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.     

E-Cadherin Is Required for Centrosome and Spindle Orientation in Drosophila Male Germline Stem Cells

Many adult stem cells reside in a special microenvironment known as the niche, where they receive essential signals that specify stem cell identity. Cell-cell adhesion mediated by cadherin and integrin plays a crucial role in maintaining stem cells within the niche. In Drosophila melanogaster, male germline stem cells (GSCs) are attached to niche component cells (i.e., the hub) via adherens junctions. The GSC centrosomes and spindle are oriented toward the hub-GSC junction, where E-cadherin-based adherens junctions are highly concentrated. For this reason, adherens junctions are thought to provide a polarity cue for GSCs to enable proper orientation of centrosomes and spindles, a critical step toward asymmetric stem cell division. However, understanding the role of E-cadherin in GSC polarity has been challenging, since GSCs carrying E-cadherin mutations are not maintained in the niche. Here, we tested whether E-cadherin is required for GSC polarity by expressing a dominant-negative form of E-cadherin. We found that E-cadherin is indeed required for polarizing GSCs toward the hub cells, an effect that may be mediated by Apc2. We also demonstrated that E-cadherin is required for the GSC centrosome orientation checkpoint, which prevents mitosis when centrosomes are not correctly oriented. We propose that E-cadherin orchestrates multiple aspects of stem cell behavior, including polarization of stem cells toward the stem cell-niche interface and adhesion of stem cells to the niche supporting cells.     

Effects of 18beta-glycyrrhetinic acid on the junctional complex and steroidogenesis in rat adrenocortical cells

Cellular junctions play important roles in cell differentiation, signal transduction, and cell function. This study investigated their function in steroid secretion by adrenal cells. Immunofluorescence staining revealed the presence of gap junctions and adherens junctions between adrenal cells. The major gap junction protein, connexin43, was seen as a linear dotted pattern of the typical gap junction plaques, in contrast to alpha-, beta-, and gamma-catenin, which were seen as continuous, linear staining of cell-cell adherens junction. Treatment with 18beta-glycyrrhetinic acid, a gap junction inhibitor, reduced the immunoreactivity of these proteins in a time- and dose-dependent manner, and caused the gap junction and adherens junction to separate longitudinally from the cell-cell contact sites, indicating the structural interdependency of these two junctions. Interestingly, 18beta-glycyrrhetinic acid stimulated a two- to three-fold increase in steroid production in these adrenal cells lacking intact cell junctions. These data raise the question of the necessity for cell communication for the endocrine function of adrenal cells. Pharmacological analyses indicated that the steroidogenic effect of 18beta-glycyrrhetinic acid was partially mediated by extracellular signal-related kinase and calcium/calmodulin-dependent kinase, a pathway distinct from the protein kinase A signaling pathway already known to mediate steroidogenesis in adrenal cells.     

ROCK and Dia have opposing effects on adherens junctions downstream of Rho

Adherens junctions (AJs) are crucial for maintaining the integrity of epithelial tissues and are often disrupted during tumour progression. Rho family proteins have been shown to regulate adherens junctions. We find that activation of the effector kinase ROCK and acto-myosin contraction disrupts AJs downstream of Rho. In contrast, signalling through the Rho effector Dia1 is required to ensure a dynamically stable interface between cells and the maintenance of adherens junction complexes. The ability of Dia1 to regulate the actin network is crucial for the localization of adherens junction components to the cell periphery.     

Remodeling the zonula adherens in response to tension and the role of afadin in this response

Morphogenesis requires dynamic coordination between cell–cell adhesion and the cytoskeleton to allow cells to change shape and move without losing tissue integrity. We used genetic tools and superresolution microscopy in a simple model epithelial cell line to define how the molecular architecture of cell–cell zonula adherens (ZA) is modified in response to elevated contractility, and how these cells maintain tissue integrity. We previously found that depleting zonula occludens 1 (ZO-1) family proteins in MDCK cells induces a highly organized contractile actomyosin array at the ZA. We find that ZO knockdown elevates contractility via a Shroom3/Rho-associated, coiled-coil containing protein kinase (ROCK) pathway. Our data suggest that each bicellular border is an independent contractile unit, with actin cables anchored end-on to cadherin complexes at tricellular junctions. Cells respond to elevated contractility by increasing junctional afadin. Although ZO/afadin knockdown did not prevent contractile array assembly, it dramatically altered cell shape and barrier function in response to elevated contractility. We propose that afadin acts as a robust protein scaffold that maintains ZA architecture at tricellular junctions.     

