Spatial, temporal and functional molecular architecture of the munc18-syntaxin interaction

Abstract

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.