Association of connexin36 with zonula occludens-1 in HeLa cells, betaTC-3 cells, pancreas, and adrenal gland

The PDZ domain-containing protein zonula occludens-1 (ZO-1), a well-established component of tight junctions, has recently been shown to interact with various connexin proteins that form gap junctions. We investigated the association of connexin36 (Cx36) with ZO-1 in various cultured cells and tissues. Punctate immunofluorescence labeling for Cx36 was detected in Cx36-transfected HeLa cells, betaTC-3 cells, pancreatic islets, and adrenal medulla. Immunofluorescence for ZO-1 was also punctate in cells and tissues, and was colocalized with Cx36 at points of cell-cell contact. Immunoprecipitation of either Cx36 or ZO-1 from cell lysates and tissue homogenates resulted in immunoblot detection of ZO-1 or Cx36, respectively, in immunoprecipitates. A 14-amino acid peptide corresponding to the carboxy-terminus of Cx36 showed binding capacity to the PDZ1 domain of ZO-1, which was eliminated after removal of the last 4 carboxy-terminus amino acids. Low micromolar concentrations of the 14-amino acid peptide produced up to 85% inhibition of Cx36 interaction with the PDZ1 domain of ZO-1. These results provide evidence for molecular interaction between Cx36 and ZO-1 in vitro, and in vivo, and suggest that the interference with Cx36/ZO-1 interaction by short carboxy-terminus peptides of Cx36 may be of value for functional studies of this interaction.     

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

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.     

Association of connexin36 with zonula occludens-1 in HeLa cells, βTC-3 cells, pancreas, and adrenal gland

The PDZ domain-containing protein zonula occludens-1 (ZO-1), a well-established component of tight junctions, has recently been shown to interact with various connexin proteins that form gap junctions. We investigated the association of connexin36 (Cx36) with ZO-1 in various cultured cells and tissues. Punctate immunofluorescence labeling for Cx36 was detected in Cx36-transfected HeLa cells, TC-3 cells, pancreatic islets, and adrenal medulla. Immunofluorescence for ZO-1 was also punctate in cells and tissues, and was colocalized with Cx36 at points of cell–cell contact. Immunoprecipitation of either Cx36 or ZO-1 from cell lysates and tissue homogenates resulted in immunoblot detection of ZO-1 or Cx36, respectively, in immunoprecipitates. A 14-amino acid peptide corresponding to the carboxy-terminus of Cx36 showed binding capacity to the PDZ1 domain of ZO-1, which was eliminated after removal of the last 4 carboxy-terminus amino acids. Low micromolar concentrations of the 14-amino acid peptide produced up to 85% inhibition of Cx36 interaction with the PDZ1 domain of ZO-1. These results provide evidence for molecular interaction between Cx36 and ZO-1 in vitro, and in vivo, and suggest that the interference with Cx36/ZO-1 interaction by short carboxy-terminus peptides of Cx36 may be of value for functional studies of this interaction.*co-1^st authors     

Connexin45 interacts with zonula occludens-1 in osteoblastic cells

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.     

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

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.     

Tight function zonula occludens-3 regulates cyclin D1-dependent cell proliferation

Coordinated regulation of cell proliferation is vital for epithelial tissue homeostasis, and uncontrolled proliferation is a hallmark of carcinogenesis. A growing body of evidence indicates that epithelial tight junctions (TJs) play a role in these processes, although the mechanisms involved are poorly understood. In this study, we identify and characterize a novel plasma membrane pool of cyclin D1 with cell-cycle regulatory functions. We have determined that the zonula occludens (ZO) family of TJ plaque proteins sequesters cyclin D1 at TJs during mitosis, through an evolutionarily conserved class II PSD-95, Dlg, and ZO-1 (PDZ)-binding motif within cyclin D1. Disruption of the cyclin D1/ZO complex through mutagenesis or siRNA-mediated suppression of ZO-3 resulted in increased cyclin D1 proteolysis and G(0)/G(1) cell-cycle retention. This study highlights an important new role for ZO family TJ proteins in regulating epithelial cell proliferation through stabilization of cyclin D1 during mitosis.     

