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Regulation of receptor signaling and membrane trafficking by beta1,6-branched n-glycans and caveolin-1/cholesterol.. Lajoie, Patrick 2008-12-31

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REGULATION OF RECEPTOR SIGNALING AND MEMBRANE TRAFFICKING BY BETA1,6-BRANCHED N-GLYCANS AND CAVEOLIN-1/CHOLESTEROL MEMBRANE DOMAIN ORGANIZATION  by PATRICK LAJOIE B.Sc. Université du Québec à Montréal, 2002  A THESIS SUBMITTED AS PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in THE FACULTY OF GRADUATE STUDIES (Anatomy)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) February 2008  © Patrick Lajoie, 2008  ABSTRACT Modification by glycosylation gives proteins a range of diverse functions reflecting their structural variability. N-glycans regulate many biological outcomes in mammalian cells under both normal and pathological conditions. They play a major role in various pathologies such as cancer and lysosomal storage diseases. Interplay between N-glycans and other regulators, such as membrane lipid domains, in the control of signaling pathways remains poorly understood. My thesis therefore focuses on how N-glycans and membrane lipid domains oppose and/or work together at different cellular levels to regulate various processes such as receptor signaling and diffusion, endocytosis and lysosomal organelle biogenesis. Mgat5 encodes for ß1,6-N-acetylglucosaminyltransferase V that produces N-glycans, the preferred ligand for galectins. In tumor cells, galectins bind glycosylated receptors at the cell surface forming a lattice, that restricts receptor endocytosis and enhances its residency at the plasma membrane. In the first part of my thesis, I report that Galectin/receptor crosslinking opposes receptor sequestration by oligomerized caveolin-1 (Cav1) domains overriding its negative regulation of epidermal growth factor receptor (EGFR) signaling, cell surface diffusion and tumor growth. These results identify Cav1 as a conditional tumor suppressor. I also demonstrate that Cav1 is a negative regulator of lipid raft-mediated endocytosis. Cav1 indirectly regulates the internalization of cholera toxin b subunit to the Golgi apparatus independently of caveolae formation. That identifies a new role for caveolin-1 outside caveolae in the regulation of raft-dependent endocytosis Finally, Mgat5 overexpression in pneumocytes is associated with the expression of a lysosomal organelle, the multilamellar body (MLB), via autophagy. MLB expression is also a characteristic of various lysosomal storage diseases. I demonstrate that cholesterol accumulation can override the need for Mgat5 overexpression in MLB formation indicating that they may form via multiple mechanisms. However, I also demonstrate that a contribution of the autophagic pathway is a common determinant of biogenesis of MLB of various lipid compositions. ii  In conclusion, Mgat5-dependent protein glycosylation and Cav1/raft domains therefore both function as regulators of plasma membrane interactions, endocytosis and lysosomal organelle biogenesis. Understanding of this interplay is crucial for the understanding of the mechanisms involve in various pathologies such as cancer and lysosomal storage diseases.  iii  TABLE OF CONTENTS ABSTRACT ....................................................................................................................... ii TABLE OF CONTENTS ................................................................................................. iv LIST OF TABLES..........................................................................................................viii LIST OF FIGURES.......................................................................................................viiii LIST OF ILLUSTRATIONS............................................................................................ x LIST OF ABBREVIATIONS .......................................................................................... xi ACKNOWLEDGMENTS.............................................................................................. xiv CO-AUTHORSHIP STATEMENT ............................................................................... xv 1. CHAPTER 1: Introduction........................................................................................... 1 1.1 Plasma membrane microdomains and cell surface protein interactions................ 2 1.1.1 Membrane domains and the regulation of protein diffusion at the cell surface ......... 2 1.1.2 Hop diffusion .............................................................................................................. 2 1.1.3 The membrane-skeleton fence model......................................................................... 3 1.1.4 The anchored-transmembrane protein pickets model................................................. 4 1.1.5 Oligomerization-induced trapping.............................................................................. 6 1.2 Magt5 and Protein glycosylation............................................................................... 6 1.2.1 Protein glycosylation in the ER and Golgi ccompartment ........................................ 6 1.2.2 The β1-6N-acetylglucosaminyltransferase V .......................................................... 10 1.3 The galectins: definition and structure.................................................................... 10 1.3.1 The galectins: definition and structure ..................................................................... 10 1.3.2 Galectin-3 (Gal-3)..................................................................................................... 10 1.3.3 Intracellular functions and secretion of Gal-3 .......................................................... 12 1.3.4 Extracellular functions of Gal-3 ............................................................................... 13 1.3.4.1 Gal3 and cell adhesion........................................................................................... 14 1.3.4.2 Gal-3 and receptor signaling.................................................................................. 15 1.3.4.3 Gal-3 and the immune response ............................................................................ 15 1.3.5 Gal-3 and cancer ....................................................................................................... 19 1.4 Lipid rafts ................................................................................................................... 23 1.4.1 Definition.................................................................................................................. 23  iv  1.4.2 Lipid raft functions ................................................................................................... 25 1.5 Caveolae and caveolin proteins ................................................................................ 28 1.5.1 Caveolins and caveolae formation............................................................................ 28 1.5.2 Cav1 and cancer........................................................................................................ 33 1.6 Endocytosis................................................................................................................. 36 1.6.1 Clathrin-mediated endocytosis ................................................................................. 36 1.6.2 Raft-dependent endocytosis...................................................................................... 38 1.6.2.1 Raft-dependent endocytosis encompasses various pathways ................................ 38 1.6.2.2 Cav1 and the regulation of raft-dependent endocytosis ........................................ 41 1.6.2.3 Signaling and raft-dependent endocytosis ............................................................. 45 1.6.2.4 Targets of raft-dependent endocytosis................................................................... 47 1.7 Protein degradation and biogenesis of lysosomal organelles................................. 47 1.7.1 The lysosomes .......................................................................................................... 48 1.7.2 Macroautophagy ....................................................................................................... 49 1.7.3 Other autophagic pathways....................................................................................... 50 1.7.4 Multilamellar bodies................................................................................................. 51 1.8 Hypotheses.................................................................................................................. 52 1.9 References................................................................................................................... 54 2. CHAPTER 2: Plasma membrane domain organization regulates EGFR signaling in tumor cells.................................................................................................... 82 2.1 Chapter summary...................................................................................................... 86 2.2 Introduction ............................................................................................................... 87 2.3 Results......................................................................................................................... 90 2.4 Discussion ................................................................................................................... 95 2.5 Materials and methods ............................................................................................ 100 2.6 Acknoweldgements .................................................................................................. 106 2.7 References................................................................................................................. 125 3. CHAPTER 3: Caveolin-1 negatively regulates cholera-toxin b subunit endocytosis to the Golgi apparatus .............................................................................. 132 3.1 Chapter summary.................................................................................................... 135 3.2 Introduction ............................................................................................................. 136  v  3.3 Results and discussion ............................................................................................. 138 3.4 Materials and methods ............................................................................................ 142 2.7 References................................................................................................................. 154 4. CHAPTER 4: The lipid composition of autophagic vacuoles regulates the expression of multilamellar bodies............................................................................... 158 4.1 Chapter summary.................................................................................................... 162 4.2 Introduction ............................................................................................................. 163 4.3 Materials and methods ............................................................................................ 167 4.4 Results....................................................................................................................... 170 4.5 Discussion ................................................................................................................. 175 4.6 Acknoweldgements .................................................................................................. 179 4.7 References................................................................................................................. 203 5. CHAPTER 5: Concluding chapter .......................................................................... 211 5.1 Cav1 as negative regulator: no need for caveolae?............................................... 211 5.2 How can Cav1 act as a negative regulator?........................................................... 213 5.3 Are Caveolae efficient endocytic structures? ........................................................ 215 5.4 Caveolin-1 and receptor signaling.......................................................................... 216 5.5 How can Mgat5 regulate caveolin-1 expression?.................................................. 218 5.6 Mgat5 and Cav1 interplay in tumor progression ................................................. 219 5.7 Caveolin-1 is a conditional tumor supressor ........................................................ 220 5. 8 Alternative signaling pathways in the absence of Magt5 .................................... 222 5.9 Mgat5 and lipid domains in lysosomal organelle biogenesis ............................... 223 5.10 The role of lipid domains in autophagy ............................................................... 225 5.11 Conclusion .............................................................................................................. 227 5.12: References ............................................................................................................. 229  vi  LIST OF TABLES Table 2.1 Percent mobile fraction and half-life of recovery for CT-B-FITC and EGFR-YFP as determined by FRAP ............................................................................... 123 Table 2.2 Percent mobile fraction and half-life of recovery for EGFR-YFP following treatment with Latrunculin A as determined by FRAP ................................................... 124 Table 4.1 Quantification of MLB expression in Mv1Lu cells and M9 clones transfected with Mgat5 before and after treatment with U18666A and 3-MA ............. 180  vii  LIST OF FIGURES Figure 1.1: The membrane-skeleton fence and anchored-transmembrane protein picket models.................................................................................................................................. 5 Figure 1.2: The biosynthesis of N-glycans......................................................................... 9 Figure 1.3: Retention of cytokine receptor at the plasma membrane by the Mgat5/galectin lattice .................................................................................................. 16 Figure 1.4: Hexosamine regulation of surface glycoproteins and responsiveness to growth and arrest cues ....................................................................................................... 17 Figure 1.5: Structure of Cav1 ........................................................................................... 32 Figure 1.6: Raft-dependent endocytosis and its regulation by Cav1................................ 44 Figure 2.1: Mgat5-/-ESC cells show enhanced responsiveness to EGF ............................ 107 Figure 2.2: Reduced Cav1 levels are associated with tumor growth in an Mgat5-/background ...................................................................................................................... 109 Figure 2.3: Cav1 regulation of EGF signaling is selective for an Mgat5-/- background ........................................................................................................ 111 Figure 2.4: Cav1 regulation of plasma membrane diffusion of CT-B-FITC and EGFR-YFP ...................................................................................................................... 113 Figure 2.5: Cav1 regulation of EGFR signaling and cell surface diffusion requires an intact scaffolding domain but not Y14 phosphorylation............................................. 115 Figure 2.6: The Mgat5/galectin lattice restricts EGFR diffusion and limits interaction with Cav1 domains .......................................................................................................... 117 Figure 2.7: The actin cytoskeleton restricts EGFR mobility .......................................... 119 Figure 2.8: Domain competition between the galectin lattice and oligomerized Cav1 microdomains regulates EGFR signaling ........................................................................ 121 Figure 3.1: Reduced caveolin expression in Mgat5-/-ESC cells is associated with increased raft-dependent CT-B uptake to the Golgi ........................................................ 144 Figure 3.2: Caveolin-1 knockdown by specific siRNA increased raft-dependent endocytosis of CT-B to the Golgi.................................................................................... 146 Figure 3.3: Indirect regulation of CT-B endocytosis by Cav1 ....................................... 148 Figure 3.4: Regulation of CT-B endocytosis by Cav1 requires an intact scaffolding domain but not its phosphorylation on tyrosine 14 ......................................................... 150  viii  Figure 3.5: Dynamin mutant inhibits CT-B transport to the Golgi, but not its internalization .................................................................................................................. 152 Figure 4.1: U18666A treatment induces the accumulation of swollen cholesterol-rich, LAMP-2 positive vacuole................................................................................................ 181 Figure 4.2: U18666A induces MLB expression............................................................. 183 Figure 4.3: U18666A induces MLB expression despite leupeptin treatment................. 185 Figure 4.4: U18666A treatment induces accumulation of swollen LAMP-2 positive vacuoles in the presence of the autophagy inhibitor 3-MA............................................. 187 Figure 4.5: Swollen vacuoles induced by U18666A in the presence of 3-MA lack concentric lamella............................................................................................................ 189 Figure 4.6: 3-MA treatment reduces MDC labelling of swollen, lysosomal vacuoles .. 191 Figure 4.7: Serum starvation stimulates of cholesterol-rich MLB ................................. 193 Figure 4.8: 3-MA treatment does not affect accessibility of LAMP-2 positive vacuoles to fluid phase endocytosis................................................................................. 195 Figure 4.9: 3-MA treatment does not affect fluid-phase endocytosis to lysosomes ...... 197 Figure 4.10: Non-lamellar, cholesterol-rich LAMP-2 positive vacuoles are acidic, lysosomal organelles........................................................................................................ 199 Figure 4.11: An autophagic contribution is required for the formation of phospholipid and cholesterol-rich MLB................................................................................................ 201 Figure 5.1: Thesis model ................................................................................................ 228  ix  LIST OF ILLUSTRATIONS Illustration 2.1: Cover image of The Journal of Cell Biology Vol. 179 No. 2 2007 ....... 83 Illustration 4.1: Cover image of The Journal of Cell Science Vol. 118 No. 9 2005 ..... 159  x  LIST OF ABBREVIATIONS AMF: Autocrine Motility Factor AP-2: Adaptor Protein 2 ARF-6: ADP Ribosylation Factor 6 BCR: B Cell Receptor Brca2: Breast Cancer Gene 2 Cav1: Caveolin-1 Cav2: Caveolin-2 Cav3: Caveolin-3 CCPs: Clathrin Coated Pits CRD: C-terminal carbohydrate Recognition Domain CT-B: Cholera Toxin B subunit CMA: Chaperone-Mediated autophagy DNA: Deoxyribonucleic Acid DOPE: L-α-dioleoylphosphatidylethanolamine DPPC: DiPalmitoylPhosphatidylCholine DRM: Detergent Resistant Membrane E2F1: E2F transcription factor 1 ECM: Extra Cellular matrix EGF: Epidermal Growth Factor EGFR: Epidermal Growth Factor Receptor EMT: Epithelial to Mesenchymal Transition ER: Endoplasmic Reticulum ERK: Extracellular signal Regulated Kinase FN: Fibronectin FOXO: Forkhead FRAP: Fluorescence Recovery After Photobleaching FRET: Fluorescence Resonance Energy Transfer Gal-3: Galectin-3 GDP: Guanosine Diphosphate GFP: Green Fluorescent Protein  xi  GlcNAc: N-acetylglucosamine GlcNAc-TV: ß1,6-N-acetylglucosaminyltransferase V GPI-AP: GlycosylPhosphatidylInositol-Anchored Proteins HPV: Human Pailloma Virus IL-2: Interleukin-2 KIF1A: Kinesin Family Member 1A KIF3C: Kinesin Family Member 3C LacCer: Lactosylceramide LacNAc: N-acetyllactosamine LAMP-2: Lysosomal Associated Protein 2 LatA: Latrunculin A LC3: Microtubule Associates Light Chain 3 LDL: Low Density Lipoprotein L-PHA: L-Phytohemagglutinin Man: Mannose MβCD: Methyl-β-Cyclodextrin MCF-7: Mammary Adenocarcinoma from Caucasian female-7 MCP: Modified Citrus Pectin MEFs: Mouse Embryonic Fibroblasts MLBs: Multilamellar Bodies MMTV: Mouse Mammary Tumor Virus mRFP: Monomeric Red Fluorescent Protein MSD: Mean Square Displacement MSK: Membrane Skeleton MUC-2: Mucin-2 ND: N-terminal Domain NPC: Niemann-Pick type C disease PIP2: Phosphatidylinositol Bisphosphate PI3K: Phosphoinositide-3-kinase PyMT: Polyoma Middle T antigen Rb: Retinoblastoma  xii  SNAP-23: Synaptosome-Associated Protein-23 SNAP-25: Synaptosome-Associated Protein-25 SNARE: Soluble NSF Attachment Receptor SV40: Simian Virus 40 ROS: Reactive Oxygen Species TCR: T Cell Receptor TfR: Transferrin Receptor TGFβ: Transforming Growth Factor beta TGFβR: Transforming Growth Factor beta Receptor UDP: Uridine Diphosphate  xiii  ACKNOWLEDGMENTS Many people have been part of my graduate studies since I started back in Montreal 5 years ago. The following is a list of the people who contributed in some way to this thesis. First, I would like to thank my supervisor, Dr Robert Nabi. With his enthusiasm, his passion and his inspiration, he was a great mentor. His patience and support as well as his crackingof-the-whip were essential for the accomplishment of the work presented here. I also want to thank him for the opportunity to come to UBC. I would like to thank, the members of my supervisory committee: Drs Wayne Vogl, Masayuki Numata and Sharon Gorski. Their comments were always helpful and appropriate. Thanks to past lab members at Université de Montréal : Ginette Gay, Phuong Le, Anaick Lagana, Mohammad Amraei, Hélène Genty, Marylin Registre, Satra Nim, and Hao Pang. They were always there to help the new graduate student I once was. Special thanks to Jacky Goetz, Pascal St-Pierre, Thao Dang, Nathalie Y and Zongjian Jia who made the trip to Vancouver with me. You made the transition easy. Thanks to the other lab members : Heather Stuart, Scott Else, Scott Strugnell (Big Papa), Maria Abramov-Newerly, Liliana Kojic, Bharat Joshi, Anat Messenberg, Trevor Scudamore, Jackie Chan, Justin Shi, Connie Chiu, Fariba Ghaidi and Lei Li. You contributed to make the Nabi lab an extraordinary place to work. Thanks to the other UBC students: Kuljeet and Moni Vaid, Dave Bates, Marcia Graves, Spencer Freeman, Dave Moniz, Robyn Lett, Kamal Garcha, Dina Karamboulas, Arthur Sampaio, Matt Cowan, Martin Williamson and Goeffroy Noel. We spent some great time together in and out of the lab (mostly out!). Finally thanks to my family who supported me for so long.  xiv  CO-AUTHORSHIP STATEMENT Lajoie P, Partridge EA., Guay G., Goetz JG., Pawling J., Lagana A., Joshi, B., Dennis JW. and Nabi IR. 2007. Plasma membrane organization regulates EGFR signaling in tumor cells, J Cell Biol, 179 (2): 341-356 I performed 80% of the experiments and data analyses presented in this paper. I designed the experiments in collaboration with Dr Nabi. I wrote the paper that was revised and edited by Dr Nabi and Dr Dennis. Emily Partridge performed the cytokine signaling experiments presented in figure 1A and B. She also did the experiments relative to the caveolin levels in tumors presented in figure 2D and the colocalization experiments presented in figure 6B. Ginette Gay performed the electron microscopy experiments presented in figure 2C. Jacky Goetz did the western blot on the effect of swainsonine and lactose on caveolin1/2 expression presented in figure 2B. Judy Pawling did the western blot presented in figure 3B. Anick Lagana did the Cav1/2 western blot presented in figure 2B. Dr Bharat Joshi generated the Y14F Cav1 mutant construct.  Lajoie P., Guay G., Dennis J.W. and Nabi I.R. 2005. The lipid composition of autophagic vacuoles regulates expression of multilamellar bodies. J Cell Sci. 118:1994-2003. I performed all the experiments and data analysis presented in this paper. Ginette Guay did the sectioning for the electron microscopy. I designed the experiments with Dr Nabi and wrote the paper that was revised and edited by Dr Nabi and Dr Dennis.  Lajoie P., Nim S., Nabi IR. 2007. Indirect regulation of cholera toxin endocytosis to the Golgi by caveolin-1, In preparation.  xv  I performed all the experiments and data analysis presented in this paper. I designed the experiments with Dr Nabi and we co-wrote the paper. S. Nim participated in the early stage of the study.  xvi  1. Introduction Sorting of proteins and lipids along the secretory pathway is a determinant of composition of biological membranes of the living cell. After their synthesis in the endoplasmic reticulum (ER), proteins transit into the Golgi apparatus where they are modified, sorted and packaged into vesicles for transport to their final destination. Transport of proteins and lipids from the Golgi to the cell surface regulates plasma membrane composition. The concept of membrane lateral heterogeneity emerged from the differential sorting of molecules to the cell surface. The plasma membrane of the cell contains regions of heterogeneity known as microdomains. The partition of lipids and proteins at the cell surface leads to the formation of different microdomains such as lipid rafts, clathrin coated pits and lectin lattices. These microdomains recruit various machineries in order to assemble complexes required for downstream signaling events. Interactions happening at the cell surface trigger internalization of signaling molecules and complexes via specific domains via different endocytic mechanisms. Internalization of cargos via these domains targets various intracellular organelles. Organelles such as early, late and recycling endosomes, multivesicular bodies and other vesicular carriers are responsible for lipid and protein intracellular trafficking. When necessary, lipids and proteins are targeted to lysosomes where they are degraded. All these compartments are dynamic and molecules flow through them maintaining constant trafficking between the secretory and endocytic pathways. In my thesis, I will discuss how protein modification via glycosylation and membrane lipid composition may act at different cellular levels. I will discuss their roles in protein interactions at the plasma membrane, in endocytosis and in the biogenesis of lysosomal organelles.  1  1.1 PLASMA MEMBRANE MICRODOMAINS AND CELL SURFACE PROTEIN INTERACTIONS 1.1.1 Membrane microdomains and the regulation of protein diffusion at the cell surface One important question in cell biology is how the interaction of proteins and lipids within various membrane domains regulate protein movement at the surface of living cells. Recent advancements in single-molecule tracking methods now allow researchers to observe the movement, recruitment and activation of single molecules in the plasma membrane of the cell (Kusumi et al., 2005). In order to characterize the behaviour of proteins at the surface of the cell, researchers have established models that allow them to illustrate and quantify protein movement at the plasma membrane. Over the years, various models intending to describe the diffusion of molecules on plasma membrane have been proposed.  The first model was derived from the fluid mosaic model and was named the twodimensional continuum fluid model. In this model, the viscosity of the liquid embedding the membrane is the dominant factor determining the movement of a given protein, independently of its size (Saffman and Delbruck, 1975). However, in living cell membranes, diffusion coefficients are smaller than in artificial membranes. Also, various membrane molecules such as receptors, form oligomers or macromolecular complexes resulting in reduced diffusion rates and even temporary immobilization (Kusumi and Hyde, 1982; Nelson et al., 1999; Roess et al., 2000). These two observations therefore indicate that the plasma membrane of the living cell cannot be considered a two-dimensional continuum fluid and that alternative models are required. 1.1.2 Hop diffusion model The plasma membrane of the cell is compartmentalized into various compartments that vary in size from 30 to 230 nm (Kusumi et al., 2005). Single-molecule tracking is usually measured in terms of mean-square displacement (MSD).  It was shown that for the  phospholipid L-α-dioleoylphosphatidylethanolamine (DOPE) 85% of the molecules undergo  2  non-random suppressed diffusion, consistent with short-term confined diffusion within a compartment and long-term movement to other compartments. A DOPE molecule is confined within a 230-nm compartment for 11 ms on average before ‘’hopping’’ to an adjacent compartment, and by repeating such temporary confinement and intercompartmental hop movement it undergoes macroscopic diffusion over many compartments (Fujiwara et al., 2002). Hop-diffusion is not only limited to lipids. Plasma membrane proteins also undergo hop-diffusion. It was shown that transferrin and alpha 2-macroglobulin receptors are confined within a compartment and then these molecules hop to an adjacent compartment where they are trapped again (Sako and Kusumi, 1994). If molecules can be trapped within a compartment, an obvious question is: what structures makes the boundaries between these confinement zones? 1.1.3 The membrane-skeleton fence model In different studies, it was shown that disruption of the actin cytoskeleton or deletion of the cytoplasmic domain of transmembrane proteins affects protein and lipid movement at the surface of the cell (Edidin, 1991; Fujiwara, 2002). From these observations, the membraneskeleton model was proposed. In this model, transmembrane protein cytoplasmic domains interact with the cytoskeleton, inducing temporary confinement zones. The transmembrane proteins then undergo hop diffusion to move to an adjacent compartment. In order to be able to undergo hop diffusion, the space between the membrane and the cytoskeleton should allow the passage of the protein transmembrane domain. This space is formed due to thermal fluctuation of these structures, when the actin filaments that form the boundaries of the compartments are temporarily dissociated and the transmembrane protein has sufficient kinetic energy to overcome the confining potential energy of the compartment barrier when it is in the compartment (Kusumi et al., 2005). It was shown that transmembrane protein band-3 diffused 10 times faster in red blood cells lacking spectrin than in normal cells (Sheetz et al., 1980). In these cells, spectrin is the major component of the cytoskeleton. Using fluorescence recovery after photobleaching (FRAP), it was shown that disruption of the cytoskeleton was able to increase lateral diffusion of sodium and potassium ATPases (Paller, 1994). Taken together, these data indicate that the membrane cytoskeleton plays a major role in regulating the diffusion of plasma membrane transmembrane proteins.  3  1.1.4 The anchored-transmembrane protein pickets model As discussed before, the membrane cytoskeleton affects movement of transmembrane proteins via interactions with their cytoplasmic domains. However, this model cannot explain what is regulating the diffusion of phospholipids in the outer leaflet of the plasma membrane. These lipids do not interact directly with the cytoskeleton. This indicates the need for an expended model. It was shown that movement of the phospholipids at the cell surface, using single molecule tracking, was independent of the extracellular domains of plasma membrane proteins and of the extracellular matrix. However, modulation of the cytoskeleton, still had an effect on DOPE diffusion (Fujiwara et al., 2002; Murase et al., 2004). Since DOPE and the cytoskeleton cannot interact directly, a new model was proposed: the anchored transmembrane protein pickets model. In this model, transmembrane proteins line up along the membrane skeleton fences and act as rows of pickets that prevent free diffusion of phospholipids. When these transmembrane proteins are lined up along the cytoskeleton over a certain threshold, they become an effective barrier to phospholipids diffusion (Kusumi et al., 2005). These pickets would not only affect diffusion of phospholipids, but also the movement of all other transmembrane proteins, indicating that they are restrained via two different mechanisms, the protein pickets and their direct interaction with the cytoskeleton (Figure 1.1).  4  Figure 1.1: The membrane-skeleton fence and anchored-transmembrane protein picket models: Interactions of proteins and lipids with the actin cytoskeleton and transmembrane protein pickets restrict diffusion at the cell surface by creating transient confinement zones. Adapted from Kusumi et al, 2005  5  1.1.5 Oligomerization-induced trapping One of the most physiologically relevant consequences of the compartmentalization of the plasma membrane by cytoskeleton fences and transmembrane pickets is the restricted diffusion of membrane molecules upon oligomerization or molecular complex formation. Monomers of membrane molecules may hop over the picket fence line with relative ease, however large complexes or oligomers would likely show a slower diffusion rate. Also, large molecular complexes are more likely to be bound or tethered to the membrane fences, even just temporarily, inducing temporary confinement of the complexes (Kusumi et al., 2005). This phenomenon probably plays a major role in signal transduction. Upon activation by ligand, many receptors are known to form oligomers. The consequent immobilization might contribute to maintaining and stabilizing the complex at the site where the signal was received. This process might be important for localizing signaling pathways at a specific site of the plasma membrane. Localization and lipid in cellular membranes is, in part, determined by their modification along the secretory pathway. One of the major modifications occurring along this pathway is the addiction of sugar residue, called glycosylation.  1.2 MGAT5 AND PROTEIN GLYCOSYLATION 1.2.1  Protein glycosylation in the ER and Golgi complex  Protein structure affects diffusion by regulating transport and interactions with other molecules. In the next section, I will focus on how protein glycosylation may affect glycoprotein behaviour. A large number of cell surface or secretory proteins contain one or more carbohydrate groups. These proteins are glycosylated and called glycoproteins. These carbohydrates are known to be involved in various biological processes. Their presence may affect protein conformation, solubility and resistance to proteolytic digestion (Fukuda, 1991). In eukaryotic cells, sugar residues are commonly linked to four different amino acid residues. They are therefore classified as O linked or O-glycans (linked to the hydroxyl group of serine, threonine or hydroxylysine) and N-linked or N-glycans (linked to the amide nitrogen  6  of asparagine). O-linked glycans on receptors and secreted glycoproteins are generally shorter with often only a few sugar residues, while N-linked glycans present a more complex arrangement (Lodish et al., 2000). The addition of sugar to protein requires energy. This energy is provided by nucleoside mono/diphosphate sugars that are brought to the Golgi from the cytoplasm. The addition of each sugar is catalyzed by a different glycosyltransferase. Mammalian cells utilize only nine sugar nucleotide donors for glycosyltransferases: UDP-glucose, UDP-galactose, UDPGlcNAc, UDP-GalNAc, UDP-xylose, UDP-glucuronic acid, GDP-mannose, GDP-fucose, and CMP-sialic acid. The glycosyltransferase catalyses the transfer of the glycosyl moiety to protein, lipid or sugar, while the released nucleotides are exported from the Golgi (Kapitonov and Yu, 1999). O-linked glycans are synthesized in the Golgi apparatus or in the rough endoplasmic reticulum (RER).  O-glycans are synthesized via the reactions catalyzed by Golgi  glycosyltransferases and sulfotransferases. The sequence of sugar addition in specific Golgi compartments is also controlled by the relative activities of glycosyltransferases acting on common acceptor substrates. If these enzymes are present in the same Golgi compartment, competition may take place, and the relative activities determine the quantities of possible products. The structure of O-glycans is very variable and as many as 8 core structures have been identified (Brockhausen, 1999). N-linked glycan biosynthesis begins in the RER from a common precursor. This precursor oligosaccharide is linked by a pyrophosphoryl residue to dolichol, a long-chain (75 – 95 carbon atoms) polyisoprenoid lipid that is firmly embedded in the ER membrane and acts as a carrier for the oligosaccharide. The structure of the mature precursor contains three glucose (Glc), nine mannose (Man), and two N-acetylglucosamine (GlcNAc) molecules and can be written as (Glc)3 (Man)9 (GlcNAc)2. This precursor is then transferred from the dolichol to the nascent polypeptide on an asparagine residue in the sequence Asn-X-Ser/Thr (Waechter and Lennarz, 1976).  7  Following the addition of the precursor to the nascent protein, 3 glucose and one mannose residues are removed from the oligosaccharide by the successive activity of three enzymes: ER glucosidases I and II and ER mannosidase. The resulting high mannose glycoprotein (Man)8 (GlcNAc)2 is then transported to the Golgi where further modifications occur during its transport from the cis to the trans-Golgi network (Schachter, 1986) (Figure 1.2). The structure of N-linked oligosaccharides is highly variable. This structural variability is dictated by tissue-specific regulation of glycosyltransferase gene expression, availability of sugar nucleotides and competition for acceptor intermediates during glycan elongation (Dennis et al., 1999). In the Golgi, mannosidase I removes three mannose residues to generate high mannose (Man)5(GlcNAc)2 N-glycans. Further modifications of these glycans lead to the synthesis of complex N-glycans. The N-glycan is transported from the cis to the trans-Golgi network. Medial Golgi-branching enzymes N-acetylglucosaminyltransferases I, II, IV and V are encoded in mammals by Mgat1, Mgat2, Mgat4a/b and Mgat5. The activity of these enzymes is dependent on the availability of their common substrate, UDP-GlcNAc (Schachter, 1986). Mgat1 and 2 enzymes each add one GlcNAc residue to generate complex branched N-glycans (Figure 1.2).The Mgat4 enzyme adds a β1,4-linked GlcNAc residue to the α1,3mannose residue while Mgat5 adds a β1,6 linked GlcNAc residue to the α1,6mannose residue. Complex N-glycans are further modified by galactosyltransferases and are variably elongated with poly N-acetyllactosamine (linear repeats of Galβ1,4 GlcNAcβ1,3) by β1,3 N-acetylglucosaminyltransferases. Sialyltransferases add sialic acid residues which become the terminus of the oligosaccharide (Dennis et al., 2002) (See Figure 1.2).  