The C. elegans zonula occludens ortholog cooperates with the cadherin complex to recruit actin during morphogenesis

The dramatic cell-shape changes necessary to form a multicellular organism require cell-cell junctions to be both pliable and strong. The zonula occludens (ZO) subfamily of membrane-associated guanylate kinases (MAGUKs) are scaffolding molecules thought to regulate cell-cell adhesion, but there is little known about their roles in vivo. To elucidate the functional role of ZO proteins in a living embryo, the sole C. elegans ZO family member, ZOO-1, was characterized. ZOO-1 localizes with the cadherin-catenin complex during development, and its junctional recruitment requires the transmembrane proteins HMR-1/E-cadherin and VAB-9/claudin, but surprisingly, not HMP-1/alpha-catenin or HMP-2/beta-catenin. zoo-1 knockdown results in lethality during elongation, resulting in the rupture of epidermal cell-cell junctions under stress and failure of epidermal sheet sealing at the ventral midline. Consistent with a role in recruiting actin to the junction in parallel to the cadherin-catenin complex, zoo-1 loss of function reduces the dynamic recruitment of actin to junctions and enhances the severity of actin filament defects in hypomorphic alleles of hmp-1 and hmp-2. These results show that ZOO-1 cooperates with the cadherin-catenin complex to dynamically regulate strong junctional anchorage to the actin cytoskeleton during morphogenesis (Lockwood, 2008).

ZO-1, ZO-2 and ZO-3, mammalian homologs of Polychaetoid

Tight junctions form an intercellular barrier between epithelial cells, serve to separate tissue compartments, and maintain cellular polarity. Paracellular sealing properties vary among cell types and are regulated by undefined mechanisms. Sequence of the full-length cDNA for human ZO-1, the first identified tight junction component, predicts a protein of 1736 aa. The N-terminal 793 aa domain is homologous to the product of DLG and to PSD-95, a 95-kDa protein located in the postsynaptic densities of rat brain. All three proteins contain both a src homology 3 region (SH3 domain), previously identified in membrane proteins involved in signal transduction, and a region homologous to guanylate kinase. ZO-1 contains an additional 943-aa C-terminal domain that is proline-rich (14.1%) and contains an alternatively spliced domain, whose expression has previously been shown to correlate with variable properties of tight junctions. The C-terminal domain of ZO-1, and its alternatively spliced region, appears to confer variable properties unique to tight junctions (Willott, 1993).

The complete cDNA sequence for canine ZO-2, a tight junction-specific protein, is presented. A single open reading frame encodes a polypeptide of 1,174 amino acids with a predicted molecular mass of 132,085 daltons. ZO-2 is a member of the membrane-associated guanylate kinase-containing (MAGUK) protein family, a family which includes an additional tight junction-associated protein, ZO-1. These proteins contain a region homologous to guanylate kinase, an SH3 domain, and variable numbers of PSD-95/discs-large/ZO-1 (PDZ) domains, shown to be involved in protein-protein interactions. ZO-2 and ZO-1 contain three PDZ domains in the N-terminal half of the molecule. Between the first and second PDZ domains, ZO-2 displays a basic region (pI = 10.27) containing 22% arginine residues. Both ZO-1 and ZO-2 have proline-rich C-terminal regions that are not homologous to other MAGUK family members. Sequence analysis of multiple ZO-2 cDNAs reveals a 36-amino acid domain in this C-terminal region present in only some of the cDNAs. Overall, ZO-2 is highly homologous to ZO-1, showing 51% amino acid identity; however, the C-terminal ends of the molecules show only 25% amino acid identity. This suggests that the C-terminal ends of ZO-1 and ZO-2 have different functions (Beatch, 1996).

