PLAKOGLOBIN, EPITHELIAL BEHAVIOR AND MESODERM INDUCTION

斑球蛋白、上皮行为和中胚层诱导

• 批准号: 2750079
• 负责人: MICHAEL William KLYMKOWSKY
• 金额: $ 20.16万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2750079
• 关键词: RNA; Xenopus; binding proteins; biological signal transduction; caldesmon; cell adhesion; cell adhesion molecules; cell differentiation; cytoskeleton; early embryonic stage; epithelium; fluorescence microscopy; gene duplication; gene expression; immunocytochemistry; immunoprecipitation; intercellular connection; mesoderm; microinjections; polymerase chain reaction; protein sequence; protein structure function; western blottings

Plakoglobin is a major structural component of cell adherence/cytoskeleton-attachment junctions. It also appears to play a key role in inductive signaling mediated by the Wnt family of proto-oncogenes and may interact with the APC tumor suppressor gene product. We propose to exploit the experimental accessibility of the Xenopus embryo to study further the role of plakoglobin in epithelial behavior and cellular differentiation. Expression of exogenous plakoglobin leads to anterior axis duplication in Xenopus. Using the injection of RNA, together with immunocytochemical methods, we will determine which regions of the embryo are most sensitive to the effects of exogenous plakoglobin and which regions of the plakoglobin molecule are required to produce axis duplication. The cytoplasmic tail of the desmosomal cadherin desmoglein-l (DsgTail) interacts with plakoglobin and suppresses its axis duplication effect. DsgTail also suppresses the effects of exogenous Xwnt8, presumably through its interaction with plakoglobin. Immunoprecipitation and RNA co- injection will be used to define the specificity of the DsgTail- plakoglobin interaction; DsgTail will also be used to study the role of endogenous plakoglobin in Wnt-mediate signaling. High levels of plakoglobin induce a gastrulation defect. This gastrulation defect is not suppressed, and may be enhanced, by the co-expression of DsgTail. Using whole-mount immunocytochemistry and in vitro studies, we will examine whether the over-expression of plakoglobin leads to changes in: i) cellular adhesion; ii) the number or distribution of adherence junctions; or iii) convergent extension, a major morphogenetic engine of vertebrate morphogenesis. At adhesion junctions or as a part of the Wnt signaling pathway, we assume that plakoglobin acts through its interactions with other proteins. To identify these proteins, we will use the yeast two- hybrid system. Co-immunoprecipitation will be used to identify plakoglobin-binding proteins (PBPs) that interact strongly with plakoglobin. Immunocytochemistry will be used to identify PBPs associated with adhesion junctions. Exogenous plakoglobin is localized to the nuclei of embryonic cells; putative PBPs that are also localized to the nucleus may be involved in the signaling functions of plakoglobin. The plakoglobin-binding region of a putative PBP may act as a dominant "interfering" mutation; we will examine the effects of expressing putative PBPs on cell adherence and axis formation. This analysis should identify PBPs involved in the structure and regulation of cellular adhesion junctions and in the processes of mesoderm induction.

白蛋白是细胞的主要结构成分 黏附/细胞骨架-附着连接。它似乎也扮演着一个关键角色 原癌基因Wnt家族在诱导信号传导中的作用 并可能与APC抑癌基因产物相互作用。我们建议 利用非洲爪哇胚胎的实验可及性进行研究 进一步探讨蛋白在上皮行为和细胞中的作用 差异化。外源性白蛋白的表达导致前部 非洲爪哇的轴复制。使用RNA注射，与 免疫细胞化学方法，我们将确定胚胎的哪些区域 对外源白蛋白的影响最为敏感 蛋白分子的某些区域是产生轴所必需的 复制。桥粒钙粘附素的胞质尾巴--桥粒粘附素-L (DsgTail)与白蛋白相互作用并抑制其轴复制 效果。DsgTail还抑制外源Xwnt8的影响，推测 通过它与蛋白的相互作用。免疫沉淀和RNA共沉淀法 注射将用于定义DsgTail的特异性- 白蛋白相互作用；DsgTail也将用于研究 内源性白蛋白在Wnt信号传导中的作用。高水平的 白蛋白导致原肠形成缺陷。这种原肠发育缺陷不是 DsgTail的共表达抑制并可能增强。vbl.使用 整装免疫细胞化学和体外研究，我们将检查 蛋白的过度表达是否会导致变化：i) 细胞黏附；ii)黏附连接的数量或分布； 或iii)收敛延伸，脊椎动物的主要形态发生引擎 形态发生。在黏附连接或作为Wnt信号的一部分 途径，我们假设白蛋白通过它与 其他蛋白质。为了鉴定这些蛋白质，我们将使用酵母两种- 混合系统。免疫共沉淀法将用于鉴定 与之强相互作用的白蛋白结合蛋白 白蛋白。免疫细胞化学将用于鉴定相关的PBPs 有粘连接头的。外源性白蛋白定位于细胞核 指胚胎细胞；推测也定位于细胞核的多氯联苯 可能参与蛋白的信号转导功能。这个 推测的PBP的白蛋白结合区可能起主导作用 “干扰”突变；我们将检查表达假定的影响 PBPS对细胞黏附和轴形成的影响。这项分析应该能确定 PBPs参与细胞黏附的结构和调节 在连接和中胚层诱导过程中。