8  Figure1.2: The N-glycan biosynthesis pathway. Abbreviations: oligosaccharyltransferase, OT; the α-glucosidases, GI, GII; the β-N-acetylglucosaminyltransferases, TI, TII, TIII, TIV, TV, T(i); the α1,2mannosidases, MI; α1,3/6mannosidases, MII, MIII; β1,4galactosyltransferases, Gal-T; α-fucosyltransferases, Fuc-T; α-sialyltransferases, SaT; sulfotransferases, SO4-T. Gene names for TI to TV are Mgat1 to Mgat5, respectively. Tun, tunicamycin. The circled numbers 1 to x represent biosynthetically related subsets of glycans. From Dennis et al., 2002  9  1.2.2  The β1-6N-acetylglucosaminyl transferase V  Tri and tetra antennary N-glycans intermediates have been shown to be preferred ligands for the addition of poly N-acetyllactosamine by β1,3GlcNAc-TI and β1,4Gal-TI (Ujita et al., 2000; van den Eijnden et al., 1988). Complex N-glycans containing N-acetyllactosamine antennae are required during mammalian development as their absence in Mgat1 knockout mice is lethal (Ioffe and Stanley, 1994; Metzler et al., 1994). Deficiencies in Mgat2 and Mgat5 acting downstream of Mgat1 lead to reduced branching of N-glycans and mutation of these loci results in a number of cellular defects (Granovsky et al., 2000; Wang et al., 2001). Mgat5 deficiency suppresses the oncogenic potency of polyomavirus middle T (PyMT) transgenic mice (Granovsky et al., 2000). The Mgat5 intermediate (β1,6 branched GlcNAc) is preferentially elongated with poly N-acetyllactosamine producing N-glycans with higher affinity for galectins than less branched structures (Hirabayashi et al., 2002). 1.3 THE MGAT5/GALECTIN LATTICE 1.3.1 The galectins: definition and structure Lectins are a unique group of proteins assigned to provide an interpreter service for the biological information encoded within specific oligosaccharide structures of glycoconjugates (Dumic et al., 2006). Lectins were first identified in plants. In 1888, Herman Stillmark identified the agglutinin of erythrocytes in extracts of castor beans (Stillmark, 1888). The ability of lectins to bind specific glycoconjugates was therefore useful to study changes in glycoprotein expression at the cell surface. It was found that lectins are not restricted to plants and are ubiquitous in nature. They play a major role in the internalization of glycoproteins and their intracellular trafficking (Ashwell and Morell, 1974; Kornfeld, 1986). They also participate in cell-cell communication by interacting with the carbohydrates expressed on the apposing cell (Sharon and Lis, 1989). Galectins are defined by a specific amino acid sequence and by recognition of β-galactoside structures (Barondes et al., 1994). To date, 14 mammalian galectins have been identified. They all share a carbohydrate recognition domain (CDR) of 130 amino acids. Galectins have  10  been classified into 3 sub-types based on the number and the organization of the CRD. The 3 sub-types are: the prototype group, the chimera group and the tandem repeat group (Hirabayashi and Kasai, 1993). The members of the prototype group (galectin-1, -2, -5, -7, 10, -11, -13, and -14) contain only one CRD and are found as dimers. Galectin-3 (Gal-3) is the only identified member of the chimera group. It contains one CRD connected to an unusually long N-terminal proline- and glycine-rich domain (Dumic et al., 2006). The members of the tandem repeat group (Galectin-4, -6, -8, -9, and -12) contain a single polypeptide chain that contains two distinct CRDs separated by a 70 amino acid sequence. These galectins can therefore bind two distinct carbohydrate epitopes. Galectins that contain a single CRD form dimers or oligomers that enable multivalent binding of carbohydrates, a crucial step in some of their biological functions (Dumic et al., 2006). Galectins are found in the cytoplasm, the nucleus, on the cell surface as well as in the extracellular space. The presence of galectins in the extracellular space is due to their secretion via a non-classical pathway, a consequence of the lack of a signal sequence for their translocation into the ER (Hughes, 1999). It was shown that secretion of Gal-3 was not inhibited by drugs affecting the classical secretory pathway involving ER to Golgi trafficking (Sato et al., 1993). If it is known that different galectins might be secreted via different pathways, these secretion pathways have yet to be characterized 1.3.2 Galectin-3 (Gal-3) The most studied member of the galectin family is Gal-3. It was discovered when it was observed that cancer cells aggregate in the presence of glycoproteins such as asialofetuin (Raz and Lotan, 1981). It has been shown to be involved in various cellular processes, under both normal and pathological conditions. Gal-3 is a 29 to 35 KDa protein that was described as mac-2 in murine macrophages (Ho and Springer, 1982). Gal-3 has a unique structure among galectin family members. Its single polypeptide chain contains 2 distinct domains: an N-terminal domain (ND) and a C-terminal carbohydrate-recognition domain. While the Cterminal CRD is involved in the binding of the lectin to carbohydrate, the N-terminal domain is responsible for its oligomerization (Hsu et al., 1992; Mehul et al., 1994). In the presence of multivalent ligands, Gal-3 assembles into pentamers via a process mediated by the ND,  11  consistent with the hypothesis that the ND is involved in Gal-3 oligomerization (Ahmad et al., 2004). The ND is also required for the full biological activity of Gal-3 (Seetharaman et al., 1998). The studies on carbohydrate-binding activity and specificity of Gal-3 identified N-acetyllactosamine (LacNAc, Galβ1,4(3)GlcNAc) as its preferential ligand (Agrwal et al., 1993; Sato and Hughes, 1992). 1.3.3 Intracellular functions and secretion of Gal-3 Its been shown that the Gal-3 sugar binding site can preferentially accommodate long oligosaccharides (Knibbs et al., 1993). Gal-3 binding to its substrate is dependent on its phosphorylation at serine 6 that regulates its binding to carbohydrates (Mazurek et al., 2000). Gal-3 is localized in the cytoplasm of the cell. Various Gal-3 cytoplasmic ligands have been identified. Gal-3 has been shown to bind Bcl-2, a protein involved in the regulation of apoptosis. Gal-3 binds Bcl-2 through its CRD and this interaction can be inhibited by competition with lactose (Yang et al., 1996). Other proteins involved in the regulation of apoptosis have been shown to interact with Gal-3 such as nuclin (Liu et al., 2004), CD95 (Fukumori et al., 2004) and Alix/AIP1 (Liu et al., 2002a). It was demonstrated that cytoplasmic Gal-3 translocates to the perinuclear membrane following apoptotic stimuli. It was proposed that this process is mediated through Gal-3 interaction with synexin, a protein involved in the regulation of apoptosis (Yu et al., 2002). Binding of Gal-3 to K-Ras (EladSfadia et al., 2004; Shalom-Feuerstein et al., 2005) and Akt (Oka et al., 2005) also identified roles for Gal-3 in the regulation of cell proliferation and survival.  Gal-3 is also localized in the nucleus where it regulates many different processes. While the ND of Gal-3 has been shown to bind various nuclear proteins, the import of Gal-3 into the nucleus is driven by the CRD and mutants containing deletions in this particular domain failed to localized to the nucleus (Gaudin et al., 2000). It is still unclear by which mechanism Gal-3 is transported to the nucleus. Cells with high expression of cytoplasmic Gal-3 show no nuclear localization indicating that specific conditions must be in place for Gal-3 import into the nucleus (Sato and Hughes, 1992). The export of Gal-3 from the nucleus is better  12  understood. Gal-3 export can be inhibited by the addition of leptomycin B, a drug that disrupts the interaction between the leucine-rich nuclear export signal and its receptor, CRM1 (chromosome maintenance region 1) (Tsay et al., 1999). In the nucleus, Gal-3 acts as a pre-mRNA splicing factor and is involved in the formation of the spliceosome (Dagher et al., 1995). In breast cancer cells, Gal-3 induces cyclin D1 promoting its activity in human breast epithelial cells independent of cell adhesion through multiple cis-elements, including the SP1 and CRE sites. These data identified a new growth promoting activity of Gal-3 through cyclin D1 induction, and suggested a function of nuclear Gal-3 in the regulation of gene transcription (Lin et al., 2002). Similarly, Gal-3 regulates transcriptional activity of the thyroid-specific transcription factor TTF-1 indicating that it might regulate proliferation of thyroid cells (Paron et al., 2003). Recently, β-catenin, a protein that like Gal-3 shuttles between the nucleus and the cytoplasm in a phosphorylationdependent manner, has been identified as a Gal-3 binding partner. Gal-3 binds to the ßcatenin/Tcf complex, colocalizes with ß-catenin in the nucleus, and induces the transcriptional activity of Tcf-4. These data support a role for Gal-3 in the regulation of Wnt/ ß-catenin pathway (Shimura et al., 2004). Gal-3 therefore possesses the ability to regulate various pathways inside the cell. However, Gal-3 is expressed at the surface of the cell where it is involved in different functions such as the regulation of cell adhesion, receptor signaling and inflammatory responses. 1.3.4 Extracellular functions of Gal-3 Recently, using fluorescence energy transfer (FRET), it has been demonstrated that Gal-3 forms oligomers that bind glycoproteins at the cell surface leading to the formation of a plasma membrane microdomain called the galectin lattice (Nieminen et al., 2007). Gal-3 oligomers at the cell surface have been shown to play a role in various cellular processes, such as cell adhesion, signal transduction, and immune response. These Gal-3 oligomers have high affinity for Mgat5-derived N-glycans (Hirabayashi et al., 2002). It was  13  shown that over expression of Mgat5 in breast carcinoma cells is associated with an increased amount of cell surface Gal-3 (Lau et al., 2007). 1.3.4.1 Gal-3 and Cell adhesion Mgat5-derived N-glycans are expressed on various proteins of the extracellular matrix (ECM) (Dennis et al., 2001). The role of Gal-3 in the modulation of cellular adhesion comes from its ability to bind glycoproteins and glycosylated constituents of the ECM such as laminin (van den Brule et al., 1995), fibronectin (Sato and Hughes, 1992), hensin (Hikita et al., 2000), elastin (Ochieng et al., 1999) and collagen IV (Ochieng et al., 1998). Transfection of cells with Mgat5 can increase the expression of β1-6 branching on integrins α5 and β1, increase cell motility and decrease cell adhesion to the substrate (Demetriou et al., 1995). It has been proposed that the size of Mgat5-derived N-glycans and their binding to galectins might affect the kinetics of interactions between different proteins involved in cell adhesion (Granovsky et al., 2000). Gal-3 has the ability to both potentiate and inhibit cell adhesion in various cell types. For example, it can stimulate the adhesion of neutrophils to laminin (Kuwabara and Liu, 1996) and to endothelial cells (Sato et al., 2002). Overexpression of Gal-3 increases the adhesion of breast cancer cells to various ECM components (Matarrese et al., 2000). However, incubation of the cells with soluble Gal-3 inhibits cellular adhesion of breast tumor cells (Ochieng et al., 1998). The effect of Gal-3 on cell adhesion is also dependent on its concentration. At low concentration, Gal-3 stimulates tumor cell motility (Le Marer and Hughes, 1996), however, at higher concentrations, increased interaction with ECM components inhibits cell migration (Matarrese et al., 2000). At specific concentration, exogenous Gal-3 added to Mgat5-expressing cells activates focal adhesion kinase (FAK) and phosphatidylinositol-3-kinase, recruits α5β1-integrin to fibrillar adhesions and stimulates cell motility. By promoting both α5β1-integrin activation and actin filament turnover, Gal-3 binding controls the translocation of fibrillar adhesion movement along actin stress fibers, fibronectin fibrils stretching and fibronectin polymerization (Lagana et al., 2006). In addition, Gal-3 can mediate homotypic cell-cell adhesion by bridging through branched, soluble  14  complementary glycoconjugates, indicating a role in aggregation of tumor cells in the circulation during metastasis (Inohara et al., 1996). 1.3.4.2 Gal-3 and receptor signaling The ability of Gal-3 to form pentamers at the cell surface leads to the formation of a suprastructure called the galectin lattice. Many cytokine receptors such as epidermal growth factor receptor (EGFR) and transforming growth factor β are N-glycosylated and may contain polylactosamine able to bind cell surface galectins. In mammary tumor cells overexpressing Mgat5, it has been proposed that these cytokine receptors are retained at the cell surface via interaction with the Mgat5-induced galectin lattice enhancing their signaling potential and preventing their downregulation by endocytosis (see Figure 1.3). Conversely, the signaling cytokine responsiveness of these receptors is greatly reduced in tumor cells lacking Mgat5 (Partridge et al., 2004). Cellular transition between growth and arrest is also highly dependent on the expression of Mgat5-derived N-glycans. The hexosamine pathway utilizes substrate (fructose-6-phosphate, glutamine, acetyl-CoA and UTP) to generate UDPGlcNAc, a limiting factor for the generation of GlcNAc branched N-glycans (Broschat et al., 2002). In Mgat5-expressing cells, glycoproteins expressing numbers of N-acetyllactosamine above (such as EGFR) and below (such as TGFβR) the threshold required for galectin binding are generated. Highly glycosylated EGFR associates with galectins, which increases signaling through the PI3K pathway and stimulates growth metabolism through the hexosamine pathway. This results in increased UDP-GlcNAc synthesis and TGFβR glycosylation and consequently association of TGFβR with the galectin lattice. The resulting increased TGFβR signaling subsequently induces growth arrest (Lau et al., 2007) (Figure 1.4).  15  Figure 1.3: Retention of cytokine receptor at the plasma membrane by the Mgat5induced galectin lattice. Following addition of Mgat5 N-glycans on the receptor in the Golgi complex, the receptor is transported to the cell surface where it is bound by the galectin lattice. This interaction reduces internalization of the receptor via endocytosis and prolongs receptor residency at the cell surface. (Adapted from Partrige et al, 2004)  16  Figure 1.4: Hexosamine regulation of surface glycoproteins and responsiveness to growth and arrest cue. Growth factor receptors have high numbers of N-glycans and, therefore, high avidities for the galectin lattice, while TGF-β receptors I and II have low multiplicities (n = 1 and n = 2). Glycoforms generated in the Golgi are above (R*) or below (R) the affinity threshold for stable association with the galectin lattice. In Mgat5−/− cells, insufficient positive feedback to growth signaling (black) results in a predominance of arrest signaling (red). In wild-type cells, (1) stimulation of RTKs in quiescent cells (2) increases PI3K signaling and promotes F-actin remodeling and preferential internalization of low-n receptors (e.g., TβR). (3) This enhances positive feedback to hexosamine/N-glycan processing, which leads ultimately to (4) increasing TβR association with galectins and autocrine arrest signaling. From Lau et al., 2007  17  1.3.4.3 Gal-3 and the immune response Gal-3 is expressed in various immune system cells including T and B cells, eosinophils, basophils and macrophages. The inflammatory response involves the sequential release of specific mediators and recruitment of circulating leukocytes (Rabinovich et al., 2004). Growing evidence now shows that galectins are involved in immune responses. However, while some members of the galectin family are associated with inhibition of the inflammatory response, others are known to initiate, amplify or sustain the signal (Rabinovich et al., 2002). However, its been proposed that the ability of galectins to crosslink various receptors at the cell surface under specific conditions might explain why the same galectin molecule may act as a negative and positive regulator of the inflammatory response depending on the nature of its interaction/binding partners (Sacchettini et al., 2001). In Gal-3 knockout mice, the inflammatory response is reduced following the induction of peritonitis by injection of thioglycollate (Colnot et al., 1998; Hsu et al., 2000). Gal-3 deficient mice sensitized with ovalbumin develop fewer eosinophils and significantly less airway hyperresponsiveness compared to similarly treated wild-type mice (Zuberi et al., 2004). Interestingly, anti-Gal-3 autoantibodies were identified in the serum of Crohn's disease patients, an autoimmune disease (Jensen-Jarolim et al., 2001). In addition, Gal-3 has been identified in synovial fluids of rheumatoid arthritis patients and is believed to play a role in the activation of synovial fibroblasts (Ohshima et al., 2003). Depletion of Mgat5-modified N-glycans by swainsonine treatment (an inhibitor of Golgi αmannosidase II) potentiates antigen-dependent T cell proliferation (Wall et al., 1988). T cell activation requires T cell receptor (TCR) clustering at the site of antigen presentation. Studies of the Mgat5-/- mouse brought new insight into the role of β1-6 branched N-glycans in the regulation of the immune response. Mgat5 deficiency was associated with lower T cell activation thresholds by directly enhancing TCR clustering. Mgat5-/- mice showed kidney autoimmune disease, enhanced delayed-type hypersensitivity and increased susceptibility to experimental autoimmune encephalomyelitis (Demetriou et al., 2001). In the Mgat5 wildtype animal, Gal-3 was associated with TCR at the cell surface, an interaction disrupted by competitive binding with lactose resulting in the production of an Mgat5-/- phenotype. These  18  data suggested that the Mgat5-induced galectin lattice restricts TCR recruitment to the site of antigen presentation (Demetriou et al., 2001). In addition, T cells from Mgat5-/- mice produce more interferon-gamma and less IL-4 compared with wild-type cells. Negative regulation of TCR signaling by β1,6GlcNAc N-glycans promotes development of proallergic Th2 cells and enhances their polarization, suggesting a mechanism for the increased autoimmune disease susceptibility observed in Mgat5-/- mice (Morgan et al., 2004). In resting T cells, partition of CD45 inside and TCR outside microdomains is positively and negatively regulated by the galectin lattice and actin cytoskeleton, respectively (Chen et al., 2007). The previously described role of Gal-3 in cellular adhesion is consistent with these observations. It has been shown that Gal-3 promotes adhesion of human neutrophils to laminin. This involves two separate mechanisms, first, the lectin bridges neutrophils to laminin, in a carbohydrate-dependent and Ca2+-, Mg2+-independent manner, and second, the lectin induces activation of neutrophils, in the presence of the divalent cations, resulting in the positive regulation of other cell adhesion molecules and enhanced adhesion to laminin. These results suggest that Gal-3 may play a role in the passage of neutrophils through the basement membrane at inflammation sites (Kuwabara and Liu, 1996). Gal-3 is therefore both a positive and a negative regulator of inflammatory responses and its effects may vary under specific conditions. 1.3.5 Gal-3 and cancer Cancer arises from a stepwise accumulation of genetic changes that liberates neoplastic cells from the homeostatic mechanisms that govern normal cell proliferation (Hahn and Weinberg, 2002). This process is called cellular trasformation. These genetic mutations lead to aberrant cell growth and tumor formation. Colonization of distant tissues by tumor cells represents the most dangerous attribute of cancer. Metastasis is defined as the progressive growth of cells at a site that is discontinuous from the primary tumor (Welch et al., 2000). Metastatic cells are a specialized subset of tumor cells within a primary tumor mass that have acquired the ability to complete the multistep metastatic cascade. In brief, these cells migrate, disseminate, extravasate, and eventually proliferate at a discontinuous secondary site(s) (Boyd, 1996).  19  There is much evidence that galectins are involved in tumor progression. Malignant transformation is associated with increased β1-6GlcNAc-branching on mature glycoproteins (Pierce and Arango, 1986; Yamashita et al., 1984). The plant lectin leukoagglutinin (L-PHA) binds Mgat5-derived N-glycans preferentially. L-PHA has successfully been used to detect the presence of these N-glycans on tissue sections (Fernandes et al., 1991). β1-6 branched Nglycans have been associated with poor prognosis and decreased survival in both breast and colorectal carcinomas (Fernandes et al., 1991; Seelentag et al., 1998). Mgat5 enzymatic activity is increased in fibroblasts and epithelial cell lines with expression of the oncogenes v-src, T24-H-ras and V-fps as well as in cells infected with polyomavirus or rous sarcoma virus (Dennis et al., 1989; Dennis et al., 1987; Pierce and Arango, 1986; Yamashita et al., 1985). Mammary tumor growth and metastases induced by the polyomavirus middle T oncogene were considerably less in Mgat5-/- mice than in transgenic littermates. The presence of Mgat5 stimulated membrane ruffling and phosphatidylinositol 3 kinase−protein kinase B activation, inducing a positive feedback loop that amplified oncogene signaling and tumor growth in vivo (Granovsky et al., 2000). Using monoclonal antibodies against Gal-3, it has been demonstrated that cell surface Gal-3 is increased in metastasic neoplasic cells compared to untransformed cell lines (Raz et al., 1986). Gal-3 protein and/or mRNA have been detected in many cancer cell lines including melanoma, fibrosarcoma, pancreatic tumor cell lines, adenocarcinoma, leukemia, colon, prostate and breast cancers (Califice et al., 2004). High expression of Gal-3 in cancer cells was also related to higher Gal-3 titre in the serum of patients presenting localized tumors compared to normal individuals and was even higher in the serum of patients presenting with metastasis (Iurisci et al., 2000). The first function of Gal-3 determined in cancer was related to cell-cell interaction. It was observed that cancer cells aggregate in the presence of glycoproteins such as asialofetuin and that protein extracts from these cells induce hemagglutination (Raz and Lotan, 1981). A monoclonal antibody against Gal-3 raised against B16 melanoma cell extracts enriched in Gal-3 inhibited the asialofetuin-induced melanoma and fibrosarcoma cell aggregation and adhesion of cancer cells (Meromsky et al., 1986). Other evidence for the role of Gal-3 in  20  cell-cell adhesion in cancer was later demonstrated by the fact that the cancer associated T antigen, which plays a role in docking breast and prostate cancer cells onto the endothelium, specifically interacts with Gal-3. T antigen bearing glycoproteins are able to mobilize Gal-3 to the surface of the endothelial cells, therefore priming them to harbour metastatic cancer cells (Glinsky et al., 2001). I discussed previously the ability of Gal-3 to bind various ECM substrates such as laminin and fibronectin. That specific capability is a determinant of Gal-3 role in cancer cell adhesion, migration and invasion. The breast carcinoma cell line BT-559 displays more rapid adhesion to laminin, fibronectin and collagen IV upon transfection with Gal-3. Also, the Gal3 transfected cells show an increased ability to invade matrigel at a rate three times faster than the parental line (Warfield et al., 1997). However, it has also been shown that addition of exogenous Gal-3 did not affect the binding of melanoma cells to laminin and therefore did not regulate adhesion of these cells to the substrate (van den Brule et al., 1995). This indicates that Gal-3 may not regulate adhesion of all cancer cells and may therefore be cell type-dependent process. It is interesting to note that Gal-3 is also able to regulate the expression of its ligands. In colon cancer, expression of Gal-3 and MUC2 intestinal mucin have been independently correlated with the malignant behaviour of colon cancer cells. The latter is a major binding partner of Gal-3. Gal-3 expression impacts on the level of expression of MUC2, giving new insight into how Gal-3 expression may be involved in cancer progression (Dudas et al., 2002). It was also shown that in human cancer, MUC-1 is able to regulate Gal-3 expression via a posttranscriptional mechanism. Gal-3 forms a bridge between MUC1 and the epidermal growth factor receptor (EGFR) and that Gal-3 is essential for EGF-mediated interactions between MUC1 and EGFR (Ramasamy et al., 2007) . Further studies have linked the expression of Gal-3 to tumor growth and the metastatic potential of cancer cells. Introduction of recombinant Gal-3 into tumorigenic, weakly metastatic UV-2237-cl-15 fibrosarcoma cells resulted in an increased incidence of experimental lung metastases in nude mice (Raz et al., 1990). However, when transfected  21  into normal fibroblasts, Gal-3 induced anchorage-independent growth in vitro but not tumorogenecity in vivo. These results indicate that expression of Gal-3 is associated with some aspects of transformation and metastasis but not with tumorogenicity per se (Raz et al., 1990). Another study showed that human breast carcinoma cells BT549 form metastatic colonies in the liver upon transfection with Gal-3 while the parental cell line that does not express endogenous Gal-3 display low metastatic potential. It was suggested that Gal-3 enhances metastatic potential via its ability to provide cancer cells with apoptotic resistance (Song et al., 2002). In addition, reduction of Gal-3 using an antisense oligonucleotide in metastatic colon cancer cells (LSLiM6, HM7) resulted in significant decrease in liver colonization while overexpression of Gal-3 in cells with low metastatic potential resulted in increased metastasis (Bresalier et al., 1998). Some reports also described a role for Gal-3 in angiogenesis. It has been reported that Gal-3 induced morphogenesis in vitro as well as angiogenesis in vivo in a dose-dependent manner. BT-549 human breast cancer cells and their Gal-3 transfected clones were injected and tumors derived from cells expressing Gal-3 presented significantly increased microvessel density (Nangia-Makker et al., 2000). It was also shown by the same group that treatment with modified citrus pectin (MCP) is able to prevent Gal-3 dependent angiogenesis (NangiaMakker et al., 2002). Finally, another identified function of Gal-3 in cancer progression is its role as an apoptotic factor. It was reported that Gal-3 can induce apoptosis of human T cells including human peripheral blood mononuclear cells and mouse activated cells. It is thus possible that Gal-3 secreted by tumor cells plays a role in the immune escape mechanism during tumor progression through the induction of apoptosis to cancer-infiltrating T cells (Fukumori et al., 2003). The galectin lattice constitutes a plasma membrane microdomain that may serve as a signaling platform for many signaling events. It is important to note that many other microdomains exist at the cell surface. In addition, I will now focus my attention on one of the most studied, however controversial, microdomains: the lipid raft.  22  1.4 THE LIPID RAFT 1.4.1 Definition The lipid raft hypothesis originated from the discovery that glycosphingolipids cluster in the Golgi apparatus before being sorted at the apical surface of polarized cells (Simons and van Meer, 1988). Further investigations showed that glycosphingolipid clusters are mostly insoluble in Triton X-100 at 4°C, forming detergent-resistant membranes (DRMs). These DRMs have a light buoyant density on sucrose gradients and are enriched in both cholesterol and glycosylphosphatidyl inosotol (GPI)-anchored proteins (Brown, 1994). Using biophysical methods, it was determined that lipid rafts are cholesterol-stabilized microdomains of 50 nm that can diffuse as one entity at the surface of living cells (Pralle et al., 2000). One crucial assessment of the raft hypothesis is that the formation of glycosphingolipid- and cholesterol-rich lipid domains can be driven solely by characteristic lipid-lipid interactions, suggesting that rafts ought to form in model membranes composed of appropriate lipids. This hypothesis was reinforced when it was confirmed by two-photon light microscopy that membrane microdomains consisting of more ordered liquid-phase enriched in glycosphingolipid, such as GM1, can form at the surface of giant unilamellar vesicles (Dietrich et al., 2001). It was postulated that lipid rafts containing a given set of proteins could change their size and composition in response to intra- or extracellular stimuli. This favours specific protein–protein interactions, resulting in the activation of signalling cascades (Simons and Toomre, 2000). The difficulties linked to the characterization of membrane microdomains led to the establishment of various models. One hypothesis was that proteins targeted to lipid rafts have a light buoyant density because they are encased in a shell of cholesterol and sphingolipid (referred to lipid shell). Lipid shells are thermodynamically stable structures that have an affinity for preexisting rafts. Hence, they target the protein they encase specifically to these membrane domains (Anderson and Jacobson, 2002). FRET experiments validated the shell hypothesis by showing that GPI-anchored proteins exist as cholesterol-sensitive microclusters of 5nm containing at most four molecules (Sharma et al., 2004b). Although the shell is proposed to be stably associated with proteins, its lipid component may interact and even interchange with non-shell lipids (Jacobson et al., 2007). It was also proposed that the  23  plasma membrane of the cell could be a mosaic of different microdomains containing fluid inclusions (Maxfield, 2002). A definition of a lipid raft that includes all these complex possibilities was formulated at the Keystone symposium on lipid rafts in 2006: Membrane rafts are small (10–200 nm), heterogeneous, highly dynamic, sterol- and sphingolipidenriched domains that compartmentalize cellular processes. Small rafts can sometimes be stabilized to form larger platforms through protein–protein and protein–lipid interactions (Pike, 2006). Most of the evidence for rafts as functional domains within the cell comes from studies of DRM-association of proteins and the effect of cholesterol modulating agents on these interactions. However, the use of detergents to identify raft-associated proteins is still controversial. It has been shown that different detergents are unlikely to reflect the same aspects of membrane organization and the effect of a given detergent may vary according to cell type (Schuck et al., 2003). While, some people think the DRMs can still give valuable information about raft heterogeneity (Pike, 2004), others do not share that opinion (Lichtenberg et al., 2005). For these reasons, the most robust analysis of lipid raft functions are those in which DRM extraction has been combined with other experimental procedures such as microscopy, which may help to visualize putative rafts. Some of the most successful studies come from studies of haematopoietic cells, especially lymphocytes and mast cells. In these particular cells, plasma membrane signaling molecules can assemble in large signaling complexes that enable them to be visualized by microscopy (Brown, 2006). The T and B cell receptors have been shown to be associated with DRMs and this interaction is cholesterol-sensitive (Cheng et al., 2001; Montixi et al., 1998). Raft components such as GM1 can be observed by microscopic techniques to cluster together upon receptor ligation in T cells (Gaus et al., 2005; Janes et al., 1999; Viola et al., 1999) and mast cells (Thomas et al., 1994). FRET analysis implicates raft-dependent interactions of the Src-family kinase Lyn with the B-cell receptor (Sohn et al., 2006). These data indicate that the definition of lipid rafts is highly dependent on the methods employed to visualize these microdomains. We can expect the definition of lipid raft to undergo continuous change as the technology available for their characterization evolves.  24  The presence of lipid rafts is not limited to the plasma membrane of the cell. Lipid raft microdomains have been localized to the ER through the identification of ER proteins erlin1 and 2 in raft fractions (Browman et al., 2006) and the sigma-1 receptor (Hayashi and Su, 2003). Due to the increase in sphingolipids and cholesterol along the secretory pathway, lipid rafts are believed to become more abundant in the Golgi complex (Eberle et al., 2002). Cholesterol-sensitive lipid raft microdomains have also been identified on the membrane of endosomes (Gagescu et al., 2000; Sobo et al., 2007) and phagosomes (Dermine et al., 2001). 1.4.2 Lipid raft functions Another role of lipid rafts in membrane behaviour is in membrane curvature. Rafts may play a role in vesicle formation by the fact that their existence requires a heterogeneous arrangement of lipids. As liquid ordered domains differ from the surrounding membrane in physical properties, there is a more or less clearly defined boundary between liquid-ordered phase and liquid-disordered phase regions that gives rise to line tension and line energy, the product of line tension and boundary length. Line tension is one of the most important parameters in the understanding of raft behaviour. At a specific point in a membrane, a large domain has a larger boundary length than a small one, and this increase in local line energy may be able to drive vesicle formation (Hanzal-Bayer and Hancock, 2007). Domain-induced budding is opposed by forces that minimize the difference between the membrane’s actual and spontaneous curvature. However, when liquid-ordered domains fuse into a larger platform, local line energy will eventually dominate so that vesicle formation becomes energetically favourable (Liu et al., 2006). One of the major roles of the lipid rafts within the cell is in the regulation of lipid and protein sorting along the secretory pathway. It has been shown that basal and apical membranes of polarized cells differ not only in their protein content but also in terms of lipid composition (Simons and Vaz, 2004). It is proposed that lipid sorting along the secretory pathway occurs by the clustering of glycosphingolipids but not glycerophospholipids via hydrogen bonds. The demonstration that segregation in the late secretory pathway led to a better understanding of spontaneous phase separation in lipid mixtures as the clustering mechanism then led to the  25  current model where sphingolipids and cholesterol accumulate along the secretory pathway by forming membrane domains similar to liquid ordered domains observed in model membranes (Hanzal-Bayer and Hancock, 2007). These domains cannot undergo retrograde transport due to their higher bending rigidity that causes partitioning away from the area of high membrane curvature and eventually from sphingolipid-enriched carriers that are delivered to the apical membrane of the polarized cell. This postulate supports the model in which these preferentially partitioned lipid microdomains affect the delivery of lipidmodified proteins to the apical membrane of the cell (Simons and Vaz, 2004). In contrast to basolateral trafficking, the apical sorting determinants with adaptors and receptors are still unclear; however it is believed that lipid rafts play a major role in the process (Delacour and Jacob, 2006). One of the best examples of involvement of rafts in apical sorting is the delivery of GPI-anchored proteins. These proteins are mainly localized at the apical surface of the cell and they are resistant to detergent extraction at 4°C, an indication of their association with sphingolipid/cholesterol-rich domains (Brown and Rose, 1992). It is known that lipid rafts also play a role in exocytosis. It was shown that actin polymerization within some Golgi-derived vesicles was membrane based. These actin tails or actin comets are observed preferentially on vesicles containing lipid raft markers and their numbers were reduced after cholesterol depletion (Rozelle et al., 2000). In addition, rafts have been implicated in vesicles trafficking along microtubules. Transport along microtubules is dependent on several motor proteins and some of them such as minus-enddirected dynein and KIFC3 and the plus-end-directed KIF1A have been found to be associated with lipid rafts (Allan et al., 2002). It was also found that the activity of some of these motors are highly dependent on phosphatidyl-inositol-4,5-bisphosphate (PIP2) concentration and rafts may therefore not only identify a specific subset of vesicles for motor attachment, but also allow motor activation by regulating PIP2 concentration (Allan et al., 2002). The role of lipid rafts in exocytosis was also confirmed by studies showing that exocytosis was inhibited by cholesterol depletion and that various SNARE proteins have been localized to DRM fractions and their clustering is cholesterol-sensitive (Salaun et al., 2005). About  26  20% of synaptosome-associated protein SNAP-25 and up to 70% of SNAP-23, involved in vesicle fusion, was found to be associated with DRM as a consequence of their palmitylation on four or five cysteine residues (Salaun et al., 2005). Raft association of other proteins involved in the exocytic pathway is less well documented. It has been reported that syntaxins may associate with lipid rafts. Syntaxin-3 has been shown to be localized in DRM, syntaxin4 is equally present in both detergent soluble and insoluble fractions, while syntaxin-2 is excluded from DRM (Delacour and Jacob, 2006). In light of what was discussed previously, it is interesting to note that syntaxin-4 is involved in basolateral trafficking while syntaxin-3 regulates apical trafficking (Simons and Vaz, 2004). Lipid rafts have also been implicated in the regulation of cell migration. One of the basic premises for a cell to move is the acquisition of cell asymmetry. Asymmetric distribution of membrane microdomains has been reported during cell migration but reports are contradictory on the location of these domains. Ganglioside monosialic acid (GM1) enriched domains accumulate at the leading edge of MCF-7 breast carcinoma cells (Manes et al., 1999) and in fibroblasts (Zhao et al., 2002) exposed to an electrical field to induce cell migration. It has been shown that leukocytes accumulate membranes patches enriched in GM3 at the leading edge and in GM1 at the trailing edge (Gomez-Mouton et al., 2001). These differences in localization of raft domains might be due to the mode of cell migration and/or be cell type dependent (Manes et al., 1999). It was proposed that viscoelastic properties of the lipid bilayers of the plasma membrane influence the actin-filament assembly in the migrating cell (Vasanji et al., 2004). It is known that assembly of branched network of filamentous actin (F-actin) is a crucial requirement for cell polarity and migration (Manes et al., 2003). Specific membrane properties regulate actin polymerization at the leading edge. Rac-1, a regulator of actin polymerization at the leading edge binds preferentially to cholesterol-enriched microdomains (del Pozo et al., 2004). Finally, lipid rafts have been associated with the recruitment of various transmembrane receptors. It was shown that the B cell receptor (BCR) rapidly translocates to lipid rafts upon cross-linking indicating a role for this cell surface microdomain in the initial step of the signaling cascade (Cheng et al., 1999).  