ZO-1 is a 210-225-kDa peripheral membrane protein associated with the cytoplasmic surfaces of the zonula occludens or tight junction. A 160-kD polypeptide, designated ZO-2, has been found to coimmunoprecipitate with ZO-1 from Madin-Darby canine kidney (MDCK) cell extracts prepared under conditions that preserve protein associations. ZO-2 was isolated by bulk coimmunoprecipitation with ZO-1. ZO-2 contains a region that is very similar to sequences in human and mouse ZO-1. This region includes both a 90-amino acid repeat domain of unknown function and guanylate kinase-like domains that are shared among members of the family of proteins that includes ZO-1, erythrocyte p55, DLG, and a synapse-associated protein from rat brain, PSD-95/SAP90. A polyclonal antiserum, raised against a unique region of ZO-2, exclusively labels the cytoplasmic surfaces of tight junctions, indicating that ZO-2 is a tight junction-associated protein. ZO-2 localizes to the region of the tight junction in a number of epithelia, including liver, intestine, kidney, testis, and arterial endothelium, suggesting that this protein is a ubiquitous component of the tight junction. Heart, a non-epithelial tissue, shows ZO-1 but no ZO-2 staining at the fascia adherens, a specialized junction of cardiac myocytes, previously shown to contain ZO-1. Thus it appears that ZO-2 is not a component of the fascia adherens, and that unlike ZO-1, this protein is restricted to the epithelial tight junction (Jesaitis, 1994)

A 130-kD protein that coimmunoprecipitates with the tight junction protein ZO-1 was bulk purified from Madin-Darby canine kidney (MDCK) cells and subjected to partial endopeptidase digestion and amino acid sequencing. Identified was a single open reading frame of 2,694 bp, coding for a protein of 898 amino acids with a predicted molecular mass of 98,414 daltons. This protein contains three PDZ domains, and in addition, a src homology (SH3) domain and a region similar to guanylate kinase, making it homologous to ZO-1 and ZO-2, as well as the Discs large tumor suppressor gene product of Drosophila, and other members of the MAGUK family of proteins. Like ZO-1 and ZO-2, the novel protein contains a COOH-terminal acidic domain and a basic region between the first and second PDZ domains. Unlike ZO-1 and ZO-2, this protein displays a proline-rich region between PDZ2 and PDZ3 and apparently contains no alternatively spliced domain. MDCK cells stably transfected with an epitope-tagged construct express the exogenous polypeptide at an apparent molecular mass of approximately 130 kD. This protein colocalizes with ZO-1 at tight junctions by immunofluorescence and immunoelectron microscopy. In vitro affinity analyses demonstrate that recombinant 130-kD protein directly interacts with ZO-1 and the cytoplasmic domain of occludin, but not with ZO-2. It is proposed that this protein be named ZO-3 (Haskins, 1998).

ZO-1, ZO-2 and ZO-3 association with junctions

ZO-1, found in the plasma membrane undercoat colocalizes at the immunofluorescence microscopic level with cadherins. Since protein ZO-1 was originally identified as a component exclusively underlying tight junctions in epithelial cells, where cadherins are not believed to be localized, the distribution of cadherins and ZO-1 were examined. In non-epithelial cells lacking tight junctions cadherins and ZO-1 colocalize, whereas in epithelial cells (e.g., intestinal epithelial cells) bearing well-developed tight junctions, cadherins and ZO-1 are clearly segregated into adherens and tight junctions, respectively. Interestingly, in epithelial cells such as hepatocytes, where tight junctions are not so well developed, ZO-1 is detected not only in the tight junction zone but also at adherens junctions. In mouse L cells transfected with cDNAs encoding N-, P-, and E-cadherins, cadherins are shown to interact directly or indirectly with ZO-1 (Itoh, 1993).

