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Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a monogenic developmental and epileptic encephalopathy with onset in early infancy that is caused by mutations in the CDKL5 gene. CDD patients often exhibit profound neurodevelopmental delay, visual and motor deficits, and autistic-like manifestations, whereas epileptic seizures typically appear as early as the third week after birth. CDKL5 is a neuron-specific serine/threonine kinase that has been implicated in different cellular processes including neurite outgrowth, microtubule remodelling, and synaptogenesis. Animal models of CDKL5 deficiency have revealed phenotypes associated with defective neurotransmission. However, the potential role of CDKL5 in presynaptic processes and synaptic vesicle (SV) membrane trafficking remains unknown. In this project, we used a novel CDKL5 KO rat model to detect potential phenotypes that are linked to loss of CDKL5 function. Using a genetically encoded fluorescent reporter, we revealed that absence of CDKL5 results in defective SV recycling in an activity-dependent manner in primary hippocampal neurons. Using a molecular replacement strategy, we showed that the kinase domain of CDKL5 was able to restore the speed of SV endocytosis indicating that the catalytic activity of CDKL5 is essential for its role in SV recycling. In agreement, we revealed that CDKL5 mutants either lacking the kinase domain or containing kinase-inactive mutations reported in CDD patients were unable to rescue this impairment suggesting that defective presynaptic processes may contribute to the CDD onset. Since the kinase activity is critical for CDKL5-mediated SV recycling, we also explored whether the phosphorylation levels of its in vitro presynaptic substrate, amphiphysin 1 (Amph1), were altered in CDKL5 KO neurons. We revealed that CDKL5 does not exert its presynaptic role by phosphorylating Amph1 at S293. At the same time, this work showed that Amph1-mediated complexes are important for SV endocytosis in presynaptic terminals. Furthermore, we mapped the Amph1 motif that interacts with a different endocytosis protein, endophilin A1, and we demonstrated that the Amph1-endophilin A1 complex is essential for SV regeneration. Finally, the phosphorylation dynamics at Amph1-S293 dictates both Amph1-mediated interactions with endophilin A1 and SV endocytosis, indicating that phosphorylation-dependent Amph1-endophilin A1 interaction is essential for optimal SV endocytosis. Overall, this study offers the first evidence of a presynaptic role of CDKL5 that is mediated through its kinase activity and creates the basis for future research on presynaptic CDKL5 that could lead to potential treatments for CDD patients.

Eukaryotic cells are subdivided into organelles with unique physicochemical compositions and functions. Establishment and maintenance of these organelles requires selective exchange of material by vesicle trafficking. Transport vesicles are formed by cytoplasmic coat protein complexes which shape the membrane, and play a further role in the selection of transmembrane cargo proteins. Transmembrane cargo receptors select cargo in the luminal compartment and bind to these coat proteins through signals in their cytoplasmic domains. Once formed, vesicles recognise and fuse with a target organelle to deliver their content. This final process is tightly regulated by small GTPases of the Rab superfamily. Here, I will describe my work investigating the role of specific Rab GTPase pathways and the KDEL cargo receptor in trafficking in the secretory and endocytic pathways in mammalian cells. There are over 60 Rabs in human cells, each of which is activated by a specific GDP-GTP exchange factor (GEF) and inactivated by a GTPase-activating protein (GAP) at a defined membrane location. For many of these Rabs the GEF and GAP were unknown at the outset of this work in which I have identified and characterised two structurally related families of Rab GEFs, the DENN and tri-longin domain proteins. The DENN family, expressed only in metazoans, which has 17 members acting on 10 different Rabs. The tri-longin family, comprises 3 heterodimeric GEF complexes: Mon1-Ccz1 for the late endosomal, lysosomal Rab7, Hps1-Hps4 (BLOC-3) which activates Rab32/Rab38 in melanogenesis and Intu-Fuz, part of CPLANE complex, which activates Rab23 at cilia. Additionally, I have shown that the unrelated Rab3GAP complex is a GEF for the ancestral Rab18 regulating the structure of the endoplasmic reticulum. In the final part of this work, I have explored how the integrity of the early secretory system depends on selective export and retrieval of proteins between the ER and the Golgi. An essential component of this system is the transmembrane cargo receptor for KDEL retrieval signals. Here I describe the structure of the KDEL receptor and mechanism by which KDEL cargo binding triggers a conformational change exposing a signal for the COP I vesicle coat.

