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Identification of Genes That Affect Acetylcholine

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Identification of Genes That Affect Acetylcholine IDENTIFICATION OF GENES THAT AFFECT ACETYLCHOLINE SIGNALING AT THE C. ELEGANS NEUROMUSCULAR JUNCTION by Shrey Patel A thesis submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Honors Bachelor of Arts in Biological Sciences with Distinction Spring 2018 © 2018 Shrey Patel All Rights Reserved IDENTIFICATION OF GENES THAT AFFECT ACETYLCHOLINE SIGNALING AT THE C. ELEGANS NEUROMUSCULAR JUNCTION by Shrey Patel Approved: __________________________________________________________ Jessica Tanis, Ph.D. Professor in charge of thesis on behalf of the Advisory Committee Approved: __________________________________________________________ Donna Woulfe, Ph.D. Committee member from the Department of Biological Sciences Approved: __________________________________________________________ Carlton Cooper, Ph.D. Committee member from the Board of Senior Thesis Readers Approved: __________________________________________________________ Paul Laux, Ph.D. Director, University Honors Program ACKNOWLEDGMENTS I would like to first thank Dr. Jessica Tanis for giving me the opportunity to conduct research towards a senior thesis. Her guidance, support, and encouragement throughout the process have been invaluable. Her mentorship has changed my views on research, helped me grow professionally and personally, and opened doors previously unimagined. I could not be more grateful. I would not be where I am without the assistance of the Tanis Lab team: Kirsten Kervin, Elaine Miller, Andy Lam, Michael Clupper, Amanda Addiego, Denis Touroutine, and Alyssa Reed. Thank you for help with laboratory techniques, input on my presentations, and for being great team members. I would like to specially thank Amanda, Kirsten, and Elaine for their contributions to this project, which has made significant progress in just one year. I could not have wished to be in another lab, for the enriching, collaborative, and friendly environment cannot be replicated. I would like to further thank Delaware Biotechnology Institute, especially Dr. Jeffrey Caplan, for their guidance with confocal imaging and analysis. Thank you to INBRE and the Undergraduate Research Program for giving me the resources to conduct research and for support through the writing process. A special thanks to my thesis committee of Dr. Tanis, Dr. Donna Woulfe, and Dr. Carlton Cooper for their patience and investment throughout the process. Lastly, I would like to thank my friends and family for their encouragement and support. I am fortunate for this opportunity and thankful to everyone involved, for my time and growth at the University of Delaware would not be the same otherwise. iii TABLE OF CONTENTS LIST OF TABLES ..................................................................................................... v LIST OF FIGURES .................................................................................................. vi ABSTRACT ............................................................................................................. vii CHAPTERS 1 INTRODUCTION ......................................................................................... 1 1.1 Neuromuscular Junction ....................................................................... 1 1.2 Congenital Myasthenic Syndromes ...................................................... 2 1.3 Caenorhabditis elegans ........................................................................ 3 1.4 Genome-wide RNAi Screen and Levamisole Sensitivity Assay .......... 5 1.5 Genes Identified in the Levamisole Sensitivity Screen ........................ 8 1.6 Objectives and Hypothesis .................................................................... 9 2 METHODS .................................................................................................. 11 2.1 Nematode Maintenance ...................................................................... 11 2.2 RNAi ................................................................................................... 13 2.3 Levamisole Assay ............................................................................... 13 2.4 Molecular Cloning .............................................................................. 14 2.5 Transgenic Worms .............................................................................. 16 2.6 Development of unc-63::YFP;eri-1 .................................................... 16 2.7 Preparing and Mounting Worms for Confocal Microscopy ............... 17 2.7.1 Microscopy for Expression Patterns and AChR Localization 17 2.7.2 Analysis of AChR Images ...................................................... 17 3 RESULTS .................................................................................................... 19 3.1 Verification of Levamisole Phenotypes .............................................. 19 3.2 Analysis of Gene Expression Patterns ................................................ 23 3.3 AChR Localization ............................................................................. 27 4 DISCUSSION .............................................................................................. 37 REFERENCES ........................................................................................................ 42 iv LIST OF TABLES Table 1: Strains of C. elegans Used in Study .......................................................... 12 v LIST OF FIGURES Figure 1: Effect of Levamisole on C. elegans ........................................................... 6 Figure 2: Levamisole Screen Schematic .................................................................... 7 Figure 3: Gene Knockdowns Resulting in Resistance to Levamisole ..................... 20 Figure 4: Gene Knockdowns Resulting in Hypersensitivity to Levamisole ............ 21 Figure 5: Gene Knockdowns that Do Not Alter Levamisole Response .................. 22 Figure 6: Constructs Developed to Study Gene Expression Patterns ...................... 24 Figure 7: Confirmation of Recombinant DNA Constructs ...................................... 25 Figure 8: Body-Wall Muscle Expressed Genes ....................................................... 26 Figure 9: Nervous System Expressed Genes ........................................................... 26 Figure 10: AChR Localization on Ventral Side of Animals with Gene Knockdowns ....................................................................................... 29 Figure 11: AChR Localization on Dorsal Side of Animals with Gene Knockdowns ....................................................................................... 30 Figure 12: AChR Puncta Abundance and Intensity on Ventral Side of Animals with Genes Knocked Down ........................................................................ 31 Figure 13: AChR Puncta Volume on the Ventral Side of Animals with Genes Knocked Down ................................................................................... 32 Figure 14: AChR Puncta Abundance and Intensity on Dorsal Side of Animals with Genes Knocked Down ........................................................................ 33 Figure 15: AChR Puncta Volume on the Dorsal Side of Animals with Genes Knocked Down ................................................................................... 34 Figure 16: Images of AChR Localization in EV and clec-1 Knockdown Animals . 35 Figure 17: AChR Localization in EV and clec-1 Knockdown Animals .................. 36 vi 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 genome- wide 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 vii 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
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