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Role and regulation of Gp78 E3 ubiquitin ligase and its ligand autocrine motility factor in mitochondrial… Wang, Peter Tien Chun 2015

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 Role and Regulation of Gp78 E3 Ubiquitin Ligase and its Ligand Autocrine Motility Factor  in Mitochondrial Dynamics and Mitochondria-Endoplasmic Reticulum Association by Peter Tien Chun Wang B.Sc., The University of British Columbia, 2009  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES  (CELL & DEVELOPMENTAL BIOLOGY)  THE UNIVERSITY OF BRITISH COLUMBIA (VANCOUVER)  JULY 2015  © Peter Tien Chun Wang, 2015  ii  Abstract  A ligand-receptor pair, autocrine motility factor (AMF) and Gp78, have been discovered to play multiple roles in mammalian cells. AMF functions as the essential glycolytic enzyme phosphoglucose isomerase in the cytoplasm, but when secreted acts a cytokine that stimulates cell motility, growth and survival. Gp78 serves as the cell surface receptor of AMF, and thus it is also known as AMFR. However, Gp78 can localize to the ER membrane and functions as an E3 ubiquitin ligase in the endoplasmic reticulum associated degradation (ERAD) pathway where it targets a wide variety of proteins for degradation. The concerted actions of AMF and Gp78 contribute to multiple aspects of cancer progression, and thus elevated levels of both proteins have been found in many types of cancers. Recently, it was discovered that AMF and Gp78 alter mitochondrial morphology and ER-mitochondria calcium coupling, processes that are essential in regulating mitochondrial metabolism and apoptosis. Furthermore, Gp78 has also been localized to ER-mitochondria contact sites where it targets the mitochondrial fusion proteins, mitofusin 1 and 2 (Mfn1/2), for degradation. In this dissertation, I show that during Gp78 induced mitophagy, autophagosome marker LC3 is recruited to mitochondria associated ER membrane. Moreover, I show that Gp78-dependent degradation of the mitofusins leads to diminished mitochondrial fusion and a perturbation of mitochondrial dynamics. I also report the ability of AMF to inhibit Gp78-induced mitochondrial fission. In my study of ER-mitochondrial association, I observed two types of ER-mitochondria contacts in HT-1080 fibrosarcoma cells: the rough and the smooth. Gp78 ubiquitin ligase activity selectively promotes rough ER-mitochondria association through the degradation of Mfn2. AMF treatment inhibits Gp78-dependent Mfn2 degradation and decreases rough ER-mitochondria contact sites. By dissecting the functions of AMF and Gp78 at the ER mitochondria contact sites, my thesis not only expands our understanding of the relationship between AMF and Gp78, it also provides novel insights into the intimate connection between the ER and mitochondria.         iii  Preface I.  Published work a). Figure 2 was adapted and published in the journal Molecular Biology of the Cell in 2013 as Figure 6B. Citation: Fu, Min., St-Pierre, P., Shankar, J., Wang, P. T. C., Joshi, B., & Nabi, I. R. (2013). Regulation of mitophagy by the Gp78 E3 ubiquitin ligase. Molecular Biology of the Cell, 24(8), 1153–62. I was credited 4th author in this paper.  b). Figure 3 was adapted and published in the Journal of Cell Science in 2013 as Figure 8. Citation: Shankar, J., Kojic, L. D., St-Pierre, P., Wang, P. T. C., Fu, M., Joshi, B., & Nabi, I. R. (2013). Raft endocytosis of AMF regulates mitochondrial dynamics through Rac1 signaling and the Gp78 ubiquitin ligase. Journal of Cell Science, 126(Pt 15), 3295–304. I was credited 4th author in this paper. c). All figures and results in Chapter 4 were published in the Journal of Cell Science in 2015. Citation: Wang, P. T. C., Garcin, P. O., Fu, M., & Masoudi, M., St-Pierre, P., Panté, N., Nabi, I.R. (2015). Distinct mechanisms controlling rough and smooth endoplasmic reticulum-mitochondria contacts Journal of Cell Science. Journal of Cell Science, (June). I was credited 1st of three co-first authors in this paper.  II.  Collaborations Chapter 4 was done in collaboration with Pierre Garcin of the Pante Lab at UBC. Pierre Garcin fixed, sectioned, prepared and imaged the samples for electron microscopy. Matthew Masoudi assisted me in analyzing a portion of the EM images.  iv  Table of contents  Abstract ........................................................................................................................................................ ii Preface ......................................................................................................................................................... iii Table of contents ......................................................................................................................................... iv List of figures .............................................................................................................................................. vii List of abbreviations .................................................................................................................................. viii Acknowledgements ..................................................................................................................................... ix Chapter 1: Introduction ................................................................................................................................ 1 1.1 AMF and Gp78 .................................................................................................................................... 1 1.1.1 Introduction to AMF/PGI ............................................................................................................. 1 1.1.2 Introduction to AMFR/GP78 ........................................................................................................ 2 1.1.3 AMF as a cytokine ........................................................................................................................ 3 1.1.4 Gp78 as the AMF receptor ........................................................................................................... 4 1.1.5 Gp78 as an E3 ubiquitin ligase ..................................................................................................... 5 1.1.6 Involvement of AMF and Gp78 in cancer progression................................................................. 6 1.2 Endoplasmic reticulum membrane domains .................................................................................... 10 1.2.1 Introduction ............................................................................................................................... 10 1.2.2 Functions of mitochondria associated ER domains ................................................................... 10 1.2.3 ER-mitochondria tethers ............................................................................................................ 11 1.2.4 Gp78 in mitochondrial dynamics and ER association ................................................................ 13 1.3 Mitophagy ......................................................................................................................................... 13 1.3.1 Introduction to mitophagy ......................................................................................................... 13 1.4 Objective of thesis............................................................................................................................. 15 Chapter 2: Gp78 induction of mitophagy .................................................................................................. 16 2.1 Background ....................................................................................................................................... 16 2.1.1 Introduction to autophagy ......................................................................................................... 16 2.1.2 Introduction to mitophagy ......................................................................................................... 17 2.1.3 Potential role of Gp78 in mitophagy .......................................................................................... 17 2.1.4 Objective .................................................................................................................................... 18 2.2 Materials and methods ..................................................................................................................... 19 v  2.3 Results ............................................................................................................................................... 20 2.3.1 LC3 localizes to Gp78-labeled ER ............................................................................................... 20 2.3.2 Gp78 recruits EGFP-LC3 to the mitochondria-associated ER ..................................................... 20 Chapter 3: Regulation of mitochondrial dynamics by AMF through Gp78 .............................................. 24 3.1 Background ....................................................................................................................................... 24 3.1.1 Introduction to mitochondrial dynamics ................................................................................... 24 3.1.2 Ubiquitin ligase Gp78 in ERAD ................................................................................................... 25 3.1.4 AMF/Gp78 and mitochondrial dynamics ................................................................................... 26 3.1.5 Objective .................................................................................................................................... 26 3.2 Materials and methods ..................................................................................................................... 27 3.3 Results ............................................................................................................................................... 28 3.3.1 AMF and Gp78 regulate mitochondrial fusion in HT-1080 cells ................................................ 28 Chapter 4: Regulation of ER-mitochondria association by AMF and Gp78 .............................................. 31 4.1. Background ...................................................................................................................................... 31 4.1.1 Introduction to ER-mitochondria association ............................................................................ 31 4.1.2 Lipid transport at ER-mitochondria contacts ............................................................................. 31 4.1.3 Calcium transfer at ER-mitochondria contact sites ................................................................... 32 4.1.4 Mitochondrial division at ER-mitochondrial contact sites ......................................................... 33 4.1.5 ER-mitochondria tethers ............................................................................................................ 33 4.1.7 Objective .................................................................................................................................... 35 4.2 Materials and methods ..................................................................................................................... 36 4.3 Results ............................................................................................................................................... 38 4.3.1 AMF-Gp78 regulation of ER-mitochondria association ............................................................. 38 4.3.2 Gp78 selectively regulates rough ER-mitochondria contacts .................................................... 39 4.3.3 AMF regulates Gp78-dependent rough ER-mitochondrial contacts ......................................... 39 4.3.4 Mfn1 inhibits smooth ER contacts, while Mfn2 inhibits rough ER contacts .............................. 40 Chapter 5: Discussion ................................................................................................................................. 49 5.1 Discussion .......................................................................................................................................... 49 5.1.1 AMF/Gp78 functions .................................................................................................................. 49 5.1.2 AMF regulation of mitochondrial dynamics through Gp78 ....................................................... 