Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.3.1.108 (TAT)
 2,389 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To investigate the in vivo interaction of syntaxin-mediated soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) assembly and insulin exocytosis in biphasic release, we examined the dynamics of insulin granule motion such as docking and fusion with the plasma membrane when the syntaxin SNARE motif (H3 domain) was transduced into living MIN6 beta cells. TAT-H3, produced by fusion of the protein transduction domain of human immunodeficiency virus-1 TAT to the syntaxin-H3 domain, was rapidly transduced into the subplasmalemmal region in living MIN6 cells. Immunoblotting analysis followed by immunoprecipitation on TAT-H3-treated MIN6 cells showed that TAT-H3 binds SNAP-25 and VAMP-2 in vivo. Transduction of MIN6 cells with TAT-H3 caused a decrease in both the first and second phase of insulin release. We therefore quantitatively analyzed approaching, docking, and fusing of green fluorescent protein-labeled single insulin granules in TAT-H3-transduced MIN6 cells by evanescent wave microscopy. Under high glucose stimulation, TAT-H3 treatment not only reduced the fusion events from previously docked granules for the first 120 s (first phase of release) but also strongly inhibited the docking and fusion from newly recruited insulin granules after this point (second phase of release). During the second phase of release we observed a marked reduction in the accumulation of newly docked insulin granules; subsequently, fusion events were significantly decreased. TAT-H3 treatment by itself, however, did not alter the number of previously docked granules without stimulation. We conclude that introduction of the H3 domain into MIN6 cells inhibits biphasic insulin release by two mechanisms. 1) In the first phase of insulin release, the H3 domain interferes with previously docked granules to be fused, and 2) in the second phase of insulin release reduced fusion events result from a marked decline of newly docked granules. Thus, syntaxin-mediated SNARE assembly modulates insulin exocytosis in biphasic insulin release in a distinct way.
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PMID:Transduction of MIN6 beta cells with TAT-syntaxin SNARE motif inhibits insulin exocytosis in biphasic insulin release in a distinct mechanism analyzed by evanescent wave microscopy. 1239 9

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.
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PMID:Site of docking and fusion of insulin secretory granules in live MIN6 beta cells analyzed by TAT-conjugated anti-syntaxin 1 antibody and total internal reflection fluorescence microscopy. 1467 8