G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
tight junction protein 1 (zona occludens 1)
G00000122 (Mus musculus)

Databases (8)

ENSG00000104067 (Ensembl human gene)
7082 (Entrez Gene)
31 (G2Cdb plasticity & disease)
TJP1 (GeneCards)
601009 (OMIM)
Marker Symbol
HGNC:11827 (HGNC)
Protein Expression
1636 (human protein atlas)
Protein Sequence
Q07157 (UniProt)

Synonyms (3)

  • DKFZp686M05161
  • MGC133289
  • ZO-1

Diseases (1)

Disease Nervous effect Mutations Found Literature Mutations Type Genetic association?
D00000272: Albuminuria N Y (17039425) Polymorphism (P) N
D00000272: Albuminuria N Y (17039425) Single nucleotide polymorphism (SNP) N


  • Evaluation of tight junction protein 1 encoding zona occludens 1 as a candidate gene for albuminuria in a Mexican American population.

    Lehman DM, Leach RJ, Johnson-Pais T, Hamlington J, Fowler S, Almasy L, Duggirala R, Stern MP and Abboud HE

    Department of Medicine/Clinical Epidemiology, University of Texas Health Science Center, San Antonio, Texas 78229, USA. lehman@uthscsa.edu

    Albuminuria, a hallmark of diabetic nephropathy, has been shown to be significantly heritable in multiple studies. Therefore, the identification of genes that affect susceptibility to albuminuria may lead to novel avenues of intervention. Current evidence suggests that the podocyte and slit diaphragm play a key role in controlling the selective sieve of the glomerular filtration barrier, and podocyte-specific genes have been identified that are necessary for maintaining its integrity. We therefore investigated the role of gene variants of tight junction protein (TJP1) which encodes another slit diaphragm-associated protein zona occludens 1 as risk factors for albuminuria in the San Antonio Family Diabetes/Gallbladder Study (SAFDGS), which consists of extended Mexican-American families with a high prevalence of type 2 diabetes. Albuminuria, defined as an albumin (mg/dl) to creatinine (mg/dl) ratio (ACR) of 0.03, which is approximately equivalent to a urinary albumin excretion (UAE) >30 mg/day, was present in a total of 14.9% of participants, and 31% had type 2 diabetes. The TJP1 exons, flanking intronic sequence, and putative proximal promoter regions were investigated in this population. Twentynine polymorphisms, including 7 nonsynonymous SNPs, were identified and genotyped in all subjects of this study for association analysis. Three sets of correlated SNPs, which include 3 exonic SNPs, were nominally associated with ACR (p value range 0.007-0.049); however, the association with the discrete trait albuminuria was not significant (p value range 0.094-0.338). We conclude that these variants in TJP1 do not appear to be major determinants for albuminuria in the SAFDGS; however, they may play a minor role in its severity in this Mexican-American population. Further examination of the TJP1 gene region in this and other cohorts will be useful to determine whether ZO-1 plays a significant role in glomerular permselectivity.

    Funded by: NIDDK NIH HHS: P50DK061597, R01-DK-42273, R01-DK-47482, R01-DK-53889; NIMH NIH HHS: MH-59490

    Experimental and clinical endocrinology & diabetes : official journal, German Society of Endocrinology [and] German Diabetes Association 2006;114;8;432-7

Literature (106)

Pubmed - other

  • The gap junction protein connexin43 interacts with the second PDZ domain of the zona occludens-1 protein.

    Giepmans BN and Moolenaar WH

    Division of Cellular Biochemistry The Netherlands Cancer Institute Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.

    Gap junctions mediate cell-cell communication in almost all tissues and are composed of channel-forming integral membrane proteins, termed connexins [1-3]. 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 [3-5], although the underlying mechanisms are poorly understood as cellular proteins that interact with connexins have yet to be identified. The carboxy-terminal cytosolic domain of Cx43 contains several phosphorylation sites and potential signalling motifs. We have used a yeast two-hybrid protein interaction screen to identify proteins that bind to the carboxy-terminal tail of Cx43 and thereby isolated the zona occludens-1 (ZO-1) protein. 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 [6-9]. The interaction of Cx43 with ZO-1 occurred through the extreme carboxyl terminus of Cx43 and the second PDZ domain of ZO-1. Cx43 associated with ZO-1 in Cx43-transfected COS7 cells, as well as endogenously in normal Rat-1 fibroblasts and mink lung epithelial cells. Confocal microscopy revealed that endogenous Cx43 and ZO-1 colocalised at gap junctions. We suggest that ZO-1 serves to recruit signalling proteins into Cx43-based gap junctions.

    Current biology : CB 

  • The PDZ2 domain of zonula occludens-1 and -2 is a phosphoinositide binding domain.

    Meerschaert K, Tun MP, Remue E, De Ganck A, Boucherie C, Vanloo B, Degeest G, Vandekerckhove J, Zimmermann P, Bhardwaj N, Lu H, Cho W and Gettemans J

    Department of Medical Protein Research, VIB, 9000 Ghent, Belgium.

    Zonula occludens proteins (ZO) are postsynaptic density protein-95 discs large-zonula occludens (PDZ) domain-containing proteins that play a fundamental role in the assembly of tight junctions and establishment of cell polarity. Here, we show that the second PDZ domain of ZO-1 and ZO-2 binds phosphoinositides (PtdInsP) and we identified critical residues involved in the interaction. Furthermore, peptide and PtdInsP binding of ZO PDZ2 domains are mutually exclusive. Although lipid binding does not seem to be required for plasma membrane localisation of ZO-1, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P (2)) binding to the PDZ2 domain of ZO-2 regulates ZO-2 recruitment to nuclear speckles. Knockdown of ZO-2 expression disrupts speckle morphology, indicating that ZO-2 might play an active role in formation and stabilisation of these subnuclear structures. This study shows for the first time that ZO isoforms bind PtdInsPs and offers an alternative regulatory mechanism for the formation and stabilisation of protein complexes in the nucleus.

    Funded by: NIGMS NIH HHS: GM68849, R01 GM068849

    Cellular and molecular life sciences : CMLS 2009;66;24;3951-66

  • Dislocation of Rab13 and vasodilator-stimulated phosphoprotein in inactive colon epithelium in patients with Crohn's disease.

    Ohira M, Oshitani N, Hosomi S, Watanabe K, Yamagami H, Tominaga K, Watanabe T, Fujiwara Y, Maeda K, Hirakawa K and Arakawa T

    Department of Gastroenterology, Osaka City University, Graduate School of Medicine, Osaka, Japan.

    Crohn's disease is associated with increased permeability of the intestine even in quiescent patients. Increased intestinal permeability may cause dysregulated immunological responses in the intestinal mucosa that leads to chronic intestinal inflammation. Tight junction proteins contribute to intestinal permeability, and functional abnormality and dislocation of such proteins may cause increased intestinal permeability. We studied the expression of tight junction proteins Rab13, vasodilator-stimulated phosphoprotein (VASP), zonula occludin-1 (ZO-1), and F-actin in the intestinal epithelium of patients with inactive inflammatory bowel disease. Surgical samples were obtained from 10 controls (without inflammatory bowel disease), 10 patients with Crohn's disease and 7 patients with ulcerative colitis. F-actin was visualized with fluorescent phalloidin. Tight junction proteins were visualized by an immunofluorescence method. Rab13, VASP, and ZO-1 were found in apical tight junctions in normal epithelium but were dislocated to the basolateral position in patients with inactive Crohn's disease, whereas the structure of F-actin was maintained in inactive mucosa. In patients with ulcerative colitis, these tight junction proteins were not dislocated. Latent dislocation of tight junction proteins in the inactive mucosa of patients with Crohn's disease may permit the invasion of gut antigens to initiate and perpetuate altered immune response.

    International journal of molecular medicine 2009;24;6;829-35

  • Localization of cortactin is associated with colorectal cancer development.

    Hirakawa H, Shibata K and Nakayama T

    Department of Tumor and Diagnostic Pathology, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523, Japan. h-hira@nagasaki-u.ac.jp

    Cortactin is a ubiquitously expressed actin filament (F-actin)-binding protein that stabilizes F-actin networks and promotes actin polymerization by activating the actin-related protein 2/3 (Arp2/3) complex. Overexpression of cortactin in cancer cells stimulate cell migration, invasion, and experimental metastasis; however, the underlying mechanism in cortactin involvement in tumor progression is not fully understood. Recently, a direct interaction between zonula occludens-1 (ZO-1) and cortactin in epithelial cells was reported. The present study aimed to further clarify the significance of the interaction between cortactin and ZO-1 in cancer progression. Cortactin expression and localization in colorectal human cancer tissues were evaluated by immunohistochemistry and immunofluorescence. Co-immunoprecipitation and immunofluorescence analysis revealed cortactin and ZO-1 interaction and localization in cancer cells. In our study, the localization of cortactin is a crucial marker for lymph node metastasis in colorectal cancer. We show how the localization of cortactin effects cancer development. A molecular interaction between cortactin and ZO-1 in migrating or polarized cancer cells was revealed. This is the first report to show the interaction of cortactin and ZO-1 in colorectal cancer progression. We conclude that localization of cortactin in cancer cells and interaction between ZO-1 and cortactin are crucial for cancer progression.

    International journal of oncology 2009;35;6;1271-6

  • Alteration of tight junction proteins is an early event in psoriasis: putative involvement of proinflammatory cytokines.

    Kirschner N, Poetzl C, von den Driesch P, Wladykowski E, Moll I, Behne MJ and Brandner JM

    Department of Dermatology and Venerology, University Hospital Hamburg-Eppendorf, Martinistrasse 52, Hamburg, Germany.

    Psoriasis is an inflammatory skin disease characterized by hyperproliferation of keratinocytes, impaired barrier function, and pronounced infiltration of inflammatory cells. Tight junctions (TJs) are cell-cell junctions that form paracellular barriers for solutes and inflammatory cells. Altered localization of TJ proteins in the epidermis was described in plaque-type psoriasis. Here we show that localization of TJ proteins is already altered in early-stage psoriasis. Occludin, ZO-1, and claudin-4 are found in more layers than in normal epidermis, and claudin-1 and -7 are down-regulated in the basal and in the uppermost layers. In plaque-type psoriasis, the staining patterns of occludin and ZO-1 do not change, whereas the claudins are further down-regulated. Near transmigrating granulocytes, all TJ proteins except for junctional adhesion molecule-A are down-regulated. Treatment of cultured keratinocytes with interleukin-1beta and tumor necrosis factor-alpha, which are present at elevated levels in psoriatic skin, results in an increase of transepithelial resistance at early time points and a decrease at later time points. Injection of interleukin-1beta into an ex vivo skin model leads to an up-regulation of occludin and ZO-1, resembling TJ protein alteration in early psoriasis. Our results show for the first time that alteration of TJ proteins is an early event in psoriasis and is not the consequence of the more profound changes found in plaque-type psoriasis. Our data indicate that cytokines are involved in alterations of TJ proteins observed in psoriasis.

    The American journal of pathology 2009;175;3;1095-106

  • Zonula occludens-1, occludin, and E-cadherin protein expression in biliary tract cancers.

    Németh Z, Szász AM, Somorácz A, Tátrai P, Németh J, Gyorffy H, Szíjártó A, Kupcsulik P, Kiss A and Schaff Z

    2nd Department of Pathology, Semmelweis University, Ulloi út 93, H-1091 Budapest, Hungary.

    The incidence of cholangiocarcinomas originating from intrahepatic and extrahepatic bile ducts, as well as of gallbladder carcinoma is increasing worldwide. The malignant transformation of biliary epithelia involves profound alterations of proteins in the intercellular junctions, among others zonula occludens-1 (ZO-1), occludin, and E-cadherin. Each plays important role in the maintenance of epithelial cell polarity and regulation of cell growth and differentiation. Our aim was to investigate ZO-1, occludin, and E-cadherin immunohistochemical reactions in tissue microarray blocks containing 57 normal and 62 neoplastic biliary tract samples. We demonstrated that the tight junction components ZO-1, occludin, and E-cadherin are downregulated in carcinomas arising from various compartments of the biliary tract (normal intrahepatic and extrahepatic bile ducts, gallbladder) as compared with their normal sites of origin. These results were confirmed by discriminant analysis yielding clear separation of the three normal sample groups from carcinomas in the corresponding locations.

    Pathology oncology research : POR 2009;15;3;533-9

  • Up-regulation of the tight-junction protein ZO-1 by substance P and IGF-1 in A431 cells.

    Ko JA, Murata S and Nishida T

    Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Ube City, Yamaguchi, Japan. jiae0831@yamaguchi-u.ac.jp

    The formation of a barrier by tight junctions is important in epithelia of various tissues. Substance P (SP) and insulin-like growth factor (IGF)-1 synergistically promote barrier function in the corneal epithelium. We have now examined the effects of SP and IGF-1 on expression of the tight-junction protein zonula occludens (ZO)-1 in A431 human epidermoid carcinoma cells. Reverse transcription-polymerase chain reaction (RT-PCR) and immunoblot analyses revealed that SP and IGF-1 increased the amounts of ZO-1 mRNA and protein in these cells in a concentration-dependent manner, with neither SP nor IGF-1 alone having such an effect. The SP- and IGF-1-induced up-regulation of ZO-1 was accompanied by phosphorylation of extracellular signal-regulated kinase (ERK), and both of these effects were blocked by PD98059, an inhibitor of ERK activation. SP and IGF-1 also increased the transepithelial electrical resistance (TER) (an indicator of barrier function) of an A431 cell monolayer in a manner sensitive to PD98059. Our results thus suggest that the synergistic induction of ZO-1 expression by SP and IGF-1 may promote barrier function in skin epithelial cells.

    Cell biochemistry and function 2009;27;6;388-94

  • Defining the human deubiquitinating enzyme interaction landscape.

    Sowa ME, Bennett EJ, Gygi SP and Harper JW

    Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.

    Deubiquitinating enzymes (Dubs) function to remove covalently attached ubiquitin from proteins, thereby controlling substrate activity and/or abundance. For most Dubs, their functions, targets, and regulation are poorly understood. To systematically investigate Dub function, we initiated a global proteomic analysis of Dubs and their associated protein complexes. This was accomplished through the development of a software platform called CompPASS, which uses unbiased metrics to assign confidence measurements to interactions from parallel nonreciprocal proteomic data sets. We identified 774 candidate interacting proteins associated with 75 Dubs. Using Gene Ontology, interactome topology classification, subcellular localization, and functional studies, we link Dubs to diverse processes, including protein turnover, transcription, RNA processing, DNA damage, and endoplasmic reticulum-associated degradation. This work provides the first glimpse into the Dub interaction landscape, places previously unstudied Dubs within putative biological pathways, and identifies previously unknown interactions and protein complexes involved in this increasingly important arm of the ubiquitin-proteasome pathway.

    Funded by: NIA NIH HHS: AG085011, R01 AG011085, R01 AG011085-16; NIDDK NIH HHS: K01 DK098285; NIGMS NIH HHS: GM054137, GM67945, R01 GM054137, R01 GM054137-14, R01 GM067945

    Cell 2009;138;2;389-403

  • Characterization of the ubinuclein protein as a new member of the nuclear and adhesion complex components (NACos).

    Aho S, Lupo J, Coly PA, Sabine A, Castellazzi M, Morand P, Sergeant A, Manet E, Boyer V and Gruffat H

    Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA, USA.

    We characterized previously a cellular protein through its interaction with cellular and viral transcription factors from the bZip family. The corresponding mRNA was detected in a wide range of cell types and the protein was highly expressed in the nucleus of human keratinocytes. On the basis of these observations, we named this protein ubinuclein.

    Results: Using a specific monoclonal antibody, we have shown in the present study that, although endogenous ubinuclein was mainly nuclear in sparse MDCK (Madin-Darby canine kidney) cells, it was exclusively present in the cell-cell junctions in confluent MDCK cultures or in polarized HT29 cells, where it co-localized with the tight junction marker ZO-1 (zonula occludens 1). In accordance with this, we have shown that ubinuclein interacted with ZO-1 in vitro and in vivo. In cultures of undifferentiated human keratinocytes, ubinuclein was essentially nuclear, but in differentiated cells, in which involucrin and periplakin reside at the apical cell membrane and at the cell-cell junctions, ubinuclein staining was observed at the lateral cell-cell borders. In human skin, ubinuclein appeared as a thread-like pattern between the upper granular cell layer and the cornified cell layer. In mouse epithelia, including bile canaliculi, bronchioli, salivary gland ducts, and oral and olfactory epithelium, ubinuclein co-localized with tight junction markers. Ubinuclein was, however, not present in endothelial cell-cell junctions. In addition, when overexpressed, ubinuclein localized to the nucleus and prevented MDCK cells from entering cytokinesis, resulting in multinucleated giant cells after several cycles of endoreplication.

    Conclusions: Ubinuclein mRNA and its corresponding protein are expressed in almost all cell types. Analyses have revealed that in most cells ubinuclein occurred in the nucleoplasm, but in cells forming tight junctions it is recruited to the plaque structure of the zonula occludens. This recruitment appeared to be dependent on cell density. Therefore ubinuclein is a new NACos (nuclear and adhesion complex component) protein.

    Funded by: NIAMS NIH HHS: R01 AR33588

    Biology of the cell 2009;101;6;319-34

  • Density-enhanced phosphatase 1 regulates phosphorylation of tight junction proteins and enhances barrier function of epithelial cells.

    Sallee JL and Burridge K

    Department of Cell and Developmental Biology and Lineberger Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA. jennifer_sallee@med.unc.edu

    Cell-cell adhesion is a dynamic process that can activate multiple signaling pathways. These signaling pathways can be regulated through reversible tyrosine phosphorylation events. The level of tyrosine phosphorylation of junctional proteins reflects the balance between protein-tyrosine kinase and protein-tyrosine phosphatase activity. The receptor-tyrosine phosphatase DEP-1 (CD148/PTP-eta) has been implicated in cell growth and differentiation as well as in regulating phosphorylation of junctional proteins. However, the role of DEP-1 in regulating tight junction phosphorylation and the integrity of cell-cell junctions is still under investigation. In this study, we used a catalytically dead substrate-trapping mutant of DEP-1 to identify potential substrates at cell-cell junctions. We have shown that in epithelial cells the trapping mutant of DEP-1 interacts with the tight junction proteins occludin and ZO-1 in a tyrosine phosphorylation-dependent manner. In contrast, PTP-PEST, Shp2, and PTPmu did not interact with these proteins, suggesting that the interaction of DEP-1 with occludin and ZO-1 is specific. In addition, occludin and ZO-1 were dephosphorylated by DEP-1 but not these other phosphatases in vitro. Overexpression of DEP-1 increased barrier function as measured by transepithelial electrical resistance and also reduced paracellular flux of fluorescein isothiocyanate-dextran following a calcium switch. Reduced DEP-1 expression by small interfering RNA had a small but significant increase in junction permeability. These data suggest that DEP-1 can modify the phosphorylation state of tight junction proteins and play a role in regulating permeability.

    Funded by: NHLBI NIH HHS: HL45100; NIGMS NIH HHS: GM29860

    The Journal of biological chemistry 2009;284;22;14997-5006

  • Flavivirus NS5 associates with host-cell proteins zonula occludens-1 (ZO-1) and regulating synaptic membrane exocytosis-2 (RIMS2) via an internal PDZ binding mechanism.

    Ellencrona K, Syed A and Johansson M

    School of Life Sciences, Södertörn University, S-14189 Huddinge, Sweden and Department of Genetics, Microbiology and Toxicology, Stockholm University, S-106 91, Stockholm, Sweden.

    Dengue virus (DENV) and tick-borne encephalitis virus (TBEV) are flaviviruses, which can cause lethal hemorrhagic fever and encephalitis, respectively. Here, we demonstrate that the TBEV-NS5 and DENV-NS5 proteins use an internal binding mechanism to target human PDZ proteins. TBEV-NS5 has high affinity to regulating synaptic membrane exocytosis-2 (RIMS2) and Scribble, whereas DENV-NS5 binds primarily to the tight junction protein zonula occludens-1 (ZO-1). Targeting of TBEV-NS5 to the plasma membrane is stabilised by ZO-1; however, DENV-NS5 co-localises with ZO-1 in the nucleus. These interactions have potential important roles in the ability of flaviviruses to manipulate cell proliferation, junction permeability and the interferon pathways.

    Biological chemistry 2009;390;4;319-23

  • Muscarinic-induced recruitment of plasma membrane Ca2+-ATPase involves PSD-95/Dlg/Zo-1-mediated interactions.

    Kruger WA, Yun CC, Monteith GR and Poronnik P

    School of Biomedical Sciences and School of Pharmacy, The University of Queensland, Brisbane QLD 4072, Australia.

