Paramecium, a unicellular ciliate, can be attracted by various chemical stimuli. Chemoattractants such as glutamate, folate, cAMP, and acetate activate different receptor mediated signal transduction pathways. The final event in these signal transductions is a hyperpolarization of membrane potential, which makes Paramecium swim smoothly and fast. There is evidence that the effecter of this hyperpolarization is the plasma membrane calcium ATPase (PMCA), that when activated, expels Ca2+ from the cell. In Paramecium three PMCA isoforms, named PMCA2, 3, and 4, have been cloned. PMCA2 is associated with lipid rafts, which is demonstrated by its resistance to cold detergent solubilization and distribution in sucrose density gradients in ultracentrifugation. PMCA3 and 4 are not associated with lipid rafts. On the cell surface, PMCAs are localized to the bases of cilia. Sterol-depletion by methyl-ß-cyclodextrin (MßCD) treatment disrupts the distribution of PMCA2 in sucrose density gradients and ciliary base-localization on the cell surface. MßCD treatment also decreases the chemoattraction to glutamate and cAMP. This indicates that PMCA2 and its association with lipid rafts are essential in the chemoattraction signal transduction pathways. Based on these results, a model of membrane domains incorporating three signal transduction pathways is proposed.

Paramecium, a ciliate, is an important model for studying Ca2+ signaling and understanding chemoreception and signal transduction. There are several proteins, such as plasma membrane calcium ATPases (PMCAs)/ calcium pumps, SERCA pumps, calmodulin and Ca2+ channels that play an important role in maintaining intracellular Ca2+ level and signaling in Paramecium. Isoform 2 of PMCA has been identified in both the cilia and pellicle membranes of Paramecium, the activity of which leads to hyperpolarization. Plasma and ciliary membrane of Paramecium is made up of a variety of sterols and sphingolipids which constitute lipid rafts, demonstrated by the presence of detergent resistant membranes and their distribution in sucrose and Optiprep density gradients. PMCAs are important markers of lipid rafts and PMCA 2 is found to be localized in lipid rafts of both the cilia and somatic membrane of Paramecium. Methyl-β-cyclodextrin treatment can remove up to 42% of sterols from pellicle membranes but only about 12% from cilia. Sterol depletion of pellicle perturbs the distribution of PMCA 2 and other raft proteins in pellicle which is not observed in cilia as evident from western blot analysis and immunomicroscopic studies. There is evidence that selection of gradient medium for study of lipid rafts and its associated proteins is very important in Paramecium. Glutamate receptors and adenylyl cyclase, the upstream molecules of the signal transduction pathway through PMCA have also been identified in cellular cilia, indicating that these raft molecules forms a platform for signaling in Paramecium cilia.

No description available.