49 5.1.3 Gp78 regulates ER-mitochondria association ............................................................................ 51 5.1.4 AMF and Gp78 regulation of rough ER contacts ........................................................................ 52 vi  5.1.5 Role of Gp78 in mitophagy ........................................................................................................ 53 5.1.6 The ER membrane is a potential source for autophagosomes .................................................. 54 5.1.7 AMF-regulation of Gp78 at ER-mitochondria contact sites ....................................................... 55 5.1.8 Potential contribution of the novel functions of AMF and Gp78 to cancer .............................. 58 Chapter 6: Conclusion ................................................................................................................................ 60 Bibliography................................................................................................................................................ 62          vii  List of figures Figure 1 Schematic of AMF and Gp78 signalling pathways and their roles in cellular functions and cancer progression ....................................................................................................................................... 9 Figure 2 EGFP-LC3 localizes to ER membrane during Gp78-induced mitophagy ..................................... 23 Figure 3 Mitochondrial fusion increases with both Gp78 knockdown and AMF treatment ................... 30 Figure 4 AMF inhibits Gp78 promotion of ER-mitochondria association ................................................. 42 Figure 5 Gp78 regulates rough but not smooth ER-mitochondria contacts ............................................. 44 Figure 6 AMF inhibits Gp78 promotion of rough ER contacts .................................................................. 46 Figure 7 Mfn1 inhibits smooth ER contacts, while Mfn2 inhibits rough ER contacts .............................. 48 Figure 8 Schematic of AMF regulation of Gp78 at ER-mitochondria contact sites .................................. 57         viii  List of abbreviations AMF  Autocrine Motility Factor AMFR  Autocrine Motility Factor Receptor CCCP  Carbonyl cyanide m-chlorophenyl hydrazine DMEM  Dulbecco modified Eagle's minimal essential medium Drp1  Dynamin related protein 1 EMT  Epithelial to mesenchymal transition FBS  Fetal bovine serum Fis1  Fission 1 protein HMG-CoA 3-hydroxy-3-methylglutaryl-coenzyme A IMM  Inner mitochondrial membrane MAM  Mitochondria associated membrane Mfn1  Mitofusin 1 Mfn2  Mitofusin 2 OMM  Outer mitochondrial membrane Opa1  Optic Atrophy protein 1 PBS-cm Phosphate buffer saline with calcium and magnesium chloride PGI  Phosphoglucose Isomerase PINK1  PTEN-induced putative kinase 1 STX17  Syntaxin 17     ix  Acknowledgements  I want to thank all the members of the Nabi Lab at UBC for the immeasurable help they provided me. It was a complete pleasure to work with a wonderful group of supportive friends. I especially want to thank Robert Nabi for his supervision and mentorship. He is an extraordinary supervisor who made me a better student and researcher. Among the lab members, I want to give extra thanks for Min Fu, Joshi Bharat, and Jay Shankar for training me in so many research techniques, and Rei Meng, Guang Guo and Matthew Masoudi for assisting me in many microscope procedures. I would like to thank the faculty and staff members of the Faculty of Cellular and Physiological Sciences at the University of British Columbia for the tremendous support, guidance, and education they provided me during my Master’s degree. Their passion for science and students is an inspiration for a student like me. I am also thankful for the graduate fellowship they offered me. I am very thankful for the great help I’ve received from my committee members, Chris Loewen and Ed Moore.   I want to thank my collaborators for my research projects. Pierre Garcin and Nelly Pante were instrumental in collecting the EM data for my thesis. I also appreciate the collaborations with Barry Young from Loewen Lab at UBC, and Michal Simon at BC Children’s Hospital. Lastly, I want to thank God for giving me the strength and peace through all the good and tough times, for he is good.    1  Chapter 1: Introduction 1.1 AMF and Gp78 AMF and Gp78 are multifunctional proteins that play a significant role in cancer progression. Studies have discovered a plethora of functions for this receptor-ligand pair in cell migration, survival, angiogenesis, and ER stress. Recently, new insights of both proteins reveal surprising connections between AMF, Gp78, and the mitochondria that expand our understanding of mitochondrial processes and ER-mitochondria association.  1.1.1 Introduction to AMF/PGI The cytokine named autocrine motility factor (AMF) was originally identified in a study to isolate a self-producing factor that stimulated cell motility. This 55kda protein promoted random and direct cell motility at concentrations less than 10nM (Liotta et al., 1986). In the same year, a neurotrophic growth factor named neuroleukin (NLK) was also discovered. Neuroleukin promotes survival in a subpopulation of neurons and mediates the differentiation of human myeloid leukemic cells (Gurney et al., 1986). Interestingly, once AMF was isolated and sequenced, it was discovered that AMF and NLK were identical proteins; furthermore, AMF/NLK bore the same sequence as phosphoglucose isomerase (PGI) (Chaput et al., 1988; Watanabe et al., 1996). PGI participates in glycolysis and glycogenesis by interconverting glucose-6-phosphate to fructose-6-phosphate. This enzyme is ubiquitously expressed and is essential for healthy cellular metabolism (Harrison, 1974; Ullrey et al., 1982). As a cytokine, AMF signalling not only stimulates cell motility, it can also act as a growth factor that promotes DNA replication and wound healing (Silletti et al., 1993). The multifunctional protein AMF has since been the focus of many studies for its contribution to cancer, metabolism and many cellular processes. In fact, many patients with gastrointestinal and kidney tumors exhibit abnormal AMF 2  secretions at detectable levels in both serum and urine, and thus AMF can be used as a prognostic marker for cancer progression (Baumann et al., 1990). This occurs even though less than 1 percent of AMF in a cell is secreted through a non-classical pathway that is currently not well understood (Lagana et al., 2000). Upon secretion, AMF binds with its cell surface receptor, the autocrine motility factor receptor (AMFR), also named Gp78.  1.1.2 Introduction to AMFR/GP78 AMFR was first purified in 1987 as a glycoprotein of Mr 78000; hence the name Gp78 (Nabi et al., 1987). In this thesis, AMFR will be referred to as Gp78. On the cell surface, Gp78 acts as a receptor that induces cell motility and proliferation upon AMF binding. However through sequence analysis and experimental studies, Gp78 was also found to exhibit E3 ubiquitin ligase activity (Shimizu et al., 1999; Fang et al. 2001). Gp78 targets a wide range of proteins for degradation including ApoB lipoprotein, HMG-CoA reductase, KAI1, and mitofusins 1 and 2 (St.Pierre et al., 2012; Liang et al., 2006; Song et al., 2005; Tsai et al., 2007; Shankar et al., 2013); thus Gp78 is a key E3 ubiquitin ligase in the endoplasmic reticulum associated degradation pathway (ERAD) (Christianson et al., 2011). Elevated expression of both AMF and Gp78 are linked to metastasis development and poor prognosis of cancer patients (Chui et al., 2008). Overexpression of Gp78 has been associated with the hyperplasia of mouse mammary glands through the degradation of KAI1, a tumour suppressor (Tsai et al., 2007; Joshi et al., 2010). The complex biology of Gp78 and AMF and their role in cancer progression has been extensively studied; however many of their functions, mechanisms of action and physiological roles have yet to be elucidated.  3  1.1.3 AMF as a cytokine Once AMF is secreted from a cell, it acts as an extracellular cytokine that signals in both an autocrine and paracrine manner. The secretion of AMF is not dependent on a signal peptide and does not utilize the classical ER/Golgi secretory pathway (Niinaka et al., 1998). Although the secretion process is not yet understood, overexpression of AMF and phosphorylation of human AMF at serine 185 are both able to induce AMF secretion from non-AMF-secreting cells (Tsutsumi et al., 2003; Haga et al., 2000). Interestingly, carbohydrate phosphate inhibitors of PGI enzymatic activity can also inhibit AMF cytokine activity, which later studies attributed to overlap of the PGI enzymatic active site with the AMF cytokine binding site (Watanabe et al., 1996; Tanaka et al., 2002). Upon interaction with its cell surface receptor Gp78, AMF is internalized through a clathrin independent, dynamin and PI3K-dependent raft-mediated pathway, and transported to the smooth ER (Benlimame et al., 1998; Kojic et al., 2007). AMF stimulated cell motility is triggered through the activation of protein kinase C, and small GTPases RhoA and Rac1. These proteins induce a signalling cascade that activates focal adhesion reorganization and cell migration (Kanbe et al., 1994; Tsutsumi et al., 2002). The story of the multifunctional protein AMF is further complicated by the discovery that in neural cells lacking the surface receptor Gp78, instead of stimulating motility, AMF acts as a maturation and survival factor (Haga et al., 2006). This result suggests AMF acts through GP78 to promote cell motility, but also induces maturation by signalling through a different receptor. Further study reveals AMF binds to a family member of EGF receptor, HER2, triggers its cleavage, and activates PI3K and mitogen-activated protein kinase (MAPK) signalling to promote trastuzumab drug resistance in breast cancer cells (Kho et al., 2012). These studies reveal the complex 4  cytokine function of AMF signals through several pathways to promote processes that drive cancer progression.     1.1.4 Gp78 as the AMF receptor Gene analysis of Gp78 reveals it is a protein of 648 amino acid with an estimated 7 transmembrane domains (Shimizu et al., 1999). The roughly 350aa C-terminal cytoplasmic tail of Gp78 includes several functional domains: a RING domain critical for E3 ligase function, a Cue domain important in the binding of ubiquitin, an E2 ubiquitin-conjugating enzyme binding site, and a P97/VC-binding domain for the retrotranslocation of substrates (Chen et al., 2006; Ballar et al., 2011). Although the domains of Gp78 ubiquitin ligase activity are well understood, the topology of Gp78 and the AMF binding mechanism are currently not clear. A monoclonal antibody called 3F3A that selectively recognizes the dephosphorylated form of Gp78 is often used in the study of Gp78 (Nabi et al., 1990; Lei Li, unpublished data). 3F3A mAb competes with AMF for Gp78 binding and can in fact stimulate cell motility in a similar fashion as AMF (Nabi et al., 1990). Study of 3F3A mAb reveals the mAb binds specifically to a dephosphorylated form of Gp78 at its C-terminal, suggesting the C-terminal of Gp78 is indeed the binding site for AMF to Gp78 (Lei Li, Mol. Biol. Cell under revision). Gp78-mediated AMF internalization to the smooth ER via a PI3K and dynamin-dependent pathway is critical for AMF-induced cell motility (Kojic et al., 2007; Shankar et al., 2013). Although the downstream signalling pathway of AMF-Gp78 are not completely understood, there is evidence that G-protein coupled receptors (GPCRs) are involved due to the observation of the ability of GPCR inhibitor pertussis toxin to inhibit AMF-Gp78 induced motility (Khan et al., 1990). Gp78 is indeed an unique protein that combines the functions of a cell surface receptor and an ER-localized E3 ubiquitin ligase. 5  1.1.5 Gp78 as an E3 ubiquitin ligase ERAD is an essential process for the maintenance ER health by degrading misfolded, excess or non-functional proteins. This key cellular process requires the recognition of target proteins, retrotranslocation from the ER to the cytoplasm if necessary, ubiquitylation, and transport to the proteasome or lysosome for degradation (Meusser et al., 2005). Among these steps, ubiquitylation not only plays an important role in targeting proteins for lysosomal and proteasomal degradation, it can also contribute to DNA repair, transcription and protein trafficking. The tagging of proteins with long chains of ubiquitin is a complex multistep pathway that requires the concerted step-wise actions of E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzymes, E3 ubiquitin ligase, and E4 ubiquitin chain elongation factors (Ande et al., 2009). Gp78 is a key component of the ERAD machinery and is a very well characterized E3 ubiquitin ligase (Fang et al., 2001; Christianson et al., 2011). As an E3 ubiquitin ligase, Gp78 recognizes and binds to its targeted substrate and transfers ubiquitin from an E2 to the substrate. In addition, it has been reported that Gp78 can also exhibit E4 activity, where Gp78 elongates the ubiquitin chain on already ubiquitylated substrates (Morito et al., 2008).  