Netrins are a small family of secreted proteins that guide growing axons during neural development by binding to the receptor DCC (Deleted in colorectal cancer). Our lab and others have previously shown that the activity of the Rho family GTPases Rac1 and Cdc42 are essential for DCC- mediated neurite outgrowth. Rac1 and Cdc42 act as molecular switches, mediating cytoskeleton remodelling when they are active and bound to GTP. Rac1 and Cdc42 are regulated positively by GEFs (guanine exchange factors) and negatively by GAPs (GTPase-activating proteins). Since DCC does not interact directly with Rac1, there should be an indirect link between DCC and Rac1. Trio is a GEF that activates Rac1 and RhoA. The orthologs of Trio in C.elegans (unc-73) and in D. melanogaster have been shown to play important roles in axon guidance, suggesting that mammalian Trio may link DCC to Rac1 activation. Here, we investigated how netrin-1 and its respective guidance receptor DCC are linked to Rac1 through studying the role of Trio in this signaling pathway. We found that Trio, Nck1, PAK1, and DCC are present in the same signaling complex, and that netrin-1-induced Rac1 activation is impaired in the absence of Trio. Trio -/- cortical neurons fail to extend neurites in response to netrin-1, while they are able to respond to glutamate. Accordingly, netrin-1-induced commissural axon outgrowth is severely impaired in Trio -/- spinal cord explants and commissural axon projections are defective in Trio -/- embryos. In addition to defects in spinal cord development, the anterior commissure is absent in Trio-null embryos, and netrin-1/DCC-dependent axonal projections that form the internal capsule and the corpus callosum are also defective in Trio -/- embryos. Thus, Trio through its ability to activate Rac1 mediates netrin-1 signaling in axon growth and guidance. / Le facteur de guidage chémotropique nétrine-1 favorise la croissance axonale à travers son récepteur DCC (Deleted in Colorectal Cancer) via l'activation de Rac1. Cependant, le facteur d'échange nucléotidique (GEF) qui lie nétrin-1/DCC à Rac1 n'a pas encore été identifié. Nous démontrons que Trio est la protéine GEF impliquée dans ce phénomène. Nous avons trouvé que Trio, Nck1, PAK1, et DCC sont présents dans le même complexe de signalisation et que l'activation de Rac1 induite par nétrine-1 est inhibée en absence de Trio. Les neurones corticaux Trio-/- échouent dans l'extension de neurites en réponse à la nétrine-1 alors qu'ils répondent à la stimulation par le glutamate. Par conséquent, l'induction de la croissance des axones commissuraux est sérieusement entravée dans les explants de moelle épinière des embryons Trio -/-. En plus du défaut dans le développement de la moelle épinière, les commissures antérieures sont absentes dans les embryons Trio-/-, et les projections axonales, qui forment la capsule interne et le corpus callosum, sont aussi affectées dans les embryons Trio -/-. Donc, par sa capacité d'activer Rac1, Trio favorise la signalisation par la nétrine-1 dans la croissance et le guidage axonale.

