Structure-Function Relationships of Immunoreceptors

描述（由申请人提供）：本研究旨在从根本上了解通过多链抗原受体激活免疫细胞的关键过程，重点关注肥大细胞上的IgE受体FceRI，作为一种在过敏性免疫应答中发挥核心作用的充分表征的原型。拟议的研究将调查FceRI信号传导中的质膜异质性，以及通常称为“脂筏”的质膜液体有序区域的参与。“具体目标1是表征FceRI与酪氨酸激酶林恩和Syk以及在IgE-FceRI通过多价配体交联以激活细胞后发生的其它信号传导蛋白之间的动态相互作用。分子间的相互作用，观察双色荧光相关光谱，这些结构基础将被探测。具体目标2是确定细胞如何在空间关系中靶向对聚集的FceRI的反应，并破译明显膜异质性的生物物理和膜运输基础。最近开发的配体的微图案化的方法揭示了质膜的内部和外部小叶组件的重组的显着差异。在更精细的尺度上形成图案应该能够以100纳米的分辨率对膜中空间靶向的分子重新分布进行成像。具体目标3是研究质膜相关的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，这些脂质相分离被假设为筏的功能后果的基础。将使用荧光共振能量转移、电子自旋共振和质谱的补充方法来评价干扰质膜稳态和功能的药物的作用。这些研究整合了物理，化学和生物学方法，为质膜结构和动力学及其在免疫细胞信号传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.