Yes-associated protein 65 localizes p62(c-Yes) to the apical compartment of airway epithelia by association with EBP50

We recently showed that the COOH terminus of the cystic fibrosis transmembrane conductance regulator associates with the submembranous scaffolding protein EBP50 (ERM-binding phosphoprotein 50 kD; also called Na(+)/H(+) exchanger regulatory factor). Since EBP50 associates with ezrin, this interaction links the cystic fibrosis transmembrane conductance regulator (CFTR) to the cortical actin cytoskeleton. EBP50 has two PDZ domains, and CFTR binds with high affinity to the first PDZ domain. Here, we report that Yes-associated protein 65 (YAP65) binds with high affinity to the second EBP50 PDZ domain. YAP65 is concentrated at the apical membrane in airway epithelia and interacts with EBP50 in cells. The COOH terminus of YAP65 is necessary and sufficient to mediate association with EBP50. The EBP50-YAP65 interaction is involved in the compartmentalization of YAP65 at the apical membrane since mutant YAP65 proteins lacking the EBP50 interaction motif are mislocalized when expressed in airway epithelial cells. In addition, we show that the nonreceptor tyrosine kinase c-Yes is contained within EBP50 protein complexes by association with YAP65. Subapical EBP50 protein complexes, containing the nonreceptor tyrosine kinase c-Yes, may regulate apical signal transduction pathways leading to changes in ion transport, cytoskeletal organization, or gene expression in epithelial cells.     

Up-regulation of inducible nitric oxide synthase and nitric oxide in Helicobacter pylori-infected human gastric epithelial cells: possible role of interferon-gamma in polarized nitric oxide secretion

Background. Nitric oxide (NO) generated by nitric oxide synthase (NOS) is known to be an important modulator of the mucosal inflammatory response. In this study, we questioned whether Helicobacter pylori infection could up‐regulate the epithelial cell inducible NOS (iNOS) gene expression and whether NO production could show polarity that can be regulated by immune mediators. Materials and Methods. Human gastric epithelial cell lines were infected with H. pylori, and the iNOS mRNA expression was assessed by quantitative RT‐PCR. NO production was assayed by determining nitrite/nitrate levels in culture supernatants. To determine the polarity of NO secretion by the H. pylori‐infected epithelial cells, Caco‐2 cells were cultured as polarized monolayers in transwell chambers, and NO production was measured. Results. iNOS mRNA levels were significantly up‐regulated in the cells infected with H. pylori, and expression of iNOS protein was confirmed by Western blot analysis. Increased NO production in the gastric epithelial cells was seen as early as 18 hours postinfection, and reached maximal levels by 24 hours postinfection. The specific MAP kinase inhibitors decreased H. pylori‐induced iNOS and NO up‐regulation. After H. pylori infection of polarized epithelial cells, NO was released predominantly into the apical compartment, and IL‐8 was released predominantly into basolateral compartment. The addition of IFN‐γ to H. pylori‐infected polarized epithelial cells showed a synergistically higher apical and basolateral NO release. Conclusion. These results suggest that apical NO production mediated by MAP kinase in H. pylori‐infected gastric epithelial cells may influence the bacteria and basolateral production of NO and IL‐8 may play a role in the tissue inflammation.     

The PDZ-interacting domain of cystic fibrosis transmembrane conductance regulator is required for functional expression in the apical plasma membrane