Scattering of MCF7 Cells by Heregulin ?-1 Depends on the MEK and p38 MAP Kinase Pathway

Heregulin (HRG) β1 signaling promotes scattering of MCF7 cells by inducing breakdown of adherens and tight junctions. Here, we show that stimulation with HRG-β1 causes the F-actin backbone of junctions to destabilize prior to the loss of adherent proteins and scattering of the cells. The adherent proteins dissociate and translocate from cell–cell junctions to the cytosol. Moreover, using inhibitors we show that the MEK1 pathway is required for the disappearance of F-actin from junctions and p38 MAP kinase activity is essential for scattering of the cells. Upon treatment with a p38 MAP kinase inhibitor, adherens junction complexes immediately reassemble, most likely in the cytoplasm, and move to the plasma membrane in cells dissociated by HRG-β1 stimulation. Subsequently, tight junction complexes form, most likely in the cytoplasm, and move to the plasma membrane. Thus, the p38 MAP kinase inhibitor causes a re-aggregation of scattered cells, even in the presence of HRG-β1. These results suggest that p38 MAP kinase signaling to adherens junction proteins regulates cell aggregation, providing a novel understanding of the regulation of cell–cell adhesion.     

Inactivation of aPKClambda results in the loss of adherens junctions in neuroepithelial cells without affecting neurogenesis in mouse neocortex

In developing mammalian telencephalon, the loss of adherens junctions and cell cycle exit represent crucial steps in the differentiation of neuroepithelial cells into neurons, but the relationship between these cellular events remains obscure. Atypical protein kinase C (aPKC) is known to contribute to junction formation in epithelial cells and to cell fate determination for Drosophila neuroblasts. To elucidate the functions of aPKClambda, one out of two aPKC members, in mouse neocortical neurogenesis, a Nestin-Cre mediated conditional gene targeting system was employed. In conditional aPKClambda knockout mice, neuroepithelial cells of the neocortical region lost aPKClambda protein at embryonic day 15 and demonstrated a loss of adherens junctions, retraction of apical processes and impaired interkinetic nuclear migration that resulted in disordered neuroepithelial tissue architecture. These results are evidence that aPKClambda is indispensable for the maintenance of adherens junctions and may function in the regulation of adherens junction integrity upon differentiation of neuroepithelial cells into neurons. In spite of the loss of adherens junctions in the neuroepithelium of conditional aPKClambda knockout mice, neurons were produced at a normal rate. Therefore, we concluded that, at least in the later stages of neurogenesis, regulation of cell cycle exit is independent of adherens junctions.     

The roles of the Na,K-ATPase beta 1 subunit in pump sorting and epithelial integrity

In epithelial MDCK cells, the Na,K-ATPase is co-localized with adherens junctions in all stages of monolayer formation starting from initiation of cell–cell contact. The Na,K-ATPase and adherens junction proteins stay partially co-localized even after internalization due to disruption of intercellular contacts by Ca²⁺ deprivation. Similar to adherens junction proteins, the Na,K-ATPase is resistant to extraction with non-ionic detergent, suggesting pump association with the cytoskeleton. In contrast, the heterodimer formed by expressed unglycosylated Na,K-ATPase β₁ subunit and the endogenous α₁ subunit is easily dissociated from the adherens junctions and cytoskeleton by detergent extraction. The MDCK cells in which half of the endogenous β₁ subunits in the lateral membrane are substituted by unglycosylated β₁ subunits display a slower rate of cell-to-cell contact formation and decreased ability to both spread over the surface and migrate. The lack of N-glycans in the Na,K-ATPase β₁ subunit results in an impairment of mature cell–cell junctions as detected by an increase in the paracellular permeability of the MDCK cell monolayers and by a decrease in resistance of adherens junction proteins to extraction by a non-ionic detergent. Therefore the N-glycans of the Na,K-ATPase β₁ subunit are important for retention of the pump at the sites of cell–cell contact. Moreover, they are important for the integrity and stability of cell–cell junctions in mature epithelia. In addition, N-glycans contribute to the formation of cell–cell contacts between surface-attached dispersed cells by mediating lamellipodia formation and stabilizing the newly formed adherens junctions.     

