Caco-2 cells were incubated with 2.5 ug/ml Brefeldin A for up to 2 hours prior to fixation. The 2 hour incubation was sometimes followed by 1 or 2 hours of chase. In the Golgi apparatus of untreated cells, CMPase enzyme activity was localized to the TGN, TPPase activity to trans saccules and NADPase activity to middle saccules. In cells treated for 5 minutes, tubulovesicular clusters (tvc's), normally seen at the cis face of Golgi stacks in control cells, expanded greatly in size. By 15 minutes, the Golgi apparatus disappeared and was replaced by numerous tubular structures some of which, TPPase and NADPase-positive, appeared to derive from the tubules of the intersaccular areas of the stacks, while others, CMPase-positive, may be derived from TGN elements. The tubules and the tvc's persisted for up to 2 hours and did not connect with the rER. Apart from occasional light reaction for TPPase activity, rER cisternae were never labeled. After a one hour chase interval, the Golgi complexes began to reform and were often associated with tvc's on their cis face. By 2 hours, the Golgi complexes were often completely reformed and exhibited normal localization of phosphatase activities.

Cubilin is a peripheral membrane protein that co-operates with the endocytic receptor megalin to mediate the endocytosis of ligands in various polarized epithelia. Megalin is expressed in the male reproductive tract where is has been implicated in the process of sperm membrane remodeling. A potential role for cubilin in the male reproductive tract has not been explored. Using RT-PCR, we found that cubilin and megalin mRNAs are expressed in the efferent ducts, corpus and cauda epididymides, and proximal and distal vas deferens. Immunohistological analysis revealed that cubilin was expressed in non-ciliated cells of the efferent ducts, principal cells of the corpus and cauda epididymides and vas deferens. Electron microscopic immunogold labeling showed cubilin in endocytic pits, endocytic vesicles and endosomes of these cells. The expression profile of cubilin in the male reproductive tract was coincident with that of megalin except in principal cells of the caput epididymidis. Double immunogold labeling showed that cubilin and megalin co-localized with one another within the endocytic apparatus and recycling vesicles of efferent duct cells. Neither protein was found in lysosomes. Injection of RAP, an antagonist of megalin interaction with cubilin, reduced the level of intracellular cubilin in cells of the efferent ducts and vas deferens. In conclusion, cubilin and megalin are co-expressed in cells of the epididymis and vas deferens and the endocytosis of cubilin in these tissues is dependent on megalin. Together, these findings highlight the potential for a joint endocytic role for cubilin and megalin in the male reproductive tract.

The Na,K-ATPase, or sodium pump, is a membrane-associated protein complex comprising two subunits, alpha and beta, both of which can exist as one of several isoforms. It generates and maintains the electrochemical Na + and K+ gradients across the plasma membrane of animal cells. These gradients are the driving force for a variety of ubiquitous and specialized cell functions, such as transport of solutes, as well as maintenance of membrane potential and cell volume. Agents that modulate the kinetic behaviour of the pump enable cells to adapt to changing needs. Distinct substrate activation profiles in various tissues presumably underlie the specialized functions of the sodium pump in these tissues. Although the tissue-specific distribution of various isoforms accounts for some of the differences in kinetic behaviour of the enzyme, other factors are also important determinants of such behaviour. This study describes the characterization of two mechanisms of sodium pump modulation. The first involves alterations in the susceptibility of the enzyme to competitive inhibition by K+ at Na+ binding sites. Studies on the alpha1 and alpha3 isoform of various tissues and cells have revealed that there exist tissue-specific components that determine the extent to which the two cations compete with each other for cytoplasmic binding sites. Specifically, pumps that are highly susceptible to K +/Na+ antagonism also bind and occlude K+ much more readily on the cytoplasmic site, and this behaviour is abrogated upon fusion of pumps into another membrane environment, that of the red blood cell. The second mechanism of regulation of pump behaviour described in this thesis involves modulation of the apparent affinity of the enzyme for ATP by the gamma subunit. This membrane protein had been previously cloned and sequenced, but very little was known about its function. This study shows that the gamma subunit is expressed uniquely in kidney tubules, and has a C-terminus-in, N-terminus-

