The cell-cell adhesion molecule EpCAM interacts directly with the tight junction protein claudin-7

We recently described that in the metastasizing rat pancreatic carcinoma line BSp73ASML the cell-cell adhesion molecule EpCAM, CD44 variant isoforms and the tetraspanins D6.1A and CD9 form a complex that is located in glycolipid-enriched membrane microdomains. This complex contains, in addition, an undefined 20 kDa protein. As such complex formation influenced cell-cell adhesion and apoptosis resistance, it became of interest to identify the 20 kDa polypeptide. This 20 kDa protein, which co-precipitated with EpCAM in BSp73ASML lysates, was identified as the tight junction protein claudin-7. Correspondingly, an association between EpCAM and claudin-7 was noted in rat and human tumors and in non-transformed tissues of the gastrointestinal tract. Co-localization of the two molecules was most pronounced at basolateral membranes, but was also observed in tight junctions. Evidence for direct protein-protein interactions between EpCAM and claudin-7 was obtained by co-immunoprecipitation after treatment of tumor cells with a membrane-permeable chemical cross-linker. The complex, which is located in glycolipid-enriched membrane microdomains, is not disrupted by partial cholesterol depletion, but claudin-7 phosphorylation is restricted to the localization in glycolipid-enriched membrane microdomains. This is the first report on an association between EpCAM and claudins in both non-transformed tissues and metastasizing tumor cell lines.     

JAM4, a junctional cell adhesion molecule interacting with a tight junction protein,MAGI-1

MAGI-1 is a membrane-associated guanylate kinase protein at tight junctions in epithelial cells. It interacts with various molecules and functions as a scaffold protein at cell junctions. We report here a novel MAGI-1-binding protein that we named junctional adhesion molecule 4 (JAM4). JAM4 belongs to an immunoglobulin protein family. JAM4 was colocalized with ZO-1 in kidney glomeruli and in intestinal epithelial cells. Biochemical in vitro studies revealed that JAM4 bound to MAGI-1 but not to ZO-1, whereas JAM1 did not bind to MAGI-1. JAM4 and MAGI-1 interacted with each other and formed clusters in COS-7 cells when coexpressed. JAM4 mediated calcium-independent homophilic adhesion and was accumulated at cell-cell contacts when expressed in L cells. MAGI-1, ZO-1, and occludin were recruited to JAM4-based cell contacts. JAM4 also reduced the permeability of CHO cell monolayers. MAGI-1 strengthened JAM4-mediated cell adhesion in L cells and sealing effects in CHO cells. These findings suggest that JAM4 together with MAGI-1 provides an adhesion machinery at tight junctions, which may regulate the permeability of kidney glomerulus and small intestinal epithelial cells.     

Multi-PDZ domain protein MUPP1 is a cellular target for both adenovirus E4-ORF1 and high-risk papillomavirus type 18 E6 oncoproteins

A general theme that has emerged from studies of DNA tumor viruses is that otherwise unrelated oncoproteins encoded by these viruses often target the same important cellular factors. Major oncogenic determinants for human adenovirus type 9 (Ad9) and high-risk human papillomaviruses (HPV) are the E4-ORF1 and E6 oncoproteins, respectively, and although otherwise unrelated, both of these viral proteins possess a functional PDZ domain-binding motif that is essential for their transforming activity and for binding to the PDZ domain-containing and putative tumor suppressor protein DLG. We report here that the PDZ domain-binding motifs of Ad9 E4-ORF1 and high-risk HPV-18 E6 also mediate binding to the widely expressed cellular factor MUPP1, a large multi-PDZ domain protein predicted to function as an adapter in signal transduction. With regard to the consequences of these interactions in cells, we showed that Ad9 E4-ORF1 aberrantly sequesters MUPP1 within the cytoplasm of cells whereas HPV-18 E6 targets this cellular protein for degradation. These effects were specific because mutant viral proteins unable to bind MUPP1 lack these activities. From these results, we propose that the multi-PDZ domain protein MUPP1 is involved in negatively regulating cellular proliferation and that the transforming activities of two different viral oncoproteins depend, in part, on their ability to inactivate this cellular factor.     