Zonula occludens-1 alters connexin43 gap junction size and organization by influencing channel accretion

Regulation of gap junction (GJ) organization is critical for proper function of excitable tissues such as heart and brain, yet mechanisms that govern the dynamic patterning of GJs remain poorly defined. Here, we show that zonula occludens (ZO)-1 localizes preferentially to the periphery of connexin43 (Cx43) GJ plaques. Blockade of the PDS95/dlg/ZO-1 (PDZ)-mediated interaction between ZO-1 and Cx43, by genetic tagging of Cx43 or by a membrane-permeable peptide inhibitor that contains the Cx43 PDZ-binding domain, led to a reduction of peripherally associated ZO-1 accompanied by a significant increase in plaque size. Biochemical data indicate that the size increase was due to unregulated accumulation of gap junctional channels from nonjunctional pools, rather than to increased protein expression or decreased turnover. Coexpression of native Cx43 fully rescued the aberrant tagged-connexin phenotype, but only if channels were composed predominately of untagged connexin. Confocal image analysis revealed that, subsequent to GJ nucleation, ZO-1 association with Cx43 GJs is independent of plaque size. We propose that ZO-1 controls the rate of Cx43 channel accretion at GJ peripheries, which, in conjunction with the rate of GJ turnover, regulates GJ size and distribution.     

ZO-1- and ZO-2-dependent integration of myosin-2 to epithelial zonula adherens

For the zonula adherens (ZA) to be established by linear arrangement of adherens junctions (AJs) in epithelial sheet cells, critical for the epithelial cell sheet formation and intercellular barrier function, myosin-2 is supposedly integrated into the ZA with the result of overlapping localization of E-cadherin/actin/myosin-2. Here, we immunofluorescently showed that myosin-2 failed to be integrated into the ZA in cultured epithelial-type ZO1(ko)/2(kd) Eph4 cells lacking ZO-1 and -2 (zonula occludens-1 and -2) by knockout and knockdown, respectively. Instead, a linearized but fragmented arrangement of AJs was formed in the way that it was positive for E-cadherin/actin, but negative for myosin-2 (designated prezonula-AJ). Transfection of full-length ZO-1 or ZO-2, or ZO-1 lacking its PDZ (PSD-95/discs large/zonula occludens-1)-1/2 domains (but not one lacking PDZ-1/2/3) into ZO1(ko)/2(kd) Eph4 cells restored the junctional integration of myosin-2 with prezonula-AJ to establish the ZA. Transfection of dominant-active RhoA or Rho-kinase (ROCK), as well as administration of lysophosphatidic acid or Y27632, which activates RhoA or inhibits ROCK, respectively, suggested that RhoA regulated the junctional integration of myosin-2 into ZA in a manner such that ROCK played a necessary but not-sufficient role. Fluorescence resonance energy transfer analyses revealed that spatiotemporal Rho-activation occurred in a ZO-1/2–dependent way to establish ZA from primordial forms in epithelial cells.     

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

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.     

Connexin43 PDZ2 binding domain mutants create functional gap junctions and exhibit altered phosphorylation

Connexin43 (Cx43) is the most abundantly expressed gap junction protein. The C-terminal tail of Cx43 is important for regulation of gap junctions via phosphorylation of specific tyrosine and serine residues and through interactions with cellular proteins. The C-terminus of Cx43 has been shown to interact with the PDZ2 domain of the tight and adherens junction associated zona occludens 1 (ZO-1) protein. Analysis of the PDZ2 binding domain of Cx43 indicated that positions -3 and -2, and the final hydrophobic amino acid at the C-terminus, are critical for ZO-1 binding. In addition, the C-termini of connexins 40 and 45, but not Cx32, interacted with ZO-1. To evaluate the functional significance of the Cx43-ZO-1 interaction, Cx43 wild type (Cx43wt) and mutants lacking either the C-terminal hydrophobic isoleucine (Cx43deltaI382) or the last five amino acids (Cx43delta378-382), required for ZO-1 binding in vitro, were introduced into a Cx43-deficient MDCK cell line. In vitro binding studies and coimmunoprecipitation assays indicated that these Cx43 mutants failed to interact with ZO-1. Confocal and deconvolution microscopy revealed that a fraction of Cx43wt colocalized with ZO-1 at the plasma membrane. A similar colocalization pattern was observed for the Cx43deltaI382 and Cx43 delta378-382 mutants, which were translocated to the plasma membrane and formed functional gap junction channels. The wt and mutant Cx43 appeared to have similar turnover rates. However, the P2 and P3 phosphoisoforms of the Cx43 mutants were significantly reduced compared to Cx43wt. These studies indicated that the interaction of Cx43 with ZO-1 may contribute to the regulation of Cx43 phosphorylation.     

