Vesicle (exosome) externalization is a mechanism for removal of obsolescent membrane proteins, such as the transferrin receptor (TFR), during reticulocyte maturation. This thesis involves further characterization of the exosomal phenomenon using avian (nucleated) and mammalian erythroid systems. Chicken reticulocytes release exosomes, indicating independence of this process from enucleation. Exosome release during chicken erythroleukemic cell (HD3) differentiation (representing pre-reticulocyte development) demonstrates that this process commences prior to the reticulocyte stage. / The heat shock protein, hsp70, has been found in exosomes from every species examined (four species, including the chicken). It is shown that hsp70 is physically, but non-covalently, associated with exosomal TFR, suggesting its possible involvement in targeting proteins into exosomes during their formation. / Exosome formation is an energy-dependent process. Our observations indicate that the primary energy substrate for avian red cells is not glucose, but glutamine, inosine and guanosine. Work with the HD3 cells (capable of glucose transport), suggests there is an early differentiation-associated switch in the chicken red cell's ability to use glucose as a major energy substrate.

Transforming growth factor-beta (TGF-beta) plays an important role in angiogenesis and wound healing. The major cell type involved in angiogenesis are microvascular endothelial cells. Endoglin, a transmembrane glycoprotein, is highly expressed on vascular endothelial cells and has been shown to bind TGF-beta1 and TGF-beta3 and inhibit TGF-beta-induced responses. Mutation in the endoglin gene has been implicated in hereditary haemorrhagic telangiectasia type 1 (HHT1), a dominantly inherited vascular disorder. The role of endoglin in regulating TGF-beta function in the microvasculature is not well understood. The initial interactions on the cell surface between endoglin and the TGF-beta receptors may be an important mechanism by which endoglin modulates TGF-beta signaling and responses. In this study, we show for the first time that on human microvascular endothelial cells, endoglin forms a heteromeric association with betaglycan, a TGF-beta receptor with which endoglin shares limited homology. This complex formation can occur in both a ligand-dependent and ligand-independent manner. In addition, we demonstrate the occurrence of three higher order complexes which contain endoglin and the TGF-beta type II and/or type I receptors in different numbers or ratios. Our results suggest that endoglin may modulate TGF-beta signal transduction by interacting with betaglycan and the TGF-beta signaling receptors on the cell surface in different ratios.

The cardinal feature of apoptosis is the activation of a family of proteases termed caspases, and in some forms, loss of mitochondrial integrity and release of cytochrome c into the cytoplasm are viewed as necessary components of cell death. In receptor-mediated apoptosis, more specifically Somatostatin (SST) receptor (SSTR)- and Fas-signaled apoptosis, an additional feature is intracellular acidification (pHi) and Protein Tyrosine Phosphatase (PTP) activity. Increased translocation and activity of PTP, more specifically SHP-1 at the cell membrane as well as the functional activation of caspase-8 are required to reduce pHi. Also, a decrease in mitochondria) membrane potential (Deltapsim) and release of cytochrome c (cyt c) are concomitant with a decrease in pHi suggesting that mitochondrial dysfunction is a consequence and not a cause of acidification. Inhibition of caspase-8 activation and prevention of SHP-1 translocation abrogates the ability of SST to induce acidification and mitochondrial disruption, whereas inhibition of other caspases such as caspase-9, or -3/7 are ineffective. Protection from cell death can also be accomplished by Bcl-2. In this model, Bcl-2 elevates resting pHi and attenuates acidification and apoptosis only when the overexpressed Bcl-2 is targeted to the endoplasmic reticulum (ER) but not the mitochondria. In conclusion, we established that mitochondrial breakdown follows acidification and that acidification is dependent on caspase-8 and SHP-1 activation. Additionally, effector caspases are induced only when there is concomitant acidification. Bcl-2 cannot prevent acidification-dependent apoptosis at the level of the mitochondria despite protecting it revealing that mitochondrial disruption is a potentiating, but not essential event in the apoptotic pathway.