A WAVE2–Arp2/3 actin nucleator apparatus supports junctional tension at the epithelial zonula adherens

The epithelial zonula adherens (ZA) is a specialized adhesive junction where actin dynamics and myosin-driven contractility coincide. The junctional cytoskeleton is enriched in myosin II, which generates contractile force to support junctional tension. It is also enriched in dynamic actin filaments, which are replenished by ongoing actin assembly. In this study we sought to pursue the relationship between actin assembly and junctional contractility. We demonstrate that WAVE2–Arp2/3 is a major nucleator of actin assembly at the ZA and likely acts in response to junctional Rac signaling. Furthermore, WAVE2–Arp2/3 is necessary for junctional integrity and contractile tension at the ZA. Maneuvers that disrupt the function of either WAVE2 or Arp2/3 reduced junctional tension and compromised the ability of cells to buffer side-to-side forces acting on the ZA. WAVE2–Arp2/3 disruption depleted junctions of both myosin IIA and IIB, suggesting that dynamic actin assembly may support junctional tension by facilitating the local recruitment of myosin.     

Major involvement of connexin 43 in seminiferous epithelial junction dynamics and male fertility

In different epithelia, cell membranes contacting one another form intercellular junctional complexes including tight, adherens and gap junctions, which could mutually influence the expression of each other. We have here investigated the role of Cx43 in the control of adherens and tight junction proteins (N-cadherin, β-catenin, occludin and ZO-1) by using conditional Sertoli cell knockout Cx43 (SCCx43KO^−/−) transgenic mice and specific anti-Cx43 siRNA. Gap junction coupling and Cx43 levels were reduced in SCCx43KO^−/− as compared to Wild-type testes. Ultrastructural analysis revealed disappearance of gap junctions, the presence of tight and adherens junctions and persistent integrity of the blood-testis barrier in SCCx43KO^−/− testis. Occludin, N-cadherin and β-catenin levels were enhanced in SCCx43KO^−/− mice as compared to Wild-type animals whereas ZO-1 levels were reduced. Cx43 siRNA blocked gap junction functionality in Sertoli cells and altered tight and adherens protein levels. The Cx43 control of tight and adherens junctions appeared channel-dependent since gap junction blockers (glycyrrhetinic acid and oleamide) led to similar results. These data suggest that the control of spermatogenesis by Cx43 may be mediated through Sertoli cell Cx43 channels, which are required, not only in cell/cell communication between Sertoli and germ cells, but also in the regulation of other junctional proteins essential for the blood-testis barrier.     

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.     

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.     

Armadillo is required for adherens junction assembly, cell polarity, and morphogenesis during Drosophila embryogenesis

Morphological and biochemical analyses have identified a set of proteins which together form a structure known as the adherens junction. Elegant experiments in tissue culture support the idea that adherens junctions play a key role in cell-cell adhesion and in organizing cells into epithelia. During normal embryonic development, cells quickly organize epithelia; these epithelial cells participate in many of the key morphogenetic movements of gastrulation. This prompted the hypothesis that adherens junctions ought to be critical for normal embryonic development. Drosophila Armadillo, the homologue of vertebrate beta-catenin, is a core component of the adherens junction protein complex and has been hypothesized to be essential for adherens junction function in vivo. We have used an intermediate mutant allele of armadillo, armadilloXP33, to test these hypotheses in Drosophila embryos. Adherens junctions cannot assemble in the absence of Armadillo, leading to dramatic defects in cell-cell adhesion. The epithelial cells of the embryo lose adhesion to each other, round up, and apparently become mesenchymal. Mutant cells also lose their normal cell polarity. These disruptions in the integrity of epithelia block the appropriate morphogenetic movements of gastrulation. These results provide the first demonstration of the effect of loss of adherens junctions on Drosophila embryonic development.     