    Efflux of cytosolic Ca2+ mediated by plasma membrane Ca2+-ATPases (PMCA) plays a key role in fine tuning the magnitude and duration of Ca2+ signaling following activation of G-protein-coupled receptors. However, the molecular mechanisms that underpin the trafficking of PMCA to the membrane during Ca2+ signaling remain largely unexplored in native cell models. One potential mechanism for the recruitment of proteins to the plasma membrane involves PDZ interactions. In this context, we investigated the role of PMCA interactions with the Na+/H+ exchanger regulatory factor 2 (NHERF-2) during muscarinic-induced Ca2+ mobilization in the HT-29 epithelial cell line. GST pull-downs in HT-29 cell lysates showed that the PDZ2 module of NHERF-2 bound to the PDZ binding motif on the C terminus of PMCA. Co-immunoprecipitations confirmed that PMCA1b and NHERF-2 associated under normal conditions in HT-29 cells. Cell surface biotinylations revealed significant increases in membrane-associated NHERF-2 and PMCA within 60 s following muscarinic activation, accompanied by increased association of the two proteins as seen by confocal microscopy. The recruitment of NHERF-2 to the membrane preceded that of PMCA, suggesting that NHERF-2 was involved in nucleating an efflux complex at the membrane. The muscarinic-mediated translocation of PMCA was abolished when NHERF-2 was silenced, and the rate of relative Ca2+ efflux was also reduced. These experiments also uncovered a NHERF-2-independent PMCA retrieval mechanism. Our findings describe rapid agonist-induced translocation of PMCA in a native cell model and suggest that NHERF-2 plays a key role in scaffolding and maintaining PMCA at the cell membrane.

    Funded by: NIDDK NIH HHS: DK061418, R01 DK061418, R01 DK061418-05, R01 DK061418-06A1

    The Journal of biological chemistry 2009;284;3;1820-30

  • Phosphorylation of Tyr-398 and Tyr-402 in occludin prevents its interaction with ZO-1 and destabilizes its assembly at the tight junctions.

    Elias BC, Suzuki T, Seth A, Giorgianni F, Kale G, Shen L, Turner JR, Naren A, Desiderio DM and Rao R

    Department of Physiology, Charles B. Stout Neuroscience Mass Spectrometry Laboratory, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA.

    Occludin is phosphorylated on tyrosine residues during the oxidative stress-induced disruption of tight junction, and in vitro phosphorylation of occludin by c-Src attenuates its binding to ZO-1. In the present study mass spectrometric analyses of C-terminal domain of occludin identified Tyr-379 and Tyr-383 in chicken occludin as the phosphorylation sites, which are located in a highly conserved sequence of occludin, YETDYTT; Tyr-398 and Tyr-402 are the corresponding residues in human occludin. Deletion of YETDYTT motif abolished the c-Src-mediated phosphorylation of occludin and the regulation of ZO-1 binding. Y398A and Y402A mutations in human occludin also abolished the c-Src-mediated phosphorylation and regulation of ZO-1 binding. Y398D/Y402D mutation resulted in a dramatic reduction in ZO-1 binding even in the absence of c-Src. Similar to wild type occludin, its Y398A/Y402A mutant was localized at the plasma membrane and cell-cell contact sites in Rat-1 cells. However, Y398D/Y402D mutants of occludin failed to localize at the cell-cell contacts. Calcium-induced reassembly of Y398D/Y402D mutant occludin in Madin-Darby canine kidney cells was significantly delayed compared with that of wild type occludin or its T398A/T402A mutant. Furthermore, expression of Y398D/Y402D mutant of occludin sensitized MDCK cells for hydrogen peroxide-induced barrier disruption. This study reveals a unique motif in the occludin sequence that is involved in the regulation of ZO-1 binding by reversible phosphorylation of specific Tyr residues.

    Funded by: NIAAA NIH HHS: R01 AA012307, R01-AA12307; NIDDK NIH HHS: R01 DK055532, R01-DK55532

    The Journal of biological chemistry 2009;284;3;1559-69

  • Ischemic injury to kidney induces glomerular podocyte effacement and dissociation of slit diaphragm proteins Neph1 and ZO-1.

    Wagner MC, Rhodes G, Wang E, Pruthi V, Arif E, Saleem MA, Wean SE, Garg P, Verma R, Holzman LB, Gattone V, Molitoris BA and Nihalani D

    Department of Medicine, Division of Nephrology, Indiana University, Indianapolis, Indiana 46202, USA.

    Glomerular injury is often characterized by the effacement of podocytes, loss of slit diaphragms, and proteinuria. Renal ischemia or the loss of blood flow to the kidneys has been widely associated with tubular and endothelial injury but rarely has been shown to induce podocyte damage and disruption of the slit diaphragm. In this study, we have used an in vivo rat ischemic model to demonstrate that renal ischemia induces podocyte effacement with loss of slit diaphragm and proteinuria. Biochemical analysis of the ischemic glomerulus shows that ischemia induces rapid loss of interaction between slit diaphragm junctional proteins Neph1 and ZO-1. To further understand the effect of ischemia on molecular interactions between slit diaphragm proteins, a cell culture model was employed to study the binding between Neph1 and ZO-1. Under physiologic conditions, Neph1 co-localized with ZO-1 at cell-cell contacts in cultured human podocytes. Induction of injury by ATP depletion resulted in rapid loss of Neph1 and ZO-1 binding and redistribution of Neph1 and ZO-1 proteins from cell membrane to the cytoplasm. Recovery resulted in increased Neph1 tyrosine phosphorylation, restoring Neph1 and ZO-1 binding and their localization at the cell membrane. We further demonstrate that tyrosine phosphorylation of Neph1 mediated by Fyn results in significantly increased Neph1 and ZO-1 binding, suggesting a critical role for Neph1 tyrosine phosphorylation in reorganizing the Neph1-ZO-1 complex. This study documents that renal ischemia induces dynamic changes in the molecular interactions between slit diaphragm proteins, leading to podocyte damage and proteinuria.

    Funded by: NIDDK NIH HHS: K01 DK072047-03, K08 DK081403, K08 DK081403-01, P50 DK061594, R01 DK069408

    The Journal of biological chemistry 2008;283;51;35579-89

  • Hepatitis C virus envelope components alter localization of hepatocyte tight junction-associated proteins and promote occludin retention in the endoplasmic reticulum.

    Benedicto I, Molina-Jiménez F, Barreiro O, Maldonado-Rodríguez A, Prieto J, Moreno-Otero R, Aldabe R, López-Cabrera M and Majano PL

    Molecular Biology Unit, Hospital Universitario de la Princesa, Madrid, Spain.

    Unlabelled: Hepatocyte tight junctions (TJ) play key roles in characteristic liver functions, including bile formation and secretion. Infection by hepatitis C virus (HCV) may cause alterations of the liver architecture and disruption of the bile duct, which ultimately can lead to cholestasis. Herein, we employed the HCV replicon system to analyze the effect of HCV on TJ organization. TJ-associated proteins occludin, claudin-1, and Zonula Occludens protein-1 (ZO-1) disappeared from their normal localization at the border of adjacent cells in Huh7 clones harboring genomic but not subgenomic replicons expressing only the nonstructural proteins. Furthermore, cells containing genomic replicons showed a cytoplasmic accumulation of occludin in the endoplasmic reticulum (ER). TJ-associated function, measured as FITC-dextran paracellular permeability, of genomic replicon-containing cells, was also altered. Interestingly, clearance of the HCV replicon by interferon-alpha (IFN-alpha) treatment and by short hairpin RNA (shRNA) significantly restored the localization of TJ-associated proteins. Transient expression of all HCV structural proteins, but not core protein alone, altered the localization of TJ-associated proteins in Huh7 cells and in clones with subgenomic replicons. Confocal analysis showed that accumulation of occludin in the ER partially co-localized with HCV envelope glycoprotein E2. E2/occludin association was further confirmed by co-immunoprecipitation and pull-down assays. Additionally, using a cell culture model of HCV infection, we observed the cytoplasmic dot-like accumulation of occludin in infected Huh7 cells.

    Conclusion: We propose that HCV structural proteins, most likely those of the viral envelope, promote alterations of TJ-associated proteins, which may provide new insights for HCV-related pathogenesis.

    Hepatology (Baltimore, Md.) 2008;48;4;1044-53

  • Different expression of occludin and ZO-1 in primary and metastatic liver tumors.

    Orbán E, Szabó E, Lotz G, Kupcsulik P, Páska C, Schaff Z and Kiss A

    2nd Department of Pathology, Semmelweis University, Budapest, Hungary.

    Tight junction (TJ) components were found to be correlated with carcinogenesis and tumor development. TJs are composed of three main integral membrane proteins; occludin, claudins and JAMs. Alteration of the TJ protein expression may play an important role in the process of cell dissociation, which is among the first steps of tumor invasion and metastasis. Reduced expression of ZO-1 has been reported to be associated with invasion of several tumors. The aim of the present study was to detect differences between occludin and ZO-1 expression in normal liver samples, HCCs and colorectal liver metastases. Expression of occludin and ZO-1 was analysed in 25 surgically removed human hepatocellular carcinomas (HCC) and 25 human colorectal liver metastases. Gene expression levels were measured by real-time RT PCR, protein expression was determined by immunohistochemistry, comparing tumors with the surrounding nontumorous parenchyma and with seven normal liver samples. Occludin and ZO-1 mRNAs showed significant downregulation in HCCs in comparison with normal liver and were also downregulated in the metastases when compared with normal liver. Occludin and ZO-1 proteins were weakly expressed on hepatocytes in normal liver, while strong expression was found on bile canaliculi. In HCCs occludin and ZO-1 did not show immunopositivity on tumor cells, while colorectal metastatic tumors revealed high levels of these molecules. HCCs and metastases are characterized by markedly different protein expression pattern of occludin and ZO-1, which phenomenon might be attributed to the different histogenesis of these tumors.

    Pathology oncology research : POR 2008;14;3;299-306

  • JunD represses transcription and translation of the tight junction protein zona occludens-1 modulating intestinal epithelial barrier function.

    Chen J, Xiao L, Rao JN, Zou T, Liu L, Bellavance E, Gorospe M and Wang JY

    Department of Surgery, University of Maryland School of Medicine, Baltimore, MD, USA.

    The AP-1 transcription factor JunD is highly expressed in intestinal epithelial cells, but its exact role in maintaining the integrity of intestinal epithelial barrier remains unknown. The tight junction (TJ) protein zonula occludens (ZO)-1 links the intracellular domain of TJ-transmembrane proteins occludin, claudins, and junctional adhesion molecules to many cytoplasmic proteins and the actin cytoskeleton and is crucial for assembly of the TJ complex. Here, we show that JunD negatively regulates expression of ZO-1 and is implicated in the regulation of intestinal epithelial barrier function. Increased JunD levels by ectopic overexpression of the junD gene or by depleting cellular polyamines repressed ZO-1 expression and increased epithelial paracellular permeability. JunD regulated ZO-1 expression at the levels of transcription and translation. Transcriptional repression of ZO-1 by JunD was mediated through cAMP response element-binding protein-binding site within its proximal region of the ZO-1-promoter, whereas induced JunD inhibited ZO-1 mRNA translation by enhancing the interaction of the ZO-1 3'-untranslated region with RNA-binding protein T cell-restricted intracellular antigen 1-related protein. These results indicate that JunD is a biological suppressor of ZO-1 expression in intestinal epithelial cells and plays a critical role in maintaining epithelial barrier function.

    Funded by: Intramural NIH HHS; NIDDK NIH HHS: DK-57819, DK-61972, DK-68491, R01 DK057819, R01 DK061972, R01 DK068491, T32 DK067872

    Molecular biology of the cell 2008;19;9;3701-12

  • Domain-swapped dimerization of ZO-1 PDZ2 generates specific and regulatory connexin43-binding sites.

    Chen J, Pan L, Wei Z, Zhao Y and Zhang M

    Department of Biochemistry, Molecular Neuroscience Center, Hong Kong University of Science and Technology, Kowloon, Hong Kong.

    PDZ domain scaffold proteins are capable of assembling macromolecular protein complexes in diverse cellular processes through PDZ-mediated binding to a short peptide fragment at the carboxyl tail of target proteins. How each PDZ domain specifically recognizes its target protein(s) remains a major conceptual question, as at least a few out of the several hundred PDZ domains in each eukaryotic genome share overlapping binding properties with any given target protein. Here, we show that the domain-swapped dimerization of zonula occludens-1 PDZ2 generates a distinct interface that functions together with the well-separated canonical carboxyl tail-binding pocket in each PDZ unit in binding to connexin43 (Cx43). We further demonstrate that the charge-charge interaction network formed by residues in the PDZ dimer interface and upstream residues of the Cx43 peptide not only provides the unprecedented interaction specificity for the complex but may also function as a phosphorylation-mediated regulatory switch for the dynamics of the Cx43 gap junctions. Finally, we provide evidence that such domain-swapped dimer assembly also occurs in other PDZ domain scaffold proteins. Therefore, our findings present a new paradigm for understanding how some PDZ domain proteins specifically bind to and regulate the functions of their target proteins.

    The EMBO journal 2008;27;15;2113-23

  • Critical regulation of TGFbeta signaling by Hsp90.

    Wrighton KH, Lin X and Feng XH

    Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX 77030, USA.

    Transforming growth factor beta (TGFbeta) controls a diverse set of cellular processes by activating TGFbeta type I (TbetaRI) and type II (TbetaRII) serine-threonine receptor kinases. Canonical TGFbeta signaling is mediated by Smad2 and Smad3, which are phosphorylated in their SXS motif by activated TbetaRI. The 90-kDa heat-shock protein (Hsp90) is a molecular chaperone facilitating the folding and stabilization of many protein kinases and intracellular signaling molecules. Here, we present evidence identifying a critical role for Hsp90 in TGFbeta signaling. Inhibition of Hsp90 function by using small-molecule inhibitors such as 17-allylamino-17-demethoxygeldanamycin (17AAG), and also at the genetic level, blocks TGFbeta-induced signaling and transcriptional responses. Furthermore, we identify TbetaRI and TbetaRII as Hsp90-interacting proteins in vitro and in vivo and demonstrate that inhibition of Hsp90 function increases TbetaR ubiquitination and degradation dependent on the Smurf2 ubiquitin E3 ligase. Our data reveal an essential level of TGFbeta signaling regulation mediated by Hsp90 by its ability to chaperone TbetaRs and also implicate the use of Hsp90 inhibitors in blocking undesired activation of TGFbeta signaling in diseases.

    Funded by: NCI NIH HHS: R01 CA108454, R01 CA108454-04, R01CA108454; NHLBI NIH HHS: P50 HL083794, P50HL083764; NIAMS NIH HHS: R01 AR053591; NIDDK NIH HHS: P50 DK064233, P50DK064233, R01 DK073932, R01DK073932; NIGMS NIH HHS: R01 GM063773, R01 GM063773-04, R01GM63773

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;27;9244-9

  • The tight junction protein complex undergoes rapid and continuous molecular remodeling at steady state.

    Shen L, Weber CR and Turner JR

    Department of Pathology, The University of Chicago, Chicago, IL 60637, USA.

    The tight junction defines epithelial organization. Structurally, the tight junction is comprised of transmembrane and membrane-associated proteins that are thought to assemble into stable complexes to determine function. In this study, we measure tight junction protein dynamics in live confluent Madin-Darby canine kidney monolayers using fluorescence recovery after photobleaching and related methods. Mathematical modeling shows that the majority of claudin-1 (76 +/- 5%) is stably localized at the tight junction. In contrast, the majority of occludin (71 +/- 3%) diffuses rapidly within the tight junction with a diffusion constant of 0.011 microm(2)s(-1). Zonula occludens-1 molecules are also highly dynamic in this region, but, rather than diffusing within the plane of the membrane, 69 +/- 5% exchange between membrane and intracellular pools in an energy-dependent manner. These data demonstrate that the tight junction undergoes constant remodeling and suggest that this dynamic behavior may contribute to tight junction assembly and regulation.

    Funded by: NCI NIH HHS: P30 CA014599, P30 CA14599; NIDDK NIH HHS: R01 DK061931, R01 DK068271, R01DK061931, R01DK068271

    The Journal of cell biology 2008;181;4;683-95

  • The second PDZ domain of zonula occludens-1 is dispensable for targeting to connexin 43 gap junctions.

    Hunter AW and Gourdie RG

    Department of Cell Biology and Anatomy, Cardiovascular Developmental Biology Center, Medical University of South Carolina, Charleston, South Carolina 29425, USA. huntera@musc.edu

    Zonula occludens (ZO)-1 is emerging as a central player in the control of gap junction (GJ) dynamics. Previously the authors reported that ZO-1 localizes preferentially to the periphery of Cx43 GJs. How ZO-1 arrives at GJ edges is unknown, but this targeting might involve we established interaction between the Cx43 C-terminus and the PDZ2 domain of ZO-1. Here the show that despite blocking the canonical PDZ2-mediated interaction by fusion of GFP to the C-terminus of Cx43, ZO-1 continued to target to domains juxtaposed with the edges of GJs comprised solely of tagged Cx43. This edge-association was not abolished by deletion of PDZ2 from ZO-1, as mutant ZO-1 also targeted to the periphery of GJs composed of either tagged or untagged Cx43. Additionally, ZO-2 was found colocalized with ZO-1 at GJ edges. These data demonstrate that ZO-1 targets to GJ edges independently of several known PDZ2-mediated interactions, including ZO-1 homodimerization, heterodimerization with ZO-2, and direct ZO-1 binding to the C-terminal residues of Cx43.

    Funded by: NHLBI NIH HHS: HL07260, HL082802, HL56728; NIGMS NIH HHS: K12GM081265

    Cell communication & adhesion 2008;15;1;55-63

  • Gap junction remodelling in human heart failure is associated with increased interaction of connexin43 with ZO-1.

    Bruce AF, Rothery S, Dupont E and Severs NJ

    National Heart and Lung Institute, Imperial College London, Guy Scadding Building, London, UK.

    Aims: Remodelling of gap junctions, involving reduction of total gap junction quantity and down-regulation of connexin43 (Cx43), contributes to the arrhythmic substrate in congestive heart failure. However, little is known of the underlying mechanisms. Recent studies from in vitro systems suggest that the connexin-interacting protein zonula occludens-1 (ZO-1) is a potential mediator of gap junction remodelling. We therefore examined the hypothesis that ZO-1 contributes to reduced expression of Cx43 gap junctions in congestive heart failure.

    Left ventricular myocardium from healthy control human hearts (n = 5) was compared with that of explanted hearts from transplant patients with end-stage congestive heart failure due to idiopathic dilated cardiomyopathy (DCM; n = 5) or ischaemic cardiomyopathy (ICM; n = 5). Immunoconfocal and immunoelectron microscopy showed that ZO-1 is specifically localized to the intercalated disc of cardiomyocytes in control and failing ventricles. ZO-1 protein levels were significantly increased in both DCM and ICM (P = 0.0025), showing a significant, negative correlation to Cx43 levels (P = 0.0029). There was, however, no significant alteration of ZO-1 mRNA (P = 0.537). Double immunolabelling demonstrated that a proportion of ZO-1 label is co-localized with Cx43, and that co-localization of Cx43 with ZO-1 is significantly increased in the failing ventricle (P = 0.003). Interaction between the two proteins was confirmed by co-immunoprecipitation. The proportion of Cx43 that co-immunoprecipitates with ZO-1 was significantly increased in the failing heart.

    Conclusion: Our findings suggest that ZO-1, by interacting with Cx43, plays a role in the down-regulation and decreased size of Cx43 gap junctions in congestive heart failure.

    Funded by: British Heart Foundation: PG/05/003

    Cardiovascular research 2008;77;4;757-65

  • Altered expression of epithelial junctional proteins in atopic asthma: possible role in inflammation.

    de Boer WI, Sharma HS, Baelemans SM, Hoogsteden HC, Lambrecht BN and Braunstahl GJ

    Department of Pulmonary Medicine, Erasmus MC, University Medical Centre, Rotterdam, The Netherlands. deboer.pim@hetnet.nl

    Epithelial cells form a tight barrier against environmental stimuli via tight junctions (TJs) and adherence junctions (AJs). Defects in TJ and AJ proteins may cause changes in epithelial morphology and integrity and potentially lead to faster trafficking of inflammatory cells through the epithelium. Bronchial epithelial fragility has been reported in asthmatic patients, but little is known about the expression of TJ and AJ proteins in asthma. We studied epithelial expression of zonula occludens-1 (ZO-1) and AJ proteins E-cadherin, alpha-catenin, and beta-catenin in bronchial biopsies from nonatopic nonasthmatic (healthy) subjects (n = 14), and stable atopic asthmatic subjects (n = 22) at baseline conditions. Immunostaining for these proteins was semi-quantified for separate cellular compartments. E-cadherin, alpha-catenin and beta-catenin were present in the cellular membrane and less in the cytoplasm. Only beta-catenin was present in the nucleus in agreement with its potential function as transcription factor. ZO-1 was present in the apicolateral membrane of superficial cells. alpha-Catenin expression was significantly lower in subjects with asthma than without and correlated inversely with numbers of eosinophils within the epithelium. ZO-1 and E-cadherin expression were significantly lower in asthmatic than in nonasthmatic subjects. Expression of beta-catenin was not different. Our results suggest that the lower epithelial alpha-catenin, E-cadherin and (or) ZO-1 expression in patients with atopic asthma contributes to a defective airway epithelial barrier and a higher influx of eosinophils in the epithelium.

    Canadian journal of physiology and pharmacology 2008;86;3;105-12

  • Toward a confocal subcellular atlas of the human proteome.

    Barbe L, Lundberg E, Oksvold P, Stenius A, Lewin E, Björling E, Asplund A, Pontén F, Brismar H, Uhlén M and Andersson-Svahn H

    Department of Biotechnology, AlbaNova University Center, Royal Institute of Technology, SE-106 91 Stockholm, Sweden.

    Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.

    Molecular & cellular proteomics : MCP 2008;7;3;499-508

  • Effect of alcohol on miR-212 expression in intestinal epithelial cells and its potential role in alcoholic liver disease.

