scholarly journals Different effects of dominant negative mutants of desmocollin and desmoglein on the cell-cell adhesion of keratinocytes

2000 ◽  
Vol 113 (10) ◽  
pp. 1803-1811
Author(s):  
Y. Hanakawa ◽  
M. Amagai ◽  
Y. Shirakata ◽  
K. Sayama ◽  
K. Hashimoto
Keyword(s):  
Cell Adhesion ◽  
Adherens Junctions ◽  
Dominant Negative ◽  
Cell Contact ◽  
Beta Catenin ◽  
Hacat Cells ◽  
Subsequent Formation ◽  
Contact Sites ◽  
E Cadherin ◽  
Cell Cell

Desmosomes contain two types of cadherin: desmocollin (Dsc) and desmoglein (Dsg). In this study, we examined the different roles that Dsc and Dsg play in the formation of desmosomes, by using dominant-negative mutants. We constructed recombinant adenoviruses (Ad) containing truncated mutants of E-cadherin, desmocollin 3a, and desmoglein 3 lacking a large part of their extracellular domains (EcaddeltaEC, Dsc3adeltaEC, Dsg3deltaEC), using the Cre-loxP Ad system to circumvent the problem of the toxicity of the mutants to virus-producing cells. When Dsc3adeltaEC Ad-infected HaCaT cells were cultured with high levels of calcium, E-cadherin and beta-catenin, which are marker molecules for the adherens junction, disappeared from the cell-cell contact sites, and cell-cell adhesion was disrupted. This also occurred in the cells infected with EcaddeltaEC Ad. With Dsg3deltaEC Ad infection, keratin insertion at the cell-cell contact sites was inhibited and desmoplakin, a marker of desmosomes, was stained in perinuclear dots while the adherens junctions remained intact. Dsc3adeltaEC Ad inhibited the induction of adherens junctions and the subsequent formation of desmosomes with the calcium shift, while Dsg3deltaEC Ad only inhibited the formation of desmosomes. To further determine whether Dsc3adeltaEC directly affected adherens junctions, mouse fibroblast L cells transfected with E-cadherin (LEC5) were infected with these mutant Ads. Both Dsc3adeltaEC and EcaddeltaEC inhibited the cell-cell adhesion of LEC5 cells, as determined by the cell aggregation assay, while Dsg3deltaEC did not. These results indicate that the dominant negative effects of Dsg3deltaEC were restricted to desmosomes, while those of Dsc3adeltaEC were observed in both desmosomes and adherens junctions. Furthermore, the cytoplasmic domain of Dsc3adeltaEC coprecipitated both plakoglobin and beta-catenin in HaCaT cells. In addition, beta-catenin was found to bind the endogenous Dsc in HaCaT cells. These findings lead us to speculate that Dsc interacts with components of the adherens junctions through beta-catenin, and plays a role in nucleating desmosomes after the adherens junctions have been established.

scholarly journals Role of Nectin in Formation of E-Cadherin–based Adherens Junctions in Keratinocytes: Analysis with the N-Cadherin Dominant Negative Mutant

2003 ◽  
Vol 14 (4) ◽  
pp. 1597-1609 ◽  
Author(s):  
Yoshinari Tanaka ◽  
Hiroyuki Nakanishi ◽  
Shigeki Kakunaga ◽  
Noriko Okabe ◽  
Tomomi Kawakatsu ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Adherens Junctions ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Adhesion Activity ◽  
Negative Mutant ◽  
E Cadherin ◽  
Cell Cell

E-Cadherin is a Ca2+-dependent cell-cell adhesion molecule at adherens junctions (AJs) of epithelial cells. A fragment of N-cadherin lacking its extracellular region serves as a dominant negative mutant (DN) and inhibits cell-cell adhesion activity of E-cadherin, but its mode of action remains to be elucidated. Nectin is a Ca2+-independent immunoglobulin-like cell-cell adhesion molecule at AJs and is associated with E-cadherin through their respective peripheral membrane proteins, afadin and catenins, which connect nectin and cadherin to the actin cytoskeleton, respectively. We showed here that overexpression of nectin capable of binding afadin, but not a mutant incapable of binding afadin, reduced the inhibitory effect of N-cadherin DN on the cell-cell adhesion activity of E-cadherin in keratinocytes. Overexpressed nectin recruited N-cadherin DN to the nectin-based cell-cell adhesion sites in an afadin-dependent manner. Moreover, overexpression of nectin enhanced the E-cadherin–based cell-cell adhesion activity. These results suggest that N-cadherin DN competitively inhibits the association of the endogenous nectin-afadin system with the endogenous E-cadherin-catenin system and thereby reduces the cell-cell adhesion activity of E-cadherin. Thus, nectin plays a role in the formation of E-cadherin–based AJs in keratinocytes.

