A role for ZO-1 and PLEKHA7 in recruiting paracingulin to tight and adherens junctions of epithelial cells

Paracingulin is a 160-kDa protein localized in the cytoplasmic region of epithelial tight and adherens junctions, where it regulates RhoA and Rac1 activities by interacting with guanine nucleotide exchange factors. Here, we investigate the molecular mechanisms that control the recruitment of paracingulin to cell-cell junctions. We show that paracingulin forms a complex with the tight junction protein ZO-1, and the globular head domain of paracingulin interacts directly with ZO-1 through an N-terminal region containing a conserved ZIM (ZO-1-Interaction-Motif) sequence. Recruitment of paracingulin to cadherin-based cell-cell junctions in Rat1 fibroblasts requires the ZIM-containing region, whereas in epithelial cells removal of this region decreases the junctional localization of paracingulin at tight junctions but not at adherens junctions. Depletion of ZO-1, but not ZO-2, reduces paracingulin accumulation at tight junctions. A yeast two-hybrid screen identifies both ZO-1 and the adherens junction protein PLEKHA7 as paracingulin-binding proteins. Paracingulin forms a complex with PLEKHA7 and its interacting partner p120ctn, and the globular head domain of paracingulin interacts directly with a central region of PLEKHA7. Depletion of PLEKHA7 from Madin-Darby canine kidney cells results in the loss of junctional localization of paracingulin and a decrease in its expression. In summary, we characterize ZO-1 and PLEKHA7 as paracingulin-interacting proteins that are involved in its recruitment to epithelial tight and adherens junctions, respectively.     

Adherens junctions: new insight into assembly,modulation and function

Adherens junctions play pivotal roles in cell and tissue organization and patterning by mediating cell adhesion and cell signaling. These junctions consist of large multiprotein complexes that join the actin cytoskeleton to the plasma membrane to form adhesive contacts between cells or between cells and extracellular matrix. The best-known adherens junction is the zonula adherens (ZA) that forms a belt surrounding the apical pole of epithelial cells. Recent studies in Drosophila have further illuminated the structure of adherens junctions. Scaffolding proteins encoded by the stardust gene are novel components of the Crumbs complex, which plays a critical role in ZA assembly.1-3 The small GTPase Rap1 controls the symmetric re-assembly of the ZA after cell division.4 Finally, the asymmetric distribution of adherens junction material regulates spindle orientation during asymmetric cell division in the sensory organ lineage.     

Balancing different types of actin polymerization at distinct sites: roles for Abelson kinase and Enabled

The proto-oncogenic kinase Abelson (Abl) regulates actin in response to cell signaling. Drosophila Abl is required in the nervous system, and also in epithelial cells, where it regulates adherens junction stability and actin organization. Abl acts at least in part via the actin regulator Enabled (Ena), but the mechanism by which Abl regulates Ena is unknown. We describe a novel role for Abl in early Drosophila development, where it regulates the site and type of actin structures produced. In Abl's absence, excess actin is polymerized in apical microvilli, whereas too little actin is assembled into pseudocleavage and cellularization furrows. These effects involve Ena misregulation. In abl mutants, Ena accumulates ectopically at the apical cortex where excess actin is observed, suggesting that Abl regulates Ena's subcellular localization. We also examined other actin regulators. Loss of Abl leads to changes in the localization of the Arp2/3 complex and the formin Diaphanous, and mutations in diaphanous or capping protein beta enhance abl phenotypes.     

Crumbs interacts with moesin and beta(Heavy)-spectrin in the apical membrane skeleton of Drosophila

The apical transmembrane protein Crumbs is necessary for both cell polarization and the assembly of the zonula adherens (ZA) in Drosophila epithelia. The apical spectrin-based membrane skeleton (SBMS) is a protein network that is essential for epithelial morphogenesis and ZA integrity, and exhibits close colocalization with Crumbs and the ZA in fly epithelia. These observations suggest that Crumbs may stabilize the ZA by recruiting the SBMS to the junctional region. Consistent with this hypothesis, we report that Crumbs is necessary for the organization of the apical SBMS in embryos and Schneider 2 cells, whereas the localization of Crumbs is not affected in karst mutants that eliminate the apical SBMS. Our data indicate that it is specifically the 4.1 protein/ezrin/radixin/moesin (FERM) domain binding consensus, and in particular, an arginine at position 7 in the cytoplasmic tail of Crumbs that is essential to efficiently recruit both the apical SBMS and the FERM domain protein, DMoesin. Crumbs, Discs lost, betaHeavy-spectrin, and DMoesin are all coimmunoprecipitated from embryos, confirming the existence of a multimolecular complex. We propose that Crumbs stabilizes the apical SBMS via DMoesin and actin, leading to reinforcement of the ZA and effectively coupling epithelial morphogenesis and cell polarity.     

