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

在神经分泌的胞吐作用期间，融合孔的形成被认为需要形成SNARE CORE复合物。然而，尚不清楚这种核心核心复合物如何引起融合孔的打开。通过与荧光蛋白结合的融合蛋白的融合蛋白的表达，我们的目的是检查细胞内游离Ca^<2+浓度（[Ca^<2+>] _ I），融合孔口的开放和SNARE CORE COLCE络合物形成之间的空间时间关系。首先，我们构建了一个实验系统，以表达用荧光蛋白标记的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蛋白类似地位于质膜上，例如内源性SNAP-25。接下来，我们构建了两光子显微镜观察系统，使我们能够同时可视化由[Ca^<2+>] _ i触发的欧米茄状结构的出现，并增加了cageD CageD-Ca^<2+化合物NP-EGTA的UV闪光照射后，更多的T Snap-25。我们已经灌注了含有近红外荧光脱氧液的细胞外溶液中的NP-EGTA载荷组织，以负面确定细胞形状。在顺序复合胞吐作用期间，SNAP-25发现横向扩散到已经胞吐作用下的秘密蔬菜的膜。该结果表明，在这种横向扩散之后，细胞较深层的囊泡膜上的核心复合物形成对于募集许多秘密蔬菜以进行有效进行大规模分泌可能至关重要。在肾上腺髓质铬蛋白细胞中也证实了顺序复合胞吐作用和SNAP-25的相似结果。我们第一次发现，铬蛋白细胞使用一种新颖的方式进行物理分泌物，即“液泡顺序”胞外生物胞外变体 - 欧米茄状结构扩大了自身，并变成了“液泡”。我们已经表明，这种膨胀是由囊泡含量的凝胶 - 溶剂过渡引起的，以加速铬蛋白细胞的分泌物。较少的