Among Gp78 substrates are HMG-CoA reductase, a critical enzyme involved in cholesterol synthesis, and its cofactor Insig-1. In the presence of low sterol levels, Gp78 targets Insig-1 for degradation, whereas at high sterol levels, Insig-1 binds to HMG-CoA reductase and recruits Gp78 to promote HMG-CoA degradation (Song et al., 2005). However, while there is clear evidence of Gp78 involvement in Insig-1 degradation, support for HMG-CoA as a Gp78 substrate is less substantial (Tsai et al., 2012). Another significant Gp78 substrate with clinical significance is KAI1, a metastasis suppressor that strengthens intercellular adhesion by 6  stabilizing E-cadherin/β-catenin complexes (Protzel et al., 2008; Abe et al., 2008). Excessive degradation of KAI1 by Gp78 led to increased metastatic potential of sarcoma cells; moreover, overexpression of Gp78 in mouse mammary glands promoted cell proliferation and hyperplasia (Tsai et al., 2007; Joshi et al., 2009). All these studies contributes to establishing Gp78 as a key player in ERAD through its ubiquitin ligase activity. 1.1.6 Involvement of AMF and Gp78 in cancer progression Increased expression of AMF and Gp78 has been associated with metastasis, tumorigenesis, and poor prognosis in cancer patients (Chui et al., 2008). In tumorigenic and metastatic breast cancer cell lines, Gp78 surface expression is significantly higher compared to dysplastic mammary epithelial cells (Kojic et al., 2007). AMF has been identified as one of the binding partners of pro-apoptotic IGFBP-3, where AMF inhibits IGFBP-3 induced apoptosis in a dose-dependent manner. On the other hand, IGFBP-3 reciprocates the inhibition by blocking the actions of AMF, potentially contributing to the anti-proliferative and pro-apoptotic effects of IGFBP-3 (Mishra et al., 2004). AMF has also been implicated in angiogenesis, an essential step in tumorigenesis for nutrition and oxygen supply, and removal of metabolic wastes in solid tumours (Funasaka et al., 2001). Upon further study, injection of HT-1080 cells overexpressing AMF into mouse dorsal air sacs promoted angiogenesis via AMF upregulation of both vascular endothelial growth factor (VEGF), a key factor in angiogenesis, and AMF surface receptor Gp78 (Funasaka et al., 2002). These studies suggest that AMF not only stimulates cell motility in an autocrine manner, it can also act in a paracrine manner to promote tumor angiogenesis.  Another key process during cancer progression is epithelial to mesenchymal transition (EMT). Cells undergoing EMT lose their epithelial phenotype after loss of their intercellular 7  junctions and polarity, and subsequently assume a motile mesenchymal phenotype. A hallmark of EMT is the loss of E-cadherin that results in the disruption of cell-cell adhesions (Hanahan et al., 2011). Interestingly, E-cadherin loss is associated with increased Gp78 expression in bladder carcinomas (Otto et al., 1997). In addition, an EMT transformed cell line of MDCK cells by the Moloney Sarcoma Virus is associated with upregulation of Gp78, downregulation of E-cadherin, increased motility, and loss of epithelial phenotype (Simard and Nabi, 1996). Furthermore, ectopic AMF expression induces EMT in normal human breast epithelial cells, whereas AMF knockdown promotes a mesenchymal-to-epithelial transformation (MET), a reversal of EMT, in aggressive human breast cancer cells (Funasaka et al., 2009). With their plethora of contributions to cancer progression, AMF and Gp78 have been subjected to extensive studies; however, there are many aspects of these proteins that we currently do not understand. A schematic of AMF and Gp78 signalling pathway and functions is shown in Figure 1.     8  Figure Legends Figure 1 Schematic of AMF and Gp78 signalling pathways and their roles in cellular functions and cancer progression.   AMF, which normally functions as a phosphoglucose isomerase in the cytoplasm, becomes a cytokine upon secretion from a cell. It signals in an autocrine or paracrine manner by binding to its cell surface receptor Gp78 to trigger cell motility, survival, and growth. Gp78 is not only a cell surface cytokine receptor, but also an E3 ubiquitin ligase that localizes to the ER, where it targets a variety of proteins for degradation as part of the endoplasmic reticulum degradation pathway. Gp78 also localizes to ER-mitochondria contact sites and targets mitochondrial fusion proteins Mfn1 and Mfn2 for degradation. The ubiquitin ligase activity of Gp78 regulates the calcium coupling between the ER and mitochondria.   9   Figure 1 Schematic of AMF and Gp78 signalling pathways and their roles in cellular functions and cancer progression    10  1.2 Endoplasmic reticulum membrane domains 1.2.1 Introduction Studies have revealed that the endoplasmic reticulum is a much more dynamic organelle than researchers previously thought. Far beyond the original concept of smooth and rough ER, new organizational domains, such as sheets and tubules, perinuclear and peripheral ER have been added to the classification (St. Pierre and Nabi, 2012). Each domain is responsible for specific functions that are critical for a cell. For example, perinuclear ER domains are the site of endoplasmic reticulum-associated degradation (ERAD) of misfolded proteins, whereas tubular peripheral smooth ER can interact with microtubules to facilitate transport of vesicles and ER reorganization (English et al., 2009). An expanding area of ER research involves deciphering the intimate interaction and association between the ER and other membrane bound organelles. These close contact sites of the ER with the Golgi, peroxisomes, plasma membrane, and mitochondria facilitates lipid transport, intra-organelle signalling, and many other critical cellular functions (Levine and Loewen, 2006). The importance of one of these ER contact domains, the mitochondria-associated membrane (MAM), is only now being fully appreciated.  1.2.2 Functions of mitochondria associated ER domains Mitochondria have long been known to be in close proximity with the ER, and indeed, direct interactions between these two organelles have been confirmed with electron microscopy (Mannella et al., 1998). The lipid raft-like regions of the ER that are in contact with mitochondria serve to facilitate a wide variety of vital cellular functions (Browman et al., 2006). For example, the ER membrane at these contact sites contains high concentrations of glycosphingolipid synthesizing enzymes, and supports direct lipid transport between the 11  mitochondria and the ER. The production and transport of lipid between the ER and mitochondria is necessary, as mitochondria cannot synthesize certain types of essential membrane lipids (Levine and Loewen, 2006; Prinz et al., 2010; Vance, 2015). An additional critical process that is dependent on ER-mitochondria interaction is calcium coupling between the ER and mitochondria. Ca2+ release from the ER and immediate uptake by mitochondria boosts oxidative phosphorylation and ATP production (Rutter at al., 2000). However, if Ca2+ release and uptake exceeds a threshold level, potentially triggered by cellular/ER stress or hypoxia, mitochondria can induce apoptosis and cell death (Deniaud et al., 2008). In addition, close membrane contacts between the two organelles can also regulate mitochondrial dynamics. Mitochondria fission and fusion events not only help to regulate mitochondria structure and distribution, they are also required for the maintenance of its health. GTPases such as Drp1 facilitate division, whereas Mfn1 and 2 promote fusion (Youle and Bliek, 2012). During mitochondrial fission, the ER membrane has been observed to physically wrap itself around mitochondria to facilitate fission either through the use of ER localized proteins or the physical force of ER itself (Friedman et al., 2011). The importance of the ER MAM domain in mitochondrial metabolism, apoptosis, lipid production and transport, and mitochondrial dynamics has made it an emerging area of study. Yet many of the components involved, signalling pathways, regulation, and biochemistry of ER-mitochondrial interactions are still unknown.  1.2.3 ER-mitochondria tethers Mitochondrial association with the ER has been observed to increase upon ER stress. The increased apposition may perhaps be an adaption of the cell to provide the ER with more 12  ATP to boost ER protein folding (Bravo et al., 2011). The dynamic interactions between ER and mitochondria not only requires tethering proteins for association, but must also be carefully regulated. One MAM regulatory protein is the B-cell receptor associated protein of 31 kDa (BAP31), a transmembrane ER protein that is activated during apoptotic induction (Breckenridge et al., 2003). Upon activation, BAP31 associates with mitochondrial membrane protein Fission-1 homologue (Fis1) and creates an MAM apoptotic platform, which subsequently leads to calcium transfer and apoptosis (Iwasawa et al., 2011). Other elusive ER-mitochondrial tethering components have only recently been identified. One of the first components discovered turned out to be a complex of the voltage dependent anion channel-1 on the mitochondria (VDAC1), mitochondrial membrane chaperone Grp78, and ER calcium release regulator IP3R1 that forms during the onset of cellular and ER stress (Szabadkai et al., 2006). VDAC1 has also been found to localize to the MAM, where it may act as a conduit for ATP transfer from the mitochondria to the ER to aid protein folding (Barmatz et al., 2004 and 2005). Moreover, at ER mitochondria contact sites, VDAC1 is crucial for mitochondria calcium transfer during apoptosis through its interaction with IP3R1 (Stefani et al., 2012).  Another protein called mitofusin 2 (Mfn2) localizes to the mitochondria and the ER, and has been implicated in both mitochondrial fusion and ER-mitochondria association (Koshiba et al., 2004). Mfn2 ablation decreased ER-mitochondrial association when observed through immunofluorescence, and prolonged calcium transfer times between the ER and mitochondria, providing evidence for the role of Mfn2 as an ER-mitochondrial tether (Brito and Scorrano, 2008). However, this observation has been disputed recently by a couple of more recent studies. In one study, upon Mfn2 ablation, increased ER-mitochondrial association was 13  observed through electron microscopy (Cosson et al., 2012). Furthermore, not only was this observed in another study, the increased ER-mitochondria calcium coupling time found in the original study has been attributed to a special adaptation of Mfn2 knockout cell lines instead of an actual loss of ER-mitochondria association (Filadi et al., 2015). The discovery of the many roles of ER-mitochondria association has also led to the identification of many regulators of this process.  1.2.4 Gp78 in mitochondrial dynamics and ER association    EM studies of cultured Hela and MDCK cells show that ER-mitochondria association primarily involves the smooth ER (Wang et al., 2000; Goetz et al., 2007). Interestingly, a subpopulation of Gp78 was localized to mitochondria associated smooth ER in a calcium sensitive manner (Wang et al., 2000). Moreover, Gp78 ubiquitin ligase activity and its regulation by AMF have been implicated in regulating ER-mitochondria calcium coupling (Fu et al., 2011). A recent study also showed the ability of AMF to regulate Gp78-dependent mitochondrial fusion (Shankar et al., 2013). These studies point to Gp78 as a potential regulator of ER-mitochondria association. Deciphering the molecular mechanisms that control these contacts and their functional purposes will provide valuable insights into many human diseases. Indeed, disruption of ER-mitochondria association has been linked to Alzheimer’s disease (Hedskog et al., 2013), type 2 diabetes (Leem and Koh, 2012), and even tumour formation (Pinton et al., 2011; Giorgi et al., 2015).  