The aim of this project was to investigate the role of lipid rafts in actin-mediated phagocytosis. Lipid rafts are defined as highly condensed membrane domains enriched in cholesterol and glycosphingolipids and are thought to participate in a range of cellular functions including actin-mediated phagocytosis. Remodelling of the actin skeleton facilitates the formation of a phagocytic membrane cup and drives the uptake of particles. Hence, actin restructuring is essential for phagocytosis. How engagement of Fc receptors triggers membrane re-organization at the site of phagocytosis and how the formation of ordered raft domains is linked to actin remodelling during phagosome maturation is currently not known. Lipid rafts potentially form platforms for local signal transduction for Fc surface receptors and secondary messengers. Raft distribution therefore would critically influence and direct their function. The hypothesis is that lipid rafts are the membrane sites on the cell surface, which enable, drive and localise actin- dependent phagocytosis. Phagocytosis in macrophages was induced with IgG-coated beads of different sizes as substrates for uptake via the Fc receptor mediated pathway. Membrane order was visualised and quantified by two-photon microscopy. Actin remodelling was imaged in parallel with confocal microscopy. Time-course and live cell imaging demonstrated that phagocytosis induces formation of highly ordered membrane domains around the phagocytic particle independently of the particle size. The high membrane order is the biophysical hallmark of lipid rafts suggested that Fc receptor cross-linking induces the coalescence of lipid rafts. Live-cell imaging further identified a temporal correlation between membrane condensation and actin restructuring at sites of phagocytosis. Membrane condensation persisted after actin detached from the sealed phagosome. Receptor clustering induced by particle binding activates Src kinases leading to tyrosine phosphorylation of ITAM motif of the receptors, activation of GTPases and actin polymerisation. Lipid raft recruitment may be driven by these events or alternatively, rafts be essential for kinase activation. Several inhibitors were used to interrupt crucial steps in the signalling cascade leading to actin restructuring. Laurdan microscopy showed that membrane order is independent of Lyn activation (inhibited with PP2), PI3K activity (inhibited with Wortmannin) and actin polymerisation (Latrunculin B). Inhibitors had differential effects on phagocytosis rates of small and large particles. Inhibition of Lyn had a more severe effect on phagocytosis of large beads than on phagocytosis of small beads. Disruption of PI3K activity with Wortmannin only inhibited phagocytosis of large but not of small particles, whereas disruption of the actin skeleton with Latrunculin B inhibited phagocytosis of small and large particles. These data suggest that membrane condensation is independent of kinase activity and occurs upstream of actin remodelling. The role of lipid rafts in phagocytosis was further investigated by modulation of sterol composition of the cell membranes. Cholesterol depletion with methyl-- cyclodextrin disrupted membrane organization at phagosomes of small and large beads and also abolished phagocytosis. However, the fluidity of the entire plasma membrane was increased upon treatment of cells with methyl--cyclodextrin suggesting that this condition was not specifically affecting phagosomal membranes. Cholesterol enrichment increased membrane condensation even further than the membrane condensation detected in control phagosomes. Incorporation of 7-keto-cholesterol (7KC) decreased membrane order of phagosomes of small beads but not of large beads. 7KC can prevent membrane condensation due to its additional keto-group, which acts as a spacer between phospholipids. Phagocytosis of large beads but not of small beads was affected by 7KC incorporation. This suggests that 7KC only moderately reduces membrane order, which diminishes but not completely abolishes phagocytosis. This might be explained by the fact that 7KC enrichment and mCD treatment diminished actin remodelling and reduced the complexity of the F-actin network. Mass spectrometry was employed to quantify the lipidome of phagosomal membranes. This is the first study that directly demonstrates that phagosomes exhibit a distinct lipid composition and were enriched in sphingomyelin (SM) but depleted of cholesterol. Furthermore, the effects of sterol modulation on lipid species abundance were investigated. Cholesterol and 7KC enrichment resulted in lower levels of PC, but higher levels of charged lipids. In addition, 7KC treatment increased SM levels. In conclusion, cross-linking of Fc receptors triggers the formation of ordered membrane domains that do not have the classical raft composition. They are cholesterol depleted but rich in sphingomyelin. The formation of these ??rafts?? occurs upstream of actin remodelling and is necessary for actin remodelling during phagocytosis.

Les cellules gonadotropes jouent un rôle clé dans la fonction de reproduction en sécrétant les hormones LH et FSH. Nous avons positionné les protéine-phosphatases 2A (PP2A) dans la voie de signalisation de la GnRH. 5% de la PP2A est localisée dans les membranes des cellules des lignées gonadotropes murines et des cellules hypophysaires ovines. Une PP2A dimérique active est co-localisée avec le récepteur de la GnRH dans les rafts de ces cellules. Son activité augmente de 40% après stimulation avec la GnRH. Nos résultats suggèrent que les PP2A pourraient réguler la signalisation induite par la GnRH, en contrôlant les MAPK. La désorganisation des rafts provoque une libération de LH en absence de GnRH, dépendante de la voie de signalisation de la GnRH. Ainsi la libération de LH serait liée à la déstructuration des rafts. Sans ligand, la voie de signalisation de la GnRH serait inhibée, et la désorganisation des rafts induirait une levée d’inhibition responsable de son activation et de la libération d’hormones. / Pituitary gonadotroph cells play a key role in the function of reproduction by secreting LH and FSH. The first aim was the positioning of PP2A in GnRH signaling pathway of. 5% of PP2A is located in the membranes of murine gonadotroph cell lines and ovine pituitary cells. An active dimeric PP2A is co-localized with the GnRH receptor in the lipid rafts of the cells membrane. PP2A activity increases by 40% after stimulation with GnRH. Our results suggest that PP2A could play a regulatory role in signaling induced by GnRH, via the control of the MAPK pathway. To better understand the underlying mechanisms of exocytosis of LH, I studied the functional relationship between lipid rafts and the release of LH. The disorganization of lipid rafts causes, in the absence of GnRH, a rapid LH release, dependant of GnRH signaling pathway. In the absence of ligand, the GnRH signaling pathway is spontaneously inhibited, and the disruption of lipid rafts would suppress this inhibition inducing the signaling activation and hormone release.

No description available.

