daf-18/PTEN mutants are hypersensitive to cholinergic drugs.
A- daf-18/PTEN encodes a lipid and protein phosphatase that hydrolyzes phosphatidylinositol (3,4,5)-trisphosphate (PIP3) to phosphatidylinositol-4,5-bisphosphate (PIP2). It is the main negative modulator of PDK and AKT activity. In daf-18/PTEN mutants, AKT-1 is overactivated leading to high levels of DAF-16/FOXO phosphorylation that prevents the translocation of this transcription factor to the nucleus. B- Gene structure of daf-18. Coding sequences are represented by blue boxes. The daf-18(e1375) mutant allele inserts a 30 bp sequence in exon IV. This insertion occurs downstream of the phosphatase catalytic domain and causes a frameshift that leads to premature truncation of the protein. This e1375 mutation partially reduces DAF-18 function. The daf-18(ok480) allele contains a 956 bp deletion that removes most of exon 3 and exon 4 and is generally considered to be a null allele. C- Schematic of C. elegans neuromuscular circuit. Red indicates GABAergic motor neurons (DD/VD) and green indicates cholinergic motor neurons (VA/VB and DA/DB). The VA and VB cholinergic motor neurons send synaptic inputs to the ventral body wall muscles and to the DD GABAergic motor neurons. The release of ACh from VA/VB neurons leads to the contraction of the ventral body wall muscles and the activation of DD GABAergic motor neurons that release GABA on the opposite side of the worm, causing relaxation of the dorsal body wall muscles. Conversely, activation of the DA and DB cholinergic motor neurons produces contraction of the dorsal body wall muscles and activates the VD GABAergic motor neurons. The VD GABAergic motor neurons release GABA, causing relaxation of the ventral body wall muscles, and thus contralateral inhibition. D-F Quantification of paralysis induced by cholinergic drugs. The assays were performed in NGM plates containing 2 mM aldicarb and 0.5 mM levamisole. Each data point represents the mean percentage of animals paralyzed ± SEM . At least four independent trials with 25-30 animals for each genotype were performed. One-way analysis of variance (ANOVA) was used to test statistical differences among strains (ns p > 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001). Strains tested are: N2 (wild-type), OAR 144 daf-18(ok480), CB1375 daf-18(e1375), GR1310 akt-1(mg144), TJ1052 age-1(hx546), VC204 akt-2(ok393), VC222 raga-1(ok386) and KQ1366 rict-1(ft7). All of these strains carry loss-of-function mutations. Furthermore, the strains denoted as “pdk-1 (lf)” and “(gf)” correspond to JT9609 pdk-1(sa680), which possesses a loss-of-function mutation, and GR1318 pdk-1(mg142), which harbors a gain-of-function mutation in the pdk-1 gene, respectively. The strain CB156 unc-25(e156) was included as a strong GABA-deficient control. G- Manual Measurement of Body length and width upon Optogenetic Stimulation of GABAergic (Left) and Cholinergic (Right) neurons. At the 2.5-second time point of light stimulation, we manually measured both the width and length of multiple animals and compared these measurements with the corresponding areas obtained from automated analysis (see Materials and Methods). The width of the worms remained relatively constant, highlighting that the alterations in body area primarily stem from changes in the animal’s length