Ponsin/SH3P12: an l-afadin- and vinculin-binding protein localized at cell-cell and cell-matrix adherens junctions

We recently isolated a novel actin filament (F-actin)-binding protein, afadin, that has two isoforms, l- and s-afadins. l-Afadin is ubiquitously expressed and specifically localized at zonula adherens (ZA) in epithelial cells and at cell-cell adherens junction (AJ) in nonepithelial cells, whereas s-afadin is abundantly expressed in neural tissue. l-Afadin has one PDZ domain, three proline-rich regions, and one F-actin-binding domain, whereas s-afadin lacks the third proline-rich region and the F-actin-binding domain. To understand the molecular mechanism of the specific localization of l-afadin at ZA in epithelial cells and at cell-cell AJ in nonepithelial cells, we attempted here to identify an l-afadin-binding protein(s) and isolated a protein, named ponsin. Ponsin had many splicing variants and the primary structures of two of them were determined. Both the two variants had three Src homology 3 (SH3) domains and turned out to be splicing variants of SH3P12. The third proline-rich region of l-afadin bound to the region of ponsin containing the second and third SH3 domains. Ponsin was ubiquitously expressed and localized at ZA in epithelial cells, at cell-cell AJ in nonepithelial cells, and at cell-matrix AJ in both types of cells. Ponsin furthermore directly bound vinculin, an F-actin-binding protein localized at ZA in epithelial cells, at cell-cell AJ in nonepithelial cells, and at cell-matrix AJ in both types of cells. Vinculin has one proline-rich region where two proline-rich sequences are located. The proline-rich region bound to the region of ponsin containing the first and second SH3 domains. l-Afadin and vinculin bound to ponsin in a competitive manner and these three proteins hardly formed a ternary complex. These results indicate that ponsin is an l-afadin- and vinculin-binding protein localized at ZA in epithelial cells, at cell-cell AJ in nonepithelial cells, and at cell-matrix AJ in both types of cells.     

Ultrastructure of the zonula adherens revealed by rapid-freeze deep-etching

The zonula adherens (ZA) in adult chicken retinal pigment epithelium was examined with cryo-electron microscopic methods. Deep-etching of the cross-fractured ZA showed globules in the intercellular space. These globules apparently correspond to the electron-dense structure seen in thin sections. Deep-etching of obliquely fractured ZA further revealed rod-like structures extending from the extracellular surface into the intercellular space. These rods (mean approximately 9 nm thick, approximately 20 nm long) were straight and sometimes divided into two or three segments. The rods typically canted at approximately 60 degrees with respect to the plasma membrane, and they were often connected to the intercellular globules at their distal ends. When the rods are compared with the isolated cadherins reported previously, it is suggested that a combination of a rod and a globule may represent an extracellular part of cadherin. Membrane particles were observed on the P-face of the ZA plasma membrane, and their distribution density was approximately seven times that of the rods. The freeze-etching also revealed a characteristic particle complex on the ZA cytoplasmic surface, which may represent the cytosolic proteins linking cadherins to actin bundles.     

E-cadherin supports steady-state Rho signaling at the epithelial zonula adherens

In simple polarized epithelial cells, the Rho GTPase commonly localizes to E-cadherin-based cell–cell junctions, such as the zonula adherens (ZA), where it regulates the actomyosin cytoskeleton to support junctional integrity and tension. An important question is how E-cadherin contributes to Rho signaling, notably whether junctional Rho may depend on cadherin adhesion. We sought to investigate this by assessing Rho localization and activity in epithelial monolayers depleted of E-cadherin by RNAi. We report that E-cadherin depletion reduced both Rho and Rho-GTP at the ZA, an effect that was rescued by expressing a RNAi-resistant full-length E-cadherin transgene. This impact on Rho signaling was accompanied by reduced junctional localization of the Rho GEF ECT2 and the centralspindlin complex that recruits ECT2. Further, the Rho signaling pathway contributes to the selective stabilization of E-cadherin molecules in the apical zone of the cells compared with E-cadherin at the lateral surface, thereby creating a more defined and restricted pool of E-cadherin that forms the ZA. Thus, E-cadherin and Rho signaling cooperate to ensure proper ZA architecture and function.     

