Compositional, biophysical, and functional consequences of membrane scrambling in immune cells

免疫细胞膜扰乱的组成、生物物理和功能后果

基本信息

批准号：
10218988
负责人：
Ilya Levental
金额：
$ 23.26万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-03-13 至 2022-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10218988
关键词：
Address Affect Antigens Apoptosis Apoptotic Automobile Driving Biomimetics Biophysics Cell Degranulation Cell fusion Cell membrane Cell model Cell physiology Cell surface Cells Cellular Membrane Cellular biology Charge Complement Complex Coupled Data Diffusion Dissociation Enzymes Exocytosis Genetic Image Immune Immune response Immune signaling In Vitro Investigation KRAS2 gene Knock-out Knowledge Lead Life Lipids Mammalian Cell Measures Mediating Mediator of activation protein Membrane Membrane Fusion Membrane Lipids Membrane Proteins Metabolism Methodology Molecular Molecular Probes Muscle Contraction Pharmacology Phenotype Phosphatidylserines Phospholipids Physiological Physiological Processes Physiology Plasma Cells Proteins Resolution Role STIM1 gene Signal Transduction Signaling Protein Stimulus Structure Surface T-Lymphocyte Technology Trees base biophysical properties cell type cost cytokine density human disease immune activation in silico insight macrophage mast cell membrane model novel novel strategies protein activation public health relevance recruit

项目摘要

Project Summary The asymmetric distribution of lipids between the two leaflets of the plasma membrane (PM) bilayer is a fundamental feature of cells across the tree of life. Establishing and maintaining disparate lipid compositions in apposing leaflets is energetically costly, implying an important physiological role for membrane asymmetry. Classically, the lipid asymmetry has been considered largely in the context of apoptosis, where the exposure of inner leaflet lipids on the cell surface marks dead cells for macrophagic engulfment. However, the non- uniform transbilayer lipid distribution of the PM is also involved in a number of other cellular contexts. For example, concentrating anionic lipids on the inner PM leaflet produces a high surface charge density and recruits positively charged proteins. Furthermore, it has become evident that a reversible loss of lipid asymmetry also occurs during healthy cell signaling, most notably in antigen-stimulated activation of a variety of immune cell types. These insights reveal a central, yet poorly understood role for both steady-state membrane asymmetry and its transient loss in immune signaling; however, they also highlight major knowledge gaps in our understanding of PM asymmetry. Specifically, the compositions and biophysical properties of the two leaflets of the PM bilayer are currently unknown, as are their changes during scrambling, either induced by apoptosis or healthy cell signaling. Finally, how asymmetry contributes to immune cell activation is almost completely not understood. We have developed a novel set of methodologies that enable us to address these questions of fundamental importance to cell biology in general, and immune signaling in particular. Based on extensive preliminary data, we propose that immune cells transiently scramble PM lipids during antigen-mediated activation to regulate charge-dependent interactions of signaling proteins with the PM. In Aim 1, we will define the changes in lipidomic and biophysical asymmetry occurring in immune cell PMs following activation by specific antigens. These changes will be compared to the robust PM scrambling induced by apoptotic stimuli. We will also probe the molecular mediators of these effects in both mast cells and T-cells. In Aim 2, we assess the functional consequences of antigen-induced PM scrambling in immune cells, and the molecular mechanisms underlying these effects. First, we will define the role of PM scrambling in immune cell activation by measuring how various functional phenotypes (including cytokine secretion, degranulation, and signaling protein activation) are affected by inhibition or knock-out of PM scrambling machinery. We will then probe the mechanistic connections between PM scrambling and immune activation by cellular, model membrane, and in silico investigations of protein-membrane interactions probing the effect of PM charge density on association of polybasic proteins with the PM. This detailed, comprehensive characterization of the compositional, biophysical, and functional asymmetry of immune cell PMs will elucidate central organizational principles in mammalian cells and how these contribute to immune physiology.

项目概要质膜（PM）双层的两个小叶之间的脂质不对称分布是整个生命树细胞的基本特征。建立和维持不同的脂质成分放置小叶的能量消耗很大，这意味着膜不对称性具有重要的生理作用。传统上，脂质不对称主要在细胞凋亡的背景下被考虑，其中暴露细胞表面上的内叶脂质标记了巨噬细胞吞噬的死亡细胞。然而，非 PM 的均匀跨双层脂质分布也涉及许多其他细胞环境。为了例如，将阴离子脂质集中在 PM 内部小叶上会产生高表面电荷密度，招募带正电荷的蛋白质。此外，很明显，脂质的可逆性损失不对称性也发生在健康细胞信号传导过程中，最明显的是在抗原刺激的多种细胞激活中免疫细胞类型。这些见解揭示了稳态和稳态的核心作用，但人们对此知之甚少。膜不对称及其免疫信号传导的短暂丧失；然而，他们也强调了主要我们对 PM 不对称性的理解存在知识差距。具体而言，组合物和生物物理 PM双层的两个小叶的特性目前未知，它们在加扰过程中的变化也是未知的，由细胞凋亡或健康细胞信号传导诱导。最后，不对称性如何影响免疫细胞激活几乎完全不被理解。我们开发了一套新颖的方法，使我们来解决这些对一般细胞生物学和免疫信号传导至关重要的问题特别的。基于大量的初步数据，我们提出免疫细胞会短暂地扰乱 PM 脂质在抗原介导的激活过程中调节信号蛋白与抗原的电荷依赖性相互作用下午。在目标 1 中，我们将定义免疫细胞中脂质组学和生物物理不对称性的变化被特定抗原激活后的 PM。这些变化将与强大的 PM 加扰进行比较由细胞凋亡刺激诱导。我们还将探讨肥大细胞中这些效应的分子介质和T细胞。在目标 2 中，我们评估了免疫中抗原诱导的 PM 扰乱的功能后果。细胞，以及这些效应背后的分子机制。首先我们来定义PM扰码的作用通过测量各种功能表型（包括细胞因子分泌、脱颗粒和信号蛋白激活）受到 PM 扰乱抑制或敲除的影响机械。然后我们将探讨 PM 加扰和免疫激活之间的机制联系通过细胞、模型膜和蛋白质-膜相互作用的计算机研究来探索效果 PM 电荷密度对多元蛋白与 PM 关联的影响。这个详细、全面免疫细胞 PM 的组成、生物物理和功能不对称性的表征将阐明哺乳动物细胞的中心组织原则以及这些原则如何促进免疫生理学。