Structure-Function Relationships of Immunoreceptors

描述(申请人提供)：这项研究的目的是通过多链抗原受体对免疫细胞激活的关键过程有一个基本的了解，它的重点是肥大细胞上的IgE受体FceRI，它是一个特征良好的原型，在过敏免疫反应中发挥核心作用。拟议的研究将调查FceRI信号中质膜的异质性，以及质膜的液体有序区域的参与，通常称为“脂筏”。具体目的1是研究FceRI与酪氨酸激酶Lyn和Syk之间的动态相互作用，以及在IgE-FceRI被多价配体交联以激活细胞后发生的其他信号蛋白。用双色荧光相关光谱观察了分子间的相互作用，并对其结构基础进行了探讨。具体目标2是确定细胞靶标如何在空间关系上对聚集的FceRI做出反应，并破译明显的膜异质性的生物物理和膜运输基础。最近发展起来的配基微图案化方法揭示了质膜内小叶和外小叶成分重组的显著差异。在更精细的尺度上进行图案化应该能够以100纳米的分辨率成像膜中空间靶向的分子再分布。具体目的3是研究质膜相关细胞骨架在调节液体有序/液体无序脂质相分离的大小和动力学中的作用，这些分离被假设为RAFT的功能后果的基础。荧光共振能量转移、电子自旋共振和质谱学的互补方法将用于评估扰乱质膜动态平衡和功能的药物的效果。这些研究结合了物理、化学和生物学的方法，为质膜结构和动力学及其在免疫细胞信号转导中的作用提供了新的见解。

DESCRIPTION (provided by applicant): This research is aimed at a fundamental understanding of key processes in immune cell activation via multichain antigen receptors, and it is focused on the receptor for IgE on mast cells, FceRI, as a well characterized prototype that plays a central role in the allergic immune response. Proposed studies will investigate plasma membrane heterogeneity in FceRI signaling, and the participation of liquid ordered regions of the plasma membrane commonly called "lipid rafts." Specific Aim 1 is to characterize dynamic interactions between FceRI and tyrosine kinases Lyn and Syk as well as other signaling proteins that occur after IgE-FceRI are crosslinked by multivalent ligands to activate the cells. Molecular interactions are observed with dual color fluorescence correlation spectroscopy, and the structural bases for these will be probed. Specific Aim 2 is to determine how the cell targets responses in spatial relationship to clustered FceRI and to decipher the biophysical and membrane trafficking bases for pronounced membrane heterogeneity. Recently developed methods for micropatterning of ligands reveal striking differences in the reorganization of inner and outer leaflet components of the plasma membrane. Patterning on finer scales should enable imaging at hundred-nanometer resolution the molecular redistributions that are spatially targeted in the membrane. Specific Aim 3 is to investigate the role of the plasma membrane-associated cytoskeleton in regulating the size and dynamics of liquid ordered/liquid disordered lipid phase separations that are hypothesized to underlie the functional consequences of rafts. Complementary methods of fluorescence resonance energy transfer, electron spin resonance, and mass spectrometry will be used to evaluate effects of agents that perturb plasma membrane homeostasis and function. These investigations integrate physical, chemical and biological approaches to provide new insights into plasma membrane structure and dynamics and their roles in immune cell signaling.