Abstract
ABSTRACTMDCK 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.
Highlights
The establishment and regulation of cell adhesion are fundamental to the development and maintenance of tissue organization in multicellular organisms
The Madin-Darby canine kidney (MDCK) clone was transduced with the GFP–tagged IIA (GFP–IIA) construct under the control of the Tet-repressible element (TRE); the resultant clones were termed GFP–IIA/IIAKO
We conclude that expression of GFP–IIA is required for assembly of the junction complex
Summary
The establishment and regulation of cell adhesion are fundamental to the development and maintenance of tissue organization in multicellular organisms. Cell–cell adhesion is mediated primarily through adherens junctions (AJs) and desmosomes. Because of the importance of cell–cell adhesion in development and tissue function in adults, it is critical to understand the mechanisms by which cells regulate the assembly and disassembly of adhesive junctions. Cadherins comprise a family of transmembrane cell-surface glycoproteins that mediate Ca2+-dependent cell–cell adhesion (Takeichi, 2014). In epithelial cells with well-developed intercellular junctions, E-cadherin is concentrated in the AJs but appears to influence other intercellular junctions such as tight junctions (TJs) and desmosomes (Gumbiner et al, 1988; Watabe et al, 1994; Amagai et al, 1995). Dysfunction of E-cadherin has been implicated in the invasiveness and carcinogenesis of tumor cells and human tumors (Vleminckx et al, 1991; Schipper et al, 1991; Berx et al, 1995)
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