Immunocytochemical study of gap junction formation

缝隙连接的形成涉及两个细胞之间的协调相互作用，并在细胞-细胞识别和特定的细胞黏附中具有重要意义。因此，在缝隙连接的形成过程中，可能存在一些细胞黏附分子的相互作用。在这个项目中，我们研究了在发育中的小鼠肝细胞中连接蛋白和最广泛、最丰富的细胞黏附分子--钙结合蛋白的动态变化。在成年肝中，几乎所有细胞都呈Cx32阳性免疫反应，免疫染色由沿细胞边界外侧的许多线状区域组成。然而，在出生时，这种染色只出现在少数肝细胞中。新生(2日龄或3日龄)肝细胞胞浆内可见强免疫反应阳性斑点。在出生后10天，免疫反应与成体大致相同。免疫电子显微镜显示，免疫金颗粒直接结合到新生肝细胞的贴壁连接和质膜下的小泡以及缝隙连接。用抗连接蛋白32和抗E-钙粘蛋白抗体进行双重免疫组化染色显示，E-钙粘蛋白存在于新生儿肝细胞中与连接蛋白32相同的粘连连接中。这些发现表明，钙粘附素可能介导细胞之间的早期黏附事件，这是连接蛋白组装成缝隙连接斑块的先决条件。

Gap junction formation involves coordinated interaction between two cells and has implications in cell-cell recognition and specific cell adnesion. Thus, there might be some interactions of cell adhesion molecules for the formation of the gap junctions. In this project we have examined the dynamics of connexins and most widespread and abundant cell adhesion molecules, cadnerins, in the developing mouse hepatocytes. In adult liver, almost all cells showed a positive immunoreaction of connexin32, and the immunostaining consisted of numerous thread-like regions along the lateral aspect of the cell borders. At birth, however, the staining was present in only a few hepatocytes. Neonatal (2-or 3-day-old) liver showed the intense immnunoreaction positive spots over the cytoplasmic compartments of the hepatocytes. At 10 days after birth the immunoreaction was approximately equal to that of the adults. Immunoelectron microscopy of connexin32 revealed that the immunogold particles bound directly to the adherens junction and the vesicles beneath the plasma membrane as well as the gap junction in neonatal hepatocytes. Double immnogold staining using anti-connexin32 and anti-E-cadherin antibodies revealed that E-cadherin is present in the same adherens junctions as connexin32 in the neonatal hepatocytes. These findings imply that cadherins may mediate an early adhesion event between cells that is a prerequisite for the assembly of connexins into gap junction plaques.