    Tang Y, Banan A, Forsyth CB, Fields JZ, Lau CK, Zhang LJ and Keshavarzian A

    Division of Digestive Disease and Nutrition, Department of Internal Medicine, Rush University Medical Center, Chicago, Illinois 60612, USA. yueming_tang@rush.edu

    Alcohol-induced gut leakiness is a key factor in alcoholic liver disease (ALD); it allows endotoxin to enter the circulation and initiate liver damage. Zonula occludens 1 (ZO-1) protein is a major component of tight junctions that regulates intestinal permeability. microRNAs (miRNAs) are recently discovered regulatory molecules that inhibit expression of their target genes.

    (i) to investigate the effect of alcohol on miRNA-212 (miR-212) and on expression of its predicted target gene, ZO-1, (ii) to study the potential role of miR-212 in the pathophysiology of ALD in man.

    Methods: Using a TaqMan miRNA assay system, we measured miR-212 expression levels in colon biopsy samples from patients with ALD and in Caco-2 cells (a human intestinal epithelial cell line) treated with or without EtOH. We measured ZO-1 protein levels using western blots. ZO-1 mRNA was assayed using real-time PCR. Intestinal barrier integrity was measured using fluorescein sulfonic acid clearance and immunofluorescent staining for ZO-1.

    Results: Ethanol increased miR-212 expression, decreased ZO-1 protein levels, disrupted tight junctions, and increased the permeability of monolayers of Caco-2 cells. An miR-212 over-expression is correlated with hyperpermeability of the monolayer barrier. miR-212 levels were higher in colon biopsy samples in patients with ALD than in healthy controls; ZO-1 protein levels were lower.

    Conclusion: These data suggest a novel mechanism for alcohol-induced gut leakiness, one in which EtOH induces miR-212 over-expression which causes gut leakiness by down-regulating ZO-1 translation. This mechanism is a potential therapeutic target for leaky gut in patients with or at risk for ALD.

    Alcoholism, clinical and experimental research 2008;32;2;355-64

  • [Methylation status of ZO-1 gene in patients with myelodysplastic syndrome].

    Kang HY, Wang C, Dou LP, Qing Y, Han XP, Sun JF, Liu CH, Lu XC, Liu Y and Yu L

    Department of Hematology, PLA General Hospital, Beijing 100853, China.

    The objective of this study was to investigate the methylation status of zonula occluden protein-1 (ZO-1) gene in patients with myelodysplastic syndrome (MDS) and to identify its roles in pathogenesis, development and classification of MDS. 85 patients with MDS and 30 healthy individuals were tested by methylation specific polymerase chain reaction (MS-PCR). The results indicated that no ZO-1 promoter methylation could be detected in healthy controls. methylation of ZO-1 gene promoter of bone marrow was found in 56.5% (48/85) MDS patients. The difference between these two kinds of subjects was statistically significant (p<0.05). The methylation status of ZO-1 gene promoter region in the subtypes of MDS was as following: RA (18/37, 48.6%), RAS (4/6, 67%), RCMD (19/30, 63%), RAEB (7/12, 58%). Every subtype of MDS patients had statistical difference from healthy people (p<0.05), but between the subtypes of MDS there were no significant statistical differences in the methylation status of ZO-1 gene, while the level of ZO-1 promoter methylation in group of RA was lower than that in other groups. It is concluded that the ZO-1 promoter region in bone marrow of MDS patient shows a hypermethylation status, which is specific for MDS. MDS is a common hematologic malignancy with clonal proliferation, it is difficult to differentiate from many other hematologic malignancies in clinical diagnosis. However, the change of ZO-1 gene methylation status is closely related to pathogenesis of MDS, therefore the ZO-1 gene as valuable diagnostic marker has important clinical significance. The ZO-1 gene may be a potential gene related to hematologic malignancies.

    Zhongguo shi yan xue ye xue za zhi 2008;16;1;70-3

  • Tight junctions in thyroid carcinogenesis: diverse expression of claudin-1, claudin-4, claudin-7 and occludin in thyroid neoplasms.

    Tzelepi VN, Tsamandas AC, Vlotinou HD, Vagianos CE and Scopa CD

    Faculty of Medicine, Department of Pathology, University Hospital, University of Patras, Patras, Greece.

    Claudins and occludin are integral constituents of tight junctions and are deregulated in a variety of malignancies. Their role in thyroid carcinogenesis has not yet been elucidated. This study investigates the expression of occludin and claudin-1, -4 and -7 in thyroid neoplasms. Ninety-one thyroid neoplasms (15 follicular adenomas, 15 follicular carcinomas, 26 papillary carcinomas, 16 papillary microcarcinomas, 8 medullary carcinomas, 3 poorly differentiated carcinomas, 8 undifferentiated carcinomas) were immunostained with antibodies against occludin and claudin-1, -4 and -7. Occludin was mainly expressed in the form of intracytoplasmic vesicles, whereas all claudins tested exhibited membranous immunostaining. Thirteen out of 15 follicular adenomas, 10/15 follicular carcinomas, 24/26 papillary carcinomas, 15/16 papillary microcarcinomas, 1/8 medullary carcinomas, 2/3 poorly differentiated carcinomas and 2/8 undifferentiated carcinomas exhibited claudin-1 expression, whereas claudin-4 was expressed in 13/15, 12/15, 23/26, 13/16, 7/8, 2/3 and 2/8 of the tumors, respectively, and claudin-7 expression was found in 67, 33, 73, 69, 25, 0 and 13% of the cases, respectively. Occludin was expressed in 100% follicular adenomas, 80% follicular carcinomas, 96% papillary carcinomas, 50% papillary microcarcinomas, 50% medullary carcinomas, 33% poorly differentiated carcinomas and 88% undifferentiated carcinomas. Occludin expression was reduced in papillary microcarcinomas, medullary carcinomas and poorly differentiated carcinomas. All claudins exhibited reduced expression in undifferentiated carcinomas. Claudin-1 was additionally reduced in medullary carcinomas and claudin-7 in follicular, medullary and poorly differentiated carcinomas. A correlation between loss of claudin-1 expression and worse disease-free survival was noted on univariate analysis. Dedifferentiation of the thyroid carcinomas is accompanied by reduction in claudin-1, -4 and -7 expression. A differential expression of tight junction proteins in the different histologic types of thyroid gland is noted. Additionally, claudin-1 expression may be an important prognostic indicator of recurrence in thyroid carcinomas.

    Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 2008;21;1;22-30

  • Domain swapping within PDZ2 is responsible for dimerization of ZO proteins.

    Fanning AS, Lye MF, Anderson JM and Lavie A

    Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7545, USA.

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

    Funded by: NIDDK NIH HHS: DK61397, R01 DK061397, R01 DK061397-05, R56 DK061397, T32 DK007739

    The Journal of biological chemistry 2007;282;52;37710-6

  • The proteasome regulates the interaction between Cx43 and ZO-1.

    Girao H and Pereira P

    Centre of Ophthalmology, Biomedical Institute for Research in Light and Image (IBILI), Faculty of Medicine, University of Coimbra, 3000-354 Coimbra, Portugal.

    Gap junction (GJ) intercellular communication (GJIC) is vital to ensure proper cell and tissue function. GJ are multimeric structures composed of proteins called connexins. Modifications on stability or subcellular distribution of connexins have a direct impact on the extent of GJIC. In this study we have investigated the role of the proteasome in regulation of connexin 43 (Cx43) internalization. Although the participation of both the proteasome and lysosome has long been suggested in Cx43 degradation, the molecular mechanisms whereby proteasome contributes to regulate Cx43 internalization and intercellular communication are still unclear. The results presented in this study envision a new mechanism whereby proteasome regulates GJIC by modulating interaction between Cx43 and ZO-1. Immunoprecipitation experiments, in the presence of proteasome inhibitors, together with immunofluorescence data indicate that the proteasome regulates interaction between Cx43 and ZO-1. Overexpression of the PDZ2 domain of ZO-1 and the expression of Cx-43 fused in frame with a V5/HIS tag, suggest that interaction between the two proteins occurs through the PDZ2 domain of ZO-1 and the C-terminus of Cx43. When interaction between Cx43 and ZO-1 is reduced, as in the presence of proteasome inhibitors, Cx43 accumulates, forming large GJ plaques at plasma membrane. Data presented in this article suggest a new pathway whereby alterations in proteasome activity may impact on GJIC as well as on non-junctional communication with extracellular environment, contributing to cell and tissue dysfunction.

    Journal of cellular biochemistry 2007;102;3;719-28

  • Zonula occludens-1 and connexin 43 expression in the failing human heart.

    Kostin S

    Core Lab for Molecular and Structural Biology, W. G. Kerckhoff Institute, Max-Planck Institute for Heart and Lung Research, Parkstrasse 1, 61231 Bad Nauheim, Germany. sawa.kostin@mpi-bn.mpg.de

    Focal disorganization of gap junctional distribution and down-regulation of the major gap junctional protein connexin 43 are typical features of myocardial remodelling in the failing human heart. Increasing evidence indicates that connexin 43 interacts with zonula-occludens-1 (ZO-1), and it has recently been shown that ZO-1 promotes the formation and growth of gap junctional plaques. In the present study, distribution patterns of ZO-1 and connexin 43 were studied in normal and in heart failure patients using double-label immunohistochemistry and confocal microscopy. ZO-1 was found to be co-localized with connexin 43 at intercalated disks. Importantly, in patients with heart failure due to dilated or ischaemic cardiomyopathy, areas of diminished connexin 43 expression were characterized by a markedly reduced ZO-1 staining. Based on these data it is concluded that in patients with heart failure, down-regulation of ZO-1 matches the diminished expression levels of connexin 43, suggesting that ZO-1 plays an important role in gap junction formation and gap junction plaque stability.

    Journal of cellular and molecular medicine 2007;11;4;892-5

  • Lipolysis products from triglyceride-rich lipoproteins increase endothelial permeability, perturb zonula occludens-1 and F-actin, and induce apoptosis.

    Eiselein L, Wilson DW, Lamé MW and Rutledge JC

    Division of Endocrinology, University of California, Davis, CA 95616, USA.

    Products generated from lipoprotein lipase-mediated hydrolysis of triglyceride-rich lipoproteins (TGRL) are reported to increase endothelial layer permeability. We hypothesize that these increases in permeability result from the active rearrangement and dissolution of the junctional barrier in human aortic endothelial cells, as well as induction of the apoptotic cascade. Human aortic endothelial cells were treated with TGRL lipolysis products generated from coincubation of human TGRL plus lipoprotein lipase. Measurement of transendothelial electrical resistance demonstrated a time-dependent decrease in endothelial barrier function in response to TGRL lipolysis products. Immunofluorescent localization of zonula occludens-1 (ZO-1) showed radial rearrangement along cell borders after 1.5 h of treatment with lipolysis products. A concurrent redistribution of F-actin from the cell body to the cell margins was observed via rhodamine phalloidin staining. Immunofluorescent imaging for occludin and vascular endothelial cadherin showed that these proteins relocalize as well, although these changes are less prominent than for ZO-1. Western analysis of cells exposed to lipolysis products for 3 h revealed the fragmentation of ZO-1, a reduction in occludin, and no change of vascular endothelial cadherin. Lipolysis products also increased caspase-3 activity and induced nuclear fragmentation. Treatments did not cause oncosis in cells at any point during the incubation. These results demonstrate that TGRL lipolysis products play an important role in the regulation of endothelial permeability, the organization of the actin cytoskeleton, the localization and expression of junctional proteins, especially ZO-1, and the induction of apoptosis.

    Funded by: NHLBI NIH HHS: HL48411, HL55667

    American journal of physiology. Heart and circulatory physiology 2007;292;6;H2745-53

  • Enhanced tight junction function in human breast cancer cells by antioxidant, selenium and polyunsaturated lipid.

    Martin TA, Das T, Mansel RE and Jiang WG

    Metastasis & Angiogenesis Research Group, Department of Surgery, Wales College of Medicine, Cardiff University, Cardiff, United Kingdom. Martinta1@cf.ac.uk

    Paracellular permeability (PCP) is governed by tight junctions (TJs) in epithelial cells, acting as cell-cell adhesion structures, the aberration of which is known to be linked to the dissociation and metastasis of breast cancer cells. This study hypothesized that the function of TJs in human breast cancer cells can be augmented by gamma linolenic acid (GLA), selenium (Se), and iodine (I) in the presence of 17-beta-estradiol, as these molecules are known to increase TJ functions in endothelial cells, using assays of trans-epithelial resistance (TER), PCP, immunofluorescence, and in vitro invasion and motility models. GLA, I, and Se individually increased TER of MDA-MB-231 and MCF-7 human breast cancer cells. The combination of all three agents also had a significant increase in TER. Addition of GLA/Se/I reduced PCP of both breast cancer cell lines. GLA/Se/I reversed the effect of 17-beta-estradiol (reduced TER, increased PCP). Immunofluorescence revealed that after treatment with Se/I/GLA over 24 h, there was increasing relocation to breast cancer cell-cell junctions of occludin and ZO-1 in MCF-7 cells. Moreover, treatment with GLA/Se/I, alone or in combination, significantly reduced in vitro invasion of MDA-MB-231 cells through an endothelial cell barrier (P < 0.0001) and reduced 17-beta-estradiol induced breast cancer cell motility (P < 0.0001). Our previous work has demonstrated that GLA, I, and Se alone, or in combination are able to strengthen the function of TJs in human endothelial cells; this has now proved to be true of human breast cancer cells. This combination also completely reversed the effect of 17-beta-estradiol in these cells.

    Journal of cellular biochemistry 2007;101;1;155-66

  • Enteropathogenic Escherichia coli, Shigella flexneri, and Listeria monocytogenes recruit a junctional protein, zonula occludens-1, to actin tails and pedestals.

    Hanajima-Ozawa M, Matsuzawa T, Fukui A, Kamitani S, Ohnishi H, Abe A, Horiguchi Y and Miyake M

    Department of Molecular Bacteriology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita-city, Osaka 565-0871, Japan.

    Enteropathogenic Escherichia coli, Shigella flexneri, and Listeria monocytogenes induce localized actin polymerization at the cytoplasmic face of the plasma membrane or within the host cytoplasm, creating unique actin-rich structures termed pedestals or actin tails. The process is known to be mediated by the actin-related protein 2 and 3 (Arp2/3) complex, which in these cases acts downstream of neural Wiskott-Aldrich syndrome protein (N-WASP) or of a listerial functional homolog of WASP family proteins. Here, we show that zonula occludens-1 (ZO-1), a protein in the tight junctions of polarized epithelial cells, is recruited to actin tails and pedestals. Immunocytochemical analysis revealed that ZO-1 was stained most in the distal part of the actin-rich structures, and the incorporation was mediated by the proline-rich region of the ZO-1 molecule. The direct clustering of membrane-targeted Nck, which is known to activate the N-WASP-Arp2/3 pathway, triggered the formation of the ZO-1-associated actin tails. The results suggest that the activation of the Arp2/3 complex downstream of N-WASP or a WASP-related molecule is a key to the formation of the particular actin-rich structures that bind with ZO-1. We propose that an analysis of the recruitment on a molecular basis will lead to an understanding of how ZO-1 recognizes a distinctive actin-rich structure under pathophysiological conditions.

    Infection and immunity 2007;75;2;565-73

  • Helicobacter pylori alters the distribution of ZO-1 and p120ctn in primary human gastric epithelial cells.

    Krueger S, Hundertmark T, Kuester D, Kalinski T, Peitz U and Roessner A

    Department of Pathology, Otto-von-Guericke University, Leipziger Str. 44, D-39120 Magdeburg, Germany. Sabine.Krueger@medizin.uni-magdeburg.de

    Helicobacter pylori infection is related to the development of diverse gastric pathologies, possibly by affecting epithelial junctional complexes that define cell polarity and play an essential role in transepithelial transport and cell-cell adhesion. Using primary gastric epithelial cell cultures, effects of H. pylori on the expression and localization of tight/adherence junction proteins and the resulting morphological changes and migratory capabilities were studied under in vivo-like conditions. Gastric epithelial cells were isolated from biopsies or gastrectomies and maintained in Quantum286 on collagen I-coated culture dishes or cover-slips. Cell cultures were characterized and further analyzed by western blot and immunofluorescent staining for ZO-1, p120ctn, and H. pylori CagA. Morphological changes and migratory response were monitored by time-lapse digital image microscopy. ZO-1 and p120ctn protein expression levels remain unaffected by H. pylori infection. Immunocytochemistry on H. pylori-infected primary cell monolayers focally showed disruption of intercellular ZO-1 staining and accumulation of ZO-1 in small vesicles. H. pylori infection recruited non-phosphorylated p120ctn to perinuclear vesicles. The fraction of phosphorylated p120ctn increased and could be detected in the nucleus, at the cell membrane, and at the leading edge of migrating cells. These alterations, triggered by H. pylori infection, are associated with an elongation phenotype and increased migration.

    Pathology, research and practice 2007;203;6;433-44

  • Altered expression of ZO-1 and ZO-2 in Sertoli cells and loss of blood-testis barrier integrity in testicular carcinoma in situ.

    Fink C, Weigel R, Hembes T, Lauke-Wettwer H, Kliesch S, Bergmann M and Brehm RH

    Institute of Veterinary Anatomy, Histology, and Embryology, University of Giessen, Giessen, Germany. cornelia.fink@vetmed.uni-giessen.de

    Carcinoma in situ (CIS) is the noninvasive precursor of most human testicular germ cell tumors. In normal seminiferous epithelium, specialized tight junctions between Sertoli cells constitute the major component of the blood-testis barrier. Sertoli cells associated with CIS exhibit impaired maturation status, but their functional significance remains unknown. The aim was to determine whether the blood-testis barrier is morphologically and/or functionally altered. We investigated the expression and distribution pattern of the tight junction proteins zonula occludens (ZO) 1 and 2 in normal seminiferous tubules compared to tubules showing CIS. In normal tubules, ZO-1 and ZO-2 immunostaining was observed at the blood-testis barrier region of adjacent Sertoli cells. Within CIS tubules, ZO-1 and ZO-2 immunoreactivity was reduced at the blood-testis barrier region, but spread to stain the Sertoli cell cytoplasm. Western blot analysis confirmed ZO-1 and ZO-2, and their respective mRNA were shown by RT-PCR. Additionally, we assessed the functional integrity of the blood-testis barrier by lanthanum tracer study. Lanthanum permeated tight junctions in CIS tubules, indicating disruption of the blood-testis barrier. In conclusion, Sertoli cells associated with CIS show an altered distribution of ZO-1 and ZO-2 and lose their blood-testis barrier function.

    Neoplasia (New York, N.Y.) 2006;8;12;1019-27

  • Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.

    Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P and Mann M

    Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark.

    Cell signaling mechanisms often transmit information via posttranslational protein modifications, most importantly reversible protein phosphorylation. Here we develop and apply a general mass spectrometric technology for identification and quantitation of phosphorylation sites as a function of stimulus, time, and subcellular location. We have detected 6,600 phosphorylation sites on 2,244 proteins and have determined their temporal dynamics after stimulating HeLa cells with epidermal growth factor (EGF) and recorded them in the Phosida database. Fourteen percent of phosphorylation sites are modulated at least 2-fold by EGF, and these were classified by their temporal profiles. Surprisingly, a majority of proteins contain multiple phosphorylation sites showing different kinetics, suggesting that they serve as platforms for integrating signals. In addition to protein kinase cascades, the targets of reversible phosphorylation include ubiquitin ligases, guanine nucleotide exchange factors, and at least 46 different transcriptional regulators. The dynamic phosphoproteome provides a missing link in a global, integrative view of cellular regulation.

    Cell 2006;127;3;635-48

  • Characterization of epithelial cell shedding from human small intestine.

    Bullen TF, Forrest S, Campbell F, Dodson AR, Hershman MJ, Pritchard DM, Turner JR, Montrose MH and Watson AJ

    Division of Gastroenterology, School of Clinical Sciences, University of Liverpool, Liverpool, UK.

    Intestinal epithelial cells migrate from the base of the crypt to the villi where they are shed. However, little is known about the cell shedding process. We have studied the role of apoptosis and wound healing mechanisms in cell shedding from human small intestinal epithelium. A method preparing paraffin sections of human small intestine that preserves cell shedding was developed. A total of 14 417 villus sections were studied. The relationship of cell shedding to leukocytes (CD45), macrophages (CD68) and blood vessels (CD34) were studied by immunohistochemistry. Apoptotic cells were identified using the M30 antibody against cleaved cytokeratin 18 and an antibody against cleaved caspase-3. Potential wound healing mechanisms were studied using antibodies against Zona Occludens-1 (ZO-1) and phosphorylated myosin light chains (MLCs). We found that 5.3% of villus sections contained a shedding cell. An eosin-positive gap was often seen within the epithelial monolayer beneath shedding cells. Shedding was not associated with leukocytes, macrophages or blood vessels. Cells always underwent apoptosis during ejection from the monolayer. Apoptotic bodies were never seen in the monolayer but morphologically normal cells that were positive for M30 or cleaved caspase-3 were often seen. ZO-1 protein was usually (41/42) localized to the apical pole of cells neighboring a shedding event. Phosphorylated MLCs could be identified in 50% of shedding events. In conclusion, cell shedding is associated with apoptosis though it remains unclear whether apoptosis initiates shedding. It is also associated with phosphorylation of MLCs; a process associated previously with wound healing.