Polarization of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel to the apical plasma membrane in epithelial cells is critical for vectorial chloride transport. Previously, we reported that the C terminus of CFTR constitutes a PDZ-interacting domain that is required for CFTR polarization to the apical plasma membrane and interaction with the PDZ domain-containing protein EBP50 (NHERF). PDZ-interacting domains are typically composed of the C-terminal three to five amino acids, which in CFTR are QDTRL. Our goal was to identify the key amino acid(s) in the PDZ-interacting domain of CFTR with regard to its apical polarization, interaction with EBP50, and ability to mediate transepithelial chloride secretion. Point substitution of the C-terminal leucine (Leu at position 0) with alanine abrogated apical polarization of CFTR, interaction between CFTR and EBP50, efficient expression of CFTR in the apical membrane, and chloride secretion. Point substitution of the threonine (Thr at position -2) with alanine or valine had no effect on the apical polarization of CFTR, but reduced interaction between CFTR and EBP50, efficient expression of CFTR in the apical membrane as well as chloride secretion. By contrast, individual point substitution of the other C-terminal amino acids (Gln at position -4, Asp at position -3 and Arg at position -1) with alanine had no effect on measured parameters. We conclude that the PDZ-interacting domain, in particular the leucine (position 0) and threonine (position -2) residues, are required for the efficient, polarized expression of CFTR in the apical plasma membrane, interaction of CFTR with EBP50, and for the ability of CFTR to mediate chloride secretion. Mutations that delete the C terminus of CFTR may cause cystic fibrosis because CFTR is not polarized, complexed with EBP50, or efficiently expressed in the apical membrane of epithelial cells.     

The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells †

Here we report on the related TBC/RabGAPs EPI64A and EPI64B and show that they function to organize the apical aspect of epithelial cells. EPI64A binds the scaffolding protein EBP50/NHERF1, which itself binds active ezrin in epithelial cell microvilli. Epithelial cells additionally express EPI64B that also localizes to microvilli. However, EPI64B does not bind EBP50 and both proteins are shown to have a microvillar localization domain that spans the RabGAP domains. CRISPR/Cas9 was used to inactivate expression of each protein individually or both in Jeg-3 and Caco2 cells. In Jeg-3 cells, loss of EPI64B resulted in a reduction of apical microvilli, and a further reduction was seen in the double knockout, mostly likely due to misregulation of Rab8 and Rab35. In addition, apical junctions were partially disrupted in cells lacking EPI64A and accentuated in the double knockout. In Caco2 loss of EPI64B resulted in wavy junctions, whereas loss of both EPI64A and EPI64B had a severe phenotype often resulting in cells with a stellate apical morphology. In the knockout cells, the basal region of the cell remained unchanged, so EPI64A and EPI64B specifically localize to and regulate the morphology of the apical domain of polarized epithelial cells.     

E3KARP mediates the association of ezrin and protein kinase A with the cystic fibrosis transmembrane conductance regulator in airway cells

Although it is generally recognized that cystic fibrosis transmembrane conductance regulator (CFTR) contains a PSD-95/Disc-large/ZO-1 (PDZ)-binding motif at its COOH terminus, the identity of the PDZ domain protein(s) that interact with CFTR is uncertain, and the functional impact of this interaction is not fully understood. By using human airway epithelial cells, we show that CFTR associates with Na(+)/H(+) exchanger (NHE) type 3 kinase A regulatory protein (E3KARP), an EBP50/NHE regulatory factor (NHERF)-related PDZ domain protein. The PDZ binding motif located at the COOH terminus of CFTR interacts preferentially with the second PDZ domain of E3KARP, with nanomolar affinity. In contrast to EBP50/NHERF, E3KARP is predominantly localized (>95%) in the membrane fractions of Calu-3 and T84 cells, where CFTR is located. Moreover, confocal immunofluorescence microscopy of polarized Calu-3 monolayers shows that E3KARP and CFTR are co-localized at the apical membrane domain. We also found that ezrin associates with E3KARP in vivo. Co-expression of CFTR with E3KARP and ezrin in Xenopus oocytes potentiated cAMP-stimulated CFTR Cl(-) currents. These results support the concept that E3KARP functions as a scaffold protein that links CFTR to ezrin. Since ezrin has been shown previously to function as a protein kinase A anchoring protein, we suggest that one function served by the interaction of E3KARP with both ezrin and CFTR is to localize protein kinase A in the vicinity of the R-domain of CFTR. Since ezrin is also an actin-binding protein, the formation of a CFTR.E3KARP.ezrin complex may be important also in stabilizing CFTR at the apical membrane domain of airway cells.     

