Nonmuscle myosin IIA is involved in recruitment of apical junction components through activation of α-catenin

Abstract

MDCK dog kidney epithelial cells express two isoforms of nonmuscle myosin heavy chain II, IIA and IIB. Using the CRISPR/Cas9 system, we established cells in which the IIA gene was ablated. These cells were then transfected with a vector that expresses GFP–IIA chimeric molecule under the control of a tetracycline-responsible element. In the absence of Dox (doxycyclin), when GFP–IIA is expressed (GFP–IIA+), the cells exhibit epithelial cell morphology, but in the presence of Dox, when expression of GFP–IIA is repressed (GFP–IIA−), the cells lose epithelial morphology and strong cell–cell adhesion. Consistent with these observations, GFP–IIA− cells failed to assemble junction components such as E-cadherin, desmoplakin, and occludin at cell–cell contact sites. Therefore, IIA is required for assembly of junction complexes. MDCK cells with an ablation of the α-catenin gene also exhibited the same phenotype. However, when in GFP–IIA− cells expressed α-catenin lacking the inhibitory region or E-cadherin/α-catenin chimeras, the cells acquired the ability to establish the junction complex. These experiments reveal that IIA acts as an activator of α-catenin in junction assembly.

The epithelial-mesenchymal transition induced by transcription factor LEF-1 is independent of β-catenin

“…Therefore, only βγ-DKO cells show morphological changes. Consistent with these observations, α-catenin KO MDCK cells showed disrupted cell-cell adhesion but the amounts of E-cadherins was not altered [22] .…”

Adherens junction regulates cryptic lamellipodia formation for epithelial cell migration

“…Isolation of cells lines using CRISPR/Cas9 plasmid-mediated gene KO For CRISPR/Cas9-mediated KO of genes, we used the pCGsapI vector developed by Takayuki Sakurai (Shinshu University, Nagano, Japan; Ozawa, 2018). The vector contains the hCas9 gene under the control of the CAG promoter and a unique cloning site, SapI, for insertion of the guide RNA under the control of the U6 promoter.…”

Myosin II isoforms play distinct roles in adherens junction biogenesis

“…Upon specific isoform expression silencing, they further proposed that NMIIA may favor the accumulation of E-cadherin in the AJ belt while NMIIB may stabilize the associated perijunctional actin ring, reinforce junctions and prevent them from disruptive forces (Smutny et al, 2010). Ozawa reported using CRISPR-Cas9 gene invalidation that NMIIA was required to assemble junctional complexes (Ozawa, 2018). Svitkina and collaborators reported an association of NMIIA with contractile actin bundle running parallel to linear AJ in endothelial cells, but failed to precisely localize NMIIB (Efimova and Svitkina, 2018).…”