Inhibition of gap junction and adherens junction assembly by connexin and A-CAM antibodies.
Inhibition of gap junction and adherens junction assembly by connexin and A-CAM antibodies.
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DOI：
10.1083/jcb.119.1.179
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发表时间：
1992-10
期刊：
影响因子：
--
通讯作者：
Johnson RG
中科院分区：
文献类型：
--
作者：
Meyer RA;Laird DW;Revel JP;Johnson RG
我们通过改变细胞与抗体Fab片段的相互作用，研究了间隙连接蛋白和细胞粘附分子的胞外结构域在间隙连接和粘附连接形成中的作用。利用免疫印迹和免疫细胞化学，我们证明Novikoff细胞含有差距连接蛋白，连接蛋白43（Cx 43），和细胞粘附分子，A-CAM（N-cadherin）。将细胞在EDTA中解离，使其恢复，并在含有从多种抗体制备的Fab片段的培养基中再聚集60分钟。我们观察到没有细胞-细胞染料转移显微注射后4分钟，在90%的细胞对与抗体的第一或第二个胞外结构域的Cx43，第二个胞外结构域的连接蛋白32（Cx 32）或A-CAM的Fab片段处理。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物或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-细胞染料转移。我们通过冷冻断裂EM观察到在用阻断细胞-细胞染料转移的Fab片段处理的细胞之间没有间隙连接，并且通过薄切片EM观察到没有粘附连接。使用冷冻断裂EM，在对照样品中约50%的细胞上发现间隙连接。我们用重新聚集的Novikoff细胞证明：（a）连接蛋白胞外结构域的功能性相互作用是间隙连接通道形成所必需的;（B）A-CAM介导的细胞相互作用是间隙连接组装所必需的;（c）A-CAM或连接蛋白胞外结构域的抗体的FaB片段阻断了粘附连接的形成。
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.