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Plasma membrane of T cells is abundant in diverse receptors and other molecules orchestrating immune responses. Numerous studies demonstrate that the localisation of proteins in the cell is non-random and that mislocalisation either in the context of plasma membrane at nanoscale or with respect to the cell interior can lead to the protein malfunction and subsequent aberrant T- cell response. In my first Ph.D. project we focused mainly on the role of the transmembrane domain length and amino acid composition, proximal sequences and the presence or absence of palmitoylation on the localisation of transmembrane adaptor proteins LAT, PAG and NTAL in T cells. We showed that plasma membrane localisation of PAG and NTAL is controlled by the amino acid composition of their TMD and is palmitoylation independent. We propose that NTAL localisation to the plasma membrane is, despite its suboptimal length, facilitated by the electrochemical asymmetry of its TMD. Among transmembrane adaptor proteins, LAT was the most interesting one. Dependency of LAT plasma membrane localisation on palmitoylation in combination with unusual amino acid composition of its TMD led us to investigate it in a separate project. My first author Ph.D. project was thus to elucidate the role of highly conserved helix-breaking amino acids,...

Les contacts cellulaires sont formés par l'association et la clusterisation des molécules d'adhésion, qui agissent comme des unités d'organisation et de signalisation, garantissant la cohérence et la plasticité des tissus. Bien que les détails moléculaires des complexes d'adhésion soient bien caractérisés, la façon dont ces unités d'adhérence supramoléculaires sont organisés et régulées dans les conditions physiologiques est méconnue. Nous avons développé et appliqué la microscopie super-résolue et une analyse quantitative pour caractériser l'organisation nanométrique de la E-cadhérine dans le tissu épithélial de l'embryon de drosophile. Les molécules individuelles de la E-cadhérine sont localisées en 3D avec une précision de 30 et 100 nm dans des directions latérale et axiale, respectivement. Nous avons constaté que la E-cadhérine existe soit sous forme de monomères ou d'oligomères, contenant jusqu'à une centaine de molécules. La distribution de taille des clusters suit une loi de puissance (sans taille caractéristique). L'analyse de la répartition de clusters à différentes étapes de la morphogénèse indique que l'état d'agrégation est dicté par la concentration de la E-cadhérine aux jonctions. Pour tenter de déterminer comment la clusterisation de la E-cadhérine est régulée, nous avons perturbé l'endocytose et l'ancrage de la E-cadhérine à l'actine, et analysé l'état de clusterisation. / Cell-cell contacts form through binding and clustering of adhesion molecules, which act as organizational and signaling units and ensure coherence and plasticity of tissues. While the molecular details of adhesion complexes are well characterized, little is known about how these supramolecular adhesion units are organized and regulated in physiological conditions. We developed and applied superresolution microscopy and quantitative analysis to characterize the nanoscale organization of E-cadherin clusters in the early epithelial tissue of Drosophilaembryos. E-cadherin molecules at adherens junctions were localized in 3D with precision of 30 and 100 nm in lateral and axial directions, respectively. We found that E-cadherin exists either as monomers or oligomeric clusters, containing up to one hundred molecules. The cluster size distribution follows a power law - referred as scale free with no characteristic size. Analysis of clustering distribution at different stages of tissue morphogenesis indicates that the state of aggregation is dictated by the junctional concentration of E-cadherin. In an attempt to determine how E-cadherin clustering might be regulated, we perturbed endocytosis and anchoring of E-cadherin to actin, and analyzed the state of E-cadherin clustering. While blocking dynamin-dependent endocytic pathways yields increases of junctional E-cadherin concentration and promotes macroscopic aggregates, RNAi knockdown of α-catenin and Par-3 reduces E-cadherin concentration and changes significantly the organization of E-cadherin.

