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L’infection par Plasmodium, parasite responsable du paludisme, débute par l’injection de sporozoïtes par un moustique du genre Anopheles. La première cible des sporozoïtes est le foie, où le parasite se développe avant l’initiation d'une phase d'infection érythrocytaire symptomatique. Dans le foie, les sporozoïtes pénètrent activement les hépatocytes en formant une vacuole parasitophore, dans laquelle le parasite se multiplie. Cette étape, appelée invasion productive, implique des facteurs parasitaires et des protéines de l’hôte, notamment CD81. Toutefois, les mécanismes mis en jeu restent méconnus. À l’aide d’une nouvelle approche génétique développée au laboratoire, nous avons produit de nouvelles souches de parasites transgéniques fluorescents, notamment chez le parasite de rongeurs P. yoelii. L’utilisation des parasites de P. yoelii GFP et d’un système cellulaire de lignées permissives ou non à l’infection, nous a permis de mieux caractériser les mécanismes cellulaires et moléculaires mis en jeu lors de l’invasion. Nous avons confirmé que l’invasion productive est précédée d’une phase de traversée cellulaire. Nous avons découvert et caractérisé la formation de vacuoles transitoires lors de cette phase de traversée cellulaire, distinctes des vacuoles parasitophores productives. Nos résultats montrent que le parasite se sert d’une perforine parasitaire, PLP1 (Perforin-Like Protein 1), pour sortir de cette vacuole transitoire et échapper à la dégradation par les lysosomes cellulaires. Une fois activés, les sporozoïtes passent d’un mode de traversée à un mode d’invasion productive. Nous avons montré que CD81 joue un rôle dans l’invasion productive. CD81 est nécessaire pour induire la sécrétion des rhoptries parasitaires, impliquées dans la formation de la jonction mobile, une structure à travers laquelle le parasite se glisse pour pénétrer dans la cellule. Nous avons pu aussi montrer qu’une autre protéine des hépatocytes, SRBI (scavenger receptor BI), définit une voie d’entrée indépendante de CD81 pour P. berghei et P. vivax. Par une approche génétique originale, nous avons pu montrer que deux protéines des micronèmes des sporozoïtes, P52 et P36, jouent un rôle majeur dans l’entrée via CD81 et SRBI, et mis à jour un lien fonctionnel entre P36 et l’entrée via SRBI. Enfin, nous avons développé plusieurs approches génétiques pour cibler le gène d’ama1 chez P. yoelii, une protéine des micronèmes impliquées dans la formation de la jonction. Nos résultats nous éclairent un peu plus sur les mécanismes d’invasion des sporozoïtes, et ouvrent des perspectives intéressantes vers le développement de nouvelles stratégies vaccinales. / Infection with the Plasmodium parasite begins with the injection of sporozoites by an Anopheles mosquito. The first target is the liver where the parasite replicates as a pre-requisite to the development of pathogenic blood stage infection. In the liver, sporozoites penetrate hepatocytes forming a parasitophorous vacuole in which the parasite multiplies. This step, the productive invasion, involves parasitic factors and host proteins, particularly CD81, but the underlying mechanisms remain largely unknown. To facilitate monitoring of sporozoite invasion, we generated novel transgenic fluorescent parasites, using a new selection strategy named GOMO (gene out marker out) in the rodent parasite P. yoelii. The use of this transgenic parasite and of host cell lines permissive or not to infection, has allowed us to better characterize the cellular and molecular mechanisms involved during invasion. We have confirmed that the productive invasion is preceded by a cell traversal phase. We discovered and characterized the formation of transient vacuoles during this step, before formation of the parasitophorous vacuole. Our results uncovered that the perforin-like protein (PLP1) mediates sporozoite egress from transient vacuoles and escape from degradation by the cell lysosomes. Once activated, the sporozoites switch from the mode of cell traversal to productive invasion. We show that CD81 plays a role in the productive invasion. CD81 is necessary to induce the secretion of rhoptries proteins, involved in the formation of the moving junction, a structure through which the parasite glides to enter the cell. We could also show that another hepatocyte protein, SR-B1 (scavenger receptor B1), defines a CD81-independent pathway for P. berghei and P. vivax infection. Using an original genetic approach, we have shown that two sporozoite micronemal proteins, P52 and P36, play a role in the entry via CD81 and SR-B1, and highlighted a functional link between P36 and entry via SR-B1. Finally, we have developed several genetic approaches to target ama1 gene in P. yoelii, which encodes a protein involved in the formation of the moving junction. Altogether, our results contribute to improve our understanding of the mechanisms of sporozoite invasion, and open interesting perspectives for the development of novel vaccine strategies.

