Observation of erocytosis by near-field microscope

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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.