Tissue barriers that restrict passage of liquids, ions, and larger solutes are essential for the development of multicellular organisms. In simple organisms this allows distinct cell types to interface with the external environment. In more complex species, the diversity of cell types capable of forming barriers increases dramatically. Although the plasma membranes of these barrier-forming cells prevent flux of most hydrophilic solutes, the paracellular, or shunt, pathway between cells must also be sealed. This function is accomplished in vertebrates by the zonula occludens, or tight junction. The tight junction barrier is not absolute but is selectively permeable and is able to discriminate between solutes on the basis of size and charge. Many tight junction components have been identified over the past 20 years, and recent progress has provided new insights into the proteins and interactions that regulate structure and function. This review presents these data in a historical context and proposes an integrated model in which dynamic regulation of tight junction protein interactions determines barrier function.

The perijunctional actomyosin ring contributes to myosin light chain kinase (MLCK)-dependent tight junction regulation. However, the specific protein interactions involved in this process are unknown. To test the hypothesis that molecular remodeling contributes to barrier regulation, tight junction protein dynamic behavior was assessed by fluorescence recovery after photobleaching (FRAP). MLCK inhibition increased barrier function and stabilized ZO-1 at the tight junction but did not affect claudin-1, occludin, or actin exchange in vitro. Pharmacologic MLCK inhibition also blocked in vivo ZO-1 exchange in wild-type, but not long MLCK ^−/− , mice. Conversely, ZO-1 exchange was accelerated in transgenic mice expressing constitutively active MLCK. In vitro, ZO-1 lacking the actin binding region (ABR) was not stabilized by MLCK inhibition, either in the presence or absence of endogenous ZO-1. Moreover, the free ABR interfered with full-length ZO-1 exchange and reduced basal barrier function. The free ABR also prevented increases in barrier function following MLCK inhibition in a manner that required endogenous ZO-1 expression. In silico modeling of the FRAP data suggests that tight junction-associated ZO-1 exists in three pools, two of which exchange with cytosolic ZO-1. Transport of the ABR-anchored exchangeable pool is regulated by MLCK. These data demonstrate a critical role for the ZO-1 ABR in barrier function and suggest that MLCK-dependent ZO-1 exchange is essential to this mechanism of barrier regulation.

Background & Aims Epithelial tight junctions are compromised in gastrointestinal disease. Processes that contribute to the resulting barrier loss include endocytic occludin removal from the tight junction and reduced occludin expression. Nevertheless, the relatively-normal basal phenotype of occludin knockout (KO) mice has been taken as evidence that occludin does not contribute to gastrointestinal barrier function. We asked whether stress could unmask occludin functions within intestinal epithelia. Methods Wildtype (WT), universal and intestinal epithelial-specific occludin KO, and villin-EGFP-occludin transgenic mice as well as WT and occludin knockdown (KD) Caco-2_BBe cell monolayers were challenged with DSS, TNBS, staurosporine, 5-FU, or TNF. Occludin and caspase-3 expression were assessed in patient biopsies. Results Intestinal epithelial occludin loss limited severity of DSS- and TNBS-induced colitis due to epithelial resistance to apoptosis; activation of both intrinsic and extrinsic apoptotic pathways was blocked in occludin KO epithelia. Promoter analysis revealed that occludin enhances CASP3 transcription and, conversely, that occludin downregulation reduces caspase-3 expression. Analysis of biopsies from Crohn’s disease and ulcerative colitis patients and normal controls demonstrated that disease-associated occludin downregulation was accompanied by and correlated with reduced caspase-3 expression. In vitro, cytokine-induced occludin downregulation resulted in reduced caspase-3 expression and resistance to intrinsic and extrinsic pathway apoptosis, demonstrating an overall protective effect of inflammation-induced occludin loss. Conclusions The tight junction protein occludin regulates apoptosis by enhancing caspase-3 transcription. These data suggest that reduced epithelial caspase-3 expression downstream of occludin downregulation is a previously-unappreciated anti-apoptotic process that contributes to mucosal homeostasis in inflammatory conditions.

Although the C-terminal cytoplasmic tail of the tight junction protein occludin is heavily phosphorylated, the functional impact of most individual sites is undefined. Here, we show that inhibition of CK2-mediated occludin S408 phosphorylation elevates transepithelial resistance by reducing paracellular cation flux. This regulation requires occludin, claudin-1, claudin-2, and ZO-1. S408 dephosphorylation reduces occludin exchange, but increases exchange of ZO-1, claudin-1, and claudin-2, thereby causing the mobile fractions of these proteins to converge. Claudin-4 exchange is not affected. ZO-1 domains that mediate interactions with occludin and claudins are required for increases in claudin-2 exchange, suggesting assembly of a phosphorylation-sensitive protein complex. Consistent with this, binding of claudin-1 and claudin-2, but not claudin-4, to S408A occludin tail is increased relative to S408D. Finally, CK2 inhibition reversed IL-13–induced, claudin-2–dependent barrier loss. Thus, occludin S408 dephosphorylation regulates paracellular permeability by remodeling tight junction protein dynamic behavior and intermolecular interactions between occludin, ZO-1, and select claudins, and may have therapeutic potential in inflammation-associated barrier dysfunction.

Polarized epithelia assemble into sheets that compartmentalize organs and generate tissue barriers by integrating apical surfaces into a single, unified structure. This tissue organization is shared across organs, species, and developmental stages. The processes that regulate development and maintenance of apical epithelial surfaces are, however, undefined. Here, using an intestinal epithelial-specific knockout (KO) mouse and cultured epithelial cells, we show that the tight junction scaffolding protein zonula occludens-1 (ZO-1) is essential for development of unified apical surfaces in vivo and in vitro. We found that U5 and GuK domains of ZO-1 are necessary for proper apical surface assembly, including organization of microvilli and cortical F-actin; however, direct interactions with F-actin through the ZO-1 actin-binding region (ABR) are not required. ZO-1 lacking the PDZ1 domain, which binds claudins, rescued apical structure in ZO-1–deficient epithelia, but not in cells lacking both ZO-1 and ZO-2, suggesting that heterodimerization with ZO-2 restores PDZ1-dependent ZO-1 interactions that are vital to apical surface organization. Pharmacologic F-actin disruption, myosin II motor inhibition, or dynamin inactivation restored apical epithelial structure in vitro and in vivo, indicating that ZO-1 directs epithelial organization by regulating actomyosin contraction and membrane traffic. We conclude that multiple ZO-1–mediated interactions contribute to coordination of epithelial actomyosin function and genesis of unified apical surfaces.