Nanostructural Dynamics of Biomembranes in Immunoreceptor Signaling

描述（由申请人提供）： T细胞反应的免疫妥协可能促进衰老过程;因此，在分子水平上理解T细胞中信号传导的时空动态是至关重要的。 我们假设富含胆固醇的膜结构域（或？筏？）促进有效T细胞信号传导的功能相关区室化，并且T细胞受体与其他信号传导蛋白和特定脂质之间的这些关键分子相互作用随着老化的进行而被破坏。 富含胆固醇的筏被假设为是短暂的和微观的，到目前为止，还没有直接观察到在体内没有显着的扰动。 这项跨学科的NIH R21提案将通过将尖端成像工具与实验方法相结合来克服这一问题，从而使我们能够定量了解分子如何在生物膜中相互作用。 为了验证我们的假设，我们提出了以下两个具体目标：具体目标1：使用单光子和双光子显微光谱以高空间和时间分辨率研究年轻和衰老T细胞模型中脂质和蛋白质诱导的功能结构域的信号传导动力学;具体目标2：使用成像质谱研究参与免疫受体信号传导的膜结构域的化学性质。对于我们对后者特异性目的的初步研究，我们将使用RBL肥大细胞中IgE受体信号传导的相关模型系统，该系统作为开发完整细胞成像质谱的强大平台，我们随后将在T细胞模型上使用。 预计结果将导致对T细胞受体信号动力学和相关质膜纳米结构如何随着T细胞衰老而变化的新见解。

DESCRIPTION (provided by applicant): Immunocompromise of the T cell response may facilitate the aging process; thus, understanding the spatio-temporal dynamics of signaling in T cells at the molecular level is crucial. We hypothesize that cholesterol-rich membrane domains (or ?rafts?) facilitate functionally relevant compartmentalization for effective T cell signaling, and these critical molecular interactions between the T cell receptor and other signaling proteins and specific lipids are disrupted as aging proceeds. Cholesterol-rich rafts are hypothesized to be transient and microscopic and, until now, have not yet been directly observed in vivo without significant perturbation. This interdisciplinary NIH R21 proposal will overcome this problem by combining cutting-edge imaging tools with experimental approaches that will allow us to understand quantitatively how molecules interact in biomembranes. To test our hypothesis, we propose the following two Specific Aims: Specific Aim 1: Investigating the signaling dynamics of lipid- and protein-induced functional domains in young and aging T cell models with high spatial and temporal resolution using one- and two-photon micro-spectroscopy; and Specific Aim 2: Investigating the chemical nature of membrane domains that participate in immunoreceptor signaling using imaging mass spectrometry. For our initial studies on the latter Specific Aim, we will use the related model system of IgE receptor signaling in RBL mast cells, which serves as a robust platform for the development of imaging mass spectrometry on intact cells that we will subsequently use on the T cell models. The projected results will lead to new insights into how T cell receptor signaling dynamics and related plasma membrane nanostructure change as T cells age.