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An ultrasensitive biosensor for high-resolution kinase activity imaging in awake mice

Nature Chemical Biology volume 17, pages 39–46 (2021)Cite this article

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Abstract

Protein kinases control nearly every facet of cellular function. These key signaling nodes integrate diverse pathway inputs to regulate complex physiological processes, and aberrant kinase signaling is linked to numerous pathologies. While fluorescent protein-based biosensors have revolutionized the study of kinase signaling by allowing direct, spatiotemporally precise kinase activity measurements in living cells, powerful new molecular tools capable of robustly tracking kinase activity dynamics across diverse experimental contexts are needed to fully dissect the role of kinase signaling in physiology and disease. Here, we report the development of an ultrasensitive, second-generation excitation-ratiometric protein kinase A (PKA) activity reporter (ExRai-AKAR2), obtained via high-throughput linker library screening, that enables sensitive and rapid monitoring of live-cell PKA activity across multiple fluorescence detection modalities, including plate reading, cell sorting and one- or two-photon imaging. Notably, in vivo visual cortex imaging in awake mice reveals highly dynamic neuronal PKA activity rapidly recruited by forced locomotion.

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Fig. 1: Identification and characterization of ExRai-AKAR2.
Fig. 2: Subcellular kinase activity detection and multiplexed imaging using ExRai-AKAR2.
Fig. 3: ExRai-AKAR2 permits robust, multi-modal detection of live-cell PKA activity.
Fig. 4: PKA activity imaging in cultured neurons using ExRai-AKAR2.
Fig. 5: ExRai-AKAR2 enables rapid and sensitive kinase activity imaging in vivo.

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Data availability

All data supporting the findings of this study are available upon reasonable request. Source data are provided with this paper.

Code availability

Custom ImageJ macros and MATLAB code used to analyze in vitro and in vivo neuronal imaging data are available upon reasonable request.

References

  1. Greenwald, E. C., Mehta, S. & Zhang, J. Genetically encoded fluorescent biosensors illuminate the spatiotemporal regulation of signaling networks. Chem. Rev. 118, 11707–11794 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Chen, Y., Saulnier, J. L., Yellen, G. & Sabatini, B. L. A PKA activity sensor for quantitative analysis of endogenous GPCR signaling via 2-photon FRET-FLIM imaging. Front Pharm. 5, 56 (2014).

    Google Scholar 

  3. Chen, Y. et al. Endogenous Gαq-coupled neuromodulator receptors activate protein kinase A. Neuron 96, 1070–1083.e5 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Tang, S. & Yasuda, R. Imaging ERK and PKA activation in single dendritic spines during structural plasticity. Neuron 93, 1315–1324.e3 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Ma, L. et al. A highly sensitive a-kinase activity reporter for imaging neuromodulatory events in awake mice. Neuron 99, 665–679.e5 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Yamaguchi, T. et al. Role of PKA signaling in D2 receptor-expressing neurons in the core of the nucleus accumbens in aversive learning. Proc. Natl Acad. Sci. USA 112, 11383–11388 (2015).

    CAS  PubMed  Google Scholar 

  7. Castro, L. R. V. et al. Striatal neurones have a specific ability to respond to phasic dopamine release. J. Physiol. 591, 3197–3214 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Yapo, C. et al. Detection of phasic dopamine by D1 and D2 striatal medium spiny neurons. J. Physiol. 595, 7451–7475 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Yagishita, S. et al. A critical time window for dopamine actions on the structural plasticity of dendritic spines. Science 345, 1616–1620 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Jongbloets, B. C., Ma, L., Mao, T. & Zhong, H. Visualizing protein kinase a activity in head-fixed behaving mice using in vivo two-photon fluorescence lifetime imaging microscopy. J. Vis. Exp. e59526 https://doi.org/10.3791/59526 (2019).

  11. Mehta, S. et al. Single-fluorophore biosensors for sensitive and multiplexed detection of signalling activities. Nat. Cell Biol. 20, 1215–1225 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Depry, C., Allen, M. D. & Zhang, J. Visualization of PKA activity in plasma membrane microdomains. Mol. Biosyst. 7, 52–58 (2011).