Eukaryotic translation elongation factor eEF1A2 is responsible for delivering aminoacylated tRNAs to the ribosome during protein synthesis. Heterozygous de novo missense mutations in EEF1A2 have been identified in individuals with epilepsy, autism and intellectual disability. The primary aim of this thesis was to assess whether these mutations operated through a loss of function, gain of function or dominant negative mechanism. To investigate this, I analysed eEF1A2 protein interactions, how these interactions varied between mutations and the functional consequences of changes in the interactome. I used affinity purification mass spectrometry to identify the interactome of eEF1A2 and compared the changes resulting from mutations in eEF1A2. Co-immunoprecipitation experiments determined that mutations could be grouped by changes in their interaction with the cognate guanine exchange factor eEF1B. One group showed complete or partial loss of binding to all four subunits of the eEF1B complex, whilst another cluster showed no change in binding compared to wildtype eEF1A2. No specific patterns of clinical features or phenotypic severity could be attributed to these groupings. My results suggested that protein synthesis would be impaired by this disrupted interaction. In vivo and in vitro protein synthesis experiments failed to detect any differences that could be attributed to the presence or absence of the D252H mutation. As no apparent differences in protein synthesis could be detected between muscle tissue from Eef1a2⁻/⁻ and Eef1a2⁺/⁺ mice or between eEF1A2⁻/⁻ and wild-type cells, it is likely that in both cases the protein synthesis assay employed was not performing adequately. Comparative analysis of Eef1a2ᴰ²⁵²ᴴ/ᴰ²⁵²ᴴ and Eef1a2⁻/⁻ mouse phenotypes determined that there is a gain of function or dominant negative element to the eEF1A2ᴰ²⁵²ᴴ mutation, highlighting that this mutation does not operate simply through a loss of function mechanism. Interactome was unable to successfully identify what this might be. In conclusion eEF1A2 mutations may operate via both a loss and gain of function mechanism. Depending on the precise mutation, eEF1A2 may be compromised in its ability to operate in protein synthesis, but at least some mutations may also result in a degree of toxicity.

The development of the central nervous system is dysregulated in neurodevelopmental disorders such as intellectual disability, autism spectrum disorder, and epilepsy. These three disorders have different clinical features, yet there is high comorbidity between them. They can be difficult to study due to their highly complex aetiologies, however there are various monogenic diseases that can cause all of them, including SYNGAP1 haploinsufficiency where the synaptic guanosine triphosphatase (GTPase)-activating protein (SYNGAP) protein levels are highly reduced; Fragile X syndrome where the fragile X mental retardation protein (FMRP) is no longer translated; and DNM1 epileptic encephalopathy where mutations in the Dynamin1 gene alter the protein function. These monogenic conditions are synaptopathies as the proteins affected play important roles in synapse stability and neurotransmission. Because of the high comorbidity between these disorders, it is hypothesised that there may be a common mechanism underlying them. We hypothesise that a deficit in presynaptic vesicle recycling may be part of a common mechanism underlying intellectual disability, autism spectrum disorder, and epilepsy especially in SYNGAP1 haploinsufficiency, Fragile X syndrome, and DNM1 epileptic encephalopathy. Using various fluorescent presynaptic activity reporters including synaptic pHluorins, tetramethylrhodamine dextran and calcium dyes to compare presynaptic activity in in vitro models of these monogenic conditions, we found differences in synaptic vesicle (SV) endocytosis in the genetically altered conditions compared to wildtype controls. We observed various SV endocytosis defects in clathrin-mediated endocytosis (CME) or activity-dependent bulk endocytosis (ADBE) in our models. We observed enhanced CME in SynGAP1 KO mouse hippocampal neurons. This enhanced SV endocytosis was accompanied by decreased SV cargo on the plasma membrane. Rat SynGAP1 KO hippocampal neurons did not display enhanced SV endocytosis, nor did neurons with the GTPase-activating (GAP) domain of SynGAP deleted. This was perhaps due to the altered time course of development between these rodent species. In mouse and rat models of Fragile X syndrome, CME was not altered compared to wildtype controls. However, in a rat model, we observed fewer nerve terminals undergoing ADBE which is the dominant SV endocytosis mode during elevated neuronal activity. De novo epileptic encephalopathy-associated mutations in DNM1 had differential effects on SV recycling through both CME and ADBE. Mouse hippocampal neurons overexpressing Dyn1R237W, Dyn1I289F and Dyn1H396D all showed less CME compared to overexpression of Dyn1WT. Moreover, fewer nerve terminals overexpressing Dyn1H396D were found to undergo ADBE. We also found that a large-conductance potassium (BK) channel opener can accelerate clathrin-mediated endocytosis and thus may be able to rescue the impaired SV endocytosis caused by these mutants. Although there is not yet a common underlying pathway at the presynaptic level between these conditions, SV recycling dysfunction is present across all of these models. Furthermore, we propose an axis of pathophysiology model where optimal SV endocytosis is required for optimised neural performance. We propose that either decreased or increased SV endocytosis can lead to the synaptic dysfunction observed in these models.

