Article Figures & Data
Figures
- Figure 1.
Depolarization selectively activates PKA signaling at the nuclear envelope. A, Sensors used in this study. In AKAR4, phosphorylation of the LRRATLVD peptide by PKA results in FHA1 phospho-peptide binding and increased cerulean-cpVenus FRET (Depry et al., 2011). In RCaMP1h, Ca2+ induces the binding of the M13 peptide by the mutant calmodulin domain (mCaM), increasing mRuby fluorescence (Hirabayashi et al., 2017). In the perinuclear-localized sensors PN-AKAR4 and PN-RCaMP1h, nesprin-1α contains five spectrin repeats (SRs) and a transmembrane KASH domain that localizes the protein to the nuclear envelope via binding to SUN domain proteins (Pare et al., 2005a). B, Grayscale CFP images of hippocampal neurons expressing AKAR4 or PN-AKAR4. Scale bar: 10 μm. C, D, Hippocampal neurons were transfected with PN-AKAR4 and PN-RCaMP1h or AKAR4 and RCaMP1h expression plasmids. Representative traces (smoothed with Prism) and pseudocolor images showing FRET (AKAR4) or intensity (RCaMP1h) responses to 40 mm KCl introduced by perfusion. Images were obtained simultaneously for AKAR4 and RCaMP1h and for PN-AKAR4 and PN-CaMP1h. Here and below, a cytosolic region of interest in the soma was measured for the non-localized AKAR4 and RcAMP1h sensors. Scale bar: 10 μm. See Figure 3 for quantification of average responses. E, Averaged trace for PN-AKAR4 response to increasing KCl concentration (10, 30, and 40 mm). Solid line and shaded area indicate mean and SEM, respectively; n = 9 from four independent experiments. F, PN-AKAR4 amplitude to different KCl concentrations. Black bars indicate mean values. Datasets were compared by one-way ANOVA and Tukey’s post hoc testing; **p ≤ 0.01, ***p ≤ 0.001.
- Figure 2.
Activity-induced perinuclear PKA activity is mAKAPα dependent. Hippocampal neurons transfected with PN-AKAR4 and PN-RCaMP1h expression plasmids and infected with adenovirus for control or mAKAP shRNA were stimulated with 40 mm KCl; n = 15 for both shRNA and include data from three experiments using separate hippocampal neuron cultures. A, Averaged traces for PN-AKAR4. B, Amplitude of PN-AKAR4 traces. C, Averaged traces for PN-RCaMP1h. D, Amplitude of PN-RCaMP1h traces. Data in A, C are mean ± SEM; black bars in B, D indicate mean values. Datasets were normally distributed and compared by unpaired t tests; ***p ≤ 0.001.
- Figure 3.
Perinuclear cAMP is regulated by L-type Ca2+ channels. Hippocampal neurons expressing PN-AKAR4 and PN-CaMP1h or AKAR4 and RCaMP1h were preincubated with 10 μm nifedipine (Nif; n = 19, 18 for parent and PN-sensors, respectively), 0.5 μm conotoxin GVIA (Con; n = 15, 36), or no inhibitor control (Ctrl; n = 14, 14) before stimulation with 40 mm KCl (bar). A, Averaged traces for PN-RCaMP1h. B, Amplitude of PN-RCaMP1h traces. C, Averaged traces for PN-AKAR4. D, Amplitude of PN-AKAR4 traces. E, Averaged traces for RCaMP1h. F, Amplitude of RCaMP1h traces. G, Averaged traces for AKAR4. H, Amplitude of AKAR4 traces. Traces show mean ± SEM and are normalized to initial baseline values (R0 or I0); black bars in B, D, F, H indicate mean values. Datasets were compared by Kruskal–Wallis and Dunn’s post hoc testing; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
- Figure 4.
Perinuclear Ca2+ is required for cAMP elevation at the nuclear envelope. A, Cyprinus carpio β-parvalbumin–nesprin-1α fusion proteins. Nesprin-1α contains five spectrin repeats (SRs) and a transmembrane KASH domain that localizes the protein to the nuclear envelope via binding to SUN domain proteins (Pare et al., 2005a). B, C, Hippocampal neurons expressing PN-RCaMP1h and either Parv-GFP-nesprin (Parv; n = 15) or control GFP-nesprin (Ctrl; n = 15) were stimulated with 40 mm KCl. D, E, Neurons expressing RCaMP1h and either Parv-GFP-nesprin (Parv; n = 26) or control GFP-nesprin (Ctrl; n = 23) were stimulated with 40 mm KCl. F, G, Neurons expressing PN-AKAR4 and either mCherry-Parv-nesprin (Parv; n = 26) or control mCherry-nesprin (Ctrl; n = 18) were stimulated with 40 mm KCl. Traces show mean ± SEM and are normalized to initial baseline values (R0 or I0); black bars in C, E, F indicate mean values. Datasets compared by Mann–Whitney U test; ***p ≤ 0.001.
- Figure 5.
Perinuclear Ca2+ regulates neurite extension. A, Images of hippocampal neurons expressing mCherry and Parv-GFP-nesprin-1α and stained with Hoechst nuclear stain in representative neurite extension assay. Scale bar: 50 μm. B, Grayscale images of mCherry fluorescence for hippocampal neurons expressing mCherry and either GFP-nesprin Parv-GFP-nesprin and cultured for 2 d in defined media ±40 mm KCl. Scale bar: 50 μm. C, Means of three independent experiments (differently colored symbols) and average mean (bars) for lengths of the longest neurite are shown; *p ≤ 0.05 as determined by matched two-way ANOVA and Tukey’s post hoc testing.
- Figure 6.
Model of cAMP signaling regulation in the perinuclear compartment. Depolarization of the plasma membrane in hippocampal neurons triggers the opening of L-type Ca2+ channels leading to increased perinuclear [Ca2+], and activation of Ca2+-dependent AC. Perinuclear cAMP binds PKA regulatory (R) subunits, activating PKA catalytic (C) subunits at mAKAPα signalosomes. PKA-phosphorylated effectors that remain to be identified regulate gene expression promoting axon extension. LTCC, L-type Ca2+ channels.
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