This thesis describes the use of a proteomics resource to define the spatial organization of the endoplasmic reticulum (ER) and to predict the function of poorly characterized proteins. By proteomics, resident proteins involved in translation and translocation (eIFs, Sec61, TRAP, and the OST complex) are enriched in rough ER. Resident chaperones and folding enzymes (PDIs, calnexin, and BiP) are in equal abundance in rough and smooth ER. The ER-associated degradation (ERAD) machinery (derlin-1, VCP/p97, sel-1L, ubiquitin conjugating enzymes, and the proteasome), and also cytosolic chaperones (Hsp90, TRiC) all colocalize with their highest enrichment in smooth ER. This suggests a model that the ER is segregated into functional subdomains, which is useful to predict functional features of poorly characterized proteins localized in the proteomics resource. Nudilin is a cytosolic-located (P body) protein affecting translation which colocalizes with translation constituents at the cytosolic face of the ER membrane. Nicalin clusters with translocon constituents and is found to associate with signal peptide peptidase as well as negatively regulate the model secretory protein tyrosinase at the translocon. Stexin also clusters with translocon constituents and associates transiently with an early N-glycosylated form of tyrosinase. Erlin-1 and TMX2 co-distribute with chaperones found throughout the rough and smooth ER and are found to associate transiently with later N-glycosylated forms of tyrosinase. The ER is spatially organized to coordinate the temporal processing of cargo. Spatial localization in the ER predicts the function of resident uncharacterized proteins. / Cette thèse décrit l’usage d’une ressource protéomique afin de définir l’organisation spatiale du réticulum endoplasmique (RE) et de prévoir la fonction de protéines peu charactérisées à ce jour. Par la protéomique, des protéines résidentes impliquées dans la translocation et la traduction (eIFs, Sec61, TRAP, et le complexe OST) sont enrichies dans le RE granuleux. Des chaperones résidentes et des enzymes de pliage (PDIs, calnexin et BiP) sont d’abondance égale dans le RE granuleux et le RE lisse. Le système de dégradation associé au RE (ERAD) (derlin-1, VCP/p97, sel-1L, enzymes de conjugaison de l’ubiquitin, protéasome) ainsi que les chaperones cytosoliques (Hsp90, TriC) sont tous colocalisés dans le RE lisse, leur enrichissement y étant le plus important. Ceci suggère que le RE est ségrégué en sous-domaines fonctionnels, ce qui est utile pour prédire les charactéristiques fonctionelles de protéines peu décrites, localisées dans la ressource protéomique. Nudilin est une protéine cytosolique (P body) affectant la traduction qui est colocalisée avec des constituants de la traduction à la surface cytosolique de la membrane du RE. Nicalin se regroupe avec des constituants du translocon et s’associe avec le signal peptide peptidase, en plus de réguler négativement la protéine de sécrétion tyrosinase au niveau du translocon. Stexin se regroupe aussi avec des constituants du translocon et s’associe de manière transitoire avec une forme N-glycosylée de tyrosinase trouvée immédiatement après sa synthèse. Erlin-1 et TMX2 se co-distribuent avec des chaperones trouvées à travers le RE lisse et granuleux et s’associent de manière transitoire avec des configurations N-glycosylée plus tardives de tyrosinase. Le RE est organisé dans l’espace afin de coordoner le traitement temporel de sa cargaison. La localisation spatialle des protéines résidentes non-charactérisées dans le RE prédit leur fonction.

