MOLECULAR DYNAMICS IN RECEPTOR-MEDIATED PHAGOCYTOSIS

受体介导的吞噬作用的分子动力学

基本信息

批准号：
2444630
负责人：
NANCY L. THOMPSON
金额：
$ 20.05万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
1986
资助国家：
美国
起止时间：
1986-07-01 至 1998-09-27
项目状态：
已结题

项目摘要

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.

拟议研究的目的是在实验和理论上定义抗体的动作和组织在不存在的情况下，底物支持的平面模型膜使用基于动态激光的特定结合细胞的存在荧光显微镜。重点将放在理解受体介导的吞噬作用的发作，其中巨噬细胞结合，摄取并破坏致病生物。在初始阶段吞噬作用，抗体与靶细胞上的抗原位点结合巨噬细胞表面上的表面和FC受体。在一组测量中，致病生物的表面将是由底物支撑的平面膜建模，含有触觉共轭磷脂，以及特异性冠状动脉的特征膜上的抗体（表面浓度，平衡结合曲线，表面关联和解离动力学，翻译和旋转迁移率和低聚状态）将被表征用于不同的抗体，膜和溶液特性。研究将是对与膜不可逆结合的抗体进行了以及在溶液和溶液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，抗体可单元与膜和抗体结合偶然地束缚到膜将特别感兴趣。这要求（例如，抗体密度，迁移率和低聚）的要求哪些与巨噬细胞相关的细胞结合并被代谢激活通过，将表征平面膜。巨噬细胞的影响对组织的结合和激活和抗体动力学将也可以检查。在一套互补的测量集中，巨噬细胞的表面将由含有纯化和重构的小鼠FC-GAMMA-RII。这些研究将需要开发与重构的形成平面膜的方法适当定向并在生理上进行的MOFC-GAMMA-RII 相关的Translationa1迁移率。为此，截断了由跨膜区域组成的MOFC-GAMMA-RII版本共轭到细胞外区域，以beta1形式细胞内区域和细胞内区域的β2形式将生成。重构受体的动力和平面膜上的抗Hapten抗体将被描述为膜和溶液特性的功能。对和在存在抗抗的情况下，结合，触发脂质体或细胞的作用触觉抗体也将被表征。拟议的研究将涉及持续发展新的动态荧光显微镜的技术，包括总版本内部反射荧光显微镜和荧光相关性光谱法。此外，开发一种新方法来观察高度荧光的单分子或颗粒结合，并且提出了从表面分离的。