L'atrophie musculaire bulbo-spinale (SBMA) est une dégénérescence lente et progressive des motoneurones causée par l'élongation du triplet nucléotidique (CAG) dans le gène codant pour le récepteur aux androgènes (RA) localisé sur le chromosome X. Dans la SBMA, ce récepteur à extension polyglutaminique (polyQ) pathogène s'accumule de manière ligand dépendante dans le cytoplasme sous forme d'agrégats mais également dans le noyau y créant des corps d'inclusions nucléaires considérés comme la marque identitaire histologique, dont le caractère cytotoxique est aujourd'hui remis en question. Nous avons développé un modèle SBMA in vitro basé sur l'expression inductible d'un RA51Q dans la lignée hybride NSC34, qui est comparé au modèle normal NSC34 exprimant un RA contenant 20Q. Nous avons démontré que l'expression du RA51Q entraîne une diminution de la viabilité ainsi qu'une altération de la croissance neuritique sans formation d'agrégats insolubles dans le noyau ou le cytoplasme des cellules. Le RA en tant que membre de la superfamille des récepteurs nucléaires est un facteur de transcription mais peut également induire des voies de signalisation non génomiques via sa localisation membranaire. Après avoir montré une localisation du RA20Q et du RA51Q dans les « lipid rafts », nous avons corrélé la diminution de la viabilité et de la pousse neuritique induite par le RA51Q à une altération de la signalisation cellulaire non génomique. Les résultats obtenus mettent en évidence une dérégulation des voies de signalisation PI3K/Akt et JNK/c-jun induite par l'expression du RA muté dans notre modèle SBMA. / Spinal Bulbar Muscular Atrophy (SBMA) is a progressive inherited motoneuron disease caused by the expansion of a trinucleotide (CAG) repeat in the gene coding for the androgen receptor (AR) located on the X chromosome. This rare disease causes muscle weaknesses, hypotonia, hyporeflexia, fasciculations of facial muscles in male patients. The androgen-dependent formation of cytoplasmic aggregates and nuclear inclusions are pathological hallmarks of this polyglutamine disease but their potential neurotoxicity is still under debate. We developed a SBMA model based on a doxycycline-inducible AR51Q expression system in the NSC34 hybrid cell line. We have shown that the expression of the mutated AR leads to a reduced viability and to an alteration of neurite outgrowth compared to cells expressing the normal AR20Q. The AR belongs to the nuclear receptor superfamily of transcription factors. However, recent data have put in evidence a membrane localization of AR initiating non-genomic signaling pathways. Because we have not observed insoluble aggregates, reduced viability and neurite outgrowth could not be correlated to AR aggregation. We hypothesized that motoneuron death is not only due to aggregate formation but also to the alteration of AR signaling pathways. We focused on a correlation between the AR localization in lipid rafts and the observed phenotypes. Our results highlight the deregulation of PI3K/Akt and JNK/c-jun signaling pathways induced by the expression of AR51Q in our SBMA model.