The functional characteristics of the tight junction protein ZO-3 were explored through exogenous expression of mutant protein constructs in MDCK cells. Expression of the amino-terminal, PSD95/dlg/ZO-1 domain-containing half of the molecule (NZO-3) delays the assembly of both tight and adherens junctions induced by calcium switch treatment or brief exposure to the actin-disrupting drug cytochalasin D. Junction formation was monitored by transepithelial resistance measurements and localization of junction-specific proteins by immunofluorescence. The tight junction components ZO-1, ZO-2, endogenous ZO-3, and occludin are mislocalized during the early stages of tight junction assembly. Similarly, the adherens junction proteins E-cadherin and beta-catenin are also delayed in their recruitment to the cell membrane, and NZO-3 expression has striking effects on actin cytoskeleton dynamics. NZO-3 expression does not alter expression levels of ZO-1, ZO-2, endogenous ZO-3, occludin, or E-cadherin; however, the amount of Triton X-100-soluble, signaling-active beta-catenin is increased in NZO-3-expressing cells during junction assembly. In vitro binding experiments show that ZO-1 and actin preferentially bind to NZO-3, whereas both NZO-3 and the carboxy-terminal half of the molecule (CZO-3) contain binding sites for occludin and cingulin. It is hypothesized that NZO-3 exerts its dominant-negative effects via a mechanism involving the actin cytoskeleton, ZO-1, and/or beta-catenin (Wittchen, 2000).

The involvement of the actin cytoskeleton in maintaining TJ integrity and regulating permeability has been well documented; this involvement is underscored by the fact that actin has multiple protein binding partners at the TJ, which themselves interact in various ways. It can be envisioned that this molecular architecture provides the means by which an actin filament network can be recruited to and organized in a functionally relevant manner at the TJ. The actin cytoskeleton is also a major structural and functional element of the AJ, and is present in a bundled actin belt around the apical periphery cell at the level of the AJ. Interestingly, in MDCK/NZO-3 cells, there is a delay in actin recruitment and formation of this perijunctional apical actin ring. Because the amino-terminal half of ZO-3 is responsible for binding F-actin, this may represent one mechanism whereby expression of this construct affects TJ and AJ assembly (Wittchen, 2000).

Not only does expression of the amino terminus of ZO-3 alter the distribution of ß-catenin, but there is also an increase in the TX-100-soluble pool of signaling-active ß-catenin. Presumably the presence of an increased level of the NZO-3 construct at early time points after calcium switch results in a downstream alteration of the E-cadherin/catenins complex at the adherens junction, releasing ß-catenin from a cytoskeletal linkage into the TX-100-soluble pool. A corresponding change in the levels of ß-catenin in the insoluble pool is not observed, although any possible change may be masked by the overall high levels of ß-catenin present in these samples. Normally cytoplasmic ß-catenin levels are strictly regulated via a ubiquitin-mediated proteolysis pathway requiring ß-catenin interaction with the cytoplasmic tumor suppressor APC. Soluble ß-catenin that escapes this targeted proteolysis is capable of translocating to the nucleus where it acts as a transcriptional transactivator in a complex with TCF/LEF family of transcription factors to direct transcription of a variety of genes that promote a proliferative phenotype. Expression of a mutant signaling-active (soluble) form of ß-catenin in MDCK cells has been shown to cause a delay in the establishment of tight confluent cell monolayers compared with control cells, and the cells appear more motile and form less compact colonies when plated at a low density. These results, taken together with data showing that NZO-3 expression delays transepithelial resistance formation after a calcium switch and results in an increased level of signaling-active, soluble ß-catenin in these cells, suggests that NZO-3 might act through ß-catenin to exert its effects on epithelial junctional complex formation. At present it is not known if this action is direct or indirect (Wittchen, 2000).

ZO-1, junction assembly and changes in ZO-1 subcellular distribution

Mammalian protein kinase C (see Drosophila PKC) is required for the proper assembly of tight junctions. Low concentrations of the specific inhibitor of PKC, calphostin C, markedly inhibit development of transepithelial electrical resistance, a functional measure of tight-junction biogenesis. The effect of PKC inhibitors on the development of tight junctions, as measured by resistance, is paralleled by a delay in the sorting of the tight-junction protein, Zona occludens 1 (ZO-1), to the tight junction. The assembly of desmosomes and the adherens junction is not detectably affected. ZO-1 is phosphorylated subsequent to the initiation of cell-cell contact, and treatment with calphostin C prevents approximately 85% of the phosphorylation increase. In vitro measurements indicate that ZO-1 may be a direct target of PKC. Membrane-associated PKC activity more than doubles during junction assembly, and immunocytochemical analysis reveals a pool of PKC zeta that appears to colocalize with ZO-1 at the tight junction. A preformed complex containing ZO-1, ZO-2, and p130, as well as 330- and 65-kDa phosphoproteins is detected by coimmunoprecipitation in both the presence and absence of cell-cell contact. Identity of the 330- and 65-kDa phosphoproteins remains to be determined, but the 65-kDa protein may well turn out to be occludin. Neither the mass of this complex nor the incorporation of ZO-1 into the Triton X-100-insoluble cytoskeleton were PKC dependent (Stuart, 1995).

