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，我采用了淡淡的显微镜和共聚焦显微镜，并使用了微杆的Nipkow磁盘扫描仪。通过这些技术，将在异爪蟾卵母细胞中表达的K-Cahnnels的Catecholamine释放出来，以动态的2段图像被捕获。结论性释放可视化Catecholamine的释放，通过首先加载荧光染料，然后将其呈硫酸含量，然后将其变成硫酸含量，然后将其视为硫酸含量。通过观察染料的荧光与配备具有高数值光圈的客观镜头的共聚焦显微镜。发现某些囊泡仅部分释放其内容物，并在没有完整释放的情况下回收。这一发现不支持thranamitters的量化量释放的广泛接受的假设。用于K通道的Messenger RNA在351位置进行了修改，因此可以用Xenpus卵母细胞表达的K渠道可以用mRNA注入的Xenpus卵母细胞（可以特异性地标记为四氨基胺甲酰胺甲酰胺型马来酰胺基胺）。）。在电压夹条件下，使用灭绝显微镜检查了卵母细胞膜上TMRM的荧光。许多荧光斑显示出反映离子通道的门控过程的亮度的电压依赖性变化（振幅为50％）。此外，随着照明的时间，这些斑点以一种量化模式突然消失，如单个荧光分子所预期的。这是在Livinf细胞中首次观察到质膜上单分子的证据。