    CAS  PubMed  Google Scholar 

  13. Komatsu, N. et al. Development of an optimized backbone of FRET biosensors for kinases and GTPases. Mol. Biol. Cell 22, 4647–4656 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 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 

  15. Miyawaki, A. et al. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887 (1997).

    CAS  PubMed  Google Scholar 

  16. Nakai, J., Ohkura, M. & Imoto, K. A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein. Nat. Biotechnol. 19, 137–141 (2001).

    CAS  PubMed  Google Scholar 

  17. Hires, S. A., Zhu, Y. & Tsien, R. Y. Optical measurement of synaptic glutamate spillover and reuptake by linker optimized glutamate-sensitive fluorescent reporters. Proc. Natl Acad. Sci. USA 105, 4411–4416 (2008).

    CAS  PubMed  Google Scholar 

  18. Steinberg, S. F. & Brunton, L. L. Compartmentation of G protein-coupled signaling pathways in cardiac myocytes. Annu. Rev. Pharmacol. Toxicol. 41, 751–773 (2001).

    CAS  PubMed  Google Scholar 

  19. Carnegie, G. K., Means, C. K. & Scott, J. D. A-kinase anchoring proteins: from protein complexes to physiology and disease. IUBMB Life 61, 394–406 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Hertel, F. & Zhang, J. Monitoring of post-translational modification dynamics with genetically encoded fluorescent reporters. Biopolymers 101, 180–187 (2013).

    Google Scholar 

  21. Surdo, N. C. et al. FRET biosensor uncovers cAMP nano-domains at β-adrenergic targets that dictate precise tuning of cardiac contractility. Nat. Commun. 8, 15031 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Allen, M. D. & Zhang, J. Subcellular dynamics of protein kinase A activity visualized by FRET-based reporters. Biochem Biophys. Res Commun. 348, 716–721 (2006).

    CAS  PubMed  Google Scholar 

  23. Ding, Y. et al. Ratiometric biosensors based on dimerization-dependent fluorescent protein exchange. Nat. Methods 12, 195–198 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Zhao, Y. et al. An expanded palette of genetically encoded Ca2+ indicators. Science 333, 1888–1891 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Qian, Y. et al. A genetically encoded near-infrared fluorescent calcium ion indicator. Nat. Methods 16, 171–174 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Herbst, K. J., Allen, M. D. & Zhang, J. Spatiotemporally regulated protein kinase A activity is a critical regulator of growth factor-stimulated extracellular signal-regulated kinase signaling in PC12 cells. Mol. Cell Biol. 31, 4063–4075 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Wu, V. et al. Illuminating the Onco-GPCRome: novel G protein-coupled receptor-driven oncocrine networks and targets for cancer immunotherapy. J. Biol. Chem. 294, 11062–11086 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Huganir, R. L. & Nicoll, R. A. AMPARs and synaptic plasticity: the last 25 years. Neuron 80, 704–717 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Liao, D., Scannevin, R. H. & Huganir, R. Activation of silent synapses by rapid activity-dependent synaptic recruitment of AMPA receptors. J. Neurosci. 21, 6008–6017 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Lu, W. et al. Activation of synaptic NMDA receptors induces membrane insertion of new AMPA receptors and LTP in cultured hippocampal neurons. Neuron 29, 243–254 (2001).

    CAS  PubMed  Google Scholar 

  31. Dana, H. et al. Sensitive red protein calcium indicators for imaging neural activity. eLife 5, e12727 (2016).

    PubMed  PubMed Central  Google Scholar 

  32. Colgan, L. A. et al. PKCα integrates spatiotemporally distinct Ca2+ and autocrine BDNF signaling to facilitate synaptic plasticity. Nat. Neurosci. 21, 1027–1037 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Leitz, J. & Kavalali, E. T. Fast retrieval and autonomous regulation of single spontaneously recycling synaptic vesicles. eLife 3, e03658 (2014).

    PubMed  PubMed Central  Google Scholar 

  34. Tang, A.-H. et al. A trans-synaptic nanocolumn aligns neurotransmitter release to receptors. Nature 536, 210–214 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Goto, A. et al. Circuit-dependent striatal PKA and ERK signaling underlies rapid behavioral shift in mating reaction of male mice. Proc. Natl Acad. Sci. USA 112, 6718–6723 (2015).