The glucocorticoid and transforming growth factor-alpha (Drosophila homolog: Spitz) regulation of growth and cell-cell contact was investigated in a mammary epithelial tumor cell line. In cell monolayers, dexamethasone coordinately suppresses DNA synthesis, stimulates monolayer transepithelial electrical resistance (TER), and decreases the paracellular leakage of inulin or mannitol across the monolayer. Constitutive production of TGF-alpha in transfected cells or exogenous treatment with TGF-alpha prevents the glucocorticoid growth suppression response and disrupts tight junction formation without affecting glucocorticoid responsiveness. DNA synthesis is not a requirement for the growth factor disruption of tight junctions. The ZO-1 tight junction protein is localized exclusively at the cell periphery in dexamethasone-treated cells; TGF-alpha causes ZO-1 to relocalize from the cell periphery back to a cytoplasmic compartment. Taken together, these results demonstrate that glucocorticoids can coordinately regulate growth inhibition and cell-cell contact of mammary tumor cells and that TGF-alpha can override both effects of glucocorticoids. These results have uncovered a novel functional cross-talk between glucocorticoids and TGF-alpha, which potentially regulates the proliferation and differentiation of mammary epithelial cells (Buse, 1995).

Under certain conditions, ZO-1 can be detected in the nucleus. Nuclear accumulation can be stimulated at sites of wounding in cultured epithelial cells. ZO-1 can be found in the nuclei of intestinal epithelial cells only along the outer tip of the villus. These results suggest that the nuclear localization of ZO-1 is inversely related to the extent and/or maturity of cell contact. The nuclear accumulation of ZO-1 may be relevant for its suggested role in membrane-associated guanylate kinase homolog signal transduction (Gottardi, 1996).

Distinct and redundant functions of Esama and VE-cadherin during vascular morphogenesis

The cardiovascular system forms during early embryogenesis and adapts to embryonic growth by sprouting angiogenesis and vascular remodeling. These processes require fine-tuning of cell-cell adhesion to maintain and reestablish endothelial contacts, while allowing cell motility. This study compared the contribution of two endothelial cell specific adhesion proteins - VE-cadherin (VE-cad/Cdh5; see Drosophila Shotgun) and Esama (Endothelial cell-selective adhesion molecule a) - during angiogenic sprouting and blood vessel fusion (anastomosis) in the zebrafish embryo by genetic analyses. Different combinations of mutant alleles can be placed into a phenotypic series with increasing defects in filopodial contact formation. Contact formation in esama mutants appear wild-type like, while esama-/-; ve-cad+/- and ve-cad single mutants exhibit intermediate phenotypes. The lack of both proteins interrupts filopodial interaction completely. Furthermore, double mutants do not form a stable endothelial monolayer, display intrajunctional gaps, dislocalization of ZO-1 (see Drosophila Polychaetoid) and defects in apical-basal polarization. In summary, VE-cadherin and Esama have distinct and redundant functions during blood vessel morphogenesis and both adhesion proteins are central to endothelial cell recognition during anastomosis (Sauteur, 2017).

