Epithelial DLD-1 Cells with Disrupted E-cadherin Gene Retain the Ability to Form Cell Junctions and Apico-basal Polarity.
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 Ca(2+)-dependent cell aggregation activity. E-cadherin re-expression in the knockout cells restored epithelial cell morphology and strong Ca(2+)-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 Ca(2+)-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.
Department of Bioscience, School of Science and Technology, Kwansei Gakuin University.
Miwako
M
Fujiwara
Kihito
K
Fujimura
Shuichi
S
Obata
Ryo
R
Yanagibashi
Tetsushi
T
Sakuma
Takashi
T
Yamamoto
Shintaro T
ST
Suzuki
D000818:Animals; D001483:Base Sequence; D015820:Cadherins / Q000172:deficiency / Q000235:genetics*; D002118:Calcium / Q000378:metabolism*; D002448:Cell Adhesion; D002449:Cell Aggregation; D045744:Cell Line, Tumor; D016764:Cell Polarity; D003851:Deoxyribonucleases / Q000378:metabolism; D051180:Desmoplakins / Q000235:genetics / Q000378:metabolism; D004285:Dogs; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D006801:Humans; D007365:Intercellular Junctions / Q000378:metabolism* / Q000648:ultrastructure; D061985:Madin Darby Canine Kidney Cells; D008969:Molecular Sequence Data; D015398:Signal Transduction; D019108:Tight Junctions / Q000378:metabolism* / Q000648:ultrastructure; D062826:Zonula Occludens-1 Protein / Q000235:genetics / Q000378:metabolism
D015820:Cadherins; C495294:DSP protein, human; D051180:Desmoplakins; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; D003851:Deoxyribonucleases; D002118:Calcium; C087310:K cadherin