The EGF receptor was shown to be mainly  27  colocalized with caveolae/raft domains, however, it moves out of this domain following activation by EGF (Mineo et al., 1999). The interleukin-2 receptor is also localized to cell surface rafts from where it is internalized (Lamaze et al., 2001). The TGF-β receptor has also been shown to be internalized via caveolae, and this process is associated with the downregulation of receptor signaling (Di Guglielmo et al., 2003). However, localization to lipid rafts has been associated with enhanced insulin receptor signaling, and this process is inhibited by cholesterol depletion (Karlsson et al., 2004). In the same way, the growth hormone receptor (GHR) as been shown to be localized to a lipid raft fraction, and these plasma membrane domains are believed to play an important role in its signaling cascade (Yang et al., 2004). The localization of receptors in raft domains has been shown to regulate their downstream signaling cascades (Cheng et al., 2001; del Pozo et al., 2004; Matveev and Smart, 2002; Stoddart et al., 2002). Lipid rafts are dynamic, and may diffuse (Pralle et al., 2000; Sprong et al., 2001) and fuse into larger and more stable structures, in response to signaling, forming larger “signal transducing platforms” (Mayor and Rao, 2004; Simons and Toomre, 2000). These larger rafts may contribute to both signal amplification and attenuation (Anderson and Jacobson, 2002; Jacobson and Dietrich, 1999; Kurzchalia and Parton, 1999; Simons and Toomre, 2000). It is believed that lipid rafts not only recruit the signaling machinery required for receptor signaling, but also the machinery required for their internalization. In the case of EGFR, it was shown that rafts may recruit signaling molecules such as Grb2 and Shc. However, EGFR is also internalized from clathrin-coated pits that form via these same raft domains. These findings suggest that rafts may represent a favourable domain for recruitment of machinery required for both signal transduction and endocytosis (Puri et al., 2005). 1.5 CAVEOLAE AND CAVEOLIN PROTEINS 1.5.1 Caveolins and caveolae formation Caveolae were first identified in the 1950’ (Palade, 1953) by electron microscopy and are defined as plasma membrane invaginations of 60 to 80 nm in diameter. Caveolae are expressed in various tissues and cell types such as smooth muscle, fibroblasts, endothelial cells and adipocytes (Parton and Simons, 2007). Caveolae have been implicated in many  28  cellular processes such as endocytosis, transcytosis, potocytosis, calcium signaling and many other signaling events (Anderson et al., 1992; Kurzchalia and Parton, 1999; Lisanti et al., 1995). Identification of caveolin-1 (Cav1) or (VIP-21), the major constituent of caveolae provided the first marker of these structures and contributed to our understanding of their functions (Kurzchalia et al., 1992; Rothberg et al., 1992). Expression of Cav1 in cells that usually lack caveolae like lymphocytes and Caco-2 cells induced caveolae expression (Fra et al., 1995; Li et al., 1998; Lipardi et al., 1998; Vogel et al., 1998; Zhao et al., 2002). Two other isoforms of caveolin are expressed in mammalian cells (Cav2 and 3) with Cav3 being muscle specific. While Cav1 and Cav3 are mostly expressed at the plasma membrane, Cav2 is localized in the Golgi apparatus and is targeted to the surface when complexed with Cav1 (Cohen et al., 2004a; Liu et al., 2002b; Parton and Richards, 2003). All three caveolin isoforms share a common topology with N and C termini in the cytoplasm and a long hairpin transmembrane domain. There are 2 isoforms of Cav1 (α and β). The domain span of the three caveolin proteins is almost the same : the amino terminal domain comprises the first 101 amino acid residues in Cav1- α and 70-86 residues in Cav1- β, Cav-2 and Cav-3. The transmembrane domain occupy 33 amino acids and the C-terminal part of the protein contains 43-44 amino acids (Williams and Lisanti, 2004). (See Figure 1.5) Caveolin proteins are found as monomers in the Golgi apparatus (Pol et al., 2005) but form high molecular weight oligomers upon transport to the plasma membrane (Ren et al., 2004). Cav1 possesses an oligomerization domain that is required for the formation of large oligomers (Schlegel and Lisanti, 2000). It appears that the first step of oligomerization occurs in the ER not long after the synthesis of the protein (Monier et al., 1996). The newlysynthesized Cav1 is then transported to the Golgi apparatus. At this step, Cav1 is still not associated with DRM (Pol et al., 2005). During its subsequent transport through the secretory pathway, Cav1 becomes associated with DRM that are characteristic of plasma membrane caveolin. It was shown that exit of Cav1 from the Golgi complex is accelerated upon addition of cholesterol (Pol et al., 2005) and inhibited after glycosphingolipid depletion (Cheng et al., 2006b). Using Cav1-GFP fusion protein, it was shown that Cav1 transits directly from the  29  Golgi complex to the plasma membrane (Tagawa et al., 2005). Based on the size of the structure exiting the Golgi, it was proposed that Cav1 oligomerizes and associates with cholesterol and glycosphingolipids to form exocytic structures similar to mature plasma membrane caveolae. These structures were named ‘exocytic caveolar carriers’ (Parton and Simons, 2007). It is not known what is driving the budding of these caveolin structures from the Golgi complex. However, it is likely that Cav1 association with cholesterol/sphingolipidenriched raft domains induced vesicle formation involving increased line tension (Parton and Simons, 2007) as discussed in the previous section. While we know that caveolae-like structures are transported from the Golgi complex to the plasma membrane, the site of mature caveolae formation remains elusive. It was first proposed that a threshold level of caveolin is necessary for the formation of plasma membrane caveolae (Fra et al., 1995). If a threshold of caveolin protein is required for the formation of caveolar structure in the Golgi complex, the slow net transit of caveolin out of the Golgi might be sufficient to allow Cav1 to reach that threshold and allow oligomerization and association with lipid rafts (Parton et al., 2006). It was demonstrated that certain antibodies may recognize the Golgi pool of Cav1 but can no longer recognize the protein at the plasma membrane, indicating a change in Cav1 conformation during its transport to the cell surface (Pol et al., 2005). Cholesterol depletion is able to restore this reactivity suggesting that Cav1 association with raft is responsible for that conformational change (Pol et al., 2005). Cav1 is required for caveolae formation; however, the driving force that induces the formation of the vesicle still remains unclear. One of the important properties of Cav1 is its ability to form oligomers. Cav1 forms higher molecular weight oligomers consisting of 14-16 Cav1 molecules (Monier et al., 1996; Sargiacomo et al., 1995). The 41 residue stretch preceding the putative transmembrane domain mediates homo-oligomerization of Cav1 (Sargiacomo et al., 1995). It is likely that Cav1 oligomerization facilitates the formation of Cav1-enriched microdomains at the cell surface. Tryptophan residues at the membrane interface could further increase cholesterol recruitment and insertion of the scaffolding domain into the membrane, achieving greater expansion of the cytoplasmic leaflet of the caveolar bulb (Parton et al., 2006). However, it was shown that Cav1 expression at the plasma membrane is not sufficient to induce caveolae formation. It appears that Cav1  30  incorporation into raft domains at the plasma membrane is a critical determinant in caveolae formation (Breuza et al., 2002). Cav1 also contains a scaffolding domain that has the ability to bind various classes of signaling proteins such as G-protein subunits, receptor and non-receptor tyrosine kinases, endothelial nitric oxide synthase (eNOS) and small GTPases (Okamoto et al., 1998; Parton and Simons, 2007). It has been shown that Cav1 has the ability to bind cholesterol via a putative cholesterol-binding domain (Murata et al., 1995). Therefore, depletion of cholesterol with cyclodextrin disrupts caveolae formation. The glycosphingolipid GD3 was highly enriched in caveolae, whereas GM3, GM1 and GD1a were present inside as well as outside the caveolae membrane (Ortegren et al., 2004). Caveolae therefore represent a sub-domain of the lipid raft microdomains that are stabilized by caveolin proteins.  31  Figure 1.5: The structure of Cav1: Cav1 contains various domains, a transmembrane domain, a scaffolding domain (CSD), an oligomerization domain (OD) and 3 palmitoylation sites. Cav1 contains a tyrosine phosphorylation site on position 14 (Y14). From Head and Insel, 2007  32  1.5.2 Caveolin-1 and cancer Cav1 has been implicated in cellular transformation, tumorogenisis and metastasis for many years, however, its role in cancer progression remains controversial. It was shown that an inverse relationship exists between Cav1 expression and cellular transformation. Upon transformation, NIH-3T3 cells show reduced Cav1 levels that inversely correlated with cell growth in soft agar (Koleske et al., 1995). Using the same soft agar assay, it was also demonstrated that overexpression of Cav1 in transformed cells can inhibit anchorageindependent growth (Engelman et al., 1997; Fiucci et al., 2002). Moreover, Cav1 knockdown in NIH-3T3 cells was sufficient to induce a transformed phenotype, allowing these cells to grow in soft agar and to form tumors in athymic mice (Galbiati et al., 1998). Work using a Cav1 null mouse provided further evidence that Cav1 may act as a transformation suppressor gene. It was shown that Cav1 null mice were more susceptible to chemical carcinogenic treatment, inducing the formation of epidermally derived tumors (Capozza et al., 2003). It was observed that epidermal hyperplasia in these mice was associated with upregulation of cyclin D1 and hyperactivation of ERK-1/2. Also, Cav-/- mouse embryonic fibroblasts (MEFs) derived from these animals were more susceptible to transformation and in vivo tumorogenesis mediated by oncogenes such as H-Ras and v-src (Williams et al., 2004). Analyses of the Cav1 null animals have demonstrated the implication of Cav1 in mammary cancer. It was reported that complete loss of Cav1 accelerates the appearance of mammary dysplastic lesions in polyoma middle T tumor-prone transgenic mice (MMTV-PyMT) (Williams et al., 2003). It was also shown that Cav1 haploinsufficiency leads to partial transformation of human breast epithelial cells. However, haploinsufficiency of Cav1 expression did not induce significant tumor formation when tested in nude mice (Zou et al., 2003). Another group has shown that Cav1 expression is able to inhibit breast cancer growth and metastasis. They showed that Cav1 was expressed in low and non-metastatic tumors but at much higher levels than in highly metastatic tumors. Also, exogenous expression of Cav1 was sufficient to suppress primary tumor growth after inoculation of cells into mammary gland. Expression of Cav1 was also shown to subsequently suppress metastasis to other organs. The same study showed that Cav1 expressing tumor cells showed reduced capacity to  33  invade matrigel, reduced response to laminin-1 and decreased metastasis to the lung and bone (Sloan et al., 2004).  All together, these data suggest that Cav1 may act as a tumor  suppressor. However, the role of Cav1 as a universal tumor suppressor gene is controversial. It has been shown that Cav1 expression is increased in metastasis-derived prostate cell lines compared to primary tumor- derived cell lines. The same group also noted that Cav1 expression was increased in breast intraductal and infiltrating ductal carcinomas as well as nodal disease (Yang et al., 1998).  Also, overexpression of Cav1 correlates with a poor prognosis and  tumor progression in oesophageal squamous cancer (Ando et al., 2007; Kato et al., 2002). Recent data also show that Cav1 expression is also associated with a poor prognosis in human breast cancer (Joshi et al, submitted). These data indicate that Cav1 may also be involved in tumor progression and metastasis. Interestingly, it was proposed that Cav1 expression may vary according to the stage of cancer development. Cav1 reduction might be required for initial transformation and tumor growth, but clonal selection for drug resistance and metastatic potential may require higher Cav1 expression (Bender et al., 2000). Studies have tried to understand the relationship between Cav1 and other oncogenes. One example of these possible interactions between Cav1 and other genes involved in cancer is the relationship between Cav1 and INK4a. Loss of the INK4a locus, encoding both p16INK4a and p19ARF cell cycle regulators, is sufficient to allow cells to become immortalized. It was shown that concomitant loss of Cav1 and INK4a results in cells with a striking proliferative advantage, demonstrating that the loss of Cav1 expression cooperates or synergizes with genetic mutations that abolish INK4a function (Williams et al., 2004). Indeed, transformation of INK4a (–/–)/Cav1(–/–) fibroblasts with oncogenes such as H-Ras and v-src renders these cells more neoplasic, forming much larger tumors in nude mice (Williams and Lisanti, 2005). Association of Cav1 with the tumor suppressor gene p53 has also been reported. In cervical carcinomas, the human papilloma virus (HPV) has been shown to play a role in malignant cellular transformation. Some HPV genes were shown to downregulate the expression of tumor suppressor p53 and Rb. The inactivation of p53 by the HPV genes was shown to be  34  able to downregulate the expression of Cav1. These data suggested that the HPV oncoprotein down-regulates caveolin-1 via inactivation of p53 and that replacement of caveolin-1 expression can partially revert HPV-mediated cell transformation (Razani et al., 2000). It was also shown that transformation of NIH-3T3 cells with Ras or Raf oncogenes is associated with downregulation of Cav1 expression. Interestingly, this decreased Cav1 expression is dependent on the p42/MAP kinase pathway and treatment of these transformed cells with MEK inhibitor PD 98059 is sufficient to restore normal Cav1 expression. The same study showed that the same treatment in v-src and v-Abl transformed cells has no effect, indicating that oncogenes may regulate Cav1 expression via multiple pathways (Engelman et al., 1999). The D7S522 locus on human chromosome 7q31.1 is commonly deleted in a variety of human cancers, including carcinomas of the breast, colon, kidney, prostate, ovary, head, and neck (Williams and Lisanti, 2005). Interestingly, it was shown that both Cav1 and Cav2 genes are localized to this locus and may therefore represent important tumor suppressor genes in this fragile region that is frequently deleted in human cancers (Engelman et al., 1998). In light of all the research done in the caveolin field in relation to cancer progression, one of the questions that remain to be answered is: is Cav1 a tumor suppressor, an oncogene, or both? Williams and Lisanti suggested that at least three mechanisms may inactivate the tumor suppressor functions of Cav1: 1- tyrosine phosphorylation, 2- serine phosphorylation and 3point mutation (Williams and Lisanti, 2005). Cav1 is phosphosphorylated on tyrosine 14 (Y14) via the recruitment of the c-src/Grb7 complex. Functionally, tyrosine 14phosphorylated Cav1 binds Grb7 and enhances both anchorage-independent growth and EGF-stimulated cell migration (Lee et al., 2001). It is also known that Cav1 may be secreted outside the cell and this process required phosphorylation on serine 80 (Schlegel et al., 2001). Mechanistically, in terms of cellular transformation, shunting Cav1 for secretion to the extracellular environment would subvert its normal intracellular tumor suppressive functions. Essentially, these changes in Cav1 membrane topology have the same consequences as a loss of Cav1 expression (Williams and Lisanti, 2005). Finally, results provide genetic evidence that a functioning Cav1 mutation may have a role in the malignant progression of human breast cancer. A mutation in Cav1 at codon 132 (P132L) was found in 16% of primary breast  35  cancers. That specific mutation of caveolin-1 had a dominant negative effect on cell transformation and invasiveness (Hayashi et al., 2001).These data provide genetic evidence that mutations in the Cav1 gene may contribute to breast cancer progression. Cell surface proteins and lipids are heterogeneously distributed within distinct plasma membrane microdomains such as the galectin lattice and lipid rafts. Localization of molecules to specific domains regulates their internalization. The next section will described one of the various pathways by which molecules enter the cell called endocytosis. 1.6 ENDOCYTOSIS Plasma membrane proteins and receptors and their ligands are internalized into the cell via endocytosis. Endocytosis can be classified into different types based on the structure of the initial carriers. In the next section, I will describe these various pathways. 1.6.1 Clathrin-mediated endocytosis In 1964, Roth and Porter described the basic aspects of internalization via clathrin-coated pits (CCPs) by studying yolk protein internalization by electron microscopy (Roth, 1964). Since then, many other cargos have been shown to be internalized via this pathway including immunoglobulin (Rodewald, 1973), low-density lipoproteins (LDL) (Anderson et al., 1976), EGF (Gorden et al., 1978) and transferrin receptors (Tf-R) (Hopkins, 1983). Two different types of clathrin-dependent endocytosis have been described: (1) constitutive endocytosis and (2) receptor-mediated endocytosis. In the first one, any receptor, bound or not to ligand are internalized. Non signaling receptors such as the Tf-R and LDL receptor have been shown to be internalized through this pathway (Anderson et al., 1982; Watts, 1985). In receptor-mediated endocytosis, receptors are internalized upon binding of ligand. Receptors internalized via this pathway include most of the growth factor receptors such as EGFR (Beguinot et al., 1984) and G-protein coupled receptors (GPCR) (Wolfe and Trejo, 2007).  36  In clathrin-mediated endocytosis, receptors are internalized via CCPs after the recognition of a specific motif within their cytoplasmic domains. This motif is recognized by the assembly polypeptide complex 2 (AP-2). It consists of two large, structurally related subunits called and 2-adaptins, a medium subunit, 2, and a small subunit, 2 (Conner and Schmid, 2003). The AP-2 complex induces clustering of receptors within CCPs and therefore favours their internalization. Clathrin is a three-legged structure, called a triskelion, formed by three clathrin heavy chains, each with a tightly associated clathrin light chain (Brodsky et al., 2001). The AP-2 complex is also involved in the recruitment of clathrin molecules and the assembly of CCPs. It recruits clathrin triskelions at the plasma membrane to ultimately form the clathrin lattice or nascent CCPs. The necessary role of AP-2 in clathrin-mediated endocytosis was confirmed when knock-down of AP-2 by siRNA was associated with > 90% decrease in the number of CCPs at the plasma membrane (Motley et al., 2003). Another protein called epsin and its binding partner Eps15 have been shown to be inserted in the membrane of the CCPs and are believed to induce membrane curvature during the formation of CCPs (Ford et al., 2002). The newly formed CCPs then pinch-off the plasma membrane, a process involving dynamin-2. Another protein, amphiphysin, functions as a linker between dynamin and clathrin coats (Takei et al., 1999). Dynamin is believed to act at the neck of the vesicles, generating the mechanical force required for vesicle fission from the plasma membrane (Warnock and Schmid, 1996). After the internalization of CCPs, the clathrin coat disassembles and the vesicles fuse with newly-formed uncoated vesicles to form sorting endosomes (also known as early endosomes). The fusion of CCPs with sorting endosomes is regulated by the GTPase Rab5 (Woodman, 2000). In the now acidic sorting endosomes, some receptors detach from their ligand (Dunn et al., 1989). As more membrane is added to the vesicles, tubular structures begin to extend from the original vacuole. These tubules pinch-off the central vacuoles to form recycling endosomes (Mayor et al., 1993). These vesicles move along the actin cytoskeleton to the plasma membrane. The receptors and membrane are this way recycled back to the plasma membrane. However, some receptors interact with a second set of cytosolic proteins that retains them in the central vacuole leading to the formation of  37  multivesicular endosomes. These will then be delivered to the lysosomes where their content will be degraded. 1.6.2 Raft-dependent endocytosis The following section has been extracted from a review published in the Journal of Cellular and Molecular Medicine (Lajoie and Nabi, 2007). 1.6.2.1 Raft-dependent endocytosis encompasses various pathways Internalization of molecules via clathrin coated pits is the best studied endocytic pathway (Benmerah and Lamaze, 2007; Perrais and Merrifield, 2005; Roth, 2006). Various other pathways, commonly referred to as clathrin-independent, have been identified and are under intense investigation. Some of these pathways are cholesterol-sensitive and therefore considered to be raft-mediated. It is important to recognize that clathrin-mediated endocytosis is also sensitive to acute depletion of cholesterol (Rodal et al., 1999; Subtil et al., 1999) and that raft recruitment precedes clathrin-dependent endocytosis for a number of ligands including EGFR, BCR and anthrax toxin (Abrami et al., 2003; Puri et al., 2005; Stoddart et al., 2002). Raft-dependent endocytic pathways will be defined by their clathrin-independence and cholesterol-sensitivity. A characteristic of some of these raft-dependent pathways is their dependence on dynamin, a molecule involved in vesicular fission from the plasma membrane (Henley et al., 1999). The formation of dynamin-dependent smooth plasma membrane vesicles, or caveolar invaginations can occur both in the presence or absence of caveolins (Le et al., 2002). Similarities between the caveolae and non-caveolin dynamin-dependent raft endocytic pathways have allowed them to be referred inclusively as caveolae/raft-dependent endocytosis (Nabi and Le, 2003). Dynamin-independent raft pathways have been described that are caveolin-independent and invoke macropinocytotic or tubular intermediates (Damm et al., 2005; Holm et al., 1995; Kirkham et al., 2005). However, macropinocytosis has also been shown to be dependent on dynamin in NIH-3T3 (Schlunck et al., 2004) and HUVEC cells (Muro et al., 2003). The dynamin dependence of macropinocytic pathways may be cell type or cargo-specific. While the heterogeneity of raft domains (Hancock, 2006) is certainly  38  indicative of higher orders of complexity and regulation of their endocytosis, to a large extent, and at least for now, raft-dependent endocytic pathways can be classified based on their caveolin- and dynamin-dependence (Figure 1.6 A). This classification is meant to reinforce similarities between raft-dependent endocytic mechanisms at the plasma membrane. Indeed, different cargoes that use similar raft endocytic mechanisms can be internalized via distinct raft domains and targeted to different intracellular sites (Marsh and Helenius, 2006; Nabi and Le, 2003). Some ligands enter the cell via a caveolae-dependent pathway. The simian virus SV40 follows a dynamin-dependent, caveolae-mediated pathway that targets a caveolin-positive endosome, the caveosome, before being delivered to the smooth endoplasmic reticulum (Pelkmans et al., 2001). When stimulated by SV40, caveolin, dynamin and actin are recruited sequentially to the caveolae (Pelkmans et al., 2001). The raft-dependent endocytic pathway of cholera toxin b-subunit (CT-B) has also been characterized as a dynamin-dependent, caveolar pathway (Henley et al., 1998; Nichols, 2002; Oh et al., 1998; Parton et al., 1994). Albumin is internalized via a dynamin-dependent pathway that requires caveolin (Minshall et al., 2000). Endocytosis of the interleukin-2 receptor occurs via a clathrin-independent, cholesterol-sensitive pathway that requires dynamin and is regulated by the RhoA GTPase (Lamaze et al., 2001). In NIH-3T3 cells, the autocrine motility factor receptor is localized to caveolae and internalization of its ligand, AMF, is cholesterol and dynamin-dependent and negatively regulated by Cav1 expression (Benlimame et al., 1998; Le et al., 2002). Acute overexpression of Cav1 has been shown to reduce the raft-dependent uptake of CT-B, AMF and SV40 (Kirkham et al., 2005; Le et al., 2002; Le and Nabi, 2003; Sharma et al., 2004a). A raft-dependent, dynamin-independent pathway has also been described for CT-B and SV40 (Damm et al., 2005; Kirkham et al., 2005) that exhibits similarity to a Cdc42-dependent pathway followed by GPI-anchored proteins (GPI-AP) and fluid phase markers (Sabharanjak et al., 2002). In fibroblasts from Cav1 knockout mice, SV40 exploits an alternate, Cav1independent pathway that is cholesterol and tyrosine kinase dependent but independent of clathrin, dynamin-2, and ARF6 (Damm et al., 2005). A similar pathway has also been described for CT-B in Cav1-/- fibroblasts where it is ARF6-dependent (Kirkham et al., 2005).  39  This pathway invokes not caveolar invaginations but the formation of uncoated tubular endocytic structures and an intracellular dynamin-dependent step for delivery to endosomes and the Golgi apparatus (Kirkham et al., 2005). Internalization of CT-B has also been shown to occur via a dynamin-independent pathway defined not by caveolin but by flotillin, another raft component (Glebov et al., 2006). Macropinocytosis, involving Rac1-dependent membrane ruffling at the plasma membrane, has also been shown to be cholesterol-sensitive, defining another dynamin-independent raft pathway (Grimmer et al., 2002; Schneider et al., 2007). CT-B therefore provides an example of an endocytic ligand internalized by several pathways including clathrin coated pits and both dynamin-dependent and independent pathways (Torgersen et al., 2001). A recent study showed that 50% of CT-B enters the cell via clathrin coated pits with the remainder internalized via dynamin-independent, caveolin-independent uncoated tubules. In the same study, the authors showed that about only 2% of the total pool of Cav1 positive caveolae contributes to the internalization of CT-B, suggesting that internalization of CT-B via caveolae represents only a minor contribution (Kirkham et al., 2005). However, CT-B internalization was found to be deficient in immortalized Cav1-/MEF-derived cell lines (Sotgia et al., 2002) contrasting with the demonstration that primary Cav1-/- MEFs show no difference in CT-B uptake compared to wild-type MEFs (Kirkham et al., 2005). In HeLa cells, depletion of flotillin by siRNA prevents its uptake via a dynaminindependent route and switches it to a dynamin-dependent route (Glebov et al., 2006). Variable cell surface expression of the CT-B receptor, GM1 ganglioside, impacts on the extent of its raft-dependent endocytosis (Pang et al., 2004). In addition, CT-B concentrations used vary significantly (from 0.05 to 10 µg/ml) between studies from different laboratories (Glebov et al., 2006; Kirkham et al., 2005; Le and Nabi, 2003; Pang et al., 2004). Interestingly, in studies defining the dynamin-independent raft pathway, both CT-B and dextran concentrations were relatively low (Kirkham et al., 2005; Mayor et al., 1994). Variable factors, ranging from expression of ligand receptors to raft components, may impact not only on the extent of CT-B uptake but also on its route of entry into different cells or clonal populations of the same cell type.  40  1.6.2.2 Cav1 and the regulation of raft-dependent endocytosis Fluorescence recovery after photobleaching (FRAP) experiments have shown that movement of Cav1 at the cell surface is restricted by cortical actin as well as through interaction with the actin-binding protein filamin (Stahlhut and van Deurs, 2000; Thomsen et al., 2002). Caveolar stability at the plasma membrane suggests that rapid, constitutive internalization and turnover of caveolae is unlikely to occur. Rapid, reversible budding of caveolae, or potocytosis, was originally suggested to regulate folate internalization (Anderson et al., 1992). More recently, TIRF microscopy was used to show that reversible caveolae budding is limited to the subplasma membrane region by the underlying actin cytoskeleton (Tagawa et al., 2005). Disruption of the actin cytoskeleton induces rapid internalization of caveolar vesicles (Conrad et al., 1995; Mundy et al., 2002; Parton, 1994). Recruitment of SV40 to caveolae induces the transient, localized breakdown of the actin cytoskeleton (Pelkmans et al., 2002). Actin depolymerization also induces internalization of tight junction proteins via a caveolae-dependent pathway (Shen and Turner, 2005). However, disruption of the actin cytoskeleton by cytochalasin D in A431 cells inhibited alkaline phosphatase uptake via caveolae (Parton et al., 1994). The submembrane actin cytoskeleton would therefore appear to be a critical regulator of the endocytic potential of caveolae. However, the extent to which an intact actin cytoskeleton promotes or restricts caveolae uptake may be cargo or cell type specific. Several raft-dependent endocytic pathways are regulated via Rho family GTPases. GPIanchored proteins have been shown to be internalized via a cdc42-regulated pathway that is independent of Rho and Rac (Sabharanjak et al., 2002). As mentioned before, the IL-2 receptor is internalized via a pathway blocked by a mutant RhoA (Lamaze et al., 2001). CTB endocytosis to the Golgi apparatus in Cos-1 cells is dependent on RhoG (Prieto-Sanchez et al., 2006). The Menkes disease ATPase (ATP7A) uptake can be inhibited by a Rac-1 dominant negative mutant (Cobbold et al., 2003). Since constituvely active Rac and RhoA have the ability to downregulate clathrin-dependent endocytosis (Lamaze et al., 1996), it appears that regulation of endocytosis via the Rho family GTPases is a complex mechanism that required further characterization of the molecular machinery involved.  41  Threshold levels of Cav1 and cholesterol regulate caveolae formation (Breuza et al., 2002; Hailstones et al., 1998). Various cholesterol modulating agents, including methyl-βcyclodextrin, nystatin and filipin, have been variously shown to inhibit both caveolae expression and raft-dependent endocytosis (Damm et al., 2005; Hailstones et al., 1998; Le et al., 2002; Parpal et al., 2001; Schnitzer et al., 1994; Sharma et al., 2004a). Caveolar endocytosis of various ligands can be significantly increased by addition of cholesterol or glycosphingolipid to human fibroblasts (Sharma et al., 2004a). Using heterokaryons expressing both Cav1-GFP and Cav1-RFP, it has been shown that cholesterol depletion increased exchange between otherwise stable Cav1 positive structures (Tagawa et al., 2005). Cav1 interacts directly with cholesterol (Fielding et al., 2002; Murata et al., 1995) and cholesterol levels in lipid raft fractions obtained from Cav1 expressing cells were 3-4 fold higher than in matched cells lacking Cav1 (Smart et al., 1996). It is possible that Cav1 regulation of raft endocytosis is linked to its ability to sequester cholesterol in raft domains (Figure 1.6 B). Caveolae budding from the plasma membrane and subsequent internalization requires dynamin II which is localized at the neck of the vesicle (Oh et al., 1998). Expression of the K44A dynamin mutant increases the number of caveolae in caveolin-expressing NIH-3T3 cells as well as the formation of morphologically similar invaginations in Ras and Abltransformed NIH-3T3 cells expressing little caveolin (Le et al., 2002). Indeed, caveolae-like structures in cells devoid of caveolin have been reported (Deckert et al., 1996). Several studies have shown that overexpression of Cav1 is associated with reduction, even inhibition of raft-dependent endocytosis (Kirkham et al., 2005; Le et al., 2002; Le and Nabi, 2003; Minshall et al., 2000). Cav1 overexpression was also found to inhibit the non-caveolar, dynamin-independent endocytosis of CT-B (Kirkham et al., 2005). Reduction of Cav1 levels in mammary tumor-derived cell lines is associated with both increased plasma membrane mobility and raft-dependent uptake of CT-B to the Golgi apparatus. Interestingly, regulation of CT-B mobility and endocytosis in these cells occurred at Cav1 levels below the threshold for caveolae formation (Lajoie, Nim and Nabi, unpublished). This suggests that Cav1 may act indirectly to regulate raft-dependent endocytosis by impacting on the composition and endocytic potential of non-caveolar raft domains (Figure 1.6B). Indeed, the idea of dynamic  42  exchange between raft domains is consistent with the ability of raft components, such as Cav1 or flotillin, to impact on the raft-dependent endocytosis of select ligands by modulating the endocytic potential of distinct raft domains.  43  Figure 1.6: Raft-dependent endocytosis and its regulation by Cav1. (A) Multiple endocytic pathways are characterized as raft-dependent and mediate the uptake of various ligands, including but not limited to those indicated. These include dynamin-dependent pathways that invoke caveolae or non-caveolin, caveolar equivalents as vesicular intermediates and that can be referred to as caveolae/raft-dependent endocytosis (Nabi and Le, 2003). Dynamin-independent pathways invoke non-caveolar tubular intermediates as well as macropinocytotic routes of uptake. While similar mechanisms control the uptake of the indicated raft-dependent ligands, they are not necessarily internalized by the same raft domains or follow similar intracellular targeting routes. (B) Cav1 may negatively regulate uptake via the dynamin-dependent, non-caveolin pathway by either stabilizing raft invaginations at the cell surface (1) or by sequestering key components, including cholesterol, dynamin and others, required for raft-dependent uptake (2). Cholesterol is not shown in the flat portion of the membrane to simplify the diagram. LacCer: lactosylceramide; CT-B: cholera toxin b subunit; GPI-AP: glycosylphosphatidylinositolanchored proteins; AMF: autocrine motility factor; IL-2: interleukin-2; SV40: simian virus 40. 44  1.6.2.3 Signaling and raft-dependent endocytosis Treatment of cells with tyrosine kinase inhibitors blocks caveolae endocytosis while addition of the phosphatase inhibitor okadaic acid triggers endocytosis (Kirkham et al., 2005; Parton et al., 1994; Pelkmans et al., 2002). Indeed, the use of the non-specific tyrosine kinase inhibitor genistein is generally recognized as a selective inhibitor of raft-dependent endocytic pathways. Cav1 is phosphorylated by Src kinase at tyrosine 14 (Glenney, 1989) however the role of Cav1 phosphorylation in raft endocytosis is still unclear. Activation of v-src in Rat-1 cells is responsible for Cav1 phosphorylation and is associated with loss of plasma membrane caveolae (Ko et al., 1998). Cav1 phosphorylation is also required for transcytosis of albumin across the endothelial cell monolayer (Tiruppathi et al., 1997). Cav1 phosphorylation on tyrosine 14 was also associated with flattening, aggregation and fusion of caveolae vesicles (Nomura and Fujimoto, 1999). It was also shown that src kinase activity was required for stimulation of caveolae internalization by glycosphingolipids and cholesterol (Sharma et al., 2004a). The predominant cellular location of tyrosine phosphorylated Cav1 is in focal adhesions. Redistribution of tyrosine phosphorylated Cav1 from focal adhesions to caveolae upon cell detachment from the extracellular matrix triggers raft-dependent endocytosis of cholesterol-enriched microdomains, or rafts, and plasma membrane depletion of Rac (del Pozo et al., 2005). In pancreatic cancer cells, EGF stimulation of Src-mediated Cav1 phosphorylation leads to a marked increase in the number of assembled caveolae at the cell surface (Orlichenko et al., 2006). However, whether Cav1 tyrosine phosphorylation is a critical regulator of caveolae internalization remains to be determined. SV40 recruitment to caveolae stimulates local tyrosine phosphorylation. Tyrosine phosphorylation inhibitors do not prevent SV40 recruitment to caveolae but do prevent recruitment of dynamin to caveolae suggesting that tyrosine phosphorylation is crucial for dynamin-dependent caveolae budding (Pelkmans et al., 2002). Similarly, the Src-dependent internalization of albumin via a Gi-coupled Src-dependent pathway (Minshall et al., 2000) requires interaction of its receptor, gp60, with Cav1 (Minshall et al., 2000; Razani et al., 2001). Reduced phosphorylation of dynamin-2 upon expression of dominant negative Src and consequent reduced association of dynamin-2 with Cav1 results in reduced albumin uptake (Shajahan et al., 2004). This suggests that tyrosine phosphorylation regulates caveolar budding by controlling dynamin recruitment to caveolae. However, the requirement for 45  tyrosine kinases in the raft-dependent uptake of AMF in cancer cells expressing low levels of Cav1 (Joshi et al, in preparation) and in the dynamin-independent raft uptake of SV40 in Cav1-/- cells (Damm et al., 2005) is indicative of further complexity for the role of tyrosine phosphorylation in raft-dependent endocytosis. An siRNA screening approach of kinase inhibitors identified a large group of 208 human kinases as regulators of SV40 entry and 39 of them were involved in caveolae/raft trafficking (Pelkmans et al., 2005). Application of a similar approach to other raft ligands may identify common and, potentially, distinct kinases that control raft-dependent endocytosis of various raft ligands. Cav1 has a well-established scaffolding function implicated in the sequestration of cytokine receptors and lipid-anchored signaling intermediates as well as cholesterol (Liu et al., 2002b; Okamoto et al., 1998). Sequestration of EGFR and TGFβR to caveolae and interaction with Cav1 is associated with inhibition of signaling capacity (Di Guglielmo et al., 2003; Mineo et al., 1999; Park et al., 2000; Razani et al., 2001). These studies were later confirmed when it was shown that Cav1 was able to induce sequestration of the receptor (Matveev and Smart, 2002) and to directly bind EGFR (Cohen et al., 2004b; Cohen et al., 2003). Moreover, the second cysteine region of EGFR contains sequences that target the receptor to caveolae/raft domains (Yamabhai and Anderson, 2002). Upon stimulation with EGF, EGFR is no longer localized in low density raft fractions, consistent with its migration from caveolae to clathrin coat pits upon stimulation (Mineo et al., 1999). Cholesterol has been proposed to indirectly regulate EGFR signaling independently of interaction with Cav1 through regulation of the cholesterol content of lipid rafts (Pike and Casey, 2002; Pike et al., 2005; Westover et al., 2003). When stimulated with a high EGF dose, EGFR is internalized via a caveolae/raftdependent pathway associated with ubiquitination of the receptor (Sigismund et al., 2005). Similarly, clathrin-dependent uptake of TGFβR is associated with subsequent signaling events via Smad2 phosphorylation in EEA1-positive endosomes while its caveolae/raftdependent internalization is associated with receptor degradation through binding to the smad7-smurf2 complex (Di Guglielmo et al., 2003). Raft-dependent endocytosis is therefore both regulated by and impacts on cell signaling.  