Progranulin is a secreted high molecular weight growth factor. While inappropriate over-expression of the progranulin gene has been associated with many cancers, haploinsufficiency leads to atrophy of the frontotemporal lobes and development of a form of dementia (FTLD-U). We have demonstrated that progranulin is abundantly expressed in mouse motor neurons in vivo and primary cultures in vitro. The cellular location of progranulin was examined using confocal microscopy. Progranulin was localised to organelles using immunocytochemistry and by examining the intracellular fate of exogenously expressed flu orescently-labelled progranulin. Taken together these studies revealed progranulin to be located within compartments of the secretory pathway. Stable transfection of the human progranulin gene into the NSC-34 cell line stimulates the appearance of dendritic structures and provides protection against apoptosis. Control cells, while expressing basal levels of progranulin do not survive in serum free conditions. This work highlights the importance of progranulin as a neuroprotective growth factor and may represent a therapeutic target for neurodegenerative diseases including FTLD-U and ALS. / La progranuline est un facteur de croissance sécrété de haut poids moléculaire. Tandis qu' une sur-expression inappropriée du gène codant la progranuline est observé dans de nombreux cancers de différentes origines, l'haploinsuffisance, quant à elle, est responsable de l'atrophie des lobes fronto-temporaux et du développement d'une forme de démence (FTLD-U). Nous avons démontré que la progranuline est exprimée abondamment dans les neurones moteurs murins in vivo et in vitro dans un modèle de culture primaire. La localisation subcellulaire de la progranuline a été examinée en microscopie confocale par immunofluorescence indirecte et confirmé par l'étude de la distribution subcellulaire d'une progranuline marquée a la eGFP. La progranuline se distribue ainsi le long des voies de secrétions. La surexpression de gène humain de la progranuline dans les cellules NSC34 stimule l'apparition de structures dendritiques et protège contre la mort cellulaire induite par la carence en sérum du milieu tandis que les cellules ne surexprimant pas la progranuline demeurent sensibles à ce stimulus. En conclusion, ce travail souligne l'importance de la progranuline comme facteur de croissance neuroprotecteur et peut représenter une cible thérapeutique de choix pour les maladies neurodégénératives.

Normal development of the placental labyrinth layer is required for proper nutrient and gas exchange between the mother and the fetus. Labyrinth layer development begins when the allantois attaches to the chorion in an event called chorioallantoic attachment. As a result of chorioallntoic attachment, the chorion and ectoplacental cone differentiate into the trophoblast cells of the placenta, and the allantois forms the embryonic vasculature of the placenta. The objectives of this thesis are to: generate an explant model that will mimic early labyrinth development, and to analyze the expression of Notch1/NICD1 during in vitro trophoblast stem cell differentiation. In Vitro culture of pre-placental tissues resulted in successful fusion between the two separate tissues, generating an explant model for chorioallantoic attachment. Notch1 was detected at 0 to 4 days of trophoblast stem cell differentiation. NICD1 localized to the cytoplasm and nucleolus of all trophoblast cell types during 0 to 6 days of differentiation. The major findings in this thesis are that chorions and allantoises cultured in vitro undergo chorioallantoic attachment, and that Notch1/NICD1 is expressed throughout trophoblast differentiation. / Le dévelopement normal de la structure labyrinthique du placenta est requis pour l'échange des gaz et des nutriments entre la mère et le fœtus. Le dévelopement du labyrinthe débute lorsque l'allantois s'attache au chorion durant l'attachmement chorio-allantoïdien. Cet attachement mène à la differentiation du chorion et de la lame ectoplacentaire en cellules trophoblastiques placentaires, alors que l'allantois formera le système vasculaire du placenta. Les objectifs de cette thèse sont : la generation d'un modèle ex vivo imitant le dévelopement primaire du labyrinthe et de l'analyse de l'expression de Notch1/NICD1 durant la différentiation des trophoblastes in vitro. Tous générant un modèle ex vivo pour l'attachement chorio-allantoïdien. Ensuite, Notch1 fut détecté entre les jours 0 et 4 de la différentiation des cellules souches trophoblastiques. NICD fut localisé dans le cytoplasme et le nucleolus de tous types de cellules trophoblastiques, durant les jours 0 à 6 de la différentiation. Cette these démontre doneques les chorions et les allantois cultivés in vitro mèneront à l'attachement chorio- allantoïdien et que Notch1/NICD1 est exprimé durant l'ensemble de la differentiation des trophoblastes.