1.3 Mitophagy 1.3.1 Introduction to mitophagy During mitochondrial oxidative phosphorylation, dangerous waste products such as peroxides, free radicals, and superoxides are produced. The production of these hazardous 14  chemicals is enhanced if respiration is constrained by hypoxic or starved conditions that are prevalent for a tumour cell. The accumulation of these reactive oxygen species can deregulate pH levels in the mitochondria, which results in mitochondrial dysfunction and, in severe cases, induction of cytotoxicity and cell death. To negate the damage, cells activate mitophagy, the autophagic degradation of damaged mitochondria (Kim et al., 2007). Autophagy is a process by which organelles and cytoplasm are sequestered and delivered to lysosomes for degradation. During mitophagy, an isolation membrane forms around the targeted mitochondria and engulf the organelle to produce an autophagosome, which then fuses with lysosomes for degradation and recycling. The origin of the isolation membrane is currently still debated, with some suggesting the source may be the mitochondria membrane itself, the plasma membrane, or the ER membrane (Kim et al., 2007; Cook et al., 2014). Some components known to be involved in mitophagy are Parkin and PINK1. Damaged mitochondria can be recognized by PINK1, a kinase that is able to detect mitochondrial stress. PINK1 subsequently recruits Parkin to the outer mitochondrial membrane (OMM). Parkin, an ubiquitin ligase active on the mitochondrial membrane ubiquitylates OMM proteins and triggers the initiation of mitophagy (Kondapalli et al., 2012). During the onset of mitophagy, LC3, a cytosolic protein commonly used as an autophagic marker, is recruited to the developing isolation membrane to facilitate its closure and complete the formation of the autophagosome (Jin and Youle, 2012). However, Parkin is not expressed in all cell types, and thus the mitophagic process must rely on other currently yet to be identified pathway(s). Interestingly, our lab has shown Gp78 can potentially trigger mitophagy upon mitochondrial damage independent of PINK1 and Parkin (Fu et al., 2013).  15  1.4 Objective of thesis As we expand our knowledge of AMF and Gp78, the roles of these proteins in regulating mitochondrial processes has emerged alongside of accumulating studies of their dysregulation in cancer (Wang et al., 2007; Huang et al., 2014). Gp78 that localizes to ER-mitochondria associated membrane domains targets Mfn1/ 2, mitochondria antiviral signaling protein (MAVs), and perhaps other yet to be identified ER-MAM proteins for degradation (Benlimame et al., 1997; Fu et al., 2013; Shankar et al., 2013; Jacobs et al., 2014). However the impact of AMF and Gp78 on mitochondrial processes are not clear. Furthermore, we have evidence of a connection between the cytokine receptor function of Gp78 and it ubiquitin ligase function, as AMF binding prevents the ability of Gp78 to degrade Mfn1 and Mfn2 (Fu et al., 2011; Shankar et al., 2013). Further understanding of AMF and Gp78 will not only provide us with novel insights into two multifunctional proteins associated with cancer, it will also provide us further understanding of the regulation of mitochondrial processes and their potential involvement in cancer progression. Thus in this thesis, I investigated the effects of AMF and Gp78 on mitochondrial processes through three objectives. I aimed to: (1) further dissect the mechanisms of Gp78-induced mitophagy upon mitochondrial damage with the autophagic marker LC3; (2) see whether Gp78 ubiquitin ligase activity and its regulation by AMF control mitochondrial dynamics (i.e. fusion and fission); (3) investigate how Gp78 promotes ER-mitochondria association and determine its regulation by AMF.    16  Chapter 2: Gp78 induction of mitophagy 2.1 Background 2.1.1 Introduction to autophagy Autophagy is an essential mechanism used to degrade damaged or redundant organelles such as ER, mitochondria, ribosomes, and the Golgi apparatus. At the onset of cellular stress such as nutrient deprivation or hypoxia, autophagy, which occurs normally at a low basal level, may be triggered to promote recycling of organelles or cellular matter to generate ATP and nutrients, and to sequester potentially toxic protein aggregates and non-functioning organelles (Levine et al., 2005). As such, autophagy initiation can lead to cell survival in response to stress; however, excessive amount of autophagy has also been found to trigger programmed cell death through the activation of caspases (Bursch et al., 2001). Initiation of autophagy begins with the inhibition of target of rapamycin (TOR), an inhibitor of autophagy during nutrient abundant conditions. At the onset of cellular stress, eukaryotic initiation factor 2α (eIF2α) kinase activates a signalling cascade that leads to the transcription of a family of genes involved in autophagy known as the ATG genes (Kim et al., 2007). These ATG proteins work in a concerted fashion to initiate autophagy, generate and mature the autophagosome from the isolation membrane, and disassemble autophagic complexes from matured autophagosomes. Once an autophagosome is formed, it fuses with lysosomes for the degradation and recycling of its contents (Noda and Inagaki, 2014). An autophagic marker that has been commonly used to detect and study autophagy is the cytosolic protein LC3. At the autophagosome, cytosolic LC3-I is cleaved to become LC3-II and attached to the membrane of the autophagosome to facilitate its formation, lengthening and closure (Tanida et al., 2004; Jin and Youle 2012).  17  2.1.2 Introduction to mitophagy Mitophagy is a form of autophagy that targets damaged mitochondria for degradation. Impaired autophagic processes have resulted in aggregation of damaged mitochondria leading to oxidative stress, underlining the significance of mitophagy in the maintenance of cellular health (Wu et al., 2009). A putative mitophagy process that has been well characterized is the PINK1-Parkin pathway. Upon mitochondrial damage and depolarization, PINK1, a mitochondrial protein that translocates to the inner mitochondrial membrane (IMM) under normal conditions through the TOM complex of the mitochondria, remains stuck on the outer mitochondrial membrane (OMM). On the OMM, PINK1 recruits the cytosolic E3 ubiquitin ligase Parkin to the mitochondria, where it ubiquitylates OMM mitochondrial proteins such as TOM20 and Omp25, and targets them for degradation through the ERAD pathway, which leads to the initiation of mitophagy (Yoshii et al., 2011). Moreover, Parkin has been found to degrade the mitochondrial fusion proteins Mfn1 and Mfn2, a process hypothesized to aid in mitophagy by preventing damaged mitochondria from fusing with healthy ones (Gegg et al., 2010). Although how ubiquitylated mitochondrial proteins initiate mitophagy is not yet clear, their actions are essential for Parkin-induced mitophagy (Eiyama et al., 2015).  2.1.3 Potential role of Gp78 in mitophagy Work done by Min Fu in the lab showed that the MAM localized E3 ubiquitin ligase Gp78 targets both Mfn1 and Mfn2 for degradation in a similar fashion to Parkin, inducing mitochondrial fragmentation (Fu et al., 2013). Upon mitochondrial depolarization with carbonyl cyanide m-chlororphenylhydrazone (CCCP) for 24 hours, the mitochondrial marker OxphosV is decreased significantly in Gp78 overexpressing cells. Furthermore, knockdown of ATG5, a critical E3 ubiquitin ligase involved in autophagosome elongation during autophagy (Pyo et al., 18  2005), prevented OxphosV loss, suggesting Gp78-induced mitochondria degradation occurs via the process of mitophagy. In addition, both siRNA knockdown of Gp78 and inhibition of Gp78 ubiquitin ligase activity by transfected mono-ubiquitin that blocks polyubiquitination resulted in inhibited mitophagy upon mitochondrial depolarization. Interestingly, in a mitofusin 1 and 2 knockdown experiment, it was discovered that Gp78 induced mitophagy is dependent on Mfn1 but not Mfn2 (Fu et al., 2013).  2.1.4 Objective  Other than the ATG5 knockdown study, we did not have concrete evidence that Gp78 is targeting mitochondria for degradation through the autophagic pathway. In this study, I employed a LC3 immunofluorescence assay to test my hypothesis. Upon the induction of autophagy, cytosolic LC3 is cleaved and conjugated to the autophagosome membrane. When observed by immunofluorescence, LC3-GFP transfected into cells will transform from a cytosolic localization to punctate spots localized to autophagosomes during autophagy (Tanida et al., 2008). If Gp78 promotion of mitochondria degradation is achieved through the activation of the autophagic pathway, it should result in LC3 recruitment to autophagosomes surrounding the mitochondria. In the event of LC3 localization to the isolation membrane in Gp78 induced mitophagy, the identity of the membrane source for the autophagosome will also be determined using ER markers.        19  2.2 Materials and methods 2.2.1 Antibodies and chemicals Anti-OxphosV Ab was from Molecular Probes (Eugene, OR). Mouse anti-Flag Ab, Rabbit anti-Flag Ab, anti-calnexin Ab  and CCCP were from Sigma-Aldrich (St. Louis, MO). Alexa-fluor 488 rabbit and 647 anti-mouse secondary Ab were from Life Technologies (Burlington, ON).  2.2.2 Cell culture, transfection, and CCCP treatment  Cos7 cells were grown on cover slips in complete Dulbecco modified Eagle's minimal essential medium (DMEM) supplemented with 10% (Fetal bovine serum (FBS), 100µg/ml penicillin, 100µg/ml streptomycin, 2mM L-glutamine and 25mM HEPES buffer at 37⁰C with 5% CO2. After 24 hours, the cells were co-transfected with Flag-Gp78 or Flag-RINGmut and EGFP-LC3 using Effectene (Qiagen, Germany) according to the manufacturer’s protocol. After 18 hours post-transfection, fresh media supplemented with 10µM of CCCP was added to the cells. Cells were incubated with CCCP for 24 hours, and subsequently fixed with 3% paraformaldehyde for 15 minutes and stored at 4⁰C in phosphate buffer saline with calcium and magnesium chloride (PBS-cm) prior to labeling.  2.2.3 Immunofluorescence  Paraformaldehyde fixed cells were permeabilized with 0.5% Triton X-100, blocked with 0.5% BSA PBS-CM, and labeled with rabbit anti-Flag and OxphosV, and subsequently with Alexa 488 anti-rabbit and 647 anti-mouse, respectively. Slides were mounted on gelvatol mounting media. Confocal images were obtained at 100x magnification with 1.4 UplanApo objective of an Olympus FV1000 (Olympic, Toyko, Japan). Colocalization was measured using the ImagePro 20  image analysis software (Media Cybernetics, Bethesda, MD). Data are presented as mean ± S.E.M. Two-tailed t-test were used to calculate statistical significance.  2.3 Results 2.3.1 LC3 localizes to Gp78-labeled ER Punctate fluorescence of EGFP-LC3 appears at the onset of mitophagy (Tanida et al., 2008). Thus to test Gp78 induced mitophagy, we transfected cells with both EGFP-LC3 and Flag-Gp78 or Flag-Ringmut, a construct with a mutated RING finger catalytic domain of Gp78 that abolishes ubiquitin ligase activity. Upon overexpression of Gp78, punctate spots of EGFP-LC3 can be observed 24 hours after CCCP induced mitochondrial depolarization (Figure 2A). Quantification shows EGFP-LC3 to be partially recruited to Flag-Gp78 labeled ER that is dramatically increased after 24 hours CCCP treatment (Figure 2B). Colocalization of EGFP-LC3 with the ER membrane protein calnexin is also significantly increased. In contrast, cells transfected with a Flag-RINGmut, do not exhibit a significant change in EGFP-LC3 localization to Flag-Gp78 and calnexin after CCCP treatment (Figure 2B). These results show Gp78 promotion of damaged mitochondrial clearance is a mitophagic process that is dependent on Gp78 ubiquitin ligase activity.   2.3.2 Gp78 recruits EGFP-LC3 to the mitochondria-associated ER  Cells transfected with Gp78 and treated for 24 hours with CCCP have reduced amount of mitochondria. The remaining fragmented mitochondria are closely associated with punctate structures labeled by EGFP-LC3 and Flag-Gp78. This effect is not observed in both untransfected and Flag-RINGmut transfected cells. The EGFP-LC3 and Flag-Gp78 associated ER is very intimately associated with the mitochondria and visually appears to nearly wrap around the organelle. As LC3 associates with the autophagophore during mitophagy (Tanida et al., 2004), 21  this observation provides evidence that suggests the source of autophagosome membrane is from the ER membrane (Figure 2C).     22  Figure Legends  Figure 2 EGFP-LC3 localizes to ER membrane during Gp78 induced mitophagy  (A) Cos7 cells were transfected with EGFP-LC3 and either Flag-Gp78 or Flag-RINGmut for 24 hours. Cells were then treated with 10µM CCCP for 24 hours and fixed with paraformaldehyde. Cells were labeled with anti-Flag (red), anti-calnexin blue, and EGFP-LC3 fluorescence (black and white; green). Bar graphs show colocalization of EGFP-LC3 and either Flag-Gp78 or calnexin (mean ± SEM, n=3; **P < 0.01; ***P < 0.005; bar, 10µM).  (B) Cos7 cells co-transfected with EGFP-LC3 and Flag-Gp78 were treated with 10µM CCCP for 24 hours and fixed with paraformaldehyde. Cells were labeled with anti-Flag (red), anti-OxphosV (blue), and EGFP-LC3 (green). Residual mitochondria in Flag-Gp78 transfected cells are closely associated with the EGFP-LC3 labeled ER. Bar, 10µM.   23  Figure 2 EGFP-LC3 localizes to ER membrane during Gp78-induced mitophagy      24  Chapter 3: Regulation of mitochondrial dynamics by AMF through Gp78 3.1 Background 3.1.1 Introduction to mitochondrial dynamics  Mitochondria are highly dynamic organelles that in a short period of time divide, fuse, move across the cell, and change shape. Fusion and fission of the mitochondria are responsible for division of mitochondria, but also govern the health of mitochondria via the exchange or segregation of damaged mitochondrial DNA, mitochondrial proteins, and lipids (Lee and Yoon, 2014). Moreover, mitochondrial fission may help separate damaged mitochondria from healthy ones during mitophagy (Eiyama et al., 2015; Fu et al., 2013). These processes are carefully regulated by a set of high molecular weight GTPases: outer mitochondrial membrane (OMM) protein Mfn1 and Mfn2, inner mitochondrial membrane protein Opa1, and dynamin related protein 1 (Drp1) (Otera et al., 2013). Mitochondrial fission is mediated in mammalian cells by Drp1, a cytosolic self-assembling GTPase. At mitochondrial division sites, Drp1 forms a T-shaped dimer or tetramer that contains a head and stalk structure. GTP induces the rearrangement of the head and stalk, which provides the force for mitochondrial membrane constriction (Smirnova et al., 2001; Ingerman et al., 2005). Mitochondrial fusion on the other hand requires Mfn1, Mfn2 and Opa1. Mfn1 and Mfn2 both are localized to OMM, share 63% sequence identity, and possess identical functional domains. A coiled-coil domain called HR2 on the mitofusins facilitates homotypic (Mfn1-Mfn1) or heterotypic (Mfn1-Mfn2) interactions between adjacent mitochondria. Fusion of mitochondria is a multistep process that requires the interactions of HR2 domains to bring the mitochondria together, and a GTP-dependent docking step that leads to fusion of the OMM (Zorzano et al., 2010; Koshiba et al., 2004). Lastly, Opa1 resides in the mitochondrial intermembrane space or attached to the IMM to facilitate fusion of 25  the IMM between mitochondria via its GTPase activity (Ishihara et al., 2006). The careful regulation of these GTPases is required to maintain a healthy population of functional mitochondria. Interestingly, Gp78 has recently been found to target both mitofusin 1 and 2 for degradation (Fu et al., 2013). 3.1.2 Ubiquitin ligase Gp78 in ERAD Gp78 is a well characterized E3 ubiquitin ligase that is a key component in a fundamental cellular process called ERAD (Christianson et al., 2012). In the ERAD pathway, proteins are targeted for degradation in an ubiquitin and proteasome dependent manner in order to recycle misfolded proteins or maintain and regulate protein levels (Meusser et al., 2005). Some of the known Gp78 substrates include T cell receptor CD3-delta, ApoB lipoprotein, HMG-CoA reductase, cystic fibrosis transmembrane conductance regulator, tumour surpressor KAI1, and mitochondrial fission proteins Mfn1 and Mfn2 (Zhong et al., 2004; Song et al., 2005; Morito et al., 2008; Tsai et al., 2007; Fu et al., 2013). Many of these substrates play significant roles in many cellular processes, and maintaining physiological levels of these proteins is crucial for the health of a cell. However, although the role of Gp78 in ERAD is well understood, very little is known about how Gp78 ubiquitin ligase activity might be regulated during ERAD. For example, excessive Gp78 ubiquitin ligase activity can decrease KAI1 levels and promote hyperplasia (Tsai et al., 2007; Joshi et al., 2010), whereas insufficient activity can lead to an accumulation of mitofusin levels and also impaired mitophagy (Fu et al., 2013). Whether or not the Gp78 ligand, AMF, can regulate Gp78 ubiquitin ligase activity remains to be investigated.  26  3.1.4 AMF/Gp78 and mitochondrial dynamics  Work done primarily by Shankar J., Kojic, L., St.Pierre P. in our lab had shown that upon binding to Gp78, AMF is internalized in a raft, dynamin and Rac1-dependent pathway with Gp78, and also downregulates cell surface levels of Gp78. AMF endocytosis also inhibits Gp78-dependent mitofusin degradation, but KAI1 degradation is unaffected. As a result of increased mitofusin levels, both mitochondrial size and speed of movement increased when observed under immunofluorescence. Moreover, in a fluorescence recovery after photobleaching (FRAP) study of mitochondrial fusion, Gp78 overexpression significantly decreased mitochondrial fusion. Interestingly mitochondrial fusion was restored upon AMF treatment on these cells. These results suggest Gp78 may regulate mitochondrial dynamics through fusion in a manner that can be inhibited by AMF.  3.1.5 Objective  Elevated mitofusin levels can have significant impact on mitochondrial dynamics; however, it was not clear if increased mitochondria fusion after AMF treatment is Gp78-dependent. The previous FRAP study on mitochondrial dynamics relied on overexpression of Gp78 in Cos7 cells; thus in this study I employed FRAP to monitor mitochondrial fusion in both Gp78 knockdown cells and AMF-treated HT-1080 fibrosarcoma cells, as HT-1080 cells express significantly higher level of endogenous Gp78 when compared to Cos7 monkey kidney cells.. This will enable the cells to respond better to AMF treatment and also allow me to knockdown Gp78. Confirmation of the involvement of endougenous Gp78 in mitochondrial dynamics after AMF treatment will uncover a novel extracellular control of mitochondrial dynamics.    27  3.2 Materials and methods 3.2.1 Reagents and constructs GFP and ds-RED tagged with a mitochondrial localization signal, called pOct-GFP and pOct-dsRed, respectively, were gifts from Heidi McBride (McGill University). Rabbit AMF/PGI and doxycycline were from Sigma-Aldrich (St. Louis, MO). Effectene transfection reagent was from Qiagen (Gemany).   3.2.2 Cell culture, transfection, and treatment  pTRIPZ HT-1080 Gp78 knockdown doxycycline inducible cells generated by Bharat Joshi in our lab were grown on coverslips in complete Roswell Park Memorial Institute media (RPMI) supplemented with 10% FBS, 100µg/ml penicillin, 100µg/ml streptomycin, 2mM L-glutamine and 25mM HEPES at 37⁰C with 5% CO2. Cells were treated with 1mg/ml of doxycycline at the time of seeding to induce Gp78 knockdown. After 24 hours, the cells were transfected with pOct-GFP using Effectene (Qiagen, Germany) according to the manufacturer’s protocol. For the serum starvation of cells, at 8 hours post-transfection, cells were washed twice with PBS and incubated in complete RPMI media without FBS for 16 hours. For AMF treatment, cells were treated with 25µg/ml of AMF in fresh RPMI medium without serum for 2 hours.    3.2.3 Fluorescence recovery after photobleaching (FRAP)  FRAP analysis of pOct-GFP transfected HT-1080 cells was performed using the 60x magnification (NA 1.35) UplanApo objective of an Olympus FV1000 with an open pinhole (800nm). Images were acquired every 5 seconds over 200 seconds and bleaching done after a single frame capture with a 250ms pulse of a 488nm laser at 85% power. Analysis was done using Olympus FV1000 software and Excel.   28  3.3 Results 3.3.1 AMF and Gp78 regulate mitochondrial fusion in HT-1080 cells  Fluorescence recovery after photobleaching (FRAP) is a technique that bleaches an area of a cell and measures the rate of recovery, which gives us a measure of particle diffusion and mobility. FRAP performed on mitochondria will provide us with the rate of mitochondrial dynamics, with slower recovery occurring when there are diminished fusion events and reduced mitochondrial mobility (Katuri et al., 2010). HT-1080 cells express high levels of endogenous Gp78, which results in low levels of mitofusins (Shankar et al., 2013). In shCN (control) HT-1080 cells, fluorescence recovery of pOct-GFP after photobleaching occurred at a significantly slower rate compared to sh6 Gp78 knockdown cells. The increase in fluorescence recovery in Gp78 knockdown cells is indicative of an increase in mitochondrial fusion and mobility. In the serum starved cohort for AMF treatments, fluorescence recovery occurred at a slower rate than in cells grown in serum supplemented media. AMF promotion of mitochondrial fusion was observed in shCN cells treated with AMF (Figure 3). These results establish Gp78 as a negative regulator of mitochondrial fusion. Furthermore, we also confirm that AMF acts as a regulator of Gp78-dependent mitochondria dynamics.    29  Figure legends Figure 3 Mitochondrial fusion increases with both Gp78 knockdown and AMF treatment (A) Doxycyclin inducible pTRIPZ shCN (control) and sh6 Gp78 knockdown HT-1080 cells were transfected with pOct-GFP and mitochondrial mobility and fusion were measured and analyzed by FRAP. Mitochondria-localized pOct-GFP was photobleached and the fluorescence recovery was measured for 200 seconds. Red squares show magnified region of the cell and yellow circles show the bleached area. Scale bars: 10µm.  (B) Upper panels: Recovery of pOct-GFP fluorescence in bleached region of shCN and sh6 Gp78 knockdown cells in serum-containing medium (percentage recovery ± s.e.m.; 8-10cells per experiment; n=3). Lower panels: recovery of pOct-GFP fluorescence in bleached region of shCN HT-1080 cells in serum free medium in the presence or absence of AMF (25µg/ml) (percentage recovery ± s.e.m.; 8-10 cells per experiment; n=3).   30   Figure 3 Mitochondrial fusion increases with both Gp78 knockdown and AMF treatment    31  Chapter 4: Regulation of ER-mitochondria association by AMF and Gp78 4.1. Background 4.1.1 Introduction to ER-mitochondria association  The first study published by Copeland and Dalton (1959) postulated close contacts between the ER and mitochondria after intimate associations of these two organelles was observed by electron microscopy. ER-mitochondria contacts have since been found to be a significant physiological process of a cell that may involve over 20% of the total mitochondrial surface (Wang et al., 2000; Rizzuto et al., 1998). Historically two types of approaches have been used to study the ER-MAM domains: microscopy and fractionation. Fluorescence microscopy has commonly been used to study these domains, although the finer details of ER-MAM domains has to be resolved by electron microscopy, as the 10-50nm distance between the ER and mitochondrial is too small to be resolved by light microscopy (Copeland and Dalton, 1959; Brito et al., 2008). Differential fractionation has been employed extensively to isolate and study ER-MAM domains. However, it is not clear if this fractionation process can isolate the MAM from all the different domains of the ER. Recently, Optiprep density gradients were used to reliably isolate MAM fractions (Rusinol et al., 1994; Wieckowski et al., 2009; Bui et al., 2010). These techniques have led to a deeper understanding of the plethora of functions and components of the MAM domain.  4.1.2 Lipid transport at ER-mitochondria contacts Lipid transport and synthesis proteins are highly enriched in MAM domains of the ER, which distinguishes it from the rest of the smooth ER (Vance, 1990). Indeed, lipid synthesis enzymes such as phosphatidylserine synthase 1 and 2, that catalyze the synthesis of phosphatidylserine (PS), predominantly localize to MAM domains and are mostly excluded from 32  the rest of the ER (Stone and Vance, 2000). Other MAM-enriched lipid synthesis proteins include diacylglycerol acyltransferase (DGAT) 1 and 2, enzymes that are responsible for catalyzing the production of triacylglycerol (Stone et al., 2009). Lipid synthesis at ER mitochondrial contact sites not only facilitates a steady production of required phospholipids to be transferred to the mitochondria, it is also responsible for the production of most cellular phosphatidylethanolamine (PE). PS is first produced on the ER and then transferred to the mitochondria, where phosphatidylserine decarboxylase in the inner mitochondrial membrane converts PS to PE, and the newly synthesized PE is transferred back to the ER. The importance of this production pathway is shown by the mitochondria defects and embryonic lethality in mouse with knockout genes for the components of this pathway (Vance et al., 2013).  