The nervous system is made up of specialized cells which receive and respond to environmental stimuli. Intercellular communication in the nervous system is achieved predominantly through chemical synaptic transmission. Within the chemical synapse, the actin cytoskeleton plays a major role in regulating synaptic activities, although the extent and clarity in our understanding of these processes is still limited. Using the genetically pliable model, Drosophila melanogaster, this thesis begins to unravel contributions of actin binding proteins to synaptic development and physiology at the larval neuromuscular junction (NMJ). Two actin binding proteins, Moesin and Nonmuscle Myosin II (NMMII) were selected for study based on previous studies implicating them in synaptic development. Combining genetics, fluorescent imaging and electrophysiological recordings this thesis unveils previously unidentified functions for Moesin and NMMII in morphology and physiology of the Drosophila NMJ. Moesin was found to help restrain synaptic growth but did not affect synaptic physiology. By correlating morphological and electrophysiological measurements in Moesin mutants, it was determined that physiology and morphology can be independently regulated at the NMJ. NMMII was used to investigate a role for actin binding proteins in physiology at the Drosophila NMJ. By using the fluorescent imaging technique, FRAP, this becomes the first research to implicate NMMII in unstimulated synaptic vesicle mobility. FRAP indicated that vesicle mobility was highly dependent on the expression level of NMMII. Electrophysiological analysis of NMMII indicated distinct mechanisms for spontaneous and evoked vesicle release. NMMII expression exhibited a positive correlation with basal synaptic transmission and was important in mobilizing vesicles for synaptic potentiation. In addition, NMMII was found to be involved in a high frequency dependent low frequency depression. This work begins to identify how vesicles traverse within boutons and suggests differential mechanisms of synaptic release, both of which are partially dependent of NMMII expression. Studying Moesin and NMMII have revealed a complex interplay between the actin cytoskeleton and synaptic function and together this research furthers our understanding of how the actin cytoskeleton regulates synaptic activity.

The nervous system is made up of specialized cells which receive and respond to environmental stimuli. Intercellular communication in the nervous system is achieved predominantly through chemical synaptic transmission. Within the chemical synapse, the actin cytoskeleton plays a major role in regulating synaptic activities, although the extent and clarity in our understanding of these processes is still limited. Using the genetically pliable model, Drosophila melanogaster, this thesis begins to unravel contributions of actin binding proteins to synaptic development and physiology at the larval neuromuscular junction (NMJ). Two actin binding proteins, Moesin and Nonmuscle Myosin II (NMMII) were selected for study based on previous studies implicating them in synaptic development. Combining genetics, fluorescent imaging and electrophysiological recordings this thesis unveils previously unidentified functions for Moesin and NMMII in morphology and physiology of the Drosophila NMJ. Moesin was found to help restrain synaptic growth but did not affect synaptic physiology. By correlating morphological and electrophysiological measurements in Moesin mutants, it was determined that physiology and morphology can be independently regulated at the NMJ. NMMII was used to investigate a role for actin binding proteins in physiology at the Drosophila NMJ. By using the fluorescent imaging technique, FRAP, this becomes the first research to implicate NMMII in unstimulated synaptic vesicle mobility. FRAP indicated that vesicle mobility was highly dependent on the expression level of NMMII. Electrophysiological analysis of NMMII indicated distinct mechanisms for spontaneous and evoked vesicle release. NMMII expression exhibited a positive correlation with basal synaptic transmission and was important in mobilizing vesicles for synaptic potentiation. In addition, NMMII was found to be involved in a high frequency dependent low frequency depression. This work begins to identify how vesicles traverse within boutons and suggests differential mechanisms of synaptic release, both of which are partially dependent of NMMII expression. Studying Moesin and NMMII have revealed a complex interplay between the actin cytoskeleton and synaptic function and together this research furthers our understanding of how the actin cytoskeleton regulates synaptic activity.

