The research presented in this dissertation consists of two primary parts. The initial focus centered on understanding the distribution of Golgi resident glycosyltransferases between the ER and Golgi at steady-state. Retrograde trafficking of these Golgi proteins has been demonstrated experimentally mandating the existence of a dynamic equilibrium between the Golgi apparatus and ER. Our published studies also included the development of a quantitative method for analysis of data collected using fluorescent microscopy. The second part of this dissertation presents results pertaining to the quantification of a unique Golgi resident protein that cycles in the late endosome bypass pathway. Using the published method of analysis and techniques developed during the initial project, the anterograde and retrograde transport kinetics of this Golgi protein were determined and used to develop a compartmental model for pH sensitive trafficking in the bypass pathway. The spatial Golgi distribution of the protein during retrograde transport to the Golgi following endosomal exit was also investigated. This research lies at the interface of experimental cell biology and quantitative computational analysis. These experiments combined more traditional experimental biological approaches with more recent computational approaches to understanding cellular mechanisms. Additionally, development of a quantitative method of analysis validated the use of fluorescent microscopy as a quantitative tool for studying intracellular proteins. / Ph. D.

À mon arrivée dans le laboratoire, les travaux réalisés indiquaient que le trafic des endosomes de recyclage (ERs) vers le Golgi était régulé par les kinases de la famille Src (KFS) et contribuait au remodelage de l’actine induit par différents stress (E4orf4, staurosporine). Or les KFS coordonnent la dynamique de l’actine et le trafic membranaire, mais les mécanismes impliqués demeurent peu caractérisés. Des évidences récentes indiquaient aussi que le trafic endosomal contribuait à la transformation cellulaire induite par des formes oncogéniques des KFS, laquelle est associée à l’assemblage de structures invasives riches en actine (podosomes). En se basant sur ces données, notre hypothèse de recherche était que le transport des ERs vers le Golgi sous le contrôle du rétromère, un régulateur majeur des voies de transport rétrograde, pourrait contribuer à la formation des podosomes lors de la transformation cellulaire induite par v-Src. Les objectifs du travail présenté dans ce mémoire étaient de caractériser les changements précoces dans le trafic endosomal dépendant du rétromère suivant l’activation de v-Src et d’adresser leur contribution dans la formation des podosomes. Mes travaux ont tiré profit de l’utilisation d’un modèle permettant l’activation rapide d’un mutant thermosensible de v-Src dans des cellules épithéliales MDCK. J’ai ainsi pu observer dans ce modèle une polarisation précoce des ERs autour des podosomes en formation. Une augmentation dans le nombre et la morphologie des endosomes rétromère-dépendants (Vps26) a été mesurée et coïncidait avec un transport accru de cargos dépendants du rétromère vers le Golgi. Notamment, l’inhibition de Rab7, un régulateur du rétromère, interfère avec la formation des endosomes Vps26-positifs et des podosomes. En conclusion, mes résultats suggèrent que v-Src active les voies de transport dépendantes du rétromère pour mobiliser des protéines de signalisation et/ou des lipides bioactifs nécessaires au remodelage polarisé de l’actine et à la formation des structures invasives.

