Epithelial DLD-1 Cells with Disrupted E-cadherin Gene Retain the Ability to Form Cell Junctions and Apico-basal Polarity

[摘要] References(34)Gene editing methods were applied to the study of E-cadherin function in epithelial cells. The E-cadherin gene in epithelial DLD-1 cells was ablated using TALEN. The resultant cells showed round fibroblast-like morphology and had almost no Ca2+-dependent cell aggregation activity. E-cadherin re-expression in the knockout cells restored epithelial cell morphology and strong Ca2+-dependent cell-cell adhesion activity, indicating that the knockout cells retained the ability to support cadherin function. The knockout cells showed partial localization of desmoplakin and ZO-1 at intercellular contact sites. The transfectants expressing mutant E-cadherin lacking the cytoplasmic domain showed clear localization of desmoplakin and ZO-1 at cell-cell contact sites, although the cells had only weak Ca2+-dependent cell adhesion activity. Electron microscopy revealed the formation of intercellular junctions and apico-basal polarity in these cells. A portion of these cells occasionally formed an epithelial-like structure after prolonged culture. When the cells were treated with blebbistatin, the localization was enhanced. However, the localization was incomplete and contained defects. Double-knockout MDCK cells for the E-cadherin and cadherin-6 genes showed similar results, suggesting that the above properties were general. The present results showed that an epithelial-like structure could be formed without E-cadherin, but that the construction of mature epithelia requires E-cadherin.

[学科分类] 分子生物学，细胞生物学和基因

[关键词] cell junction;CRISPR/Cas9;E-cadherin;epithelial cells;TALEN