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Graf1 Regulates Myo6 Dependent Mitochondrial Actin Remodeling

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Graf1 Regulates Myo6 Dependent Mitochondrial Actin Remodeling GRAF1 REGULATES MYO6 DEPENDENT MITOCHONDRIAL ACTIN REMODELING Zachary Opheim A thesis submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Master of Science in the Department of Pathology and Laboratory Medicine in the School of Medicine. Chapel Hill 2019 Approved by: Joan Taylor Chris Mack Jon Homeister © 2019 ZACHARY OPHEIM ALL RIGHTS RESERVED ii ABSTRACT Zachary Opheim: GRAF1 Regulates MYO6 Dependent Mitochondrial Actin Remodeling (Under the direction of Joan Taylor) Cardiomyocytes are long lived cells that require a constant supply of ATP generated by mitochondria. Mitochondrial dysfunction results in various diseases, highlighting the importance of mitochondrial quality control. Here we demonstrate that GRAF1 regulates mitochondrial quality via interaction with MYO6, a known regulator of mitophagy. Recent studies revealed MYO6 promotes the formation of actin “cages” around damaged mitochondria in response to stress. We show GRAF1 and MYO6 co- localize to depolarized mitochondria. Additionally, we reveal GRAF1 and MYO6 interact and is dependent upon actin polymerization. Knockdown of GRAF1 results in clustered mitochondrial network morphology following mitochondrial depolarization and subsequent recovery. Furthermore, we observe a lack of MYO6 dissociation from mitochondria following knockdown of GRAF1. In addition, GRAF1 was found to promote mitochondrial function in growth conditions requiring mitochondrial dependent oxidative phosphorylation. Our novel findings suggest that GRAF1 regulates the dissociation of actin cages around damaged mitochondria to promote their eventual degradation. iii TABLE OF CONTENTS LIST OF FIGURES……………………………………………………………………………..vi LIST OF ABBREVIATIONS…………………………………………………………………...vii CHAPTER 1: INTRODUCTION………………………………………………………………..1 1.1 Autophagy……………………………………………………………………………1 1.2 Mitochondrial control pathways……………………………………………………3 1.3 PINK1/Parkin-mediated mitophagy…………………………………………….....4 1.4 Mitochondrial dynamics and mitophagy……………………………………….....5 1.5 Mitophagy and the cardiovascular system……………………………………….7 1.6 RhoA and actin dynamics……………………………………………………….....8 1.7 Actin, myosins, and autophagy……………………………………………………9 1.8 MYO6……………………………………………………………………………….10 1.9 GRAF1……………………………………………………………………………...11 CHAPTER 2: RESULTS………………………………………………………………………13 2.1 GRAF1 regulates cardiomyocyte viability and forms rings around mitochondria……………………………………………………………………………13 2.2 GRAF1 colocalizes and interacts with MYO6………………………………….13 iv 2.3 GRAF1 restores mitochondrial morphology following mitochondrial insult…15 2.4 GRAF1 regulates MYO6 dissociation from mitochondria……………………..16 2.5 GRAF1 regulates mitochondrial function during OXPHOS…………………...17 CHAPTER 3: DISCUSSION………………………………………………………………….18 CHAPTER 4: METHODS……………………………………………………………………..32 REFERENCES…………………………………………………………………………………36 v LIST OF FIGURES Figure 1 - Mitochondrial Control Pathways…………………………………………………23 Figure 2 - GRAF1 regulates cardiomyocyte cell viability and forms rings around mitochondria……………………………………………………………………………………24 Figure 3 - MYO6 colocalizes with GRAF1 on Parkin positive structures………………..25 Figure 4 - GRAF1 interacts with MYO6 and is dependent on actin polymerization…….26 Figure 5 - GRAF1 restores mitochondrial morphology following CCCP treatment……..27 Figure 6 - GRAF1 restores mitochondrial morphology in cardiomyocytes………………28 Figure 7 - GRAF1 regulates MYO6 dissociation from mitochondria……………………..29 Figure 8 - GRAF1 regulates mitochondrial function………………………………………..30 Figure 9 - Schematic model of GRAF1 and MYO6 dependent actin cages…………….31 vi LIST OF ABBREVIATIONS ATG Autophagy related protein CCCP Carbonyl m-chlorophenyl hydrazine DFCP-1 Double FYVE domain containing protein 1 DMSO Dimethyl sulfoxide DRP-1 Dynamin related protein 1 EAD Endosome assisted degradation ER Endoplasmic reticulum ESCRT Endosomal sorting complexes required for transport FIS1 Mitochondrial fission protein 1 GAP GTP-ase activating protein GEF Guanine exchange factor I/R Ischemia reperfusion LC3 Microtubule associated protein 1 light chain 3 LIR LC3 interacting motif MDV Mitochondrial derived vesicle MFN Mitofusin vii MI Myocardial infarction MYO1C Myosin IC MYO6 Myosin VI NMM2A Non-muscle myosin IIA NRCM Neonatal rat cardiomyocyte OMM Outer mitochondrial membrane OPA1 Optic atrophy protein 1 OXPHOS Oxidative phosphorylation PDH Pyruvate dehydrogenase PINK1 PTEN induced kinase 1 PI3K Phosphatidylinositol 3-kinase sIR Simulated ischemia reperfusion UBD Ubiquitin binding domain ULK1 Unc51-like autophagy activating kinase viii CHAPTER 1: INTRODUCTION Heart failure is a leading cause of death in the United states