Interaction of the human somatostatin receptor 3 with the multiple PDZ domain protein MUPP1 enables somatostatin to control permeability of epithelial tight junctions

The presence of heterotrimeric G-proteins at epithelial tight junctions suggests that these cellular junctions are regulated by so far unknown G-protein coupled receptors. We identify here an interaction between the human somatostatin receptor 3 (hSSTR3) and the multiple PDZ protein MUPP1. MUPP1 is a tight junction scaffold protein in epithelial cells, and as a result of the interaction with MUPP1 the hSSTR3 is targeted to tight junctions. Interaction with MUPP1 enables the receptor to regulate transepithelial permeability in a pertussis toxin sensitive manner, suggesting that hSSTR3 can activate G-proteins locally at tight junctions.

Structured summary:

MINT-6800756, MINT-6800770: MUPP1 (uniprotkb:O75970) and hSSTR3 (uniprotkb:P32745) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-6800587:hSSTR3 (uniprotkb:P32745) physically interacts (MI:0218) with MUPP1 (uniprotkb:O55164) by pull down (MI:0096)MINT-6800562:hSSTR3 (uniprotkb:P32745) physically interacts (MI:0218) with MUPP1 (uniprotkb:O75970) by two hybrid (MI:0018)MINT-6800622:hSSTR3 (uniprotkb:P32745) physically interacts (MI:0218) with PIST (uniprotkb: Q9HD26), Hsp70 (uniprotkb:P08107), Maguk p55 (uniprotkb: Q8N3R9), MAGI3 (uniprotkb:Q5TCQ9), ZO-2 (uniprotkb:Q9UDY2), ZO-1 (uniprotkb:Q07157) and MUPP1 (uniprotkb:O55164) by pull down (MI:0096)MINT-6800607, MINT-6801122:hSSTR3 (uniprotkb:P32745) physically interacts (MI:0218) with MUPP1 (uniprotkb:O75970) by anti bait coimmunoprecipitation (MI:0006)     

Interaction of junctional adhesion molecule with the tight junction components ZO-1, cingulin, and occludin

Junctional adhesion molecule (JAM) is an integral membrane protein that has been reported to colocalize with the tight junction molecules occludin, ZO-1, and cingulin. However, evidence for the association of JAM with these molecules is missing. Transfection of Chinese hamster ovary cells with JAM (either alone or in combination with occludin) resulted in enhanced junctional localization of both endogenous ZO-1 and cotransfected occludin. Additionally, JAM was coprecipitated with ZO-1 in the detergent-insoluble fraction of Caco-2 epithelial cells. A putative PDZ-binding motif at the cytoplasmic carboxyl terminus of JAM was required for mediating the interaction of JAM with ZO-1, as assessed by in vitro binding and coprecipitation experiments. JAM was also coprecipitated with cingulin, another cytoplasmic component of tight junctions, and this association required the amino-terminal globular head of cingulin. Taken together, these data indicate that JAM is a component of the multiprotein complex of tight junctions, which may facilitate junction assembly.     

The Src homology 3 domain is required for junctional adhesion molecule binding to the third PDZ domain of the scaffolding protein ZO-1

Tight junctions are cell-cell contacts that regulate the paracellular flux of solutes and prevent pathogen entry across cell layers. The assembly and permeability of this barrier are dependent on the zonula occludens (ZO) membrane-associated guanylate kinase (MAGUK) proteins ZO-1, -2, and -3. MAGUK proteins are characterized by a core motif of protein-binding domains that include a PDZ domain, a Src homology 3 (SH3) domain, and a region of homology to guanylate kinase (GUK); the structure of this core motif has never been determined for any MAGUK. To better understand how ZO proteins organize the assembly of protein complexes we have crystallized the entire PDZ3-SH3-GUK core motif of ZO-1. We have also crystallized this core motif in complex with the cytoplasmic tail of the ZO-1 PDZ3 ligand, junctional adhesion molecule A (JAM-A) to determine how the activity of different domains is coordinated. Our study shows a new feature for PDZ class II ligand binding that implicates the two highly conserved Phe(-2) and Ser(-3) residues of JAM. Our x-ray structures and NMR experiments also show for the first time a role for adjacent domains in the binding of ligands to PDZ domains in the MAGUK proteins family.     