Identification of EBP50: A PDZ-containing Phosphoprotein that Associates with Members of the Ezrin-Radixin-Moesin Family

Members of the ezrin-radixin-moesin (ERM) family of membrane–cytoskeletal linking proteins have NH₂- and COOH-terminal domains that associate with the plasma membrane and the actin cytoskeleton, respectively. To search for ERM binding partners potentially involved in membrane association, tissue lysates were subjected to affinity chromatography on the immobilized NH₂-terminal domains of ezrin and moesin, which comprise the ezrin-radixin-moesin–association domain (N-ERMAD). A collection of polypeptides at 50–53 kD from human placenta and at 58-59 kD from bovine brain bound directly to both N-ERMADs. The 50–53-kD placental proteins migrated as a major 50-kD species after phosphatase treatment, indicating that the heterogeneity is due to different phosphorylation states. We refer to these polypeptides as ERM-binding phosphoprotein 50 (EBP50). Sequence analysis of human EBP50 was used to identify an ~2-kb human cDNA that encodes a 357-residue polypeptide. Recombinant EBP50 binds tightly to the N-ERMADs of ezrin and moesin. Peptide sequences from the brain candidate indicated that it is closely related to EBP50. EBP50 has two PSD-95/DlgA/ZO-1–like (PDZ) domains and is most likely a homologue of rabbit protein cofactor, which is involved in the protein kinase A regulation of the renal brush border Na⁺/H⁺ exchanger. EBP50 is widely distributed in tissues, and is particularly enriched in those containing polarized epithelia. Immunofluorescence microscopy of cultured cells and tissues revealed that EBP50 colocalizes with actin and ezrin in the apical microvilli of epithelial cells, and immunoelectron microscopy demonstrated that it is specifically associated with the microvilli of the placental syncytiotrophoblast. Moreover, EBP50 and ezrin can be coimmunoprecipitated as a complex from isolated human placental microvilli. These findings show that EBP50 is a physiologically relevant ezrin binding protein. Since PDZ domains are known to mediate associations with integral membrane proteins, one mode of membrane attachment of ezrin is likely to be mediated through EBP50.     

A CD317/tetherin–RICH2 complex plays a critical role in the organization of the subapical actin cytoskeleton in polarized epithelial cells

CD317/tetherin is a lipid raft–associated integral membrane protein with a novel topology. It has a short N-terminal cytosolic domain, a conventional transmembrane domain, and a C-terminal glycosyl-phosphatidylinositol anchor. We now show that CD317 is expressed at the apical surface of polarized epithelial cells, where it interacts indirectly with the underlying actin cytoskeleton. CD317 is linked to the apical actin network via the proteins RICH2, EBP50, and ezrin. Knocking down expression of either CD317 or RICH2 gives rise to the same phenotype: a loss of the apical actin network with concomitant loss of apical microvilli, an increase in actin bundles at the basal surface, and a reduction in cell height without any loss of tight junctions, transepithelial resistance, or the polarized targeting of apical and basolateral membrane proteins. Thus, CD317 provides a physical link between lipid rafts and the apical actin network in polarized epithelial cells and is crucial for the maintenance of microvilli in such cells.     

Epstein-Barr virus infection of polarized tongue and nasopharyngeal epithelial cells

Epstein-Barr virus (EBV) initially enters the body through the oropharyngeal mucosa and subsequently infects B lymphocytes through their CD21 (CR2) complement receptor. Mechanisms of EBV entry into and release from epithelial cells are poorly understood. To study EBV infection in mucosal oropharyngeal epithelial cells, we established human polarized tongue and pharyngeal epithelial cells in culture. We show that EBV enters these cells through three CD21-independent pathways: (i) by direct cell-to-cell contact of apical cell membranes with EBV-infected lymphocytes; (ii) by entry of cell-free virions through basolateral membranes, mediated in part through an interaction between beta1 or alpha5beta1 integrins and the EBV BMRF-2 protein; and (iii) after initial infection, by virus spread directly across lateral membranes to adjacent epithelial cells. Release of progeny virions from polarized cells occurs from both their apical and basolateral membranes. These data indicate that multiple approaches to prevention of epithelial infection with EBV will be necessary.     

