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Vol. 16, Issue 10, 5040-5052, October 2005
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-induced Endocytosis of Tight Junction Proteins: Myosin II-dependent Vacuolarization of the Apical Plasma Membrane
* 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;
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, 142290 Pushchino, Russia;
Department of Pathology, The University of Chicago, Chicago, IL 60637; and
|| Welsh School of Pharmacy, University of Wales, Cardiff CF10 3XF, Wales, United Kingdom
Submitted March 8, 2005;
Revised June 28, 2005;
Accepted July 19, 2005
Monitoring Editor: Keith Mostov
Disruption of epithelial barrier by proinflammatory cytokines such as IFN-
represents a major pathophysiological consequence of intestinal inflammation. We have previously shown that IFN- increases paracellular permeability in model T84 epithelial cells by inducing endocytosis of tight junction (TJ) proteins occludin, JAM-A, and claudin-1. The present study was designed to dissect mechanisms of IFN--induced endocytosis of epithelial TJ proteins. IFN- treatment of T84 cells resulted in internalization of TJ proteins into large actin-coated vacuoles that originated from the apical plasma membrane and resembled the vacuolar apical compartment (VAC) previously observed in epithelial cells that lose cell polarity. The IFN- dependent formation of VACs required ATPase activity of a myosin II motor but was not dependent on rapid turnover of F-actin. In addition, activated myosin II was observed to colocalize with VACs after IFN- exposure. Pharmacological analyses revealed that formation of VACs and endocytosis of TJ proteins was mediated by Rho-associated kinase (ROCK) but not myosin light chain kinase (MLCK). Furthermore, IFN- treatment resulted in activation of Rho GTPase and induced expressional up-regulation of ROCK. These results, for the first time, suggest that IFN- induces endocytosis of epithelial TJ proteins via RhoA/ROCK-mediated, myosin II-dependent formation of VACs.
Abbreviations used: Arp 3, actin-related protein 3; IFN-, interferon-; JAM, junctional adhesion molecule; MLC, myosin light chain; MLCK, myosin light chain kinase; MNMM, mammalian nonmuscle myosin; MYPT, myosin phosphatase target subunit; PFA, paraformaldehyde; ROCK, Rho-associated kinase; TJ, tight junction; VAC, vacuolar apical compartment; VASP, vasodilator-stimulated phosphoprotein.
Address correspondence to: Asma Nusrat (anusrat{at}emory.edu).
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