Submitted by Renata Fontoura (comunicaicc@fiocruz.br) on 2014-11-26T15:13:12Z No. of bitstreams: 2 Dissertação Claudia Maria do Nascimento Moreira - 01.pdf: 4994656 bytes, checksum: c9acb588dbfcaa318394a1f54b594a32 (MD5) Dissertação Claudia Maria do Nascimento Moreira - 01.pdf: 4994656 bytes, checksum: c9acb588dbfcaa318394a1f54b594a32 (MD5) / Made available in DSpace on 2014-11-26T15:15:37Z (GMT). No. of bitstreams: 2 Dissertação Claudia Maria do Nascimento Moreira - 01.pdf: 4994656 bytes, checksum: c9acb588dbfcaa318394a1f54b594a32 (MD5) Dissertação Claudia Maria do Nascimento Moreira - 01.pdf: 4994656 bytes, checksum: c9acb588dbfcaa318394a1f54b594a32 (MD5) Previous issue date: 2013 / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil / O complexo adaptador 1 (AP-1) atua na formação do revestimento de vesículas com clatrina na rede trans-Golgi em células eucariontes. O conhecimento sobre o complexo AP-1 em tripanosomatídeos é escasso, mas já foi demonstrada sua importância na infectividade de Leishmania mexicana em macrófagos, assim como na viabilidade de Trypanosoma brucei. O Trypanosoma cruzi é um protozoário flagelado pertencente à família Trypanosomatidae, sendo o agente etiológico da doença de Chagas, a qual afeta milhões de pessoas no mundo. Neste contexto, esta dissertação teve como objetivo identificar os genes que codificam as quatro subunidades do complexo AP-1 no genoma de T. cruzi, analisar sua expressão e localização subcelular nesse parasita. Busca em banco de dados genômicos permitiu identificar as sequências codificantes para todas as subunidades do complexo AP-1, sendo obtidos os seguintes números de acesso gênicos: AP1-γ: XP_818958.1; AP1-β: XP_820334.1; AP1-µ:XP_818899.1; AP1-σ: XP_804127.1. Foram produzidos anticorpos em camundongos contra as proteínas recombinantes de todas as subunidades do complexo AP-1. Análise da especificidade dos anticorpos foi realizada por western blot e a localização subcelular das proteínas foi feita por imunofluorescência em microscopia de epifluorescência e microscopia confocal a laser. Resultados negativos foram obtidos com o antisoro contra a subunidade AP1-σ. Anticorpos obtidos contra as subunidades AP1-µ (policlonal) e AP1-β (monoclonal) reconheceram polipetídeos de tamanho compatível ao peso molecular da proteína endógena em extratos de formas epimastigotas, porém não reconheceram as proteínas por imunofluorescência. O antisoro policlonal obtido contra a subunidade AP1-γ reconheceu em extratos de diferentes formas evolutivas de T. cruzi (epimastigotas, tripomastigotas e amastigotas) um polipeptídeo de peso molecular compatível com o predito em banco de dados (~90 kDa). Imunolocalização demonstrou reação positiva pontual em região compatível com a do complexo de Golgi deste protozoário: entre núcleo e cinetoplasto de formas tripomastigotas e entre cinetoplasto e bolsa flagelar de epimastigotas e amastigotas. Colocalização da AP1-γ com a GTPase Rab7 (marcador de complexo de Golgi em T. cruzi) confirmou a localização desta subunidade no complexo de Golgi desse parasita. Nossos resultados demonstram que as subunidades do complexo AP-1 são conservadas e expressas em T. cruzi e que pelo menos a subunidade AP1-γ possui localização celular em complexo de Golgi, similar ao que é descrito em outras células eucariontes. / The adaptor complex 1 (AP-1) acts in the formation of clathrin-coated vesicles at the transGolgi network of eukaryotic cells. Knowledge about the AP-1 complex in trypanosomatids is scarce, but it has been already demonstrated its importance in the infectivity of Leishmania mexicana in macrophages, as well as the viability of Trypanosoma brucei. Trypanosoma cruzi is a protozoan flagellate that belongs to the family Trypanosomatidae, being the etiologic agent of Chagas disease, which