EXTRACELLULAR MATRIX AND EPITHELIAL LUMEN MORPHOGENESIS

细胞外基质和上皮管腔形态发生

• 批准号: 3447953
• 负责人: HARLAN G HALL
• 金额: $ 5.2万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-09-30 至 1986-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3447953
• 关键词: antibody formation; apical membrane; biomaterials; cell membrane; chemotaxis; chromatography; colchicine; connective tissue development; cytochalasins; cytoskeleton; electron microscopy; extracellular matrix; gel electrophoresis; gel filtration chromatography; histogenesis; immunoprecipitation; microscopy; monoclonal antibody; mucopolysaccharides; radiotracer

Two epithelial cell lines, MDCK and NMuMG, growing as monolayers on type I collagen gels, respond to an overlay of additional collagen by altering their polarity and reorganizing to form lumina. This provides a model system for studying the mechanisms of a morphogenetic event, specifically the role of extracellular matrix (ECM) material in the determination of cellular polarity and organization of epithelia. Lumen formation is a biological assay which may be correlated with detectable biochemical and cytological activities. The inducibility, availability, accessibility and relative simplicity gives this model many advantages over other epithelial morphogenetic systems. This proposal questions 1) the specificity of the ECM material that induces cell polarity and lumen formation, that is, whether the effect is specific to collagen, 2) what macromolecular synthesis, particularly that of ECM material, accompanies and is required for lumen formation, and 3) if the mechanism of polarity determination and lumen formation entails transmembrane associations of the cytoskeleton with the ECM and restructuring of the cytoskeleton. ECM components such as glycosaminoglycans (GAGs), fibronectin, laminin as well as other compounds will be added either in solution, coupled to dextrans, or incorporated into gels of hydroxyethylmethylmethacrylate and tested for their ability to induce lumen formation. Synthesis of ECM components will be followed by metabolic incorporation of radioisotopes. GAGs will be characterized by two dimensional cellulose acetate electrophoresis and column chromatography and proteins by two dimensional gel electrophoresis. Immunological techniques will be utilized for the identification of specific ECM and cytoskeletal proteins. Differential solubilization will be used to fractionate the cells to explore possible polarized transmembrane associations of the cytoskeleton with the ECM. The upper and lower collagen gels will be analyzed separately to follow changes in the associations that may occur during the course of lumen formation. Light and electron microscopic immunolocalization methods will be used to visually follow the polarization of the ECM and cytoskeletal proteins and their possible redistribution during lumen formation. To determine if there is a functional role for the cytoskeleton in lumen formation, the effects of colchicine, taxol and cytochalasin B will be tested.

两种上皮细胞系，MDCK和NMuMG，在I型细胞上生长为单层 胶原蛋白凝胶，通过改变胶原蛋白的结构， 它们的极性和重组形成光腔。 这提供了一个模型 研究形态发生事件机制的系统，特别是 细胞外基质（ECM）材料在确定 细胞极性和上皮组织。 管腔形成是一种 生物测定，其可能与可检测的生物化学和 细胞学活动。 的诱导，可用性，可访问性和 相对简单使得该模型比其他上皮细胞模型具有许多优点。 形态发生系统 这一建议质疑1）的具体性， 诱导细胞极性和管腔形成的ECM材料，即， 该效果是否是特定于胶原蛋白，2）什么大分子 合成，特别是ECM材料的合成，伴随着并且是必需的 对于管腔形成，以及3）如果极性确定的机制和 管腔的形成需要细胞骨架的跨膜结合， ECM和细胞骨架的重组。 ECM组件，例如 糖胺聚糖（GAG）、纤连蛋白、层粘连蛋白以及其它化合物 将在溶液中加入，与葡聚糖偶联，或掺入 甲基丙烯酸羟乙基甲酯的凝胶，并测试其 诱导管腔形成。 ECM组分的合成之后， 放射性同位素的代谢结合。 GAG的特征在于： 二维醋酸纤维素电泳和柱层析 和蛋白质。 免疫 技术将用于识别特定的ECM， 细胞骨架蛋白 将使用差异增溶， 破碎细胞以探索可能的极化跨膜 细胞骨架与ECM的关联。 上部和下部 将分别分析胶原凝胶，以跟踪 在管腔形成过程中可能发生的关联。 光 和电子显微镜免疫定位方法将用于 视觉上跟随ECM和细胞骨架蛋白的极化， 它们在管腔形成期间可能的重新分布。 以确定是否 细胞骨架在管腔形成中具有功能性作用， 将测试秋水仙碱、紫杉醇和细胞松弛素B的作用。

项目成果

Characterization of the intermediate filament proteins of murine mammary gland epithelial cells. Response to collagen substratum.

小鼠乳腺上皮细胞中间丝蛋白的表征。

• DOI: 10.1016/0014-4827(86)90343-5
• 发表时间: 1986
• 期刊: Experimental cell research
• 影响因子: 3.7
• 作者: Hall,HG;Bissell,MJ
• 通讯作者: Bissell,MJ