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。这些技术进步将被用于研究对过敏反应至关重要的关键蛋白质-蛋白质相互作用。这种三维分子跟踪的新方法使用四个双聚焦共焦体积元件和X，Y和Z的主动反馈，每5毫秒一次，以跟踪活细胞内的3D动态分子运动。作为一个模型系统，我们建议按照三维时空动态Fc？RI-IgE。作为过敏反应的关键介质，该系统对人类健康具有重要意义。通过使用IgE-量子点（QD）探针能够追踪这种高亲和力IgE受体。我们的3D分子跟踪方法的优势包括在高背景环境中跟踪QD的能力，以及在跟踪分子时对分子进行时间分辨光谱的能力。在这里，我们建议显着提高上下文信息和这种新开发的3D跟踪方法的时空分辨率。此外，这些实质性的技术改进将被用来探索关键步骤涉及的Fc？RI-IgE信号传导和经由内吞作用的下调。本提案的具体目标是：目标1。提高3D跟踪显微镜的上下文和时空分辨率。该目标包括方法，同时图像的关键GFP标记的复合物在3D分子跟踪QD标记的Fc？RI-IgE，使用闪烁抑制量子点获取更长的3D轨迹，以及提高我们3D跟踪方法的时空分辨率的方法。目标二。应用这些新的进展来分析的分子机制，管理贩运和信号能力的内化Fc？RI.本目标探讨的时空动态Fc？RI-IgE复合物与肌动蛋白、微管、网格蛋白以及激酶林恩和Syk相互作用。