Abstract
To determine the site of insulin exocytosis in the pancreatic beta cell plasma membrane, we analyzed the interaction between the docking/fusion of green fluorescent protein-tagged insulin granules and syntaxin 1 labeled by TAT-conjugated Cy3-labeled antibody (Ab) using total internal reflection fluorescence microscopy (TIRFM). Monoclonal Ab against syntaxin 1 was labeled with Cy3 then conjugated with the protein transduction domain of HIV-1 TAT. TAT-conjugated Cy3-labeled anti-syntaxin 1 Ab was transduced rapidly into the subplasmalemmal region in live MIN6 beta cells, which enabled us to observe the spatial organization and distribution of endogenous syntaxin 1. TIRFM imaging revealed that syntaxin 1 is distributed in numerous separate clusters in the intact plasma membrane, where insulin secretory granules were docked preferentially to the sites of syntaxin 1 clusters, colocalizing with synaptosomal-associated protein of 25 kDa (SNAP-25) clusters. TIRFM imaging analysis of the motion of single insulin granules demonstrated that the fusion of insulin secretory granules stimulated by 50 mm KCl occurred exclusively at the sites of the syntaxin 1 clusters. Cholesterol depletion by methyl-beta-cyclodextrin treatment, in which the syntaxin 1 clusters were disintegrated, decreased the number of docked insulin granules, and, eventually the number of fusion events was significantly reduced. Our results indicate that 1) insulin exocytosis occurs at the site of syntaxin 1 clusters; 2) syntaxin 1 clusters are essential for the docking and fusion of insulin granules in MIN6 beta cells; and 3) the sites of syntaxin 1 clusters are distinct from flotillin-1 lipid rafts.
Highlights
Protein receptors (SNAREs)1 are expressed in pancreatic  cells and play an important role in the insulin exocytotic process [2, 5, 6]
Site of Insulin Exocytosis that syntaxin 1 is distributed in separate clusters in the intact plasma membrane where insulin granules are preferentially docked and fused, indicating that syntaxin 1 clusters are requisite for effective insulin exocytosis
T-SNAREs Are Distributed in Separate Clusters in the Plasma Membrane of MIN6  Cells—To analyze the localization of t-SNAREs and their association with insulin granules in the plasma membrane of MIN6  cells, green fluorescent protein (GFP)-tagged insulintransfected cells were fixed, immunostained with anti-syntaxin 1 and anti-SNAP-25 antibodies, and observed by both EPIF microscopy and total internal reflection microscopy (TIRFM) of the same cell
Summary
Protein receptors (SNAREs)1 are expressed in pancreatic  cells and play an important role in the insulin exocytotic process [2, 5, 6]. TIRF illuminates fluorophores close to the plasma membrane (within ϳ100 nm) [10], allowing us to observe with high resolution the single insulin granules approaching, docking, and fusing with the plasma membrane and the spatial localization of endogenous t-SNAREs such as syntaxin 1 and SNAP-25 in the plasma membrane of live cells.
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