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signal transduction in cells analyzed by imaging and protein rotation

通过成像和蛋白质旋转分析细胞中的信号转导

基本信息

批准号：
07458165
负责人：
KAWATO Suguru
金额：
$ 4.74万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

We have examined Ca^<2+> signaling, LDL uptake and the activity of cytochrome P450scc in individual rat brain glial cells and bovine adrenocortical fasciculata cells in order to investigate the molecular mechanisms of steroid hormonc synthesis. With fluorescence video-enhanced microscopy imaging, we have demonstrated that the Ca^<2+> signaling occurred in individual Calcium Green-1 loaded cells upon stimulation with neurotransmitters (scrotonin, glutamate, histamine) to astroglial cells and adrenocorticotropin (ACTH) to adrenal cells at physiological concentration. We observed three patterns of Ca^<2+> signaling which were 1)Ca^<2+> oscillations, 2)step increase in Ca^<2+> concentration, and 3)Ca^<2+> oscillations superimposed on a step-like increase in Ca^<2+>. The results indicated that the Ca^<2+> signaling is the second messenger.Traffic of cndosomes which contain DiI labeled LDL in astroglial cells and adrcnal cells was observed in the time scale of 30 min with video-cnhanced microscopy Some localization of LDL in the cytoplasm was observed for astrocytes. For oligodendrocytes, incorporated LDL was distributed over the entire cytoplasmic region of both cell body and multiple branched cell processes.confocal laser scanning microscopy.Confocal imaging of the activity of cytochrome P450scc was performed in adrenocortical cells. Upon conversion of cholesterol-resorufin by P450scc to pregncnolonc and resorufin, the P450scc activity was observed by the strong fluorescence spots and weak fluorescence patches of resorufin.

我们检测了单个大鼠脑胶质细胞和牛肾上腺皮质束细胞的钙信号转导、低密度脂蛋白摄取和细胞色素P450scc活性，以探讨类固醇激素合成的分子机制。通过荧光视频增强显微成像，我们证明了在生理浓度的神经递质(肌萎缩素、谷氨酸、组胺)刺激星形胶质细胞和促肾上腺皮质激素(ACTH)刺激肾上腺细胞时，单个钙绿-1负载细胞发生了钙信号转导。我们观察到了Ca^&lt；2+&gt；信号的三种模式，即1)Ca^&lt；2+&gt；2)阶跃升高，以及3)Ca^&lt；2+&gt；振荡叠加Ca^&lt；2+&gt；结果表明，Ca~(2+)~(2+)是第二信使。在视频增强显微镜下，在30min的时间尺度上观察到星形胶质细胞和肾上腺细胞内含有DiI标记的低密度脂蛋白的胞体的运输，在星形胶质细胞的胞浆中也观察到了一些低密度脂蛋白的定位。对于少突胶质细胞，掺入的低密度脂蛋白分布在胞体和多个分支细胞突起的整个胞浆区。激光共聚焦扫描显微镜，肾上腺皮质细胞色素P450scc活性的共聚焦成像。当P450scc将胆固醇-间苯二酚转化为孕激素和间苯二酚时，通过间苯二酚的强荧光斑点和弱荧光斑块观察到P450scc的活性。