Thrombosis, the major cause of heart attack and strokes,1 is triggered by localized clotting of the blood as the result of deregulated platelet aggregation. During the repair of vascular injury, clotting usually occurs when platelets adhere to each other at the site of vascular injury in order to stop bleeding.2 Distinct protein receptors and adhesive ligands together with the blood flow conditions govern this process. One of the negative regulators in platelet aggregation is Disabled-2 (Dab2), a modular protein that is released upon platelet activation to the extracellular platelet surface.3 Dab2 inhibits platelet aggregation through its phosphotyrosine-binding (PTB) domain by competing with fibrinogen for ï ¡IIï ¢3 integrin binding on the activated platelet surface.3 Sulfatides are also found on the platelet surface,4 interacting with adhesive and coagulation proteins5-7 and, thus, they are thought to play a major role in haemostasis and thrombogenesis. Here, we show that the Dab2 PTB domain specifically interacts with sulfatides through two conserved basic motifs. The sulfatide-binding site overlaps with that of phosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P2) in the PTB domain. Whereas sulfatides recruit the Dab2 PTB domain to the platelet surface, thus sequestering the protein from thrombin-mediated platelet aggregation, the phosphoinositide mediates its internalization. Experimental data support the hypothesis that two pools of Dab2 co-exist at the platelet surface and that the balance between them controls the extent of the clotting response. / Master of Science

Degradation of membrane proteins in the vacuole/lysosome is dependent on their prior sorting into the multivesicular body (MVB) pathway. This sorting process involves incorporation of proteins into vesicles that are formed by budding of the limiting membrane of the endosome into the lumen of the organelle. The MVB sorting process on the whole is highly conserved from yeast to human, and depends on the Vps27/Hrs, ESCRT-I, -II, and -III protein complexes functioning sequentially on the endosomal membrane, as well as on additional factors, such as the ubiquitinating enzyme Rsp5/Nedd4. It has now been established that ubiquitin serves as a sorting signal for many cargoes into the MVB pathway. In this thesis work, we provide evidence that Bro1 is not required for protein ubiquitination or early steps of endocytosis, but functions at the late endosome level as an integral component of the MVB pathway. Similarly to its human homologue Alix, Bro1 interacts with components of the ESCRT-I and ESCRT-III complexes. The putative role of Bro1/Alix in bridging an interaction between ESCRT-I and –III might be important to strengthen an association of these protein complexes to allow efficient sorting of cargo proteins. Deficiency in Bro1 results in recycling of the endocytosed Gap1 permease back to the plasma membrane, a process coupled to deubiquitination of the permease. This recycling is a non-classical phenotype for cells impaired in MVB pathway thus suggesting Bro1 to have a particular role in this sorting process. Furthermore, the conserved C-terminal proline-rich domain (PRD) of Bro1 is specifically important for MVB sorting of cargo proteins that are subject to ubiquitination. We show Bro1 (via its PRD) to play a highly important role in recruitment of the deubiquitinating enzyme Doa4 to the endosome. Consistent with this, Bro1 is required for deubiquitination of cargo proteins, a step occurring just before cargo incorporation into the endosomal vesicles, and similarly to Doa4, for ubiquitin recycling. In contrast to previous interpretations, we show that Doa4 has a direct role in sorting of ubiquitinated cargo proteins into the MVB pathway. We propose that Doa4 – via its association to Bro1 - achieves this role by catalyzing deubiquitination of cargo proteins and/or some components of the MVB sorting machinery. We further show Bro1 to interact with the ubiquitin ligase Rsp5, which, in addition to being required for cargo protein ubiquitination at the plasma membrane, apparently contributes to multiple steps of endocytosis and MVB sorting. Also the Bro1-Rsp5 interaction is dependent on the C-terminal PRD region of Bro1. We propose that this interaction is conserved. A role for ubiquitin in regulation of the MVB sorting machinery is emerging: the function of factors recognizing and sorting ubiquitinated cargo proteins in the MVB pathway is suggested to be coupled to their cycling between ubiquitinated and deubiquitinated stages. A growing body of evidence indicates that ubiquitin ligases of the Rsp5/Nedd4 family play a central role in this regulation. We speculate the Bro1/Alix protein, through its ability to simultaneously interact with factors of the MVB sorting machinery and with ubiquitinating and deubiquitinating enzymes to play a central role in the successive rounds of ubiquitination and deubiquitination of specific factors along the MVB pathway.

