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Visulization of a single molecule in a living cell

活细胞中单个分子的可视化

基本信息

批准号：
08557003
负责人：
TERAKAWA Susumu
金额：
$ 6.02万
依托单位：
Hamamatsu University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

The purpose of this project is to visualize real-time images of molecules in living cells. To test this, I employed the evanescence microscopy and the confocal microscopy with a microlens-attached Nipkow disk scanner. By these techniques, catecholamine release from chromaffin vesicles and gating activity of K-cahnnels expressed in Xenopus oocytes were captured as dynamic 2-dimesnional images.Exocytotic release of catecholamine was visualized by first loading a fluorescent dye, quinacrine, into chromaffin vesicles, and then by observing the fluorescence of the dye with the confocal microscope equipped with an objective lens of a high numerical aperture. It was found that some vesicles release their contents only partially and recycle without a full release. This findings do not support the widely accepted hypothesis of quantal release of tranamitters.Messenger RNA for K-channel was modified at 351 position, so that K-channel expressed in Xenpus oocytes injected with the mRNA can be specifically labeled with tetramthyl rhodamine maleimide (TMRM). Under the voltage clamp condition, the fluorescence of TMRM on the oocyte membrane was examined with the evanescence microscope. Many fluorescent spots showed voltage dependent changes in brightness (50% in amplitude with 100mV depolarization) that reflects the gating process of the ion channel. Moreover, with the time of illumination these spots disappeared abruptly in a mode of quantal bleaching, as expected for a single fluorescent molecule. This is evidence for the first observation of single molecules on the plasma membrane in a livinf cell.

该项目的目的是可视化活细胞中分子的实时图像。为了测试这一点，我采用了渐逝显微镜和共焦显微镜与微透镜连接Nipkow磁盘扫描仪。通过将荧光染料奎纳克林（quinacrine）加载到嗜铬囊泡中，然后在配有高数值孔径物镜透镜的共聚焦显微镜下观察染料的荧光，获得了嗜铬囊泡释放的儿茶酚胺和爪蟾卵母细胞表达的K通道的门控活性的动态二维图像。有人发现，一些囊泡释放其内容物只有部分和循环没有完全释放。本研究对K通道的信使RNA进行了351位修饰，使其能被四甲基罗丹明马来酰亚胺（TMRM）特异性标记。在电压钳条件下，用隐失显微镜观察TMRM在卵母细胞膜上的荧光。许多荧光点显示出亮度的电压依赖性变化（100mV去极化时幅度为50%），这反映了离子通道的门控过程。此外，随着光照时间的推移，这些斑点以量子漂白的方式突然消失，正如对单个荧光分子所预期的那样。这是首次在活细胞质膜上观察到单分子的证据。