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Two problems have hampered the use of light microscopy for structural studies of cellular organelles for a long time: the limited spatial resolution, and the difficulty of obtaining true structural boundaries from complex intensity curves. The advent of modern super-resolution light-microscopy techniques and their combination with objective image segmentation now provide us with the means to bridge the gap between light and electron microscopy in cell biological applications. This study provides the first comparative correlative analysis of threedimensional (3D) structures obtained by 4Pi-microscopy with those of transmission electron microscopy (TEM). The distribution within the cisternae of isolated Golgi stacks of the cargo protein PC3-pEGFP is here mapped in 3D by both 4Pi-microscopy and TEM for a detailed comparative analysis of their imaging capabilities. A high correlation was seen for these structures, demonstrating the particular accuracy of 4Pi-microscopy. Furthermore, for the first time, the transport of a cargo molecule (VSVG-pEGFP) through individual Golgi stacks (labelled by GalT-venusYFP) »s visualized at the 4Pi-microscope super-resolution level.

The ability of cells to export proteins is essential for numerous cellular functions. Proteins for export are synthesised in the endoplasmic reticulum (ER) and move through the Golgi complex to the trans-Golgi network (TGN), from where they are shipped to various final destinations including the plasma membrane (PM) (apical or basolateral, in polarized cells), early and late endosomes, and secretory granules. The membranous carriers involved in the trafficking step from the TGN directly to the PM are much larger than vesicles, and are pleiomorphic rather than vesicular. They also do not possess an external coat, as has been seen with other types of carriers (e.g. clathrin, COPI and COPII vesicles). At present, the molecular mechanisms of the formation of these large pleiomorphic carriers are completely unknown. I built up a thematic interactome to look at interactions between the many players involved in TGN-to-PM trafficking, and I have added some newly discovered interesting interactions through use of a two-hybrid matrix system. I thus focused my attention on some key elements such as the Arf1 and Arl1 interacting protein Arfaptin, the TRAnsport Protein Particle (TRAPP) complex, protein kinase D (PKD) and C-terminal binding protein/VBFA-ribosylated substrate (Ctbpl-S/BARS) since these were revealed as being important nodes into this thematic interactome. Arfaptin is a protein specifically recruited to the TGN through a BAR domain which can interact with membranes, possibly bending them; thus Arfaptin might be relevant in the initial formation of carriers. We have discovered that PKD (a protein involved in fission) phosphorylates and displaces Arfaptin from the Golgi complex, regulating its action in time, since the fission-mediated action of PKD occurs later.

The molecular machinery responsible for the generation of transport carriers that move cargo from the Golgi complex to the plasma membrane relies on a tight physical and functional interplay between proteins and lipids. Among the lipid-binding proteins composing this machinery, we previously identified the four-phosphate adaptor proteins FAPP1 and FAPP2 (FAPPs), the pleckstrin homology domain of which binds phosphatidylinositol 4-phosphate and the small GTPase ARFl.

The rab family of small, ras-like GTPases regulate membrane trafficking events in the secretory and endocytic pathways. They appear to be involved in all stages of vesicular transport, from vesicle budding and cargo selection to motility, docking and membrane fusion. Through a cycle of GTP binding and hydrolysis, rab-effector proteins are recruited to membrane sub-domains in a temporally and spatially specific manner. Several rab proteins localise to the Golgi apparatus, the organelle consisting of stacked, flattened, membrane-bound cisternae through which newly-synthesised proteins are transited and modified, and where proteins and lipids are sorted and packaged for transport to other subcellular destinations. The structure of the Golgi is maintained by a matrix of proteins, many of which have now been shown to be rab-effector proteins. This thesis focuses on Golgi-localised rab proteins and their effector proteins. The cis-Golgi-localised rab protein, rabl, is shown to interact with the Golgi matrix/golgins GM130 and p115 while rab2 binds GM130 as well as a novel tethering factor named golgin-45. siRNA-mediated depletion of these rabs and golgins revealed them to be important for the maintenance of Golgi structure and suggested that p115 is primarily recruited to Golgi membranes by its interaction with rab1 rather than its association with GM130. A search for additional rab1 effectors revealed potential interactions between rab1 and the phosphoinositide- binding/metabolising proteins centaurin?2 and MTMR6. A search for novel effectors of the trans-Golgi-localised rab protein, rab6, was also made, revealing specific interactions with the dynactin subunit p150glued and the dynactin/dynein accessory proteins BicD1 and BicD2. These interactions are proposed to mediate the recruitment of dynactin/dynein to membranous cargo and control minus-end directed, microtubule-dependent vesicle motility. An additional rab6 effector, GOPC, was also identified, which may be responsible for cargo recognition and sorting. Finally, the cis-Golgi rab-effector and matrix protein, GM130, is shown to have a hitherto unsuspected role in the activation of a family of Golgi-localised Ste20 kinases. The implications of all these interactions for Golgi structure and function is discussed.