scholarly journals Quantitative analysis of cadherin-catenin-actin reorganization during development of cell-cell adhesion.

1996 ◽  
Vol 135 (6) ◽  
pp. 1899-1911 ◽  
Author(s):  
C L Adams ◽  
W J Nelson ◽  
S J Smith
Keyword(s):  
Cell Adhesion ◽  
Time Lapse ◽  
Cell Contact ◽  
Beta Catenin ◽  
Actin Reorganization ◽  
Cell Contacts ◽  
Spatial Ordering ◽  
Triton X ◽  
E Cadherin ◽  
Cell Cell

Epithelial cell-cell adhesion requires interactions between opposing extracellular domains of E-cadherin, and among the cytoplasmic domain of E-cadherin, catenins, and actin cytoskeleton. Little is known about how the cadherin-catenin-actin complex is assembled upon cell-cell contact, or how these complexes initiate and strengthen adhesion. We have used time-lapse differential interference contrast (DIC) imaging to observe the development of cell-cell contacts, and quantitative retrospective immunocytochemistry to measure recruitment of proteins to those contacts. We show that E-cadherin, alpha-catenin, and beta-catenin, but not plakoglobin, coassemble into Triton X-100 insoluble (TX-insoluble) structures at cell-cell contacts with kinetics similar to those for strengthening of E-cadherin-mediated cell adhesion (Angres, B., A. Barth, and W.J. Nelson. 1996. J. Cell Biol. 134:549-557). TX-insoluble E-cadherin, alpha-catenin, and beta-catenin colocalize along cell-cell contacts in spatially discrete micro-domains which we designate "puncta," and the relative amounts of each protein in each punctum increase proportionally. As the length of the contact increases, the number of puncta increases proportionally along the contact and each punctum is associated with a bundle of actin filaments. These results indicate that localized clustering of E-cadherin/catenin complexes into puncta and their association with actin is involved in initiating cell contacts. Subsequently, the spatial ordering of additional puncta along the contact may be involved in zippering membranes together, resulting in rapid strengthening of adhesion.

scholarly journals Novel Kelch-like Protein, KLEIP, Is Involved in Actin Assembly at Cell-Cell Contact Sites of Madin-Darby Canine Kidney Cells

2004 ◽  
Vol 15 (3) ◽  
pp. 1172-1184 ◽  
Author(s):  
Takahiko Hara ◽  
Hiroshi Ishida ◽  
Razi Raziuddin ◽  
Stephan Dorkhom ◽  
Keiju Kamijo ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Cell Contact ◽  
Kidney Cells ◽  
Actin Assembly ◽  
Madin Darby Canine Kidney ◽  
Contact Sites ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
E Cadherin ◽  
Cell Cell

Dynamic rearrangements of cell-cell adhesion underlie a diverse range of physiological processes, but their precise molecular mechanisms are still obscure. Thus, identification of novel players that are involved in cell-cell adhesion would be important. We isolated a human kelch-related protein, Kelch-like ECT2 interacting protein (KLEIP), which contains the broad-complex, tramtrack, bric-a-brac (BTB)/poxvirus, zinc finger (POZ) motif and six-tandem kelch repeats. KLEIP interacted with F-actin and was concentrated at cell-cell contact sites of Madin-Darby canine kidney cells, where it colocalized with F-actin. Interestingly, this localization took place transiently during the induction of cell-cell contact and was not seen at mature junctions. KLEIP recruitment and actin assembly were induced around E-cadherin–coated beads placed on cell surfaces. The actin depolymerizing agent cytochalasin B inhibited this KLEIP recruitment around E-cadherin–coated beads. Moreover, constitutively active Rac1 enhanced the recruitment of KLEIP as well as F-actin to the adhesion sites. These observations strongly suggest that KLEIP is localized on actin filaments at the contact sites. We also found that N-terminal half of KLEIP, which lacks the actin-binding site and contains the sufficient sequence for the localization at the cell-cell contact sites, inhibited constitutively active Rac1-induced actin assembly at the contact sites. We propose that KLEIP is involved in Rac1-induced actin organization during cell-cell contact in Madin-Darby canine kidney cells.

scholarly journals Identification of a Drosophila homologue of alpha-catenin and its association with the armadillo protein.

