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Regulation of Wnt Receptor Activation by the Tumor Suppressor Apc REGULATION OF WNT RECEPTOR ACTIVATION BY THE TUMOR SUPPRESSOR APC By Vicente Kenyi Saito-Diaz Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Cell and Developmental Biology May, 2017 Nashville, Tennessee Approved: William Tansey, Ph.D. Andrea Page-McCaw, Ph.D. Jin Chen, M.D., Ph.D. Scott Hiebert, Ph.D. Ethan Lee, M.D., Ph.D. Copyright © 2017 by Vicente Kenyi Saito-Diaz All Rights Reserved ii For my mother. Y para Mollie, por su apoyo incondicional. iii ACKNOWLEDGEMENTS The first person I need to thank is my mentor, Dr. Ethan Lee. His patience, guidance, and support has made this work possible. I will always be amazed by his passion for science and his ability to think outside the box. Despite the inherent difficulties of the scientific endeavors, he is always excited about his work. I hope I can follow his example and share the excitement of science wherever I go. If I can call myself a scientist today, it is mainly because of him. Also, I would like to thank my committee members, Bill Tansey, Jin Chen, Andrea Page-McCaw, and Scott Hiebert. Your guidance and insight helped me realize of my true potential. It is thanks to your support and patience that I am writing these words today. I want to give special thanks to my chairman, Bill. I am very thankful for your wise words and your confidence in me. I would like to thank my lab mates, former and current. They always helped me, gave me words of advice, and kept me sane throughout this years. Specially, I want to thank Dr. Curtis Thorne. Although we overlapped for a short period of time, he introduced me to the awesomeness of the Lee lab and gave me one of the best pieces of advice I received during graduate school. I would like to thank Leah and Aja who played pivotal roles in this work. I would also want to thank to Dr. Laura Lee and her lab. For many years, they were the voice of reason and provided the very needed outside perspective which helped my project at many levels. There is a person who played a fundamental role in my life, Dr. Abel Alcazar- Roman. He selected me to participate in a little experiment called Research Experience iv for Peruvian Undergraduates. His decision changed my life. I am honored to have met such a generous person. His energy and desire to change our home country, Peru, rubbed on me and allowed me to grow and do things I would never knew I could. I consider him a friend and a brother. I would like to thank Dr. Jose Luis Aguilar, Anita Arce, Pilar García, and Ivan Lozada. They were my first mentors and guided my first steps as a scientist. I was lucky to have worked with such an amazing group. Also, I would like to thank Dr. Daniela Drummond-Barbosa. She had faith that a Peruvian undergrad with no experience working in developmental biology would be an asset to her lab. I worked in her lab for one summer and the experience showed me a whole new world. It was an incredible experience. I would specially like to thank my parents and my sister whose unconditional support have been invaluable throughout my life. My mother, who like water turning a rock into a sculpture, very patiently shaped me into the person I am today. Although, she is not with me anymore, she is still the driving force that pushes me to become a better person every day. I also want to thank my sister, who supported me during many difficult times of my life, and believes that I will win a Nobel Prize someday. And of course, I want to thank my beautiful wife, Mollie. Every day with you is a fun new adventure and I am thrilled to share many more with you. You take me to places I don’t even dream about. Without you, I would have never run two half-marathons or adopted our puppy, Nelson. You make me a better person. Thank you. v TABLE OF CONTENTS Page DEDICATION .................................................................................................................. iii ACKNOWLEDGEMENTS ............................................................................................... iv LIST OF FIGURES .......................................................................................................... ix LIST OF ABBREVIATIONS ............................................................................................ xi Chapter I. INTRODUCTION TO THE WNT SIGNALING PATHWAY .......................................... 1 Introduction ................................................................................................................. 1 The cell: the basic unit of life ....................................................................................... 1 History of the Wnt pathway ......................................................................................... 2 The current model of the Wnt pathway ....................................................................... 5 Surface receptor activation.......................................................................................... 6 Inhibition of the -catenin destruction complex ......................................................... 18 Activation of a Wnt-specific nuclear transcriptional complex ..................................... 28 The scaffold protein APC .......................................................................................... 32 The interaction between cells and their extracellular environment ............................ 35 Regulation of the Wnt pathway by endocytosis ......................................................... 43 II. MATERIALS AND METHODS .................................................................................. 47 Cell lines.................................................................................................................... 47 Drosophila RNAi lines, Gal4 driver, and progenitor cell reporter ............................... 47 Transfections ............................................................................................................. 48 Immunoblots and immunoprecipitation ...................................................................... 48 Generation of the mAb7E5 antibody ......................................................................... 49 Generation of RKO APCKO ........................................................................................ 49 Generation of stable cell lines ................................................................................... 50 Sucrose density gradients ......................................................................................... 50 Endocytosis assays ................................................................................................... 51 Microscopy ................................................................................................................ 51 Live cell imaging ........................................................................................................ 52 Line scan analysis ..................................................................................................... 53 Drosophila RNAi experiments ................................................................................... 53 vi Drosophila immunohistochemistry ............................................................................ 54 Auxin-dependent degradation assay ......................................................................... 55 Reporter assay .......................................................................................................... 55 RNA isolation and qRT-PCR ..................................................................................... 56 Statistical analysis ..................................................................................................... 56 Colocalization analysis .............................................................................................. 57 III.INHIBITION OF THE WNT PATHWAY BY A MONOCLONAL ANTIBODY AGAINST THE CO-RECEPTOR LRP6 .......................................................................................... 58 Introduction ............................................................................................................... 58 Results ...................................................................................................................... 59 Development of an antibody that inhibits LRP6 ......................................................... 59 mAb7E5 inhibits ligand-mediated Wnt activation ...................................................... 60 mAb7E5 binds specifically to LRP6 ........................................................................... 61 mAb7E5 inhibits Wnt signaling in breast cancer cells ............................................... 69 Discussion ................................................................................................................. 72 IV. APC INHIBITS LIGAND-INDEPENDENT WNT SIGNALING BY THE CLATHRIN ENDOCYTIC PATHWAY .............................................................................................. 75 Introduction ............................................................................................................... 75 Results ...................................................................................................................... 76 LRP6 is required for Wnt pathway activation in APC mutant cells but not in cells with mutant -catenin ......................................................................................................
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