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.

Synaptic vesicle recycling is the reuse of synaptic membrane material and proteins after vesicles have been exocytosed at the pre-synaptic terminal of a neuronal synapse. The discovery of the mechanisms by which recycling operates is a subject of active research. Within small mammalian central nervous system nerve terminals, two studied mechanisms of recovery are clathrin-mediated endocytosis and activity-dependent bulk endocytosis. Research into the comparative kinetics and mechanisms underlying these endocytosis mechanisms commonly involves time-series fluorescence microscopy of in vitro cultures. Synaptic proteins are tagged with fluorescent markers, or the synaptic vesicles are labelled with fluorescent dye. The change in fluorescence levels of individual synapses over time in response to stimuli is used to understand synaptic activity. The image analysis of these time-series images frequently requires substantial manual effort to extract the changing synaptic fluorescence intensity levels over time. This work focusses on two closely interlinked areas, the development of improved automated image analysis tools to facilitate the analysis of microscopy image data, and computational simulations to leverage the data obtained from these experiments to gain mechanistic insight into the underlying processes involved in synaptic vesicle recycling. The imaged properties of synapses within the time-series images are characterised, in terms of synapse movement during the course of an experiment. This characterisation highlights the properties which risk adding error to the extracted fluorescence intensity data, as analysis generally requires segmentation of regions of interest with fixed size and location. Where possible, protocols to optimise the manual selection of synapses in the image are suggested. The manual selection of synapses within time-series images is a common but time consuming and difficult task. It requires considerable skill on the part of the researcher to select synapses from noisy images without introducing error or bias. Automated tools for either general image segmentation or for segmentation of synapse-like puncta do exist, but have mixed results when applied to time-series experiments. This work introduces the use of knowledge of the experiment protocol into the segmentation process. The selection of synapses as they respond to known stimuli is compared against other current segmentation methods, and tools to perform this segmentation are provided. This use of synapse activity improves the quality of the segmented set of synapses over existing segmentation tools. Finally, this work builds a number of computational models, to allow published individual data points to be aggregated into a coherent view of overall synaptic vesicle recycling. The first is FM-Sim, a stochastic hybrid model of overall synapse recycling as is expected to occur during the course of an experiment. This closed system model handles the processes of exocytosis and endocytosis. It uses Bayesian inference to fit model parameters to experimental data. In particular, it uses the experimental protocol to separate the mechanisms and rates that may contribute to the observed experimental data. The second is a mathematical model of one aspect of synaptic vesicle recycling of particular interest - homoeostasis of plasma membrane integrity on the presynaptic terminal. This model provides bounds on efficiency of the studied endocytosis mechanisms at recovery of plasma membrane area during and after neuronal stimulus. Both the image analysis and the computational simulations demonstrated in this work provide useful tools and insights into current research of synaptic vesicle recycling and the role of activity-dependent bulk endocytosis. In particular, the utility of adding time-dependent experimental protocol knowledge to both the image analysis tools and the computational simulations is shown.

Synaptic vesicle (SV) recycling in the presynapse is essential for the maintenance of neurotransmission. During mild stimulation clathrin-mediated endocytosis (CME) dominates, however during intense stimulation activity-dependent bulk endocytosis (ADBE) is the dominant form of membrane retrieval. The aim of this thesis was to determine how the signalling molecule GSK3 controlled ADBE, with the hypothesis that this enzyme was required at multiple stages of this endocytosis mode. I also hoped to identify a specific cargo for ADBE. I found that during intense action potential stimulation, a localised calcium increase is necessary for the activation of Akt, which inhibited GSK3. This activation was mediated via a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. Furthermore, I found that phosphatidylinositol 4-kinaseIIα (PI4KIIα), a molecule whose abundance is regulated by GSK3, had a key role in ADBE. Specifically, I found that the absence of PI4KIIα accelerated CME but inhibited ADBE and that PI4KIIα controls CME and ADBE via distinct mechanisms. The PI4KIIα study revealed potential cross-talk between CME and ADBE. To determine whether modulation of either endocytosis mode impacts on the other, the retrieval of genetically-encoded reporters of SV cargo was monitored during intense stimulation during inhibition of either CME or ADBE. The recovery of almost all SV cargo was unaffected by ADBE inhibition but was arrested by abolishing CME. In contrast, VAMP4-pHluorin retrieval was perturbed by inhibiting ADBE and not by blocking CME. Knockdown of VAMP4 also arrested ADBE, indicating that in addition to being the first identified ADBE cargo, it is also essential for this endocytosis mode to proceed.