Los ovocitos ováricos totalmente crecidos de anfibios están fisiológicamente arrestados en la profase de la primera división meiótica. La progesterona, a través de un proceso denominado maduración, induce el desarresto ovocitario que se traduce en la ruptura de la vesícula germinal (GVBD). Se acepta generalmente que la hormona desencadena la maduración por un mecanismo no genómico que involucra la inhibición de la adenilil ciclasa y la disminución del AMPc intracelular a través de la unión a un receptor ligado a la membrana plasmática. Como respuesta al estímulo hormonal se sintetiza la oncoproteína c-Mos cuya actividad biológica está mediada por la cascada de las proteínas activadas por mitógenos (MAPK). En conjunto con la activación de reguladores del ciclo celular estos caminos convergen en la activación del factor promotor de la maduración (MPF). Los rafts de membrana, en particular la estructura invaginada de las caveolae, podrían proporcionar un ambiente óptimo para la interacción entre la progesterona y el ovocito en la maduración meiótica. Sin embargo, las bases moleculares y los mecanismos de la posible participación de los microdominios caveolares en la transducción de señales de la maduración aún no han sido completamente dilucidados. En este trabajo de tesis se analizó el efecto de la maduración inducida por la progesterona sobre el contenido y la composición de los lípidos neutros y polares de las plaquetas vitelinas, organelas del ovocito de Bufo arenarum que cumplen un rol importante durante la embriogénesis. Se realizó un análisis cuantitativo de los lípidos y de las proteínas de las membranas de baja densidad aisladas de ovocitos ováricos y la identificación bioquímica y el estudio biofísico de los microdominios tipo-caveolares, como un requisito para comprender mejor sus funciones regulatorias. La metil-β-ciclodextrina (MβCD) se usó como herramienta para modular el colesterol celular con el fin de evaluar la participación de los rafts de membrana en la maduración inducida por progesterona y en la inducida por ceramida, disparador de la reiniciación de la meiosis en otras especies de anfibios. En particular, se indagó la vía de las MAPK en la señalización de la maduración meiótica. Se demostró que los lípidos de las plaquetas vitelinas están involucrados activamente en la reiniciación del ciclo meiótico lo que apoya la hipótesis de un rol dinámico de estas organelas. La maduración produjo una disminución en el contenido total de fosfolípidos fundamentalmente por la caída de fosfatidilcolina, un fosfolípido considerado esencial para que se complete la meiosis. Los principales cambios en el perfil de ácidos grasos se observaron en esfingomielina, en ácido fosfatídico y en los diacilgliceroles, lípidos bioactivos implicados en caminos de señalización celular. El tratamiento hormonal disminuyó el nivel de la esfingomielina plaquetaria lo que podría vincularse con su rol como precursor de ceramidas. Se pusieron a punto distintos métodos de aislamiento de microdominios de membrana y se logró obtener una fracción de membranas livianas en ausencia de detergentes. Se determinó que dicha fracción deriva de la membrana plasmática, está enriquecida en colesterol y en el gangliósido GM1 y presenta un nivel importante de esfingomielina. Las membranas livianas muestran un enriquecimiento en una caveolina de 21 kDa indicando la existencia de estructuras tipo-caveolares en el ovocito de Bufo arenarum. Además, están asociadas significativamente con las moléculas señales, c-Src y H-Ras. En estas membranas se encontró una banda proteica que, por espectrometría de masa, se identificó como la cadena pesada de la miosina no muscular planteando la posibilidad de una relación con el citoesqueleto. La depleción de colesterol, mediada por MβCD, afectó principalmente el nivel de colesterol de las membranas livianas alterando el orden lipídico y la localización de los marcadores moleculares de rafts, caveolina, c-Src y GM1, e inhibiendo la maduración de manera dosis-dependiente lo que sugiere que estos microdominios de membrana están involucrados en la inducción hormonal. La repleción de colesterol indicó una recuperación de la habilidad para madurar de los ovocitos tratados especialmente en la concentración de MβCD 25 mM en la cual la reversibilidad fue cercana al valor control. Se demostró que la ceramida es un inductor efectivo de la maduración que afecta la distribución de los marcadores moleculares de rafts en las fracciones de membrana. Por el contrario, la progesterona no parece afectar la integridad de los microdominios de membrana. En concordancia con la inhibición de la GVBD, el tratamiento con MβCD retardó la fosforilación en tirosinas y la activación de la p42 MAPK en la maduración inducida por progesterona. La presencia de los marcadores de rafts, caveolina, GM1, c-Src y H-Ras, y el hallazgo de moléculas señales de la cascada de las MAPK funcionalmente asociadas a las membranas livianas, sugieren que esta fracción enriquecida en microdominios tipo-caveolares puede, en parte, recrear eficazmente la señalización de la maduración. / Amphibian full-grown ovarian oocytes are physiologically arrested at the first meiotic prophase. Progesterone, through a mechanism called maturation, induces meiotic resumption represented by germinal vesicle breakdown (GVBD). It is generally accepted that progesterone hormone triggers maturation through a nongenomic mechanism that involves the inhibition of adenylyl cyclase and the reduction of intracellular cAMP by association with a plasma membrane receptor. As a response to the hormonal stimuli oncoprotein c-Mos is synthesized and its biological activity is mediated by the mitogen-activated protein kinase (MAPK) cascade. Together with the activation of cell cycle regulators, both pathways converge in the activation of the M-phase promoting factor (MPF). Membrane rafts, particularly the invaginated structure of caveolae, seems to provide an optimal environment for hormone binding leading to meiotic maturation. However, the molecular bases and the mechanisms of the posible caveolar microdomain involvement in maturation signal transduction pathways have not been fully elucidated to date. In the present thesis, the effect of progesterone-induced maturation on the quantity and composition of neutral and polar lipids of yolk platelets, organelles from Bufo arenarum oocyte that play an important role during embryogenesis, were analyzed. A quantitative analysis of lipids and proteins of low-density membranes isolated from ovarian oocytes and the biochemical identification and a biophysical study of caveolae-like microdomains were performed as a requisite to further understand how these domains carry out their regulatory functions. Methyl-β-cyclodextrin (MβCD) was thus used for cellular cholesterol modulation in order to assess the membrane raft involvement in maturation induced by progesterone and by ceramide, the latter being trigger of meiosis reinitiation in other amphibian species. We demonstrated that lipids from yolk platelets are actively involved in the resumption of the meiotic cell cycle supporting the hypothesis of a dynamic role for these organelles. Phospholipid content decreased mainly as a result of a fall at the level of phosphatidylcholine, a phospholipid considered crucial for the completion of meiosis. Fatty acid composition registered significant changes in sphingomyelin, phosphatidic acid and diacylglycerols, bioactive lipids involved in cellular signaling pathways. Hormonal treatment induced a decrease at sphingomyelin level that could be related to its role as ceramide precursor. Different isolation methods were assayed to obtain membrane microdomains and a light membrane fraction was obtained in the absence of detergents. Light membranes derive from the plasma membrane, show an enrichment in cholesterol and GM1 ganglioside, and evidence an important level of sphingomyelin. The finding of a 21 kDa caveolin enriched in light membranes indicates the presence of caveolae-like structures in Bufo arenarum oocytes. In support of this finding, signaling molecules as c-Src and H-Ras are significantly associated to this fraction. A protein band was found in these membranes and it was identified as a non-muscle myosin heavy chain by mass spectrometry suggesting possible membrane-cytoskeleton interactions. Cholesterol depletion mediated by MβCD affected mainly light membranes cholesterol level disturbing lipid order and localization of rafts markers, caveolin, c-Src, and GM1 and inhibiting maturation in a dose-dependent manner, thus suggesting that these membrane microdomains are involved in hormonal induction. Cholesterol repletion showed a recovery of the ability of MβCD-treated oocytes to mature, particularly at the 25 mM concentration at which reversibility was close to control level. We also demonstrated that ceramide is an effective inducer of maturation that affects the distribution of raft markers among membrane fractions. On the contrary, progesterone seems not to affect membrane microdomain integrity. In agreement with GVBD inhibition, MβCD treatment delayed tyrosine phosphorylation and p42 MAPK activation in progesterone-induced maturation. The presence of the rafts markers, caveolin, GM1, c-Src, and H-Ras, and the finding of signaling molecules from the MAPK cascade functionally associated to light membranes suggest that this fraction enriched in caveolae-like microdomains could efficiently recreate, at least in part, maturation signaling.

