Regulation of epithelial permeability by the actin cytoskeleton

The actin cytoskeleton is a dynamic structure necessary for cell and tissue organization, including the maintenance of epithelial barriers. The epithelial barrier regulates the movement of ions, macromolecules, immune cells and pathogens, and is thus essential for normal organ function. Disruption in the epithelial barrier has been shown to coincide with alterations of the actin cytoskeleton in several disease states. These disruptions primarily manifest as increased movement through the paracellular space, which is normally regulated by tight junctions. Despite extensive research demonstrating a direct link between the actin cytoskeleton and epithelial permeability, our understanding of the physiological mechanisms that link permeability and tight junction structure are still limited. In this review we explore the role of the actin cytoskeleton at tight junctions and present several areas for future study

Zonula Occludens-1 and -2 Are Cytosolic Scaffolds That Regulate the Assembly of Cellular Junctions

The integrity of the tight junction barrier in epithelial and endothelial cells is critical to human health, but we still lack a detailed mechanistic knowledge of how the barrier is formed during development or responds to pathological and pharmacological insults. This limits our understanding of barrier dysfunction in disease and slows the development of therapeutic strategies. Recent studies confirm the long-maintained but previously unsupported view that the zonula occludens (ZO) proteins ZO-1 and ZO-2 are critical determinants of barrier formation. However, ZO proteins can also be components of adherens junctions, and recent studies suggest that ZO proteins may also promote the assembly and function of these junctions during epithelial morphogenesis. We review these studies and outline several recent observations that suggest that one role of ZO proteins is to regulate cytoskeletal dynamics at cell junctions. Finally, we propose a model by which the functional activities of ZO proteins in the adherens junction and tight junction are differentiated by a novel regulatory motif known as the U6 or acidic motif

The Unique-5 and -6 Motifs of ZO-1 Regulate Tight Junction Strand Localization and Scaffolding Properties

The proper cellular location and sealing of tight junctions is assumed to depend on scaffolding properties of ZO-1, a member of the MAGUK protein family. ZO-1 contains a conserved SH3-GUK module that is separated by a variable region (unique-5), which in other MAGUKs has proven regulatory functions. To identify motifs in ZO-1 critical for its putative scaffolding functions, we focused on the SH3-GUK module including unique-5 (U5) and unique-6 (U6), a motif immediately C-terminal of the GUK domain. In vitro binding studies reveal U5 is sufficient for occludin binding; U6 reduces the affinity of this binding. In cultured cells, U5 is required for targeting ZO-1 to tight junctions and removal of U6 results in ectopically displaced junction strands containing the modified ZO-1, occludin, and claudin on the lateral cell membrane. These results provide evidence that ZO-1 can control the location of tight junction transmembrane proteins and reveals complex protein binding and targeting signals within its SH3-U5-GUK-U6 region. We review these findings in the context of regulated scaffolding functions of other MAGUK protein

Dimerization of the Scaffolding Protein ZO-1 through the Second PDZ Domain

The tight junction protein ZO-1 is known to link the transmembrane proteins occludin, claudins, and JAMs to many cytoplasmic proteins and the actin cytoskeleton. Although specific roles for ZO-1 at the tight junction are unknown, it is widely assumed that ZO-1, together with its homologs ZO-2 and ZO-3, serves as a platform to scaffold various transmembrane and cytoplasmic tight junction proteins. Thus the manner in which the zonula occludens (ZO) proteins multimerize has implications for the protein networks they can coordinate. The purpose of our study was to determine whether ZO-1 forms homodimers and to determine the protein interaction region. Using laser light scattering and analytical centrifugation, we show that protein sequences corresponding to the NH(2)-terminal half of ZO-1 form stable homodimers with a submicromolar equilibrium dissociation constant. Analysis of the molecular weight of different truncated forms of ZO-1 revealed that the second PDZ domain is both necessary and sufficient for dimerization. This interaction does not use the beta-finger motif described for other PDZ dimers. Furthermore, ZO-1 does not dimerize via an Src homology 3 to Guk domain interaction as was demonstrated previously for MAGUKs, like PSD-95. Results from immunoprecipitation experiments with polarized Madin-Darby canine kidney epithelial cells stably transfected with full-length GFP-ZO-1 indicate that a substantial portion of ZO-1 forms homodimers in vivo. As described previously, ZO-1 also forms heterodimers with ZO-2 and ZO-3. We conclude that the dimerization of ZO proteins is unlike that of other MAGUKs and that the previously unrecognized ZO-1 homodimers may allow formation of protein networks distinct from those of heterodimers with ZO-2 and ZO-3

