose acceptance behavior in Drosophila. Neuron 73, 941–950 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Inagaki, H.K. et al. Visualizing neuromodulation in vivo: TANGO-mapping of dopamine signaling reveals appetite control of sugar sensing. Cell 148, 583–595 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Nusbaum, M.P., Blitz, D.M., Swensen, A.M., Wood, D. & Marder, E. The roles of co-transmission in neural network modulation. Trends Neurosci. 24, 146–154 (2001).

    CAS  PubMed  Google Scholar 

  6. Takemura, S.Y. et al. A visual motion detection circuit suggested by Drosophila connectomics. Nature 500, 175–181 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Sheng, M. & Greenberg, M.E. The regulation and function of c-fos and other immediate early genes in the nervous system. Neuron 4, 477–485 (1990).

    CAS  PubMed  Google Scholar 

  8. Reijmers, L.G., Perkins, B.L., Matsuo, N. & Mayford, M. Localization of a stable neural correlate of associative memory. Science 317, 1230–1233 (2007).

    CAS  PubMed  Google Scholar 

  9. Koya, E. et al. Targeted disruption of cocaine-activated nucleus accumbens neurons prevents context-specific sensitization. Nat. Neurosci. 12, 1069–1073 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Guenthner, C.J., Miyamichi, K., Yang, H.H., Heller, H.C. & Luo, L. Permanent genetic access to transiently active neurons via TRAP: targeted recombination in active populations. Neuron 78, 773–784 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Kawashima, T. et al. Functional labeling of neurons and their projections using the synthetic activity-dependent promoter E-SARE. Nat. Methods 10, 889–895 (2013).

    CAS  PubMed  Google Scholar 

  12. Fujita, N. et al. Visualization of neural activity in insect brains using a conserved immediate early gene, Hr38. Curr. Biol. 23, 2063–2070 (2013).

    CAS  PubMed  Google Scholar 

  13. Masuyama, K., Zhang, Y., Rao, Y. & Wang, J.W. Mapping neural circuits with activity-dependent nuclear import of a transcription factor. J. Neurogenet. 26, 89–102 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Rhoads, A.R. & Friedberg, F. Sequence motifs for calmodulin recognition. FASEB J. 11, 331–340 (1997).

    CAS  PubMed  Google Scholar 

  15. Luan, H., Peabody, N.C., Vinson, C.R. & White, B.H. Refined spatial manipulation of neuronal function by combinatorial restriction of transgene expression. Neuron 52, 425–436 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Pfeiffer, B.D. et al. Refinement of tools for targeted gene expression in Drosophila. Genetics 186, 735–755 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Nassel, D.R. Insulin-producing cells and their regulation in physiology and behavior of Drosophila. Can. J. Zool. 90, 476–488 (2012).

    Google Scholar 

  18. Hamada, F.N. et al. An internal thermal sensor controlling temperature preference in Drosophila. Nature 454, 217–220 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Peersen, O.B., Madsen, T.S. & Falke, J.J. Intermolecular tuning of calmodulin by target peptides and proteins: differential effects on Ca2+ binding and implications for kinase activation. Protein Sci. 6, 794–807 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Potter, C.J., Tasic, B., Russler, E.V., Liang, L. & Luo, L. The Q system: a repressible binary system for transgene expression, lineage tracing, and mosaic analysis. Cell 141, 536–548 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Riabinina, O. et al. Improved and expanded Q-system reagents for genetic manipulations. Nat. Methods 12, 219–222 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Bloomquist, B.T. et al. Isolation of a putative phospholipase C gene of Drosophila, norpA, and its role in phototransduction. Cell 54, 723–733 (1988).

    CAS  PubMed  Google Scholar 

  23. Estes, P.S., Ho, G.L.Y., Narayanan, R. & Ramaswami, M. Synaptic localization and restricted diffusion of a Drosophila neuronal synaptobrevin—green fluorescent protein chimera in vivo. J. Neurogenet. 13, 233–255 (2000).

    CAS  PubMed  Google Scholar 

  24. Wong, A.M., Wang, J.W. & Axel, R. Spatial representation of the glomerular map in the Drosophila protocerebrum. Cell 109, 229–241 (2002).

