The basement membrane protein laminin-332 (laminin-5) mediates both stable cell adhesion and rapid cell migration, and thus has the potential to either restrain or promote tumor cell metastasis. The major cellular receptors for laminin-332 are integrin α3β1, which mediates rapid tumor cell migration, and integrin α6β4, which often mediates stable cell attachment. Tetraspanin protein CD151 interacts directly with both α3β1 and α6β4 integrins and with other tetraspanins, thereby promoting α3β1 and α6β4 association with tetraspanin-enriched microdomains on the cell surface. To explore the possibility of selectively modulating tumor cell responses to laminin-332, we re-expressed a series of CD151 mutants in epidermoid carcinoma cells with near total, RNAi- mediated silencing of endogenous CD151. CD151's interactions with its integrin partners or its interactions with other tetraspanins were selectively disrupted by specific mutations in the CD151 large extracellular loop (EC2 domain) or in intracellular CD151 palmitoylation sites, respectively. CD151- integrin association and CD151-tetraspanin association were both important for α3β1 integrin- dependent initial adhesion and rapid migration on laminin-332. Remarkably, however, only CD151-integrin association was required for stable, α6β4 integrin-dependent cell attachment on laminin-332. In gap-filling assays, where CD151-silenced cells moved more rapidly than WT cells, again, only CD151-integrin association was required to restrict movement into the gap, suggesting that both α3β1 and α6β4 integrin must be able to associate with CD151 in order restrict group motility. In addition, we found that a QRD amino acid motif in the CD151 EC2 domain that had been thought to be crucial for CD151-integrin interaction is not essential for CD151-integrin association or for CD151's ability to promote several different integrin functions. These new data suggest potential strategies for selectively modulating migratory cell responses to laminin-332, while leaving stable cell attachment on laminin-332 intact.

Cancer metastasis is a multi-stage process initiated by the cancer cell acquiring the ability to migrate. The protein profile of such a cell undergoes dramatic changes including changes in integrin expression. Integrins play a major role in cell adhesion, motility, differentiation, blood clotting, tissue organization and cell growth as well as cancer cell migration, invasion and metastasis. Integrin a6, which can pair with integrin b4 or b1 is a laminin receptor and is detected in epithelial cells. Earlier studies have reported uPA mediated integrin a6 cleavage in prostate cancer resulting in loss of the ligand binding domain. Site-directed mutagenesis studies have identified the cleavage site to be at R594R595 located in the "stalk" region of the integrin a6. Prostate cancer cells PC3N-a6-RR cells, bearing a R594R595 to A594A595 mutation, engineered to express the uncleavable form of integrin a6 were found to migrate 6.4 folds lesser on Laminin-1 as compared to the PC3N-a6-WT cells which expressed the wild-type integrin a6. This result suggests that integrin a6 cleavage enhances migration. Prostate cancer is known to metastasize to the bone. Injection of the PC3N-a6-WT cells in mouse femurs resulted in increased bone destruction and pain behavior when compared to the femurs injected with PC3N-a6-RR cells indicating that the integrin a6 cleavage could affect and modify the bone microenvironment. An observation that complete conversion of integrin a6 to a6p was not observed in cell lines even in presence of excess uPA suggested a regulatory mechanism. Integrins are known to associate with many proteins including tetraspanins, which are transmembrane proteins, that function as protein adapters. Integrin a6 was found to be refractory to uPA mediated cleavage when complexed with tetraspanin CD151. The amount of integrin a6 available for cleavage increased when CD151 levels were decreased by CD151 siRNA treatment. These results suggest that the integrin a6 available and unavailable for cleavage can be modulated by interaction with CD151 and hence affect the migratory potential of the cell. Collectively these data suggest that integrin a6 cleavage can enhance cell migration, initiate signals to modify the tumor microenvironment and can be regulated by interaction with tetraspanin CD151.