46  Raft-dependent endocytosis therefore includes various endocytic routes, distinct from clathrin-mediated endocytosis that can be classified based on their dependence on Cav1 and dynamin. These pathways share sensitivity to cholesterol depletion as well as to other more selective regulators whose cell-specific expression may impact on the endocytic pathway followed by multiple raft-dependent ligands. By impacting indirectly on raft domain organization, various raft components, including cholesterol, Cav1 and flotillin, regulate raftdependent endocytosis. Cav1 acts as a determinant of raft-dependent endocytosis by stabilizing rafts at the cell surface, via receptor recruitment or through sequestration of cholesterol and other critical determinants of raft-dependent endocytosis. Further study of raft-dependent endocytosis should lead to the further classification and identification of specific regulators of the endocytic potential of these varied pathways. Open questions that remain to be addressed are whether and how cargo impacts on raft-dependent endocytosis. 1.6.2.4 Targets of raft-dependent endocytosis Two raft endocytic cargos, CT-B and SV40 have been show to be targeted to a intracellular organelle called the caveosome (Nichols, 2002; Parton et al., 1994; Pelkmans et al., 2001). The caveosome is a distinct organelle, which unlike endosomes possesses a neutral pH and is labelled by Cav1 (Nichols, 2002). SV40 transits via the caveosome on its way to the ER (Pelkmans et al., 2001). On the other hand, CT-B is internalized to the Golgi apparatus (Kirkham et al., 2005; Le and Nabi, 2003; Nichols, 2002). Another cargo, the autocrine motility factor is internalized to the ER without any know intermediate step (Le et al., 2002; Le and Nabi, 2003). It was also shown that Cav1 can be targeted intracellularly to the lipid droplets, indicating that these structures might be targeted via raft-dependent endocytosis (Le Lay et al., 2006). It was shown that caveolae and endosomes may merge together via a rab5dependent pathway, indicating that clathrin dependent and independent pathways can interact together (Pelkmans et al., 2004). 1.7  PROTEIN  DEGRADATION  AND  BIOGENESIS  OF  LYSOSOMAL  ORGANELLES Proteins from the extracellular space internalized via endocytosis as well as endogenous proteins are continuously degraded by proteases showing different dynamics. This need for  47  protein degradation comes from the fact that the intracellular environment is constantly producing agents with harmful properties. Degradation of the irreversibly damaged proteins is then a necessary process that involves various pathways. The degradation pathways are similar between cells, however, their prevalence may vary according to cell type and cellular conditions (Cuervo, 2004). 1.7.1 The lysosomes The lysosome is the final destination for molecules targeted for degradation from the extracellular space or from within the cell (Kornfeld and Mellman, 1989). It is a membranebound organelle that contains up to 40 degradative enzymes called acid hydrolases. To enable the activity of these enzymes, the interior of the lysosome is maintained at a pH of 4.8. These enzymes are proteases, nucleases, glycosidases and lipases that are required for the degradation of lysosomal contents. The membrane of the lysosome protects the cytoplasm from the degradative activity of the lysosomal enzymes. However, degradation products such as nucleotides, sugars and amino acids can be transported from the lysosome to the cytoplasm. Lysosomal associated membrane proteins (LAMPs) are highly glycosylated, which is believed to protect them from lysosomal degradation (Fukuda, 1991). Newly synthesized acid hydrolases are transported from the Golgi complex to the early endosomes following their binding to the mannose-6-phosphate receptor (M6PR). It was shown that CCPs bud from the Golgi network and are delivered to the endosomes. This process involved the adaptor proteins AP and GGAs (Golgi-localized, γ-ear-containing, ADP ribosylationfactor-binding proteins) (Doray et al., 2002). It appears that AP-3 is the major adaptor protein involved in the traffic of lysosomal membrane proteins to the lysosome (Dell'Angelica et al., 1999; Le Borgne et al., 1998). AP-1 has also been localized to CCPs budding from the Golgi and containing lysosomal membrane proteins (Honing et al., 1996). It was shown that LAMPs are targeted to the plasma membrane before their delivery to lysosomes via endocytosis (Nabi et al., 1991). Lysosomal membrane proteins and lysosomal phosphatase contain a 10-20 amino acids cytoplasmic tail containing a tyrosine based motif that is required for their targeting to the lysosome (Hunziker and Geuze, 1996). Mutations in this motif have been shown to cause redistribution of lysosomal membrane proteins to the  48  cell surface (Ihrke et al., 2000). It is believed that late endosomes fuse with lysosomes to form hybrid organelles. Reformation of lysosomes from the hybrid organelle is believed to involve recruitment of clathrin and AP-2 adaptors and this could help remove endosomal membrane proteins that are not present in lysosomes (Luzio et al., 2000). 1.7.2 Macroautophagy The delivery of material to the lysosomes not only occurs via endocytosis. Intracellular proteins and organelles are also degraded via a pathway called macroautophagy. Macroautophagy involves the sequestration of complete portions of the cytoplasm including soluble proteins but also complete organelles, into a double membrane vesicle known as the autophagosome (Seglen et al., 1996). After fusion with lysosomes, the autophagosome acquires the hydrolases required for the degradation of its content. Macroautophagy is activated during nutrient starvation and becomes the major source of amino acids during the first hours of starvation (Cuervo, 2004). For many years, it was believed that the isolating membrane from which the autophagosome derives comes from the endoplasmic reticulum (Dunn, 1990). However, there is now evidence for de novo formation of this membrane. In yeast, newly identified proteins revealed their arrangement in a vaculoar structure called the preautophagosome.  This represents the starting point of the isolation membrane that  elongates to form the autophagosome (Noda et al., 2002). The formation of the autophagosome requires two forms of conjugation, lipid-protein and protein-protein (Ohsumi, 2001).  The first event involves the conjugation of a single lipid molecule  (phosphatidylethanolamine) to the C-terminus of a protein known as LC3 (microtubuleassociated protein light chain 3). In the second step, a protein, Atg12, binds to another protein, Atg5. This step is required for the targeting of LC3 to the limiting membrane. Protein phosphorylation also plays a major role in the formation of the autophagosome. The presence of the type III phosphatidylinositol (PI) 3-kinase to the preautophagosome is necessary for the localization of the conjugation system to the forming unit. Once the two sides of the autophagosome fuse together, there is a second fusion with the lysosome to form an autolysosome (Cuervo, 2004). However, interaction of the autophagosome with other members of the endocytic pathway have been reported (Gordon and Seglen, 1988), indicating that these two distinct pathways may interact with each other at various points.  49  The regulation of autophagy is a complex process that involves various regulators. Most of the regulation occurs at the initial sequestration step. This step is dependent on PI3K and can be inhibited by various PI3K inhibitors such as 3-methyladenine (3-MA) and wortmanin (Munafo and Colombo, 2001). Regulation of macroautophagy also involves the nutrient sensor kinase, the mammalian target of rapamycin (mTor) . When mTor is not stimulated by nutrient or inhibited by specific inhibitors such as rapamycin, autophagosome formation is favoured (Yang et al., 2005). It was also shown that cholesterol depletion can induce autophagy implicating a possible role for lipid rafts in this process (Cheng et al., 2006a). Autophagy is therefore a tightly regulated pathway whose activation may vary according to cell type and environment. 1.7.3 Other autophagic pathways Macroautophagy is not the only form of autophagy. Microautophagy represents another mechanism by which complete regions of the cytoplasm are delivered to lysosomes. However, in this case, the engulfing membrane is the lysosomal membrane itself, eliminating the need for the formation of an intermediate structure such as the autophagosome. Microautophagy was first identified by electron microscopy as the presence of lysosome-like organelles containing multiple vesicles in their lumen. These multivesicular bodies are different from the ones generated by endocytosis as they contain lysosomal enzymes and are therefore acidic. Interestingly, microautophagy is not affected by nutrient deprivation indicating that this pathway is distinct from macroautophagy (Cuervo, 2004). Another pathway by which cytosolic proteins are targeted to lysosomes was discovered more recently. This pathway is known as chaperone-mediated autophagy (CMA). The major characteristic of CMA is that substrate proteins are directly translocated through the lysosomal membrane into the lumen. All substrate proteins contain a motif, related to the pentapeptide KFERQ, required for targeting to the lysosomal compartment (Dice, 1990). This particular motif is recognized by the chaperone Hsc73. The substrate/chaperone complex is then targeted to the lysosomal membrane where it binds a receptor protein, the lysosomal associated protein type 2a (Lamp2a) (Cuervo and Dice, 1996). It is believed that association of the complex with multiple molecules of Lamp2a creates a discontinuity in the membrane that allows the translocation of the substrate.  Unknown mechanisms then  50  dissociate chaperone, receptor and substrate to allow its degradation. The receptor is then reinserted into the lysosomal membrane (Cuervo, 2004). CMA is activated under stress conditions such as prolonged nutrient deprivation. During starvation, macroautophagy is activated in order to supply the cell with amino acids for protein synthesis. However, after 12 hours of starvation, macroautophagy activity declines in order to prevent the cell from “eating itself’’. CMA is then activated and becomes the major source of amino acids via the degradation of specific cytosolic proteins (Cuervo and Dice, 1998). 1.7.4 Multilamellar bodies Delivery of protein and lipids to lysosomal degradative compartments via multiple pathways such as autophagy and endocytosis consequently impacts on the composition of these compartments. These processes give rise to various types of lysosomal organelles that vary in both lipid and protein composition. Multilamellar bodies (MLBs) are lysosomal organelles containing multiple concentric membrane layers. MLB size varies from 100 to 2400 nm and they are found in various cell types under both normal and pathological conditions. Their principal functions are the storage and the secretion of lipids (Schmitz and Muller, 1991). MLBs are phospholipid-rich organelles composed of 95% dipalmitoylphosphatidylcholine (DPPC). In type II pneumocytes, MLBs are responsible for the storage and secretion of the pulmonary surfactant that prevents alveolae from collapsing during respiration. MLB formation in pneumocytes is dependent on the expression of the surfactant protein B (SP-B) indicating that MLB protein content is a determinant of their expression (Clark et al., 1995). SP-B is synthesized as a pro-protein which is cleaved within late endosomes/multivesicular bodies (MVBs) compartments before its delivery to MLBs (Voorhout et al., 1992). In normal mouse pneumocytes, multivesicular bodies fuse with MLBs (Hallman, 2004). However, in SP-B -/- mouse, no mature MLBs are detected and MVBs fuse with vesicular inclusion bodies (Stahlman et al., 2000). The lysosomal nature of MLBs was demonstrated by the localization of various lysosomal hydrolases to these organelles (de Vries et al., 1985; DiAugustine, 1974; Golfischer et al., 1968). In the lung, MLB formation is therefore dependent on the lipid and protein composition of late lysosomal organelles.  51  In mink type II pneumocytes, it was shown that MLBs are formed via autophagy. It was proposed that autophagy may represent an efficient way to deliver phospholipids to lysosomal organelles (Hariri et al., 2000). Electron microscopy studies of fetal lung also demonstrated coordinated loss of cytoplasmic glycogen granules and the formation of MLBs (Campiche et al., 1963; O'Hare and Sheridan, 1970), supporting a role for autophagy in the incorporation of cytoplasmic components into MLBs. MLBs are also a major characteristic of various lysosomal storage diseases where aberrant accumulation of lipids in lysosomes is observed (Gieselmann, 1995). In Niemman-Pick type C (NPC) disease, accumulation of cholesterol in lysosomes induces the formation of cholesterol-rich MLBS (Blanchette-Mackie, 2000). Recently, it was shown that NPC fibroblasts display increased autophagy, supporting the hypothesis that autophagy is involved in the formation of these organelles in lysosomal storage diseases (Liao et al., 2007; Pacheco et al., 2007). Transfection of mink type II pneumocytes with Mgat5 was able to induce MLB formation. It was suggested that protein glycosylation may protect lysosomal proteins from degradation and therefore contribute to MLB formation (Hariri et al., 2000). In lysosomal storage diseases, deficiency in lysosomal galactosidases and sialidases is associated with MLB formation indicating that modified oligosaccharides within lysosomal organelles can induce lamellar body formation under pathological conditions (Allegranza et al., 1989; Alroy et al., 1985; Amano et al., 1983). MLB formation may therefore be regulated via both protein glycosylation and lipid composition under normal and pathological conditions. 1.8 HYPOTHESES Protein glycosylation and lipids regulate many biological outcomes in mammalian cells under both normal and pathological conditions. However, interplay between N-glycans and other regulators, such as membrane lipid domains, in the control of signaling pathways remains poorly understood. The basic hypothesis of my thesis is that glycosylation products and lipid domains oppose or act together in order to regulate signaling and trafficking at  52  different cellular levels including the cell surface, endocytic compartments and lysosomal organelles. The second chapter of my thesis represents the continuation of the work presented in Partridge et al., 2004. In this paper, it was demonstrated that the Mgat5-induced galectin lattice sequesters EGFR, preventing its downregulation via endocytosis and enhancing its signaling potential. Interestingly, the PyMT-Mgat5-/- mice exhibit reduced tumor growth compared to wild type littermates (Granovsky et al., 2000). It was observed that Mgat5 null cells derived from mammary tumors have differential expression of Cav1 and response to EGF. I therefore postulated that in the absence of the Mgat5-induced galectin lattice, Cav1 may regulate receptor signaling and diffusion at the cell surface. Chapter 2 presents a study on how competitive recruitment of EGFR to different plasma membrane domains, the Mgat5induced galectin lattice and Cav1 oligomers, regulates receptor signaling diffusion and tumor growth. In chapter 3, I investigate the role of Cav1 in raft-dependent endocytosis. Cav1 has been descibed as a negative regulator of raft-dependent endocytosis (Le and Nabi, 2002). Using tumor cells expressing various levels of Cav1 I studied the role of Cav1 in raft-dependent endocytosis. I postulate that Cav1 can act as a negative regulator of endocytosis even at expression levels below the threshold required for caveolae formation Finally, chapter 4 is a study, which follows the previous publication of the Nabi lab showing that Mgat5 overexpression is associated with the biogenesis of MLBs in pnemocytes (Hariri et al., 2000). I postulate that MLBs, a lysosomal organelle, can form via multiple mechanisms and test the effect of cholesterol on MLB formation via autophagy. 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Critical role for galectin-3 in airway inflammation and bronchial hyperresponsiveness in a murine model of asthma. Am J Pathol. 165:2045-53.  81  CHAPTER 2  Plasma membrane domain organization regulates EGFR signaling in tumor cells  A version of this chapter has been accepted for publication in the Journal of Cell biology, 82 Lajoie et al, 2007, Plasma membrane domain organization regulates EGFR signaling in tumor cells, J Cell Biol, 178 (2) 341-356  Illustration 2.1: Cover image, The Journal of Cell Biology Vol. 179 No.2 2007  83  Plasma membrane domain organization regulates EGFR signaling in tumor cells Patrick Lajoie1, Emily A. Partridge2, Ginette Guay3, Jacky G. Goetz1,3, Judy Pawling2, Annick Lagana4, Bharat Joshi1, James W. Dennis2,5 and Ivan R. Nabi1* 1.  Department of Cellular and Physiological Sciences Life Sciences Institute University of British Columbia 2350 Health Sciences Mall Vancouver, British Columbia, Canada V6T 1Z3  2.  Samuel Lunenfeld Research Institute Mount Sinai Hospital 600 University Ave. R988 Toronto, Ontario, Canada M5G 1X5  3.  Dept. of Pathology and Cell Biology Université de Montréal Montreal, Quebec, Canada H3C 3J7  4.  Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, Québec, Canada H4P 2R2  5.  Departments of Medical Genetics & Microbiology and of Laboratory Medicine and Pathology University of Toronto, Toronto, Ontario, Canada  Condensed title: Plasma membrane domain competition for EGFR #Characters: 37,211 Manuscript number: 200611106RR  84  *Corresponding author: Dr. Ivan R. Nabi Department of Cellular and Physiological Sciences Life Sciences Institute University of British Columbia 2350 Health Sciences Mall Vancouver, BC Canada  V6T 1Z3  85  2.1 CHAPTER SUMMARY Macromolecular complexes exhibit reduced diffusion in biological membranes however the physiological consequences of this characteristic of plasma membrane domain organization remain elusive. We report that competition between the galectin lattice and oligomerized caveolin-1 microdomains for EGFR recruitment regulates EGFR signaling in tumor cells. In mammary tumor cells deficient for Golgi β1,6N-acetylglucosaminyltransferase V (Mgat5), a reduction in EGFR binding to the galectin lattice allows increased association with stable caveolin-1 cell surface microdomains that suppresses EGFR signaling. Depletion of caveolin1 enhances EGFR diffusion, responsiveness to EGF, and relieves Mgat5-deficiency imposed restrictions on tumor cell growth. In Mgat5+/+ tumor cells, EGFR association with the galectin lattice reduces first-order EGFR diffusion rates and promotes receptor interaction with the actin cytoskeleton. Importantly, EGFR association with the lattice opposes sequestration by caveolin-1 overriding its negative regulation of EGFR diffusion and signaling. Caveolin-1 is therefore a conditional tumor suppressor, whose loss is advantageous when β1,6GlcNAc branched N-glycans are below a threshold for optimal galectin lattice formation.  86  2.2 INTRODUCTION The plasma membrane of the cell is organized into cell surface domains that include clathrin coated pits, lipid rafts and caveolae. Lipid rafts have been proposed to be transient and dynamic nanodomains of <10 nm in size (Hancock, 2006; Mayor and Rao, 2004). Caveolae are invaginated lipid raft macrodomains (50-150 nm) whose stability at the plasma membrane is attributable in large part to the formation of highly stable oligomers of their coat protein, caveolin-1 (Parton et al., 2006; Thomsen et al., 2002; van Deurs et al., 2003). Clathrin coated pits (100-150 nm) internalize rapidly upon formation at the same plasma membrane site and their lateral cell surface mobility is enhanced by actin cytoskeleton depolymerization (Gaidarov et al., 1999). The membrane skeleton, associated with the cytoplasmic face of the plasma membrane, is composed of a meshwork of actin filaments and associated proteins that form “fences” and “pickets” that restrict molecular diffusion and partition the membrane into compartments that vary in size from fifty to several hundred nanometers in size. Large scale movements require traversing of these compartmental boundaries, via a process called hop-diffusion, providing an explanation for the reduced diffusion of macromolecular complexes in biological membranes (Edidin et al., 1991; Heuser and Kirschner, 1980; Jacobson et al., 1995; Kusumi et al., 2005b; Morone et al., 2006). However, identification of physiological processes regulated by plasma membrane domain compartmentalization remains limited. Molecular crosslinking of raft components has been proposed to generate stabilized domains that promote transmembrane signaling and interaction with the cytoskeleton (Kusumi et al., 2004; Simons and Toomre, 2000). Clustering of the GPI-anchored receptor CD59 was recently shown to result in transient recruitment of Gαi2 and Lyn and immobilization through binding to F-actin, termed stimulation-induced temporary arrest of lateral diffusion (STALL); recruitment of PLCγ to CD59 clusters undergoing STALL results in local IP3-Ca2+ signaling events (Suzuki et al., 2007a; Suzuki et al., 2007b). Single particle tracking analysis has shown that movement of cell surface bound murine polyoma-like virus particles is actinrestricted, cholesterol-dependent and not associated with caveolae or clathrin-coated pits (Ewers et al., 2005). However, transient anchorage of crosslinked GPI-anchored proteins was found to be dependent on caveolin in addition to cholesterol and Src family kinases (Chen et  87  al., 2006). Ras clustering upon activation supports a role for macromolecular complex formation in signal transduction (Murakoshi et al., 2004; Plowman et al., 2005). Glycan-based domains generated by galectin binding to cell surface glycoproteins have been proposed  (Brewer  et  al.,  2002).  The  Mgat5  gene  encodes  β1,6N-  acetylglucosaminyltransferase V (GlcNAc-TV), a Golgi processing enzyme that modifies Nglycans generating high-affinity ligands for galectins (Demetriou et al., 2001). The galectins are a family of β-galactoside-binding proteins with affinities for N-glycans proportional to GlcNAc-branching (Hirabayashi et al., 2002) that can crosslink glycoproteins to form molecular lattices (Ahmad et al., 2004). Close interactions of galectin-3 on the cell surface have recently been shown by FRET demonstrating that galectin-3 can oligomerize to form a lattice (Nieminen et al., 2007). We have shown that binding of Mgat5-modified N-glycans on EGF and TGF-β receptors to galectin-3 opposes receptor loss to constitutive endocytosis and thereby sensitizes cells to cytokines. Blocking clathrin coated pit endocytosis with potassium depletion and lipid raft endocytosis with nystatin rescued cytokine EGF and TGF-β sensitivity in Mgat5-/- tumor cells (Partridge et al., 2004). This suggested that galectin binding protects receptors from negative regulation through interaction with clathrin-coated pits and lipid rafts. Herein, we demonstrate that reduction of EGFR lateral mobility by Mgat5-dependent, galectin-mediated crosslinking limits interaction of the receptor with stable inhibitory domains of oligomerized caveolin. Our data indicates that recruitment to positive regulatory Mgat5/galectin-dependent macromolecular complexes limits large-scale macrodiffusion of EGFR effectively competing with receptor recruitment to other plasma membrane domains. Mgat5 gene expression and its β1,6GlcNAc-branched N-glycan products increase with oncogenic transformation in human cancers of the breast and colon, and contribute directly to tumor progression and metastasis in mice (Dennis et al., 2002). In transgenic mice expressing the PyMT transgene under the control of the mouse mammary tumor virus (MMTV) long terminal repeat, Mgat5-/- mice show delayed tumor development, and considerably fewer lung metastases compared to their Mgat5+/+ littermates (Granovsky et al., 2000). In contrast  88  to the Mgat5-/- background, MMTV-PyMT mammary tumorigenesis is accelerated in Cav1-/mice (Capozza et al., 2003; Williams et al., 2003). Cav1 has been shown to act as a negative regulator of growth signalling (Parton and Simons, 2007; Razani et al., 2000) that, via its scaffolding domain (Okamoto et al., 1998; Smart et al., 1999), recruits EGF, PDGF and TGFβ receptors to caveolae and suppresses responsiveness to these cytokines (Di Guglielmo et al., 2003; Matveev and Smart, 2002; Razani et al., 2001). The CAV1 gene maps to a tumor suppressor locus (D7S522; 7q31.1) frequently deleted in human carcinomas including breast cancer (Williams and Lisanti, 2005). Up to 16% of human breast cancers express a CAV1 P132L mutation that correlates with breast tumor progression and acts as a dominant-negative for scaffold domain-dependent growth suppression (Hayashi et al., 2001; Lee et al., 2002). However, contrasting with its apparent tumor suppressor function, Cav1 expression is associated with a poor prognosis in multiple tumor types, including breast (Savage et al., 2007; Suzuoki et al., 2002; Yang et al., 1999). The demonstration here that Mgat5 expression overrides the tumor suppressor function of Cav1 identifies the latter as a “conditional” tumor suppressor. The Mgat5-dependent galectin-glycoprotein lattice is a positive signalling environment that regulates EGFR mobility and acts dominantly to protect receptors from negative regulation and immobilization through interaction with oligomerized Cav1. Mgat5-dependent expression of the galectin lattice relieves Cav1 suppression in Mgat5+/+ PyMT mammary tumor cells and responsiveness to EGF is rescued in Mgat5-/- tumor cells by reducing Cav1 levels below a threshold. Our results demonstrate competitive recruitment of EGFR to the extracellular galectin lattice and stable caveolin-1 microdomains, and show that the integrity of these domains determines signaling potential and tumor progression.  89  2.3 RESULTS Reduced Cav1 expression is associated with increased EGFR signaling and tumor growth in Mgat5-/- tumor cells The majority of PyMT Mgat5-/- tumors are small (<2 cm3), however a minority (5-10%) of breast tumors in PyMT Mgat5-/- mice display an acceleration of growth, suggesting ‘escape’ from the suppressive effects of the Mgat5-deficiency (Granovsky et al., 2000). Mgat5-/- and “escaper” Mgat5-/-ESC tumor cell lines were established from small and large tumors, respectively, of MMTV-PyMT Mgat5-/- mice. Compared to Mgat5+/+ mammary carcinoma cells, the Mgat5-/- cell line is markedly less sensitive to EGF and TGFβ and is deficient in EMT and fibronectin matrix deposition (Lagana et al., 2006; Partridge et al., 2004). In contrast to Mgat5-/- cells, Mgat5-/-ESC cells display levels of responsiveness to EGF comparable to that of wild-type Mgat5+/+ cells (Figure 2.1A). However, responsiveness to TGF-β in both Mgat5-/- and Mgat5-/-ESC cells is impaired relative to Mgat5+/+ cells (Figure 2.1B). Furthermore, both Mgat5-/- and Mgat5-/-ESC cells are deficient in EMT and fibronectin matrix deposition compared to Mgat5+/+ cells (Figure 2.1C). Therefore, the phenotypic rescue of Mgat5-/-ESC cells, that is permissive for tumor growth in the absence of Mgat5, is associated with increased EGFR signaling but not TGFβ signaling, EMT and FN remodeling associated with Mgat5-dependent invasive tumor cell phenotypes. Both Mgat5-/- cell lines show reduced expression of Cav1 relative to Mgat5+/+ cells by both quantitative immunofluorescence and western blot (Figure 2.2 A,B). However, Cav1, and total Cav, levels were reduced to a significantly greater extent (p<0.05) in Mgat5-/-ESC than in Mgat5-/- cells (Figure 2.2 A,B). Stable infection of Mgat5-/- and Mgat5-/-ESC cells with an Mgat5 expression vector generated rescued Mgat5-/- (Rescue) and Mgat5-/-ESC (ESC-Rescue) cell lines that present restored β1-6GlcNAc-branched N-glycan expression, as verified by labelling with L-PHA-FITC that binds the β1-6 branch of N-glycans (Figure 2.2A). However, Cav1 expression was restored to wild-type levels only upon rescue of Mgat5-/- but not Mgat5/-ESC  cells (Figure 2.2A,B). In Mgat5+/+ cells, Cav1/2 levels were reduced by swainsonine, an  α-mannosidase II inhibitor that blocks N-glycan branching, and by β-lactose, a competitive inhibitor of galectin binding at the cell surface (Figure 2.2B). Expression of Mgat5 and the N-glycan processing pathway can therefore impact on Cav1. This suggests that Mgat5 and  90  the expression of β1,6GlcNAc-branched N-glycans results in positive feedback to increase Cav1 levels. The failure of Mgat5 to restore Cav1 expression in Mgat5-/-ESC cells suggests that additional genetic modifications may occur in the larger “escaper” Mgat5-/- tumors that block Cav1 up-regulation and suppression of growth. Electron microscopic analysis of the cells shows a dramatic reduction in the number of cell surface associated caveolae in both Mgat5-/- and Mgat5-/-ESC cells relative to Mgat5+/+ or Rescue cells, that express elevated Cav1 levels, without affecting the number of clathrin coated pits (Figure 2.2C). Cav1 is required for caveolae formation (Fra et al., 1995) suggesting that the threshold level of Cav1 required for caveolae formation is not attained in either Mgat5-/- cell line. To probe for possible functional interactions between Cav1 and the Mgat5-dependent lattice in tumor cells, we compared Cav1 protein levels and mammary tumor volume in MMTV-PyMT Mgat5+/- and MMTV-PyMT Mgat5-/- mice at 12 weeks of age. Cav1 levels in Mgat5-/- tumor lysates correlated inversely with increased tumor growth, while no correlation with tumor size was observed in Mgat5+/- tumors (Figure 2.2D). Therefore, reduced Cav1 is observed following either chemical disruption of the galectin lattice in Mgat5+/+ mammary tumor cells in vitro and spontaneously with tumor progression in PyMT Mgat5-/- tumors in vivo. Cav1 regulates EGFR mobility and activation in the absence of the galectin lattice To determine whether lower endogenous Cav1 levels in Mgat5-/-ESC cells were permissive for cytokine responsiveness, Mgat5-/-ESC cells were infected with an adenoviral vector for expression of Cav1 (Figure 2.3A). Overexpression of Cav1 protein levels in Mgat5-/-ESC cells suppressed EGF-dependent Erk1/2 phosphorylation and nuclear translocation to levels comparable to untreated Mgat5-/- cells (Figure 2.3A). Cav1 adenoviral infection of Mgat5+/+ cells, Rescue and ESC-Rescue cells did not inhibit EGF responsiveness (Figure 2.3A). This suggests that expression of β1,6GlcNAc branched N-glycans protects EGFR from negative regulation by Cav1. This was confirmed by reducing Cav1 with siRNA in Mgat5-/- cells that enhanced the EGF response, but had no such effect in Mgat5+/+, Mgat5-/-ESC or Rescue cell lines (Fig 2.3B). Thus, Mgat5 expression in tumor cells blocks the ability of Cav1 to act as a suppressor of EGFR signaling.  