Tube formation, or tubulogenesis, is an elaborate form of epithelial morphogenesis that includes processes such as cell migration and cell shape changes. The embryonic Drosophila heart, or dorsal vessel, is an excellent model of tubulogenesis and more specifically the signaling mechanisms required for cell migration and lumen formation. Similar to vertebrate heart formation, Drosophila heart tubulogenesis begins with the collective migration of cardioblasts that meet at the midline and adhere at specialised junctions, enclosing a lumen between them. Roundabout, and its ligand Slit, are required to restrict cell-to-cell adhesions to the junctional domains of contralateral cardioblasts, as well as maintain the integrity of the lumen. The localisation patterns of Robo, and other luminal cell surface receptors important for lumen formation are significantly modified throughout heart formation. Initial receptor expression is broadly distributed over the cardioblast surface. Receptors are then relocalised to specific cell surface domains by late embryonic development. The mechanisms by which Robo and other cell surface receptors are localised have yet to be determined. Endocytosis is a promising mechanism by which cell surface receptors are targeted and trafficked to cell surface domains. Specifically, vesicular trafficking proteins, such as Rab GTPases, are molecular switches that regulate endocytic events. Here, we investigated the roles of Rab5, Rab11, and Sec6 during heart formation. Of these, only Rab5, a regulator of the early endosome, was required for lumen formation. Particularly, gain of function, loss of function, and overexpression of rab5 resulted in reduced lumen phenotype, characterised by lumen pockets rather than a continuous lumen along the anterior-posterior axis. Perturbed Rab5 function also resulted in the mislocalisation of Robo at the basal domain. Live imaging showed that expression of rab5 dominant negative, constitutively active, and overexpression constructs did not perturb apical membrane motility of migrating cardioblasts in the developing heart. / Thesis / Master of Science (MSc)

Mit fortschreitendem Durchschnittsalter der Bevölkerung gewinnen altersbedingte Krankheiten immer mehr an Signifikanz. Demenz und Einschränkungen der Beweglichkeit wirken sich auf Individuen sowie auf Familien aus, da die progressive Abnahme kognitiver und physischer Fähigkeiten ihren Tribut von der Lebensqualität Betroffener sowie den Pflegenden fordert. Morbus Parkinson (PD) ist eine neurodegenerative Erkrankung, welche sich durch Symptome des Bewegungsapparates äußert, bedingt durch degenerative Prozesse im Mittelhirn, und welche mit Veränderungen des Gemütszustandes, Verhaltens sowie Depression und Demenz fortschreiten können. PD betrifft in der Regel ältere Personen, jedoch wurden Gene verschiedener zellulärer Funktionen identifiziert, deren Mutation zu einer frühen oder gar juvenilen Ausprägung der Krankheit führen kann. Ein Hauptakteur in PD ist α-Synuclein (ASYN), ein kleines Protein welches in PD-typischen Proteinablagerungen gefunden wurde. Die zelluläre Funktion von ASYN ist immernoch unbekannt, Mutation oder Überexpression jedoch können zu einem hypermorphen Phänotyp führen und die Verbindung zu PD ist daher unumstritten. Studien haben gezeigt, dass ASYN mit Proteintransportwegen und der Aufstellung der Transportmaschinerie interferiert. Genetische Rasterstudien identifizierten Modulatoren von ASYN-Toxizität in Genclustern des Vesikeltransports. Ebenso konnten Studien in Hefe zeigen, dass Überexpression von ASYN diverse Transportwege stört, besonders zu beachten ist hier der ER-zu-Golgi Transportweg, welcher kritisch für Posttranslationale Modifikationen verschiedener Proteine ist. Des Weiteren greift ASYN-Pathologie störend in die Homöostase von Rab GTPasen ein, eine Proteinfamilie involviert in Vesikeltransport, manche deren Mitglieder ASYN-Toxizität reduzieren können. In dieser Studie zeigen wir in einer Rasteruntersuchung mit Rab GTPasen in einem Säugerzellmodell von ASYN-Proteinanreichungen, dass die ASYN-Pathologe zu einer weitreichenden Störung von Rab GTPase assoziierten Transportwegen führt. Wir identifizieren zwei unterschiedliche endosomale Stoffwechselwege welche beim Auftreten von ASYN-Proteinablagerungen fehlreguliert werden: der endosomale-lysosomale-Proteintransportweg welcher das frühe Endosom beinhaltet, sowie den trans-Golgi Netzwerk (TGN) Transportweg. Die kleinen Rab GTPasen Rab5A, Rab7 und Rab8A haben fundamentale Auswirkungen auf die Formation von ASYN-Proteinansammlungen, Sekretion und Toxizität. Wir zeigen dass Rab8A in der Lage ist ASYN-Proteinansammlungen zu modulieren und agiert protektiv in Bezug auf zelluläre Toxizitätslevel in unserem Modell. Rab5A, ein Protein des frühen Endosoms, fehllokalisiert mit Formation der Ablagerungen, während das lysosomale Rab7 die Anzahl der Ablagerungen erhöht, aber nicht ihnen kolokalisiert. Des Weiteren benutzen wir Größenexklusionschromatographie (SEC) und Enzyme Linked Immunosorbent Assay (ELISA) um zu zeigen, dass Rab5A und Rab7 in Abhängigkeit ihres Aktivitätszustandes die Partikelgröße von ASYN ändert und die Sekretion moduliert. Die abschließende Bewertung eines Tiermodells welches humanes ASYN pan-neuronal überexpremiert zeigte, dass lysosomales Rab7 und die Protease Cathepsin D (CatD) in Hirnregionen verantwortlich für Bewegung, Motivation und Gedächtnis herausreguliert sind. Unsere Arbeit sowohl in Säugerzellkultur sowie in transgenen Tieren deutet darauf hin, dass die ASYN-Pathologie Auswirkungen auf das endosomale Transportsystem hat, aber zeigt auch die Fähigkeit von Proteinen, welche mit diesem Transportsystem assoziiert sind, die Toxizität von ASYN zu modulieren. Daher schließen wir, dass Anomalien in der Transportmaschinerie von Endosomen, welche durch Fehlregulation ASYN verursacht wurden, zur Entstehung der PD Pathologie beitragen.