Binding of CAP70 to inducible nitric oxide synthase and implications for the vectorial release of nitric oxide in polarized cells

In this article we analyze the mechanisms by which the C-terminal four amino acids of inducible nitric oxide synthase (NOS2) interact with proteins that contain PDZ (PSD-95/DLG/ZO-1) domains resulting in the translocation of NOS2 to the cellular apical domain. It has been reported that human hepatic NOS2 associates to EBP50, a protein with two PDZ domains present in epithelial cells. We describe herein that NOS2 binds through its four carboxy-terminal residues to CAP70, a protein that contains four PDZ modules that is targeted to apical membranes. Interestingly, this interaction augments both the cytochrome c reductase and .NO-synthase activities of NOS2. Binding of CAP70 to NOS2 also results in an increase in the population of active NOS2 dimers. In addition, CAP70 participates in the correct subcellular targeting of NOS2 in a process that is also dependent on the acylation state of the N-terminal end of NOS2. Hence, nonpalmitoylated NOS2 is unable to progress toward the apical side of the cell despite its interaction with either EBP50 or CAP70. Likewise, if we abrogate the interaction of NOS2 with either EBP50 or CAP70 by fusing the GFP reporter to the carboxy-terminal end of NOS2 palmitoylation is not sufficient to confer an apical targeting.     

Interaction of carboxyl-terminal peptides of cytosolic-tail of apactin with PDZ domains of NHERF/EBP50 and PDZK-1/CAP70

The C-terminal PDZ-binding motifs are required for polarized apical/basolateral localization of many membrane proteins. Ezrin–radixin–moesin (ERM) proteins regulate the organization and function of specific cortical structures in polarized epithelial cells by connecting filamentous (F)-actin to plasma membrane proteins through EBP50. Previous work showed that the membrane phosphoprotein apactin (an 80-kDa type I membrane protein derived from pro-Muclin) is associated with the acinar cell apical actin cytoskeleton and that this association is modulated by changes in the phosphorylation state of the apactin cytosolic tail. The carboxyl-terminal amino acids of apactin (–STKL–COOH) are predicted to form a type I PDZ-binding domain, similar to that of CFTR (–DTRL–COOH). Pairwise sequence comparison between NHERF/EBP50 and PDZK1/CAP70 PDZ domains reveals significant identity among the 83 amino-acid residues (12–92) of EBP50 and CAP70 (241–323), which are involved in the interaction with the carboxyl-terminal peptides (STKL–COOH and phosphomimetics) of apactin. Hence, the specificity and affinity of interactions are identical between them, which is corroborated with the two hybrid results. Substitution of all the four-carboxyl-terminal amino acids in the wild type to Ala reduces the interaction. Only the carbonyl oxygen and amide nitrogen of Ala are found to be involved in hydrogen bonding. Further, truncation of the wild carboxyl-terminal peptide to RGQPP–COOH, showed very low affinity of interaction with PDZ1 domain. Only the atom O^ε1 of Gln-2 hydrogen bonds with N^ε2 of His72 of PDZ domain. Ser-3 amino acid in wild type apactin protein (STKL–COOH) is not involved in hydrogen bonding with PDZ1 domain. However, substitution of Ser-3 to Asp-3 in PDTKL–COOH peptide increases the affinity of interaction of PDTKL–COOH with PDZ1 domain. Thus, carboxyl-terminal Asp(D) -3, Thr(T) -2, Lys(K) -1 and Leu(L) 0 are involved in numerous interactions with PDZ1 domains of NHERF/EBP50 and PDZK1/CAP70.     

