REGULATORY MECHANISMS OF SEMAPHORIN/PLEXIN/MICAL-MEDIATED F-ACTIN DISASSEMBLY AND CELLULAR REMODELING APPROVED BY SUPERVISORY COMMITTEE Jonathan Terman, Ph.D. (Advisor) Jane Johnson, Ph.D. (Chair) Neal Alto, Ph.D. Helmut Kramer, Ph.D. DEDICATION I dedicate this dissertation to all the people who have guided me through this process and who have been instrumental in my success. Specifically, I am appreciative of my mentor, Dr. Jonathan Terman, who encouraged me at every step and helped me become a critical scientific thinker. His guidance on my dissertation research has been invaluable to me. I would also like to express my deep gratitude to my committee members: Dr. Jane Johnson, Dr. Neal Alto, and Dr. Helmut Kramer for their useful suggestions over the years which have helped direct and shape my research. Furthermore, I am incredibly thankful for my wonderful colleagues in the Terman lab over the years – Chris Spaeth, Laura Alto, Gias Ahmed, Jimok Yoon, Gizem Yesilyurt, Heng Wu, Ruei-Jiun Hung, and Taehong Yang – who have always been willing to listen to my ideas and discuss them with me, explain concepts or teach techniques to me, or help me in any way. I truly could not ask for better people to work with every day. I am also extremely grateful for my family - for my parents, Steve and Vicky, and my sister, Sarah, who have always supported me and believed in me. Finally, I am especially grateful for my husband, Ryan, who has been a source of endless support, encouragement, love, and inspiration, and for my daughter, Corinne, who has brought me so much joy and happiness. I cannot thank you enough for all you have done to ensure my success. REGULATORY MECHANISMS OF SEMAPHORIN/PLEXIN/MICAL-MEDIATED F-ACTIN DISASSEMBLY AND CELLULAR REMODELING by SHANNON KAY GOOD RICH DISSERTATION Presented to the Faculty of the Graduate School of Biomedical Sciences The University of Texas Southwestern Medical Center at Dallas In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY The University of Texas Southwestern Medical Center at Dallas Dallas, Texas May, 2017 Copyright by Shannon Kay Good Rich, 2017 All Rights Reserved REGULATORY MECHANISMS OF SEMAPHORIN/PLEXIN/MICAL-MEDIATED F-ACTIN DISASSEMBLY AND CELLULAR REMODELING Publication No. Shannon Kay Good Rich The University of Texas Southwestern Medical Center at Dallas, 2017 Supervising Professor: Jonathan R. Terman, Ph.D. Dynamic changes to the actin cytoskeleton modify the shape of cells and their membranous extensions, and underlie diverse developmental and functional events in multiple tissues including migration, navigation, and connectivity. Semaphorins, together with their Plexin receptors, are a large family of extracellular cues that trigger complex cytoskeletal rearrangements to direct these cellular phenomena, but the mechanisms regulating their effects are poorly understood. Emerging evidence identifies Mical, a conserved oxidoreductase (Redox) enzyme, as a critical component in Semaphorin/Plexin signaling through its post-translational oxidation of F-actin, which promotes actin instability and disassembly. How this Mical-mediated redox regulation of actin dynamics is locally v positioned and coordinated with the activity of other actin regulatory proteins to achieve specific, targeted effects on the cytoskeleton remains unknown. Therefore, as a part of my dissertation research, I used a genetic assay to begin to address these questions and search for proteins that could alter Semaphorin/Plexin/Mical signaling effects on the cytoskeleton. In this dissertation, I present my discovery of a functional interplay between Mical and two critical new interactors – cofilin, a well-known ubiquitous F-actin regulatory protein, and Sisyphus, an unconventional class XV myosin. With regards to cofilin, my in vivo genetic/functional assays reveal that cofilin activity is required for and enhances Semaphorin/Plexin/Mical-dependent cytoskeletal rearrangements and morphological changes. Additionally, in vitro biochemical assays demonstrate that cofilin preferentially binds Mical-oxidized actin and accelerates its disassembly. Together, these findings indicate that cofilin and Mical act as a functional pair in both neuronal and non-neuronal cells to rapidly and efficiently disassemble actin filaments. Similarly, my results reveal that Sisyphus is necessary and sufficient for triggering Semaphorin/Plexin/Mical-dependent F-actin disassembly/cellular remodeling. Moreover, using in vivo functional assays, I find that Sisyphus uses its myosin motor activity and the first MyTH4 domain of its C-terminal tail region to modify the subcellular localization of Mical. In this way, Sisyphus spatially controls Mical-dependent F-actin disassembly/cellular remodeling. Therefore, both