MECHANISMS OF EPITHELIUM FORMATION AND STABILIZATION

上皮形成和稳定的机制

• 批准号: 3198724
• 负责人: EILEEN D ADAMSON
• 金额: $ 23.71万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-03-11 至 1995-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3198724
• 关键词: affinity chromatography; cell adhesion; cell adhesion molecules; cell cell interaction; clone cells; cytoskeletal proteins; cytoskeleton; embryo /fetus cell /tissue; epithelium; gene expression; genetic manipulation; genetic regulatory element; immunoprecipitation; integrins; molecular cloning; northern blottings; phenotype; phosphorylation; posttranscriptional RNA processing; protein structure function; southern blotting; transfection; vinculin; western blottings

Close interaction between cells (compaction) is needed for the formation of the junctions that bind cells together to form an epithelium. The process of epithelium formation can be studied in a model system using F9 embryonal carcinoma (EC) cells. In aggregate cultures of the undifferentiated stem cells, the cells compact tightly, and after four days of induction by retinoic acid in the medium, an outer epithelial layer is observed. A mutant F9 cell line (F9att-5.51), does not compact in suspension cultures and will not differentiate into epithelial structures. The mutant line will not grow as a nomolayer because it does not attch to substrates. Although the membrane glycoprotein uvomorulin (UM, E-cadherin) is reduced in the mutant, the introduction of excess UM into the mutant cells did not rescue the mutant behavior and therefore cannot account for the defects in this line. We have now identified a component in the compaction mechanism that is likely to be the primary cause of all the adhesive defects in the mutant cell line. The cytoskeletal protein vinculin is completely absent from mutant cells. Vinculin is known to be involved both in cell-cell interactions and specifically in zonula adherens junctions in epithelial cells as well as in cell-substrate adhesion plaques. The absence of this protein could therefore account for the adhesive defects in 5.51 cells. This will be tested by the transfection of an expression vector for vinculin and for deleted versions that can be used to determine the roles of the domains of vinculin in adhesion. We will identify and characterize the 5' regulatory sequences of the gene. The second major aim is to study the other components in multistep adhesion processes by cell fractionation and affinity chromatography using the contrasting F9 cell lines that already exist and those that will be constructed during the research perioc. This proposal exploits the exostence of the mutant cell line to explore the structure-function domains of vinculin and the interactions between adhesive components in the processes that ensure epithelial function and stability. Knowledge of adhesive components is important in addressing problems of metastases in carcinoma as well as understanding the regulation of the cytoskeleton and how it relates to cell behavior.

形成需要细胞之间的紧密相互作用（压实） 将细胞结合在一起形成上皮的连接。 这 上皮形成的过程可以在模型系统中研究 F9 胚胎癌 (EC) 细胞。 在总体文化中 未分化的干细胞，细胞紧密紧密，四次后 培养基中的视黄酸诱导天数，外上皮 观察到层。 突变型 F9 细胞系 (F9att-5.51)，不致密 悬浮培养，不会分化为上皮细胞 结构。 突变株系不会生长为单分子层，因为它确实 不附着在基材上。 虽然膜糖蛋白 uvomorulin （UM，E-钙粘蛋白）在突变体中减少，引入过量的 UM 进入突变细胞并不能挽救突变行为，因此 无法解释该行的缺陷。 我们现在已经确定了一个 压实机制中的组件可能是主要的 突变细胞系中所有粘附缺陷的原因。 这 突变细胞中完全不存在细胞骨架蛋白纽蛋白。 已知纽蛋白参与细胞间相互作用和 特别是在上皮细胞的小带粘附连接以及 在细胞-基质粘附斑块中。缺乏这种蛋白质可能会 因此解释了 5.51 细胞中的粘附缺陷。 这将是 通过转染纽蛋白表达载体和 已删除的版本可用于确定域的角色 粘连蛋白的粘附。 我们将识别并表征 5' 基因的调控序列。 第二个主要目标是研究 细胞分级分离多步粘附过程中的其他成分 和使用对比 F9 细胞系的亲和层析 已经存在的以及将在研究期间建造的 期间。 该提案利用突变细胞系的存在来 探索纽蛋白的结构-功能域及其相互作用 确保上皮过程中的粘合成分之间 功能和稳定性。 了解粘合剂成分很重要 解决癌症转移问题以及 了解细胞骨架的调节及其与 细胞行为。