Transglutaminases are enzymes that stabilize extracellular matrices by catalyzing the formation of protease-resistant isopeptide crosslinks between and within their substrate proteins. Two transglutaminase isoforms, tissue transglutaminase (tTG) and factor XIIIa (FXIIIa), are expressed in skeletal tissues. Tissue transglutaminase has been previously localized in bone to osteoblasts, the osteoid layer and the pericellular matrix of osteocytes. Factor XIIIa has been localized to mineralizing chondrocytes, however, its expression in osteoblasts has not been reported yet. In order to understand the role of transglutaminases in hard tissue formation, we have investigated tTG and FXIIIa expression and activity during osteoblast differentiation and mineralization using the well-characterized MC3T3-E1 preosteoblast cell line. We show that tTG and FXIIIa are expressed at all time points examined in the cell cultures, and that their expression and activity does not appear to be significantly changed by the differentiation or mineralization state of the cells. We show that tTG localizes homogeneously throughout the cells and the extracellular matrix, whereas FXIIIa distribution is polarized to one side of the cells at earlier time points, but becomes incorporated into the matrix produced by differentiating cells in mature cultures. These expression profiles suggest a dual role, initially in cell adhesion and attachment and, later, in protein assembly, where the transglutaminases likely stabilize the matrix and assist in the formation of an extracellular matrix competent for subsequent mineralization. The identification and characterization of two transglutaminase isoforms in osteoblasts suggest a potentially overlapping roles for these two enzymes in the MC3T3-E1 cell culture system.

Human telomerase is a ribonucleoprotein complex, minimally composed of a catalytic subunit, hTERT and an RNA component, hTR that contains the template used to synthesize telomere DNA. Telomerase is present in over 80% of all cancer cells. Therefore, an understanding of telomerase regulation is important for cancer research. This thesis work was focused upon the study of telomerase regulation by telomerase associated proteins and alternatively spliced hTERT mRNA variants. Tandem affinity purification (TAP) and immunopurifications with anti-hTERT antibody were used to visualize potential telomerase associated proteins. No specific proteins were identified in TAP-hTERT transfected cells. hTERT was purified from 293 telomerase positive cells, and proteins specifically associated with hTERT were visualized. Insertion 3 and 4 alternatively spliced hTERT mRNA variants were detected in telomerase positive cell lines by RT-PCR and Southern blot analysis. The gamma-deletion alternatively spliced hTERT mRNA variant was detected in Huh-7 liver cells. These results provide information and tools important for a greater understanding of telomerase.

Apoptosis is a programmed cell death process essential for development and homeostasis and its deregulation is involved in many diseases. The caspases are a family of cysteine proteases present as inactive pro-enzymes in all cell types and activated following apoptotic stimulation. These proteases cleave a particular subset of protein substrates which results in the organized break down of the cells which shrink and are cleared through phagocytosis. In this thesis, many apoptotic processes are discussed. First, the CEACAM1-L cell-cell adhesion molecule is identified as a caspase-3 substrate during apoptosis. This cleavage event alters CEACAM1-L adhesion properties which might affect apoptotic cell clearance. Second, the brain development defect phenotype of Caspase-3-null mice is investigated. The mouse strain specificity of that mutation allowed the identification of caspase-7 as a compensating enzyme for the loss of caspase3 in precursor neurons. Finally, new functions are elucidated for Hippi, a pro-apoptotic molecule involved in the neurodegenerative Huntington's disease. Hippi-targeted deletion revealed its role in node cilia assembly and in Sonic hedgehog signaling during early mouse embryo development. Altogether, these findings provide new insights into several aspects of the apoptosis program.