Telomeres are nucleoprotein structures that protect the ends of chromosomes from recombination and fusion. Telomeres are prefentially maintained by the enzyme telomerase. Telomerase is a reverse transcriptase, minimally composed of two core components, a catalytic subunit, TElomerase Reverse Transcriptase (TERT) and an RNA subunit, Telomerase RNA (TR) that carries the template for the replenishment of the telomeres. Telomerase is present throughout evolution, from ciliates and yeasts to vertebrates. Likely because it performs reverse transcription to maintain telomeres in these organisms, telomerases are remarkably conserved in terms of structure as well as in term of function. Nevertheless, some species-specific differences exist between telomerases from different model organisms. Studying these differences can deepen our knowledge of the telomerase enzyme, critical for human diseases such as cancer. The conservation and distinction between telomerases was explored by studying the functional conservation of a pseudoknot structure between Tetrahymena and human TRs and the response of mouse and human cells to the mutations of the subunits of telomerase. The hypothesis that the pseudoknot domains of the Tetrahymena and human TRs (tTR and hTR, respectively) may be functionally interchangeable was tested by constructing a chimeric TR (htTR) where the hTR pseudoknot domain was exchanged for tTR pseudoknot domain. htTR exhibited a weak, non processive telomerase activity in vitro, and was defective in telomere elongation, demonstrating that the Tetrahymena and human pseudoknots are not fully functionally interchangeable. Then, the importance of telomerase for cellular proliferation in mouse compared to human cells was investigated by studying the cellular consequences of mutating each core subunit of the mouse telomerase. First, the TERT-DN, a dominant-negative catalytically-inactive mutant of mTERT, was stably introduced into a mouse cell line (CB17) whose telomere / Les télomères sont des structures nucléoprotéiques qui protègent les extrémités des chromosomes contre la recombinaison et la fusion. Les télomères sont maintenus de manière préférentielle par l'enzyme télomérase. La télomérase est une transcriptase inverse, composée minimalement de deux constituants essentiels, la sous-unité catalytique TElomerase Reverse Transcriptase (TERT) et la sous-unité ARN, Telomerase RNA (TR) qui comprend la matrice pour la génération des télomères. La télomérase est présente à travers l'évolution, des ciliés au vertébrés en passant par les levures. Vraisemblablement parce que l'enzyme utilise un mécanisme de transcription inverse pour maintenir les télomères dans ces organismes, l'enzyme est remarquablement conservée, tant en terme de structure qu'en terme de fonction. Néanmoins, quelques différences propres aux espèces existent entre les télomérases d'organismes modèles. Étudier ces différences peut approfondir notre connaissance de la télomérase, une enzyme cruciale pour des maladies humaines tel que le cancer. La conservation et la distinction entre les télomérases furent explorées en étudiant la conservation fonctionnelle d'une structure de pseudonœud entre les sous-unités TRs de Tétrahyména et de l'humain, et en étudiant la réponse des cellules murines et humaines à des mutations dans les deux sous-unités essentielles de la télomérase. L'hypothèse que les domaines pseudonoeuds de l'ARN de la télomérase de Tétrahyména et de l'humain (tTR et hTR, respectivement) soient fonctionnellement interchangeables a été testée en construisant un TR chimérique (htTR) où le domaine pseudonoeud de hTR a été échangé avec le domaine pseudonoeud de tTR. htTR a démontré une faible activité non processive in vitro et était défectif en terme d'élongation des télomères, démontrant que les pseudonoeuds de Tétrahyména et de l'humain ne sont pas complètement fonctionnel

Gap junctions are membrane channels between closely apposed cells that allow for intercellular exchange of small molecules. These channels are formed from a family homologous proteins known as connexins (Cx). In the present study we studied connexin trafficking within the cell with particular emphasis on 1/ the mechanisms of gap junction formation and removal and on 2/ the early events associated with connexin biosynthesis. The mechanisms of gap junction formation and removal were studied by treating Normal Rat Kidney and BICR-MlR$ sb{ rm k}$ rat mammary tumour cells with ilimaquinone (IQ), a novel and reversible inhibitor of protein secretion. The effects of IQ treatment on Cx43 maturation and gap junction assembly were then examined. The events associated with biosynthesis were examined through localization of Cx32 messenger RNA to either free or membrane-bound polyribosomes in rat liver hepatocytes through Northern blot analysis. In these studies we observed that IQ inhibited gap junction formation without inhibiting trafficking of Cx43 to the cell surface. Upon removal of the drug, gap junction plaques reformed. We concluded that additional factors other than phosphorylation are necessary for Cx43 assembly into gap junctions and this process is independent of microtubules. Preliminary studies revealed Cx32 mRNA in populations of free and membrane-bound polyribosomes suggesting that Cx32 is inserted into the endoplasmic reticulum membrane both post- and co-translationally.

