Endocytosis effectuates a critical interface between the eukaryotic cell and its apposing environment. It is, subsequently, paramount for many physiologically important processes and encompasses a diverse array of mechanisms and pathways. The classical endocytic routes mediated by clathrin and caveolin are the best understood and the molecular roles of their major regulators, such as dynamin, adaptor proteins and various lipid species, are the most comprehensively described. Recent identification of an assortment of constitutive, noncaveolar, clathrin-independent endocytic (CIE) pathways has expanded the endocytic system. Unlike the classical endocytic pathways, little is known about the guiding parameters of CIE routes. Consequently, it is not possible to understand the important cellular roles these pathways may be fulfilling. This study has begun to characterise the very basic parameters governing the morphologically striking Clathrin-Independent Carrier (CLIC) pathway. Development of a diverse molecular toolkit has now allowed the quantitation of endocytic capacity provided by CLICs, the visualisation of subtle sorting components of the CLIC pathway, the isolation of novel CLIC cargo and regulators, and has linked this mechanism to the critical cellular processes of cellular migration and membrane repair. Calculation of the individual capacity of endocytic routes provides important information about the contribution of each pathway to total plasma membrane (PM) uptake and turnover. Quantitation of the volume, surface area and number of structures forming per minute in this study shows that CLICs provide the vast majority of constitutive endocytosis, up to four times the capacity of the clathrin mediated endocytic (CME) pathway. As the equivalent of the entire PM area could pass through the CLIC pathway within 12 minutes it is evident that CLICs are fundamental housekeepers of bulk membrane internalisation. Thus, they are likely to be central regulators of PM homeostasis and turnover. High-resolution tomography, in conjunction with analysis of CLIC cargo trafficking, identifies these carriers as complex, pleiomorphic structures that sort the bulk of membrane to early endosomes and recycle cargo back to the cell surface. Such vast internalisation combined with an ability to rapidly recycle components quickly attributes the CLIC pathway as a complex sorting station. Isolation of novel cargo and regulators has identified a striking array of proteins now associated with the CLIC pathway for the first time. A significant proportion of identified targets localise to lipid-rafts and recycle from the PM, facets consistent with association to the CLIC pathway. Numerous targets have also been directly implicated in clathrin-independent endocytosis by independent groups. Verification of selected cargo, such as CD44, Thy-1 and myoferlin, showing specific internalisation through the CLIC pathway, has provided insight into the sorting ability of the CLIC pathway and links to adhesion turnover and membrane recycling. Consistent with a role in cellular adhesion turnover, it was found that CLICs become polarised within migrating cells. This has shown the first instance of spatial separation between three major endocytic routes, CLICs, caveolae and CME and highlights the important and coordinated roles of multiple endocytic pathways during physiologically significant processes. The specific internalisation of paxillin, Thy-1 and CD44 through CLICs at the leading edge of migrating cells suggests that CLICs rapidly turnover adhesion components for dynamic extracellular sensation during directional cell migration. Indeed, specific ablation of the CLIC pathway significantly impedes cellular migration, implying coordination with CME at the leading edge. This study has defined numerous parameters of the CLIC pathway, developing the current understanding of this poorly defined route and places the CLIC pathway as a unique player during critical cellular processes.

The connection between T cell activation, plasma membrane order and actin filament dynamics was the main focus of this study. Laurdan and di-4-ANEPPDHQ, membrane order sensing probes, were shown to report only on lipid packing rather than being influenced by the presence of membrane-inserted peptides justifying their use in membrane order studies. These dyes were used to follow plasma membrane order in live cells at 37°C. Disrupting actin filaments had a disordering effect while stabilizing actin filaments had an ordering effect on the plasma membrane, indicating there is a basal level of ordered domains in resting cells. Lowering PI(4,5)P2 levels decreased the proportion of ordered domains strongly suggesting that the connection of actin filaments to the plasma membrane is responsible for the maintaining the level of ordered membrane domains. Membrane blebs, which are detached from the underlying actin filaments, contained a low fraction of ordered domains. Aggregation of membrane components resulted in a higher proportion of ordered plasma membrane domains and an increase in cell peripheral actin polymerization. This strongly suggests that the attachment of actin filaments to the plasma membrane induces the formation of ordered domains. Limited cholesterol depletion with methyl-beta-cyclodextrin triggered peripheral actin polymerization. Cholesterol depleted cells showed an increase in plasma membrane order as a result of actin filament accumulation underneath the membrane. Moderate cholesterol depletion also induced membrane domain aggregation and activation of T cell signaling events. The T cell receptor (TCR) aggregation caused redistribution of domains resulting in TCR patches of higher order and the bulk membrane correspondingly depleted of ordered domains. This suggests the preexistence of small ordered membrane domains in resting T cells that aggregate upon cell activation. Increased actin polymerization at the TCR aggregation sites showed that actin polymerization is strongly correlated with the changes in the distribution of ordered domains. The distribution of the TCR in resting cells and its colocalization with actin filaments is cell cycle dependent. We conclude that actin filament attachment to the plasma membrane, which is regulated via PI(4,5)P2, plays a crucial role in the formation of ordered domains. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 4: Manuscript.</p>

