IDENTIFICATION OF GENES THAT AFFECT ACETYLCHOLINE SIGNALING AT THE C. ELEGANS NEUROMUSCULAR JUNCTION
Date
2018-05
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
Acetylcholine (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.
Description
Keywords
Biological Sciences, acetylcholine signaling, c. elegans neuromuscular junction, genes