Claudin-8 interacts with multi-PDZ domain protein 1 (MUPP1) and reduces paracellular conductance in epithelial cells.

The claudin family is a set of integral membrane proteins found at cell-cell interactions in tight junctions. To identify proteins that interact with claudin-8, we used the yeast two-hybrid system to search for binding partners. Using the C-terminal 37 amino acids of claudin-8 as bait, we screened a human kidney cDNA library and identified multi-PDZ domain protein 1 (MUPP1) as a claudin-8 binding protein. MUPP1 contains 13 PDZ domains and binds to claudin-8 though its PDZ9 domain. When MDCK cells were transfected with epitope-tagged claudin-8 or MUPP1, both molecules were concentrated at cell-cell junctions. The interaction of claudin-8 and MUPP1 in vivo was confirmed by co-immunolocalization and co-immunoprecipitation in MDCK cells. Expression of claudin-8-myc increased transepithelial electrical resistance (TER) and reduced paracellular flux using FITC-dextran as a tracer. Over-expression of FLAG-MUPP1 in MDCK cells also reduced the epithelial paracelhular conductance. Our results indicate that claudin-8 and MUPP1 interact in tight junctions of epithelial cells and are involved in the tight junction barrier function.     

Junctional adhesion molecule (JAM) binds to PAR-3: a possible mechanism for the recruitment of PAR-3 to tight junctions

At tight junctions (TJs), claudins with four transmembrane domains are incorporated into TJ strands. Junctional adhesion molecule (JAM), which belongs to the immunoglobulin superfamily, is also localized at TJs, but it remains unclear how JAM is integrated into TJs. Immunoreplica electron microscopy revealed that JAM showed an intimate spatial relationship with TJ strands in epithelial cells. In L fibroblasts expressing exogenous JAM, JAM was concentrated at cell-cell adhesion sites, where there were no strand-like structures, but rather characteristic membrane domains free of intramembranous particles were detected. These domains were specifically labeled with anti-JAM polyclonal antibody, suggesting that JAM forms planar aggregates through their lateral self-association. Immunofluorescence microscopy and in vitro binding assays revealed that ZO-1 directly binds to the COOH termini of claudins and JAM at its PDZ1 and PDZ3 domains, respectively. Furthermore, another PDZ-containing polarity-related protein, PAR-3, was directly bound to the COOH terminus of JAM, but not to that of claudins. These findings led to a molecular architectural model for TJs: small aggregates of JAM are tethered to claudin-based strands through ZO-1, and these JAM aggregates recruit PAR-3 to TJs. We also discuss the importance of this model from the perspective of the general molecular mechanisms behind the recruitment of PAR proteins to plasma membranes.     

Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions

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 revealed that chick full- length occludin introduced into human and bovine epithelial cells was correctly delivered to and incorporated into preexisting TJ. Further transfection studies with various deletion mutants showed that the domain E, especially its COOH-terminal approximately 150 amino acids (domain E358/504), was necessary for the localization of occludin at TJ. Secondly, domain E was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and this fusion protein was shown to be specifically bound 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 narrowed down the sequence necessary for the ZO-1/ZO-2 association into the domain E358/504. Furthermore, this region directly associated with the recombinant ZO-1 produced in E. coli. We 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.     

Neutrophil migration across tight junctions is mediated by adhesive interactions between epithelial coxsackie and adenovirus receptor and a junctional adhesion molecule-like protein on neutrophils

Neutrophil (polymorphonuclear leukocytes [PMN]) transepithelial migration during inflammatory episodes involves a complex series of adhesive interactions and signaling events. Previous studies have shown that key adhesive interactions between leukocyte CD11b/CD18 and basally expressed fucosylated glycoproteins followed by binding to desmosomal-associated JAM-C are key elements of the transmigration response. Here we provide the first evidence that PMN-expressed junctional adhesion molecule-like protein (JAML) regulates transmigration via binding interactions with epithelial coxsackie and adenovirus receptor (CAR). Experiments with a JAML fusion protein revealed specific binding of JAML to epithelial CAR expressed at tight junctions in T84 cell monolayers and normal human colonic mucosa. Furthermore, JAML-CAR binding is mediated via the membrane distal immunoglobulin (Ig) loop of CAR and the membrane proximal Ig loop of JAML. PMN bound to immobilized CAR but not JAML in a divalent cation-independent manner. Lastly, in assays of PMN transepithelial migration, JAML/CAR fusion proteins and their antibodies significantly inhibited transmigration in a specific manner. Taken together, these results indicate that JAML and CAR are a novel pair of adhesion molecules that play an important role in modulating PMN migration cross epithelial tight junctions. These findings add a new element to a multistep model of PMN transepithelial migration and may provide new targets for anti-inflammatory therapies.     