affects millions of people worldwide. In this context, this study aimed to identify the genes encoding the four subunits of the AP-1 complex in the genome of T. cruzi and analyze their expression and subcellular localization in this parasite. Search in genomic database identified gene sequences coding for all subunits of the AP-1 complex, the following accession numbers being obtained: AP1-γ: XP_818958.1; AP1-β: XP_820334.1; AP1-µ: XP_818899 .1; AP1-σ: XP_804127.1. Antibodies were produced in mice against recombinant proteins of all subunits of the AP-1 complex. Analysis of the specificity of the antibodies was performed by western blot and subcellular localization of the proteins by immunofluorescence was done by epifluorescence microscopy and confocal laser microscopy. Negative results were obtained with the antiserum against subunit AP1-σ. Antibodies raised against the AP1-µ (polyclonal) and AP1-β (monoclonal) subunits recognized polypeptides with size compatible to the molecular weight of the endogenous protein in extracts from epimastigotes, but no proteins could be localized by fluorescence microscopy. The antiserum polyclonal obtained against the AP1-γ subunit recognized a polypeptide in extracts of different evolutionary forms of T. cruzi (epimastigotes, trypomastigotes and amastigotes) of molecular weight consistent with that predicted in database (~90 kDa). Immunolocalization showed punctual positive reaction in the region compatible with the Golgi complex of this protozoan: between nucleus and kinetoplast of trypomastigotes and between kinetoplast and flagellar pocket of epimastigotes and amastigotes. Colocalization of AP1-γ with the GTPase Rab7 (a marker of the Golgi complex in T. cruzi) confirmed the location of this subunit in the Golgi apparatus of this parasite. Our results demonstrate that the subunits of the AP-1 complex are conserved and expressed in T. cruzi and that at least the AP1-γ subunit has cellular localization in the Golgi apparatus, similar to that described in other eukaryotic cells.

Clathrin coated vesicles were the first type of coated vesicle to be characterised. The coat consists of two components, clathrin and adaptor (or AP) complexes, the AP-1 complex is associated with the clathrin coated vesicles that bud from the TGN and the AP-2 complex is associated with the clathrin coated vesicles that bud from the plasma membrane. A new type of adaptor-like complex was discovered in our laboratory and was published in 1996. The complex has been shown to consist of two known proteins, beta3B and mu3B, and two unknown proteins of 160kD and 22kD. Unlike the conventional adaptor complexes this complex is not associated with clathrin. The aim of this thesis was to complete the characterisation of the adaptor-like complex and to establish its function. My studies have shown that, the adaptor-like complex consist of an alpha/gamma like subunit, delta, a beta subunit (beta3A/B), a mu subunit (mu3A/B) and a sigma subunit (sigma3A/B). We named the adaptor-like complexAP-3, by analogy with the AP-1 and AP-2 complexes. The AP-3 complex is localised to perinuclear and more peripheral membranes in non-neuronal cells, with little overlap with endocytic markers. The beta subunit of the AP-3 complex is the major target for phosphorylation. Analysis of mice with mutations in the beta3A subunit, and in the delta subunit of the AP-3 complex, have revealed that the beta subunit is required for the stability of the mu subunit and that the delta subunit is essential for the stability of the whole complex. Further analysis of the mutant mice indicated that the mice lack significant levels of functional AP-3 complex. Studies on fibroblasts generated from these mice revealed that the AP-3 complex plays a role in the trafficking of LAMPI to lysosomes.