    Funded by: NIDDK NIH HHS: R01 DK61931, R01 DK68271, R21 DK074976

    Laboratory investigation; a journal of technical methods and pathology 2006;86;10;1052-63

  • Dual roles of tight junction-associated protein, zonula occludens-1, in sphingosine 1-phosphate-mediated endothelial chemotaxis and barrier integrity.

    Lee JF, Zeng Q, Ozaki H, Wang L, Hand AR, Hla T, Wang E and Lee MJ

    Gheens Center on Aging, University of Louisville Health Sciences Center, Louisville, Kentucky 40202, USA.

    In this report, sphingosine-1-phosphate (S1P), a serum-borne bioactive lipid, is shown to activate tight-junction-associated protein Zonula Occludens-1 (ZO-1), which in turn plays a critical role in regulating endothelial chemotaxis and barrier integrity. After S1P stimulation, ZO-1 was redistributed to the lamellipodia and cell-cell junctions via the S1P1/G(i)/Akt/Rac pathway. Similarly, both endothelial barrier integrity and cell motility were significantly enhanced in S1P-treated cells through the G(i)/Akt/Rac pathway. Importantly, S1P-enhanced barrier integrity and cell migration were abrogated in ZO-1 knockdown cells, indicating ZO-1 is functionally indispensable for these processes. To investigate the underlying mechanisms, we demonstrated that cortactin plays a critical role in S1P-induced ZO-1 redistribution to the lamellipodia. In addition, S1P significantly induced the formation of endothelial tight junctions. ZO-1 and alpha-catenin polypeptides were colocalized in S1P-induced junctional structures; whereas, cortactin was not observed in these regions. Together, these results suggest that S1P induces the formation of two distinct ZO-1 complexes to regulate two different endothelial functions: ZO-1/cortactin complexes to regulate chemotactic response and ZO-1/alpha-catenin complexes to regulate endothelial barrier integrity. The concerted operation of these two ZO-1 complexes may coordinate two important S1P-mediated functions, i.e. migration and barrier integrity, in vascular endothelial cells.

    Funded by: NHLBI NIH HHS: R01HL067330, R01HL071071

    The Journal of biological chemistry 2006;281;39;29190-200

  • Evaluation of tight junction protein 1 encoding zona occludens 1 as a candidate gene for albuminuria in a Mexican American population.

    Lehman DM, Leach RJ, Johnson-Pais T, Hamlington J, Fowler S, Almasy L, Duggirala R, Stern MP and Abboud HE

    Department of Medicine/Clinical Epidemiology, University of Texas Health Science Center, San Antonio, Texas 78229, USA. lehman@uthscsa.edu

    Albuminuria, a hallmark of diabetic nephropathy, has been shown to be significantly heritable in multiple studies. Therefore, the identification of genes that affect susceptibility to albuminuria may lead to novel avenues of intervention. Current evidence suggests that the podocyte and slit diaphragm play a key role in controlling the selective sieve of the glomerular filtration barrier, and podocyte-specific genes have been identified that are necessary for maintaining its integrity. We therefore investigated the role of gene variants of tight junction protein (TJP1) which encodes another slit diaphragm-associated protein zona occludens 1 as risk factors for albuminuria in the San Antonio Family Diabetes/Gallbladder Study (SAFDGS), which consists of extended Mexican-American families with a high prevalence of type 2 diabetes. Albuminuria, defined as an albumin (mg/dl) to creatinine (mg/dl) ratio (ACR) of 0.03, which is approximately equivalent to a urinary albumin excretion (UAE) >30 mg/day, was present in a total of 14.9% of participants, and 31% had type 2 diabetes. The TJP1 exons, flanking intronic sequence, and putative proximal promoter regions were investigated in this population. Twentynine polymorphisms, including 7 nonsynonymous SNPs, were identified and genotyped in all subjects of this study for association analysis. Three sets of correlated SNPs, which include 3 exonic SNPs, were nominally associated with ACR (p value range 0.007-0.049); however, the association with the discrete trait albuminuria was not significant (p value range 0.094-0.338). We conclude that these variants in TJP1 do not appear to be major determinants for albuminuria in the SAFDGS; however, they may play a minor role in its severity in this Mexican-American population. Further examination of the TJP1 gene region in this and other cohorts will be useful to determine whether ZO-1 plays a significant role in glomerular permselectivity.

    Funded by: NIDDK NIH HHS: P50DK061597, R01-DK-42273, R01-DK-47482, R01-DK-53889; NIMH NIH HHS: MH-59490

    Experimental and clinical endocrinology & diabetes : official journal, German Society of Endocrinology [and] German Diabetes Association 2006;114;8;432-7

  • Interaction of zonula occludens-1 (ZO-1) with alpha-actinin-4: application of functional proteomics for identification of PDZ domain-associated proteins.

    Chen VC, Li X, Perreault H and Nagy JI

    Department of Chemistry, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada.

    The use of recombinant "bait" proteins to capture protein-binding partners, followed by identification of protein interaction networks by mass spectrometry (MS), has gained popularity and widespread acceptance. We have developed an approach using recombinant PDZ protein interaction modules of the membrane-associated guanylate kinase (MAGUK) protein zonula occludens-1 (ZO-1) to pull-down and screen for proteins that interact with these modules via their PDZ domain binding motifs. Identification of proteins by MS of pull-down material was achieved using a vacuum-based chromatography sample preparation device designed for matrix-assisted laser desorption/ionization (MALDI) MS. MS analysis of tryptic fragments in pull-down material revealed a number of potential ZO-1 interacting candidates, including the presence of peptides corresponding to the cortical membrane scaffolding protein alpha-actinin-4. Interaction of alpha-actinin-4 with ZO-1 was confirmed by coimmunoprecipitation of these two proteins from cultured cells, as well as from brain, liver, and heart, and by immunoblot detection of alpha-actinin-4 after pull-down with the first PDZ domain of ZO-1. In contrast, the highly homologous alpha-actinin family member, alpha-actinin-1, displayed no association with ZO-1. Immunofluorescence showed colocalization of alpha-actinin-4 with ZO-1 in cultured HeLa and C6 glioma cells, as well as in a variety of tissues in vivo, including brain, heart, liver, and lung. This study demonstrates the utility of MS-based functional proteomics for identifying cellular components of the ZO-1 scaffolding network. Our finding of the interaction of ZO-1 with alpha-actinin-4 provides a mechanism for linking the known protein recruitment and signaling activities of ZO-1 with alpha-actinin-4-associated plasma membrane proteins that have regulatory activities at cell-cell and cell-extracellular matrix contacts.

    Journal of proteome research 2006;5;9;2123-34

  • Comparative structural analysis of the Erbin PDZ domain and the first PDZ domain of ZO-1. Insights into determinants of PDZ domain specificity.

    Appleton BA, Zhang Y, Wu P, Yin JP, Hunziker W, Skelton NJ, Sidhu SS and Wiesmann C

    Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080, USA.

    We report a structural comparison of the first PDZ domain of ZO-1 (ZO1-PDZ1) and the PDZ domain of Erbin (Erbin-PDZ). Although the binding profile of Erbin-PDZ is extremely specific ([D/E][T/S]WV(COOH)), that of ZO1-PDZ1 is similar ([R/K/S/T][T/S][W/Y][V/I/L](COOH)) but broadened by increased promiscuity for three of the last four ligand residues. Consequently, the biological function of ZO-1 is also broadened, as it interacts with both tight and adherens junction proteins, whereas Erbin is restricted to adherens junctions. Structural analyses reveal that the differences in specificity can be accounted for by two key differences in primary sequence. A reduction in the size of the hydrophobic residue at the base of the site(0) pocket enables ZO1-PDZ1 to accommodate larger C-terminal residues. A single additional difference alters the specificity of both site(-1) and site(-3). In ZO1-PDZ1, an Asp residue makes favorable interactions with both Tyr(-1) and Lys/Arg(-3). In contrast, Erbin-PDZ contains an Arg at the equivalent position, and this side chain cannot accommodate either Tyr(-1) or Lys/Arg(-3) but, instead, interacts favorably with Glu/Asp(-3). We propose a model for ligand recognition that accounts for interactions extending across the entire binding site but that highlights several key specificity switches within the PDZ domain fold.

    The Journal of biological chemistry 2006;281;31;22312-20

  • Uncovering quantitative protein interaction networks for mouse PDZ domains using protein microarrays.

    Stiffler MA, Grantcharova VP, Sevecka M and MacBeath G

    Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, USA.

    One of the principal challenges in systems biology is to uncover the networks of protein-protein interactions that underlie most biological processes. To date, experimental efforts directed at this problem have largely produced only qualitative networks that are replete with false positives and false negatives. Here, we describe a domain-centered approach--compatible with genome-wide investigations--that enables us to measure the equilibrium dissociation constant (K(D)) of recombinant PDZ domains for fluorescently labeled peptides that represent physiologically relevant binding partners. Using a pilot set of 22 PDZ domains, 4 PDZ domain clusters, and 20 peptides, we define a gold standard dataset by determining the K(D) for all 520 PDZ-peptide combinations using fluorescence polarization. We then show that microarrays of PDZ domains identify interactions of moderate to high affinity (K(D) < or = 10 microM) in a high-throughput format with a false positive rate of 14% and a false negative rate of 14%. By combining the throughput of protein microarrays with the fidelity of fluorescence polarization, our domain/peptide-based strategy yields a quantitative network that faithfully recapitulates 85% of previously reported interactions and uncovers new biophysical interactions, many of which occur between proteins that are co-expressed. From a broader perspective, the selectivity data produced by this effort reveal a strong concordance between protein sequence and protein function, supporting a model in which interaction networks evolve through small steps that do not involve dramatic rewiring of the network.

    Funded by: NIGMS NIH HHS: 1 R01 GM072872-01, 5 T32 GM07598-25, R01 GM072872, R01 GM072872-04, T32 GM007598

    Journal of the American Chemical Society 2006;128;17;5913-22

  • Kidney claudin-19: localization in distal tubules and collecting ducts and dysregulation in polycystic renal disease.

    Lee NP, Tong MK, Leung PP, Chan VW, Leung S, Tam PC, Chan KW, Lee KF, Yeung WS and Luk JM

    Department of Surgery, Queen Mary Hospital, The University of Hong Kong, Pokfulam, Hong Kong, SAR, China.

    Tight junction (TJ) constitutes the barrier by controlling the passage of ions and molecules via paracellular pathway and the movement of proteins and lipids between apical and basolateral domains of the plasma membrane. Claudins, occludin, and junctional adhesion molecules are the major three transmembrane proteins at TJ. This study focuses a newly identified mammalian TJ gene, claudin-19, in kidneys. Mouse claudin-19 composes of 224 amino acids and shares 98.2% and 95% amino acid homology with rat and human, respectively; the most evolutionary-related claudins are claudin-1 and -7, which share approximately 75% DNA sequence homology with claudin-19. Claudin-19 is abundantly expressed in the mouse and rat kidneys among the organs examined by Northern blots, and to a much less extent, also found in brain by RT-PCR. Claudin-19 and zonula occludens-1 (ZO-1) are localized at junctional regions of Madin-Darby canine kidney (MDCK) cells by immunofluorescent microscopy. In addition, ZO-1 is found in the claudin-19-associated protein complexes in MDCK cells by co-immunoprecipitation. Using aquaporin-1 and aquaporin-2 antibodies as markers for different renal segment, strong expression of claudin-19 was observed in distal tubules of the cortex as well as in the collecting ducts of the medulla. To less extent, claudin-19 is also present in the proximal tubules (cortex) and in the loop of Henle (medulla). Furthermore, intense claudin-19 immunoreactivity is found co-localized with the ZO-1 in kidneys from postnatal day 15, day 45, and adult rats and mice. Similar localizations of claudin-19 and ZO-1 are also observed in human kidneys. Since these renal segments are mainly for controlling the paracellular cation transport, it is suggested that claudin-19 may participate in these processes. In human polycystic kidneys, decreased expression and dyslocalization of claudin-19 are noticed, suggesting a possible correlation between claudin-19 and renal disorders. Taken together, claudin-19 is a claudin isoform that is highly and specifically expressed in renal tubules with a putative role in TJ homeostasis in renal physiology.

    FEBS letters 2006;580;3;923-31

  • Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes.

    Kimura K, Wakamatsu A, Suzuki Y, Ota T, Nishikawa T, Yamashita R, Yamamoto J, Sekine M, Tsuritani K, Wakaguri H, Ishii S, Sugiyama T, Saito K, Isono Y, Irie R, Kushida N, Yoneyama T, Otsuka R, Kanda K, Yokoi T, Kondo H, Wagatsuma M, Murakawa K, Ishida S, Ishibashi T, Takahashi-Fujii A, Tanase T, Nagai K, Kikuchi H, Nakai K, Isogai T and Sugano S

    Life Science Research Laboratory, Central Research Laboratory, Hitachi, Ltd., Kokubunji, Tokyo, 185-8601, Japan.

    By analyzing 1,780,295 5'-end sequences of human full-length cDNAs derived from 164 kinds of oligo-cap cDNA libraries, we identified 269,774 independent positions of transcriptional start sites (TSSs) for 14,628 human RefSeq genes. These TSSs were clustered into 30,964 clusters that were separated from each other by more than 500 bp and thus are very likely to constitute mutually distinct alternative promoters. To our surprise, at least 7674 (52%) human RefSeq genes were subject to regulation by putative alternative promoters (PAPs). On average, there were 3.1 PAPs per gene, with the composition of one CpG-island-containing promoter per 2.6 CpG-less promoters. In 17% of the PAP-containing loci, tissue-specific use of the PAPs was observed. The richest tissue sources of the tissue-specific PAPs were testis and brain. It was also intriguing that the PAP-containing promoters were enriched in the genes encoding signal transduction-related proteins and were rarer in the genes encoding extracellular proteins, possibly reflecting the varied functional requirement for and the restricted expression of those categories of genes, respectively. The patterns of the first exons were highly diverse as well. On average, there were 7.7 different splicing types of first exons per locus partly produced by the PAPs, suggesting that a wide variety of transcripts can be achieved by this mechanism. Our findings suggest that use of alternate promoters and consequent alternative use of first exons should play a pivotal role in generating the complexity required for the highly elaborated molecular systems in humans.

    Genome research 2006;16;1;55-65

  • Membrane-type 1 matrix metalloproteinase expression is regulated by zonula occludens-1 in human breast cancer cells.

    Polette M, Gilles C, Nawrocki-Raby B, Lohi J, Hunziker W, Foidart JM and Birembaut P

    Institut National de la Sante et de la Recherche Medicale UMRS 514, Laboratory of Histology, IFR 53, CHU Maison Blanche, Reims, France. myriam.polette@univ-reims.fr

    The acquisition of a migratory/invasive phenotype by tumor cells is characterized by the loss of cell-cell adhesion contacts and the expression of degradative properties. In this study, we examined the effect of the disorganization of occludin/zonula occludens (ZO)-1 tight junction (TJ) complexes on the expression of membrane-type 1 matrix metalloproteinase (MT1-MMP). We first compared the expression of MT1-MMP and the localization of occludin/ZO-1 complexes in breast tumor cell lines displaying various degrees of invasiveness. We showed that the expression of MT1-MMP in invasive breast tumor cell lines correlates with the absence of occludin and with a cytoplasmic localization of ZO-1. In contrast, noninvasive cell lines displayed a membrane staining for both ZO-1 and occludin and did not express MT1-MMP. In vivo, cytoplasmic ZO-1 and MT1-MMP could be detected in invasive tumor clusters of human breast carcinomas. We then used RNA interference strategy to inhibit ZO-1 expression in invasive BT549 cells and to evaluate the effect of ZO-1 down-regulation on MT1-MMP expression. We observed that ZO-1 small interfering RNA transfection down-regulates MT1-MMP mRNAs and proteins and subsequently decreases the ability of tumor cells to invade a reconstituted basement membrane in a Boyden chamber assay. Inversely, transfection of expression vectors encoding wild-type ZO-1 or the NH2-terminal fragment of ZO-1 comprising the PSD95/DLG/ZO-1 domains in BT549 activated a human MT1-MMP promoter luciferase reporter construct and increased cell invasiveness. Such transfections concomitantly activated the beta-catenin/TCF/LEF pathway. Our results therefore show that ZO-1, besides its structural role in TJ assembly, can intervene in signaling events promoting tumor cell invasion.

    Cancer research 2005;65;17;7691-8

  • Influence of protein kinase C on transcription of the tight junction elements ZO-1 and occludin.

    Weiler F, Marbe T, Scheppach W and Schauber J

    Department of Medicine, Division of Gastroenterology, University of Wuerzburg, Wuerzburg, Germany. weiler_f@klinik.uni-wuerzburg.de

    Tight junctions as an epithelial barrier against paracellular diffusion have mainly been investigated on the protein level with particular respect to subcellular localization. In this study, real-time PCR has been established to investigate the influence of protein kinase C (PKC) modulation on the transcription of tight junction elements occludin and ZO-1 in the cell line T84. Activation of PKC by the phorbol ester TPA induced ZO-1 and occludin transcription, whereas PKC inhibition lead to decreased expression levels. Activation of PKC exerted its effect on transcript level directly. PKC signal was partially transduced via MEK1/MEK2 but depended strongly on MAPK independent pathways probably involving nuclear localized PKC, whereas p38 signaling was not implicated. TPA induced loss of function concomitant with a dislocation of ZO-1 and occludin could be prevented by inhibition of MEK1 by PD98059. Overall ZO-1 and occludin seem to be identically regulated in colonic epithelium on the transcript level.

    Journal of cellular physiology 2005;204;1;83-6

  • HIV-1 gp120 proteins alter tight junction protein expression and brain endothelial cell permeability: implications for the pathogenesis of HIV-associated dementia.

    Kanmogne GD, Primeaux C and Grammas P

    Department of Pharmacology, Center for Neurovirology and Neurodegenerative Disorders, University of Nebraska Medical Center, Omaha, Nebraska 68198-5215, USA. gkanmogne@unmc.edu

    Breakdown of the blood-brain barrier (BBB) is commonly seen in patients with HIV-associated dementia (HAD) despite the lack of productive infection of the brain endothelium. It is likely that secreted viral products play a major role in BBB damage and the development of HAD. The objective of this study is to determine the effects of gp120 proteins on brain endothelial cell permeability and junctional protein expression. Our results showed that treatment of cultured human brain endothelial cells with gp120 for 24 hours results in increased permeability of the endothelial monolayer. Also, gp120 proteins caused disruption and downregulation of the tight junction proteins ZO-1, ZO-2, and occludin in these cells. Other junctional proteins such as claudin-1 and claudin-5 were unaffected by gp120 treatment. These data demonstrate that HIV gp120 proteins alter both the functional and molecular properties of the BBB, which could increase trafficking of HIV, infected cells, and toxic humoral factors into the central nervous system and contribute to the pathogenesis of HAD.

    Funded by: NIMH NIH HHS: 1K01MH068214-1

    Journal of neuropathology and experimental neurology 2005;64;6;498-505

  • Up-regulated expression of zonula occludens protein-1 in human melanoma associates with N-cadherin and contributes to invasion and adhesion.

    Smalley KS, Brafford P, Haass NK, Brandner JM, Brown E and Herlyn M

    Wistar Institute, 3601 Spruce St., Philadelphia, Pennsylvania 19104, USA.

    During the process of malignant transformation, nascent melanoma cells escape keratinocyte control through down-regulation of E-cadherin and instead communicate among themselves and with fibroblasts via N-cadherin-based cell-cell contacts. The zonula occludens (ZO) protein-1 is a membrane-associated component of both the tight and adherens junctions found at sites of cell-cell contact. In most cancers, levels of ZO-1 are typically down-regulated, leading to increased motility. Here we report the novel observation that ZO-1 expression is up-regulated in melanoma cells and is located at adherens junctions between melanoma cells and fibroblasts. Immunofluorescence and co-immunoprecipitation studies showed co-localization of ZO-1 with N-cadherin. Down-regulation of ZO-1 in melanoma cells through RNA interference produced marked changes in cell morphology--leading to a less-dendritic, more rounded phenotype. Consistent with a role in N-cadherin-based adhesion, RNAi-treated melanoma cells were less adherent and invasive when grown in a collagen gel. These data provide the first evidence that increased ZO-1 expression in melanoma contributes to the oncogenic behavior of this tumor and further illustrate that protein products of genes, such as ZO-1, can function in either a pro- or anti-oncogenic manner when expressed in different cellular contexts.

    Funded by: NCI NIH HHS: CA 10815, CA 25874, CA 76674, CA 93372, P01 CA025874, P30 CA010815, P50 CA093372, R01 CA076674

    The American journal of pathology 2005;166;5;1541-54

  • Canonical transient receptor potential channel 4 (TRPC4) co-localizes with the scaffolding protein ZO-1 in human fetal astrocytes in culture.

    Song X, Zhao Y, Narcisse L, Duffy H, Kress Y, Lee S and Brosnan CF

    Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.