cofilin and Sisyphus function to promote Mical-mediated F-actin disassembly; thereby, they act as critical regulators of Semaphorin/Plexin/Mical-mediated effects on cytoskeletal and morphological dynamics. Thus, my findings unveil novel molecular and biochemical mechanisms that orchestrate cellular, developmental, and neural biology. vi TABLE OF CONTENTS PRIOR PUBLICATIONS ..................................................................................................... ix LIST OF FIGURES ............................................................................................................... x LIST OF TABLES ............................................................................................................... xiii LIST OF DEFINITIONS ..................................................................................................... xiv CHAPTER ONE General Introduction ................................................................................. 1 CHAPTER TWO F-actin dismantling through a Redox-driven synergy between Mical and cofilin ......................................................................................................................... 20 Abstract .......................................................................................................................... 20 Introduction .................................................................................................................... 21 Results ............................................................................................................................ 23 Discussion ...................................................................................................................... 31 Figures ............................................................................................................................ 34 Materials and Methods ................................................................................................... 61 CHAPTER THREE The Class XV Myosin Sisyphus Spatially Targets Mical to Direct Semaphorin/Plexin-mediated Actin Disassembly and Cellular Remodeling ............ 74 Abstract .......................................................................................................................... 74 Introduction .................................................................................................................... 75 Results ............................................................................................................................. 77 Discussion ...................................................................................................................... 88 Figures ............................................................................................................................ 95 Materials and Methods ................................................................................................. 122 vii CHAPTER FOUR Summary and Conclusions................................................................. 133 BIBLIOGRAPHY ............................................................................................................... 140 viii PRIOR PUBLICATIONS Sephton, C.F., Good, S.K., Atkin, S., Dewey, C.M., Mayer, P., Herz, J., and Yu, G. (2010). TDP-43 Is a Developmentally Regulated Protein Essential for Early Embryonic Development. J. Biol. Chem. 285, 6826–6834. Dewey, C.M., Cenik, B., Sephton, C.F., Dries, D.R., Mayer, P., Good, S.K., Johnson, B.A., Herz, J., and Yu, G. (2011). TDP-43 Is Directed to Stress Granules by Sorbitol, a Novel Physiological Osmotic and Oxidative Stressor. Mol. Cell. Biol. 31, 1098–1108. Grintsevich, E.E.*, Yesilyurt, H.G.*, Rich, S.K., Hung, R.-J., Terman, J.R., and Reisler, E. (2016). F-actin dismantling through a redox-driven synergy between Mical and cofilin. Nat Cell Biol 18, 876–885. (*Co-first authors) ix LIST OF FIGURES FIGURE 1.1. Mical-mediated Redox regulation of actin controls Semaphorin/Plexin F-actin Disassembly .................................................................................................. 19 FIGURE 2.1. Mical/F-actin dynamics are modulated by cofilin ........................................ 34 FIGURE 2.2 Cofilin slows F-actin oxidation by Mical but accelerates filament disassembly ........................................................................................................................ 36 FIGURE 2.3 Further characterization of the interaction of Mical and cofilin in modulating F-actin disassembly and the quantification of Mical-oxidized actin .............. 38 FIGURE 2.4 Further characterization of Mical-oxidized actin using a limited proteolysis assay with subtilisin and an antibody directed against the Met-44 residue