L’invasion cellulaire est un phénomène pendant lequel les cellules présentent des changements importants dans leur forme et acquièrent la capacité à dégrader les structures qui les entourent afin de migrer au sein d’un organisme. L’invasion est cruciale pour la stabilité physiologique d’un organisme car elle intervient à différentes étapes de notre vie notamment lors du développement embryonnaire ou pendant une réaction immunitaire. Cependant ce mécanisme est également majeur lors de la progression tumorale. Dans nos organismes, les cellules sont entourées par la matrice extracellulaire (MEC) qui doit être remodelée ou dégradée lors du processus d’invasion. Cette dégradation est réalisée par des structures cellulaires spécialisées, les invadopodes, qui sont des sites où sont libérés les protéines spécialisées dans la dégradation de la MEC, parmi lesquelles MT1-MMP joue un rôle prépondérant. Les capacités à envahir et à dégrader la MEC d’une cellule dépendent donc fortement de la libération de MT1-MMP aux sites de dégradation. Les mécanismes qui permettent le transport de MT1-MMP aux zones d’intérêts sont encore mal compris. Dans ce contexte, nous avons mis en évidence que les Flotillines sont d’importants régulateurs de l’adressage et la libération de MT1-MMP à ces sites de dégradations. Les Flotillines 1 et 2 sont des protéines ubiquitaires très conservées. La quantité de Flotillines est augmentée dans de nombreux cancers invasifs et ceci est considéré comme un marqueur de mauvais pronostic. Lors de mon projet, nous avons mis en évidence que l’augmentation de la quantité de Flotillines dans des cellules normales de différentes origines, est suffisante pour induire une forte invasion cellulaire in vivo et in vitro (respectivement dans des modèles de xénogreffes chez le poisson zèbre dans modèles de sphéroïdes 3D dans des matrices de collagènes). De même, la suppression des Flotillines dans des cellules cancéreuses est associée à une diminution de leurs capacités invasives in vivo et in vitro. Ces résultats s’expliquent par le fait que les Flotillines régulent le trafic intracellulaire de MT1-MMP et augmentent sa libération aux sites de dégradation favorisant ainsi l’invasion cellulaire. / Tumor cell invasion and consecutive metastasis formation are the main cause of death in cancer patients. Invading tumor cells are surrounded by stroma and extracellular matrix (ECM) that is remodeled or degraded during the metastatic process. ECM degradation is mediated by specialized organelles called invadosomes. Their function strongly depends on matrix metalloproteinases (MMPs) that degrade ECM. Among all the MMPs, MT1-MMP plays a major role the invasive behavior of metastatic cells.Flotillin 1 and 2 are two ubiquitous and highly conserved membrane proteins that can assemble in large oligomers, known to participate in membrane proteins clustering and endocytosis. Flotillins are overexpressed in many invasive cancers and considered as markers of poor prognosis, results we confirmed using several sarcoma and carcinoma models. During my project we identified Flotillins as regulators of MT1-MMP trafficking and cell invasion.We used a dual reciprocal approach consisting of the overexpression of Flotillins in non tumoral cells and of down-regulation of Flotillins in metastatic cells. We showed that flotillins downregulation in invasive cancer cells dramatically inhibit their invasive properties as monitored in vitro using a 3D-collagen invasion assay and in vivo using zebrafish xenografts. Reciprocally, ectopic overexpression of Flotillins in non-tumoral cells is sufficient to induce their invasive behavior in vitro and in vivo. This increase of invasion is mainly due to a higher ability to degrade the matrix in a MT1-MMP-dependent manner. Finally, we showed that Flotillins are critical regulators of the trafficking and the release of MT1-MMP at the degradation site.