and accounts for 610,000 deaths per year1. Half of the deaths from heart failure are attributed to myocardial infarction (MI). Damage from MI results in cell death of irreplaceable cardiomyocytes due to ischemia from blockage of the coronary blood supply. Cardiomyocytes constantly work to supply the body with oxygen and nutrients and are one of the most metabolically active cell types. Furthermore, cardiomyocytes rely on oxygen-dependent ATP production from mitochondria as their main source of energy. Upon ischemia, nutrients and oxygen supply are blocked to cardiomyocytes. Ischemic cardiomyocytes undergo a rapid decline of intracellular ATP concentration and increased intracellular calcium concentration, resulting in mitochondrial dysfunction and depolarization2. These findings highlight the importance of mitochondrial function in vulnerability to and recovery from MI and other cardiovascular diseases. Therefore, understanding the mechanism regulating mitochondrial health and function may allow for promising therapeutic targets to provide cardioprotection in the setting of MI or ischemia/reperfusion injury (I/R). 1.1 Autophagy Autophagy is a conserved intracellular degradation/recycling system fundamental for cellular homeostasis and metabolism. Autophagy is important for cell survival during nutrient stress as degraded amino and fatty acids recycled from cellular constituents such as organelles and macromolecules by autophagy can be used to generate ATP 1 and other necessary cellular components3. Autophagy is activated by a multitude of stimuli, including nutrient deprivation, hypoxia, oxidative stress, and protein aggregates. Initiation and various steps of autophagy are regulated by autophagy-related (ATG) proteins in a sequential manner. Autophagosome biogenesis begins with the activation of the unc51-like autophagy activating kinase 1 (ULK1) comlplex. Once activated, the ULK complex phosphorylates the class-III phosphatidylinositol 3-kinase (PI3K) complex 4,5. The PI3K complex is responsible for generating phostatidylinositol 3-phosphate on emerging membrane sources termed omegasomes, which are marked by double FYVE domain- containing protein (DFCP1). Omegasomes are cupped shaped membrane extensions from the endoplasmic reticulum (ER) that mark the site of autophagosome formation4. The Atg8-phosphatidylethanolamine ubiquitin pathway is responsible for the lipidation of microtubule-associated protein 1 light chain 3 (LC3), a critical component of the autophagy machinery. Further downstream, the C-terminus of LC3 is cleaved, generating LC3-I which is further modified by conjugation with phosphatidylethanolamine to form LC3-II. Once processed, LC3-II is localizes to both membranes of the autophagosome and facilitates linkage of the autophagic machinery with the expanding autophagosome4. Finally, the autophagosome fuses with the lysosme where cargo is degraded. Autophagy was initially described as a non-specific, bulk degradative process, however recent studies indicate selective forms of autophagy that specifially target protein aggregates, bacteria, or organelles such as the ER, peroxisomes and mitochondria for degradation6,7. Mitochondrial autophagy, or mitophagy is the selective 2 degradation of damaged mitochondria and is a critical quality control mechanism, especially for highly metabolic and post-mitotic cells such as cardiomyocytes. 1.2 Mitochondrial control pathways To ensure proper mitochondrial homeostasis and function, cells utilize various pathways to regulate mitochondrial quality depending on the extent of mitochondrial insult. Several “first-line” of defense mechanisms exist to maintain mitochondrial function before complete degradation of the entire mitochondria ensues. Upon low levels of oxidative stress, mitochondrial derived vesicles (MDV) are recruited to discrete sites on the mitochondria (Fig. 1A) in a PTEN Induced Kinase I (PINK1) dependent manner, however this mechanism occurs at a more localized level than PINK1/Parkin- mediated mitophagy (described in detail below)8. Experimental evidence suggests any oxidized mitochondrial proteins can serve as cargo for MDVs, however the main targets are VDAC (membrane pore protein), core2 (complex III subunit) and the matrix protein pyruvate dehydrogenase (PDH)9. Finally, MDVs deliver oxidized cargo to be degraded in the lysosome or peroxisome depending on cargo content10. Another mitochondrial quality control pathway termed “piecemeal mitophagy” targets specific regions of damaged mitochondria (Fig. 1B). Localized damaged regions of individual mitochondria are targeted by ER-specific structures enriched with the omegasome marker DFCP-111. The omegasome contact sites serve as a recruitment signal for LC3 which in turn leads to autophagosomal engulfment along oxidized portions of mitochondria11.
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