Src is a 60 kDa tyrosine kinase that is expressed in most of animal tissues. Src has three splice variants, C-src, which is ubiquitously expressed, and N1- and N2-src, which are neuronal specific splice variants. The srcs are differentially spliced at their SH3 domains, therefore the hypothesis is that this splicing allows them to have different binding partners and perform different roles in neurons. The aim of this project is to identify new interactions for the three src splice variants in neurons and their possible functional roles. The SH3 domains, kinase active truncated proteins ( 80) and kinase dead mutant full length versions of the three splice variants of src were cloned from a rat brain cDNA library into bacterial expression vectors. GST-pull downs from nerve terminal lysates showed that different src splice variants had different binding partners. These partners were identified by mass spectrometry and confirmed by western blotting. C-src binding partners included dynamin, synapsin, and synaptojanin, while N2-src binding partners included synaptophysin, Munc18-1, and NSF. The interaction between N2-src and Munc 18-1 was characterized further; however a number of in vitro interaction assays and kinase assays showed that Munc 18-1 may not be a direct binding partner for N2-src or substrate. N1-src displayed a stimulation-dependent interaction with dynamin I. This was shown to be phosphorylation-dependent in contrast to C-src binding. The major phosphorylation sites on dynamin I, S774 and S778, were not involved in the regulation of N1-src binding. The binding site for N1-src on dynamin I was different to C-src, with extensive mutagenesis studies suggesting that the interaction site is at the tail of the dynamin I xa splice variant, which has an additional two phosphorylation sites.

>Magister Scientiae - MSc / The yeast Saccharomyces cerevisiae is considered a good host used for heterologous protein production due to the organism’s microbial safety, rapid growth and eukaryotic post- translational processing. As a fermentative organism, S. cerevisiae is thus not only a useful platform for the production of biopharmaceuticals and industrial enzymes, but also a promising organism for second-generation biofuel production. Substantial effort has been focused on alleviating the many bottlenecks in recombinant gene expression, as well as in the secretory pathway to enhance heterologous protein titres. It was recently shown that highly active endocytosis could decrease the overall secreted protein titre in the supernatant. In this study, we aimed to block endocytotic and vacuolar complexes to ultimately disrupt, or impair, the endocytotic and vacuolar mechanisms of proteolysis and test the effect that this would have on secreted heterologous protein titres. This was accomplished by knocking out various genes involved in endocytosis and transforming the strains with genes encoding various hydrolases including β-glucosidase (Bgl), xylanase (Xyn2), endoglucanase (Eg2) and cellobiohydrylase (Cbh1). Our study demonstrated that genetic blocking of endocytotic mechanisms as well as vacuolar complexes could theoretically improve heterologous protein secretion in S. cerevisiae. Endoglucanase (Eg2) titres displayed improvement of 26% and 30% in strains which had the RVS161 and VRP1 genes deleted and xylanase titres displayed an improvement of 71% and 143% in strains with the END3 and SSA4 gene deletions. Several of the gene knockouts tested improved Xyn2 and Eg2 titres but the effect of the different gene targets varied widely. A double knock-out strain with deletions in CLC1 and RVS161 secreted 104% more Eg2 than its parental control strain on a per dry cell weight basis, a significant synergistic improvement. Other double knock-out strains displayed additive or similar activities when compared to their controls. Cbh1 secretion could not be improved through the gene deletions tested in our study and Bgl activity could not be measured in our transformants. These results demonstrate the different relationships of various heterologous proteins with various components of the secretion machinery and may also imply how endocytosis as well as vacuolar complexes affect the level of secreted protein.

