Cell polarity development and protein trafficking in hepatocytes lacking E-cadherin/beta-catenin-based adherens junctions

Using a mutant hepatocyte cell line in which E-cadherin and beta-catenin are completely depleted from the cell surface, and, consequently, fail to form adherens junctions, we have investigated adherens junction requirement for apical-basolateral polarity development and polarized membrane trafficking. It is shown that these hepatocytes retain the capacity to form functional tight junctions, develop full apical-basolateral cell polarity, and assemble a subapical cortical F-actin network, although with a noted delay and a defect in subsequent apical lumen remodeling. Interestingly, whereas hepatocytes typically target the plasma membrane protein dipeptidyl peptidase IV first to the basolateral surface, followed by its transcytosis to the apical domain, hepatocytes lacking E-cadherin-based adherens junctions target dipeptidyl peptidase IV directly to the apical surface. Basolateral surface-directed transport of other proteins or lipids tested was not visibly affected in hepatocytes lacking E-cadherin-based adherens junctions. Together, our data show that E-cadherin/beta-catenin-based adherens junctions are dispensable for tight junction formation and apical lumen biogenesis but not for apical lumen remodeling. In addition, we suggest a possible requirement for E-cadherin/beta-catenin-based adherens junctions with regard to the indirect apical trafficking of specific proteins in hepatocytes.     

Restoration of tight junction structure and barrier function by down-regulation of the mitogen-activated protein kinase pathway in ras-transformed Madin-Darby canine kidney cells

In the Madin-Darby canine kidney epithelial cell line, the proteins occludin and ZO-1 are structural components of the tight junctions that seal the paracellular spaces between the cells and contribute to the epithelial barrier function. In Ras-transformed Madin-Darby canine kidney cells, occludin, claudin-1, and ZO-1 were absent from cell-cell contacts but were present in the cytoplasm, and the adherens junction protein E-cadherin was weakly expressed. After treatment of the Ras-transformed cells with the mitogen-activated protein kinase kinase (MEK1) inhibitor PD98059, which blocks the activation of mitogen-activated protein kinase (MAPK), occludin, claudin-1, and ZO-1 were recruited to the cell membrane, tight junctions were assembled, and E-cadherin protein expression was induced. Although it is generally believed that E-cadherin-mediated cell-cell adhesion is required for tight junction assembly, the recruitment of occludin to the cell-cell contact area and the restoration of epithelial cell morphology preceded the appearance of E-cadherin at cell-cell contacts. Both electron microscopy and a fourfold increase in the transepithelial electrical resistance indicated the formation of functional tight junctions after MEK1 inhibition. Moreover, inhibition of MAPK activity stabilized occludin and ZO-1 by differentially increasing their half-lives. We also found that during the process of tight junction assembly after MEK1 inhibition, tyrosine phosphorylation of occludin and ZO-1, but not claudin-1, increased significantly. Our study demonstrates that down-regulation of the MAPK signaling pathway causes the restoration of epithelial cell morphology and the assembly of tight junctions in Ras-transformed epithelial cells and that tyrosine phosphorylation of occludin and ZO-1 may play a role in some aspects of tight junction formation.     

FRAP Analysis Reveals Stabilization of Adhesion Structures in the Epidermis Compared to Cultured Keratinocytes

Proper development and tissue maintenance requires cell-cell adhesion structures, which serve diverse and crucial roles in tissue morphogenesis. Epithelial tissues have three main types of cell-cell junctions: tight junctions, which play a major role in barrier formation, and adherens junctions and desmosomes, which provide mechanical stability and organize the underlying cytoskeleton. Our current understanding of adhesion function is hindered by a lack of tools and methods to image junctions in mammals. To better understand the dynamics of adhesion in tissues we have created a knock-in ZO-1-GFP mouse and a BAC-transgenic mouse expressing desmoplakin I-GFP. We performed fluorescence recovery after photobleaching (FRAP) experiments to quantify the turnover rates of the tight junction protein ZO-1, the adherens junction protein E-cadherin, and the desmosomal protein desmoplakin in the epidermis. Proteins at each type of junction are remarkably stable in the epidermis, in contrast to the high observed mobility of E-cadherin and ZO-1 at adherens junctions and tight junctions, respectively, in cultured cells. Our data demonstrate that there are additional mechanisms for stabilizing junctions in tissues that are not modeled by cell culture.     