ZO-1 protein interactions

ZO-1 is concentrated at the cadherin-based cell adhesion sites in non-epithelial cells. cDNAs encoding the full-length ZO-1, its amino-terminal half (N-ZO-1), and carboxyl-terminal half (C-ZO-1) were introduced into mouse L fibroblasts expressing exogenous E-cadherin (EL cells). The full-length ZO-1 as well as N-ZO-1 are concentrated at cadherin-based cell-cell adhesion sites. In good agreement with these observations, N-ZO-1 is specifically coimmunoprecipitated from EL transfectants expressing N-ZO-1 (NZ-EL cells) with the E-cadherin/alpha, beta catenin complex. In contrast, C-ZO-1 is localized along actin stress fibers. Recombinant N-ZO-1 can bind directly to alpha catenin, but not to beta catenin or the cytoplasmic domain of E-cadherin. The dissociation constant between N-ZO-1 and alpha catenin is approximately 0.5 nM. In contrast to this, recombinant C-ZO-1 cosediments with actin filaments in vitro, with a dissociation constant of approximately 10 nM. The cadherin-based cell adhesion activity of NZ-EL cells was compared with that of parent EL cells. Cell aggregation assay has revealed no significant differences among these cells, but the cadherin-dependent intercellular motility, i.e., the cell movement in a confluent monolayer, is significantly suppressed in NZ-EL cells. It is concluded that in nonepithelial cells, ZO-1 works as a cross-linker between cadherin/catenin complex and the actin-based cytoskeleton through direct interaction with alpha catenin and actin filaments at its amino- and carboxyl-terminal halves, respectively, and that ZO-1 is a functional component in the cadherin-based cell adhesion system (Itoh, 1997).

Gap junctions mediate cell-cell communication in almost all tissues and are composed of channel-forming integral membrane proteins, termed connexins. Connexin43 (Cx43) is the most widely expressed and the most well-studied member of this family. Cx43-based cell-cell communication is regulated by growth factors and oncogenes, although the underlying mechanisms are poorly understood because cellular proteins that interact with connexins have yet to be identified. The carboxy-terminal cytosolic domain of Cx43 contains several phosphorylation sites and potential signaling motifs. A yeast two-hybrid protein interaction screen has been used to identify proteins that bind to the carboxy-terminal tail of Cx43; in this way, the zona occludens-1 (ZO-1) protein was isolated. ZO-1 is a 220 kDa peripheral membrane protein containing multiple protein interaction domains including three PDZ domains and a Src homology 3 (SH3) domain. The interaction of Cx43 with ZO-1 occurs through the extreme carboxyl terminus of Cx43 and the second PDZ domain of ZO-1. Cx43 associates with ZO-1 in Cx43-transfected COS7 cells, as well as endogenously in normal Rat-1 fibroblasts and mink lung epithelial cells. Confocal microscopy reveals that endogenous Cx43 and ZO-1 colocalize at gap junctions. It is suggested that ZO-1 serves to recruit signaling proteins into Cx43-based gap junctions (Giepmans, 1998).

The interaction of cadherin-catenin complex with the actin-based cytoskeleton through alpha-catenin is indispensable for cadherin-based cell adhesion activity. E-cadherin-alpha-catenin fusion molecules show cell adhesion and cytoskeleton binding activities when expressed in nonepithelial L cells. Deletion mutants of E-cadherin-alpha-catenin fusion molecules lacking various domains of alpha-catenin were constructed and introduced into L cells. Detailed analysis has identified three distinct functional domains of alpha-catenin: a vinculin/alpha-actinin-binding domain, a ZO-1-binding domain, and an adhesion-modulation domain. Furthermore, cell dissociation assays reveal that the fusion molecules containing the ZO-1-binding domain, in addition to the adhesion-modulation domain, confer the strong state of cell adhesion activity on transfectants, although those lacking the ZO-1-binding domain confer only the weak state. The disorganization of actin-based cytoskeleton by cytochalasin D treatment shifts the cadherin-based cell adhesion from the strong to the weak state. In the epithelial cells, where alpha-catenin is not precisely colocalized with ZO-1, the ZO-1-binding domain does not completely support the strong state of cell adhesion activity. These studies show that the interaction of alpha-catenin with the actin-based cytoskeleton through the ZO-1-binding domain is required for the strong state of E-cadherin-based cell adhesion activity (Imamura, 1999).