Les exosomes sont des vésicules membranaires de 30 à 100 nm de diamètre, formées dans les endosomes multivésiculaires et sécrétées par la plupart des cellules. Les propriétés biophysiques et biochimiques des exosomes ainsi que les mécanismes permettant leur biogénèse et sécrétion ont fait l’objet de nombreuses études. Cependant, ces derniers sont encore méconnus, limitant l'analyse des fonctions des exosomes in vivo. Au moins deux mécanismes ont été proposés pour la biogénèse des exosomes : un mécanisme nécessiterait l’action de protéines impliquées dans le tri endosomal, les ESCRT (« endosomalsorting complex required for transport »). Un autre mécanisme serait indépendant de leur fonction. La sécrétion des exosomes, une fois générés dans les endosomes, requiert la petite GTPase, Rab27a, comme montré dans un modèle cellulaire humain. Mes travaux de thèse ont porté sur l’étude des mécanismes moléculaires impliqués dans la biogénèse et la sécrétion des exosomes. Une première étude visant à analyser la fonction de Rab27a dans des cellules murines, m’a permis de mettre en évidence l’existence de différentes populations d’exosomes, dont la sécrétion dépend ou non de Rab27a. Une deuxième étude a eu pour objectif d’analyser l’implication des ESCRT dans la biogénèse des exosomes dans des cellules HeLa CIITA. Le criblage d’une librairie d’ARN d’interférence dirigés contre les différentes protéines ESCRT, a permis l’identification de 7 molécules potentiellement impliquées dans cette voie : HRS, STAM1, TSG101, leur inactivation induisant la diminution de la sécrétion des exosomes. L’inactivation de CHMP4C, VPS4B,VTA1 et ALIX, au contraire, l’augmente. L’inhibition de l’expression de ces candidats suivie de l’analyse des exosomes sécrétés a démontré l’hétérogénéité des vésicules sécrétées, et une modification de leur taille et de leur composition protéique par rapport aux cellules contrôle. Plus particulièrement, l’inactivation d’ALIX induit une augmentation de lasécrétion d‘exosomes de plus grande taille, et l’enrichissement sélectif en molécules de CMH de classe II. En accord, j’ai montré que les cellules inactivées pour ALIX, aussi bien des cellules HeLa que des cellules dendritiques humaines ont une plus forte expression de CMH de classe II à la surface et dans des compartiments intracellulaires. Ces résultats suggèrent l’implication de certains membres de la famille ESCRT dans la voie de biogenèse et sécrétion des exosomes, ainsi qu’un rôle potentiel d’Alix dans le trafic des molécules CMH de classe II, et dans la modulation de la composition protéique des exosomes. / Exosomes are small membrane vesicles with sizes ranging from 30 to 100 nm in diameter, which are formed in multivesicular endosomes and secreted by most cell types. Numerous studies have focused on the biophysical and biochemical properties of exosomes, as well as the mechanisms of biogenesis and secretion of these vesicles. However, these aspects are not fully understood, which limits the analysis of the functions of exosomes in vivo. At least two mechanisms have been proposed for the biogenesis of exosomes : one would rely on the function of proteins involved in endosomal sorting, the ESCRT family (for “endosomal sorting complex required for transport”). Another mechanism would be independent of their activity. Once exosomes are formed in endosomes, their secretion requires the small GTPase RAB27A, as shown in a human cell line. The objective of my PhD project was to gain insights into the molecular mechanisms that drive exosome biogenesis and secretion. A first study performed to analyze the function of Rab27a in murine cells allowed me to show the existence of different populations of exosomes, dependent or not on Rab27a for their secretion. A second study was aimed at analyzing the involvement of ESCRT proteins in exosome biogenesis in HeLa-CIITA cells. Seven molecules potentially involved in this process were identified on the basis of the screening of an RNA interference library directed against the different ESCRT proteins: the inactivation of HRS, STAM1 and TSG101 induced a decrease in exosome secretion, whereas the down regulation of CHMP4C, VPS4B, VTA1 and ALIX increased it. Gene expression of the different candidate proteins was inhibited and exosomes secreted by these cells were analyzed: we showed the heterogeneity of the secreted vesicles, as well as an alteration of their size and protein composition, as compared to control cells. In particular, the inactivation of ALIX induced an increase in the secretion of larger vesicles, and the selective enrichment of these vesicles in MHC class II molecules. Accordingly, I showed that both HeLa-CIITA and human primary dendritic cells inactivated for ALIX possess a higher expression of MHC class II molecules at the cell surface and in intracellular compartments. These results suggest that some members of the ESCRT family are involved in the exosome biogenesis and secretion pathway, and propose a potential role of ALIX in the trafficking of MHC class II molecules and in the modulation of the protein composition of exosomes.

