Site of Docking and Fusion of Insulin Secretory Granules in Live MIN6 β Cells Analyzed by TAT-conjugated Anti-syntaxin 1 Antibody and Total Internal Reflection Fluorescence Microscopy

Abstract

To determine the site of insulin exocytosis in the pancreatic ␤ 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 antisyntaxin 1 Ab was transduced rapidly into the subplasmalemmal region in live MIN6 ␤ 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 synaptosomalassociated 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-␤-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 ␤ cells; and 3) the sites of syntaxin 1 clusters are distinct from flotillin-1 lipid rafts.Insulin is stored in large dense core vesicles in pancreatic ␤ cells and is released by exocytosis when glucose levels rise (1). We (2) and others (3, 4) have demonstrated previously that soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) 1 are expressed in pancreatic ␤ cells and play an important role in the insulin exocytotic process (2, 5, 6). However, it is not known how t-SNAREs are structurally organized and distributed in the pancreatic ␤ cell plasma membrane and how they spatially interact with the insulin granule during docking and fusion. A recent study described the distribution and spatial organization of t-SNAREs using the procedure of plasma membrane sheets derived from PC12 cells (7, 8), but technical difficulties have prevented such a study in pancreatic ␤ cells.Recently, we developed an approach using a green fluorescent protein (GFP)-tagged insulin granule system combined with total internal reflection microscopy (TIRFM) (9). Using this system, we were able to observe the motion of single insulin granules such as in docking and fusion in the exocytotic process during physiological stimulation. TIRF illuminates fluorophores close to the plasma membrane (within ϳ100 nm) (10), allowing us to observe with high resolution not only the single insulin granules approaching, docking, and fusing with the plasma membrane but also...

Myosin-Va Regulates Exocytosis through the Submicromolar Ca2+-dependent Binding of Syntaxin-1A

“…This possibility is strongly supported by a very recent report that movements of insulin-containing dense-core secretory vesicles along the cortical actin network depend on myosin-Va and are essential for regulated exocytosis (Varadi et al, 2005). In addition, syntaxin-1 is localized close to the site of exocytosis (Stanley et al, 2003;Ohara-Imaizumi et al, 2004), where it could participate in the process of exocytosis by interacting with myosin-Va.…”

“…This possibility is strongly supported by a very recent report that movements of insulin-containing dense-core secretory vesicles along the cortical actin network depend on myosin-Va and are essential for regulated exocytosis (Varadi et al, 2005). In addition, syntaxin-1 is localized close to the site of exocytosis (Stanley et al, 2003;Ohara-Imaizumi et al, 2004), where it could participate in the process of exocytosis by interacting with myosin-Va.A previous report indicated that hippocampal slices of dilute lethal mice, which lack myosin-Va, do not show a The anti-myosin-Va neck antibody does not alter myosin-Va-based sliding velocity. Velocity measurements were carried out as described in Figure 2D in the presence of 1:200 anti-myosin-Va neck antibody (bottom; n ϭ 80) or normal IgG (middle; n ϭ 66) at pCa ϭ 6.…”

Enlargeosome, an Exocytic Vesicle Resistant to Nonionic Detergents, Undergoes Endocytosis via a Nonacidic Route

“…Interestingly, this treatment had been reported to inhibit all types of regulated exocytosis investigated so far (Lang et al, 2001;Ohara-Imaizumi et al, 2004;Salaun et al, 2004). In PC12-27 cells, depletion was amply effective, as revealed by the blockade of the cholera toxin endocytosis.…”

Transcellular Neuroligin-2 Interactions Enhance Insulin Secretion and Are Integral to Pancreatic β Cell Function

“…The functional equivalent of the presynaptic active sites of secretion in ␤ cells are microdomains (alternatively referred to as "excitosomes," "Ca 2ϩ microdomains," or "secretory microdomains"), where the submembrane secretory apparatus assembles beneath the plasma membrane in close proximity to voltage-gated calcium channels (43)(44)(45)(46). Because these microdomains seem to be especially important for early phase insulin secretion, we next asked whether NL-2 affected early phase insulin secretion more than late phase (47)(48)(49).…”