Degradation of membrane proteins in the vacuole/lysosome is dependent on their prior sorting into the multivesicular body (MVB) pathway. This sorting process involves incorporation of proteins into vesicles that are formed by budding of the limiting membrane of the endosome into the lumen of the organelle. The MVB sorting process on the whole is highly conserved from yeast to human, and depends on the Vps27/Hrs, ESCRT-I, -II, and -III protein complexes functioning sequentially on the endosomal membrane, as well as on additional factors, such as the ubiquitinating enzyme Rsp5/Nedd4. It has now been established that ubiquitin serves as a sorting signal for many cargoes into the MVB pathway. In this thesis work, we provide evidence that Bro1 is not required for protein ubiquitination or early steps of endocytosis, but functions at the late endosome level as an integral component of the MVB pathway. Similarly to its human homologue Alix, Bro1 interacts with components of the ESCRT-I and ESCRT-III complexes. The putative role of Bro1/Alix in bridging an interaction between ESCRT-I and –III might be important to strengthen an association of these protein complexes to allow efficient sorting of cargo proteins. Deficiency in Bro1 results in recycling of the endocytosed Gap1 permease back to the plasma membrane, a process coupled to deubiquitination of the permease. This recycling is a non-classical phenotype for cells impaired in MVB pathway thus suggesting Bro1 to have a particular role in this sorting process. Furthermore, the conserved C-terminal proline-rich domain (PRD) of Bro1 is specifically important for MVB sorting of cargo proteins that are subject to ubiquitination. We show Bro1 (via its PRD) to play a highly important role in recruitment of the deubiquitinating enzyme Doa4 to the endosome. Consistent with this, Bro1 is required for deubiquitination of cargo proteins, a step occurring just before cargo incorporation into the endosomal vesicles, and similarly to Doa4, for ubiquitin recycling. In contrast to previous interpretations, we show that Doa4 has a direct role in sorting of ubiquitinated cargo proteins into the MVB pathway. We propose that Doa4 – via its association to Bro1 - achieves this role by catalyzing deubiquitination of cargo proteins and/or some components of the MVB sorting machinery. We further show Bro1 to interact with the ubiquitin ligase Rsp5, which, in addition to being required for cargo protein ubiquitination at the plasma membrane, apparently contributes to multiple steps of endocytosis and MVB sorting. Also the Bro1-Rsp5 interaction is dependent on the C-terminal PRD region of Bro1. We propose that this interaction is conserved. A role for ubiquitin in regulation of the MVB sorting machinery is emerging: the function of factors recognizing and sorting ubiquitinated cargo proteins in the MVB pathway is suggested to be coupled to their cycling between ubiquitinated and deubiquitinated stages. A growing body of evidence indicates that ubiquitin ligases of the Rsp5/Nedd4 family play a central role in this regulation. We speculate the Bro1/Alix protein, through its ability to simultaneously interact with factors of the MVB sorting machinery and with ubiquitinating and deubiquitinating enzymes to play a central role in the successive rounds of ubiquitination and deubiquitination of specific factors along the MVB pathway.