The scaffolding protein EBP50 regulates microvillar assembly in a phosphorylation-dependent manner

The mechanisms by which epithelial cells regulate the presence of microvilli on their apical surface are largely unknown. A potential regulator is EBP50/NHERF1 (ERM-binding phosphoprotein of 50 kD/Na(+)-H(+) exchanger regulatory factor), a microvillar scaffolding protein with two PDZ domains followed by a C-terminal ezrin-binding domain. Using RNAi and expression of RNAi-resistant EBP50 mutants we systematically show that EBP50 is necessary for microvillar assembly and requires that EBP50 has both a functional first PDZ domain and an ezrin-binding site. Expression of mutants mimicking Cdc2 or PKC phosphorylation are nonfunctional in microvillar assembly. Biochemical analysis reveals that these mutants are defective in PDZ1 accessibility when PDZ2 is occupied, and can be rendered functional in vivo by additional mutation of PDZ2. EBP50 is not necessary for mitotic cell microvilli, and PKC activation causes a rearrangement of microvilli on cells due to phosphorylation-dependent loss of EBP50 function. Thus, EBP50 is a critical factor that regulates microvilli assembly and whose activity is regulated by signaling pathways and occupation of its PDZ2 domain.     

NHERF1/EBP50 head-to-tail intramolecular interaction masks association with PDZ domain ligands

Loss of cell polarity is one of the initial alterations in the development of human epithelial cancers. Na(+)/H(+) exchanger regulatory factor (NHERF) homologous adaptors 1 and 2 are membrane-associated proteins composed of two amino (N)-terminal PDZ domains and an ezrin-radixin-moesin (ERM)-binding (EB) carboxyl (C)-terminal region. We describe here an intramolecular conformation of NHERF1/EBP50 (ERM-binding phosphoprotein 50) in which the C-terminal EB region binds to the PDZ2 domain. This novel head-to-tail conformation masked the interaction of both PDZ domains with PDZ domain-specific ligands, such as PTEN and beta-catenin. An EB region composite structure comprising an alpha-helix ending in a PDZ-binding motif imparted opposite effects to NHERF1 associations, mediating binding to ERM proteins and inhibiting binding of PDZ domain ligands. The PDZ domain inhibition was released by prior association of ezrin with the EB region, a condition that occurs in vivo and likely disrupts NHERF1 head-to-tail interaction. In contrast, NHERF2 did not present a regulatory mechanism for protein complex formation. Functionally, NHERF1 is required to organize complexes at the apical membranes of polarized epithelial cells. The regulation of NHERF1 interactions at the apical membrane thus appears to be a dynamic process that is important for maintaining epithelial-tissue integrity.     

Mechanism of inducible nitric oxide synthase exclusion from mycobacterial phagosomes

Mycobacterium tuberculosis is sensitive to nitric oxide generated by inducible nitric oxide synthase (iNOS). Consequently, to ensure its survival in macrophages, M. tuberculosis inhibits iNOS recruitment to its phagosome by an unknown mechanism. Here we report the mechanism underlying this process, whereby mycobacteria affect the scaffolding protein EBP50, which normally binds to iNOS and links it to the actin cytoskeleton. Phagosomes harboring live mycobacteria showed reduced capacity to retain EBP50, consistent with lower iNOS recruitment. EBP50 was found on purified phagosomes, and its expression increased upon macrophage activation, paralleling expression changes seen with iNOS. Overexpression of EBP50 increased while EBP50 knockdown decreased iNOS recruitment to phagosomes. Knockdown of EBP50 enhanced mycobacterial survival in activated macrophages. We tested another actin organizer, coronin-1, implicated in mycobacterium-macrophage interaction for contribution to iNOS exclusion. A knockdown of coronin-1 resulted in increased iNOS recruitment to model latex bead phagosomes but did not increase iNOS recruitment to phagosomes with live mycobacteria and did not affect mycobacterial survival. Our findings are consistent with a model for the block in iNOS association with mycobacterial phagosomes as a mechanism dependent primarily on reduced EBP50 recruitment.     

