Des forces intermoléculaires sur lesquelles nous avons peu de connaissances préalables sont souvent essentielles à la stabilité et à l'évolution des assemblages biologiques. Dans cette thèse, nous nous concentrons sur deux de ces forces qui sont impliquées de façon critique dans la déformation soit des biopolymères, soit des membranes. Nous déduisons ces forces en conciliant la géométrie d'une telle déformation avec des modèles mécaniques simples. Dans la première partie de la thèse, nous examinons la force d'attraction entre les molécules d'ADN médiées par des cations multivalents. Cette attraction est nécessaire pour compenser la rigidité de l'ADN lors du confinement de grandes quantités d'ADN dans des environnements relativement petits, tels que les noyaux des spermatozoïdes. In vitro, les cations multivalents causent la condensation de l'ADN en faisceaux toroïdaux denses. Grâce à des données sur la géométrie de ces faisceaux, nous pouvons étudier la compétition entre les forces attractives et la rigidité de l'ADN. Nous inférons telles forces et proposons que la courbure toroïdale affaiblisse l'adhésion entre les molécules d'ADN. Dans la deuxième partie de la thèse, nous nous intéressons à la force de liaison d'un complexe protéique de remodelage membranaire, ESCRT-III, aux membranes cellulaires. Les protéines ESCRT-III s'assemblent en polymères qui remodèlent la membrane au cours de nombreux processus cellulaires, allant du bourgeonnement du VIH à la cytokinèse. Le mécanisme par lequel les polymères ESCRT-III déforment les membranes n'est toujours pas clair. In vitro, les polymères ESCRT-III peuvent transformer des vésicules membranaires sphériques en tubes hélicoïdaux. Nous proposons que les tubes hélicoïdaux résultent du positionnement particulier des sites de liaison membranaire sur la surface des polymères ESCRT-III. De plus, nous déduisons la force de liaison entre les monomères ESCRT-III et la membrane à partir de la géométrie des tubes hélicoïdaux. / Inter-molecular forces on which we have poor prior knowledge are often essential for the stability and evolution of biological assemblies. In this thesis, we focus on two such forces that are critically involved in the deformation of either biopolymers or membranes. We infer these forces by reconciling the geometry of such deformation with simple mechanical models. In the first part of the thesis, we consider the attractive force between DNA molecules mediated by multivalent cations. This attraction is required to compensate DNA bending rigidity when packaging large quantities of DNA in comparatively small environments, such as the nuclei of sperm cells. In vitro, multivalent cations drive DNA condensation into dense toroidal bundles. Geometrical data on DNA toroidal bundles give access to the competition between inter-helical attraction and DNA bending rigidity. From these data, we infer inter-helical forces and argue that the toroid curvature weakens the adhesion between DNA molecules. In the second part of the thesis, we turn to the binding force of a membrane remodeling protein complex, ESCRT-III, to cellular membranes. ESCRT-III proteins assemble into membrane-remodeling polymers during many cellular processes, ranging from HIV budding to cytokinesis. The mechanism by which ESCRT-III polymers deform membranes is still unclear. In vitro, ESCRT-III polymers can reshape spherical membrane vesicles into helical tubes. We argue that helical tubes result from the peculiar positioning of membrane-binding sites on the surface of ESCRT-III polymers. Furthermore, we infer the binding force between ESCRT-III and membrane from the geometry of helical tubes.

The maturation of early endosomes into multivesicular bodies (MVBs) and subsequent trafficking to lysosomes is an important event for the control and silencing of endocytosed membrane receptors. The endosomal-sorting complex required for transport (ESCRT) proteins appear to play a key role in this event. Phosphatidylinositol lipids including PtdIns(3,5)P2 have been implicated in the MVB-lysosomal pathway and an ESCRT-III component CHMP3 binds to this lipid in vitro. The purpose of this thesis was to investigate the link between ESCRT proteins, PtdIns(3,5)P2 and endo-lysosomal trafficking. Firstly, a protein expressed by Salmonella, which is a phosphatase that acts on PtdIns(3,5)P2, was investigated as a potential tool for manipulating cellular PtdIns(3,5)P2 levels. Our results suggest that it is potentially a useful tool for this purpose and that expression of SopB perturbs endosome to lysosome trafficking. These findings provide further evidence for a role of PtdIns(3,5)P2 in endo-lysosomal trafficking.