91  To explore the effects of Cav1 on cell surface raft dynamics, we measured the lateral diffusion rate of GM1-bound cholera toxin b-subunit (CT-B) at the cell surface using fluorescence recovery after photobleaching (FRAP), at room temperature to limit internalization of GM1-bound CT-B (Figure 2.4A, Table 2.1) (Nichols et al., 2001). Mgat5-/ESC  cells exhibited increased CT-B diffusion relative to the other cell lines. Both the rate of  diffusion and fractional recovery of CT-B into the FRAP region was increased in Mgat5-/-ESC cells suggesting that Cav1 contributes to reduced mobility of CT-B. Indeed, surface diffusion of CT-B in Mgat5-/-ESC cells was reduced upon transfection with Cav1-mRFP. Conversely, CT-B diffusion was enhanced by Cav1 siRNA treatment of Mgat5+/+ and Mgat5-/- cells and presented a profile equivalent to that of Mgat5-/-ESC cells. Therefore, the increased Cav1 expression in Mgat5-/- cells relative to Mgat5-/-ESC cells correlates inversely with raft dynamics. As observed for CT-B, EGFR-YFP showed enhanced cell surface diffusion and reduced immobile fraction in Mgat5-/-ESC cells relative to Mgat5-/- cells by FRAP analysis (Figure 2.4B, Table 2.1). Cav1 transfection of Mgat5-/-ESC cells reduced the diffusion rate and increased the immobile fraction, while Cav1 siRNA transfection of Mgat5-/- cells had the opposing effect. In contrast, modulation of Cav1 levels in Mgat5+/+ cells by either adenoviral Cav1 expression or Cav1 siRNA did not impact on EGFR-YFP diffusion (Figure 4B, Table I). Therefore, by limiting exchange of EGFR-YFP between the bleached zone and the rest of the plasma membrane, Cav1 restricts EGFR mobility, but only in the absence of the Mgat5/galectin lattice. In both Mgat5+/+ and Mgat5-/- cells, Cav1 migrates at the bottom of a 5-40% sucrose gradient (Monier et al., 1995; Sargiacomo et al., 1995) in contrast to monomeric RhoA that migrates in lower density fractions (Figure 2.5A). In blue native gels, Cav1 migrates at approximately 500 kD in both cell lines (Figure 2.5B), corresponding to the high molecular mass oligomers of Cav1 reported previously (Monier et al., 1995; Sargiacomo et al., 1995). Therefore, in spite of reduced expression of Cav1 and caveolae, Cav1 still forms stable oligomers in Mgat5-/- cells. In order to compare the behavior of Cav1 at different expression levels, Mgat5-  92  /-ESC  cells were transfected with Cav1-mRFP and diffusion of Cav1 tested by FRAP (Figure  2.5C). The transfected cells were subsequently fixed and labeled for Cav1, in order to compare transfected Cav1-RFP expression levels to endogenous Cav1 levels in Mgat5-/- and Mgat5+/+ cells. Even when expressed at low levels, below those of Mgat5+/+ cells, Cav1 remained highly immobile. Interestingly, the Cav1 mobile fraction, but not t1/2 of recovery, increased significantly (p<0.01) with Cav1 intensity. These results indicate that even when expressed at low levels Cav1 still forms stable oligomers at the plasma membrane. Mgat5-/-ESC cells were transfected with mutants of Cav1, either a Y14F mutation of the tyrosine phosphorylation site or a F92A/V94A mutation of the scaffolding domain (Li et al., 1996; Nystrom et al., 1999). Cav1-dependent inhibition of cell surface diffusion of both CTB and EGFR-YFP (Figure 2.5D, Table 2.1) and of EGFR signaling (Figure 5E) in Mgat5-/-ESC cells is independent of Y14F phosphorylation but requires an intact scaffolding domain. Colocalization of EGFR and Cav1 is similarly increased in Mgat5-/-ESC cells transfected with wild-type Cav1 and Cav1-Y14F, however the Cav1 scaffolding domain mutant shows reduced colocalization with EGFR (Figure 2.5F). Recruitment to the galectin lattice and EGFR diffusion The mobile EGFR-YFP fraction was significantly greater (p<0.01) and the rate of recovery significantly faster (p<0.01) after photobleaching in Mgat5+/+ cells treated with lactose than in untreated or sucrose treated cells, and comparable to that observed in Mgat5-/-ESC cells (Figure 2.6A, Table 2.1). Lactose treatment of Mgat5+/+ also reduces Cav1 levels (Figure 2.2B). To determine whether reduced Cav1 levels are responsible for increased EGFR-YFP mobility, Cav1 transfected Mgat5+/+ cells were treated with lactose. Cav1 overexpression increased recruitment of EGFR to the immobile fraction but did not affect the first-order diffusion rate of EGFR compared to cells treated with lactose alone (Figure 2.6A, Table 2.1). Disrupting the lattice with lactose did not alter CT-B diffusion in Mgat5+/+ cells, suggesting that GM1 is not restricted by galectins (Table 2.1). Mgat5 retroviral rescue of Mgat5-/- cells did not impact on EGFR-YFP mobility however rescue of Mgat5-/-ESC cells, that did not restore Cav1 levels, reduced the rate of recovery and the mobile fraction of EGFR-YFP  93  (Figure 2.6A, Table 2.1). This confirms that galectin binding to N-glycans restricts the rate of diffusion of EGFR-YFP. Colocalization of EGFR with Cav1 is increased in Mgat5+/+ cells relative to Mgat5-/- cells and disruption of galectin binding in Mgat5+/+ cells with lactose, but not sucrose, increases EGFR colocalization with Cav1 to levels observed in Mgat5-/- cells (Figure 2.6B). In order to test Cav1 association with EGFR in live cells, Mgat5-/-ESC and ESC-Rescue cells were cotransfected with Cav1-CFP and EGFR-YFP and time lapse videos acquired every 10 seconds over 5 minutes. Cells were fixed and labeled for Cav1 to determine relative Cav1 levels and average colocalization of Cav1-CFP and EGFR-YFP determined over the course of the movie (Figure 6C, Supplementary movies 1-4). In ESC-Rescue cells, Cav1 association with EGFR was significantly lower (p<0.01) than in Mgat5-/-ESC cells, irrespective of Cav1 levels. These data are consistent with the competitive exchange of EGFR between two cell surface domains, a signaling-competent Mgat5-dependent lattice and a negative regulatory Cav1enriched microdomain. The actin cytoskeleton restricts the mobility of EGFR-YFP in Mgat5+/+ cells To assess the role of the actin-based membrane skeleton on EGFR-YFP diffusion, the actin cytoskeleton was disrupted by treatment with latrunculin A (LatA). Phalloidin labeling of LatA treated cells shows a loss of actin stress fibers and reduction of total F-actin in Mgat5+/+ and Mgat5-/- cell lines (Figure 2.7 A). Disruption of the actin cytoskeleton with LatA significantly increased (p<0.05) the mobile fraction of EGFR-YFP in Mgat5+/+ cells (Figure 2.7B, Table 2.2). The effect of LatA on EGFR stabilization in Mgat5+/+ was also observed in Mgat5+/+ cells overexpressing Cav1 or transfected with Cav1 siRNA (Figure 2.7B, Table 2.1). LatA treatment had no effect on the rate of diffusion or the immobile fraction of EGFR in Mgat5+/+ cells treated with lactose or in either Mgat5-/- cell line (Figure 2.7C). Latticeassociated EGFR therefore shows preferential interaction with the actin cytoskeleton relative to EGFR in the absence of the lattice. However, disruption of the actin cytoskeleton with LatA does not alter the first-order rate of EGFR diffusion. This suggests that the galectin lattice and F-actin act together to regulate the mobility of the non-Cav1 associated fraction of EGFR.  94  2.4 DISCUSSION Competition between the galectin lattice and Cav1 domains regulates EGFR signaling In this study, we show that negative regulation of EGFR through recruitment to Cav1 microdomains is opposed by expression of β1,6GlcNAc-branched N-glycans. Mgat5 and Cav1 therefore interact to regulate growth signaling and tumor progression. The galectin lattice impedes the diffusion rate of EGFR, confirming our earlier report that the lattice represents a surface microdomain that limits EGFR downregulation by endocytosis (Partridge et al., 2004). Lactose-mediated disruption of the galectin lattice in wild-type Mgat5+/+ cells increases EGFR first-order diffusion while restoration of the lattice by Mgat5 expression in Mgat5-/-ESC cells restricts EGFR diffusion independently of Cav1 expression. Cav1 overexpression reduces EGFR in the mobile fraction but does not suppress the effect of lactose on the first-order rate of diffusion of EGFR dynamics. The Cav1 microdomain and galectin lattice are therefore distinct cell surface domains that differentially regulate the distribution and dynamics of EGFR. Deletion of the EGFR cytoplasmic domain did not impact on EGFR lateral mobility leading to the suggestion that extracellular interactions constrain the lateral diffusion of EGFR (Livneh et al., 1986). Mgat5 deficiency and lactose competition have previously been shown to inhibit galectin binding to EGFR (Partridge et al., 2004), identifying a requirement for extracellular galectin binding to N-glycans in the regulation of EGFR diffusion at the cell surface. We envisage that recruitment to the galectin lattice reduces the propensity of EGFR to perform hop diffusion. Disruption of the actin cytoskeleton with LatA increased the mobile fraction but not the rate of diffusion of EGFR-YFP, distinguishing its action from lactose-mediated disruption of the lattice. While we cannot exclude the possibility of incomplete disruption of the submembrane actin cytoskeleton by LatA (Lagana et al., 2006), our data are consistent with a role for membrane protein density as a key regulator of diffusion rates in biological membranes (Frick et al., 2007; Fujiwara et al., 2002). Importantly, the effect of actin cytoskeleton disruption was not observed in Mgat5-/- cells or Mgat5+/+ cells treated with lactose where the galectin lattice is reduced. Therefore, galectinbound EGFR is also stabilized by the actin cytoskeleton. It is likely that galectin cross-links  95  EGFR to other actin-associated membrane glycoproteins generating actin-stabilized signaling domains (Figure 8). It is important to note that the scale of measurement, with respect to both time and domain size, using FRAP is dramatically larger than that measured by single particle tracking (Kusumi et al., 2005a). It is therefore not clear whether the Cav1-dependent immobilization of EGFR that we have measured by FRAP is equivalent to the cholesterol-dependent transient anchorage observed by single particle tracking (Chen et al., 2006; Dietrich et al., 2002; Ewers et al., 2005; Pralle et al., 2000; Smith et al., 2006). The immobile fraction detected by FRAP in our study reflects those EGFR-YFP molecules whose interaction with other plasma membrane components, such as Cav1, the galectin lattice or the actin cytoskeleton, constrains their ability to exchange freely with fluorescent EGFR-YFP outside the bleached region. This may not necessarily reflect stable, or even direct, molecular interactions, but rather the preferential recruitment of EGFR to microdomains that restrict protein exchange across membrane barriers (Figure 8). Cav1 microdomains Activation of EGFR has been shown to occur in non-caveolar raft domains that associate with nascent coated pits (Puri et al., 2005). Similarly, in Mgat5+/+ cells, blocking coated pit endocytosis by K+ depletion leads to precocious activation of Erk and growth signaling, that can be suppressed by disruption of rafts with nystatin (Partridge et al., 2004). Negative regulation of EGFR diffusion and signaling by Cav1 oligomers is consistent with the previously reported stable interaction of EGFR with Cav1 and caveolae (Couet et al., 1997; Matveev and Smart, 2002; Mineo et al., 1999). However, the ability of Cav1 to form immobile oligomers that associate with and regulate EGFR diffusion and signalling at levels below the threshhold for caveolae formation argues that oligomerized Cav1 can functionally sequester EGFR independently of caveolae formation. Indeed, in endothelial cells, overexpression of Cav1 inhibits eNOS activity without increasing caveolae expression suggesting that a pool of Cav1 outside of caveolae may be responsible (Bauer et al., 2005; Parton and Simons, 2007).  96  Freeze-etch studies have identified a striated caveolin coat on flat membrane domains as well as caveolae (Rothberg et al., 1992) and threshold levels of Cav1 in cell surface domains are required for caveolae formation (Breuza et al., 2002). Furthermore, in contrast to the caveolae-rich basolateral surface of MDCK cells, the apical surface expresses Cav1 but no caveolae (Verkade et al., 2000). Caveolin forms stable oligomers (Monier et al., 1995; Sargiacomo et al., 1995) and the caveolin coat of vesicular transporters is highly stable (Pelkmans et al., 2004). Cav1 regulation of EGFR signaling and dynamics in Mgat5-/- cells that express few caveolae suggests that the regulatory function of Cav1 is dependent on Cav1 oligomerization but not necessarily on caveolae formation. Cav1 oligomers show a reduced mobility relative to larger (more intense) Cav1 structures (Figure 2.5 C), perhaps reflecting increased dynamics and exchange of Cav1 in caveolae. However, in the absence of Mgat5 expression, Cav1 at varying expression levels functions equivalently to regulate the diffusion of CT-B and EGFR as well as EGFR signaling. In blue native gel analysis, Cav1 in Mgat5-/- cells migrates as a sharp band indicative of a highly stable oligomeric configuration. Similar SDS stable oligomers were predicted to contain 15 caveolin molecules (Monier et al., 1995). This is considerably less than the predicted 145 Cav1 molecules per caveolae (Parton et al., 2006) and consistent with the reduced intensity and size of the Cav1 spots detected in cells expressing reduced levels of Cav1, such as Mgat5-/- cells. The stable interaction of EGFR with Cav1 oligomers argues that these domains form a stable platform for recruitment of receptors and other interacting proteins. While the spatial relationship of Cav1 oligomers, the galectin lattice and the membrane skeleton remains uncertain, we suggest that the reduced mobility of both Cav1 oligomers and the galectin lattice is due to the reduced ability of proteins and lipids recruited to these macromolecular domains to undergo hop-diffusion (Figure 2.8). Cav1 is a conditional tumor suppressor Spontaneous down-regulation of Cav1 in Mgat5-/- tumor cells argues that these conditions select for relief from Cav1-mediated negative regulation of signaling at the cell surface. In Mgat5+/+ cells, inhibition of the lattice with swainsonine or lactose treatment reduced Cav1  97  expression, while Mgat5 rescue of Mgat5-/- cells restored Cav1 levels. This suggests that β1,6GlcNAc branching is an upstream regulator of Cav1. The inability of Mgat5 rescue to restore Cav1 levels in Mgat5-/-ESC cells is suggestive of Cav1 loss due to a stable genetic change. Moreover, the inverse correlation between Cav1 levels and tumor size in Mgat5-/tumors suggests that reducing Cav1 expression is one mechanism that can relieve growth restriction imposed by Mgat5 deficiency. PyMT Mgat5-/- mice display a dramatic reduction in the incidence of tumor metastasis, even in those animals that develop ‘escaper’ fast-growth tumors (Granovsky et al., 2000). In this regard, while responsiveness to EGF is largely restored by Cav1 suppression in Mgat5-/-ESC cells, it does not rescue the lattice-dependent deficiency in TGFβ signaling, EMT or FN fibrillogenesis. This suggests that Mgat5 and β1,6GlcNAc-branched N-glycans play additional roles, distinct from Cav1 regulation, in tumor cell polarity, motility and invasion (Cheung and Dennis, 2007). In contrast to Mgat5-/- tumor cells, EGFR diffusion and responsiveness to EGF are not altered by either overexpression or partial knock-down of Cav1 expression in Mgat5+/+ cells. This suggests that β1,6GlcNAc branched N-glycans and lattice retention of EGFR override negative regulation by Cav1. Although Mgat5-deficient tumor cells are partially depleted of surface EGFR due to constitutive endocytosis (Partridge et al., 2004), reducing Cav1 levels in the cells appears to compensate by increasing the availability of EGFR to liganddependent activation. We conclude that, unlike Mgat5-/- cells where Cav1 expression is significantly higher (p<0.05), Cav1 levels in Mgat5-/-ESC cells are below the threshold required for suppression, leaving an estimated 10-15,000 surface EGFR available for optimal activation of the MAPK activation (Lau et al., 2007; Partridge et al., 2004). Surface residency of EGFR in the lattice is therefore permissive for ligand activation and limits both constitutive endocytosis (Partridge et al., 2004) and sequestration by inhibitory, immobile Cav1 domains (Figure 8). Thus, Cav1 depletion enhances the availability of surface EGFR on Mgat5-/- cells, thereby removing a negative regulator of growth (Figure 8). We suggest that Cav1 loss compensates for ~five-fold decreased in surface EGFR numbers observed in Mgat5-/- cells (Partridge et al., 2004), and is thus epistatic for EGF sensitivity.  98  There are only 400-800 TGFβ receptors per cell with a short surface half-life (t1/2 ~ 2h) compared to >105 surface EGFRs with a t1/2 > 6h. TβRII has few N-glycans (n=2) compared to EGFR (n=8) and is therefore relatively more dependent on the branching of its N-glycans for residency in the lattice at the cell surface (Lau et al., 2007). In contrast, EGFR has both greater affinity for the lattice due to higher N-glycan number and greater sensitivity to regulation by Cav1 microdomains. Our data therefore argue that affinity for the galectin lattice and Cav1-enriched microdomains partners with receptor endocytosis rates to determine receptor availability to ligand. Finally, elevated Mgat5 expression in advanced tumors may render the suppressor function of Cav1 redundant by maintaining receptor tyrosine kinases in the galectin lattice within a physical spacing that precludes association with Cav1 microdomains. These results provide support for Cav1 as a conditional tumor suppressor, whose loss is advantageous when β1,6GlcNAc branched N-glycans are below a threshold for optimal lattice formation. Depletion of Cav1 or increased Mgat5 expression both support early-stage tumor growth; importantly, expression of the latter will permit the elevated Cav1 levels associated with poor prognosis in some tumor types.  99  2.5 MATERIALS AND METHODS Mice and cell lines Transgenic mice deficient in Mgat5 expression were crossed onto polyoma virus middle T (PyMT) transgenic mice on a 129sv x FVB background (Granovsky et al., 2000). Mammary tumor samples were dissected from Mgat5+/- and Mgat5-/- mice and snap-frozen on dry ice for subsequent protein extraction. Cell lines were established from solid mammary carcinoma samples dissected from either Mgat5+/+ or Mgat5-/- genotypes. The cell lines used herein are designated Mgat5+/+, Mgat5+/+(2.8), Mgat5-/- and Mgat5-/-ESC. Mgat5-/- and Mgat5-/-ESC cells genetically rescued by infection with a pMX-PIE retroviral vector for expression of murine Mgat5, designated Rescue and ESC-Rescue, respectively, were selected by growth in medium containing 1 µg/ml puromycin (Partridge et al., 2004). All cell lines were grown in complete medium containing DMEM (Dulbecco’s modified Eagles’s medium) supplemented with 10% FBS, non-essential amino acids, glutamine, vitamins and penicillin/streptomycin in a 5% CO2/air incubator at 37˚C. For signal transduction experiments, cells were rinsed twice and incubated overnight in serum-free DMEM at 37oC prior to performing the experiment. Disruption of the actin cytoskeleton was performed by treating cells with 0.5 µM Latruculin A in complete DMEM for 20 min at 37oC prior to experiments. Constructs, transfection, adenoviral infection and siRNA EGFR-YFP was obtained from Z. Wang (University of Alberta, Edmonton, Canada), pOCTdsRED from H. McBride (University of Ottawa, Ottawa, Canada), and myc-tagged Cav1 wild-type and the F92A/V94A scaffolding domain mutant from M. J. Quon (NIH, Bethesda, USA). Human Cav1 was inserted into pRFP-N1 and tyrosine(Y)14 mutated to phenylalanine (F) by utilizing PCR based overlapping extension technique (Forward: 5’ G GGA ATT CTA GCA TGT CTG GGG GCA AAT ACG TAG ACT CGG AGG GAC ATC TCT TCA CC 3’, Reverse: 5’ GGG ATC CCC AGA TCC TCT TCT GAG ATG AG 3’). Cells were transfected using Effectene (Qiagen) 24 hours prior to experiments. Adenovirus expressing myc-tagged Cav1 under control of the tetracycline-regulated promoter was used to infect cells for 48 hours as previously described (Le et al., 2002; Zhang et al., 2000). Infected cells were visualized using anti-myc (Santa Cruz SC-40) antibody. To  100  knockdown Cav1 expression, cells were cultured in complete medium for two days prior to transfection with specific mouse Cav1 siRNA oligonucleotides or with control siRNA (Dharmacon Inc.). Briefly, cells were rinsed twice with serum-free DMEM without antibiotics, transfected with siRNA for 4 hours using Dharmafect 3 transfection reagent, washed 2X with complete DMEM and then incubated in complete media for 48 hours. Western Blotting Cells were lysed in TNTE (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton-X100, 1 mM EDTA, and Protease Inhibitor Cocktail (Sigma P8340)). Lysate protein levels were quantified using the BCA Protein Assay Reagents A and B (Pierce). Western blots of 30 µg total protein were probed with polyclonal antibodies against Cav1, Cav1/2 (Transduction Laboratories 610060 and 611338, respectively), γ-tubulin (clone GTU-88, Sigma T6557) or β-actin (Sigma A5316) followed by the appropriate HRP-conjugated secondary antibodies and chemiluminescence. Band intensity was quantified by densitometry with Scion image analysis software. Blue native gels were performed as described previously (Ren et al., 2004). Briefly, cells were lysed at 4 °C in lysis buffer (500 mM 6-amino caproic acid, 2mM EDTA, 25 mM Bistris, pH 7.0) containing 120 mM N-Octyl-glucoside for 30 min. Lysates were clarified by centrifugation at 13,200 rpm for 10 min. Supernatant were mixed with 1/10 volume of sample buffer containing 5% R-250 Coomassie blue and 1/10 volume glycerol. Proteins were separated on linear 4-15% acrylamide gels run at 100 V at 4 °C until the dye reached the middle of the gel. Blue cathode buffer (50 mM Tricine, 15 mM Bistris and 0.02% R-250 Coomassie blue) was then replaced with clear cathode buffer (with no Coomassie blue) and gels run at 200 V until the dye reached the bottom of the gels. Proteins were then transferred to PVDF membrane and processed for immunoblotting with Cav1 polyclonal antibody (Santa Cruz sc-894). Electron microscopy Cells were rinsed with 0.1 mM sodium cacodylate, pH 7.3, fixed for one hour with 2% glutaraldehyde at 4oC, rinsed with cacodylate buffer, scraped from the Petri dish, pelleted and  101  post-fixed with 2% osmium tetroxide at 4oC. The cells were dehydrated and embedded in LR-White resin. Ultra thin sections were prepared, contrasted with uranyl acetate and lead citrate and visualized with a Zeiss CM902 or a Hitachi H7600 transmission electron microscope. Smooth caveolar invaginations and clathrin-coated vesicles within 100 nm of the plasma membrane were counted per cell profile as previously described (Le et al., 2002). Nuclear translocation of Erk and Smad2/3 Cells were plated in 96-well plates at 5000 cells/well or on cover slips and serum starved for 24 h, then stimulated with EGF or TGF-β1 in DMEM plus 0.2% FBS. After various times with cytokine, cells were fixed for 10 min with 3.7% formaldehyde at 20oC, washed with PBS plus 1% FBS, and permeabilized using 100% MeOH for 2 minutes. The cells were washed 3 times and blocked in PBS plus 10% FBS for one hour at 37oC. Mouse anti-pErk1/2 (Thr202/Tyr204) (Sigma M-8159) or mouse anti-Smad2/3 (S66220) (Transduction Laboratories) was added at 1/1000 in PBS plus 10% FBS and incubated overnight at 4oC. The cells were washed 3 times with PBS plus 1% FBS and Alexa Fluor 488 labeled antimouse Ig (Molecular Probes) added at 1/1000 with Hoechst (1/2000) for 1 hour at 20oC. After washing 3 times, the plates were scanned using an ArrayScan automated fluorescence microscope (Cellomics Inc). The difference in nuclear and cytoplasmic staining intensity was determined individually for 100 cells per well, and substraction of total nuclear intensity values from cytoplasmic intensity values was used to represent the change in activation following addition of cytokine. The S.E of the mean (n=100) was generally < 4% at each assay point. Alternatively, cells were plated on cover slips for 24 hours and transfected with either myctagged Cav1, Cav1Y14F or Cav1F92AV94A or with Cav1 siRNA or control siRNA for 2 days. Cells were serum starved for 24 hours prior to stimulation with 100 ng/ml EGF for 5 min and fixed and labeled with mouse anti-phospho-Erk1/2 and either rabbit anti-myc (Santa Cruz sc-789) or rabbit anti-Cav1 (Santa Cruz sc-894) followed by Hoescht staining. Confocal images of cells mounted in Celvol 205 (Celanese Ltd.) were acquired on an Olympus Fluoview 1000 confocal microscope with a UPlanapochromat 1.35 NA 60X objective with equivalent acquisition settings the mean intensity of nuclear p-Erk quantified by creating a  102  mask based on Hoescht staining using ImagePro Plus software (Mediacybernetics Inc.). Data from 3 independent experiments (>36 cells/condition) were compiled and normalized to Mgat5+/+ cells stimulated for 5 min with EGF. Fluorescence recovery after photobleaching (FRAP) FRAP analysis of cells incubated with CT-B for 3 minutes or transfected with EGFR-YFP was performed in regular culture media without phenol red at room temperature. Images were acquired on an Olympus FV1000 confocal microscope with a UPlanapochromat 1.35 NA 60X objective and fully opened pinhole. Photobleaching of CT-B-FITC was performed using 10 scans with the 488 nm laser at full power within a square area 20 pixels wide. EGFR-YFP photobleaching experiments were performed using 20 scans of a 405 nm Olympus SIM scanner laser at full power within a circular ROI of 27 pixel diameter. To study the effect of Cav1 on CT-B diffusion, cells were transfected with Cav1-mRFP, Cav1Y14F-mRFP, Cav1F92AV94A-mRFP or Cav1 siRNA 2 days prior to experiments. Disruption of the galectin lattice was performed by treating the cells with 20 mM β-lactose or sucrose for 2 days. To study EGFR diffusion, cells were plated for 6 hours and then transfected with EGFR-YFP. The next day, cells were transfected with Cav1 or control siRNA or infected with Cav1 adenovirus. After 4 hours, the media was changed to complete DMEM or complete DMEM containing 20 mM β-lactose or sucrose for 2 days. Recovery data (6-8 cells from each of 3 independent experiments) were analyzed with Graphpad Prizm software using non-linear regression with a bottom to (bottom+span) algorithm. Half-life of recovery and mobile fraction were calculated as previously described (Reits and Neefjes, 2001). For experiments using myc-tagged Cav1 constructs, cells were cotransfected with either EGFRYFP or pOCT-dsRED in order to visualize the transfected cells. Similarly, recovery data for transfected Cav1-mRFP in Mgat5-/-ESC cells was obtained by FRAP analysis at room temperature as previously described. Images were acquired with equivalent acquisition settings and in order to compared Cav1-mRFP intensity to endogenous Cav1 levels, Mgat5+/+, Mgat5-/- and Cav1-mRFP transfected Mgat5-/-ESC cells were fixed and labelled in parallel with Cav1 polyclonal antibody (Santa Cruz sc-894). A graph of Cav1 intensity vs Cav1-mRFP intensity was generated for fixed Mgat5-/-ESC cells and a linear regression performed to determine the intensity of Cav1-mRFP, relative to endogenous Cav1 levels in  103  Mgat5+/+ cells, in the bleach zone of live cells that underwent FRAP analysis. FRAP data are presented in function of normalized Cav1-mRFP intensity for both the mobile fraction and half time of recovery. r2 values were calculated from a linear regression performed with GraphPad Prizm software. Quantitative immunofluorescence L-PHA and Cav1 expression levels were quantified from fluorescent images of cells mounted in Celvol 205 (Celanese Ltd.) acquired with the 60X (1.35 NA) UPlanapochromat objective of an Olympus Fluoview 1000 confocal microscope. Quantification of L-PHA-FITC levels (mean  density  of  fluorescence)  was  performed  using  ImagePro  Plus  software  (MediaCybernetics Inc.) from confocal images acquired with equivalent acquisition settings. Values from 3 independent experiments were normalized to the intensity of Mgat5+/+ cells and significance determined by a Student t-test. To measure EGFR colocalization with Cav1, cells were plated on glass coverslips and preincubated for 48 h with 20 mM β-lactose or sucrose in complete DMEM, fixed and labeled with rabbit anti-EGFR (sc-03), mouse antiCav1 (Molecular Probes T-2767) and Hoechst. Images were acquired with a 100X (NA 1.4) Olympus Planapochromat objective of a Deltavision Restoration Microscope and 64-layer stacks acquired and deconvolved with softWoRx image analysis software (Applied Precision). Colocalization was quantified in Adobe Photoshop by defining a 'box' of set dimensions and scoring the incidence of yellow stain within this box from 6 randomly selected regions within the cytoplasm of the cell. Alternatively, cells transfected with myctagged Cav1 constructs were labeled with anti-myc (Upstate, 05-724), anti-EGFR (sc-03). From confocal images of cells mounted in Celvol 205 (Celanese Ltd.) acquired with the 60X objective (1.35 NA) of an Olympus FV1000 confocal microscope, the relative intensity of myc-Cav1 associated with EGFR labeling was determined using the colocalization coefficient of ImagePro Plus imaging software (Media Cybernetics Inc.). To quantify EGFR-Cav1 association in live cells, Mgat5-/-ESC and ESC-Rescue cells were cotransfected with Cav1-CFP and EGFR-YFP. Time lapse images of cells were acquired every 10 seconds for 5 minutes in regular culture media without phenol red at room temperature with the 60X UPlanapochromat objective (1.35 NA) of an Olympus FV1000 confocal  104  microscope. Cav1 acquisition settings were kept constant and high and low Cav1-CFP expressing cells determined relative to endogenous Cav1 levels in Mgat5+/+ cells. The Pearson’s coefficient for Cav1-CFP and EGFR-YFP was calculated from two random regions of the cell for each individual time frame and the average Pearson’s coefficient over time determined from 3 independent experiments (n>15) using ImagePro Plus software (Media Cybernetics Inc.). Cav1 oligomerization gradients Cav1 oligomerization was determined using velocity sucrose gradient centrifugation as previously described (Monier et al, 1996). Briefly, cells were grown in 100 mm Petri dishes and lysed on ice in 500 µl of lysis buffer (25 mM MES pH 6.5, 150 mM NaCl, 60 mM Noctylgucoside and protease inhibitor cocktail). This lysate was overlaid on top of 4.2 ml of a 5%-30% discontinous sucrose gradient prepared in the same lysis buffer. The gradients were centrifuged in a SW55 rotor (Beckman) for 6 hours at 53,000 rpm. 12 equal fractions of 392 µl were collected from the top of the gradient and an equal volume of each fraction analyzed by SDS-PAGE and transferred onto nitrocellulose membranes for immunoblotting with antiCav1 (Santa Cruz sc-894) or anti-Rho A (Santa Cruz sc-418) antibodies. Online supplemental material Time-lapse video microscopy: Videos 1-4 correspond to Figure 6 C. Mgat5-/-ESC and ESCRescue cells were transfected with Cav1-CFP (red) and EGFR-YFP (green). Movies are of Mgat5-/-ESC cells expressing high (Video 1) and low (Video 2) Cav1 levels and ESC-Rescue cells expressing high (Video 3) and low (Video 4) Cav1 levels.  105  2.6 ACKNOWLEDGEMENTS We thank Zhixiang Wang for the EGFR-YFP construct, Michael J. Quon for the wild-type and scaffolding domain mutant Cav1 constructs and Heidi McBride for the pOct-dsRed. This research was supported by a grant from the Canadian Institutes for Health Research (CIHR) to IRN and JWD and a CIHR studentship to EP. PL is a research student of the Terry Fox Foundation through an award from the National Cancer Institute of Canada. JGG holds a doctoral fellowship from the Ministère de la Recherche et des Technologies for his doctoral studies to be submitted jointly to the Université de Montréal and the Université Louis Pasteur de Strasbourg (UMR CNRS 7034). Satra Nim participated in early stages of this study.  106  A ERK nuclear translocation (Arbitrary units)  300 200 100 0 300  0  10 20 30 40 50 60 Mgat5-/-  200 100 0  -100  0  Smad Nuclear Translocation (%)  B  10 20 30 40 50 60  300  Mgat5-/-ESC  200 100 0  0  10 20 30 40 50 60 Time (min)  100 Mgat5+/+ Mgat5-/Mgat5-/-ESC  50  0 0  10  20  30  40  50  60  Time (min)  Mgat5+/+  Mgat5-/-  Mgat5-/-ESC  Fibronectin Hoechst  E-Cadherin Hoechst  C  +EGF 5 min  Mgat5+/+  Figure 2.1 107  Figure 2.1: Mgat5-/-ESC cells show enhanced responsiveness to EGF. (A) Scan array determination of p-Erk nuclear translocation in Mgat5+/+, Mgat5-/- and Mgat5-/-ESC cells following stimulation with EGF (100 ng/ml) for the indicated times of incubation. Representative p-Erk labeling of cells upon EGF stimulation for 5 minutes are shown. (B) Scan array determination of Smad nuclear translocation in Mgat5+/+, Mgat5-/- and Mgat5-/-ESC cells following stimulation with TGFβ (100 ng/ml) for the indicated times of incubation. (C) In contrast to Mgat5+/+ cells, Mgat5-/- and Mgat5-/-ESC tumor cells display E-cadherin labeled (green) epithelial cell-cell adherens junctions (top row) and little fibronectin organized into fibrils (green). Cell nuclei are labeled with Hoechst (blue). Bar = 20 µm.  108  -/-  75  **  a t5 Re /scu Mg e a t5 -/-E ES SC CRe scu e  Cav1/2:β-actin  60  **  40  ** **  20  14  Caveolae Clathrin coated pits and vesicles  12 10 8 6  *  *  0,8  +β -la  W  0,4 0  150  Mgat5+/- R=0.00047 Mgat5-/- R=0.6  125 100 75 50 25  scu e  SC -/-E  at5  (2.6) (2.8) Mgat5+/+  Mg  0  -/-  2  Re  4  Mg at5  Mgat5-/-ESC  Number per cell profile  D Mgat5-/Mgat5-/-(22.9)  .6)  at5 Mg  ES  80  C Mgat5+/+  *  1,2  0  0  c  .6)  ue  C  Re  sc  ES  C-  -/-  ue  at5  sc  Mg  Re  Mg  Mg  Mg  .8)  at5  -/-  .6)  (2  (2  at5  Mg  Mg  at5  +/+  ES  -/at5  Mg  **  *  Cav1/2 (22 kDa)  *  Cav1:Tubulin  20  **  **  Cav1:β-actin  40  +/+  C  .8) (2  Mg  at5  -/-  .6) (2  +/+  at5  +/+  Mg  at5 Mg  Cav-1/2 (% intensity)  80  (2.6) (2.8) Mgat5+/+  ß-actin (42 kDa)  100  100 60  0  (2.6) (2.8) Mgat5+/+  ß-actin (42 kDa)  120  ** **  25  Cav1 (22 kDa)  Cav1/2 (22 kDa)  *  50  (2  0  75  (2  25  **  100  +/+  50  *  125  a t5 Re /scu Mg e a t5 -/-E ES SC CRe scu e  L-PHA Hoechst  100  Cav1 Hoechst  B  Cav1 intensity (%)  L-PHA intensity (%)  Cav1 Hoechst  Mgat5-/-ESC ESC-Rescue  Rescue  125  +S  Mgat5  .6)  (2.8)  L-PHA Hoechst  (2.6)  (2  A  0  0.0  2.5  5.0  7.5  10.0 12.5  Tumor Volume (cm)  Figure 2.2  109  Figure 2.2: Reduced Cav1 levels are associated with tumor growth in an Mgat5-/background. (A) Mgat5+/+, Mgat5-/-, Rescue, Mgat5-/-ESC and ESC-Rescue cells were grown on coverslips for 48 hours and stained with L-PHA-FITC (green, top row) or Cav1 (green, bottom row). Cell nuclei are stained with Hoechst (blue). Quantification of L-PHA and Cav1 intensity is shown as a bar graph (n=3; >25 cells per condition). Bar = 20 µm. (B) Equal protein amounts of cell lysates from Mgat5+/+(2.6) and (2.8), Mgat5-/- and Mgat5-/-ESC and, as indicated, Rescue and ESC-Rescue cells were western blotted with Cav1/2 or Cav1-specific antibodies and quantified by densitometry. Mgat5+/+ were treated with 1 µM swainsonine (SW) or 20 mM β-lactose (β-lac) for 48 hours, blotted for Cav1/2 and β-actin and quantified by densitometry. (C) Representative electron micrographs of the plasma membrane of Mgat5+/+, Mgat5-/- and Mgat5-/-ESC cells. Quantification revealed that both Mgat5-deficient cell lines present reduced expression of caveolae but not clathrin coated pits. Bar = 0.2 µm. (D) Spontaneous MMTV-PyMT mammary carcinomas were dissected from 12 week-old Mgat5+/- and Mgat5-/- mice and subjected to quantitative western blotting of Cav1 expression levels. Blots were also probed with γ-tubulin as a loading control and levels of Cav1 normalized to γ-tubulin levels as determined by densitometry. Results are plotted against tumor volume. *p<0.05, **p<0.005 relative to Mgat5+/+, unless otherwise indicated.  110  A  myc-Cav1 Hoechst  Mgat5-/-  Control + Cav1  120  nuclear p-ERK (%)  Mgat5+/+  p-Erk  100 80 60  *  40  Mgat5-/-ESC  20 0  Mgat5+/+ Rescue  Mgat5-/-  Cav-1 (22 kDa)  +EGF  v1 CT L Ca v1 CT L  Ca  v1 CT L Ca v1 CT L  Ca  p-Erk Mgat5-/-  -  +  Mgat5-/-  -  +  -  +  -  Rescue  +  -  Mgat5-/ESC  +  ESC Rescue  Control + Cav1 siRNA + Ctl siRNA  120  γ-tub (48 kDa) siRNA:  +  Mgat5+/+  Mgat5-/ESC  Cav1 Hoechst  *  100  nuclear p-ERK (%)  B  +EGF +Cav1 siRNA  -  EGF :  80 60 40 20 0  EGF :  -  +  -  +  Mgat5+/+ Mgat5-/-  Rescue  -  +  Mgat5-/ESC  -  +  ESC Rescue  Figure 2.3  111  Figure 2.3: Cav1 regulation of EGF signaling is selective for an Mgat5-/- background. (A) Mgat5+/+, Mgat5-/-, Rescue, Mgat5-/-ESC and ESC-Rescue cells were infected with adenoviruses coding for myc-tagged Cav1 prior to stimulation with EGF (100 ng/ml) for 5 min. Cells were fixed and stained for p-Erk (green) and Cav1 (red) and nuclei identified by Hoechst staining (blue). Quantification of untreated and EGF treated cells presenting nuclear p-Erk is shown as a bar graph (n=3; >36 cells per condition). (B) Mgat5+/+, Rescue, Mgat5-/and Mgat5-/-ESC cells were treated with Cav1 siRNA, non-specific (CTL) siRNA or left untreated (none) and then blotted for Cav1 and γ-tubulin (γ-tub). Mgat5+/+, Mgat5-/-, Rescue, Mgat5-/-ESC and ESC-Rescue cells were transfected with non-specific (CTL) siRNA or siRNA coding for Cav1 prior to stimulation with EGF (100 ng/ml) for 5 min. Cells were fixed and stained for p-Erk (green) and Cav1 (red), nuclei were identified by Hoechst staining (blue) and representative confocal images of untreated and Cav1 siRNA treated Mgat5-/- cells are shown. The percentage of cells presenting nuclear p-Erk was quantified in untreated and EGF treated cells, either untransfected (white bars) or transfected with Cav1 (black bars) or control (grey bars) siRNA, is shown as a bar graph (n=3; >48 cells per condition). Bar = 20 µm; *p<0.01 relative to control untransfected cells.  112  CT-B-FITC  Fluorescence intensity (% of control)  A  B  100 Mgat5+/+ 75 50  0  Ctl  Fluorescence intensity (%) of control)  +Cav1 siRNA  +Cav1  Ctl  Time (sec)  0.0 2.5 5.0 7.5 10.0 12.5 15.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0  Mgat5+/+  100  C  Ctl  +Cav1  +Cav1  EGFR-YFP  Mgat5-/- +Cav1 siRNA  +Cav1 siRNA  75  Mgat5-/-ESC +Cav1 siRNA Ctl  +Cav1  50  +Cav1  25 0  +Cav1 siRNA  +Cav1 siRNA  25  Mgat5-/-ESC  Mgat5-/-  +Cav1  Ctl 0  50  Ctl 100 150 200 250 0  Pre-bleach  50  Time (sec)  100 150 200 250 0  Bleach T=0  50  100 150 200 250  Post-bleach T=240 s  255  EGFR-YFP  0  Figure 2.4  113  Figure 2.4: Cav1 regulation of plasma membrane diffusion of CT-BFITC and EGFR-YFP. (A). Mgat5+/+, Mgat5-/- and Mgat5-/-ESC cells were incubated with 5 µg/ml FITC-CT-B at room temperature and a portion of the cytoplasm bleached and imaged for fluorescent recovery. Percent intensity (±SEM) of FITC-CT-B in the bleached zone during recovery is shown for one representative experiment (n=6 cells) for Mgat5+/+ (left), Mgat5-/- (center) and Mgat5-/-ESC (right) cells either untransfected (red) or transfected with Cav1 siRNA (blue) or Cav1-mRFP (+Cav1; black), as indicated. (B) Alternatively, Mgat5+/+ (left), Mgat5-/- (center) and Mgat5-/-ESC (right) cells transfected with EGFR-YFP (red) and subsequently transfected with Cav1 siRNA (blue) or infected with Cav1 adenovirus (+Cav1; black) were maintained at room temperature and a portion of the cytoplasm bleached and imaged for fluorescent recovery. Percent intensity (±SEM) of FITC-CT-B in the bleached zone during recovery is shown for one representative experiment (n=6 cells). See Table I for quantitative values for all conditions tested. (C) Representative images of an EGR-YFP transfected cell are shown pre-bleach, immediately after bleaching (T=0), and following recovery (240 sec). Bar = 20 µM.  114  B 12 11 10 9  8 7 6  5 4  3 2 1  Mgat5+/+  Bottom  Low Cav1  100  CT-B-FITC  75  125  r2 = 0.1929  100  Ctl +F92A/V94A  50  75 50  25  +Cav1  25  0  0  0  60 40  12.5 15  Ctl  +F92A/V94A  50 0  100 200 300 400 Cav1-mRFP intensity (% of Mgat5+/+(2.6))  500  100 75  * *  +Y14F  +Cav1  25 0  50  100  150  200  250  time (sec) +Cav1 Y14F +Cav1 F92A/V94A  +Cav1 WT  Cav1/EGFR  F  nuclear p-ERK  7.5 10 time (min)  75  20 0  +Y14F  EGFR-YFP  100  r2 = 0.0007  5  2.5  80  0  D  25 0  29  Ctl WT Y14F F92A/ V94A  Figure 2.5  Cav1/EGFR overlay  High Cav1  Half time of recovery (sec) Mobile Fraction (%)  Mgat5-/-ESC  66  Top  D  50  272  Rho A (24 kDa)  Mgat5+/+  E  545  Cav1 (22 kDa)  Mgat5-/-  C  Mg at5 +/ + Mg at5 -/-  A  60 40  *  20 0  WT Y14F F92A/ V94A  115  Figure 2.5: Cav1 regulation of EGFR signaling and cell surface diffusion requires an intact scaffolding domain but not Y14 phosphorylation. (A) n-Octylglucoside lysates of Mgat5+/+ and Mgat5-/- cells were analyzed by velocity sucrose gradient centrifugation and fractions immunoblotted for Cav1 and monomeric Rho A, as indicated. Fraction 1 is the top of the gradient and fraction 12 the bottom. (B) n-Octylglucoside lysates of Mgat5+/+ and Mgat5-/- cells were separated on blue native gels and blotted for Cav1. (C) Mgat5-/-ESC cells were transfected with Cav1-mRFP and Cav1 diffusion was assessed by FRAP. Cav1 intensity in the bleached zone was determined by comparison of Cav1 labeling intensity and RFP fluorescence in fixed cells and normalized to Cav1 intensity in Mgat5+/+ cells. Representative confocal images of high and low Cav1-mRFP expressing cells are presented. Mobile fraction (top graph) and half time of recovery (bottom graph) in function of Cav1 expression are presented. (D) Mgat5-/-ESC cells were cotransfected with myc-tagged Cav1 wild-type (WT), Y14F mutant or F92A/V94A scaffolding domain mutant as well as pOCT-dsRed to identify transfected cells and then incubated with CT-B-FITC at room temperature. Alternatively, myc-tagged Cav1 and mutants were cotransfected with EGFR-YFP. Percent intensity (±SEM) in the bleached zone for CT-B-FITC and EGFR-YFP during recovery is shown for one representative experiment (n=6 cells). See Table 1 for quantitative values for all conditions tested. (E) Mgat5-/-ESC cells were transfected with myc-tagged Cav1 wild-type (WT), Y14F mutant or F92A/V94A scaffolding domain mutant. Cells stimulated with EGF (100 ng/ml) for 5 min were fixed and stained with anti-p-Erk, and anti-myc to identify myctagged Cav1 labeled cells. Nuclei were identified by Hoechst staining. Quantification of pErk nuclear translocation from confocal images is shown as a bar graph (n=3; >24 cells per condition) *p<0.05. (F) Mgat5-/-ESC cells transfected with myc-tagged Cav1 wild-type (WT), Y14F mutant or F92A/V94A scaffolding domain mutant were immunofluorescently labeled with anti-EGFR (green) and anti-myc to localize myc-Cav1 (red) in the absence of ligand. Inset boxes (red) are enlarged to reveal incidence of co-localization. The percent of EGFR spots that overlap with Cav1 is presented in graphic form (*p<0.05). Bar = 20 µm.  116  +Lac  A 100  Hoechst/EGFR/Cav1  control  +Lac +Cav1 Ctl +lactose  Mgat5-/-  25 0 100  Mgat5-/-ESC  75 50  ESC-Rescue  7.5  *  5.0  2.5  0.0  co  control  Mgat5+/+ Mgat5-/-  nt ro l la ct os su e cr os e  Rescue  75 50  Cav1/EGFR overlap (%)  0 100  Mgat5+/+  25  Mgat5-/-  Fluorescence intensity (%) of control)  75 50  B  25 0  0  50 100 150 200 250 Time (sec)  Cav1-CFP/EGFR-YFP High Cav1  Low Cav1  High Cav1  Low Cav1  Low Cav1 High Cav1 Cav1 intensity(%)  125  2’  0  1’  2’  0  1’  2’  0  1’  2’  3’  4’  5’  3’  4’  5’  3’  4’  5’  3’  4’ 5’  *  50  *  25 0 0.4 0.3  *  0.2 0.1 0  -R  es  /-E  C  ES  t5 ga M  Mgat5-/-ESC  *  cu e  1’  75  SC  0  100  Cav1/EGFR overlay (Pearson’s coeff.)  