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, although bPAC activation inside the dCirlKO background didn’t further lower action existing frequenciesScholz et al. eLife 2017;6:e28360. DOI: ten.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 dCirlTA 1 s x 900 HzeCurrent (pA) 60 40 20Control (dCirlRescue) PhasicdCirlN-RFP/TAdCIRLN-RFPdCirlN-RFP/HAFigure five. Differential effect of GPS mutations on mechanosensitivity. (a) Structure in the dCIRL GPS area. The GPS separates NTF from CTF in proteolyzable aGPCRs. The C-terminal cleavage component includes the Stachel sequence, a potent receptor agonist in a lot of aGPCRs (light blue). Magenta: conserved, mutated residues that are needed for GPS cleavage. (b) Western blot of entire fly protein extracts containing wildtype or proteolysisdefective GPS variants of dCIRL probed against an mRFP tag in the NTF. The dCIRL-GPSwt sample displays only a fragment corresponding for the cleaved NTF (ca. 106 kDa; filled circle), while the two GPS mutants include a band representing the full-length receptor (ca. 218 kDa; open circle). (c) SIM photos of dCIRLN-RFP fusion proteins with wildtype and proteolysis-resistant GPS in lch5. The protein is trafficked into dendrites and cilia, no matter autoproteolytic cleavage. Scale bar five mm. (d) Receptor current recordings (typical of 8 sweeps) of lch5 neurons under TTX inhibition highlight the divergent effects with the GPS mutations on mechanosensitivity (dark blue, dCirlHA; light blue, dCirlTA). (e) Quantification of tonic and phasic receptor current 2-Phenylacetamide medchemexpress elements. Regardless of abrogating GPS cleavage, the response profile in the dCirlHA receptor variant is unaffected (900 Hz, phasic: p=0.464, tonic: p=0.460, Student’s t-test vs. dCirlRescue). In contrast, altering the initial residue from the Stachel sequence in dCirlTA mutants abolishes the receptor’s mechanosensory function, resulting inside 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: ten.7554/eLife.28360.considerably (Figure 6a ). Conversely, pharmacological inhibition of adenylyl cyclase activity particularly rescued dCirlKO neuron function (Figure 6d). These observations indicate that increased cAMP levels attenuate the mechanosensory response and recommend 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 straight visualize neuronal cAMP dynamics in the course of mechanical stimulationScholz et al. eLife 2017;6: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 5 9 13 Stimulus frequency (x 100 Hz)eight ofResearch articleNeurosciencea4 s x 900 HzControlb900 Hz 10x 1 s 1 scFrequency (Hz)wt dCirlKO Control 100 60 20 two four six 8 10 Time (s)50 pA 1s4 s x 900 HzFrequency (Hz) + Photostim.900 Hz 10x 1 s 1 s100 60 20 2 4 six 8 ten Time (s)eight mW/mm2 Control dCirlKO 100 60 20 1 1 five 9 13 5 9 13 Stimulus frequency (x 100 Hz)dFrequency (Hz)+ SQ22536 ns one hundred 60Figure six. cAMP signaling by dCIRL. (a) Example present recordings from wildtype lch5 neurons through only mechanical (upper panel) and c.