Doctor of Philosophy / Department of Biology / Rollie J. Clem / Members of the protein super family of tetraspanins are best defined by a simple structure comprising four transmembrane domains, two extracellular loops of unequal size, and short cytoplasmic regions. Despite their small size, tetraspanins are able to participate in multiple functions, as diverse as B cell activation, cancer metastasis, and viral infection. To compensate for a lack of intrinsic enzymatic activity, tetraspanins have gained the fascinating ability of associating with numerous different proteins. In addition, tetraspanins interact with each other forming a network on the plasma membrane: the tetraspanin web. In this way, functionally related proteins binding to different tetraspanins can be brought into close vicinity, thereby enhancing signaling pathways. The tetraspanin web is a dynamic environment and its regulation has grasped the attention of several research groups in the past few years. Particularly, several tetraspanins have been found to be palmitoylated, a post-translational modification attaching a palmitic acid to cysteine residues in a reversible manner. Palmitoylation is thought to be important for the integrity of the tetraspanin web. We examined the effect of disrupting putative palmitoylation sites on the tetraspanin CD81 by mutating its juxtamembrane cysteines. By flow cytometry, we observed a decrease in the detection of mutant CD81 at the cell surface. This was not due to defects in protein trafficking or antibody affinity, and might reflect an abnormal CD81 distribution in a membrane environment that prevents the exposure of the epitope recognized by the CD81 antibody. Immunoblotting analysis revealed a novel CD81 processing event that was impaired in the mutant CD81 proteins compared to wild-type. Finally, co-immunoprecipitation assays showed a reduction in binding of tetraspanin CD9 and Ig superfamily member EWI-2 to mutant CD81. Taken together, these results suggest the importance of juxtamembrane cysteines (via palmitoylation or protein conformational changes) in protein interactions of CD81 within the tetraspanin web. Although 33 tetraspanins are expressed in humans, less than half of them have been well studied. Among the “orphan” tetraspanins awaiting further examination is Tspan-2. Here, we provide the first elements for the characterization of mammalian Tspan-2 by investigating expression patterns, N-glycosylation status, and association with other tetraspanins.

Angiogenesis is the process of new blood vessel sprouting from pre-existing vessels and is responsible for generating the majority of nascent vessels during development, tissue regeneration and disease. During angiogenesis, sprouting endothelial cells (ECs) are organised into leading 'tip' cells (TCs) and trailing 'stalk' cells (SCs). This hierarchal organisation of TCs and SCs is essential for the coordinated collective migration of ECs during sprouting. However, the precise mechanisms that define TC verses SC behaviour and identity remains uncertain. Transcriptomic analysis of sprouting vessels in zebrafish embryos led to the identification of a novel TC-associated gene, transmembrane 4 L six family member 1 (tm4sf1). We find that tm4sf1 expression is tightly spatiotemporally restricted to migrating TCs during intersegmental vessel (ISV) sprouting in zebrafish. Furthermore, TC tm4sf1 expression is controlled by the vascular endothelial growth factor receptor (Vegfr) - Notch signalling axis. Morpholino oligonucleotide (MO)-mediated knockdown of tm4sf1 reveals a subtle delay in ISV sprouting upon loss of tm4sf1 expression. Moreover, using multiplexed, real-time imaging approaches and in-depth analysis of TC and SC behaviours at single cell resolution, we reveal that the delay in ISV sprouting is specifically due to reduced TC motility. Furthermore, we find that tm4sf1 functions to induce TC motility in the leading daughter cell following TC mitosis, to rapidly re-establish post-mitotic TC behaviour. Generation of tm4sf1 mutant zebrafish lines using both transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR) confirms that Tm4sf1 modulates TC behaviour. Additionally, mechanistic studies in human ECs reveal that tm4sf1 regulates VEGFR-mediated signalling upon VEGF-stimulation, which subsequently controls cell migration and expression of the TC determinants, DLL4 and VEGFR2. Hence, our results suggest that tm4sf1 is a novel modulator of the TC-SC hierarchy and collective EC migration during ISV sprouting. Overall, these findings have potential therapeutic implications since tm4sf1 may be a promising target for the manipulation of pathological angiogenesis in disease.

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.

No description available.

Les tétraspanines sont des glycoprotéines membranaires impliquées dans une variété de processus physiologiques et pathologiques. En cancérologie, de nombreuses études cliniques et expérimentales ont établi un lien entre le niveau d’expression de certaines tétraspanines et la formation de métastases. Ce travail porte sur la tétraspanine Co-029/tpan8 et son rôle dans le processus métastatique dans le cancer du côlon. Afin d’appréhender ce rôle, un modèle cellulaire a été développé dans le laboratoire à partir de la lignée Isreco1 où nous avons surexprimé de manière stable le Co-029/tspan8. Sur ce modèle nous avons étudié dans un premier temps la migration cellulaire, élément capital participant à la genèse des métastases. Nous avons montré que le Co-029/tspan8 coordonne la motilité cellulaire en fonction de plusieurs signaux issus de la membrane (intégrines, complexe E-cadhérine/p120ctn, EGFR, MET). Par ailleurs, nous avons montré dans une première étude clinique que l'expression de Co-029/tspan8 dans la tumeur serait de pronostic péjoratif. Nous avons également montré que l’anticorps anti-Co029 (Ts29.2) produit dans notre laboratoire induit une réduction significative de la croissance tumorale in vivo dans la souris nude. Nos travaux suggèrent que Co-029/tspan8 serait à la fois un marqueur de pronostic et une cible potentielle pour l'utilisation thérapeutique des anticorps monoclonaux dans le cancer du côlon. / Tetraspanins are membrane glycoproteins involved in a variety of physiological and pathological processes. In cancerology, many clinical and experimental studies have established a link between expression’s level of some tetraspanins and metastasis. This work focuses on the tetraspanin Co-029/tspan8 and its implication in the metastatic process in colon carcinoma. We developed a cellular model from primary colon carcinoma cell line Isreco1 in which we stably overexpressed Co-029/tspan8. In this model we studied, first, cell migration, an essential process for metastasis formation. We have shown that Co-029/tspan8 coordinates cell motility according to several signals from the membrane (integrins, E-cadherin/p120ctn complex, EGFR, MET). Moreover, in a first clinical study the Co-029/tspan8 expression in primary tumors seems to correlate with poor prognosis. We also showed that the antibody anti-Co029 (Ts29.2) produced in our laboratory induced a significant inhibition of tumor growth in nude mice. Our study suggests Co-029/tspan8 as a marker of prognosis and a potential target for monoclonal antibody therapy in colon cancer.

