Dans les années 2000, la Dre Johanne Tremblay et son équipe identifient un gène régulé négativement par le calcium extracellulaire dans les glandes parathyroïdiennes de rat hypertendu (SHR). Initialement nommé Hypertension-related calcium regulated gene (HCaRG), puis COMM domain-containing 5 (COMMD5), ce gène codant pour une petite protéine de 24,67 kDa fait partie d’une famille de 10 protéines ayant une structure carboxy-terminale homologue nommée domaine COMM. De nombreux rôles ont été associés à cette famille de protéines et l’analyse expressionnelle dans différents types de cancers montre une modulation, laissant penser qu’elles auraient un rôle oncogénique ou suppresseur de tumeurs. Les études ont démontré que COMMD5 entraine une maturation des jonctions cellulaires, une diminution de la prolifération et favorise la migration cellulaire. La surexpression de COMMD5 dans les tubules proximaux de rein accélère la réparation suite à un dommage aigu en limitant d’une part la prolifération tout en favorisant la migration et la re-différenciation cellulaire. Partant de ces observations, nous avons focalisé nos études sur le développement du carcinome à cellules rénales, une pathologie affectant 300 000 personnes chaque année dans le monde. L’hypothèse que nous avons émise était que COMMD5 puisse potentiellement jouer un rôle anti-oncogénique dans le cancer du rein en contrôlant la prolifération et la différenciation cellulaires. Afin de vérifier notre hypothèse, nous avons étudié le rôle de COMMD5 dans le maintien de l’intégrité épithéliale des cellules via la régulation de la transition épithélio-mésenchymateuse (EMT) et le contrôle du récepteur du facteur de croissance épidermique (EGFR). Nos résultats ont démontré que COMMD5 est diminuée dans les carcinomes rénaux et est corrélée avec la survie des patients. La présence du facteur de transcription induit par l’hypoxie 1 (HIF1α), exprimé dans la majorité des tumeurs solides rénales a induit une diminution de COMMD5. La perte de COMMD5 dans les cellules de tubules proximaux de reins humains (HK-2) a favorisé la dé-différenciation et la tumorigénicité des cellules, médiées par l’activation de la transition épithélio-mésenchymateuse. De plus, cette perte de COMMD5 a entrainé également une réorganisation du cytosquelette d’actine ayant pour conséquence la dérégulation endosomale du récepteur de l’EGF et favorisant une activation prolongée. Dans les carcinomes rénaux, la surexpression de COMMD5 a diminué la prolifération cellulaire suivie d’une re-différenciation grâce à deux mécanismes. D’une part, COMMD5 a régulé négativement la protéine HIF1α, induisant ainsi une transition mésenchymo-épithéliale (MET), tout en séquestrant le facteur de transcription SNAIL dans le cytoplasme. D’autre part, COMMD5 contrôle négativement l’expression transcriptionnelle des récepteurs ErbB par une hyperméthylation de leurs promoteurs. Dans son ensemble, les résultats innovant de cette thèse démontrent que COMMD5 est un gène ayant des caractéristiques anti-oncogéniques en contrôlant la différenciation cellulaire via le mécanisme de transition épithélio-mésenchymateux et la régulation de l’expression des récepteurs ErbB. / In the 2000s, Dr. Johanne Tremblay and her team identified a gene that was negatively regulated by extracellular calcium in hypertensive rat parathyroid glands. Originally named Hypertension-related calcium regulated gene (HCaRG) and renamed COMM Domaincontaining 5 (COMMD5), this gene encoding a small protein of 24.67 kDa is part of a family of 10 proteins sharing a homologous structure in the carboxy-terminal position named COMM domain. Many roles have been associated and expressional analysis of different types of cancer shows a modulation suggesting that they have an oncogenic or tumor suppressor roles. Studies have shown that COMMD5 induces maturation of the cell junctions, decreased cell proliferation and promotes migration. The overexpression of COMMD5 in the renal proximal tubules accelerates repair by promoting cell proliferation and ultimately induces cell migration and redifferentiation after acute injury. Based on these observations, we focused on the development of renal cell carcinoma, a disease affecting 300,000 people each year worldwide. Our hypothesis is that COMMD5 plays a tumor suppressor role in kidney cancer by controlling cell proliferation and differentiation. To test our hypothesis, we investigated the role of COMMD5 in maintaining the epithelial integrity of cells through the regulation of epithelial to mesenchymal transition (EMT) and the control of epidermal growth factor receptors (EGFR). The results showed that COMMD5 is decreased in kidney carcinomas resulting of a great negative indicator of the survival prognostic. The presence of hypoxia-inducible factor 1a (HIF1α), expressed in the majority of solid tumors, leads to a decrease of COMMD5 in the proximal tubule cells (HK-2). Inhibition of COMMD5 promotes dedifferentiation and tumorigenicity of cells mediated by epithelial to mesenchymal transition. The loss of COMMD5 induces a reorganization of the actin cytoskeleton resulting in endosomal dysregulation of the EGFR receptor and promoting its activation. In renal cell carcinoma, COMMD5 overexpression decreases cell proliferation and induces their redifferentiation by two mechanisms: firstly, COMMD5 induces an inhibition of the HIF1α protein expression resulting in a mesenchymal to epithelial transition and sequesters the SNAIL transcription factor in the cytoplasm; secondly, COMMD5 negatively regulates the transcriptional expression of the ErbB receptors. Taken together, these results of this thesis show that COMMD5 is a gene showing tumor suppressor characteristics by controlling cellular differentiation and by regulating the expression of ErbB receptors.