    Members of the mammalian transient receptor potential (TRP) family form cation-permeable channels at the plasma membrane implicated in capacitative calcium influx after activation by either second-messenger-mediated pathways or store depletion, or both. This study shows that with the use of RT-PCR, Western blotting, and immunohistochemistry, resting astrocytes express TRPC4 at the cell membrane, particularly at sites of cell-to-cell contact. By confocal imaging and immunoelectron microscopy, we detected co-localization of TRPC4 with the scaffolding protein zonula occludens 1 (ZO-1), and demonstrated that immunoprecipitation with antibodies to ZO-1 brought down TRPC4, and vice-versa. It has been proposed that the targeting of TRPC4 to the cell membrane is dependent on the interaction of the C-terminal TRL motif with PDZ domains. Using transfection of astrocytes with myc-tagged TRPC4 or TRL-motif truncated TRPC4 (deltaTRL), we found that deltaTRL localized predominantly to a juxtanuclear compartment, whereas the wild-type protein showed cell surface distribution. Deletion of the TRL motif also reduced plasma membrane expression as assessed by cell surface biotinylation experiments. Using GST fusion proteins, we found that TRPC4 interacted with the PDZ1 domain of ZO-1 and that this was also dependent on the TRL motif. Thus, our data demonstrate that the PDZ-interacting domain of TRPC4 controls its cell surface localization. These data implicate TRPC4 in the regulation of calcium homeostasis in astrocytes, particularly as part of a signaling complex that forms at junctional sites between astrocytes.

    Funded by: NIGMS NIH HHS: T32GM07288; NINDS NIH HHS: NS046620, NS07098, NS11920, NS40137

    Glia 2005;49;3;418-29

  • Association of ARVCF with zonula occludens (ZO)-1 and ZO-2: binding to PDZ-domain proteins and cell-cell adhesion regulate plasma membrane and nuclear localization of ARVCF.

    Kausalya PJ, Phua DC and Hunziker W

    Institute of Molecular and Cell Biology, Epithelial Cell Biology Laboratory, Singapore 138673, Singapore.

    ARVCF, an armadillo-repeat protein of the p120(ctn) family, associates with classical cadherins and is present in adherens junctions, but its function is poorly understood. Here, we show that ARVCF interacts via a C-terminal PDZ-binding motif with zonula occludens (ZO)-1 and ZO-2. ARVCF and ZO-1 partially colocalize in the vicinity of the apical adhesion complex in polarized epithelial Madin-Darby canine kidney cells. ARVCF, ZO-1, and E-cadherin form a complex and are recruited to sites of initial cell-cell contact in sparse cell cultures. E-cadherin binding and plasma membrane localization of ARVCF require the PDZ-binding motif. Disruption of cell-cell adhesion releases ARVCF from the plasma membrane and an increased fraction of the protein localizes to the nucleus. Nuclear localization of ARVCF also requires the PDZ-binding motif and can be mediated by the PDZ domains of ZO-2. Thus, the interaction of ARVCF with distinct PDZ-domain proteins determines its subcellular localization. Interactions with ZO-1 and ZO-2, in particular, may mediate recruitment of ARVCF to the plasma membrane and the nucleus, respectively, possibly in response to cell-cell adhesion cues.

    Molecular biology of the cell 2004;15;12;5503-15

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • Nephrin forms a complex with adherens junction proteins and CASK in podocytes and in Madin-Darby canine kidney cells expressing nephrin.

    Lehtonen S, Lehtonen E, Kudlicka K, Holthöfer H and Farquhar MG

    Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, USA.

    Mutations in the NPHS1 gene encoding nephrin lead to congenital nephrotic syndrome of the Finnish type. Nephrin is a key component of the glomerular slit diaphragms between epithelial foot processes, but its role in the pathogenesis of this disease is poorly understood. To further clarify the molecular mechanisms involved we investigated the interactions between nephrin and other components of the foot processes and filtration slits, especially adherens junction proteins, and searched for novel nephrin interacting proteins. Using co-immunoprecipitation and pull-down assays we show here that nephrin forms a multiprotein complex with cadherins and p120 catenin and with three scaffolding proteins, ZO-1, CD2AP, and CASK, in kidney glomeruli and when expressed in Madin-Darby canine kidney cells. CASK was identified as a novel binding partner of nephrin by mass spectrometry and was localized to podocytes in the glomerulus. CASK is a scaffolding protein that participates in maintenance of polarized epithelial cell architecture by linking membrane proteins and signaling molecules to the actin cytoskeleton. Our results support a model whereby the glomerular slit diaphragms are composed of cell adhesion molecules of the immunoglobulin and cadherin superfamilies that are connected to each other and to the actin cytoskeleton and signaling networks via the cytoplasmic scaffolding proteins CASK, CD2AP, and ZO-1.

    Funded by: NIDDK NIH HHS: DK 17724, R01 DK017724, R37 DK017724

    The American journal of pathology 2004;165;3;923-36

  • The specific fates of tight junction proteins in apoptotic epithelial cells.

    Bojarski C, Weiske J, Schöneberg T, Schröder W, Mankertz J, Schulzke JD, Florian P, Fromm M, Tauber R and Huber O

    Institute of Clinical Chemistry and Pathobiochemistry, Charité - Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany.

    The polarized morphology of epithelial cells depends on the establishment and maintenance of characteristic intercellular junctions. The dramatic morphological changes observed in apoptotic epithelial cells were ascribed at least in part to the specific fragmentation of components of adherens junctions and desmosomes. Little, however, is known about tight junctions during apoptosis. We have found that after induction of apoptosis in epithelial cells, tight junction proteins undergo proteolytic cleavage in a distinctive manner correlated with a disruption of tight junctions. The transmembrane protein occludin and, likewise, the cytoplasmic adaptor proteins ZO-1 and ZO-2 are fragmented by caspase cleavage. In addition, occludin is cleaved at an extracellular site by a metalloproteinase. The caspase cleavage site in occludin was mapped C-terminally to Asp(320) within the C-terminal cytoplasmic domain. Mutagenesis of this site efficiently blocked fragmentation. In the presence of caspase and/or metalloproteinase inhibitors, fragmentation of occludin, ZO-1 and ZO-2 was blocked and cellular morphology was almost fully preserved. Interestingly, two members of the claudin family of transmembrane tight junction proteins exhibited a different behavior. While the amount of claudin-2 protein was reduced similarly to occludin, ZO-1 and ZO-2, claudin-1 was either fully preserved or was even increased in apoptotic cells.

    Journal of cell science 2004;117;Pt 10;2097-107

  • Influence of retinoic acid on adhesion complexes in human hepatoma cells: a clue to its antiproliferative effects.

    Ara C, Devirgiliis LC and Massimi M

    Department of Basic and Applied Biology, University of L'Aquila, L'Aquila, Italy.

    Retinoic acid exerts antiproliferative and differentiative effects in normal and transformed in vitro hepatocytes. In order to verify whether these effects are related to a modulation of adhesion molecules, we used Western blot analysis and immunofluorescence microscopy to investigate the E-cadherinl/beta-catenin complex, the main system of adherens junctions, and the occludin/ZO-1 complex present in the tight junctions in HepG2 cells cultured in the presence or absence of retinoic acid. Results showed that retinoic acid treatment increases the amount of beta-catenin bound to E-cadherin by decreasing its tyrosine-phosphorylation level. Similar results were obtained with the tight junction system, in which the amount of occludin/ZO-1 complex is increased by a similar mechanism that reduced the level of ZO-1 phosphorylation on tyrosine. Immunofluorescence images also confirm these results, showing the localization on the cell surface of both adhesion complexes. Their insertion into the plasma membrane could be suggestive of an optimal reassembly and function of adherens and tight junctions in hepatoma cells, indicating that retinoic acid, besides inhibiting cell proliferation, improves cell-cell adhesion, sustaining or inducing the expression of a more differentiated phenotype.

    Cell communication & adhesion 2004;11;1;13-23

  • Connexin47, connexin29 and connexin32 co-expression in oligodendrocytes and Cx47 association with zonula occludens-1 (ZO-1) in mouse brain.

    Li X, Ionescu AV, Lynn BD, Lu S, Kamasawa N, Morita M, Davidson KG, Yasumura T, Rash JE and Nagy JI

    Department of Physiology, Faculty of Medicine, University of Manitoba, 730 William Avenue, Winnipeg, Manitoba, Canada R3E 3J7.

    Gap junctions between glial cells in mammalian CNS are known to contain several connexins (Cx), including Cx26, Cx30 and Cx43 at astrocyte-to-astrocyte junctions, and Cx29 and Cx32 on the oligodendrocyte side of astrocyte-to-oligodendrocyte junctions. Recent reports indicating that oligodendrocytes also express Cx47 prompted the present studies of Cx47 localization and relationships to other glial connexins in mouse CNS. In view of the increasing number of connexins reported to interact directly with the scaffolding protein zonula occludens-1 (ZO-1), we investigated ZO-1 expression and Cx47/ZO-1 interaction capabilities in brain, spinal cord and Cx47-transfected HeLa cells. From counts of over 9000 oligodendrocytes labeled by immunofluorescence in various brain regions, virtually all of these cells were found to express Cx29, Cx32 and Cx47. Oligodendrocyte somata displayed robust Cx47-immunopositive puncta that were co-localized with punctate labeling for Cx32 and Cx43. By freeze-fracture replica immunogold labeling, Cx47 was abundant on the oligodendrocyte-side of oligodendrocyte/astrocyte gap junctions. By immunofluorescence, labeling for Cx47 along myelinated fibers was sparse in most brain regions, whereas Cx29 and Cx32 were previously found to be concentrated along these fibers. By immunogold labeling, Cx47 was found in numerous small gap junctions linking myelin to astrocytes, but not within deeper layers of myelin. Brain subcellular fractionation revealed a lack of Cx47 enrichment in myelin fractions, which nevertheless contained an enrichment of Cx32 and Cx29. Oligodendrocytes were immunopositive for ZO-1, and displayed almost total Cx47/ZO-1 co-localization. ZO-1 was found to co-immunoprecipitate with Cx47, and pull-down assays indicated binding of Cx47 to the second PDZ domain of ZO-1. Our results indicate widespread expression of Cx47 by oligodendrocytes, but with a distribution pattern in relative levels inverse to the abundance of Cx29 in myelin and paucity of Cx29 in oligodendrocyte somata. Further, our findings suggest a scaffolding and/or regulatory role of ZO-1 at the oligodendrocyte side of astrocyte-to-oligodendrocyte gap junctions.

    Funded by: NCRR NIH HHS: S10 RR015706; NINDS NIH HHS: NS31027, NS39040, R01 NS044010, R01 NS044395

    Neuroscience 2004;126;3;611-30

  • A novel claudin 16 mutation associated with childhood hypercalciuria abolishes binding to ZO-1 and results in lysosomal mistargeting.

    Müller D, Kausalya PJ, Claverie-Martin F, Meij IC, Eggert P, Garcia-Nieto V and Hunziker W

    Charité Children's Hospital, Department of Pediatric Nephrology, Berlin, Germany. dominik.mueller@charite.de

    Mutations in the gene coding for the renal tight junction protein claudin 16 cause familial hypomagnesemia with hypercalciuria and nephrocalcinosis, an autosomal recessive disorder of renal Ca(2+) and Mg(2+) handling that progressively leads to chronic renal failure, with nephrolithiasis having been reported in heterozygous carriers. Screening a cohort of 11 families with idiopathic hypercalciuria identified a novel homozygous mutation in the claudin 16 gene in two families. In contrast to classical symptoms of familial hypomagnesemia with hypercalciuria and nephrocalcinosis, the patients displayed serious but self-limiting childhood hypercalciuria with preserved glomerular filtration rate. The mutation results in inactivation of a PDZ-domain binding motif, thereby disabling the association of the tight junction scaffolding protein ZO-1 with claudin 16. In contrast to wild-type claudin 16, the mutant no longer localizes to tight junctions in kidney epithelial cells but instead accumulates in lysosomes. Thus, mutations at different intragenic sites in the claudin 16 gene may lead to particular clinical phenotypes with a distinct prognosis. Mutations in claudin 16 that affect interaction with ZO-1 lead to lysosomal mistargeting, providing-for the first time, to our knowledge-insight into the molecular mechanism of a disease-associated mutation in the claudin 16 gene.

    American journal of human genetics 2003;73;6;1293-301

  • Human ENS regulates the intestinal epithelial barrier permeability and a tight junction-associated protein ZO-1 via VIPergic pathways.

    Neunlist M, Toumi F, Oreschkova T, Denis M, Leborgne J, Laboisse CL, Galmiche JP and Jarry A

    Institut National de la Santé et de la Recherche Médicale U 539, Faculté de Médecine, 1, rue Gaston Veil, 44035 Nantes Cedex, France.

    Although the enteric nervous system (ENS) has been shown to regulate various mucosal functions, its role in the physiological control of the human intestinal epithelial barrier is unknown. The aim of this study was to investigate whether the ENS is able to modulate epithelial barrier permeability and a key tight junction-associated protein, zonula occludens-1 (ZO-1). Therefore, we developed a co-culture model, consisting of human submucosa containing the submucosal neuronal network and human polarized colonic epithelial monolayers (HT29-Cl.16E or Caco-2). Submucosal neurons were activated by electrical field stimulation (EFS). Permeability was assessed by measuring the flux of paracellular permeability markers (FITC-dextran or FITC-inulin) across epithelial monolayers. Expression of ZO-1 was determined by immunofluorescence, quantitative immunoblot analysis, and real time RT-PCR. Using the coculture model, we showed that EFS of submucosal neurons resulted in a reduction in FITC-dextran or FITC-inulin fluxes, which was blocked by TTX. In HT29-Cl.16E, the effect of submucosal neuron activation was blocked by a VIP receptor antagonist (VIPra) and reproduced by VIP. Furthermore, ZO-1 expression (mRNA, protein) assessed in HT29-Cl.16E, was significantly increased after submucosal neuron activation by EFS. These effects on ZO-1 expression were blocked by TTX and VIPra and reproduced by VIP. In conclusion, our results strongly suggest a modulatory role of VIPergic submucosal neuronal pathways on intestinal epithelial barrier permeability and ZO-1 expression.

    American journal of physiology. Gastrointestinal and liver physiology 2003;285;5;G1028-36

  • The junctional adhesion molecule (JAM) family members JAM-2 and JAM-3 associate with the cell polarity protein PAR-3: a possible role for JAMs in endothelial cell polarity.

    Ebnet K, Aurrand-Lions M, Kuhn A, Kiefer F, Butz S, Zander K, Meyer zu Brickwedde MK, Suzuki A, Imhof BA and Vestweber D

    Institute of Cell Biology, ZMBE, University of Münster, Germany. ebnetk@uni-muenster.de

    Tight junctions play a central role in the establishment of cell polarity in vertebrate endothelial and epithelial cells. A ternary protein complex consisting of the cell polarity proteins PAR-3 and PAR-6 and the atypical protein kinase C localizes at tight junctions and is crucial for tight junction formation. We have recently shown that PAR-3 directly associates with the junctional adhesion molecule (JAM), which suggests that the ternary complex is targeted to tight junctions of epithelial cells through PAR-3 binding to JAM. The expression of JAM-related proteins by endothelial cells prompted us to test whether recruitment of the ternary complex in endothelial cells can occur through binding to JAM-2, JAM-3, endothelial cell-selective adhesion molecule (ESAM) or coxsackie- and adenovirus receptor (CAR). Here we show that the two JAM-related proteins JAM-2 and JAM-3 directly associate with PAR-3. The association between PAR-3 and JAM-2/-3 is mediated through the first PDZ domain of PAR-3. In agreement with the predominant expression of JAM-2 and JAM-3 in endothelial cells, we found that PAR-3 is expressed by endothelial cells in vivo and is localized at cell contacts of cultured endothelial cells. PAR-3 associates with JAM-2/-3 but not with the JAM-related Ig-superfamily members ESAM or CAR. In addition, we show that the tight junction-associated protein ZO-1 associates with JAM-2/-3 in a PDZ domain-dependent manner. Using ectopic expression of JAM-2 in CHO cells, we show that the junctional localization of JAM-2 is regulated by serine phosphorylation and that its clustering at cell-cell contacts recruits endogenous PAR-3 and ZO-1. Our findings suggest that JAM-2 affects endothelial cell junctions by its regulated clustering at intercellular contacts, and they support a role for JAM-2, and possibly JAM-3, in tight junction formation of endothelial cells.

    Journal of cell science 2003;116;Pt 19;3879-91

  • Neph1 and nephrin interaction in the slit diaphragm is an important determinant of glomerular permeability.

    Liu G, Kaw B, Kurfis J, Rahmanuddin S, Kanwar YS and Chugh SS

    Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA.

    Neph1-deficient mice develop nephrotic syndrome at birth, indicating the importance of this protein in the development of a normal glomerular filtration barrier. While the precise subcellular localization of Neph1 remains unknown, its relationship with other components of the glomerular filtration barrier is of great interest in this field. In this paper, we localize the expression of Neph1 to the glomerular slit diaphragm by immunogold electron microscopy in rodents and describe its direct interaction with two other components of the slit diaphragm, nephrin and ZO-1. Both native and recombinant Neph1 associate with each other as dimers and multimers and interact with nephrin via their extracellular segments. Disruption of the Neph1-nephrin interaction in vivo by injecting combinations of individual subnephritogenic doses of anti-Neph1 and anti-nephrin results in complement- and leukocyte-independent proteinuria with preserved foot processes. This disruption modestly reduces Neph1 and nephrin protein expression in podocytes and dramatically reduces ZO-1 protein expression via the interaction of ZO-1 PDZ domains with the cytoplasmic tail of Neph1, independent of changes in mRNA expression of all three genes. The interaction between nephrin and Neph1 is specific and not shared by either protein with P-cadherin, another integral slit diaphragm protein. The interaction between nephrin and Neph1 therefore appears to be an important determinant of glomerular permeability.

    Funded by: NIDDK NIH HHS: DK-28492, DK-60635, DK-61275, K08 DK061275, R01 DK028492, R01 DK060635, R37 DK028492

    The Journal of clinical investigation 2003;112;2;209-21

  • The protein tyrosine phosphatase Pez is a major phosphatase of adherens junctions and dephosphorylates beta-catenin.

    Wadham C, Gamble JR, Vadas MA and Khew-Goodall Y

    Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Adelaide, SA 5000, Australia.

    Cell-cell adhesion regulates processes important in embryonal development, normal physiology, and cancer progression. It is regulated by various mechanisms including tyrosine phosphorylation. We have previously shown that the protein tyrosine phosphatase Pez is concentrated at intercellular junctions in confluent, quiescent monolayers but is nuclear in cells lacking cell-cell contacts. We show here with an epithelial cell model that Pez localizes to the adherens junctions in confluent monolayers. A truncation mutant lacking the catalytic domain acts as a dominant negative mutant to upregulate tyrosine phosphorylation at adherens junctions. We identified beta-catenin, a component of adherens junctions, as a substrate of Pez by a "substrate trapping" approach and by in vitro dephosphorylation with recombinant Pez. Consistent with this, ectopic expression of the dominant negative mutant caused an increase in tyrosine phosphorylation of beta-catenin, demonstrating that Pez regulates the level of tyrosine phosphorylation of adherens junction proteins, including beta-catenin. Increased tyrosine phosphorylation of adherens junction proteins has been shown to decrease cell-cell adhesion, promoting cell migration as a result. Accordingly, the dominant negative Pez mutant enhanced cell motility in an in vitro "wound" assay. This suggests that Pez is also a regulator of cell motility, most likely through its action on cell-cell adhesion.

    Molecular biology of the cell 2003;14;6;2520-9

  • Lens connexins alpha3Cx46 and alpha8Cx50 interact with zonula occludens protein-1 (ZO-1).

    Nielsen PA, Baruch A, Shestopalov VI, Giepmans BN, Dunia I, Benedetti EL and Kumar NM

    Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

    Connexin alpha1Cx43 has previously been shown to bind to the PDZ domain-containing protein ZO-1. The similarity of the carboxyl termini of this connexin and the lens fiber connexins alpha3Cx46 and alpha8Cx50 suggested that these connexins may also interact with ZO-1. ZO-1 was shown to be highly expressed in mouse lenses. Colocalization of ZO-1 with alpha3Cx46 and alpha8Cx50 connexins in fiber cells was demonstrated by immunofluorescence and by fracture-labeling electron microscopy but showed regional variations throughout the lens. ZO-1 was found to coimmunoprecipitate with alpha3Cx46 and alpha8Cx50, and pull-down experiments showed that the second PDZ domain of ZO-1 was involved in this interaction. Transiently expressed alpha3Cx46 and alpha8Cx50 connexins lacking the COOH-terminal residues did not bind to the second PDZ domain but still formed structures resembling gap junctions by immunofluorescence. These results indicate that ZO-1 interacts with lens fiber connexins alpha3Cx46 and alpha8Cx50 in a manner similar to that previously described for alpha1Cx43. The spatial variation in the interaction of ZO-1 with lens gap junctions is intriguing and is suggestive of multiple dynamic roles for this association.

    Funded by: NEI NIH HHS: EY-13605, EY1792, P30 EY001792, R01 EY013605, R01 EY014232

    Molecular biology of the cell 2003;14;6;2470-81

  • Tight junction-related protein expression and distribution in human corneal epithelium.

    Ban Y, Dota A, Cooper LJ, Fullwood NJ, Nakamura T, Tsuzuki M, Mochida C and Kinoshita S

    Department of Ophthalmology, Nantan General Hospital, 25 Yagiueno, Yagi-cho, Funai-gun, 629-0197, Kyoto, Japan. yban@ophth.kpu-m.ac.jp

    Purpose: To investigate the expression and cellular distribution of the tight junction-related proteins occludin, claudin and ZO-1 in human corneal epithelium.

