Thesis (Ph. D.)--University of Virginia, 2005. / Includes bibliographical references (leaves 175-196). Also available online through Digital Dissertations.

Le CD95 appartient à la famille du TNF-R. Il est capable de déclencher un signal apoptotique et joue un rôle prépondérant dans le maintien de l’homéostasie du système immunitaire et dans l’élimination de cellules infectées ou transformées. Suite à la fixation de son ligand le CD95L ou d’un anticorps agoniste, le CD95 recrute la protéine FADD, qui à son tour agrège les caspases initiatrices (i.e., caspase -8 et -10). Le complexe formé par le CD95, FADD et les caspases -8/10 est appelé DISC, pour Deah Inducing Signaling Complex. Une fois le DISC formé, l’agrégation des caspases initiatrices entraîne leur activation, et la mort de la cellule par apoptose. Ce signal médié par CD95, peut être modulé par la distribution ou l’exclusion du récepteur vis à vis des microdomaines membranaires ou radeaux lipidiques. Ces domaines de la membrane plasmique sont des structures membranaires enrichies en sphingolipides et cholestérol. La relocalisation de CD95 dans ces radeaux lipidiques permet d’amplifier le signal de mort. L’activation de la voie phosphatidylinositol 3-kinase (PI3K)/Akt, est connue pour sa capacité à protéger les cellules tumorales du signal apoptotique médié par CD95, et à induire des mouvements latéraux de CD95 à la membrane. Nos travaux montrent que l’inhibition de la voie PI3K/Akt induit i) la relocalisation du CD95 dans les microdomaines et ii) l’induction du signal apoptotique CD95 indépendamment de la présence du ligand CD95L. Ainsi, nous mettons en évidence que la voie PI3K/Akt est capable d’augmenter le seuil d’activation du signal CD95 en agissant en amont de la formation du DISC ou de l’interaction CD95/CD95L, en maintenant le récepteur exclu des microdomaines. / CD95 belongs to the TNF-R superfamily, and it triggers an apoptotic signal. CD95 plays a key role in homeostasis of the immune system and in the elimination of infected and transformed cells. Upon CD95L binding, CD95 recruits FADD, which in turn aggregates initiator caspases (i.e., caspase-8 and -10). The complex CD95, FADD and caspase-8/10 is called DISC for Death Inducing Signaling Complex. At the DISC level, caspase aggregation leads to their activation and death of the cells through apoptosis. The CD95-mediated apoptotic signal is modulated by microdomains, or lipid rafts, which are plasma membrane sub-domains enriched in sphygolipids and cholesterol. Thereby, partition of CD95 into lipid rafts promotes the apoptotic signal. Activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is known to prevent the CD95-mediated apoptotic signal in malignant cells, and to control lateral mobility of CD95 at the plasma membrane. Herein, we showed that inhibition of PI3K signal induced i) the distribution of CD95 into lipid rafts and ii) the subsequent induction of the CD95-mediated apoptotic signal through a CD95L independent manner. In conclusion, we pinpointed that PI3K/Akt signaling pathway inhibits the CD95 signal by acting upstream DISC formation and even upstream the CD95-CD95L interaction through the exclusion of the death receptor from the microdomains.

The plasma membrane (PM) of live cells has a striking phospholipid asymmetry between bilayer leaflets, yet the purpose of this fundamental structure remains elusive. It is also unknown whether and how this phospholipid asymmetry impacts on the lateral organization of the PM, such as microdomains or lipid rafts that are thought to facilitate specific protein-protein interactions. Here, we generated asymmetric giant unilamellar vesicles (GUVs) and found that microdomain formation is inhibited by outer leaflet very long acyl chain (24:0) sphingomyelin (SM), the primary sphingolipid species in mammalian cells. Interestingly, although cholesterol is believed to associate favourably with SM, molecular dynamic (MD) simulations of asymmetric membranes indicate a strong preference for cholesterol in the inner leaflet when 24:0 SM is in the outer leaflet, as well as, interdigitation of 24:0 SM acyl chain across the centre of the bilayer. We thus hypothesized that the outer leaflet-localized 24:0 SM interdigitates across the leaflets of the bilayer and facilitates cholesterol enrichment in the inner leaflet. Indeed, we obtained evidence that, in asymmetric unilamellar vesicles with 24:0 SM exclusively in the outer leaflet, 75-80% of cholesterol was partitioned into the inner leaflet, which was correlated with the disappearance of microdomains in GUVs. Importantly, in live cell PM, where 24:0 sphingolipids are the predominant species and exclusively in the outer leaflet, cholesterol was similarly enriched in the cytoplasmic leaflet. SM with shorter acyl chains such as 16:0, a minor species in mammalian cells, failed to generate cholesterol asymmetry and promoted microdomains in both symmetric and asymmetric GUVs. Furthermore, we generated live mammalian cells with either 16:0 or 24:0 SM and analyzed submicron domains in these cells, using density-dependent FRET of GPI-anchored proteins. Indeed, 16:0 SM cells are capable of forming submicron domains. The 24:0 SM cells, by contrast, are nearly devoid of submicron domains, as are unmodified control cells. Moreover, we silenced ceramide synthase 2 (CerS2), the enzyme that generates very long acyl chain sphingolipids. We found that silencing CerS2 alters diffusional properties of membrane proteins, consistent with enhanced microdomain formation. Together, our results establish a surprising and central role of very long acyl chain sphingolipids in regulating membrane lateral organization, including in the native plasma membrane, by creating cholesterol asymmetry. We propose that sphingolipid asymmetry functions to dynamically regulate microdomains in live cells.