1993 ◽  
Vol 121 (5) ◽  
pp. 1133-1140 ◽  
Author(s):  
H Oda ◽  
T Uemura ◽  
K Shiomi ◽  
A Nagafuchi ◽  
S Tsukita ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Cell Lines ◽  
Chromosome Band ◽  
Cell Contact ◽  
Beta Catenin ◽  
Contact Sites ◽  
Adhesion System ◽  
A Cell ◽  
Drosophila Embryos ◽  
Cell Cell

The cadherin cell adhesion system plays a central role in cell-cell adhesion in vertebrates, but its homologues are not identified in the invertebrate. alpha-Catenins are a group of proteins associated with cadherins, and this association is crucial for the cadherins' function. Here, we report the cloning of a Drosophila alpha-catenin gene by low stringent hybridization with a mouse alpha E-catenin probe. Isolated cDNAs encoded a 110-kD protein with 60% identity to mouse alpha E-catenin, and this protein was termed D alpha-catenin. The gene of this protein was located at the chromosome band 80B. Immunostaining analysis using a mAb to D alpha-catenin revealed that it was localized to cell-cell contact sites, expressed throughout development and present in a wide variety of tissues. When this protein was immunoprecipitated from detergent extracts of Drosophila embryos or cell lines, several proteins co-precipitated. These included the armadillo product which was known to be a Drosophila homologue of beta-catenin, another cadherin-associated protein in vertebrates, and a 150-kD glycoprotein. These results strongly suggest that Drosophila has a cell adhesion machinery homologous to the vertebrate cadherin-catenin system.

scholarly journals Changes in E-cadherin rigidity sensing regulate cell adhesion

2017 ◽  
Vol 114 (29) ◽  
pp. E5835-E5844 ◽  
Author(s):  
Caitlin Collins ◽  
Aleksandra K. Denisin ◽  
Beth L. Pruitt ◽  
W. James Nelson
Keyword(s):  
Cell Adhesion ◽  
Signaling Molecules ◽  
Membrane Dynamics ◽  
Cell Contact ◽  
Adhesion Assay ◽  
Cell Periphery ◽  
Actin Organization ◽  
Rigidity Sensing ◽  
E Cadherin ◽  
Cell Cell

Mechanical cues are sensed and transduced by cell adhesion complexes to regulate diverse cell behaviors. Extracellular matrix (ECM) rigidity sensing by integrin adhesions has been well studied, but rigidity sensing by cadherins during cell adhesion is largely unexplored. Using mechanically tunable polyacrylamide (PA) gels functionalized with the extracellular domain of E-cadherin (Ecad-Fc), we showed that E-cadherin–dependent epithelial cell adhesion was sensitive to changes in PA gel elastic modulus that produced striking differences in cell morphology, actin organization, and membrane dynamics. Traction force microscopy (TFM) revealed that cells produced the greatest tractions at the cell periphery, where distinct types of actin-based membrane protrusions formed. Cells responded to substrate rigidity by reorganizing the distribution and size of high-traction-stress regions at the cell periphery. Differences in adhesion and protrusion dynamics were mediated by balancing the activities of specific signaling molecules. Cell adhesion to a 30-kPa Ecad-Fc PA gel required Cdc42- and formin-dependent filopodia formation, whereas adhesion to a 60-kPa Ecad-Fc PA gel induced Arp2/3-dependent lamellipodial protrusions. A quantitative 3D cell–cell adhesion assay and live cell imaging of cell–cell contact formation revealed that inhibition of Cdc42, formin, and Arp2/3 activities blocked the initiation, but not the maintenance of established cell–cell adhesions. These results indicate that the same signaling molecules activated by E-cadherin rigidity sensing on PA gels contribute to actin organization and membrane dynamics during cell–cell adhesion. We hypothesize that a transition in the stiffness of E-cadherin homotypic interactions regulates actin and membrane dynamics during initial stages of cell–cell adhesion.