The PLEKHA7–PDZD11 complex regulates the localization of the calcium pump PMCA and calcium handling in cultured cells

The plasma membrane calcium ATPase (PMCA) extrudes calcium from the cytosol to the extracellular space to terminate calcium-dependent signaling. Although the distribution of PMCA is crucial for its function, the molecular mechanisms that regulate the localization of PMCA isoforms are not well understood. PLEKHA7 is implicated by genetic studies in hypertension and the regulation of calcium handling. PLEKHA7 recruits the small adapter protein PDZD11 to adherens junctions, and together they control the trafficking and localization of plasma membrane associated proteins, including the Menkes copper ATPase. Since PDZD11 binds to the C-terminal domain of b-isoforms of PMCA, PDZD11 and its interactor PLEKHA7 could control the localization and activity of PMCA. Here, we test this hypothesis using cultured cell model systems. We show using immunofluorescence microscopy and a surface biotinylation assay that KO of either PLEKHA7 or PDZD11 in mouse kidney collecting duct epithelial cells results in increased accumulation of endogenous PMCA at lateral cell–cell contacts and PDZ-dependent ectopic apical localization of exogenous PMCA4x/b isoform. In HeLa cells, coexpression of PDZD11 reduces membrane accumulation of overexpressed PMCA4x/b, and analysis of cytosolic calcium transients shows that PDZD11 counteracts calcium extrusion activity of overexpressed PMCA4x/b, but not PMCA4x/a, which lacks the PDZ-binding motif. Moreover, KO of PDZD11 in either endothelial (bEnd.3) or epithelial (mouse kidney collecting duct) cells increases the rate of calcium extrusion. Collectively, these results suggest that the PLEKHA7–PDZD11 complex modulates calcium homeostasis by regulating the localization of PMCA.     

p120‐catenin differentially regulates cell migration by Rho‐dependent intracellular and secreted signals

The adherens junction protein p120‐catenin is implicated in the regulation of cadherin stability, cell migration and inflammatory responses in mammalian epithelial tissues. How these events are coordinated to promote wound repair is not understood. We show that p120 catenin regulates the intrinsic migratory properties of primary mouse keratinocytes, but also influences the migratory behavior of neighboring cells by secreted signals. These events are rooted in the ability of p120‐catenin to regulate RhoA GTPase activity, which leads to a two‐tiered control of cell migration. One restrains cell motility via an increase in actin stress fibers, reduction in integrin turnover and an increase in the robustness of focal adhesions. The other is coupled to the secretion of inflammatory cytokines including interleukin‐24, which causally enhances randomized cell movements. Taken together, our results indicate that p120‐RhoA‐GTPase‐mediated signaling can differentially regulate the migratory behavior of epidermal cells, which has potential implications for chronic wound responses and cancer.     

Cortactin Scaffolds Arp2/3 and WAVE2 at the Epithelial Zonula Adherens

Cadherin junctions arise from the integrated action of cell adhesion, signaling, and the cytoskeleton. At the zonula adherens (ZA), a WAVE2-Arp2/3 actin nucleation apparatus is necessary for junctional tension and integrity. But how this is coordinated with cadherin adhesion is not known. We now identify cortactin as a key scaffold for actin regulation at the ZA, which localizes to the ZA through influences from both E-cadherin and N-WASP. Using cell-free protein expression and fluorescent single molecule coincidence assays, we demonstrate that cortactin binds directly to the cadherin cytoplasmic tail. However, its concentration with cadherin at the apical ZA also requires N-WASP. Cortactin is known to bind Arp2/3 directly (Weed, S. A., Karginov, A. V., Schafer, D. A., Weaver, A. M., Kinley, A. W., Cooper, J. A., and Parsons, J. T. (2000) J. Cell Biol. 151, 29–40). We further show that cortactin can directly bind WAVE2, as well as Arp2/3, and both these interactions are necessary for actin assembly at the ZA. We propose that cortactin serves as a platform that integrates regulators of junctional actin assembly at the ZA.     

AIP1 acts with cofilin to control actin dynamics during epithelial morphogenesis

During epithelial morphogenesis, cells not only maintain tight adhesion for epithelial integrity but also allow dynamic intercellular movement to take place within cell sheets. How these seemingly opposing processes are coordinated is not well understood. Here, we report that the actin disassembly factors AIP1 and cofilin are required for remodeling of adherens junctions (AJs) during ommatidial precluster formation in Drosophila eye epithelium, a highly stereotyped cell rearrangement process which we describe in detail in our live imaging study. AIP1 is enriched together with F-actin in the apical region of preclusters, whereas cofilin displays a diffuse and uniform localization pattern. Cofilin overexpression completely rescues AJ remodeling defects caused by AIP1 loss of function, and cofilin physically interacts with AIP1. Pharmacological reduction of actin turnover results in similar AJ remodeling defects and decreased turnover of E-cadherin, which also results from AIP1 deficiency, whereas an F-actin-destabilizing drug affects AJ maintenance and epithelial integrity. Together with other data on actin polymerization, our results suggest that AIP1 enhances cofilin-mediated actin disassembly in the apical region of precluster cells to promote remodeling of AJs and thus intercellular movement, but also that robust actin polymerization promotes AJ general adhesion and integrity during the remodeling process.     