L’entérotoxine stable à la chaleur STb est produite par les Escherichia coli entérotoxinogènes (ETEC). Son rôle dans la diarrhée post-sevrage porcine est établi. L’internalisation de STb a été observée dans des cellules épithéliales intestinales humaines et de rat. Cependant, le mécanisme d’internalisation n’est pas totalement compris, particulièrement dans le jéjunum porcin, la cible in vivo de STb. Par la cytométrie en flux, nous avons examiné l’internalisation de STb couplée à un marqueur fluorescent dans les cellules épithéliales intestinales porcines IPEC-J2 et les fibroblastes murins NIH3T3. Nos résultats révèlent que l’internalisation de STb est températureindépendante dans les IPEC-J2 tandis qu’elle est température-dépendante dans les NIH3T3, où la réorganisation de l’actine est aussi nécessaire. Toutefois, les niveaux de sulfatide, le récepteur de STb, sont semblables à la surface des deux lignées. Le sulfatide est internalisé à 37°C de façon similaire entre les deux types cellulaires. La rupture des lipid rafts, les microdomaines membranaires contenant le sulfatide, par la méthyl-βcyclodextrine ou la génistéine, n’affecte pas l’internalisation de STb dans les deux lignées. Notre étude indique que le mécanisme d’internalisation de STb est dépendant du type cellulaire. L’activité de la cellule hôte peut être requise ou non. Le récepteur de STb, le sulfatide, n’est pas directement impliqué dans ces mécanismes. L’internalisation activité cellulaire-dépendante suggère une endocytose, nécessitant la réorganisation de l’actine mais pas les lipid rafts. L’internalisation de STb est donc un processus complexe dépendant du type cellulaire, qu’il apparait plus relevant d’étudier dans des modèles cellulaires représentatifs des conditions in vivo. / Heat-stable enterotoxin b (STb) is one of the toxins produced by enterotoxigenic Escherichia coli (ETEC) and its role in swine post-weaning diarrhea is well established. Internalization of STb in intestinal human and rat epithelial cells has been shown by previous studies. However, the uptake mechanism is still not fully understood, especially in porcine jejunum epithelium, the in vivo STb target. Using flow cytometry, we studied internalization of fluorescently-labelled STb in porcine epithelial intestinal IPEC-J2 and murine fibroblast NIH3T3 cell lines. Our results revealed that STb is internalized in both cell lines. Toxin uptake is not dependent on the temperature in IPEC-J2 cells, whereas it is in NIH3T3 fibroblasts. Actin reorganization is only required for STb internalization in NIH3T3 cells. However, membrane sulfatide, the toxin receptor, is similarly present in both cell lines and similarly internalized with time at 37°C. Disruption of lipid rafts, known to contain sulfatide, with inhibitors (methyl-βcyclodextrin or genistein), did not affect toxin uptake in both cell lines. Altogether, these data indicate that STb internalization mechanisms are cell-type dependent. Moreover, uptake can depend on host cell activity or not. Sulfatide, the toxin receptor, is not directly involved in these mechanisms. Uptake independent on cell activity occurs in porcine intestinal epithelium. The cell activity-dependent uptake suggests an endocytosis, which requires actin rearrangement and is not mediated by lipid rafts. STb internalization is therefore a complex process varying upon cell type, which should preferentially be studied in cellular models representative of in vivo conditions, such as porcine cell lines.