Autophagy requires the biogenesis of autophagosomes (APs), which are large multilamellar vesicles that sequester cytoplasmic substrates and undergo a maturation process that ultimately leads to their fusion with lysosomes. Previous studies have suggested that local production of phosphatidylinositol-3-phosphate (PI3P) by class III phosphatidylinositol 3-kinase (PI3K) (i.e., Vps34) is required for AP biogenesis at specialized sites of the endoplasmic reticulum called "omegasomes". Although Vps34 is the sole source of PI3P in budding yeast, mammalian cells can produce PI3P through alternate pathways, including direct synthesis by the class II PI3Ks; however, the physiological relevance of these alternate pathways in the context of autophagy is unknown. To address this question, we generated Vps34 knock-out mouse embryonic fibroblasts (MEFs) and analyzed the impact of Vps34 deletion on autophagy in mammalian cells. Using a novel higher affinity 4x-FYVE finger PI3P-binding probe, we found a Vps34-independent pool of PI3P accounting for ~35% of the total amount of this lipid species by biochemical analysis. Importantly, WIPI-1, an autophagy-relevant PI3P probe, still formed some puncta upon starvation-induced autophagy in the Vps34 knock-out MEFs. Additional characterization of autophagy by electron microscopy as well as protein degradation assays showed that while Vps34 is important for starvation-induced autophagy there is a significant component of functional autophagy occurring in the absence of Vps34. Given these findings, class II PI3Ks (α and β isoforms) were examined as potential positive regulators of autophagy. Depletion of class II PI3Ks reduced recruitment of WIPI-1 and LC3 to AP nucleation sites and caused an accumulation of the autophagy substrate, p62, which was exacerbated upon the concomitant ablation of Vps34. Our studies indicate that while Vps34 is the main PI3P source during autophagy, class II PI3Ks also significantly contribute to PI3P generation and regulate AP biogenesis. In addition, we used a lipidomic approach to capture the lipid profile of cells in the presence and absence of Vps34 under steady-state and during starvation-induced autophagy. Lipidomics is an emerging powerful tool with the potential to identify new interconnected metabolic lipid networks as well as generate new hypotheses. Here, we identified a new relationship between Vps34 and cholesterol homeostasis. Additionally, we identified specific changes in lysolipids during autophagy. Lastly, we investigated whether the retromer complex plays a role in autophagy. Retromer is a protein complex that binds PI3P on the endosomal membrane and mediates retrograde trafficking of transmembrane proteins from the endosome to the trans-Golgi network. Recent studies have shown a downregulation of this complex associated with sporadic Alzheimer's disease (AD) and have demonstrated aberrant trafficking and processing of APP, a pathological feature of AD, as a result of retromer deficiency. Because retromer is important for maintaining endo-lysosomal system function, we hypothesized that it promote efficient autophagy and may contribute to the dysfunctional autophagy observed in AD when impaired. Using standard autophagy assays, such as assessing LC3 conjugation and puncta formation, our preliminary studies suggest a negative regulatory role for retromer in autophagy. Additionally, we observed a strong association of retromer with Atg9, an autophagy-related gene transmembrane protein that is believe to traffic lipids to the growing autophagosome membrane and recycle autophagy proteins from this compartment.