Human immunodeficiency virus type 1 (HIV-1) is a human retrovirus of the lentivirus subgroup which primarily infects T cells and macrophages, and causes acquired immune deficiency syndrome (AIDS). Since its emergence in the early 1980s, HIV-1 has caused a global pandemic which is still responsible for over one million deaths per year, primarily in sub-Saharan Africa. HIV-1 has been the subject of intense study for over three decades, which has resulted not only in major advances in cell biology, but also in numerous drug treatments that effectively control the infection. However, cessation of treatment always results in reemergence of the infection due to the ability of HIV-1 (and other lentiviruses) to establish a persistent quiescent infection known as latency. The elimination of latently-infected cells is the primary goal of current research towards a cure for HIV-1, alongside efforts to develop vaccines, which have thus far been fruitless. The spread of HIV-1 to susceptible target cells (which express the receptor CD4 and a co-receptor; CXCR4 or CCR5) can take place when antigen-presenting cells, such as dendritic cells, capture virus particles and then pass them on to target cells, without themselves becoming infected. Alternatively, productively infected T cells or macrophages can spread HIV-1 either by shedding virus particles to the milieu, which are then stochastically acquired by target cells, or through transient contacts between infected and uninfected cells known as virological synapses (VSs). VS-mediated cell-to-cell transmission is thought to be highly efficient due to the release of virus directly onto (or very near to) a target cell, and some evidence suggests that the VS is a privileged site which allows the virus to evade neutralizing antibodies and drugs. However, and most importantly, it is of central interest to us because the same transient cell adhesions that facilitate virus transfer can also result in the fusion of the two cells to form a syncytium, due to the presence of the viral fusogen Env and its receptor and co-receptor on either side of the VS. While T cell syncytia can be found in vivo, they remain small, and it appears that the majority of VSs resolve without fusion. The regulation of HIV-1-induced cell-cell fusion and the fate of those syncytia are the focus of the work presented here. A family of host transmembrane proteins, the tetraspanins, which regulate cell-cell fusion in other contexts (e.g. the fusion of myoblasts to form and maintain myotubes), were found to inhibit HIV-1-induced cell-cell fusion. Our investigations have further characterized this regulation, concluding that tetraspanins allow cells to reach the fusion intermediate known as hemifusion before their ability to repress fusion takes effect. In parallel, because syncytia are nevertheless found both in infected individuals and in a humanized mouse model for HIV-1, we also became interested in whether small T cell-based syncytia were able to participate in HIV-1 spread by transmitting virus to target cells. Using a simple three dimensional in vitro culture system which closely recapitulates those in situ observations, we found that small syncytia can contact target cells and transmit virus without fusing with them. Overall, these studies further our understanding of HIV-1-induced syncytia and reveal a previously unrecognized role for these entities as active participants in HIV-1 spread.