Il est maintenant reconnu que la ségrégation latérale des composants de la membrane plasmique, ou compartimentation membranaire, joue un rôle important dans la régulation de la fonction de nombreuses protéines membranaires. Les tétraspanines constituent une famille de protéines intégrales à quatre régions transmembranaires possédant une capacité unique à s’associer entre elles et avec des nombreuses autres molécules de surface pour former un réseau d'interactions moléculaires, le « tetraspanins web ». Il a été proposé que via l’organisation de ce réseau, les tétraspanines joueraient un rôle dans la compartimentation membranaire des protéines auxquelles elles s’associent. Elles jouent également un rôle dans la compartimentation cellulaire en contrôlant le trafic de certaines des protéines associées. Le laboratoire a précédemment démontré que six tétraspanines conservées (Tspan5, 10, 14, 15, 17 et 33) de la sous-Famille des TspanC8 (caractérisées par la présence de huit cystéines dans le grand domaine extracellulaire) interagissent directement avec la métalloprotéase ADAM10 et régulent sa sortie du réticulum endoplasmique. Cette protéase ancrée à la membrane est responsable du clivage protéolytique de l'ectodomaine de diverses molécules de surface et est indispensable pour l'activation de la voie de signalisation NOTCH. Nous démontrons que Tspan5 et Tspan14 sont des régulateurs positifs de la voie de signalisation Notch et que Tspan15 est un régulateur négatif, agissant en amont de la γ-Sécrétase. Cette régulation négative est associée à un changement d’environnement membranaire ainsi qu’à une dynamique différente d’ADAM10 comme le montre un suivi de la protéase à l’échelle de la molécule unique. Par ailleurs, l’analyse par spectrométrie de masse quantitative a montré que la capacité d’ADAM10 à s’associer à des composants connus du réseau de tétraspanines était influencée par son interaction avec ces tétraspanines. Enfin, nous avons identifié une région de la cellule enrichie en CD9 qui contenait également ADAM10 lorsque Tspan5 est exprimée, mais ce n’est pas le cas avec Tspan15. Cette étude démontre que les différentes TspanC8 influencent différemment la capacité d’ADAM10 à cliver certains de ses substrats et illustre la capacité des tétraspanines à réguler l’activité de leurs protéines partenaires en contrôlant leur compartimentation membranaire. / It is now recognized that the lateral segregation of components of the plasma membrane or membrane compartmentalization, play an important role in the regulation of many membrane proteins functions. Tetraspanins are a family of integral membrane proteins with four transmembrane domains with a unique ability to associate with one another and with many other surface molecules to organize a molecular interactions network, the “tetraspanins web”. It was suggested that via the organization of this network, the tetraspanin play a role in membrane compartmentalization of proteins to which they associate. Tetraspanins also play a role in cellular compartmentalization by controlling the trafficking of some associated proteins. In the lab, it has been shown that six conserved tetraspanin (Tspan5, 10, 14, 15, 17 et 33) of the TspanC8 subgroup (characterized by the presence of eight cysteines in the large extracellular domain) interact directly with the metalloproteinase ADAM10 et mediates its exit from the endoplasmic reticulum. This membrane-Anchored metalloprotease mediated ectodomain shedding of various integral molecules and is essential for Notch signaling pathway activation. We demonstrate that both Tspan5 and Tspan14 are positive regulators of Notch signaling pathway, whereas Tspan15 appears as negative regulator, acting in upstream γ-Secretase. This downregulation is associated with a modification of membrane environment as well as different dynamics of ADAM10, as shown by monitoring of protease using single molecule tracking. Furthermore, quantitative mass spectrometry analysis revealed that ADAM10 ability to associate with known components of “tetraspanin web” was influenced by its interaction with these tetraspanins. Finally, we have identified a CD9 enriched cell area which also contained ADAM10 when Tspan5 is expressed but not with Tspan15.This study demonstrates that different TspanC8 influence differently the ADAM10 ability to cleave some of its substrates and illustrate the ability of tetraspanins to regulate the activity of their proteins partners by controlling their membrane compartmentalization.