The present study examines the endocytosis of testicular sulfated glycoprotein-1 (SGP-1) by the nonciliated cells of the efferent ducts. SGP-1 is a 70 KDa protein secreted by the Sertoli cells. Once secreted in the seminiferous lumen, the protein binds to the tail of spermatozoa. In the efferent ducts, it is endocytosed by the nonciliated cells, presumably via a receptor-mediated process. Because the initial steps of receptor-mediated endocytosis result from the binding of a ligand's terminal oligosaccharide to a receptor on the ceil surface, several monosaccharides were injected into the lumen of the rete testis to study their effect on the endocytosis of SGP-1 in the efferent duct. The labeling density of various endocytic compartments was estimated and compared in untreated and treated animals with various sugars. The following sugars were tested: glucose, galactose, mannose, mannose 6-phosphate, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid. The findings suggest that, via its sialic acid binding domain, SGP-1 may bind to glycolipids on the tail of spermatozoa, remove them from the membrane forming a lipo-protein complex. The complex may then be endocytosed by the nonciliated cells, via a receptor that would recognize SGP-1's terminal sialic acid residues, and be delivered to the lysosomes to be degraded. (Abstract shortened by UMI.)

Regulators of G-protein Signaling (RGSs) are negative regulators of G-protein Coupled Receptor (GPCR) mediated signaling that function to limit the lifetime of receptor-activated Galpha proteins. Heterologously expressed mammalian RGSs can functionally complement a yeast mutant lacking its RGS containing gene SST2. Here we show that four mammalian RGSs differentially inhibit the activation of a FUS1-LacZ reporter gene by the STE2 encoded GPCR in yeast with the apparent rank order potency: RGS1 > RGS16 > RGS2 > RGS5. In order to examine the role of the GPCR in modulating RGS function, we functionally expressed the human somatostatin receptor 5 (SSTR5) in yeast. / The ability of RGSs to inhibit SSTR5 signaling was further assessed in cells expressing modified Gpa1 proteins. / Yeast have also been shown to be a useful model organism for the study of the localization of mammalian RGS proteins. We have constructed a series of vectors that allow us to express proteins fused to a Green Fluorescent Protein (GFP). (Abstract shortened by UMI.)

Natriuretic peptides form a family of structurally-related peptides known to regulate salt and water homeostasis and to cause vasodilation. Synthesis of atrial (ANP), brain (BNP), and C-type (CNP) natriuretic peptides occurs mainly in the heart and brain and has been identified recently in the female reproductive tract. The expression of ANP and CNP, as well as their cognate guanylyl cyclase receptors (NPR-A and NPR-B, respectively), have been detected in the rat ovary. / We have shown previously that the expression of the natriuretic peptides and their receptors, in the rat ovary, is modulated by the estrous cycle. Since estrogen and the gonadotropins (luteinizing hormone (LH) and follicle-stimulating hormone (FSH)) are important regulators of follicular development and ovarian function we hypothesized that expression of the natriuretic peptide system is modulated by these hormones. In order to test this hypothesis, the expression of the natriuretic peptide system (peptide and receptor) was evaluated in ovarian cells (granulosa and residual tissue cells) obtained from immature female rats treated with either diethylstilbestrol (DES), an estrogen analogue, or equine chorionic gonadotropin (eCG), a gonadotropin which possesses both LH and FSH activity. (Abstract shortened by UMI.)

Regulators of G protein signaling (RGS) are proteins that negatively regulate G protein-coupled receptor signaling. Although a conserved core domain is necessary and sufficient for their GTPase accelerating protein (GAP) activity, many RGSs possess C- and N-terminal protein-binding motifs that augment GAP activity and participate in other cellular regulatory mechanisms. / Human RGS 1 functionally complements a Saccharomyces cerevisiae mutant lacking the RGS homologue Sst2p. We demonstrate that deletion of the N-terminus or RGS domain negatively affects this ability in Sst2p-deficient strains, whereas deletion of the C-terminal 10 residues of RGS 1 does not. Coexpression of the N-terminus and RGS domains restores complementation of Sst2p to that of wild type. The conservative replacement of sequential residues spanning the N-terminus of RGS1 causes little loss of function. These results suggest that the N-terminal and RGS domains of RGS I function in concert to effect signaling and that the C-terminal 10 residues of RGS I are not required for this activity. Further, residues present in the N-terminus are not highly conserved suggesting that overall structure, rather than individual residues or motifs, may be important for function.