Epithelial barrier assembly requires coordinated activity of multiple domains of the tight junction protein ZO-1

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

Domain Swapping within PDZ2 Is Responsible for Dimerization of ZO Proteins

ZO-1 is a multidomain protein involved in cell-cell junctions and contains three PDZ domains, which are necessary for its function in vivo. PDZ domains play a central role in assembling diverse protein complexes through their ability to recognize short peptide motifs on other proteins. We determined the structure of the second of the three PDZ domains of ZO-1, which is known to promote dimerization as well as bind to C-terminal sequences on connexins. The dimer is stabilized by extensive symmetrical domain swapping of β-strands, which is unlike any other known mechanism of PDZ dimerization. The canonical peptide-binding groove remains intact in both subunits of the PDZ2 dimer and is created by elements contributed from both monomers. This unique structure reveals an additional example of how PDZ domains dimerize and has multiple implications for both peptide binding and oligomerization in vivo

ZO-1 recruitment to -catenin - a novel mechanism for coupling the assembly of tight junctions to adherens junctions

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

A Laminin G-EGF-Laminin G Module in Neurexin IV Is Essential for the Apico-Lateral Localization of Contactin and Organization of Septate Junctions

Septate junctions (SJs) display a unique ultrastructural morphology with ladder-like electron densities that are conserved through evolution. Genetic and molecular analyses have identified a highly conserved core complex of SJ proteins consisting of three cell adhesion molecules Neurexin IV, Contactin, and Neuroglian, which interact with the cytoskeletal FERM domain protein Coracle. How these individual proteins interact to form the septal arrays that create the paracellular barrier is poorly understood. Here, we show that point mutations that map to specific domains of neurexin IV lead to formation of fewer septae and disorganization of SJs. Consistent with these observations, our in vivo domain deletion analyses identified the first Laminin G-EGF-Laminin G module in the extracellular region of Neurexin IV as necessary for the localization of and association with Contactin. Neurexin IV protein that is devoid of its cytoplasmic region is able to create septae, but fails to form a full complement of SJs. These data provide the first in vivo evidence that specific domains in Neurexin IV are required for protein-protein interactions and organization of SJs. Given the molecular conservation of SJ proteins across species, our studies may provide insights into how vertebrate axo-glial SJs are organized in myelinated axons

Cortactin is necessary for E-cadherin–mediated contact formation and actin reorganization

Classical cadherin adhesion molecules are key determinants of cell–cell recognition during development and in post-embryonic life. A decisive step in productive cadherin-based recognition is the conversion of nascent adhesions into stable zones of contact. It is increasingly clear that such contact zone extension entails active cooperation between cadherin adhesion and the force-generating capacity of the actin cytoskeleton. Cortactin has recently emerged as an important regulator of actin dynamics in several forms of cell motility. We now report that cortactin is recruited to cell–cell adhesive contacts in response to homophilic cadherin ligation. Notably, cortactin accumulates preferentially, with Arp2/3, at cell margins where adhesive contacts are being extended. Recruitment of cortactin is accompanied by a ligation-dependent biochemical interaction between cortactin and the cadherin adhesive complex. Inhibition of cortactin activity in cells blocked Arp2/3-dependent actin assembly at cadherin adhesive contacts, significantly reduced cadherin adhesive contact zone extension, and perturbed both cell morphology and junctional accumulation of cadherins in polarized epithelia. Together, our findings identify a necessary role for cortactin in the cadherin–actin cooperation that supports productive contact formation