Ultracytochemical study of gap junctions under low electron beam damage condition

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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.