The majority of glycosylphosphatidylinositol (GPI)-anchored proteins enter mammalian cells via the clathrin-, and dynamin-independent GPI-enriched early endosomal compartment/ Clathrin-independent carrier endocytic (GEEC/ CLIC) pathway. Using artificially lipid-anchored proteins, we have examined how the ‘anchor' structure of these proteins affects their endocytic trafficking after initial internalization from the plasma membrane. In the first part of this thesis, I show that soluble proteins, anchored to cell-inserted saturated and unsaturated phosphatidylethanolamine (PE)-polyethylene glycol (PEGs), distribute within the cell identical to the GPI-anchored folate receptor α, all of which colocalize significantly with markers of the ERC, but not with markers of the late endosomes/lysosomes. Soluble proteins, tightly bound to saturated PE-PEG anchors in CHO cells recycled back to the plasma membrane with a half life (t1/2) of 25-30 minutes, similarly to the folate receptor α. By contrast, proteins bound to unsaturated PE-PEG anchors recycled back to the plasma membrane with a t1/2 of 7-9 minutes, similarly to the transferrin receptor (TfR). These results support a potential role for membrane rafts in differential endocytic recycling of GPI-anchored proteins from transmembrane proteins (such as TfR) in these cells. Earlier reports indicated that GPI-anchored proteins are targeted to late endosomes in BHK cells, in contrast to CHO cells, and suggested that this phenomenon is due to differential association with membrane rafts in each cell line (Fivaz et al., 2002; Sabharanjak et al., 2002; Kalia et al., 2006). In the second part of this thesis, I studied the possible role of membrane rafts in this differential endocytic sorting using our artificially lipid-anchored proteins in BHK cells. Our results showed that, in BHK cells, endocytosed proteins artificially tethered to either saturated or unsaturated PE-PEG ‘anchors' are again distributed within the cell esse / La majorité des protéines liées au glycosylphosphatidylinositol (GPI) entrent dans les cellules mammifères par le biais du compartiment de l'endosome précoce par l'intermédiaire d'une voie indépendante des clathrines et dynamines enrichie en GPI et du transporteur endocytosomique indépendant des clathrines (GEEC/ CLIC). En utilisant des protéines attachées artificiellement à des lipides, nous avons étudié la manière avec laquelle ces « ancres lipidiques » influencent le trafic endocytosomique des protéines suite à l'internalisation initiale à partir de la membrane plasmique. Dans la première partie de cette thèse, je démontre que les protéines solubles, lorsque attachées à du phosphatidylethanolamine (PE)-polyethyleneglycol (PEGs) directement injecté dans les cellules de manière saturante ou non-saturante, suit une distribution intracellulaire identique à celle du récepteur du folate α lié au GPI, c'est-à-dire qu'ils colocalisent presque parfaitement avec des marqueurs de la compartiment de recyclage d'endocytose (CRE), mais non avec ceux de l'endosome tardif/lysosomes. Les protéines solubles liées solidement aux ancres de PE/PEG saturés dans les cellules CHO se recyclent vers la membrane plasmique avec une demi-vie (t1/2) de 25-30 minutes, comme c'est le cas pour le récepteur folate α. Par contre, la t1/2 des protéines liées au PE/PEG non-saturé n'est que de 7-9 minutes, tel qu'observé pour le récepteur de la transferrine (TfR). Ces résultats suggèrent un rôle potentiel pour les ‘radeaux' membranaires dans le recyclage différentiel des protéines liées au GPI à partir de protéines transmembranaires (comme TfR) dans ces cellules. Des publications antérieures ont indiqué que, contrairement à ce qui a été observé dans les cellules CHO, les protéines liées au GPI sont ciblées vers l'endosome tardif dans les cellules BHK et suggère donc que ce phénomène est dû à leur association différentielle aux « r