Biogenesis of the multivesicular body (MVB) organelle is an important process for regulation of signalling in the cell. Signal receptors embedded within the outer MVB membrane can be sorted into intralumenal vesicles which bud away from the cytosol to within the MVB preventing further signalling. Sorting of receptors, invagination of the membrane and release of vesicles into the MVB lumen are mediated by the endosomal sorting complexes required for transport (ESCRT) along with a range of accessory proteins including histidine domain protein tyrosine phosphatase (HD-PTP). HD-PTP is a multidomain protein which makes several interactions with ESCRT partners, including ESCRT-0, ESCRT-I and ESCRT-III. This thesis focusses specifically on the interaction between HD-PTP CC domain and Ubap1 (ESCRT-I), and the two interactions of HD-PTP Bro and PRR domains with STAM2 (ESCRT-0) SH3 and Core domains. To address the structure of HD-PTP, multiple techniques were used: X-ray crystallography, which gives high resolution structural information; small angle X-ray scattering (SAXS), which gives low resolution data for large non-crystallisable units in their solution state; and double electron-electron resonance (DEER) spectroscopy, which gives high resolution nanometre-range distance constraints between cysteines labelled with methanethiosulfonate spin label (MTSL). It was shown by X-ray crystallography that HD-PTP has an elongated CC domain, in stark contrast to its homologues ALIX and Bro1 which both have V-shaped CC domains. The CC domain showed limited flexibility both by SAXS and DEER. Further investigation showed that there was no significant conformational change upon binding its ESCRT-I partner Ubap1. The multidomain structure of HD-PTP Bro1-CC-PRR was described by SAXS, showing that these domains form an extended arrangement in solution. In addition, SAXS was also used to analyse the structure of these domains in complex with STAM2 (ESCRT-0), which showed that STAM2 is simultaneously tethered by the Bro1 domain and PRR. The Bro-CC-PRR portion of HD-PTP, has 9 cysteines, so with the aim of measuring local structural information in the CC domain alone, alternative spin labelling methods were investigated. Use of a bromoacrylaldehyde spin label (BASL), instead of MTSL, allowed more selective labelling of surface exposed cysteines, and avoided labelling most of the cysteines in the Bro1 domain. This novel method allowed the shape of the CC domain to be monitored during STAM2 binding and showed that there is no induced conformational change.

Ubiquitination is a post-translational modification tht mediates sorting of integral membrane proteins to lysosomes for their degradation. ESCRTs (Endosomal Sorting Complex Required For Transport) bind and sequester ubiquitinated membrane proteins and direct them into multivesicular bodies (MVBs). ESCRTs themselves become covalently ubiquitinated, simply by virtue of non-covalently binding Ub. However, it is unclear whether this regulates a critical aspect of ESCRT function. In yeast, many MVB cargo proteins are ubiquitinated by the HECT-type Ub-ligase Rsp5, sometimes via the action of Rsp5 adaptor proteins. While many Rsp5 targets are modified by polyubiquitination, it remains unclear whether polyubiquitination is a necessary signal for their incorporation into MVBs. Despite years of research, these and related questions have been difficult to resolve because it is technically quite challenging to control the level of a given protein's ubiquitination. The aim of this research was to develop a novel technique, which can render proteins resistant to ubiquitination. The technique involved the fusion of the Ub-peptidase to a protein of interest via a flexible linker, essentially creating a "DUb module". The intent of this module would be to cleave any Ub form the target protein, essentially immunizing it from the effects of ubiquitination. This novel method was used in combination with several conventional methods to examine the role of ubiquitination within the endocytic pathway and in particular focus on the questions of what type of ubiquitin signal was sufficient for sorting into MVB vesicles and whether ubiquitination of ESCRTs was required for their sorting activity. We found that a single Ub was sufficient for membrane protein entry into MVBs in the absence of ESCRT ubiquitination.