Previous studies have demonstrated that receptor tyrosine kinases (RTKs) use common intracellular signal transduction pathways. This is remarkable due to the divergent cellular responses elicited from different RTKs. The insulin receptor (IR) is involved in blood glucose homeostasis whereas the epidermal growth factor receptor (EGFR) has been linked to liver regeneration. We therefore used rat liver, an organ enriched in both EGF and insulin receptors, to study the specificity of signal transduction in vivo. / Upon EGF administration, the EGFR at the plasma membrane (PM) was tyrosine phosphorylated on the major in vivo activation site, Y 1173, and recruited the adaptor proteins SHC and GRB2. Following ligand-mediated endocytosis, the activated EGFR was associated on endosomal membranes with the signaling complex SHC/GRB2 and the guanine nucleotide exchange factor, SOS. EGF administration also led to tyrosine phosphorylation of the cytosolic proteins focal adhesion kinase (FAK) and SHC. These observations were associated with increased MAP kinase activity and the transcription of c-myc, c-fos and c-jun. / In response to insulin, IR kinase activity and autophosphorylation was observed at the PM but not after IR internalization into endosomes. This is postulated to be due to rapid degradation of insulin in the endosomal lumen allowing for the dephosphorylation of the IR by protein tyrosine phosphatase(s). To evaluate the role of endosomal degradation in IR signaling, an insulin analog, termed H2, was characterized for its clearance and processing in liver. Although liver uptake of H2 was similar to that of insulin, its clearance was slower. This correlated with reduced ligand dissociation from internalized IR as well as slower degradation kinetics. / In response to H2, IR traffic was delayed in the endosomal apparatus. This occurred with increased IR autophosphorylation and tyrosine kinase activity in this compartment. H2 but not insulin induced JNK activity and c-jun transcription. Insulin stimulated MAP kinase and glycogen synthase (GS) activities in rat liver. H2, however, was less efficient in inducing MAP kinase activity than insulin and GS activity was not observed. Impaired GS activity in response to H2 correlated with increased PKC activity. The above observations of insulin and H2 were not due to changes in SHC nor IRS-1 signaling. / These studies indicate that the modification of RTKs at the level of the endosome alters receptor traffic and specificity of signal transduction pathways and support the hypothesis that RTK endocytosis plays a role in the regulation of RTK signaling.

Multiple hormones and growth factors regulate mammary epithelial and breast cancer cell growth and differentiation. While prolactin (PRL) is a differentiation hormone, epidermal growth factor (EGF) is an important mitogen. Here we examined the PRL and EGF crosstalk mechanism in mammary epithelial cells. Our results indicate that PRL and EGF exhibit antagonistic properties that impacts mammary epithelial cell proliferation and differentiation. While EGF blocked PRL-induced cellular differentiation, PRL suppressed EGF-induced cell growth. EGF was shown to block PRL-induced cellular differentiation without inhibiting PRL proximal signaling cascade such as Jak2 and Stat5a tyrosine phosphorylation. We identified Grb2 as a novel substrate of the PRLR/Jak2 complex. Moreover, the tyrosine phosphorylation of Grb2 was shown to be an essential mechanism utilized by PRL to inhibit EGF signals to MAPK activation and cell growth. Together these results define a novel signaling mechanism underlying PRL and EGF crosstalk in mammary epithelial and breast cancer cell growth and differentiation. / La PRoLactine (PRL) et Epidermal Growth Factor (EGF) sont des facteurs importants pour la croissance cellulaire, la différenciation et la carcinogenèse mammaire. Tandis qu'EGF est un mitogène puissant, PRL est critique pour l'induction de la différenciation de cellules épithéliales mammaires. Tandis qu'EGF bloque la différenciation cellulaire causée par PRL, PRL supresse la croissance cellulaire dûe à EGF. D'un côté, on a montré qu'EGF bloque la différenciation cellulaire sans inhiber la signalisation proximale de PRL. De l'autre, le traitement avec PRL inhibe l'activation de MAP Kinases par EGF et la prolifération cellulaire. De plus, le traitement avec PRL entraîne une augmentation significative de la phosphorylation en ou sur les tyrosines de Grb2. Pour confirmer ces résultats, nous avons utilisé Grb2YF, une forme de Grb2 sans sites de phosphorylations de tyrosines. En présence de PRL et Grb2YF, nous avons observé un rétablissement de l'activation de Ras et des MAP Kinases. Nos données suggèrent que la PRL réduit l'activation des MAP Kinases et la prolifération cellulaire induite par EGF grâce à la phosphorylation des sites tyrosines de Grb2 empêchant ainsi l'interaction avec Sos. Ensemble ces résultants définissent un nouveau mécanisme de signalisation qui met en évidence la communication entre PRL et EGF dans la croissance et différenciation des cellules épithéliales mammaires et cancérigènes.

