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The LIN-12/Notch signaling pathway is highly conserved in all animals, and is crucial for proper development. It is a key pathway in specifying cell fate in many cellular contexts, and dysregulation of the pathway can have deleterious consequences. Therefore, understanding how LIN-12/Notch signaling is regulated in different contexts has been a main area of interest in the field. Previous studies in different model organisms have identified many modes of regulation of the signaling pathway, one of which is endocytosis of the ligand and receptor. Here, I further investigated the role of endocytosis in LIN-12/Notch signaling in multiple developmental contexts in Caenorhabditis elegans. Work in Drosophila and vertebrates had previously established that ligand-mediated activation of Notch requires ubiquitination of the intracellular domain of the transmembrane ligand and the activity of the endocytic adaptor Epsin in the signaling cell. The consensus in the field is that Epsin-mediated endocytosis of mono-ubiquitinated ligand generates a pulling force that exposes a cleavage site in Notch for an ADAM protease, a critical step in signal transduction. In contrast, in this thesis, I examined two different transmembrane ligands in several different cell contexts and found that activation of LIN-12/Notch and the paralogous GLP-1/Notch in C. elegans does not require either Epsin-mediated endocytosis or ubiquitination of the intracellular domain of the ligand. Results obtained by a collaborator indicate that C. elegans ligand and receptor interactions are tuned to a lower force threshold than are Drosophila ligand and receptor interactions, potentially accounting for these differences. I also looked at the role of endocytosis in regulating LIN-12 signaling in the context of vulval development. The cell fate pattern of six vulval precursor cells (VPCs) is mediated by EGFR and LIN-12/Notch signaling. Previous work using multicopy transgenes in fixed specimens indicated that LIN-12 is post-translationally downregulated via endocytosis in response to EGFR activation in the VPC named P6.p, an event that appeared essential for ligands to activate LIN-12/Notch in neighboring VPCs. In this thesis, I manipulate the endogenous lin-12 gene and examine live specimens to show that LIN-12 appears to be regulated transcriptionally in P6.p and evidence that there may be additional potential endocytic motifs that may regulate LIN-12 in this context.

Glutamate is the major excitatory neurotransmitter in the mammalian brain and acts at the same time as one of the most powerful neurotoxins. In order to ensure a continuous communication between nerve cells, glutamate transporters are crucial for both the efficient removal of transmitter and the buffering of glutamate in the synaptic cleft during synaptic transmission. The buffering of glutamate subsequently influences the activation of different receptor classes in a spatio-temporal manner. In this context the rapid translocation of glutamate transporters between plasma membrane and intracellular compartments (membrane trafficking) is an interesting regulatory aspect for changes in cell surface localization. This highly dynamic mechanism is well-established for different glutamate receptor classes and associated with synaptic plasticity. In this thesis membrane trafficking of glial and neuronal glutamate transporters and its underlying regulative endo- and exocytic mechanisms were investigated in detail. Thereby a novel tyrosine-based adaptor protein complex 2 (AP2) binding motif - Y V N G G F - in the cytoplasmic C-terminus of glutamate transporter subtype EAAC1 was identified. The interaction between AP2 and its binding motif facilitates clathrin-mediated endocytosis of EAAC1, which is constitutively recycled between plasma membrane and endosomal structures under basal conditions. Additionally the activity of tyrosine kinases could be linked to the plasma membrane localization of EAAC1, suggesting the regulation of AP2-EAAC1 interaction by phosphorylation of the tyrosine residue within the identified binding motif. Furthermore it could be shown that cholesterol directly influences both endocytosis of EAAC1 and transporter functionality. Altogether the data offers new insights into modulatory mechanisms underlying glutamatergic neurotransmission and elucidation in regards to diseases of the central nervous system associated with glutamate toxicity.

Les tétraspanines sont des protéines à quatre domaines transmembranaires ayant la capacité d’interagir avec des partenaires et de les intégrer au sein d’un réseau dynamique d’interactions nommé tetraspanin web. Parmi elles, les TspanC8 représentent une sous-famille partageant un même partenaire, ADAM10.Cette protéase, essentielle au développement, est responsable du clivage de nombreux substrats dont le récepteur Notch, plusieurs cadhérines et facteurs de croissance. ADAM10 est également impliquée dans la régulation de plusieurs pathologies, telles que la maladie d’Alzheimer ou des cancers.Les précédentes recherches du laboratoire ont montré que les TspanC8 régulent la fonction d’ADAM10 à travers son expression et sa compartimentation membranaire. Les travaux exposés dans cette thèse montrent que plusieurs TspanC8 régulent également l’activité d’ADAM10 à travers une endocytose et une stabilité différentielle et en explorent les mécanismes sous-jascents. Ils démontrent également l’existence d’une compétition entre TspanC8 pour l’association avec ADAM10, et portent une réflexion sur l’utilisation de tags et sur les biais expérimentaux qui peuvent en découler. / Tetraspanins are a family of proteins containing four-transmembrane domains that can interact with partners to include them in a dynamic network of interactions named tetraspanin web. Among them, the TspanC8 tetraspanin subfamily is known to share one partner, the ADAM10 metalloprotease.The ADAM10 protease is essential to development and is responsible for the shedding of a number of substrates, including the Notch receptor ectodomain, several cadherins and growth factors. ADAM10 is also implicated in the regulation of several pathologies including Alzheimer’s disease and carcinogenesis.Previous studies from our laboratory show that TspanC8 family members regulate the function of ADAM10 through its expression and its membrane compartmentalisation.Data presented in this thesis demonstrate that several TspanC8s also regulate ADAM10’s activity through differential stability and endocytosis, and explore the subjascent mechanisms. We also show that TspanC8 family members compete for association with ADAM10; thus, we bring elements of reflexion the use of tags and subsequent experimental bias.

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京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20566号 / 医博第4251号 / 新制||医||1022(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 小柳 義夫, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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