P1 prophage is stably maintained in E. coli as a low-copy-number plasmid. Stable maintenance of P1 is dependent on the function of the plasmid encoded partition system, parABS. ParA is the partition ATPase, ParB is the partition-site binding protein, and parS is the partition site. The concerted action of these proteins results in dynamic movement of the plasmid over the bacterial nucleoid, which results in its stable maintenance. Plasmid movement has been proposed to be caused by interactions between parS bound ParB and nucleoid bound ParA. In this thesis, I have identified a complex of ParA, ParB, and DNA that is capable of promoting plasmid stability. ParA, ParB, DNA interactions required the ATP bound conformation of ParA. The ParA-ParB-DNA complex was dynamically regulated by nucleotide hydrolysis, which promoted complex disassembly. Complex formation resulted from the cooperative binding of ParA and ParB to DNA. ParA-ParB and ParB-DNA interactions were both necessary for complex formation. ParA-ParB-DNA complex size was regulated by ParB stimulation of ParA-ATP hydrolysis. Microscopy demonstrated that complexes resulted in the association of multiple DNA molecules due to protein binding. The properties of complex assembly, dynamics, and DNA grouping lead me to propose a model where associations between ParA bound to the bacterial nucleoid and the partition complex mediated plasmid movement and localization.

P1 prophage is stably maintained in E. coli as a low-copy-number plasmid. Stable maintenance of P1 is dependent on the function of the plasmid encoded partition system, parABS. ParA is the partition ATPase, ParB is the partition-site binding protein, and parS is the partition site. The concerted action of these proteins results in dynamic movement of the plasmid over the bacterial nucleoid, which results in its stable maintenance. Plasmid movement has been proposed to be caused by interactions between parS bound ParB and nucleoid bound ParA. In this thesis, I have identified a complex of ParA, ParB, and DNA that is capable of promoting plasmid stability. ParA, ParB, DNA interactions required the ATP bound conformation of ParA. The ParA-ParB-DNA complex was dynamically regulated by nucleotide hydrolysis, which promoted complex disassembly. Complex formation resulted from the cooperative binding of ParA and ParB to DNA. ParA-ParB and ParB-DNA interactions were both necessary for complex formation. ParA-ParB-DNA complex size was regulated by ParB stimulation of ParA-ATP hydrolysis. Microscopy demonstrated that complexes resulted in the association of multiple DNA molecules due to protein binding. The properties of complex assembly, dynamics, and DNA grouping lead me to propose a model where associations between ParA bound to the bacterial nucleoid and the partition complex mediated plasmid movement and localization.

Toxoplasma gondii, l'agent causal de la toxoplasmose appartient au phylum des Apicomplexes. Comme son nom l'indique, le parasite possède un complexe unique d'organites sécrétoires apicaux, les micronèmes, rhoptries et le conoïde, qui jouent un rôle essentiel dans l’invasion de la cellule hôte et la survie du parasite. T. gondii est devenu un modèle populaire de biologie cellulaire et aussi un outil de référence pour l'étude de l’organisation ultra-structurale et des différentes fonctions des autres parasites du phylum Apicomplexa tel que Plasmodium, l’agent causal de la malaria. Cette thèse porte sur deux facteurs essentiels à la survie du parasite : le complexe adapteur de la clathrine AP1 et la petite GTPase Rab11A qui jouent un rôle crucial dans la régulation de certaines voies du trafic intracellulaire de T. gondii. Ainsi, nos travaux ont permis de démontrer un rôle pour AP1 dans le triage différentiel et le transport vésiculaire des protéines MIC et ROP depuis le Trans-Golgi-Network (TGN) et les compartiments endosomaux, respectivement. D’autre part, nos résultats ont révélé un rôle original de AP1 dans la division parasitaire aux stages tardifs de la cytokinèse. Nous avons également identifié