Expression of a dominant negative cadherin mutant inhibits proliferation and stimulates terminal differentiation of human epidermal keratinocytes

1996 ◽  
Vol 109 (13) ◽  
pp. 3013-3023 ◽  
Author(s):  
A.J. Zhu ◽  
F.M. Watt
Keyword(s):  
Cell Adhesion ◽  
Terminal Differentiation ◽  
Intercellular Adhesion ◽  
Dominant Negative ◽  
Epidermal Keratinocytes ◽  
Dominant Negative Mutant ◽  
Beta Catenin ◽  
Human Epidermal Keratinocytes ◽  
Negative Mutant ◽  
E Cadherin

Cell adhesion molecules are not only required for maintenance of tissue integrity, but also regulate many aspects of cell behaviour, including growth and differentiation. While the regulatory functions of integrin extracellular matrix receptors in keratinocytes are well established, such functions have not been investigated for the primary receptors that mediate keratinocyte intercellular adhesion, the cadherins. To examine cadherin function in normal human epidermal keratinocytes we used a retroviral vector to introduce a dominant negative E-cadherin mutant, consisting of the extracellular domain of H-2Kd and the transmembrane and cytoplasmic domains of E-cadherin. As a control a vector containing the same construct, but with the catenin binding site destroyed, was prepared. High levels of expression of the constructs were achieved; the dominant negative mutant, but not the control, formed complexes with alpha-, beta- and gamma-catenin. In cells expressing the dominant negative mutant there was a 5-fold decrease in the level of endogenous cadherins and a 3-fold increase in the level of beta-catenin. Cell-cell adhesion and stratification were inhibited by the dominant negative mutant and desmosome formation was reduced. Expression of the mutant resulted in reduced levels of the alpha 2 beta 1 and alpha 3 beta 1 integrins and increased cell motility, providing further evidence for cross-talk between cadherins and the beta 1 integrins. In view of the widely documented loss of E-cadherin in keratinocyte tumours it was surprising that the dominant negative mutant had an inhibitory effect on keratinocyte proliferation and stimulated terminal differentiation even under conditions in which intercellular adhesion was prevented. These results establish a role for cadherins in regulating keratinocyte growth and differentiation and raise interesting questions as to the relative importance of cell adhesion-dependent and -independent mechanisms.

E-cadherin mediated cell adhesion recruits SAP97 into the cortical cytoskeleton

1998 ◽  
Vol 111 (8) ◽  
pp. 1071-1080 ◽  
Author(s):  
S.M. Reuver ◽  
C.C. Garner
Keyword(s):  
Cell Adhesion ◽  
Model Systems ◽  
Molecular Organization ◽  
Cell Contact ◽  
Adhesion Sites ◽  
Associated Proteins ◽  
Adhesion Complex ◽  
Cortical Cytoskeleton ◽  
E Cadherin ◽  
Cell Cell

Members of the SAP family of synapse-associated proteins have recently emerged as central players in the molecular organization of synapses. In this study, we have examined the mechanism that localizes one member, SAP97, to sites of cell-cell contact. Utilizing epithelial CACO-2 cells and fibroblast L-cells as model systems, we demonstrate that SAP97 is associated with the submembranous cortical cytoskeleton at cell-cell adhesion sites. Furthermore, we show that its localization into this structure is triggered by E-cadherin. Although SAP97 can be found in an E-cadherin/catenin adhesion complex, this interaction seems to be mediated by the attachment of SAP97 to the cortical cytoskeleton. Our results are consistent with a model in which SAP97 is recruited to sites of cell-cell contact via an E-cadherin induced assembly of the cortical cytoskeleton.

