La petite protéine G Arf6 et son facteur d'échange EFA6 sont impliquées dans de nombreux processus cellulaires tels que le remodelage du cytosquelette d’actine, le transport vésiculaire et mise en place de la polarité épithéliale. Elles jouent également un rôle dans la voie d'endocytose dépendante de la clathrine. Ce travail de thèse nous a permis d’identifier différents mécanismes régulant l’interaction d’EFA6 avec ses différents partenaires. Nous avons pu mettre en évidence une interaction directe entre le domaine N-BAR de l’endophiline et le domaine Sec7 d’EFA6. Nous avons démontré que la courbure membranaire était un facteur régulant cette interaction. EFA6 est capable d’interagir et de recruter l’endophiline sur une membrane lipidique plane alors qu’en présence de vésicules courbées le complexe protéique ne se forme pas. Nous observons également que l’endophiline stimule l’activité d’échange nucléotidique d’EFA6 sur Arf6. Dans un second temps nous avons démontré, dans une étude menée par le Dr Cherfils, que l’activité catalytique d’EFA6 était régulée par une boucle de rétrocontrôle négatif exercée spécifiquement par la protéine Arf6-GTP. Celle-ci induit une diminution de l’activité d’échange d’EFA6 probablement grâce à sa capacité à interagir avec le domaine PH-C-terminal d’EFA6. Enfin, nous avons mis en évidence un repli intramoléculaire entre le domaine C-terminal et le domaine PH d’EFA6 qui semble contrôler l’interaction de cette extrémité C-terminale avec différents partenaires dont la β-arrestine et de façon surprenante la protéine Arf6 dans sa forme inactive. / The small G protein Arf6 and its exchange factor EFA6 control numerous cellular processes such as actin cytoskeleton remodeling, vesicular transport and apico-basal cell polarity. They are also involved in clathrin-dependent endocytosis. In this work we identify different mechanisms by which EFA6 interaction with its various partners is regulated. We have highlighted a direct interaction between the N-BAR domain of endophilin and the Sec7 domain of EFA6. We demonstrated that this interaction is regulated by the membrane curvature. EFA6 interacts and recruits endophilin on a flat lipid membrane whereas the protein complex does not occur in the presence of curved vesicules. We showed that endophilin stimulates the nucleotidic exchange activity of EFA6 on Arf6. Next we demonstrated that the catalytic activity of EFA6 is regulated by a negative feedback loop specifically mediated by the Arf6-GTP. We observed in the presence of Arf6-GTP a decrease of EFA6 catalytic activity and we showed that this effect was due to an interaction between Arf6-GTP and PH-C-terminal domain of EFA6. Finally we demonstrated an intramolecular folding between the C-terminal domain and the PH domain of EFA6 that controls the interaction of the C-terminus domain with various partners including β-arrestin and surprisingly the inactive GDP form of Arf6.

Os fungos filamentosos passam por um crescimento polarizado, desde a germinação ao alongamento das hifas, até formar um complexo micélio. A região apical do crescimento polarizado do fungo apresenta dois tipos diferentes de vesículas, entre elas, as microvesículas. As ADP-ribosylation factors (ARFs), são proteínas monoméricas ligadoras de GTP e pertence ao grupo de proteínas da superfamília Ras. Essas proteínas são divididas em cinco famílias: ARF, RAB, RAN, RAS e RHO que formam um conjunto de sub-sistemas que são responsáveis, entre outras funções, pela regulação do transporte de vesículas no interior da célula fúngica, entre outras funções, como transduções de sinais e regulação do tráfego vesicular na região de crescimento apical, o spitzenkörper. São proteínas de ancoramento e de marcação de vesículas, envolvidas no tráfego, catálise e fusão por meio de sinalização de membrana-alvo para as vesículas de transporte transmembrana. As ARF são importantes para o crescimento das hifas, além de participar da montagem de vesículas por meio de endocitoses, do transporte destas vesículas entre as organelas e na exocitose. Adicionalmente, as ARFs sofrem o processo de N-miristoilação, uma irreversível lipidação proteica em que o miristato do miristoil CoA é covalentemente ligado a uma glicina secundária da proteína alvo, aumentando a sua hidrofobicidade. Além desta regulação, as ARFs são moduladas pela ação das ARF-GAP (GTPase Activating Protein) e ARF-GEFs (Guanine nucleotide Exchange Factor). Neste trabalho foi proposta a deleção de três ARFs preditivamente miristoiladas (arfA, arfB and arlA), além de dupla-deleção com ?gcsA (ARF-GAP) e a caracterização genotípica e fenotípica das ADP ribosylation fator no fungo filamentoso patogênico Aspergillus fumigatus. Como caracterização das linhagens deletadas, notou-se que arfA demonstra ser essencial para A. fumigatus, enquanto que o fungo foi capaz de se desenvolver na ausência de arfB, arlA e duplo mutantes com ?gcsA. Porém, de forma alternada nas linhagens mutantes, houve redução do diâmetro da colônia, desestruturação de conidióforos, polarização dicotômica e redução de corpos lipídicos na região de crescimento apical. Além das alterações da parede celular que implicou em altações na carga de superfície, formação de biofilme e virulência. Testes de sensibilidades, bem como as análises de níveis de expressão gênica frente a a compostos danosos a eucariotos e antifúngicos evidenciaram que as ARFs e GcsA estão envolvidas em reparos a danos frente a diferentes alvos citoplasmáticos. Ainda, a localização das ARFs fusionadas com GFP (Green Flourescence Protein) em A. fumigatus evidenciou que ArfB está nas regiões apicais das hifas e conidióforos, enquanto ArlA está distribuído em todo citoplasma. Portanto as ARFs em A. fumigatus estão envolvidas nos processos básicos do fungo, como: o crescimento, a virulência e a reprodução / The filamentous fungi undergo polarized growth, from germination to hyphae