8 ABSTRACT Rab GTPases (Rabs) are the most populous branch of eukaryotic Ras GTPase superfamily. In active GTP-binding conformation, they serve as key instruments in defining transient membrane identity and through various effectors regulate formation, transport, conversion, and fusion of membrane vesicles. This is important for upkeep of compartmentalized structure of eukaryotic cells and for facilitating both endo- and exocytic processes. Rabs are converted into GDP-binding conformation by interactions with Rab GTPase activating proteins (Rab GAPs) that possess ability to significantly speed up weak intrinsic GTP hydrolytic activity of Rabs. Through this process, Rab GAPs can limit scope of the Rabs' activity and lay out spatiotemporal boundaries for varying Rab populations. In this thesis, I tried to characterize a Rab GAP, GAP2, seemingly necessary for standard development of thale cress plants. Besides TBC catalytic domain, GAP2 (product of At2g39280 gene) possesses a C-terminal coiled-coil motif, which was previously found to interact with Rab GTPases. Experiments aiming to complement T-DNA insertion mutant in GAP2, elucidate GAP2 intracellular localization, novel interacting partners, and character of interaction with the Rabs discovered in the pilot study were undertaken. The results suggest that...

Rab GTPases are small signaling molecules that play an important role in vesicle trafficking in eukaryotic cells. Correct signaling through small GTPases allows orchestration of vesicle transport among cellular organelles and also to the cell wall providing cell wall material for cell growth and elongation. Engagement of Rab GTPases in the regulation of endomembrane trafficking is one of the evolutionary conserved aspects of secretion regulation. The network of Rab GTPases interaction includes also various downstream effectors. One of them is the exocyst complex involved in vesicle docking at the plasma membrane. It is a complex composed of eight different subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84). Exocyst was discovered as Sec4p Rab GTPase effector in yeast and also data from animal models describe the Sec15 exocyst subunit as the Rab-interacting partner, but data from plants are missing. On the other hand, numerous studies identified exocyst role in tip growth of pollen tube and root hairs, seed coat formation, cell plate and cell wall formation, hypocotyl elongation, and importantly also PIN auxin efflux carriers recycling and polar auxin transport. There are two paralogues of SEC15 in the Arabidopsis genome, SEC15a and SEC15b, the previous one already shown to be...

