Signal termination processes of GPCRs are well established, unlike processes that regulate the assembly and intracellular trafficking of these signaling complexes. Bimolecular fluorescence complementation was used to study GPCR dimer formation in two projects. Firstly, the importance of Rab GTPases on the cell surface expression and signaling of two chemokine receptors expressed on prostate cancer cells was examined. Rab GTPases necessary for CXCR4 and CCR2 cell surface expression and signaling were different from those necessary for the CXCR4/CCR2 heterodimer. Therefore, this project emphasizes the importance of studying heterodimers as unique entities from their constituent receptors. Secondly, interactions between molecular chaperones and two coreceptors necessary for HIV infection – CCR5, a chemokine GPCR, and the main HIV receptor, CD4, a glycoprotein – were investigated. Further emphasizing the unique characteristics of GPCR dimers, this project found that molecular chaperones interact differently with CCR5 homodimers, when compared to CCR5/CD4 heterodimers.

The membrane trafficking system mediates delivery of macromolecules and metabolites to discrete intracellular compartments from their site of uptake or synthesis. For many pathogens the trafficking system has a special relevance as it is responsible for maintaining the host-pathogen interface, i.e., the cell surface. Both the surface and the underlying trafficking apparatus are intimately connected with immune evasion in many parasites including those belonging to the highly divergent order Kinetoplastida. Kinetoplastid parasites are etiological agents of several neglected tropical diseases such as African sleeping sickness, Chagas disease, and Leishmaniasis. Newly available sequences of many kinetoplastid genomes were used to reconstruct evolution of trafficking across this lineage, using three central paralogous trafficking families: Rabs, SNAREs and Rab-GAPs, which have defined roles in specific trafficking events. Further, proteomics was used to analyse a representative SNARE complex to explore compositional conservation between kinetoplastids and Opistokhonts. Overall there is little evidence for large scale expansions or contractions of these protein families, excluding a direct association with parasitism or changes to host range, host immunosophistication or transmission mechanisms. The data indicate a stepwise sculpting of the trafficking system where the large repertoire of the basal bodonids is mainly retained by the cruzi group, while extensive lossses characterise other lineages, particularly the African trypanosomes and phytomonads. Kinetoplastids possess several lineage-specific Rabs but all retain a core canonical Rab set; by contrast there is little novelty within the SNARE family even though certain canonical endosomal SNAREs appear to show a considerable degree of sequence divergence. Proteomics suggests that SNARE complex composition is largely conserved. The major changes in Rab and SNARE repertoires are associated with endosomal and late exocytic pathways, which is consistent with the considerable evolution of surface proteomes. Therefore, despite the absence of a transition per se associated with parasitism, adaptation of membrane trafficking is likely under active selection where it meets the host environment.

Crosspresentation allows antigen presenting cells to present peptides from exogenously derived antigens onto MHC Class I for presentation to CD8+ T cells. Though this pathway shares key players with the Classical Class I and Class II pathways, several questions remain. A genomewide siRNA screen was performed to look for genes that selectively affected the crosspresentation or the Class II pathways. Among the genes identified in the screen was the Rab GTPase Rab39a. Rab39a was required for efficient crosspresentation but was dispensable for the presentation of endogenously expressed antigen. Both TAP-dependent and independent antigen required Rab39a for efficient presentation. Rab39a localized to late endosomes and phagosomes, though interestingly it was not required for the Class II pathway. Analysis of phagosomes from Rab39a KO or rescued cells has shown that in the presence of Rab39a, phagosomes were enriched for the open form of MHC Class I as well as TAP1, a member of the peptide loading complex. The enriched open form of MHC-I was peptide receptive, suggesting that it could contribute to crosspresentation. Phagosomes from Rab39a positive cells had reduced degradative capability and had increased levels of Sec22b, a SNARE protein reported to deliver ER-golgi sourced cargo to phagosomes. Furthermore, inhibition of ER-golgi transport via brefeldin A abolished the phenotype conferred by Rab39a. Thus, we hypothesize that Rab39a mediates the delivery of ER-golgi derived cargo to the antigen containing phagosome. This delivery allows peptide receptive MHC-I, as well as the peptide loading complex to reach the antigen, thereby facilitating crosspresentation.