Influenza A virus neuraminidase protein enhances cell survival through interaction with carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) protein

The influenza virus neuraminidase (NA) protein primarily aids in the release of progeny virions from infected cells. Here, we demonstrate a novel role for NA in enhancing host cell survival by activating the Src/Akt signaling axis via an interaction with carcinoembryonic antigen-related cell adhesion molecule 6/cluster of differentiation 66c (C6). NA/C6 interaction leads to increased tyrosyl phosphorylation of Src, FAK, Akt, GSK3β, and Bcl-2, which affects cell survival, proliferation, migration, differentiation, and apoptosis. siRNA-mediated suppression of C6 resulted in a down-regulation of activated Src, FAK, and Akt, increased apoptosis, and reduced expression of viral proteins and viral titers in influenza virus-infected human lung adenocarcinoma epithelial and normal human bronchial epithelial cells. These findings indicate that influenza NA not only aids in the release of progeny virions, but also cell survival during viral replication.     

A novel protein with homology to the junctional adhesion molecule. Characterization of leukocyte interactions

We have cloned a novel cDNA belonging to the Ig superfamily that shows 44% similarity to the junctional adhesion molecule (JAM) and maps to chromosome 21q21.2. The open reading frame of JAM2 predicts a 34-kDa type I integral membrane protein that features two Ig-like folds and three N-linked glycosylation sites in the extracellular domain. A single protein kinase C phosphorylation consensus site and a PDZ-binding motif are present in the short intracellular tail. Heterologous expression of JAM2 in Chinese hamster ovary cells defined a 48-kDa protein that localizes predominantly to the intercellular borders. Northern blot analysis showed that JAM2 is preferentially expressed in the heart. JAM2 homotypic interactions were demonstrated by the ability of JAM2-Fc to capture JAM2-expressing Chinese hamster ovary cells. We further showed that JAM2, but not JAM1, is capable of adhering to the HSB and HPB-ALL lymphocyte cell lines. Neutralizing mouse anti-JAM2 polyclonal antibodies provided evidence against homotypic interactions in this assay. Biotinylation of HSB cell membranes revealed a 43-kDa counter-receptor that precipitates specifically with JAM2-Fc. These characteristics of JAM2 led us to hypothesize a role for this novel protein in adhesion events associated with cardiac inflammatory conditions.     

Nectin/PRR: an immunoglobulin-like cell adhesion molecule recruited to cadherin-based adherens junctions through interaction with Afadin, a PDZ domain-containing protein

We have isolated a novel actin filament-binding protein, named afadin, localized at cadherin-based cell-cell adherens junctions (AJs) in various tissues and cell lines. Afadin has one PDZ domain, three proline-rich regions, and one actin filament-binding domain. We found here that afadin directly interacted with a family of the immunoglobulin superfamily, which was isolated originally as the poliovirus receptor-related protein (PRR) family consisting of PRR1 and -2, and has been identified recently to be the alphaherpes virus receptor. PRR has a COOH-terminal consensus motif to which the PDZ domain of afadin binds. PRR and afadin were colocalized at cadherin-based cell-cell AJs in various tissues and cell lines. In E-cadherin-expressing EL cells, PRR was recruited to cadherin-based cell-cell AJs through interaction with afadin. PRR showed Ca2+-independent cell-cell adhesion activity. These results indicate that PRR is a cell-cell adhesion molecule of the immunoglobulin superfamily which is recruited to cadherin-based cell-cell AJs through interaction with afadin. We rename PRR as nectin (taken from the Latin word "necto" meaning "to connect").     