un partenaire de AP1, la protéine unique de T. gondii possédant un domaine ENTH : EpsL (pour Espin-Like Protein). Dans les autres Eucaryotes, les protéines epsines sont connues pour activer la formation des vésicules à clathrine en co-opération avec les complexes AP1 et AP2. Nos résultats ont effectivement démontré un rôle de EpsL, similaire à AP1, pour la biogénèse des rhoptries et micronèmes. Nous avons, dans un deuxième temps, examiné les différentes fonctions de la petite GTPase Rab11A. Notre étude par vidéo-microscopie, semble indiquer que Rab11A régule le transport de vésicules depuis le TGN vers la périphérie cellulaire et en particulier, les pôles basal et apical du parasite. Après sur-production de la forme mutée inactive de Rab11A, nous avons démontré un nouveau rôle de la protéine dans la sécrétion des protéines membranaires de surface et dans l'exocytose des granules denses, lors de l'invasion de la cellule hôte mais aussi durant la réplication parasitaire. Finalement, des expériences de pull-down ont permis d’identifier un partenaire intéressant liant Rab11A seulement sous sa forme activée, la protéine unique de T. gondii contenant un domaine HOOK (TgHOOK), que nous avons caractérisée au niveau fonctionnel. Nos résultats suggèrent que TgHOOK régule le transport des vésicules positives pour Rab11A d’une manière dépendante des microtubules. Par conséquent, cette dernière étude a permis de révéler de nouveaux aspects encore inexplorés, bien qu’essentiels, des mécanismes régulant la sécrétion de molécules à la surface parasitaire. / Toxoplasma gondii, the causative agent for the disease Toxoplasmosis belongs to the phylum Apicomplexa. As the name implies, the parasite possesses a unique complex of apical secretory organelles namely the micronemes, rhoptries and conoid, which favor host cell invasion and intracellular survival. T.gondii has become a popular cell biology model and also a reference tool for studying the structure and functions of other important parasites that belong to the same phylum, such as plasmodium, but also higher eukaryotes. The recent advances in dissecting protein trafficking pathways have led to a better understanding of the biogenesis of apical organelles and also to the identification of crucial protein molecules that could determine the fate of the parasite. This thesis focuses on two different molecules, the Clathrin Adaptor complex AP1 and the small GTPase Rab11A that play a crucial role in distinct trafficking pathways of the parasite contributing to a wide range of functions. First, we reveal a role of AP1 in the differential sorting of microneme and rhoptry proteins at the Tran-Golgi-Network and endosomal level, respectively. Accordingly, depletion of AP1 leads to a defect in apical organelle biogenesis. In addition, we reveal an original role of AP1 in parasite division by regulating late stages of cytokinesis. We also identified and studied a partner of AP1, the unique ENTH domain containing protein of the parasite, EpsL (for Espin-like protein). In other Eukaryotes, epsin proteins are well known regulators of clathrin-mediated vesicular budding in co-operation with AP1 and AP2. We demonstrated that EpsL shares similar functions to AP1 in regulating rhoptry and microneme formation. We next worked on the small GTPase Rab11A and defined the dynamics of the protein within the parasite by live imaging. In addition to its known role in cytokinesis, we unravelled a novel function for the molecule in the secretion of surface membrane proteins and the exocytosis of dense granules during both, parasite invasion and replication. Further, pull down experiments on active Rab11A helped us fish an interesting partner molecule, the unique HOOK-domain containing protein that we functionally characterized for the first time in T.gondii. Our data suggest a role of Rab11A in microtubule-dependent transport of vesicules in a HOOK-regulated manner. Therefore, our study provides novel molecular insights into a yet unexplored but essential aspect of constitutive secretion in the parasite.