4.1.3 Calcium transfer at ER-mitochondria contact sites Calcium transfer between the ER and mitochondria plays a crucial role in regulating the bioenergetics of a cell and mediating programmed cell death (Rutter et al., 2000). ER localized inositol triphosphate receptor (IP3R) is a calcium channel that releases Ca2+ from the ER to the mitochondria at a low basal level in resting cells to control ATP production in the mitochondria. This is an essential process, as the absence of mitochondrial IP3R-dependent Ca2+ signalling compromises cellular metabolism (Cardenas et al., 2010). The same receptor has been implicated in promoting cell death. Cellular and ER stress can lead to prolonged Ca2+ release from IP3R to the mitochondria and induce an accumulation of Ca2+ in the mitochondrial matrix. If unabated, the accumulation will trigger proapoptotic pathways that subsequently lead to mitochondrial permeability changes, loss of electrochemical gradient, localization of apoptosis regulator Bax to mitochondria, and the release of cytochrome C (Deniuad et al., 2008). In 33  addition, cytochrome C released from the mitochondria can interact with IP3R on the ER and further promote ER-mitochondria Ca2+ transfer in a positive feedback manner (Boehning et al., 2003). Interestingly, during ER stress, cytosolic chaperone Grp78 interacts with IP3R on the ER and voltage dependent anion channel (VDAC) on the mitochondrial to promote ER mitochondrial association, possibly serving to provide more ATP for protein folding (Szabadkai et al., 2006). These intimate connections between the ER and mitochondria are integral to cellular metabolism, ER stress and apoptosis, highlighting the importance of the interaction between these two organelles.  4.1.4 Mitochondrial division at ER-mitochondrial contact sites The ER has also been postulated to facilitate mitochondrial division. ER tubules are able to wrap itself around the mitochondria and mediate constriction prior to the recruitment of mitochondrial fission protein Drp1 (Friedman et al., 2011). Upon contact between ER tubules and mitochondria at the site of division, an ER snare protein syntaxin 17 (STX17) localizes to the raft-like domain of the MAM. At these contact sites, STX17 interacts with Drp1 and facilitates mitochondrial fission by determining Drp1 localization and activity (Arasaki et al., 2015). Interestingly, during starvation, STX17 localizes to ER-mitochondria contact sites that are not involved in division, and promotes the formation of autophagosomes, indicative of the potential role of the ER-MAM domain in biogenesis of autophagosomes (Hamasaki et al., 2013).  4.1.5 ER-mitochondria tethers Although the function of ER-MAM domains has been studied extensively, only a few components of the tethers and regulatory processes have been clearly elucidated. The dynamic nature of ER mitochondria association that may increase or decrease depending on cellular 34  conditions indicates the existence of reversible regulated tethers (Csordas et al., 2006). Among them, as previously described, is the cytosolic chaperone Grp78. By interacting with mitochondrial VDAC during the onset of ER stress and subsequently forming a complex with IP3R on the ER, mitochondria are brought in close apposition with the ER. Accordingly, Grp78 knockdown reduces calcium transfer from the ER to mitochondria at the onset of ER stress (Szabadkai et al., 2006). Another regulator of MAM is B-cell receptor associated protein of 31 Kda (Bap31), a transmembrane ER protein that associates with class I MHC molecules and mediate its folding and export (Abe et al., 2009). During the induction of apoptosis, Bap31 is cleaved by caspase 8, and the resulting fragment promotes pro-apoptotic calcium release from the ER (Breckenridge et al., 2003). Further study revealed that mitochondrial fission protein Fis1 interacts and forms a complex with Bap31 on the ER, and facilitates its cleavage to produce a pro-apoptotic Bap31 fragment. The promotion of ER-mitochondria association by Fis1-Bap31 complexes increases calcium signalling and activates the mitochondria for apoptosis (Iwasawa et al., 2011). Interestingly, free cytosolic calcium levels have also been reported to regulate ER-mitochondria association (Goetz et al., 2007). A new ER-mitochondrial tethering complex named ER membrane protein complex (EMC) has been recently identified in yeast, but has also been found to be conserved in mammalian cells. EMC proteins interact with the Tom5 mitochondrial protein to promote ER-mitochondria contacts and lipid transfer between the two organelles (Lahiri et al., 2014). Finally, it is believed that mitofusin 2 (Mfn2) act as a physical tether between mitochondrial and ER, although this observation has been disputed (Brito et al., 2008; Pizzo et al., 2015).     35  E3 ubiquitin ligase Gp78 has been localized to ER-mitochondria contact sites and targets both Mfn1 and Mfn2 for degradation. (Goetz et al., 2007; Shankar et al., 2013). AMF has also been shown to selectively alter Gp78 ubiquitin ligase activity by inhibiting the ability of Gp78 to target mitofusins for degradation but not KAI1 (Shankar et al., 2013). In a recent study, Gp78 ubiquitin ligase activity has been found to play a significant role in regulating ER-mitochondria calcium coupling (Fu et al., 2011). As calcium transfer is altered upon changes in ER-mitochondria association, these results provide evidence for AMF and Gp78-regulation of ER-mitochondria association (Brito et al., 2008). Furthermore, our lab has found Gp78 overexpression in Cos7 cells increased colocalization of the mitochondria marker OxphosV and dephosphorylated ER-localized Gp78 labeled by the Gp78 antibody 3F3A (Min Fu, unpublished data). The relationship between Gp78 degradation of the mitofusins and regulation of ER-mitochondria contacts remains an interesting question. 4.1.7 Objective  We postulate that Gp78 may play a role in regulating ER-mitochondria contact sites through the degradation of the mitofusins, and thereby affect ER mitochondria calcium transfer. Furthermore, Gp78-dependent ER-mitochondria association could potentially be regulated by the extracellular cytokine AMF. In Gp78 overexpressing cells, we observed increased colocalization of mitochondria label with the Gp78-labeled ER. However, it was unclear if the observed effect can be attributed to an increase in ER-mitochondria association or perhaps the indirect result of elevated Gp78 levels, which led to higher colocalization. Moreover, contacts between the ER and mitochondria are within 50nm, which lay beyond the resolution of a confocal microscope. In order to further understand ER-mitochondria 36  association, techniques that provide higher resolution are required. In this study, I aimed to examine the effects of Gp78 on ER-mitochondria association by using an independent ER-MAM marker, syntaxin 17 (STX17) for immunofluorescence and also with electron microscopy. To further understand the regulation and mechanisms involved, I will study the potential effects and roles of both AMF and the mitofusins on Gp78-dependent ER-mitochondria association.  4.2 Materials and methods 4.2.1 Antibodies and reagents  Rat IgM anti-Gp78 mAb 3F3A was as described (Nabi et al., 1990). OxphosV antibodies were from Molecular Probes (Eugene, OR) or Abcam (Cambridge, MA), M2 anti-Flag and syntaxin 17 Ab were from Sigma-Aldrich (St. Louis, MO), Mfn1 and Mfn2 Ab were from Santa Cruz, anti-rat IgM secondary Ab from Jackson ImmunoResearch Laboratories, anti-mouse and anti-rabbit secondary Ab from Molecular Probes (Burlington, ON).  4.2.2 Cell Culture, constructs and treatments  Cos7 and HT-1080 cells were grown on cover slips in complete DMEM and RPMI respectively, supplemented with 10% FBS, 100µg/ml penicillin, 100µg/ml streptomycin, 2mM L-glutamine and 25mM HEPES at 37⁰C with 5% CO2. After 24 hours, the Cos7 cells were double transfected with Flag-Gp78 or Flag-RINGmut using Effectene (Qiagen, Germany) according to the manufacturer’s protocol. Stable HT-1080 clones expressing control scrambled shCtl and Gp78-targeted shGp78 shRNAs in doxycycline-inducible pTRIPZ plasmid were maintained in medium containing 2mg/ml puromycin and 1mg/ml doxycycline to induce Gp78 knockdown. Cos7 cells were grown for 24 hours post-transfection and HT-1080 cells grown for 48 hours 37  after induction before being fixed with either pre-cooled methanol acetone or 3% paraformaldehyde respectively, for 15 minutes and stored at 4⁰C in PBS-CM prior to labeling. For AMF treatments, both Cos7 and HT-1080 cells were starved overnight and incubated with 25µg/ml AMF for 2 hours.  4.2.3 Immunofluorescence  Flag-Gp78 transfected Cos7 cells fixed by methanol-acetone were labeled with rat 3F3A, mouse OxphosV, and rabbit syntaxin-17 primary antibodies, and Alexa 488, 568, and 647 secondary antibodies. Peripheral ER 3F3A labeling identified Flag-Gp78 transfected cells. HT-1080 cells fixed by 3% paraformaldehyde were permeabilized with 0.5% Triton X-100 for 10 minutes, and labeled with mouse OxphosV, and rabbit syntaxin-17 primary antibodies, and Alexa 488 and 647 secondary Ab. 3D image stacks of syntaxin-17 and OxphosV labels were obtained with 60x (NA 1.4) Zeiss planapochromat objective of a III Yokogawa spinning disk confocal microscope. The percentage of syntaxin-17 label that overlapped a 3D mask of OxphosV labeled mitochondria volumes was quantified using Slidebook image analysis software (III). Data are presented as mean ± S.E.M. Two-tailed t-test were used to calculate statistical significance..  4.2.4 Electron microscopy  Sample preparation and imaging with electron microscopy was done in collaboration with Pierre Garcin from the Nelly Pante lab (UBC). HT-1080 cells were grown on Aclar film (Pelco), chilled, washed with PBS, and fixed with 2% glutaraldehyde in 0.1M sodium cacodylate on ice. Samples were then incubated with 1% osmium tetroxide in cacodylate buffer, stained 38  with 0.1% uranyl acetate for an hour, and progressively dehydrated through an ethanol series, followed by 100% acetone treatment, and embedding in Epon 812 (Fluka). 50-60nm thick sections were cut with a Leica Ultramicrotome and stained with 2% uranyl acetate and 2% lead citrate, and visualized on a FEI Tecnai G2 Spirit transmission electron microscope (acceleration voltage: 120kV). Mitochondria perimeter/area and length of smooth/rough ER interfaces were measured to obtain the interface percentage using ImagePro 6.0 software. Widths of smooth/rough ER-mitochondria interfaces were determined by measuring the shortest distance between the ER membrane and the mitochondrial outer membrane at two sites of each contact. Data are presented as mean ± S.E.M. Two-tailed t-test were used to calculate statistical significance.  4.3 Results 4.3.1 AMF-Gp78 regulation of ER-mitochondria association  To test whether Gp78 regulates ER-mitochondria association, Cos7 cells were transfected with Flag-Gp78 or a Ring finger mutant of Gp78 (Flag-Ringmut). In cells labeled with syntaxin17 (STX17), a marker for ER-mitochondria contact sites, and mitochondrial OxphosV, 3D spinning disk confocal stacks show close association of STX17-labeled ER and mitochondria. Gp78 overexpression significantly increased this association, whereas Gp78 Ring finger mutant lacking ubiquitin ligase activity did not alter the extent of association (Figure 4). Upon AMF treatment, the amount of ER-mitochondria contacts were not altered in both untransfected cells and Flag-Ringmut transfected cells; however, the ability of Gp78 overexpression to promote ER-mitochondria association is inhibited (Figure 4). These results provide evidence that Gp78 promotes ER-mitochondria association, whereas AMF negatively regulates Gp78-induced ER-mitochondria association.  39  4.3.2 Gp78 selectively regulates rough ER-mitochondria contacts   Electron microscopy was used to provide sufficient resolution to resolve ER-mitochondria contacts. High resolution images of HT-1080 fibrosarcoma cells that were taken by Pierre Garcin in the Pante Lab (UBC) revealed two types of ER-mitochondria contacts: smooth and the rough. Smooth ER contacts are typically shorter and closely opposed to mitochondria (8.0 ± 0.5nm), while rough ER contacts are wider (50.5 ± 1.7nm), longer and have ribosomes facing the mitochondria surface (Figure 5A). In HT-1080 cells, rough ER contacts cover approximately twice the mitochondrial surface area as smooth ER contacts. Gp78 knockdown in shGp78 inducible cells (same as sh6 in chapter 3) significantly increased mitochondria size compared to control shCTL cells, likely due to the increase in mitochondria fusion caused by elevated mitofusin levels. Gp78 knockdown did not affect smooth ER contacts, but reduced rough ER contacts by almost half (Figure 5B).  