Au cours de l'endocytose, les cargos de membrane plasmique (PM) sont internalisés puis dirigés vers l'endosome précoce (EE), les corps multivésiculaires (MVBs), puis le lysosome/vacuole pour dégradation. Le ciblage des protéines vers les zones invaginées des MVBs requiert l'action des complexes ESCRTs. Chez la levure comme chez les mammifères, les mutants déficients pour ces complexes présentent un compartiment endosomal anormal (Cl E) et une accumulation à la PM de diverses protéines. Nous avons montré que chez S. cerevisiae la stabilisation de la perméase à uracile (Fur4p) à la PM dans les mutants ESCRTs résulte de son recyclage vers la PM après internalisation. Fur4p ne traverse pas les compartiments Golgiens lors de son recyclage depuis le compartiment Cl E. Cette voie de recyclage est distincte de celle empruntée par la v-SNARE Snc1p. Fur4p est également capable de recycler depuis l'EE et suit alors la même voie de recyclage que Snc1p. Ceci suggère une complexité inattendue des voies de recyclage chez la levure.<br />Nous nous sommes aussi intéressés à Irs4p et Tax4p, 2 protéines à domaine EH, un domaine trouvé jusqu'alors dans des protéines impliquées dans l'endocytose ou le recyclage. Irs4p et Tax4p ne sont pas impliquées dans ces processus mais interviennent de façon redondante au cours de l'autophagie, un transport catabolique vésiculaire de fractions de cytoplasme ou d'organelles vers le lysosome/vacuole. Irs4p et Tax4p interagissent physiquement avec la machinerie d'autophagie et sont partiellement localisées à la structure Pré-autophagosomale, d'où émanent les vésicules d'autophagie. Ces résultats étendent donc la panoplie des fonctions des protéines à domaine EH.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

and key words Mouse polyomavirus-derived virus-like particles composed from major capsid protein VP1 (MPyV VP1-VLPs) are interesting structures for use as a delivery system of various cargos into cells. VP1 protein self-assembles into icosahedral particles of 45 nm in diameter that are hollow highly regular nanoparticles. In this work, model small molecule cargo, Cyclodextrin-Based Bimodal Fluorescence/MRI Contrast Agent, was encapsidated into MPyV VP1-VLPs. The cargo was stably associated with VLPs and was delivered into mammalian cells using these VLPs. To prevent VLPs entrapment in endolysosomal compartments and increase the potential of VLPs applications, MPyV VP1 protein was modified by insertion of histidine-tag (6 histidine long sequence surrounded by glycine and serine) sequences into VP1 surface loop DE, because histidine modification of synthetic systems had enhancing effect on endosome escape and cargo delivery. With the use of in Bac-to-Bac® baculovirus expression system His-VP1 protein was expressed in insect cells and a variety of VP1-assemblies was obtained: long tubules and small 20nm VLPs formed from VP1 with 4 histidine-tags in DE loop, and novel VP1 nanostructure, which we named nano-jumpers, formed from VP1 with 2 histidine-tags. Nonetheless the endosome escape properties of...

Shiga toxin and cholera toxin are members of the AB5 family of protein exotoxins. The A subunit is the enzymatic subunit, whereas the pentameric B subunit binds cell surface receptors and carries the A subunit to the endoplasmic reticulum (ER) where it can be released into the cytosol. The B-subunits (STxB or CTxB) mediate toxin traffic along the retrograde pathway from the plasma membrane to the ER via early/recycling endosomes and the Golgi apparatus. It is unknown if STxB requires transport through the Golgi, or if it is just kinetically favorable. It is also unknown if the recycling endosome (RE) plays a role in the retrograde transport of STxB and CTxB. The first goal of this dissertation research was to demonstrate that transport through the Golgi is required for STxB to reach the ER. Using aluminum fluoride treatment, a simple temperature block, and cytoplast studies, I show that Golgi transport is necessary for STxB to reach the ER. The second goal of this dissertation research was to tease apart how STxB and CTxB move through early and recycling endosomes as well as elucidate a mechanism of how STxB exits endosomes en route to the Golgi. The role of the RE in STxB and CTxB transport is unclear. I used transferrin colocalization and temperature block studies to show that STxB and CTxB traffic through the RE. I then used HRP ablation of the RE to show that STxB requires the RE to reach the Golgi. I also examined the role of an RE-specific protein, EHD1, in exit of STxB from the RE. EHD1 has been previously shown to regulate recycling Tfn exit from the RE but its role in STxB transport is unknown. Expression of a dominant negative form of EHD1 arrested STxB at the RE and prevented it from reaching the Golgi. Together, these results suggest that STxB and CTxB transit the RE, STxB requires a functional RE for normal retrograde trafficking, and that STxB exit from the RE is regulated by EHD1.

