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

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聚焦体积单元和每5毫秒一次的X， Y和Z主动反馈来跟踪活细胞内部的三维动态分子运动。作为一个模型系统，我们建议遵循Fc - i - ige的三维时空动态。该系统作为过敏反应的关键中介，与人体健康有着重要的关系。通过使用IgE-量子点（QD）探针，可以跟踪这种高亲和力的IgE受体。我们的3D分子跟踪方法的优点包括在高背景环境中跟踪量子点的能力，以及在跟踪分子时对分子进行时间分辨光谱的能力。在此，我们建议显著提高这种新开发的3D跟踪方法的上下文信息可用性和时空分辨率。此外，这些实质性的技术改进将用于探索Fc？i - ige信号通路及胞吞作用的下调。本建议的具体目标是：目标1。提高三维跟踪显微镜的上下文和时空分辨率。本课题包括在QD标记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内化Fc？RI。本文旨在探讨Fc？i - ige复合物与肌动蛋白，微管，网格蛋白，Lyn和Syk激酶相互作用。

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.