In den vergangenen Jahren sind Lipide, Membranen und deren Organisationsformen mehr und mehr in den Fokus der biologischen Forschung gerückt. Es wurde vorgeschlagen, dass in zellulären Membranen selbstassemblierende, submikroskopische Aggregate aus Sphingolipiden, Cholesterol und bestimmten Proteinen existieren und man vermutet, dass insbesondere Viren diese “Lipid Rafts” für ihren Zusammenbau nutzen und auf diese Art ihre Proliferationseffizienz erhöhen. Gleichwohl sind die genaue biologische Funktion und auch die molekulare Basis der Assoziation bestimmter Protein mit Lipid Rafts auch weiterhin unbekannt. In der vorliegenden Arbeit wurde Fluoreszenz-Lebenszeit-Mikroskopie genutzt, um die Lipid-Raft-Anreicherung des HIV-1 Glycoproteins gp41 zu untersuchen. Förster-Resonanz-Energietransfer zwischen fluoreszenzmarkierten viralen und Raft-Marker-Proteinen wurde gemessen, um deren gemeinsame, lokale Aufkonzentrierung in Lipid Rafts nachzuweisen. Durch Verwendung verschiedener Deletions- und Mutationsvarianten des Proteins konnte nicht nur seine Lipid-Raft-Präferenz demonstriert, sondern auch das Cholesterol-Bindemotiv (CRAC) als entscheidender Faktor der lateralen Sortierung identifiziert werden. Wir haben in diesem Kontext auch eine systematische Zell-zu-Zell-Variabilität in unseren Daten bemerkt, die einen zugrundeliegenden zellbiologischen Mechanismus der Membranorganisation nahelegt. Mithilfe von Fluoreszenz-Polarisations-Mikroskopie konnte zudem eine klare CRAC-Abhängigkeit der gp41-Oligomerisierung aufgezeigt werden. Die von uns gewonnenen Daten erlauben einen tieferen Einblick in die molekulare Basis und die biologischen Folgen der cholesterol-abhängigen lateralen Proteinorganisation für Virusassemblierungsprozesse an biologischen Membranen. / In recent years, there has been a considerable interest in the molecular organization of biological membranes. It has been hypothesized that self-assembling, freely diffusing, submicroscopic domains consisting of sphingolipids, cholesterol and certain proteins exist and the prevailing view is that those lipid rafts serve as platforms for specific molecular interactions by the preferential exclusion and inclusion of proteins. It was presumed, that in particular viruses make use of plasma membrane lipid rafts to augment the infection process and spread efficiently. However, the exact biological function and physical basis of protein partitioning into microdomains remains an outstanding question in virus biology. In the present study, fluorescence lifetime imaging microscopy was used to study lipid raft partitioning of the HIV-1 glycoprotein gp41 by detecting Foerster Resonance Energy Transfer between fluorescently labeled viral and raft marker proteins in living cells. Plasma membrane microdomain association of gp41 was demonstrated and by introducing systematic mutations and truncations in different gp41 motifs, the cholesterol recognition amino acid consensus (CRAC) was identified as the crucial determinant of the lateral sorting. Interestingly, we observed a systematic cell-to-cell variability in our raft related data that suggests underlying cell-biological mechanisms of membrane organization. Moreover, fluorescence polarization microscopy revealed a distinct CRAC requirement for gp41 oligomerization whereas other properties, such as intracellular distribution and expression efficiency were clearly demonstrated to be CRAC independent. Our data provide further insight into the molecular basis and biological implications of the cholesterol dependent lateral protein sorting for virus assembly processes at cellular plasma membranes.

Der Viruspartikelzusammenbau hängt von der Anreicherung viraler Untereinheiten in spezifischen Domänen der PM ab. Es wird vorgeschlagen, dass Membran-Rafts – geordnete, sphingomyelin- und cholesterinreiche Mikrodomänen in der PM – als lokale Rekrutierungsstellen dienen. Hämagglutinin (HA) ist ein homotrimeres Glykoprotein in der Hülle des Influenzavirus. Es dient der Bindung an die Wirtszelle und der Fusion mit dem Endosom. Es wird angenommen, dass HA bei der Abschnürung der Viruspartikel von der Zelle mitwirkt. Zwei Hauptbeobachtungen führten zu der Hypothese, dass sich HA in Lipid-Mikrodomänen einlagert: HA wurde biochemisch in Detergens-resistenten Membranen nachgewiesen und die Virushülle ist mit raftbildenden Lipiden angereichert. Um die Rolle der HA-Transmembrandomäne für die Lipid-Raft-Inkorporation aufzuklären, wurde ein Konstrukt entwickelt, das den C-Terminus von HA mit dem gelb fluoreszierenden Protein YFP fusioniert, und die Transmembrandomäne, nicht