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.

Alix est une protéine adaptatrice impliquée dans la régulation du système endolysosomal grâce à son interaction avec des protéines comme CIN85 et les endophilines, protéines participant à l'endocytose de récepteurs ou les protéines des complexes ESCRT nécessaires à la maturation des compartiments endosomaux, Alix pourrait également contrôler la mort neuronale en se fixant à la protéine ALG-2. Le but de mon projet de thèse a été de tester si Alix intervient dans la mort des motoneurones au cours du développement in vivo et de définir les mécanismes moléculaires sous-jacents. J'ai montré que l'expression d'une protéine tronquée, correspondant à la moitié C-terminale d'Alix (Alix-CT), protège les motoneurones cervicaux de la mort cellulaire programmée précoce (PCD). Cette protection dépend de la liaison d'Alix-CT à ALG-2 et à ESCRT-I mettant en évidence l'implication du complexe Alix/ALG-2 avec les protéines ESCRT dans la mort naturelle des motoneurones : Alix ferait le lien entre la voie endolysosomale et la machinerie de mort cellulaire. J'ai montré que la délétion du site de liaison à CIN85, connue pour participer à l'endocytose du récepteur au TNF, le TNFR1, dans Alix-CT, inhibe sa capacité protectrice sur la PCD. La suite de mon travail démontre qu'Alix fonctionne en aval de la signalisation du TNFR1 à la fois in vivo et in vitro. Alix/ALG-2 interagit avec le TNFR1 au niveau des endosomes et pourrait permettre la formation d'une plateforme d'activation de la caspase-8 qui serait recrutée au niveau des « endosome de mort » contenant le TNFR1.

Endosomes are dynamic vesicular structures which transport cargo molecules internalized into the cell via endocytosis. Endosomal trafficking of cargo involves a large number of individual endosomes that regularly interact with each other via fusion and fission and thus form a dynamic network wherein endocytosed cargo is sorted and transported to various other intracellular compartments. In this study we present a general theoretical framework that takes into account individual endosomes and several key microscopic interaction processes among them. By combining theory with quantitative experiments, we seek to address the fundamental question of how the behaviour of the endosomal network emerges from the interactions among many individual endosomes of different sizes and cargo contents. Our theory is based on distributions of endosomes of various sizes and cargo amount. We compare our theory to experimental time course distributions of LDL, a degradative cargo, in a population of early endosomes. Early endosomes display a broad distribution of cargo with a characteristic power law, which we show is a consequence of stochastic fusion events of cargo carrying early endosomes. A simple model can quantitatively describe time-dependent statistics of LDL distributions in individual early endosomes. From fits of the theory to experimental data we can determine key parameters of endosomal trafficking such as the endosome fusion rate and the fluxes of cargo into and out of the network. Our theory predicts several experimentally confirmed scaling behaviours, which arise as a result of endosome fusion. Our theory provides a link between the dynamics at individual endosome level and average properties of the endosomal network. We show from our theory that some features of the endosomal distributions, which arise from interactions among individual endosomes, are sensitive to alterations in chosen parameters. This provides a direct means to study perturbation experiments wherein the cargo distribution can vary in response to changes of the endocytic system. Our analysis provides a powerful tool for the study of genetic and chemical perturbations that may alter specific systems properties and for extracting various kinetic rates involved in endosomal trafficking from only still images at different points.