Le transport intracellulaire du cholestérol et sa distribution correcte au niveau des différentes membranes sont essentiels pour assurer de nombreuses fonctions cellulaires. Malgré l’importance de ce transport les mécanismes de sa régulation restent encore mal connus. L’objectif de cette thèse était de mieux caractériser les acteurs du transport intracellulaire du cholestérol. Dans ce contexte, nous nous sommes intéressés à deux acteurs de ce transport : la dynamine et les Rab GTPases. Dans la première partie de la thèse nous avons utilisé le dynasore, un inhibiteur pharmacologique de la dynamine pour étudier le rôle de la dynamine dans le contrôle du transport endolysosomal dans les cellules HeLa et les macrophages humains. Nous avons ainsi confirmé le rôle de la dynamine dans la sortie du compartiment endolysosomal et la régulation de l’homéostasie du cholestérol. Dans la deuxième partie de la thèse, nous avons étudié le rôle de Rab7 et de Rab9 dans le transport du cholestérol en utilisant la technique d’ARN interférence ainsi que l’expression de mutants dominant négatifs. Nous avons montré qu’en plus de son rôle classique dans les étapes tardives du transport du cholestérol, Rab7 contrôle les étapes précoces du transport endosomal. Enfin, nous avons évalué le rôle de Rab7 dans notre modèle de macrophages humains surchargés. Nous avons mis en évidence un effet limité de l’inactivation de Rab7 sur le contrôle de l’homéostasie du cholestérol mais à l’inverse un effet majeur pour l’efflux du cholestérol vers l’apo AI. En conclusion, notre étude a permis de mieux caractériser le transport vésiculaire du cholestérol et de démontrer son importance dans la régulation de l’homéostasie intracellulaire en cholestérol. Nos résultats permettent également d’établir le rôle critique de Rab7 dans le trafic des LDL au niveau des endosomes précoces. / Intracellular transport of cholesterol and its distribution within cellular membranes are essential to maintain correct cellular functions. Despite the importance of this transport, mechanisms that regulate cholesterol transport still poorly defined. The objectives of this thesis were to better characterize the actors of intracellular cholesterol trafficking. In this context, we focused our interest on two known actors of intracellular transport : dynamin and Rab GTPases. In the first part of this thesis, we used dynasore, a pharmacological dynamin inhibitor, to study the role of dynamin in the control of endolysosomal transport in HeLa cells and human macrophages. We confirmed the role of dynamin in endolysosomal sorting and cholesterol homeostasis regulation. In the second part of this thesis, we studied the role of Rab7 and Rab9 in the regulation of cholesterol transport using RNA interference and dominant negative mutants. We showed that in addition to it classical role in late steps of cholesterol transport, Rab7 controls also early steps of endosomal trafficking. Finally, we evaluated the role of Rab7 in our model of loaded human macrophages. We showed a weak impact of Rab7 inactivation on cholesterol homeostasis but a major effect on cholesterol efflux to apo AI. In conclusion, in this study we have better characterized the vesicular transport of cholesterol and demonstrated its importance in cholesterol intracellular homeostasis. Our results also establish that Rab7 plays a critical role in the sorting of LDL at the early endosome.

Rab GTPases are major regulatory proteins of vesicle traffic and thus responsible for membrane identity, vesicle targeting and vesicle fusion. The angiosperm Rab GTPase family is grouped into eight clades (Rab-A to Rab-H) that are broadly conserved in animals and yeasts. It has been proposed that the Rab-A clade has diversified in land plants giving rise to six plant- specific structural subclasses, Rab-A1 to Rab-A6. Previous work suggests that the Arabidopsis Rab-A2 and Rab-A3 proteins define a novel endosomal compartment that lies on a pathway between the Golgi and the plasma membrane. In dividing cells, the Rab-A2/A3 compartment is implicated in biosynthetic traffic to the cell plate but it is unclear what traffics through this compartment in non-dividing cells. In this project, I investigated a range of membrane trafficking pathways in Arabidopsis thaliana. These were probed for dependency on RAB-A2a function, using the dominant negative approach combined with fluorescent marker technology. The data presented in this thesis suggests that RAB-A2a acts on a protein recycling pathway that is used by PIN2:GFP.