<p dir="ltr"><i>Legionella pneumophila</i> strains harboring wild-type <i>rpsL</i> such as Lp02<i>rpsL</i>_WT cannot replicate in mouse bone marrow-derived macrophages (BMDMs) due to induction of extensive lysosome damage and apoptosis. The mechanism of this unique infection-induced cell death remains unknown. Using a genome-wide CRISPR/Cas9 screening, we identified <i>Hmg20a </i>and <i>Nol9</i> as host factors important for restricting strain Lp02<i>rpsL</i>_WT in BMDMs. Depletion of <i>Hmg20a</i> protects macrophages from infection-induced lysosomal damage and apoptosis, allowing productive bacterial replication. The restriction imposed by <i>Hmg20a</i> was mediated by repressing the expression of several endo-lysosomal proteins, including the small GTPase Rab7. We found that SUMOylated Rab7 is recruited to the bacterial phagosome via SulF, a Dot/Icm effector that harbors a SUMO-interacting motif (SIM). Moreover, overexpression of Rab7 rescues intracellular growth of strain Lp02<i>rpsL</i>_WT in BMDMs. Our results establish that <i>L. pneumophila</i> exploits the lysosomal network for the biogenesis of its phagosome in BMDMs.</p>

No description available.

Chez la levure Saccharomyces cerevisiae, la croissance est orientée et nécessite l’apport de membranes et d’enzymes pour la synthèse de la paroi cellulaire. La régulation du transport des vésicules permettant cet apport est assuré par les GTPases de la famille Ypt/Rab. Sec4p, une Ypt/Rab GTPase, est impliquée dans l’exocytose en assurant la spécificité de l’ancrage des vésicules post-golgiennes envoyées aux sites de croissance. La régulation de son activité GTPase est essentielle pour sa fonction.Nous nous intéressons aux protéines Gyp5p et Gyl1p, deux membres de la famille des protéines activatrices des GTPases Ypt/Rab chez S. cerevisiae. Le laboratoire a montré l’implication du complexe Gyp5p-Gyl1p dans l’exocytose polarisée vraisemblablement par la régulation de Sec4p. Notre étude a montré une interaction directe in vitro de ces deux protéines, ainsi qu’une interdépendance pour une bonne localisation du complexe aux sites de croissance polarisée, c'est-à-dire au sommet du bourgeon durant la croissance apicale et au cou du bourgeon durant la cytocinèse. Cette localisation dépend de deux formines, d’éléments du polarisome et des câbles d’actine. De plus, nous avons montré par des expériences d’immunofluorescence et de microscopie électronique en collaboration avec J.-M. Verbavatz (iBiTec-S, CEA), que ces protéines sont transportées sur des vésicules de sécrétion jusqu’aux sites de croissance polarisée.Notre étude de l’interaction du complexe Gyp5p-Gyl1p avec Rvs167p, une protéine à domaine BAR (Bin1-Amphiphysin-Rvs167p) a montré que Gyp5p et Gyl1p sont nécessaires pour la bonne localisation de Rvs167p au sommet du petit bourgeon et que ces complexes se forment principalement dans des fractions enrichies en membrane plasmique. Pour mieux caractériser ces interactions, nous avons réalisé une mutation de la proline 473 dans le domaine SH3 de Rvs167p et des délétions des séquences riches en proline de Gyp5p et Gyl1p. Ces mutations entraînent un défaut d’interaction de Rvs167p avec Gyp5p et Gyl1p et la perte de la localisation de Rvs167p au sommet du petit bourgeon. Afin de comprendre la fonction de ces interactions, nous avons réalisé des expériences de microscopie électronique et des tests de sécrétion de l’endo-β-1,3-glucanase, Bgl2p dans une souche Δrvs167. Nous avons mis en évidence une accumulation de vésicules de sécrétion au niveau du petit bourgeon et un défaut de sécrétion de Bgl2p à 13°C dans cette souche. De plus, nous avons également observé une accumulation de vésicules de sécrétion dans une souche exprimant Rvs167p mutée pour la proline 473 et un défaut de sécrétion de Bgl2p dans une souche exprimant Gyp5p et Gyl1p dépourvues de leurs séquences riches en proline. Ces résultats montrent que Rvs167p joue un rôle dans l’exocytose polarisée au stade du petit bourgeon et que cette fonction dépend de son recrutement par Gyp5p et Gyl1p au sommet du petit bourgeon. / In Saccharomyces cerevisiae, growth is oriented and requires the contribution of membranes and enzymes for the synthesis of the cell wall. Regulation of vesicles transport allowing this contribution is provided by the Ypt/Rab GTPases family. Sec4p, a Ypt/Rab GTPase, is involved in exocytosis by controlling the tethering of post-Golgi vesicles at sites of growth. Regulation of Sec4p GTPase activity by is essential for its function.We studied the proteins Gyp5p and Gyl1p, two members of the Ypt/Rab GTPases activiting proteins (RabGAP) family in S. cerevisiae. Gyp5p and Gyl1p interact with Sec4p and are involved in the control of exocytosis at the small-bud stage. Our study showed that Gyp5p and Gyl1p interact directly in vitro and are interdependent for their correct localization to the sites of polarized growth, e.g. the bud tip during apical growth and the bud neck during cytokinesis. We showed that the localization of Gyp5p and Gyl1p to the sites of polarized growth depends on the formins Bni1 and Bnr1, but also on polarisome components and actin cables. Moreover, we showed by immunofluorescence and electron microscopy (in collaboration with J.-M. Verbavatz), that Gyp5p and Gyl1p are transported onto secretory vesicles to access the sites of polarized growth.We studied the interaction of Gyp5p and Gyl1p with Rvs167p, a BAR domain (Bin1-Amphiphysin-Rvs167p) protein and showed that Gyp5p and Gyl1p are necessary for the recruitment of Rvs167p to the small-bud tip. Both the mutation of the proline 473 in the SH3 domain of Rvs167p and the deletion of the proline-rich regions of Gyp5p and Gyl1p disrupt the interaction of Rvs167p with Gyp5p and Gyl1p and impair the localization of Rvs167p to the tips of small buds. Electron microscopy experiments unraveled an accumulation of secretory vesicles in small buds of rvs167Δcells and β-1,3-endoglucanase Bgl2p secretion assays showed Bgl2p secretion defects in cultures enriched in small buds at 13°C. In addition, an accumulation of secretory vesicles was observed in Rvs167pP473L strain, and Bgl2p secretion defect were found in strains expressing Gyp5p and Gyl1p deleted of their proline-rich sequences. These results show that Rvs167p plays a role in polarized exocytosis at the small bud stage and that its function in exocytosis depends on its recruitment to the tip of small buds by the RabGAP proteins Gyp5p and Gyl1p.

