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.

Our study aims to investigate the role of Syntaxin-3 in glucose stimulated insulin secretion (GSIS) and how it regulates the recruitment to plasma membrane and/or exocytotic fusion of insulin granules. We examined endogenous Syn-3 function by down-regulating its expression using siRNA/lenti-shRNA, which impaired GSIS. Although Syn-3 depleted cells showed no change in the number and fusion of docked granules, there was a reduction in newcomer granules and their subsequent exocytotic fusion. We then examined the effects of overexpressing Syn-3-WT, which enhanced biphasic GSIS. Since open conformation (OF) Syn-1A was reported to enhance exocytosis by promoting SNARE complex formation, we constructed OF Syn-3. Exogenous OF Syn-3 had no effect on secretion as it is unable to be trafficked to insulin granules. Taken together, we conclude that Syn-3 facilitates mobilization of newcomer insulin granules to the plasma membrane, to contribute to both first and second phase of GSIS in pancreatic beta cells.

Our study aims to investigate the role of Syntaxin-3 in glucose stimulated insulin secretion (GSIS) and how it regulates the recruitment to plasma membrane and/or exocytotic fusion of insulin granules. We examined endogenous Syn-3 function by down-regulating its expression using siRNA/lenti-shRNA, which impaired GSIS. Although Syn-3 depleted cells showed no change in the number and fusion of docked granules, there was a reduction in newcomer granules and their subsequent exocytotic fusion. We then examined the effects of overexpressing Syn-3-WT, which enhanced biphasic GSIS. Since open conformation (OF) Syn-1A was reported to enhance exocytosis by promoting SNARE complex formation, we constructed OF Syn-3. Exogenous OF Syn-3 had no effect on secretion as it is unable to be trafficked to insulin granules. Taken together, we conclude that Syn-3 facilitates mobilization of newcomer insulin granules to the plasma membrane, to contribute to both first and second phase of GSIS in pancreatic beta cells.

Macrophages serve key roles in host defence by initiating inflammatory responses to infection and/or injury. They contribute to innate immunity by secreting a range of pro-inflammatory cytokines (e.g. TNF and IL-6) upon activation as well as by phagocytosing pathogens and dead cells, which is necessary for the resolution of inflammation and effective wound repair. Macrophages also contribute to adaptive immunity by functioning as antigen presenting cells.Colony stimulating factor 1 (CSF-1) is the major growth factor governing the differentiation, proliferation and survival of macrophages. Although not as well appreciated, CSF-1 also regulates some of the immune functions of macrophages, such as cytokine secretion and phagocytosis. However, the mechanisms by which CSF-1 governs the immune functions of macrophages are poorly understood. Cytokine secretion, phagocytosis and antigen presentation involve various vesicle trafficking and membrane fusion events, processes in which SNARE proteins play vital roles. Thus, the hypothesis tested in this thesis was that CSF-1 modulates the immune functions of macrophages by regulating the expression and/or activity of SNARE proteins that regulate endocytic and exocytic processes.In this study, the endosomal SNARE protein syntaxin 7 was identified, via microarray analysis, as a CSF-1 inducible gene in primary mouse macrophages. Syntaxin 7 has previously been detected in phagosomal membranes in macrophages. Furthermore, syntaxin 7 has recently been implicated in the secretion of cytokines (e.g. TNF) from macrophages by forming a novel complex with syntaxin 6, Vti1b and VAMP3.

The voltage-gated K+ channel Kv2.1 has been intimately linked with neuronal apoptosis. After ischemic, oxidative, or inflammatory insults, Kv2.1 mediates a pronounced, delayed enhancement of K+ efflux, generating an optimal intracellular environment for caspase and nuclease activity, key components of programmed cell death. This apoptosis-enabling mechanism is initiated via Zn2+-dependent dual phosphorylation of Kv2.1, increasing the interaction between the channel's intracellular C-terminus domain and the SNARE(soluble N-ethylmaleimide-sensitive factor activating protein receptor) protein syntaxin 1A. Subsequently, an upregulation of de novo channel insertion into the plasma membrane leads to the critical enhancement of K+ efflux in damaged neurons. Here, we investigated whether a strategy designed to interfere with the cell death-facilitating properties of Kv2.1, specifically its interaction with syntaxin 1A, could lead to neuroprotection following ischemic injury in vivo. The minimal syntaxin 1A-binding sequence of Kv2.1 C terminus (C1aB) was first identified via a far-Western peptide screen and used to create a protherapeutic product by conjugating C1aB to a cell-penetrating domain. The resulting peptide (TAT-C1aB) suppressed enhanced whole-cell K+ currents produced by a mutated form of Kv2.1 mimicking apoptosis in a mammalian expression system, and protected cortical neurons from slow excitotoxic injury in vitro, without influencing NMDA-induced intracellular calcium responses. Importantly, intraperitoneal administration of TAT-C1aB in mice following transient middle cerebral artery occlusion significantly reduced ischemic stroke damage and improved neurological outcome. These results provide strong evidence that targeting the proapoptotic function of Kv2.1 is an effective and highly promising neuroprotective strategy.

VPS33B is a Sec1/Munc18 protein required for the biogenesis of α-granules in megakaryocytes, which give rise to platelets. Mutations in VPS33B cause arthrogryposis, renal dysfunction and cholestasis (ARC) syndrome. Platelets from ARC patients completely lack α-granules, causing a bleeding disorder. VPS33B plays a role in vesicular fusion events through its interaction with the SNARE proteins, though no such interactions have been identified. Here, it is shown that VPS33B interacts with STX6, a member of the syntaxin subfamily of SNAREs. The introduction of ARC mutations into VPS33B completely abrogated binding to STX6. Confocal microscopy studies revealed STX6 co-localizes well with markers of the α-granule biogenesis pathway. This implies a role for the interaction of VPS33B with STX6 in α-granule biogenesis. Based on the known structure of STX6 and that predicted of VPS33B, suggests a novel and unique mode of binding between VPS33B and STX6 compared to other identified SM-STX pairs.