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Cette thèse porte sur la description de l'assemblage des virus dans le cadre de la physique statistique ainsi que sur les méthodes de mesure de cet assemblage utilisant les marqueurs fluorescents. Nous nous y attachons à décrire la dynamique de l'agrégation des protéines aux échelles de la population et du virus unique. Nous proposons deux méthodes pour mesurer les grandeurs physiques associées : taille et forme de la structure finale d'une part, taux d'agrégation au cours de la croissance d'autre part. Dans ce travail, nous nous sommes intéressés à la description physique de l'auto-assemblage des protéines virales. La physique de l'auto-assemblage in-vitro des virus sphériques, dont la structure est déterminée par l'agencement régulier de leurs constituants protéiques, a été théoriquement et expérimentalement caractérisée auparavant par des modèles d'agrégation. Les modèles existants décrivaient l'assemblage à quantité de composants viraux fixée dans un système ferme à partir des constituants élémentaires du virus. In-vivo, la situation est bien entendu différente. Abstraction faite de la grande complexité du milieu cellulaire, les virus s'échappent de la cellule une fois formés pour aller infecter de nouvelles cellules. De plus, la quantité de constituants est sans cesse modifiée par la fabrication ou la dégradation des protéines virales. Enfin les méthodes de mesures utilisées in-vitro ne sont généralement plus envisageables in-vivo. Nous avons donc étudié les effets d'un flux de matière dans système ouvert via le calcul de l'état stationnaire, et via la résolution numérique des équations d'évolution des populations d'agrégats qui décrivent la cinétique d'agrégation des protéines virales. Dans ce cadre, nous avons mis en valeur le lien entre la description de l'état général du système en termes de populations et le devenir individuel d'un virus en formation pour le suivi duquel des méthodes expérimentales existent. Nous nous sommes alors attachés à proposer un traitement approprié de telles données expérimentales pour déterminer les valeurs des paramètres physiques du modèle / In this thesis work, we study the self-assembly of viral particles and focus on the analysis of measurements based on fluorescence labeling of viral proteins. We propose a theoretical model of the dynamic of viral proteins self-assembly at the cell membrane based on previous models developed to describe the in-vitro assembly of spherical viruses. We study the evolution of the populations in the successive stages of viral budding as well as the evolution of single particle within this framework. We also provide various data analysis to measure the physical values involved in the process: rate of aggregation during the bud growth, size and shape of the eventual structure. Viruses are biological objects unable to replicate without infecting an host cell since they lack part of the molecular machinery mandatory for genetic code replication and proteins production. Originally aimed at controlling the diseases they cause, the study of viruses is now rich of applications in medical and technological field (gene therapy, phage therapy, targeted therapy, bio-templating, cargo specific encapsulation, etc.). The existent models describing the self-assembly of viral proteins have successfully captured many features observed in the in-vitro experiments. We study the expected evolution when an open system is considered with an input flux of proteins and an output flux of released virion, characteristic of the in-vivo situation. We derive the population distribution at steady state and numerically study their dynamic under constant viral protein input flux. We also study the case of a single bud evolution which can be followed by its fluorescence emission. We study the possibility to estimate shape parameters at the single viral particle level such as radius and completion for the human immunodeficiency virus (HIV) from single molecule localization superresolution microscopy. These techniques known as (f)PALM or (d)STORM, record labeled proteins position with a precision of few to tens of nanometers. We propose an approach base on the maximum likelihood statistical method which is tested on both real and simulated images of fully formed particles. Our results suggest that it can offer a precision on the determination of the global structure finner than the positioning precision of the single proteins. This efficiency is however tempered when the parameter of interest does not affect the figures of merit to which the method is sensitive such as the apparent area and the image contours

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CD4 co-receptor of main T cell receptor (TCR) is essential for proper development of T lymphocytes and their function in adaptive immune responses. It is believed that CD4 stabilizes the interaction of TCR with antigenic ligand, peptide-MHC, and thereby improves T cell-dependent responses during immune reaction. CD4 is transmembrane glycoprotein with a number of structural motifs in its intracellular domain which do not dramatically affect its sorting to the plasma membrane but can influence its local organization at nanoscale. CD4 was shown to transiently accumulate in the immunological synapse formed between T cell and antigen-presenting cell. Such accumulation is rapidly followed by its internalization and/or delocalization outside the synapse. This is in contrast with TCR which accumulates strongly in the immunological synapse and is later found enriched in the central area of this structure. It is therefore unclear how TCR and its CD4 co-receptor function together when binding to their common ligand during the initiation of signaling in T cells. We aim to study localization of CD4 at nanoscale using advanced fluorescence microscopy techniques achieving significant improvements in resolution. In this work, CD4 and its mutant variants, potentially causing its different localization at the...