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

项目摘要 脂类在质膜(PM)双层的两个叶之间的不对称分布是 生命之树上细胞的基本特征。建立和维护不同的脂类成分 相反的小叶是能量昂贵的，这意味着膜不对称的一个重要的生理作用。 传统上，脂质不对称在很大程度上是在细胞凋亡的背景下被考虑的，在这种情况下，暴露 细胞表面的内叶脂质标志着死亡细胞被巨噬细胞吞噬。然而，非- PM的均匀跨双层脂质分布也涉及到许多其他细胞环境。为 例如，将阴离子脂集中在内部PM小叶上产生高表面电荷密度和 招募带正电的蛋白质。此外，很明显，可逆性的脂类损失 在健康的细胞信号传递过程中也会发生不对称，最显著的是在抗原刺激的各种 免疫细胞类型。这些见解揭示了这两个稳定状态的核心但鲜为人知的角色 膜不对称及其在免疫信号中的暂时性丢失；然而，它们也突出了主要的 我们对PM不对称性的理解存在知识差距。具体地说，其组成和生物物理 PM双层的两个小叶的性质目前未知，它们在加扰期间的变化也是未知的， 要么由细胞凋亡诱导，要么由健康细胞信号诱导。最后，不对称对免疫细胞的影响 激活几乎完全不被理解。我们开发了一套新的方法，使 我们要解决这些对一般细胞生物学和免疫信号具有根本重要性的问题 很特别。基于大量的初步数据，我们认为免疫细胞瞬间扰乱了PM的脂质 在抗原介导的激活过程中调节信号蛋白与 下午。在目标1中，我们将定义免疫细胞中发生的脂体和生物物理不对称性的变化。 经前综合症被特定抗原激活后的经前综合征。这些变化将与强大的首相争夺战相提并论 由细胞凋亡刺激诱导。我们还将探索这些效应在两个肥大细胞中的分子介体。 和T细胞。在目标2中，我们评估了抗原诱导的PM扰乱在免疫中的功能后果 细胞，以及这些效应背后的分子机制。首先，我们将定义PM争夺战的角色 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)受抑制或敲除PM扰乱的影响 机械设备。然后，我们将探索PM扰乱和免疫激活之间的机制联系 通过细胞、模型膜和电泳学研究蛋白质-膜相互作用的影响 质膜电荷密度对多元蛋白质与质膜结合的影响这份详细的、全面的 免疫细胞PM的组成、生物物理和功能不对称性的特征将被阐明 哺乳动物细胞的中央组织原则以及这些原则如何对免疫生理学做出贡献。

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.