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.

形成需要紧密相互作用（压实）（压实） 将细胞结合在一起形成上皮的连接。 这 可以在模型系统中研究上皮形成过程 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，四个 视黄酸在培养基中诱导的天数，外皮 观察到层。 突变的F9细胞系（F9ATT-5.51），不紧凑 在悬浮文化中，不会区分上皮 结构。 突变系列不会作为Nomolayer增长，因为它确实会 不对底物进行。 虽然膜糖蛋白紫vomorin （UM，E-钙粘蛋白）在突变体中减少，引入过量的UM 进入突变细胞没有拯救突变体行为，因此 无法说明这一行中的缺陷。 我们现在已经确定了 压实机制中的组成部分可能是主要的 突变细胞系中所有粘合缺陷的原因。 这 突变细胞完全不存在细胞骨架蛋白质蛋白。 已知Vinculin既参与细胞 - 细胞相互作用，又参与 特别是在上皮细胞中的Zonula粘附连接处 在细胞覆盖斑块中。这种蛋白质的不存在 因此，说明了5.51个细胞中的粘附缺陷。 这将是 通过转染的表达载体的Vinculin和 删除的版本可用于确定域的作用 粘附中的Vinculin。 我们将识别和表征5' 基因的调节序列。 第二个主要目的是研究 通过细胞分馏中多步粘附过程中的其他组件 和亲和力色谱法使用对比的F9细胞系 已经存在，以及将在研究期间构建的 周期 该建议利用了突变细胞系的扩展为 探索Vinculin的结构功能域和相互作用 在过程中的粘合成分之间确保上皮 功能和稳定性。 对粘合剂成分的了解很重要 在解决癌中转移的问题以及 了解细胞骨架的调节及其与 细胞行为。