The dynamic rearrangement of cell-cell junctions such as tight junctions and adherens junctions is a critical step in various cellular processes, including establishment of epithelial cell polarity and developmental patterning. Tight junctions are mediated by molecules such as occludin and its associated ZO-1 and ZO-2; adherens junctions are mediated by adhesion molecules such as cadherin and its associated catenins. The transformation of epithelial cells by activated Ras (see Drosophila Ras) results in the perturbation of cell-cell contacts. The ALL-1 fusion partner from chromosome 6 (AF-6) has been identified as a Ras target. AF-6 has the PDZ domain, which is thought to localize AF-6 at the specialized sites of plasma membranes, such as cell-cell contact sites. The roles of Ras and AF-6 were investigated in the regulation of cell-cell contacts. AF-6 accumulates at the cell-cell contact sites of polarized MDCKII epithelial cells and has a distribution similar to that of ZO-1 but somewhat different from those of catenins. Immunoelectron microscopy reveals a close association between AF-6 and ZO-1 at the tight junctions of MDCKII cells. Native and recombinant AF-6 interacts with ZO-1 in vitro. ZO-1 interacts with the Ras-binding domain of AF-6; this interaction was inhibited by activated Ras. AF-6 accumulates with ZO-1 at the cell-cell contact sites in cells lacking tight junctions such as Rat1 fibroblasts and PC12 rat pheochromocytoma cells. The overexpression of activated Ras in Rat1 cells results in the perturbation of cell-cell contacts, followed by a decrease of the accumulation of AF-6 and ZO-1 at the cell surface. These results indicate that AF-6 serves as one of the peripheral components of tight junctions in epithelial cells and cell-cell adhesions in nonepithelial cells, and that AF-6 may participate in the regulation of cell-cell contacts, including tight junctions, via direct interaction with ZO-1 downstream of Ras (Yamamoto, 1997).

Occludin is an integral membrane protein localizing at tight junctions (TJ) with four transmembrane domains and a long COOH-terminal cytoplasmic domain (domain E) consisting of 255 amino acids. Immunofluorescence and laser scan microscopy reveals that chick full-length occludin introduced into human and bovine epithelial cells is correctly delivered to and incorporated into preexisting TJ. Transfection studies with various deletion mutants show that the domain E, especially its COOH-terminal approximately 150 amino acids (domain E358/504), is necessary for the localization of occludin at TJ. Domain E was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and this fusion protein specifically binds to a complex of ZO-1 (220 kD) and ZO-2 (160 kD) among various membrane peripheral proteins. In vitro binding analyses using glutathione-S-transferase fusion proteins of various deletion mutants of domain E narrows down the sequence necessary for the ZO-1/ZO-2 association into the domain E358/504. Furthermore, this region directly associates with the recombinant ZO-1 produced in E. coli. It is concluded that occludin itself can localize at TJ and directly associate with ZO-1. The coincidence of the sequence necessary for the ZO-1 association with that for the TJ localization suggests that the association with underlying cytoskeletons through ZO-1 is required for occludin to be localized at TJ (Furuse, 1994).

Postsynaptic density-95 (PSD-95/SAP-90) is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins that assemble protein complexes at synapses and other cell junctions. MAGUKs comprise multiple protein-protein interaction motifs, including PDZ, SH3 and guanylate kinase (GK) domains, and these binding sites mediate the scaffolding function of MAGUK proteins. Synaptic binding partners for the PDZ and GK domains of PSD-95 have been identified, but the role of the SH3 domain remains elusive. The SH3 domain of PSD-95 mediates a specific interaction with the GK domain. The GK domain lacks a poly-proline motif that typically binds to SH3 domains; instead, SH3/GK binding is a bi-domain interaction that requires both intact motifs. Although isolated SH3 and GK domains can bind in trans, experiments with intact PSD-95 molecules indicate that intramolecular SH3/GK binding dominates and prevents intermolecular associations. SH3/GK binding is conserved in the related Drosophila MAGUK protein Discs large, but is not detectable for C. elegans LIN-2. Many previously identified genetic mutations of MAGUKs in invertebrates occur in the SH3 or GK domains, and all of these mutations disrupt intramolecular SH3/GK binding (McGee, 1999).