The plasma membrane of eukaryotic cells contains cholesterol and glycosphingolipids enriched nanodomains known as lipid rafts; which are believed to exist in a liquid ordered (lo) state. Methyl-beta-cyclodextrin (MBCD) is used to deplete cellular cholesterol and a widespread assumption is that MBCD preferentially targets cholesterol in lipid rafts. To analyze this in T cells a progressive cholesterol extraction protocols was established. At 37ºC, MBCD treatment does not lead to the preferential loss of cholesterol from TX-DRMs. At 0ºC only 35% of total cholesterol could be extracted demonstrating that less than 35% of the cell’s cholesterol is found in the plasma membrane. Moreover, incubation of cells at 0ºC causes loss of plasma membrane cholesterol and an increase in cholesteryl esters. The increase in cholesterol esters upon cold exposure is linked to the cholesterol concentration induced activation of ACAT enzyme which converts cholesterol to cholesteryl esters. Cholesterol concentration specific activation of ACAT and conversion of cholesterol to cholesteryl esters during the loading of cholesterol onto T cells by MBCD was also observed. By using MBCD for progressive cholesterol depletion from T cells at 37ºC, the effect of cholesterol depletion on T cell signaling was addressed. At 10-20% cholesterol depletion levels, tyrosine phosphorylation is increased and ERK is activated. Peripheral actin polymerization, cell spreading and membrane protrusions are also triggered by limited cholesterol depletion. Upon limited cholesterol depletion aggregation of lipid rafts in the plasma membrane was observed. The aggregation of lipid rafts upon cholesterol depletion does not dependent on the signaling proteins such as Src-kinases. Upon cholesterol depletion there is an increase in overall plasma membrane order, indicative of more ordered domains forming at the expense of disordered domains. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: In press.</p>

The plasma membrane of eukaryotic cells contains cholesterol and glycosphingolipids enriched nanodomains known as lipid rafts; which are believed to exist in a liquid ordered (lo) state. Methyl-beta-cyclodextrin (MBCD) is used to deplete cellular cholesterol and a widespread assumption is that MBCD preferentially targets cholesterol in lipid rafts. To analyze this in T cells a progressive cholesterol extraction protocols was established. At 37ºC, MBCD treatment does not lead to the preferential loss of cholesterol from TX-DRMs. At 0ºC only 35% of total cholesterol could be extracted demonstrating that less than 35% of the cell’s cholesterol is found in the plasma membrane. Moreover, incubation of cells at 0ºC causes loss of plasma membrane cholesterol and an increase in cholesteryl esters. The increase in cholesterol esters upon cold exposure is linked to the cholesterol concentration induced activation of ACAT enzyme which converts cholesterol to cholesteryl esters. Cholesterol concentration specific activation of ACAT and conversion of cholesterol to cholesteryl esters during the loading of cholesterol onto T cells by MBCD was also observed. By using MBCD for progressive cholesterol depletion from T cells at 37ºC, the effect of cholesterol depletion on T cell signaling was addressed. At 10-20% cholesterol depletion levels, tyrosine phosphorylation is increased and ERK is activated. Peripheral actin polymerization, cell spreading and membrane protrusions are also triggered by limited cholesterol depletion. Upon limited cholesterol depletion aggregation of lipid rafts in the plasma membrane was observed. The aggregation of lipid rafts upon cholesterol depletion does not dependent on the signaling proteins such as Src-kinases. Upon cholesterol depletion there is an increase in overall plasma membrane order, indicative of more ordered domains forming at the expense of disordered domains. / At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: In press.

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. 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.

