Assuntos
Controle de Doenças Transmissíveis , Centros Comunitários de Saúde , Serviços de Saúde Comunitária , Planejamento em Desastres , Vítimas de Desastres/psicologia , Vítimas de Desastres/reabilitação , Terremotos , Doenças Transmitidas por Alimentos/prevenção & controle , Política Nutricional , Apoio Social , Feminino , Humanos , Japão , MasculinoRESUMO
To define the roles of α-catenin in cell-cell adhesion, the E-cadherin, α-catenin, ß-catenin, and/or plakoglobin genes were inactivated in F9 teratocarcinoma cells. An E-cadherin-α-catenin fusion protein (Eα) restored full cell-adhesion function and organized the actin-based cytoskeleton and ZO-1, an actin filament binding protein, in F9 cells lacking all endogenous cadherin-catenin complex components. There were two types of cadherin-based cell-adhesion junctions in parental F9 cells, those with ZO-1 and those without ZO-1, and only junctions with ZO-1 were associated with thick actin bundles. Additionally, ZO-1 localized to most Eα-based cell-adhesion junctions. These data demonstrated that Eα supported cadherin-based cell adhesion and recruited actin bundles and ZO-1 to cell-cell contact sites in the absence of cytoplasmic α-catenin. Moreover, the C-terminal half of α-catenin was involved in the formation of cell-adhesion junctions with ZO-1.
Assuntos
Caderinas/metabolismo , Citoesqueleto/ultraestrutura , alfa Catenina/metabolismo , Actinas/ultraestrutura , Animais , Caderinas/genética , Adesão Celular , Linhagem Celular Tumoral , Deleção de Genes , Proteínas de Membrana/análise , Camundongos , Fosfoproteínas/análise , Proteína da Zônula de Oclusão-1 , alfa Catenina/genéticaRESUMO
P120 plays an essential role in cadherin turnover. The molecular mechanism involved, however, remains only partially understood. Here, using a gene trap targeting technique, we replaced the genomic sequence of p120 with HA-tagged p120 cDNA in mouse teratocarcinoma F9 cells. In the p120 knock-in (p120KI) cells, we found that the expression level of p120 was severely reduced and that the expression level of other components of the cadherin-catenin complex was also reduced. The stable expression of various p120 mutants in p120KI cells revealed that the armadillo repeat domain of p120 is sufficient to restore the expression level of E-cadherin. In p120KI cells, internalized E-cadherin was frequently detected as large aggregates. Transient expression of wild-type p120 and mutant p120 lacking the N-terminal region induced both relocalization of E-cadherin at the cell-cell boundaries and the disappearance of cytoplasmic E-cadherin aggregates. Transient expression of mutant p120 lacking the C-terminal region, however, only induced a small increase in E-cadherin signals at the cell-cell boundary. In these cells, the cytoplasmic E-cadherin signals became brighter and the expressed mutant p120 was incorporated in the E-cadherin aggregates. These results suggested the novel function of the p120 C-terminal region in regulating the trafficking of cytoplasmic E-cadherin.
Assuntos
Caderinas/metabolismo , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Animais , Cateninas , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Citoplasma/metabolismo , Imunofluorescência , Lisossomos/metabolismo , Camundongos , Camundongos Knockout , Fosfoproteínas/genética , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , gama Catenina/metabolismo , delta CateninaRESUMO
F9 teratocarcinoma cells in which beta-catenin and/or plakoglobin genes are knocked-out were generated and investigated in an effort to define the role of beta-catenin and plakoglobin in cell adhesion. Loss of beta-catenin expression only did not affect cadherin-mediated cell adhesion activity. Loss of both beta-catenin and plakoglobin expression, however, severely affected the strong cell adhesion activity of cadherin. In beta-catenin-deficient cells, the amount of plakoglobin associated with E-cadherin dramatically increased. In beta-catenin/plakoglobin-deficient cells, the level of E-cadherin and alpha-catenin markedly decreased. In these cells, E-cadherin formed large aggregates in cytoplasm and membrane localization of alpha-catenin was barely detected. These data confirmed that beta-catenin or plakoglobin is required for alpha-catenin to form complex with E-cadherin. It was also demonstrated that plakoglobin can compensate for the absence of beta-catenin. Moreover it was suggested that beta-catenin or plakoglobin is required not only for the cell adhesion activity but also for the stable expression and cell surface localization of E-cadherin.
Assuntos
Adesão Celular/efeitos dos fármacos , beta Catenina/metabolismo , gama Catenina/metabolismo , Animais , Caderinas/metabolismo , Adesão Celular/fisiologia , Imuno-Histoquímica , Camundongos , Modelos Genéticos , Transdução de Sinais , Transfecção , Células Tumorais Cultivadas , alfa Catenina/metabolismo , beta Catenina/farmacologia , gama Catenina/farmacologiaRESUMO
Epithelium formation is a common event in animal morphogenesis. It has been reported that F9 cells differentiate into visceral endoderm-like epithelial cells when cell aggregates are cultured in the presence of retinoic acid. The present investigation set out to determine whether this in vitro model could be used under monolayer culture conditions, which is suitable for a detailed analysis of epithelial differentiation. We performed comparative gene expression analyses of F9 cells grown under aggregate and monolayer culture conditions prior to and following treatment with retinoic acid. Under these conditions, induction in the expression of differentiation marker genes was confirmed, even in monolayer cultures. Junctional complex and apical membrane formation, both of which are characteristic of epithelial cells, were also observed under monolayer culture conditions. Because of the merit of monolayer culture condition, we found that apical membrane and junctional complex formation are strictly regulated during epithelial differentiation. It was also revealed that F9 cells differentiated into epithelial cells predominantly on the fourth and fifth day following retinoic acid induction. These results showed that a monolayer culture of F9 cells represents a viable in vitro model that can be employed to elucidate mechanisms pertaining to epithelium formation.