The mechanisms by which altered processing, distribution and secretion of proteolytic enzymes occur, facilitating degradation of the extracellular matrix in invasive and metastatic cells, are not fully understood. Studies on the MCF-10 A breast epithelial cell line and its premalignant, c-Ha-ras-transfected MCF-10AneoT counterpart have shown that the ras-transfected cell line has a more alkaline pH. The objective of this study was to determine which organelles of the endosome-lysosome route were alkalinized and shifted to the cell periphery after ras-transfection. Antibodies to the hapten 2,4-dinitrophenyl (DNP), required for pH studies, were raised in rabbits and chickens using DNP-ovalbumin (DNP-OVA) as immunogen. Cationised DNP-OVA (DNP-catOVA) was also inoculated to increase antibody titres. Anti-hapten and carrier antibody titres were assessed. In rabbits, cationisation seems useful to increase anti-DNP titres if a non-self carrier protein (OVA) is used. In chickens, cationisation of DNP-OVA seems necessary to produce a sustained anti-OVA (anti-self) response (implying a potential strategy for cancer immunotherapy). Oregon Green® 488 dextran pulse-chase uptake and fluorescent microscopy, and (2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP) uptake, immunolabelling for DNP (a component of DAMP) and unique markers for the early endosome (early endosome antigen-I, EEAI), the late endosome (cation-independent mannose-6-phosphate receptor, CI-MPR) and the lysosome (small electron dense morphology and lysosome-associated membrane protein-2, LAMP-2) and electron mlcroscopy was performed. The pH of late endosomes and lysosomes in the ras-transfected MCF-10AneoT cell line were found to be relatively alkalinised and Iysosomes shifted toward the cell periphery. The acidic pH of late endosomes is required to release precursor cysteine and aspartic proteases from their receptors (e.g. CI-MPR), process the precursors to active proteases and to allow receptor recycling. The more alkaline pH observed potentially explains the altered processing of proteases in rastransfected cells. Alkalinisation ofthe cytosol may affect the cytoskeleton responsible for, among other things, the positioning and trafficking of various organelles, causing relocation of Iysosomes toward the cell periphery and actin depolymerisation. This may enable fusion of Iysosomes with the plasma membrane and the release of proteolytic enzymes, facilitating the observed invasive phenotype. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

Toxoplasma gondii, comme Plasmodium falciparum appartiennent au phylum des Apicomplexes. Ce groupe de parasites ont comme dénominateurs communs, trois organites apicaux : rhoptries, micronèmes et granules denses contenant des facteurs indispensables pour la reconnaissance, l’entrée et la survie du parasite au sein de la cellule hôte. Le récepteur transmembranaire de type 1 appelé TgSORTLR ("Toxoplasma gondii Sortilin-Like Receptor") est nécessaire à la biogenèse des organelles de sécrétion rhoptrie et micronème (Sloves et al., 2012). Le domaine C-terminale de la TgSORTLR, lie TgVps26 et TgVps35 deux protéines appartement au complexe Rétromère essentiel au recyclage protéique chez les mammifères et S. cerevisiae. Nous avons construit le premier interactome du CRC de T. gondii et des autres Apicomplexes. Contrairement aux mammifères, le Rétromère de T. gondii est composé du CRC (Complexe de Reconnaissance du Cargo) TgVps35-TgVps26-TgVsp29 et l’absence du dimère de Sorting Nexin (SNX). Nous avons identifié plusieurs protéines connues de l’ELC (Endosomal-like compartment) ainsi que des protéines parasitaires spécifiques. La déplétion conditionnelle de TgVps35 démontre que le complexe Rétromère n’est pas seulement crucial pour la biogenèse des rhoptries, micronèmes et granules denses, mais aussi pour l’architecture et l’intégrité du parasite. Nous avons montré que le recyclage de la TgSORTLR entre l’ELC et le TGN (Tans-Golgi-Network) est essentiel au trafic des protéines de sécrétion rhoptries et micronèmes. Par ailleurs nous avons décrit deux nouvelles protéines hypothétiques TgHP12 et TgHP03 pouvant être impliquées respectivement dans le trafic vers l’ELC et vers la membrane plasmique. Afin nous avons identifié et caractérisé une protéine chimérique TgHP25 avec les domaines BAR et SBF2, pouvant être impliquée dans la biogenèse de l’organite rhoptrie. En somme notre travail souligne l’importance du recyclage protéique et l’implication de protéines spécifiques dans la maturation des organites et l’intégrité du parasite. / Toxoplasma gondii, like Plasmodium falciparum are belong to the Apicomplexan phylum. This group of parasites have as a common denominator, three apical organelles: rhoptries, micronemes and dense granules containing the essential factors for recognition, entry and survival into the host cell. The Toxoplasma gondii Sortilin-Like Receptor (TgSORTLR), is essential for the biogenesis of apical secretory organelles rhoptries and micronemes (Sloves et al., 2012). The C-terminal tail of TgSORTLR specifically binds to TgVps26 and TgVps35 proteins, two components of a pentameric complex called retromer (RC), and known to play an essential role in retrograde transport in yeast and mammals. We now report the first retromer-trafficking interactome in T. gondii and other apicomplexan parasites. In contrast to yeast and mammals, T. gondii RC harbors a singular architecture typified by a Vps35-Vps26-Vps29 trimer complex and the absence of the dimer of sorting nexins. Rather, we identified several known endosomal-like compartment (ELC) proteins and unrelated parasite-specific proteins. The conditional ablation of TgVps35 demonstrates that the Retromer complex is not only crucial for the biogenesis of rhoptries, micronemes and dense granules but also for maintaining a proper parasite architecture and integrity. We showed that the recycling of TgSORTLR between ELC and Trans-Golgi Network (TGN), is essential for proper protein trafficking to secretory organelles rhoptries and micronemes. Furthermore, we will describe two novel parasite-specific proteins TgHP12 and TgHP03, whose functions are likely related to ELC and plasma membrane. So we identified and characterized a chimeric protein TgHP25 with the BAR and SBF2 domains, may be involved in the biogenesis of the organelle rhoptrie. In short our work emphasizes the importance of protein recycling and involvement of specific proteins in the maturation of organelles and integrity of the parasite.