The molecular mechanisms of putative anti-inflammatory nutrients, i.e., fish oil, curcumin and limonin, were investgated with respect to CD4+ T cell function. Initially, using a DO11.10 mouse model which exhibits a transgenic T cell receptor specific to OVA 323-339 peptide, we demonstrated that dietary fish oil suppresses antigen-specific Th1 clonal expansion in vivo. Following immunization, the accumulation of adoptively transferred transgenic cells in wild type recipient mouse lymph nodes was suppressed. In addition, cell division analysis by carboxyfluorescein succinimidyl ester (CFSE) revealed that both total cell number in lymph nodes as well as cell division were decreased by fish oil. Since n-3 polyunsaturated fatty acids (PUFA), active long chain fatty acids in fish oil, elicit favorable effects on a variety of cell types, e.g., anti-tumor effect on colonocytes, amelioration of coronary heart disease and anti-inflammatory effects involving T cells, B cells, dendritic cells and macrophages, we postulated that a fundamental mechanism of action may explain the multiple effects observed. In a series of experiments described herein, we demonstrated that n-3 PUFA alters the formation/location of membrane subdomains, referenced to as lipid rafts. Specifically, lipid raft formation at the immunological synapse (IS) in CD4+ T cells was suppressed following membrane enrichment with n-3 PUFA. The alteration of lipid rafts down-regulated the localization of select signaling proteins, including F-actin, PKC and PLC-1, and phosphorylation of PLC-1 at the IS. Consequently, CD4+ T cell proliferation was suppressed as assessed by CFSE analysis and radioactive thymidine incorporation. Phytochemicals have been used for chemopreventive and chemotherapeutic purposes. We examined the putative anti-inflammatory effects of curcumin (1%) and limonin (0.02%) with respect to CD4+ T cell function. Dietary curcumin and limonin suppressed NF-B activation in CD4+ T cells. In addition, CD4+ T cell proliferation was modulated by 2% curcumin. We further investigated the combined therapeutic potential of phytochemicals and fish oil, containing n-3 PUFA. Interestingly, fish oil and limonin together significantly (P<0.05) suppressed T cell proliferation, whereas feeding either fish oil or limonin alone showed little effect. In summary, our data indicated that dietary fish oil alters proximal signaling of T cells by perturbing lipid raft formation. Curcumin and limoin are capable of suppressing NF-B in T cells, thereby exhibiting a synergistic effect when combined with fish oil. Further studies are required to elucidate the relationship of dietary dose of active compoments with respect to mechanism of actions.

The Mevalonate Pathway: A Potential Therapeutic Target for JAK2-driven Myeloproliferative Neoplasms Lori Nicole Griner Abstract Myeloproliferative neoplasms (MPNs) are diseases of hematopoietic stem cell origin and are characterized by uncontrolled growth of cells of the myeloid compartment. The Philadelphia chromosome negative classical MPNs, including polycythemia vera, essential thrombocythemia, and myelofibrosis, are diseases of dysregulated JAK2 signaling. In fact, the majority of MPN patients have activating mutations in JAK2 (e.g JAK2-V617F), a tyrosine kinase that contributes to the growth and survival of myeloid cells. While MPNs were first described over sixty years ago, a significant need remains to develop therapeutic strategies for them. Inhibitors of JAK2 are currently being developed, and one inhibitor, ruxolitinib, was recently approved for certain MPN patients. Ruxolitinib has made profound impacts on improving splenomegaly and constitutional symptoms in MPN patients, but it and other JAK2 inhibitors have not significantly reduced the JAK2 mutant allele burden, and thus such inhibitors have not induced remission in these patients. The current consensus in the MPN field supports JAK inhibition for the treatment of patients, but a further understanding of MPNs and JAK2 signaling, as well as improved JAK2 inhibitors, may be necessary for treating MPN patients. The work described in this dissertation has uncovered novel requirements for JAK2-V617F-driven signaling and transformation. We demonstrate that JAK2-V617F co-localizes with lipid rafts, cholesterol-rich microdomains within the plasma membrane that function to serve as platforms for signaling complex formation. Signaling complex formation is a necessary component for dysregulated signaling induced by JAK2-V617F. We provide evidence that cholesterol altering-lipid raft disrupting agents attenuate JAK2-V617F-driven signaling. We also show that cholesterol-lowering statins are effective at downregulating JAK2 signaling and inducing apoptosis in JAK2-V617F-driven cell lines. Importantly, we show that statins, inhibitors of the mevalonate pathway, inhibit the growth of primary MPN cells, while the same statin doses have no effect on healthy controls. Impressively, we demonstrate that statins cooperate with multiple JAK inhibitors, including ruxolitinib, to inhibit cell growth and induce apoptosis of JAK2-V617F-driven cells. This report establishes statin-mediated inhibition of the mevalonate pathway as a potential approach to improve MPN therapeutics. We propose future studies with statins and JAK2 inhibitors in the treatment of MPNs.