Les anomalies congénitales de la glycosylation (CDG) sont des maladies génétiques rares caractérisées par une glycosylation aberrante des protéines. Récemment, un sous-groupe de CDG dues à des perturbations de l'homéostasie de l’appareil de Golgi a fait son apparition. En 2012, notre équipe a identifié TMEM165 comme étant une protéine golgienne impliquée dans les CDG, mais dont les fonctions biologiques et cellulaires demeurent inconnues. Au cours de mon doctorat, nous avons montré que l'homéostasie golgienne du Mn2+ était altérée en absence de TMEM165. Alors que de forts défauts de glycosylation, en particulier des défauts de galactosylation, ont été observés dans des cellules déficientes en TMEM165, nous avons découvert que la supplémentation en Mn2+ était suffisante pour rétablir une glycosylation normale. De façon intéressante, nous avons également démontré que ce défaut de glycosylation pouvait également être supprimé par une supplémentation en galactose. Fort de cette observation, la supplémentation orale en galactose a été testée chez des patients déficients en TMEM165 et il a été prouvé que ce traitement améliorait significativement les paramètres biochimiques et cliniques. De plus, nous avons mis en évidence que la stabilité de TMEM165 était altérée en présence d'une concentration élevée de Mn2+. En effet, nous avons montré que l'exposition à des concentrations élevées de Mn2+ entraînait une dégradation lysosomale rapide de TMEM165. Dans l'ensemble, notre étude montre que TMEM165 est (i) un acteur clé de la glycosylation golgienne en régulant finement l'homéostasie du Mn2+ et (ii) une nouvelle protéine de l’appareil de Golgi sensible au manganèse. / Congenital Disorders of Glycosylation (CDG) are severe inherited diseases in which aberrant protein glycosylation is a hallmark. From this genetically and clinically heterogeneous group, a significant subgroup due to Golgi homeostasis defects is emerging. Our team previously identified TMEM165 as a Golgi protein involved in CDG. But despite strong efforts, the biological and cellular functions of TMEM165 were not known so far. During my thesis, we highlighted that Golgi Mn2+ homeostasis was impaired due to TMEM165 deficiency. While strong glycosylation defects, especially galactosylation defects, were observed in TMEM165 depleted cells, we discovered that Mn2+ supplementation was sufficient to fully restore a normal glycosylation. Interestingly, we also demonstrated that the observed glycosylation defects in mammalian cells could be overcome by galactose supplementation. Strong of this observation, oral galactose supplementation in TMEM165 deficient patients was assayed and this treatment was proven to significantly improve biochemical and clinical parameters. Moreover, we highlighted TMEM165 as a novel Golgi protein whose stability is altered in the presence of high manganese concentration. Indeed, we showed that exposure to high Mn2+ concentrations led to a rapid lysosomal degradation of TMEM165. Altogether, our study points TMEM165 as (i) a key player in Golgi glycosylation by finely regulating Golgi Mn2+ homeostasis and (ii) a novel Golgi protein sensitive to manganese.