Super-resolution spatial, temporal and functional characterisation of voltage-gated calcium channels involved in exocytosis

The process of information transfer between neurons or endocrine cells is one of the most important, intricate and temporally precise processes in the body. Exocytosis, which is central to the process of excitation-­‐secretion coupling, is triggered by calcium signalling through voltage-­‐gated calcium channels. Super-­‐resolution imaging offers the possibility to fully understand the spatial relationship between the SNARE proteins involved in exocytosis, vesicles and the associated voltage-­‐gated calcium channels. In this thesis the focus is on exploring the trigger for exocytosis, specifically the spatial and functional role that voltage-­‐gated calcium channels play in this process. Super-­‐ resolution imaging techniques have been applied to measure the interaction between Cav2.2 calcium channels and the syntaxin1a SNARE protein, where binding was found to affect the overall channel distribution. A novel method of caged dye conjugated ω-­‐ conotoxin GVIA binding was developed for live cell single molecule imaging of Cav2.2 calcium channels. An innovative approach to analyse channel functionality and the distribution of calcium events at the plasma membrane was developed to create a temporal-­‐spatial map of calcium activity across the cell. These developments, combined with newly developed techniques in optical patching and simultaneous calcium and vesicle imaging reveal the functional relationship of voltage-­‐gated calcium channel and exocytosis at unprecedented spatial and temporal scales

Activation of conventional protein kinase C (PKC) is critical in the generation of human neutrophil extracellular traps

BACKGROUND: Activation of NADPH oxidase is required for neutrophil extracellular trap (NET) formation. Protein kinase C (PKC) is an upstream mediator of NADPH oxidase activation and thus likely to have a role in NET formation. METHODS: Pharmacological inhibitors were used to block PKC activity in neutrophils harvested from healthy donor blood. RESULTS: Pan PKC inhibition with Ro-31-8220 (p<0.001), conventional PKC inhibition with Go 6976 (p<0.001) and specific PKCβ inhibition with LY333531 (p<0.01) blocked NET formation in response to PMA. Inhibition of novel and atypical PKC had no effect. LY333531 blocked NET induction by the diacylglycerol analogue OAG (conventional PKC activator) (p<0.001). CONCLUSIONS: Conventional PKCs have a prominent role in NET formation. Furthermore PKCβ is the major isoform implicated in NET formation