The vesicle-mediated degradation of low-density lipoprotein (LDL) is an essential cellular function due to its role in cellular biosynthesis of membranes and steroids. Using multi-color single particle tracking fluorescence microscopy, the intracellular degradation of LDL was probed in live, intact cells. Unique to these experiments is the direct observation of LDL degradation using an LDL-based probe that increases fluorescence intensity upon degradation. Specifically, individual LDL particles were labeled with multiple fluorophores resulting in a quenched fluorescent signal. The characteristics of the vesicle responsible for degradation were determined and the vesicle dynamics involved in LDL degradation were quantified. Visualization of early endosomes, late endosomes and lysosomes was accomplished by fluorescently labeling vesicles with variants of GFP. Transient colocalization of LDL with specific vesicles and the intensity of the LDL particle were measured simultaneously. These studies, which are the first to directly observe the degradation of LDL within a cell, strive to completely describe the endo-lysosomal pathway and quantify the dynamics of LDL degradation in cells.

Ligand-activated signal transduction is a process critical to cell survival and function as it serves as a means of communication between the cells and their environment. Endocytosis is generally thought to down-regulate incoming signals by reducing the surface availability of receptors. However, increasing evidence in many systems suggests a notion which is referred to as the „signalling endosome" hypothesis - that endocytosis can also actively contribute to signalling apart from clearance of activated receptors and thereby attenuation of signalling. The functional aspect of signalling endosomes has been well-characterized in several pathways including RTK and TGF-β signalling. There are, however, various other signalling pathways where the active mechanism of endocytotic regulation is yet to be understood. In this study, we probe this aspect in the cytokine signalling system, where the receptors are known to internalize but the significance of such internalization and precise mechanism is unclear. My thesis aims to elucidate the function and molecular details of internalization of cytokine receptor using interleukin-4 receptor (IL-4R) signalling as a model. IL-4 and IL-13 ligands can induce assembly of three distinct complexes: IL4 induced IL-4Rα – IL-2Rγ (type I), IL-4 induced IL-4Rα – IL-13Rα1 (type II) or the IL-13 induced IL-13Rα1-IL-4Rα (type II). The formation of any of these complexes triggers signalling through the JAK/STAT pathway. However, models of how the oligomerization of the transmembrane receptors and activation takes place are very diverse and lack a clear molecular and biophysical understanding of the underlying receptor dynamics. Previous results of the lab had shown that the affinities between subunits are low, precluding complex formation at the plasma membrane at physiological concentrations. In addition, IL-4R subunits localize in to endosomal structures adjacent to the plasma membrane. It had already been shown that the shared IL-4R subunit IL-2Rγ is internalized by a specific, actin dependent, Rac1/Pak1 regulated endocytosis route in the IL-2 context. We could show that pharmacological suppression of this endocytosis pathway also prevented IL-4 induced JAK/STAT signalling, placing endocytosis upstream of signalling. Here I show using immuno-EM techniques that these endosomal structures are multivesicular bodies. Importantly, I could show that receptor subunits are highly enriched in the limiting membrane of these endosomes relative to the adjacent plasma membrane. Using quantitative loading assays I could furthermore demonstrate that this enrichment is achieved by constitutive internalization of receptors from the cell surface into cortical endosomes. The trafficking kinetics of the receptor subunits is independent of ligand occupancy. Pharmacological inhibition shows that receptors and ligand traffic via the previously identified Rac1/Pak1 pathway. Finally, Vav2 was identified as a candidate Guanine Exchange Factor (GEF) that may regulate Rac1 activity and thereby control the actin polymerization cascade driving IL-4R endocytosis. Immunoprecipitations showed that Vav2 interacts both with the cytoplasmic tail region of the receptors and the receptor associated 2 kinase JAK3. Vav2 may thus couple the receptor/JAK complexes to the Rac1/Pak1 mediated endocytosis route. Taken together, our results suggests that stable „signalling endosomes‟ adjacent to the plasma membrane act as enrichment centres, where ligand and receptor concentrations are locally increased by constitutive trafficking. The confined environment of the endosome then compensates for the weak affinities between the ligand and receptor and facilitates ligand-mediated receptor dimerization. Importantly, overexpression of both type II IL-4R subunits renders signal transduction resistant to endocytosis inhibition, strongly suggesting that the critical factor effecting signalling is sufficient concentration, which the endosomes facilitate achieving. The endosomes are thus dispensable as signalling scaffolds when the receptors are in sufficient concentration, where activated receptors could interact with downstream pathway components. Endocytosis thus provides a crucial means for the signalling process to overcome the thermodynamic hurdles for receptor oligomerization. In conclusion, our data propose a novel, purely thermodynamic role of endosomes in regulating cytokine receptor signalling not seen in any other signalling pathway.

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.