aber die N-terminale Ektodomäne von HA enthält. In transfizierten Säugetierzellen wurde der Förster-Resonanz-Energie-Transfer (FRET) zwischen diesem Konstrukt und einem GPI-verankerten cyan fluoreszierenden Protein CFP (Raft-Marker) durch Fluoreszenz-Lebenszeit-Mikroskopie (FLIM) gemessen. Die Ergebnisse zeigen, dass sich HA-Konstrukte in Cholesterin-abhängigen Lipiddomänen anreichern, was durch eine erhöhte FRET-Effizienz nachgewiesen wurde. Zudem führen ein Cholesterinentzug aus der PM und die Deletion hochkonservierter Palmitylierungsstellen zu einer signifikanten Verringerung selbiger; sehr gering war diese zwischen dem HA-Konstrukt und einem Nicht-Raft-Marker. Darüberhinaus konnte durch ortsspezifische Mutagenese gezeigt werden, dass die verwendeten Konstrukte disulfidbrückenverbundene Oligomere bilden, welche Voraussetzung für den Transport der Konstrukte an die PM sind. Zeitaufgelöste Anisotropiemessungen ergaben für diese ein starkes Homo-FRET-Signal, welches die Oligomerisierungshypothese bestätigt. / Numerous enveloped viruses bud from the host cell plasma membrane (PM). Assembly of the new viral particles depends on the accumulation of the viral subunits at specific sites of the cell membrane. Lipid domains or rafts enriched of sphingomyelin and cholesterol were suggested as sites for local recruitment of viral components. Hemagglutinin (HA), a homotrimeric glycoprotein embedded in the envelope of influenza virus, mediates binding of the virus to the host cell and fusion between the viral envelope and the endosomal membrane. HA might play an important role in budding of the viral particles from the host cell. Two observations led to the suggestion that HA entraps in lipid microdomains. First, HA was rescued in DRM fractions, second the viral envelope was found to be enriched in lipids generally forming rafts. To elucidate the role of the HA transmembrane domain in lipid raft localization we expressed constructs harboring the transmembrane domain and the cytoplasmic tail but lacking the N-terminal ectodomain of HA in the PM of mammalian cells. We studied energy transfer (FRET) between these constructs and a GPI anchored CFP as a raft marker by fluorescence lifetime imaging microscopy (FLIM). Our results suggest that HA constructs are indeed sorted into cholesterol-dependent lipid domains since cholesterol depletion of the PM caused a significant decrease of FRET efficiency. Likewise, deletion of the three highly conserved palmitoylation sites of HA is also accompanied by a reduction of FRET efficiency. Site directed mutagenesis demonstrated that TMD-HA constructs form disulfide linked oligomers and that oligomerization is fundamental for the transport to the PM. This result was corroborated by time resolved anisotropy measurements that revealed strong homoFRET between TMD-HA-YFP molecules, thus indicating protein clustering. Accordingly, trimerization of full length HA is fundamental for stability and the subsequent delivery of the protein to the cell surface.

Die Degranulation von Mastzellen soll durch Glycosphingolipide, welche mit der Zellmembran wechselwirken inhibiert werden. Der Sphingosingrundkörper wurde in zehn-stufigen Synthese ausgehend von N-Boc-Serin, aufgebaut. Die anschließende Glycosylierung erfolgte nach der Trichloracetimidatmethode in sehr guten Ausbeuten und stellte den Schlüsselschritt dar. Durch die Variation von unter Anderem der Amidseitenkette, der Glycosylkopfgruppe und des Sphingosingrundkörpers wurde eine Vielzahl an Derivaten für das Screening im Degranulationsassay bereitgestellt. / The present dissertation covers the synthesis of glycosphingolipids which interact with the cell membrane in order to inhibit the degranulation of mast cells. The sphingosin body was synthesized in ten steps starting from N-Boc-Serin. The key step, the glycosylation was achieved using the trichloracetimidat method. The variation of the amid sidechain, the gylcosyl headgroup and the sphingosin body created a number of derivatives that were tested in the degranulation assay.