No description available.

No description available.

Eukaryotic cells depend on cargo uptake into the endocytic membrane system, which comprises a functionally interconnected network of endosomal compartments. The establishment and maintenance of such diverse compartments in face of the high rates of exchange between them, poses a major challenge for obtaining a molecular understanding of the endocytic system. Rab-GTPases have emerged as architectural key element thereof: Individual family members localize selectively to endosomal compartments, where they recruit a multitude of cytoplasmic effector proteins and coordinate them into membrane sub-domains. Such &amp;quot;Rab-domains&amp;quot; constitute modules of molecular membrane identity, which pattern the endocytic membrane system into a mosaic of Rab-domains. The main objective of this thesis research was to link such &amp;quot;static&amp;quot; mosaic-view with the highly dynamic nature of the endosomal system. The following questions were addressed: How are neighbouring Rab-domains coordinated? Are Rab-domains stable or can they undergo assembly and disassembly? Are the dynamics of Rab-domains utilized in cargo transport? The first part of this thesis research focused on the organization of Rab-domains in the recycling pathway. Utilizing Total Internal Reflection (TIRF) microscopy, Rab11-, but neither Rab4- nor Rab5-positive vesicles were observed to fuse with the plasma membrane. Rab4-positive membranes, however, could be induced to fuse in presence of Brefeldin A. Thus, these experiments complete the view of the recycling pathway by the following steps: a) Rab11-carriers likely mediate the return of recycling cargo to the surface; b) such carriers are presumably generated in an Arf-dependent fission reaction from Rab4-positive compartments. Rab11-chromatography was subsequently carried out in the hope of identifying Rab11-effectors functioning at the Rab4-Rab11 domain interface. An as yet uncharacterized ubiquitin ligase was identified, which selectively interacts with both Rab4 and Rab11. Contrary to expectations, however, the protein (termed RUL for *R*ab interacting *U*biquitin *L*igase) does not function in recycling,but appears to mediate trafficking between Golgi/TGN and endosomes instead.In order to address the dynamics of Rab-domains, fluorescently tagged Rab-GTPases were imaged during cargo transport reactions in living cells. Herefore high-speed/long-term imaging procedures and novel computational image analysis tools were developed. The application of such methodology to the analysis of Rab5-positive early endosomes showed that a) The amount of Rab5 associated with individual endosomes fluctuates strongly over time; b) such fluctuations can lead to the &amp;quot;catastrophic&amp;quot; loss of the Rab5-machinery from membranes; c) Rab5 catastrophe is part of a functional cycle of early endosomes, involving net centripetal motility, continuous growth and increase in Rab5 density. Next, the relevance of Rab5 catastrophe with respect to cargo transfer into either the recycling- or degradative pathway was examined. Recycling cargo (transferrin) could be observed to exit Rab5-positive early endosomes via the frequent budding of tubular exit carriers. Exit of degradative cargo (LDL) from Rab5-positive endosomes did not involve budding, but the rapid loss of Rab5 from the limiting membrane.Rab5-loss was further coordinated with the concomitant acquisition of Rab7, suggesting &amp;quot;Rab conversion&amp;quot; as mechanism of transport between early- and late endosomes.Altogether, this thesis research has shown that first, Rab-machineries can be acquired and lost from membranes. Second, such dynamics provide a molecular mechanism for cargo exchange between endosomal compartments. Jointly, these findings lead to the concept of Rab-domain dynamics modulation in /trans/ between neighbouring domains as mechanistic principle behind the dynamic organization of membrane trafficking pathways.

