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 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.