Thesis (Ph. D.)--Case Western Reserve University, 2004. / [School of Medicine] Department of Pathology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Dissertation (Ph.D.)--University of Toledo, 2010. / "Submitted to the Graduate Faculty In partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Science." Title from title page of PDF document. "A Dissertation entitled"--at head of title. Non-Latin script record Bibliography: p. 68-101.

Orientadores: Eneida de Paula, Cleyton Crepaldi Domingues / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T17:11:37Z (GMT). No. of bitstreams: 1 Casadei_BrunaRenata_M.pdf: 5692711 bytes, checksum: 9dcfea152b622eb3bd7d81b793794e3e (MD5) Previous issue date: 2011 / Resumo: A visão atual sobre membranas biológicas abarca descrições cada vez mais complexas devido, principalmente, à descoberta de novos papéis atribuídos aos lipídios e sua heterogeneidade. Em particular a associação de esfingolipídios e colesterol, acrescida de proteínas específicas, constitui a base da formação de domínios membranares conhecidos como lipid rafts. Rafts são microdomínios funcionais de biomembranas e estão envolvidos em diversos processos biológicos como reconhecimento celular, endocitose, transdução de sinal, entre outros processos. Uma estratégia experimental para estudar esses domínios é a preparação de frações de membrana parcialmente resistentes ao tratamento com detergentes, a baixa temperatura (4°C). Neste trabalho demonstramos pela primeira vez o preparo e caracterização de frações resistentes a detergente (DRMs) extraídas de membranas de eritrócito humano, a partir do tratamento com os detergentes não iônicos polioxietileno 20-oleil éter (Brij 98) e polioxietileno 20-cetil éter (Brij 58), seguida de separação por ultracentrifugação em gradiente de sacarose. Essas DRMs foram obtidas a 4°C e 37°C, a partir de membranas intactas e com conteúdo reduzido de colesterol (após tratamento com metil-beta-ciclodextrina) e foram comparadas com DRMs de Triton X-100 (TX-100) em relação ao tamanho, conteúdo proteico e lipídico e grau de organização da bicamada. As frações de DRM de Brij mostraram-se enriquecidas em colesterol e fosfolipídios com ácidos graxos de cadeia saturada (em especial ácido lignocérico das esfingomielinas), características consistentes com lipid rafts. No entanto, DRMs de TX-100 apresentaram maior proporção de esfingomielinas/glicerofosfolipídios que as frações obtidas com Brij, além de menor proporção de fosfatidiletanolamina, um lipídio preferencial da monocamada interna da membrana de eritrócitos. Em relação à solubilização proteica, a membrana do eritrócito foi mais resistente ao tratamento com Brij 98 do que aos outros dois detergentes. Flotilina-2 e estomatina foram encontradas nas frações de DRMs de Brij 98 e Brij 58, porém a redução do conteúdo de colesterol da membrana eritrocitária resultou numa menor associação da flotilina-2 àquelas DRMs. Resultados de Ressonância Paramagnética Eletrônica, com uso de marcadores de spin do tipo doxil-estearato não acusaram variação significativa no grau de empacotamento dos lipídios nas bicamadas de DRMs de Brij 98 e Brij 58 em relação à membrana eritrocitária, diferentemente do observado em DRMs de TX-100 e do que seria esperado para domínios lipídicos na fase líquido-ordenada. Em conclusão, DRMs de eritrócitos humanos, com características compatíveis aos domínios funcionais de biomembranas (rafts), foram obtidas tanto a 4ºC quanto a 37ºC; essas frações resistentes aos Brij apresentaram tamanho, composição proteica e lipídica e grau de empacotamento da bicamada diferente das DRMs de TX-100, indicando um processo de extração diferencial dos componentes da membrana eritrocitária induzida por esses detergentes / Abstract: Depiction of biological membranes has been turning more and more complex lately due to the new roles assigned to their lipid components and their heterogeneity. The association between sphingolipids and cholesterol is the basis of lipid rafts formation. Rafts are functional microdomains of biological membranes which have been associated to different biological processes such as cellular recognition, endocytosis and signal transduction, among others. An useful experimental approach to study lipid rafts is the preparation of membrane fractions partially resistant to detergents under low temperature (4ºC). In this work we report the isolation of detergent resistant membranes (DRMs) from human erythrocytes treated with the non-ionic detergents polioxyethylene 20-oleoyl ether (Brij 98) and polioxyethylene 20-cetyl ether (Brij 58) followed by sucrose gradient ultracentrifugation. Such DRMs were obtained at 4°C and 37°C, from cholesterol-depleted (treated with methyl-beta-cyclodextrin) and intact erythrocyte membranes and they compared to DRMs obtained with Triton X-100 (TX-100) regarding