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Mechanisms of BAR Protein Function in Brush Border Assembly by Meagan Marie Postema Dissertation Submitted to the Faculty Of

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Mechanisms of BAR Protein Function in Brush Border Assembly by Meagan Marie Postema Dissertation Submitted to the Faculty Of Mechanisms of BAR Protein Function in Brush Border Assembly By Meagan Marie Postema 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 September 30, 2019 Nashville, TN Approved: Kathleen L. Gould, Ph.D. Irina Kaverina, Ph.D. Robert J. Coffey, M.D. Matthew J. Tyska, Ph.D. For Izzy and Teddy ii ACKNOWLEDGEMENTS First and foremost, I would like to thank my wonderful mentor, Matt Tyska, for the tremendous amount of support and guidance he has provided me. Because of the countless hours Matt has spent helping me improve my writing and presentation skills and discussing my project, I am a better writer, public speaker, and critical thinker. Matt is a tremendous teacher, and his passion and enthusiasm for science is contagious; it has been a privilege to have trained with such an amazing scientist and mentor. I am also thankful for my supportive and encouraging committee members, Drs. Kathy Gould, Bob Coffey, and Irina Kaverina. They have always been accessible when I need them and have provided much needed advice, guidance and recommendation letters. Furthermore, I must acknowledge the Vanderbilt Microtubules and Motors Club and Epithelial Biology Center for thoughtful discussions on my research. As well as the Nikon Center of Excellence and the Electron Microscopy Core, especially Evan Krystofiak who has helped me perfect my SEM technique and provided guidance on KO tissue experiments. I also thank the CDB staff members, especially Susan and Kristi, who work diligently to keep our department running and are always available when needed. I must also acknowledge Tyska lab members, as I am beyond fortunate to have worked with such wonderful and amazing people. Past lab members Nathan and Meredith were exceptionally helpful mentors and provided the guidance I needed to excel with my project. Moreover, my experience as a graduate student would not have been the same without the current lab members Suli, Caroline, Bella, Leslie, Angelo, Colbie and Gill. I will forever be grateful for all the amazing conversations, lunches, and coffee breaks I’ve had with my lab mates; they provided many necessary distractions, as well as incredible iii support over the last few years. I am confident that the friends I have made in the lab will be friends for life, and I look forward to seeing their many future successes. I must acknowledge my friends in the department and IGP program who have provided an enormous amount of support and love over the last five years. Also, my “murderino” group who have become lifelong friends and have immensely enriched my time living in Nashville. Finally, I must thank my family for the love and support they have provided, without them I would not be where I am today. My parents have always been there for me and I’m grateful for the number of times they have come to Nashville to visit me over the last few years. Additionally, I need to thank my younger sister, Melanie, my beautiful and bright nieces, Grace and Caroline, and my older sister, Nichole, who have all been extremely supportive through this entire process and I am continually grateful for their love and understanding. iv TABLE OF CONTENTS Page DEDICATION ................................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................... iii TABLE OF CONTENTS ................................................................................................. v LIST OF FIGURES .......................................................................................................... viii LIST OF ABBREVIATIONS ............................................................................................. x Chapter I. INTRODUCTION ...................................................................................................... 1 Polarized epithelial cells ........................................................................................... 1 Epithelial cell classifications ................................................................................. 2 Apical surface specializations ............................................................................. 4 Enterocytes as model to study apical specializations ........................................ 6 Mechanisms of microvillar protrusions ........................................................... 8 Actin Assembly ........................................................................................................ 9 Nucleating proteins ............................................................................................... 10 Capping protein and EPS8 ................................................................................... 12 Bundling proteins .................................................................................................. 13 BAR domain-containing proteins ......................................................................... 14 I-BAR domain-containing proteins.................................................................. 16 F-BAR domain-containing proteins ................................................................ 18 Membrane binding and higher order assemblies of BAR domains ............... 19 Auxiliary domains and regulatory mechanisms of BAR proteins ................... 22 BAR domain curvature sensing vs. curvature generation ............................. 24 Cellular roles for BAR domain proteins .................................................................... 26 Cell migration ........................................................................................................ 27 Endocytosis .......................................................................................................... 29 Contractile ring assembly ..................................................................................... 31 EHEC colonization................................................................................................ 32 Summary ................................................................................................................... 35 II. MATERIALS AND METHODS ................................................................................ 37 v Cell culture and organoids ....................................................................................... 37 Transfections and lentivirus production ................................................................... 37 Cloning and constructs ............................................................................................. 38 Immunofluorescence ................................................................................................ 40 Frozen tissue preparation ........................................................................................ 42 Western blot analysis ............................................................................................... 43 Pulldown assays ....................................................................................................... 44 Light microscopy ....................................................................................................... 44 Electron microscopy ................................................................................................ 45 Image analysis .......................................................................................................... 46 Statistical analysis ................................................................................................... 47 Animal studies ......................................................................................................... 47 III. IRTKS ELONGATES EPITHELIAL MICROVILLI USING EPS8-DEPENDENT AND INDEPENDENT MECHANISMS ................................................................. 48 Summary ................................................................................................................... 48 Introduction ............................................................................................................... 49 Results ...................................................................................................................... 52 IRTKS localizes to the tips of microvilli in differentiating epithelial cells ............. 52 IRTKS tracks the distal tips of growing microvilli ................................................. 53 IRTKS tip targeting is driven by the N-terminal I-BAR domain ........................... 57 IRTKS promotes microvillar elongation using its SH3 and WH2 domains ......... 57 EPS8 colocalizes with IRTKS and interacts with its SH3 domain ...................... 60 IRTKS promotes EPS8 enrichment at the tips of microvilli ................................. 61 EPS8 promotes microvillar elongation ................................................................ 63 IRTKS uses EPS8-dependent and independent mechanisms to elongate microvilli ............................................................................................................ 66 Discussion ................................................................................................................. 72 Conclusion ................................................................................................................ 76 IV. PACSIN2-DEPENDENT APICAL ENDOCYTOSIS REGULATES
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