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.     

Association of Rho-associated protein kinase 1 with E-cadherin complexes is mediated by p120-catenin

The dynamic functional linkage of cadherins with the underlying actin cytoskeleton is tightly regulated to achieve proper cell-cell adhesion. p120-catenin (p120) regulates both cadherin stability and actin dynamics, but the relationship between these two functions remains unclear. Using a novel proteomic approach called reversible cross-link immunoprecipitation, or ReCLIP, we previously identified a physical interaction between p120 and Rho-associated protein kinase 1 (ROCK1), a major effector of RhoA. In this paper, we show that a discrete fraction of cellular ROCK1 coimmunoprecipitates with p120 and precisely colocalizes to adherens junctions (AJs). Manipulation of AJs using a calcium-switch assay and cadherin-blocking antibodies indicates direct recruitment of ROCK1 to newly forming junctions. Importantly, we find that p120 links ROCK1 to the cadherin complex, as ROCK1 coimmunoprecipitates with wild-type but not p120-uncoupled E-cadherin. Moreover, depletion of ROCK1 using short-hairpin RNA results in dramatic mislocalization of the cadherin complex and junctional actin. These data are consistent with a model in which p120 dynamically regulates Rho-GTPase activity at the cadherin complex through transient interaction with several of its up- and downstream effectors, including ROCK1.     

Binding between the Junctional Proteins Afadin and PLEKHA7 and Implication in the Formation of Adherens Junction in Epithelial Cells

Adherens junction (AJ) is a specialized cell-cell junction structure that plays a role in mechanically connecting adjacent cells to resist strong contractile forces and to maintain tissue structure, particularly in the epithelium. AJ is mainly comprised of cell adhesion molecules cadherin and nectin and their associating cytoplasmic proteins including β-catenin, α-catenin, p120^ctn, and afadin. Our series of studies have revealed that nectin first forms cell-cell adhesion and then recruits cadherin to form AJ. The recruitment of cadherin by nectin is mediated by the binding of α-catenin and p120^ctn to afadin. Recent studies showed that PLEKHA7 binds to p120^ctn, which is associated with E-cadherin, and maintains the integrity of AJ in epithelial cells. In this study, we showed that PLEKHA7 bound to afadin in addition to p120^ctn and was recruited to the nectin-3α-based cell-cell adhesion site in a manner dependent on afadin, but not on p120^ctn. The binding of PLEKHA7 to afadin was required for the proper formation of AJ, but not for the formation of tight junction, in EpH4 mouse mammary gland epithelial cells. These results indicate that PLEKHA7 plays a cooperative role with nectin and afadin in the proper formation of AJ in epithelial cells.     

Expanded functions in the apical cell domain to regulate the growth rate of imaginal discs

The Drosophila expanded (ex) gene encodes a product (Ex) that shares homology with the Protein 4.1 family of proteins, many of which are enriched at specific lateral cell junctions and the apical cellular domain. Ex colocalizes with actin in the apical domain of imaginal disc epithelial cells, where it partially overlaps the distribution of phosphotyrosine (PY)-containing proteins. This suggests that Ex is present in or associated with adherens junctions. Genetic studies show that Ex is necessary for proper regulation of final cell number in adult wings and for the formation of eyes, distal leg, and distal antennal segments. We have generated mitotic clones that lack Ex using the twin spot technique, and demonstrated that the primary function of Ex is to regulate cell proliferation. Overexpressing Ex protein results in a decrease in final cell number in wings, suggesting a direct relationship between Ex function and proliferation rate. Dev. Genet. 20:103–110, 1997. © 1997 Wiley-Liss, Inc.     

