Kishimoto Takuya, Liu Ting-Ting, Hatakeyama Hiroyasu, Nemoto Tomomi, Takahashi Noriko, Kasai Haruo
Department of Cell Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki 444-8787, Japan.
J Physiol. 2005 Nov 1;568(Pt 3):905-15. doi: 10.1113/jphysiol.2005.094003. Epub 2005 Sep 8.
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.
我们使用双光子激发成像和细胞外极性示踪剂（TEP成像），在与玻璃盖玻片相邻的PC12细胞基部区域研究了PC12细胞的胞吐作用。TEPIQ（基于双光子细胞外极性示踪剂成像的定量分析）分析显示，大多数胞吐作用是由平均直径为220nm的大致密核心囊泡（LVs）介导的，并且即使通过释放笼锁Ca2+化合物使胞质Ca2+浓度大幅增加来诱导胞吐作用，LVs的胞吐作用发生得也很缓慢，平均延迟约为7秒。我们还发现，97%的胞吐LVs停留在质膜上，72%的融合孔保持开放构象超过30秒，76%引发了位于胞质溶胶更深位置的囊泡的顺序复合胞吐作用。通过用FM1-43（一种极性膜示踪剂）对二氨基联苯胺进行光转化的电子显微镜研究，证实了PC12细胞的顺序复合胞吐作用。我们的数据表明，刺激前LVs与质膜的对接不一定会加速融合反应，而胞吐LVs的对接及其稳定性促进了顺序复合胞吐作用，从而允许深层囊泡的动员。
