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Fig. 1
bPAC light and dark activity. A cAMP concentrations from whole Xenopus oocytes kept in the dark (dark) or after illumination with 0.3 mW mm-2 473 nm for 30 s (light). Data are mean ± SEM, n = 3 experiments with 5 oocytes each. ***p < 0.0001, *p < 0.05, Dunnett’s multiple comparisons vs control following one-way ANOVA (p < 0.0001). B Endogenous PDE activity in oocyte extracts. At time 0 minutes 0.15 μM cAMP was added to the soluble extracts in the absence (control) or presence of 1 mM IBMX (+ IBMX). n = 3 experiments, extracts pooled from 15 oocytes. C cAMP production of Venus-bPAC containing soluble extract after adding ATP into the reaction buffer in the absence and presence of 1 mM IBMX under dark condition. Note that the control oocyte (black in C) did not produce any cAMP after adding ATP. D Light (473 nm) intensity dependence of mean normalized cAMP production by Venus-bPAC or bPAC at 473 nm. Km = 26.2 μW mm-2 and 24.8 μW mm-2, respectively. E Working mechanism of the PKA activity FRET sensor Booster-PKA. F Representative ratio images (mKate2/mKoκ) of the hippocampal neurons expressing Booster-PKA alone or together with bPAC. Shown at right are individual measurements, median, and interquartile range. ***p < 0.0001, **p < 0.001, unpaired t tests. n = 20, 10, 7, and 18
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Fig. 2
Reducing bPAC dark activity. A Model of bPAC structure (5MBK, 5M2A), a parallel homodimer (chain A and B colored in light and dark shades), marked with the “FAD” chromophore, ATP substrate, and residues of interest. Asterisks indicate residues from the other chain of the dimer. Green spheres represent the two catalytic Mg2+ ions. B Point mutations of key residues and their effect on dark and light cAMP concentration in whole oocytes as in Fig. 1A. All bPAC mutants are either tagged with Venus at the N-terminus (S27A, L123R, F198Y, F198W, H266W, and T267Y) or eYFP at the C-terminus (K197A, K197A/D201A, and R278A). C Normalized cAMP production at different time delays in the dark after 500 ms, 473 nm, 0.3 mW mm-2 light stimulation fitted with a mono-exponential function (bPAC(wt) τ = 10.1 s, Venus-bPAC(F198Y) τ = 4.5 s. D Effect of membrane anchoring on cAMP concentration of whole oocytes in dark and light conditions as in Fig. 1A. E Cytoplasmic and membrane distribution of bPAC variants indicated by Venus fluorescence. Data in B, D, and E are individual values and mean ± SEM. n = 3–6. Control = non-injected oocytes. ***p < 0.0001, *p < 0.05, ns = not significant, Dunnett’s multiple comparisons vs control following one-way ANOVA (p < 0.0001)
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Fig. 3
Behavioral assessment of bPAC variants in Drosophila. A Mechanical nociceptors (C4da sensory neurons) expressing bPACs (grey, ppk-GAL4 driver). The soluble R278A and F198Y variants and Glyco-Venus-bPAC(S27A) are located in the cell body and dendrites. CD8-Venus-bPAC(wt) is confined to the somatic region of C4da neurons. B Nocifensive behavior in groups of larvae expressing bPAC variants in C4da neurons in the dark (black bar) or blue light (blue bar). Note that control larvae exhibited no nocifensive behavior except in response to von Frey filament stimulation and larvae expressing unmodified bPAC showed nocifensive behavior in the dark
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Fig. 4
Development of membrane-targeted PACmn and characteristics of the light-evoked responses in expressing hippocampal neurons. A cAMP concentrations of whole oocytes expressing soluble or membrane-targeted (Lyn) bPAC variants in dark and light conditions as in Fig. 1A. Mean ± SEM, n = 3 experiments with 5 oocytes each. ***p < 0.0001, *p < 0.05, Dunnett’s multiple comparisons vs control. B Cytosol and membrane distribution of bPAC variants in oocytes indicated by Venus fluorescence, as in Fig. 2D. C PACmn construct design and hippocampal slice culture electroporation strategy. D Confocal projection images of hippocampal neurons co-expressing PACmn (2xLyn-Venus-bPAC(F198Y)) and mKate2, together with close-up of an apical dendrite. Merged image PACmn (yellow) mKate2 (magenta) colocalization appears white. E Whole-cell responses to somatic current injections from −400 pA to 700 pA in a PACmn expressing neuron in the dark (resting membrane potential −70 mV). F Membrane resistance and G holding current of non-transfected (NT), PACmn + mCNG, or bPAC(wt) + mCNG—expressing neurons when clamping the membrane voltage at −70 mV. H Photocurrents evoked by five consecutive 470 nm light flashes (50 ms, 1 mW mm-2, interval 100 s) in a PACmn + mCNG expressing neuron. I Representative currents recorded from PACmn and mCNG expressing hippocampal neurons in response to 50 ms 470-nm light pulses of varying intensity and the light intensity-response relationship fitted with a quadratic equation. Currents were normalized to the maximum current recorded for each neuron. J Representative currents recorded from PACmn and mCNG expressing hippocampal neurons in response to 1 mW mm-2 470 nm light pulses of varying duration (5 ms to 15 s). At right is the light duration-response relationship (total charge). The maximum charge from each neuron was set to 100%