Deregulation of autophagy plays an important role in the pathogenesis of diseases such as cancer, neuronal degenerative or cardiovascular disease. Autophagy is a process to engulf the cytoplasmic contents into autophagosome and deliver them for lysosomal degradation. Its major function is to clear unfolded protein or damage organelles for maintaining proper metabolic homeostasis and normal cell physiological activities. Autophagy and multivesicular bodies, MVBs, cooperate to regulate the turnover of intracellular macromolecule, defective organelles and signaling receptor. Endosomal sorting complex required for transport, ESCRT, is important for the formation of MVBs, which regulates membrane receptor recycling, protein sorting and vesicular trafficking. Tumor Susceptibility Gene 101(TSG101) is a member of ESCRT-I that plays an important role on MVBs formation and maintaining ESCRT function. Previous report indicated that autophagosome accumulation upon deprivation of TSG101, implying possible role of TSG101 during autophagic process. In this study, we observed the increase of TSG101 and autophagic marker proteins, such as LC3-II and ATG upon nutrient starvation. Furthermore, knockdown TSG101 in cervical carcinoma HeLa cell resulted in the elevation of LC3-II, ATG3 and ubiquitinated protein aggregates marker protein p62, which is congruous to other reports. However, in neuroblastoma SH-SY5Y cell, transfection of siRNA led to the decrease of LC-II and ubiquitinated protein level. These results indicated that TSG101 might be critical for autophagy and the maintenance of steady-state level of cellular ubiquitinated proteins. Ectopic upregulatory expression of HA-TSG101 led to the increase of LC3-II in both cell type. The elevation of ATG3 level is also observed in HeLa cell. Therefore, we speculated that TSG101 might be important for the formation of autophagosome, but our data did not exclude the possible role of TSG101 in regulation of the fusion of autophagosome and lysosome, because the increase of ATG3 indicated ectopic HA-TSG101 might facilitate the execution of autophagic flow. In addition, we have established GFP-LC3 expression cell lines. Our imaging data showed the colocalization of TSG101 and GFP-LC3 in both cytoplasm and nucleus that might be an interesting research topic for investigation the role of TSG101 in autophagic pathway.

CHMP2B est une sous-unité du complexe ESCRT-III, un complexe cytosolique très conservé, responsable du remodelage des membranes biologique, dans divers processus cellulaires. Des mutations de CHMP2B sont associées à une forme familiale de démence frontotemporale. Une étude précédente a mis en évidence que les mutants pathogènes de CHMP2B altèrent la morphologie des épines dendritiques, un phénomène potentiellement à l'origine de la maladie. Ce travail de recherche a pour objectif de décrire le rôle de CHMP2B, et du complexe ESCRT-III, dans la structure et le fonctionnement des épines dendritiques. Dans des lignées cellulaires, nous avons démontré que CHMP2B a la propriété de s'associer préférentiellement à la membrane plasmique, de se polymériser en filaments hélicoïdaux et de former de longs et fins tubes membranaires. Ce résultat indique que CHMP2B est directement impliqué dans le remodelage de la membrane plasmique. Dans les neurones, CHMP2B se concentre dans des régions sous-membranaires proches de la PSD. Une analyse biochimique a montré que CHMP2B et CHMP4B sont associées à d'autres sous-unités, pour former un complexe ESCRT-III postsynaptique particulièrement stable. Nous avons identifié par spectrométrie de masse que ce complexe interagit également avec des protéines d'échafaudage postsynaptiques et des protéines de remodelage du cytosquelette d'actine. La déplétion de CHMP2B par RNAi, dans des neurones en culture, affecte la complexité de l'arborisation dendritique, la morphologie des épines dendritiques et empêche le gonflement des épines associé à la LTP. Des expériences de récupération, avec des mutants pontuels, indiquent que le rôle de CHMP2B dans le maintien de l'arborisation dendritique est dépendant à la fois de de son association avec ESCRT-III et la bicouche phospholipidique. Nous proposons une nouvelle fonctionnalité pour un complexe ESCRT-III contenant CHMP2B, dans les processus de remodelage de la membrane postsynaptique associés à la maturation et à la plasticité des épines dendritiques. / CHMP2B is a subunit of ESCRT-III, a highly conserved cytosolic protein machinery, responsible for