Portal de Periódicos da CAPES

Sequential compound exocytosis of large dense‐core vesicles in PC12 cells studied with TEPIQ (two‐photon extracellular polar‐tracer imaging‐based quantification) analysis

2005; Wiley; Volume: 568; Issue: 3 Linguagem: Inglês

10.1113/jphysiol.2005.094003

1469-7793

Takuya Kishimoto, Tingting Liu, Hiroyasu Hatakeyama, Tomomi Nemoto, Noriko Takahashi, Haruo Kasai,

Advanced Fluorescence Microscopy Techniques

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 approximately 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.