4.3.3 AMF regulates Gp78-dependent rough ER-mitochondrial contacts  In contrast to Cos7 cells, that normally express low basal levels of Gp78 (Figure 4), treatment of HT-1080 cells, that express high levels of endogenous Gp78, with AMF led to a significant decrease in colocalization of syntaxin-17 labeled ER and mitochondria (Figure 6A). When cells were starved overnight to activate the signalling pathway of AMF, mitochondria in ShGp78 knockdown cells did not increase in size when compared to that of shCTL; however, AMF treatment increased mitochondrial size in shCTL but not shGp78 cells (Figure 6B). Paralleling the reduction of rough ER contacts in HT-1080 Gp78 knockdown cells, AMF treatment also reduced rough ER contacts in shCTL cells and did not affect smooth ER contacts. AMF affected neither rough nor smooth ER contacts in shGp78 cells (Figure 6B). AMF-regulation 40  of Gp78 ubiquitin ligase activity therefore selectively controls rough ER-mitochondria association. 4.3.4 Mfn1 inhibits smooth ER contacts, while Mfn2 inhibits rough ER contacts  As Gp78 targets both Mfn1 and Mfn2 for degradation, elevated levels of mitofusins are present in shGp78 knockdown cells (Figure 7A). Parallel to the results from electron microscopy, analysis of the colocalization of syntaxin-17 labeled ER and mitochondria in HT-1080 shGp78 knockdown cells reveals decreased ER-mitochondria association. Mfn1 or Mfn2 siRNA knockdown did not affect ER-mitochondria association in shCtl HT-1080 cells. However, both induced a significant increase in contacts in shGp78 cells (Figure 7A). EM analysis showed that Mfn1 and Mfn2 knockdown reduced mitochondrial size as expected. Mfn1 knockdown did not affect rough ER-mitochondria contacts in both shCtl and shGp78 cells, but significantly increased smooth ER-mitochondria contacts in both cell lines. Mfn2 knockdown on the other hand did not have a significant effect on either type of contacts for shCtl cells, but reversed the loss of rough ER-mitochondria contacts in shGp78 cells (Figure 7B). These observations suggest that in HT-1080 cells, Mfn1 and Mfn2 mediate distinct mechanisms that inhibit the formation of smooth and rough ER-mitochondria contacts, respectively.    41  Figure legends Figure 4 AMF inhibits Gp78 promotion of ER-mitochondria contacts (A) 3D reconstructed images of Cos7 cells transfected with either Flag-Gp78 or Flag-RINGmut for 24 hours. Cells were starved overnight, treated with AMF 25µg/ml, and fixed with methanol-acetone. Cells were labeled with mitochondria OxphosV (red), and syntaxin-17 (green). Bar graphs show 3D volume colocalization of syntaxin-17 labeled ER and OxphosV labeled mitochondria (Mean ± S.E.M.; n=25-30; **p<0.01; Bar: 10 µm).   42   Figure 4 AMF inhibits Gp78 promotion of ER-mitochondria association      43  Figure Legends Figure 5 Gp78 regulates rough but not smooth ER-mitochondria association (A) Electron microscopy images of shCTL and shGp78 HT-1080 cells. Insets show rough (top) and smooth (bottom) ER-mitochondria close contacts. Bar graph shows average width of rough and smooth contacts (Mean ± S.E.M.; n=50; bar: 500 nm; insets: 200 nm).  (B) Bar graphs show average mitochondrial area for shCTL and shGp78 HT-1080 cells (Mean ± S.E.M.; n>167; ***p<0.001) and percentage of mitochondrial membrane interfacing with rough or smooth ER contact sites (Mean ± S.E.M.; n>167; ***p<0.001).    44       Figure 5 Gp78 regulates rough but not smooth ER-mitochondria contacts 45  Figure Legend Figure 6 AMF inhibits Gp78 promotion of rough ER contacts in HT-1080 cells (A) 3D reconstructed immunofluorescent images of HT1080 cells treated with AMF (+AMF; 2 hours) and labeled for syntaxin 17 (green) and OxphosV (red). Bar graph shows percentage of mitochondrial volume overlapped by syntaxin 17 (Mean ± S.E.M.; n= 25-30; *p<0.05). (B) Representative electron microscopic images of serum-starved shCTL HT-1080 cells untreated and treated with AMF for 2h (Bar: 500 nm). Bar graph shows average mitochondrial perimeter of AMF treated, serum starved shCTL and shGp78 HT-1080 cells. Bar graph shows percentage of mitochondrial membrane interfacing with rough or smooth ER contact sites after AMF treatment of shCTL and shGp78 HT-1080 cells (Mean ± S.E.M.; n>162; **p<0.01; ***p<0.001).   46     Figure 6 AMF inhibits Gp78 promotion of rough ER contacts 47  Figure Legends Figure 7 Mfn1 inhibits smooth ER contacts, while Mfn2 inhibits rough ER contacts  (A) 3D reconstructed confocal stacks of syntaxin-17 (green) and OxphosV (red) labeled shCTL and shGp78 cells transfected with control (CTL), Mfn1 or Mfn2 siRNA (Bar: 10um). Western blot of Mfn1, Mfn2 and β-actin in shCTL and shGp78 cells transfected with Mfn1 and Mfn2 siRNA. Bar graph shows percentage of mitochondrial volume overlapped by syntaxin-17 (Mean ± S.E.M.; n=25-30; **p<0.01, ***p<0.001).  (B) Electron microscopy images of shCTL cells transfected with control or Mfn1 siRNA and shGp78 cells transfected with control or Mfn2 siRNA (Bar: 1 µm). Bar graphs show average mitochondrial perimeter and percentage of mitochondrial membrane interfacing with rough or smooth ER contact sites for shCTL and shGp78 HT-1080 cells transfected with control, Mfn1 or Mfn2 siRNA (Mean ± S.E.M.; n>150; ** p<0.01, ***p<0.001).     48    Figure 7 Mfn1 inhibits smooth ER contacts, while Mfn2 inhibits rough ER contacts 49  Chapter 5: Discussion 5.1 Discussion 5.1.1 AMF/Gp78 functions Since the initial discovery of AMF (Liotta et al., 1986), progressive research has revealed the complex biology of this dual function glycolytic enzyme and motility inducing protein. As a cytoplasmic enzyme also known as phosphoglucose isomerase (PGI), AMF participates in a critical step of carbohydrate metabolism by facilitating the interconversion of glucose-6-phosphate to fructose-6-phosphate. Indeed, PGI knockout mouse proved to be embryonic lethal, and mutations that produced diminished PGI activity lead to human diseases (Harrison, 1974; West et al., 1990; Kanno et al., 1996). When released from the cell and acting as a cytokine, AMF binds with its cell surface receptor Gp78, and is internalized through a clathrin independent, dynamin and PI3K-dependent raft mediated pathway, and transported to the smooth ER (Benlimame et al., 1998; Kojik et al., 2007). Through Gp78 signalling in a protein kinase 3 and Rho-GTPase-dependent pathway, AMF can trigger cell motility, survival, growth, maturation, and epithelial-to-mesenchymal conversion (Silletti et al., 1993; Funasaka et al., 2009; Haga et al. 2006; Fu et al., 2011; Kanbe et al., 1994). The great plethora of functions of AMF and Gp78 highlight the importance of both proteins in maintaining normal cellular health; however, also due to these functions, dysregulation of these processes can have severe consequences. As a result, AMF and Gp78 are implicated in many types of cancers (Chui et al., 2008; Kojic et al., 2007).  5.1.2 AMF regulation of mitochondrial dynamics through Gp78 Adding to the complexity of the story, similar to AMF, Gp78 also possesses two very different functions: as cell surface receptor and an ER transmembrane E3 ubiquitin ligase 50  (Shimizu et al., 1999; Fang et al. 2001). Gp78 ubiquitin ligase activity plays a key role in ERAD, a process that is essential in maintaining proper levels of certain proteins and the degradation of many non-functional or damaged proteins (Meusser et al., 2005). Some substrates that Gp78 targets for degradation through ubiquitination include tumour suppressor KAI1, HMG-CoA, and Apo lipo B protein (Liang et al., 2006; Song et al., 2005; Tsai et al., 2007). Gp78 activity must be regulated carefully, as dysregulation can result in excessive degradation of KAI1, leading to hyperplasia (Joshi et al., 2010), while loss of Gp78 can hamper response to ER stress (Fu et al., 2011). Interestingly, Gp78 mediated ubiquitylation of substrates transpires at the peripheral smooth ER, where ER-mitochondrial association typically occurs (St.Pierre et al., 2012; Goetz et al., 2007). We identified both mitochondrial fusion proteins mitofusin 1 and 2 as Gp78 substrates. These two OMM proteins facilitates mitochondria fusion, a process governing mitochondrial dynamics that is essential for mitochondrial division, health and mitophagy (Lee and Yoon, 2014; Eiyama et al., 2015). The management of Mfn1 and Mfn2 levels is required to maintain proper mitochondrial dynamics. Here in this study, we report Gp78 as a novel regulator of mitochondrial dynamics through the degradation of Mfn1 and Mfn2. Loss of Gp78 that leads to an accumulation of mitofusins is accompanied by increased mitochondrial fusion. Interestingly, AMF interaction with Gp78 inhibits Gp78 degradation of mitofusins, which also results in elevated levels of mitochondrial fusion similar to that of Gp78 knockdown. This is the first report of extracellular regulation of a key component of ERAD. However, the mechanism by which AMF regulates Gp78 activity remains unclear. Gp78-dependent degradation of KAI1 is unaffected by AMF treatment, which shows that AMF does not generally inhibit all Gp78 ubiquitin ligase activity. As Gp78 potentially targets mitofusin for degradation at the ER-51  mitochondria interface, AMF control of Gp78 mitofusin degradation can be attributed to a change in ER-mitochondria association.  5.1.3 Gp78 regulates ER-mitochondria association Many central cellular processes have long been known to be dependent on MAM domains. Lipid transfer at these domains is responsible for providing the mitochondria with necessary phospholipids and the production of PE (Vance et al., 2013). Calcium transfer from the ER to the mitochondria at the MAM will not only boost mitochondrial respiration and ATP production in the time of need, it also facilitates apoptosis through the release of cytochrome C (Rutter et al., 2000; Deniuad et al., 2008). Recent studies have also shown ER-mitochondria contact sites to be sites of mitochondrial division (Friedman et al., 2011; Arasaki et al., 2015). Certain ER-mitochondria association regulators have been identified. Both Grp78 during ER stress, and Bax31 during apoptosis, promote ER-mitochondria association through complex formation with mitochondrial protein VDAC and FIS1, respectively (Szabadkai et al., 2006; Iwasawa et al., 2011). A tether for the ER and mitochondria is the EMC complex that facilitates lipid transfer between the two organelles (Lahiri et al., 2014). Another proposed ER-mitochondria tether is Mfn2 (Scorrano et al., 2008). However, the function of Mfn2 as a tether has been recently challenged (Cosson et al., 2012; Filadi et al., 2015). In a previous study, we found that AMF and Gp78 alter ER-mitochondria calcium transfer (Fu et al., 2011), thus we began this study to look at the potential impact AMF and Gp78 on ER-mitochondria association. In this study we show that Gp78 ubiquitin ligase activity promotes ER-mitochondria association. Furthermore, AMF treatment reverses this effect and induces ER-mitochondria disassociation 52  similar to that of Gp78 knockdown. These results establish AMF and Gp78 as regulators of ER-mitochondria association.  5.1.4 AMF and Gp78 regulation of rough ER contacts Upon examination of the juxtaposition of ER and mitochondria through electron microscopy, two distinct types of contacts are observed in HT-1080 fibrosarcoma cells. Rough ER contacts that are roughly 50nm wide with ribosomes on the ER facing the mitochondrial surface are widely observed in these cells. Whereas, smooth contacts appear much more intimate at roughly 10nm wide and are typically much shorter than rough ER contacts. In a study on linkages between the ER and mitochondria in rat liver cells, smooth and rough ER-mitochondria contacts were also observed (Csordas et al., 2006). Interestingly, in our study, loss of Mfn1 or Mfn2 both led to an increase in ER-mitochondria contacts. Mfn1 did not alter rough ER, but emerged as a regulator of smooth ER contacts. On the other hand, Mfn2 knockdown significantly restored rough ER contacts in Gp78 knockdown cells that have reduced rough ER contacts. We speculate that the inability of Mfn2 knockdown to increase rough ER contacts in shCTL HT-1080 cells (control) is due to pre-existing low levels of Mfn2 caused by elevated expression of Gp78 in these cells (Fig 4.4A). These results are in accordance with recent knockdown studies of Mfn2 that found that Mfn2 knockdown generally increased ER-mitochondria association (Cosson et al., 2012; Filadi et al., 2015). In this study we establish a Gp78 as a novel promoter of ER-mitochondria association that may be achieved through the degradation of Mfn2, a negative regulator of ER-mitochondrial interaction.  