membrane remodeling in diverse cellular mechanisms. Mutations in CHMP2B are responsible for a familial form of frontotemporal dementia. A previous study highlighted that FTD-related mutants of CHMP2B impair the morphological maturation of dendritic spines, a process that may underlie neurodegeneration in this disease. The goal of this research work id directed towards understanding the role of CHMP2B and ESCRT-III in dendritic spines structure and function. In cell lines, we demonstrated that CHMP2B associates preferentially with the plasma membrane, polymerizes in helical filaments and forms long and thin membrane protrusions. This result indicates that CHMP2B is directly involved in plasma membrane remodeling. In neurons, CHMP2B concentrates in specific sub-membrane microdomains close to the PSD. Biochemical analysis revealed that CHMP2B and CHMP4B associate with other subunits to form a remarkably stable postsynaptic ESCRT-III complex. Mass-spectrometry indicated that this complex also interacts with postsynaptic scaffolds and proteins involved in actin cytoskeleton remodelling. RNAi depletion of CHMP2B, in cultured neurons, alters stability of dendrite branching and morphology of dendritic spines, and impairs spine head growth, normally associated with LTP. Rescue experiments, with point mutants, indicated that CHMP2B activity in dendrite branching is dependent on its capacity to both bind phospholipids and oligomerization with ESCRT-III. We propose a novel functionality for an ESCRT-III complex containing CHMP2B, in maturation-dependent and plasticity-dependent processes of dendritic spine morphogenesis.

La mise en place de l’asymétrie droite/gauche (D/G) est fondamentale au développement normal des organismes bilatériens. Cependant, les mécanismes essentiels à la cassure de symétrie ainsi que leur conservation au cours de l’évolution sont encore mal compris. Chez le poisson-zèbre l’organisateur de l’asymétrie D/G ou vésicule de Kupffer (KV), consiste en une sphère remplie d’un liquide mis en mouvement de façon circulaire antihoraire pas le battement de cils motiles. Nous avons observé dans la lumière de la KV la présence de vésicules positives pour la protéine ESCRT Chmp1b. De plus, une diminution du nombre de ces vésicules est associée à l’apparition de défauts de latéralité, ce qui suggère un rôle potentiel de ces vésicules dans la mise en place de l’asymétrie D/G. Nous montrons que malgré sa forme circulaire, le flux de la KV peut transporter de façon asymétrique des vésicules. Deuxièmement, nous avons également montré que la fonction de la protéine Myo1D, impliquée dans l’asymétrie D/G chez la drosophile, est conservée chez le poisson-zèbre. La perte de Myo1D a un impact sur les signaux les plus précoces d’initialisation de la latéralité. De plus, Myo1D interagit de façon fonctionnelle avec la protéine Vangl2, un composant de la voie PCP contrôlant le positionnement et l’orientation des cils de la KV. Ces découvertes établissent ainsi que le système Myo1D représente une machinerie conservée au cours de l’évolution pour l’établissement d’une latéralité chez les animaux. / The proper establishment of Left/Right (L/R) asymmetry is fundamental to normal development in bilaterians. However, the initial mechanisms involved in symmetry breaking and their conservation throughout the course of evolution remain poorly understood. In zebrafish, the L/R organizer region or Kupffer’s vesicle (KV) consists as a fluid filled cavity, whose circular counterclockwise motion is driven by the motion of motile cilia. We have observed the presence of vesicles containing the ESCRT component Chmp1b in the lumen of the KV. Hence, the diminution of their number is associated with laterality defect, suggesting a potential role for those vesicles in L/R asymmetry establishment. We also participate to show that in spite of its overall circular geometry, the KV flow can ensure the directional transport of vesicles in the organ lumen. Secondly, we are also showing that the function of the Myo1D protein, implicated in L/R asymmetry establishment in drosophila, is conserved in zebrafish. The loss of Myo1D leads to an impaired early lateralization. Moreover, Myo1D functionally interact with the protein Vangl2, a component of the PCP pathway, which controls the tilt and positioning of KV cilia. These findings are supportive of a mechanism according to which Myo1D system represents a conserved molecular machinery essential for L/R asymmetry establishment in bilaterians.