    CAS  PubMed  Google Scholar 

  36. Paukert, M. et al. Norepinephrine controls astroglial responsiveness to local circuit activity. Neuron 82, 1263–1270 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Reimer, J. et al. Pupil fluctuations track rapid changes in adrenergic and cholinergic activity in cortex. Nat. Commun. 7, 13289 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Polack, P.-O., Friedman, J. & Golshani, P. Cellular mechanisms of brain state-dependent gain modulation in visual cortex. Nat. Neurosci. 16, 1331–1339 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Fu, Y. et al. A cortical circuit for gain control by behavioral state. Cell 156, 1139–1152 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Lee, A. M. et al. Identification of a brainstem circuit regulating visual cortical state in parallel with locomotion. Neuron 83, 455–466 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. McDougal, D. H. & Gamlin, P. D. Autonomic control of the eye. Compr. Physiol. 5, 439–473 (2015).

    PubMed  PubMed Central  Google Scholar 

  42. Mohr, M. A. et al. jYCaMP: an optimized calcium indicator for two-photon imaging at fiber laser wavelengths. Nat. Methods 17, 694–697 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Abdelfattah, A. S. et al. Bright and photostable chemigenetic indicators for extended in vivo voltage imaging. Science 365, 699–704 (2019).

    CAS  PubMed  Google Scholar 

  44. Piatkevich, K. D. et al. A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters. Nat. Chem. Biol. 14, 352–360 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Patriarchi, T. et al. Ultrafast neuronal imaging of dopamine dynamics with designed genetically encoded sensors. Science 360, eaat4422 (2018).

    PubMed  PubMed Central  Google Scholar 

  46. Feng, J. et al. A genetically encoded fluorescent sensor for rapid and specific in vivo detection of norepinephrine. Neuron 102, 745–761.e8 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Lee, S. J., Chen, Y., Lodder, B. & Sabatini, B. L. Monitoring behaviorally induced biochemical changes using fluorescence lifetime photometry. Front Neurosci. 13, 766 (2019).

    PubMed  PubMed Central  Google Scholar 

  48. Lee, S. J. et al. Cell-type specific asynchronous modulation of PKA by dopamine during reward based learning. Preprint at bioRxiv, https://doi.org/10.1101/839035 (2019).

  49. Zhang, J., Hupfeld, C. J., Taylor, S. S., Olefsky, J. M. & Tsien, R. Y. Insulin disrupts beta-adrenergic signalling to protein kinase A in adipocytes. Nature 437, 569–573 (2005).

    CAS  PubMed  Google Scholar 

  50. Zhang, J., Ma, Y., Taylor, S. S. & Tsien, R. Y. Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering. Proc. Natl Acad. Sci. USA 98, 14997–15002 (2001).

    CAS  PubMed  Google Scholar 

  51. Lu, T.-W. et al. Two PKA RIα holoenzyme states define ATP as an isoform-specific orthosteric inhibitor that competes with the allosteric activator, cAMP. Proc. Natl Acad. Sci. USA 116, 16347–16356 (2019).

    CAS  PubMed  Google Scholar 

  52. Drobizhev, M., Molina, R. & Hughes, T. Characterizing the two-photon absorption properties of fluorescent molecules in the 680-1300 nm spectral range. Bio-Protocol 10, e3498 (2020).

    PubMed  PubMed Central  Google Scholar 

  53. Barnett, L. M., Hughes, T. E. & Drobizhev, M. Deciphering the molecular mechanism responsible for GCaMP6m’s Ca2+-dependent change in fluorescence. PLoS ONE 12, e0170934 (2017).

    PubMed  PubMed Central  Google Scholar 

  54. Molina, R. S. et al. Understanding the fluorescence change in red genetically encoded calcium ion indicators. Biophys. J. 116, 1873–1886 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Ward, W. W. in Green Fluorescent Protein 39–65 (John Wiley & Sons, Inc., 2005); https://doi.org/10.1002/0471739499.ch3

  56. Drobizhev, M., Makarov, N. S., Tillo, S. E., Hughes, T. E. & Rebane, A. Describing two-photon absorptivity of fluorescent proteins with a new vibronic coupling mechanism. J. Phys. Chem. B 116, 1736–1744 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Miyamoto, S. et al. PHLPP-1 negatively regulates Akt activity and survival in the heart. Circ. Res 107, 476–484 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Goldschmidt, H. L. et al. DGKθ catalytic activity is required for efficient recycling of presynaptic vesicles at excitatory synapses. Cell Rep. 14, 200–207 (2016).