Zonula occludens-1 and -2 regulate apical cell structure and the zonula adherens cytoskeleton in polarized epithelia

The structure and function of both adherens (AJ) and tight (TJ) junctions are dependent on the cortical actin cytoskeleton. The zonula occludens (ZO)-1 and -2 proteins have context-dependent interactions with both junction types and bind directly to F-actin and other cytoskeletal proteins, suggesting ZO-1 and -2 might regulate cytoskeletal activity at cell junctions. To address this hypothesis, we generated stable Madin-Darby canine kidney cell lines depleted of both ZO-1 and -2. Both paracellular permeability and the localization of TJ proteins are disrupted in ZO-1/-2-depleted cells. In addition, immunocytochemistry and electron microscopy revealed a significant expansion of the perijunctional actomyosin ring associated with the AJ. These structural changes are accompanied by a recruitment of 1-phosphomyosin light chain and Rho kinase 1, contraction of the actomyosin ring, and expansion of the apical domain. Despite these changes in the apical cytoskeleton, there are no detectable changes in cell polarity, localization of AJ proteins, or the organization of the basal and lateral actin cytoskeleton. We conclude that ZO proteins are required not only for TJ assembly but also for regulating the organization and functional activity of the apical cytoskeleton, particularly the perijunctional actomyosin ring, and we speculate that these activities are relevant both to cellular organization and epithelial morphogenesis.     

Structural changes in the carboxyl terminus of the gap junction protein connexin 40 caused by the interaction with c-Src and zonula occludens-1

c-Src can disrupt the connexin 43 (Cx43) and zonula occludens-1 (ZO-1) interaction, leading to down-regulation of gap junction intercellular communication. Previously, the authors characterized the interaction of domains from these proteins with the carboxyl terminus of Cx43 (Cx43CT) and found that binding of the c-Src SH3 domain to Cx43CT disrupted the Cx43CT/ZO-1 PDZ-2 domain complex. Because Cx43 and Cx40 form heteromeric connexons and display similar mechanisms of pH regulation, the authors addressed whether Cx40CT interacts with these domains in a similar manner as Cx43CT. Nuclear magnetic resonance (NMR) data indicate that Cx40CT is an intrinsically disordered protein. NMR titrations determined that PDZ-2 affected the last 28 Cx40CT residues and SH3 shifted numerous amino-terminal Cx40CT residues. Finally, the Cx40CT/PDZ-2 complex was unaffected by SH3 and both domains interacted simultaneously with Cx40CT. This result differs from when the same experiment was performed with Cx43CT, suggesting different mechanisms of regulation exist between connexin isoforms, even when involving the same molecular partners.     

Connexin45 interacts with zonula occludens-1 and connexin43 in osteoblastic cells

The relative expression of connexin43 and connexin45 modulates gap junctional communication and production of bone matrix proteins in osteoblastic cells. It is likely that changes in gap junction permeability are determined by the interaction between these two proteins. Cx43 interacts with ZO-1, which may be involved in trafficking of Cx43 or facilitating interactions between Cx43 and other proteins. In this study we sought to identify proteins that associate with Cx45 by coprecipitation in non-denaturing conditions. Cx45 was isolated with a 220-kDa protein that we identified as ZO-1. Under the same conditions, Cx43 also was isolated with anti-Cx45 antiserum from Cx45-transfected ROS cells (ROS/Cx45 cells). Cx43 antiserum could also coprecipitate ZO-1 in the transfected and untransfected ROS cells. Double label immunofluorescence studies showed that ZO-1, Cx43, and Cx45 colocalized at appositional membranes in ROS/Cx45 cells suggesting that all three proteins are normally associated in the cells. Additionally, we found that in vitro translated ZO-1 binds to the carboxyl-terminal of Cx45 indicating that there is a direct interaction between the carboxyl-terminal of Cx45 and ZO-1. These studies demonstrate that ZO-1 interacts with Cx45 as well as with Cx43, and suggest that the interaction of connexins with ZO-1 may play a role in regulating the composition of the gap junction and may modulate connexin-connexin interactions.     

