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

双光子显微镜研究的神经分泌细胞模型中钙依赖性胞吐作用的横截面图像分析

基本信息

批准号：
17590195
负责人：
NEMOTO Tomomi
金额：
$ 2.24万
依托单位：
National Institute for Physiological Sciences
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590195/
关键词：
cell physiology calcium two-photon microscopy secretory gland laser exocytosis biophysics SNARE 生理学外分泌腺

项目摘要

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

在神经分泌的胞吐过程中，融合孔的形成被认为需要SNARE核心复合物的形成。然而，仍然不清楚这种SNARE核心复合物如何诱导融合孔开放。通过SNARE蛋白与荧光蛋白的融合蛋白的表达，我们旨在研究细胞内游离Ca^<2+>浓度（[Ca^<2+>]_i）、融合孔开放和SNARE核心复合物形成之间的时空关系。首先，我们构建了一个实验系统来表达荧光蛋白标记的t-SNARE蛋白SNAP-25的融合蛋白。对PC 12细胞的组织化学和免疫荧光研究表明，这种外源荧光SNAP-25蛋白类似于内源SNAP-25定位在质膜上。我们接下来构建了双光子显微观察系统，使我们能够同时观察到由[Ca^<2+>]_i增加和荧光激发的omega样结构的出现。 ...更多信息在负载的笼状-Ca ^2+化合物NP-EGTA的UV闪光光解之后测试SNAP-25。我们已经在含有近红外荧光葡聚糖的细胞外溶液中灌注了NP-EGTA负载的组织以负性地确定细胞形状。在连续复合胞吐过程中，已发现SNAP-25侧向扩散到已经经历胞吐的分泌囊泡的膜。这一结果表明，在这种侧向扩散之后，在细胞深层内的囊泡膜处形成SNARE核心复合物对于募集许多分泌囊泡以有效地进行大量分泌可能是非常关键的。在肾上腺髓质嗜铬细胞中也证实了顺序复合胞吐和SNAP-25的类似结果。我们首次发现嗜铬细胞通过一种新的方式进行生理分泌，即“空泡顺序”胞吐--许多欧米茄样结构自我膨胀，形成“空泡”。我们已经表明，这种膨胀是由囊泡内容物的凝胶-溶胶转变，以加速嗜铬细胞的分泌。少