Regulation of adherens junctions and epithelial paracellular permeability: a novel function for polyamines

Maintenance of intestinal mucosal epithelial integrity requires polyamines that are involved in the multiple signaling pathways controlling gene expression and different epithelial cell functions. Integrity of the intestinal epithelial barrier depends on a complex of proteins composing different intercellular junctions, including tight junctions, adherens junctions, and desmosomes. E-cadherin is primarily found at the adherens junctions and plays a critical role in cell-cell adhesions that are fundamental to formation of the intestinal epithelial barrier. The current study determined whether polyamines regulate intestinal epithelial barrier function by altering E-cadherin expression. Depletion of cellular polyamines by alpha-difluoromethylornithine (DFMO) reduced intracellular free Ca2+ concentration ([Ca2+]cyt), decreased E-cadherin expression, and increased paracellular permeability in normal intestinal epithelial cells (IEC-6 line). Polyamine depletion did not alter expression of tight junction proteins such as zona occludens (ZO)-1, ZO-2, and junctional adhesion molecule (JAM)-1. Addition of exogenous polyamine spermidine reversed the effects of DFMO on [Ca2+]cyt and E-cadherin expression and restored paracellular permeability to near normal. Elevation of [Ca2+]cyt by the Ca2+ ionophore ionomycin increased E-cadherin expression in polyamine-deficient cells. In contrast, reduction of [Ca2+]cyt by polyamine depletion or removal of extracellular Ca2+ not only inhibited expression of E-cadherin mRNA but also decreased the half-life of E-cadherin protein. These results indicate that polyamines regulate intestinal epithelial paracellular barrier function by altering E-cadherin expression and that polyamines are essential for E-cadherin expression at least partially through [Ca2+]cyt.     

Catenins and zonula occludens-1 form a complex during early stages in the assembly of tight junctions

We characterized the role of the E-cadherin adhesion system in the formation of epithelial tight junctions using the calcium switch model. In MDCK cells cultured in low (micromolar) calcium levels, the tight junctional protein Zonula Occludens-1 (ZO-1) is distributed intracellularly in granular clusters, the larger of which codistribute with E-cadherin. Two hours after activation of E-cadherin adhesion by transfer to normal (1.8 mM) calcium levels, ZO-1 dramatically redistributed to the cell surface, where it localized in regions rich in E-cadherin. Immunoprecipitation with ZO-1 antibodies of extracts from cells kept in low calcium and 2 h after shifting to 1.8 mM Ca2+ demonstrated the association of ZO-1 with alpha-, beta-, and gamma- catenins. E-cadherin was not detected in the ZO-1 immunoprecipitates but it was found in beta-catenin immunoprecipitates that excluded ZO-1, suggesting that the binding of ZO-1 to catenins may weaken the interaction of these proteins with E-cadherin. Immunofluorescence and immunoelectron microscopy confirmed a close association of beta-catenin and ZO-1 at 0 and 2 h after Ca2+ switch. 48 h after Ca2+ switch, upon complete polarization of the epithelium, most of the ZO-1 had segregated from lateral E-cadherin and formed a distinct, separate apical ring. The ZO-1-catenin complex was not detected in fully polarized monolayers. MDCK cells permanently transformed with Moloney sarcoma virus, which expresses low levels of E-cadherin, displayed clusters of cytoplasmic ZO-1 granules and very little of this protein at the cell surface. Upon transfection with E-cadherin into Moloney sarcoma virus-MDCK cells, ZO-1 redistributed to E-cadherin-rich lateral plasma membrane but later failed to segregate into mature tight junctions. Our experiments suggest that catenins participate in the mobilization of ZO-1 from the cytosol to the cell surface early in the development of tight junctions and that neoplastic transformation may block the formation of tight junctions, either by decreasing the levels of E-cadherin or by preventing a late event: the segregation of tight junction from the zonula adherens.     