LAT1, also named SLC7A5, is a human plasma membrane transporter covalently associated with the glycoprotein CD98 (SLC3A2). It catalyses the uptake of several essential amino acids, including leucine, thereby contributing to the activation of the mTORC1 (mechanistic Target of Rapamycin Complex 1) kinase complex. This control of mTORC1 by LAT1 is notably important for the rapid proliferation of tumour cells. Moreover, LAT1 overexpression has been observed in numerous cancers, and is often associated with a poor prognosis. Despite the importance of LAT1 in normal and tumour cells, the mechanisms controlling the intracellular traffic of this transporter, and particularly its endocytosis, are still poorly known.Many plasma membrane transporters are downregulated by endocytosis, in response to various signals. In yeast, endocytosis of plasma membrane permeases is triggered by their ubiquitylation mediated by the ubiquitin ligase Rsp5, acting in association with adaptors of the α-arrestin family. Ubiquitin also plays an important role in endocytosis of many human transporters. The main objective of this thesis work was to characterize the mechanisms mediating endocytosis of LAT1 in HeLa tumour cells.In the first part of this work, we studied the effect of FTY720, an analog of sphingoid bases with antitumor properties, on a HeLa T-Rex stable cell line expressing a LAT1-GFP construct. We also analysed the effect of FTY720 on several yeast plasma membrane permeases. Our results show that FTY720 induces a loss of activity and the endocytosis of permeases in yeast and of LAT1 in HeLa cells. Yeast permease internalization is associated with their ubiquitylation by the Rsp5 ubiquitin ligase. Moreover, FTY720 decreases TORC1/mTORC1 activity. Based on these results, we propose a model for the effect of FTY720 in yeast: this drug, by decreasing the activity of numerous nutrient transporters, leads to TORC1 inhibition. This loss of TORC1 activity promotes, via a still unknown mechanism, the Rsp5-dependent ubiquitylation and endocytosis of permeases. A similar mechanism could be responsible for FTY720-induced endocytosis of LAT1 in HeLa cells.In the second part of this work, we further investigated the mechanisms involved in LAT1 endocytosis. We found that LAT1 is internalized and degraded upon activation of protein kinase C (PKC) by the phorbol ester PMA. This treatment also elicited the ubiquitylation of LAT1 via the Nedd4-2 ubiquitin ligase. Finally, we identified the lysines 19, 25 and 30 in the N-terminal tail of LAT1 as the major sites of its ubiquitylation in response to PMA, and we showed that these lysines are important for PMA-induced downregulation. Future work will determine the importance of this control mechanism of LAT1 in the context of normal and tumour cells. / Ce travail porte sur l’étude du transporteur d’acides aminés humain LAT1, également appelé SLC7A5. LAT1, situé à la membrane plasmique où il est associé à la protéine CD98 (SLC3A2), est le transporteur principal de leucine dans les cellules et joue un rôle central dans l’activation de mTORC1 par cet acide aminé. Ce transporteur est surexprimé dans les cellules de nombreuses tumeurs, dans lesquelles il est souvent associé à un mauvais pronostic. Le trafic intracellulaire de LAT1 et, en particulier, son endocytose sont très peu connus. Mon travail de thèse a eu pour but d’étudier l’endocytose de LAT1 et les facteurs impliqués dans ce mécanisme. Dans la première partie de cette recherche, nous avons étudié l’effet du FTY720, un analogue de bases sphingoïdes, sur une lignée