In dieser Arbeit beleuchten wir die subzelluläre Lokalisation von Flotillin unter Einfluss von Cholesterin. Wir zeigen, dass die zelluläre Lokalisation abhängig vom Cholesterinniveau der Zelle ist. Eine Cholesterindepletion bringt Flotillin in die Plasmamembran, sowie umgekehrt eine Überversorgung mit Cholesterin Flotillin in cholesterinhaltige, endosomale Strukturen führt. Dabei ist die Umverteilung abhängig von der Integrität des Zytoskeletts. Außerdem zeigt die vorliegende Arbeit, dass die Umverteilung von Flotillin von der Plasmamembran hin zu endosomalen, intrazellulären Kompartimenten abhängig vom Vorhandensein von zwei putativen Cholesterinbindungs-/Interaktionsdomänen ist. Aus den gewonnenen Daten ergeben sich weiterhin Hinweise, dass Cholesterin an Flotillin gebunden in das späte Endosomen transportiert wird. In weiterführenden Versuchen unserer Gruppe zeigte sich, dass die exosomale Freisetzung von Cholesterin bei ansteigenden zellulären Cholesterinkonzentrationen erhöht wird und dass die exosomale Cholesterinfreisetzung von Flotillin abhängig ist. Die Daten deuten auf eine mögliche Rolle von Flotillin und Exosomen bei der zellulären Cholesterinhomöostase hin.

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Orientador: Eneida de Paula / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-12T15:46:25Z (GMT). No. of bitstreams: 1 Paula_Eneidade_D.pdf: 7752367 bytes, checksum: 4ac3295742d441a15e315a3d1c9c2c3d (MD5) Previous issue date: 2009 / Resumo: Detergentes constituem uma das ferramentas mais importantes no estudo de membranas biológicas. A eficiência de um detergente em solubilizar biomembranas e suas proteínas depende de suas propriedades físico-químicas e a solubilização parcial pode resultar em membranas resistentes a detergentes (DRMs). A resistência a detergentes dessas frações membranares constituídas principalmente de colesterol, esfingolipídios e proteínas específicas foi analisada nesse trabalho, usando membranas de eritrócitos humanos. Embora não haja evidências experimentais suficientes pra afirmar que DRMs correspondam aos microdomínios naturais existentes em biomembranas, conhecidos como lipid rafts e que também são enriquecidos em colesterol e esfingolipídios, DRMs são bons modelos para o estudo daqueles. A obtenção de DRMs com uso do detergente octaetilenoglicol mono lauril éter (C12E8) a baixa temperatura (4oC) é descrita pela primeira vez. Os detergentes zwiteriônicos ASB-14, ASB-16 e CHAPS também foram testados, mas falharam no isolamento de DRMs com baixa densidade. DRMs obtidas com C12E8 e com Triton X-100 apresentaram um aumento da razão colesterol/proteína de pelo menos três vezes em relação à membrana original. A proteína flotilina-2, considerada um marcador de lipid rafts, foi detectada em DRMs isoladas com Triton X-100 e em DRMs com C12E8 isoladas a partir de células depletadas de colesterol. Proteínas do citoesqueleto também foram encontradas em DRMs, exceto quando as células foram previamente depletadas de colesterol. Resultados de ressonância paramagnética eletrônica com uso de marcadores de spin do tipo doxil-estearato revelaram maior grau de organização das cadeias acila dos lipídios de DRMs em relação à membrana original, independentemente do detergente utilizado. Nossos resultados também mostraram que DRMs de eritrócitos podem ser também obtidos na temperatura fisiológica (37°C) como mesmo conteúdo de colesterol presente em DRMs isoladas a 4°C. A necessidade do uso de carbonato de sódio no protocolo para obtenção de DRMs sugere fortemente a existência de uma associação eletrostática entre DRMs e o citoesqueleto eritrocitário. / Abstract: Detergents constitute an important tool for the study of cell membranes. The solubilization efficiency of a specific detergent depends upon its physicochemical properties so that detergent-resistant membranes (DRMs) can be obtained as a result of the partial solubilization of biological membranes. In this study DRMs which are structures enriched in cholesterol, sphingolipids and specific proteins, were isolated and characterized from human erythrocyte membranes. Although there are no evidences to support that DRMs correspond to the natural existing microdomains of natural membranes, known as lipid rafts which are also cholesterol and sphingolipid-enriched structures, DRMs are good models