L’entérotoxine stable à la chaleur STb est produite par les Escherichia coli entérotoxinogènes (ETEC). Son rôle dans la diarrhée post-sevrage porcine est établi. L’internalisation de STb a été observée dans des cellules épithéliales intestinales humaines et de rat. Cependant, le mécanisme d’internalisation n’est pas totalement compris, particulièrement dans le jéjunum porcin, la cible in vivo de STb. Par la cytométrie en flux, nous avons examiné l’internalisation de STb couplée à un marqueur fluorescent dans les cellules épithéliales intestinales porcines IPEC-J2 et les fibroblastes murins NIH3T3. Nos résultats révèlent que l’internalisation de STb est températureindépendante dans les IPEC-J2 tandis qu’elle est température-dépendante dans les NIH3T3, où la réorganisation de l’actine est aussi nécessaire. Toutefois, les niveaux de sulfatide, le récepteur de STb, sont semblables à la surface des deux lignées. Le sulfatide est internalisé à 37°C de façon similaire entre les deux types cellulaires. La rupture des lipid rafts, les microdomaines membranaires contenant le sulfatide, par la méthyl-βcyclodextrine ou la génistéine, n’affecte pas l’internalisation de STb dans les deux lignées. Notre étude indique que le mécanisme d’internalisation de STb est dépendant du type cellulaire. L’activité de la cellule hôte peut être requise ou non. Le récepteur de STb, le sulfatide, n’est pas directement impliqué dans ces mécanismes. L’internalisation activité cellulaire-dépendante suggère une endocytose, nécessitant la réorganisation de l’actine mais pas les lipid rafts. L’internalisation de STb est donc un processus complexe dépendant du type cellulaire, qu’il apparait plus relevant d’étudier dans des modèles cellulaires représentatifs des conditions in vivo. / Heat-stable enterotoxin b (STb) is one of the toxins produced by enterotoxigenic Escherichia coli (ETEC) and its role in swine post-weaning diarrhea is well established. Internalization of STb in intestinal human and rat epithelial cells has been shown by previous studies. However, the uptake mechanism is still not fully understood, especially in porcine jejunum epithelium, the in vivo STb target. Using flow cytometry, we studied internalization of fluorescently-labelled STb in porcine epithelial intestinal IPEC-J2 and murine fibroblast NIH3T3 cell lines. Our results revealed that STb is internalized in both cell lines. Toxin uptake is not dependent on the temperature in IPEC-J2 cells, whereas it is in NIH3T3 fibroblasts. Actin reorganization is only required for STb internalization in NIH3T3 cells. However, membrane sulfatide, the toxin receptor, is similarly present in both cell lines and similarly internalized with time at 37°C. Disruption of lipid rafts, known to contain sulfatide, with inhibitors (methyl-βcyclodextrin or genistein), did not affect toxin uptake in both cell lines. Altogether, these data indicate that STb internalization mechanisms are cell-type dependent. Moreover, uptake can depend on host cell activity or not. Sulfatide, the toxin receptor, is not directly involved in these mechanisms. Uptake independent on cell activity occurs in porcine intestinal epithelium. The cell activity-dependent uptake suggests an endocytosis, which requires actin rearrangement and is not mediated by lipid rafts. STb internalization is therefore a complex process varying upon cell type, which should preferentially be studied in cellular models representative of in vivo conditions, such as porcine cell lines.

The structure and functions of lipid rafts and the mechanisms of intracellular membrane trafficking are major topics in current cell biological research. Rafts have been proposed to act as sorting platforms during biosynthetic transport, especially along pathways that deliver proteins to the apical membrane of polarised cells. Based on this, the aim of this work was to contribute to the understanding of apical sorting in epithelial cells. The study of how lipid rafts are structured has been hampered by the scarcity of techniques for their purification. Rafts are thought to be partially resistant to solubilisation by mild detergents, which has made the isolation of detergent-resistant membranes (DRMs) the primary method to characterise them biochemically. While a growing number of detergents is being used to prepare DRMs, it is not clear what can be inferred about the native structure of cell membranes from the composition of different DRMs. This issue was addressed by an analysis of DRMs prepared with a variety of mild detergents. The protein and lipid content of different DRMs from two cell lines, Madin-Darby canine kidney (MDCK) and Jurkat cells, was compared. It was shown that the detergents differed considerably in their ability to selectively solubilise membrane proteins and lipids. These results make it unlikely that different DRMs reflect the same underlying principle of membrane organisation. Another obstacle for understanding apical sorting is that the evidence implicating certain proteins in this process has come from various disparate approaches. It would be helpful to re-examine the putative components of the apical sorting machinery in a single experimental system. To this end, a retroviral system for RNA interference (RNAi) in MDCK cells was established. Efficient suppression of thirteen genes was achieved by retroviral co-expression of short hairpin RNAs and a selectable marker. In addition, the system was extended to simultaneously target two genes, giving rise to double knockdowns.Retroviral RNAi was applied to deplete proteins implicated in apical sorting. Surprisingly, none of the knockdowns analysed caused defects in surface delivery of influenza virus hemagglutinin, a common marker protein for apical transport. Therefore, none of the proteins examined is absolutely required for transport to the apical membrane of MDCK cells. Cells may adapt to the depletion of proteins involved in membrane trafficking by activating alternative pathways. To avoid such adaptation, a visual transport assay was established. It is based on the adenoviral expression of fluorescent marker proteins whose surface transport can be followed microscopically as soon as RNAi has become effective. With this assay, it should now be possible to screen the knockdowns for defects in surface transport. Taken together, this work has provided a number of experimental tools for the study of membrane trafficking in epithelial cells. First, the biochemical analysis of DRMs highlighted that DRMs obtained with different detergents are unlikely to correspond to distinct types of membrane microdomains in cell membranes. Second, the retroviral RNAi system should be valuable for defining the function of proteins, not only in membrane transport, but also in processes like epithelial polarisation. Third, the visual assay for monitoring the surface transport of adenovirally expressed marker proteins should be suitable to detect defects in polarised sorting.

