Imaging ERK and PKA Activation in Single Dendritic Spines during Structural Plasticity  Shen Tang, Ryohei Yasuda  Neuron  Volume 93, Issue 6, Pages 1315-1324.e3 (March 2017) DOI: 10.1016/j.neuron.2017.02.032 Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Design and Functional Expression of ERK Sensors in HeLa Cells (A) Schematic representation of FRET-based ERK sensor, EKARet-cyto. The sensor is composed of phospho-Thr binding domain (WW domain), EV linker, ERK substrate peptide and docking site, sREACh with S208F/R223F/V224L mutations, and EGFP. ERK phosphorylation triggers the association of the WW domain with the substrate peptide, increasing FRET between EGFP and sREAChet. The three-residue mutations in sREACh enhanced FRET signal. (B) Schematic representation of EKAREV-cyto and EKARet-cyto. (C and D) HeLa cells expressing EKAREV-cyto (C) or EKARet-cyto (D) were stimulated with 100 ng/mL EGF treatment, followed by inhibition with 50 μM U0126. Cells were imaged at indicated time points. (E) EGF-induced lifetime change of EKAREV-cyto (n = 12), EKARsg-cyto (n = 4), EKARet-cyto (n = 4), and negEKARet-cyto (n = 18). (F) Fluorescence lifetime changes in HeLa cells expressing different ERK sensors after EGF administration (12 min). ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (ANOVA followed by Tukey’s test). Error bars indicate SEM. See also Figure S1. Neuron 2017 93, 1315-1324.e3DOI: (10.1016/j.neuron.2017.02.032) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Spatiotemporal Dynamics of ERK Activation in Dendritic Structures (A) Fluorescence lifetime images of EKARet-cyto dendritic structures before and after PMA and U0126 application. (B) Time course of EKARet-cyto activity in spines (average of five spines; error bars indicate SEM) and their parent dendrite in (A) in response to bath application of PMA and, subsequently, U0126. On average, fluorescence lifetime changed by 0.128 ± 0.020 ns (spines, four neurons) and 0.128 ± 0.018 ns (dendrites) in response to PMA. (C) Fluorescence lifetime images of EKARet-nuc in the nucleus before and after PMA application. Neuron 2017 93, 1315-1324.e3DOI: (10.1016/j.neuron.2017.02.032) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 ERK Activation and Effect of AP5 during sLTP of Single Dendritic Spines Induced with Two-Photon Glutamate Uncaging (A) Representative fluorescence lifetime images of ERK activation during glutamate uncaging. Stimulated spine was marked with the white arrowhead. (B) ERK activation during sLTP along the stimulated dendrite as a function of the distance from the base of the stimulated spine (n = 12/4; spines/neurons) (stimulated spine, red circle; dendrites, black circles). (C) Average time course of fluorescence lifetime change of EKARet-cyto in stimulated spines (red line), adjacent dendrites (0 μm, blue line), adjacent spines (green line), and dendritic segments 13 μm away from the stimulated spine (black line) during spine structural plasticity induced with two-photon glutamate uncaging. Same dataset as in (B). The inset shows a closer view of the initial 6 min. (D) Quantification of fluorescence lifetime change in (B) and (C) at indicated time points. (E) Average time course of fluorescence lifetime change of EKARet-cyto in the stimulated spines and the adjacent dendrite in the absence (n = 5/3) or presence (n = 11/3) of AP5. (F) Averaged time course of volume change in the stimulated spines and adjacent dendrites in the absence (n = 5/3) or presence (n = 11/3) of AP5 correlated to (E). The glutamate uncaging was indicated as the black bar in (E) and (F). (G–I) Quantification of peak fluorescence lifetime change of EKARet-cyto (G) correlated to (E), spine volume change during the transient phase (6 min; H), and spine volume change during the sustained phase (average of 27–36 min; I) of sLTP in the stimulated spines and adjacent dendrites in the absence (n = 5/3; spines/neurons) or presence (n = 11/3; spines/neurons) of AP5 correlated to (F). Spine volume changes were measured as changes in fluorescence intensity of EKARet-cyto in the stimulated spines. All data were presented as mean ± SEM (error bars). Statistical significance was tested with one-way ANOVA followed by Tukey’s test or t test (ns p > 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). Error bars indicate SEM. See also Figures S2 and S3. Neuron 2017 93, 1315-1324.e3DOI: (10.1016/j.neuron.2017.02.032) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Design