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. 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.

Gene therapy is the delivery of genetic material to cells for a therapeutic effect. Retroviruses are one of the most common viral vectors used for gene therapy, especially lung gene therapy. However the lung has many physical and immunological barriers to gene transfer vectors, and currently, too few cells are genetically modified for the effective treatment of lung diseases such as cystic fibrosis. One of the main reasons for low cell transduction is the lack of commonly-used receptors for gene therapy vectors on the apical surface of polarized epithelial cells. The objective of this project was to determine how to incorporate proteins into the lentiviral lipid bilayer in order to develop a recombinant retrovirus that can efficiently deliver genes to polarized epithelial cells via their apical membranes. We analyzed the process of incorporating heterologous viral and non-viral proteins into lentiviruses and determined key factors that allowed for successful protein incorporation into the lentiviral lipid bilayer. We found that lipid rafts segregated raft proteins, and for a protein to be incorporated into virus particles, it must be colocalized with lentivirus-associated rafts. When cells were treated with the cholesterol-extracting compound, methyl-beta-cyclodextrin, previously sequestered viral and non-viral raft proteins were then colocalized and non-viral proteins were incorporated into lentiviruses. We also created a lentivirus pseudotyped with envelope proteins from human parainfluenza type 3 (HPIV3), which naturally targets polarized epithelial cells of the lung. Lentiviruses were able to incorporate HPIV3 glycoproteins, hemagglutinin-neuraminidase (HN) and fusion (F), and were able to transduce polarized cells in a manner consistent with lentiviral-mediated transduction via sialated receptors for HPIV3, however titers were too low for clinical use. We increased protein expression of HN and found that while expression, envelope incorporation, and titer increased, lentiviruses still incorporated too few envelope proteins for efficient transduction. We determined low envelope incorporation rates were due to lack of interactions with Gag, and increasing active and passive interactions with Gag enhanced HN and F incorporation into lentiviruses. Overall, this research is significant because it provides insight into viral assembly and protein incorporation for the generation of pseudotyped lentiviruses for human gene transfer.

The connection between T cell activation, plasma membrane order and actin filament dynamics was the main focus of this study. Laurdan and di-4-ANEPPDHQ, membrane order sensing probes, were shown to report only on lipid packing rather than being influenced by the presence of membrane-inserted peptides justifying their use in membrane order studies. These dyes were used to follow plasma membrane order in live cells at 37°C. Disrupting actin filaments had a disordering effect while stabilizing actin filaments had an ordering effect on the plasma membrane, indicating there is a basal level of ordered domains in resting cells. Lowering PI(4,5)P2 levels decreased the proportion of ordered domains strongly suggesting that the connection of actin filaments to the plasma membrane is responsible for the maintaining the level of ordered membrane domains. Membrane blebs, which are detached from the underlying actin filaments, contained a low fraction of ordered domains. Aggregation of membrane components resulted in a higher proportion of ordered plasma membrane domains and an increase in cell peripheral actin polymerization. This strongly suggests that the attachment of actin filaments to the plasma membrane induces the formation of ordered domains. Limited cholesterol depletion with methyl-beta-cyclodextrin triggered peripheral actin polymerization. Cholesterol depleted cells showed an increase in plasma membrane order as a result of actin filament accumulation underneath the membrane. Moderate cholesterol depletion also induced membrane domain aggregation and activation of T cell signaling events. The T cell receptor (TCR) aggregation caused redistribution of domains resulting in TCR patches of higher order and the bulk membrane correspondingly depleted of ordered domains. This suggests the preexistence of small ordered membrane domains in resting T cells that aggregate upon cell activation. Increased actin polymerization at the TCR aggregation sites showed that actin polymerization is strongly correlated with the changes in the distribution of ordered domains. The distribution of the TCR in resting cells and its colocalization with actin filaments is cell cycle dependent. We conclude that actin filament attachment to the plasma membrane, which is regulated via PI(4,5)P2, plays a crucial role in the formation of ordered domains. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 4: Manuscript.