Enhanced Cav1 intensity  Equivalent Cav1 aquisition  C  ESC-Rescue  Figure 2.6 117  Figure 2.6 The Mgat5/galectin lattice restricts EGFR diffusion and limits interaction with Cav1 domains. (A) Percent intensity (±SEM) in the bleached zone of EGFR-YFP during recovery is shown for one representative experiment (n=6 cells) for Mgat5+/+ cells (left), either untreated (Ctl) or treated with 20 mM lactose (+Lac) for 48 hours, or infected with Cav1 adenovirus and treated with 20 mM lactose for 48 hours (+Lac+Cav1). Percent intensity (±SEM) in the bleached zone of EGFR-YFP during recovery is shown for one representative experiment (n=6 cells) for Mgat5-/- and Rescue cells (left) and Mgat5-/-ESC and ESC-Rescue cells (right). See Table 1 for quantitative values for all conditions tested. (B) Mgat5+/+ and Mgat5-/- cells either untreated or pretreated for 48 h with 20 mM lactose or sucrose (images not shown) were immunofluorescently labeled for EGFR (red) and Cav1 (green) in the absence of ligand. Higher magnification images are presented to reveal incidence of co-localization. The percent of EGFR spots that overlap with Cav1 is presented in graphic form. *p<0.01 relative to untreated cells. (C) Time lapse images of Mgat5-/-ESC and ESC-Rescue cells cotransfected with Cav1-CFP and EGFR-YFP were acquired every 10 seconds for 5 minutes. Merged images of representative cells expressing high and low Cav1CFP levels are displayed with equivalent Cav1 acquisition settings and enhanced Cav1 intensity. Images are shown for t=0 and higher magnifications are shown every minute for 5 minutes. Cav1-CFP intensity was quantified relative to Mgat5+/+ cells (top graph) and average Pearson’s colocalization coefficients determined from the time lapse movies are presented for high and low Cav1 expressing cells (bottom graph). Videos are included in the Supplemental material. *p<0.01. Bars = 20 µm.  118  untreated  + Lat A  Mgat5+/+  A  B  C  Mgat5+/+  100  + LatA  75  Fluorescent intensity (%)  Mgat5-/-  25 0  50  100  Mgat5-/-ESC  +β-Lac + LatA 50  25  100  150  200  250  + Cav1 siRNA +LatA  50  100  100  ESC  150  200  time (sec)  250  0  150  200  250  ESC + LatA  Mgat5-/- + LatA Mgat5-/-  25  0  50  50  25  Hoechst/Actin  0  75  + Cav + Cav1 siRNA  50  0 100  + Cav1 +LatA  75  0  + β-Lac  75  Control  50  0  Mgat5+/+  100  0  50  100  150  200  250  time (sec)  Figure 2.7  119  Figure 2.7 The actin cytoskeleton restricts EGFR mobility. (A) Mgat5+/+, Mgat5-/- and Mgat5-/-ESC cells, either untreated (Control) or treated with latrunculin A (+LatA) for 20 minutes were fixed and stained with phalloidin-Alexa568 (red) and Hoechst (blue). (B) Percent intensity (±SEM) in the bleached zone of transfected EGFR-YFP during recovery is shown for one representative experiment (n=6 cells) of untreated and latrunculin A treated (+LatA) Mgat5+/+ cells (top) or for untreated and latrunculin A treated Mgat5+/+ cells infected with Cav1 adenovirus (+Cav1) or transfected with Cav1 siRNA (+ Cav1 siRNA) (bottom). (C) Percent intensity (±SEM) in the bleached zone of transfected EGFR-YFP during recovery is shown for one representative experiment (n=6 cells) of Mgat5+/+ cells pretreated for 48 hours with 20 mM lactose with (+Lac+LatA) or without (+Lac) Lat A (top) as well as untreated and latrunculin A treated (+LatA) Mgat5-/- and Mgat5-/-ESC (ESC) cells (bottom). See Table II for quantitative values for all conditions tested.  120  +Mgat5/ galectin lattice -Mgat5/ galectin lattice  A  D  +Cav1  B  -Cav1  E  EGFR (reduced signaling potential) EGFR (enhanced signaling potential) Actin filament Membrane (glyco)protein  +LatA C  F  Gal-3 EGFR oligomers Cav1 oligomers Caveolae  Figure 2.8  121  Figure 2.8: Domain competition between the galectin lattice and oligomerized Cav1 microdomains regulates EGFR signaling. In Mgat5 expressing cells, EGFR is recruited to galectin lattice domains that limit EGFR diffusion, promote interaction with the actin-based membrane skeleton and limit interaction with negative regulatory oligomerized Cav1 microdomains (A) such that reduction of Cav1 expression impacts on neither EGFR diffusion nor signaling (B). In the absence of the Mgat5/galectin lattice, EGFR freely diffuses across MSK boundaries and is recruited to Cav1 oligomers, as well as caveolae, that negatively regulate signaling (D) and Cav1 downregulation restores EGFR signaling (E). EGFR in the galectin lattice stably interacts with the membrane skeleton (A,B) and depolymerization of the actin cytoskeleton increases EGFR exchange within the galectin lattice but does not enhance the rate of EGFR diffusion or interaction with Cav1 microdomains (C,F). Diagram inspired from (Morone et al., 2006).  122  EGFR-YFP  Table 2.1: Percent mobile fraction and half-life of recovery for CT-B-FITC and EGFRYFP as determined by FRAP Mgat5+/+  Mgat5-/-  Rescue  Mgat5-/-ESC  ESCRescue  Mf (%) t1/2 (sec)  Mf (%) t1/2 (sec)  Mf (%) t1/2 (sec)  Mf (%) t1/2 (sec)  Mf (%) t1/2 (sec)  Control  64.8 ± 5.2 33.9± 1.9  60.8 ± 5.9 42.0 ± 3.3  68.6 ± 4.1 34.6± 4.6  83.2 ± 3.0* 21.0 ± 4.1*  68.3 ±4.3 35.5 ±3.7  +Cav1 adeno  57.2 ± 4.3 34.3 ± 3.2  63.5 ± 4.7 46.5 ± 3.7  63.5 ± 5.2 32.5 ± 4.0  51.1 ± 2.8* 37.2 ± 4.2*  66.5 ± 5.1 35.7 ± 4.1  + Cav1 wt  56.5 ± 4.9* 35.6 ± 4.7*  +Cav1 Y14F  53.6 ± 3.6* 36.5 ± 5.1*  +Cav1 F92A/V94A  78.7 ± 4.2 24.6 ± 3.8  +Cav1 siRNA  71.9 ± 5.0 33.6 ± 5.6  +Ctl siRNA  63.3 ± 4.2 33.1 ± 2.1  64.8 ± 4.8. 36.5 ± 5.1  80.1 ± 4.1* 23.3 ± 3.6*  63.6 ± 4.8 36.4 ± 5.0  65.1 ± 4.2. 34.5 ± 4.1  85.4 ± 2.7* 22.4 ± 3.1*  65.8 ± 4.6. 35.5 ± 4.1  57.6 ± 4.7* 46.2 ± 4.4*  79.2 ± 5.1 22.9 ± 2.7  61.6 ± 4.4 42.6 ± 4.0  78.6 ± 3.6* 24.4 ± 4.3* 66.3 ± 4.4 32.5 ± 4.2  85.2 ± 4.6* 21.7 ± 4.7*  76.7 ± 4.1* 24.5 ± 4.0* 65.3 ± 5.1 37.2 ±4.8  Mf (%) t1/2 (min)  Mf (%) t1/2 (min)  Mf (%) t1/2 (min)  Mf (%) t1/2 (min)  Mf (%) t1/2 (min)  26.9 ± 5.6 1.7 ± 0.1 24.5 ± 5.6 1.8 ± 0.3  22.6 ± 5.2 1.8 ± 0.2 24.3 ± 3.1 1.6 ± 0.2  23.5 ± 4.2 1.7 ± 0.1 24.5 ± 5.0 1.8 ± 0.2  65.8 ± 4.8* 1.3 ± 0.2** 31.8 ± 6.1* 1.7 ± 0.1** 36.2 ± 5.0* 1.7 ± 0.2** 34.3 ± 5.3* 1.8 ± 0.2**  62.2 ± 5.1* 1.2 ± 0.3** 26.9 ± 5.6* 1.8 ± 0.2**  +Sucrose  82.1 ± 4.7* 21.2 ± 2.2* 60.2 ± 7.1 36.5 ± 3.4  +Lactose +Cav1  67.3 ± 4.0† 19.7 ± 1.3*  +Lactose  Control +Cav1-RFP  CT-B-FITC  91.0 ± 2.6* 29.7 ± 4.1* 59.3 ± 5.7 42.2 ± 3.2  + Cav1 WT +Cav1 Y14F +Cav1 F92A/V94A +Cav1 siRNA +Ctl siRNA +Lactose +Sucrose  58.2 ± 3.7 1.3 ± 0.2 60.0 ± 5.4* 1.1 ± 0.2** 28.8 ± 5.2 1.8 ± 0.2 23.3 ± 4.2 1.8 ± 0.3 24.0 ± 4.8 1.6 ± 0.2  71.9 ± 5.7* 1.2 ± 0.2** 22.4 ± 5.1 1.9 ± 0.3 23.4± 4.2 1.9 ± 0.2 23.4 ± 4.6 1.8 ± 0.3  66.8 ± 5.2* 1.1 ± 0.2** 21.9 ± 4.3 1.7 ± 0.1 22.6 ± 4.1 1.9 ± 0.3 22.4 ± 5.1 1.8 ± 0.2  61.5 ± 3.7 1.2 ± 0.2 62.4 ± 4.2* 1.3 ± 0.1** 66.9 ± 5.1* 1.2 ± 0.3** 62.7 ± 3.9* 1.2 ± 0.2**  64.7 ± 4.1 1.3 ± 0.2 66.3 ± 3.7* 1.2 ± 0.2** 62.4 ± 4.3* 1.2 ± 0.3** 64.0 ± 4.9* 1.2 ± 0.3**  *p<0.01, **p<0.05; in italics compared to Mgat5+/+(2.6) and in bold compared to control †p<0.01 compared to Mgat5+/+(2.6) treated with lactose data presented ± S.E.M.  123  Table 2.2: Percent mobile fraction and half-life of recovery for EGFR-YFP following treatment with Latrunculin A as determined by FRAP. EGFR-YFP - Latrunculin A  Mgat5+/+  Mgat5-/Mgat5-/-ESC  Control +Cav1 adeno +Cav1siRNA +Ctl siRNA +Lactose +Sucrose Control Control  + Latrunculin A  Mf (%)  t1/2 (min)  Mf (%)  t1/2 (min)  65.3 ± 4.2 63.6 ± 5.1 66.2 ± 4.1 64.1 ± 4.2 80.3 ± 4.5 63.1± 4.6  36.4 ± 3.7 34.4 ± 4.4 35.3 ± 4.2 34.5 ± 4.3 24.5 ± 3.1 35.9 ± 3.4  77.4 ± 4.6* 76.4 ± 3.2* 74.5 ± 3.4* 78.6 ± 3.8* 77.4 ± 4.8 75.0 ± 4.1*  37.7± 4.2 41.5 ± 5.2 36.7 ± 4.6 39.6 ± 4.4 23.2 ± 3.5 36.6± 3.0  62.4 ± 4.3  40.4 ± 4.5  64.5 ± 3.8  38.7 ± 3.8  78.4 ± 5.3  22.6 ± 3.7  76.6 ± 4.9  24.5 ± 4.2  ± S.E.M.; *p<0.05 compared to absence to Latrunculin A  124  2.7 REFERENCES Ahmad, N., H.J. Gabius, S. Andre, H. Kaltner, S. Sabesan, R. Roy, B. Liu, F. Macaluso, and C.F. Brewer. 2004. Galectin-3 precipitates as a pentamer with synthetic multivalent carbohydrates and forms heterogeneous cross-linked complexes. The Journal of biological chemistry. 279:10841-7. Bauer, P.M., J. Yu, Y. Chen, R. Hickey, P.N. Bernatchez, R. Looft-Wilson, Y. 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Caveolin-1 inhibits epidermal growth factor-stimulated lamellipod extension and cell migration in metastatic mammary adenocarcinoma cells (MTLn3). 130  Transformation suppressor effects of adenovirus-mediated gene delivery of caveolin1. J. Biol. Chem. 275:20717-25.  131  CHAPTER 3 Caveolin-1 negatively regulates cholera-toxin b subunit endocytosis to the Golgi apparatus  A version of this chapter will be submitted for publication. Lajoie et al., 2008. Caveolin-1 132 negatively regulates cholera-toxin b subunit endocytosis to the Golgi apparatus  CAVEOLIN-1 NEGATIVELY REGULATES CHOLERA-TOXIN B SUBUNIT ENDOCYTOSIS TO THE GOLGI APPARATUS  Patrick Lajoie1, Satra Nim2, and Ivan R. Nabi1*  1.  Department of Cellular and Physiological Sciences Life Sciences Institute University of British Columbia 2350 Health Sciences Mall Vancouver, British Columbia, Canada V6T 1Z3  2.  Département de Pathologie et Biologie Cellulaire Université de Montréal CP6128 Succursale centre-ville Montreal, Québec, Canada, H3C 3J7  Condensed title: Caveolin-1 regulation of cholera toxin b endocytosis  133  *Corresponding author: Dr. Ivan R. Nabi Department of Cellular and Physiological Sciences Life Sciences Institute University of British Columbia 2350 Health Sciences Mall Vancouver, BC Canada  V6T 1Z3  134  3.1 CHAPTER SUMMARY GM1-bound cholera toxin b subunit (CT-B) enters the cells via various endocytic routes including clathrin-dependent and dynamin-dependent and independent raft pathways. Using our Mgat5 model with cells expressing various level of caveolin-1 (Cav1), we have shown that Cav1 negatively regulates CT-B diffusion at the cell surface independently of expression of caveolae. Golgi N-acetylglucosaminyltransferase V (Mgat5) generates galectin-binding β1,6GlcNAc-branched N-glycans on receptors that promote surface residency and cytokine responsiveness. Cav1 levels in mammary tumor cells derived from PyMT transgenic mice are significantly lower in Mgat5-/- cells compared to Mgat5+/+ cells. Loss of Cav1 expression in Mgat5-/- is associated with increased CT-B uptake to the Golgi apparatus via a clathrinindependent, cholesterol-sensitive pathway. However, expression of Cav1 to a level below the threshold required for caveolae formation is sufficient to prevent CT-B uptake in Mgat5-/cells and siRNA-mediated Cav1 knockdown increased CT-B uptake in Cav1 expressing cells.  Cav1 negative regulation of CT-B internalization is independent of Cav1  phosphorylation and requires an intact scaffolding domain. 3D reconstructions of cells expressing mutant dynamin K44A suggest that dynamin does not block initial internalization of CT-B but rather acts downstream on its transport to the Golgi apparatus. Cav1 and CT-B do not colocalize, arguing for an indirect regulation of raft-endocytosis by Cav1. These results suggest a role for Cav1 oligomers in the regulation of raft-dependent endocytosis outside caveolae.  135  3.2 INTRODUCTION Lipid rafts are small heterogeneous membrane domains enriched in cholesterol and sphingolipids involved in various biological processes (Pike, 2006). Raft microdomains have been characterized as detergent resistant membrane (DRMs) with light buoyancy on sucrose gradients. Partition of various molecules into lipid rafts at the plasma membrane has been shown to favor their internalization via a mechanism called raft-dependent endocytosis (Kirkham and Parton, 2005; Nabi and Le, 2003). Raft-dependent endocytic pathways have been characterized mostly by their sensitivity to cholesterol depletion and their clathrin independence (Lajoie and Nabi, 2007). Caveolae are a subdomain of lipid rafts that have been implicated in many cellular processes such as endocytosis, transcytosis, potocytosis, calcium signaling and many other signaling events (Anderson et al., 1992; Kurzchalia and Parton, 1999; Lisanti et al., 1995).Various studies have shown a role for caveolae in the internalization of different cargo such as the simian virus 40 (SV40), lactosylceramide and albumin (Minshall et al., 2000; Pelkmans et al., 2001; Sharma et al., 2003). Caveolin-1 is the major component of plasma membrane caveolae. It was shown that a threshold expression level of Cav1 is required for the formation of caveolae (Fra et al., 1995). Expression of Cav1 in cells that usually lack caveolae induces caveolae expression (Fra et al., 1995; Li et al., 1998; Vogel et al., 1998).  In Cav1-/- cells, cargo known to enter the cell via caveolae, such as SV40 and the cholera toxin b subunit can be internalized via raft-dependent pathways independently of caveolae (Damm et al., 2005; Torgersen et al., 2001). It was shown that CT-B and SV40 (Damm et al., 2005; Glebov et al., 2006; Kirkham et al., 2005) can be internalized via a dynaminindependent raft pathway and these pathways may involve tubular carriers (Kirkham et al., 2005). It was proposed that Cav1 stabilized rafts at the cell surface reducing their endocytic potential (Nabi and Le, 2003). Conversely, it was shown that overexpression of Cav1 can prevent internalization of ligands such as CT-B and the autocrine motility factor (Kirkham et al., 2005; Kojic et al., 2007; Le et al., 2000; Le et al., 2002) (Sharma et al., 2004). However, other cargos such as glycosphingolipids are internalized via caveolae in a dynamin-dependent  136  manner (Puri et al., 2001; Shajahan et al., 2004). These data indicate that raft-dependent endocytosis may invoke many different endocytic routes.  Using Mgat5-/- cells lines expressing various Cav1 levels, we have shown that Cav1 regulates cell surface diffusion of CT-B. We therefore used this model to study the regulation of CT-B endocytosis by Cav1. The loss of Cav1 expression in Mga5null cells was associated with increased CT-B uptake to the Golgi apparatus. Conversely, siRNA mediated Cav1 knock-down increased CT-B uptake in Cav1 expressing cells. Interestingly, expression of Cav1 below the threshold required for caveolae formation negatively regulated CT-B uptake indicating that Cav1 regulatory activity is independent of caveolae formation. Moreover, CTB did not colocalize with Cav1 suggesting that the regulation of endocytosis involves an indirect mechanism. Our results support a role for Cav1 oligomers outside caveolae in the stabilization of raft domains at the cell surface and the reduction their endocytic potential.  137  3.3 RESULTS AND DISCUSSION Cav1 negatively regulates raft-dependent endocytosis of CT-B to the Golgi Mgat5-/- cells show a reduction of about 50% of Cav1 expression when compared to Mgat5+/+ cells. However, Mgat5-/-ESC cells display poorly detectable Cav1 expression (Figure 3.1A). Both Mgat5 null cell lines were rescued with a retrovirus encoding for Mgat5. When rescued, Mgat5-/- cells (Rescue) showed restored Cav1 expression to the level observed in the Mgat5+/+ cells while Mgat5-/-ESC cells (ESC-Rescue) show negligible increased in Cav1 expression (Figure 1 A). When labeled at 4°C, all cell lines displayed the same amount of cell surface GM1 expression (Figure 3.1 B). However, Mgat5-/-ESC cells showed increased endocytosis of CT-B to the Golgi apparatus (Figure 3.1 C). These results are consistent with our previous results that showed that loss of Cav1 was associated with increased cell surface diffusion of CT-B in Mgat5-/-ESC cells (Lajoie et al., 2007). ESC-Res cells displayed the same level of CT-B uptake than Mgat5-/-ESC cells indicating that Mgat5 expression is not involve in the regulation of CT-B internalization (Figure 1 C) Internalization of CT-B in Mgat5-/-ESC cells was sensitive to cholesterol depletion and inhibited by treatment with methyl-β-cyclodextrin (MβCD). CT-B endocytosis to the Golgi was dynamin-dependent and inhibited by the dynamin mutant K44A (Figure 3.1 C). It was not affected by the clathrin-hub mutant, therefore identifying the endocytic pathway as raftdependent. It was shown previously that overexpression of Cav1 was able to inhibit CT-B internalization (Kirkham et al., 2005; Le et al., 2000). Consistent with these results, CT-B internalization was greatly reduced in Mgat5-/-ESC cells infected with Cav1 adenovirus. Conversely, siRNA-mediated knock-down of Cav1 in Mgat5+/+ and Mgat5-/- cells was associated with decreased Cav1 expression (Figure 3.2A) significant increased uptake of CTB to the Golgi indicating that Cav1 is negatively regulating CT-B uptake in these cells (Figure 3.2A). Uptake of CT-B in Mgat5-/- cells following Cav1 siRNA treatment was still dynamin and cholesterol dependent and clathrin-independent and therefore raft-dependent (Figure 3.2 C).  138  We have shown before that Mgat5-/- cells express Cav1 at a level below the threshold required for caveola formation (Lajoie et al., 2007). These results suggest that Cav1 may act as a negative regulator independently of its role in caveolae formation. Functions of Cav1 outside caveolae are still unclear. It was shown that in endothelial cells, overexpression of Cav1 is associated with inhibition of eNOS signaling (Bauer et al., 2005). However, the overexpression did not correlate with increased caveolae formation. It was then proposed that Cav1 may act as a reservoir of Cav1 molecules that may be released under specific conditions (Parton and Simons, 2007). Our results therefore support a role for negative regulation by Cav1 outside caveolae.  Indirect regulation of CT-B endocytosis by Cav1 is independent of Cav1 phosphorylation and requires an intact scaffolding domain The ability of Cav1 to bind both cholesterol and dynamin-2 indicates that direct interaction between Cav1 and CT-B is not required for its negative regulatory activity. Interestingly, CTB poorly colocalized with Cav1 in both Mgat5+/+ and Mgat5-/- cells following endocytosis for 30 minutes. Mgat5-/-ESC cells transfected with Cav1-mRFP also display rare colocalization between CT-B and Cav1 supporting an indirect effect of Cav1 on CT-B endocytosis (Figure 3.3). Taken together, these data support a role for Cav1 outside caveolae in the negative regulation of CT-B internalization via raft-dependent/dynamin-independent endocytosis. The effect of Cav1 is therefore indirect and may involved sequestration of Cav1 binding partners such as dynamin-2 and cholesterol. Cav1 is phosphorylated on tyrosine 14 and Cav1 phosphorylation was shown to play a role in endocytosis via caveolae. In rat-1 cells, Cav1 phosphorylation is associated with loss of cell surface caveolae (Ko et al., 1998). Cav1 phosphorylation on tyrosine 14 was also associated with fusion of caveolae vesicles (Nomura and Fujimoto, 1999). Src kinase activity was associated with Cav1 phosphorylation and transcytosis of albmunin in endothelial cells  139  (Tiruppathi et al., 1997). It was also shown that Src kinase activity is required for internalization of caveolae follwing stimulation by cholesterol and glycosphingolipids (Sharma et al., 2004). It was proposed that integrin-mediated adhesion regulates the presence of raft domains on the plasma membrane by regulating internalization through a caveolindependent pathway involving changes in phosphocaveolin localization (del Pozo et al., 2005). The arrival of SV40 particles into caveolae also stimulates Cav1 phosphorylation (Pelkmans et al., 2002). However, it remains unclear whether Cav1 phosphorylation of tyrosine 14 represents a critical regulator of caveolae/raft-dependent endocytosis. We show that Cav1 is able to negatively regulate CT-B endocytosis in Mgat5-/- cells. We have shown that these cells do not express the phosphorylated form of Cav1(Goetz et al., 2007), indicating that negative regulation of raft endocytosis by Cav1 may be independent of its phosphorylation. In order to confirm this hypothesis, we have transfected Mgat5-/-ESC cells with a Cav1 containing a Y14F mutation of the tyrosine phosphorylation site. Indeed, Mgat5/-ESC  cells transfected with this mutant displayed a reduced internalization of CT-B to the  Golgi as observed in cells expressing the wild-type form of Cav1 (Figure 3.4 A, B). Cav1 also contains a scaffolding domain that has been shown to be required for its interaction with various receptors (Couet et al., 1997; Garcia-Cardena et al., 1997; Nystrom et al., 1999) and the formation of Cav1 oligomers (Sargiacomo et al., 1995). To test whether the Cav1 scaffolding domain was required for the inhibition of CT-B endocytosis, we transfected the Mgat5-/-ESC cells with a F92A/V94A mutation of the scaffolding domain. Interestingly, the cells transfected with the mutant did not display significant reduction in CT-B internalization (Figure 3.4 A, B) suggesting that the scaffolding domain is critical for the Cav1 negative regulatory activity. Indeed, in endothelial cells it was shown that, unlike the wild-type Cav1, this specific mutation was not inhibiting eNOS signalling (Bernatchez et al., 2005). It is possible that alteration of the scaffolding domain inhibits Cav1 interactions with crucial components of the machinery requierd for CT-B endocytosis. Cav1 is known as a cholesterol binding protein (Murata et al., 1995). Cav1 may negatively regulate endocytosis by sequestring cholesterol away from other raft domain and the scaffolding domains mutation may possible affect this funtion. The same way, Cav1 is known to recuit dynamin-2 (Yao et  140  al., 2005) and we can postulate that the scaffolding domain may play a role in this interaction. Cav1 may therefore regulate endocytosis by recruiting dynamin-2 away from rafts.  Dynamin-2 is involved in the intracellular transport of CT-B to the Golgi It was shown previously that CT-B endocytosis to the Golgi is Cav1 dependent (Kirkham et al., 2005; Le and Nabi, 2003). However, introduction of the dynamin-2 mutant K44A did not prevent internalization of CT-B but rather blocked the intracellular transport of the toxin to the Golgi complex (Kirkham et al., 2005). To adress this question we performed 3D reconstructions of Mgat5-/-ESC cells infected with the dynamin K44A adenovirus follwing internalization of CT-B. Consistent with the previous study by Kirkam et al. we found that CT-B was still internalized in dynamin K44A infected cells but that it did not colocalize with the GM130 labeled Golgi (Figure 3.5). These results confirm that dynamin-2 is therefore acting on the intracellular transport of CT-B. It was shown that internalization of CT-B via a dynamin-independent pathway involves its transport via flotillin-1 positive endosomes (Glebov et al., 2006). These structures may therefore be involved in the initial internalization steps of CT-B internalization. However, dynamin-2 may regulate CT-B later transport to the Golgi. Taken together, these data support a role for Cav1 outside caveolae in the negative regulation of CT-B internalization via raft-dependent/dynamin-independent endocytosis. The effect of Cav1 is therefore indirect and may involve sequestration of Cav1 binding partners such as dynamin-2 and cholesterol.  141  3.4 MATERIAL AND METHODS Cholera toxin-B subunit endocytosis: To follow CT-B endocytosis, 5 µg/ml CT-B-FITC was incubated with the cells for 15 or 30 minutes at 37oC and after rinsing with medium, the cells were fixed with 3% paraformaldehyde in PBS and labeled with primary mouse monoclonal antibody against the Golgi marker GM130 (Transduction Laboratories) and Cav1 polyclonal antibody (Santa Cruz sc-984). Where indicated, the cells were treated for 30 minutes at 37˚C with 5 mM methyl-β-cyclodextrin (mβCD) before incubation with CTb-bFITC in the presence of mβCD. Species-specific secondary antibodies conjugated to Alexa 568 or Alexa647 (Molecular Probes) were used.  Images were acquired with the 63X  Planapoochromat objective using the 488, 568 and 633 nm laser lines of an Olympus FV1000 confocal microscope. CT-B-FITC endocytosis to the Golgi apparatus was quantified from confocal images of cells labeled for FITC-CTX-B and GM130 by determining, 6 individual images per sample and the proportion of cellular FITC-CTX-B labeling that overlapped with the GM130 labeled Golgi apparatus using Image Pro PLs software (mediacybernetics inc.). Adenoviral infections: adenoviruses expressing either the tetracycline-regulated chimeric transcription activator (tTA), or HA-tagged wild-type dynamin-1, HA-tagged dynK44A mutant, and myc-tagged caveolin-1 under the control of the tetracycline-regulated promoter were as previously described (Le et al., 2002). Cells were plated on coverslips for 24 hours, infected with the different viruses and process for CT-B-FITC uptake 48 hours postinfection. Cells were then fixed with 3% paraformaldehyde and labeled for the appropriate tag (anti HA, anti-T7 tag and anti-myc). Transfections: Cells were cultured in complete DMEM for 24 prior to transfection with specific mouse caveolin-1 siRNA oligonucleotides or with control siRNA (dharmacon inc.). Briefly, cells were transfected with Dharmafect3 tranfection reagent a final concentration of 0.1µM of SiRNA in complete DMEM. Cells were processed for CT-B-FITC 48 hours posttransfection. Alternatively, cells were transfected with various Cav1 constructs using effectene transfection reagent. Cells were transfected with Cav1-wt, Cav1-Y14F and Cav1  142  F92A/V94A for 24 hours and process for CT-B-FITC uptake experiments. Cells were fixed and labeled with anti-myc and anti-GM130. 3D reconstructions: Cells were grown on coverslips and infected with dynamin K44A adenovirus. Cells were processed for CT-B-FITC endocytosis,fixed and labeled for GM130, HA and hoescht. Z-stacks were aquired using the 60X Planapoochromat objective of a FV1000 confocal microscope using a 0.5 microns step size. 3D reconstructions were obtnained using Image Pro Plus 3D constructor software (Mediacybernetics inc.).  143  A  B  Surface GM1  100 75 50 25  Cav1/2 intensity  0 125  Mgat5+/+ (2.6)  Mgat5+/+ (2.8)  Mgat5-/-  Rescue  Mgat5-/-ESC  ESC-Rescue  100 75  *  50  * *  25  t5 -/R es ES C ES C -R  *  CT-B uptake to Golgi (%)  C  *  C  ES  *  ES  es  t5 ga  R  -/-  8 2. M  6  0  C  *  10  2.  CT-B/Golgi overlap (%)  20  -R  2. 8 M  ga  2. 6  0  0,25 0,2 0,15 0,1  *  *  0,05  *  0 ctl  mbcd  Dynamin WT  Dynamin K44A  ClathrinHub  Caveolin-1  Figure 3.1  144  Figure 3.1: Reduced caveolin expression in Mgat5-/-ESC cells is associated with increased raft-dependent CT-B uptake to the Golgi. (a) Mgat5+/+ (2.6 and 2.8), Mgat5-/-, Rescue, Mgat5-/-ESC, and ESC-Rescue cells were probed for cell surface expression of GM1 with CT-B at 4°C (top), labelled with Cav1/2 antibody and caveolin intensity quantified (middle) and with FITC-CT-B for 15 and 30 minutes and CT-B delivery to the Golgi quantified (bottom). (b) Merge images are presented for CT-B endocytosis (green) for 30 minutes and then fixed and labeled for GM130, a Golgi marker (red). (c) Infection of Mgat5-/- 22.10 cells with adenoviruses coding for Cav1 reduced CT-B endocytosis to the Golgi. Bars = 20µm  145  A  CT-B  GM130  CT-B/GM130  Cav1  Untreated  + Cav1 siRNA  + Ctl siRNA  CT-B uptake to Golgi (%)  Cav1/2 intensity (AU)  B  CC  18000 16000 14000 12000 10000 8000 6000 4000 2000 0  0,18 0,16 0,14 0,12  *  0,1  **  0,08 0,06 0,04 0,02 0  0.25  Cav siRNA  ****  0.20  ***  0.10  -  +  + mβCD  + Dyn K44A  + Cla Hub  0.10 0.05 0  siRNA:  -  Cav1  Mgat5+/+  CTL  -  Cav1  Mgat5-/-  CTL  -  Mgat5-/ESC  Figure 3.2 146  Figure 3.2: Caveolin-1 knock-down by specific siRNA increased raft-dependent endocytosis of CT-B to the Golgi. Mgat5+/+ and Mgat5-/- cells were treated with nonspecific or Cav1 siRNA and processed for CT-B endocytosis for 30 min. (a) Cells were then fixed and labelled for Cav1 and GM130 and merge images are presented. (b) Quantification of CT-B uptake to the Golgi following treatment with Cav1 SiRNA. Treatment of Mgat5+/+ and Mgat5-/- with Cav1 SiRNA reduced Cav1 expression to the level of Mgat5-/-ESC cells. CTB uptake to the Golgi was increased in both Mgat5+/+ and Mgat5-/- cells in response to Cav1 knock-down. (c) Infection of Mgat5-/- cells treated with Cav1 SiRNA with adenoviruses and treatment with MβCD defines CT-B-FITC uptake as raft-dependent. Bars = 20µm  147  A  Mgat5+/+  CT-B/Cav1 Mgat5-/-  B  Mgat5-/-ESC  +Cav1-wt  Figure 3.3  148  Figure 3.3: Indirect regulation of CT-B endocytosis by Cav1. (a)Mgat5+/+ and Mgat5-/and cells were plated on coverslips and processed for CT-B-FITC (green) endocytosis for 30 min at 37°C. Cells were fixed and labelled for Cav1 (red). (b) Mgat5-/-ESC cells were transfected with Cav1-mRFP vector processed for CT-B-FITC (green) endocytosis for 30 min at 37°C. Cells were fixed and labelled for Cav1 (red). Higher magnification for regions boxed in red are shown to reveal the incidence of colocalization. Bars = 20µm  149  A  CT-B/Golgi Mgat5-/-ESC  Cav1  +Wt  +Y14F  CT-B/Golgi overlay  B  +F92A/V94A 0.25 0.20 0.15  *  0.10  *  0.05 0.00  Ctl  +wt  +Y14F +V92A/ F94A  Figure 3.4  150  Figure 3.4: Regulation of CT-B endocytosis by Cav1 requires an intact scaffolding domain but not its phosphorylation on tyrosine 14. (a) Mgat5-/-ESC cells were transfected with myc-tagged Cav1 wild type, Y14F mutant or F92A/V94A scaffolding domain mutant. Cells were processed for CT-B-FITC (green) endocytosis for 30 min at 37°C, fixed and labelled with anti-myc and anti-GM130 and the CT-B-FITC/Golgi overlay quantified. (b) Quantification of CT-B uptake to the Golgi in Mgat5-/-ESC cells transfected with various Cav1 constructs. Bars = 20µm  151  Control  Mgat5-/-ESC  +Dynamin K44A  CT-B-FITC/GM130/Hoescht  Figure 3.5  152  Figure 3.5: Dynamin mutant inhibits CT-B transport to the Golgi, but not its internalization. Mgat5-/-ESC cells were infected with adenovirus encoding the dynamin mutant K44A. 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Expression of caveolin-1 and polarized formation of invaginated caveolae in Caco-2 and MDCK II cells. J Cell Sci. 111 ( Pt 6):825-32. Yao, Q., J. Chen, H. Cao, J.D. Orth, J.M. McCaffery, R.V. Stan, and M.A. McNiven. 2005. Caveolin-1 interacts directly with dynamin-2. J Mol Biol. 348:491-501.  157  CHAPTER 4  The lipid composition of autophagic vacuoles regulates the expression of multilamellar bodies  A version of this chapter has been published in the Journal of Cell Science. Lajoie et al, 158 2005. The lipid composition of autophagic vacuoles regulates the expression of multilamellar bodies. J Cell Sci. 118:1994-2003  Illustration 4.1: Cover image, The Journal of Cell Science Vol. 118 No. 9 2005  159  The lipid composition of autophagic vacuoles regulates the expression of multilamellar bodies  Patrick Lajoie1, 2, Ginette Guay2, James W. Dennis3 and Ivan R. Nabi1, 2* 1  Department of Cellular and Physiological Sciences, University of British Columbia,  Vancouver, British Columbia, Canada, V6T 1Z3, 2Département de pathologie et biologie cellulaire, Université de Montréal, Montréal, Québec, Canada H3C 3J7 and 3Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada M5G 1X5  Running title: Biogenesis of multilamellar bodies Keywords : multilamellar bodies, cholesterol, lysosomes, phosphatidylinositol-3 kinase, autophagy, autophagic vacuoles  160  *Corresponding author: Dr. Ivan R. Nabi Department of Cellular and Physiological Sciences University of British Columbia 2177 Wesbrook Mall Vancouver, British Columbia Canada  V6T 1Z3  161  4.1 CHAPTER SUMMARY Multilamellar bodies (MLBs) are responsible for surfactant secretion in type II alveolar cells but also accumulate in other cell types under pathological conditions, including cancer and lysosomal storage diseases such as Niemann-Pick C (NPC), a congenital disease where defective cholesterol transport lead to its accumulation in lysosomes. Mv1Lu type II alveolar cells transfected with Golgi β1,6 N-acetylglucosaminyltransferase V (Mgat5), enhancing the polylactosamine content of complex-type N-glycans, exhibit stable expression of MLBs whose formation requires lysosomal proteolysis within dense autophagic vacuoles (Hariri et al, Mol. Biol. Cell 11:255-268, 2000). MLBs of Mgat5 transfected Mv1Lu cells are phospholipid-rich and cholesterol-poor. In Mv1Lu cells treated with the NPC-mimicking drug U18666A, cholesterol-rich MLBs accumulate independently of both Mgat5 expression and lysosomal proteolysis. Inhibition of autophagy by blocking the PI3K pathway with 3methyladenine prevents MLB formation and results in the accumulation of non-lamellar, acidic  lysosomal  vacuoles.  3-methyladenine  inhibited  the  accumulation  of  monodansylcadaverine, a phospholipid-specific marker for autophagic vacuoles, but did not block endocytic access to the lysosomal vacuoles. Induction of autophagy via serum starvation resulted in an increased size of cholesterol-rich MLBs. While expression of MLBs in the Mv1Lu cell line can be induced by modulating lysosomal cholesterol or protein glycosylation, an autophagic contribution of phospholipids is critical for the formation of concentric membrane lamella within late lysosomal organelles.  162  4.2 INTRODUCTION Multilamellar bodies are lysosomal organelles containing multiple concentric membrane layers. MLBs vary in size from 100 to 2400 nm and are found in various cell types where their principal functions are storage and secretion of lipids (Schmitz and Muller, 1991). In lung alveolar type II cells, MLBs are responsible for the secretion of the surfactant film that prevents alveola from collapse during respiration (Hatasa and Nakamura, 1965). Deficient expression of the hydrophobic surfactant protein B (SP-B) results in the formation of immature MLBs and secretion of non-functional surfactant (Foster et al., 2003). Abnormal MLBs have also been observed in familial desquamative and non-specific interstitial pneumonitis associated with mutations in surfactant protein C (SP-C) gene (Thomas et al., 2002). Another protein, the ATP-binding cassette transporter A3 (ABCA3) is also localized to lamellar bodies of alveolar type II cells and is critical for the formation of MLBs and expression of surfactant (Mulugeta et al., 2002; Shulenin et al., 2004). MLB formation in type II alveolar cells is therefore critically dependent on the protein composition of the organelle. The lipid composition of MLBs is 95% dipalmitoyl phophatidylcholine (DPPC), a neutral phospholipid that represents the major active component of surfactant (Schmitz and Muller, 1991). Lung surfactant is composed of 80% glycerophospholipid, 10% cholesterol and 10% protein; the amount of cholesterol within surfactant can increase relatively to phospholipid under certain conditions including exercise (Doyle et al., 1994) and hyperpnea (Orgeig et al., 2003). In lung MLBs, cholesterol is localized primarily to the limiting membrane of the organelle (Orgeig, 2001) and whether MLBs are the source of surfactant cholesterol remains uncertain (Hass and Longmore, 1979; Orgeig et al., 1995). Augmentation of cellular cholesterol stimulates MLB expression, the accumulation of cholesterol within MLBs, and the uptake of palmitic acid, the precursor of DPPC, in alveolar type II cells (Kolleck et al., 2002). Extracellular cholesterol is therefore internalized and stored in the MLBs of type II alveolar cells but whether it impacts on their biogenesis remains poorly understood. The presence of MLBs is also associated with various lysosomal storage diseases, including gangliosidosis, Tay-sachs, Fabry’s and Niemann-Pick, associated with deficiencies in various  163  lysosomal degradative enzymes and aberrant lysosomal accumulation of lipids (BlanchetteMackie, 2000; Gieselmann, 1995; Pentchev et al., 1987; Platt et al., 1997). Cholesterol accumulation is closely related to the expression of MLBs in the Niemann-Pick lysosomal storage diseases. While Niemann-Pick A and B are associated with sphingomyelinase deficiency, Niemann Pick C and D are due to impaired intracellular cholesterol trafficking (Kolodny, 2000; Pagano et al., 2000). Patients with Niemann-Pick C disease are deficient for expression of the NPC1 protein implicated in the regulation of intracellular cholesterol traffic (Blanchette-Mackie, 2000; Ory, 2000; Pentchev et al., 1987). A class of drugs (class 2 amphiphiles such as U18666A) impairs cellular cholesterol traffic and results in the accumulation of cholesterol in late endosomes, lysosomes and MLBs mimicking NiemannPick C disease (Butler et al., 1992; Lange et al., 1998; Liscum et al., 1989). Fibroblasts from patients with sphingolipid storage diseases present defective lipid transport and sorting (Pagano et al., 2000). In many of these diseases, altered cholesterol homeostasis leads to perturbations in lipid traffic (Puri et al., 1999). Increased cellular cholesterol alters sphingolipid trafficking resulting in its delivery not to the Golgi from the plasma membrane but rather to endolysosomal compartments such that altered trafficking of sphingolipids can be considered a diagnostic tool for the identification of sphingolipid-storage diseases (Chen et al., 1999). U18666A–mediated cholesterol accumulation can be reduced by overexpression of Rab 7 and 9 GTPases suggesting that the modulation of endosomal lipid composition can impact on the delivery of material to lysosomes (Choudhury et al., 2002; Kobayashi et al., 1999; Pagano et al., 2000). Cholesterol accumulation in lysosomal organelles has further been proposed to result in the trapping of lipid raft components in late endocytic structures that could result in the formation of MLBs (Lusa et al., 2001; Simons and Gruenberg, 2000). Transfection of the Mv1Lu mink lung alveolar type II cell line, that does not express MLBs, with β1-6-N-acetyl-glucosaminyl-transferase V (Mgat5) results in the stable expression of cytoplasmic MLBs implicating glycosylation of lysosomal glycoproteins in MLB expression (Hariri et al., 2000). Interestingly, defects in galactosidases (galactosidosis) and sialidases (sialidosis) as well as accumulation of polylactosamine are associated with MLB accumulation in lysosomal storage diseases (Allegranza et al., 1989; Amano et al., 1983;  164  Berra et al., 1986; DeGasperi et al., 1990). L-PHA labelling of Mgat5 generated β1-6 GlcNAc-branched N-glycans in MLBs has also been described in melanomas and other cancers (Handerson and Pawelek, 2003). Multilamellar bodies are therefore a ubiquitous organelle expressed under various physiological and pathological conditions. The similar morphology and lysosomal nature of this organelle in various cell types argues that common mechanisms must necessarily regulate its formation. Studies of MLB biogenesis in the Mgat5-transfected type II alveolar Mv1Lu cells showed that MLB formation could be prevented by treatment with the protease inhibitor, leupeptin, resulting in the accumulation of dense autophagic vacuoles, and that inhibition of autophagy with 3-methyladenine (3-MA) also blocked MLB expression (Hariri et al., 2000). Similarly, lysosomal protein degradation is required for MLB formation in primary human lung type II alveolar cells (Guttentag et al., 2003). Early electron microscopic studies of fetal lung describing the coordinate loss of glycogen with the accumulation of multilamellar bodies (Campiche et al., 1963; O'Hare and Sheridan, 1970) and the presence of cytoplasmic glycogen in lamellar bodies in type II epithelial cells in fetal lung further support a role for autophagy in MLB formation (Stahlman et al., 2000; Weaver et al., 2002). However, in spite of the known ability of cholesterol accumulation to stimulate MLB formation in lysosomal storage disease models, the impact of lysosomal cholesterol on the biogenetic mechanisms underlying MLB formation remains undetermined. MLBs in Mgat5 transfected Mv1Lu cells are phospholipid-rich and cholesterol-poor and we have used the NPC-mimicking amphiphilic drug U18666A (3β-(2-diethylaminoethoxy)androstenone) to study the role of lysosomal cholesterol accumulation on the biogenesis of MLBs in the Mv1Lu type II alveolar cell line. U18666A stimulates MLB formation in both parental and Mgat5-transfected Mv1Lu cells. It further transforms the dense autophagic vacuoles that accumulate following extended treatment with the lysosomal protease inhibitor leupeptin into MLBs. Cholesterol therefore induces MLB formation in Mv1Lu type II alveolar cells independently of both Mgat5 expression and lysosomal degradation. While inhibition of autophagy with the PI3 kinase inhibitor 3-methyladenine prevents the expression of concentric lamella in the U18666A-induced cholesterol-rich lysosomal  165  vacuoles, stimulation of autophagy by serum starvation results in an increased size of MLBs presenting the concentric lamellar morphology typical of MLBs. Phospholipid-rich MLBs as well as U18666A-induced cholesterol-rich MLBs are accessible to fluid phase uptake after 4 hours and labelled for lysotracker red identifying them as late lysosomal organelles or autolysosomes. Similarly, the non-lamellar vacuoles induced in the presence of 3-MA are late lysosomal organelles but exhibit significantly reduced labelling for the autophagic vacuole marker monodansylcadaverine. Multiple factors, including cholesterol content, glycosylation and autophagy, can therefore contribute to the formation of concentric lamella within secondary lysosomes and autolysosomes.  