Griepp E B, Dolan W J, Robbins E S, Sabatini D D
J Cell Biol. 1983 Mar;96(3):693-702. doi: 10.1083/jcb.96.3.693.
Measurements of the transepithelial electrical resistance correlated with freeze-fracture observations have been used to study the process of tight junction formation under various experimental conditions in monolayers of the canine kidney epithelial cell line MDCK. Cells derived from previously confluent cultures and plated immediately after trypsin- EDTA dissociation develop a resistance that reaches its maximum value of several hundred ohms-cm(2) after approximately 24 h and falls to a steady-state value of 80-150 ohms- cm(2) by 48 h. The rise in resistance and the development of tight junctions can be completely and reversibly prevented by the addition of 10 mug/ml cycloheximide at the time of plating, but not when this inhibitor is added more than 10 h after planting. Thus tight junction formation consists of separable synthetic and assembly phases. These two phases can also be dissociated and the requirement for protein synthesis after plating eliminated if, following trypsinization, the cells are maintained in spinner culture for 24 h before plating. The requirement for protein synthesis is restored, however, if cells maintained in spinner culture are treated with trypsin before plating. Actinomycin D prevents development of resistance only in monolayers formed from cells derived from sparse rather than confluent cultures, but new mRNA synthesis is not required if cells obtained from sparse cultures are maintained for 24 h in spinner culture before plating. Once a steady-state resistance has been reached, its maintenance does not require either mRNA or protein synthesis; in fact, inhibition of protein synthesis causes a rise in the resistance over a 30-h period. Following treatments that disrupt the junctions in steady- state monolayers recovery of resistance also does not require protein synthesis. These observations suggest that proteins are involved in tight junction formation. Such proteins, which do not turn over rapidly under steady-state conditions, are destroyed by trypsinization and can be resynthesized in the absence of stable cell-cell or cell-substratum contact. Messenger RNA coding for proteins involved in tight junction formation is stable except when cells are sparsely plated, and can also be synthesized without intercellular contacts or cell-substratum attachment.
通过将跨上皮电阻测量结果与冷冻断裂观察结果相关联，已被用于研究犬肾上皮细胞系MDCK单层在各种实验条件下紧密连接形成的过程。从先前汇合培养物中获得的细胞，在胰蛋白酶-EDTA解离后立即接种，会产生电阻，该电阻在约24小时后达到数百欧姆·厘米²的最大值，并在48小时前降至80 - 150欧姆·厘米²的稳态值。在接种时添加10微克/毫升的环己酰亚胺可完全且可逆地阻止电阻升高和紧密连接的形成，但在接种后超过10小时添加该抑制剂则无效。因此，紧密连接的形成包括可分离的合成阶段和组装阶段。如果在胰蛋白酶消化后，细胞在接种前在旋转培养中维持24小时，这两个阶段也可以解离，并且接种后对蛋白质合成的需求可以消除。然而，如果在接种前用胰蛋白酶处理在旋转培养中维持的细胞，对蛋白质合成的需求会恢复。放线菌素D仅在由稀疏而非汇合培养物来源的细胞形成的单层中阻止电阻的发展，但如果从稀疏培养物中获得的细胞在接种前在旋转培养中维持24小时，则不需要新的mRNA合成。一旦达到稳态电阻，其维持既不需要mRNA也不需要蛋白质合成；事实上，在30小时内抑制蛋白质合成会导致电阻升高。在破坏稳态单层中连接的处理后，电阻的恢复也不需要蛋白质合成。这些观察结果表明蛋白质参与紧密连接的形成。这种在稳态条件下不会快速周转的蛋白质，会被胰蛋白酶消化破坏，并且可以在没有稳定的细胞-细胞或细胞-基质接触的情况下重新合成。编码参与紧密连接形成的蛋白质的信使RNA是稳定的，除非细胞稀疏接种，并且也可以在没有细胞间接触或细胞-基质附着的情况下合成。
