Assembly of the tight junction: the role of diacylglycerol.
Assembly of the tight junction: the role of diacylglycerol.
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DOI：
10.1083/jcb.123.2.293
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发表时间：
1993-10
期刊：
影响因子：
--
通讯作者：
Anderson JM
中科院分区：
文献类型：
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作者：
Balda MS;Gonzalez-Mariscal L;Matter K;Cereijido M;Anderson JM
细胞外Ca 2+触发MDCK细胞中紧密连接（TJ）的组装和封闭。这些事件由G蛋白、磷脂酶C、蛋白激酶C（PKC）和钙调蛋白调节。在目前的工作中，我们观察到，1，2-二辛酰甘油（diC 8）促进TJ在低细胞外Ca 2+中的组装，如TJ相关蛋白ZO-1易位到质膜、在冷冻断裂复制品中观察到的连接原纤维的形成、细胞间隙对[3 H]甘露醇的渗透性降低以及肌动蛋白丝向细胞周边的重组所证明的，通过使用罗丹明-鬼笔环肽的荧光显微镜观察。相比之下，低Ca 2+中的diC 8不会诱导Ca依赖性粘附蛋白E-钙粘蛋白（uvomorulin）的重新分布。细胞外抗体E-钙粘蛋白块连接的形成通常诱导加入钙2+。diC 8抵消了这种抑制，表明PKC可能在由E-钙粘蛋白介导的细胞-细胞粘附激活的信号通路中。此外，我们还发现了一种新的130 kD的磷蛋白，它与ZO-1/ZO-2复合物共免疫沉淀。虽然TJ的组装和密封可能涉及PKC的激活，但ZO-1、ZO-2和130-kD蛋白的磷酸化水平在加入Ca 2+或PKC激动剂后没有改变。这三种蛋白质的复合物甚至在低细胞外Ca 2+中也存在，这表明Ca 2+或diC 8的加入触发了预先形成的TJ亚复合物的易位和组装。
Extracellular Ca2+ triggers assembly and sealing of tight junctions (TJs) in MDCK cells. These events are modulated by G-proteins, phospholipase C, protein kinase C (PKC), and calmodulin. In the present work we observed that 1,2-dioctanoylglycerol (diC8) promotes the assembly of TJ in low extracellular Ca2+, as evidenced by translocation of the TJ-associated protein ZO-1 to the plasma membrane, formation of junctional fibrils observed in freeze-fracture replicas, decreased permeability of the intercellular space to [3H]mannitol, and reorganization of actin filaments to the cell periphery, visualized by fluorescence microscopy using rhodamine-phalloidin. In contrast, diC8 in low Ca2+ did not induce redistribution of the Ca-dependent adhesion protein E-cadherin (uvomorulin). Extracellular antibodies to E-cadherin block junction formation normally induced by adding Ca2+. diC8 counteracted this inhibition, suggesting that PKC may be in the signaling pathway activated by E-cadherin-mediated cell-cell adhesion. In addition, we found a novel phosphoprotein of 130 kD which coimmunoprecipitated with the ZO-1/ZO-2 complex. Although the assembly and sealing of TJs may involve the activation of PKC, the level of phosphorylation of ZO-1, ZO-2, and the 130-kD protein did not change after adding Ca2+ or a PKC agonist. The complex of these three proteins was present even in low extracellular Ca2+, suggesting that the addition of Ca2+ or diC8 triggers the translocation and assembly of preformed TJ subcomplexes.