Observation of erocytosis by near-field microscope

将GFP偶联突触素-I(SYT-I-GFP)基因稳定导入PC12细胞，可在PC12细胞内观察到SYT-I-GFP囊泡。电压钳PC12细胞的去极化使SYT-I-GFP的荧光强度降低。通过检测SYT-I-GFP荧光变化的特点，揭示了SYT-I-GFP荧光减弱与胞吐过程有关的机制。荧光强度从-20 mV开始呈电压依赖性，在+0 mV时达到最大值。高钾溶液(60 MM)或钙离子载体(离子霉素)作用于PC12细胞后，SYT-I-GFP荧光强度也降低。用全内反射荧光显微镜观察细胞膜下(距底物100 nm)Syt-I-GFP荧光强度的变化，发现细胞内钙离子浓度([Ca^&lt；2+&gt；]i)从100 nm开始荧光减弱，1μM时荧光强度达到最大。刺激活体PC12细胞使[Ca~(2+)]_i增加，可导致SYT-I-GFP荧光点的暗点丢失。用分泌颗粒的膜标志物FM4-对细胞进行染色，同时检测SYT-I-GFP和FM4-的荧光强度变化。FM4-和SYT-I-GFP荧光点共存，高K刺激时两者均减少。时延分析表明，SYT-I-GFP比FM4-快0.4秒。这些结果表明，PC12细胞内钙离子通过电压依赖性钙离子通道内流，导致SYT-I-GFP荧光减弱，并伴随着囊泡的胞吐。

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.