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Characterization of the Teneurin C-Terminal Associated Peptide (TCAP) and Latrophilin Ligand-Receptor Pair in an Immortalized Skeletal Muscle Cell Line

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Characterization of the Teneurin C-Terminal Associated Peptide (TCAP) and Latrophilin Ligand-Receptor Pair in an Immortalized Skeletal Muscle Cell Line Characterization of the Teneurin C-terminal Associated Peptide (TCAP) and Latrophilin Ligand-Receptor Pair in an Immortalized Skeletal Muscle Cell Line by Thomas Dodsworth A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Cell and Systems Biology University of Toronto Ⓒ Copyright by Thomas Dodsworth (2019) Characterization of the Teneurin C-terminal Associated Peptide (TCAP) and Latrophilin Ligand-Receptor Pair in an Immortalized Skeletal Muscle Cell Line Thomas Dodsworth Master of Science Graduate Department of Cell and Systems Biology University of Toronto 2019 Abstract Teneurin C-terminal associated peptide (TCAP) is an ancient and conserved bioactive peptide that is evolutionarily related to corticotropin releasing factor (CRF). Recently, synthetic TCAP-1 was shown to increase cellular energy availability and alter contractile performance in rodent skeletal muscle. However, the exact receptor signalling mechanism through which this occurs is unknown. Based on evidence of their interaction in vitro, we hypothesized that TCAP-1 signals through latrophilins—a family of Adhesion G-protein coupled receptors—to elicit its effects in muscle. To test this, I knocked-down and knocked-out latrophilins in the immortalized mouse myoblast C2C12 cell line by small interfering RNA (siRNA) and CRISPR/Cas9 gene editing methods, respectively, and examined the efficacy of TCAP-1 in knockdown and knockout cells. We determined that latrophilin-1 is necessary for TCAP-1-mediated increases in intracellular calcium, NADH turnover, and PGC-1a expression. This establishes, for the first time, that the TCAP-latrophilin ligand-receptor pair has a functional role in skeletal muscle. ii Acknowledgements My completion of this degree would not have been possible without the support of these incredible individuals. First, I would like to thank my supervisor Dr. David Lovejoy for being an incredibly supportive mentor through this entire experience. You have instilled in me a passion for research that I never knew I had, and I am so grateful for the opportunity to be a part of your lab. I would like to thank the members of my thesis committee, Dr. Les Buck and Dr. Junchul Kim, for their support and feedback throughout this degree, and to Dr. Marius Locke for agreeing to serve as an examiner. To Dr. Dalia Barsyte-Lovejoy, who never ceases to amaze me with her knowledge of molecular biology—your help with this project has been immeasurable. I extend my gratitude to everyone in the Lovejoy lab who made this experience fulfilling. To Dr. David Hogg, thank you for your training and guidance, and for always breaking the silence on quiet days in the lab. To Dr. Andrea D’Aquila, thank you for passing along the reins of this project, and for always keeping me entertained in line for coffee. To Mia Husić, I would not have been able cross the finish line without your level-headed pep talks over lunch. To David Wosnick, I’m glad to have shared this experience with someone as resilient and persevering as you—I’ll see you on the other side. To Norzin Shrestha, thank you for brightening my day whenever we pass each other in the hall, and to my undergraduate student Fernando Jurado Soria, thank you for your assistance with this project and for always being a positive spirit in the lab. I would like to thank everyone in CSB who has lent a hand along the way. To the Buck, Mitchell and Chang labs, and to Dr. Pauline Wang at CAGEF—thank you for allowing me to use your equipment and for helping me troubleshoot. To Peggy Salmon, Chris Garside and everyone I had the pleasure of TA-ing with—thank you for making it a positive experience. To everyone who works in Ramsay Wright, especially those I see here late at night: I am inspired by your dedication, and it has been a pleasure to work alongside you. I thank my parents, my sisters and my friends in Toronto and abroad who have listened to me rant and rave about the ups and downs of this program for the past 2 years—I promise that I’ll be less annoying now. And lastly, I send my gratitude to the staff at Second Cup, Tim Hortons and the AC, who have kept me caffeinated for the past 2 years—you are the true unsung heroes of this degree. iii Table of Contents Abstract .......................................................................................................................................... ii Acknowledgements ....................................................................................................................... iii Table of Contents .......................................................................................................................... iv List of Figures & Tables ................................................................................................................. vi List of Abbreviations ................................................................................................................... viii Chapter 1: Introduction 1.1. Discovery, Structure and Expression of the Teneurins .......................................................... 