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Vol. 16, Issue 7, 3289-3300, July 2005

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

Mica Ohara-Imaizumi ^*, Toshihisa Ohtsuka  , Satsuki Matsushima , Yoshihiro Akimoto ^||, Chiyono Nishiwaki ^*, Yoko Nakamichi ^*, Toshiteru Kikuta ^*, Shintaro Nagai ^*, Hayato Kawakami ^||, Takashi Watanabe , and Shinya Nagamatsu ^*

^* Department of Biochemistry, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan; Department of Clinical Pathology, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan; ^|| Department of Anatomy, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan; and KAN Research Institute, Shimogyo-ku, Kyoto 600-8815, Japan

Submitted September 20, 2004; Revised April 25, 2005; Accepted May 3, 2005
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 cells and is localized close to insulin granules docked on the plasma membrane-facing blood vessels. Total internal reflection fluorescence microscopy 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. Total internal reflection fluorescence 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 small interfering RNA reduced the glucose-evoked insulin release. These data suggest that the CAZ-related protein ELKS functions in insulin exocytosis from pancreatic cells.

Abbreviations used: BSA, bovine serum albumin; CAZ, cytomatrix at the active zone; CCD, charge-coupled device; EPIF, epifluorescence; GFP, green fluorescent protein; KRB, Krebs-Ringer buffer; mAb, monoclonal antibody; pAb, polyclonal antibody; PBS, phosphate-buffered saline; PSF, point spread function; PTD, protein transduction domain; SNAP-25, synaptosomal associated protein of 25 kDa; TIRFM, total internal reflection fluorescence microscopy; t-SNARE, target-soluble N-ethylmaleimide–sensitive factor attachment protein receptor.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Present address: Department of Clinical and Molecular Pathology, Toyama Medical and Pharmaceutical University, Sugitani, Toyama 930-0152, Japan.

Address correspondence to: Shinya Nagamatsu (shinya{at}kyorinu.ac.jp).

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