the size, protein and lipid content and membrane fluidity. Brij DRMs were found to be enriched in cholesterol and phospholipids containing saturated fatty acids (especially those from sphingomyelin), features commonly associated to lipid rafts. Nevertheless, TX-100 DRMs presented higher sphingomyelin/phospholipid ratios and lower phosphatidylethanolamine content (a glycerophospholipid mainly present in the inner leaflet) than the Brij's. As for protein solubilization, erythrocyte membranes were more resistant to Brij 98 than to the other two detergents. Flotillin-2 and stomatin were found in both Brij 98 and Brij 8 DRMs. However, flotillin-2 was partially solubilized when DRMs were prepared from cholesterol-depleted erythrocyte membrane. Electron paramagnetic resonance experiments, with doxyl stearic acid spin labels incorporated in the DRMs showed no significant changes in the bilayer compactness of Brij 98 and 58 DRMs in comparison to intact membrane, a unexpected result for lipid domains existing in a liquid-ordered phase, and in contrast to results previously observed with TX-100 DRMs. Altogether, these results show the isolation of DRMs from human erythrocytes treated with Brij 98 and Brij 58 at low (4°C) and physiological temperature (37°C). These detergent-resistant membrane fractions, obtained with Brij 98 and 58 presented some lipid rafts features, although with differences in protein and lipid contents, size and membrane fluidity in comparison to TX-100 DRMs. Besides, these results suggest that TX-100, Brij 98 and Brij 58 induced a different solubilization process on the erythrocyte membrane / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Dissertation summary The role of membrane microdomains and transmembrane adaptor proteins PRR7 and SCIMP in the regulation of immunoreceptor signaling Matouš Hrdinka How do the plasma membrane microdomains and transmembrane adaptor proteins (TRAPs) influence the outcome of immunoreceptor signaling? These have been the important questions of molecular immunology. In spite of the years of intensive research, these problems remain incompletely understood. The plasma membrane is a highly dynamic heterogeneous bilayer spontaneously organized into microdomains of various size, composition, and lifetime. The lipid rafts are one example of such microdomains and have been implicated in many biological processes, including immunoreceptor signaling. Because rafts are enriched in many signaling proteins, they are believed to function as platforms for signal initiation and propagation. The TRAPs are important organizers and regulators of immunoreceptor signaling. For example, LAT is indispensable in T cell receptor (TCR) signaling and T cell development, PAG for the regulation of Src family tyrosine kinases (SFKs), and NTAL is a multifunctional negative and positive regulator. The presence of these TRAPs in lipid rafts seems to be crucial for their functions, however, is still a matter of debate. Moreover, other so far...

Membrane rafts are small plasma membrane domains that contain high levels of cholesterol and sphingolipids. They have been implicated in processes as diverse as signal transduction, endocytosis and cholesterol trafficking. Traditional methods for the biochemical preparation of lipid rafts involve the extraction of membranes with nonionic detergents followed by the separation of a low-density, detergent-resistant membrane fraction on density gradients. Because of concerns regarding the possible introduction of artifacts through the use of detergents, several methods were developed for the isolation of lipid rafts that do not involve detergent extraction. / In this study, we compared three different biochemical methodologies of membrane rafts preparation from purified rat liver PM. Only detergent-resistant membranes (DRMs) fulfill the requirements of membrane rafts. We subsequently found using the low dose of EGF (1 ug/100 g BW); the content of EGFR in PM-DRMs did not changed significantly following EGF administration. When a higher dose of EGF (5 ug/100 g BW) is administrated we observed a rapid and almost complete disappearance of EGFR (around 80%) from both PM and DRMs fractions. Interestingly, following the administration of a low or high dose of EGF, the pool of EGFR in the PM-DRMs fraction becomes highly Tyr-phosphorylated. In accordance with the higher level of EGFR Tyr-Phosphorylation, EGF induced an augmented recruitment of Grb2 and Shc proteins to PM-DRMs compared with whole PM. / Furthermore neither high nor low dose of EGF affects the caveolin content in DRMs and PM. These observations suggest that EGFR located in DRM are competent for signaling and non-caveolae PM rafts are involved in the compartmentalization and presumably internalization of the EGFR.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 107-117).