stable de cellules HeLa TRex exprimant LAT1-GFP, et nous l’avons comparé à son effet dans la levure. Nous avons observé que ce composé induisait une diminution de l’activité et l’endocytose de nombreux transporteurs de nutriments chez la levure ainsi que de LAT1 dans les cellules HeLa. De plus, l’internalisation des perméases de levure en réponse au FTY720 est associée à leur ubiquitylation, via l’ubiquitine ligase Rsp5. Nous avons également montré que ce composé diminue l’activité du complexe kinase TOR1/mTOR1. Ces différents résultats nous ont amené à l’hypothèse suivante :le FTY720, en induisant une perte d’activité de nombreux transporteurs de nutriments chez la levure, inhibe TORC1. Cette perte d’activité de TORC1 activerait, via un mécanisme encore inconnu, l’ubiquitylation et l’endocytose Rsp5-dépendante des perméases. Un mécanisme similaire pourrait être responsable de l’endocytose de LAT1 dans les cellules HeLa.Dans une seconde partie, nous avons étudié les facteurs impliqués dans l’endocytose de LAT1 en réponse à un second composé, le PMA, un activateur de la PKC. Nous avons montré que ce composé induit l’internalisation et l’ubiquitylation de LAT1 via l’activation de la PKC. Nous avons trouvé que cette modification dépend de l’ubiquitine ligase Nedd4-2. De plus, les lysines 19, 25 et 30 semblent être les cibles principales de l’ubiquitine ligase. En effet, une diminution importante de l’ubiquitylation et de la localisation intracellulaire de LAT1 en réponse au PMA est observée dans un mutant LAT1K19R,K25R,K30R. D’autres études seront nécessaires pour évaluer l’importance de ce mécanisme de régulation de LAT1 dans les cellules saines et cancéreuses. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Lee Ming Tsung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 160-177). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Abbreviations --- p.vi / Table of contents --- p.vii / List of Figures --- p.xii / List of Tables --- p.xiv / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Nutritional values --- p.2 / Chapter 1.3 --- Medicinal values --- p.3 / Chapter 1.3.1 --- Anti-tumor effect --- p.3 / Chapter 1.3.2 --- Anti-viral and anti-caries effect --- p.4 / Chapter 1.3.3 --- Immunopotentiating effect --- p.4 / Chapter 1.3.4 --- Hypocholesterolaemic effect --- p.5 / Chapter 1.4 --- Life cycle and morphology --- p.6 / Chapter 1.5 --- Growth requirements --- p.9 / Chapter 1.5.1 --- Nutritional factors --- p.9 / Chapter 1.5.2 --- Physical and chemical factors --- p.10 / Chapter 1.6 --- Application of L. edodes --- p.12 / Chapter 1.7 --- Endocytosis --- p.13 / Chapter 1.7.1 --- Different types of endocytosis --- p.13 / Chapter 1.7.1.1 --- Phagocytosis --- p.14 / Chapter 1.7.1.2 --- Pinocytosis --- p.15 / Chapter 1.7.1.3 --- Receptor-mediated endocytosis --- p.15 / Chapter 1.7.2 --- The Endocytic Pathway --- p.17 / Chapter 1.7.3 --- Endocytosis in fungi --- p.20 / Chapter 1.7.4 --- Rab GTPases --- p.21 / Chapter 1.7.4.1 --- Control of the active and inactive state of Rab proteins --- p.22 / Chapter 1.7.4.2 --- Regulation of docking and fusion of membrane in endosomal trafficking --- p.23 / Chapter 1.7.4.3 --- Rab7 GTPase --- p.26 / Chapter 1.8 --- Aims of the project --- p.28 / Chapter Chapter 2 --- Protein-protein Interaction Study of Le.Rab7 by in vivo and in vitro Interaction Assay --- p.29 / Chapter 2.1 --- Introduction --- p.29 / Chapter 2.2 --- Materials and Methods --- p.36 / Chapter 2.2.1 --- Yeast two-hybrid screening --- p.36 / Chapter 2.2.1.1 --- Confirmation of the clones Le.Rab7-pGBK.T7 --- p.36 / Chapter 