Abstract
Unravelling the dynamic molecular interplay behind complex physiological processes such as neuronal plasticity requires the ability to both detect minute changes in biochemical states in response to physiological signals and track multiple signalling activities simultaneously. Fluorescent protein-based biosensors have enabled the real-time monitoring of dynamic signalling processes within the native context of living cells, yet most commonly used biosensors exhibit poor sensitivity (for example, due to low dynamic range) and are limited to imaging signalling activities in isolation. Here, we address this challenge by developing a suite of excitation ratiometric kinase activity biosensors that offer the highest reported dynamic range and enable the detection of subtle changes in signalling activity that could not be reliably detected previously, as well as a suite of single-fluorophore biosensors that enable the simultaneous tracking of as many as six distinct signalling activities in single living cells.
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Acknowledgements
The authors are grateful to L. Looger, G. Yellen, M. Matsuda, T. Kitaguchi and R. Campbell for generously providing plasmids and to S. Taylor for providing purified PKA catalytic subunit. We also wish to thank E. Greenwald for helping with the bleed-through analysis and correction, as well as E. Lopez Ortega for helping with subcloning and neuronal imaging experiments. This work was supported by Brain Initiative Grant R01 MH111516 (to R.L.H. and J.Z.) and by R35 CA197622, R01 DK073368 and R01 GM111665 (to J.Z.). Y.Z. is supported by the National Natural Science Foundation of China (grant 31771125), the Ministry of Science and Technology of the People’s Republic of China (grant 2017YFE0103400) and The One Thousand Talents Plan-Recruitment Program for Young Professionals.
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S.M. and J.Z. conceived of the project. S.M. designed ExRai-AKAR and generated all of the biosensors used in the study, with assistance from B.T.; A.M. performed in vitro characterization of purified biosensors; S.M. and J.-f.Z. performed live-cell imaging in HeLa, HEK293T, NIH3T3 and PC12 cells; Y.Z., R.H.R. and R.L.H. devised the neuronal experiments; Y.Z. and R.H.R. performed live-cell imaging in primary cortical neurons; R.L.H. and J.Z. supervised the project and coordinated the experiments; S.M., Y.Z., R.H.R., J.-f.Z. and A.M. analysed the data; and S.M., Y.Z., R.H.R., R.L.H. and J.Z. wrote the manuscript.
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Supplementary Figure 1 Additional characterization of ExRai-AKAR.
(a) Normalized excitation spectra of unphosphorylated ExRai-AKAR (1 μM) measured in buffer with the indicated pH values (see Methods). (b) Plot of the maximum intensity of the 380 nm excitation peak (teal) and the 488 nm excitation peak at each pH. Non-linear regression was performed to determine the pKa value for each excitation peak. (c) Plot of the ratio of the 480 nm and 380 nm excitation peaks obtained at each pH. Data points in b and c represent the mean ± SEM of three independent determinations. (d) The ExRai-AKAR response is reversible. Average time-course showing the 480/380 excitation ratio change in HeLa cells expressing ExRai-AKAR and treated with 50 μM Fsk and 100 μM IBMX (Fsk/IBMX) followed by 20 μM H89. The curve is plotted as the 480/380 excitation ratio normalized with respect to time 0 (R/R0). The solid line represents the mean, and the shaded area represents SEM. n=6 cells from a single experiment; experiment was repeated 10 times with similar results. (e-g) Individual single-cell traces showing the non-normalized PKA-stimulated changes in (e) 480 nm-excited fluorescence intensity, (f) 380 nm-excited fluorescence intensity, and (g) 480/380 excitation ratio in HeLa cells expressing ExRai-AKAR and treated with Fsk/IBMX. n = 8 cells from the same representative experiment shown in Fig. 1; experiment was repeated 11 times with similar results
Supplementary Figure 2 Investigating compartmentalized PKA signaling in growth factor-stimulated PC12 cells.
