Three-Dimensional Molecular Tracking of IgE-Fc{epsilon}RI in Live Cells

活细胞中 IgE-Fc{epsilon}RI 的三维分子追踪

• 批准号: 8889188
• 负责人: JAMES H WERNER
• 金额: $ 40.7万
• 依托单位: LOS ALAMOS NAT SECTY-LOS ALAMOS NAT LAB
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8889188
• 关键词: 3-Dimensional; Actins; Affinity; Antigens; Automobile Driving; Binding; Biological Models; Blinking; Cell membrane; Cell surface; Cells; Clathrin; Complex; Development; Diffusion; Dimensions; Down-Regulation; Dynamin; Electron Microscopy; Elements; Endocytic Vesicle; Endocytosis; Endosomes; Environment; Fc epsilon RI; Feedback; Fluorescence Microscopy; Genetic Transcription; Green Fluorescent Proteins; Hand; Health; Histamine Release; Human; IgE; IgE Receptors; Image; Individual; Kinetics; Label; Life; Light; Measurement; Measures; Mediating; Mediator of activation protein; Membrane; Methods; Microscope; Microtubules; Molecular; Molecular Analysis; Motion; Motor; Phosphorylation; Phosphotransferases; Photons; Play; Process; Protein Tyrosine Kinase; Proteins; Quantum Dots; Receptor Aggregation; Recruitment Activity; Resolution; Role; Side; Signal Transduction; Specificity; Spectrum Analysis; Staging; System; Testing; Time; Vesicle; Vesicle Transport Pathway; Work; allergic response; antigen binding; base; charge coupled device camera; coated pit; fluorescence imaging; improved; inhibitor/antagonist; insight; mast cell; millisecond; molecular dynamics; movie; novel strategies; protein complex; protein protein interaction; receptor; signal processing; spatiotemporal; temporal measurement; trafficking

DESCRIPTION (provided by applicant): This work substantially expands the technical capabilities of newly developed three-dimensional molecular tracking methods. These technical advances will be exploited to study key protein-protein interactions important for the allergic response. This new approach to three dimensional molecular tracking uses four overlaping confocal volume elements and active feedback in X, Y, and Z once every 5 milliseconds to follow 3D dynamic molecular motion inside of live cells. As a model system, we propose to follow the 3D spatio- temporal dynamics of Fc?RI-IgE. As the key mediator for the allergic responses, this system has significant relevance to human health. Tracking of this high affinity IgE receptor is enabled through the use of IgE- quantum dot (QD) probes. Advantages of our approach to 3D molecular tracking include the ability to follow QDs in high background environments and the ability to perform time-resolved spectroscopy on the molecules while they are being followed. Here, we propose to significantly improve the contextual information available and the spatio-temporal resolution of this newly developed 3D tracking method. Moreover, these substantial technical improvements will be used to explore crucial steps involved in Fc?RI-IgE signaling and down- regulation via endocytosis. The specific aims of this proposal are: Aim 1. To increase the contextual and spatio-temporal resolution of the 3D tracking microscope. This Aim includes methods to simultaneous image key GFP labeled complexes during 3D molecular tracking of QD labeled Fc?RI-IgE, the acquisition of longer 3D trajectories using blinking-suppressed quantum dots, and methods to increase the spatio-temporal resolution of our 3D tracking methods. Aim 2. To apply these new advances to the analysis of molecular mechanisms that govern trafficking and signaling competency of internalized Fc?RI. This Aim explores the spatio-temporal dynamics of Fc?RI-IgE complexes interacting with actin, mircotubules, clathrin, and the kinases Lyn and Syk.

描述（由申请人提供）：这项工作极大地扩展了新开发的三维分子追踪方法的技术能力。这些技术进步将用于研究对过敏反应很重要的关键蛋白质-蛋白质相互作用。这种三维分子跟踪的新方法使用四个重叠的共焦体积元件以及每 5 毫秒一次的 X、Y 和 Z 主动反馈，以跟踪活细胞内的 3D 动态分子运动。作为模型系统，我们建议遵循 Fc?RI-IgE 的 3D 时空动态。作为过敏反应的关键介质，该系统与人类健康具有重要意义。通过使用 IgE 量子点 (QD) 探针可以跟踪这种高亲和力 IgE 受体。我们的 3D 分子追踪方法的优点包括能够在高背景环境中追踪量子点，以及在追踪分子时对分子执行时间分辨光谱的能力。在这里，我们建议显着提高这种新开发的 3D 跟踪方法的可用上下文信息和时空分辨率。此外，这些实质性的技术改进将用于探索 FcRI-IgE 信号传导和通过内吞作用下调所涉及的关键步骤。该提案的具体目标是： 目标 1. 提高 3D 跟踪显微镜的上下文和时空分辨率。该目标包括在 QD 标记的 Fc?RI-IgE 的 3D 分子跟踪过程中同时对 GFP 标记的复合物进行图像关键的方法、使用闪烁抑制量子点采集更长的 3D 轨迹，以及提高我们的 3D 跟踪方法的时空分辨率的方法。目标 2. 将这些新进展应用于分析控制内化 Fc?RI 运输和信号传导能力的分子机制。该目标探索 Fc?RI-IgE 复合物与肌动蛋白、微管、网格蛋白以及激酶 Lyn 和 Syk 相互作用的时空动力学。