Apical membrane targeting and trafficking of the human proton-coupled transporter in polarized epithelia

The human proton-coupled folate transporter (hPCFT) is a recently discovered intestinal transporter involved in folate uptake in epithelia (and possibly other cells). Little is currently known about the structure-function relationship of the different domains of this transporter, particularly which regions are important for substrate transport as well as targeting of the transporter to the apical cell surface of polarized cells. Here we have investigated the role of the COOH-terminal domain and a well-conserved sequence separating transmembrane (TM) domains TM2 and TM3 (DXXGRR; amino acids 109-114) speculated by others to be important for transport function. Using live cell imaging approaches, we show that 1) an hPCFT-yellow fluorescent protein construct is functionally expressed at the apical membrane domain and is localized differentially to the human reduced folate carrier; 2) the predicted cytoplasmic COOH-terminal region of hPCFT is not essential for apical targeting or transporter functionality; 3) mutations that ablate a consensus beta-turn sequence separating predicted TM2 and TM3 abolished apical [(3)H]folic acid uptake as a consequence of endoplasmic reticulum retention of mutant, likely misfolded, transporters; and 4) cell surface delivery of hPCFT is disrupted by microtubule depolymerization or by overexpression of the dynactin complex dynamitin (p50). For the first time, our data present information regarding structure-function and membrane targeting of the hPCFT polypeptide, as well as the mechanisms that control its steady-state expression in polarized cells.     

The Cytoplasmic Tail of Rhodopsin Acts as a Novel Apical Sorting Signal in Polarized MDCK Cells

All basolateral sorting signals described to date reside in the cytoplasmic domain of proteins, whereas apical targeting motifs have been found to be lumenal. In this report, we demonstrate that wild-type rhodopsin is targeted to the apical plasma membrane via the TGN upon expression in polarized epithelial MDCK cells. Truncated rhodopsin with a deletion of 32 COOH-terminal residues shows a nonpolar steady-state distribution. Addition of the COOH-terminal 39 residues of rhodopsin redirects the basolateral membrane protein CD7 to the apical membrane. Fusion of rhodopsin''s cytoplasmic tail to a cytosolic protein glutathione S-transferase (GST) also targets this fusion protein (GST–Rho39Tr) to the apical membrane. The targeting of GST–Rho39Tr requires both the terminal 39 amino acids and the palmitoylation membrane anchor signal provided by the rhodopsin sequence. The apical transport of GST–Rho39Tr can be reversibly blocked at the Golgi complex by low temperature and can be altered by brefeldin A treatment. This indicates that the membrane-associated GST–Rho39Tr protein may be sorted along a yet unidentified pathway that is similar to the secretory pathway in polarized MDCK cells. We conclude that the COOH-terminal tail of rhodopsin contains a novel cytoplasmic apical sorting determinant. This finding further indicates that cytoplasmic sorting machinery may exist in MDCK cells for some apically targeted proteins, analogous to that described for basolaterally targeted proteins.     

Gp135/podocalyxin and NHERF-2 participate in the formation of a preapical domain during polarization of MDCK cells

Epithelial polarization involves the segregation of apical and basolateral membrane domains, which are stabilized and maintained by tight junctions and membrane traffic. We report that unlike most apical and basolateral proteins in MDCK cells, which separate only after junctions have formed, the apical marker gp135 signifies an early level of polarized membrane organization established already in single cells. We identified gp135 as the dog orthologue of podocalyxin. With a series of domain mutants we show that the COOH-terminal PSD-95/Dlg/ZO-1 (PDZ)-binding motif is targeting podocalyxin to the free surface of single cells as well as to a subdomain of the terminally polarized apical membrane. This special localization of podocalyxin is shared by the cytoplasmic PDZ-protein Na+/H+ exchanger regulatory factor (NHERF)-2. Depleting podocalyxin by RNA interference caused defects in epithelial polarization. Together, our data suggest that podocalyxin and NHERF-2 function in epithelial polarization by contributing to an early apical scaffold based on PDZ domain-mediated interactions.     

