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.

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.