The present dissertation aims to shed light on the molecular machinery involved in the process of shell formation (thecagenesis) in Arcella (Arcellinida : Amoebozoa). Arcellinida are single-celled testate amoebae organisms, characterized by the presence of an outer shell (test or carapace); it is a monophyletic lineage of Amoebozoa, sister group to a naked amoeboid lineage. No homologous structure to shell is present in the sister group of Arcellinida, thus it is considered an evolutionary novelty. The origin and evolution of the shell in Arcellinida are currently open questions; deciphering its formation process is a key step to address these questions. During each reproductive process by budding division, these organisms build a new shell. In the span of more than a century, several authors have described the thecagenesis process on Arcellinida, primarily focusing on the genus \\textit, based on cyto-morphological evidence. Conversely, the absence of molecular data has impaired advances on describing the molecular aspects of shell formation. In this study, we designed and applied a molecular framework to identify candidate genes and develop a molecular model for the shell formation process in Arcella; we based this framework on single-cell RNA-sequencing, gene expression profiling, Gene Ontology analysis, and comparative analysis of cyto-morphological with newly generated molecular data. We identify and propose a set of 539 genes as the candidate genes for shell formation, based on expression profiling and biological process assignment. We propose a model for the the shell formation process, which describes the mechanistic aspect of this process, hypothetically based on a molecular machinery conserved in Eukaryotes. Additionally, we identified a massive expansion of the Rab GTPase family, a protein likely to be involved on the process of shell formation. In the lights of the present study, we briefly discuss possible evolutionary scenarios involved on the origin and evolution of the shell and present future perspectives; we propose the shell of Arcellinida as a prosperous model to study the origin and evolution of evolutionary novelties, as well as other evolutionary questions / A presente dissertação tem como objetivo lançar luz sobre a maquinaria molecular envolvida no processo de formação de teca (tecagênese) em \\textit (Arcellinida: Amoebozoa). Arcellinida são amebas tecadas unicelulares, caracterizadas pela presença de uma teca (carapaça ou concha) externa; é uma linhagem monofilética de Amoebozoa, grupo irmão de alguns organismos amebóides nus. Nenhuma estrutura homóloga à carapaça está presente no grupo irmão de Arcellinida, sendo considerada como uma novidade evolutiva. A origem e evolução da carapaça em Arcellinida são questões em aberto; Decifrar seu processo de formação é um passo fundamental para abordar essas questões. Durante todo processo reprodutivo, por divisão por brotamento, estes organismo constroem uma nova concha. No decorrer de mais de um século, vários autores descreveram o processo de tecagênese nestes organismos, focando principalmente no gênero \\textit, baseados em evidências cito-morfológicas. Enquanto isso, a ausência de dados moleculares impede avanços na descrição dos aspectos moleculares da formação de conchas. Neste estudo, projetamos e aplicamos uma \\textit molecular para identificar genes candidatos e desenvolver um modelo molecular para o processo de formação de teca em \\textit; Baseamos este \\textit em sequenciamento de RNA \\textit, perfil de expressão gênica, análise de \\textit{Gene Ontology} e análise comparativa de dados cito-morfológicos e moleculares. Nós identificamos e propomos um conjunto de 539 genes como genes candidatos para a formação de carapaça, com base no perfil de expressão e na atribuição de processos biológica. Propomos um modelo para o processo de formação de carapaça, que descreve o aspecto mecanicista deste processo, hipoteticamente baseado em um mecanismo molecular conservado em Eucariotos. Além disso, identificamos uma expansão maciça da família gênica das Rab GTPase, gene provavelmente envolvida no processo de formação de carapaça. À luz do presente estudo, discutimos brevemente possíveis cenários evolutivos envolvidos na origem e evolução da teca e apresentamos perspectivas futuras; propomos a teca dos Arcellinida como próspero modelo para estudar a origem e evolução das novidades evolutivas, bem como outras questões evolutivas