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Fig. 5
PACmn raises cAMP in hippocampal neurons. A Sample currents elicited by a 50-ms 470 nm light pulse (arrow) in neurons expressing either bPAC(wt) or PACmn together with mCNG. B Photocurrent amplitude and C slope recorded in neurons expressing PACmn or bPAC(wt) together with mCNG in response to 50 ms 470-nm light pulses of varying intensity. D Time from stimulation to onset, peak, and time from the peak to decay to ½ peak of responses to 50 ms, 1 mW mm-2 470 nm light pulses. E Currents recorded from a neuron expressing PACmn together with mCNG in response to 50 ms, 1 mW mm-2 470-nm light pulses (arrows), before and 15 min after wash-in of forskolin (FSK; 100 μM) and IBMX (75 μM). Plotted are median and interquartile range
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Fig. 6
PACmn activates PKA when illuminated without altering basal activity in hippocampal neurons. A Resting FRET ratios (soma) in hippocampal neurons expressing Booster-PKA alone or together with bPAC variants. Note that only PACmn does not change resting PKA activity. Shown are median and interquartile range. ***p < 0.0001, ns = not significant, Dunnett’s multiple comparisons vs sensor only following one-way ANOVA (p < 0.0001). n = 52, 43, 31, 29, and 18 somata. B Representative ratio images (mKate2/mKoκ) of the hippocampal neurons (soma and dendrite with spines) expressing BoosterPKA before and after foskolin (FSK). C PKA activity in Booster-PKA expressing hippocampal neurons. Bar indicates time of FSK application. D, E Representative ratio images (mKate2/mKoκ) of hippocampal neurons expressing Booster-PKA together with bPAC(wt) (D) or PACmn (E) before and after being illuminated for 2 s with 1 mW mm-2 470-nm blue light. F PKA activity in Booster-PKA neurons co-expressing bPAC(wt) or PACmn. Arrow indicates a 2 s blue light pulse. In both C and F, the solid lines are from the soma, dashed lines from the dendrites and spines. n (somata): 10 (Booster-PKA), 7 (+bPAC(wt)), 11 (+PACmn); n (dendrites and spines): 10 (Booster-PKA), 18 (+bPAC(wt)), and 14 (+PACmn). Shading indicates SEM. Results presented in A are from experiments done using a different microscope; thus, the difference in resting ratios in comparison to C and F
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Fig. S1. bPAC variants in HeLa229 cells and hippocampal neurons.
A) Human HeLa229 cells (ATCC CCL-2.1TM) were cultured in 10% (v/v) heat inactivated FBS (Sigma-Aldrich) RPMI1640 + GlutaMAXTM medium (GibcoTM). The cells were grown in a humidified atmosphere containing 5% (v/v) CO2 at 37 °C. For microscopy, HeLa229 cells were grown on coverslips in 12-well plates and, upon adhesion, transfected with plasmids encoding CD8-Venus-bPAC(wt), Glyco-Venus-bPAC(wt) and PACmn (2xLyn-Venus-bPAC(F198Y)) using Viromer® RED (230155; Biozym, Oldendorf, Germany) according to manufacturer’s instructions. 24 h post transfection, the cells were fixed with 4% PFA and then mounted onto glass-slides using 2.5% Mowiol-DABCO (Carl Roth, Karlsruhe, Germany). Images were acquired on a Zeiss (Oberkochen, Germany) ELYRA S.1 SR-SIM structured illumination platform using a PLAN-Apochromat 63x oil-immersion objective with a numerical aperture of 1.4. Reconstruction of super-resolution images was performed using the ZEN image-processing platform with a SIM module. A single plane from the bottom of the cells is shown. Note the improved plasma membrane localization of PACmn. B) Live hippocampal neurons expressing Venus-tagged bPAC variants. Maximum intensity projections of confocal LSM images.
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Fig. S2. Expression of bPAC variants in Drosophila motoneurons
Expression of bPAC variants (green) in Drosophila motoneurons (ok6-GAL4 driver) labelled with HRP (horseradish peroxidase, magenta). Ventral nerve cord (VNC, arrowheads indicate axons), neuromuscular junction (NMJ). Note that the soluble and Glyco-Venus-bPAC(S27A) are expressed throughout the motoneurons, whereas CD8-Venus-bPAC(wt) is absent from the NMJ.
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Fig. S3. Motoneuron morphology of Drosophila larvae.
Imaging by confocal microscopy shows that neither the size of the NMJ (motoneurons labelled with the membrane marker anti-HRP), nor the number of presynaptic active zones (AZs, labelled with anti-Brp) are altered in bPAC expressing larvae [ok6-GAL4>UAS-bPAC and ok6-GAL4>UAS-Venus-bPAC(F198Y)] raised in the dark.
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Fig. S4. Expression of Lyn-Venus-bPAC(wt) and 2xLyn-Venus-bPAC(wt) in Xenopus oocytes.
Oocytes were injected with the indicated amount of cRNAs of Lyn-Venus-bPAC(wt) and 2xLyn-Venus-bPAC(wt). After 3 days of expression, oocytes were imaged by confocal microscopy. Shown are single planes through the center of the oocyte.
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