Differential Roles for Actin Polymerization and a Myosin II Motor in Assembly of the Epithelial Apical Junctional Complex

Abstract

Differentiation and polarization of epithelial cells depends on the formation of the apical junctional complex (AJC), which is composed of the tight junction (TJ) and the adherens junction (AJ). In this study, we investigated mechanisms of actin reorganization that drive the establishment of AJC. Using a calcium switch model, we observed that formation of the AJC in T84 intestinal epithelial cells began with the assembly of adherens-like junctions followed by the formation of TJs. Early adherens-like junctions and TJs readily incorporated exogenous G-actin and were disassembled by latrunculin B, thus indicating dependence on continuous actin polymerization. Both adherens-like junctions and TJs were enriched in actin-related protein 3 and neuronal Wiskott-Aldrich syndrome protein (N-WASP), and their assembly was prevented by the N-WASP inhibitor wiskostatin. In contrast, the formation of TJs, but not adherens-like junctions, was accompanied by recruitment of myosin II and was blocked by inhibition of myosin II with blebbistatin. In addition, blebbistatin inhibited the ability of epithelial cells to establish a columnar phenotype with proper apico-basal polarity. These findings suggest that actin polymerization directly mediates recruitment and maintenance of AJ/TJ proteins at intercellular contacts, whereas myosin II regulates cell polarization and correct positioning of the AJC within the plasma membrane. INTRODUCTIONTwo major functions of epithelia in multicellular organisms are protection from the external environment and preservation of the unique biochemical composition within different body compartments (Schock and Perrimon, 2002). These functions require restriction of the passage of solutes and macromolecules (Madara, 1998) that depends on three types of specialized plasma membrane structures termed tight junction (TJ), adherens junction (AJ), and desmosomes (Tsukita et al., 2001;Matter and Balda, 2003). TJs and AJs are closely positioned at the apical-most aspect of the lateral plasma membrane and are functionally coupled in the regulation of paracellular permeability. Thus, the TJ and AJ are collectively referred to as the apical junctional complex (AJC). Both TJs and AJs represent multiprotein complexes consisting of integral membrane proteins and peripheral proteins associated with the cytosolic side of the plasma membrane (Yap et al., 1997;Tsukita et al., 2001;Pokutta and Weis, 2002;Matter and Balda, 2003). It is generally thought that integral membrane proteins of TJs and AJs interact with partners on the opposing cell plasma membrane in a trans manner providing mechanical forces for cell-cell adhesion and creating a physical barrier that limits diffusion of solutes and macromolecules. The transmembrane proteins of TJs include occludin, members of the claudin protein family and two immunoglobulin (Ig)-like proteins, junctional adhesion molecule (JAM)-A and coxsackie adenovirus receptor (Tsukita et al., 2001;Gonzalez-Mariscal et al., 2003;Matter and Balda, 2003). The major integral membrane component...

Abl tyrosine kinases regulate cell–cell adhesion through Rho GTPases

“…The Abl kinases regulate multiple cytoskeletal processes such as membrane ruffling, chemotaxis, and cell spreading (29). Dynamic regulation of the actin cytoskeleton is required for the formation, stability, and dissolution of adherens junctions (5,6,29,30). Therefore, we tested whether Abl/Arg kinase inhibition could alter the cytoskeletal architecture of confluent cells.…”

“…The extracellular domain of the cadherins cluster in a calcium-dependent manner and trigger association of the cadherin cytoplasmic domain with ␤-catenin, which in turn binds to ␣-catenin, a protein that links the cadherincatenin complex to the actin cytoskeleton (1). Disruption of the actin cytoskeleton, genetic inactivation of actin regulatory proteins, or disruption of the link between the cadherin-catenin complex and the actin cytoskeleton all result in a loss of adherens junctions (4)(5)(6). Adherens junction formation, stability, and dissolution are processes regulated by Rho family GTPases (7).…”

ZO-1- and ZO-2-Dependent Integration of Myosin-2 to Epithelial Zonula Adherens

“…When wild-type ZO1(wt)/2(wt) Eph4 cells were treated with latrunculin A or blebbistatin, myosin-2 and actin became dissociated from the ZA, with faint traces of E-cadherin persisting (Supplemental Figure 1S). On treatment with either latrunculin A or blebbistatin, myosin-2 signals were separated from the ZA that was further disrupted in a manner as evidenced by the disappearance of immunofluorescence signals for E-cadherin (Ivanov et al, 2005).…”

Rab13 Small G Protein and Junctional Rab13-binding Protein (JRAB) Orchestrate Actin Cytoskeletal Organization during Epithelial Junctional Development

“…Adhesion requires several components as follows: specific transmembrane adhesion molecules that mediate binding to an external surface; cytoskeletal filaments that attach to the cytoplasmic faces of the adhesion site; and a submembrane plaque that interconnects the two (3)(4)(5)(6). A dense band of actin and myosin encircles epithelial cells in the plane of cell-cell junctions; this band plays a crucial role in the assembly and disassembly of cell-cell associations (7)(8)(9). During development, the assembly of spot-like primordial adherens junctions occurs in parallel with the formation of thick F-actin bundles at the free border (arcs), followed by maturation of the primordia into belt-like adherens junctions (10 -12).…”