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

描述(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来研究对过敏反应至关重要的关键蛋白质-蛋白质相互作用。这种新的三维分子跟踪方法使用四个重叠的共焦体积元素，并每5毫秒在X、Y和Z方向上进行一次主动反馈，以跟踪活细胞内的三维动态分子运动。作为一个模型系统，我们建议遵循Fc？Ri-IgE的三维时空动力学。作为过敏反应的关键介质，该系统对人类健康具有重要的意义。对这种高亲和力的IgE受体的跟踪是通过使用IgE量子点(QD)探针实现的。我们的3D分子跟踪方法的优势包括能够在高背景环境中跟踪量子点，以及能够在分子被跟踪时对其执行时间分辨光谱。在这里，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和时空分辨率。此外，这些实质性的技术改进将被用于探索Fc？RI-IgE信号转导和通过内吞作用下调调控的关键步骤。该方案的具体目标是：目标1。提高3D跟踪显微镜的背景分辨率和时空分辨率。这一目标包括在QD标记的Fc？RI-IgE的3D分子跟踪过程中同时成像关键的GFP标记的络合物的方法，使用眨眼抑制的量子点获取更长的3D轨迹的方法，以及提高我们的3D跟踪方法的时空分辨率的方法。目的2.将这些新进展应用于内化Fc？RI转运和信号传递能力的分子机制分析。本研究的目的是探索Fc？RI-IgE复合体与肌动蛋白、微管、网状蛋白以及Lyn和Syk激酶相互作用的时空动力学。 公共卫生相关性：这项工作旨在跟踪活细胞内关键受体分子(Fc？RI-IgE复合体)的三维空间和时间动态。由于该受体是过敏反应的关键介质，因此它与人类健康具有重要的相关性。通过3D和实时跟踪这些受体及其与其他细胞蛋白质的相互作用，我们将获得对这一重要信号过程的有价值的见解。

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. PUBLIC HEALTH RELEVANCE: This work aims to follow the three dimensional spatial and temporal dynamics of key receptor molecules (Fc?RI-IgE complexes) inside of live cells. As this receptor is the key mediator of the allergic response, it has significant relevance to human health. By following these receptors and their interactions with other cellular proteins in 3D and in real-time, we will gain valuable insight into this important signaling process.