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

描述(由申请人提供)：该项目的长期目标是加强对免疫系统细胞对过敏原(抗原)存在的反应的最早事件的了解。在过敏原侵袭的超敏反应中最初的效应细胞是肥大细胞。除了启动这些信号通路的机制外，这些细胞中导致脱颗粒和分泌激素介质导致炎症和过敏症状的信号通路总体上是众所周知的。人们普遍认为，与高亲和力的IgE受体Fc5RI结合的IgE抗体的多价抗原交联是刺激该受体脱颗粒所必需的。然而，这种交联性事件是如何在内质膜小叶上被识别的，还不是很清楚。因此，跨膜信号转导的生物物理阐明有可能有助于确定治疗干预病理性免疫细胞信号的策略。大量文献强调膜内成分的异质性在细胞信号传递中的作用。动态组成的横向异质性是生物膜非理想混合特性的必然结果，然而，组成波动或结构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实验区之间的概念划分。我们正在使用一种新的方法来检测质膜的异质性，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。第一个目的是通过1H MAS核磁共振和荧光成像表征质膜中流体结构域形成的生物物理依赖于质膜的组成和各种额外的生理相关的控制参数，包括受体交联。这一特征将使我们为实现目标#2做好准备，在该目标中，我们将通过共聚焦荧光显微镜定量检查信号蛋白如何分布在膜域之间的分子细节。由此得到的分配系数将允许对当前的跨膜信号转导模型进行关键的重新检查，并推动通过动态成分异质性来微调信号保真度的数学模型的发展。在目标#3中，我们将比较控制质膜囊泡中结构域形成的条件与活细胞中的细胞信号能力，并将澄清膜成分是否处于混合/分离转变附近是放大对刺激的免疫反应的重要原则。过敏影响着5000多万美国人，并给医疗保健系统带来巨大痛苦和成本[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，因此可能有助于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.