    CAS  PubMed  Google Scholar 

  59. Pnevmatikakis, E. A. & Giovannucci, A. NoRMCorre: an online algorithm for piecewise rigid motion correction of calcium imaging data. J. Neurosci. Methods 291, 83–94 (2017).

    CAS  PubMed  Google Scholar 

  60. Telford, W. G., Babin, S. A., Khorev, S. V. & Rowe, S. H. Green fiber lasers: an alternative to traditional DPSS green lasers for flow cytometry. Cytometry 75A, 1031–1039 (2009).

    Google Scholar 

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Acknowledgements

The authors are grateful to R. Campbell (University of Alberta) for generously providing B-GECO1 and NIR-GECO1 and to S. S. Taylor (UC San Diego) for providing purified PKA catalytic subunit. We also wish to thank D. Schmitt, along with E. Griffis and D. Bindels from the UC San Diego Nikon Imaging Center, for assistance with confocal imaging, as well as R. C. Johnson and O. Martinez for helping with subcloning, T.W. Jung for scientific illustrations, and J. Heller Brown and C. Brand for helping with cardiac myocyte experiments. Work in J.Z.’s laboratory is supported by the National Institutes of Healthy (NIH) (grant nos. R35 CA197622 and R01 DK073368) and the Air Force Office of Scientific Research (FA9500-18-1-0051). Work by R.L.H. and J.Z. was also supported by the NIH Brain Initiative (grant no. R01 MH111516). L.T. was supported by NIH (grant no. DP2 MH107056). The work of M.D., T.E.H. and R.S.M. was supported by NIH (grant nos. U01 NS094246 and U24 NS109107). R.S.M. also acknowledges support from an NIH Ruth L. Kirschstein National Research Service Award (grant no. F31NS108593).

Author information

Author notes

  1. Richard H. Roth

    Present address: Department of Neurosurgery, Stanford University School of Medicine, Palo Alto, CA, USA

  2. These authors contributed equally: Jin-Fan Zhang, Bian Liu, Ingie Hong.

Authors and Affiliations

  1. Department of Pharmacology, University of California, San Diego, La Jolla, CA, USA

    Jin-Fan Zhang, Albert Mo, Brian Tenner, Wei Lin, Jason Z. Zhang, Sohum Mehta & Jin Zhang

  2. Department of Bioengineering, University of California, San Diego, La Jolla, CA, USA

    Jin-Fan Zhang, Jason Z. Zhang & Jin Zhang

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

    Bian Liu, Ingie Hong, Richard H. Roth & Richard L. Huganir

  4. The Kavli Neuroscience Discovery Institute, The Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Bian Liu, Ingie Hong, Richard H. Roth & Richard L. Huganir

  5. Department of Cell Biology and Neuroscience, Montana State University, Bozeman, MT, USA

    Rosana S. Molina, Mikhail Drobizhev & Thomas E. Hughes

  6. Department of Biochemistry and Molecular Medicine, University of California, Davis, Sacramento, CA, USA

    Lin Tian

  7. Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA

    Jin Zhang

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Contributions

S.M. and J.Z. conceived the project. A.M. and B.T. constructed the linker library and performed lysate screening with support from L.T. J.F.Z. characterized ExRai-AKAR2 fluorescence in vitro. J.F.Z. and S.M. performed live-cell imaging in HeLa, HEK293T and PC12 cells. J.Z.Z. performed live-cell imaging in neonatal cardiomyocytes. J.F.Z. and W.L. carried out microplate reader assays. B.T. and J.F.Z. performed flow cytometry measurements. B.L., I.H., R.H.R. and R.L.H. devised neuronal studies. B.L. performed live-cell imaging in cultured hippocampal neurons. R.S.M., M.D. and T.E.H. characterized ExRai-AKAR2 two-photon excitation in vitro. I.H. and R.H.R. performed two-photon imaging in awake head-fixed mice. R.L.H., S.M. and J.Z. supervised the project and coordinated experiments. J.F.Z., B.L., I.H., S.M., A.M., B.T. and J.Z.Z. analyzed the data. S.M., B.L., I.H., J.F.Z., R.L.H. and J.Z. wrote the manuscript.

Corresponding authors

Correspondence to Richard L. Huganir, Sohum Mehta or Jin Zhang.

