Les REMORINES du groupe 1 sont des protéines spécifiques des plantes, localisées dans la membrane plasmique. Nous avons montré que StREM1.3 (REM) constitue un marqueur des radeaux lipidiques, des domaines membranaires du plasmalemme enrichis en stérols et sphingolipides. De plus, REM se trouve enrichie dans les plasmodesmes (PD), des canaux ancrés dans la paroi qui assurent les communications intercellulaires. Nous avons mis en évidence pour la première fois le rôle physiologique de REM dans la plante, cette protéine est capable de ralentir la propagation virale du Potato Virus X (PVX) et d’autres virus. Par ailleurs, l’activité antivirale de REM est régulée par phosphorylation et conduit à une modification de la taille du pore des PD par dépôt de callose. Des candidats protéiques ont été sélectionnées et leur validation fonctionnelle a été initiée in planta par des approches de transgénèse, en expression transitoire et sur des plantes transgéniques soumises à des infections virales pour étudier la propagation des virus. Des approches de biochimie d’interaction des protéines, et d’imagerie ont également été envisagés. Le sujet de cette thèse vise à appréhender les mécanismes de l’interaction de REM avec ses partenaires dans la membrane lors de l’infection virale, en se focalisant sur les interactions protéines-protéines lors de la réponse au PVX. Nous nous intéresserons plus particulièrement aux protéines des PD et des radeaux membranaires qui sont très probablement ciblées lors de cette interaction avec les virus. / Group 1 REMORINs are plant-specific proteins located at the plasma membrane. We have shown that StREM1.3 (REM) is a marker of lipid rafts, plasma membrane domains enriched in sterols and sphingolipids. In addition, REM is enriched in plasmodesmata channels (PD) which are anchored within the cell wall and enable intercellular communication between virtually all plant cells. We have demonstrated for the first time the physiological role of REM in plants, this protein is able to reduce the viral cell-to-cell movement of Potato Virus X (PVX) and other viruses. Moreover, the antiviral activity of REM is regulated by phosphorylation and leads to a modification of the pore size of PD via the accumulation of callose, a sugar polymer, around the neck regions of PD. In order to understand how REM is able to induce the accumulation of callose in these specific regions, a large set of proteins have been selected and the deciphering of their functions have been initiated in planta by transgenic approaches, in transient expression and on transgenic plants, which will be subjected to viral infections to study the spread of viruses. Protein interaction, biochemistry and imaging approaches were also used to study this question. This thesis aims at understanding the mechanisms of the REM interaction with its membrane partners during viral infection, focusing on the protein-protein interactions during the response to PVX. We will focus more particularly on PD proteins and membrane rafts that are most likely targeted during this interaction with viruses

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In order to maintain a multicellular organism cells need to interact and communicate with each other. Signalling cascades such as the Bone Morphogenic Protein (BMP) and Hedgehog (Hh) signalling pathways therefore play essential roles in development and disease. Intercellular signalling also underlies the function of stem cell niches, signalling microenvironments that regulate behaviour of associated stem cells. Range and intensity of the niche signal controls stem cell proliferation and differentation and must therefore be strictly regulated. The testis and ovary of the fruit fly Drosophila melanogaster are established models of stem cell niche biology. In the apical tip of the testis, germ line stem cell (GSCs) and somatic cyst stem cells (CySCs) are arranged around a group of postmitotic somatic cells termed hub. While it is clear which signals regulate GSC maintenance it is unclear how these signals are spatially regulated. Here I show that BMP signalling is specifically activated at the interface of niche and stem cells. This local activation is possible because the transport of signalling and adhesion molecules is coupled and directed towards contact sites between niche and stem cells. I further show that the generation of the BMP signal in the wing disc follows the same mechanism. Hh signalling controls somatic stem cell populations in the Drosophila ovary and the mammalian testis. However, it was unknown what role Hh might play in the fly testis, where the components of this signalling cascade are also expressed. Here I show that overactivation of Hh signalling leads to an increased proliferation and an expansion of the cyst stem cell compartment. Finally, while the major components of the Hh signalling pathway are known, detailed knowledge of how signal transduction is implemented at the cell biological level is still lacking. Here, I show that localisation of the key signal transducer Smo to the plasma membrane is sufficient for phosphorylation of its cytoplasmic tail and downstream pathway activation. Using advanced, microscopy based biophysical methods I further demonstrate that Smo clustering is, in contrast to the textbook model, independent of phosphorylation.