PKC-δ binds to E-cadherin and mediates EGF-induced cell scattering

EGF is known to affect adherens junctions and disrupt cell-cell adhesion in a variety of carcinomas but the underlying mechanisms are not completely understood. Using human tumor epithelial cells overexpressing EGFR we demonstrated that EGF-induced cell scattering was mediated by protein kinase C-delta (PKC-δ). PKC-δ knockdown by siRNA significantly inhibited EGF-induced internalization of E-cadherin into the cytoplasm and blocked cell scattering. EGF phosphorylated PKC-δ at Y311 and ectopic expression of the mutant Y311F prevented PKC-δ binding to E-cadherin and EGF-induced cell scattering. Moreover, depletion of Src using siRNA decreased EGF-induced phosphorylation of PKC-δ at Y311 and blocked scattering. Finally, EGF reduced expression of the tight junction protein, occludin, and this effect was also mediated by PKC-δ through Src. In summary, PKC-δ mediated the effects of EGF on adherens and tight junctions thereby playing an important role in cell-cell adhesion with possible wider implications in tumor metastasis or epithelial-to-mesenchymal transition.     

Cadherin function in junctional complex rearrangement and posttranslational control of cadherin expression

The role ofE-cadherin, a calcium-dependent adhesion protein, in organizing andmaintaining epithelial junctions was examined in detail by expressing afusion protein (GP2-Cad1) composed of the extracellular domain of anonadherent glycoprotein (GP2) and the transmembrane and cytoplasmicdomains of E-cadherin. All studies shown were also replicated using ananalogous cell line that expresses a mutant cadherin construct (T151)under the control of tet repressor. Mutant cadherin was expressed at~10% of the endogenous E-cadherin level and had no apparent effecton tight junction function or on distributions of adherens junction,tight junction, or desmosomal marker proteins in establishedMadin-Darby canine kidney cell monolayers. However, GP2-Cad1accelerated the disassembly of epithelial junctional complexes anddelayed their reassembly in calcium switch experiments. Inducingexpression of GP2-Cad1 to levels approximately threefold greater thanendogenous E-cadherin expression levels in control cells resulted in adecrease in endogenous E-cadherin levels. This was due in part toincreased protein turnover, indicating a cellular mechanism for sensingand controlling E-cadherin levels. Cadherin association with cateninsis necessary for strong cadherin-mediated cell-cell adhesion. In cellsexpressing low levels of GP2-Cad1, protein levels and stoichiometry ofthe endogenous cadherin-catenin complex were unaffected. Thus effectsof GP2-Cad1 on epithelial junctional complex assembly and stabilitywere not due to competition with endogenous E-cadherin for cateninbinding. Rather, we suggest that GP2-Cad1 interferes with the packingof endogenous cadherin-catenin complexes into higher-order structuresin junctional complexes that results in junction destabilization.     

A novel four transmembrane spanning protein, CLP24. A hypoxically regulated cell junction protein.