The intramolecular interactions described here are reminiscent of recent studies showing that intramolecular SH3 domain associations mediate autoinhibition of Src and Tec family tyrosine kinases. In the Src family kinase, Hck (the SH3 motif) binds to and blocks the catalytic activity of the adjacent tyrosine kinase domain. This intramolecular SH3 domain interaction within Hck is displaced, and tyrosine kinase activity is restored when the Hck SH3 domain binds to an appropriate protein ligand in trans. By analogy, the intramolecular SH3 domain interaction within PSD-95 may regulate the GK domain. While this interaction does not alter GK domain binding to either GKAP or MAP1A, the SH3 domain may regulate an as yet unidentified catalytic activity of the GK domain. It is also possible that the intramolecular SH3/GK interaction mediates functional interactions between the SH3 domain and other unknown proteins: studies with Dlg in Drosophila have suggested that the GK domain may act as a negative regulator of Dlg in controlling cell proliferation. PDZ domains within PSD-95 can negatively regulate GK binding activity, though the PDZ domains themselves do not bind to GK. Since the SH3 domain is interposed between the PDZ and GK domains, the SH3/GK interaction described here could play a role in autoinhibition of GK binding by PDZ domains. Alternatively, while the data best support a model in which intramolecular associations between the SH3 and GK domains predominate, it does not eliminate the possibility that other factors present in vivo may facilitate intermolecular interactions that could contribute to the scaffolding functions of PSD-95 (McGee, 1999).

Occludin is a transmembrane protein of the tight junction that functions in creating both an intercellular permeability barrier and an intramembrane diffusion barrier. Creation of the barrier requires the precise localization of occludin, and a distinct family of transmembrane proteins called claudins, into continuous linear fibrils visible by freeze-fracture microscopy. Conflicting evidence exists regarding the relative importance of the transmembrane and extracellular versus the cytoplasmic domains in localizing occludin in fibrils. Recent studies suggest that the structural basis for occludin's sealing properties may arise from the ability of this protein to form adhesive contacts with proteins on adjacent cells. To specifically address whether occludin's COOH-terminal cytoplasmic domain is sufficient to target it into tight junction fibrils, or whether extracellular adhesive contacts serve this function, chimeras with the transmembrane portions of connexin 32 were created. Despite the gap junction targeting information present in their transmembrane and extracellular domains, these connexin-occludin chimeras localize within fibrils associated with tight junctions when expressed in MDCK cells, as assessed by immunofluorescence and immunogold freeze-fracture imaging. Localization of chimeras at tight junctions depends on the COOH-terminal ZO-binding domain and not on the membrane proximal domain of occludin. Furthermore, neither endogenous occludin nor claudin is required for targeting to ZO-1-containing cell-cell contacts, since in normal rat kidney fibroblasts targeting of chimeras again requires only the ZO-binding domain. These results suggest an important role for cytoplasmic proteins, presumably ZO-1, ZO-2, and ZO-3, in localizing occludin in tight junction fibrils. Such a scaffolding and cytoskeletal coupling function for ZO MAGUKs is analogous to that of other members of the MAGUK family (Mitic, 1999).

Epithelial tight junctions regulate paracellular diffusion and restrict the intermixing of apical and basolateral plasma membrane components. A Y-box transcription factor, ZONAB (ZO-1-associated nucleic acid-binding protein), has been identified that binds to the SH3 domain of ZO-1, a submembrane protein of tight junctions. ZONAB localizes to the nucleus and at tight junctions, and binds to sequences of specific promoters containing an inverted CCAAT box. In reporter assays, ZONAB and ZO-1 functionally interact in the regulation of the ErbB-2 promoter in a cell density-dependent manner. In stably transfected overexpressing cells, ZO-1 and ZONAB control expression of endogenous ErbB-2 and function in the regulation of paracellular permeability. These data indicate that tight junctions directly participate in the control of gene expression and suggest that they function in the regulation of epithelial cell differentiation (Balda, 2000).