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 FYVE domain is a lipid binding domain found in approximately 27 different mammalian proteins. It specifically interacts with the lipid, PI(3)P, which is enriched on early endosomes. Consequently, many FYVE domain-containing proteins localize to the endosome, however the ability of FYVE domains to target to endosomal membranes is variable, despite high sequence conservation. Here we describe the structural requirements necessary for endosomal localization and liposome avidity. As FYVE domains are lipid binding domain, many FYVE domain-containing proteins have been implicated in membrane trafficking. We performed an RNAi screen of FYVE domain-containing proteins to identify general regulators of endocytosis in Caenorhabditis elegans. In this screen, we identified the EEA1, a known regulator of endocytosis and two novel genes: WDF2 and KIAA1643. Initial characterization of WDF2 suggest that its function is conserved in humans. Of all the FYVE domain-containing proteins, we have been particularly interested in SARA (Smad Anchor for Receptor Activation); a protein implicated in the TGFβ signaling pathway. This protein contains a binding domain for the TGFβ mediated transcription factor, Smad2/3, and a FYVE domain. It was the presence of the FYVE domain, an endosomal targeting signal, in SARA that lead us to hypothesize that endocytosis might be a necessary step in TGFβ signaling. SARA localizes to the early endosome; the TGFβ receptors also internalize into these endosome. When this internalization is prohibited, there is correlative decrease in Smad2/3 phosphorylation, Smad2 nuclear translocation and TGFβ mediated transcription. Overexpression of a dominant negative SARA construct and SARA siRNA oligonucleotides inhibit TGFβ signaling. We conclude that TGFβ receptor signaling to Smad2/3 occurs on the endosome and this signaling requires SARA. Receptor mediated endocytosis has been classically thought of as an important mechanism for attenuating signaling pathways. We have redefined the role of endocytosis to include the necessary and positive regulation of specific signaling pathways. We have also extended our insights into the biological role of the endosome as a compartment specialized for the assembly and propagation of specific extracellular signals.

L'utilisation des oligonucléotides antisens PMO ou PNA, pour corriger les erreurs d'épissage par blocage stérique, constitue une nouvelle stratégie prometteuse pour réguler l'expression génétique. Ces ON peuvent mener au traitement de maladies comme la β-thalassémie, la dystrophie musculaire de Duchenne (DMD) ou les cancers. Cependant leur développement clinique requiert un système de délivrance efficace. Les peptides cationiques (CPPs) sont caractérisés par leur capacité à s'internaliser dans les cellules eucaryotes. Cependant leur efficacité à promouvoir la délivrance cytoplasmique et nucléaire des ON est limitée par leur séquestration dans des vésicules d'endocytose, ce qui est à l'origine de la dégradation du matériel internalisé. Nous avons contribué à l'étude du trafic intracellulaire et de l'activité dans un essai de correction d'épissage de plusieurs familles de CPPs capables de délivrer efficacement des analogues d'ON à des doses non toxiques et en absence d'agents endosomolytiques. Nos études mécanistiques indiquent que ces constructions (covalentes ou non covalentes) CPP-ON sont endocytées par la voie clathrine, que la ségrégation dans les endosomes reste une limitation et qu'il existe une bonne corrélation entre leur activité biologique et leur capacité à déstabiliser les membranes endosomales. / The use of antisense oligonucleotides PMO or PNA to correct splicing errors by steric- block represents a new promising therapeutic strategy. These ONs lead to the treatment of diseases such as β-thalassemia, Duchenne muscular dystrophy (DMD) or cancers. However their functional success requires efficient delivery. Cationic cell penetrating peptides (CPPs) are characterized by their ability to be internalized in eukaryotic cells. However their efficiency in promoting cytoplasmic and nuclear delivery of ON has been hampered by endocytic sequestration and subsequent degradation of internalized material in endocytic vesicles, which is responsible for the degradation of internalized material. We have contributed to the study of intracellular trafficking and activity (using splicing correction assay) of several families of CPPs capable of delivering effective analogs ON at nontoxic doses and in the absence of agents endosomolytic. Our mechanistic studies indicate that these constructs (covalent or noncovalent) CPP-ON are internalized through clathrin, that segregation in endosomes remains a limitation and that there is good correlation between biological activity and their ability to destabilize endosomal membranes.

