Super-resolution Single Molecule Localization Microscopy of the Exocytotic Machinery Underlying Insulin Secretion
Poster Jun 12, 2016
Deirdre M. Kavanagh, Alison Dun, Rory R. Duncan, and Colin Rickman.
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. Combining SMLM with diffraction-limited microscopy we have examined the co-location of t-SNARE proteins and insulin vesicles in HIT-T15 cells. Using GFP tagged insulin, or immunostaining for insulin, we were able to observe the relative spatial distribution of vesicles to individual t-SNARE molecules. Applying a Bayesian approach of quantitative cluster analysis, we systematically compared t-SNARE membrane clustering following normal and elevated glucose exposure. We observed that t-SNARE proteins form clusters of low and high molecular density with a non-random spatial distribution on the plasma membrane. Furthermore, we do not observe t-SNAREs clustered directly underneath insulin vesicles, noting only partial overlap between the two. Overall, these results provide new insight into the molecular organization of t-SNARE proteins at the plasma membrane of β-cells.
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