Inhibition of gap junction and adherens junction assembly by connexin and A-CAM antibodies

We examined the roles of the extracellular domains of a gap junction protein and a cell adhesion molecule in gap junction and adherens junction formation by altering cell interactions with antibody Fab fragments. Using immunoblotting and immunocytochemistry we demonstrated that Novikoff cells contained the gap junction protein, connexin43 (Cx43), and the cell adhesion molecule, A-CAM (N-cadherin). Cells were dissociated in EDTA, allowed to recover, and reaggregated for 60 min in media containing Fab fragments prepared from a number of antibodies. We observed no cell-cell dye transfer 4 min after microinjection in 90% of the cell pairs treated with Fab fragments of antibodies for the first or second extracellular domain of Cx43, the second extracellular domain of connexin32 (Cx32) or A-CAM. Cell-cell dye transfer was detected within 30 s in cell pairs treated with control Fab fragments (pre-immune serum, antibodies to the rat major histocompatibility complex or the amino or carboxyl termii of Cx43). We observed no gap junctions by freeze-fracture EM and no adherens junctions by thin section EM between cells treated with the Fab fragments that blocked cell-cell dye transfer. Gap junctions were found on approximately 50% of the cells in control samples using freeze-fracture EM. We demonstrated with reaggregated Novikoff cells that: (a) functional interactions of the extracellular domains of the connexins were necessary for the formation of gap junction channels; (b) cell interactions mediated by A-CAM were required for gap junction assembly; and (c) Fab fragments of antibodies for A-CAM or connexin extracellular domains blocked adherens junction formation.     

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.     

Activation of Both MAP Kinase and Phosphatidylinositide 3-Kinase by Ras Is Required for Hepatocyte Growth Factor/Scatter Factor–induced Adherens Junction Disassembly

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates the motility of epithelial cells, initially inducing centrifugal spreading of colonies followed by disruption of cell–cell junctions and subsequent cell scattering. In Madin–Darby canine kidney cells, HGF/SF-induced motility involves actin reorganization mediated by Ras, but whether Ras and downstream signals regulate the breakdown of intercellular adhesions has not been established. Both HGF/SF and V12Ras induced the loss of the adherens junction proteins E-cadherin and β-catenin from intercellular junctions during cell spreading, and the HGF/SF response was blocked by dominant-negative N17Ras. Desmosomes and tight junctions were regulated separately from adherens junctions, because they were not disrupted by V12Ras. MAP kinase, phosphatidylinositide 3-kinase (PI 3-kinase), and Rac were required downstream of Ras, because loss of adherens junctions was blocked by the inhibitors PD098059 and LY294002 or by dominant-inhibitory mutants of MAP kinase kinase 1 or Rac1. All of these inhibitors also prevented HGF/SF-induced cell scattering. Interestingly, activated Raf or the activated p110α subunit of PI 3-kinase alone did not induce disruption of adherens junctions. These results indicate that activation of both MAP kinase and PI 3-kinase by Ras is required for adherens junction disassembly and that this is essential for the motile response to HGF/SF.     

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.     

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.     

An RPTPα/Src family kinase/Rap1 signaling module recruits myosin IIB to support contractile tension at apical E-cadherin junctions

Cell–cell adhesion couples the contractile cortices of epithelial cells together, generating tension to support a range of morphogenetic processes. E-cadherin adhesion plays an active role in generating junctional tension by promoting actin assembly and cortical signaling pathways that regulate myosin II. Multiple myosin II paralogues accumulate at mammalian epithelial cell–cell junctions. Earlier, we found that myosin IIA responds to Rho-ROCK signaling to support junctional tension in MCF-7 cells. Although myosin IIB is also found at the zonula adherens (ZA) in these cells, its role in junctional contractility and its mode of regulation are less well understood. We now demonstrate that myosin IIB contributes to tension at the epithelial ZA. Further, we identify a receptor type-protein tyrosine phosphatase alpha–Src family kinase–Rap1 pathway as responsible for recruiting myosin IIB to the ZA and supporting contractile tension. Overall these findings reinforce the concept that orthogonal E-cadherin–based signaling pathways recruit distinct myosin II paralogues to generate the contractile apparatus at apical epithelial junctions.