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MBC in Press, published online ahead of print May 11, 2005
Mol. Biol. Cell 10.1091/mbc.E04-09-0816

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Submitted on September 20, 2004
Revised on April 25, 2005
Accepted on May 3, 2005

ELKS, a Protein Structurally Related to the Active Zone-associated Protein CAST, Is Expressed in Pancreatic {beta} Cells and Functions in Insulin Exocytosis: Interaction of ELKS with Exocytotic Machinery Analyzed by Total Internal Reflection Fluorescence Microscopy

Mica Ohara-Imaizumi,* Toshihisa Ohtsuka,{dagger} Satsuki Matsushima,{ddagger} Yoshihiro Akimoto,{sect} Chiyono Nishiwaki,* Yoko Nakamichi,* Toshiteru Kikuta,* Shintaro Nagai,* Hayato Kawakami,{sect} Takashi Watanabe,{ddagger} and Shinya Nagamatsu*

Departments of *Biochemistry, {ddagger}Clinical Pathology, and {sect}Anatomy, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan; {dagger}KAN Research Institute, Shimogyo-ku, Kyoto 600-8815, Japan

Monitoring Editor: Suzanne Pfeffer

The cytomatrix at the active zone (CAZ) has been implicated in defining the site of Ca2+-dependent exocytosis of neurotransmitters. Here, we demonstrate the expression and function of ELKS, a protein structurally related to the CAZ protein CAST, in insulin exocytosis. The results of confocal and immunoelectron microscopic analysis showed that ELKS is present in pancreatic {beta} cells and is localized close to insulin granules docked on the plasma membrane-facing blood vessels. Total internal reflection fluorescence microscopy (TIRFM) imaging in insulin-producing clonal cells revealed that the ELKS clusters are less dense and unevenly distributed than syntaxin 1 clusters, which are enriched in the plasma membrane. Most of the ELKS clusters were on the docking sites of insulin granules that were colocalized with syntaxin 1 clusters. TIRF images of single-granule motion showed that the fusion events of insulin granules mostly occurred on the ELKS cluster, where repeated fusion was sometimes observed. When the Bassoon-binding region of ELKS was introduced into the cells, the docking and fusion of insulin granules were markedly reduced. Moreover, attenuation of ELKS expression by siRNA reduced the glucose-evoked insulin release. These data suggest that the CAZ-related protein ELKS functions in insulin exocytosis from pancreatic {beta} cells.

Address correspondence to: Shinya Nagamatsu (shinya{at}kyorin-u.ac.jp)




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