Les cellules épithéliales forment un tissu de cellules étroitement juxtaposées qui assure une barrière physique et chimique entre les compartiments internes et externes du corps. L’intégrité de ces tissus est donc essentielle. Au cours du développement et de la vie adulte, le tissu doit grandir ou se régénérer, ce qui implique de nombreuses divisions cellulaires. La dernière étape de la division, la cytodiérèse, met en jeu la formation d’un anneau contractile qui, en se fermant, va séparer les cellules sœurs. Une fois complètement fermé, il donne naissance au midbody, juste sous le niveau des jonctions adhérentes, au sein des jonctions septées, chez la drosophile. L’ultime étape, l’abscission, permet la séparation physique définitive et l’isolation cytoplasmique des cellules sœurs. Si de nombreuses études ont décrit ces processus dans les cellules isolées, peu de choses sont connues quant à la cytodiérèse des cellules épithéliales. Ce travail de thèse a permis de mettre en évidence que malgré le recrutement de tous les effecteurs et régulateurs de l’abscission, celle-ci est retardée dans les cellules épithéliales. Des expériences de photo-conversion de KAEDE ont montré que l’abscission est liée à l’entrée en mitose des cellules épithéliales. La question de l’intégrité du tissu et notamment de la barrière de perméabilité a ensuite été investigué. Nous avons montré que les cellules voisines formaient des protrusions de membrane restant connectées au midbody tout au long de sa lente migration vers le pôle basal des cellules. Les expériences de FRAP menées sur les jonctions bicellulaires et tri-cellulaires des jonctions septées ont permis de montrer que celles-ci se formaient juste sous les jonctions adhérentes et toujours au-dessus du midbody, participant ainsi à la migration de ce dernier vers le pôle basal. Les contacts maintenus avec les voisines ainsi que l’assemblage polarisé des jonctions septées participent au maintien de l’intégrité du tissu au cours des divisions de cellules épithéliales. / Epithelial cells are closely juxtaposed to form a tissue playing a physical and chemical barrier between external and internal body compartments. Thus, tissue integrity is essential. During development and adult life, epithelia has to growth and regenerate meaning a lot of divisions. At the end of cell division, cytokinesis occurs, implying the formation of a contractile ring which contracts to separate daughter cells. In Drosophila, once totally closed, the contractile ring gives rise to the midbody, just below adherens junctions, in the septate junctions layer. Last step of cytokinesis, abscission, permits the final cut and the cytoplasmic isolation of daughter cells. If cytokinesis is well described in isolated cells, little is known about epithelial cells cytokinesis. This work shows that whereas all abscission regulators and effectors are recruited, abscission is delayed in epithelial cells. KAEDE photo-conversion assays show that abscission is linked to epithelial cells mitosis entry. Then we investigate how permeability barrier is maintained during cell division. We show that neighboring cells present finger-like protrusions contacting the midbody all along the midbody is moving basally across septate junctions. FRAP experiments on bicellular and tricellular septate junctions show that they form just below adherens junctions and always above the midbody, leading to its basal migration. Contacts maintained with neighbors and polarized assembly of septate junctions participate to the maintenance of tissue integrity throughout epithelial cells divisions.

