CELL-MATRIX INTERACTIONS DURING MORPHOGENESIS

形态发生过程中的细胞-基质相互作用

• 批准号: 2198581
• 负责人: Stuart A. Newman
• 金额: $ 20.58万
• 依托单位: NEW YORK MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-04-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2198581
• 关键词: cartilage development; cell adhesion; cell differentiation; chick embryo; circular dichroism; embryo /fetus tissue /cell culture; embryogenesis; extracellular matrix; fibronectins; fluorescence polarization; gene expression; heparan sulfate; heparin; histogenesis; limb bone; mesenchyme; protein engineering; proteoglycan; site directed mutagenesis; spectrometry; vertebrate embryology

The molecular mechanisms of mesenchymal morphogenesis in the embryonic vertebrate limb will be analyzed using a multi-leveled approach. Interaction between cell surface heparan sulfate and the amino-terminal domain (NTD) of extracellular fibronectin has been shown previously to play an essential role in promoting precartilage cell condensation and subsequent cartilage differentiation. Therefore we will design site-specific reagents that interfere with this interaction in living cell cultures in order to determine the precise residues on fibronectin required for skeletal morphogenesis. The strategy consists of using protein engineering to produce mutant forms of the fibronectin-NTD. Sites chosen for mutagenesis will include those predicted to interact with heparin/heparan sulfate. The mutated proteins will be tested in several positive assays for changed ability to interact with heparin: (i) matrix-driven translocation, an in vitro assay for fibronectin-dependent morphogenesis in model extracellular matrices. Domains of fibronectin required for activity in this assay have previously correlated well with domains required for mesenchymal morphogenesis; (ii) circular dichroism, and (iii) polarization of fluorescence, spectroscopic techniques that can be used to probe fibronectin-NTD conformation in the presence and absence of heparin. Sites in the fibronectin-NTD the mutagenesis of which alters interactions of the protein with heparin in these assays will be the basis for designing oligopeptide and antibody reagents for use in tissue cultures of developing limb mesenchyme. Inhibition of mesenchymal condensation and cartilage-specific gene expression by these reagents will identify sites in fibronectin required for normal development, and help define the molecular mechanism of tissue morphogenesis in the embryonic limb.

胚胎中间充质形态发生的分子机制 将使用多级方法分析脊椎动物的肢体。 细胞表面硫酸盐与氨基末端之间的相互作用 细胞外纤连蛋白的结构域（NTD）先前已显示为 在促进前细胞凝结中起着至关重要的作用 随后的软骨分化。 因此我们将设计 干扰这种相互作用的现场特定试剂 细胞培养物以确定纤连蛋白的精确残基 骨骼形态发生所需。 该策略包括使用 蛋白质工程产生纤连蛋白NTD突变形式。 选择诱变的站点将包括被预测相互作用的站点 与肝素/肝硫酸盐。 突变的蛋白质将在 与肝素互动的能力变化的几种积极测定： （i）基质驱动的易位，一种体外测定 模型细胞外基质中的纤连蛋白依赖性形态发生。 该测定中需要的纤连蛋白域 以前与间充质所需的域相关 形态发生； （ii）圆二色性和（iii）极化 荧光，光谱技术可用于探测 在存在和不存在肝素的情况下，纤连蛋白-NTD构象。 纤连蛋白-NTD中的位点，其变化的诱变 在这些测定中，蛋白质与肝素的相互作用将是 设计用于组织的寡肽和抗体试剂的基础 发展肢体间充质的培养物。 抑制间充质 这些试剂的凝结和软骨特异性基因表达 将确定正常发育所需的纤连蛋白的位点，以及 有助于定义组织形态发生的分子机制 胚胎肢体。