INVESTIGATING FACTORS THAT REGULATE THE DIRECT DRP1-MFF INTERACTION by RYAN W CLINTON Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Advisor: Jason A Mears, Ph.D. Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY August 2018 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of Ryan W Clinton Candidate for the Doctor of Philosophy Degree (Thesis Advisor) Jason A. Mears, Ph.D. (Committee Chair) Philip Kiser, Ph.D. (Committee Member) Rajesh Ramachandran, Ph.D. (Committee Member) Derek Taylor, Ph.D. (Committee Member) Edward W. Yu, Ph.D. Date of Defense May 31st, 2018 *We also certify that written approval has been obtained for any proprietary material contained therein. ii DEDICATION This work is dedicated to pursuing curiosity, seeking new knowledge, to the wonderful friends that I’ve made during my time in Cleveland, and to my ever-supportive family. iii TABLE OF CONTENTS DEDICATION iii LIST OF TABLES viii LIST OF FIGURES ix ACKNOWLEDGEMENTS xii LIST OF ABBREVIATIONS xiii ABSTRACT 1 Chapter 1: Introduction 3 1.1 Mitochondrial membrane architecture and organization 4 1.2 Functions of the mitochondrial membranes 6 1.3 Mitochondrial dynamics 8 1.4 Mitochondrial autophagy 10 1.5 Mitochondria in apoptotic signaling 11 1.6 The dynamin family GTPases 12 1.7 Non-mitochondrial dynamin family proteins 14 1.8 Mitochondrial fusion dynamin family proteins 16 1.9 Dynamin-related protein 1 18 1.10 Mitochondrial fission factor 22 1.11 Other Drp1 partner proteins 24 iv 1.12 Post-Translational regulation of Drp1 and Mff 26 1.13 Methods for assessing membrane protein function and assembly in vitro 28 1.14 Foundation and experimental framework 29 Figures and legends 31 Chapter 2: Using Scaffold Liposomes to Reconstitute Lipid-proximal 36 Protein-protein Interactions in vitro 2.1 Abstract 37 2.2 Introduction 37 2.3 Protocol 40 2.3.1 Scaffold liposome preparation 40 2.3.2 Use of scaffold liposomes for protein binding analysis 42 2.3.3 Use of scaffold liposomes for enzymatic assay 44 2.4 Representative results 46 2.5 Discussion 49 2.6 Acknowledgements 52 Figures and Legends 53 v Chapter 3: Dynamin-Related Protein 1 Oligomerization in Solution Impairs 59 Functional Interactions with Membrane-Anchored Mitochondrial Fission Factor 3.1 Abstract 60 3.2 Introduction 61 3.3 Results 64 3.4 Discussion 77 3.5 Materials and Methods 83 3.6 Acknowledgements 90 Figures and Legends 91 Chapter 4: Mff Interacts with the Stalk of Drp1 Via a Novel VD-Occluded 104 Interface to Promote Drp1 Assembly and Membrane Constriction 4.1 Abstract 105 4.2 Introduction 105 4.3 Results 108 4.4 Discussion 124 4.5 Materials and Methods 128 4.6 Acknowledgements 138 vi Figures and Legends 140 Chapter 5: Conclusions and Future Directions 159 5.1 Summary 160 5.2 Recapitulating membrane fission in vitro 162 5.3 Direct effects of Mff post-translational modification on the 166 Drp1-Mff complex 5.4 Structures of a Drp1-Mff complex 167 5.5 Discerning the specific role of Mff in mitochondrial fission 171 5.6 Identifying proteins and lipids in the Mff microenvironment in vivo 174 5.7 Conclusions 175 Figures and Legends 176 References 179 vii LIST OF TABLES Table 4.1: Hydroxyl Radical Footprinting Oxidation Rates of Drp1 Peptides 157 Table 4.2: Hydroxyl Radical Footprinting Oxidation Rates of Drp1 Amino 158 Acid Side Chains viii LIST OF FIGURES Figure 1.1: Mitochondrial membrane architecture and dynamics 31 Figure 1.2: Characteristic domains and structural features of 33 dynamin family proteins Figure 1.3: The mitochondrial fission apparatus differs between 34 yeast and higher eukaryotes Figure 2.1: Lipid preparation schematic 53 Figure 2.2: Methods to assess protein assembly 54 Figure 2.3: Structural assessment of Drp1 recruitment 56 Figure 2.4: Scaffold Liposome Enzymatic assay 57 Figure 3.1: The variable domain (VD) of Drp1 is a 91 negative-regulator of Mff-induced self-assembly Figure 3.2: Coupling of Mff to topology-enforcing liposomes 93 enhances Drp1 stimulation Figure 3.3: The VD is not essential for mitochondrial targeting 95 and subsequent fission in MEF cells Figure 3.4: Mutations that alter the multimeric equilibrium of 97 Drp1 interfere with Mff-induced self-assembly Figure 3.5: Removal of the VD rescues the R376E defect in 99 Mff-induced assembly ix Figure 3.6: Oligomerization of Mff cytosolic domains promotes 101 Mff-induced Drp1 self-assembly Figure 3.7: Mff selectively promotes oligomerization of 103 assembly-competent Drp1 dimers Figure 4.1: Drp1 polymers constrict Mff-decorated 140 liposomes upon addition of GTP Figure 4.2: Mff builds Drp1 polymers on membranes 142 via a stalk interface Figure 4.3: Sequence conservation analysis highlights 144 Drp1 stalk loops as potential Mff-interaction sites Figure 4.4: Mutation of stalk loop 1CS disrupts 146 Drp1-Mff interaction in vitro Figure 4.5: Mutation of L1CS, not L3S disrupts 148 Drp1-Mff interactions Figure 4.6: Drp1TSN functions