Figure 7. dCIRL reduces cAMP levels in sensory neurons in response to mechanical stimulation.

(a) Schematic structure of the cAMP sensor Epac1-camps, which changes its conformation and fluorescence property upon binding of cAMP. Corresponding pseudocolor FRET images (YFP/CFP ratios) of Ich5 neurons (iav-GAL4>UAS-Epac1-camps) at low and high cAMP concentrations. Scale bar 10 µm. (b) Absolute FRET values (YFP/CFP ratios) recorded in control and dCirl^KO Ich5 neurons, corresponding to the region of interest depicted in (a). In order to ensure a dynamic sensor range, 0.5 µM FSK was first added to the preparation (Maiellaro et al., 2016). Mechanical stimulation (900 Hz, pink bar) decreases cAMP levels in control but not in dCirl^KO Ich5 neurons. At the end of the experiment, maximal FRET responses are induced by 10 µM FSK and 100 µM IBMX (3-Isobutyl-1-methylxanthin), a non-selective phosphodiesterase inhibitor. (c) Average time course of piezo-induced FRET changes in control and dCirl^KO Ich5 neurons. Data are expressed as percentages of the low forskolin response and presented as mean ± SEM. ΔFRET at 70 s: Control: 16.28 ± 4.05%, n = 14; dCirl^KO: 0.147 ± 3.78%, n = 6 larvae. Number denotes p value of comparison at 70 s with a Student’s t-test. See also Figure 7—figure supplements 1 and 2.

DOI: http://dx.doi.org/10.7554/eLife.28360.012

Figure 7—figure supplement 1. Basal cAMP levels in ChO neurons.

Activation of adenylyl cyclase by low FSK concentrations triggers similar cAMP accumulations in control and dCirl^KO neurons. FSK-induced ΔFRET values are expressed as percentages of the maximal FRET response and presented as mean ± SEM. Control: 66.17 ± 6.77%, n = 12; dCirl^KO: 52.33 ± 10.89%, n = 6; p=0.276, Student’s t-test.

DOI: http://dx.doi.org/10.7554/eLife.28360.013

Figure 7—figure supplement 2. A synthetic peptide mimicking dCIRL’s tethered agonist stimulates Gα_i coupling.

(a) Total and cell surface expression of various dCirl constructs. COS-7 cells were transfected with 1 µg (total expression)/0.5 µg (cell surface expression) of empty vector (pcDps or pHLSec) or plasmid encoding either dCirl constructs, the C. elegans Latrophilin homolog lat-1 or the human ADP receptor P2Y₁₂ which served as controls. Expression levels were measured 48 hr post transfection using ELISA on lysed cells (total expression) or intact cells (cell surface expression). Data are displayed as x-fold of empty vector and displayed as mean ± SEM of at least three independent experiments, each performed in triplicate. (b) Peptide-stimulated cAMP response of dCIRL. COS-7 cells transfected with 0.2 µg empty control vector (pcDps) or plasmid encoding dCirl were stimulated with 1 mM putative Stachel sequence-derived peptide of 12 aa length and cAMP levels were measured by cAMP accumulation assay. A scrambled peptide of the same length and amino acid composition (scrambled P12) served as negative control. To control for peptide specificity, the human vasopressin type 2 receptor (V₂R) was used, which does not respond to the peptides tested. Basal cAMP levels (empty vector, no peptide) are 3.2 ± 0.7 nM. Data are normalized to respective non-stimulated controls and are given as mean ± SEM. (c) dCIRL does not signal via a G_q protein signaling cascade upon treatment with Stachel sequence-derived peptides. COS-7 cells transfected with 0.2 µg empty control vector (pcDps) or plasmid encoding dCirl were stimulated with 1 mM peptide and IP accumulation assays were performed. Neither the potentially activating peptide nor the scrambled peptide show any effect on dCIRL. The rat muscarinic M3 acetylcholine receptor M₃R was used as a control for peptide specificity. Basal IP levels (empty vector, no peptide) are 434 ± 52 nM. Data normalized to respective non-stimulated controls are given as mean ± SEM of four independent experiments, each performed in triplicate. (d) dCIRL is activated by P12 to mediate a G_i protein signal. To measure functional coupling of dCIRL to Gα_i upon stimulation with P12, a chimeric Gα_qi4 protein was applied to reroute a G_i-mediated signal to a Gα_q protein cascade. 0.2 µg of plasmid containing pcDps or dCirl were co-transfected with 0.02 µg plasmid with the chimeric protein DNA into COS-7 cells. IP accumulation assays were performed detecting Gα_q-mediated increase in IP levels upon treatment with 1 mM peptide P12 or scrambled P12. A significant increase in IP was detected upon P12 challenge. The human P2Y₁₂ receptor served as a control for peptide specificity. Basal IP levels (empty vector, no peptide): 385 ± 32 nM. Data normalized to respective non-stimulated controls are given as mean ± SEM of four independent experiments, each performed in triplicate. p values < 0.05 are indicated above the respective columns, all others were > 0.05.

DOI: http://dx.doi.org/10.7554/eLife.28360.014