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Investigating the Mechanism of UBR-1, an Arg/N-End Rule Pathway E3 Ligase That Modulates Caenorhabditis Elegans Motor Behaviour

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Investigating the Mechanism of UBR-1, an Arg/N-End Rule Pathway E3 Ligase That Modulates Caenorhabditis Elegans Motor Behaviour Investigating the Mechanism of UBR-1, an Arg/N-end Rule Pathway E3 Ligase that Modulates Caenorhabditis elegans Motor Behaviour by Min Wu A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Molecular Genetics University of Toronto © Copyright by Min Wu 2019 Investigating the Mechanism of UBR-1, an Arg/N-end Rule Pathway E3 Ligase that Modulates Caenorhabditis elegans Motor Behaviour Min Wu Master of Science Department of Molecular Genetics University of Toronto 2019 Abstract The Arg/N-end rule pathway regulates protein half-lives in eukaryotes. UBR-1 E3 ligase serves as a substrate recognition component of this pathway in C. elegans, which recognizes N-terminal degradation signals of substrates and mediates their ubiquitination. The functional loss of UBR-1 results in a unique motor defect: animals’ posterior body fails to establish normal bending due to synchronized A-type motor neuron activation during backward locomotion. This motor defect was found to be caused by aberrant glutamate metabolism. Specifically, removing a glutamate synthesizing enzyme GOT-1 could restore ubr-1 mutants’ bending during reversals. Locomotory patterns are regulated by sensorimotor circuits. These findings connect aberrant glutamate levels with neural circuit dysfunction in the UBR-1-dependent modulation of motor outputs. Extending from these findings, my studies further reveal UBR-1’s main sites of action to be premotor interneurons that regulate backward locomotion. Results of my studies help decipher a physiological role of UBR-1 in the nervous system. ii Acknowledgments I am very grateful to have been mentored by my supervisor Dr. Mei Zhen. She has been guiding me to become a better thinker and researcher with her rich experience, keen scientific sense, and extraordinary enthusiasm for science. Without her, this project would not be possible. She encouraged and led me through difficulties in this project. I thank her for all these very rewarding experiences. I thank my committee members, Dr. Peter Roy and Dr. William Ryu, for their very helpful criticism, directions, and suggestions. I thank Dr. Jyothsna Chitturi for her important work on C. elegans UBR-1, which provides the essential basis for this project. I thank Dr. Wesley Hung for his contributions throughout this project. His technical support and expertise walked me through many difficulties in this project. I thank Kevin Zhang for his work on the ubr-1 suppressor screens. I thank all other members of the Zhen Lab, Dr. Ben Mulcahy, Dr. Shangbang Gao, Dr. Yan Li, Ying Wang, Jun Meng, Yangning Lu, Daniel Witvliet, many undergraduate students and past members, who provided me with technical support, helpful discussions and encouragement. I thank Dr. Quan Wen, who led me into this field of science and gave me lots of inspirations. I thank Dr. Janine Harper for reading and editing the first draft of this thesis. Last but not least, I thank my parents for their unconditional love and support. To you I dedicate this thesis. iii List of Specific Contributions I was solely responsible for experiments, data collection and figure preparation for all figures in Chapter 2-4, unless specified as otherwise. Confocal images in Figures 8 & 12 were taken by Dr. Wesley Hung. Behaviour recordings in Figure 10 were acquired by Dr. Jyothsna Chitturi. iv Table of Contents Acknowledgments.......................................................................................................................... iii List of Specific Contributions ........................................................................................................ iv Table of Contents .............................................................................................................................v List of Tables ............................................................................................................................... viii List of Figures ................................................................................................................................ ix Chapter 1 ..........................................................................................................................................1 Introduction .................................................................................................................................1 1.1 Proteolysis ............................................................................................................................1 1.1.1 The ubiquitin-proteasome system ............................................................................2 1.1.2 The N-end rule pathway in eukaryotes ....................................................................6 1.1.3 The N-end rule pathway in prokaryotes...................................................................8 1.1.4 The UBR family E3 ubiquitin ligases ....................................................................12 1.2 The mechanisms of Ubr1 in different model organisms ....................................................14 1.2.1 The discovery and characterization of Ubr1 in S. cerevisiae .................................14 1.2.2 The Johanson-Blizzard syndrome (JBS), a multisystem disorder caused by loss-of-function mutations in human UBR1 ..........................................................16 1.2.3 The mouse models .................................................................................................17 1.2.4 The cell culture model ...........................................................................................20 1.2.5 The C. elegans model ............................................................................................21 1.3 The locomotion of C. elegans ............................................................................................22 1.3.1 The models for the mechanism of locomotion ......................................................23 1.3.2 The neural mechanism underlying backward locomotion .....................................26 1.3.3 The neural mechanism underlying forward locomotion ........................................28 1.4 Objectives ..........................................................................................................................30 Chapter 2 ........................................................................................................................................31 v Materials and Methods ..............................................................................................................31 2.1 Molecular biology and C. elegans strains ..........................................................................31 2.1.1 Strains, constructs and oligos .................................................................................31 2.1.2 UBR-1 RING domain replacement allele ..............................................................31 2.2 Locomotory behaviour assay .............................................................................................37 2.2.1 Image acquisition ...................................................................................................37 2.2.2 Motor activity quantification .................................................................................38 2.3 Fluorescence microscopy ...................................................................................................39 2.4 Optogenetic cell ablation ...................................................................................................39 2.5 Calcium imaging ................................................................................................................39 2.6 Statistical analysis ..............................................................................................................40 Chapter 3 ........................................................................................................................................41 Results .......................................................................................................................................41 3.1 ubr-1 mutants exhibit reduced body bending during reversals ..........................................41 3.2 UBR-1 is expressed in multiple tissues .............................................................................43 3.3 UBR-1 is critically required in mechanosensory neurons and premotor INs to affect the reversal motor pattern ..................................................................................................43 3.4 Reduced bending results from synchronization of A-MNs’ intrinsic activities ................47 3.5 Reduced bending results from premotor IN-mediated dysregulation of the coordination of A-MNs’ intrinsic activities .......................................................................50 3.6 Neuronal toxicity and aberrant glutamate signaling may account for the reduced bending phenotype in ubr-1 mutants .................................................................................53 Chapter 4 ........................................................................................................................................56 Conclusions and Future Perspectives ........................................................................................56 4.1 Conclusions ........................................................................................................................56 4.2 Future Perspectives ............................................................................................................57
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