ZO-1 mRNA and protein expression during tight junction assembly in Caco-2 cells.
ZO-1 mRNA and protein expression during tight junction assembly in Caco-2 cells.
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DOI：
10.1083/jcb.109.3.1047
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发表时间：
1989-09
期刊：
影响因子：
--
通讯作者：
Mooseker MS
中科院分区：
文献类型：
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作者：
Anderson JM;Van Itallie CM;Peterson MD;Stevenson BR;Carew EA;Mooseker MS
我们以前确定和特点ZO-1作为一种外周膜蛋白，特别是与细胞质表面的紧密连接。在这里，我们描述了部分cDNA序列编码大鼠和人类ZO-1和它们的使用，研究在Caco-2人肠上皮细胞系的紧密连接的装配的识别。用单克隆抗体筛选，从大鼠GtII表达文库中分离到一个大鼠cDNA。多克隆抗体的cDNA编码的融合蛋白，这些抗体的几个属性支持这种cDNA编码ZO-1。ZO-1 mRNA在大鼠和Caco-2细胞中表达，主要转录物约为7.5 kb。为了破坏紧密连接并研究随后的组装过程，将Caco-2细胞在无Ca++/Mg++的旋转培养基中悬浮生长48小时，在此期间它们失去细胞-细胞接触，变圆，并通过免疫荧光显微镜显示ZO-1的扩散和斑点定位。在含Ca++/Mg++的培养基中以汇合密度重新铺板数小时内，附着的细胞在细胞-细胞接触处显示ZO-1的离散定位。在2天内，完全融合的单层形成，ZO-1定位在一个连续的垫圈样的方式包围所有的细胞。ZO-1 mRNA水平在旋转培养的细胞中最高，并且在培养2-3周后，在重新铺板时迅速下降并稳定在初始水平的约10%。ZO-1蛋白质水平在无接触细胞中最低，在同一时期上升了五到八倍。相反，蔗糖酶-异麦芽糖酶（一种顶端膜水解酶）的mRNA水平仅在汇合单层形成后增加。因此，在这种紧密连接的接触依赖性组装模型中，既有细胞-细胞接触后开始的快速组装，也有涉及ZO-1 mRNA和蛋白质水平表达变化的长期调节。
We previously identified and characterized ZO-1 as a peripheral membrane protein specifically associated with the cytoplasmic surface of tight junctions. Here we describe the identification of partial cDNA sequences encoding rat and human ZO-1 and their use to study the assembly of tight junctions in the Caco-2 human intestinal epithelial cell line. A rat cDNA was isolated from a lambda-gtll expression library by screening with mAbs. Polyclonal antibodies were raised to cDNA-encoded fusion protein; several properties of these antibodies support this cDNA as encoding ZO-1. Expression of ZO-1 mRNA occurs in the rat and Caco-2 cells with a major transcript of approximately 7.5 kb. To disrupt tight junctions and study the subsequent process of assembly, Caco-2 cells were grown in suspension for 48 h in Ca++/Mg++- free spinner medium during which time they lose cell-cell contacts, become round, and by immunofluorescence microscopy show diffuse and speckled localization of ZO-1. Within hours of replating at confluent density in Ca++/Mg++-containing media, attached cells show discrete localization of ZO-1 at cell-cell contacts. Within 2 d, fully confluent monolayers form, and ZO-1 localizes in a continuous gasket-like fashion circumscribing all cells. ZO-1 mRNA levels are highest in cells in spinner culture, and upon replating rapidly fall and plateau at approximately 10% of initial levels after 2-3 wk in culture. ZO-1 protein levels are lowest in contact-free cells and rise five- to eightfold over the same period. In contrast, mRNA levels for sucrase- isomaltase, an apical membrane hydrolase, increase only after a confluent monolayer forms. Thus, in this model of contact-dependent assembly of the tight junction, there is both a rapid assembly beginning upon cell-cell contact, as well as a long-term modulation involving changes in expression of ZO-1 mRNA and protein levels.