High-mobility group box 1 (HMGB1) protein regulates tumor-associated cell migration through the interaction with BTB domain

High-mobility group box 1 (HMGB1) was shown to be strongly implicated in high incidences of metastasis and the poor clinical pathologic conditions found in various human tumors. In this study, we explored the possible mechanism of HMGB1 in tumor metastases in vitro, using a human carcinoma cell system. BTB, as a negative regulator of cell cycle progression, was identified as a HMGB1 interacting partner. The ectopic expression of HMGB1 activates cell growth by suppressing BTB-induced cell death, decreasing Bax and p53 expression, while enhancing Bcl-xL, Bcl-2, cyclin D1, and NF-κB expression. HMGB1 activates the FAK/PI3K/mTOR signaling cascade, and BTB prominently inhibits HMGB1-induced oncogenesis. The effect of HMGB1 on FAK/mTOR signaling was also confirmed through the silencing of HMGB1 expression. These insights provide evidence that HMGB1 enhances cell proliferation and suppresses apoptosis. Collectively, our results show an underlying mechanism for an HMGB1-associated promotion of carcinoma cells.     

The cell polarity protein ASIP/PAR-3 directly associates with junctional adhesion molecule (JAM).

The establishment and maintenance of cellular polarity are critical for the development of multicellular organisms. PAR (partitioning-defective) proteins were identified in Caenorhabditis elegans as determinants of asymmetric cell division and polarized cell growth. Recently, vertebrate orthologues of two of these proteins, ASIP/PAR-3 and PAR-6, were found to form a signalling complex with the small GTPases Cdc42/Rac1 and with atypical protein kinase C (PKC). Here we show that ASIP/PAR-3 associates with the tight-junction-associated protein junctional adhesion molecule (JAM) in vitro and in vivo. No binding was observed with claudin-1, -4 or -5. In fibroblasts and CHO cells overexpressing JAM, endogenous ASIP is recruited to JAM at sites of cell-cell contact. Over expression of truncated JAM lacking the extracellular part disrupts ASIP/PAR-3 localization at intercellular junctions and delays ASIP/PAR-3 recruitment to newly formed cell junctions. During junction formation, JAM appears early in primordial forms of junctions. Our data suggest that the ASIP/PAR-3-aPKC complex is tethered to tight junctions via its association with JAM, indicating a potential role for JAM in the generation of cell polarity in epithelial cells.     

Carcinoembryonic antigen (CEA) inhibits NK killing via interaction with CEA-related cell adhesion molecule 1

The NK killing activity is regulated by activating and inhibitory NK receptors. All of the activating ligands identified so far are either viral or stress-induced proteins. The class I MHC proteins are the ligands for most of the inhibitory NK receptors. However, in the past few years, several receptors have been identified that are able to inhibit NK killing independently of class I MHC recognition. We have previously demonstrated the existence of a novel inhibitory mechanism of NK cell cytotoxicity mediated by the homophilic carcinoembryonic Ag (CEA)-related cell adhesion molecule 1 (CEACAM1) interactions. In this study, we demonstrate that CEACAM1 also interacts heterophilically with the CEA protein. Importantly, we show that these heterophilic interactions of CEA and CEACAM1 inhibit the killing by NK cells. Because CEA is expressed on a wide range of carcinomas and commonly used as tumor marker, these results represent a novel role for the CEA protein enabling the escape of tumor cells from NK-mediated killing. We further characterize, for the first time, the CEACAM1-CEA interactions. Using functional and binding assays, we demonstrate that the N domains of CEACAM1 and CEA are crucial but not sufficient for both the CEACAM1-CEACAM1 homophilic and CEACAM1-CEA heterophilic interactions. Finally, we suggest that the involvement of additional domains beside the N domain in the heterophilic and homophilic interactions is important for regulating the balance between cis and trans interactions.     