Die Degranulation von Mastzellen soll durch Glycosphingolipide, welche mit der Zellmembran wechselwirken inhibiert werden. Der Sphingosingrundkörper wurde in zehn-stufigen Synthese ausgehend von N-Boc-Serin, aufgebaut. Die anschließende Glycosylierung erfolgte nach der Trichloracetimidatmethode in sehr guten Ausbeuten und stellte den Schlüsselschritt dar. Durch die Variation von unter Anderem der Amidseitenkette, der Glycosylkopfgruppe und des Sphingosingrundkörpers wurde eine Vielzahl an Derivaten für das Screening im Degranulationsassay bereitgestellt. / The present dissertation covers the synthesis of glycosphingolipids which interact with the cell membrane in order to inhibit the degranulation of mast cells. The sphingosin body was synthesized in ten steps starting from N-Boc-Serin. The key step, the glycosylation was achieved using the trichloracetimidat method. The variation of the amid sidechain, the gylcosyl headgroup and the sphingosin body created a number of derivatives that were tested in the degranulation assay.

No description available.

Cells in the body communicate with each other in order to cooperate efficiently. This communication is in part achieved by regulated secretion of signaling molecules, which when released from a cell may activate receptors present at the plasma membrane of an adjacent cell. Such signals affect both cell fate and behavior. Dysregulated signaling may lead to disease, including cancer. This thesis is focused on how exocytosis and subsequent activation and trafficking of receptors can be regulated, and what the consequences of this regulation may be for cell migration. Actin filaments are important transport structures for secretory vesicle trafficking. In Paper 1, actin polymerization was shown to induce formation of ordered lipid domains in the plasma membrane. Accordingly, actin filaments may thus create and stabilize specific membrane domains that enable docking of vesicles containing secretory cargo. The RhoGEF FGD5 regulates Cdc42 which can result in cytoskeletal rearrangements. In Paper II, FGD5 was shown to be selectively expressed in blood vessels and required for normal VEGFR2 signaling. FGD5 protected VEGFR2 from proteasome-mediated degradation and was essential for endothelial cells to efficiently respond to chemotactic gradients of VEGFA. The exocyst component EXOC7 is essential for tethering secretory vesicles to the plasma membrane prior to SNARE-mediated fusion. In Paper III, EXOC7 was required for trafficking of VEGFR2-containing vesicles to the inner plasma membrane and VEGFR2 presentation at the cell surface. The ability of tumor cells to escape the primary tumor and establish metastasis is in part dependent on their capacity to migrate. In Paper IV, a method based on time-lapse microscopy and fluorescent dyes was created to analyze single cancer cell migration in mixed cancer cell cultures, and in particular the influence of different types on neighboring cells was assessed. In conclusion, these studies have enhanced our understanding of the mechanisms behind cellular trafficking, and may be applied in the future to develop more specific therapeutics to treat cancer and other diseases associated with abnormal angiogenesis and cellular migration.