    CAS  PubMed  Google Scholar 

  25. Clark, D.A., Bursztyn, L., Horowitz, M.A., Schnitzer, M.J. & Clandinin, T.R. Defining the computational structure of the motion detector in Drosophila. Neuron 70, 1165–1177 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Gaudry, Q., Hong, E.J., Kain, J., de Bivort, B.L. & Wilson, R.I. Asymmetric neurotransmitter release enables rapid odor lateralization in Drosophila. Nature 493, 424–428 (2013).

    CAS  PubMed  Google Scholar 

  27. Friggi-Grelin, F. et al. Targeted gene expression in Drosophila dopaminergic cells using regulatory sequences from tyrosine hydroxylase. J. Neurobiol. 54, 618–627 (2003).

    CAS  PubMed  Google Scholar 

  28. Alekseyenko, O.V., Lee, C. & Kravitz, E.A. Targeted manipulation of serotonergic neurotransmission affects the escalation of aggression in adult male Drosophila melanogaster. PLoS ONE 5, e10806 (2010).

    PubMed  PubMed Central  Google Scholar 

  29. Cole, S.H. et al. Two functional but noncomplementing Drosophila tyrosine decarboxylase genes. J. Biol. Chem. 280, 14948–14955 (2005).

    CAS  PubMed  Google Scholar 

  30. Wu, Q. et al. Developmental control of foraging and social behavior by the Drosophila neuropeptide Y-like system. Neuron 39, 147–161 (2003).

    CAS  PubMed  Google Scholar 

  31. Park, D., Veenstra, J.A., Park, J.H. & Taghert, P.H. Mapping peptidergic cells in Drosophila: where DIMM fits in. PLoS ONE 3, e1896 (2008).

    PubMed  PubMed Central  Google Scholar 

  32. Fridell, Y.W., Sanchez-Blanco, A., Silvia, B.A. & Helfand, S.L. Targeted expression of the human uncoupling protein 2 (hUCP2) to adult neurons extends life span in the fly. Cell Metab. 1, 145–152 (2005).

    CAS  PubMed  Google Scholar 

  33. Joiner, M.A. & Griffith, L.C. Mapping of the anatomical circuit of CaM kinase–dependent courtship conditioning in Drosophila. Learn. Mem. 6, 177–192 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Shohat-Ophir, G., Kaun, K.R., Azanchi, R., Mohammed, H. & Heberlein, U. Sexual deprivation increases ethanol intake in Drosophila. Science 335, 1351–1355 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Géminard, C., Rulifson, E.J. & Leopold, P. Remote control of insulin secretion by fat cells in Drosophila. Cell Metab. 10, 199–207 (2009).

    PubMed  Google Scholar 

  36. Rajan, A. & Perrimon, N. Drosophila cytokine unpaired 2 regulates physiological homeostasis by remotely controlling insulin secretion. Cell 151, 123–137 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Crocker, A., Shahidullah, M., Levitan, I.B. & Sehgal, A. Identification of a neural circuit that underlies the effects of octopamine on sleep:wake behavior. Neuron 65, 670–681 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Roeder, T. Tyramine and octopamine: Ruling behavior and metabolism. Annu. Rev. Entomol. 50, 447–477 (2005).

    CAS  PubMed  Google Scholar 

  39. Monastirioti, M., Linn, C.E. Jr. & White, K. Characterization of Drosophila tyramine beta-hydroxylase gene and isolation of mutant flies lacking octopamine. J. Neurosci. 16, 3900–3911 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Chen, T.W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Kitamoto, T. Conditional modification of behavior in Drosophila by targeted expression of a temperature-sensitive shibire allele in defined neurons. J. Neurobiol. 47, 81–92 (2001).

    CAS  PubMed  Google Scholar 

  42. Broughton, S.J. et al. Longer lifespan, altered metabolism, and stress resistance in Drosophila from ablation of cells making insulin-like ligands. Proc. Natl. Acad. Sci. USA 102, 3105–3110 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Schrödel, T., Prevedel, R., Aumayr, K., Zimmer, M. & Vaziri, A. Brain-wide 3D imaging of neuronal activity in Caenorhabditis elegans with sculpted light. Nat. Methods 10, 1013–1020 (2013).