166  4.3 MATERIALS AND METHODS Chemicals Filipin complex, leupeptin, 3-MA, MDC, and gelvatol were purchased from Sigma (St-Louis, MO) and Alexa 488 goat anti-mouse IgG, Alexa 568 goat anti-mouse IgG, Alexa 647 goat anti-mouse IgG secondary antibodies, Lysotracker Red, Sytox green and FITC-dextran (10,000 MW, lysine-fixable) from Molecular Probes (Eugene, OR). U18666A was purchased from Biomol (Plymouth Meeting, PA), G418 from Invitrogen (Burlington, ON) and Nile Red from ICN Biomedicals (Costa Mesa, CA.). Cell Culture Normal Mv1Lu mink lung epithelial cells, the M9 clone of Mv1Lu cells transfected with Mgat5 (Demetriou et al., 1995) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with glutamine, vitamins, non-essential amino acids (Invitrogen, Burlington, ON) and 10% FBS (Medicorp, Laval, Qc) in an air/5% CO2 atmosphere at constant humidity at 37oC. To maintain the phenotype of Mgat5-transfected Mv1Lu cells, the medium was supplemented with G418 at a final concentration of 600 µg/ml (Hariri et al., 2000). Cells were plated at a density of 40,000 cells/cm2 and the medium was replaced every two days. To induce formation of cholesterol-rich MLBs, medium was supplemented with U18666A (1:10,000 of a 10 mg/ml stock solution in ethanol) for 24 hours. Leupeptin was added to the medium at a concentration of 2 µg/ml. 3-MA was dissolved directly in the medium at a final concentration of 10 mM and then sterile filtered (Hariri et al., 2000). Immunofluorescence Normal and Mgat5 transfected Mv1Lu cells were grown on glass coverslips for 6 days and then processed for immunofluorescence as previously described (Hariri et al., 2000). Cells were fixed with 3% paraformaldehyde for 15 minutes, washed with PBS supplemented with 0.1 mM Ca2+ and 1 mM Mg2+ (PBS-CM) and then incubated for 15 minutes with PBS-CM supplemented with 0.2% BSA to reduce non-specific binding and 0.07 % saponin to permeabilize cellular membranes. AC17 anti-LAMP-2 antibody (Nabi et al., 1991; Nabi and Rodriguez-Boulan, 1993) was used to determine the cellular distribution of LAMP-2 using Alexa 488 goat anti-mouse IgG as secondary antibody. Intracellular cholesterol was  167  visualized by filipin labelling (1:25 of a stock solution of 10 mg/ml in DMSO). Phospholipid content was determined by mounting coverslips in gelvatol containing Nile Red (1:1000 of a saturated solution). To follow fluid phase endocytosis, cells were incubated for various periods of time with lysine-fixable FITC-Dextran (5 mg/ml) in cell media. Cells were then washed with PBS-CM and processed for immunofluorescence as described above. Visualisation of acidic organelles was performed by incubation of cells for 10 min with 0.1 µM Lysotracker Red added to the medium prior to fixation and labelling with filipin. Labelled cells were viewed with a 100X NeoFluor objective of a Zeiss Axiophot fluorescent microscope equipped with UV, FITC, and rhodamine filter sets, a QImaging Retiga CCD camera and Northern Eclipse imaging software (Empix Imaging, Mississuaga, ON) or the 100X planapochromat objective of a Leica TCS-SP1 confocal microscope equipped with Argon (488), Krypton (568) and HeNe (633) lasers. Quantification of endocytosis was performed by counting the percentage of swollen LAMP-2/Nile Red positive vacuoles labelled for FITC-dextran from 10 confocal images per condition. Presented data was compiled from three independent experiments. MDC labelling was performed as previously described (Biederbick et al., 1995; Munafo and Colombo, 2001). Images were obtained with the 60X PlanApo objective of an Olympus-PTI microscope system equipped with DeltaRam V monochromator and Cascade BF CCD camera. Equivalent acquisition settings and display parameters were used for all samples from a single experiment. Quantification was performed by measuring the average intensity of MDC labelling within large vacuoles (1.4 µm to 8 µm), labelled in a second fluorescent channel for Nile Red, using Image Master software. Presented data, compiled from three independent experiments, were normalized to the mean MDC intensity values of MLBs from control M9 cells and a two-tailed Student’s t test was performed. Induction of autophagy by serum starvation was performed on cells grown on coverslips for 6 days and treated with U18666A for 24 hours. Cells were washed twice with serumcontaining or serum-free DMEM containing U18666A and incubated for 0.5, 1, 3 or 6 hours before fixation and labelling with flilipin and Sytox green. Images were obtained with the  168  60X PlanApo objective of an Olympus FV1000 confocal microscope. The surface area of filipin positive structures, of a diameter of 0.5 to 5 µm that corresponded to MLB size (Table 1), was determined using Image Master software. Overlapping vacuoles were manually segmented where possible and only single, isolated filipin positive structures were quantified. The number of cells present in each field was determined by counting Sytox green labeled nuclei. Equivalent acquisition settings and display parameters were used for all samples from a single experiment and data from 3 different experiments were compiled and a two-tailed Student’s t test performed. Electron Microscopy Cells were rinsed with 0.1 mM sodium cacodylate, pH 7.3 and then fixed for one hour with 2% glutaraldehyde at 4oC. After fixation, the cells were rinsed with cacodylate buffer, scraped from the Petri dish, pelleted and post-fixed with 2% osmium tetroxide at 4oC. The cells were dehydrated and then embedded in LR-White resin. Ultra thin sections were prepared and contrasted with uranyl acetate and lead citrate to enhance contrast. The sections were visualized with a Zeiss CEM902 electron microscope. Quantification of the expression of MLBs and of swollen lysosomal vacuoles in the 3-MA experiments was determined by circumscribing the cytoplasm (excluding the nucleus) and the MLBs and non-lamellar lysosomal vacuoles from 6 images at 3000× magnification and determining the area of the circumscribed regions. MLBs were defined as membrane-bound cytoplasmic organelles presenting at least three distinct circumferential concentric membrane lamellae. MLBs were composed either completely of concentric lamella or of concentric lamella surrounding a single dense core (Hariri et al., 2000). Swollen lysosomal vacuoles were defined by the presence of multiple internal structures surrounded by a limiting membrane and could be morphologically distinguished from MLBs.  169  4.4 RESULTS MLB expression in type II alveolar cells following U18666A-mediated lysosomal cholesterol accumulation To assess the phospholipid and cholesterol distribution of the MLBs expressed upon Mgat5 transfection of Mv1Lu cells, both parental Mv1Lu and the M9 clone of Mgat5-transfected Mv1Lu cells (Demetriou et al., 1995; Hariri et al., 2000) were labelled for Nile Red, a phospholipid-specific dye that labels MLBs of alveolar type II cells (Gonzales et al., 2001; Guttentag et al., 2003), and for filipin, a cholesterol-specific dye. While both Nile Red and filipin labelling did not associate with the LAMP-2 positive lysosomes of Mv1Lu cells (Fig. 4.1A-D), the multiple large LAMP-2-positive lysosomal structures that correspond to MLBs in the M9 clone of Mgat5-transfected Mv1Lu alveolar cells (Hariri et al., 2000) were labelled for Nile Red indicating that they are phospholipid-rich (Fig. 4.1E-H). Labelling of cholesterol with filipin shows that the lumen of the MLBs is not labelled although a peripheral filipinpositive ring is frequently observed to circumscribe the Nile Red positive MLBs (Fig. 4.1G and Q-T), consistent with the fact that in type II alveolar cells, cholesterol is mainly localized to the limiting membrane of the organelles (Orgeig and Daniels, 2001; Punnonen et al., 1988). Treatment of both Mv1Lu and Mgat5-transfected M9 cells with U18666A resulted in the expression of large LAMP-2-positive vacuoles labelled for both Nile Red and filipin, reflecting the ability of U18666A to induce cholesterol accumulation in both lysosomes and MLBs, respectively (Fig. 4.1I-P). By electron microscopy, Mv1Lu cells do not present MLBs while M9 cells present multiple cytoplasmic MLBs (Fig. 4.2A,B and Table 4.1), as previously reported (Hariri et al., 2000). In both cell lines, numerous MLBs are observed upon treatment with U18666A (Fig. 2C,D and Table I) indicating that lysosomal accumulation of cholesterol results in MLB formation independently of Mgat5 expression levels. In Mv1Lu cells, lysosomal cholesterol accumulation due to U18666A treatment therefore results in de novo formation of MLBs. In M9 cells, U18666A treatment increases the number of MLBs and the cytoplasmic area covered by MLBs (Table 4.1) indicating that  170  cholesterol accumulation occurs within preexisting phospholipid-rich MLBs without disrupting their lamellar morphology but also induces new MLBs. Cholesterol accumulation overrides the role of lysosomal degradation but not autophagy in MLB formation MLBs are lysosomal organelles and express various lysosomal hydrolases (de Vries et al., 1985; DiAugustine, 1974; Hatasa and Nakamura, 1965; Hook and Gilmore, 1982). The previously demonstrated ability of leupeptin treatment to prevent MLB formation in Mgat5 transfected Mv1Lu cells (Hariri et al., 2000) led us to determine whether U18666A could induce MLB formation in leupeptin treated M9 cells. After 96 hours in the presence of leupeptin, Mv1Lu and M9 cells express large LAMP-2/Nile Red positive vacuoles and treatment with U18666A for 24 hours results in the formation of vacuoles strongly labelled for filipin, essentially identical to that observed in cells not treated with leupeptin (data not shown). By electron microscopy, treatment of Mv1Lu cells with leupeptin for 4 days induced the accumulation of dense autophagic vacuoles (AVd) (Fig. 4.3A). In M9 cells, leupeptin treatment resulted in the disappearance of MLBs and their replacement by large AVd (Fig. 4.3B), as previously reported (Hariri et al., 2000). Treatment of leupeptin-treated cells with U18666A for the final 24 hours resulted in the induction of MLBs and disappearance of dense autophagic vacuoles in both cell types (Fig. 4.3C,D). U18666A-mediated accumulation of cholesterol therefore results in the formation of concentric lamellar structures within autophagic vacuoles independently of protein degradation. 3-MA inhibits autophagy at the sequestration step (Seglen and Gordon, 1982) and was previously shown to prevent MLB formation in Mgat5 transfected Mv1Lu cells (Hariri et al., 2000). By immunofluorescence, M9 cells incubated with 3-MA for 3 days no longer express large LAMP-2/Nile Red positive vacuoles and Nile Red labelling presents a diffuse cytoplasmic distribution (Fig. 4.4E-H), similar to that of untreated (Fig. 4.1) and 3-MA treated Mv1Lu cells (Fig. 4.4A-D). Treatment of 3-MA treated Mv1Lu and M9 cells with U18666A for 24 hours induces the formation of large LAMP-2/Nile Red/filipin positive vacuoles (Fig. 4I-P). As previously reported (Hariri et al., 2000), 3-MA treatment of both  171  Mv1Lu and Mgat5-transfected M9 cells results in the appearance of vacuoles that do not present internal concentric lamella and that are morphologically distinct from MLBs (Fig. 4.5A,D). U18666A treatment of 3-MA treated Mv1Lu and M9 cells resulted in the expression of non-lamellar vacuoles morphologically similar to those induced by 3-MA in the absence of U18666A but larger in size (Fig. 4.5B,C,E,F and Table 4.1). In the presence of U18666A, the non-lamellar vacuoles represent the only cytoplasmic structures that could correspond  to  the  filipin-labelled  LAMP-2-positive  vacuoles  observed  by  immunofluorescence labelling (Table 4.1). The non-lamellar vacuoles observed in the presence of 3-MA therefore correspond to U18666A-induced cholesterol-rich lysosomal vacuoles. 3-MA treatment therefore prevents both the expression of MLBs in M9 cells and the ability of cholesterol accumulation to reorganize the internal membranes of lysosomal and autophagic vacuoles into the concentric, circumferential lamella typical of MLBs. The MLBs of untreated and U18666A treated M9 cells (Fig. 4.6A,C) are labelled for monodansylcadaverine (MDC), a marker for autophagic vacuoles (Biederbick et al., 1995; Munafo and Colombo, 2001). Following 3-MA treatment, the intensity of MDC labelling is significantly reduced in both untreated and U18666A treated cells (Fig. 4.6B,D). The continued presence of large, swollen vacuoles labelled for Nile Red in U18666A, 3-MA treated cells allowed us to quantify vacuole associated MDC labelling. As seen in Fig. 4. 6E, untreated and U18666A treated cells present a similar mean MDC intensity that is significantly reduced upon 3-MA treatment reflecting the ability of 3-MA to inhibit autophagy in this cell line as previously described (Hariri et al., 2000). Serum starvation stimulates macroautophagy in various cell lines (Munafo and Colombo, 2001; Susan and Dunn, 2001). Starvation of both untransfected and Mgat5 transfected Mv1Lu cells following U18666A treatment leads to an increase in the size of filipin positive vacuoles after 3 and 6 hours as well as an increase in the total area covered by filipin positives vacuoles per cell (Fig. 4.7D,E). The vacuoles of serum-starved cells still present a multilamellar morphology (data not shown). Stimulation of macroautophagy is therefore associated with increased size and expression of MLBs.  172  3-MA does not affect fluid phase endocytosis to lysosomes 3-MA is an inhibitor of PI3 kinase (PI3K) (Blommaart et al., 1997) and the PI3K product PtdIns 3-phosphate (PI(3)P) is known to be involved in endocytic trafficking as well as the autophagic process (Petiot et al., 2000; Simonsen et al., 2001; Wurmser et al., 1999). Inhibition of PI3K activity has also been shown to induce the redistribution of lysosomal glycoproteins from lysosomes to mannose-6-phosphate receptor-negative, acid hydrolasenegative late endosomal compartments (Reaves et al., 1996). We therefore undertook to determine whether 3-MA treatment was preventing endocytosis in Mgat5 transfected Mv1Lu cells and whether the U18666A LAMP-2 positive vacuoles are late endocytic or lysosomal structures. Uptake of the fluid phase marker FITC-dextran to phospholipid and cholesterolrich MLBs was followed in untreated and 3-MA treated cells. As can be seen in Figure 4.8, FITC-dextran is not present in LAMP-2-positive phospholipid- and cholesterol-rich MLBs of M9 cells after 30 minutes endocytosis but accumulates in these structures after 4 hours. Similar results were observed for Mv1Lu and M9 cells treated with U18666A in the absence or presence of 3-MA (Fig. 4.8). Quantification revealed that delivery of FITC-dextran to LAMP-2/Nile Red labelled lysosomal vacuoles was essentially identical irrespective of the presence of U18666A and/or 3-MA (Fig. 4.8S). Labelling of the majority of the vacuoles after 4 hours uptake identifies them as late lysosomal structures, consistent with the derivation of MLBs from dense autophagic vacuoles (Hariri et al., 2000). Inhibition of autophagy in the presence of 3-MA (Hariri et al., 2000), argues that U18666A is acting on secondary lysosomes of Mv1Lu and M9 cells. The equivalent rate of delivery of FITCdextran to the MLBs of untreated M9 cells and the non-lamellar cholesterol-rich vacuoles induced by U18666A in the presence of 3-MA indicates that 3-MA is not inhibiting fluid phase endocytosis (Fig. 4.8S). Similarly, 3-MA did not prevent FITC-dextran endocytosis to LAMP-2 positive lysosomes in M9 cells (Fig. 4.9). Lysotracker is an uncharged compound that is freely permeant to cell membranes and accumulates in acidic compartments by a process involving diffusion and trapping by protonation (Haller et al., 1996). MLBs have a pH of 6.1 or less that is maintained by an energy-dependent process (Chander et al., 1986). Both phospholipid- and cholesterol-rich MLBs are positive for Lysotracker Red labelling (Fig. 4.10). The LAMP-2 positive vacuoles  173  observed in 3-MA and U18666A treated cells are also labelled by Lysotracker Red (Fig. 10J,K,L). 3-MA does not therefore interfere with the endocytic accessibility and acidic lysosomal nature of the large cholesterol-rich vacuoles induced by U18666A.  174  4.5 DISCUSSION Multiple mechanisms modulate the expression of MLBs in the Mv1Lu type II alveolar cell line MLB expression is usually lost upon long term culture and in established cell lines of type II alveolar cells (Diglio and Kikkawa, 1977; Guttentag et al., 2003; Kawada et al., 1990; Rannels et al., 1987; Sannes, 1991; Tanswell et al., 1991). MLB expression in the mink Mv1Lu type II alveolar cell line can be induced by expression of β1-6GlcNAc-branched Nglycans (Hariri et al., 2000). MLBs were also induced in the Mv1Lu type II alveolar cell line by treatment with U18666A, a compound that promotes lysosomal cholesterol accumulation, independently of Mgat5 transgene expression. U18666A treatment has been shown to induce MLB formation in CHO cells (Lusa et al., 2001) but not in HeLa cells (Tomiyama et al., 2004), MDCK or mammary carcinoma cells (data not shown). Lysosomal cholesterol accumulation is therefore not sufficient to induce MLB formation and other cell-type specific factors are required. Nevertheless, the ability of both Mgat5 and cholesterol to independently promote MLB expression in the Mv1Lu type II alveolar cell line shows that multiple factors can modulate the expression of this organelle. The ability of cholesterol to override the need for lysosomal degradation in phospholipid-rich MLB formation demonstrates that the biogenesis of this organelle is a complex process that can follow different pathways. Varied lysosomal contents and biogenetic pathways are therefore permissive for MLB formation, consistent with the expression of these morphologically identical structures under normal physiological conditions in type II alveolar cells as well as pathologically in cancer and lysosomal storage diseases of different genetic origins. Mgat5 and expression of β1-6GlcNAc-branched N-glycans products are upregulated with tumor progression (Dennis et al., 1999) and "coarse vesicles" presenting a lamellar morphology are revealed by L-PHA, a probe for branched N-glycans, in various cancers (Handerson and Pawelek, 2003). The expression of β1-6GlcNAc-branched N-glycans on lysosomal glycoproteins may promote their expression or stability thereby contributing to lamella expression. Knockout of LAMP-2 results in the mistargeting of lysosomal hydrolases and accumulation of autophagic vacuoles (Eskelinen et al., 2002; Tanaka et al., 2000). LAMP-1 and -2 are major cellular carriers of polylactosamine (Carlsson and Fukuda, 1990) and the impact of their glycosylation on lysosome biogenesis and autophagy remain to be  175  determined. N-glycans on cytokine receptors bind to galectins in an Mgat5-dependent manner and form a multivalent lattice that retains the receptors at the cell surface. The Mgat5-modified N-glycans increase surface residency of receptors, lowering the response thresholds to multiple cytokines, including TGF-β, EGF, PDGF and IGF (Partridge et al., 2004). In this manner autocrine signaling through PI3 kinase is potentiated in Mgat5expressing cells, and may contribute to MLB formation in both Mgat5-transfected Mv1Lu type II alveolar cells as well as in tumor cells that express increased levels of Mgat5 (Handerson and Pawelek, 2003; Hariri et al., 2000). An autophagic contribution promotes lamella formation in lysosomal organelles Autophagy is involved in both sphingolipid and glycoprotein metabolism (Ghidoni et al., 1996) and, conversely, sphingolipid ceramide can modulate autophagy in HT-29 cells (Scarlatti et al., 2004). 3-MA and other inhibitors of PI3K activity block autophagy (Blommaart et al., 1997; Hariri et al., 2000; Munafo and Colombo, 2001; Seglen and Gordon, 1982). Our previous demonstration that 3-MA inhibits both autophagy and the formation of MLBs in Mgat5 transfected Mv1Lu cells (Hariri et al., 2000) was confirmed in this study by the disappearance of phospholipid-rich MLBs in 3-MA treated cells (Figs. 4, 5). 3-MA did not prevent the accumulation of lysosomal cholesterol but the swollen, lysosomal structures no longer expressed the concentric lamella typical of MLBs. The similar morphology of these swollen, lysosomal structures with the smaller inclusion bodies seen upon 3-MA treatment in the absence of U18666A suggests that these two structures are similar lysosomal organelles that vary in size due to accumulation of cholesterol. As seen in Table 1, 3-MA treatment of U18666A treated cells resulted in essentially the complete transition from expression of MLBs to non-lamellar lysosomal vacuoles. The large size of U18666A-induced vacuoles enabled us to identify and compare the multilamellar and non-lamellar lysosomal structures in untreated and 3-MA treated cells by light microscopy. Inhibitors of PI3 kinase also block vesicular transport from late endosomes to lysosomes (Punnonen et al., 1994) and impair both early endosome fusion and maturation of lysosomes (Mousavi et al., 2003; Reaves et al., 1996; Simonsen et al., 1998). More recently, PI3K  176  signalling has been shown to more specifically regulate endosomal sorting (Petiot et al., 2003). The large non-lamellar LAMP-2 positive, cholesterol-rich vacuoles formed upon addition of U18666A to 3-MA are labelled by fluid-phase uptake of FITC-dextran after 4 hours but not 30 minutes and are positive for lysotracker indicating that they are acidic, late lysosomal organelles. The non-lamellar vacuoles that accumulate in the presence of 3-MA and U18666A are therefore not prelysosomal compartments but correspond to late or secondary lysosomes. Furthermore, 3-MA does not affect endocytic accessibility of fluid phase material to these late, lysosomal organelles. Biosynthetic trafficking of LAMP to lysosomes can include transit via early endosomes and/or the plasma membrane (Nabi et al., 1991; Peden et al., 2004) and recycling of LAMPs permits uptake of anti-LAMP antibodies to lysosomes (Lippincott-Schwartz and Fambrough, 1987; Nabi et al., 1991; Williams and Fukuda, 1990). As observed for fluid phase uptake of FITC-dextran, anti-LAMP-2 antibodies internalized by recycling LAMP-2 access MLBs and non-lamellar vacuoles after 2-4 hours (data not shown). Multivesicular bodies or endosomes have been observed to fuse with MLBs in type II alveolar cells (Chevalier and Collet, 1972; Voorhout et al., 1993; Vorbroker et al., 1995) and sorting of endosomal content to the internal vesicles of multivesicular bodies may represent a requisite aspect of MLB formation (Piper and Luzio, 2001; Stahl and Barbieri, 2002; Weaver et al., 2002). Similarly, fusion between various elements of the endolysosomal and autophagic pathways is well-documented (Fig. 11). Formation of nascent autophagic vacuoles is followed rapidly by acquisition of hydrolytic enzymes by fusion with pre-existing lysosomes (Lawrence and Brown, 1992; Lawrence and Brown, 1993; Lee et al., 1989; Liou et al., 1997) and early autophagic vacuoles fuse with endosomes before fusing with lysosomes (Gordon and Seglen, 1988). Interaction between autophagic vacuoles and both endosomal and lysosomal compartments therefore necessarily contributes to the composition of autolysosomes (Fig. 11). Autophagic vacuoles have long been noted for the presence of whorl-like membrane structures (Schmitz and Muller, 1991). MDC is a fluorescent probe whose specificity for autophagic vacuoles (Biederbick et al., 1995; Munafo and Colombo, 2001) is based, unlike  177  other lysomotropic agents, on its ability to act as a solvent polarity probe exhibiting enhanced fluorescence upon interaction with membrane lipids (Niemann et al., 2000). MDC should therefore selectively labels dense phospholipid containing organelles such as autophagic vacuoles and MLBs. LC3 is a specific marker for the isolation membrane of autophagosomes and autophagosomes (Kabeya et al., 2000; Mizushima, 2004; Mizushima et al., 2001). MDC positive structures negative for LC3 are induced by nutrient starvation and MDC would therefore appear to be a marker for mature autophagic vacuoles or autolysosomes (Munafo and Colombo, 2001; Gutierrez et al., 2004), consistent with this report. Reduced labelling of cholesterol-rich U18666A-induced vacuoles with MDC in the presence of 3-MA (Fig. 4.6) is therefore indicative of the reduced delivery of phospholipids to these organelles via autophagy thereby altering the lipid composition of these organelles and preventing their reorganization into concentric lamella, as observed by EM. The ability of serum starvation to enhance the size of MLBs (Fig. 7) further demonstrates that macroautophagy regulates the expression of these organelles. This study shows that autophagic delivery of phospholipids to late lysosomal organelles promotes MLB expression in the Mv1Lu type II alveolar cell line and, indeed, argues that MLBs are autolysosomes. However, autoradiographic studies have shown that phospholipids are delivered to MLBs of type II alveolar cells in intact lung directly from the Golgi apparatus (Chevalier and Collet, 1972). Furthermore, MLBs are absent from Mv1Lu cells, as in most type II alveolar derived cells in culture (Diglio and Kikkawa, 1977; Guttentag et al., 2003; Kawada et al., 1990; Rannels et al., 1987; Sannes, 1991; Tanswell et al., 1991), and the ubiquitous expression of MLBs in various other cell types argues that multiple mechanisms may contribute to MLB expression. Indeed, we are able to induce MLB expression in Mv1Lu cells by modulating both protein glycosylation and lysosomal cholesterol content. Whether autophagy plays a critical or regulatory role in MLB formation and surfactant expression in lung type II alveolar cells remains to be determined. Nevertheless, regulation of the lipid composition of late, lysosomal organelles by autophagy promotes the expression of MLBs and may modulate expression of these organelles under both physiological and pathological conditions.  178  4.6 ACKNOWLEDGEMENTS This study was supported by a grant from the Canadian Institutes of Health Research (CIHR). IRN is the recipient of a CIHR Investigator award.  179  Table 4.1: Quantification of MLB expression in Mv1Lu cells and M9 clones transfected with Mgat5 before and after treatment with U18666A and 3-MA. Multilamellar bodies (MLBs) Non-lamellar lysosomal vacuoles Cell type Number Size range % cell Number Size range % cell area area (median) (median) Mv1Lu control Mv1Lu +U18666A  1  (µm) 4.6  0.13  -  (µm) -  -  130  0.3-6.2 (1.6)  4.35±0.8  -  -  -  Mv1Lu +3MA  0  0  0  62  0.5-3.5 (1.3)  1.1±0.5  Mv1Lu +U18666A +3MA M9 control  7  0.2-5.4 (1.3)  0.02±0.05  86  0.8-6.0 (1.7)  3.5±0.7  107  0.1-6.7 (1.7) 0.1-7.8 (1.9)  5.1 ±1.6  -  -  -  7.4 ±0.9  -  -  -  0.1-5.8 (1.2) 0.1-6.1 (1.5)  0.4±0.3  76  1.26±0.7  0.4±0.3  100  0.1-3.3 (1.1) 0.1-7.4 (1.9)  M9 +U18666A  169  M9 +3MA  22  M9 +U18666A +3MA  9  6.5±0.5  180  A  LAMP-2  B  Nile Red  C  Filipin  D  Merge  Mv1Lu E  F  G  H  I  J  K  L  Mv1Lu +U18666A MI  N  O  P  M9 +U18666A Q  R  S  T  M9  M9  Figure 4.1  181  Figure 4.1: U18666A treatment induces the accumulation of swollen cholesterol-rich, LAMP-2 positive vacuoles. Untransfected Mv1Lu cells (A-D, I-L) and Mgat5 transfected M9 clones (E-H, M-Q) were cultured in regular medium for 6 days (A-H, Q) and select cultures incubated with U18666A for the final 24 hours (I-P). The cells were then fixed and triple labelled with anti-LAMP-2 antibody (A, E, I, M), Nile Red (B, F, J, N) and filipin (C, G, K, O). Merged images (D, H, L, P) show LAMP-2 in green, Nile Red in red, and filipin in blue. In untreated M9 cells, filipin labelling is concentrated at the limiting membrane of the vacuoles (Q-T). In the presence of U18666A, the LAMP-2/Nile Red-positive vacuoles (arrows) accumulate cholesterol and are all filipin positive after 24h. Bars: 5 µm.  182  A  B  Mv1Lu C  M9 D  Mv1Lu +U18666A  M9 +U18666A  Figure 4.2  183  Figure 4.2: U18666A induces MLB expression. Mv1Lu (A, C) and Mgat5 transfected M9 (B, D) cells were incubated with U18666A for 24 hours (C and D) and processed for electron microscopy. Cytoplasmic MLBs are present in M9 cells as well as in both untransfected Mv1Lu and M9 clones following treatment with U18666A. Bars: 2.5 µm.  184  A  B  Mv1Lu C  M9 D  Mv1Lu +U18666A  M9 +U18666A  Figure 4.3  185  Figure 4.3: U18666A induces MLB expression despite leupeptin treatment. Mv1Lu (A, C) and Mgat5 transfected M9 (B, D) cells were incubated with leupeptin (2 µg/ml) for 96 hours and U18666A was added to select cultures (C, D) for the final 24 hours before processing for electron microscopy. In the absence of U18666A, leupeptin induces the accumulation of dense autophagic vacuoles however U18666A still induces MLB expression in leupeptin treated cells. Bars: 2.5 µm.  186  LAMP-2  Nile Red  Merge  Filipin  A  B  C  D  Mv1Lu +3-MA E  F  G  H  I  J  K  L  Mv1Lu +U18666A+3-MA M  N  O  P  M9 +3-MA  M9 +U18666A+3-MA  Figure 4.4  187  Figure 4.4: U18666A treatment induces accumulation of swollen LAMP-2 positive vacuoles in the presence of the autophagy inhibitor 3-MA. Untransfected Mv1Lu (A-D, IL) and Mgat5 transfected M9 (E-H, M-P) cells were grown in media supplemented with 10 mM 3-MA for 3 days and select cultures were incubated with U18666A for the final 24 hours (I-P). The cells were fixed and then triple labelled with anti-LAMP-2 (A, E, I and M), Nile Red (B, F, J and N) and filipin (C, G, K and O). Merged images (D, H, L and P) show LAMP-2 in green, Nile Red in red, and filipin in blue. Large phospholipid-rich, LAMP-2 positive vacuoles are not observed following 3-MA treatment but U18666A is still able to induce the formation of swollen, filipin-positive lysosomal vacuoles. Bar: 5 µm.  188  A  B  C  Mv1Lu  Mv1Lu +U18666A  Mv1Lu +U18666A  D  E  F  M9  M9 +U18666A  M9 +U18666A  Figure 4.5  189  Figure 4.5:  Swollen vacuoles induced by U18666A in the presence of 3-MA lack  concentric lamella. Untransfected Mv1Lu (A-C) and Mgat5 transfected M9 (D-F) cells were incubated with 10 mM 3-MA for 3 days and select cultures treated with U18666A for 24 hours (B,C,E and F) prior to processing for electron microscopy. 3-MA treatment results in the disappearance of MLBs in M9 cells and expression of membrane-bound, non-lamellar inclusion bodies that lack concentric lamella. Bars: A, C, E and F: 0.5 µm, B and D 2 µm.  190  B  M9 C  M9 +3-MA D  M9 +U24  M9 +U24 + 3-MA  Intensity of MDC labelling (%)  A  120 M9 M9 +U18666A M9 +U18666A +3-MA  100 80 60 40  p= 0.0016 *  20 0  Figure 4.6  191  Figure 4.6: 3-MA treatment reduces MDC labelling of swollen, lysosomal vacuoles. M9 cells were grown in regular medium (A and C) or in media supplemented with 10 mM 3-MA for 3 days (B and D) and select cultures were incubated with U18666A for the final 24 hours (C and D). Cells were incubated with MDC and Nile Red for 10 minutes and images of MDC labelling of unfixed cells are shown. E) Mean MDC labelling intensity of lysosomal vacuoles positive for Nile Red was quantified in untreated M9 cells as well as in U18666A treated M9 cells in the presence or absence of 3-MA (mean ± S.D. of 3 independent experiments). MLBs present in untreated M9 cells and cells treated with U18666A are strongly labelled for MDC however MDC labelling of swollen, lysosomal vacuoles formed in the presence of 3-MA treatment is significantly reduced. Bar: 10 µm.  192  A  B  C  Mv1Lu U24 0  Mv1Lu U24 +6h  Mv1Lu U24 +6h -serum  Mv1Lu  + Serum - Serum  E  *  *  11 0,5 0,5 00  ***  8 8 7  66 5  44 3  22 1  0  30 min  1h  3h  6h  2,5 2,5 22 1.5 1,5  M9  + Serum - Serum  *  *  11 0,5 0,5 00 9  Total area of vacuoles per cell (µm2)  Total area of vacuoles per cell (µm2)  9  00  Average area of vacuoles (µm2)  2,5 2,5 22 1,5 1.5  Average area of vacuoles (µm2)  D  *  88 7  66  **  5  44 3  22 1  00  0  30 min  1h  3h  6h  Figure 4.7  193  Figure 4.7: Serum starvation stimulates expression of cholesterol-rich MLBs. Untransfected Mv1Lu cells were grown for 6 days and U18666A was added for the final 24 hours (A). Select cultures were then washed and incubated for 6 hours in serum-containing (B) or serum-free (C) media and labeled with filipin. The expression of filipin-labeled MLBs was quantified from both untransfected Mv1Lu (D) and Mgat5 transfected M9 cells (E) incubated in serum-containing (empty bars) or serum-free (filled bars) media for 0.5, 1, 3 and 6 hours labeled with filipin and the nuclear dye Sytox green. The average area of filipinlabeled vacuoles and the total area covered by these vacuoles per cell were quantified (mean ± S.E.M. of 3 independent experiments). *p< 0.005, **p<0.01, ***p<0.05 Bar : 10 µm  194  30 min A  LAMP-2  B  4h  FITC-dextran  C  Merge  ctlLAMP-2 30' D  E  FITC-dextran  F  Merge  Ctl  G  H  I  J  K  L  M  N  O  P  Q  R  U24  U24 +3-MA  S % LAMP-2/NileRed vacuoles positive for FITC-dextran  90  M9 untreated  80 70  M9+U18666A  60 50  M9+U18666A +3-MA  40 30  Mv1Lu+U18666A  20 10 0 30min  1h  2h  4h  Mv1Lu+U18666A +3-MA  Time of endocytosis  Figure 4.8  195  Figure 4.8: 3-MA treatment does not affect accessibility of LAMP-2 positive vacuoles to fluid phase endocytosis. M9 cells either untreated (CTL, A-F), treated with U18666A for 24 hours (U24h; G-L) or grown for 3 days in the presence of 10mM 3-MA with U18666A added for the final 24 hours (U24h + 3-MA; M-R) were incubated for 30 minutes (A-C, G-I, M-O) or 4 hours (D-F, J-L, P-R) in the presence of 5 mg/ml FITC-dextran prior to labelling for LAMP-2. LAMP-2 labelling (A, D, G, J, M, P) is shown in red and FITC-dextran labelling (B, E, H, K, N, Q) in green and merged images are presented in C, F, I, L, O and R. The percentage of LAMP-2/Nile Red-positive swollen vacuoles containing FITC-dextran at 0.5, 1, 2 and 4 hours of FITC-dextran endocytosis was quantified from Mv1Lu and M9 cells treated or not with 3-MA for 3 days and/or U18666A for the final 24 hours, as indicated (S). For all treatment conditions, FITC-dextran does not accumulate in swollen LAMP-2 positive vacuoles at 30 minutes and the majority are accessible after only 4 hours endocytosis even in the presence of 3-MA. Bar: 5 µM.  196  LAMP-2 A  B  30 min D  E  FITC-dextran  C  Merge  F  4h  Figure 4.9  197  Figure 4.9: 3-MA treatment does not affect fluid-phase endocytosis to lysosomes. M9 cells were grown in the presence of 3-MA for 3 days and then incubated with FITC-dextran for 30 minutes (A-C) or 4 hours (D-F) prior to labelling for LAMP-2 (A and D). FITCdextran labelling (B, E) is shown in green and LAMP-2 in red in the merged images (C and F). FITC-dextran does not accumulate in lysosomal structures at 30 minutes but reaches them after 4 hours of endocytosis. Bar: 5 µM.  198  Filipin  Lysotracker A  Control D  B  C  E  F  ctl  +3-MA G  +U18666A J  Merge  H  I  K  L  +U18666A+3-MA  Figure 4.10  199  Figure 4.10: Non-lamellar, cholesterol-rich LAMP-2 positive vacuoles are acidic, lysosomal organelles. M9 cells were grown in regular medium (A-C, G-I) or in media supplemented with 10 mM 3-MA for 3 days (D-F, J-L) and select cultures were incubated with U18666A for the final 24 hours (G-L). Viable cells were labelled with Lysotracker Red (A, D, G and J) and after fixation with filipin (B, E, H and K). Merged images (C, F, I and L) show Lysotracker Red in red and filipin in blue. The large cholesterol-rich swollen lysosomal vacuoles induced by U18666A are still acidic in the presence of 3-MA. Bar: 5 µM.  200  A  isolation membrane  plasma membrane  B  isolation membrane  plasma membrane  autophagosome endosome  endosome  +3-MA autolysosome  lysosome +U  +U +U  leupeptin  lysosome  +U  phospholipidrich MLB  cholesterolrich MLB  Non-lamellar lysosomal vacuole  Figure 4.11  201  Figure 4.11: An autophagic contribution is required for the formation of phospholipid and cholesterol-rich MLBs. A) Extensive interaction occurs between the endocytic (black) and autophagic (red) pathways. Maturation of autolysosomes into MLBs requires lysosomal degradation and is inhibited by leupeptin. 