Ultracytochemical study of gap junctions under low electron beam damage condition

本项目在低温电子显微镜下，利用超细胞化学技术研究了在细胞间通讯中起重要作用的间隙连接的超微结构。我们得到的数据如下：我们开发了一种超细胞化学方法来检测钙调素（钙结合蛋白之一）结合位点的细胞内定位，结果表明，大鼠肝脏和心脏缝隙连接的细胞质表面有钙调素结合位点，这表明缝隙连接的功能可能受钙离子的调节。通过低温电镜观察，阴性染色分离的间隙连接在连接子分布模式和连接子平均直径方面与常规电镜下观察到的图像有很大的不同。在冷冻电镜下，Ca^<++>- atp酶在离体心脏间隙连接上的定位在连接子颗粒周围，具有非常精确和均匀的分布模式，而在转换电镜下，则观察到粗糙和聚集的反应产物。这些结果表明，在低温电子显微镜下观察孤立的间隙结对于检测近原生结构是非常有用的。

In this project, the ultrastructure of gap junctions which play an important role in cell-to cell communication was investigated by using ultracytochemical thchnique under a cryo-electron microscope. The data we obtained are as follows:1. By an ultracytochemical method that we developed to detect the intracellular localization of calmodulin (one of the calcium-binding proteins) binding sites, it was demonstrated that rat liver and heart gap junctions have calmodulin binding sites on their cytoplasmic surface, suggesting that the function of gap junctions may be regulated by calcium ions.2. The cryo-electron microscopic observation of negatively-stained isolated gap junctions revealed a quite different image as compared with that observed under a conventional electron microscope in terms of the connexon distribution pattern and the average diameter of connexons.3. The localization of Ca^<++>-ATPase on isolated heart gap junctions under a cryoelectron microscope was found around connexon particles with a very precise and uniform distribution pattern, whereas coarse and aggregated reaction products were observed under a convertional electron microscope. These results indicate that isolated gap junction observation under a cryo-electron microscope can be considered very useful in detecting near-to-native structure.