The pre-symptomatic stage of neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) occurs several decades before the clinical onset. Changes in the lysosomal network, i.e. the autophagosomal, endosomal and lysosomal vesicular system, are among the first alterations observed. There are currently no treatments to slow or cure neurodegenerative diseases, and there is a great need for discovery of treatment targets in cellular pathways where pathology pre-dates the neuronal death. It is also crucial to be able to diagnose neurodegenerative diseases earlier, both to enable early intervention treatment and aid in selecting clinical trial populations before the patient has widespread pathology. This thesis aims at investigating the potential of lysosomal network proteins as biomarkers and therapeutic targets in neurodegenerative disease. A targeted search for lysosomal network proteins was performed in cerebrospinal fluid (CSF) from AD patients, and seven proteins: early endosomal antigen 1 (EEA1), lysosomal-associated membrane proteins 1 and 2 (LAMP-1, LAMP-2), lysozyme, microtubule-associated protein 1 light chain 3 (LC3), Rab3 and Rab7, were elevated. The levels of EEA1, LAMP-1, LAMP-2, LC3, lysozyme and Rab3 were also measured in CSF from parkinsonian syndrome patients: PD, clinically diagnosed 4-repeat tauopathy, pathologically confirmed corticobasal degeneration (CBD) and pathologically confirmed progressive supranuclear palsy (PSP) patients. LAMP-1 and LAMP-2 were decreased in PD. LC3 and lysozyme levels were increased in 4-repeat tauopathy patients. EEA1 was decreased and lysozyme increased in PSP, and LAMP-1, LAMP-2, LC3 and lysozyme were increased in CBD. The lysosomal network proteins had different CSF protein profiles in all the parkinsonian syndromes, as well as in AD. It should be emphasized that only a select few of the lysosomal network proteins were observed to be changed, rather than a general change in lysosomal network proteins, which implicates the involvement of these seven proteins in specific pathological processes. The most interesting candidates, LAMP-2 and lysozyme, were selected for further study for their involvement in the pathology of AD. Lysozyme was found to co-localise with Aβ plaques in AD patients and overexpression prolonged survival and improved the activity in a Drosophila model of AD. Lysozyme was found to alter the aggregation pathway of Aβ1-42, to counteract the formation of toxic Aβ species and to protect from Aβ1-42 induced cell toxicity. Aβ1-42 in turn was found to increase the expression of lysozyme in both neuronal and glial cells. These data suggest that lysozyme levels rise in AD as a compensatory response which is protective against Aβ associated toxicity. LAMP-2 mRNA and protein were found increased in brain areas relevant for AD pathology and various cellular models showed complex involvement of LAMP-2 in Aβ related pathology, with extensive crosstalk between LAMP-2 and Aβ. Exposure to oligomeric Aβ1-42 caused an upregulation of LAMP-2 and in turn, overexpression of LAMP-2 caused a reduction in secreted levels of Aβ1-42, as well as changing the generation pattern of Aβ and affecting clearance and secretion of Aβ1-42. These data indicate that the increased levels of LAMP-2 in AD could be an attempt to regulate Aβ generation and secretion. In summary, this thesis reports that utilising lysosomal network proteins as biomarkers and novel therapeutic targets for neurodegenerative diseases holds great promise.

No description available.