ABSTRACT We followed the localization of GFP-tagged myosin I (Myo5), septin (Cdc12) and rhodamin-stained actin during bud and shmoo formation in opaque-phase cells of Candida albicans, and monitored the mating-associated processes of cell fusion, zygote budding, septum formation, daughter cell development and the dynamics of nuclear migration and division. The localization of Myo5p, Cdc12p and actin during budding in opaque and white cells is similar. In pheromone-stimulated cells, these proteins localize in shmoos in patterns consistent with hyphal formation in white-phase cells. MTLa cells generate shmoos 5-7 hours earlier than MTLα cells in mixed populations. In the daughter cell generated after mating, Cdc12p, Myo5p and actin localize as they do under vegetative budding conditions. Intriguingly, isogenicity for the mating type locus is involved in the positioning of the nuclear division; in MTLa cells the nucleus divides within the mother cell up to 70% of the time, rather than across the mother-bud neck. / RÉSUMÉ Nous avons déterminé la localisation de l'actine ainsi que de la myosine I (Myo5) et de la septine (Cdc12) modifiées avec la protéine fluorescente verte (GFP) chez Candida albicans en phase opaque (reproductive) et blanche (végétative). Plus particulièrement, nous avons porté notre attention sur les processus associé à la reproduction tel le bourgeonnement et l'expansion cellulaire (shmoo), la conjugaison et la fusion cellulaire, le bourgeonnement et le développement des zygotes (cellules -filles issues de la conjugaison). Nous avons aussi observé la configuration subcellulaire de Myo5p, Cdc12p et de l’actine lors de la migration et de la division nucléaire en phase blanche. Que ce soit en phase opaque ou en phase blanche, la localisation de Myo5p, Cdc12p et de l'actine reste similaire lors du bourgeonnement. Lors d’une stimulation à la phéromone, en phase opaque, ces trois protéines ont le même patron d’organisation cellulaire que lors de la formation d'hyphes en phase blanche. Les cellules de type MTLa produisent des shmoo entre 5 et 7 heures plus tôt que les cellules de type MTLα dans une population mixte. Dans les zygotes, Cdc12p, Myo5p et l'actine ont la même localisation que celle observée dans les cellule-filles issues du bourgeonnement en mode végétatif. Étonnamment, l'isogénicité du locus génétique déterminant le type sexuel de la cellule influence la position du noyau lors de la division; Ainsi, dans 70% des cas, le noyau des cellules de type MTLa se divise à l'intérieur de la cellule-mère plutôt qu'au travers du col entre la cellule-mère et le bourgeon.

Alendronate (ALN) is effective in the treatment of osteoporosis. However, ALN has been recently associated with an increased risk of serious atrial fibrillation. We investigated the effects of ALN on cytosolic free calcium concentration ([Ca2+]i) in cardiomyocytes. HL-1 atrial cardiomyocytes were loaded with fura-2 and examined using microspectrofluorimetry. ALN (10-8, 10-7, 10-6 M) induced transitory high frequency oscillations of [Ca2+]i with greater frequency for 10-8 M ALN (61 ± 10 mHz) compared to 10-6 ALN (42 ± 4 mHz). In cells treated with 10-6 M ALN responses to subsequent application of caffeine were delayed, and exhibited a decrease in the rate and amplitude of [Ca2+]i increase. Long term (48 h) exposure to 10-8 M Alendronate resulted in delay of caffeine-induced Ca2+ transients and decreased rate of [Ca2+]i increase, followed by oscillations in [Ca2+]i of 54 ± 8 mHz versus those observed at higher concentrations of Alendronate (35 ± 5 mHz). Changes in calcium dynamics were accompanied by significant changes in the expression of sarcoendoplasmic reticulum ATPase (SERCA2a), calsequestrin and calreticulin. / Alendronate est efficace dans le traitement de l’ostéoporose. Alendronate a récemment été associe avec une risque élevé de fibrillation auriculaire sérieux. On a examine les effets d’Alendronate sur la de calcium cytosolique ([Ca2+]i) dans les cellules musculaires cardiaques. Les cellules musculaires cardiaques HL-1 étaient chargés avec Fura-2 et examinés par microspectrofluorimetrie. Alendronate (10-8, 10-7, 10-6 M) ont provoqués des oscillations de [Ca2+]i fugaces et haute fréquences à 10-8 M Alendronate (61 ± 10 mHz) comparé aux concentrations plus hautes (42 ± 4 mHz). Dans les cellules traits avec 10-6 M ALN, la réponse à l’application de caféine était avec délai, et a manifesté un diminution dans la rythme et amplitude d’augmentation de [Ca2+]i. L’exposition a l’ALN à long terme (48 h) ont provoqué un délai des élévations de calcium transitoires, et un diminution du rythme d’augmentation de [Ca2+]i suites par les oscillations de [Ca2+]i, caractérisés par un augmentation de fréquences avec 10-8 M Alendronate (54 ± 8 mHz) compare aux concentrations plus hautes (35 ± 5 mHz). Le changement des dynamiques de calcium étaient accompagnés par les changements considérables dans l’expression d’ATPase (SERCA2a), calsequestrin et calreticulin du réticulum sarcoendoplasmique.

