Cross-sectional image analysis of calcium dependent exocytosis in neurosecretion cell models studied by two-photon microscopy

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During exocytosis in the neurosecretion, the formation of fusion pore is thought to require the formation of SNARE core complex. It remains unclear, however, how such SNARE core complexes induce fusion pore opening. By the expression of fusion proteins of SNARE proteins combined with fluorescent proteins, we have aimed to examine spatiotemporal relationship between intracellular free Ca^<2+> concentration ([Ca^<2+>]_i), fusion pore opening and SNARE core complex formation. At first, we have constructed an experimental system to express fusion protein of a t-SNARE protein SNAP-25 tagged with a fluorescent protein. Histchemical and immunofluorescent studies on PC12 cells have shown that such exogenous fluorescent SNAP-25 proteins were localized similarly on the plasma membrane like endogenous SNAP-25. We next constructed two-photon microscopic observation system that enables us to visualize simultaneously appearance of omega-like structure triggered by [Ca^<2+>]_i increase and fluorescen … More t SNAP-25 after UV flash photolysis of loaded caged-Ca^<2+> compounds NP-EGTA. We have perfused the NP-EGTA loaded tissues in an extracellular solution containing near-infrared-fluorescent dextrans to determine cell shapes negatively. During sequential compound exocytosis, SNAP-25 has found to diffuse laterally to the membrane of secretory vesicles which had already underwent exocytosis. This result suggests that SNARE core complex formation at the vesicle membrane within the deeper layers in the cell after such the lateral diffusion might be quite critical for the recruitment of many secretory vesicles to carry out effectively massive secretions. Similar results on sequential compound exocytosis and SNAP-25 have been confirmed also in adrenal medullary chromaffin cells. For the first time, we have found that chromaffin cells carry out physiological secretions by using a novel manner, "vacuolar sequential" exocytosis-many omega-like structures expanded themselves and became "vacuoles". We have shown that such expansions were caused by a gel-sol transition of vesicle contents to accelerate secretions in chromaffin cells. Less