2.2.1.1.1 --- Bacterial transformation --- p.36 / Chapter 2.2.1.1.2 --- PCR screening for positive transformants --- p.38 / Chapter 2.2.1.1.3 --- Plasmid preparation and confirmation of transformants --- p.38 / Chapter 2.2.1.1.4 --- Sequencing --- p.39 / Chapter 2.2.1.2 --- Confirmation of Le.Rab7 protein expression in yeast --- p.40 / Chapter 2.2.1.2.1 --- Yeast transformation --- p.40 / Chapter 2.2.1.2.2 --- Yeast protein extraction --- p.40 / Chapter 2.2.1.2.3 --- Western Blotting --- p.41 / Chapter 2.2.1.3 --- Yeast Two-hybrid screening by Yeast-mating --- p.42 / Chapter 2.2.1.4 --- Identification of Preys --- p.44 / Chapter 2.2.1.4.1 --- PCR screening for clones grown on plates --- p.44 / Chapter 2.2.1.4.2 --- Colony lift filter assay --- p.45 / Chapter 2.2.1.4.3 --- Sequencing --- p.47 / Chapter 2.2.1.5 --- Confirmation of interaction by Co-transformation assay --- p.47 / Chapter 2.2.1.5.1 --- Plasmid preparation of positive clones --- p.47 / Chapter 2.2.1.5.2 --- Transformation and bacterial plasmid preparation --- p.48 / Chapter 2.2.1.5.3 --- Yeast two-hybrid screening by co-transformation --- p.48 / Chapter 2.2.1.5.4 --- Colony lift filter assay --- p.50 / Chapter 2.2.2 --- Rapid Amplification of cDNA 5'ends --- p.51 / Chapter 2.2.2.1 --- RNA preparation --- p.51 / Chapter 2.2.2.1.1 --- Strains and culture conditions --- p.51 / Chapter 2.2.2.1.2 --- RNA extraction --- p.51 / Chapter 2.2.2.2 --- 5' RACE --- p.52 / Chapter 2.2.2.2.1 --- RNA processing --- p.52 / Chapter 2.2.2.2.2 --- Reverse transcription --- p.53 / Chapter 2.2.2.2.3 --- Nested PCR for 5'RLM-RACE --- p.53 / Chapter 2.2.2.3 --- "Gel analysis of products, TA cloning of RACE product and sequencing" --- p.54 / Chapter 2.2.2.4 --- Cloning of full-length Le.Rab5 --- p.54 / Chapter 2.2.3 --- In vitro protein-protein interaction assay --- p.55 / Chapter 2.2.3.1 --- Plasmid extraction from E.coli --- p.55 / Chapter 2.2.3.2 --- In vitro translation --- p.56 / Chapter 2.2.3.3 --- In vitro co-immunoprecipitation --- p.56 / Chapter 2.3 --- Results --- p.57 / Chapter 2.3.1 --- Yeast two-hybrid analysis by yeast mating assay --- p.57 / Chapter 2.2.1.1 --- Confirmation of the clones Le.Ra67-pGBKT7 --- p.57 / Chapter 2.3.1.1.1 --- PCR screening for positive transformants --- p.57 / Chapter 2.3.1.1.2 --- Plasmid preparation and confirmation of transformants --- p.58 / Chapter 2.3.1.1.3 --- Sequencing --- p.59 / Chapter 2.2.1.2 --- Confirmation of protein expression in yeast --- p.60 / Chapter 2.3.1.2.1 --- Yeast transformation --- p.60 / Chapter 2.3.1.2.2 --- SDS-PAGE and Western blotting of Le.Rab7 in yeast --- p.61 / Chapter 2.2.1.3 --- Yeast two-hybrid screening by yeast mating assay --- p.62 / Chapter 2.2.1.4 --- Identification of Preys --- p.63 / Chapter 2.3.1.4.1 --- PCR screening for clones grown on plates --- p.63 / Chapter 2.3.1.4.2 --- Colony lift assay --- p.65 / Chapter 2.3.1.4.3 --- Sequencing --- p.67 / Chapter 2.3.2 --- Confirmation of interactions by co-transformation assay --- p.70 / Chapter 2.2.2.1 --- Yeast two-hybrid analysis by co-transformation assay --- p.70 / Chapter 2.2.2.2 --- Colony lift filter assay --- p.70 / Chapter 2.2.2.3 --- Selection of prey plasmids for in vitro binding assay --- p.72 / Chapter 2.3.3 --- Rapid amplification of cDNA ends (RACE) --- p.76 / Chapter 2.2.3.1 --- TA cloning of RACE product and sequencing --- p.76 / Chapter 2.2.3.2 --- Cloning of full-length Le.Rab5 --- p.79 / Chapter 2.3.4 --- In vitro protein-protein interaction assay --- p.80 / Chapter 2.4 --- Discussion --- p.82 / Chapter Chapter 3 --- Temporal and