The eIF2α kinases PERK and PKR are well characterized regulators of protein synthesis. When they are activated in response to various cellular stresses, they inhibit translation initiation by phosphorylating eIF2α on a key residue. Recently, PKR has been shown to regulate the expression of specific proteins independent of its ability to inhibit protein synthesis, and we wished to identify additional targets of this particular function. The cellular levels of cyclin D1, a cell cycle control protein that regulates progression through G1, decrease under conditions that activate various eIF2α kinases. We demonstrate that this is not due to the inhibition of cyclin D1 synthesis, but rather the induction of cyclin D1 degradation by the 26S proteasome. This degradation is ubiquitin-dependent, and although cyclin D1 synthesis is unaffected eIF2α phosphorylation is required. Our work provides evidence of a functional cross-talk between the eIF2α kinases and the degradation of specific proteins during periods of acute stress. PKR has also been shown to regulate the function of the transcription factors Stat1 and Stat3, which mediate the cellular response to a wide variety of extracellular signals. In order for these proteins to accumulate in the nucleus and drive gene transcription, they must first be phosphorylated on a conserved tyrosine residue. The transcriptional activity of Stat proteins is tightly regulated, and therefore the phosphorylation and subsequent dephosphorylation events are critically important. Herein we demonstrate that the tyrosine phosphorylation of Stat1 and Stat3 is compromised by the catalytic activity of PKR, and that this proceeds through the nuclear phosphatase TC-PTP. This decrease in tyrosine phosphorylation abrogates the transcriptional activity of Stat1 and Stat3 normally observed in response to IFN-γ and IL-6 stimulation, respectively. Phosphorylation of both TC-PTP and eIF2α by PKR is required for the inhibition of Stat1 / Les kinases PERK et PKR sont des régulateurs reconnus de la synthèse de protéines. En réponse à différents stress cellulaires, elles empêchent l’initiation de la traduction en phosphorylant un résidu-clé du module alpha de la protéine eIF2. Récemment, PKR a été montrée de régler l'expression des protéines spécifiques independent de sa capacité d'empêcher la synthèse de protéine, et nous avons souhaité identifier les cibles additionnelles de cette fonction particulière. Les niveaux cellulaires de cycline D1, une protéine de contrôle qui dirige la progression du cycle cellulaire à travers G1, diminuent lorsque PERK et PKR sont activés. Nous démontrons que ceci ne découle pas de l’inhibition de la synthèse de cycline D1, mais plutôt de l’induction de sa dégradation par le protéasome 26S. Cette dégradation dépend de l’ubiquitine et requiert la phosphorylation d’eIF2 au module alpha, bien que la synthèse de cycline D1 ne soit pas affectée. Nos travaux fournissent des preuves concluantes d’un échange fonctionnel entre les kinases eIF2α et la dégradation de protéines exprimées durant les périodes de stress aiguë.Il a été démontré que PKR régule aussi la fonction des facteurs de transcription Stat1 et Stat3, qui aiguillonnent la réponse cellulaire à une grande variété de signaux extracellulaires. La phosphorylation de ces protéines sur un résidu tyrosine mène à leur exportation et à leur accumulation dans le noyau cellulaire, d’où elles stimulent la transcription génique. L’activité transcriptionnelle des protéines Stat est strictement contrôlée et chaque événement de phosphorylation ou de déphosphorylation revêt une importance cruciale. Nous révélons que la phosphorylation d’un résidu tyrosine de Stat1 et Stat3 est compromise par l’activité catalytique de PKR et que la phosphatase nucléaire TC-PTP en est la responsable. Cette diminution des niveaux de phosphorylation du rési

The pathway of membrane insertion and assembly of a signal anchor sequence specific for the outer mitochondrial membrane has been investigated. Signal anchor protein insertion into the outer membrane, in vitro, was found to overlap with a general import pathway followed by the outer membrane protein, porin, as well as matrix-targeted proteins. However, signal anchor protein insertion did not require a postreceptor import step involved in porin insertion and matrix protein translocation. Also, in contrast to the membrane insertion of porin, signal anchor protein insertion did not require nucleoside triphosphates for transfer of bound precursor to the membrane assembled form. Following outer membrane integration, a hybrid protein containing the signal anchor sequence of yeast Tom70 was found to assemble into homodimers. Dimerization was mediated by the transmembrane domain. A sequence motif containing alanine residues clustered on one face of the predicted membrane-spanning $ alpha$-helix was important for dimerization, perhaps allowing favourable close packing of the transmembrane helices.