The eukaryotic cell is organized into several membranous subcellular compartments that contribute to the spatial segregation of the many cell physiological functions. One of these organelles, the endoplasmic reticulum (ER), is a continuous compartment, emanating from the nuclear envelope, through the rough ER, and ending with the reticulated smooth ER network. Each domain of the organelle is distinct for its morphology, as well as the many diverse functions that it performs. In this thesis, a survey of a proteomics resource for both the rough and smooth domains of the ER reveals that the organelle is further divided into spatial and functional subdomains. Furthermore, the assignment of novel proteins that were uncovered by proteomics to these various functional clusters can provide insight into their functions and guide future functional studies. Lastly, this thesis demonstrates the importance of the distinct morphology of the smooth ER network in maintaining basic physiological processes. Taken together, these data demonstrate that while the ER is a single, continuous, subcellular compartment, it is highly complex in its spatial, functional, and morphological organization. / La cellule eucaryote est constituée de plusieurs structures spécialisées dénommées organites qui contribuent à la démarcation des fonctions physiologiques de la cellule. L'un de ces organites, le réticulum endoplasmique (RE), est une structure continue qui provient de l'enveloppe nucléaire, devient le RE rugueux, et se termine avec le réseau réticulé RE lisse. Chaque division de l'organite est unique pour sa morphologie, ainsi que ses fonctions nombreuses et diverses. Dans cette thèse, une analyse d'une ressource protéomique révèle que les deux domaines du RE sont encore divisés en de nouvelles sections régionales et fonctionnelles. De plus, l'attribution de nouvelles protéines découvertes par la protéomique à ces divisions fonctionnelles peut donner un aperçu de leurs fonctions et guider de nouvelles études. Enfin, cette thèse démontre l'importance de la morphologie distincte du réseau RE lisse pour le maintient de certains processus physiologiques de base. Le tout démontre que même si le RE est en effet un compartiment continu, son organisation régionale, fonctionnelle et morphologique est très complexe.