Physical stress such as the surface roughness of the implants may activate the NFkB signalling pathway in macrophages. This activation is intimately related to the mechanism(s) by which the macrophage interacts with the surface through serum proteins and/or the formation of membrane rafts. This thesis examines the role of surface topography on activation of the NFkB signalling pathway in macrophages. We examined the effect of implant surface topography on activating the NFkB signalling pathway in the RAW 264.7 macrophage cell line. We also examined the effect surface roughness had on the adhesion of the macrophages using the different media. To finish, we observed the effect the different media and the surface roughness had on the morphology of the macrophages by Scanning Electron Microscopy. Activation of the NFkB pathway was surface topography dependent. The Smooth surface showed the highest level of activation followed by the Etched then the SLA. Addition of suboptimal concentrations of LPS mildly enhanced the response by signalling through the Toll receptor. Activation of NFKB occurred in the absence of fetal calf sera, although to a lesser extent. All three surfaces had very few cells with nuclear translocation at the 5 minutes time point with no significant statistical differences between the surfaces. After 30 minutes, translocation reached comparable levels to those surfaces tested with complete medium. Disruption of the lipid rafts affected the triggering and signalling of the NFkB pathway. This inhibitory effect was concentration and time dependent. Smooth surfaces bound more macrophages in the 30 minutes assay. Fetal calf serum appeared to be very critical for adhesion and spreading of the macrophages on the various surfaces examined. Removal of cholesterol did not affect adhesion or spreading on their respective surfaces. We have clearly demonstrated that the lipid rafts along with surface topography play a role in the activation on NFKB. This in-vitro study has demonstrated that surface topography modulated activation of the NFKB signalling pathway in a time-dependent manner. However, at present, it is unclear through which receptor(s) / surface structure the signal pathway is initiated.