(a) Domain structures of ExRai-AKAR-NES and ExRai-AKAR-Kras. (b, c) Representative average excitation ratio time-courses of (b) ExRai-AKAR-NES (n = 5 cells) and (c) ExRai-AKAR-Kras (n = 4 cells) in HeLa cells treated with Fsk/IBMX. Inset: representative fluorescence images showing the (b) cytosolic or (c) plasma membrane distribution of probe fluorescence in the individual excitation channels. Curves are plotted as 480/380 excitation ratio normalized with respect to time 0 (R/R0). Solid lines represent the mean, and the shaded areas represent SEM. Scale bar, 30 μm. (d-i) Growth factor-induced PKA responses in PC12 cells monitored using ExRai-AKAR. (d, e) Excitation ratio time-courses of the ExRai-AKAR-Kras response in PC12 cells stimulated with (d) 200 ng/mL NGF (n = 8 cells pooled from 4 experiments) or (e) 100 ng/mL EGF (n = 7 cells pooled from 4 experiments). Inset: Representative images showing the plasma membrane distribution of probe fluorescence in the individual excitation channels. Scale bars, 10 μm. (f-i) Excitation ratio time-courses of the ExRai-AKAR responses in PC12 cells stimulated with (f, h) 200 ng/mL NGF (n = 17 cells pooled from 8 experiments) or (g, i) 100 ng/mL EGF (n = 16 cells pooled from 6 experiments) and recorded in the (f, g) nucleus or (h, i) cytosol. All curves are plotted as 480/380 excitation ratio normalized with respect to time 0 (R/R0). Thick lines indicate the average responses, and thin lines depict individual single-cell traces. Experiments in b and c were each repeated 3 times with similar results
Supplementary Figure 3 Non-normalized response curves for additional cpFP-based sensors.
(a, b) Individual single-cell traces showing the non-normalized changes in 480/380 excitation ratio in (a) ExRai-CKAR-expressing HeLa cells treated with 100 ng/mL PMA (n = 5 cells from the same representative experiment shown in Fig. 3b) and (b) in ExRai-AktAR-expressing NIH3T3 cells treated with 50 ng/mL PDGF (n = 15 cells pooled from 12 experiments). (c, d) Individual single-cell traces showing non-normalized changes in fluorescence intensity in (c) sapphireAKAR-expressing HeLa cells treated with 50 μM Fsk and 100 μM IBMX (Fsk/IBMX; n = 5 cells from the same representative experiment shown in Fig. 4c) and (d) in sapphireCKAR-expressing HeLa cells treated with 100 ng/mL PMA (n = 4 cells from the same representative experiment shown in Fig. 4d). (e, f) Individual single-cell traces showing non-normalized changes in fluorescence intensity in (e) blueAKAR-expressing HeLa cells treated with Fsk/IBMX (n = 4 cells from the same representative experiment shown in Fig. 4g) and (f) in blueCKAR-expressing HeLa cells treated with PMA (n = 5 cells from the same representative experiment shown in Fig 4h). Experiments were repeated (a) 5, (c) 6, (d) 5, (e) 5, and (f) 6 times with similar results
Supplementary Figure 4 Characterization of red-shifted single-fluorophore activity sensors.
(a) Initial unsuccessful attempt to generate a red-shifted kinase activity sensor based on cpRFP. Top: Domain structure of redAKAR prototype containing cp-mRuby sandwiched between a PKA substrate sequence and FHA1 domain. Bottom: Average time-course of RFP fluorescence intensity in HEK293T cells transfected with redAKAR and treated with 50 μM Fsk. The solid line indicates the mean, and the shaded area indicates SEM. Curve is plotted as RFP fluorescence intensity normalized with respect to time 0 (F/F0). n = 14 cells from a representative experiment. Inset: Representative epifluorescence image showing very dim RFP fluorescence in HEK293T cells transfected with redAKAR. Scale bar, 30 μm. (b) The RAB-EKARev response is reversible. Average time-course showing the change in RFP fluorescence intensity in HEK293T cells expressing RAB-EKARev and treated with 100 ng/mL EGF followed by 20 μM U0126 to reverse the response. The curve is plotted as RFP fluorescence intensity normalized with respect to time 0 (F/F0). The solid line represents the mean, and the shaded area represents SEM. n = 18 cells from a representative experiment. (g) Individual single-cell traces showing non-normalized RFP fluorescence intensity changes in (left) RAB-EKARev-expressing HEK293T cells treated with 100 ng/mL EGF (n = 10 cells from the same representative experiment shown in Fig. 5c), (middle) in RAB-AKARev-expressing HeLa cells treated with Fsk/IBMX (n = 6 cells from the same representative experiment shown in Fig. 5d), and (right) in RAB-ICUE-expressing HEK293T cells treated with Fsk/IBMX (n = 6 cells from the same representative experiment shown in Fig. 5e). Experiments were repeated (b) 9, (c) 7, (d) 3, and (e) 4 times with similar results; experiment in a was repeated twice with similar results
Supplementary Figure 5 Additional representative curves and bleed-through analysis for three-parameter imaging in HeLa cells.