Interplay between components of a novel LIM kinase-slingshot phosphatase complex regulates cofilin

Slingshot (SSH) phosphatases and LIM kinases (LIMK) regulate actin dynamics via a reversible phosphorylation (inactivation) of serine 3 in actin-depolymerizing factor (ADF) and cofilin. Here we demonstrate that a multi-protein complex consisting of SSH-1L, LIMK1, actin, and the scaffolding protein, 14-3-3zeta, is involved, along with the kinase, PAK4, in the regulation of ADF/cofilin activity. Endogenous LIMK1 and SSH-1L interact in vitro and co-localize in vivo, and this interaction results in dephosphorylation and downregulation of LIMK1 activity. We also show that the phosphatase activity of purified SSH-1L is F-actin dependent and is negatively regulated via phosphorylation by PAK4. 14-3-3zeta binds to phosphorylated slingshot, decreases the amount of slingshot that co-sediments with F-actin, but does not alter slingshot activity. Here we define a novel ADF/cofilin phosphoregulatory complex and suggest a new mechanism for the regulation of ADF/cofilin activity in mediating changes to the actin cytoskeleton.     

LIM kinase 1 and cofilin regulate actin filament population required for dynamin-dependent apical carrier fission from the trans-Golgi network

The functions of the actin cytoskeleton in post-Golgi trafficking are still poorly understood. Here, we report the role of LIM Kinase 1 (LIMK1) and its substrate cofilin in the trafficking of apical and basolateral proteins in Madin-Darby canine kidney cells. Our data indicate that LIMK1 and cofilin organize a specialized population of actin filaments at the Golgi complex that is selectively required for the emergence of an apical cargo route to the plasma membrane (PM). Quantitative pulse-chase live imaging experiments showed that overexpression of kinase-dead LIMK1 (LIMK1-KD), or of LIMK1 small interfering RNA, or of an activated cofilin mutant (cofilin S3A), selectively slowed down the exit from the trans-Golgi network (TGN) of the apical PM marker p75-green fluorescent protein (GFP) but did not interfere with the apical PM marker glycosyl phosphatidylinositol-YFP or the basolateral PM marker neural cell adhesion molecule-GFP. High-resolution live imaging experiments of carrier formation and release by the TGN and analysis of peri-Golgi actin dynamics using photoactivatable GFP suggest a scenario in which TGN-localized LIMK1-cofilin regulate a population of actin filaments required for dynamin-syndapin-cortactin–dependent generation and/or fission of precursors to p75 transporters.     

Enterocytic differentiation is modulated by lipid rafts-dependent assembly of adherens junctions

Integrity of the epithelial barrier is determined by apical junctional complexes which also participate in the signalling pathways inducing intestinal cell differentiation. Lipid rafts (LR) have been proposed to play a role in the organization and the function of these intercellular complexes. This study investigated potential mechanisms by which LR could participate in the establishment of adherens junctions (AJ) and the initiation of enterocytic differentiation.We showed that the differentiation of epithelial cells in rat colons correlates with the emergence of LR. Using HT-29 cells we demonstrated that during the differentiation process, LR are required for the recruitment and the association of p120ctn to E-cadherin. Silencing of flotillin-1, a LR component, alters the recruitment of AJ proteins in LR and delays the expression of differentiation markers. Furthermore, the ability of p120ctn/E-cadherin complexes to support cell differentiation is altered in HT-29 Rac1N17 cells. These results show a contributory role of LR in the enterocytic differentiation process, which serve as signalling platforms for Rac1-mediated organization of AJ. A better understanding of the mechanism involved in the establishment of junctional complex and their role in enterocytic differentiation provides new insights into the regulation of intestinal homeostasis.     

Differential effects of substrate-analogue inhibitors on nitric oxide synthase dimerization

Cigarette smoking has been linked to almost all major types of cancer. Emerging evidence suggests that smoking initiates transformed cell growth and migration by disrupting cell-cell interactions in the polarized mucosal epithelium. Together with other adherens junction proteins, p120-catenin (p120ctn) maintains cell-cell adhesion through its direct interaction with E-cadherin (E-cad). Mislocalization and/or loss of p120ctn have been reported in all lung cancer subtypes and are related to poor prognosis. Here, we showed that p120ctn modulates smoke-induced cell migration via the EGFR/Src-P pathway. Chemical blockade of EGFR/Src signaling inhibited smoke-induced activation of cofilin (an actin severing protein) and promoted cell migration in the presence of p120ctn but had little effect on blocking migration in the absence of p120ctn. These data suggested that smoke-induced cell migration was mediated via an EGFR/Src-dependent signaling pathway in cells that expressed p120ctn, but upon loss of p120ctn, migration continued to occur via an alternative, EGFR/Src-independent pathway. Thus, gradual loss of membrane p120ctn with lung cancer progression may contribute to reduced effectiveness of conventional chemotherapies, such as those directed against EGFR.