Interaction with decay-accelerating factor facilitates coxsackievirus B infection of polarized epithelial cells

All coxsackie B (CB) viruses can initiate infection by attaching to the coxsackievirus and adenovirus receptor (CAR). Although some CB isolates also bind to decay-accelerating factor (DAF), the role of DAF interaction during infection remains uncertain. We recently observed that CAR in polarized epithelial cells is concentrated at tight junctions, where it is relatively inaccessible to virus. In the experiments reported here we found that, unlike CAR, DAF was present on the apical surface of polarized cells and that DAF-binding isolates of CB3 and CB5 infected polarized epithelial cells more efficiently than did isolates incapable of attaching to DAF. Virus attachment and subsequent infection of polarized cells by DAF-binding isolates were prevented in the presence of anti-DAF antibody. Serial passage on polarized cell monolayers selected for DAF-binding virus variants. Taken together, these results indicate that interaction with DAF on the apical surface of polarized epithelial cells facilitates infection by a subset of CB virus isolates. The results suggest a possible role for DAF in infection of epithelial cells at mucosal surfaces.     

The keratin-binding protein Albatross regulates polarization of epithelial cells

The keratin intermediate filament network is abundant in epithelial cells, but its function in the establishment and maintenance of cell polarity is unclear. Here, we show that Albatross complexes with Par3 to regulate formation of the apical junctional complex (AJC) and maintain lateral membrane identity. In nonpolarized epithelial cells, Albatross localizes with keratin filaments, whereas in polarized epithelial cells, Albatross is primarily localized in the vicinity of the AJC. Knockdown of Albatross in polarized cells causes a disappearance of key components of the AJC at cell–cell borders and keratin filament reorganization. Lateral proteins E-cadherin and desmoglein 2 were mislocalized even on the apical side. Although Albatross promotes localization of Par3 to the AJC, Par3 and ezrin are still retained at the apical surface in Albatross knockdown cells, which retain intact microvilli. Analysis of keratin-deficient epithelial cells revealed that keratins are required to stabilize the Albatross protein, thus promoting the formation of AJC. We propose that keratins and the keratin-binding protein Albatross are important for epithelial cell polarization.     

Characterization of intermediate filaments and their structural organization during epithelium formation in pigmented epithelial cells of the retina in vitro

Summary Retinal pigmented epithelial cells of chicken have circumferential microfilament bundles (CMBs) at the zonula adherens region. Isolated CMBs are polygons filled with a meshwork composed primarily of intermediate filaments; they show three major components of 200000, 55000, and 42000 daltons in SDS-gel electrophoresis. Here we have characterized the 55000-dalton protein immunochemically and ultrastructurally. Immunoblotting and immunofluorescence microscopy have shown that the 55000-dalton protein is an intermediate filament protein, vimentin.Vimentin filaments changed their distribution during differentiation of pigmented epithelial cells in culture. The protein in the elongated cells showed a fibroblast-type pattern of intermediate filaments. During epithelium formation, the filaments were uniformly distributed and formed a finer meshwork at the apical level. In pigmented epithelial cells that differentiated and matured in culture, vimentin and actin exhibited their characteristic behavior after treatment with colcemid. In the central to basal region of the cell, intermediate filaments formed thick perinuclear bundles. In the apical region, however, intermediate filaments changed in organization from a nonpolarized meshwork to a polarized bundle-like structure. Simultaneously, new actin bundles were formed, running parallel to the intermediate filaments. This suggests that there is some interaction between microfilaments and intermediate filaments in the apical region of these cells.