In order to maintain a multicellular organism cells need to interact and communicate with each other. Signalling cascades such as the Bone Morphogenic Protein (BMP) and Hedgehog (Hh) signalling pathways therefore play essential roles in development and disease. Intercellular signalling also underlies the function of stem cell niches, signalling microenvironments that regulate behaviour of associated stem cells. Range and intensity of the niche signal controls stem cell proliferation and differentation and must therefore be strictly regulated. The testis and ovary of the fruit fly Drosophila melanogaster are established models of stem cell niche biology. In the apical tip of the testis, germ line stem cell (GSCs) and somatic cyst stem cells (CySCs) are arranged around a group of postmitotic somatic cells termed hub. While it is clear which signals regulate GSC maintenance it is unclear how these signals are spatially regulated. Here I show that BMP signalling is specifically activated at the interface of niche and stem cells. This local activation is possible because the transport of signalling and adhesion molecules is coupled and directed towards contact sites between niche and stem cells. I further show that the generation of the BMP signal in the wing disc follows the same mechanism. Hh signalling controls somatic stem cell populations in the Drosophila ovary and the mammalian testis. However, it was unknown what role Hh might play in the fly testis, where the components of this signalling cascade are also expressed. Here I show that overactivation of Hh signalling leads to an increased proliferation and an expansion of the cyst stem cell compartment. Finally, while the major components of the Hh signalling pathway are known, detailed knowledge of how signal transduction is implemented at the cell biological level is still lacking. Here, I show that localisation of the key signal transducer Smo to the plasma membrane is sufficient for phosphorylation of its cytoplasmic tail and downstream pathway activation. Using advanced, microscopy based biophysical methods I further demonstrate that Smo clustering is, in contrast to the textbook model, independent of phosphorylation.:Summary 1 List of publications 3 1 Introduction 9 Aims of the thesis 15 2 Generation of a local BMP signal in testis and wing disc 17 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 2.1.1 Stem cells and stem cell niches . . . . . . . . . . . . . . 19 2.1.2 The Drosophila testis stem cell niche . . . . . . . . . . 20 2.1.3 BMP signalling in the fly . . . . . . . . . . . . . . . . . 23 2.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 2.2.1 The BMP niche signal is transduced locally at adherens junctions 25 2.2.2 Generation of the local BMP niche signal . . . . . . . . 30 2.2.3 Exocyst involvement in long-range BMP signalling . . 34 2.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 3 Hedgehog pathway overactivation in the testicular niche 41 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 3.1.1 The role of Hedgehog in the fly . . . . . . . . . . . . . 43 3.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 3.2.1 Overexpression of Hh increases the CySC number and expands their range 45 3.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 4 Visualization of Smo phosphorylation and biophysical detection of Smo clustering 49 4.1 Introduction (part I) . . . . . . . . . . . . . . . . . . . . . . . 51 4.1.1 Hedgehog signalling in the fly . . . . . . . . . . . . . . 51 4.1.2 Reception and transduction of the Hh signal by Ptc and Smo 54 4.2 Results (part I) . . . . . . . . . . . . . . . . . . . . . . . . . . 56 4.2.1 A fluorescent reporter for Drosophila Smo tail phosphorylation 56 4.2.2 Smo phosphorylation and localisation in the salivary gland 61 4.2.3 Smo localisation in cultured insect cells . . . . . . . . . 63 4.2.4 Smo membrane localisation and phosphorylation . . . . 65 4.3 Introduction (part II) . . . . . . . . . . . . . . . . . . . . . . . 67 4.3.1 Fluorescence correlation spectroscopy (FCS) . . . . . . 67 4.3.2 Dual-color fluorescence cross-correlation spectroscopy (FCCS) 72 4.3.3 Artefacts in FCS/FCCS . . . . . . . . . . . . . . . . . 73 4.4 Results (part II) . . . . . . . . . . . . . . . . . . . . . . . . . . 79 4.4.1 Smo clustering measured by FCCS . . . . . . . . . . . 79 4.5 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82