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The endosomal trafficking system is a network of highly coordinated cellular pathways that control the growth and function of cells. The coordination of secretion and endocytosis in cells is one of the primary drivers of polarized growth, where new plasma membrane and cell wall components are deposited at the growing apex. In plants, one of the cell types exhibiting polarized growth are the root hairs. Root hairs are regulated extensions of epidermal cells called trichoblasts and are essential for anchorage, absorption of water and nutrients, and plant-microbe interactions. In this thesis, I characterize a previously undescribed protein involved in retromer function and endosomal trafficking pathways that regulate tip growth in root hairs of Arabidopsis thaliana. The large retromer complex functions in recycling receptors in endosomal trafficking pathways essential for diverse developmental programs including cell polarity, programmed cell death, and shoot gravitropism in the model plant, Arabidopsis thaliana. I have characterized VPS26C, a novel member of the large retromer complex, that is essential in maintaining root hair growth in Arabidopsis. We used Bimolecular Fluorescence Complementation (BiFC) analysis to demonstrate thatVPS26C interacts with previously characterized core retromer subunits VPS35A and VPS29. Genetic analysis also indicates that vps26c suppresses the root hair growth and cell wall organization phenotypes of a null mutant of the SNARE VTI13 that localizes to early endosomes and the vacuole membrane, indicating a crosstalk between the VPS26C-retromer and VTI13-dependent vesicular trafficking pathways. Phylogenetic analysis was used to show that VPS26C genes are present in most angiosperms but appear to be absent in monocot genomes. Moreover, using a genetic complementation assay, we have demonstrated that VPS26C shares deep conservation of biochemical function with its human ortholog (DSCR3/VPS26C). We also used an affinity purification-based proteomic analysis to identify proteins associated with VTI13 in young seedlings. Preliminary results suggest that a number of proteins linked to cell plate organization in plants are associated with the VTI13 proteome, emphasizing the potential role of this pathway in new cell wall biosynthesis/organization. Additionally, we have identified endoplasmic reticulum (ER)-body proteins, involved in plant defense response pathways, suggesting that either the VTI13 endosomal trafficking pathway is functioning in plant defense responses, or the ER-body proteins have additional independent function(s) in Arabidopsis roots that depend on VTI13. In summary, I have described a novel retromer complex essential for polarized growth in Arabidopsis. VPS26C is an ancient gene and shares sequence and functional homology between human and Arabidopsis. vps26c is a genetic suppressor of the vti13- dependent root hair growth and cell wall organization pathways. Proteomic analysis of VTI13 endosomes in young seedlings suggests that a number of proteins associated with cell plate formation are associated with VTI13 compartments, supporting the genetic analysis described here and serves as a starting point to further describe the role of this pathway in controlling polarized growth in plants.

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.

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.

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.