Platelet endothelial cell adhesion molecule 1 (PECAM-1) and its interactions with glycosaminoglycans: 1. Molecular modeling studies

Platelet endothelial cell adhesion molecule 1 (PECAM-1) has many functions, including its roles in leukocyte extravasation as part of the inflammatory response and in the maintenance of vascular integrity through its contribution to endothelial cell-cell adhesion. PECAM-1 has been shown to mediate cell-cell adhesion through homophilic binding events that involve interactions between domain 1 of PECAM-1 molecules on adjacent cells. However, various heterophilic ligands of PECAM-1 have also been proposed. The possible interaction of PECAM-1 with glycosaminoglycans (GAGs) is the focus of this study. The three-dimensional structure of the extracellular immunoglobulin (Ig) domains of PECAM-1 were constructed using homology modeling and threading methods. Potential heparin/heparan sulfate-binding sites were predicted on the basis of their amino acid consensus sequences and a comparison with known structures of sulfate-binding proteins. Heparin and other GAG fragments have been docked to investigate the structural determinants of their protein-binding specificity and selectivity. The modeling has predicted two regions in PECAM-1 that appear to bind heparin oligosaccharides. A high-affinity binding site was located in Ig domains 2 and 3, and evidence for a low-affinity site in Ig domains 5 and 6 was obtained. These GAG-binding regions were distinct from regions involved in PECAM-1 homophilic interactions.     

Platelet endothelial cell adhesion molecule 1 (PECAM-1) and its interactions with glycosaminoglycans: 2. Biochemical analyses

Platelet endothelial cell adhesion molecule 1 (PECAM-1) (CD31), a member of the immunoglobulin (Ig) superfamily of cell adhesion molecules with six Ig-like domains, has a range of functions, notably its contributions to leukocyte extravasation during inflammation and in maintaining vascular endothelial integrity. Although PECAM-1 is known to mediate cell adhesion by homophilic binding via domain 1, a number of PECAM-1 heterophilic ligands have been proposed. Here, the possibility that heparin and heparan sulfate (HS) are ligands for PECAM-1 was reinvestigated. The extracellular domain of PECAM-1 was expressed first as a fusion protein with the Fc region of human IgG1 fused to domain 6 and second with an N-terminal Flag tag on domain 1 (Flag-PECAM-1). Both proteins bound heparin immobilized on a biosensor chip in surface plasmon resonance (SPR) binding experiments. Binding was pH-sensitive but is easily measured at slightly acidic pH. A series of PECAM-1 domain deletions, prepared in both expression systems, were tested for heparin binding. This revealed that the main heparin-binding site required both domains 2 and 3. Flag-PECAM-1 and a Flag protein containing domains 1-3 bound HS on melanoma cell surfaces, but a Flag protein containing domains 1-2 did not. Heparin oligosaccharides inhibited Flag-PECAM-1 from binding immobilized heparin, with certain structures having greater inhibitory activity than others. Molecular modeling similarly identified the junction of domains 2 and 3 as the heparin-binding site and further revealed the importance of the iduronic acid conformation for binding. PECAM-1 does bind heparin/HS but by a site that is distinct from that required for homophilic binding.     

Absence of the tight junctional protein AF-6 disrupts epithelial cell-cell junctions and cell polarity during mouse development.

BACKGROUND: The establishment, maintenance and rearrangement of junctions between epithelial cells are extremely important in many developmental, physiological and pathological processes. AF-6 is a putative Ras effector; it is also a component of tight and adherens junctions, and has been shown to bind both Ras and the tight-junction protein ZO-1. In the mouse, AF-6 is encoded by the Af6 gene. As cell-cell junctions are important in morphogenesis, we generated a null mutation in the murine Af6 locus to test the hypothesis that lack of AF-6 function would cause epithelial abnormalities. RESULTS: Although cell-cell junctions are thought to be important in early embryogenesis, homozygous mutant embryos were morphologically indistinguishable from wild-type embryos through 6.5 days post coitum (dpc) and were able to establish all three germ layers. The earliest morphological abnormalities were observed in the embryonic ectoderm of mutant embryos at 7.5 dpc. The length of the most apical cell-cell junctions was reduced, and basolateral surfaces of those cells were separated by multiple gaps. Cells of the embryonic ectoderm were less polarized as assessed by histological criteria and lateral localization of an apical marker. Mutant embryos died by 10 dpc, probably as a result of placental failure. CONCLUSIONS: AF-6 is a critical regulator of cell-cell junctions during mouse development. The loss of neuroepithelial polarity in mutants is consistent with a loss of efficacy of the cell-cell junctions that have a critical role in establishing apical/basolateral asymmetry.