(a) Representative single-cell time-courses showing the changes in BFP, RFP, and T-sapphire (TsR) fluorescence intensity in HeLa cells co-expressing blueAKAR (blue), RAB-EKARev (red), and sapphireCKAR (teal) upon sequential treatment with 50 μM Fsk and 100 μM IBMX (Fsk/IBMX), 100 ng/mL EGF, and 100 ng/mL PMA. (b) Representative single-cell time-courses showing the changes in BFP, RFP, and T-sapphire fluorescence intensity in HeLa cells co-expressing blueAKAR (blue), RAB-ICUE (red), and sapphireCKAR (teal) upon sequential treatment with Fsk/IBMX and PMA. (c) Alternative three-parameter imaging of PKA, cAMP, and PKC. Representative single-cell traces of the change in TsR, RFP, and BFP fluorescence intensity in HeLa cells co-expressing sapphireAKAR (teal), RAB-ICUE (red), and blueCKAR (blue) upon sequential treatment with Fsk/IBMX and PMA (n = 14 cells total across 6 independent experiments). All curves are plotted as fluorescence intensity normalized with respect to time 0 (F/F0). (d) To analyze fluorescence bleed-through, HeLa cells were individually transfected with sapphireCKAR, blueAKAR, RAB-EKARev, or RAB-ICUE, and images were acquired in the TsR, BFP, and RFP channels for cells expressing each construct. Representative scatter plots show the pixel intensity in each channel as a function of the pixel intensity in the probe channel, and line traces show the pixel intensity in the probe channel alone. Obvious bleed-through of fluorescence intensity was only detected in the TsR channel in cells expressing blueAKAR, and the bleed-through correction factor was calculated by determining the slope of the TsR vs BFP pixel intensity graph for blueAKAR-expressing cells. (e, f) Representative single-cell traces of TsR channel intensity in HeLa cells transfected only with blueAKAR before (dashed line) and after (solid line) BFP bleed-through correction. (g) Representative fluorescence images for each construct in each channel. Scale bars, 30 μm. Single-cell traces in a and b are derived from the same datasets as Figs. 6a (5 independent experiments) and 6b (3 independent experiments), respectively. Experiments in d-g were performed 5 times with similar results
Supplementary Figure 6 Characterization of single-fluorophore kinase sensors in cultured rat cortical neurons and HeLa cells.