ZO-1 is an actin filament (F-actin)-binding protein that localizes to tight junctions and connects claudin to the actin cytoskeleton in epithelial cells. Claudins are four-transmembrane domain proteins that constitute the backbone of tight junction strands. Claudins constitute a new gene family, the 'claudin' family, which includes at least 16 members. Interestingly, most of the claudin family members end in YV at their COOH termini and thus are good candidates for the binding partners for PDZ domains. In nonepithelial cells that have no tight junctions, ZO-1 localizes to adherens junctions (AJs) and may connect cadherin to the actin cytoskeleton indirectly through ß- and alpha-catenins as one of many F-actin-binding proteins. Nectin is an immunoglobulin-like adhesion molecule that localizes to AJs and is associated with the actin cytoskeleton through afadin, an F-actin-binding protein. Ponsin is an afadin- and vinculin-binding protein that also localizes to AJs. The nectin-afadin complex has a potency to recruit the E-cadherin-ß-catenin complex through alpha-catenin in a manner independent of ponsin. Whether nectin recruits ZO-1 to nectin-based cell-cell adhesion sites has been examined by the use of cadherin-deficient L cell lines stably expressing various components of the cadherin-catenin and nectin-afadin systems, and alpha-catenin-deficient F9 cell lines. Nectin shows a potency to recruit not only alpha-catenin but also ZO-1 to nectin-based cell-cell adhesion sites. This recruitment of ZO-1 is dependent on afadin but independent of alpha-catenin and ponsin. These results indicate that ZO-1 localizes to cadherin-based AJs through interactions not only with alpha-catenin but also with the nectin-afadin system (Yokoyama, 2001).

Mammalian homologs of the Drosophila polarity proteins Stardust, Discs Lost (now redefined as Drosophila Patj), and Crumbs have been identified as Pals1, Pals1-associated tight junction protein (PATJ), and human Crumbs homolog 1 (CRB1), respectively. PATJ, Pals1, and CRB1 can form a tripartite tight junction complex in epithelial cells and PATJ recruits Pals1 to tight junctions. The Pals1/PATJ interaction is not crucial for the ultimate targeting of PATJ itself to tight junctions. This prompted an examination to see if any of the 10 post-synaptic density-95/Discs Large/zona occludens-1 (PDZ) domains of PATJ could bind to the carboxyl termini of known tight junction constituents. The 6th and 8th PDZ domains of PATJ were found to be able to interact with the carboxyl termini of zona occludens-3 (ZO-3) and claudin 1, respectively. PATJ missing the 6th PDZ domain mislocalizes away from cell contacts. Surprisingly, deleting the 8th PDZ domain has little effect on PATJ localization. Finally, reciprocal co-immunoprecipitation experiments revealed that full-length ZO-3 can associate with PATJ. Hence, the PATJ/ZO-3 interaction is likely important for recruiting PATJ and its associated proteins to tight junctions (Roh, 2002)

The PDZ domain-containing protein zonula occludens-1 (ZO-1) selectively localizes to the cytoplasmic basis of the slit diaphragm, a specialized cell-cell contact in between glomerular podocytes necessary to prevent the loss of protein in the urine. However, the function of ZO-1 at the slit diaphragm has remained elusive. Deletion of Neph1, a slit diaphragm protein of the immunoglobulin superfamily with a cytoplasmic PDZ binding site, causes proteinuria in mice. This study demonstrates now that Neph1 binds ZO-1. This interaction was mediated by the first PDZ domain of ZO-1 and involved the conserved PDZ domain binding motif present in the carboxyl terminus of the three known Neph family members. Furthermore, Neph1 co-immunoprecipitates with ZO-1 from lysates of mouse kidneys, demonstrating that this interaction occurs in vivo. Both deletion of the PDZ binding motif of Neph1 as well as threonine-to-glutamate mutation of the threonine within the binding motif abrogated binding of ZO-1, suggesting that phosphorylation may regulate this interaction. ZO-1 binding was associated with a strong increase in tyrosine phosphorylation of the cytoplasmic tail of Neph1 and dramatically accelerated the ability of Neph1 to induce signal transduction. Thus, these data suggest that ZO-1 may organize Neph proteins and recruit signal transduction components to the slit diaphragm of podocytes (Huber, 2003).

Cortactin, a Z0-1 interacting protein

Continued: polychaetoid Evolutionary homologs part 2/2

polychaetoid: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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