Nanostructural Dynamics of Biomembranes in Immunoreceptor Signaling

描述（由申请人提供）：T细胞反应的免疫抑制可能促进衰老过程；因此，在分子水平上理解T细胞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）促进了有效T细胞信号传导的功能相关区隔化，并且T细胞受体与其他信号蛋白和特定脂质之间的这些关键分子相互作用随着年龄的增长而被破坏。富含胆固醇的木筏被假设是短暂的和微观的，直到现在，还没有在没有显著扰动的情况下直接观察到体内。这个跨学科的NIH R21提案将通过结合尖端成像工具和实验方法来克服这个问题，这将使我们能够定量地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：利用单光子和双光子微光谱学研究高时空分辨率的年轻和衰老T细胞模型中脂质和蛋白质诱导的功能域的信号动力学；特异性目标2：利用成像质谱法研究参与免疫受体信号传导的膜结构域的化学性质。对于后一种特异性Aim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，这将为完整细胞成像质谱的开发提供一个强大的平台，我们随后将在T细胞模型上使用。预计的结果将为T细胞受体信号动力学和相关的质膜纳米结构如何随着T细胞老化而变化提供新的见解。

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.