Currently, the physiological differences in the function of these contacts are not understood. Mfn1 knockdown that inhibits mitochondrial fusion and promotes smooth ER 53  contacts also prevents Gp78 induced mitophagy (Fu et al., 2013); however, increased mitochondrial fusion has also been found to protect mitochondria from mitophagy (Rombold et al., 2011). Smooth ER contacts may be an inhibitor of mitophagy; nonetheless, this argues that the control of ER-mitochondria contacts may also be involved in the mitophagic process. Rough ER contacts on the other hand have ribosomes facing the mitochondrial surface and may be associated with translocon-mediated ER-mitochondria calcium coupling (Flourakis et al., 2006). Translocon regulates ER calcium stores through the passive release of calcium into the cytoplasm, a process essential for ER stress, apoptosis, and cellular metabolism (Hammadi et al., 2013). Gp78 has also been reported to interact with many components of the translocon complex, including derlin-1, VIMP, and PNGase (Ballar et al., 2007; Li et al., 2006; Ye et al., 2005). Whether selective regulation of rough ER contacts by AMF and Gp78 contributes to translocon-mediated calcium coupling remains to be determined. Perhaps the ability of AMF to promote cell survival and apoptosis resistance is achieved through the promotion of ER-mitochondria disassociation leading to perturbed pro-apoptotic ER-mitochondria calcium coupling.  5.1.5 Role of Gp78 in mitophagy The impact of Gp78 on mitochondria moves beyond mitochondrial dynamics and ER association. We discovered that Gp78 plays an important role in the promotion of mitophagy, an autophagic process that is essential for clearing and recycling damaged mitochondria. Impaired mitophagy can lead to dangerous accumulation of reactive oxidative species especially when the cell is exposed to nutrient deprivation or hypoxia, conditions often experienced by tumour cells (Gomes and Scorrano, 2013). In a fashion similar to Parkin, a well-54  established regulator of mitophagy, Gp78 ubiquitylates and targets Mfn1 and Mfn2 for degradation to regulate mitochondria fusion, mobility, and mitochondrial autophagy. Gp78 promotes the clearance of damaged and depolarized mitochondria through the autophagic pathway, as confirmed by the inhibition of Gp78-dependent mitophagy by ATG5 knockdown, and the recruitment of LC3 to the mitochondria associated ER membrane. Gp78 induced mitophagy does not require Parkin, as the process proceeds undisturbed in Parkin-null Hela cells. Moreover, unlike Parkin, Gp78 induces mitophagy through a pathway independent of PINK1. Furthermore, Parkin induced mitophagy requires Mfn2 but not Mfn1 (Chen and Dorn, 2013), whereas we found Gp78 induced mitophagy requires Mfn1 but not Mfn2. These results reveal a novel mitophagic pathway that Gp78 employs to degrade damaged mitochondria independently from Parkin. It has been hypothesized that Parkin targeting of mitofusins for degradation during mitophagy prevents mitochondrial fusion to help isolate damaged mitochondria (Twig et al., 2008; Eiyama et al., 2015). It is likely that Gp78 mediated mitophagy also depends on this process; however, the basis for the requirement of Mfn1 but not Mfn2 for Gp78 mediated mitophagy is not clearly understood.  5.1.6 The ER membrane is a potential source for autophagosomes Autophagy requires the engulfment of cytoplasm or organelle by double membrane vesicles called autophagosomes. Interestingly, this study provides insight into the origin of the initial autophagic membrane, also called isolation membrane or phagophore. Although this topic has been well scrutinized, the origin remains uncertain, as ER, mitochondria, Golgi and plasma membrane have all been implicated as membrane contributors (Tooze and Yoshimori, 2010; Cook et al., 2014). Recent advances have shown direct contacts between the initial 55  phagophore and the ER; moreover, autophagosomes derive from phosphatidylinositol 3-phosphate enriched membrane compartments that are connected to the ER (Axe et al., 2008; Hayashi-Nishino., 2009). The recruitment of EGFP-LC3 to calnexin and Flag-Gp78 labeled ER during the process of mitophagy supports the ER membrane as a source for the autophagic isolation membrane. This suggests that upon the detection of damaged mitochondria, Gp78 at the mitochondria-associated ER domain initiates mitophagy in a Mfn1-dependent manner leading to the recruitment of autophagic proteins and LC3. This is followed by the formation of the autophagosome from the ER membrane, which surrounds the mitochondria and subsequently leads to the clearance and recycling of the damaged mitochondria through lysosomal fusion. 5.1.7 AMF-regulation of Gp78 at ER-mitochondria contact sites Gp78 targeting of the mitofusins for degradation at ER-mitochondria contact sites leads to reduced levels of mitofusin on the mitochondria that promotes mitochondrial fission and rough ER association. AMF binding to Gp78 at the cell surface triggers a selective inhibition of Gp78-induced mitofusin degradation, which leads to their accumulation on the mitochondria. The elevated levels of mitofusins reverse the effect of Gp78 on the mitochondria, which promote mitochondrial fusion and negatively regulate rough ER association (Figure 8). Whether AMF-regulation of Gp78 play a role in Gp78-dependent mitophagy remains to be determined; however, we speculate there might be a significant impact, as mitofusin levels are crucial for proper induction of mitophagy (Chen and Dorn, 2013; Fu et al., 2013).      56  Figure Legends Figure 8 Schematic of AMF regulation of Gp78 at ER-mitochondria contact sites  At the ER-mitochondrial contact sites, Gp78 targets Mfn1 and Mfn2 for degradation, resulting in diminished levels of both proteins on the OMM. Upon AMF treatment, Gp78 is prevented from degrading the mitofusins, which results in an accumulation of both proteins on the mitochondria. This promotes the disassociation of rough ER-mitochondria contacts and mitochondrial fusion. Whether or not AMF regulates Gp78-induced mitophagy remains to be further investigated.    57  Figure 8 Schematic of AMF regulation of Gp78 at ER-mitochondria contact sites    58  5.1.8 Potential contribution of the novel functions of AMF and Gp78 to cancer  AMF and Gp78 contribute to a wide range of physiological effects that promote tumour progression. AMF has the ability to inhibit IGFBP-3 induced apoptosis, promote angiogenesis in tumour cells, stimulate cell motility, and trigger EMT (Mishra et al., 2004; Funasaka et al., 2002; Simard and Nabi, 1996). Gp78 on the other hand degrades tumour suppresser KAI1, protects against ER stress, and induces hyperplasia when overexpressed (Fu et al., 2011; Joshi et al., 2010). It is therefore no surprise that the elevated expression of both proteins have been highly associated with cancer and poor prognosis (Gong et al., 2005; Chui et al., 2008). In these studies, we establish AMF and Gp78 as regulators of mitochondrial dynamics, mitophagy, and ER-mitochondria association. Whether these physiological roles of AMF and Gp78 also play a role in cancer progression remains to be determined. Interestingly, decreased expression of Mfn2 and slightly elevated levels of mitochondrial fission protein Drp1 are reported in lung cancer cell lines. Overexpression of Mfn2 and Drp1 knockdown in these cancer cells led to diminished tumour volume, reduction in cell proliferation, and spontaneous apoptosis (Rehman et al., 2012). In breast cancer cells, higher levels of Drp1 and lower levels of Mfn1 has been reported. Mfn1 silencing and overexpression of Drp1, which resulted in mitochondrial fragmentation, enhanced the metastatic abilities of these cancer cells (Zhao et al., 2013). Contrarily, upregulation of mitochondrial fusion protein Opa1 that produced elongated mitochondria led to decreased sensitivity to apoptosis and promoted cell survival (Lee et al., 2004). These results indicate that mitochondrial dynamics play an important role in cancer cell development, survival, and invasiveness. Gp78 targets both Mfn1 and Mfn2 for degradation, which produces mitochondrial fragmentation; whereas, AMF treatment promotes mitochondrial fusion and elongation of the mitochondria. It would be interesting to see if these 59  effects on the mitochondria contribute to the pro-survival, growth, and the motility inducing effects of AMF and Gp78.     60  Chapter 6: Conclusion  In this dissertation, I aimed to further dissect and understand the role of two proteins intimately associated with cancer. Since AMF was discovered over 20 years ago (Liotta et al., 1986), the large numbers of studies that followed unlocked a plethora of functions and roles of this cytokine. Yet there are still many things about AMF that remain unclear. For one, its cell surface receptor Gp78 also functions as an ubiquitin ligase as a part of the ERAD pathway. Whether or not AMF binding with Gp78 triggering internalization of the receptor-ligand complex affect the ubiquitin ligase activity of Gp78 is not known. Moreover, based on immunofluorescence confocal microscopy and calcium studies, Gp78 has been postulated to play a role in regulating mitochondrial dynamics (Fu et al., 2011). With these questions in mind, I decided to investigate the regulation of AMF on GP78, and their physiological effects on the mitochondria.   What I found in these studies expanded our understanding of both AMF, Gp78, mitochondrial dynamics and ER association. A novel pathway of mitophagy that is dependent on Gp78 instead of the well-established Parkin pathway was identified. This novel process utilizes Mfn1, and contrary to Parkin induced mitophagy, is independent of Pink1 and Mfn2. Our results highlight the importance of ubiquitin ligases in the induction of mitophagy. Moving onto mitochondrial dynamics, i.e. fusion and fission, we establish Gp78 regulation of such processes through the targeted degradation of Mfn1 and Mfn2. The ability of AMF to inhibit Gp78-dependent degradation of mitofusins increases mitochondrial fusion events, allowing us to report the first instance of an extracellular regulation of a component of ERAD. As we moved the investigation towards ER-mitochondrial association, the mechanism for AMF selective 61  regulation of Gp78 substrates became clear. Gp78 ubiquitin ligase activity, perhaps through the degradation of the mitofusins, led to an increase in ER-mitochondrial association. Interestingly, in the HT-1080 fibrosarcoma cells we studied, we found two distinct types of ER-mitochondrial contacts using electron microscopy. Gp78 may promote rough ER-mitochondria contacts through the degradation of Mfn2, while leaving smooth ER contacts in these cells unaffected. Upon AMF treatment, the amount of rough ER-mitochondria contacts decreases, providing the underlying mechanisms for inhibition of Gp78-dependent mitofusin degradation, and prolonged calcium coupling time after AMF treatment. We also found Mfn1 did not affect rough ER-mitochondria contacts, but instead inhibited smooth ER-mitochondria contacts.    These findings provide many new insights to the field of AMF and Gp78, and ER-mitochondria research. However, there are still many questions left to be answered about AMF and its regulation of Gp78. Even though we understand that Gp78 has a profound impact on the mitochondria, the physiological purposes of such regulation remain unknown. As ER-mitochondria contacts affects calcium coupling, lipid transport, apoptosis, and mitochondrial division, it would be interesting to see if Gp78 indeed plays a role in regulating such cellular processes. Moreover, with the upregulation of Gp78 and AMF in many types of cancers, we wonder if the effects of Gp78 on mitochondria play a significant role in cancer progression. Damaged mitochondria as a result from hypoxia and lack of nutrients must be cleared through mitophagy or else the cell may risk damage and death. Moreover, both mitochondrial fusion and fission have been reported to play a significant role in tumour growth. Deeper understanding of both AMF and its regulation on Gp78 will provide us with novel insights towards the biology of cancer.  62  Bibliography Abe F., Nancy Van Ladasky, P., J. J. A., & Edidin, M. (2009). Interaction of Bap31 and MHC class I molecules and their traffic out of the endoplasmic reticulum. 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