elongation, to form a mycelial complex. The apical region of the polarized growth of the fungus presents two different types of vesicles, among them, the microvesicles. ADP-ribosylation factors (ARFs) are monomeric GTP-binding proteins and belong to a group of superfamily Ras proteins. These proteins are divided into five families: ARF, RAB, RAN, RAS and RHO that form a set of subsystems that are responsible, over others things, for the regulation of vesicle transport within the fungal cell, among other functions, such as signal transduction and regulation of the vesicular traffic in the apical growth region, the Spitzenkörper. They are anchoring and vesicle marking proteins involved in trafficking, catalysis and fusion by means of target membrane signaling to the transmembrane transport vesicles. ARFs are important for the growth of hyphae, besides participating in vesicle assembly through endocytosis, the transport of these vesicles between the organelles and exocytosis. In addition, the ARFs undergo the N-myristoylation process, an irreversible protein lipidation in which the myristoyl CoA myristate is covalently linked to a secondary glycine of the target protein, increasing its hydrophobicity. In addition to this regulation, the Arfs are modulated by the action of Arf-GAP (GTPase Activating Protein) and ARF-GEFs (Guanine nucleotide Exchange Factor). In this work, the deletion of three myristoylated ARFs (arfA, arfB and arlA), as well as double-deletion with ?gcsA (ARF-GAP) and phenotypic and genotypic characterization of ADP ribosylation fator in the pathogenic fungus Aspergillus fumigatus was proposed. As a characterization of the deleted strains, arfA shown to be essential for A. fumigatus, whereas the fungus was able to develop in the absence of arfB, arlA and double mutants with ?gcsA. However, in the mutant strains, there was a decrease in colony diameter, deconjugation of conidiophores, dichotomous polarization and reduction of lipid bodies in the apical growth region. In addition, cell wall changes were registered that implied in surface charge elevations, biofilm formation and virulence. In tests of sensitivities, as well as the analysis of levels of gene expression against compounds harmful to eukaryotes and antifungals showed that ARFs and GcsA (Arf-GAP) are involved in damage repair against different cytoplasmic targets. Furthermore, the location of the GFP-fused GFPs (Green Flourescence Protein) in A. fumigatus evidenced that ArfB is in the apical regions of the hyphae and conidiophores, while ArlA is diffuse in every cytoplasm. Therefore, the ARFs in A. fumigatus are involved in the basic processes of the fungus, such as growth, virulence and reproduction

La bactérie Myxococcus xanthus forme des structures multicellulaires appelées corps fructifères pour résister à des conditions de carence nutritive. La formation de ces structures implique un système chimiotactique particulier, le système Frz, qui régule le changement de direction des cellules, provoqué par la relocalisation simultanée des deux appareils de motilité (A) et (S) d’un pôle à l’autre de la cellule. Au cours de ma thèse, j’ai travaillé sur la connexion entre le système chimiotactique Frz et ses protéines cibles MglAB dans le contrôle de l’inversion de la polarité. L’axe de polarité des cellules est établi par MglA, une petite protéine G de la famille Ras, qui constitue un embranchement vers la régulation des deux appareils de motilité au pôle avant, et son inhibiteur MglB localisé au pôle arrière. Nous avons montré qu’en interagissant directement et spécifiquement avec le cytosquelette, MglA contrôle l’assemblage et le désassemblage de la machinerie de motilité A. Par une approche évolutive, nous avons élucidé l’architecture modulaire du système Frz et l’implication de quatre domaines régulateurs pour connecter le système Frz aux protéines MglAB, filtrer et amplifier le signal. Nous proposons un mécanisme d’inversion de la polarité dans lequel l’action indépendante de deux RRs à chaque pôle de la cellule perturbe les interactions entre une petite protéine G et son inhibiteur apparenté pour convertir un axe de polarité stable en un oscillateur biochimique. La régulation de la direction de mouvement chez M. xanthus pourrait donc constituer un cas émergent de couplage entre des régulateurs de type procaryotes et eucaryotes. / The bacterium Myxococcus xanthus forms multicellular structures called fruiting bodies to resist to starvation conditions. Fruiting body formation implies a chemosensory-like system, the Frz system which regulates directional changes through the simultaneous pole-to-pole relocalization of two motility systems, (A) and (S). During my PhD, I have worked on the connection between the Frz chemosensory-like system and the downstream regulators MglA and MglB in the control of polarity inversion. The cell polarity axis is established by (i) a Ras-like small G protein, MglA, which constitutes a branch node in the regulation of A and S motility systems at the leading cell pole, and (ii) its cognate inhibitor MglB that localizes at the lagging cell pole. We showed that MglA interacts directly and specifically with the cytoskeleton to promote assembly and disassembly of the A-motility machinery. Using an evolutionary approach, we elucidated the modular architecture of the Frz system and the implication of four regulatory domains to (i) connect the Frz system to the MglAB proteins, (ii) filter and (iii) amplify the signal. We now propose a mechanism for polarity inversion in which the independent action of two response regulators at each cell pole perturbs the interactions between a small-G-protein and its cognate inhibitor to trigger the conversion of a stable polarity axis into a biochemical oscillator. The regulation of directional movement in M. xanthus is an interesting emergent coupling between prokaryotes and eukaryotes regulators.