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: 8689897
• 负责人: JAMES H WERNER
• 金额: $ 40.8万
• 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 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8689897
• 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; Transport Vesicles; Vesicle; 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; 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）：这项工作大大扩展了新开发的三维分子跟踪方法的技术能力。这些技术进步将被利用，以研究关键的蛋白质蛋白质相互作用对过敏反应很重要。对三维分子跟踪的这种新方法使用四个重焦共聚焦体积元素和X，Y和Z中的主动反馈，每5毫秒一次遵循3D动态分子运动，内部的活细胞内部。作为模型系统，我们建议遵循FC？RI-IGE的3D时空动力学。作为过敏反应的关键调解人，该系统与人类健康具有重要意义。通过使用IgE-Quantum Dot（QD）探针来启用这种高亲和力IgE受体的跟踪。我们使用3D分子跟踪方法的优点包括在高背景环境中跟踪QD的能力以及在遵循分子时对分子进行时间分辨光谱的能力。在这里，我们建议显着改善这种新开发的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通过内吞作用涉及FC？RI-IGE信号传导和下调调节的关键步骤。该提案的具体目的是：目标1。增加3D跟踪显微镜的上下文和时空分辨率。该目的包括在QD标记的FC？ri-igE的3D分子跟踪过程中同时图像钥匙GFP标记的复合物的方法，使用闪烁的量子点获得更长的3D轨迹，以及增加我们3D跟踪方法的时空分辨率的方法。目的2。将这些新的进步应用于分子机制的分析，这些分子机制负责内在FC？ri的运输和信号传导能力。这个目的探讨了与肌动蛋白，mircotubules，clathrin和激酶Lyn和Syk相互作用的FC？RI-IGE复合物的时空动力学。