Assembly of Epithelial Tight Junctions Is Negatively Regulated by Par6

AbstractEpithelial cells display apical-basal polarity, and the apical surface is segregated from the basolateral membranes by a barrier called the tight junction (TJ). TJs are constructed from transmembrane proteins that form cell-cell contacts—claudins, occludin, and junctional adhesion molecule (JAM)—plus peripheral proteins such as ZO-1 [1, 2]. The Par proteins (partitioning-defective) Par3 and Par6, plus atypical protein kinase C (aPKC) function in the formation or maintenance of TJs [3–5] and more generally in metazoan cell polarity establishment [6, 7]. Par6 contains a PDZ domain and a partial CRIB (Cdc42/Rac interactive binding) domain and binds the small GTPase Cdc42. Here, we show that Par6 inhibits TJ assembly in MDCK II epithelial cells after their disruption by Ca2+ depletion but does not inhibit adherens junction (AJ) formation. Transepithelial resistance and paracellular diffusion assays confirmed that assembly of functional TJs is delayed by Par6 overexpression. Strikingly, the isolated, N-terminal fragment of PKCζ, which binds Par6, also inhibits TJ assembly. Activated Cdc42 can disrupt TJs [3–5], but neither a dominant-negative Cdc42 mutant nor the CRIB domain of γPAK (p21-activated kinase), which inhibits Cdc42 function, observably inhibit TJ formation. These results suggest that Cdc42 and Par6 negatively regulate TJ assembly in mammalian epithelial cells