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I have shown that Cav1 may negatively regulate both EGFR signaling and cell surface diffusion (Lajoie et al., 2007) and raft-dependent endocytosis of CT-B to the Golgi (Lajoie et al, 2007 in preparation). Caveolae have been extensively studied over the years and have been shown to play many roles within the cell. However, if the role of Cav1 in caveolae formation has been well characterized, the potential roles of Cav1 outside caveolae remain poorly understood. Using the Mgat5-/- cells and Mgat5-/-ESC expressing low Cav1 levels, I have demonstrated that Cav1 may act as a negative regulator at low expression levels. Electron microscopy studies of Mgat5-/- cells have revealed that these cells express very few caveolae. It is unlikely that the relatively rare caveolae present at the plasma membrane of these cells are responsible for the inhibition of both EGFR signaling and diffusion and CT- B uptake. The conclusions of work presented in this thesis therefore support a role for Cav1 independently of caveolae formation. Other groups have reported evidence for a role for non-caveolar Cav1. One of the best examples comes from studies of an endothelial cell specific Cav1 transgenic mouse. Endothelial cells from this animal display higher Cav1 expression associated with inhibition of eNOS activation (Bauer et al., 2005). However, it is surprising that increased Cav1 expression in these cells does not translate to an increase in plasma membrane caveolae compared to wild-type cells. It is therefore possible that the effects mediated by the Cav1 increase are due to the non-caveolar pool of Cav1. Also, another group showed that integrins interact with Cav1, and that interaction is likely to occur outside of caveolae (Wary et al., 1996). In the manuscripts presented previously in this thesis, I have used Cav1 mutated on tyrosine 14 (Y14F) that cannot be phosphorylated. This mutant has showed the potential to inhibit  211  both EGFR signaling and diffusion and raft-dependent endocytosis of CT-B. To this day, we do not know if this Cav1 mutant is able to induce caveolae formation upon transfection in Mgat5 null cells that do not express caveolae. I am in the process of generating stable cell lines expressing this mutant that will enable me to perform electron microscopy studies that will help us to address this question. It has previously been shown that EGF treatment stimulates Cav1 phosphorylation and induces caveolae formation at the plasma membrane. Upon transfection with the Y14F mutant, EGF treatment no longer induces caveolae formation suggesting that Cav1 phosphorylation is a critical determinant of caveolae expression at the surface of the cell, at least in the presence of EGF (Orlichenko et al., 2006). It is therefore possible that in our Mgat5 model, the high expression of caveolae in Mgat5+/+ cells is due to their increased EGF sensitivity. Unlike both Mgat5 null cell lines, the Mgat5+/+ cells express the phosphorylated form of Cav1 that may be required for caveolae formation. Moreover, Cav1 phosphorylation can be increased by the addition of exogenous galectin-3 indicating a potential link between the Mgat5/galectin lattice and Cav1 phosphorylation (Goetz et al, 2007). Considering these data, expression of the Y14F mutant might inhibit caveolae formation. That would support the idea that caveolae are not required for negative regulation by Cav1. However, expression of the Y14F Cav1 mutant in Cav1-/- cells has been shown to induce normal caveolae (del Pozo et al., 2005). Caveolae formation may be dependent on various factors and specific conditions. These conditions may also vary according to the cell type. The negative regulatory activity of Cav1 is possibly a mechanism distinct from caveolae expression. We have shown that mutations in the Cav1 scaffolding domain disrupt the ability of Cav1 to act as a negative regulator. Here again, we do not know if mutations in the scaffolding domain inhibit caveolae formation. The scaffolding domain has been shown to be involved in the attachment of Cav1 to the plasma membrane (Schlegel et al., 1999). Mutations in this domain should therefore affect its insertion into the membrane and consequently impair caveolae formation. We also do not know if that specific mutant is being phosphorylated. More investigations are therefore required in order to characterize the role of Cav1 phosphorylation in caveolae biogenesis.  212  The threshold of Cav1 expression level required for inhibition of cytokine signaling and raft endocytosis might be different than the one required for caveolae formation. Based on our results, expression of sufficient level of Cav1 oligomers at the plasma membrane is the crucial determinant for its negative regulatory activity. Caveolae formation may therefore follow its proper regulation that may include different elements such as Cav1 phosphorylation. Cav1 regulation of caveolae formation and Cav1 negative regulatory activity of receptor signaling and endocytosis should therefore be considered as two separate mechanisms. This does not mean that caveolae are not involved in the regulation of receptor signaling and endocytosis. Caveolae have been shown to contribute to the internalization of various ligands and receptors (Di Guglielmo et al., 2003; Matveev and Smart, 2002; Mineo et al., 1999; Park et al., 2000; Parton and Simons, 2007). However, their expression might not be an absolute requirement for the negative regulatory activity of Cav1. The work presented in this thesis has shown that Cav1 is able to regulate EGFR signaling independently of caveolae. These data identify a new role for Cav1, outside caveolae. These finding may contribute to reinforce the growing idea that Cav1 plays a role independently of caveolae formation. 5.2 How can Cav1 act as a negative regulator? We have discussed the role of Cav1 as a negative regulator and shown that it may act outside caveolae. If Cav1 acts outside caveolae, how is it working? Cav1 has been shown to bind cholesterol through a putative cholesterol binding domain (Murata et al., 1995). Cholesterol binding to Cav1 is essential to the association of Cav1 with raft domains and for its oligomerization and later transport to the Golgi complex and to the plasma membrane (Parton et al., 2006). Displacement of cholesterol from lipid rafts by ceramide has been shown to reduce raft cholesterol content and was associated with reduced Cav1 levels in raft fractions (Yu et al., 2005). These results indicate that cholesterol is a crucial element involved in the proper targeting of Cav1 to lipid raft domains. We have demonstrated that Cav1 can negatively regulate raft-dependent uptake of both cholera toxin and autocrine motility factor (AMF) (Kojic et al., 2007; Le et al., 2002; Nabi and Le, 2003). It is therefore possible that upon overexpression, Cav1 will contribute to  213  sequestering cholesterol within lipid rafts. In chapter 1, I have discussed the variable nature and content of lipid raft domains. Based on recent studies from the Nabi lab, it seems unlikely that Cav1 positive raft domains are involved in efficient internalization of both CTB and AMF (Kojic et al., 2007). It is more likely that Cav1 expression stabilizes raft domains at the plasma membrane and therefore prevents their internalization via endocytosis (Lajoie and Nabi, 2007; Nabi and Le, 2003). Cholesterol is essential for raft endocytosis and these pathways are sensitive to cholesterol depletion (Lajoie and Nabi, 2007). I have demonstrated that raft mobility at the cell surface is reduced upon expression of Cav1 (Lajoie et al., 2007) indicating that Cav1 expression alters raft behaviour. It is possible that equilibrium exists between Cav1 and cholesterol level. Overexpressing one or the other may switch that equilibrium and therefore favour a different regulation of raft movement and internalization. It has been shown that increasing the plasma membrane sphingolipid content using natural and synthetic glycosphingolipids and elevation of cholesterol using cyclodextrin/cholesterol complexes increases caveolar endocytosis (Sharma et al., 2004). I am now investigating the role of cholesterol in the negative regulation of CT-B endocytosis in the Mgat5 cell lines. I propose to use cholesterol-cyclodextrin complex to deplete and increase plasma membrane cholesterol and test the effects on CT-B endocytosis. In biological membranes, the ratio between cholesterol and phospholipids is maintained slightly below 1:1 (Lange et al., 1989). It has been demonstrated that when in excess and therefore free from interaction with phospholipids, cholesterol shows a higher chemical activity (McConnell and Radhakrishnan, 2003). This pool has been described as active cholesterol (Lange et al., 2004; Lange et al., 2005; McConnell and Radhakrishnan, 2003; Puri et al., 1999). Cav1 may therefore regulate cholesterol-dependent processes, such as raft endocytosis, through sequestration of active cholesterol. Dynamin-2 represents another molecule that plays a major role in endocytosis of various ligands via both clathrin-dependent and independent pathways. It is known that Cav1 and dynamin-2 directly interact (Yao et al., 2005). Dynamin-2 is essential for the internalization  214  of many endocytic carriers. It localises at the neck of endocytic vesicles, generating the mechanical force required for fission from the plasma membrane (Oh et al., 1998). In the model I proposed, Cav1 stabilizes lipid rafts at the plasma membrane reducing their endocytic potential (Lajoie and Nabi, 2007; Nabi and Le, 2003). We have also demonstrated that expression of a mutant dynamin-2 is able to induce caveolae-like structures in cells lacking Cav1 (Le et al., 2002). Taken together, these data indicate that Cav1 and dynamin-2 are both key regulators of raft-dependent endocytosis. CT-B can be internalized via both dynamin-2 dependent and independent pathways (Glebov et al., 2006; Kirkham et al., 2005; Torgersen et al., 2001). However, recent data have shown that dynamin-2 might not prevent CT-B internalization but rather act on its subsequent transport to the Golgi complex (Kirkham et al., 2005). Our results support this hypothesis, since 3D reconstructions of Mgat5-/-ESC cells expressing the dynamin-2 mutant K44A still show intracellular CT-B labelling that does not colocalize with the Golgi complex following endocytosis for 30 minutes. This supports a role for dynamin-2 in CT-B endocytosis as previously shown (Kirkham et al., 2005). Since Cav1 can bind dynamin-2 directly, it is therefore possible that Cav1 overexpression will inhibit CT-B endocytosis by sequestering dynamin-2 in Cav1 positive rafts, preventing it from acting on the internalization of lipid rafts. Experiments testing whether or not overexpression of the wild-type form of dynamin-2 is able to rescue inhibition by Cav1 should help to test this hypothesis. 5.3 Are Caveolae efficient endocytic structures? I have discussed previously the role of Cav1 in the regulation of raft-dependent endocytosis. It was previously proposed that Cav1 acts as a negative regulator of endocytosis by stabilizing raft domains at the cell surface (Nabi and Le, 2003). FRAP experiments also demonstrated that Cav1 is highly immobile at the plasma membrane (Kirkham et al., 2005; Torgersen et al., 2001). Caveolae are also stabilized at the cell surface by filamin which binds Cav1 and the actin cytoskeleton (Stahlhut and van Deurs, 2000). If caveolae represent such stable structures, how can they be dynamic and efficient endocytic carriers? Endocytosis of different cargo such as CT-B, SV40 and albumin via caveolae has been presented as proof that caveolae mediate endocytosis. However, CT-B has been shown to be efficiently  215  internalized in cells devoid of caveolae (Torgersen et al., 2001). In the same way, SV40 can be internalized via pathways independently of caveolae (Damm et al., 2005). It was proposed that a pool of caveolae may show local short-range mobility and moreover, a few caveolae 'bypass' these restraints and become internalized – either because they have not been sufficiently stabilized or as a means of general caveolin/caveolae turnover (Hommelgaard et al., 2005). Therefore it was proposed that oligomeric complexes of caveolin-1 confer permanent structural stability to caveolar vesicles that transiently interact with endosomes to form subdomains and release cargo selectively by compartment-specific cues (Pelkmans et al., 2004). 5.4 Cav1 and receptor signaling I have discussed the role of Cav1 in the regulation of raft-dependent endocytosis. However, as I have demonstrated in chapter 2, Cav1 may also suppress receptor signaling (Lajoie et al., 2007). Caveolae have been described as potential signaling platforms where receptors and other machinery required for signal transduction are recruited. This assessment is based on the interaction of various receptors with the scaffolding domain of Cav1 (Li et al., 1996). However, it was also shown that this domain is involved in the attachment of Cav1 to the membrane (Schlegel et al., 1999). Therefore, antibodies against this region recognize Cav1 present in the Golgi apparatus but not at the cell surface suggesting that the scaffolding domain epitope is masked into the plasma membrane (Pol et al., 2005). Based on these observations, it is possible that the binding of receptors to caveolae-localized Cav1 is not the crucial determinant of receptor signaling. It was also shown that upon treatment with EGF, the EGF receptor moves outside caveolae (Mineo et al., 1999). This supports the idea that receptor signaling might occur outside caveolae even for receptors that have been localized to these structures. Various endocytic pathways have been shown to be dependent on Cav1 expression. Besides EGFR, activation of eNOS (Bernatchez et al., 2005), the receptor for advanced glycation end products (RAGE) (Reddy et al., 2006) and adenylyl cyclase (Swaney et al., 2006) are all dependent on Cav1. However, the role of caveolae in these processes remains to be determined.  216  Using the Mgat5-/- cells, I have demonstrated that EGFR signaling can be inhibited by Cav1 in the absence of caveolae. I also have demonstrated that these low Cav1 expression levels are also associated with reduced lipid raft mobility. It was proposed that small lipid rafts are mobile and diffuse at the cell surface to form bigger rafts that act as signaling platforms for signal transduction (Pralle et al., 2000). It has been shown by others that EGFR can be recruited into raft domains at the cell surface from which originate clathrin coated pits and where other signaling molecules are recruited (Puri et al., 2005). In my model, Cav1 may prevent the assembly of these raft-signaling platforms by impairing raft movement at the cell surface. EGFR signaling has been linked to its internalization via clathrin coated pits (CCPs) (Wilde et al., 1999). It was demonstrated that blocking endocytosis via CCPs is able to stimulate EGFR signaling by preventing its downregulation (Partridge et al., 2004). These results support the idea that EGFR signaling occurs in CCPs before their internalization. In my model, it would be interesting to determine the localization of EGFR by electron microscopy. To date, it remains to be determined if EGFR is localized in lipid rafts upon its sequestration by the Mgat5/galectin lattice. In addition, we do not know if the expression of Cav1 is affecting EGFR localization to lipid rafts. Using GM1 staining in electron microscopy, the distribution of these raft microdomains could be observed as well as the amount of EGFR localized in lipid rafts. It would also be interesting to determine the effect of Cav1 expression on EGFR endocytosis. It has been shown before that expression of the Mgat5/galectin lattice opposes receptor internalization by maintaining receptors at the cell surface (Partridge et al., 2004). This is in part based on the observation that receptors localize more to early endosomes in Mgat5-/cells than in Magt5+/+ cells. However, these experiments were never performed using the Mgat5-/-ESC cells that do not express Cav1. Since these cells do not present either the galectin lattice or Cav1, why do they show increased EGFR signaling? In chapter 2, I argue that loss of the negative regulatory activity of Cav1 is responsible for that phenomenon. However, the mechanism underlying the process is unknown. Since inhibition of signaling does not appear  217  to require caveolae, it is unlikely that the effect of Cav1 is due to the internalization of EGFR via caveolae.  It is therefore possible that Cav1 acts the same way as the Mgat5  galectin/lattice, but with an opposite effect. Cav1 could prevent internalization of EGFR via CCPs but at the same time, prevents its localization to signaling competent raft domains. To answer that question, it would be pertinent to measure cell surface EGFR expression following EGF treatment in Mgat5-/-ESC cells with or without transfection with Cav1. 5.5 How can Mgat5 regulate Cav1 expression? In our cell model, I show that rescuing the Mgat5-/- cells with a retrovirus encoding Mgat5 is able to rescue Cav1 expression to a level similar to what is observed in Mgat5+/+ cells. I also demonstrate that disruption of the galectin lattice with β-lactose and swainsonine is associated with a significant decrease in Cav1 expression. Taken together, these data suggest that the Mgat5/galectin lattice is able to regulate Cav1 expression. The transcription regulation of Cav1 is still unclear. It has been shown that Cav1 transcription can be regulated by FOXO (Forkhead) transcription factors. When active, FOXO factors can bind to DNA in promoter sequences and subsequently regulate gene expression. It was demonstrated that caveolin-1 expression was increased upon induction or over-expression of FOXO factors at both mRNA and protein levels and this increased Cav1 expression was associated with decreased sensitivity to EGF (van den Heuvel et al., 2005). Interestingly, the FOXO transcriptional activity is dependent on the PI3K/AKT signaling pathway. AKT terminates transcription by initiating nuclear export of FOXO proteins. It phosphorylates FOXO consequently masking the nuclear localization signal, interfering with DNA-binding, disrupting the association with the co-activator p300-CBP (CREB-binding protein) and mediating the interaction with 14-3-3 proteins (Brunet et al., 1999). The result is inhibition of FOXO nuclear functions and removal of FOXO from the nucleus. FOXO that has been exported from the nucleus as a result of AKT-dependent phosphorylation is rapidly degraded (Seoane et al., 2004). Smad proteins activated by TGF-β form a complex with FOXO proteins to turn on the growth inhibitory gene p21Cip1 (Massague et al., 2005). Since Mgat5-/- cells exhibit increased Akt phosphorylation (Mendelsohn et al, 2007) and decreased TGF-β responsiveness (Partridge et al., 2004), it is tempting to postulate that downregulation  218  of these signaling pathways may affect the activity of Cav1 transcription factors such as FOXO. This could explain the downregulation of Cav1 observed in Mgat5-/- cells. 5.6 Mgat5 and Cav1 interplay in tumor progression Oncogenic activation of Erk/PI3K pathways in tumor cells promotes autocrine TGF-ß signaling and epithelial-to-mesenchymal transition (EMT) (Ozdamar et al., 2005; Thiery, 2003). However, PyMT Mgat5–/– cells maintain cell-cell adhesion junctions and columnar epithelium morphology while Mgat5+/+ cells show loss of cell–cell adhesions (Partridge et al., 2004). It was shown that adenoviral reintroduction of Mgat5 in Mgat5–/– cells is able to restore the mesenchymal phenotype (Lau et al., 2007). The fact that rescued cells also show increased Cav1 expression lead me to think that Cav1 might have a role in this process, as a downstream effector of Mgat5. Also, I have observed that the ESC-Rescue cells do not seem to undergo EMT and do not show restored Cav1 expression supporting a role for Cav1 in EMT. This hypothesis should therefore be tested by reintroducing Cav1 in ESC-Rescue cells and verifying the expression of E-cadherin labelled junctions. In human tumors overexpressing EGFR, acute EGF treatment induces caveolae-dependent endocytosis of E-cadherin junctions. Antisense RNA-mediated reduction of caveolin-1 expression in EGFR-overexpressing tumor cells recapitulated these EGF-induced effects and enhanced invasion into collagen gel (Lu et al., 2003). It is therefore possible that the inability of ESC-Rescue cells to undergo EMT is due to their lack of Cav1 expression. It is pertinent to test whether or not Cav1 knockdown by siRNA in Mgat5+/+ cells can affect their ability to undergo EMT. EMT is also dependent on other growth factors such as TGF-β. TGF-ß can induce EMT of carcinoma cells, which leads to invasion and metastasis (Derynck et al., 2001; Zavadil and Bottinger, 2005). I have shown that both Mgat5 null cell lines are defective in terms of response to TGF-ß. This result suggests that Cav1, at least at levels below the threshold for caveolae formation, is not associated with reduced TGF-ß signaling. It has been demonstrated that, in Mgat5 expressing cells, TGF-β receptor glycosylation is dependent on the increased growth and metabolism generated by their hyperresponsiveness to EGF. This  219  increased growth and metabolism generated through the hexosamine pathway causes increased production of UDP-GlcNAc and therefore increased glycosylation of TGF-β receptor (Lau et al., 2007). In Mgat5-/-ESC cells, it is unknown if the restored EGF signaling due to the loss of Cav1 leads to increased growth and metabolism. However, in the scenario where these cells would present increased UDP-GlcNAc synthesis, the absence of Mgat5 should render impossible the transfer of GlcNAc residue to glycoproteins. We can therefore expect that TGF-β remains poorly glycosylated in these cells, even if they show restored EGF signaling. In this case, TGF-β would not associate with the galectin lattice and the cells would remain poorly responsive to the cytokine. These cells should therefore not undergo EMT. Since EMT has been linked to the ability of breast tumor cells to enter the circulation and seed metastasis (Kang and Massague, 2004) I can postulate that the Mgat5-/-ESC cells will present a reduced ability to form metastasis, even if the loss of Cav1 allows them to grow to a larger volume.  The role of Cav1 in tumor progression is not limited only to EMT. It is well documented that Cav1 plays a major role in tumor cell motility. It has been shown that Mgat5+/+ cells and Rescue cells are significantly more motile then the Mgat5-/- cells and that migration of these cells is galectin dependent (Lagana et al., 2006). Also we now demonstrate that Mgat5-/-ESC cells are not motile and that motility is not rescued in ESC-Rescue cells (Goetz et al., 2007). Again, these results suggest that Cav1 may have a role in Mgat5-dependent cell migration. In order to address that it would be interesting to transfect ESC-Rescue cells with Cav1 and assess their motility. I have also demonstrated that Cav1 phosphorylation was a crucial determinant of cell migration (Joshi et al, in preparation) and we should therefore do the same experiment after transfecting the cells with the Y14F Cav1 mutant. 5.7 Cav1 is a conditional tumor suppressor It is important to understand that cancer progression is not dictated by only one factor, but is rather determined by a combination of various elements. It has been previously argued by others that Cav1 could be a tumor suppressor based on the work in Cav1 null mice. These mice show greater susceptibility to dysplastic lesions and tumor formation when crossed with  220  the MMTV-PyMT mouse (Williams et al., 2003). However, many studies have shown that Cav1 may be associated with tumor progression in various types of cancer (Ando et al., 2007; Kato et al., 2002; Yang et al., 1998). In my study, I have identified that Cav1 is a conditional tumor suppressor and its activity can be overridden by the expression of the Mgat5/galectin lattice (Lajoie et al., 2007). In the literature, other examples of genes that act as a conditional tumor suppressor have been presented. Germline mutations of BRCA2 are responsible for about one-third of the cases of hereditary breast cancer (Ford et al., 1998). However, it has been shown that no tumors arise in mice carrying conditional BRCA2 alleles, mammary and skin tumors developed frequently in females carrying conditional Brca2 and Trp53 alleles (Jonkers et al., 2001). These results indicated that BRCA2 is a conditional tumor suppressor that acts synergistically with p53. E2F1 is another gene that has been shown to act as both a tumor suppressor and an oncogene. Cancer cells often contain mutations that lead to the loss of retinoblastoma tumor suppressor (Rb) function and the activation of E2F-dependent transcription (Dyson, 1998). This results in the deregulation of cell proliferation and increased sensitivity to apoptosis. However, studies have shown that E2F1 can have both positive and negative effects on tumor growth whether it is absent or overexpressed (Johnson, 2000). E1A, another viral oncoprotein that targets Rb family members, has also been demonstrated to have both oncogenic and tumorsuppressive properties. The ability of E1A to transform cells depends on its ability to bind Rb and activate E2F1 (Raychaudhuri et al., 1991). These examples show that genes involved in cancer progression are interacting with other genes and these interplays define their role as tumor suppressor or oncogene. We have demonstrated that Cav1 may act as a tumor suppressor, but this role can be overridden by the expression of Mgat5. This constitutes an important advance in our understanding of the role of Cav1 in cancer progression.  221  5. 8 Alternative signaling pathways in the absence of Magt5 PyMT mammary carcinoma cells in an Mgat5-/- background are less responsive to cytokines than PyMT Mgat5+/+ cells, and yet fail to growth arrest upon serum withdrawal. They display increased glucose transport, reactive oxygen species (ROS) production and activated ERK and Akt (Mendhelson et al., 2007). These effects can be reversed by supplementing the hexosamine pathway via the addition of GlcNAc to the cells. GlcNAc supplementation increases triantennary N-glycan production and rescues EGFR and TGF-β signaling in Mgat5-/- cells (Lau et al., 2007). The Mgat5+/+ and Mgat5-/- tumor cells therefore display different growth phenotypes with the first one relying on growth factors and the latter on ROS signaling. It remains unknown if the Mgat5-/-ESC cells display the same phenotype upon serum deprivation. It would therefore be interesting to determine if Cav1 may play a role in the increased ROS signaling in the absence of Mgat5. Since siRNA-knockdown of Cav1 in Mgat5-/- cells restores EGF signaling, Cav1 expression might play a role in the control of ROS production by restoring partial dependence on growth factors to Mgat5-/- cells. The work presented in this thesis constitutes a significant advance in our understanding of the role of Cav1 in tumor progression. However, one question remains. Why are tumors in the Mgat5 null background associated with the loss of caveolae? It can be postulated that Cav1 and caveolae are associated with the regulation of some signaling pathways associated with tumor growth and cancer progression. One of the best examples is the negative regulation of eNOS by Cav1 (Bernatchez et al., 2005; Goligorsky et al., 2002). eNOS is known to generate nitric oxide that counterbalances elevated intracellular ROS levels and to escape oxidative stress. It was suggested that tumor cells may present elevated eNOS activity in order to protect themselves against oxidative stress-induced apoptosis (Wartenberg et al., 2003). As described previously, Mgat5 null tumor cells growth relies on ROS signaling. We know that upon serum starvation, Mgat5-/- cells continue to growth due to increase of ROS production, but without undergoing apoptosis (Mendelshon et al., 2007). It is therefore possible that the reduction of Cav1 and loss of caveolae relieves the negative regulation of eNOS, protecting these cells against oxidative stress. Whether or not Cav1 is able to regulate eNOS activity in  222  the absence of caveolae remains to be determined. If so, differences should be observed in term of eNOS activity and ROS production between Mgat5-/- and Mgat5-/-ESC cells. My cell model therefore represents a powerful tool to study the impact of Cav1 outside caveolae. 5.9 Mgat5 and lipid domains in lysosomal organelle biogenesis In my thesis, I have shown that Mgat5-dependent protein glycosylation and lipid domains such as lipid rafts act at different levels within the cell, such as the plasma membrane and on the internalization of molecules from the cell surface. I have also demonstrated that Mgat5 overexpression is associated with the increased formation of multilamellar bodies via autophagy (Hariri et al., 2000). I have also demonstrated that the lipid composition of late lysosomal organelles may affect the formation of MLBs (Lajoie et al., 2005). This demonstrates that these two components can have different functions at various cellular levels. If Mgat5 expression can stimulate expression of MLBs, the role of glycosylation in this process is still unclear. It is proposed that glycosylation may protect proteins against degradation and therefore contribute to the formation of the concentric lamellae present in MLBs (Hariri et al., 2000). β1-6 branched N-glycans on erythropoietin have been shown to enhance cytokine half-life and activity in vivo (Takeuchi and Kobata, 1991). The LAMP glycoproteins are the major proteins of the lysosomal membrane and the major protein carriers of polylactosamine chains (Carlsson et al., 1988). It was proposed that heavy glycosylation of these proteins protects them against degradation by the lysosomal hydrolases (Fukuda, 1991). Removal of N-glycans from LAMP proteins has been associated with increased degradation of the proteins (Barriocanal et al., 1986). It is now clear that LAMP proteins play a major role in the biogenesis of lysosomes and the regulation of autophagy. In LAMP-2-deficient mice, autophagic vacuoles accumulate in many tissues, including liver, pancreas, muscle, and heart. It has been proposed that the accumulation of autophagic vacuoles in LAMP-2 null cells is due to a defect in lysosomal biogenesis (Eskelinen et al., 2002). However, it has never been shown that glycosylation of lysosomal proteins may affect lysosome biogenesis. In order to asses this question, we could  223  use the Mgat5 tumor cell model to study the impact of Mgat5-dependent glycosylation on lysosomal organelles. Using an inducible LAMP-2-GFP construct, we would be able to measure the half-life of LAMP-2 positive organelles in both Mgat5+/+ and Mgat5-/- cells. This would determine whether glycosylation affects the stability of these organelles. One interesting experiment would be to determine if protein glycosylation affects endocytic delivery of LAMP proteins to the lysosomes. It has been shown that LAMP proteins can transit via the plasma membrane before being delivered to the lysosomes (Nabi et al., 1991). Cell surface immunoprecipitation approaches could therefore be used in order to study LAMP-2 trafficking in the Mgat5 cell lines. LAMP proteins polylactosamine glycosylation is dependent on the protein resident time in the Golgi complex (Nabi and Dennis, 1998). It is therefore possible that the Golgi residence time of the protein may be different due to the absence of Mgat5. Differential trafficking of lysosomal proteins from the Golgi may consequently affect lysosomal organelle biogenesis. Lysosome biogenesis could therefore be studied using a FRAP approach, monitoring the formation of LAMP-2 positive organelles. These experiments could establish a link between LAMPs proteins glycosylation and lysosome biogenesis. To date, no available data demonstrate a clear role for protein glycosylation in the formation of the autophagosome. Danon disease (lysosomal glycogen storage disease with normal acid maltase) is characterized by a cardiomyopathy, myopathy and variable mental retardation. Mutations in the coding sequence of the lysosomal-associated membrane protein 2 (LAMP2) were shown to cause a LAMP-2 deficiency in patients with Danon disease (Saftig et al., 2001). This disease is also associated with accumulation of autophagic vacuoles (Nishino, 2006). It is therefore believed that the impaired autophagic degradation observed in the LAMP-2 mouse is due to deficient interaction of autophagosomes with lysosomes. The fusion between these organelles is crucial for the acquisition of the hydrolytic enzymes required for the degradation of the autophagosome content (Lawrence and Brown, 1992). In order to study the relationship between protein glycosylation and lysosome-autophagosome fusion we could transfect the various Mgat5 cell lines with LC3-GFP (a marker of autophagosome) and measure colocalization with LAMP-2. If protein glycosylation affects  224  the fusion between the two organelles, we would observe differences in the overlap between the two markers. The Mgat5-dependent pathways regulating autophagy are still unclear. Mgat5+/+ cells display increased ERK signaling and reduced Akt signaling compared to Mgat5-/- cells (Mendhelson et al., 2007). Reduction of Akt signaling is associated with inhibition of the mTor/S6 kinase pathway and with induction of autophagy in tumor cells (Aoki et al., 2007). It has been shown that upon serum starvation Mgat5-/- cells display increased production of ROS, which is associated with increased growth and metabolism (Mendhelson et al., 2007). In the absence of nutrients, autophagy is an efficient source of nutrients for the cell. Increased ROS production in Mgat5-/- cells leads to hyperactivation of the Akt/MAPK pathway during starvation. Therefore, autophagy could be impaired in Mgat5-/- cells. It would therefore be interesting to study the activation of the mTor pathway in the Mgat5 cell lines. It is possible that regulation of signaling pathways such as PI3K, Akt/MAPK and mTor by Mgat5 control the autophagic response in these cells. These experiments could potentially identify Magt5 as a regulator of autophagy. Ultimately, the availability of the LC3-GFP and Mgat5 knock out mice could represent a model for studying the Mgat5dependent regulation of autophagy in vivo. Using these mice models, the formation of autophagosomes in different organs under starvation conditions could be compared, in the presence and absence of Mgat5. 5.10 The role of lipid domains in autophagy I have shown that accumulation of cholesterol within autophagic vacuoles can induce the formation of MLBs. Cholesterol-rich MLBs are a major characteristic of Niemann-Pick type C (NPC) disease in which cholesterol aberrantly accumulates in the lysosomes (BlanchetteMackie, 2000). Recently, it has been shown that cholesterol accumulation in this particular pathology was associated with increased basal autophagy in NPC fibroblasts. NPC deficiency activates basal autophagy through increased expression of Beclin-1, a highly conserved member of the class III PI3K complex that is critical for the formation of autophagosomes (Pacheco et al., 2007). This was the first convincing study showing a link between lysosomal  225  cholesterol accumulation and autophagy. However, more experiments showing that Beclin-1 knock-down in these cells could reduce cholesterol accumulation in lysosomes are required to directly link the increased autophagy to the pathology. It has also been shown that cholesterol depletion is involved in the induction of autophagy. Depletion of plasma membrane cholesterol by MβCD induces the formation of autophagosomes via PI3K-dependent mechanisms. It was proposed that disruption of cell surface lipid rafts may inhibit Akt signaling, leading to the suppression of mTor signaling and induction of autophagy (Cheng et al., 2006). This is one of the first reports suggesting that lipid rafts might be involved in autophagy. Another group has also demonstrated that LAMP-2a, the receptor involved in chaperone-mediated autophagy (CMA) is localized in lipid rafts on the lysosomal membrane and the lipid composition of these microdomains regulates the activation of CMA (Kaushik et al., 2006). These data support a role for lipid rafts in the regulation of autophagic pathways. Indeed, it was shown before that autophagy can be stimulated by sphingolipids such as ceramide (Scarlatti et al., 2004). Over the years, it has been shown that lipid rafts affect multiple signaling pathways (Simons and Toomre, 2000). In light of these findings, it is not surprising that modulation of raft domains have an impact on autophagy. Interestingly, a possible role for Cav1 in autophagy is currently unknown. Our work supports a role for Cav1 in the regulation of raft diffusion and endocytosis. I have also discussed the ability of Cav1 to bind cholesterol. Since raft components are now being implicated in the autophagic pathway, it would be interesting to test whether regulation of raft behaviour by Cav1 may impact on autophagy.  226  5.11 Conclusion Globally, we have demonstrated that Mgat5-mediated glycosylation is involved in the regulation of various biological processes at different cellular levels. However, pathways regulated by Mgat5 are also controlled by another important regulator, the membrane lipid domains. We first demonstrated that competitive recruitment of cell surface EGFR to the Mgat5/galectin lattice and the Cav1 domains is a determinant of receptor signaling and tumor growth. Both the Mgat5 galectin lattice and lipid domains are also involved in the regulation of internalization of molecules via specific endocytic pathway. Finally, these two domains also regulate biogenesis of lysosomal organelles. The consequences of proteins glycosylation and the roles of lipid domains such as rafts and caveolae have been extensively studied over the years. However, as shown in this thesis, we have developed a model that allows us to study the complex interplay between these to regulators. This model gives us the tools to understand how these entities oppose and/or work together under both normal and pathological conditions. I strongly believe that such integrative studies are the key to understand the complex biology of the cell in different pathologies such as cancer.  227  Galectin lattice  Cav1 scaffolds CT-B  p-ERK  EGFR  p-ERK  Early endosome  Late endosome Lysosome  Autophagosome  MLB  Figure 5.1: Thesis model: Mgat5-dependent protein glycosylation and Cav1/cholesterol domains therefore function as regulators of plasma membrane interactions, endocytosis and lysosomal organelle biogenesis.  228  5.12 References Ando, T., H. Ishiguro, M. Kimura, A. Mitsui, Y. Mori, N. Sugito, K. Tomoda, R. Mori, K. Harada, T. Katada, R. Ogawa, Y. Fujii, and Y. Kuwabara. 2007. The overexpression of caveolin-1 and caveolin-2 correlates with a poor prognosis and tumor progression in esophageal squamous cell carcinoma. Oncol Rep. 18:601-9. Aoki, H., Y. Takada, S. Kondo, R. Sawaya, B.B. Aggarwal, and Y. Kondo. 2007. 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