Figure 4

Cegar-3Dq is functional upon stimulation with CNO in C. elegans.

(A–D) Expression and protein localisation of cegar-3Dq::yfp is indistinguishable from gar-3::yfp, both driven by the gar-3 promoter. Expression was detected in the pharynx (A, B; 1./3. row: DIC, 2./4. row: fluorescent image) and in the male tail (C, D; 1./3. row: DIC, 2./4. row: fluorescent image). Localisation of the DREADD appears to be similar to the one of GAR-3. Scale bars = 10 μm. (E) Analyses of expression levels by quantification of intensities reveals that cegar-3Dq::yfp expression is significantly weaker than gar-3 expression. For quantification, images of the pharynx from different cegar-3Dq::yfp expressing strains were taken and analysed in comparison with images from nematodes carrying gar-3::yfp. (F) Protraction rate in male nematodes containing the cegar-3Dq construct. Males were stimulated with 100 mM Oxo M, 10 mM CCh, 2 mM CNO or H₂O as negative control. Subsequently, spicule protraction was scored. Wild-type worms (pha-1(e2123); him-5 (e1490)), gar-3 (pha-1(e2123); him-5 (e1490) gar-3(gk305)) and transgenic gar-3; Ex[gar-3(+)] males served as controls. Data are shown as mean ± SD. n.s. not significant; **p < 0.01; ***p < 0.001; n ≥ 300. (G) Spicule protraction rates are dependent on the concentration of CNO. Male nematodes were incubated with the indicated concentrations of CNO and spicule protraction was scored subsequently. Nematodes deficient for gar-3 (pha-1(e2123); him-5 (e1490) gar-3(gk305)) served as negative control. Data are shown as mean ± SD, **p < 0.01; ***p < 0.001; n ≥ 200. Indicated below each set of columns is the schematic model related to the respective data. (H) Treatment duration does not have an effect on spicule protraction rate after 3 minutes. Male nematodes were incubated with 2 mM CNO and spicule protraction was scored. As negative control, nematodes deficient for gar-3 (pha-1(e2123); him-5 (e1490) gar-3(gk305)) were used. Data are shown as mean ± SD, **p < 0.01; ***p < 0.001; n ≥ 200. (I) The effect of DREADD activation is reversible. Transgenic gar-3; Ex[gar-3(+)] males were stimulated with 2 mM CNO. After 3 minutes the spicule protraction rate was determined (time point termed “0 minutes after CNO withdrawal”) and nematodes were withdrawn from the drug by transferring them into M9. Subsequently, spicule protraction was measured at distinct time points. Data are shown as mean ± SD, n ≥ 150.