    Methods: Light and electron immunohistochemistry was used to determine tissue distribution of occludin, claudin-1 and ZO-1 in the human corneal epithelium. Reverse transcription-polymerase chain reaction was used to reveal claudin mRNA expression in human corneal epithelium.

    Results: In transverse sections, occludin and ZO-1 were localized at the apical cell-cell junctions between superficial cells in stratified corneal epithelium. Both basal and basolateral membranes of superficial cells were stained by the claudin-1 antibody, but no apical membrane staining was observed. In en face sections, claudin-1 and ZO-1 antibodies showed as bands that corresponded to the junctional complex. Claudin-1 staining of superficial cell cytoplasm was also observed. Occludin staining was seen at the junctional complex, where it was not continuous, but dotted along the cell junctions. The transcripts for claudin-1 and several other claudin isotypes, such as -2, -3, -4, -7, -9 and -14 were identified.

    Conclusion: Not only occludin, but also some claudins act as integral transmembrane proteins in the corneal epithelium. ZO-1 is a component of the corneal epithelial tight junction, as it is in most epithelial cells.

    Experimental eye research 2003;76;6;663-9

  • The carboxyl terminus of Neph family members binds to the PDZ domain protein zonula occludens-1.

    Huber TB, Schmidts M, Gerke P, Schermer B, Zahn A, Hartleben B, Sellin L, Walz G and Benzing T

    Renal Division, University Hospital Freiburg, Hugstetter Str. 55, 79106 Freiburg, Germany.

    The PSD95/Dlg/ZO-1 (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. We demonstrate 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, our data suggest that ZO-1 may organize Neph proteins and recruit signal transduction components to the slit diaphragm of podocytes.

    The Journal of biological chemistry 2003;278;15;13417-21

  • Rab8B GTPase and junction dynamics in the testis.

    Lau AS and Mruk DD

    Population Council, Center for Biomedical Research, New York, New York 10021, USA.

    Throughout spermatogenesis, germ cells migrate from the basal to the adluminal compartment while remaining attached to Sertoli cells via actin-based adherens and intermediate filament-based anchoring junctions. However, the events that trigger deadhesion and adhesion remain largely unknown. As part of our continued effort in elucidating the mechanism of germ cell movement, we have examined the role of Rab8B, a GTPase probably participating in intracellular trafficking events at the site of the adherens junction. By RT-PCR Rab8B mRNA was found in the brain, testis, heart, kidney, and spleen. Immunohistochemical studies revealed that Rab8B was concentrated predominantly in the basal compartment, localizing to a similar site at which immunoreactive E-cadherin was found. Additional experiments demonstrated that Rab8B associated with the actin, intermediate filament, and microtubule cytoskeletal networks. When Sertoli cells were cultured at high density or germ cells were cocultured with Sertoli cells, Rab8B increased significantly during junction assembly. Moreover, inclusion of germ cell-conditioned medium in Sertoli cell cultures resulted in stimulation of Rab8B expression. Conversely, treatment of adult rats with 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide reduced Rab8B mRNA and protein levels, coinciding with the time of germ cell loss from the epithelium. Taken collectively, these studies suggest that Rab8B participates in adherens junction dynamics in the testis.

    Endocrinology 2003;144;4;1549-63

  • Dedifferentiation and decreased expression of adhesion molecules, E-cadherin and ZO-1, in colorectal cancer are closely related to liver metastasis.

    Kaihara T, Kusaka T, Nishi M, Kawamata H, Imura J, Kitajima K, Itoh-Minami R, Aoyama N, Kasuga M, Oda Y, Hattori M and Fujimori T

    Dept. of Surgical and Molecular Pathology, Dokkyo University School of Medicine, Tochigi, Japan.

    Carcinoma cells with high metastatic potential often show a dedifferentiated phenotype at the primary site. In this study, a total of 48 cases (24 primary tumors of colorectal cancer (Pr-CRC) with liver metastasis, 24 without) were examined for E-cadherin and ZO-1 expression by immunohistochemical staining, and for their dedifferentiated phenotype. The expression levels of E-cadherin and ZO-1 were markedly decreased in the cancer cells of tumors with liver metastasis. Moreover, dedifferentiation of cancer cells, which was evaluated by the modified Gleason score, was also related to liver metastasis. However, none of the conventional clinicopathologic parameters of invasion, except lymph node metastasis, showed any relationship with liver metastasis. These results indicate that dedifferentiation and a decreased expression of E-cadherin and ZO-1 are closely related to liver metastasis.

    Journal of experimental & clinical cancer research : CR 2003;22;1;117-23

  • Redifferentiation and ZO-1 reexpression in liver-metastasized colorectal cancer: possible association with epidermal growth factor receptor-induced tyrosine phosphorylation of ZO-1.

    Kaihara T, Kawamata H, Imura J, Fujii S, Kitajima K, Omotehara F, Maeda N, Nakamura T and Fujimori T

    Department of Surgical and Molecular Pathology, Dokkyo University School of Medicine, 880 Kita-kobayashi, Mibu, Shimotsuga, Tochigi 321-0293.

    Tubular gland structures of colorectal cancer (CRC) have been demonstrated to undergo dedifferentiation at the primary site, and then the gland structures are re-formed in the liver metastases. In this study, we examined the degree of differentiation of the gland structure of 48 cases of CRCs (24 cases with synchronous liver metastasis, 24 cases without metastasis) by the modified Gleason grading system. We also investigated the role of ZO-1, one of the tight junction proteins, in the morphological changes, i.e., dedifferentiation and redifferentiation, of CRCs at the primary site and liver metastases. Liver-metastasized CRCs (2.47+/-0.37) showed a lower score in the modified Gleason grading system than the corresponding primary tumors (3.28+/-0.36) did, i.e., the tumor cells had undergone redifferentiation at liver metastases. ZO-1 was expressed at the apical cell borders of normal colorectal epithelium, the luminal side of which has tubular gland structures. In comparison with this normal epithelium, the ZO-1 expression level was frequently reduced in primary CRC with liver metastasis (20.8%) and ZO-1 was reexpressed in liver metastasized cancers (79.2%). Furthermore, it was demonstrated by an immunoprecipitation-western blotting analysis on 5 cases of CRC with liver metastasis that ZO-1 bound to epidermal growth factor receptor (EGFR) irrespective of the phosphorylation status of EGFR, and that EGFR associated ZO-1 was highly tyrosine-phosphorylated only in the primary CRC, but was dephosphorylated in the liver-metastasized cancers. Our observations suggest that tyrosine phosphorylation of ZO-1 leads to down-regulation of the function of ZO-1 and dedifferentiation of the glands in CRCs, and these phenomena contribute to liver metastases, and redifferentiation of the glands occurs in the liver metastases.

    Cancer science 2003;94;2;166-72

  • Tyrosine phosphorylation and dissociation of occludin-ZO-1 and E-cadherin-beta-catenin complexes from the cytoskeleton by oxidative stress.

    Rao RK, Basuroy S, Rao VU, Karnaky KJ and Gupta A

    Department of Physiology, University of Tennessee Health Sciences Center, 894 Union Avenue, Memphis, TN 38163, U.S.A. rkrao@physio1.utmem.edu

    The oxidative-stress-induced alteration in paracellular junctional complexes was analysed in Caco-2 cell monolayer. Oxidative stress induced a rapid increase in tyrosine phosphorylation of occludin, zonula occludens (ZO)-1, E-cadherin and beta-catenin. An oxidative-stress-induced decrease in transepithelial electrical resistance was associated with a redistribution of occludin-ZO-1 and E-cadherin-beta-catenin complexes from the intercellular junctions. Genistein, a tyrosine kinase inhibitor, prevented the oxidative-stress-induced decrease in resistance and redistribution of protein complexes. Occludin, ZO-1, E-cadherin and beta-catenin in the Triton-insoluble cytoskeletal fraction were reduced by oxidative stress, which was prevented by genistein. Oxidative stress also reduced the co-immunoprecipitation of ZO-1 with occludin, which was prevented by genistein. Co-immunoprecipitation of beta-catenin with E-cadherin was unaffected by oxidative stress or genistein. ZO-1, E-cadherin and beta-catenin in the plasma membrane or membrane-cytoskeleton were either slightly reduced or unaffected by oxidative stress or genistein. These results show that oxidative stress induces tyrosine phosphorylation and cellular redistribution of occludin-ZO-1 and E-cadherin-beta-catenin complexes by a tyrosine-kinase-dependent mechanism.

    Funded by: NIAAA NIH HHS: R01-AA12307; NIDDK NIH HHS: R01 DK055532, R01-DK55532

    The Biochemical journal 2002;368;Pt 2;471-81

  • The human plasma proteome: history, character, and diagnostic prospects.

    Anderson NL and Anderson NG

    Plasma Proteome Institute, Washington, DC 20009-3450, USA. leighanderson@plasmaproteome.org

    The human plasma proteome holds the promise of a revolution in disease diagnosis and therapeutic monitoring provided that major challenges in proteomics and related disciplines can be addressed. Plasma is not only the primary clinical specimen but also represents the largest and deepest version of the human proteome present in any sample: in addition to the classical "plasma proteins," it contains all tissue proteins (as leakage markers) plus very numerous distinct immunoglobulin sequences, and it has an extraordinary dynamic range in that more than 10 orders of magnitude in concentration separate albumin and the rarest proteins now measured clinically. Although the restricted dynamic range of conventional proteomic technology (two-dimensional gels and mass spectrometry) has limited its contribution to the list of 289 proteins (tabulated here) that have been reported in plasma to date, very recent advances in multidimensional survey techniques promise at least double this number in the near future. Abundant scientific evidence, from proteomics and other disciplines, suggests that among these are proteins whose abundances and structures change in ways indicative of many, if not most, human diseases. Nevertheless, only a handful of proteins are currently used in routine clinical diagnosis, and the rate of introduction of new protein tests approved by the United States Food and Drug Administration (FDA) has paradoxically declined over the last decade to less than one new protein diagnostic marker per year. We speculate on the reasons behind this large discrepancy between the expectations arising from proteomics and the realities of clinical diagnostics and suggest approaches by which protein-disease associations may be more effectively translated into diagnostic tools in the future.

    Molecular & cellular proteomics : MCP 2002;1;11;845-67

  • Molecular cloning, functional expression, and tissue distribution of a novel human gap junction-forming protein, connexin-31.9. Interaction with zona occludens protein-1.

    Nielsen PA, Beahm DL, Giepmans BN, Baruch A, Hall JE and Kumar NM

    Department of Cell Biology, Scripps Research Institute, La Jolla, California 92037, USA. petern@scripps.edu

    A novel human connexin gene (GJA11) was cloned from a genomic library. The open reading frame encoded a hypothetical protein of 294 amino acid residues with a predicted molecular mass of 31,933, hence referred to as connexin-31.9 (Cx31.9) or alpha 11 connexin. A clone in GenBank containing the Cx31.9 gene localized to chromosome 17q21.2. Northern analysis of Cx31.9 showed a major 4.4-kilobase transcript, which was expressed at varying levels in all tissues analyzed. Two monoclonal antibodies generated against different domains of Cx31.9 recognized a 30-33-kDa protein from cells overexpressing Cx31.9. Immunofluorescence of overexpressing cells indicated the presence of Cx31.9 between adjacent cells, consistent with its localization to gap junctions. Double voltage clamp analyses of Cx31.9-overexpressing cells, and of paired Xenopus oocytes injected with Cx31.9 cRNA, demonstrated junctional currents indicative of gap junction channel formation. In contrast to previously characterized connexins, Cx31.9 showed no voltage-dependent gating within a physiologically relevant range. Cx31.9 was detected in human tissues by immunoblot analysis, and immunofluorescence localized Cx31.9 expression to vascular smooth muscle cells. Furthermore, it was demonstrated that Cx31.9 interacted with ZO-1. Thus, Cx31.9 represents a novel connexin gene that in vivo generates a protein with unique voltage gating properties.

    Funded by: NEI NIH HHS: EY12142

    The Journal of biological chemistry 2002;277;41;38272-83

  • Evidence for a functional interaction between cingulin and ZO-1 in cultured cells.

    D'Atri F, Nadalutti F and Citi S

    Department of Molecular Biology, University of Geneva, 1211 Geneva 4, Switzerland.

    Cingulin, a protein component of the submembrane plaque of tight junctions (TJ), contains globular and coiled-coil domains and interacts in vitro with several TJ and cytoskeletal proteins, including the PDZ protein ZO-1. Overexpression of Xenopus cingulin in transfected Xenopus A6 cells resulted in the disruption of endogenous ZO-1 localization, suggesting that cingulin functionally interacts with ZO-1. Glutathione S-transferase pull-down experiments showed that a conserved ZO-1 interaction motif (ZIM) at the NH(2) terminus of cingulin is required for cingulin-ZO-1 interaction in vitro. An NH(2)-terminal region of cingulin, containing the ZIM, was sufficient, when fused to coiled-coil sequences, to target transfected cingulin to junctions. However, deletion of the ZIM did not abolish junctional localization of transfected cingulin in A6 cells, suggesting that cingulin can be recruited to TJ through multiple protein interactions. Interestingly, the ZIM was required for cingulin recruitment into ZO-1-containing adherens junctions of Rat-1 fibroblasts, indicating that cingulin junctional recruitment does not require the molecular context of TJ. Cingulin coiled-coil sequences enhanced the junctional accumulation of expressed cingulin head region in A6 cells, but purified recombinant cingulin did not form filaments under physiological conditions in vitro, suggesting that the cingulin coiled-coil domain acts primarily by promoting dimerization.

    The Journal of biological chemistry 2002;277;31;27757-64

  • Interferon-beta counteracts inflammatory mediator-induced effects on brain endothelial cell tight junction molecules-implications for multiple sclerosis.

    Kuruganti PA, Hinojoza JR, Eaton MJ, Ehmann UK and Sobel RA

    Department of Pathology, Stanford University School of Medicine, California 94305, USA.

    To elucidate mechanisms of endothelial cell (EC) dysfunction in CNS inflammatory responses and beneficial effects of interferon-beta (IFN-gamma) in multiple sclerosis (MS), we analyzed effects of individual and combinations of soluble inflammatory mediators on the intracellular localization of the EC tight junction-associated molecules zonula occludens-1 and -2 (ZO-1 and ZO-2) in human brain ECs. The cytoplasm in the majority of cells in control EC cultures was clear; ZO-1 and ZO-2 were localized peripherally near sites of cell contact and associated with submembranous cytoplasmic filaments. H2O2 induced reversible time- and concentration-dependent translocation of ZO-1 and ZO-2 to a random distribution within EC cytoplasm and retraction of EC borders. For low concentrations, these effects were accompanied by less prominent submembranous filaments but not by evidence of cytotoxicity, increased cell death or altered amounts of ZO-1. Tumor necrosis factor-beta induced similar alterations but interferon-y did not. Co-treatment with either cytokine increased H2O2 effects whereas IFN-beta reversed H2O2-induced effects. In control white matter samples, EC cytoplasm was clear and ZO-1 was located on cell borders. In inflammatory/demyelinating lesions, EC ZO-1 was diffuse, indicating that the alterations induced in vitro mimic those in active MS lesions. These findings suggest that in MS patients, IFN-beta treatment may counteract inflammatory mediator effects on CNS EC tight junction molecules, thereby preserving EC barrier function.

    Journal of neuropathology and experimental neurology 2002;61;8;710-24

  • The coxsackievirus and adenovirus receptor is a transmembrane component of the tight junction.

    Cohen CJ, Shieh JT, Pickles RJ, Okegawa T, Hsieh JT and Bergelson JM

    Division of Immunologic and Infectious Diseases, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA. cohenc@email.chop.edu

    The coxsackievirus and adenovirus receptor (CAR) mediates viral attachment and infection, but its physiologic functions have not been described. In nonpolarized cells, CAR localized to homotypic intercellular contacts, mediated homotypic cell aggregation, and recruited the tight junction protein ZO-1 to sites of cell-cell contact. In polarized epithelial cells, CAR and ZO-1 colocalized to tight junctions and could be coprecipitated from cell lysates. CAR expression led to reduced passage of macromolecules and ions across cell monolayers, and soluble CAR inhibited the formation of functional tight junctions. Virus entry into polarized epithelium required disruption of tight junctions. These results indicate that CAR is a component of the tight junction and of the functional barrier to paracellular solute movement. Sequestration of CAR in tight junctions may limit virus infection across epithelial surfaces.

    Funded by: NHLBI NIH HHS: HL07443, HL51818, HL54734, P01 HL051818, T32 HL007443; NIAID NIH HHS: T32 AI007278, T32 AI07278

    Proceedings of the National Academy of Sciences of the United States of America 2001;98;26;15191-6

  • Use of surface plasmon resonance for real-time analysis of the interaction of ZO-1 and occludin.

    Schmidt A, Utepbergenov DI, Krause G and Blasig IE

    Institute of Molecular Pharmacology, Robert-Rössle-Strasse 10, 13125 Berlin, Germany.

    Surface plasmon resonance (SPR) spectroscopy was applied to study in real time, the interaction between the tight junction proteins ZO-1 and occludin. To imitate the morphology of tight junctions, a cytosolic tail of mouse occludin was immobilised at the sensor and guanylate kinase-like domain (Guk) was allowed to pass over the modified chip surface. The Guk domain of ZO-1 (residues 644-812) was found to bind to the cytoplasmic, carboxy-terminal region of occludin (residues 378-521). This interaction was systematically characterised with respect to the concentrations of both proteins and the binding conditions. Under the given experimental conditions, association and dissociation showed saturation kinetics, with affinity in micromolar range: k(a) = 4.14 +/- 0.52 x 10(3) M(-1) s(-1), k(d) = 3.04 +/- 0.38 x 10(-3) s(-1), K(D) = 639 +/- 51 nM. The results support the hypothesis that the Guk domain of ZO-1 is involved in the recruitment of the transmembrane protein occludin at tight junctions by interacting with the cytosolic carboxy-terminal sequence of occludin, located far from the cell membrane. We demonstrate the use of SPR spectroscopy as an effective approach for characterisation of the interactions of junction proteins.

    Biochemical and biophysical research communications 2001;288;5;1194-9

  • Altered expression and localization of the tight junction protein ZO-1 in primary and metastatic pancreatic cancer.

    Kleeff J, Shi X, Bode HP, Hoover K, Shrikhande S, Bryant PJ, Korc M, Büchler MW and Friess H

    Department of Visceral and Transplantation Surgery, University of Bern, Switzerland.

    Introduction: ZO-1 is a tight junction membrane protein that plays a critical role in cell-cell interaction, proliferation, and differ entiation.

    Aim: To localize and evaluate the expression of ZO-1 in the normal human pancreas, in pancreatic ductal adenocarcinoma (PDAC), and in chronic pancreatitis (CP).

    Northern and Western blot analysis revealed ZO-1 expression in all six tested pancreatic cancer cell lines. Expression of ZO-1 mRNA was increased sixfold in PDAC samples in comparison with normal samples (p = 0.04). Confocal microscopy revealed the presence of ZO-1 in the apical and apicolateral areas of ductular cells in the normal pancreas. Similarly, in CP, ZO-1 was localized at apical and apicolateral areas of small proliferating ductular cells and large metaplastic ducts. In PDAC, however, ZO-1 expression was observed irrespective of whether the cancer cells formed duct-like structures or exhibited a diffuse infiltrating pattern. Metastatic pancreatic cancer cells within lymph nodes displayed variable staining patterns, ranging from apical and apicolateral to a diffuse membranous staining.

    Conclusion: These observations suggest that ZO-1 is overexpressed in PDAC and raise the possibility that this overexpression may confer a metastatic advantage to pancreatic cancer cells.

    Funded by: NCI NIH HHS: CA-40162, CA-66263

    Pancreas 2001;23;3;259-65

  • Connexin45 directly binds to ZO-1 and localizes to the tight junction region in epithelial MDCK cells.

    Kausalya PJ, Reichert M and Hunziker W

    Institute of Molecular and Cell Biology, Epithelial Cell Biology Laboratory, Singapore.

    Zonula occludens protein 1 (ZO-1) is a cytosolic tight junction protein that tethers transmembrane proteins such as occludin, claudin and junctional adhesion molecule to the actin cytoskeleton. The interaction between ZO-1 and claudin or junctional adhesion molecule occurs via the amino-terminal PSD95/Dlg/ZO-1 (PDZ) domains in ZO-1. A yeast two-hybrid screen to search for proteins that interact with the PDZ domains of ZO-1 identified connexin (Cx) 45. Cx45 interacts with the PDZ domains of ZO-1 and ZO-3, but not ZO-2, via a short C-terminal PDZ binding motif (SVWI). In transfected epithelial Madin-Darby canine kidney cells, Cx45 co-localizes with endogenous ZO-1 at or near tight junctions and co-precipitation experiments show that Cx45 and ZO-1 directly interact. Inactivating the C-terminal PDZ-binding motif in Cx45 affects its co-precipitation and co-localization with ZO-1. The growing number of connexins (i.e. Cx43 and Cx45) that can associate with ZO proteins indicate that ZO proteins may play a more general role in organizing gap junctions and/or in recruiting signaling molecules that regulate intercellular communication.

    FEBS letters 2001;505;1;92-6

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

    Itoh M, Sasaki H, Furuse M, Ozaki H, Kita T and Tsukita S

    1Department of Cell Biology, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan.