Basal-like triple negative breast cancers (TNBCs) display poor prognostic features with larger tumor size, higher tumor grade, and an increased risk for lymph node and distant metastasis as well as tumor recurrence. Transforming growth factor beta (TGFβ) is a key regulator of the cellular processes by which breast cancer cells from the primary tumor metastasize to distant organs. However, the molecular mechanisms underlying TGFβ's pro-metastatic effects remain to be fully elucidated. Here, we investigated the role of TGFβ signaling pathway in regulating cell migration, invasion and cancer stem cell self-renewal capacity, which are the initial and critical steps in breast cancer metastasis. Our studies initially identified a novel function for the cell cycle regulator p21 and its binding partner acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-induced TNBC cell migration and invasion in vitro as well as tumor invasiveness in vivo. As p21 can interact with different cyclin and CDK complexes, we investigated whether other cell cycle regulators are also involved in TGFβ-induced tumor progression. We found that TGFβ promotes physical interaction and nuclear co-localization between cyclin D1 and p21. The co-expression of cyclin D1 and p21 proteins promote tumor growth and locally invasive tumors. In addition, we found that TGFβ can activate cyclin D1/CDK4 complex to promote cancer stem cell activity and self-renewal capacity in TNBC. Together, we have defined p21, cyclin D1 and CDK4 as key downstream regulators of TGFβ tumor-promoting functions. / Le cancer du sein triple-négatif (CSTN) de type basal démontre des signes cliniques caractéristiques d'un mauvais pronostique, tels qu'une taille et un grade plus élevés des tumeurs, un risque augmenté de développer des métastases lymphatiques et distantes, ainsi qu'un plus grand risque de récurrence de la maladie. Le TGFβ est un régulateur clé des procédés cellulaires par lesquels les cellules cancéreuses du cancer du sein se détachent de la tumeur primaire pour former des métastases aux organes distants. Cependant, les mécanismes moléculaires par lesquels le TGFβ accomplit son rôle pro-métastatique restent à être élucidés. Ici, nous investiguons le rôle du TGFβ dans la régulation de la migration cellulaire, l'invasion, et la capacité des cellules souches à se renouveler, qui sont les étapes initiales et critiques à la formation de métastases dans le cancer du sein. Notre étude a tout d'abord identifié une nouvelle fonction pour le régulateur de cycle cellulaire p21 et son partenaire associé, l'acetyltransférase pCAF, comme étant des régulateurs de transcription dont la présence est critique pour la migration et l'invasion cellulaire in vitro et l'invasion tumorale in vivo médiées par le TGFβ pour le CSNT. Le p21 pouvant interagir avec différents complexes de cycline et CDK, nous avons investigué si d'autres régulateurs du cycle cellulaire sont aussi impliqués dans la progression tumorale médiée par le TGFβ. Nous avons trouvé que le TGFβ promeut l'interaction physique et la co-localisation nucléaire entre la cycline D21 et p21. La co-expression de la cycline D1 et de p21 promeut la croissance tumorale et l'invasion locale des tumeurs. De plus, nous avons trouvé que le TGFβ peut activer le complexe cycline D1/CDK4 pour promouvoir l'activité des cellules souches cancéreuses, ainsi que leur capacité de renouvèlement dans le CSNT. Ces résultats nous ont permis de définir p21, la cycline D1, et CDK4, comme des régulateurs clés en aval du TGFβ dans ses fonctions pro-métastatiques

The formation and maintenance of boundaries between embryonic regions are crucial to preserve the organization of the embryo during development. One important parameter that needs to be regulated at the interface between cells of two different tissues is cell-cell adhesion. In the present study, we attempted to understand and characterize the dynamics of cadherin-mediated cell-cell adhesion at the ectoderm-mesoderm boundary of the Xenopus gastrula. At this boundary, it has been recently shown that cells undergo cycles of attachments and detachments as the result of ephrin/Eph signaling (Rohani, Canty, Luu, Fagotto, & Winklbauer, 2011). Using three-dimensional live confocal microscopy, we found that the total levels of cadherin at the membrane do not change reproducibly during local repulsive events between ectodermal and mesodermal cells. Cadherin clusters, however, appear during contact establishment and often begin to fade away prior to the start of cell detachments. While the role of acto-myosin contraction in this process is still not fully elucidated, we found that cortical actin was upregulated during cell detachments, and that phospho-myosin was enriched at the endogenous ectoderm-mesoderm boundary. Furthermore, our data suggest that there may be two pools of activated myosin, and that the one that is most prominent at the boundary is the one involved in global cortical tension. / La formation et le maintien de frontières entre les différentes régions embryonnaires jouent un role primordial pour préserver l'organisation de l'embryon au cours du développement. L'adhérence intercellulaire est l'un des paramètres qui doivent être régulés aux interfaces entre les cellules de différents tissus. Dans la présente étude, nous avons tenté de comprendre et de caractériser la dynamique des cadhérines à la frontière entre l'ectoderme et le mésoderme dans la gastrula de Xénope. A cette frontière, il a été démontré que les cellules effectuent des cycles d'attachements puis de détachements, dictés par la voie de signalisation éphrine/Eph (Rohani et al., 2011). Par le biais de microscopie confocale tridimensionnelle, nous avons constaté que la quantité de cadhérines à la membrane ne variait pas au cours des instances de répulsions locales entre les cellules de l'ectoderme et du mésoderme. Les "clusters" de cadhérines, en revanche, apparaissent dès l'initiation du contact intercellulaire et semblent diminuer d'intensité progressivement, avant que les cellules ne se détachent complètement. Bien que le rôle de la contraction d'actine-myosine ne soit pas encore totalement élucidé, nous montrons que l'actine corticale augmente pendant et après les détachements cellulaires, et que la phospho-myosine est régulée à la hausse à la frontière ectoderme-mésoderme endogène. De plus, nos données suggèrent qu'il pourrait y avoir deux "pools" de myosine, et que celle qui se démarque à la frontière est celle qui est responsable de la tension corticale globale des cellules.