ABSTRACT Epithelia within tubular organs form and expand lumens. Failure of these processes can result in serious developmental anomalies. Although tight junction assembly is crucial to epithelial polarization, the contribution of specific tight junction proteins to lumenogenesis is undefined. Here, we show that ZO-1 (also known as TJP1) is necessary for the formation of single lumens. Epithelia lacking this tight junction scaffolding protein form cysts with multiple lumens and are defective in the earliest phases of polarization, both in two and three dimensions. Expression of ZO-1 domain-deletion mutants demonstrated that the actin-binding region and U5-GuK domain are crucial to single lumen development. For actin-binding region, but not U5-GuK domain, mutants, this could be overcome by strong polarization cues from the extracellular matrix. Analysis of the U5-GuK binding partners shroom2, α-catenin and occludin showed that only occludin deletion led to multi-lumen cysts. Like ZO-1-deficiency, occludin deletion led to mitotic spindle orientation defects. Single lumen formation required the occludin OCEL domain, which binds to ZO-1. We conclude that ZO-1–occludin interactions regulate multiple phases of epithelial polarization by providing cell-intrinsic signals that are required for single lumen formation.

ABSTRACT Known proteins associated with the cell-adhesion protein E-cadherin include catenins and proteins involved in signaling, trafficking and actin organization. However, the list of identified adherens junction proteins is likely to be incomplete, limiting investigation into this essential cell structure. To expand the inventory of potentially relevant proteins, we expressed E-cadherin fused to biotin ligase in MDCK epithelial cells, and identified by mass spectrometry neighboring proteins that were biotinylated. The most abundant of the 303 proteins identified were catenins and nearly 40 others that had been previously reported to influence cadherin function. Many others could be rationalized as novel candidates for regulating the adherens junction, cytoskeleton, trafficking or signaling. We further characterized lipoma preferred partner (LPP), which is present at both cell contacts and focal adhesions. Knockdown of LPP demonstrated its requirement for E-cadherin-dependent adhesion and suggested that it plays a role in coordination of the cell–cell and cell–substrate cytoskeletal interactions. The analysis of LPP function demonstrates proof of principle that the proteomic analysis of E-cadherin proximal proteins expands the inventory of components and tools for understanding the function of E-cadherin.

Summary The formation of a barrier between epithelial cells is a fundamental determinant of cellular homeostasis, protecting underlying cells against pathogens, dehydration and damage. Assembly of the tight junction barrier is dependent upon neighboring epithelial cells binding to one another and forming adherens junctions, but the mechanism for how these processes are linked is poorly understood. Using a knockdown and substitution system, we studied whether ZO-1 binding to α-catenin is required for coupling tight junction assembly to the formation of adherens junctions. We found that preventing ZO-1 binding to α-catenin did not appear to affect adherens junctions. Rather the assembly and maintenance of the epithelial barrier were disrupted. This disruption was accompanied by alterations in the mobility of ZO-1 and the organization of the actin cytoskeleton. Thus, our study identifies α-catenin binding to ZO-1 as a new mechanism for coupling the assembly of the epithelial barrier to cell-to-cell adhesion.

Summary Tight junctions (TJs) regulate the paracellular movement of ions, macromolecules and immune cells across epithelia. Zonula occludens (ZO)-1 is a multi-domain polypeptide required for the assembly of TJs. MDCK II cells lacking ZO-1, and its homolog ZO-2, have three distinct phenotypes: reduced localization of occludin and some claudins to the TJs, increased epithelial permeability, and expansion of the apical actomyosin contractile array found at the apical junction complex (AJC). However, it is unclear exactly which ZO-1 binding domains are required to coordinate these activities. We addressed this question by examining the ability of ZO-1 domain-deletion transgenes to reverse the effects of ZO depletion. We found that the SH3 domain and the U5 motif are required to recruit ZO-1 to the AJC and that localization is a prerequisite for normal TJ and cytoskeletal organization. The PDZ2 domain is not required for localization of ZO-1 to the AJC, but is necessary to establish the characteristic continuous circumferential band of ZO-1, occludin and claudin-2. PDZ2 is also required to establish normal permeability, but is not required for normal cytoskeletal organization. Finally, our results demonstrate that PDZ1 is crucial for the normal organization of both the TJ and the AJC cytoskeleton. Our results establish that ZO-1 acts as a true scaffolding protein and that the coordinated activity of multiple domains is required for normal TJ structure and function.

The function of occludin remains elusive. Proposed roles include maintenance of tight junction barriers, signaling and junction remodeling. To investigate a potential role in mediating cytokine-induced changes in barrier properties, we measured barrier responses to interferon-γ plus TNFα in control, occludin-overexpressing and occludin knockdown MDCK II monolayers. MDCK cells show a complex response to cytokines characterized by a simultaneous increase in the transepithelial electrical resistance and a decrease in the barrier for large solutes. We observed that overexpression of occludin increased and occludin knockdown decreased sensitivity to cytokines as assessed by both these parameters. It is known that caveolin-1 interacts with occludin and is implicated in several models of cytokine-dependent barrier disruption; we found that occludin knockdown altered the subcellular distribution of caveolin-1 and that partitioning of caveolin into detergent-insoluble lipid rafts was influenced by changing occludin levels. Knockdown of caveolin decreased the cytokine-induced flux increase, whereas the increase in the electrical barrier was unaltered; the effect of double knockdown of occludin and caveolin was similar to that of occludin single knockdown, consistent with the possibility that they function in the same pathway. These results demonstrate that occludin is required for cells to transduce cytokine-mediated signals that either increase the electrical barrier or decrease the large solute barrier, possibly by coordinating the functions of caveolin-1.