    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.

    The Journal of cell biology 2001;154;3;491-7

  • HGF regulates tight junctions in new nontumorigenic gastric epithelial cell line.

    Hollande F, Blanc EM, Bali JP, Whitehead RH, Pelegrin A, Baldwin GS and Choquet A

    Laboratoire de Biochimie des Membranes, Faculté de Pharmacie, 15 av. C. Flahault, 34060 Montpellier cedex, France. fhollande@ww3.pharma.univ-montp1.fr

    The regulation of intercellular adhesion by hepatocyte growth factor (HGF) was examined on a novel nontumorigenic gastric epithelial cell line (IMGE-5) derived from H-2Kb-tsA58 transgenic mice. IMGE-5 cells constitutively expressed cytokeratin 18 and HGF receptors. Under permissive conditions (33 degrees C + interferon-gamma), IMGE-5 cells proliferated rapidly but did not display membrane expression of adherens and tight junction proteins. Under nonpermissive conditions, their proliferation was decreased and they displayed a strong, localized membrane expression of E-cadherin/beta-catenin and occludin/ZO-1. HGF treatment largely prevented the targeting of ZO-1 to the tight junction and induced a significant decrease of the transepithelial resistance measured across a confluent IMGE-5 cell monolayer. HGF rapidly increased the tyrosine phosphorylation of ZO-1 and decreased its association with occludin in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent manner. PI 3-kinase was also involved in HGF-induced migration of IMGE-5 cells. Our results demonstrate that 1) HGF prevents the appearance of ZO-1 in the membrane during epithelial cell differentiation; 2) HGF causes partial relocalization of ZO-1 to the cytoplasm and nucleus and concomitantly stimulates cell dissociation and migration; and 3) IMGE-5 cells offer a useful model for the study of gastric epithelial cell differentiation.

    American journal of physiology. Gastrointestinal and liver physiology 2001;280;5;G910-21

  • Expression of ZO-1 and occludin in normal human placenta and in hydatidiform moles.

    Marzioni D, Banita M, Felici A, Paradinas FJ, Newlands E, De Nictolis M, Mühlhauser J and Castellucci M

    Institute of Normal Human Morphology, Faculty of Medicine, University of Ancona, Via Tronto, 10/A, I-60020 Ancona, Italy.

    Zonula occludens-1 (ZO-1) and occludin are key molecules in cell-cell contacts. They are tight junction constituents and therefore play a pivotal role in tissue differentiation and organogenesis. In the present report we have investigated the expression of ZO-1 and occludin in normal human placentae and in hydatidiform moles using immunohistochemical and Western blot analyses. In normal placentae, ZO-1 and occludin were mainly localized in the apical part of the syncytium, in cell-cell contacts between syncytium and villous cytotrophoblastic cells as well as between the latter. Extravillous cytotrophoblast of cell islands and cell columns was positive for ZO-1 and occludin in the cell layers proximally located to the villous stroma whereas the cytotrophoblastic cells, distally located from the villous stroma, were totally negative. Furthermore, fetal vessels showed a positive staining pattern for ZO-1 throughout gestation, whereas a positive reaction for occludin was produced mainly at term. A striking result was the altered expression of ZO-1 and occludin in partial and complete moles. In 11 moles, these two molecules were not expressed at all, while in the other nine, their expression was only cytoplasmic in syncytium and villous cytotrophoblastic cells. These findings suggest that ZO-1 and occludin participate in normal placental development, maintaining the organization and functions of different tissue components. The down-regulation and/or dysregulation of these two molecules may be related to phenotypic changes associated with epithelial cell transformation of the chorionic villi in partial and complete moles.

    Molecular human reproduction 2001;7;3;279-85

  • Connexin-43 interactions with ZO-1 and alpha- and beta-tubulin.

    Giepmans BN, Verlaan I and Moolenaar WH

    Division of Cellular Biochemistry, The Netherlands Cancer Institute and Center for Biomedical Genetics, Amsterdam. bgiep@nki.nl

    Gap junctions are composed of connexins that form transmembrane channels between adjacent cells. The C-terminal tail of connexin-43 (Cx43), the most widely expressed connexin member, has been implicated in the regulation of Cx43 channel gating. Interestingly, channel-independent processes regulated by Cx43 have also been postulated. In our studies to elucidate the mechanism of Cx43 channel gating by growth factors and to explore additional functions of gap junctions, we have identified three interacting partners of the C-terminal tail of Cx43 (Cx43CT). (i) the c-Src tyrosine kinase, which phosphorylates Cx43CT and is involved in G protein-mediated inhibition of Cx43 gap junctional communication. (ii) the ZO-1 'scaffold' protein, which might recruit signaling proteins into Cx43-based gap junctions. (iii) microtubules (consisting of alpha/beta-tubulin dimers), which extend with their distal ends to Cx43-based gap junctions, suggesting that Cx43 gap junctions may play a novel role in regulating microtubule stability in contacted cells. Here we show that Cx43 binds alpha-tubulin equally well as beta-tubulin. In addition, we show that the second, but not the first, PDZ domain of ZO-1 binds directly to Cx43, and we confirm that the very C-terminal isoleucine residue of Cx43 is critical for ZO-1 binding.

    Cell communication & adhesion 2001;8;4-6;219-23

  • Connexin45 interacts with zonula occludens-1 in osteoblastic cells.

    Laing JG, Manley-Markowski RN, Koval M, Civitelli R and Steinberg TH

    Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA. laing@id.wustl.edu

    Connexin43 (Cx43) and Cx45 are co-expressed in a number of different tissues. Studies demonstrated that Cx45 transfected ROS (ROS/Cx45) cells, were less permeable to low molecular weight dyes than untransfected ROS cells, that have gap junctions made of Cx43. This suggests that there may be a functionally important interaction between Cx43 and Cx45 in these cells. One way in which these proteins may interact is by associating with the same set of proteins. In order to isolate connexin interacting proteins, we isolated Cx45 from Cx45 transfected ROS cells (ROS/Cx45 cells) under mild detergent conditions. These studies showed that Cx45 co-purified with the tight junction protein, ZO-1. Immunofluorescence studies of ROS/Cx45 cells simultaneously stained with polyclonal Cx45 antibody and a monoclonal ZO-1 antibody showed that Cx45 and ZO-1 colocalized in ROS/Cx45 cells. Furthermore we found that ZO-1 could bind to peptides derived from the carboxyl terminal of Cx45 that had been covalently bound to an agarose resin. These data suggests that Cx45 and ZO-1 directly interact in ROS/Cx45 cells.

    Cell communication & adhesion 2001;8;4-6;209-12

  • Human pre-mRNA cleavage factor II(m) contains homologs of yeast proteins and bridges two other cleavage factors.

    de Vries H, Rüegsegger U, Hübner W, Friedlein A, Langen H and Keller W

    Department of Cell Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Germany.

    Six different protein factors are required in vitro for 3' end formation of mammalian pre-mRNAs by endonucleolytic cleavage and polyadenylation. Five of the factors have been purified and most of their components cloned, but cleavage factor II(m) (CF II(m)) remained uncharacterized. We have purified CF II(m) from HeLa cell nuclear extract by several chromatographic steps. During purification, CF II(m) activity separated into two components, one essential (CF IIA(m)) and one stimulatory (CF IIB(m)) for the cleavage reaction. CF IIA(m) fractions contain the human homologs of two yeast 3' end processing factors, Pcf11p and Clp1p, as well as cleavage factor I(m) (CF I(m)) and several splicing and transcription factors. We report the cloning of hClp1 and show that it is a genuine subunit of CF IIA(m). Antibodies directed against hClp1 deplete cleavage activity, but not polyadenylation activity from HeLa cell nuclear extract. hClp1 interacts with CF I(m) and the cleavage and polyadenylation specificity factor CPSF, suggesting that it bridges these two 3' end processing factors within the cleavage complex.

    The EMBO journal 2000;19;21;5895-904

  • Cortactin localization to sites of actin assembly in lamellipodia requires interactions with F-actin and the Arp2/3 complex.

    Weed SA, Karginov AV, Schafer DA, Weaver AM, Kinley AW, Cooper JA and Parsons JT

    Department of Microbiology and Cancer Center, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.

    Cortactin is an actin-binding protein that is enriched within the lamellipodia of motile cells and in neuronal growth cones. Here, we report that cortactin is localized with the actin-related protein (Arp) 2/3 complex at sites of actin polymerization within the lamellipodia. Two distinct sequence motifs of cortactin contribute to its interaction with the cortical actin network: the fourth of six tandem repeats and the amino-terminal acidic region (NTA). Cortactin variants lacking either the fourth tandem repeat or the NTA failed to localize at the cell periphery. Tandem repeat four was necessary for cortactin to stably bind F-actin in vitro. The NTA region interacts directly with the Arp2/3 complex based on affinity chromatography, immunoprecipitation assays, and binding assays using purified components. Cortactin variants containing the NTA region were inefficient at promoting Arp2/3 actin nucleation activity. These data provide strong evidence that cortactin is specifically localized to sites of dynamic cortical actin assembly via simultaneous interaction with F-actin and the Arp2/3 complex. Cortactin interacts via its Src homology 3 (SH3) domain with ZO-1 and the SHANK family of postsynaptic density 95/dlg/ZO-1 homology (PDZ) domain-containing proteins, suggesting that cortactin contributes to the spatial organization of sites of actin polymerization coupled to selected cell surface transmembrane receptor complexes.

    Funded by: NCI NIH HHS: CA29243, CA40042, F32 CA075695, P01 CA040042, R01 CA029243, R37 CA029243; NIGMS NIH HHS: GM38542, R01 GM038542, R01 GM038542-11S1

    The Journal of cell biology 2000;151;1;29-40

  • Junctional adhesion molecule interacts with the PDZ domain-containing proteins AF-6 and ZO-1.

    Ebnet K, Schulz CU, Meyer Zu Brickwedde MK, Pendl GG and Vestweber D

    Institute of Cell Biology, ZMBE, University of Muenster, D-48149 Muenster, Germany.

    We have identified the PDZ domain protein AF-6 as an intracellular binding partner of the junctional adhesion molecule (JAM), an integral membrane protein located at cell contacts. Binding of AF-6 to JAM required the presence of the intact C terminus of JAM, which represents a classical type II PDZ domain-binding motif. Although JAM did not interact with the single PDZ domains of ZO-1 or of CASK, we found that a ZO-1 fragment containing PDZ domains 2 and 3 bound to JAM in vitro in a PDZ domain-dependent manner. AF-6 as well as ZO-1 could be coprecipitated with JAM from endothelial cell extracts, demonstrating the association of the endogenously expressed molecules in vivo. Targeting of JAM to sites of cell contacts could be affected by the loss of the PDZ domain-binding C terminus. Full-length mouse JAM co-distributed with endogenous AF-6 in human Caco-2 cells at sites of cell contact independent of whether adjacent cells expressed mouse JAM as an extracellular binding partner. In contrast, truncated JAM lacking the PDZ domain-binding C terminus did not co-distribute with endogenous AF-6, but was restricted to cell contacts between cells expressing mouse JAM. Our results suggest that JAM can be recruited to intercellular junctions by its interaction with the PDZ domain-containing proteins AF-6 and possibly ZO-1.

    The Journal of biological chemistry 2000;275;36;27979-88

  • Human and Xenopus cingulin share a modular organization of the coiled-coil rod domain: predictions for intra- and intermolecular assembly.

    Citi S, D'Atri F and Parry DA

    Department of Molecular Biology, University of Geneva, Geneva, 1205, Switzerland. Sandra.Citi@molbio.unige.ch

    The complete nucleotide and derived amino acid sequences of Homo sapiens cingulin cDNA (5143 bp) were determined by sequencing two distinct EST clones that showed significant sequence homology to Xenopus laevis cingulin. Protein sequence analysis indicates that the molecule contains two chains and has a tripartite structure with N-terminal (head) domains, a coiled-coil rod domain (length, 120 nm), and short C-terminal (tail) domains. Human and Xenopus cingulin heads are only 33% identical, yet a human cingulin N-terminal fragment still interacts with canine ZO-1 and ZO-2 in vitro. The rod domain contains two A and two B subdomains, though it lacks the third B subdomain present in Xenopus cingulin. The heptad substructures of Xenopus and human cingulins were further characterized by computer analysis and indicated that the two-stranded coiled-coil structure contained chains that were parallel and in axial register. Fast Fourier transform analysis and a scoring technique designed to recognize potential interactions between different supramolecular arrangements suggests that cingulin dimers may further assemble through antiparallel interactions between the last approximately 100 amino acids of the coiled-coil region. Cingulin mRNA ( approximately 5.2 kb) was detected by Northern blotting in epithelial tissues. A human cingulin EST was mapped to chromosome 1q21 using the UniGene database.

    Journal of structural biology 2000;131;2;135-45

  • MAGUK proteins: structure and role in the tight junction.

    González-Mariscal L, Betanzos A and Avila-Flores A

    Department of Physiology, Biophysics and Neurosciences, Center of Research and Advanced Studies (CINVESTAV), Ap. Postal 14-740, Mexico DF, 07000, Mexico. lorenza@fisio.cinvestav.mx

    ZO-1, ZO-2 and ZO-3 are tight junction (TJ)-associated proteins that belong to the MAGUK family. In addition to the presence of the characteristic MAGUK modules (PDZ, SH3 and GK), ZOs have a distinctive carboxyl terminal with splicing domains, acidic- and proline-rich regions. The modular organization of these proteins allows them to function as scaffolds, which associate to transmembrane TJ proteins, the cytoskeleton and signal transduction molecules. ZOs shuttle between the TJ and the nucleus, where they may regulate gene expression.

    Seminars in cell & developmental biology 2000;11;4;315-24

  • Proteomic analysis of NMDA receptor-adhesion protein signaling complexes.

    Husi H, Ward MA, Choudhary JS, Blackstock WP and Grant SG

    Centre for Genome Research, Centre for Neuroscience, University of Edinburgh, West Mains Road, Edinburgh EH9 3JQ, UK.

    N-methyl-d-aspartate receptors (NMDAR) mediate long-lasting changes in synapse strength via downstream signaling pathways. We report proteomic characterization with mass spectrometry and immunoblotting of NMDAR multiprotein complexes (NRC) isolated from mouse brain. The NRC comprised 77 proteins organized into receptor, adaptor, signaling, cytoskeletal and novel proteins, of which 30 are implicated from binding studies and another 19 participate in NMDAR signaling. NMDAR and metabotropic glutamate receptor subtypes were linked to cadherins and L1 cell-adhesion molecules in complexes lacking AMPA receptors. These neurotransmitter-adhesion receptor complexes were bound to kinases, phosphatases, GTPase-activating proteins and Ras with effectors including MAPK pathway components. Several proteins were encoded by activity-dependent genes. Genetic or pharmacological interference with 15 NRC proteins impairs learning and with 22 proteins alters synaptic plasticity in rodents. Mutations in three human genes (NF1, Rsk-2, L1) are associated with learning impairments, indicating the NRC also participates in human cognition.

    Nature neuroscience 2000;3;7;661-9

  • Cingulin contains globular and coiled-coil domains and interacts with ZO-1, ZO-2, ZO-3, and myosin.

    Cordenonsi M, D'Atri F, Hammar E, Parry DA, Kendrick-Jones J, Shore D and Citi S

    Department of Biology, University of Padova, 35121 Padova, Italy.

    We characterized the sequence and protein interactions of cingulin, an M(r) 140-160-kD phosphoprotein localized on the cytoplasmic surface of epithelial tight junctions (TJ). The derived amino acid sequence of a full-length Xenopus laevis cingulin cDNA shows globular head (residues 1-439) and tail (1,326-1,368) domains and a central alpha-helical rod domain (440-1,325). Sequence analysis, electron microscopy, and pull-down assays indicate that the cingulin rod is responsible for the formation of coiled-coil parallel dimers, which can further aggregate through intermolecular interactions. Pull-down assays from epithelial, insect cell, and reticulocyte lysates show that an NH(2)-terminal fragment of cingulin (1-378) interacts in vitro with ZO-1 (K(d) approximately 5 nM), ZO-2, ZO-3, myosin, and AF-6, but not with symplekin, and a COOH-terminal fragment (377-1,368) interacts with myosin and ZO-3. ZO-1 and ZO-2 immunoprecipitates contain cingulin, suggesting in vivo interactions. Full-length cingulin, but not NH(2)-terminal and COOH-terminal fragments, colocalizes with endogenous cingulin in transfected MDCK cells, indicating that sequences within both head and rod domains are required for TJ localization. We propose that cingulin is a functionally important component of TJ, linking the submembrane plaque domain of TJ to the actomyosin cytoskeleton.

    The Journal of cell biology 1999;147;7;1569-82

  • Direct binding of three tight junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of claudins.

    Itoh M, Furuse M, Morita K, Kubota K, Saitou M and Tsukita S

    Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.

    ZO-1, ZO-2, and ZO-3, which contain three PDZ domains (PDZ1 to -3), are concentrated at tight junctions (TJs) in epithelial cells. TJ strands are mainly composed of two distinct types of four-transmembrane proteins, occludin, and claudins, between which occludin was reported to directly bind to ZO-1/ZO-2/ZO-3. However, in occludin-deficient intestinal epithelial cells, ZO-1/ZO-2/ZO-3 were still recruited to TJs. We then examined the possible interactions between ZO-1/ZO-2/ZO-3 and claudins. ZO-1, ZO-2, and ZO-3 bound to the COOH-terminal YV sequence of claudin-1 to -8 through their PDZ1 domains in vitro. Then, claudin-1 or -2 was transfected into L fibroblasts, which express ZO-1 but not ZO-2 or ZO-3. Claudin-1 and -2 were concentrated at cell-cell borders in an elaborate network pattern, to which endogenous ZO-1 was recruited. When ZO-2 or ZO-3 were further transfected, both were recruited to the claudin-based networks together with endogenous ZO-1. Detailed analyses showed that ZO-2 and ZO-3 are recruited to the claudin-based networks through PDZ2 (ZO-2 or ZO-3)/PDZ2 (endogenous ZO-1) and PDZ1 (ZO-2 or ZO-3)/COOH-terminal YV (claudins) interactions. In good agreement, PDZ1 and PDZ2 domains of ZO-1/ZO-2/ZO-3 were also recruited to claudin-based TJs, when introduced into cultured epithelial cells. The possible molecular architecture of TJ plaque structures is discussed.

    The Journal of cell biology 1999;147;6;1351-63

  • Protein interactions at the tight junction. Actin has multiple binding partners, and ZO-1 forms independent complexes with ZO-2 and ZO-3.

    Wittchen ES, Haskins J and Stevenson BR

    Department of Cell Biology, University of Alberta, Edmonton T6G 2H7, Canada.

    Defining how the molecular constituents of the tight junction interact is a prerequisite to understanding tight junction physiology. We utilized in vitro binding assays with purified recombinant proteins and immunoprecipitation analyses to define interactions between ZO-1, ZO-2, ZO-3, occludin, and the actin cytoskeleton. Actin cosedimentation studies showed that ZO-2, ZO-3, and occludin all interact directly with F-actin in vitro, indicating that actin is engaged in multiple interactions at the tight junction. Low speed sedimentation analyses demonstrated that neither ZO-2, ZO-3, nor occludin act as F-actin cross-linking proteins, and further evidence indicates that these proteins do not bind to actin filament ends. The binding interactions of ZO-2, ZO-3, and occludin were corroborated in vivo by immunofluorescence colocalization experiments which showed that all three proteins colocalized with actin aggregates at cell borders in cytochalasin D-treated Madin-Darby canine kidney cells. Exploration of other tight junction protein interactions demonstrated that ZO-2 binds directly to both ZO-1 and occludin. Contrary to previous beliefs, our immunoprecipitation results indicate that ZO-1, ZO-2, and ZO-3 exist in situ primarily as independent ZO-1.ZO-2 and ZO-1.ZO-3 complexes rather than a trimeric ZO-1.ZO-2.ZO-3 grouping. These studies elucidate direct binding interactions among tight junction-associated proteins, giving insight into their organization as a multimolecular structure.

    The Journal of biological chemistry 1999;274;49;35179-85

  • In vivo interaction of AF-6 with activated Ras and ZO-1.

    Yamamoto T, Harada N, Kawano Y, Taya S and Kaibuchi K

    Division of Signal Transduction, Nara Institute of Science and Technology, Ikoma, Nara, 630-0101, Japan.

    AF-6 contains two putative Ras-associating domains (RA domains) which are seen in several Ras effectors such as RalGDS and RIN1. We previously showed that an AF-6 fragment containing the amino-terminal (N-terminal) RA domain directly binds to activated Ras and ZO-1 in vitro. In this study, we showed that a single amino acid mutation in the N-terminal RA domain of AF-6 abolished the interaction of AF-6 with activated Ras and that the sites of this critical amino acid residue were similar to those for Raf-1 and RalGDS. The overexpression of the N-terminal RA domain of AF-6 inhibited the Ras-dependent c-fos promoter/enhancer stimulation in NIH3T3 cells. Endogenous AF-6 was coimmunoprecipitated with activated Ras from Rat1 cells expressing activated Ras. Moreover, we showed that AF-6 was coimmunoprecipitated with ZO-1 from Rat1 cells. Taken together, these results indicate that the Ras-interacting region on AF-6 is structurally similar to that on Raf-1 and on RalGDS and that AF-6 interacts with activated Ras and ZO-1 in vivo.

