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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All data supporting the findings of this study are available upon reasonable request. Source data are provided with this paper.
Custom ImageJ macros and MATLAB code used to analyze in vitro and in vivo neuronal imaging data are available upon reasonable request.
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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).
The authors declare no competing interests.
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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
(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
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
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
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. Source data
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. Source data
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.
(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 (2020). https://doi.org/10.1038/s41589-020-00660-y