Single Molecule Imaging to Quantify FcεRI Signaling Dynamics

描述(申请人提供)：肥大细胞激活的一个关键角色是高亲和力的Ig E受体，FcεRI。虽然关于FcεRI通过多价抗原交联所启动的事件链知道得很多，但我们对蛋白质动力学如何促进信号传递的理解存在着根本的差距。我们的长期目标是了解蛋白质相互作用的动态和随机行为如何影响信号传播。这项建议的目的是量化FcεRI信号早期事件中的动态蛋白质相互作用。我们的中心假设是，蛋白质-蛋白质相互作用的持续时间调节信号结果。这项拟议研究的基本原理是，了解蛋白质-蛋白质相互作用动力学在信号中的作用是理解细胞如何塑造免疫反应的强度和质量的下一步。我们将整合多种成像模式，包括新颖的和成熟的，以捕获和量化管理FcεRI膜相关信号的早期事件，以实现三个特定目标：1)确定促进FcεRI激活的相互作用寿命和扩散动力学；2)量化Syk接头蛋白与信号复合体结合的动力学；以及3)确定Fc？RI和Lat信号斑块之间的簇稳定性和相互作用。这种方法是创新的，因为我们将开发在分子水平上对生化事件进行成像的方法，使我们能够获得有关使用传统生物化学技术无法确定的信号事件的动态信息。这项拟议的研究意义重大，因为我们提议获得的定量信息以前从未被直接测量过，并将为细胞生物学领域带来新的视角。此外，这里描述的多色单分子成像技术的发展将适用于许多其他基本的生物学问题。我们获得的有关FCεRI信令的信息将有助于填补我们在FCεRI如何启动信令方面的知识空白。最终，我们希望这些信息将为以蛋白质相互作用和定位为目标的药物设计开辟新的途径。

DESCRIPTION (provided by applicant): A key player in mast cell activation is the high affinity IgE receptor, FcεRI. While much is known about the chain of events initiated by crosslinking of FcεRI through multivalent antigen, there is a fundamental gap in our understanding of how protein dynamics facilitate signaling. Our long term goal is to understand how the dynamic and stochastic behavior of protein-protein interactions influences signal propagation. The objective of this proposal is to quantify dynamic protein interactions during early events in FcεRI signaling. Our central hypothesis is that the duration of protein-protein interactions modulates the signaling outcome. The rationale for the proposed research is that understanding the role of protein-protein interaction dynamics in signaling is the next step in understanding how the cell shapes the strength and quality of an immune response. We will integrate multiple imaging modalities, both novel and established, to capture and quantify early events that govern FcεRI membrane associated signaling in order to achieve three specific aims: 1) To determine the interaction lifetime and diffusional dynamics that facilitate FcεRI activation; 2) To quantify the kinetics of Syk adaptor protein binding to the signaling complex; and 3) To determine the cluster stability of LAT and the interplay between Fc?RI and LAT signaling patches. The approach is innovative because we will develop methodology for imaging biochemical events at the molecular level that will allow us to obtain dynamic information about signaling events that cannot be determined using traditional biochemistry techniques. The proposed research is significant because the quantitative information that we propose to obtain has not been directly measured before and will bring new perspectives to the cell biology community. Furthermore, the development of multi-color single molecule imaging techniques described here will be applicable to many other fundamental biological questions. The information we gain about FcεRI signaling will help to fill the gaps in our knowledge of how FcεRI initiates signaling. Ultimately, we expect that this information will open new avenues for drug design that target protein interactions and localization.