comparably to Drp1WT 150 Figure 4.7: Drp1TSN is deficient in Mff binding and mitochondrial fission in cells 152 Figure 4.8: Hydroxyl radical footprinting reveals a VD 154 occluded surface on the stalk of Drp1 Figure 4.9: Loop 1CS oxidation is comparable between 156 Drp1WT and Drp1G363D, an assembly-defective dimer mutant Figure 5.1: Mff recruits Drp1 to membranes to form a functional 176 x membrane remodeling copolymer Figure 5.2: AMPK phosphomimetic Mff mutants are deficient in Drp1 177 assembly and stimulation Figure 5.3: Drp1-Mff complexes for cryo-EM study 178 xi ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Jason Mears, for his advice over the years and for helping me to develop and cultivate my ability to critically and logically interpret and assess scientific data. I would also like to thank Dr. Charles Hoppel along with the other members of the Center for Mitochondrial Diseases for the perspective that I gained at our weekly meetings. In particular, I would like to thank Dr. Srinivasan Dasarathy for his advice at one of these meetings which has helped me get the most out of any scientific presentation: don’t be afraid to ask questions if you don’t understand something, or you will just be wasting your time. I would also like to thank all of the members of my thesis committee for giving me valuable advice and perspective on my research project over the years to guide it as it developed. Finally, I would like to acknowledge all members of the Mears lab, Dr. Chris Francy and Dr. Frances Alvarez taught me about Drp1 and mitochondria when I first joined the lab, and I hope that I’ve been able to pass on a similar foundation to the new members of Jason’s lab. I would also like to thank my supervisor at Blue Sky Biotech, Dr. Edward Esposito. Without his guidance so early in my career, and the standards of excellence that he taught me to expect from myself and others, I wouldn’t be sitting here writing this dissertation right now. Finally, I would like to thank my family, especially my wife Jesi who has always inspired me to improve, and who helped me to keep persevering when it felt like my experiments were rebelling against me over the years. xii LIST OF ABBREVIATIONS CC Coiled-coil CL Cardiolipin Cryo-EM Cryo-electron microscopy BSE Bundled signaling element Drp1 Dynamin related Protein 1 ETC Electron Transport Chain EM Electron microscopy GC Galactosyl Ceramide GED GTPase effector domain GMP-PCP β,γ-Methyleneguanosine 5’-triphosphate GTP Guanosine triphosphate GUV Giant unilamellar vesicle HDX Hydrogen-deuterium exchange IMS Intermembrane space MEF Mouse embryonic fibroblast Mff Mitochondrial fission factor xiii Mfn1/2 Mitofusin 1/2 MiD49/51 Mitochondrial dynamics proteins of 49/51 kDa MIM Mitochondrial inner membrane MOM Mitochondrial outer membrane mtDNA Mitochondrial DNA MMP Mitochondrial membrane potential Opa1 Optic atrophy protein 1 PA Phosphatidic acid PC Phosphatidylcholine PE Phosphatidylethanolamine PH Pleckstrin homology PRD Proline-rich domain PS Phosphatidylserine SL Scaffold liposome SUPER Supported bilayers with excess membrane reservoir TM Transmembrane VD Variable domain xiv Investigating Factors that Regulate the Direct Drp1-Mff Interaction Abstract by RYAN W CLINTON The complex processes of mitochondrial fission and fusion are opposing functions whose proper balance ensures optimal mitochondrial function in eukaryotic cells. Aberrant mitochondrial morphology where one of these mitochondria-shaping processes dominates the other are commonly found in diverse pathologies, highlighting the importance of maintaining appropriate rates of both fission and fusion. Both of these competing processes are mediated by members of the dynamin superfamily of membrane-remodeling GTPases. Scission of the mitochondrial membranes is carried out by an ancient member of this protein superfamily called dynamin-related protein 1 (Drp1). While its function is required for fission, Drp1 alone is unable to mediate this complex process, and requires interaction with one or more partner proteins of the mitochondrial outer membrane to ensure fission. Chordates express several such proteins whose genetic interaction with Drp1 has been proven to be crucial for maintenance of appropriate mitochondrial morphology. These include mitochondrial fission protein 1 (Fis1), mitochondrial fission factor (Mff), and mitochondrial dynamics proteins of 49 and 51 kilodaltons (MiD 49/51). Of these proteins, the first that was proposed to contribute significantly to the maintenance of mitochondrial morphology in man was Mff. Due to its relatively recent discovery, its specific role(s) in this function remain unclear. To address this lack of knowledge, the primary objective of these studies was to better understand the various factors that control the association of Drp1 and Mff, and to shed light on the regulatory mechanisms that underlie this interaction.