Obligate intracellular pathogens must acquire host cell-derived nutrients to facilitate their survival. One such bacterial pathogen, Anaplasma phagocytophilum, replicates within neutrophils and non-phagocytic cells in a bacterial-modified, host cell-derived vacuole. The bacterium exploits host cell vesicular trafficking pathways to route nutrients to its vacuole and utilizes Rab GTPases, guanine nucleotide-dependent, vesicular trafficking regulators, to do so. We previously discovered that the A. phagocytophilum vacuolar membrane is decorated with a specific subset of Rab GTPases - Rab1, Rab4A, Rab10, Rab11, Rab14, Rab22A and Rab35. Rab1 is exclusively found on the endoplasmic reticulum (ER) and thus its localization suggests that the bacterium intercepts the ER. Rab10, which is found on the ER, trans-Golgi and recycling endosomes, localizes to the vacuolar membrane in a guanine nucleotide-independent and bacterial protein synthesis-dependent manner. This suggests that a bacterial-encoded protein is binding to and recruiting Rab10. In this study, we determined that A. phagocytophilum hijacks two very nutrient-rich sources in the secretory pathway - trans-Golgi- and endoplasmic reticulum-derived vesicles. A. phagocytophilum localizes perinuclearly adjacent to the Golgi apparatus during infection. A. phagocytophilum and Anaplasma marginale, an intravacuolar bovine pathogen, also localize near the smooth ER and rough ER in both mammalian and tick host cells. These results are supported by transmission electron microscopy analyses of infected cells. Membrane markers for the rough ER label the peripheries of A. marginale and A. phagocytophilum organisms in both mammalian and tick host cells, which suggests that they are translocated into the pathogen vacuole. Furthermore, membrane markers for trans-Golgi-derived vesicles, including endogenous Rab10, label the periphery of intravacuolar A. phagocytophilum organisms. Markers for the trans-Golgi and the ER co-fractionate with A. phagocytophilum in density gradient centrifugation studies. siRNA knockdown of Rab10 pronouncedly reduces delivery of trans-Golgi markers into the pathogen-occupied vacuole, significantly reduces infection, and impedes bacterial conversion to the bacterium’s dense-cored form. These results suggest that trans-Golgi recruitment is Rab10 dependent and is critical for bacterial development. We identified an outer membrane A. phagocytophilum moonlighting protein, uridine monophosphate kinase that specifically binds GST-Rab10 in affinity chromatography assays and interacts with Rab10 in vivo. We hypothesize that this surface protein is mediating the interaction of the bacteria with intravacuolar trans-Golgi derived vesicles. This interaction could be critical for the delivery of essential nutrients. Taken together, these data suggest that nutritional virulence mechanisms of A. phagocytophilum and A. marginale target the host secretory pathway. Additionally, they suggest a novel mechanism whereby pathogens translocate nutrient rich vesicles into the pathogen vacuole, thus delivering essential nutrients right to their front door.

