Single molecule localization microscopy (SMLM) of the exocytotic machinery underlying insulin secretion.

Insulin secretion requires the fusion of insulin-containing vesicles with the plasma membrane through the process of exocytosis. Membrane fusion is driven by the action of SNARE proteins. In β-cells, various SNARE isoforms exist; these include: syntaxin1a, 3 and 4 and SNAP-23 and SNAP-25 (t-SNAREs) at the plasma membrane, together with VAMP2 on the vesicular membrane. Type 2 diabetes (T2DM) occurs when β-cells can no longer compensate for the prolonged high elevations of glucose, leading to insufficient insulin secretion. Single molecule localization microscopy (SMLM) utilizes photoswitchable fluorescent probes to control the density of fluorescent emitters. By imaging photoswitching events repeatedly over thousands of frames we can build up a precise map of fluorophore positions. Using SMLM this project aims to understand the plasma membrane organisation of tSNARE proteins underlying insulin secretion under normal physiological conditions and how this is altered in Type 2 related hyperglycemia.