Spatial expression of Le.Rab7，Le.Rab5 and Le.RACKl --- p.87 / Chapter 3.1 --- Introduction --- p.87 / Chapter 3.2 --- Materials and Methods --- p.93 / Chapter 3.2.1 --- Northern blot analysis --- p.93 / Chapter 3.2.1.1 --- RNA fractionation by formaldehyde gel electrophoresis --- p.93 / Chapter 3.2.1.2 --- Northern blotting --- p.94 / Chapter 3.2.1.2.1 --- Transfer of RNAs --- p.94 / Chapter 3.2.1.2.2 --- Probe preparation --- p.95 / Chapter 3.2.1.2.3 --- "Hybridization, Stringency washes and Signal detection" --- p.96 / Chapter 3.2.2 --- Quantitative RT-PCR --- p.97 / Chapter 3.2.2.1 --- cDNA synthesis from different developmental stages --- p.97 / Chapter 3.2.2.1.1 --- RNA preparation extraction --- p.97 / Chapter 3.2.2.1.2 --- DNase I treatment --- p.97 / Chapter 3.2.2.1.3 --- Reverse transcription --- p.98 / Chapter 3.2.2.2 --- Real time PCR --- p.98 / Chapter 3.2.2.2.1 --- Primer design and verification --- p.98 / Chapter 3.2.2.2.2 --- Real time PCR reaction and data analysis --- p.100 / Chapter 3.2.3 --- In situ RNA-RNA hybridization --- p.101 / Chapter 3.2.3.1 --- Preparation of samples and probes --- p.101 / Chapter 3.2.3.1.1 --- Tissue preparation --- p.101 / Chapter 3.2.3.1.2 --- RNA probe synthesis --- p.101 / Chapter 3.2.3.2 --- Hybridization and Signal development --- p.102 / Chapter 3.2.3.3 --- Image viewing --- p.103 / Chapter 3.3 --- Results --- p.105 / Chapter 3.3.1 --- Northern blot analysis --- p.105 / Chapter 3.3.2 --- Quantitative RT-PCR assays --- p.109 / Chapter 3.3.3 --- In situ RNA-RNA hybridization --- p.113 / Chapter 3.4 --- Discussion --- p.119 / Chapter Chapter 4 --- Existence of endocytosis and Protein localization of Le.Rab7 in L. edodes --- p.123 / Chapter 4.1 --- Introduction --- p.123 / Chapter 4.2 --- Materials and Methods --- p.127 / Chapter 4.2.1 --- Tracing the endocytie pathway using FM4-64 dye --- p.127 / Chapter 4.2.1.1 --- Strains and culture conditions --- p.127 / Chapter 4.2.1.2 --- FM4-64 internalization in mycelium and gill tissue of L. edodes --- p.127 / Chapter 4.2.2 --- Drug treatment effect on the internalization of FM4-64 dye --- p.128 / Chapter 4.2.3 --- Double labeling with AM4-64 and anti-Le.Rab7 antibody --- p.129 / Chapter 4.2.3.1 --- Synthesis of Le.Rab7 antibody --- p.129 / Chapter 4.2.3.1.1 --- Customization of Le.Rab7 antiserum --- p.129 / Chapter 4.2.3.1.2 --- Validation of anti-Le.Rab7 polyclonal antiserum --- p.129 / Chapter 4.2.3.2 --- Double immunofluorescence labeling --- p.130 / Chapter 4.2.4 --- Immunohistochemistry of young and mature fruiting body --- p.131 / Chapter 4.2.4.1 --- Tissue preparation --- p.131 / Chapter 4.2.4.2 --- Immunohistochemical staining --- p.132 / Chapter 4.2.4.3 --- Image viewing --- p.133 / Chapter 4.3 --- Results --- p.134 / Chapter 4.3.1 --- Presence of endocytosis in L .edodes --- p.134 / Chapter 4.3.2 --- Validation of active transport of FM4-64 --- p.137 / Chapter 4.3.3 --- Dye internalization at specific structures in L. edodes --- p.138 / Chapter 4.3.4 --- Presence of Le.Rab7 protein in the endosomal structures along the endocytic pathway --- p.142 / Chapter 4.3.5 --- Presence of Le.Rab7 protein in the pre- and hymenophore of fruiting body --- p.145 / Chapter 4.4 --- Discussion --- p.148 / Chapter Chapter 5 --- General discussion --- p.152 / References --- p.160

