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

摘要

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.