A novel hypoxically regulated intercellular junction protein (claudin-like protein of 24 kDa, CLP24) has been identified that shows homology to the myelin protein 22/epithelial membrane protein 1/claudin family of cell junction proteins, which are involved in the modulation of paracellular permeability. The CLP24 protein contains four predicted transmembrane domains and a C-terminal protein-protein interaction domain. These domains are characteristic of the four transmembrane spanning (tetraspan) family of proteins, which includes myelin protein 22, and are involved in cell adhesion at tight, gap and adherens junctions. Expression profiling analyses show that CLP24 is highly expressed in lung, heart, kidney and placental tissues. Cellular studies confirm that the CLP24 protein localizes to cell-cell junctions and co-localizes with the beta-catenin adherens junction-associated protein but not with tight junctions. Over-expression of CLP24 results in decreased adhesion between cells, and functional paracellular flux studies confirm that over-expression of the CLP24 protein modulates the junctional barrier function. These data therefore suggest that CLP24 is a novel, hypoxically regulated tetraspan adherens junction protein that modulates cell adhesion, paracellular permeability and angiogenesis.     

Caveolae Mediate Growth Factor-induced Disassembly of Adherens Junctions to Support Tumor Cell Dissociation

Remodeling of cell–cell contacts through the internalization of adherens junction proteins is an important event during both normal development and the process of tumor cell metastasis. Here we show that the integrity of tumor cell–cell contacts is disrupted after epidermal growth factor (EGF) stimulation through caveolae-mediated endocytosis of the adherens junction protein E-cadherin. Caveolin-1 and E-cadherin closely associated at cell borders and in internalized structures upon stimulation with EGF. Furthermore, preventing caveolae assembly through reduction of caveolin-1 protein or expression of a caveolin-1 tyrosine phospho-mutant resulted in the accumulation of E-cadherin at cell borders and the formation of tightly adherent cells. Most striking was the fact that exogenous expression of caveolin-1 in tumor cells that contain tight, well-defined, borders resulted in a dramatic dispersal of these cells. Together, these findings provide new insights into how cells might disassemble cell–cell contacts to help mediate the remodeling of adherens junctions, and tumor cell metastasis and invasion.     

Rnd3/RhoE induces tight junction formation in mammary epithelial tumor cells

Glucocorticoid hormones stimulate adherens and tight junction formation in Con8 mammary epithelial tumor cells through a multistep process in which the membrane organization of structural apical junction proteins and tight junction sealing is controlled by specific signal transduction components. We have previously shown that dexamethasone stimulation of apical junction formation requires down-regulation of the small GTPase RhoA. Here we identified Rnd3/RhoE, a GTPase-deficient Rho family member and RhoA antagonist, as a key regulator of apical junction dynamics. Exogenously expressed Rnd3/RhoE co-localized with actin at the cell periphery and induced the localization of the adherens junction protein beta-catenin and the tight junction protein ZO-1 to sites of cell-cell contact, and led to the formation of highly sealed tight junctions. Treatment with glucocorticoids was not required to achieve complete apical junction remodeling. Consistent with Rnd3/RhoE acting as an antagonist of RhoA, expression of Rnd3/RhoE rescued the disruptive effects of constitutively active RhoA on apical junction organization. Our results demonstrate a new role for the Rho family member Rnd3/RhoE in regulating the assembly of the apical junction complex and tight junction sealing.     

Expression of junction-associated proteins differentiates mouse intestinal M cells from enterocytes

The intestinal lymphoid follicles and associated structures are specialised antigen sampling and inductive sites of the mucosal immune system. The overlying follicle-associated epithelium (FAE) includes the specialised antigen sampling M cells that are also exploited as a route of pathogen invasion. In this immunohistochemical study we analysed the junctional complexes of the mouse intestinal FAE. Protein expression at this site resembled that of other simple epithelia. Specifically, claudin-1/3 and ZO-1 were detected in the tight junctions, E-cadherin, alpha-, beta- and gamma-catenin, vinculin, alpha-actinin and polymerised actin were associated with the adherens junctions and the desmosomes were labelled with a desmosomal protein probe. These markers failed to reveal cell type-associated variations in the tight junctions and desmosomes. In contrast, M cell adherens junctions were distinguished by enhanced expression of beta-catenin, alpha-actinin, polymerised actin and, in some areas, E-cadherin. In addition, M cell junctions exhibited increased expression of intercellular adhesion molecule-1 and phosphotyrosine, and the M cell apical surfaces displayed characteristic patterns of beta-catenin, alpha-actinin and actin expression. We have thus partially defined the junctional complexes of mouse intestinal FAE and identified M cell-specific characteristics that may further explain the biology and function of this unique cell type.     

