Specific substrates composed of collagen and fibronectin support the formation of epithelial cell sheets by MDCK cells lacking α-catenin or classical cadherins.

The effect of the extracellular matrix substrates on the formation of epithelial cell sheets was studied using MDCK cells in which the α-catenin gene was disrupted. Although the mutant cells did not form an epithelial cell sheet in conventional cell culture, the cells formed an epithelial cell sheet when they were cultured on or in a collagen gel; the same results were not observed when cells were cultured on collagen-coated cover glasses or culture dishes. Moreover, the cells cultured on the cell culture inserts coated with fibronectin, Matrigel, or vitronectin formed epithelial cell sheets, whereas the cells cultured on cover glasses coated with these proteins did not form the structure, implying that the physical and chemical features of the substrates exert a profound effect on the formation of epithelial cell sheets. MDCK cells lacking the expression of E- and K-cadherins displayed similar properties. When the mutant MDCK cells were cultured in the presence of blebbistatin, they formed epithelial cell sheets, suggesting that myosin II was involved in the formation of these sheets. These cell sheets showed intimate cell-cell adhesion, and electron microscopy confirmed the formation of cell junctions. We propose that specific ECM substrates organize the formation of basic epithelial cell sheets, whereas classical cadherins stabilize cell-cell contacts and promote the formation of structures.

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.

Desmocollin-2 alone forms functional desmosomal plaques, with the plaque formation requiring the juxtamembrane region and plakophilins.

The role of the juxtamembrane region of the desmocollin-2 cytoplasmic domain in desmosome formation was investigated by using gene knockout and reconstitution experiments. When a deletion construct of the desmocollin-2 juxtamembrane region was expressed in HaCaT cells, the mutant protein became localized linearly at the cell-cell boundary, suggesting the involvement of this region in desmosomal plaque formation. Then, desmocollin-2 and desmoglein-2 genes of epithelial DLD-1 cells were ablated by using the CRISPR/Cas9 system. The resultant knockout cells did not form desmosomes, but re-expression of desmocollin-2 in the cells formed desmosomal plaques in the absence of desmoglein-2 and the transfectants showed significant cell adhesion activity. Intriguingly, expression of desmocollin-2 lacking its juxtamembrane region did not form the plaques. The results of an immunoprecipitation and GST-fusion protein pull-down assay suggested the binding of plakophilin-2 and -3 to the region. Ablation of plakophilin-2 and -3 genes resulted in disruption of the plaque-like accumulation and linear localization of desmocollin-2 at intercellular contact sites. These results suggest that the juxtamembrane region of desmocollin-2 and plakophilins are involved in the desmosomal plaque formation, possibly through the interaction between this region and plakophilins.

Protocadherin-9 involvement in retinal development in Xenopus laevis.

Biological roles of most protocadherins (Pcdhs) are a largely unsolved problem. Therefore, we cloned cDNA for Xenopus laevis protocadherin-9 and characterized its properties to elucidate the role. The deduced amino acid sequence was highly homologous to those of mammalian protocadherin-9 s. X. laevis protocadherin-9 expressed from the cDNA in L cells showed basic properties similar to those of mammalian Pcdhs. Expression of X. laevis protocadherin-9 was first detected in stage-31 embryos and increased as the development proceeded. In the later stage embryos and the adults, the retina strongly expressed protocadherin-9, which was mainly localized at the plexiform layers. Injection of morpholino anti-sense oligonucleotide against protocadherin-9 into the fertilized eggs inhibited eye development; and eye growth and formation of the retinal laminar structure were hindered. Moreover, affected retina showed abnormal extension of neurites into the ganglion cell layer. Co-injection of protocadherin-9 mRNA with the morpholino anti-sense oligonucleotide rescued the embryos from the defects. These results suggest that X. laevis protocadherin-9 was involved in the development of retina structure possibly through survival of neurons, formation of the lamina structure and neurite localization.