The ESCRT machinery has a well established role within the endocytic pathway. Studies conducted in Drosophila have identified ESCRT proteins as important regulators of epithelial cell polarity and growth. Consequently ESCRTs have been classified as potential tumour suppressors. Alterations in the expression of various ESCRT components have been observed in human cancers. However, the possible link between ESCRT proteins, mammalian epithelial cell polarity and tumourigenesis has not been investigated. This thesis demonstrates for the first time that the ESCRT-I protein, Tsg101, is required for maintenance of mammalian epithelial cell organisation and polarity. siRNA knockdown of Tsg101 in the human Caco-2 cell line results in the formation of a multilayered epithelium with compromised apicobasal polarity. In addition, Tsg101 depletion impairs differentiation of the epithelial sheet and formation of polarised 3D Caco-2 cysts. Depletion of Tsg101 also results in intracellular accumulation of the tight junction protein, claudin-1. This is shown to be constitutively endocytosed and recycled in Caco-2 epithelial monolayers, suggesting that ESCRT-I is required for claudin-1 recycling to tight junctions. Tsg101 knockdown also impairs epithelial barrier formation and enhances Caco-2 migratory ability. This suggests that tight junction integrity is impaired and may contribute to the loss of Caco-2 cell organisation and polarity observed upon Tsg101 depletion. Finally, Tsg101 depleted Caco-2 cells appear to overproliferate, forming multilayered regions of the epithelial sheet. However, multilayered cells are eventually eliminated via apoptosis. Preliminary results suggest that inhibition of this apoptotic response enhances the aberrant epithelial phenotype, suggesting that the ability to evade apoptosis may be an important factor in determining the tumourigenic potential of ESCRT-I depletion. Therefore, results presented in this thesis suggest that the role of ESCRT-I as a tumour suppressor is conserved from Drosophila to mammals.

Cell-penetrating peptides are a class of peptides which have achieved a lot of recognition due to their vector abilities. Since their discovery over a decade ago, there has been an uncertainty concerning the mechanism by which they are internalized into the cells. Early studies claimed the uptake to be receptor- and energy independent, whereas more recent studies have shifted the general view to a more endocytotic belief, without prior binding to a receptor. As an increasing amount of reports emerges claiming the uptake to be endocytic, there is still a discrepancy concerning which endocytic mechanism that is responsible for the internalization and how to exploit the endocytic machinery for improved delivery. The main aim of this thesis was to elucidate the internalization mechanism for a series of cell-penetrating peptides derived from naturally occurring proteins, such as the prion protein which is thought to be the infectious particle in prion disorders. Furthermore, applications in gene regulation and improvement of delivery efficacy by induction of endosomolysis were examined. The results obtained confirm the uptake of cell-penetrating peptides to be endocytic; however the internalization mechanism appears to be peptide dependent where macropinocytosis is the most widespread endocytic component responsible for the internalization. The results further demonstrate that the biological response can be increased manifold by the induction of endosomolysis, either by using lysosomotropic agents or peptides able to alter their secondary structure upon protonation with concomitant endosomolysis. Altogether the results prove that enhanced delivery using cell-penetrating peptides can be achieved by exploiting the intrinsic endocytic mechanisms involved in the translocation process.