The cell junction protein VAB-9 regulates adhesion and epidermal morphology in C. elegans

Epithelial cell junctions are essential for cell polarity, adhesion and morphogenesis. We have analysed VAB-9, a cell junction protein in Caenorhabditis elegans. VAB-9 is a predicted four-pass integral membrane protein that has greatest similarity to BCMP1 (brain cell membrane protein 1, a member of the PMP22/EMP/Claudin family of cell junction proteins) and localizes to the adherens junction domain of C. elegans apical junctions. Here, we show that VAB-9 requires HMR-1/cadherin for localization to the cell membrane, and both HMP-1/alpha-catenin and HMP-2/beta-catenin for maintaining its distribution at the cell junction. In vab-9 mutants, morphological defects correlate with disorganization of F-actin at the adherens junction; however, localization of the cadherin-catenin complex and epithelial polarity is normal. These results suggest that VAB-9 regulates interactions between the cytoskeleton and the adherens junction downstream of or parallel to alpha-catenin and/or beta-catenin. Mutations in vab-9 enhance adhesion defects through functional loss of the cell junction genes apical junction molecule 1 (ajm-1) and discs large 1 (dlg-1), suggesting that VAB-9 is involved in cell adhesion. Thus, VAB-9 represents the first characterized tetraspan adherens junction protein in C. elegans and defines a new family of such proteins in higher eukaryotes.     

PICK-1: a scaffold protein that interacts with Nectins and JAMs at cell junctions

Nectin adhesion molecules are involved in the early steps of cell junction formation. Later during the polarisation process, Nectins are components of epithelial adherens junctions where they are indirectly associated with the E-cadherin/Catenins complex via the adaptator AF-6. To have a better understanding of Nectin-based cell junctions, we looked for some new Nectins' partners. We demonstrate that the scaffold molecule PICK-1, involved in the clustering of junctional receptors in synaptic junctions, interacts directly with Nectins in a PSD-95/Dlg/ZO-1 domain-dependent manner and is localised at adherens junctions in epithelial cells. Finally, we observed that protein interacting with C-kinase-1 (PICK-1) also interacts directly with the junctional adhesion molecules, and we suggest that PICK-1 could be involved in the regulation of both adherens and tight junctions in epithelial cells.     

Flotillins Directly Interact with γ-Catenin and Regulate Epithelial Cell-Cell Adhesion

Flotillin-1 and flotillin-2 are two homologous, membrane raft associated proteins. Although it has been reported that flotillins are involved in cell adhesion processes and play a role during breast cancer progression, thus making them interesting future therapeutic targets, their precise function has not been well elucidated. The present study investigates the function of these proteins in cell-cell adhesion in non-malignant cells. We have used the non-malignant epithelial MCF10A cells to study the interaction network of flotillins within cell-cell adhesion complexes. RNA interference was used to examine the effect of flotillins on the structure of adherens junctions and on the association of core proteins, such as E-cadherin, with membrane rafts. We here show that the cadherin proteins of the adherens junction associate with flotillin-2 in MCF10A cells and in various human cell lines. In vitro, flotillin-1 and flotillin-2 directly interact with γ-catenin which is so far the only protein known to be present both in the adherens junction and the desmosome. Mapping of the interaction domain within the γ-catenin sequence identified the Armadillo domains 6–8, especially ARM domain 7, to be important for the association with flotillins. Furthermore, depletion of flotillins significantly influenced the morphology of the adherens junction in human epithelial MCF10A cells and altered the association of E-cadherin and γ-catenin with membrane rafts. Taken together, these observations suggest a functional role for flotillins, especially flotillin-2, in cell-cell adhesion in non-malignant epithelial cells.     