for the study of rafts. DRMs from erythrocytes obtained with 8 polyoxyethylene lauryl ether (C12E8) at low temperature (4oC) are described here for the first time. The zwitterionic detergents ASB-14, ASB-16 and CHAPS failed to isolate these low buoyant density fractions. Triton X-100 and C12E8 DRMs presented a cholesterol/protein mass ratio 3 times higher than in the whole membrane. Flotillin-2, a marker of lipid rafts, was confined within the DRM fractions obtained with Triton X-100 and it was partially associated with C12E8 DRMs when erythrocyte cells were previously cholesterol-depleted. Association of membrane-skeleton proteins with DRMs was also observed, except when DRMs were prepared from cholesterol-depleted cells. Results of electron paramagnetic resonance through the use of doxyl stearate spin labels revealed that DRMs are highly ordered structures in respect to the original membrane and that their acyl chain packing is not different if prepared with either Triton X-100 or C12E8. We have also found that DRMs from erythrocytes can be isolated at physiological temperature (37°C), presenting the same cholesterol content as in DRMs prepared at 4°C. The fact that we were only able to prepare DRMs when sodium carbonate was used stronglysuggests the existence of an electrostatic association between DRMs and the membrane-skeleton. / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Flotillin-1 és una proteïna associada a membrana plasmàtica implicada en processos de trànsit de vesícules, reordenació del citoesquelet i transducció de senyals. Estudis previs en el laboratori han demostrat que Flotillin-1 és capaç de translocar-se a nucli en resposta a un estímul mitogènic i afavorir la proliferació de diverses línies cel·lulars. Els mecanismes mitjançant els quals provoca aquests efectes són desconeguts i objecte del present estudi.D'una banda demostrem que Flotillin-1 és un factor regulador de la cinasa Aurora B, una proteïna que intervé en el control de la mitosi i més concretament en el anaphase checkpoint. El knock-down de Flotillin-1 provoca events mitòtics aberrants, acompanyats del descens tant en l'expressió d'Aurora B com de la seva activitat mesurada com els nivells de fosforilació de la histona H3. Flotillin-1 interacciona amb Aurora B i evita la seva degradació per la via del proteasoma.D'altra banda, Flotillin-1 interacciona amb el receptor transmembrana Notch1, implicat en nombrosos processos de regulació de proliferació, diferenciació, apoptosi, etc. Flotillin-1 regula la localització subcel·lular de Notch1 així com la seva capacitat com activador transcripcional. La depleció o mutació de Flotillin-1 dificulta l'entrada de Notch1 a nucli i l'expressió dels gens diana de les famílies Hes/Hrt. En conjunt, es presenta a Flotillin-1 com una proteïna capaç d'actuar a diferents nivells i regular processos i vies de senyalització cel·lular que li confereixen un paper com a regulador de la proliferació cel·lular. / Flotillin-1 is a protein associated to plasma membrane involved in vesicle trafficking, cyotskeleton reorganization and signal transduction. Previous findings in our laboratory has shown that Flotillin-1 is able to translocate the nucleus under mitogenic stimulus and increase proliferation rates of several cell lines. The mechanisms of action are unknown and object of the present study.First, we show that Flotillin-1 is a regulator factor of the mitotic kinase Aurora B, a protein involved in control of mitosis and, specifically, in the anaphase checkpoint. The knock-down of Flotillin-1 causes aberrant mitotic events, decrease in Aurora B levels and its activity, measured as protein levels of phosporilated histone H3. Flotillin-1 interacts with Aurora B and avoid its degradation by the proteasome pathway.In addition, Flotillin-1 interacts with the transmembrane receptor Notch1, involved in many regulatory processes of proliferation, differentiation, apoptosis, etc. Flotillin-1 regulates the subcellular localization of Notch1 and its activity as transcriptional activator. The mutation or depletion of Flotillin-1 difficult the entry of Notch1 in the nucleus and the expression of its target genes Hes/ HRT. Overall, Flotillin-1 is a protein capable of acting at different levels, processes and signaling pathways in order to be a regulator of cell proliferation.