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Extended data

Extended Data Fig. 1 Identification and characterization of ExRai-AKAR2.

a, Maximum 480 nm/405 nm excitation ratio changes (ΔR/R) of ExRai-AKAR linker variants in HeLa cells stimulated with 50 μM Fsk and 100 μM IBMX. The best-performing candidate was designated ExRai-AKAR2 (P = 0.0002, unpaired two-tailed Student’s t-test with Welch’s correction). From left to right: n = 34, 27, 27, 31, 31, 49, 29, 27, 23, 30, 40, 30, 41, 24, 32, 43, 43, 40, 32, 45, and 30 cells combined from 3 independent experiments each. b, Domain structures of ExRai-AKAR1 and ExRai-AKAR2. c, ExRai-AKAR2 is modestly but significantly brighter than ExRai-AKAR1 in both excitation channels (****P < 0.0001; unpaired two-tailed Student’s t-test with Welch’s correction). n = 227 (ExRai-AKAR1) and 136 cells (ExRai-AKAR2) imaged in multiple fields across 4 and 5 separate experiments, respectively. Data are plotted as log-transformed intensity values. Brightness increases for each channel were calculated by subtracting the average log intensity of ExRai-AKAR1 from that of ExRai-AKAR2 and then reversing the log transformation (10x). For example, the average log intensities at 480-nm excitation for ExRai-AKAR2 and ExRai-AKAR1 were 3.129 and 2.883, respectively. Given that 10(3.129–2.883) = 1.762, we conclude that ExRai-AKAR2 has ~76% higher 480 nm-excited intensity, on average, than ExRai-AKAR1. d, ExRai-AKAR2 exhibits significantly larger fluorescence intensity changes in both excitation channels versus ExRai-AKAR1 in response to PKA stimulation (****P < 0.0001; unpaired two-tailed Student’s t-test with Welch’s correction). n = 68 (ExRai-AKAR1) and 70 (ExRai-AKAR2) cells combined from 3 independent experiments. e,f, ExRai-AKAR2 exhibits a dramatically higher (e) maximum 488 nm/405 nm excitation ratio change (ΔR/R) and (f) signal-to-noise ratio compared with ExRai-AKAR1 in HeLa cells stimulated with Fsk/IBMX (****P < 0.0001; unpaired two-tailed Student’s t-test with Welch’s correction). Data in f are pooled from 3 (ExRai-AKAR2) and 4 (ExRai-AKAR1) experiments. Error bars indicate mean±s.e.m. ExRai-AKAR2 data in d, e are reproduced from Fig. 1c–e.

Source data

Extended Data Fig. 2 Comparing ExRai-AKAR2 performance with previous PKA sensors.

(a, b) Side-by-side comparison of ExRai-AKAR2 with existing intensity-ratio-based PKA sensors. a, Average time-course showing the enhanced ratio response (ΔR/R0) of ExRai-AKAR2 (n = 18) versus ExRai-AKAR1 (n = 11), AKAR4 (n = 18), and AKAR3-EV (n = 18) in HeLa cells stimulated with 50 μM Fsk and 100 μM IBMX (Fsk/IBMX). Solid lines indicate average responses; shaded areas, s.d. b, Summary of the maximum ratio changes (ΔR/R) for ExRai-AKAR2 (n = 44), ExRai-AKAR1 (n = 39), AKAR4 (n = 38), and AKAR3-EV (n = 36) following Fsk/IBXM stimulation. Representative pseudocolor images below the graph depict the raw emission (AKAR4, AKAR3-EV) or excitation ratio (ExRai-AKAR1, ExRai-AKAR2) before (upper) and after (lower) Fsk/IBMX stimulation. Warmer colors indicate higher ratios. Scale bars, 10 μm. Data are representative of (a) or combined from (b) 2 independent experiments. Error bars in b indicate mean±s.e.m. c, Fsk dose response of ExRai-AKAR2. HeLa cells expressing ExRai-AKAR2 (n = 48) were successively stimulated with the indicated concentrations of Fsk, followed by 100 μM IBMX. Data are plotted as ΔR/ΔRmax = (R[Fsk]−R0)/(RIBMX − R0), where R[Fsk] is the maximum ratio recorded after the addition of a given Fsk dose, RIBMX is the maximum ratio recorded following IBMX addition at the end of the experiment, and R0 is the ratio recorded immediately prior to the first drug addition (for example, t = 0). Data are combined from 2 independent experiments. Solid and dashed lines indicate the median and quartiles, respectively. ****P < 0.0001 vs. 0; two-tailed Wilcoxon signed-rank test.

