Observation of erocytosis by near-field microscope

建立了稳定转染GFP偶联突触蛋白- 1 （Syt-I-GFP）基因的方法，以观察活体PC12中含有Syt-I-GFP的囊泡。电压箝位PC12细胞的去极化降低了Syt-I-GFP的荧光强度。通过观察Syt-I-GFP荧光变化的性质，揭示Syt-I-GFP荧光下降与胞吐过程的关系。荧光衰减与电压有关，从-20 mV开始，在+0 mV时达到最大衰减。当高钾溶液（60 mM）或钙离子载体（离子霉素）作用于PC12细胞时，Syt-I-GFP荧光强度也降低。荧光下降从细胞内钙离子浓度（[Ca^<2+>]_i）的100 nM处开始，在1μM处达到最大。用全内反射荧光显微镜（TIRFM）检测细胞膜下（距底物100nm处）Syt-I-GFP荧光强度的变化。刺激活PC12细胞增加[Ca^<2+>]_i，导致Syt-I-GFP荧光点暗点缺失。用分泌颗粒的膜标记物FM4-64染色细胞，同时检测Syt-I-GFP和FM4-64的荧光强度变化。在高钾刺激下，FM4-64和Syt-I-GFP荧光点共定位，两者均降低。时间推移分析表明，Syt-I-GFP比FM4-64快0.4秒。这些结果表明，Ca^<2+>通过电压依赖性Ca^<2+>通道内流导致Syt-I-GFP荧光降低，这一过程随后是PC12细胞囊泡的胞外分泌。

Stable transfection of GFP conjugated synaptotagmin-I (Syt-I-GFP) gene was established to visualize Syt-I-GFP containing vesicles in the living PC12. Depolarizations of the voltage clamped PC12 cells decreased the fluorescence intensity of Syt-I-GFP.The properties of the changes in Syt-I-GFP fluorescence was examined to reveal the mechanism of Syt-I-GFP fluorescence decrease in relation to exocytosis process. The fluorescence decrease was voltage dependent started from -20 mV and reached a maximum decrease at +0 mV.The intensity of Syt-I-GFP fluorescence also decreased when a high potassium solution (60 mM) or calcium ionophore (ionomycin) was applied to the PC12 cells. The fluorescence decrease started from 100 nM of intracellular calcium ion concentration ([Ca^<2+>]_i) and reached a maximum level at 1μM.Syt-I-GFP fluorescence intensity changes underneath the cell membrane (100 nm from the substrate) was examined with a total internal reflection fluorescence microscope (TIRFM). Stimulation of live PC12 cells which increases [Ca^<2+>]_i led to dark punctuate loss of Syt-I-GFP fluorescence spots. Staining the cells with FM4-64 a membrane marker of secretory granules, the fluorescence intensity changes from both Syt-I-GFP amd FM4-64 were examined simultaneously. FM4-64 and Syt-I-GFP fluorescence spots colocalized and both were decreased when high K stimulation was applied. The time lapse analysis showed that Syt-I-GFP decreased 0.4 sec faster than the FM4-64. These results suggest that Ca^<2+> influx through voltage dependent Ca^<2+> channels cause the Syt-I-GFP fluorescence decrease and this process is followed by the exocytosis of vesicles in PC12 cells.