    Biochemical and biophysical research communications 1999;259;1;103-7

  • Characterization of ZO-2 as a MAGUK family member associated with tight as well as adherens junctions with a binding affinity to occludin and alpha catenin.

    Itoh M, Morita K and Tsukita S

    Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan.

    ZO-2, a member of the MAGUK family, was thought to be specific for tight junctions (TJs) in contrast to ZO-1, another MAGUK family member, which is localized at TJs and adherens junctions (AJs) in epithelial and nonepithelial cells, respectively. Mouse ZO-2 cDNA was isolated, and a specific polyclonal antibody was generated using corresponding synthetic peptides as antigens. Immunofluorescence microscopy with this polyclonal antibody revealed that, similarly to ZO-1, in addition to TJs in epithelial cells, ZO-2 was also concentrated at AJs in nonepithelial cells such as fibroblasts and cardiac muscle cells lacking TJs. When NH2-terminal dlg-like and COOH-terminal non-dlg-like domains of ZO-2 (N-ZO-2 and C-ZO-2, respectively) were separately introduced into cultured cells, N-ZO-2 was colocalized with endogenous ZO-1/ZO-2, i.e. at TJs in epithelial cells and at AJs in non-epithelial cells, whereas C-ZO-2 was distributed along actin filaments. Consistently, occludin as well as alpha catenin directly bound to N-ZO-2 as well as the NH2-terminal dlg-like portion of ZO-1 (N-ZO-1) in vitro. Furthermore, immunoprecipitation experiments revealed that the second PDZ domain of ZO-2 was directly associated with N-ZO-1. These findings indicated that ZO-2 forms a complex with ZO-1/occludin or ZO-1/alpha catenin to establish TJ or AJ domains, respectively.

    The Journal of biological chemistry 1999;274;9;5981-6

  • The tight junction protein ZO-1 establishes a link between the transmembrane protein occludin and the actin cytoskeleton.

    Fanning AS, Jameson BJ, Jesaitis LA and Anderson JM

    Department of Internal Medicine, Yale School of Medicine, New Haven, Connecticut 06510, USA. alan.fanning@yale.edu

    The tight junction protein ZO-1 belongs to a family of multidomain proteins known as the membrane-associated guanylate kinase homologs (MAGUKs). ZO-1 has been demonstrated to interact with the transmembrane protein occludin, a second tight junction-specific MAGUK, ZO-2, and F-actin, although the nature and functional significance of these interactions is poorly understood. To further elucidate the role of ZO-1 within the epithelial tight junction, we have introduced epitope-tagged fragments of ZO-1 into cultured MDCK cells and identified domains critical for the interaction with ZO-2, occludin, and F-actin. A combination of in vitro and in vivo binding assays indicate that both ZO-2 and occludin interact with specific domains within the N-terminal (MAGUK-like) half of ZO-1, whereas the unique proline-rich C-terminal half of ZO-1 cosediments with F-actin. Consistent with these observations, we found that a construct encoding the N-terminal half of ZO-1 is specifically associated with tight junctions, whereas the unique C-terminal half of ZO-1 is distributed over the entire lateral surface of the plasma membrane and other actin-rich structures. In addition, we have identified a 244-amino acid domain within the N-terminal half of ZO-1, which is required for the stable incorporation of ZO-1 into the junctional complex of polarized MDCK cells. These observations suggest that one functional role of ZO-1 is to organize components of the tight junction and link them to the cortical actin cytoskeleton.

    Funded by: NCI NIH HHS: CA66263; NIDDK NIH HHS: DK34989, DK45134; ...

    The Journal of biological chemistry 1998;273;45;29745-53

  • Cortactin associates with the cell-cell junction protein ZO-1 in both Drosophila and mouse.

    Katsube T, Takahisa M, Ueda R, Hashimoto N, Kobayashi M and Togashi S

    Neurogenetics Research Project, Mitsubishi Kasei Institute of Life Sciences, Minamiooya 11, Machida-shi, Tokyo 194-8511, Japan.

    Cortactin is an actin filament-binding protein localizing at cortical regions of cells and a prominent substrate for Src family protein-tyrosine kinases in response to multiple extracellular stimuli. Human cortactin has been identified as a protein product of a putative oncogene, EMS1. In this report, we describe the identification of a Drosophila homolog of cortactin as a molecule that interacts with Drosophila ZO-1 using yeast two-hybrid screening. Drosophila cortactin is a 559-amino acid protein highly expressed in embryos, larvae, and pupae but relatively underexpressed in adult flies. Deletion and substitution mutant analyses revealed that the SH3 domain of Drosophila cortactin binds to a PXXP motif in the proline-rich domain of Drosophila ZO-1. Colocalization of these proteins at cell-cell junction sites was evident under a confocal laser-scanning microscope. In vivo association was confirmed by coimmunoprecipitation of cortactin and ZO-1 from Drosophila embryo lysates. We also demonstrate an association for each of the murine homologs by immunoprecipitation analyses of mouse tissue lysates. Our previous work has demonstrated the involvement of ZO-1 in a signaling pathway that regulates expression of the emc gene in Drosophila. The potential roles of the cortactin.ZO-1 complex in cell adhesion and cell signaling are discussed.

    The Journal of biological chemistry 1998;273;45;29672-7

  • Occludin is concentrated at tight junctions of mouse/rat but not human/guinea pig Sertoli cells in testes.

    Moroi S, Saitou M, Fujimoto K, Sakakibara A, Furuse M, Yoshida O and Tsukita S

    Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan.

    Occludin is the only integral membrane protein identified to date as a component of tight junctions (TJs). Here, we examined the distribution and expression of occludin in murine testis bearing well-developed TJ. In the adult mouse testis, occludin was concentrated at TJ strands, which are located at the most basal regions of lateral membranes of Sertoli cells. In immunoblotting, occludin showed a characteristic multiple banding pattern, suggesting that occludin is highly phosphorylated in the testis. In 1-wk-old mouse testis, occludin was distributed diffusely at the lateral membranes of Sertoli cells, and even at this stage, highly phosphorylated occludin was detected. With development, occludin gradually became concentrated at the most basal regions of Sertoli cells. The same results were obtained in rat, but unexpectedly occludin was not detected in human or guinea pig Sertoli cells by immunofluorescence microscopy as well as by immunoblotting. Inasmuch as TJs are also well developed in Sertoli cells of these species, we concluded that, at least in the testes of these species, there are some Sertoli cell-specific isoforms of occludin or other TJ-associated integral membrane proteins that differ from occludin.

    The American journal of physiology 1998;274;6;C1708-17

  • ZO-3, a novel member of the MAGUK protein family found at the tight junction, interacts with ZO-1 and occludin.

    Haskins J, Gu L, Wittchen ES, Hibbard J and Stevenson BR

    Department of Cell Biology and Anatomy, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.

    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. A resulting 19-amino acid sequence provided the basis for screening canine cDNA libraries. Five overlapping clones contained 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. Sequence analysis showed that this protein contains three PSD-95/SAP90, discs-large, ZO-1 (PDZ) domains, a src homology (SH3) domain, and a region similar to guanylate kinase, making it homologous to ZO-1, ZO-2, 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 expressed the exogenous polypeptide at an apparent molecular mass of approximately 130 kD. Moreover, this protein colocalized with ZO-1 at tight junctions by immunofluorescence and immunoelectron microscopy. In vitro affinity analyses demonstrated that recombinant 130-kD protein directly interacts with ZO-1 and the cytoplasmic domain of occludin, but not with ZO-2. We propose that this protein be named ZO-3.

    The Journal of cell biology 1998;141;1;199-208

  • Involvement of ZO-1 in cadherin-based cell adhesion through its direct binding to alpha catenin and actin filaments.

    Itoh M, Nagafuchi A, Moroi S and Tsukita S

    Department of Cell Biology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan.

    ZO-1, a 220-kD peripheral membrane protein consisting of an amino-terminal half discs large (dlg)-like domain and a carboxyl-terminal half domain, is concentrated at the cadherin-based cell adhesion sites in non-epithelial cells. We introduced cDNAs encoding the full-length ZO-1, its amino-terminal half (N-ZO-1), and carboxyl-terminal half (C-ZO-1) into mouse L fibroblasts expressing exogenous E-cadherin (EL cells). The full-length ZO-1 as well as N-ZO-1 were concentrated at cadherin-based cell-cell adhesion sites. In good agreement with these observations, N-ZO-1 was 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 was localized along actin stress fibers. To examine the molecular basis of the behavior of these truncated ZO-1 molecules, N-ZO-1 and C-ZO-1 were produced in insect Sf9 cells by recombinant baculovirus infection, and their direct binding ability to the cadherin/catenin complex and the actin-based cytoskeleton, respectively, were examined in vitro. Recombinant N-ZO-1 bound directly to the glutathione-S-transferase fusion protein with alpha catenin, but not to that with beta catenin or the cytoplasmic domain of E-cadherin. The dissociation constant between N-ZO-1 and alpha catenin was approximately 0.5 nM. On the other hand, recombinant C-ZO-1 was specifically cosedimented with actin filaments in vitro with a dissociation constant of approximately 10 nM. Finally, we compared the cadherin-based cell adhesion activity of NZ-EL cells with that of parent EL cells. Cell aggregation assay revealed no significant differences among these cells, but the cadherin-dependent intercellular motility, i.e., the cell movement in a confluent monolayer, was significantly suppressed in NZ-EL cells. We conclude 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.

    The Journal of cell biology 1997;138;1;181-92

  • Large-scale concatenation cDNA sequencing.

    Yu W, Andersson B, Worley KC, Muzny DM, Ding Y, Liu W, Ricafrente JY, Wentland MA, Lennon G and Gibbs RA

    A total of 100 kb of DNA derived from 69 individual human brain cDNA clones of 0.7-2.0 kb were sequenced by concatenated cDNA sequencing (CCS), whereby multiple individual DNA fragments are sequenced simultaneously in a single shotgun library. The method yielded accurate sequences and a similar efficiency compared with other shotgun libraries constructed from single DNA fragments (> 20 kb). Computer analyses were carried out on 65 cDNA clone sequences and their corresponding end sequences to examine both nucleic acid and amino acid sequence similarities in the databases. Thirty-seven clones revealed no DNA database matches, 12 clones generated exact matches (> or = 98% identity), and 16 clones generated nonexact matches (57%-97% identity) to either known human or other species genes. Of those 28 matched clones, 8 had corresponding end sequences that failed to identify similarities. In a protein similarity search, 27 clone sequences displayed significant matches, whereas only 20 of the end sequences had matches to known protein sequences. Our data indicate that full-length cDNA insert sequences provide significantly more nucleic acid and protein sequence similarity matches than expressed sequence tags (ESTs) for database searching.

    Funded by: NHGRI NIH HHS: 1F32 HG00169-01, F32 HG000169, F33 HG000210, P30 HG00210-05, R01 HG00823

    Genome research 1997;7;4;353-8

  • A "double adaptor" method for improved shotgun library construction.

    Andersson B, Wentland MA, Ricafrente JY, Liu W and Gibbs RA

    Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, 77030, USA.

    The efficiency of shotgun DNA sequencing depends to a great extent on the quality of the random-subclone libraries used. We here describe a novel "double adaptor" strategy for efficient construction of high-quality shotgun libraries. In this method, randomly sheared and end-repaired fragments are ligated to oligonucleotide adaptors creating 12-base overhangs. Nonphosphorylated oligonucleotides are used, which prevents formation of adaptor dimers and ensures efficient ligation of insert to adaptor. The vector is prepared from a modified M13 vector, by KpnI/PstI digestion followed by ligation to oligonucleotides with ends complementary to the overhangs created in the digest. These adaptors create 5'-overhangs complementary to those on the inserts. Following annealing of insert to vector, the DNA is directly used for transformation without a ligation step. This protocol is robust and shows three- to fivefold higher yield of clones compared to previous protocols. No chimeric clones can be detected and the background of clones without an insert is <1%. The procedure is rapid and shows potential for automation.

    Funded by: NHGRI NIH HHS: R01 HG00823

    Analytical biochemistry 1996;236;1;107-13

  • Localization of the tight junction protein gene TJP1 to human chromosome 15q13, distal to the Prader-Willi/Angelman region, and to mouse chromosome 7.

    Mohandas TK, Chen XN, Rowe LB, Birkenmeier EH, Fanning AS, Anderson JM and Korenberg JR

    Department of Pathology, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire 03756, USA.

    The gene encoding the tight junction (zonula occludens) protein, TJP1, was mapped to human chromosome 15q13 by fluorescence in situ hybridization (FISH) using a cDNA probe. The Jackson Laboratory backcross DNA panel derived from the cross (C57BL/6JEi x SPRET/Ei) F1 females x SPRET/Ei males was used to map the mouse Tjp1 to chromosome 7 near position 30 on the Chromosome Committee Map, a region with conserved homology to human chromosome 15q13. FISH studies on metaphases from patients with the Prader-Willi (PWS) or the Angelman syndrome (AS) showed that TJP1 maps close but distal to the PWS/AS chromosome region.

    Funded by: NHGRI NIH HHS: HG00037, HG00941; NIDDK NIH HHS: DK45134

    Genomics 1995;30;3;594-7

  • Epidermal growth factor induces tyrosine phosphorylation and reorganization of the tight junction protein ZO-1 in A431 cells.

    Van Itallie CM, Balda MS and Anderson JM

    Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520-8019, USA.

    Addition of epidermal growth factor (EGF) to A431 human epidermal carcinoma cells results in actin reorganization and phosphorylation of several cytoskeletal proteins. In the present study, we found that EGF treatment of this cell line also results in the redistribution and tyrosine phosphorylation of ZO-1. In normal polarized epithelial cells, ZO-1 is restricted to the cytoplasmic surface of the most apical of the intercellular junctions, the tight junction. In contrast, ZO-1 in the majority of unstimulated A431 cells in small subconfluent islands colocalizes with actin along the lateral cell membranes and in rare microspikes and membrane ruffles. Exposure to EGF results in a transient redistribution of actin into an apically positioned ring. ZO-1 becomes highly focused at apical sites of cell contact and co-localizes with the newly formed band of perijunctional actin. Coincidently, ZO-1 and another tight junction protein, ZO-2, become transiently phosphorylated on tyrosine residues, as determined by anti-phosphotyrosine immunoblotting. Pre-treatment of A431 cells with cytochalasin D, which disrupts normal microfilament organization, does not affect EGF-dependent phosphorylation of the EGF receptor. However, cytochalasin D pretreatment blocks both the EGF-induced ZO-1 rearrangement and tyrosine phosphorylation, suggesting that these responses are dependent on an intact actin microfilament system. We speculate that the transient tyrosine phosphorylation of ZO-1 in response to EGF treatment may be involved in remodeling of intercellular junctions in A431 cells.

    Funded by: NIDDK NIH HHS: DK34989, DK45134, P01-DK38979

    Journal of cell science 1995;108 ( Pt 4);1735-42

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

    Furuse M, Itoh M, Hirase T, Nagafuchi A, Yonemura S, Tsukita S and Tsukita S

    Department of Information Physiology, National Institute for Physiological Sciences, Okazaki, Aichi, Japan.

    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.

    The Journal of cell biology 1994;127;6 Pt 1;1617-26

  • The tight junction protein ZO-1 is homologous to the Drosophila discs-large tumor suppressor protein of septate junctions.

    Willott E, Balda MS, Fanning AS, Jameson B, Van Itallie C and Anderson JM

    Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06510.

    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 are homologous to the product of the lethal(1)discs-large-1 (dlg) tumor suppressor gene of Drosophila, located in septate junctions, and to a 95-kDa protein located in the postsynaptic densities of rat brain, PSD-95. All three proteins contain both a src homology region 3 (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 was previously shown to correlate with variable properties of tight junctions. dlg mutations result in loss of apical-basolateral epithelial cell polarity and in neoplastic growth. These results suggest a protein family specialized for signal transduction on the cytoplasmic surface of intercellular junctions. These results also provide biochemical evidence for similarity between invertebrate septate and vertebrate tight junctions. The C-terminal domain of ZO-1, and its alternatively spliced region, appears to confer variable properties unique to tight junctions.

    Funded by: NIDDK NIH HHS: DK07356, DK34989, DK45134

    Proceedings of the National Academy of Sciences of the United States of America 1993;90;16;7834-8

  • The 220-kD protein colocalizing with cadherins in non-epithelial cells is identical to ZO-1, a tight junction-associated protein in epithelial cells: cDNA cloning and immunoelectron microscopy.

    Itoh M, Nagafuchi A, Yonemura S, Kitani-Yasuda T, Tsukita S and Tsukita S

    Department of Information Physiology, National Institute for Physiological Sciences, Aichi, Japan.

    We previously identified a 220-kD constitutive protein of the plasma membrane undercoat which colocalizes at the immunofluorescence microscopic level with cadherins and occurs not only in epithelial M., S. Yonemura, A. Nagafuchi, Sa. Tsukita, and Sh. Tsukita. 1991. J. Cell Biol. 115:1449-1462). To clarify the nature and possible functions of this protein, we cloned its full-length cDNA and sequenced it. Unexpectedly, we found mouse 220-kD protein to be highly homologous to rat protein ZO-1, only a part of which had been already sequenced. This relationship was confirmed by immunoblotting with anti-ZO-1 antibody. As protein ZO-1 was originally identified as a component exclusively underlying tight junctions in epithelial cells, where cadherins are not believed to be localized, we analyzed the distribution of cadherins and the 220-kD protein by ultrathin cryosection immunoelectron microscopy. We found that in non-epithelial cells lacking tight junctions cadherins and the 220-kD protein colocalize, whereas in epithelial cells (e.g., intestinal epithelial cells) bearing well-developed tight junctions cadherins and the 220-kD protein are clearly segregated into adherens and tight junctions, respectively. Interestingly, in epithelial cells such as hepatocytes, which tight junctions are not so well developed, the 220-kD protein is detected not only in the tight junction zone but also at adherens junctions. Furthermore, we show in mouse L cells transfected with cDNAs encoding N-, P-, E-cadherins that cadherins interact directly or indirectly with the 220-kD protein. Possible functions of the 220-kD protein (ZO-1) are discussed with special reference to the molecular mechanism for adherens and tight junction formation.

    The Journal of cell biology 1993;121;3;491-502

  • Two classes of tight junctions are revealed by ZO-1 isoforms.

    Balda MS and Anderson JM

    Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06510.

    The tight junction forms the intercellular barrier separating tissue compartments. The characteristics of this barrier are remarkably diverse among different epithelia and endothelia and are not explained by our limited knowledge of its molecular composition. Two isoforms of the 220-kDa tight junction protein ZO-1 result from alternative RNA splicing and differ by an internal 80-amino acid domain, termed alpha (E. Willott, M. S. Balda, M. Heintzman, B. Jameson, and J. M. Anderson. Am. J. Physiol. 262 (Cell Physiol. 31): C1119-C1124, 1992). Using antibodies specific for each isoform and double-labeled immunofluorescence microscopy, we observed that the ZO-1 alpha- isoform is restricted to junctions of endothelial cells and highly specialized epithelial cells of both seminiferous tubules (Sertoli cells) and renal glomeruli (podocytes); in contrast, the ZO-1 alpha+ isoform is expressed in cells of all other epithelia examined. Both immunoblotting and ribonuclease protection analysis confirmed this pattern of expression. This distribution does not correlate with differences in junctional resistance or ultrastructural complexity. Instead, we observe a correlation with junctional plasticity; ZO-1 alpha- is expressed in structurally dynamic junctions, whereas ZO-1 alpha+ is expressed in those which are less dynamic. This is the first molecular distinction among tight junctions and reveals a fundamental dichotomy with implications for how the paracellular barriers of endothelia and epithelia are regulated.

    Funded by: NIDDK NIH HHS: DK-34989, DK-45134

    The American journal of physiology 1993;264;4 Pt 1;C918-24

  • Localization and differential expression of two isoforms of the tight junction protein ZO-1.

    Willott E, Balda MS, Heintzelman M, Jameson B and Anderson JM

    Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06510.

    ZO-1 is a peripheral membrane protein of approximately 225 kDa located on the cytoplasmic side of all tight junctions. ZO-1 cDNA sequencing disclosed the presence of a 240-bp sequence in only some of the ZO-1 cDNAs studied. This 240-bp region encoded an inframe insertion of 80 amino acids, named motif-alpha. Expression of the predicted transcripts in normal rat and human tissues and in human epithelial cell lines (Caco-2, T84, Hep G2) was shown by reverse transcription of RNA and then DNA amplification. Immunoblot analysis showed both protein isoforms were present; however, in different cell lines, their amounts differed markedly relative to each other. Immunolocalization at light and ultrastructural levels, using antibodies generated against motif-alpha or shared sequences flanking it, indicated both forms localized indistinguishably to tight junctions. These observations demonstrate the existence and variable expression of ZO-1 isoforms and raise the question whether these isoforms contribute to tight junction diversity in different epithelia.

    Funded by: NIDDK NIH HHS: DK-34989, DK-38979

    The American journal of physiology 1992;262;5 Pt 1;C1119-24

Gene lists (8)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000015 G2C Homo sapiens Human NRC Human orthologues of mouse NRC adapted from Collins et al (2006) 186
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000034 G2C Homo sapiens Pocklington H3 Human orthologues of cluster 3 (mouse) from Pocklington et al (2006) 30
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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