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Vol. 16, Issue 10, 4519-4530, October 2005

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Myosin-Va Regulates Exocytosis through the Submicromolar Ca²+-dependent Binding of Syntaxin-1A

Michitoshi Watanabe ^*  , Kazushige Nomura ^*   ||, Akihiro Ohyama   ^¶ #, Ryoki Ishikawa ^@, Yoshiaki Komiya , Kohei Hosaka ^**, Emiko Yamauchi , Hisaaki Taniguchi , Nobuyuki Sasakawa , Konosuke Kumakura , Tatsuo Ushiki , Osamu Sato ^|||| ¶¶, Mitsuo Ikebe ^||||, and Michihiro Igarashi ^* 

^* Division of Molecular and Cellular Biology, Niigata University, Niigata, Niigata 951-8510, Japan; Division of Microscopic Anatomy and Bio-Imaging, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Niigata 951-8510, Japan; Division of Center for Trans-disciplinary Research, Niigata University, Niigata, Niigata 951-8510, Japan; Department of Molecular and Cellular Neurobiology, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan; ^|| Department of Orthopedic Surgery, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan; ^¶ Department of Anesthesiology and Reanimatology, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan; ^@ Department of Molecular and Cellular Pharmacology, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan; ^** Basic Sciences for Medicine, Gunma University School of Health Sciences, Maebashi, Gunma 371-8514, Japan; Division of Enzyme Physiology, Institute for Enzyme Research, University of Tokushima, Tokushima, Tokushima 770-8503, Japan; Life Science Institute, Sophia University, Chiyoda-ku, Tokyo 102-8554, Japan; and ^|||| Department of Physiology, University of Massachusetts Medical School, Worcester, MA 01655-0127

Submitted March 25, 2005; Revised June 30, 2005; Accepted July 11, 2005
Monitoring Editor: Anthony Bretscher

Myosin-Va is an actin-based processive motor that conveys intracellular cargoes. Synaptic vesicles are one of the most important cargoes for myosin-Va, but the role of mammalian myosin-Va in secretion is less clear than for its yeast homologue, Myo2p. In the current studies, we show that myosin-Va on synaptic vesicles interacts with syntaxin-1A, a t-SNARE involved in exocytosis, at or above 0.3 µM Ca²⁺. Interference with formation of the syntaxin-1A–myosin–Va complex reduces the exocytotic frequency in chromaffin cells. Surprisingly, the syntaxin-1A-binding site was not in the tail of myosin-Va but rather in the neck, a region that contains calmodulin-binding IQ-motifs. Furthermore, we found that syntaxin-1A binding by myosin-Va in the presence of Ca²⁺ depends on the release of calmodulin from the myosin-Va neck, allowing syntaxin-1A to occupy the vacant IQ-motif. Using an anti-myosin-Va neck antibody, which blocks this binding, we demonstrated that the step most important for the antibody's inhibitory activity is the late sustained phase, which is involved in supplying readily releasable vesicles. Our results demonstrate that the interaction between myosin-Va and syntaxin-1A is involved in exocytosis and suggest that the myosin-Va neck contributes not only to the large step size but also to the regulation of exocytosis by Ca²⁺.

Abbreviations used: AFM, atomic force microscopy; CaM, calmodulin; CaMKII, Ca²⁺/CaM-dependent protein kinase II; GST, glutathione S-transferase; RU, resonance units.

These authors contributed equally to this work.

^# Present address: Department of Ophthalmology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan.

^¶¶ Present address: Department of Pharmacology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan.

Address correspondence to: Michihiro Igarashi (tarokaja{at}med.niigata-u.ac.jp).

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