    PubMed  Google Scholar 

  44. Ma, H. et al. gammaCaMKII shuttles Ca(2)(+)/CaM to the nucleus to trigger CREB phosphorylation and gene expression. Cell 159, 281–294 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Fosque, B.F. et al. Neural circuits. Labeling of active neural circuits in vivo with designed calcium integrators. Science 347, 755–760 (2015).

    CAS  PubMed  Google Scholar 

  46. Schmidt, D. & Cho, Y.K. Natural photoreceptors and their application to synthetic biology. Trends Biotechnol. 33, 80–91 (2015).

    CAS  PubMed  Google Scholar 

  47. Bath, D.E. et al. FlyMAD: rapid thermogenetic control of neuronal activity in freely walking Drosophila. Nat. Methods 11, 756–762 (2014).

    CAS  PubMed  Google Scholar 

  48. Palmer, A.E. et al. Ca2+ indicators based on computationally redesigned calmodulin-peptide pairs. Chem. Biol. 13, 521–530 (2006).

    CAS  PubMed  Google Scholar 

  49. Zhou, C., Rao, Y. & Rao, Y. A subset of octopaminergic neurons are important for Drosophila aggression. Nat. Neurosci. 11, 1059–1067 (2008).

    CAS  PubMed  Google Scholar 

  50. Nässel, D.R. & Winther, A.M. Drosophila neuropeptides in regulation of physiology and behavior. Prog. Neurobiol. 92, 42–104 (2010).

    PubMed  Google Scholar 

  51. Tian, L. et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat. Methods 6, 875–881 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Montigiani, S., Neri, G., Neri, P. & Neri, D. Alanine substitutions in calmodulin-binding peptides result in unexpected affinity enhancement. J. Mol. Biol. 258, 6–13 (1996).

    CAS  PubMed  Google Scholar 

  53. Bischof, J., Maeda, R.K., Hediger, M., Karch, F. & Basler, K. An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases. Proc. Natl. Acad. Sci. USA 104, 3312–3317 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Berdnik, D., Chihara, T., Couto, A. & Luo, L. Wiring stability of the adult Drosophila olfactory circuit after lesion. J. Neurosci. 26, 3367–3376 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Sweeney, L.B. et al. Temporal target restriction of olfactory receptor neurons by Semaphorin-1a/PlexinA-mediated axon-axon interactions. Neuron 53, 185–200 (2007).

    CAS  PubMed  Google Scholar 

  56. Smith, H.K. et al. Inducible ternary control of transgene expression and cell ablation in Drosophila. Dev. Genes Evol. 206, 14–24 (1996).

    CAS  PubMed  Google Scholar 

  57. Lai, S.L., Awasaki, T., Ito, K. & Lee, T. Clonal analysis of Drosophila antennal lobe neurons: diverse neuronal architectures in the lateral neuroblast lineage. Development 135, 2883–2893 (2008).

    CAS  PubMed  Google Scholar 

  58. Hombría, J.C., Brown, S., Hader, S. & Zeidler, M.P. Characterization of Upd2, a Drosophila JAK/STAT pathway ligand. Dev. Biol. 288, 420–433 (2005).

    PubMed  Google Scholar 

  59. Katsov, A.Y. & Clandinin, T.R. Motion processing streams in Drosophila are behaviorally specialized. Neuron 59, 322–335 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Kim, D.E., Chivian, D. & Baker, D. Protein structure prediction and analysis using the Robetta server. Nucleic Acids Res. 32, W526–W531 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Wu, J.S. & Luo, L. A protocol for dissecting Drosophila melanogaster brains for live imaging or immunostaining. Nat. Protoc. 1, 2110–2115 (2006).

    CAS  PubMed  Google Scholar 

Download references

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

  1. Howard Hughes Medical Institute and Department of Biology, Stanford University, Stanford, California, USA

    Xiaojing J Gao, Jiefu Li & Liqun Luo

  2. The Solomon H. Snyder Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Olena Riabinina & Christopher J Potter

  3. Department of Neurobiology, Stanford University, Stanford, California, USA

    Thomas R Clandinin & Liqun Luo

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  1. Xiaojing J Gao
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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.

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

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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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