La protéine Nef des virus de l’immunodéficience humaine (VIH-1 et VIH-2) joue un rôle essentiel dans la physiopathologie de l’infection et induction du SIDA. La capacité de Nef à perturber le trafic intracellulaire de protéines membranaires, et notamment du récepteur CD4, circulant entre les compartiments de la voie d’endocytose pourrait rendre compte de son importance comme facteur de virulence au cours de l’infection naturelle. Les mécanismes responsables des perturbations de la voie d’endocytose induites par Nef au cours de l’infection ne sont pas totalement élucidés, mais il est admis qu’elles résultent d’interactions avec les complexes adaptateurs (AP) associés à la clathrine et participant au transport vésiculaire entre les différents compartiments de la voie d’endocytose. Notre objectif était de déterminer les mécanismes par lesquels Nef influe positivement sur le pouvoir infectieux du VIH-1 en interagissant avec la machinerie cellulaire de la voie d’endocytose. Notre programme s’est organisé autour de deux axes principaux: le premier a consisté à étudier l’implication respective des différents types de complexes AP (AP-1, -2 et -3) sur les perturbations du fonctionnement de la voie d’endocytose induites par Nef en analysant son impact sur le niveau d’expression de surface de CD4; le deuxième axe a consisté à évaluer l’impact de l’interaction de Nef avec les complexes AP sur les capacités infectieuses des particules virales. Le rôle respectif des différents complexes AP dans ces fonctions de Nef a donc été étudié après déplétion de l’expression des complexes AP-1, AP-2 et AP-3 par une approche d’ARN interférence. Les résultats obtenus montrent que contrairement à certaines données de la littérature, la déplétion des complexes AP de la voie d’endocytose ne semble pas avoir un impact majeur sur la capacité de Nef à moduler l’expression de surface de CD4, même si une légère diminution de l’activité de Nef a pu être révélée dans notre étude réalisée sur des cellules HeLa-CD4 transduites par les shRNA ciblant les complexes AP-2. Inversement, nos résultats confirment que la déplétion des complexes AP-1, AP-2 et AP-3 dans les cellules productrices des particules virales se traduit par une diminution importante des propriétés infectieuses de ces particules sur lesquelles l’impact positif de Nef n’est plus alors capable de se manifester. En conclusion, ce travail a donc permis de montrer que les complexes AP de la voie d’endocytose sont indispensables pour que Nef puisse exercer son rôle positif sur le pouvoir infectieux du VIH-1. Il est maintenant important de confirmer ces résultats en analysant le rôle fonctionnel des complexes AP sur les activités de Nef dans les cibles cellulaires naturelles du VIH-1, lymphocytes et macrophages. / Nef protein of human immunodeficiency virus (HIV-1 and HIV-2) plays an essential role in the pathophysiology of infection and induction of AIDS. The ability of Nef to disrupt intracellular trafficking of membrane proteins, including the CD4 receptor, moving between the compartments of the endocytic pathway could account for its importance as a virulence factor during natural infection. The mechanisms responsible for disruption of the endocytic pathway induced by Nef during infection are not fully understood, but it is accepted that they arise from interactions with adaptor complexes (AP) associated with clathrin and participant in vesicular transport between the different compartments of the endocytic pathway. Our objective was to determine the mechanisms by which Nef positively affects the infectivity of HIV-1 by interacting with the cellular machinery of the endocytic pathway. Our program has been organized around two main axes: the first was to investigate the respective involvement of different types of complexes (AP-1, -2 and -3) on the Nef induced disruption of the endocytic pathway by analyzing its impact on the level of surface expression of CD4; the second axis was to evaluate the impact of the interaction of Nef with AP complexes on the infectious capacity of the viral particles. The respective roles of the different AP complexes in these functions of Nef has been studied after depletion of the expression of complex AP-1, AP-2 and AP-3 by RNA interference approach. The results show that, contrary to some literature data, depletion of AP complex endocytic pathway does not appear to have a major impact on the ability of Nef to modulate the surface expression of CD4, although a slight decreased activity of Nef could be revealed in our study on HeLa-CD4 cells transduced with the shRNA targeting complex AP-2. Conversely, our results confirm that the depletion of complex AP-1, AP-2 and AP-3 in the cells producing viral particles resulted in a significant decrease in infectious properties of these particles on which the positive impact of Nef is no longer able to manifest. In conclusion, this work has shown that complex AP of endocytic pathway are essential for Nef to exercise its positive role in the infectivity of HIV-1. It is now important to confirm these findings by analyzing the functional role of AP complexes on the activities of Nef in the natural cellular targets of HIV-1, lymphocytes and macrophages.