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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) in the lateral membranes not facing the glass-cover slip. Upon photolysis of a caged Ca2+ compound, TEP imaging with FM1-43 (a polar membrane tracer) detected massive exocytosis of vesicles with a time constant of about 1 s. TEPIQ (two-photon extracellular polar-tracer imaging-based quantification) analysis revealed that the diameter of vesicles was small (55 nm). Extensive exocytosis of small vesicles (SVs) was shown to be mediated by the transient opening of a fusion pore with a diameter less than about 1.6 nm, and to be followed by direct (‘kiss-and-run’) endocytosis and translocation of the endocytic vesicles (EVs) deep into the cytoplasm. These processes were unaffected by GTP-
-S. In contrast, constitutive endocytic vesicles exhibited a diameter of 90 nm, took up molecules with a diameter of > 12 nm, and their formation was blocked by GTP--S. Electron-microscopic investigation with photoconversion of diaminobenzidine using FM1-43 confirmed an abundance of EVs with a diameter of 54 nm in stimulated cells. They rapidly translocated into the cytosol, and fused with endosomal organelles. The number of SV exocytosis events vastly exceeded the number of SVs morphologically docked at the plasma membrane. Simultaneous capacitance and FM1-43 measurements indicated that TEP imaging detected most SV exocytosis, and the fusion pore was closed within 2 s. Thus, we have, for the first time, directly visualized massive exocytosis of small vesicles in a non-synaptic preparation, and have revealed their fusion-pore mediated exocytosis and endocytosis.
(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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