Source data

Extended Data Fig. 3 Detecting local PKA signaling with subcellularly targeted ExRai-AKAR2.

a, Domain structures of ExRai-AKAR2 constructs targeted to the plasma membrane, outer mitochondrial membrane, and ER membrane. b, Representative confocal fluorescence images showing the plasma membrane, mitochondrial, and ER localization of pmExRai-AKAR2, mitoExRai-AKAR2, and erExRai-AKAR2, respectively, in both excitation channels (Ex488, Ex405). For mito- and erExRai-AKAR2, images of the red fluorescence signal (Ex561) from MitoTracker RED and ER-Tracker RED, respectively, are also shown. Merged images (far right) depict the overlay of the Ex488 (yellow), Ex405 (cyan), and Ex561 (magenta) channels. Images are representative of 2 independent experiments per condition. c–e, Time-course plots showing all individual traces corresponding to the raw 480/405 excitation ratio responses of pmExRai-AKAR2 (left), mitoExRai-AKAR2 (middle), and erExRai-AKAR2 (right), along with representative epifluorescence images of both excitation channels (below) illustrating ROI selection (dashed white lines), for the experiments shown in Fig. 2a–c. Thick lines indicate mean responses, and thin lines depict individual single-cell traces. Scale bars in b–c, 10 μm. f, Summary of the maximum excitation ratio changes (ΔR/R) for pmExRai-AKAR2 (PM; n = 46 cells from 3 experiments), mitoExRai-AKAR2 (Mito; n = 43 cells from 4 experiments), and erExRai-AKAR2 (ER; n = 35 cells from 3 experiments) in HeLa cells stimulated with Fsk/IBMX. Error bars in d indicate mean±s.e.m.

Source data

Extended Data Fig. 4 ExRai-AKAR2 is a more sensitive FACS probe than ExRai-AKAR1.

HEK293T cells transfected with ExRai-AKAR1 were analyzed via flow cytometry before and after stimulation with 50 μM Fsk and 100 μM IBMX as described in the Methods. a, Contour plot showing the 488 nm- and 405 nm-excited fluorescence intensities of transfected cells without (teal) and with (green) Fsk/IBMX stimulation. b, Frequency distribution of 488 nm/405 nm excitation ratio illustrating the population shift caused by Fsk/IBMX stimulation (****P < 0.0001; Kolmogorov-Smirnov test). Data are representative of 3 independent experiments. Overlaid gray and black dashed lines depict the frequency distributions for ExRai-AKAR2 transfected cell populations before and after Fsk/IBMX treatment, respectively (redrawn from Fig. 3a). c, Table summarizing the input values and results of the sensitivity index (SI) calculation (see Methods). ExRai-AKAR2 shows 2-fold higher sensitivity compared with ExRai-AKAR1.

Source data

Extended Data Fig. 5 Imaging PKA activity in neonatal rat ventricular myocytes using ExRai-AKAR2.

a, Representative images of 480 nm-excited (Ex480) and 380 nm-excited (Ex380) fluorescence from a neonatal rat ventricular myocyte (NRVM) expressing ExRai-AKAR2. Scale bar, 10 μm. b,c, Time-lapse epifluorescence imaging of ExRai-AKAR2 excitation ratio changes in NRVMs stimulated with (b) 100 nM Iso or (c) 50 μM Fsk and 100 μM IBMX. Thick lines indicate mean responses, and thin lines depict individual single-cell traces. d, Summary of maximum 480 nm/380 nm excitation ratio changes. Bars represent mean±s.e.m. n = 13 (Iso) and 20 (Fsk/IBMX) cells from 3 independent experiments for b–d.