Exogenous expression of the amino-terminal half of the tight junction protein ZO-3 perturbs junctional complex assembly

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

Behavior of tight-junction, adherens-junction and cell polarity proteins during HNF-4alpha-induced epithelial polarization

We previously reported that expression of tight-junction molecules occludin, claudin-6 and claudin-7, as well as establishment of epithelial polarity, was triggered in mouse F9 cells expressing hepatocyte nuclear factor (HNF)-4alpha [H. Chiba, T. Gotoh, T. Kojima, S. Satohisa, K. Kikuchi, M. Osanai, N. Sawada. Hepatocyte nuclear factor (HNF)-4alpha triggers formation of functional tight junctions and establishment of polarized epithelial morphology in F9 embryonal carcinoma cells, Exp. Cell Res. 286 (2003) 288-297]. Using these cells, we examined in the present study behavior of tight-junction, adherens-junction and cell polarity proteins and elucidated the molecular mechanism behind HNF-4alpha-initiated junction formation and epithelial polarization. We herein show that not only ZO-1 and ZO-2, but also ZO-3, junctional adhesion molecule (JAM)-B, JAM-C and cell polarity proteins PAR-3, PAR-6 and atypical protein kinase C (aPKC) accumulate at primordial adherens junctions in undifferentiated F9 cells. In contrast, CRB3, Pals1 and PATJ appeared to exhibit distinct subcellular localization in immature cells. Induced expression of HNF-4alpha led to translocation of these tight-junction and cell polarity proteins to beltlike tight junctions, where occludin, claudin-6 and claudin-7 were assembled, in differentiated cells. Interestingly, PAR-6, aPKC, CRB3 and Pals1, but not PAR-3 or PATJ, were also concentrated on the apical membranes in differentiated cells. These findings indicate that HNF-4alpha provokes not only expression of tight-junction adhesion molecules, but also modulation of subcellular distribution of junction and cell polarity proteins, resulting in junction formation and epithelial polarization.     

DLG-1 Is a MAGUK Similar to SAP97 and Is Required for Adherens Junction Formation

Cellular junctions are critical for intercellular communication and for the assembly of cells into tissues. Cell junctions often consist of tight junctions, which form a permeability barrier and prevent the diffusion of lipids and proteins between cell compartments, and adherens junctions, which control the adhesion of cells and link cortical actin filaments to attachment sites on the plasma membrane. Proper tight junction formation and cell polarity require the function of membrane-associated guanylate kinases (MAGUKs) that contain the PDZ protein-protein interaction domain. In contrast, less is known about how adherens junctions are assembled. Here we describe how the PDZ-containing protein DLG-1 is required for the proper formation and function of adherens junctions in Caenorhabditis elegans. DLG-1 is a MAGUK protein that is most similar in sequence to mammalian SAP97, which is found at both synapses of the CNS, as well as at cell junctions of epithelia. DLG-1 is localized to adherens junctions, and DLG-1 localization is mediated by an amino-terminal domain shared with SAP97 but not found in other MAGUK family members. DLG-1 recruits other proteins and signaling molecules to adherens junctions, while embryos that lack DLG-1 fail to recruit the proteins AJM-1 and CPI-1 to adherens junctions. DLG-1 is required for the proper organization of the actin cytoskeleton and for the morphological elongation of embryos. In contrast to other proteins that have been observed to affect adherens junction assembly and function, DLG-1 is not required to maintain cell polarity. Our results suggest a new function for MAGUK proteins distinct from their role in cell polarity.     