How proliferating cells maintain the copy number and overall size of their organelles is not clear. In the budding yeast Saccharomyces cerevisiae the G1 cyclins Cln1,2,3p control initiation of cell division by regulating the activity of the cyclin-dependent kinase (Cdk) Cdc28p. We show that Cln3p controls vacuolar (lysosomal) biogenesis and segregation. First, loss of Cln3p, but not Cln1p or Cln2p, resulted in vacuolar fragmentation. Although the vacuoles of cln3Δ cells were fragmented, together they occupied a large space, which accounted for a significant fraction of the overall cell size increase in cln3Δ cells. Second, cytosol prepared from cells lacking Cln3p had reduced vacuolar homotypic fusion activity in cell-free assays. Third, vacuolar segregation was perturbed in cln3Δ cells. Our findings reveal a novel role for a eukaryotic G1 cyclin in cytoplasmic organelle biogenesis and segregation. Furthermore we show that the scaffold protein Bem1p, a critical regulator of Cdc42p activity, is a downstream effector of Cln3p/Cdc28p complex. The Cdc42p GTPase is known to be required for vacuole fusion. Our results suggest that Ser72 on Bem1p is phosphorylated by Cdc28p in a Cln3p-dependent manner to promote vacuole fusion. Replacing Ser72 with Asp, to mimic phosphorylation at an optimal Cdkconsensus site located in the first SH3 domain of Bem1p, suppressed vacuolar fragmentation in cells lacking Cln3p. Using in vivo and in vitro assays, we found that Cln3p was unable to promote vacuole fusion in the absence of Bem1p or in the presence of a non-phosphorylatable Bem1p-Ser72Ala mutant. Furthermore, activation of Cdc42p also suppressed vacuolar fragmentation in the absence of Cln3p. Our results provide a mechanism that links cyclin-dependent kinase activity with vacuole fusion through Bem1p and the Cdc42p GTPase cycle.

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Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

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Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Afin de mieux comprendre les mécanismes du trafic cellulaire, les processus de transport des substrats vacuolaires à travers le tonoplaste, le stockage des métabolites et leur dégradation, une analyse globale et exhaustive du protéome vacuolaire d'Arabidopsis thaliana a été réalisée. La connaissance de la localisation subcellulaire des protéines permet de mieux comprendre la fonction des organelles et la compartimentation du métabolisme des plantes. Mais la description précise du protéome d'un organite nécessite d'identifier clairement les véritables protéines résidantes du compartiment étudié. Une tâche si précise est complexe puisqu'elle nécessite la mise en place d'une préparation d'organites purs et homogènes. Pour y parvenir, un protocole de purification de vacuoles à partir de protoplastes isolés de cellules en culture sur un gradient de densité de Ficoll a été amélioré. La combinaison de plusieurs approches de protéomique a permis d'identifier les protéines présentes dans les fractions vacuolaires soluble et membranaire de façon quantitative et fonctionnelle. Les différentes approches ont ainsi mis en évidence des associations et mécanismes moléculaires complexes qui régissent les différentes activités vacuolaires. Cette protéothèque de référence constitue une base pour étudier la dynamique du protéome vacuolaire en réponse à plusieurs stress incluant les métaux lourds. Plusieurs méthodes sans a priori et ciblée ont été proposé afin d'étudier l'impact du cadmium sur la vacuole, ce compartiment cellulaire clé de la détoxication. / To better understand the mechanisms governing cellular traffic, transport process of substrates across the tonoplast, storage of various metabolites and their ultimate degradation, a comprehensive and thorough analysis of Arabidopsis thaliana vacuolar proteome was performed. Protein subcellular localization knowledge is an important step toward assigning functions of organelles and plant metabolism compartmentation. But confident description of proteome organelle content requires clear identification of the true resident proteins of the studied compartment. This task involves pitfalls and requires that either organelle preparations are free of contaminants or that techniques are used to discriminate between genuine organelle residents and contaminating proteins. To achieve this, vacuoles purification protocol from protoplasts on a Ficoll density gradient has been improved. The combination of several proteomic approaches attempt to present soluble and membrane vacuolar proteins in a quantitative and functional manner. Different approaches have thus shown associations and complex molecular mechanisms that govern the various vacuolar activities. The constitute proteins library provides references to study the vacuolar proteome dynamics in response to different stresses including heavy metals. Many methods without a priori or targeted were proposed to study the impact of cadmium on the vacuole, the key cell compartment of detoxification. Proteomics provides powerful tools for characterizing the protein contents of vacuoles during cadmium stress.