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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.

拟议研究的目标是通过实验和从理论上定义抗体的运动和组织衬底支撑的平面模型膜，在没有和存在特定结合的细胞，使用基于激光的动态荧光显微镜。重点将放在了解受体介导的吞噬作用的开始，巨噬细胞在其中结合，吞噬并摧毁病原体。在…的初始阶段吞噬作用，抗体结合到靶细胞上的抗原部位。与巨噬细胞表面的Fc受体结合。在一组测量中，病原体的表面将是由含有半抗原的基质支撑的平面膜模拟- 共轭磷脂及其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齐聚状态)将被表征适用于不同的抗体、膜和溶液性质。研究将会是两者都是针对不可逆转地结合到膜上的抗体进行的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，抗体以单价结合在膜上，抗体将二价结合到膜上将是特别有意义的。这个要求(例如，抗体密度、流动性和寡聚化) 巨噬细胞相关细胞与哪些细胞结合，并被代谢激活由此，我们将对平面膜进行表征。巨噬细胞的作用结合和激活对抗体的组织和动态将也要接受检查。在一组互补的测量中，巨噬细胞的表面将由衬底支撑的平面膜模拟，该平面膜包含纯化的和重组小鼠Fc-γ-RII。这些研究将需要重组平面膜制备方法的研究进展 MOFC-伽马-RII，定向正确，生理上经历翻译活动的相关程度1。要做到这一点，截断由跨膜区组成的mOFC-γ-RII的版本结合到胞外区，结合到β1形式的胞内区，以及胞内区的Beta2形式将会被生成。重组受体的动力学和平面膜上的抗半抗原抗体的特征是膜的功能和溶液的性质。和的要求结合，半抗原化脂质体或细胞，在存在抗- 半抗原抗体，也将被表征。拟议的研究将涉及继续开发新的动态荧光显微镜技术，包括TOTAL版本内反射荧光显微镜与荧光关联光谱学。此外，一种新的观察方法的发展高荧光的单分子或粒子，因为它们结合和提出了从曲面解离的方法。