Introdução: In vivo ou in vitro a exposição de fibroblastos a alta concentração de glicose promove um aumento do estresse oxidativo e consequentemente prejudica a migração celular, assim como a maturação da adesão. Além disso, a elevada concentração de glicose reduz a expressão de diferentes integrinas na superfície celular devido alterações na síntese do receptor e sua reciclagem. Objetivo: Avaliar os efeitos da elevada concentração de glicose no tráfego de vesículas contendo EEA1 (endossomos primários), Rab4 (via rápida da reciclagem), Rab11 (via lenta de reciclagem) e Rab7 (endossomos de degradação) em fibroblastos NIH3T3. Métodos: células foram cultivadas em meio contendo baixa concentração de glicose (LG, 5 mM) ou em alta concentração (HG 25 mM) durante 21 dias antes de realizar os experimentos. EEA1, Rab4, Rab11 e Rab7 expressão e distribuição foram avaliados por western blotting e imunofluorescência, respectivamente. Resultados: Células expostas à alta concentração não apresentaram diferenças na expressão e distribuição das proteínas EEA1 e Rab7, enquanto a expressão de Rab11 foi reduzida em 30%. Conclusão: a alta concentração de glicose altera a via lenta da reciclagem contendo Rab11, afetando potencialmente a reciclagem de integrinas e outros receptores e a sua expressão na superfície celular. / Background: In vivo or in vitro exposure of fibroblasts to high glucose concentrations (HG) promotes oxidative stress and consequently impairs cell migration, also inhibiting adhesion maturation. Additionally, HG reduces the expression of different integrins on the cell surface, potentially due to altered receptor synthesis and recycling. Aim: to evaluate the effects of HG on the trafficking vesicles containing EEA1 (early endosomes), Rab4 (fast recycling pathway) and Rab7 (endocytic degradation pathway) on NIH3T3 fibroblasts. Methods: cells were cultured under low glucose (LG, 5 mM) or HG (25 mM) concentrations during 21 days before the assays. EEA1, Rab4 and Rab7 expression and distribution were evaluated by western blotting and immunofluorescence, respectively. Results: HG did not affect proteins EEA1 and Rab7 expression and distribution, whereas Rab11 expression was reduced by 30%. The number of vesicles containing Rab11 was also significantly reduced in HG cells. Conclusion: high glucose alters the slow recycling endocytic pathway via Rab11, potentially affecting integrins and other receptors synthesis and expression on the cell surface.

Introdução: In vivo ou in vitro a exposição de fibroblastos a alta concentração de glicose promove um aumento do estresse oxidativo e consequentemente prejudica a migração celular, assim como a maturação da adesão. Além disso, a elevada concentração de glicose reduz a expressão de diferentes integrinas na superfície celular devido alterações na síntese do receptor e sua reciclagem. Objetivo: Avaliar os efeitos da elevada concentração de glicose no tráfego de vesículas contendo EEA1 (endossomos primários), Rab4 (via rápida da reciclagem), Rab11 (via lenta de reciclagem) e Rab7 (endossomos de degradação) em fibroblastos NIH3T3. Métodos: células foram cultivadas em meio contendo baixa concentração de glicose (LG, 5 mM) ou em alta concentração (HG 25 mM) durante 21 dias antes de realizar os experimentos. EEA1, Rab4, Rab11 e Rab7 expressão e distribuição foram avaliados por western blotting e imunofluorescência, respectivamente. Resultados: Células expostas à alta concentração não apresentaram diferenças na expressão e distribuição das proteínas EEA1 e Rab7, enquanto a expressão de Rab11 foi reduzida em 30%. Conclusão: a alta concentração de glicose altera a via lenta da reciclagem contendo Rab11, afetando potencialmente a reciclagem de integrinas e outros receptores e a sua expressão na superfície celular. / Background: In vivo or in vitro exposure of fibroblasts to high glucose concentrations (HG) promotes oxidative stress and consequently impairs cell migration, also inhibiting adhesion maturation. Additionally, HG reduces the expression of different integrins on the cell surface, potentially due to altered receptor synthesis and recycling. Aim: to evaluate the effects of HG on the trafficking vesicles containing EEA1 (early endosomes), Rab4 (fast recycling pathway) and Rab7 (endocytic degradation pathway) on NIH3T3 fibroblasts. Methods: cells were cultured under low glucose (LG, 5 mM) or HG (25 mM) concentrations during 21 days before the assays. EEA1, Rab4 and Rab7 expression and distribution were evaluated by western blotting and immunofluorescence, respectively. Results: HG did not affect proteins EEA1 and Rab7 expression and distribution, whereas Rab11 expression was reduced by 30%. The number of vesicles containing Rab11 was also significantly reduced in HG cells. Conclusion: high glucose alters the slow recycling endocytic pathway via Rab11, potentially affecting integrins and other receptors synthesis and expression on the cell surface.

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