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1 Department of Cell Physiology, National Institute for Physiological Sciences, and Graduate University of Advanced Studies (SOKENDAI), Myodaiji, Okazaki 444-8787, Japan
2
Center for Disease Biology and Integrative Medicine, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan
3
Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
4
Genome Research Center, National Yang-Ming University, Taipei, Taiwan
We investigated exocytosis of PC12 cells using two-photon excitation imaging and extracellular polar tracers (TEP imaging) at the basal region of PC12 cells adjacent to the glass cover slip. TEPIQ (two-photon extracellular polar-tracer imaging-based quantification) analysis revealed that most exocytosis was mediated by large dense-core vesicles (LVs) with a mean diameter of 220 nm, and that exocytosis of LVs occurred slowly with a mean latency of 
7 s even though exocytosis was induced with large increases in cytosolic Ca2+ concentration by uncaging of a caged-Ca2+ compound. We also found that 97% of exocytic LVs remained poised at the plasma membrane, 72% maintained their fusion pores in an open conformation for more than 30 s, and 76% triggered sequential compound exocytosis of vesicles that were located deeper in the cytosol. Sequential compound exocytosis by PC12 cells was confirmed by electron microscopic investigation with photoconversion of diaminobenzidine by FM1-43 (a polar membrane tracer). Our data suggest that pre-stimulus docking of LVs to the plasma membrane does not necessarily hasten the fusion reaction, while docking and resulting stability of exocytic LVs facilitates sequential compound exocytosis, and thereby allowing mobilization of deep vesicles.
(Received 1 July 2005;
accepted after revision 1 September 2005;
first published online 8 September 2005)
Corresponding author H. Kasai: Department of Cell Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki 444-8787, Japan. Email: hkasai{at}nips.ac.jp
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