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

项目摘要 脂质在质膜（PM）双层的两个小叶之间的不对称分布是 生命之树中细胞的基本特征。建立和维持不同的脂质组成 在贴壁小叶是能量昂贵的，这意味着膜不对称的重要生理作用。 经典地，脂质不对称性已经在很大程度上被认为是在细胞凋亡的背景下，其中暴露 细胞表面的内小叶脂质标记死亡细胞以供巨噬细胞吞噬。然而，非- PM的均匀跨双层脂质分布也涉及许多其它细胞环境。为 例如，在内部PM小叶上浓缩阴离子脂质产生高表面电荷密度， 招募带正电荷的蛋白质。此外，已经明显的是，脂质的可逆损失 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各种 免疫细胞类型。这些见解揭示了一个中心，但知之甚少的作用，为稳态 膜的不对称性及其在免疫信号传导中的瞬时损失;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，组合物和生物物理性质 PM双层的两个小叶的性质目前是未知的，它们在扰乱期间的变化也是未知的， 由细胞凋亡或健康细胞信号传导诱导。最后，不对称性如何有助于免疫细胞 激活几乎完全不被理解。我们开发了一套新颖的方法， 我们要解决这些问题的根本重要性，细胞生物学的一般，免疫信号， 特别的。基于广泛的初步数据，我们提出免疫细胞瞬时争夺PM脂质 在抗原介导的活化过程中调节信号蛋白与细胞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发生的脂质组学和生物物理不对称性的变化 特异性抗原激活后的PM。这些变化将与鲁棒PM加扰进行比较 由凋亡刺激诱导。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加扰的作用 通过测量各种功能表型（包括细胞因子分泌， 脱粒和信号蛋白活化）受到PM扰乱的抑制或敲除的影响 机械.然后，我们将探讨PM扰乱和免疫激活之间的机械联系 通过细胞、模型膜和计算机研究蛋白质-膜相互作用， PM电荷密度对多元蛋白与PM的缔合的影响。这份详尽全面的 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 哺乳动物细胞中的核心组织原则以及这些原则如何有助于免疫生理学。

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.