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，目前尚不清楚这种SNARE核心复合物如何通过与荧光结合的SNARE蛋白的表达来诱导融合孔开放。蛋白质，我们的目的是检查细胞内游离 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带有荧光蛋白标记的 t-SNARE 蛋白 SNAP-25 的融合蛋白，对 PC12 细胞的组织化学和免疫荧光研究表明，这种外源荧光 SNAP-25 蛋白类似地定位在细胞上。接下来，我们构建了双光子显微观察系统，使我们能够同时观察由质膜样内源性 SNAP-25 触发的 omega 样结构的出现。负载笼状 Ca^<2+> 化合物 NP-EGTA 的紫外闪光光解后，[Ca^<2+>]_i 增加并发出荧光……更多 t SNAP-25 我们已在细胞外溶液中灌注了负载 NP-EGTA 的组织。含有近红外荧光葡聚糖以确定阴性细胞形状的过程中，SNAP-25 已发现横向扩散到已经存在的分泌囊泡膜上。这一结果表明，在这种横向扩散之后，在细胞更深层的囊泡膜上形成 SNARE 核心复合物，对于招募许多分泌囊泡以有效地进行大量分泌来说可能非常关键。胞吐作用和SNAP-25也在肾上腺髓质嗜铬细胞中得到证实，我们首次发现嗜铬细胞通过一种新型的方式进行生理分泌。方式，“液泡顺序”胞吐作用-许多类似omega的结构本身膨胀并变成“液泡”，我们已经证明这种膨胀是由囊泡内容物的凝胶-溶胶转变引起的，以加速嗜铬细胞的分泌。