At the neuronal synapse, neurotransmitter-filled synaptic vesicles (SVs) fuse with the presynaptic plasma membrane during activity. Following exocytosis, SVs must be retrieved for neurotransmission to be maintained. Several modes of SV recycling have been identified. During mild neuronal activity, clathrin-mediated endocytosis has been regarded as the dominant SV retrieval mode, however the recently identified ultrafast endocytosis mode may also be important in this condition. During elevated activity, activity-dependent bulk endocytosis (ADBE) is the dominant SV retrieval pathway. In ADBE, large invaginations are formed from the plasma membrane, which then undergo scission to create bulk endosomes. In a second distinct step, SVs bud from these endosomes and specifically repopulate the reserve SV pool. However, since its first identification, only few molecules have been shown to participate in ADBE. The aim of this PhD was to identify novel molecules and elucidate the molecular mechanism of ADBE. To achieve this, two independent biochemical approaches were designed to purify and enrich bulk endosomes from primary neuronal cultures. In the first approach, bulk endosomes and SVs were labelled with a dye, FM1-43, using a strong stimulus. Cells were broken mechanically and the post nuclear supernatant, that contains all intracellular organelles, was collected. The supernatant was then subjected to subcellular fractionation using discontinuous Nycodenz gradients. This stimulated sample was always processed in parallel with a basal sample, where no neuronal stimulus was applied, in order to visualise activity dependent FM loading. After different fractionation protocols were applied, bulk endosomes were efficiently separated from SVs, as revealed by tracking fluorescence in different fractions. The fractionation results were further validated by electron microscopy, where bulk endosomes and SVs were labelled with horseradish peroxidase and purified using the established protocol. Immunoblotting against selected SV cargo proteins from stimulated bulk endosome and SV samples, indicated the specific and preferential localisation of VAMP4 on bulk endosomes, in contrast to other SV cargo. The molecular identity of bulk endosomes was also approached by submitting the bulk endosome fractions to semi-quantitative mass spectrometry. This analysis revealed many different proteins that were identified in bulk endosome samples and quantification approaches further indicated proteins that can be localised on bulk endosomes and have a potential role in ADBE. A second magnetic isolation approach was designed, to purify bulk endosomes using a completely different methodology. In this case, bulk endosomes were specifically labelled with iron nanoparticles, which are preferentially taken up by bulk endosomes since they are larger than SVs. The cells were broken as before and post nuclear supernatant was acquired. In this case, the supernatant was submitted to magnetic isolation that separated iron beads labelled structures from all other intracellular organelles. An extensive immunoblotting analysis of magnetic bulk endosomes validated that VAMP4 and syndapin I, two essential ADBE proteins, were enriched in these purified samples. These magnetic bulk endosomes were also analysed using semi-quantitative MS and revealed many proteins with a potential role in ADBE. Significant overlap between the two independent methods was observed, further validating these approaches. Combining these two methods with bioinformatics tools allowed the identification of the molecular signature of ADBE as well as novel key candidates for this process. Specific molecules were investigated for their role in ADBE and SV recycling using a variety of different real-time fluorescent imaging assays. A major focus was on rab small GTPases. High molecular weight dextran uptake was used to specifically study the role of these proteins in ADBE, as it preferentially reports uptake via larger bulk endosomes. A pH sensitive chimeric protein, synaptophysin-pHluorin, was used to investigate the role of these proteins in CME. Additional imaging assays were used to answer emerging questions regarding the function and localisation of these targets in the presynapse. Using these approaches, rab11A and rab35 were found to promote ADBE and accelerate clathrin-mediated endocytosis. This effect was specific to high intensity stimulation, while SV exocytosis was not affected. Further research on the role of both novel and established ADBE molecules will provide key future insights into the mechanism of both bulk endosome generation/scission and subsequent SV reformation. A very promising group is rab proteins and now evidence for their implication in SV recycling is presented here. Identification and characterisation of new targets will allow to investigate the role of ADBE in neurotransmission in both physiology and pathophysiology.

