Biophysics of fluid lipid/protein membrane domains and immune cell signaling

描述（由申请人提供）：本项目的长期目标是增强对免疫系统细胞对过敏原（抗原）存在的反应中最早事件的理解。在过敏原入侵的过敏反应中，最初的效应细胞是肥大细胞。这些细胞中导致脱粒和分泌激素介质引起炎症和过敏性超敏反应症状的信号传导途径总体上已得到充分理解，但启动这些途径的机制除外。广泛接受的是，通过与高亲和力IgE受体Fc 5 RI结合的IgE抗体的多价抗原交联对于刺激该受体的脱粒是必需的。然而，这种交联事件是如何在内质膜小叶被识别的，还不清楚。因此，跨膜信号转导的生物物理学阐明有可能有助于识别用于治疗性干扰病理性免疫细胞信号传导的策略。大量的文献强调了膜内组成不均一性在细胞信号传导中的作用。动态成分的横向异质性是生物膜的非理想混合特性的必然结果，然而，成分波动或域的功能重要性尚不清楚。在这一重要研究领域取得进展的一个主要障碍是脂质模型膜和细胞膜研究的大型实验领域之间的概念划分。我们正在使用一种新的方法来检查质膜异质性，包括从免疫细胞获得的微米级质膜囊泡的光学显微镜和光谱表征。我们的初步数据表明，这些囊泡的膜可以分离成横向共存的流体域。目的#1是通过1H MAS NMR和荧光成像表征质膜中流体域形成的生物物理学，这取决于质膜组成和各种其他生理相关的控制参数，包括受体交联。这种表征将使我们为实现目标#2做好准备，在目标#2中，我们将通过共聚焦荧光显微镜定量检查信号蛋白如何在膜结构域之间分布的分子细节。由此产生的分配系数将允许关键的重新检查目前的跨膜信号转导模型，以及激励发展的数学模型的微调信号保真度的动态组成的异质性。在目标#3中，我们将比较质膜囊泡中结构域形成的条件与活细胞中的细胞信号传导能力，并将阐明在混合/分层过渡附近保持平衡的膜组成是否是放大对刺激的免疫应答的重要原则。过敏症影响超过5000万美国人，并给医疗保健系统造成重大痛苦和成本[1]。缺乏治愈治疗的部分原因是缺乏对过敏原如何刺激免疫系统反应的了解。我们的研究有可能阐明过敏原入侵后最早的分子事件的机制方面，因此可以帮助确定预防和治疗过敏性超敏反应的策略。

DESCRIPTION (provided by applicant): The long-term objective of this project is to enhance the understanding of the earliest events in the response of immune system cells towards the presence of allergens (antigens). The initial effector cells in hypersensitive reactions to allergen invasion are mast cells. The signaling pathways in these cells that lead to degranulation and secretion of hormonal mediators causing inflammation and the symptoms of allergic hypersensitivity are well-understood overall, except for the mechanisms involved in initiating these pathways. It is widely accepted that cross-linking by multivalent antigen of IgE antibodies that are bound to the high affinity IgE receptor Fc5RI is necessary for stimulating degranulation by this receptor. How this cross-linking event is recognized at the inner plasma membrane leaflet, however, is not well understood. Biophysical elucidation of transmembrane signal transduction therefore has the potential to contribute to identifying strategies for therapeutic interference with pathological immune cell signaling. A large literature exists emphasizing the role of intra-membrane compositional heterogeneities in cell signaling. Dynamic compositional lateral heterogeneity is a necessary consequence of the non-ideal mixing properties of biological membranes, however, the functional importance of compositional fluctuations, or domains, is unclear. A major hindrance for progress in this important field of research is the conceptual division between the large experimental areas of lipid model membrane and cellular membrane research. We are using a novel approach to examine plasma membrane heterogeneity, consisting of optical microscopic and spectroscopic characterization of micron-sized plasma membrane vesicles obtained from immune cells. Our preliminary data indicate that the membranes of these vesicles can segregate into laterally coexisting fluid domains. Aim #1 is to characterize, by 1H MAS NMR and fluorescence imaging, the biophysics of fluid domain formation in plasma membranes depending on plasma membrane composition and a variety of additional physiologically relevant control parameters, including receptor crosslinking. This characterization will prepare us to achieve Aim #2 where we will quantitatively examine, by confocal fluorescence microscopy, the molecular details of how signaling proteins distribute among membrane domains. The resulting partition coefficients will allow for critical re-examination of current models for transmembrane signaling transduction, as well as motivate the development of mathematical models for the fine tuning of signaling fidelity by dynamic compositional heterogeneities. In Aim #3, we will compare the conditions governing domain formation in plasma membrane vesicles to cell signaling capacities in live cells and will clarify whether membrane composition poised near a mixing/demixing transition is an important principle in amplifying immune response to stimulation. Allergies affect more than 50 million Americans and cause significant suffering and costs to the health care system[1]. The absence of curing treatments is due in part to the lack of understanding how allergens stimulate immune system responses. Our research has the potential to elucidate mechanistic aspects of the earliest molecular events following allergen invasion and could therefore help identify strategies for preventing and treating allergic hypersensitivity.