IDENTIFICATION OF GENES THAT AFFECT ACETYLCHOLINE SIGNALING AT THE C. ELEGANS NEUROMUSCULAR JUNCTION

Jessica TanisAcetylcholine (ACh) signaling at the neuromuscular junction (NMJ) is required for muscle contraction. Many muscular disorders arise from genetic mutations altering either ACh release or abundance of postsynaptic acetylcholine receptors (AChRs). Identifying the genes that affect ACh signaling will lead to a better understanding of such disorders. Caenorhabditis elegans serves as an excellent model organism for the study of signaling at the neuromuscular junction as the body-wall muscles are functionally similar to vertebrate skeletal muscles. A C. elegans genomewide RNA interference (RNAi) screen identified 156 gene knockdowns that caused either resistance or hypersensitivity to the AChR agonist levamisole. The altered levamisole sensitivity suggests that these genes are involved in ACh signaling. Based on predicted function and homology, we hypothesize that thirteen of the 156 genes identified in the screen affect AChR trafficking and/or abundance at the NMJ. Our first goal was to confirm the levamisole phenotype resulting from knockdown of these genes. We performed time course assays and found that knockdown of mca-3, arf-3, sec-12, and nsf-1 led to levamisole resistance, while knockdown of cogc-4, epn-1, tag- 53, clec-1, and F54D7.2 resulted in levamisole hypersensitivity. Our second goal was to determine which of these genes were expressed in the body-wall muscles. Prior studies had suggested that mca-3, arf-3, F54D7.2, epn-1, sym-4, erd-2, unc-73, tag-53, and clec-1 were muscle-expressed, however, the expression patterns for D1081.4, cogc-4, nsf-1, and sec-12 were unknown. We made constructs consisting of the regulatory and promoter sequences followed by mCherry for each respective gene and then created transgenic worms. Confocal imaging showed that cogc-4 and sec-12 were expressed in the body-wall muscles, while D1081.4 and nsf-1 were expressed in neurons. Our final goal was to determine if knockdown of arf-3, sec-12, cogc-4, epn- 1, mca-3, tag-53, and clec-1 affect AChR localization. We grew worms that expressed an AChR tagged with YFP on the RNAi clones and used confocal imaging to quantitate AChR::YFP localization on both the ventral and dorsal sides of animals. Average intensity of AChR::YFP, average volume of each puncta, total number of puncta per 50 μm and total volume of the puncta per 50 μm were analyzed. While knockdown of each gene studied had a significant effect on AChR localization, suggesting that these genes play some role in AChR localization, knockdown of clec-1 had the greatest impact. Knockdown of clec-1 increased AChR::YFP fluorescence, puncta volume, and total volume of AChRs on the ventral side, but decreased AChR::YFP fluorescence, AChR puncta, volume of each puncta, and total volume of AChRs on the dorsal side. Since 82% of clec-1 knockdown animals showed decreased AChR abundance on the dorsal side, future studies will focus on determining the mechanism by which CLEC-1 regulates AChR localization.Biological Science