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      A C. elegans genome-wide RNAi screen for altered levamisole sensitivity identifies genes required for muscle function

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          Abstract

          At the neuromuscular junction (NMJ), postsynaptic ionotropic acetylcholine receptors (AChRs) transduce a chemical signal released from a cholinergic motor neuron into an electrical signal to induce muscle contraction. To identify regulators of postsynaptic function, we conducted a genome-wide RNAi screen for genes required for proper response to levamisole, a pharmacological agonist of ionotropic L-AChRs at the Caenorhabditis elegans NMJ. A total of 117 gene knockdowns were found to cause levamisole hypersensitivity, while 18 resulted in levamisole resistance. Our screen identified conserved genes important for muscle function including some that are mutated in congenital myasthenic syndrome, congenital muscular dystrophy, congenital myopathy, myotonic dystrophy, and mitochondrial myopathy. Of the genes found in the screen, we further investigated those predicted to play a role in endocytosis of cell surface receptors. Loss of the Epsin homolog epn-1 caused levamisole hypersensitivity and had opposing effects on the levels of postsynaptic L-AChRs and GABA A receptors, resulting in increased and decreased abundance, respectively. We also examined other genes that resulted in a levamisole-hypersensitive phenotype when knocked down including gas-1, which functions in Complex I of the mitochondrial electron transport chain. Consistent with altered ATP synthesis impacting levamisole response, treatment of wild-type animals with levamisole resulted in L-AChR–dependent depletion of ATP levels. These results suggest that the paralytic effects of levamisole ultimately lead to metabolic exhaustion.

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          THE GENETICS OF CAENORHABDITIS ELEGANS

          Methods are described for the isolation, complementation and mapping of mutants of Caenorhabditis elegans, a small free-living nematode worm. About 300 EMS-induced mutants affecting behavior and morphology have been characterized and about one hundred genes have been defined. Mutations in 77 of these alter the movement of the animal. Estimates of the induced mutation frequency of both the visible mutants and X chromosome lethals suggests that, just as in Drosophila, the genetic units in C.elegans are large.
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            Calcium signaling.

            Calcium ions (Ca(2+)) impact nearly every aspect of cellular life. This review examines the principles of Ca(2+) signaling, from changes in protein conformations driven by Ca(2+) to the mechanisms that control Ca(2+) levels in the cytoplasm and organelles. Also discussed is the highly localized nature of Ca(2+)-mediated signal transduction and its specific roles in excitability, exocytosis, motility, apoptosis, and transcription.
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              Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans.

              Genetic interference mediated by double-stranded RNA (RNAi) has been a valuable tool in the analysis of gene function in Caenorhabditis elegans. Here we report an efficient induction of RNAi using bacteria to deliver double-stranded RNA. This method makes use of bacteria that are deficient in RNaseIII, an enzyme that normally degrades a majority of dsRNAs in the bacterial cell. Bacteria deficient for RNaseIII were engineered to produce high quantities of specific dsRNA segments. When fed to C. elegans, such engineered bacteria were found to produce populations of RNAi-affected animals with phenotypes that were comparable in expressivity to the corresponding loss-of-function mutants. We found the method to be most effective in inducing RNAi for non-neuronal tissue of late larval and adult hermaphrodites, with decreased effectiveness in the nervous system, in early larval stages, and in males. Bacteria-induced RNAi phenotypes could be maintained over the course of several generations with continuous feeding, allowing for convenient assessments of the biological consequences of specific genetic interference and of continuous exposure to dsRNAs.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                G3 (Bethesda)
                Genetics
                g3journal
                G3: Genes|Genomes|Genetics
                Oxford University Press
                2160-1836
                April 2021
                13 March 2021
                13 March 2021
                : 11
                : 4
                : jkab047
                Affiliations
                [1 ] Department of Physiology, Perelman School of Medicine, University of Pennsylvania , Philadelphia, PA 19104, USA
                [2 ] Department of Biological Sciences, University of Delaware , Newark, DE 19716, USA
                Author notes

                Shrey Pate and Erin M. Smith contributed equally to this work.

                Present address: Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19716, USA
                Present address: Drexel University College of Medicine, Philadelphia, PA 19129, USA
                Present address: Center for Advanced Study of Human Paleobiology, The George Washington University, Washington, DC 20052, USA
                Present address: WuXi AppTec, Philadelphia, PA 19112, USA
                Corresponding author: Department of Biological Sciences, University of Delaware, 105 The Green, 233 Wolf Hall, Newark, DE 19716, USA. E-mail: jtanis@ 123456udel.edu
                Author information
                https://orcid.org/0000-0002-7993-1013
                Article
                jkab047
                10.1093/g3journal/jkab047
                8049432
                33713125
                fa1b71d2-f4bc-4e99-b916-be42822dc017
                © The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 11 November 2020
                : 9 February 2021
                : 9 February 2021
                : 09 February 2021
                Page count
                Pages: 11
                Funding
                Funded by: NIH-NIAMS;
                Award ID: F32 AR060128
                Funded by: NIH-NIGMS IDeA Network of Biomedical Research Excellence (INBRE);
                Award ID: P20 GM103446
                Funded by: Core Center Access Award;
                Award ID: S10 RR027273
                Award ID: LSM880 (S10 OD016361
                Funded by: NIH-NIGMS;
                Award ID: P20 GM103446
                Funded by: NSF, DOI 10.13039/100000001;
                Award ID: IIA-1301765
                Funded by: State of Delaware;
                Categories
                Mutant Screen Report
                AcademicSubjects/SCI01180
                AcademicSubjects/SCI01140
                AcademicSubjects/SCI00010
                AcademicSubjects/SCI00960

                Genetics
                c. elegans,levamisole,acetylcholine,gaba,endocytosis,epn-1,gas-1,atp
                Genetics
                c. elegans, levamisole, acetylcholine, gaba, endocytosis, epn-1, gas-1, atp

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