ZO-1 function in the assembly of tight junctions in MDCK epithelial cells

Zonula occludens-1 (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 hours in tight junction formation in MDCK epithelial cells, but mature junctions appear 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 discernable 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 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. INTRODUCTION Epithelia form the archetypal polarized tissue of the Metazoa (Knust and Bossinger, 2002; Gibson and Perrimon, 2003; Nelson, 2003). Typically, they create a barrier between the extracellular environment and the interior of the organism. Epithelia organize into layered sheets of cells with apical-basal polarity and strong cell-cell adhesions. The tight junction is the most apical adhesion between epithelial cells and is a complex structure that functions both as a barrier to the paracellular diffusion of ions and molecules, and as a fence to separate the apical plasma membrane from the basolateral domain in polarized epithelia (D'Atri and Citi, 2002; Gonzalez-Mariscal et al., 2003; Matter and Balda, 2003). Tight junctions are fine-tuned to perform specific