Meyer R A, Laird D W, Revel J P, Johnson R G
Department of Genetics and Cell Biology, University of Minnesota, St. Paul 55108.
J Cell Biol. 1992 Oct;119(1):179-89. doi: 10.1083/jcb.119.1.179.
We examined the roles of the extracellular domains of a gap junction protein and a cell adhesion molecule in gap junction and adherens junction formation by altering cell interactions with antibody Fab fragments. Using immunoblotting and immunocytochemistry we demonstrated that Novikoff cells contained the gap junction protein, connexin43 (Cx43), and the cell adhesion molecule, A-CAM (N-cadherin). Cells were dissociated in EDTA, allowed to recover, and reaggregated for 60 min in media containing Fab fragments prepared from a number of antibodies. We observed no cell-cell dye transfer 4 min after microinjection in 90% of the cell pairs treated with Fab fragments of antibodies for the first or second extracellular domain of Cx43, the second extracellular domain of connexin32 (Cx32) or A-CAM. Cell-cell dye transfer was detected within 30 s in cell pairs treated with control Fab fragments (pre-immune serum, antibodies to the rat major histocompatibility complex or the amino or carboxyl termii of Cx43). We observed no gap junctions by freeze-fracture EM and no adherens junctions by thin section EM between cells treated with the Fab fragments that blocked cell-cell dye transfer. Gap junctions were found on approximately 50% of the cells in control samples using freeze-fracture EM. We demonstrated with reaggregated Novikoff cells that: (a) functional interactions of the extracellular domains of the connexins were necessary for the formation of gap junction channels; (b) cell interactions mediated by A-CAM were required for gap junction assembly; and (c) Fab fragments of antibodies for A-CAM or connexin extracellular domains blocked adherens junction formation.
我们通过改变细胞与抗体Fab片段的相互作用，研究了缝隙连接蛋白和细胞粘附分子的细胞外结构域在缝隙连接和黏着连接形成中的作用。利用免疫印迹和免疫细胞化学技术，我们证明诺维科夫细胞含有缝隙连接蛋白连接蛋白43（Cx43）和细胞粘附分子A-CAM（N-钙黏蛋白）。细胞在乙二胺四乙酸（EDTA）中解离，恢复后，在含有从多种抗体制备的Fab片段的培养基中重新聚集60分钟。在用针对Cx43的第一或第二细胞外结构域、连接蛋白32（Cx32）的第二细胞外结构域或A-CAM的抗体的Fab片段处理的细胞对中，90%的细胞对在显微注射4分钟后未观察到细胞间染料转移。在用对照Fab片段（免疫前血清、针对大鼠主要组织相容性复合体或Cx43的氨基或羧基末端的抗体）处理的细胞对中，在30秒内检测到细胞间染料转移。在用阻断细胞间染料转移的Fab片段处理的细胞之间，通过冷冻断裂电子显微镜未观察到缝隙连接，通过超薄切片电子显微镜未观察到黏着连接。使用冷冻断裂电子显微镜在对照样品中约50%的细胞上发现了缝隙连接。我们用重新聚集的诺维科夫细胞证明：（a）连接蛋白的细胞外结构域的功能相互作用是缝隙连接通道形成所必需的；（b）缝隙连接组装需要由A-CAM介导的细胞相互作用；（c）针对A-CAM或连接蛋白细胞外结构域的抗体的Fab片段阻断了黏着连接的形成。
