Structure-Function Relationships of Immunoreceptors

描述(申请人提供)：这项研究旨在阐明由质膜免疫受体启动的、在空间和时间上高度协调的细胞信号的集体分子事件。主要关注肥大细胞上的高亲和力IgE受体Fc5RI，它在过敏免疫反应中发挥核心作用，是这些研究的一个有价值的模型系统。这些研究的目的是了解发生在质膜环境中的蛋白质和脂类之间的结构相互作用，这些相互作用会被IgE-Fc5RI的抗原交联改变，并导致跨膜触发细胞内信号级联反应。中心假设是，交联型IgE-Fc5RI、Lyn激酶和其他早期信号成分之间的相互作用在膜的异质结构中协调，包括它与细胞骨架的相互作用。建议的研究将探讨动态膜结构和相互作用在Fc5RI信号中的作用，特别是有序膜结构域(通常称为“筏”)的参与。具体目标1将继续开发用于微米和纳米尺度上的抗原图案化的化学修饰底物，包括使用热敏聚合物来快速触发抗原暴露。空间控制的，微米级的共聚的IgE-Fc5RI和细胞骨架的组装将被作为参与内化和吞噬启动的结构进行研究。具体目标2将利用扫描电子显微镜和超高分辨率荧光显微镜在纳米尺度上研究IgE-Fc5RI介导的信号组装的空间调节和动力学，近场光学显微镜的孔径将被纳米化，作为测量单个膜组件运动和相互作用动力学的一种新方法。结合数据分析，将开发一个理论框架来描述簇状受体如何通过膜同步它们的激活状态。具体目标3将侧重于利用高分辨率显微镜和类相特性的电子自旋共振测量，在空间上调节IgE-Fc5RI介导的信号的膜结构组件中的脂质的参与。Fc5RI和有序性与无序性之间的接近程度的变化将通过荧光共振能量转移来测量。将评估胆固醇在质膜内外小叶上的分布，以及这种分布的扰动如何影响细胞信号事件。这些研究结合了不同的物理、化学和细胞生物学方法，为质膜结构和动力学及其在免疫受体信号转导中的作用提供了新的见解。 与公共健康相关：真核细胞的异质质膜微粒维持蛋白质和脂类相互作用的稳定状态，支持基本细胞功能，同时作为选择性屏障，准备对环境刺激做出反应。劫持或破坏这些高度协调的膜相互作用涉及许多疾病状态，包括病原性感染、神经变性和一些癌症。近年来，质膜在空间调控受体介导的信号事件中的复杂作用越来越受到重视。一个突出的例子是肥大细胞上的IgE受体Fc5RI，它在过敏免疫反应中发挥着核心作用。我们研究的直接目标是在分子水平上阐明发生在动态质膜结构域内的结构相互作用，这些结构相互作用被IgE-Fc5RI的抗原交联改变，并导致跨膜触发细胞内信号级联和免疫细胞反应。更广泛地说，质膜参与细胞反应的详细特征将为治疗应用中的干预提供新的机会，并更清楚地理解健康和疾病的细胞生物学。

DESCRIPTION (provided by applicant): This research is aimed at elucidating the collective molecular events of cellular signaling that are initiated by plasma membrane immunoreceptors and are highly orchestrated in space and time. Focus is primarily on the high affinity receptor for IgE, Fc5RI, on mast cells, which plays a central role in the allergic immune response and serves as a valuable model system for these studies. The goal of these studies is to understand structural interactions of proteins and lipids occurring within the plasma membrane environment that are altered by antigen crosslinking of IgE-Fc5RI and result in transmembrane triggering of the intracellular signaling cascade. The central hypothesis is that interactions between crosslinked IgE-Fc5RI, Lyn kinase, and other early signaling components are coordinated within the heterogeneous structure of the membrane including its interactions with the cytoskeleton. Proposed studies will investigate the roles of dynamic membrane structure and interactions in Fc5RI signaling, and, in particular, the participation of ordered membrane domains (commonly called "rafts"). Specific Aim 1 will continue to develop chemically modified substrates for patterning antigen on micro- and nanometer lengths scales, including use of thermosensitive polymers for rapidly triggered exposure of antigen. Spatially controlled, micron scale assemblies of co- clustered IgE-Fc5RI and cytoskeleton will be investigated as structures involved in initiation of internalization and phagocytosis. Specific Aim 2 will examine spatial regulation and dynamics of IgE-Fc5RI-mediated signaling assemblies on the nanoscale with scanning electron microscopy and super high-resolution fluorescence microscopy, and apertures for near-field optical microscopy will be nanofabricated as a novel approach for measuring motional and interactional dynamics of individual membrane components. Together with data analysis, a theoretical framework will be developed to describe how clustered receptors synchronize their activation state via the membrane. Specific Aim 3 will focus on participation of lipids in the membrane structural assemblies that spatially regulate IgE-Fc5RI mediated signaling using high resolution microscopies and electron spin resonance measurements of phase-like properties. Changes in proximity between Fc5RI and order- vs disorder- preferring lipids will be measured with fluorescence resonance energy transfer. Cholesterol distribution across the outer and inner leaflets of the plasma membrane will be assessed, and how perturbations of this distribution affect cell signaling events will be evaluated. These investigations integrate diverse physical, chemical and cell biological approaches to provide new insights into plasma membrane structure and dynamics and their roles in immunoreceptor signaling. PUBLIC HEALTH RELEVANCE: The heterogeneous plasma membrane millieu of eukaryotic cells maintains a steady state of protein and lipid interactions that support basal cell function and, while serving as a selective barrier, is poised to respond to environmental stimuli. Hijacking or disrupting these highly orchestrated membrane interactions is involved in numerous disease states, including pathogenic infection, neurodegeneration, and some cancers. In recent years the intricate participation of the plasma membrane in spatially regulating receptor-mediated signaling events has become increasingly appreciated. A prominent example is the receptor for IgE, Fc5RI, on mast cells, which plays a central role in the allergic immune response. The immediate goal of our studies is molecular level elucidation of the structural interactions occurring within dynamic plasma membrane domains that are altered by antigen crosslinking of IgE-Fc5RI and result in transmembrane triggering of the intracellular signaling cascade and immune cell responses. More generally, a detailed characterization of plasma membrane participation in cellular responses will provide new opportunities for intervention in therapeutic applications and a clearer understanding of the cell biology of health and disease.