Catenins in Xenopus embryogenesis and their relation to the cadherin-mediated cell-cell adhesion system

Development ◽  
1993 ◽  
Vol 118 (2) ◽  
pp. 629-640 ◽  
Author(s):  
S. Schneider ◽  
K. Herrenknecht ◽  
S. Butz ◽  
R. Kemler ◽  
P. Hausen
Keyword(s):  
Cell Adhesion ◽  
Plasma Membranes ◽  
Early Embryo ◽  
Xenopus Embryo ◽  
Free Form ◽  
Beta Catenin ◽  
Metabolic Labelling ◽  
Adhesion System ◽  
E Cadherin ◽  
Cell Cell

In the course of an analysis of cell-cell adhesion in the Xenopus embryo, antibodies directed against alpha- and beta-catenin were applied to investigate their relation to the cadherins occurring early in this system. The results demonstrate that alpha- and beta-catenin are provided maternally and increase in amount throughout embryogenesis. Immunoprecipitations indicate that both of the catenins are complexed to U-cadherin in the early phase of embryogenesis and to E-cadherin, when it appears during gastrulation. An excess of alpha-catenin occurs in free form in the early embryo, whereas all of the beta-catenin seems to be complexed to cadherin. Synthesis of the two components throughout early embryogenesis and their binding to newly synthesized cadherins were demonstrated by metabolic labelling. The spatial distribution of alpha-catenin was analysed by immunohistology. During cleavage alpha-catenin is deposited evenly along the plasma membranes within the embryo, while the cell peripheries at the surface of the embryo remain devoid of alpha-catenin. At later stages, the pattern of alpha-catenin distribution becomes more complex. Quantitative differences in the intensity of staining along the plasma membranes in the different regions of the embryo can be distinguished. Particularly the appearance of E-cadherin in the gastrula ectoderm is accompanied by conspicuous depositions of alpha-catenin along the respective plasma membranes in this layer. All cells in the later embryo, apart from the neural crest cells, carry alpha-catenin on their plasma membranes indicating the universal character of cadherin-mediated cell-cell adhesion in the Xenopus embryo.

scholarly journals Immediate and Delayed Effects of E-Cadherin Inhibition on Gene Regulation and Cell Motility in Human Epidermoid Carcinoma Cells

2005 ◽  
Vol 25 (20) ◽  
pp. 9138-9150 ◽  
Author(s):  
Henriette Andersen ◽  
Jakob Mejlvang ◽  
Shaukat Mahmood ◽  
Irina Gromova ◽  
Pavel Gromov ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Cell Motility ◽  
Family Members ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Short Term ◽  
Mesenchymal Transition ◽  
Negative Mutant ◽  
E Cadherin ◽  
Cell Cell

ABSTRACT The invasion suppressor protein, E-cadherin, plays a central role in epithelial cell-cell adhesion. Loss of E-cadherin expression or function in various tumors of epithelial origin is associated with a more invasive phenotype. In this study, by expressing a dominant-negative mutant of E-cadherin (Ec1WVM) in A431 cells, we demonstrated that specific inhibition of E-cadherin-dependent cell-cell adhesion led to the genetic reprogramming of tumor cells. In particular, prolonged inhibition of cell-cell adhesion activated expression of vimentin and repressed cytokeratins, suggesting that the effects of Ec1WVM can be classified as epithelial-mesenchymal transition. Both short-term and prolonged expression of Ec1WVM resulted in morphological transformation and increased cell migration though to different extents. Short-term expression of Ec1WVM up-regulated two AP-1 family members, c-jun and fra-1, but was insufficient to induce complete mesenchymal transition. AP-1 activity induced by the short-term expression of Ec1WVM was required for transcriptional up-regulation of AP-1 family members and down-regulation of two other Ec1WVM-responsive genes, S100A4 and igfbp-3. Using a dominant-negative mutant of c-Jun (TAM67) and RNA interference-mediated silencing of c-Jun and Fra-1, we demonstrated that AP-1 was required for cell motility stimulated by the expression of Ec1WVM. In contrast, Ec1WVM-mediated changes in cell morphology were AP-1-independent. Our data suggest that mesenchymal transition induced by prolonged functional inhibition of E-cadherin is a slow and gradual process. At the initial step of this process, Ec1WVM triggers a positive autoregulatory mechanism that increases AP-1 activity. Activated AP-1 in turn contributes to Ec1WVM-mediated effects on gene expression and tumor cell motility. These data provide novel insight into the tumor suppressor function of E-cadherin.

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