Cryoelectron Microscopy and Cytochemistry of Gap Junctions

在这个项目中，我们展示了缝隙连接的以下细胞化学方面。酶细胞化学：酶活性定位于缝隙连接膜的细胞质一侧。缝隙连接电荷的细胞化学显示：缝隙连接的细胞质一侧显示出高密度的阴离子部位分布。蛋白激酶C(PKC)在缝隙连接上的免疫细胞化学：佛波酯，12-O-十四烷酰佛波醇-13-乙酸酯(TPA)，是最有效的肿瘤促进剂之一，已知会抑制培养细胞之间的代谢合作。此外，已知TPA在体内和体外都能激活蛋白激酶C。在这个项目中，我们已经证明了TPA处理导致胞浆PKC移位到缝隙连接。我们的结果表明，PKC在TPA诱导的代谢协同抑制中起着至关重要的作用。缝隙连接蛋白(Cx32)的免疫细胞化学：我们研究了Cx32在新生大鼠肝细胞内的分布。免疫电子显微镜显示，小鼠抗连接蛋白32的单抗可直接与6日龄大鼠肝细胞的贴壁连接区和缝隙连接区结合。我们的发现表明，黏附连接介导了肝细胞之间的早期黏附事件，这是缝隙连接连接蛋白32组装的先决条件。

In this project, we have demonstrated the following cytochemical aspects of gap junctions.1. Enzyme cytochemistry : Ca^<++>-ATPase activity is localized on the cytoplasmic side of gap junctional membranes.2. Cytochemical demonstration of charge on gap junctions : Cytoplasmic side of gap junctions shows the high density distribution of anionic sites.3. Immunocytochemistry of Protein Kinase C (PKC) on gap junctions : The phorbol ester, 12-O-Tetradecanoyl Phorbol-13-Acetate (TPA), one of the most potent tumor-promoting agents, is known to inhibit metabolic cooperation between cultured cells. In addition, TPA is also known to activate protein kinase C both in vivo and in vitro. In this project we have demonstrated that TPA-treatment causes a translocation of cytosolic PKC to the gap junction. Our results imply that PKC have a crucial role in TPA-induced inhibition of metabolic cooperation.4. Immunocytochemistry of gap junction protein (connexin 32) : We have studied the intracellular distribution of connexin 32 in newly born rat hepatocytes. Immunoelectron microscopy demonstrated that the mouse monoclonal antibody against connexin 32 binds directly to the adherent junction region as well as the gap junction in 6 day-old rat hepatocytes. Our findings imply that the adherent junction mediates an early adhesion event between hepatocytes that is a prerequisite for the assembly of connexin 32 of gap junctions.