Le bourgeonnement est l'étape finale du cycle viral du virus VIH. Les particules virales vont devoir modifier la topologie de la membrane plasmique afin de promouvoir leur libération dans le milieu extracellulaire ; cette étape est réalisée par le recrutement de protéines ESCRT (en particulier CHMP4 et CHMP2) au point de bourgeonnement. A ce jour, les détails moléculaires de ce recrutement sont méconnus. Lethal Giant Discs (LgD) a été décrite dans la littérature comme un régulateur du traffic endosomal, et une interaction avec CHMP4B a été proposée pour l'orthologue humain CC2D1A. Un point majeur de ce travail aura été de caractériser l'interaction CC2D1A.CHMP4B, mais également de mieux comprendre l'organisation de la protéine. En particulier j'ai résolu la structure d'un fragment de LgD à 2.4 Å, comprenant une région hélicale et un domaine C2 en c-terminal. En outre, nous montrons que CC2D1A inhibe la capacité de CHMP4B à polymériser in vitro. A partir d'une structure cristallographique de CHMP4B et de données biochimiques, nous montrons que le site d'interaction de CC2D1A sur CHMP4B est impliqué dans la polymérisation de CHMP4B, et important pour la fonction de la protéine dans le contexte du bourgeonnement du HIV. Un projet parallèle m'a également conduit à définir un protocole de purification de la protéine CHMP2B recombinante sous forme monomérique, cet isoforme ayant été récemment impliqué dans la formation de structures tubulaires à la membrane plasmique et dans des activités de scission membranaire. En particulier, j'ai pû caractériser la protéine en présence de liposomes et préciser de nouveaux partenaires cellulaires. / Budding is the final step of HIV infection. Viral particles will have to modify the topology of the plasma membrane in order to achieve their correct release from the infected cell, by recruiting ESCRT proteins at the budding point, and among them CHMP4 and CHMP2 isoforms. So far, the molecular details of this recruitment are not precisely known.. Lethal Giant Discs (LgD) has been descibed in the litterature as a regulator of endosomal trafficking, and an interaction with CHMP4B has been proposed. A major point of this research is to propose a structural basis for this interaction, as well as a better understanding of the role and general organization of LgD/CC2D1A. The crystal structure of a LgD fragment (comprising a predicted coiled-coil motif and a c-terminal C2 domain) was solved in our lab at 2.4 A. Moreover, we show that CC2D1A impairs in vitro the ability of CHMP4B to polymerize. Based on a crystallographic structure of CHMP4B and biochemical data, we also show that the binding site of CC2D1A on CHMP4B is itself involved in polymerization, in the context of HIV budding. As a side project, I've also set up a protocole to obtain pure monomeric CHMP2B, which has been shown to polymerize at the plasma membrane, and I've characterized the protein in the presence of liposomes, along with new partners.

The ability of cell-penetrating peptides to cross plasma membranes has been explored for various applications, including the delivery of bioactive molecules to inhibit disease-causing cellular processes. The uptake mechanisms by which cell-penetrating peptides enter cells depend on the conditions, such as the cell line the concentration and the temperature. To be used as therapeutics, each novel cell-penetrating peptide needs to be fully characterized, including their physicochemical properties, their biological activity and their uptake mechanism. Our group has developed a series of highly performing, non-toxic cell-penetrating peptides, all derived from the original sequence of transportan 10. These analogs are called PepFects and NickFects and they are now a diverse family of N-terminally stearylated peptides. These peptides are known to form noncovalent, nano-sized complexes with diverse oligonucleotide cargoes. One bottleneck that limits the use of this technology for gene therapy applications is the efficient release of the internalized complexes from endosomal vesicles. The general purpose of this thesis is to reveal the mechanisms by which our in house designed peptides enter cells and allow the successful transport of biofunctional oligonucleotide cargo. To reach this goal, we used both biophysical and cell biology methods. We used spectroscopy methods, including fluorescence, circular dichroism and dynamic light scattering to reveal the physicochemical properties. Using confocal and transmission electron microscopy we observed and tracked the internalization and intracellular trafficking. Additionally we tested the biological activity in vitro and the cellular toxicity of the delivery systems. We conclude that the transport vectors involved in this study are efficient at perturbing lipid membranes, which correlates with their remarkable capacity to transport oligonucleotides into cells. The improved and distinct capacities to escape from endosomal vesicles can be the result of their different structures and hydrophobicity. These findings extend the knowledge of the variables that condition intracellular Cell-penetrating peptide mediated transport of nucleic acids, which ultimately translates into a small step towards successful non-viral gene therapy.

No description available.

No description available.