Synapses are the communication junctions of the nervous system and contain protein machinery necessary for cognitive functions such as learning and memory. Postsynaptic density protein-95 (PSD-95) is a key scaffolding molecule at the PSD of synapses, yet its sub-synaptic organisation in the mammalian brain remains poorly understood. This thesis presents the use of genetically labelled PSD-95 with super-resolution imaging to resolve its nano-architecture in the mouse brain. To visualize PSD-95, two knock-in mouse lines were generated where the fluorescent proteins eGFP or mEos2 was fused to the carboxyl terminus of the endogenous PSD- 95 protein (PSD-95-eGFP or PSD-95-mEos2). Methods were developed by which fixed tissue sections of PSD-95-eGFP mice were examined using gated-stimulated emission depletion (g-STED) microscopy and PSD-95-mEos2 sections were examined with photoactivatable localisation microscopy (PALM) and quantitative image analysis was developed for both methods. From these platforms it was demonstrated that PSD-95 has a two tiered organisation: it is assembled into nanoclusters (NCs) approximately 140 nm diameter, which form part of the greater envelope of the PSD within synapses. Synapse subtypes were observed as characterised by the number of NCs per PSD. Using double colour g- STED microscopy. It was then asked whether PSD-95 nano-architecture remained the same across different sub-regions of the brain. A survey of PSD-95 was performed from seven different sub-regions of the hippocampus, quantifying ~110,000 NCs within ~70,000 PSDs from across the two super-resolution platforms. It was found that synapses displayed structural diversity both within and between different brain subregions as a function of the number of NCs per PSD. PSD-95 NCs were structurally conserved across the hippocampus, but showed molecular diversity in the abundance of PSD-95 molecules within. The findings of this thesis are: 1) genetic labelling of endogenous proteins combined with super-resolution microscopy is a powerful tool to study synaptic protein organisation in tissue. 2) Synaptic structural diversity in the brain is underlined by the number of PSD-95 NC units per synapse 3) PSD-95 NCs are structurally conserved but molecularly diverse synaptic units of synapses throughout the brain. These findings suggest that cognitive processing at the synapse is based upon a conserved, fundamental, molecular architecture.

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.

Synaptophysin (Syp) is a major integral synaptic vesicle (SV) protein; there are 31 copies of Syp per vesicle, which totals up to 10% of the total SV protein content. Despite being the major SV protein, little is known about the interaction partners of Syp and as a result there has been no clear role attributed to it. One key feature of Syp is that its cytoplasmic C-terminus contains 10 pentapeptide repeats, nine of which are initiated by a tyrosine residue. Syp is the major tyrosine phospho-protein on SVs. The kinase thought to phosphorylate Syp in vivo is the ubiquitously expressed non-receptor kinase C-Src. There are two splice variants of C-Src, N1- and N2-Src, which are only expressed in neuronal tissues. Although the 3 Srcs are structurally similar, they differ by a small insert of amino acids into their SH3 domains (the N-Src loop). Examination of the amino acid sequence of the cytosolic C-terminus of Syp revealed a putative type one SH3 domain interaction motif. A screen using SH3 domains of synaptic proteins as bait in GST-pull downs from nerve terminal lysate allowed an inventory of potential interaction partners of Syp to be created. Reciprocal experiments using the C-terminal of Syp as bait confirmed many of these interactions. Single point mutations of the SH3 interaction motif on Syp highlighted that syndapin and C-Src bound to Syp via this motif. These binding mutants were inserted in Syp superecliptic synaptophluorin (SypHy) to determine the functional consequences of these interactions. These mutants did not affect the trafficking of Syp when expressed in cortical neurons derived from Syp knockout mice. However, the SH3 interaction motif was fundamental for the retrieval of VAMP (vesicle associated membrane protein) when expressed in Syp knockout cultures. Importantly, this role is not mediated through a direct interaction with VAMP with the SH3 interaction motif implicating either syndapin, C-Src or both in Syp-dependent VAMP retrieval. The 3 different Srcs had different methods of interaction with Syp, and in vitro protein kinase assays the ability of the three Src splice variants to phosphorylate Syp was assessed. Key differences in both speed and efficiency of Syp phosphorylation was observed for the different Src splice variants. Mutagenesis of either all 9 tyrosine residues, only previously identified sites resulted in changes in Syp interactions in GST-pull down assays from nerve terminal lysates. To investigate the role of Syp phosphorylation in the SV lifecycle, the tyrosine pentapeptide repeats were truncated from the C-terminal of Syp in both a mCerulean tagged Syp and SypHy. The experiments showed that these potential tyrosine phosphorylation sites were not involved in the trafficking of Syp but key in the retrieval of VAMP from the plasma membrane during the SV lifecycle. I have indentified an SH3 interaction motif on the C-terminal of Syp that is critical in forming a complex of proteins that are responsible for the retrieval of VAMP during the SV lifecycle. Further experiments have shown that this key interaction is potentially phosphorylation dependent. My preliminary mass spectrometry analysis has provided a catalogue of proteins that can potentially interact with Syp, identifying proteins that may bind to either the Syp C-terminus SH3 interaction motif or to other regions in a phosphorylation dependent manner. This has provided a list of potential candidate proteins for the VAMP retrieval complex.