La polyprotéine de structure Gag du VIH-1 est responsable de l’assemblage des particules virales dans les cellules infectées. Au niveau moléculaire, cette protéine s’oligomérise en formant des complexes Gag-Gag autour de deux plates-formes moléculaires, d'une part l'ARN génomique via son domaine NCp7 (NucleoCapsid protein 7) et d'autre part, la membrane plasmique via son domaine MA (Matrice). De plus, lors du trafic de Gag dans la cellule, Gag détourne les protéines ESCRT comme TSG101 et ALIX de la machinerie cellulaire afin de bourgeonner et d’être libérées dans le milieu extracellulaire. Dans cette thèse, nous avons étudié le rôle du domaine NCp7 seul ou au sein de Gag (GagNC) dans les interactions Gag-Gag et Gag-TSG101 en utilisant des approches biochimiques et de la microscopie de fluorescence quantitative. Les résultats ont montré que l'absence du domaine NCp7 affecte l’oligomerisation de Gag qui s’accumule alors dans le cytoplasme sous forme d’agrégats de taille importante. Par ailleurs, le trafic intracellulaire de Gag est affecté par les mutations dans le domaine GagNC avec une augmentation importante de temps nécessaire à Gag pour arriver à la membrane plasmique. Enfin, nous avons montré que GagNC i) renforce l’interaction entre le domaine p6 de Gag et TSG101 et ii) par sa fonction dans le trafic de Gag, est responsable de la localisation de TSG101 à la PM. Sur la base de ces résultats, des études sont maintenant en cours pour développer des tests afin d’identifier des molécules possédant un potentiel anti virale. / The Gag structural polyprotein of HIV-1 orchestrates viral particle assembly in producer cells, in a process that requires two platforms, the genomic RNA on the one hand and a membrane with a lipid bilayer, on the other. During its transportation from translating ribosomes to plasma membrane, Gag hijacks cellular proteins of the cytoskeleton and the ESCRT proteins like TSG101, Alix, etc., to egress viral particles. However, a number of questions remain to be answered before they are clearly apprehended. In this thesis, , we studied the role of the NC domain alone or as part of Gag (GagNC) in Gag-Gag and Gag-TSG101 interactions, which are essential for the assembly and budding of HIV-1 particles using quantitative fluorescent microscopy and biochemical approach. Results, showed that the absence of NC domain lead to (1) an accumulation of Gag as large aggregates that are dispersed in the cytoplasm, (2) a decrease of Gag-Gag condensation and (3) a delay for Gag-Gag complexes in reaching the PM, (4) improved interaction between Gag and TSG101, and (5) by its virtue in Gag trafficking docks TSG101 to the PM. This regulatory effect of NCp7 domain in either TSG101 or Gag or both protein- regulated pathways during virus budding can be exploited to develop inhibitors targeting HIV-1.

Mutations in MCOLN1, which encodes the cation channel protein TRPML1, result in the neurodegenerative lysosomal storage disorder Mucolipidosis type IV. Mucolipidosis type IV patients show lysosomal dysfunction in many tissues and neuronal cell death. The orthologue of TRPML1 in Caenorhabditis elegans is CUP-5; loss of CUP-5 results in lysosomal dysfunction in many tissues and death of developing intestinal cells that results in embryonic lethality. We previously showed that a null mutation in the ATP-Binding Cassette transporter MRP-4 rescues the lysosomal defect and embryonic lethality of cup-5(null) worms. Here we show that reducing levels of the Endosomal Sorting Complex Required for Transport (ESCRT)-associated proteins DID-2, PHI-33, and ALX-1/EGO-2, which mediate the final de-ubiquitination step of integral membrane proteins being sequestered into late endosomes, also almost fully suppress cup-5(null) mutant lysosomal defects and embryonic lethality. Indeed, we show that MRP-4 protein is hypo-ubiquitinated in the absence of CUP-5 and that reducing levels of ESCRT-associated proteins suppresses this hypo-ubiquitination. Thus, increased ESCRT-associated de-ubiquitinating activity mediates the lysosomal defects and corresponding cell death phenotypes in the absence of CUP-5.