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Extended Data Fig. 6 Imaging PKA activity using ExRai-AKAR2 in cultured hippocampal neurons.

a–c, Time-course plots showing all individual traces of the PKA-induced change in ExRai-AKAR2 fluorescence in hippocampal neurons stimulated with 50 μM Fsk and 2 μM rolipram (Rol) at 488-nm (a) and 405-nm (b) laser excitation, along with the raw 488 nm/405 nm excitation ratio (c). Thick lines indicate mean responses, and thin lines depict individual single-cell traces. d, Summary of the maximum Fsk/Rol-stimulated ExRai-AKAR responses in cultured hippocampal neurons. Error bars represent mean±s.e.m. Data in d correspond to time-courses shown in Fig. 3a. n = 54 (ExRai-AKAR2), 63 (ExRai-AKAR2[T/A]), 41 (ExRai-AKAR2 + H89) and 40 (ExRai-AKAR1) cells. ****P < 0.0001 vs. ExRai-AKAR2, Welch’s ANOVA followed by Dunnett’s test for multiple comparisons. e, Representative confocal fluorescence images of the 488 nm (Ex488) and 405 nm (Ex405) channels for hippocampal neurons expressing ExRai-AKAR2 (left) or ExRai-AKAR2[T/A] (right), illustrating the selection of ROIs (dashed white lines) for experiments reported in Fig. 4a–c. Scale bars, 10 μm. f, Plot of ExRai-AKAR2-expressing neurons showing heterogeneous PKA responses of individual neurons treated with 1 μM isoproterenol, representing 36 neurons from one of three independent experiments shown in Fig. 4b.

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Extended Data Fig. 7 Correlated Ca2+ and PKA dynamics during LTP in cultured hippocampal neurons.

a, Representative confocal fluorescence images of the 561 nm (jRGECO1a, red), 488 nm (Ex488, green), and 405 nm (Ex405, blue) channels for hippocampal neurons co-transfected with ExRai-AKAR2 and jRGECO1a, for experiments reported in Fig. 4d. Scale bar, 20 μm. b, Flow chart showing how Ca2+ transients were identified and how ROIs were drawn. c, (upper) Average responses of jRGECO1a (red) and ExRai-AKAR2 (green) for the neuron shown in (a). (lower) Color-coded time-courses of the jRGECO1a (left) and ExRai-AKAR2 (right) responses for 100 representative Ca2+ transients presented as raster plots. Each row represents one ROI. d, Responses of jRGECO1a (red) and ExRai-AKAR2 (green) in all 7 hippocampal neurons aligned to the peaks of Ca2+ transients. e, Relationship of relative PKA response and calcium influx from 1108 events in 7 neurons. Pearson correlation is not significant (P = 0.098, R2 = 0.0025). f, Aligning average responses from jRGECO1a (red) and ExRai-AKAR2 (R488/405, green) to randomly selected time points in the recording shows that PKA transients are specifically triggered by Ca2+ spikes. Three randomizations were performed using the 7 cells from 3 separate experiments. Solid lines indicate mean responses from 7 cells; shaded areas, s.d.

Extended Data Fig. 8 In vivo imaging of PKA activity using ExRai-AKAR2.

(a, b) Two-photon characterization of ExRai-AKAR2. a, Two-photon excitation spectra of purified ExRai-AKAR2 in the unphosphorylated (gray curve) and phosphorylated (green curve) states. The spectra are presented in molecular two-photon brightness values, F2, measured in GM (see Methods for details). Each spectrum consists of two overlapping bands – one belonging to the neutral form (peaking at 800 nm) and another belonging to the anionic form (peaking near 940 nm). n = 3 independent experiments. b, Ratio of the F2 values for phosphorylated and unphosphorylated ExRai-AKAR2. The ratio shows two peaks: at 930 nm (5.4) and 980 nm (5.1). The presence of two peaks is explained by a slight shift of the anionic two-photon absorption band upon phosphorylation (from 948 to 936 nm). Although excitation at 930 nm is the best for two-photon imaging, the whole range from 905 to 1000 nm provides a good contrast with F2(phospho)/F2(unphopsho) >4.6. c, Sub-second response latencies for ExRai-AKAR2 following the onset of forced locomotion. Data from Fig. 5e are re-plotted here at higher temporal frequency. Significant deviations from baseline are detected as early as 350 ms (P = 0.0116, one-tailed one-sample t-test against baseline=0; P = 0.0364, paired one-tailed t-test with Welch’s correction against last baseline point at t = 0). Error bars indicate s.d.

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Zhang, JF., Liu, B., Hong, I. et al. An ultrasensitive biosensor for high-resolution kinase activity imaging in awake mice. Nat Chem Biol 17, 39–46 (2021). https://doi.org/10.1038/s41589-020-00660-y

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