Transient loss of plasma membrane asymmetry in mammalian cells: mechanisms and function

哺乳动物细胞质膜不对称性的瞬时丧失：机制和功能

• 批准号: 10302320
• 负责人: Milka Doktorova
• 金额: $ 6.64万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10302320
• 关键词: Achievement; Affect; Affinity; Antigens; Apoptosis; Apoptotic; Appearance; Behavior; Binding; Biochemical; Biological Assay; Biological Process; Biophysics; Calcium Spikes; Cell Degranulation; Cell Membrane Permeability; Cell membrane; Cells; Characteristics; Charge; Data; Diffusion; Disease; Dissociation; Electrostatics; Environment; Enzymes; Eukaryotic Cell; Face; Fluorescence Microscopy; Goals; Health; Imaging Techniques; Immune; Immune signaling; In Situ; In Vitro; Integral Membrane Protein; Interphase Cell; Investigation; Investments; Knowledge; Link; Lipids; Mammalian Cell; Measurement; Mediating; Mediator of activation protein; Membrane; Membrane Lipids; Membrane Proteins; Methodology; Microscopy; Molecular; Monitor; Movement; Mutagenesis; Pathology; Peripheral; Phagocytosis; Pharmacology; Phosphatidylserines; Physiological; Proliferating; Protein Isoforms; Proteins; Regulation; Research; Resolution; Resources; Rest; Role; Signal Transduction; Signaling Protein; Structure; Surface; Testing; biophysical properties; biophysical tools; cancer cell; cell type; density; extracellular; flexibility; fluidity; genetic manipulation; immune activation; in silico; insight; lipidomics; macrophage; mast cell; nanoscale; novel; physical property; ras Oncogene; response; segregation; study characteristics

PROJECT SUMMARY / ABSTRACT A central feature of the plasma membrane (PM) of eukaryotic cells is the asymmetric distribution of various lipid types across the two membrane leaflets. Maintaining such transbilayer organization requires investment of significant energetic resources, implying an important functional role for interleaflet lipid segregation. A prominent example are charged lipids like phosphatidylserine (PS): normally restricted to the cytoplasmic PM leaflet in healthy cells, their permanent exposure on the exoplasmic face marks the cell for phagocytosis by macrophages. The paradigm has recently been challenged by observations that PS temporarily flips to the outer PM leaflet during antigen-induced activation in healthy immune cells. The purpose and mechanisms of this phenomenon are wholly unknown, nor is it clear whether this redistribution is specific for PS, or rather leads to wholesale lipidomic and biophysical scrambling of the two PM leaflets. To investigate these intriguing questions, we propose to study the characteristics, regulatory mechanisms, and functional roles of this reversible lipid scrambling during immune cell activation. In Aim 1, we will use advanced lipidomics and imaging techniques to characterize the changes in membrane lipid organization during immune cell activation. We hypothesize that antigen activation induces transient, highly localized, and non-specific lipid scrambling, in contrast to the canonical permanent PS exposure associated with apoptosis. In Aim 2, we will identify the molecular mechanisms responsible for this effect. Using super-resolution microscopy, we will monitor the nanoscale organization of various lipid translocators, flipped lipids, and key machinery in the immune signaling cascade. We expect to observe spatial segregation and confinement of these key molecules into specialized PM domains. Finally, in Aim 3 we will examine the functional implications of lipid scrambling by evaluating the effect of PM asymmetry on protein-membrane interactions. We hypothesize that activation-induced scrambling reduces the charge density of the inner PM leaflet, leading to dissociation of charge-dependent signaling proteins from the membrane. To test this hypothesis, we will use fluorescence microscopy in situ—as well as biophysical tools in vitro and in silico—to examine the membrane binding affinity of polybasic-domain containing proteins, including the oncogene Ras, in symmetric and asymmetric membranes. The successful execution of these aims will produce important and novel insights into physiologically regulated changes in membrane organization and its role in immune cell activation. Moreover, these studies will provide the first detailed lipidomic and biophysical characterization of both unperturbed and functionally modified PM asymmetry. The findings may be extensible to cell activation in a variety of other contexts, and are thus expected to have far-reaching impacts by revealing new potential targeting for understanding and treating pathologies.

项目总结/摘要 真核细胞质膜的一个重要特征是各种脂质的不对称分布 两个膜瓣之间的类型。维持这种跨双层组织需要投资 显著的能量来源，意味着小叶间脂质分离的重要功能作用。一个突出 例如带电荷的脂质，如磷脂酰丝氨酸（PS）：通常仅限于细胞质PM小叶， 对于健康的细胞，它们永久暴露在外质面上标志着细胞被巨噬细胞吞噬。 最近观察到PS暂时翻转到外部PM瓣叶，这一范例受到挑战 在健康免疫细胞中抗原诱导的活化过程中。这一现象的目的和机制 是完全未知的，也不清楚这种再分配是否是专门为PS，或者更确切地说，导致批发 两个PM瓣叶的脂质组学和生物物理学混乱。为了研究这些有趣的问题，我们 建议研究这种可逆脂质的特性、调节机制和功能作用 在免疫细胞激活过程中混乱。在目标1中，我们将使用先进的脂质组学和成像技术， 表征免疫细胞活化期间膜脂质组织的变化。我们假设 抗原活化诱导瞬时的、高度局部化的和非特异性的脂质混乱，与 典型的永久PS暴露与凋亡相关。在目标2中，我们将确定 负责这种影响的机制。使用超分辨率显微镜，我们将监测纳米尺度的 组织的各种脂质易位，翻转脂质，和关键机制的免疫信号级联。 我们希望观察到空间隔离和限制这些关键分子到专门的PM域。 最后，在目标3中，我们将通过评估PM的影响来研究脂质混乱的功能含义。 蛋白质-膜相互作用的不对称性。我们假设激活诱导的混乱减少了 内PM小叶的电荷密度，导致电荷依赖性信号蛋白从 膜的为了验证这一假设，我们将使用荧光显微镜原位以及生物物理工具， 体外和计算机模拟-检测含有多碱性结构域的蛋白质的膜结合亲和力，包括 对称和不对称膜中的癌基因Ras。这些目标的成功实现将 产生重要的和新的见解生理调节的变化，膜组织及其 在免疫细胞活化中的作用。此外，这些研究将提供第一个详细的脂质组学和生物物理 未扰动和功能修改的PM不对称性的表征。这些发现可能是可扩展的 在各种其他情况下的细胞激活，因此预计将通过揭示 新的潜在靶点，以了解和治疗病理。