Morphology, cell-cell interactions, and migratory activity of IAR-2 epithelial cells transformed with the RAS oncogene: contribution of cell adhesion protein E-cadherin

The destruction of stable cell-cell adhesion and the acquisition of the ability to migrate are consistent stages of neoplastic evolution of tumor cells of epithelial origin. We studied the morphologic and mi gration characteristics of epithelial cells of Iar1162 and IAR1170 clones derived from a mixed culture of on cogene N-RasV12-transformed cell line IAR-2. It was found that the mutant oncogene RAS can cause two types of morphological changes in IAR-2 epithelial cells. Cells of one type (IAR1162 clones) underwent epithelial-mesenchymal transition: they stopped to express E-cadherin, acquired fibroblast-like morphology, and did not form tight junctions. Cells of the other type (IAR1170 clones) retained a morphology close to the morphology of nontransformed progenitor cells, formed E-cadherin-based adherens junctions and tight junctions, and formed a monolayer in confluent culture. However, in both IAR1162 and IAR1170 cells, the mutant oncogene RAS caused the destruction of marginal actin bundle and the reorganization of cell-cell adherens junctions. RAS-transformed IAR1162 and IAR1170 epithelial cells acquired the ability to migrate on a flat substrate as well as through narrow pores in membranes of migration chambers. A videomicroscopic study of transformed epithelial cell cultures demonstrated the instability of cell-cell contacts and the independent nature of cell migration. IAR 1170 epithelial cells, which had E-cadherin-based adherens junctions, were also able to move as a group (collective migration). 1162D3 cells, which lost the ability to express endogenous E-cadherin as a result of Ras-transformation, were transfected with a plasmid carrying the CDH1. As a result of transfection, clones of cells with different levels of expression of exogenous E-cadherin were obtained. The high level of expression of exogenous E-cadherin in transformed epithelial cells led to a decrease in the rate of migration on a two-dimensional substrate of the cells that were in contact with neighboring cells but almost had no effect on the migration of single cells, at the same time increasing the number of cells that migrated through the pores in migration chambers. Thus, the destruction of marginal actin bundle and the change in the spatial organization of cell-cell adherens junctions, irrespective of the presence or absence of E-cadherin, was accompanied by destruction of stable cell-cell adhesion and the appearance of locomotor activity in Ras-transformed epithelial cells. The retaining of E-cadherin in cell-cell adhesion junctions affects the locomotor activity of transformed epithelial cells and plays an important role in their collective migration.     

Adjudin-mediated junction restructuring in the seminiferous epithelium leads to displacement of soluble guanylate cyclase from adherens junctions

A plethora of evidence supports the role of cyclic nucleotides in junction restructuring. For instance, studies have shown cGMP to be a key regulator of junction assembly and disassembly in different in vitro and in vivo systems. In this study, we examine the role of soluble guanylate cyclase (sGC) in junction restructuring in the seminiferous epithelium of the rat testis. First, the interaction of soluble guanylate cyclase beta1 (sGCbeta1; sGC is a heterodimer comprised of an alpha and a beta subunit) with proteins that constitute adherens and tight junctions in the testis was demonstrated. By immunoprecipitation, sGCbeta1 was found to associate with occludin, JAM-A, and ZO-1, as well as with cadherin, catenin, nectin, afadin, ponsin, and espin, suggestive of its role in cell junction dynamics. These results were corroborated in part by immunohistochemistry experiments, which revealed that the localization of sGCbeta1 was largely restricted to the site of the apical and basal ectoplasmic specialization. Next, the role of sGC in junction dynamics was addressed by using an in vivo model of junction restructuring. Administration of Adjudin--a chemical entity known to specifically perturb adhesion between Sertoli and germ cells (i.e., round and elongate(ing) spermatids and most spermatocytes)--resulted in a approximately 1.5-fold increase in sGCbeta1, coinciding with the loss of germ cells from the epithelium. More importantly, the ability of sGCbeta1 to associate with cadherin increased approximately three-fold during Adjudin-mediated restructuring of Sertoli-germ cell junctions, whereas its interaction with tight junction proteins (i.e., occludin and ZO-1) decreased. Taken collectively, these results suggest that sGC participates in the remodeling of cell junctions during spermatogenesis.