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Membrane Microdomains As Platforms for Extra-Cellular Fusions

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Membrane Microdomains As Platforms for Extra-Cellular Fusions Loyola University Chicago Loyola eCommons Dissertations Theses and Dissertations 2019 Membrane Microdomains as Platforms for Extra-Cellular Fusions Michael Hantak Follow this and additional works at: https://ecommons.luc.edu/luc_diss Part of the Virology Commons Recommended Citation Hantak, Michael, "Membrane Microdomains as Platforms for Extra-Cellular Fusions" (2019). Dissertations. 3337. https://ecommons.luc.edu/luc_diss/3337 This Dissertation is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion in Dissertations by an authorized administrator of Loyola eCommons. For more information, please contact [email protected]. This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License. Copyright © 2019 Michael Hantak LOYOLA UNIVERSITY CHICAGO MEMBRANE MICRODOMAINS AS PLATFORMS FOR EXTRA-CELLULAR FUSIONS A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL IN CANDIDACY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY PROGRAM IN MICROBIOLOGY AND IMMUNOLOGY BY MICHAEL HANTAK CHICAGO, ILLINOIS MAY 2019 Copyright by Michael Hantak, 2019 All rights reserved. ACKNOWLEDGEMENTS I would like to thank my parents, my brother Joey, and my partner Matt, for their unwavering support and encouragement to pursue my dreams. I also owe a tremendous amount of gratitude to my mentor, Dr. Tom Gallagher, for the opportunity to learn in his lab. Tom’s enthusiasm for science has been truly contagious, and his expertise and feedback have made me become a better scientist, communicator, and educator. I would also like to thank my lab mates Dr. Jung-Eun Park, Dr. James Earnest, Arlene Barlan, Enya Qing, Natalie Nidetz, Dr. Mayukh Sarkar, and Ryan Sweeney. Their technical expertise was essential to my success in the lab, and my work has been shaped for the better by their creativity and curiosity. I would also like to thank Drs. Stan Perlman, Paul McCray, Kun Li, Keith Jones, and Lauren Haar for their collaboration with my project. I am very grateful for my experiences working in the labs of Dr. Brian J. Wilkinson (Illinois State University-Normal, IL) and Dr. Devkumar Mustafi (University of Chicago- Chicago, IL). Dr. Wilkinson and Dr. Mustafi introduced me to laboratory science, and their mentorship has been an instrumental part of my training. Furthermore, I would like to thank my dissertation committee, Drs. Ed Campbell, Francis Alonzo, Seth Robia, and Susan Uprichard, for their continued feedback and generosity with their time. I would also like to thank the staff, students and faculty in the Department of Microbiology and Immunology and across campus for creating a collaborative learning environment that laid the foundation for my success. iii Finally, this work would not be possible without: funding support from the T32 Immunology Training Grant to Dr. Knight; generous donations from the FALK Foundation and the Doyle Fund; a Fellowship from the Arthur J. Schmitt Foundation; and the expertise of Pat Simms and Ashley Hess at the flow cytometry core. TABLE OF CONTENTS ACKNOWLEDGEMENTS .................................................................................... iii LIST OF FIGURES ............................................................................................. viii CHAPTER ONE: REVIEW OF THE LITERATURE 1 Extra-Cellular Membrane Fusion ........................................................................1 Steps in Membrane Fusion .................................................................................2 Viral Membrane Fusion ......................................................................................3 Coronavirus Membrane Fusion .......................................................................4 CoV Entry Routes ............................................................................................5 Extracellular Vesicles .........................................................................................8 EVs in Intercellular Communication .................................................................9 Extracellular Vesicle Heterogeneity ............................................................... 10 Extracellular Vesicle Cargo Delivery and Membrane Fusion ......................... 10 Cellular Membrane Fusion ............................................................................... 12 Monocyte Membrane Fusion ......................................................................... 13 Cellular Membrane Fusion Catalysts ............................................................. 14 Facilitators of Membrane Fusion ...................................................................... 16 Tetraspanins ..................................................................................................... 17 Tetraspanin-Enriched Microdomains ............................................................. 19 Tetraspanins in Virus Entry ........................................................................... 20 Tetraspanins in Non-Viral Membrane Fusion ................................................ 20 Inhibitors of Membrane Fusion ......................................................................... 21 Interferon-Induced Transmembrane Proteins ................................................... 22 IFITM Activities during Enveloped Virus Entry ............................................... 23 Non-Viral IFITM Activities .............................................................................. 25 Purpose of Dissertation .................................................................................... 26 CHAPTER TWO: MATERIALS AND METHODS ................................................ 29 Cells and Reagents .......................................................................................... 29 Plasmids and Cloning ....................................................................................... 30 Transfections .................................................................................................... 31 Generation of CD9 Knockout Cells ................................................................... 32 Pseudoviruses .................................................................................................. 32 Isolation of Tetraspanin-Enriched Membranes ................................................. 33 Recombinant Adenovirus Production ............................................................... 34 Transduction and Infection of Mice ................................................................... 34 EV Isolation ...................................................................................................... 35 Transmission Electron Microscopy ................................................................... 35 Western Blot Analyses ..................................................................................... 35 Quantifying EV Concentration .......................................................................... 36 Quantifying EV Cargo Secretion by Luciferase Assay ...................................... 37 Quantifying Intercellular Transfer of DSPs ....................................................... 37 vi Monitoring Intercellular Transfer of DSPs by Fluorescence Microscopy........... 38 Analysis of EV-Cell Binding .............................................................................. 39 Production of IFITM3 KO Cells ......................................................................... 39 Quantifying IFNβ-Mediated Blockade of EV Cargo Transfer ............................ 40 In Vitro EV-Virus and EV-EV Fusion Assays .................................................... 40 Fluorescence Microscopic Analyses of EVs ..................................................... 41 Production of ifitm5-Encoding Lentiviral Particles and Cell Lines ..................... 42 Immunofluorescence Microscopy of RAW Cells ............................................... 42 Osteoclast Formation Assay ............................................................................. 43 Osteoclast Differentiation Assay ....................................................................... 43 Viral Syncytial Formation Assay ....................................................................... 43 Statistical Analysis ............................................................................................ 44 CHAPTER THREE: RESULTS ........................................................................... 45 SECTION 1: TEMs as Platforms for Virus-Cell Membrane Fusion ................... 45 The Tetraspanin CD9 Facilitates MERS-CoV Entry ...................................... 45 CD9 Holds DPP4 in TEMs ............................................................................. 47 CD9 Brings DPP4 and TMPRSS2 into Close Proximity ................................ 48 CD9 and TMPRSS2 Are Critical for MERS-CoV Infection in vivo .................. 50 SECTION 2: TEMs as Platforms for Extracellular Vesicle Membrane Fusions 53 Membrane Fusion Drives EV Content Transfer ............................................. 53 Tetraspanins Promote EV Release and Enable Cargo Transfer ................... 57 Tetraspanins Act at the Level of EV Cargo Delivery to Enable EV Transfers 61 Additional Enablers of EV-mediated Cargo Transfer ..................................... 62 Type I Interferons Block EV Cargo Transfer through IFITM3 ........................ 64 IFITM3 Blocks EV Cargo Transfer at the Level of Membrane Fusion............ 66 IFITM3
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