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Vol. 17, Issue 4, 1922-1932, April 2006

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Zonula Occludens-1 Function in the Assembly of Tight Junctions in Madin-Darby Canine Kidney Epithelial Cells

Center for Cell Signaling, Department of Microbiology, University of Virginia School of Medicine, Charlottesville, VA 22908-0577

Submitted July 19, 2005; Revised January 6, 2006; Accepted January 12, 2006
Monitoring Editor: Asma Nusrat

Zonula occludens (ZO)-1 was the first tight junction protein to be cloned and has been implicated as an important scaffold protein. It contains multiple domains that bind a diverse set of junction proteins. However, the molecular functions of ZO-1 and related proteins such as ZO-2 and ZO-3 have remained unclear. We now show that gene silencing of ZO-1 causes a delay of 3 h in tight junction formation in Madin-Darby canine kidney (MDCK) epithelial cells, but mature junctions seem functionally normal even in the continuing absence of ZO-1. Depletion of ZO-2, cingulin, or occludin, proteins that can interact with ZO-1, had no discernible effects on tight junctions. Rescue of junction assembly using murine ZO-1 mutants demonstrated that the ZO-1 C terminus is neither necessary nor sufficient for normal assembly. Moreover, mutation of the PDZ1 domain did not block rescue. However, point mutations in the Src homology 3 (SH3) domain almost completely prevented rescue. Surprisingly, the isolated SH3 domain of ZO-1 could also rescue junction assembly. These data reveal an unexpected function for the SH3 domain of ZO-1 in regulating tight junction assembly in epithelial cells and show that cingulin, occludin, or ZO-2 are not limiting for junction assembly in MDCK monolayers.

Abbreviations used: GUK, guanylate kinase-like; PDZ, PSD-95, Discs large, ZO-1; YFP, yellow fluorescent protein; ZO-1, zonula occludens-1.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Ian G. Macara (igm9c{at}virginia.edu).

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