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Published online May 14, 2007
doi:10.1083/jcb.200608132
The Journal of Cell Biology, Vol. 177, No. 4, 695-705
The Rockefeller University Press, 0021-9525 $30.00
© 2007 Ohara-Imaizumi et al.
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Article

Imaging analysis reveals mechanistic differences between first- and second-phase insulin exocytosis



Mica Ohara-Imaizumi1, Tomonori Fujiwara2, Yoko Nakamichi1, Tadashi Okamura5, Yoshihiro Akimoto3, Junko Kawai1,6, Satsuki Matsushima4, Hayato Kawakami3, Takashi Watanabe4, Kimio Akagawa2, and Shinya Nagamatsu1

1 Department of Biochemistry, 2 Department of Cell Physiology, 3 Department of Anatomy, and 4 Department of Clinical Pathology, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan
5 Division of Animal Models, Department of Infectious Diseases, Research Institute, International Medical Center of Japan, Tokyo 162-8655, Japan
6 Department of Medicine, Metabolism and Endocrinology, Juntendo University School of Medicine, Tokyo, 113-8421, Japan

Correspondence to Shinya Nagamatsu: shinya{at}kyorin-u.ac.jp

The mechanism of glucose-induced biphasic insulin release is unknown. We used total internal reflection fluorescence (TIRF) imaging analysis to reveal the process of first- and second-phase insulin exocytosis in pancreatic ß cells. This analysis showed that previously docked insulin granules fused at the site of syntaxin (Synt)1A clusters during the first phase; however, the newcomers fused during the second phase external to the Synt1A clusters. To reveal the function of Synt1A in phasic insulin exocytosis, we generated Synt1A-knockout (Synt1A–/–) mice. Synt1A–/– ß cells showed fewer previously docked granules with no fusion during the first phase; second-phase fusion from newcomers was preserved. Rescue experiments restoring Synt1A expression demonstrated restoration of granule docking status and fusion events. Inhibition of other syntaxins, Synt3 and Synt4, did not affect second-phase insulin exocytosis. We conclude that the first phase is Synt1A dependent but the second phase is not. This indicates that the two phases of insulin exocytosis differ spatially and mechanistically.

Abbreviations used in this paper: [Ca2+]i, intracellular Ca2+ concentration; CCD, charge-coupled device; KRB, Krebs-Ringer buffer; PTD, protein transduction domain; SNAP-25, synaptosome-associated protein of 25 kD; TIRF, total internal reflection fluorescence; TIRFM, TIRF microscopy; VAMP, vesicle-associated membrane protein; WT, wild-type.


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