Amphiphysin (Amph) is a multi-domain protein that has been implicated in synaptic vesicle (SV) endocytosis. In vertebrates, Amph1 associates with SVs and binds to known endocytic proteins, such as dynamin and clathrin. Overexpression of the vertebrate Amph1 SH3 domain is sufficient to inhibit SV endocytosis in the lamprey synapse. However, these in vitro and overexpression studies may not reflect Amph function in vivo. To investigate Amph function in vivo, I used Drosophila melanogaster as a model organism. I discovered that Drosophila Amph was broadly expressed throughout all developmental stages and was also highly expressed in specialized membranes such as the postsynaptic membrane at the larval neuromuscular junction and the t-tubule membranes of muscles. amph mutants were viable and had normal synaptic transmission, results that were inconsistent with a role for Amph in SV endocytosis. However, amph mutants had impaired locomotion, which may reflect a defect in the t-tubule network, a membrane system that is specialized to couple muscle membrane excitation to muscle contraction. To further explore this idea, I undertook a structure-function approach to ask if different Amph functional domains could rescue the t-tubule and locomotory defects observed in amph mutants. Partial rescue was observed for most constructs, suggesting that Amph function was dependent on more than one domain. To further elucidate how Amph functions at the t-tubule network, I used different in vitro methods to investigate novel protein partners for Amph. A GST pull-down approach identified actin as a potential Amph partner, consistent with studies in yeast. However, I could not confirm a direct interaction between Amph and actin in Drosophila. Another candidate partner was the actin-nucleating protein, Wiskott Aldrich Syndrome Protein, WASP. Although WASP and Amph could be coimmunoprecipitated in vitro, WASP was not expressed at the t-tubule membrane, and Wasp mutants had normal t-tubule morphology. Clearly, Amph is essential for normal t-tubule morphology and future work is needed to further define the function of Amph at the t-tubule network.

N-Methyl-D-aspartate receptors (NMDARs) are a principal subtype of excitatory ligandgated ion channel with prominent roles in physiology and disease in the mammalian central nervous system (CNS). Activation of NMDARs requires binding of both glutamate and glycine. Apart from its co-agonist action, glycine can also prime NMDARs for subsequent internalization upon binding of both glutamate and glycine. However, the molecular basis responsible for mediating and regulating glycine priming and NMDAR endocytosis is largely unknown. In my thesis, I discovered the principle that although NMDAR gating and priming share a common requirement for glycine binding, the molecular constraints for gating are distinct from those for priming through two mutations of the glycine binding site in GluN1 subunit of the NMDAR that, while maintaining gating of NMDARs, eliminate glycine priming of the receptors. One of the molecular signatures of glycine priming is recruitment of the endocytic adaptor protein AP-2. I have characterized the two regions in GluN2 subunits required for enhanced AP-2 association. This unexpected result suggests binding of glycine initiates a conformational change transmitted from GluN1 to GluN2 allowing for docking of endocytic machinery. Furthermore, I have discovered that naturally occurring splice variants of GluN1 subunit, containing a 21 amino acid sequence in the N-terminus domain (N1) cassette, abrogate glycine stimulated AP-2 recruitment and glycine-primed NMDAR internalization. These findings imply that there are distinct populations of native NMDARs in the CNS – those lacking N1 that show glycine-primed internalization and those containing N1 that are not primable. Collectively, my thesis work demonstrates a dramatic all-or-none priming effect with splice variants of NMDARs, a highly unexpected discovery providing novel insight into the molecular mechanisms and physiological role of glycine priming. Ultimately, elucidating principles and mechanisms of glycine priming lay the foundation for new types therapeutic approaches for CNS disorders, approaches without the deleterious consequences of directly blocking NMDARs.