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Neurotransmission is essential for neuronal communication. At the presynapse, synaptic vesicles (SVs) undergo exocytosis to release neurotransmitter in response to incoming action potentials, and endocytosis to maintain the supply of SVs needed for further rounds of exocytosis. A key event during SV endocytosis is the efficient sorting and localisation of SV proteins at the plasma membrane. This ensures that nascent SVs that are formed have the correct molecular composition to participate in subsequent exocytic events. The sorting of SV proteins at the plasma membrane is usually facilitated by adaptor proteins (e.g. AP-2) which recognise binding motifs present on key SV proteins and facilitate their internalisation during endocytosis. In addition to this, certain SV proteins possess the ability to chaperone each other as part of an endocytic transport complex throughout the SV recycling process. In conjunction with AP-2-facilitated sorting, the transport of complexed SV proteins during endocytosis provides further mechanistic insight into how SVs are generated with consistent high fidelity for functional viability. Using pHluorins as a tool to visualise SV protein trafficking in hippocampal cultures, the relationship between two key SV proteins, synaptic vesicle protein 2A (SV2A) and synaptotagmin I (SYT1), was investigated. SYT1 predominantly acts as the Ca2+ sensor for fast synchronous release at the presynapse, whilst the exact function of SV2A remains unknown to this day. In this study, the ablation of the AP-2 binding site in SV2A (Y46A) resulted in increased SYT1 surface expression and accelerated SYT1 retrieval compared to WT SV2A. No additive defects were observed when a second point mutation (T84A) was introduced to SV2A that disrupts the phosphorylation-dependent interaction between SV2A and SYT1, thus confirming that SYT1 localisation and retrieval is dependent on normal SV2A retrieval by AP-2. The hypothesis that disruption of the SV2A-SYT1 interaction may provide an underlying mechanism for motor onset seizures in epilepsy was also investigated. An epilepsy-related mutation (R383Q) in SV2A also resulted in increased SYT1 surface expression and accelerated SYT1 retrieval mirroring the defects caused by the Y46A mutation. Introduction of Y46A or T84A mutation into SV2A R383Q resulted in no additive defects compared to the single mutant, suggesting that the observed defects in SYT1 localisation and retrieval kinetics in the epilepsy-related mutant may be caused by the ablation of normal SV2A internalisation. GST pulldown assays, mass spectrometry and western blotting data indicate that presence of the mutation disrupts normal binding of the SV2A cytosolic loop with actin, tubulin and certain subunits of V-ATPase. Finally, a link between SV2A-dependent presynaptic dysfunction and epilepsy was examined through studies utilising the anti-epileptic drug, levetiracetam (LEV). SV2A contains a binding site for LEV, suggesting that it may act as a carrier for the drug into the presynapse. Hippocampal neuronal cultures were treated with LEV at various concentrations in the presence of specific patterns of neuronal activity. No observed effects of the drug on synaptophysin, vesicular glutamate transporter 1 (VGLUT1) and SYT1 recycling were observed, suggesting that LEV is unlikely to function as a modulator of excitatory presynaptic activity or by influencing SV2A function. In conclusion, this work demonstrates that SV2A is essential for accurate SYT1 trafficking and a link has been established between defective SV2A internalisation and subsequent downstream effects on SYT1 localisation and retrieval during SV recycling.

BACKGROUND Heart disease and renal dysfunction are often mutually reinforcing conditions, although the factors underlying this relationship are not fully understood. Cardiac remodelling resulting from disease is partly caused by the reactivation of developmental programmes. By unravelling the mechanisms that drive kidney development and function, it may be possible to gain novel insight into remodelled kidney states that are linked to disease. In this study, we have investigated the interplay between renal and cardiovascular systems during nephrogenesis at the level of the blood filter. METHODOLOGY Owing to the optical transparency and rapid external development of the embryo, the zebrafish provides a research model for advanced imaging technologies, allowing us to visualise structures located deep within living specimens. Here, we combine deep-tissue live imaging and novel functional assays to study development and function of the pronephric kidney - the first and most basic kidney to form in the embryo. RESULTS Using two-photon excitation microscopy, we have successfully established methodology for performing, deep-tissue, time-lapse imaging in living embryos of the two primary cell types forming the kidney – endothelial and epithelial cells. Fluorescence angiograms were performed using supra-vital dye agents to visualise circulatory flow in relation to the pronephric vasculature and the process of blood-filtration. Observations from live imaging studies, supported by immunostaining, were used to create a comprehensive model of the developing glomerular morphology, and interactions at the endothelial-epithelial cell interface, where glomerular epithelial primordia merge around the vascular component of the pronephric kidney. To investigate renal function, we devised a novel assay of pronephric filtration, by tracking the accumulation of injected fluorescent tracers within the excreted filtrate of embryos. This allowed us to relate our time-lapse observations to maturation of the glomerulus, and the evolution of perm-selective function. Finally, we explored methods of mechanically obstructing blood-flow in order to investigate whether altered hemodynamic forces would influence pronephric development. We found that in those embryos with severely disrupted circulatory flow, the glomerular morphology was affected. CONCLUSIONS In summary, the combination of these techniques has allowed us to visualise the multi-cellular organisation of the pronephric kidney over time, which has previously been limited to primarily fixed-tissue approaches. A detailed model of pronephric development has been developed, which could ultimately be used to dissect the molecular mechanisms underlying embryonic kidney development.

Cellular communication requires the transport of chemical messengers between intracellular compartments and from cell to cell. The regulated exocytosis of a secretory vesicle at the plasma membrane involves the merger of two bilayers, with markedly different lipid composition, within a millisecond time scale. The spatial and temporal control of the protein and lipid complement at these fusion sites is essential. A highly conserved family of proteins are known to drive this fusion event; SNAP-25 and syntaxin-1 (t-SNAREs) associate at the plasma membrane in a 1:1 stoichiometry to provide a binding site for the vesicle-membrane protein synaptobrevin (v-SNARE). The formation of this complex and subsequent fusion requires accessory proteins for efficient calcium-triggered exocytosis; which of these proteins facilitate the initial attachment of vesicle to the plasma membrane prior to fusion is still under debate. Specific sites for vesicle fusion have been proposed and the organisation of lipids and proteins at these fusion sites has been extensively investigated with limited spatial and temporal resolution; however the presence of raft-forming lipids at these sites as well as the arrangement of SNARE proteins at the molecular level is still under contention. The data presented within this thesis aims to elucidate the protein and lipid environment at the fusion site using super-resolution microscopy and advanced vesicle tracking. Under diffraction-limited microscopy the t-SNAREs are visualised as 200 nm homogenous clusters; however I have used single molecule localisation microscopy to reveal a more complex heterogeneous molecular arrangement. Quantification of lipid order exclusively at the plasma membrane provided insight into the influence of cholesterol-induced lipid arrangement on SNAP-25 localisation. In addition the t-SNARE interaction was investigated using TCSPC-FLIM identifying two lipid-order-dependent conformations in distinct clusters at the plasma membrane. Extensive vesicle tracking at optimum sampling rates demonstrated the ‘sampling’ behaviour of LDCVs and allowed characterisation of vesicle fusion sites. In summary I find that vesicles exhibit preference for residence and probably fusion at regions of plasma membrane with a low t-SNARE density; these proteins appear to exert control over exocytosis by adopting alternative conformations that are under cholesterol-induced regulation.