Les protéines ERMs (Ezrine, radixine et moésine) jouent un rôle central in cellulo, dans de nombreux processus cellulaires tels que les infections, la migration et la division cellulaire. Parmi celles-ci, la moésine est plus particulièrement impliquée dans la formation de la synapse immunologique, l’infection virale et bactérienne, et les métastases cancéreuses. D’un point de vue structural, les ERM peuvent être en conformation inactive (replies sur elles-mêmes) ou actives (ouvertes), ce qui permet leur interaction a la fois avec les constituants du cytosquelette (actine et tubuline) via leur domaine C-terminal et la membrane plasmique via leur domaine FERM. La liaison a la membrane plasmique se fait principalement et spécifiquement via un lipide de la famille des phosphoinositides, le phosphatidyl 4,5 bisphosphate (PIP2). De plus, les protéines peuvent être phosphorylées, ce qui contribue à leur ouverture structurale. Cependant, le rôle de la phosphorylation sur les interactions ERM/membrane et ERM/cytosquelette, bien que beaucoup étudié in cellulo, est peu compris au niveau moléculaire.Le but de cette thèse est précisément d’étudier, au niveau moléculaire et à l’aide de systèmes biomimétiques, les interactions entre des protéines recombinantes et des membranes biomimétiques contenant du PIP2. Pour cela, nous avons mis au point des membranes lipidiques sous forme de vésicules unilamellaires (petites ou larges) et de bicouches lipidiques supportées, qui permettent de caractériser les interactions entre protéines et membranes par des techniques biophysiques complémentaires, notamment la cosédimentation quantitative, la microscopie et spectroscopie de fluorescence, et la microbalance à cristal de quartz. Dans une première partie, nous avons étudié le rôle de la double phosphorylation de la moésine (réalisée par mutation sur site spécifique) sur les interactions moésine/membrane biomimétique, en comparaison de la protéine sauvage, les protéines recombinantes et les mutants ayant été produites et purifiées au laboratoire.Nos résultats mettent en évidence une interaction spécifique et coopérative pour le double mutant phosphomimétique alors que cette interaction est simple dans le cas de la protéine sauvage. Dans une seconde partie, nous avons employé les bicouches lipidiques supportées contenant le PIP2 pour étudier les mécanismes molécules d’adsorption de la protéine virale Gag et de ses mutants. Les méthodologies développées dans ce travail de thèse ouvrent des perspectives en biophysique moléculaires car elles sont facilement transposables à l’étude d’autres protéines sur des membranes lipidiques modèles contenant des phosphoinositides.Mots clés: Ezrine-Radixine-Moésine, phosphoinositides, PIP2, interactions protéine-lipide, membrane lipidique biomimétique, protéine virale Gag, cytosquelette. / ERM (ezrin, radixin, moesin) proteins play a central role in cellulo in a large number of physiological and pathological processes, including cell infection, migration and cell division. Among the ERMs, moesin is particularly involved in the formation of the immunological synapse, viral and bacterial infection, and cancer metastasis. From a structural point of view, ERMs can be in inactive (closed) conformation or active (open), which enable them to interact on one side with the cytoskeleton (actin and tubulin) via their C-terminal domain and on the other side with the plasma membrane via their FERM domain. Binding to the plasma membrane is mediated via a specific lipid of the phosphoinositide family, the phosphatidylinositol(4,5)bisphosphate (PIP2). In addition, ERM can be phosphorylated, which contribute to their structural opening. To date, the role of the phosphorylation in ERM/membrane and ERM/cytoskeleton interactions, although widely studied in cellulo, remains poorly understood at the molecular level.The aim of this PhD thesis is precisely to study, at the molecular level and using biomimetic systems, interactions between recombinant proteins and biomimetic membranes containing PIP2. To this end, we have engineered lipid membranes in the form of large and small unilamellar vesicles and supported lipid bilayers. These biomimetic membranes are used to characterize interactions between proteins and membranes by complementary biophysical techniques, notably quantitative cosedimentation, fluorescence microscopy and spectroscopy, and quartz crystal microbalance with dissipation monitoring. In a first part, we studied the role of double phosphorylation on moesin, achieved via a site-specific mutation on threonine residues, on moesin/biomimetic membrane interactions, in comparison to the wild type protein. The recombinant proteins and mutants were produced in our laboratory.Our results show that there is a specific and cooperative interaction for the double phosphomimetic mutant while interactions is 1:1 in the case of the wild type protein. In a second part, we used supported lipid bilayers containing PIP2 to study the molecular adsorption mechanism of the viral protein Gag and of its mutants. The methodologies that were developed in this work open perspectives in molecular biophysics since they are easily adaptable to other proteins on model lipid membranes containing phosphoinositidesKeywords: Ezrin-Radixin-Moesin, phosphoinositides, PIP2, protein/lipid interactions, biomimetic lipid membrane, Gag viral protein, cytoskeleton.

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