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Vol. 16, Issue 6, 2636-2650, June 2005
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* Epithelial Pathobiology Research Unit, Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 30322;
Department of General Surgery, University of Muenster, 48149 Muenster, Germany
Submitted January 19, 2005;
Revised March 16, 2005;
Accepted March 21, 2005
Monitoring Editor: Sandra Schmid
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.
Abbreviations used: AJ, adherens junction; AJC, apical junctional complex; Arp2/3, actin-related proteins 2/3; CSB, cytoskeleton-stabilizing buffer; HBSS, HEPES-buffered Hank's balanced salt solution; HCM, high calcium medium; JAM, junctional adhesion molecule; LCM, low calcium medium; MNMM, mammalian nonmuscle myosin; N-WASP, neuronal Wiskott-Aldrich syndrome protein; RMLC, regulatory myosin light chain; TJ, tight junction; TX-100, Triton X-100.
Address correspondence to: Andrei I. Ivanov (aiivano{at}emory.edu).
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