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J. Biol. Chem., Vol. 279, Issue 9, 8403-8408, February 27, 2004

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

Mica Ohara-Imaizumi, Chiyono Nishiwaki, Toshiteru Kikuta, Konosuke Kumakura, Yoko Nakamichi, and Shinya Nagamatsu¶

From the Department of Biochemistry (II), Kyorin University School of Medicine, Shinkawa 6-20-2, Mitaka, Tokyo 181-8611, and the Life Science Institute, Sophia University, Kioi-cho 7-1, Chiyoda, Tokyo 102-8554, Japan

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 anti-syntaxin 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 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--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.

Received for publication, August 13, 2003 , and in revised form, November 13, 2003.

^* This work was supported by Grants-in-aid for Scientific Research C14570130 (to M. O.-I.) and B15390108 (to S. N.), by Exploratory Research Grant 14657043 (to S. N.) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology, and by a grant from the Japan Private School Promotion Foundation (to S. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Movie 1.

^¶ To whom correspondence should be addressed: Dept. of Biochemistry (II), Kyorin University School of Medicine, Shinkawa 6-20-2, Mitaka, Tokyo 181-8611, Japan. Fax: 81-422-47-5538; E-mail: shinya{at}kyorin-u.ac.jp.

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