VPS33B is a Sec1/Munc18 protein required for the biogenesis of α-granules in megakaryocytes, which give rise to platelets. Mutations in VPS33B cause arthrogryposis, renal dysfunction and cholestasis (ARC) syndrome. Platelets from ARC patients completely lack α-granules, causing a bleeding disorder. VPS33B plays a role in vesicular fusion events through its interaction with the SNARE proteins, though no such interactions have been identified. Here, it is shown that VPS33B interacts with STX6, a member of the syntaxin subfamily of SNAREs. The introduction of ARC mutations into VPS33B completely abrogated binding to STX6. Confocal microscopy studies revealed STX6 co-localizes well with markers of the α-granule biogenesis pathway. This implies a role for the interaction of VPS33B with STX6 in α-granule biogenesis. Based on the known structure of STX6 and that predicted of VPS33B, suggests a novel and unique mode of binding between VPS33B and STX6 compared to other identified SM-STX pairs.

The KATP channel is a key regulator of the pancreatic β-cell, effectively linking metabolic status to electrical activity. Syntaxin-1A has been previously reported by our lab to both bind and inhibit the KATP channel via the nucleotide-binding folds (NBFs). The purpose of this thesis project was to elucidate the precise regions within the NBFs responsible for the Syn-1A- KATP interaction. In vitro binding assays revealed that Syn-1A associates with the Walker domains of both NBF1 and NBF2. Furthermore, site directed mutagenesis of the conserved lysine in Walker A of both NBFs abolishes Syn-1A affinity for this region. Electrophysiological recordings indicate that channel inhibition was mediated primarily through interaction with NBF1-Walker B and both Walkers of NBF2. Based on these results, we propose a model by which Syn-1A acts as an inhibitory clamp on the KATP channel, effectively buffering minor fluctuations in ATP/ADP concentration to prevent unnecessary channel activity.

The KATP channel is a key regulator of the pancreatic β-cell, effectively linking metabolic status to electrical activity. Syntaxin-1A has been previously reported by our lab to both bind and inhibit the KATP channel via the nucleotide-binding folds (NBFs). The purpose of this thesis project was to elucidate the precise regions within the NBFs responsible for the Syn-1A- KATP interaction. In vitro binding assays revealed that Syn-1A associates with the Walker domains of both NBF1 and NBF2. Furthermore, site directed mutagenesis of the conserved lysine in Walker A of both NBFs abolishes Syn-1A affinity for this region. Electrophysiological recordings indicate that channel inhibition was mediated primarily through interaction with NBF1-Walker B and both Walkers of NBF2. Based on these results, we propose a model by which Syn-1A acts as an inhibitory clamp on the KATP channel, effectively buffering minor fluctuations in ATP/ADP concentration to prevent unnecessary channel activity.

Indiana University-Purdue University Indianapolis (IUPUI) / Type 2 diabetes involves defects in glucose-stimulated insulin secretion (GSIS) from the pancreatic beta cells in combination with defects in peripheral (muscle and adipose) tissue glucose uptake. Both GSIS and glucose uptake are regulated by Syntaxin 4 (Syn4)-Munc18c complexes. Importantly, reports link obesity and Type 2 diabetes in humans with changes in protein levels of Munc18c and Syn4; yet the molecular mechanisms underlying this requirement remain unclear. The central hypothesis proposed is that Syn4-Munc18c complexes are modulated by post-translational modifications and novel interactions. Toward this, we found that Syn4-Munc18c complexes are regulated by tyrosine phosphorylation of Munc18c at Y219 in beta cells. Munc18c tyrosine phosphorylation disrupts Syn4-Munc18c complexes, which leads to an increase in Munc18c associating with the double C2 domain protein Doc2β. Disruption of Syn4-Munc18c upon tyrosine phosphorylation results in an increase in Syn4-SNARE complex formation and GSIS from beta cells. Similarly, tyrosine phosphorylation of Munc18c at Y219 and also Y521, disrupts its association with Syn4 in insulin-stimulated 3T3L1 adipocytes and skeletal muscle. In vitro kinase assays further suggested that the insulin receptor tyrosine kinase targeted Y521 of Munc18c. Further investigations using 3T3L1 adipocytes and skeletal muscle extracts indicate that Munc18c interacts with the insulin receptor tyrosine kinase in an insulin-dependent manner, resulting in phosphorylation of Munc18c, coordinate with the timing of its dissociation from Syn4. Finally, we found that stimulus-induced changes occurred also with Syn4, most notably in the islet beta cells. Syn4-mediated insulin release requires F-actin remodeling to mobilize insulin granules to the plasma membrane. Our studies reveal that Syn4 directly associates with F-actin in MIN6 beta cells, and that the disruption of this complex correlates with increases in glucose-stimulated insulin secretion. Future studies will focus upon the potential link between Syn4, F-actin remodeling with Munc18c, to further gain understanding of the requirements for Syn4-Munc18c complexes in insulin secretion. In sum, given the parallels of Munc18c tyrosine phosphorylation in regulating Syn4-Munc18c interaction and exocytosis in beta cells and peripheral tissues, manipulations of this complex may have therapeutic potential as a strategy to treat Type 2 diabetes.