Research Doctorate - Doctor of Philosophy (PhD) / Membrane fusion events are a fundamental aspect of cellular biology and underpin important processes such as organ formation and fertilisation. Within the latter, proteins that are expressed on the egg surface which are responsible for mediating sperm recognition, binding and fusion to the egg, are yet to be fully determined. Evidence does however suggest that egg surface glycophosphatidylinositol (GPI)-anchored proteins play a role in sperm binding, whilst another class of proteins, known as tetraspanins, appear to be important in downstream events of membrane fusion. Of the tetraspanins, CD9 and CD81 have been identified as fulfilling roles in membrane fusion; identifications are however yet to obtained for the important GPI-anchored protein(s). This research aimed to identify and characterise egg surface proteins implicated in sperm-egg interaction, and embodied attempts to both identify the important GPI-anchored protein(s) as well as expand upon tetraspanin studies through investigations into mice lacking the tetraspanin CD151. Throughout this research, it was hypothesised that membrane fusion events of fertilisation parallelled those of enveloped virus – host cell fusion, for which rearrangement of surface protein thiols is essential. In vitro binding and fusion experiments were utilised as functional bioassays in the investigation of factors affecting sperm-egg interaction, such as tetraspanin deletion and the xenobiotic modification of cell surface thiols, while mass spectrometry (MS)-based proteomics and bioinformatics-based analyses were employed to compile oocyte protein databases and to identify candidate proteins responsible for mediating sperm-egg interaction, such as GPI-anchored proteins. It was determined that exposing oocytes to compounds with a capacity to alkylate cell surface thiols strongly inhibited sperm-egg binding. Additionally, while CD151 deletion had no effect on sperm-egg binding, the downstream events of membrane fusion were significantly impaired. Ovaries from CD151 null mice also exhibited abnormal phenotypes. In addition, a total of 11 identifications were obtained in the search for the GPI-anchored proteins expressed within eggs, however only 6 of these were deemed to be potential mediators of sperm-egg interaction. In conclusion, the experiments outlined herein demonstrate a novel inhibitory effect for specific xenobiotics on sperm-egg interaction, and correlate the inhibitory action of these compounds with their capacity to reduce cell surface thiol labelling. A novel role for CD151 in the mediation of sperm-egg fusion was also discovered, while at the same time the important GPI-anchored protein(s) implicated in sperm-egg binding may be among 6 identified potential candidates. Together the findings reiterate the consensus that oocytes possess a cell surface protein complex responsible for mediating sperm binding and fusion as separate events, and in light of the demonstrated importance of surface thiols, that events of sperm-egg membrane fusion parallel those of enveloped virus – host cell fusion.

Research Doctorate - Doctor of Philosophy (PhD) / Membrane fusion events are a fundamental aspect of cellular biology and underpin important processes such as organ formation and fertilisation. Within the latter, proteins that are expressed on the egg surface which are responsible for mediating sperm recognition, binding and fusion to the egg, are yet to be fully determined. Evidence does however suggest that egg surface glycophosphatidylinositol (GPI)-anchored proteins play a role in sperm binding, whilst another class of proteins, known as tetraspanins, appear to be important in downstream events of membrane fusion. Of the tetraspanins, CD9 and CD81 have been identified as fulfilling roles in membrane fusion; identifications are however yet to obtained for the important GPI-anchored protein(s). This research aimed to identify and characterise egg surface proteins implicated in sperm-egg interaction, and embodied attempts to both identify the important GPI-anchored protein(s) as well as expand upon tetraspanin studies through investigations into mice lacking the tetraspanin CD151. Throughout this research, it was hypothesised that membrane fusion events of fertilisation parallelled those of enveloped virus – host cell fusion, for which rearrangement of surface protein thiols is essential. In vitro binding and fusion experiments were utilised as functional bioassays in the investigation of factors affecting sperm-egg interaction, such as tetraspanin deletion and the xenobiotic modification of cell surface thiols, while mass spectrometry (MS)-based proteomics and bioinformatics-based analyses were employed to compile oocyte protein databases and to identify candidate proteins responsible for mediating sperm-egg interaction, such as GPI-anchored proteins. It was determined that exposing oocytes to compounds with a capacity to alkylate cell surface thiols strongly inhibited sperm-egg binding. Additionally, while CD151 deletion had no effect on sperm-egg binding, the downstream events of membrane fusion were significantly impaired. Ovaries from CD151 null mice also exhibited abnormal phenotypes. In addition, a total of 11 identifications were obtained in the search for the GPI-anchored proteins expressed within eggs, however only 6 of these were deemed to be potential mediators of sperm-egg interaction. In conclusion, the experiments outlined herein demonstrate a novel inhibitory effect for specific xenobiotics on sperm-egg interaction, and correlate the inhibitory action of these compounds with their capacity to reduce cell surface thiol labelling. A novel role for CD151 in the mediation of sperm-egg fusion was also discovered, while at the same time the important GPI-anchored protein(s) implicated in sperm-egg binding may be among 6 identified potential candidates. Together the findings reiterate the consensus that oocytes possess a cell surface protein complex responsible for mediating sperm binding and fusion as separate events, and in light of the demonstrated importance of surface thiols, that events of sperm-egg membrane fusion parallel those of enveloped virus – host cell fusion.