Clase bPAC (Stierl et al., 2011) (iav-GAL4UAS-bPAC). Photoinduced cAMP 60-81-1 In stock elevation in wildtype lch5 quenched neuronal activity for the level observed in dCirlKO mutants, whilst bPAC activation within the dCirlKO background did not additional 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 5. Differential impact of GPS mutations on mechanosensitivity. (a) Structure of the dCIRL GPS region. The GPS separates NTF from CTF in proteolyzable aGPCRs. The C-terminal cleavage component contains the Stachel sequence, a potent receptor agonist in a lot of aGPCRs (light blue). Magenta: 58880-19-6 manufacturer conserved, mutated residues that happen to be necessary for GPS cleavage. (b) Western blot of whole 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), even though the two GPS mutants contain 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, irrespective of autoproteolytic cleavage. Scale bar five mm. (d) Receptor present recordings (average of 8 sweeps) of lch5 neurons beneath 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 existing elements. In spite of abrogating GPS cleavage, the response profile on 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 within a dCirlKO response profile (900 Hz, phasic: p=0.030, tonic: p=0.023, Student’s t-test vs. dCirlRescue). Information are presented as imply SEM, n = eight larvae per genotype. DOI: ten.7554/eLife.28360.substantially (Figure 6a ). Conversely, pharmacological inhibition of adenylyl cyclase activity specifically rescued dCirlKO neuron function (Figure 6d). These observations indicate that improved cAMP levels attenuate the mechanosensory response and suggest that dCIRL modulates neuronal activity by suppressing cAMP production. Next, we employed the FRET-based cAMP sensor Epac1-camps (Maiellaro et al., 2016; Nikolaev et al., 2004) to straight visualize neuronal cAMP dynamics for the duration of mechanical stimulationScholz et al. eLife 2017;6:e28360. DOI: ten.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 five 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 Handle one hundred 60 20 two 4 6 eight 10 Time (s)50 pA 1s4 s x 900 HzFrequency (Hz) + Photostim.900 Hz 10x 1 s 1 s100 60 20 2 4 six eight 10 Time (s)eight mW/mm2 Handle dCirlKO one hundred 60 20 1 1 5 9 13 5 9 13 Stimulus frequency (x 100 Hz)dFrequency (Hz)+ SQ22536 ns 100 60Figure 6. cAMP signaling by dCIRL. (a) Example present recordings from wildtype lch5 neurons for the duration of only mechanical (upper panel) and c.