High Resolution Characterization of Receptor Signaling and Regulation

项目摘要 这项持续研究的目的是在微米和纳米尺度上表征集体分子事件 由质膜受体或离子通道启动的细胞信号 在空间和时间上精心编排。肥大细胞受IgE受体的刺激，在 免疫反应是研究细胞的基本机制的有价值的模型系统 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基础上：1) 以更高的分辨率描述受刺激细胞信号的启动，重点放在关键但微妙的地方 发生在细胞膜上的事件；2)将先进的技术和精炼的假设转化为 在模型细胞中发展到与神经退行性疾病相关的细胞过程中断。 创新方面包括将荧光相关光谱(ImFCS)的成像模式改进为 测量异质质膜内信号相关成分的动态相互作用。 ImFCS提供的大数据集和稳健的分析为多组分扩散提供了精确的值 和瞬时限制参数，揭示了选择性探测器经历的微妙相互作用。这和 其他定量荧光显微镜将用于评估构成和刺激形成 在细胞内被破坏的激酶复合体、膜运输和线粒体动力学 病理学。特定目标1将实施ImFCS来测量跨膜蛋白的扩散特性 以及锚定在质膜内外小叶上的蛋白质，以揭示独特的环境 这些探针在IgE受体抗原交联启动跨膜反应前后所经历的 发信号。我们将在几秒钟的时间刻度内衡量受刺激的变化，并协作调整理论 根据Ig E-FcεRI团簇大小随时间的变化解释我们结果的模型与早期 信号事件。特定目标2将合作研究模型细胞，以获得对细胞的机械性洞察- 与神经退行性变相关的病理基础。在现有表型的基础上，我们将继续 评估α-突触核蛋白突变体的结构特征，这些突变体扰乱膜运输并 帕金森氏病患者的线粒体活性。我们将使用微图案表面和ImFCS来产生新的 了解小胶质细胞介导的β纤维在阿尔茨海默病和阿尔茨海默病中出错的水解性 在髓鞘过少疾病中被破坏的磷脂酰肌醇激酶复合体。

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.