Occludin is a functional component of the tight junction.
Occludin is a functional component of the tight junction.
复制标题
DOI：
--
复制
发表时间：
1996-09
影响因子：
4
通讯作者：
Karin M. McCarthy;I. Skare;Michael C. Stankewich;M. Furuse;S. Tsukita;R. Rogers;R. Lynch;E. Schneeb
中科院分区：
文献类型：
--
作者：
Karin M. McCarthy;I. Skare;Michael C. Stankewich;M. Furuse;S. Tsukita;R. Rogers;R. Lynch;E. Schneeb
闭合蛋白在哺乳动物紧密连接活性中的作用通过用免疫学可检测的鸡闭合蛋白“标记"闭合蛋白池来检查。这通过首先用Lac阻遏物基因转染MDCK细胞来实现。然后将HygR克隆用插入Lac操纵子构建体中的鸡闭合蛋白cDNA转染。将所得HygR/NeoR克隆接种在多孔插入物上并使其形成紧密连接。一旦达到稳态跨上皮电阻，加入异丙基-β-D-硫代半乳糖苷以诱导鸡闭合蛋白表达。用Oc-2单克隆抗体免疫标记的单层共聚焦激光扫描显微镜显示，鸡occludin精确定位于预先形成的紧密连接。当稀疏的文化保持在低钙+培养基中，鸡occludin和犬ZO-1共定位于点状网站在细胞质中，这表明他们的协会在相同的泡状结构。在低钙培养基中，这两种蛋白质也共同定位于偶尔的细胞对之间的接触部位，在质膜上形成一个突出的酒吧。在将异丙基-β-D-硫代半乳糖苷加入到先前已达到稳态跨上皮电阻的单层中的两小时内，通过蛋白质印迹法可检测到鸡闭合蛋白;这与某些细胞的细胞膜处鸡闭合蛋白的局部免疫荧光染色相吻合。在加入诱导剂后5小时，跨上皮电阻开始逐渐升高，高于对照稳态值，31小时后达到新的稳态值，高于基线30-40%。去除异丙基-β-D-硫代半乳糖苷后，鸡occludin表达缓慢下降，直到72小时后在蛋白质印迹中不再检测到;在此期间，跨上皮电阻也恢复到基线值。虽然蛋白质印迹的光密度分析表明，鸡occludin的存在下，E-钙粘蛋白或ZO-1的表达没有检测到的影响，不能排除的可能性，ZO-1可能是一个限制因素，鸡occludin在细胞表面的表达。为了测试鸡occludin的表达是否影响紧密连接组装的过程，在加入Ca 2+以刺激紧密连接组装之前，用异丙基-β-D-硫代半乳糖苷处理低Ca 2+培养基中的单层24或48小时。鸡occludin没有改变跨上皮电阻发展的速率，然而，稳态值比未补充诱导剂的对照单层高30-40%。通过冷冻断裂分析，平行紧密连接链的数量从对照中的三条模式转变为表达鸡闭合蛋白的细胞中的四条，并且紧密连接网络的平均宽度从175 +/-11 nm增加到248 +/-16 nm。接种诱导表达鸡闭合蛋白的汇合单层后两天，甘露醇通量相对于对照单层降低至不同程度。随着与诱导剂的继续孵育，甘露醇通量在第11天增加至50%，并且TER上升至高于对照的45%。在去除异丙基-β-D-硫代半乳糖苷后，这两种变化都是可逆的。这些数据是一致的概念，occludin有助于电屏障功能的紧密连接，并可能形成水孔紧密连接链。
Occludin's role in mammalian tight junction activity was examined by 'labeling' the occludin pool with immunologically detectable chick occludin. This was accomplished by first transfecting MDCK cell with the Lac repressor gene. HygR clones were then transfected with chick occludin cDNA inserted into a Lac operator construct. The resulting HygR/NeoR clones were plated on porous inserts and allowed to form tight junctions. Once steady state transepithelial electrical resistance was achieved, isopropyl- beta-D-thiogalactoside was added to induce chick occludin expression. Confocal laser scanning microscopy of monolayers immunolabeled with Oc-2 monoclonal antibody revealed that chick occludin localized precisely to the preformed tight junctions. When sparse cultures were maintained in low Ca2+ medium, chick occludin and canine ZO-1 co-localized to punctate sites in the cytoplasm suggesting their association within the same vesicular structures. In low calcium medium both proteins also co-localized to contact sites between occasional cell pairs, where a prominent bar was formed at the plasma membrane. Chick occludin was detectable by western blot within two hours of adding isopropyl- beta-D-thiogalactoside to monolayers that had previously achieved steady state transepithelial electrical resistance; this coincided with focal immunofluorescence staining for chick occludin at the cell membrane of some cells. A gradual rise in transepithelial electrical resistance, above control steady state values, began five hours after addition of the inducing agent reaching new steady state values, which were 30-40% above baseline, 31 hours later. Upon removal of isopropyl- beta-D-thiogalactoside chick occludin expression declined slowly until it was no longer detected in western blots 72 hours later; transepithelial electrical resistance also returned to baseline values during this time. While densitometric analysis of western blots indicated that the presence of chick occludin had no detectable effect on E-cadherin or ZO-1 expression, the possibility cannot be excluded that ZO-1 might be a limiting factor in the expression of chick occludin at the cell surface. To test whether expression of chick occludin affected the process of tight junction assembly, monolayers in low Ca2+ medium were treated with isopropyl- beta-D-thiogalactoside for 24 or 48 hours, before Ca2+ was added to stimulate tight junction assembly. Chick occludin did not alter the rate at which transepithelial electrical resistance developed, however, steady state values were 30-40% above control monolayers not supplemented with the inducing agent. By freeze fracture analysis, the number of parallel tight junction strands shifted from a mode of three in controls to four strands in cells expressing chick occludin and the mean width of the tight junction network increased from 175 +/- 11 nm to 248 +/- 16 nm. Two days after plating confluent monolayers that were induced to express chick occludin, mannitol flux was reduced to a variable degree relative to control monolayers. With continued incubation with the inducing agent, mannitol flux increased on day 11 to 50%, and TER rose to 45% above controls. Both of these changes were reversible upon removal of isopropyl- beta-D-thiogalactoside. These data are consistent with the notion that occludin contributes to the electrical barrier function of the tight junction and possibly to the formation of aqueous pores within tight junction strands.