The neuron-specific K+/Cl- cotransporter 2 (KCC2) is one of the major cation Cl- cotransporter (CCC) proteins in the central nervous system that controls Cl- homeostasis via mediating Cl- extrusion. KCC2 has been established as an essential protein in promoting the maturation of synaptic inhibition during development and controlling neuronal excitibility in the adult central nervous system (CNS). Although various mechanisms are known to regulate KCC2 gene expression, accumulating evidence has shown KCC2 activity can be altered on much shorter time scales in a manner of 10-20 minutes. This suggested post-translational mechanisms, which take place much more rapidly than gene expression, may also be an important means by which KCC2 function can be regulated. However, what these post-translational mechanisms are and how they contribute to the control of KCC2 function remain elusive. In this thesis, I set out to characterize the molecular mechanisms governing the post-translational regulation of KCC2, including its endocytosis, quaternary assembly, and cell surface expression. Specifically, my results suggest: 1) Endogenous KCC2 interacts with the clathrin-mediated endocytosis (CME) machinery; 2) KCC2 is constitutively endocytosed via a CME-dependent mechanism; 3) A di-leucine motif, 657LL658, is essential for both KCC2 constitutive endocytosis and the binding of KCC2 to the CME adaptor protein AP-2 complex; 4) Two regions within KCC2 proximal and central carboxyl terminus respectively mediate KCC2 dimerization, and in particular mutating the 657LL658 residues to alanines completely abolishes KCC2 dimerization; 5) The 657LL658 sequence is highly conserved amongst closely related K+/Cl- cotransporter proteins, but absent from the more distant Na+/Cl- cotransporters controlling Cl- uptake, suggesting an evolutionarily conserved mechanism may regulate the constitutive endocytosis and dimerization of functionally homologous KCC members; and 6) KCC2 expression at the neur / Le co-transporteur d’ions K+ et Cl- (KCC2), spécifique aux neurones, est l’un des plus importants co-transporteurs de cations chlorure (CCC) du système nerveux central. Il contrôle l’homéostasie de la cellule en régulant l’extrusion d’ions chlorure. KCC2 est reconnu comme étant une protéine essentielle à la maturation de l’inhibition synaptique au cours du développement et au contrôle de l’excitabilité des neurones dans le système nerveux central de l’adulte. Bien que les mécanismes régulant l’expression de KCC2 soient connus, de nouvelles données suggèrent que l’activité de KCC2 peut aussi être modifiée sur de plus courtes périodes, de l’ordre de 10 à 20 minutes. Ceci suggère qu’un contrôle post-traductionnel, agissant beaucoup plus rapidement qu’un contrôle transcriptionnel, pourrait aussi être important dans la régulation de l’activité de KCC2. Toutefois, la nature de ces mécanismes post-traductionnels et la façon par laquelle ils contribuent à la fonction de KCC2 demeurent inconnus.Dans cette thèse, je vise à caractériser les mécanismes moléculaires qui gouvernent la régulation post-traductionnelle de KCC2, incluant son expression à la surface cellulaire, son endocytose et son assemblage quaternaire. Plus spécifiquement, mes résultats suggèrent que: 1) Le KCC2 endogène interagit avec la machinerie d’endocytose par clathrines (CME) ; 2) Le KCC2 est endocyté constitutivement par un mécanisme dépendant des clathrines; 3) Le motif di-leucine 657LL658 est essentiel non seulement à l’endocytose constitutive de KCC2, mais aussi à l’interaction entre KCC2 et le complexe protéique adaptateur AP-2; 4) Deux régions à l’intérieur de KCC2, l’une proximale et l’autre centrale à l’extrémité C-terminale, sont responsables de la dimérisation de KCC2 qui peut être abrogée par la substitution des leucine 657 et 658 par des alanines; 5) La séquence 657LL658 est très conservée par