Clase bPAC (Stierl et al., 2011) (iav-GAL4UAS-bPAC). Photoinduced cAMP elevation in wildtype lch5 quenched neuronal activity for the level observed in dCirlKO mutants, whilst bPAC activation within the dCirlKO background did not additional decrease action present frequenciesScholz et al. eLife 2017;6:e28360. DOI: 10.7554/eLife.dCdCdCirl K-RBSxCesO7 ofResearch articleNeuroscienceaR T H S V C S C N H LcNTF -2 +1 GPS dCirlN-RFPHRPRFP acTub MergeCTFb250 150GPSHA GPSTA GPSwt50 TubulinddCirlRescue dCirlKO dCirlHA SB-612111 web dCirlTA 1 s x 900 HzeCurrent (pA) 60 40 20Control (dCirlRescue) PhasicdCirlN-RFP/TAdCIRLN-RFPdCirlN-RFP/HAFigure 5. Differential effect of GPS mutations on mechanosensitivity. (a) Structure from the dCIRL GPS area. The GPS separates NTF from CTF in proteolyzable aGPCRs. The C-terminal cleavage component consists of the Stachel sequence, a potent receptor agonist in a lot of aGPCRs (light blue). Magenta: conserved, mutated residues which can be important for GPS cleavage. (b) Western blot of whole fly protein extracts containing wildtype or proteolysisdefective GPS variants of dCIRL probed against an mRFP tag within the NTF. The dCIRL-GPSwt sample displays only a fragment corresponding towards the cleaved NTF (ca. 106 kDa; filled circle), even though the two GPS mutants include a band representing the full-length receptor (ca. 218 kDa; open circle). (c) SIM images of dCIRLN-RFP fusion proteins with wildtype and proteolysis-resistant GPS in lch5. The protein is trafficked into dendrites and cilia, regardless of autoproteolytic cleavage. Scale bar five mm. (d) Receptor existing recordings (average of 8 sweeps) of lch5 neurons beneath TTX inhibition highlight the divergent effects of your GPS mutations on mechanosensitivity (dark blue, dCirlHA; light blue, dCirlTA). (e) Quantification of tonic and phasic receptor existing components. Despite abrogating GPS cleavage, the response profile from the dCirlHA receptor variant is unaffected (900 Hz, phasic: p=0.464, tonic: p=0.460, Student’s t-test vs. dCirlRescue). In contrast, changing the very first residue of the Stachel sequence in dCirlTA mutants abolishes the receptor’s mechanosensory function, resulting in a dCirlKO response profile (900 Hz, phasic: p=0.030, tonic: p=0.023, Student’s t-test vs. dCirlRescue). Data are presented as imply SEM, n = 8 larvae per genotype. DOI: 10.7554/eLife.28360.considerably (Figure 6a ). Conversely, pharmacological inhibition of adenylyl cyclase activity specifically rescued dCirlKO neuron function (Figure 6d). These observations indicate that elevated cAMP levels attenuate the mechanosensory response and suggest that dCIRL modulates neuronal activity by suppressing cAMP production. Subsequent, we employed the FRET-based cAMP sensor Epac1-camps (Maiellaro et al., 2016; Nikolaev et al., 2004) to directly visualize neuronal cAMP dynamics for the duration of mechanical stimulationScholz et al. eLife 2017;six:e28360. DOI: 10.7554/eLife.Tethered agonist (Stachel)T N F A I L M D V V D E H Q HTonic 20 1020 pA 400 ms1 5 9 13 1 five 9 13 Stimulus frequency (x 100 Hz)8 ofResearch articleNeurosciencea4 s x 900 HzControlb900 Hz 10x 1 s 1 scFrequency (Hz)wt dCirlKO Manage one hundred 60 20 2 4 6 8 ten Time (s)50 pA 1s4 s x 900 HzFrequency (Hz) + Photostim.900 Hz 10x 1 s 1 s100 60 20 two 4 six eight ten Time (s)8 mW/mm2 Manage dCirlKO one hundred 60 20 1 1 5 9 13 5 9 13 Stimulus frequency (x 100 Hz)dFrequency (Hz)+ SQ22536 ns one hundred 60Figure 6. cAMP signaling by dCIRL. (a) Instance present recordings from wildtype lch5 neurons through only mechanical (upper panel) and c.