(a) Average time-course of ExRai-AKAR (Ex488) and dsRed responses in the cell soma following Fsk stimulation. Insets: Representative ExRai-AKAR fluorescence images before (-Fsk) and after (+Fsk) stimulation and maximal responses (F/F0) (****p<0.0001, two-tailed paired Student’s t-test; n = 19 neurons pooled from 3 experiments]). Data are shown as dot plots; bars represent mean ± SEM. (b) Average time-course of RAB-EKARev and GFP responses in the cell soma following DHPG stimulation. Insets: Representative RAB-EKARev fluorescence images before (-DHPG) and after (+DHPG) stimulation and maximal responses (F/F0) (P = 0.0117, paired Student’s t-test; n = 6 neurons pooled from 3 experiments). Data are shown as dot plots; bars represent mean ± s.e.m. (c) Average time-course of ExRai-AKAR (Ex488), RAB-EKARev, and BCaMP responses in the cell soma following Fsk stimulation. Inset: maximal responses (F/F0) of ExRai-AKAR, RAB-EKARev, and BCaMP (p = 0.0005, ****p<0.0001, Kruskal-Wallis test; n = 68 neurons pooled from 6 experiments). (d) Average time-course of ExRai-AKAR (488 nm excitation), RAB-EKARev, and BCaMP responses in the cell soma following DHPG stimulation. Inset: Maximal responses (F/F0) of ExRai-AKAR, RAB-EKARev, and BCaMP (****p<0.0001, Kruskal-Wallis test; n = 104 neurons pooled from 10 experiments). Box-and-whisker plots depict the median, interquartile range, min, max, and mean (+). All data points are shown. Maximum responses are calculated with respect to the initial intensity at time 0 (F/F0). (e) Domain structures of Lyn-sapphireAKAR and sapphireAKAR-NLS. (f, g) Average time-courses of (f) Lyn-sapphireAKAR (n = 5 cells) and (g) sapphireAKAR-NLS (n = 6 cells) responses in HeLa cells treated with 50 μM Fsk and 100 μM IBMX (Fsk/IBMX). (h) Domain structure of Lyn-RAB-EKARev and RAB-EKARev-NLS. (i, j) Average time-courses of (i) Lyn-RAB-EKARev (n = 9 cells) and RAB-EKARev-NLS (n = 18 cells) in HeLa cells treated with 100 ng/mL EGF. Data in f, g, i, and j are pooled from two independent experiments; representative images show probe localization. Curves are normalized with respect to the average baseline intensity (a-d) or to time 0 (F/F0) (f, g, i, and j). Solid lines represent the mean; shaded areas, SEM. Scale bars, 10 μm. See Supplementary Table 1 for bar graph source data
Supplementary Figure 7 Additional representative curves and bleed-through analysis for four- and six-parameter imaging in HeLa cells.
(a) Representative single-cell time-courses showing the changes in T-sapphire (TsR), YFP, BFP, and RFP fluorescence intensity in HeLa cells co-expressing sapphireAKAR (teal), Flamindo2 (yellow), blueCKAR (blue), and RCaMP (red) upon sequential treatment with 50 μM Fsk and 100 μM IBMX (Fsk/IBMX), 100 ng/mL PMA, and 1 μM ionomycin (iono). (b, c) Representative single-cell time-courses of the changes in TsR, YFP, BFP, and RFP fluorescence intensity in HeLa cells co-expressing Lyn-sapphireAKAR (teal), sapphireAKAR-NLS (light blue), Flamindo2 (yellow), RAB-EKARev-NLS (red), Lyn-RAB-EKARev (pink), and B-GECO1 (blue) upon sequential treatment with Fsk/IBMX, EGF, and 100 μM histamine, showing cells both without (b) and with (c) EGF-induced calcium transients. All curves are plotted as fluorescence intensity normalized with respect to time 0 (F/F0). (d) To analyze fluorescence bleed-through, HeLa cells were individually transfected with sapphireAKAR, blueCKAR, Flamindo2, or RCaMP, and images were acquired in the TsR, BFP, YFP, and RFP channels for cells expressing each construct. Representative scatter plots show the pixel intensity in each channel as a function of the pixel intensity in the probe channel, and line traces show the pixel intensity in the probe channel alone. Obvious bleed-through of fluorescence intensity was only detected in the TsR channel in cells expressing blueCKAR, and the bleed-through correction factor was calculated by determining the slope of the TsR vs BFP pixel intensity graph for blueCKAR-expressing cells. (e) Representative single-cell traces of TsR channel intensity in a HeLa cell transfected only with blueCKAR before (dashed line) and after (solid line) BFP bleed-through correction. (f) Representative fluorescence images for each construct in each channel. Scale bars, 30 μm. Single-cell traces in a and b, c are derived from the same datasets as Figures 6e (12 independent experiments) and 6f (10 independent experiments), respectively. Experiments in d-f were repeated 5 times with similar results
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Mehta, S., Zhang, Y., Roth, R.H. et al. Single-fluorophore biosensors for sensitive and multiplexed detection of signalling activities. Nat Cell Biol 20, 1215–1225 (2018). https://doi.org/10.1038/s41556-018-0200-6
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DOI: https://doi.org/10.1038/s41556-018-0200-6
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