1 1.2. Discovery and Structure of TCAP ......................................................................................... 3 1.3. Expression and Processing of TCAP ..................................................................................... 5 1.4. Actions of Teneurin and TCAP in the Central Nervous System ........................................... 7 1.5. Actions of Teneurin and TCAP in Reproductive Tissues ...................................................... 9 1.6. Actions of Teneurins and TCAP in Skeletal Muscle ............................................................. 9 1.7. Evidence for Teneurin and TCAP Receptors ....................................................................... 10 1.8. Discovery, Structure and Expression of the Latrophilins ..................................................... 11 1.9. Ligands and Signalling of Latrophilin .................................................................................. 14 1.10. Description of the Teneurin/TCAP-Latrophilin System ..................................................... 15 1.11. Evolution of the Teneurin/TCAP-Latrophilin System ....................................................... 17 1.12. Skeletal Muscle Cell Biology & Use of Immortalized Muscle Cell Lines ........................ 19 1.13. Thesis Rationale & Research Aims .................................................................................... 20 Chapter 2: Methods 2.1. Cell Culture .......................................................................................................................... 23 2.2. RNA Extraction .................................................................................................................... 24 2.3. Reverse Transcription and Polymerase Chain Reaction ....................................................... 25 2.4. Cloning and Sequencing of Polymerase Chain Reaction Products ...................................... 27 2.5. Quantitative Reverse Transcription Polymerase Chain Reaction ......................................... 27 2.6. Protein Extraction & Western Blot ....................................................................................... 28 2.7. Small Interfering RNA Transfection .................................................................................... 29 iv 2.8. T7 Endonuclease Assay ........................................................................................................ 29 2.9. Live-Cell Calcium Imaging .................................................................................................. 30 2.10. Resazurin-Resorufin Fluorescence Assay .......................................................................... 30 2.11. Statistical Analysis ............................................................................................................ 31 Chapter 3: Results 3.1. Characterization of TCAP-3 mRNA in the Adult Mouse Brain by 5’RACE PCR ............... 32 3.2. Expression of Latrophilins in C2C12 cells ........................................................................... 34 3.3. Establishing Methods of Latrophilin-1 and 3 siRNA Knockdown ...................................... 35 3.4. Intracellular Calcium Dynamics in TCAP-1-treated Latrophilin-1 and 3 Knockdowns ...... 36 3.5. Development of Latrophilin-1 CRISPR/Cas9 Knockouts .................................................... 36 3.6. Intracellular Calcium Dynamics in TCAP-1-treated Latrophilin-1 Knockouts ................... 40 3.7. Resorufin Fluorescence in TCAP-1-treated Latrophilin-1 CRISPR/Cas9 Knockouts ......... 40 3.8. Myosin Heavy Chain Expression in TCAP-1-treated Tibialis Anterior Muscle Tissue ...... 44 3.9. Myosin Heavy Chain I and Peroxisome Proliferator-activated Receptor-g Coactivator 1a Expression in TCAP-1-treated C2C12 cells ........................................................................ 45 3.10. Peroxisome Proliferator-activated Receptor-g Coactivator 1a Expression in TCAP-1- treated Latrophilin-1 CRISPR/Cas9 Knockouts ................................................................ 47 Chapter 4: Discussion 4.1. Independent Transcription of TCAP .................................................................................... 48 4.2. Small Interfering RNA Knockdown of Latrophilins ............................................................ 50 4.3. CRISPR/Cas9 Knockout of Latrophilins .............................................................................
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