Regulation of soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNARE) mediated exocytosis is dependent upon four key proteins; the vesicular SNARE synaptobrevin, target SNAREs SNAP-25 and syntaxin and the Sec1/Munc18 (SM) protein munc18-1. Despite the munc18-1-syntaxin interaction being central to regulated vesicle exocytosis the spatial and temporal pattern of their molecular distribution and interaction in neuroendocrine and neuronal cells remains undefined. Using in vitro and molecular approaches this thesis shows that disruption of the munc18- 1-syntaxin-N-terminal interaction results in significant changes in syntaxin localisation, membrane-proximal vesicle dynamics and fusion efficiency within neuroendocrine cells. Using the super-resolution techniques Ground State Depletion-Individual molecule return (GSDIM) Microscopy and Photoactivation Localisation Microscopy (PALM) this thesis has demonstrated that the spatial distribution of single munc18-1 molecules is non-random and that few munc18-1 molecules are required for exocytosis to proceed in neuroendocrine cells. Furthermore, targeted disruption of the N-terminal interaction resulted only in a reorganisation of interaction with syntaxin with no change in the molecular spatial pattern of secretory vesicles, syntaxin or munc18-1. Single molecule imaging PALM (sptPALM) enabled the investigation of the complex spatio-temporal behaviours of single munc18-1 molecules in living neuroendocrine cells. Spatially resolved maps of single munc18-1 molecules demonstrated that munc18-1 exhibits a caged motion within areas of the plasma membrane and were found to move between molecular storage depots distinct from vesicle docking sites. To explore the precise spatial and temporal sequence of interactions between syntaxin and munc18-1 in living neurons, super-resolution imaging techniques PALM and sptPALM were employed. Two kinetically and spatially distinct populations of munc18-1 molecules co-exist within a living neuron and munc18-1 requires syntaxin to traffic efficiently in axons but not for its retention in nerve terminals. Moreover, Fluorescence Correlation Spectroscopy (FCS) revealed that the majority of munc18-1 molecules do not interact with syntaxin in nerve terminals and the diffusion rate of syntaxin is significantly slowed down upon neuronal depolarisation.

Excessive release of neurotransmitter is a characteristic of epileptogenic cells. A number of lines of evidence implicate defects in the synaptic vesicle cycle as a cause of this excessive release. Synaptic vesicles are retrieved by more than one route in central nerve terminals. During mild stimulation the dominant synaptic vesicle retrieval pathway is classical clathrin mediated endocytosis. During elevated neuronal activity retrieval of synaptic vesicle membrane by bulk endocytosis is the predominant retrieval method. As it is triggered by strong stimulation, bulk endocytosis may be of importance in retrieval during epilepsy, however little is currently known about this pathway. In order to investigate the role of bulk endocytosis, we sought to establish a cell culture model of epilepsy, to develop an assay to distinguish retrieval by bulk endocytosis, and to use these tools to look at the molecular players controlling this form of endocytosis. Characterisation of bulk endocytosis through the development of tailored assay systems has revealed that bulk endocytosis is a fast event that is triggered during strong stimulation. Bulk endocytosis provides the nerve terminal with an appropriate mechanism to meet the demands of synaptic vesicle retrieval during periods of intense synaptic vesicle exocytosis. Inhibition of a dephosphorylation specific dynamin I-syndapin I interaction by competitive peptides inhibits activity dependent bulk endocytosis, implicating this interaction in a role in this method of synaptic vesicle retrieval. Having characterised the strength of stimulation needed to activate bulk endocytosis, and the speed at which it occurs, we also investigated the effects of known anti-epileptogenic drugs on bulk endocytosis in our central nerve terminal model system.