CELL SURFACE DYNAMICS OF IGG FC RECEPTOR INTERACTIONS

IGG FC 受体相互作用的细胞表面动力学

• 批准号: 2877667
• 负责人: NANCY L. THOMPSON
• 金额: $ 10万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-28 至 2000-09-27
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2877667
• 关键词: antibody receptor; antigen antibody reaction; fluorescence microscopy; fluorescence spectrometry; immunoglobulin G; lipid bilayer membrane; macrophage; membrane activity; membrane lipids; molecular dynamics; phagocytosis; receptor binding; tissue /cell culture

The objective of the proposed research is to experimentally and theoretically define the motions and organizations of antibodies at substrate-supported planar model membranes, both in the absence and presence of specifically bound cells, using dynamic, laser-based fluorescence microscopy. Emphasis will be placed on understanding the onset of receptor-mediated phagocytosis, in which macrophages bind, ingest and destroy pathogenic organisms. During the initial phase of phagocytosis, antibodies bind both to antigenic sites on the target cell surface and to Fc receptors on the macrophage cell surface. In one set of measurements, the surfaces of pathogenic organisms will be modeled by substrate-supported planar membranes containing hapten- conjugated phospholipids, and the characteristics of hapten-specific antibodies on the membranes (surface concentrations, equilibrium binding curves, surface association and dissociation kinetics, translational and rotational mobilities, and oligomerization states) will be characterized for different antibody, membrane and solution properties. Studies will be conducted both for antibodies that are irreversibly bound to the membranes and for antibodies that are in equilibrium between solution and the membranes. In the latter case, the dynamic conversion between antibodies in solution, antibodies bound monovalently to membranes, and antibodies bound bivalently to membranes will be of particular interest. The requirements (e.g., antibody density, mobility, and oligomerization) for which macrophage-related cells bind to, and are metabolically activated by, planar membranes will be characterized. The effects of macrophage binding and activation on the organization and dynamics of antibodies will also be examined. In a complementary set of measurements, the surfaces of macrophages will be modeled by substrate-supported planar membranes containing purified and reconstituted mouse Fc-gamma-RII. These studies will require the development of methods for forming planar membranes with reconstituted moFc-gamma-RII that is properly oriented and undergoes physiologically relevant degrees of translationa1 mobility. To do this, truncated versions of moFc-gamma-RII that consist of the transmembrane region conjugated to the extracellular region, to the beta1 form of the intracellular region, and to the beta2 form of the intracellular region will be generated. The dynamics of the reconstituted receptors and of anti-hapten antibodies at the planar membranes will be characterized as a function of membrane and solution properties. The requirements for and effects of bound, haptenated liposomes or cells, in the presence of anti- hapten antibodies, will also be characterized. The proposed research will involve the continued development of new techniques in dynamic fluorescence microscopy, including versions of total internal reflection fluorescence microscopy and fluorescence correlation spectroscopy. In addition, the development of a new method for observing highly fluorescent single molecules or particles as they bind and dissociate from surfaces is proposed.

拟议研究的目的是通过实验和 从理论上定义抗体的运动和组织， 基底支撑的平面模型膜，在不存在和 存在特异性结合的细胞，使用动态的，基于激光的 荧光显微镜重点将放在理解 受体介导的吞噬作用的开始，其中巨噬细胞结合，摄取 消灭病原体初始阶段 吞噬作用，抗体结合到靶细胞上的抗原位点 表面和巨噬细胞表面上的Fc受体。 在一组测量中，病原生物体的表面将被 由含有半抗原的基底支撑的平面膜模拟， 结合磷脂，和半抗原特异性的特性， 膜上的抗体（表面浓度、平衡结合 曲线，表面缔合和解离动力学，平移和 旋转迁移率和低聚状态）将被表征 用于不同的抗体、膜和溶液性质。研究将 对于不可逆地结合到膜上的抗体， 而对于在溶液和抗体之间处于平衡的抗体， 膜。在后一种情况下，抗体之间的动态转换 在溶液中，抗体将单链抗体结合到膜上， 结合到膜上的生物素将是特别感兴趣的。的 要求（例如，抗体密度、迁移率和寡聚化）， 与巨噬细胞相关的细胞结合并被代谢激活 将表征平面膜。 巨噬细胞的作用 结合和激活抗体的组织和动力学将 也要检查。 在一组互补的测量中，巨噬细胞的表面将 通过含有纯化的和 重组小鼠Fc-γ-RII。这些研究将需要 形成具有重构膜的平面膜的方法的开发 moFc-γ-RII，其被适当定向并在生理上经历 相关的预防性流动程度。 为此， 由跨膜区组成的moFc-γ-RII版本 与细胞外区域缀合，与细胞外区域的β 1形式缀合， 胞内区域，以及胞内区域的β 2形式 将被生成。重组受体的动力学和 平面膜上的抗半抗原抗体将被表征为 膜的功能和溶液性质。的要求和 结合的、半抗原化的脂质体或细胞的作用， 半抗原抗体也将被表征。 拟议的研究将涉及新的持续发展， 技术在动态荧光显微镜，包括版本的总 内反射荧光显微术和荧光关联 谱此外，开发一种新的观察方法， 高荧光单分子或颗粒， 提出了从表面分离。