Rufener L , Bedoni N , Baur R , Rey S , Glauser DA , Bouvier J , Beech R , Sigel E , Puoti A .

P40 OD010440 NIH HHS

	Figure 2. ACR-23 protein during C.elegans development.A) L2 larva expressing a acr-23::gfp transgene. Twisted morphology is caused by the transformation marker rol-6(su1006). B) Same worm expressing myo-3::mCherry. C) and D) Expression in the adult. Bar, 50 µm. E) Analysis of ACR-23::GFP expression in response to monepantel after 24 hours of exposure. Loading and general protein expression were tested with α-tubulin and LET-512, respectively.
	Figure 3. Choline concentration dependence.A) Current traces from a choline concentration response curve obtained from a Xenopus oocyte expressing ACR-23 receptors. The bars indicate the time period of choline perfusion. Choline concentrations are indicated above the bars. B) Current amplitude measured in Xenopus oocytes expressing a wild-type ACR-23 receptor (filled circles) or a mutant ACR-23 receptor (filled squares) after choline perfusion. The mutant ACR-23 subunit corresponding to allele acr-23(cb101) bears a D112N substitution in the extracellular loop. Mean ± SD of experiments carried out with 4–5 oocytes from two batches are shown.
	Figure 4. Monepantel is a direct agonist of ACR-23 channel.A) Current traces obtained from a Xenopus oocyte expressing ACR-23 upon perfusion with monepantel. The bars indicate the time period of monepantel perfusion. Monepantel concentrations are indicated above the bars. B) Averaged monepantel current amplitudes measured with monepantel (filled circles), monepantel sulfone (open circles) and with monepantel (filled triangles) and monepantel sulfone (open triangles) supplemented with 0.3 mM choline. One point is in brackets as the measured current reached the linear limit of the amplifier (20 µA), preventing a precise measurement. The effect of monepantel on mutant ACR-23(cb101) is shown by the filled squares. Mean ± SEM of experiments carried out with 3–6 oocytes from two batches are shown.
	Figure 5. Effect of ric-3 on the response to monepantel.A) Dose response to monepantel. The average number of progeny reaching adulthood after 3 days is plotted versus the concentration of monepantel. ric-3(md158) mutants are partially resistant to low doses of monepantel. B) ric-3(md158) adult grown on 1 µM monepantel (bottom) compared to a sibling grown in absence of the drug (top). The reduced length is certainly caused by muscle contraction due to monepantel. White arrows and arrowheads point at the heads and vulvae, respectively. C) ACR-23::GFP protein levels in wild-type (lanes 1,2) and ric-3 (lanes 3,4) animals after 2 hours of exposure to 10 µM monepantel (lanes 2,4). α-Tubulin was used to verify equal loading. D) Growth of wild-type and mutant strains on monepantel. Light gray (0 µM), dark gray (20 µM) and black (50 µM) bars represent the growth rate after 9 days of culture. In the absence of monepantel, wild-type (N2) worms grow much faster than any of the mutants tested. Genotypes are indicated. Values for growth rates are defined in the methods section (1, poor growth; 5, robust growth). acr-23 single mutants grew to level 5 in less than 5 days, whereas ric-3; acr-23 double mutants reached level 2 in absence of monepantel, and level 4 in presence of the drug. E) Growth rates after 9 days on the inactive R-enantiomer of monepantel. F) ric-3(md158); acr-23(cb27) adult grown in absence of monepantel. G) Adult ric-3(md158); acr-23(cb27) sibling grown on 20 µM monepantel. In both panels (F and G), one germline arm is outlined. Arrowheads and arrows point at oocytes and early embryos, respectively. The outlined germline arm shows robust production of gametes (panel G). Scale bar, 50 µm.
	Figure 1. Effects of monepantel on C. elegans and rescue of mutant acr-23.A) Adult N2 hermaphrodite with hatched embryos on 60 µM monepantel. L1 and L2 larvae are paralyzed and coiled (carcass of mother is outlined). B) Higher resolution of an arrested L2 larva. The germline is outlined. C) acr-23(cb27) animals carrying an extrachromosomal array containing wild-type acr-23 grown on standard NGM. Transgenic animals have a rolling phenotype (red arrowhead; non transgenic worms are denoted by a black arrowhead in C and D). D) In presence of monepantel, only non-rolling siblings are mobile and fertile. Transgenic animals are immobilized and dying or dead. Panels C) and D) show plates with the progeny of one single rolling parent incubated for 6 days at 20°C. E) Phenotypes of homozygotic and heterozygotic acr-23(cb27). eDf43 is a deficiency that spans acr-23 and neighboring genes. Depicted adult worms are one heterozygote, with dominant pharyngeal GFP expression (nT1g), and one non-glowing acr-23(cb27) homozygote, both grown on 20 µM monepantel. White arrows indicate all embryos present in the uterus. Similar results were obtained with acr-23(ok2804)/nT1g heterozygotes (not shown). Asterisks indicate the head. Bar, 50 µm. F) acr-23 mutants make fewer reversals. The rate of reversal events, where worms shift from forward to backward locomotion or vice versa, was measured in starved worms crawling on food-free NGM plates at 20°C. n is the number of movies analyzed, each tracking 15 to 60 individuals over at least 1 minute. *, P-value = 6.4E-5 versus N2 by Student's T-test (two-tailed). Results are expressed as mean with s.e.m as error bars.

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