and Characterization of PSD-PDZ1-2 Tethered EKARet-PSD in Response to Glutamate Uncaging (A) Schematic representation of EKARet-PSD. (B) Fluorescence lifetime images of EKARet-PSD expressed in a CA1 pyramidal neuron during sLTP induced with two-photon glutamate uncaging at a spine (white arrowhead). The stimulation causes ERK activation in adjacent spines (white arrows). (C) Averaged time courses of fluorescence lifetime change in puncta at various distances from the stimulated spines (n = 7/4; puncta/neurons). The inset to (C) shows a closer view of the initial 6 min. Error bars indicate SEM. See also Figures S1, S4, and S8. Neuron 2017 93, 1315-1324.e3DOI: (10.1016/j.neuron.2017.02.032) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 Design and Characterization of PKA Sensors in HeLa Cells and Neurons (A) Schematic representation of FRET pair-based PKA sensors: AKAR3EV-cyto and AKARet-cyto. (B) Fluorescence lifetime images of AKAR3EV-cyto and AKARet-cyto in HeLa cells before and after forskolin and IBMX administration. (C) Averaged time courses of fluorescence lifetime change of AKAR3EV-cyto (n = 6), AKARet-cyto (n = 4), and negAKARet-cyto (n = 4) in response to forskolin and IBMX application. (D) Fluorescence lifetime images of AKARet-cyto before and after forskolin and IBMX administration in neurons. (E) Averaged time courses of fluorescence lifetime change of AKARet-cyto in spines (n = 28/4; spines/neurons) and their parent dendrites (n = 4/4; dendrites/neurons). Data are presented as mean ± SEM (error bars). See also Figures S1 and S5. Neuron 2017 93, 1315-1324.e3DOI: (10.1016/j.neuron.2017.02.032) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 Spatiotemporal PKA Activation after Glutamate Uncaging at a Single Spine (A) Fluorescence lifetime images of AKARet-cyto in a secondary dendrite of a CA1 pyramidal neuron during sLTP. (B) PKA activation along the dendrite at various distances from the stimulated spine (n = 11/3) (stimulated spine, red circle; dendrites, black circles). (C) Average time course of fluorescence lifetime change of AKARet-cyto in stimulated spine (red line), adjacent dendrite (0 μm, blue line), adjacent spine (green line), and dendritic segments 13 μm away from the stimulated spine (black line) during spine structural plasticity induced with two-photon glutamate uncaging (n = 11/3), corresponding to (A) and (B). The inset to (C) shows a closer view of the initial 6 min. (D) Quantification of fluorescence lifetime change in (C) at indicated time points. (E) Averaged time courses of fluorescence lifetime changes of AKARet-cyto in the stimulated single spines, adjacent dendrites during sLTP induced in a single spine (n = 7/3). AP5 eliminated PKA activation (n = 11/3). (F) Volume change of the stimulated spines and adjacent dendrites measured as changes in fluorescence intensity of AKARet-cyto (n = 7/3; spines/neurons), AP5 eliminated spine volume change (n = 11/3; spines/neurons), correlated to (C). (G–I) Quantification of peak fluorescence lifetime change of AKARet-cyto (G) correlated to (E), spine volume change during the transient phase (4 min; H), and spine volume change during the sustained phase (average of 27–36 min; I) of sLTP in the stimulated spines and adjacent dendrites in the absence (n = 7/3; spines/neurons) or presence (n = 11/3; spines/neurons) of AP5 correlated to (F). All data were presented as mean ± SEM (error bars). Statistical significance was tested with one-way ANOVA followed by Tukey’s test or t test (ns p > 0.05, ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). See also Figures S6 and S7. Neuron 2017 93, 1315-1324.e3DOI: (10.1016/j.neuron.2017.02.032) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 Design and Characterization of PSD-PDZ1-2 Tethered AKARet-PSD during Glutamate Uncaging (A) Schematic representation of AKARet-PSD. (B) Fluorescence lifetime images of AKARet-PSD during sLTP in a secondary dendrite of a CA1 pyramidal neuron induced in a single dendritic spine with two-photon glutamate uncaging (white arrowhead). The stimulation causes PKA activation in adjacent spines (white arrows). (C) Averaged time courses of fluorescence lifetime changes in puncta at various distances from the stimulated spines (n = 9/4; puncta/neurons). Data were presented as mean ± SEM (error bars). See also Figures S1, S8, and S9. Neuron 2017 93, 1315-1324.e3DOI: (10.1016/j.neuron.2017.02.032) Copyright © 2017 Elsevier Inc. Terms and Conditions