The PDZ domains of zonula occludens-1 induce an epithelial to mesenchymal transition of Madin-Darby canine kidney I cells. Evidence for a role of beta-catenin/Tcf/Lef signaling

The integrity of cell-cell contacts such as adherens junctions (AJ) and tight junctions (TJ) is essential for the function of epithelia. During carcinogenesis, the increased motility and invasiveness of tumor cells reflect the loss of characteristic epithelial features, including cell adhesion. While beta-catenin, a component of AJ, plays a well characterized dual role in cell adhesion and signal transduction leading to epithelial cell transformation, little is known about possible roles of tight junction components in signaling processes. Here we show that mutants of the TJ protein zonula occludens protein-1 (ZO-1), which encode the PDZ domains (ZO-1 PDZ) but no longer localize at the plasma membrane, induce a dramatic epithelial to mesenchymal transition (EMT) of Madin-Darby canine kidney I (MDCKI) cells. The observed EMT of these MDCK-PDZ cells is characterized by a repression of epithelial marker genes, a restricted differentiation potential and a significantly induced tumorigenicity. Intriguingly, the beta-catenin signaling pathway is activated in the cells expressing the ZO-1 PDZ protein. Ectopic expression of the adenomatous polyposis coli tumor suppressor gene, known to down-regulate activated beta-catenin signaling, reverts the transformed fibroblastoid phenotype of MDCK-PDZ cells. Thus, cytoplasmic localization of the ZO-1 PDZ domains induces an EMT in MDCKI cells, most likely by modulating beta-catenin signaling.     

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

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.     

Fusion of GFP to the carboxyl terminus of connexin43 increases gap junction size in HeLa cells

The pattern of gap junctional coupling between cells is thought to be important for the proper function of many types of tissues. At present, little is known about the molecular mechanisms that control the size and distribution of gap junctions. We addressed this issue by expressing connexin43 (Cx43) constructs in HeLa cells, a connexin-deficient cell line. HeLa cells expressing exogenously introduced wild-type Cx43 formed small, punctate gap junctions. By contrast, cells expressing Cx43-GFP formed large, sheet-like gap junctions. These results suggest that the GFP tag, which is fused to the carboxyl terminus of Cx43, alters gap junction size by masking the carboxyl terminal amino acids of Cx43 that comprise a zonula occludins-1 (ZO-1) binding site. We are currently testing this hypothesis using deletion and dominant-negative constructs that directly target the interaction between Cx43 and ZO-1.     

Fusion of GFP to the Carboxyl Terminus of Connexin43 Increases Gap Junction Size in HeLa Cells

The pattern of gap junctional coupling between cells is thought to be important for the proper function of many types of tissues. At present, little is known about the molecular mechanisms that control the size and distribution of gap junctions. We addressed this issue by expressing connexin43 (Cx43) constructs in HeLa cells, a connexin-deficient cell line. HeLa cells expressing exogenously introduced wild-type Cx43 formed small, punctate gap junctions. By contrast, cells expressing Cx43-GFP formed large, sheet-like gap junctions. These results suggest that the GFP tag, which is fused to the carboxyl terminus of Cx43, alters gap junction size by masking the carboxyl terminal amino acids of Cx43 that comprise a zonula occludins-1 (ZO-1) binding site. We are currently testing this hypothesis using deletion and dominant-negative constructs that directly target the interaction between Cx43 and ZO-1.     

Fusion of GFP to the Carboxyl Terminus of Connexin43 Increases Gap Junction Size in HeLa Cells

The pattern of gap junctional coupling between cells is thought to be important for the proper function of many types of tissues. At present, little is known about the molecular mechanisms that control the size and distribution of gap junctions. We addressed this issue by expressing connexin43 (Cx43) constructs in HeLa cells, a connexin-deficient cell line. HeLa cells expressing exogenously introduced wild-type Cx43 formed small, punctate gap junctions. By contrast, cells expressing Cx43-GFP formed large, sheet-like gap junctions. These results suggest that the GFP tag, which is fused to the carboxyl terminus of Cx43, alters gap junction size by masking the carboxyl terminal amino acids of Cx43 that comprise a zonula occludins-1 (ZO-1) binding site. We are currently testing this hypothesis using deletion and dominant-negative constructs that directly target the interaction between Cx43 and ZO-1.