Physical stress such as the surface roughness of the implants may activate the NFkB signalling pathway in macrophages. This activation is intimately related to the mechanism(s) by which the macrophage interacts with the surface through serum proteins and/or the formation of membrane rafts. This thesis examines the role of surface topography on activation of the NFkB signalling pathway in macrophages. We examined the effect of implant surface topography on activating the NFkB signalling pathway in the RAW 264.7 macrophage cell line. We also examined the effect surface roughness had on the adhesion of the macrophages using the different media. To finish, we observed the effect the different media and the surface roughness had on the morphology of the macrophages by Scanning Electron Microscopy. Activation of the NFkB pathway was surface topography dependent. The Smooth surface showed the highest level of activation followed by the Etched then the SLA. Addition of suboptimal concentrations of LPS mildly enhanced the response by signalling through the Toll receptor. Activation of NFKB occurred in the absence of fetal calf sera, although to a lesser extent. All three surfaces had very few cells with nuclear translocation at the 5 minutes time point with no significant statistical differences between the surfaces. After 30 minutes, translocation reached comparable levels to those surfaces tested with complete medium. Disruption of the lipid rafts affected the triggering and signalling of the NFkB pathway. This inhibitory effect was concentration and time dependent. Smooth surfaces bound more macrophages in the 30 minutes assay. Fetal calf serum appeared to be very critical for adhesion and spreading of the macrophages on the various surfaces examined. Removal of cholesterol did not affect adhesion or spreading on their respective surfaces. We have clearly demonstrated that the lipid rafts along with surface topography play a role in the activation on NFKB. This in-vitro study has demonstrated that surface topography modulated activation of the NFKB signalling pathway in a time-dependent manner. However, at present, it is unclear through which receptor(s) / surface structure the signal pathway is initiated.

Physical stress such as the surface roughness of the implants may activate the NFkB signalling pathway in macrophages. This activation is intimately related to the mechanism(s) by which the macrophage interacts with the surface through serum proteins and/or the formation of membrane rafts. This thesis examines the role of surface topography on activation of the NFkB signalling pathway in macrophages. We examined the effect of implant surface topography on activating the NFkB signalling pathway in the RAW 264.7 macrophage cell line. We also examined the effect surface roughness had on the adhesion of the macrophages using the different media. To finish, we observed the effect the different media and the surface roughness had on the morphology of the macrophages by Scanning Electron Microscopy. Activation of the NFkB pathway was surface topography dependent. The Smooth surface showed the highest level of activation followed by the Etched then the SLA. Addition of suboptimal concentrations of LPS mildly enhanced the response by signalling through the Toll receptor. Activation of NFKB occurred in the absence of fetal calf sera, although to a lesser extent. All three surfaces had very few cells with nuclear translocation at the 5 minutes time point with no significant statistical differences between the surfaces. After 30 minutes, translocation reached comparable levels to those surfaces tested with complete medium. Disruption of the lipid rafts affected the triggering and signalling of the NFkB pathway. This inhibitory effect was concentration and time dependent. Smooth surfaces bound more macrophages in the 30 minutes assay. Fetal calf serum appeared to be very critical for adhesion and spreading of the macrophages on the various surfaces examined. Removal of cholesterol did not affect adhesion or spreading on their respective surfaces. We have clearly demonstrated that the lipid rafts along with surface topography play a role in the activation on NFKB. This in-vitro study has demonstrated that surface topography modulated activation of the NFKB signalling pathway in a time-dependent manner. However, at present, it is unclear through which receptor(s) / surface structure the signal pathway is initiated. / Dentistry, Faculty of / Graduate