High Resolution Characterization of Receptor Signaling and Regulation

项目摘要 这项持续的研究旨在表征微观和纳米尺度上的集体分子事件， 由质膜受体或离子通道启动， 在空间和时间中被编排。肥大细胞受IgE受体刺激，在免疫调节中发挥关键作用。 免疫反应，作为一个有价值的模型系统，用于研究细胞的基本机制， 在嘈杂的环境中对特定的刺激做出反应。拟议的研究将建立在既定的推力：1） 以越来越高的分辨率描绘刺激细胞信号传导的启动，重点关注关键但微妙的 2）将我们所掌握的先进技术和完善的假设转化为现实， 已经在模型细胞中发展到破坏与神经变性疾病相关的细胞过程。 创新方面包括推进荧光相关光谱（ImFCS）的成像模式， 测量异质质膜内信号相关组分的动态相互作用。 ImFCS提供的大型数据集和强大的分析功能可为多组分扩散提供精确的数值 和瞬态约束参数，揭示了选择性探针所经历的微妙的相互作用。这个和 其它定量荧光显微术将用于评价 激酶复合物，膜运输和线粒体动力学，这是破坏细胞 病理学具体目标1将实施ImFCS以测量跨膜蛋白的扩散特性 和蛋白质锚定到质膜内部和外部小叶揭示不同的环境 这些探针在IgE受体抗原交联以启动跨膜 发信号。我们将以秒为单位测量刺激的变化， 模型来解释我们的结果在IgE-FcεRI簇大小的时间依赖性变化方面与早期 信号事件。Specific Aim 2将合作研究模型细胞，以获得对细胞机制的深入了解。 基于与神经变性相关的病理学。基于已建立的表型，我们将继续 评估破坏性调节膜运输的α-突触核蛋白变体的结构特征， 帕金森病的线粒体活动。我们将使用微图案化表面和ImFCS来产生新的 了解小胶质细胞介导的Aβ原纤维水解在阿尔茨海默病中出错， 磷脂酰肌醇激酶复合物在髓鞘形成不足疾病中被破坏。

Project Summary This continuing research is aimed at characterizing, at micro- and nanoscales, the collective molecular events of cellular signaling that are initiated by plasma membrane receptors or ion channels and are highly orchestrated in space and time. Mast cells, which are stimulated by IgE-receptors and play a pivotal role in immune responses, serve as a valuable model system for investigating basic mechanisms by which cells respond to specific stimuli in a noisy environment. Proposed research will build on established thrusts: 1) Delineate with increasing resolution the initiation of stimulated cell signaling with a focus on critical but subtle events occurring at cellular membranes; 2) Translate the advanced technologies and refined hypotheses we have developed in model cells to disruption of cellular processes associated with neurodegenerative diseases. Innovative aspects include advancing an imaging modality of fluorescence correlation spectroscopy (ImFCS) to measure dynamic interactions of signaling-related components within the heterogeneous plasma membrane. The large data sets and robust analytics afforded by ImFCS yield precise values for multi-component diffusion and transient confinement parameters, revealing subtle interactions experienced by selective probes. This and other quantitative fluorescence microscopies will be used to evaluate constitutive and stimulated formation of kinase complexes, membrane trafficking, and mitochondrial dynamics, which are disrupted in cellular pathologies. Specific Aim 1 will implement ImFCS to measure diffusion properties of transmembrane proteins and proteins anchored to plasma membrane inner and outer leaflets to reveal distinctive environments experienced by these probes before and after antigen-crosslinking of IgE-receptors to initiate transmembrane signaling. We will measure stimulated changes in seconds timescale and collaboratively adapt theoretical models to interpret our results in terms of time-dependent changes of IgE-FcεRI cluster size as related to early signaling events. Specific Aim 2 will collaboratively investigate model cells to gain mechanistic insight into cell- based pathologies associated with neurodegeneration. Building on established phenotypes, we will continue to evaluate structural features of α-synuclein variants that disruptively modulate membrane trafficking and mitochondrial activities in Parkinson’s disease. We will use micro-patterned surfaces and ImFCS to yield new understanding of microglia-mediated hydrolysis of Aβ fibrils that goes awry in Alzheimer’s disease and phosphatidyl inositol kinase complexes that are disrupted in hypomyelination diseases.