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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要真核细胞质膜（PM）的一个中心特征是各种脂质的不对称分布维持这种跨双层组织需要投资重要的能量资源，意味着叶间脂质分离的重要功能作用。例如带电脂质，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。该范式最近受到观察结果的挑战，即 PS 暂时翻转到外部 PM 传单在抗原诱导的健康免疫细胞激活过程中，这种现象的目的和机制。完全未知，也不清楚这种重新分配是否特定于 PS，或者更确切地说会导致批发为了研究这些有趣的问题，我们对两个 PM 传单进行了脂质组学和生物物理学的混杂。拟研究这种可逆脂质的特性、调节机制和功能作用在目标 1 中，我们将使用先进的脂质组学和成像技术来解决免疫细胞激活过程中的混乱问题。表征免疫细胞激活过程中膜脂组织的变化。抗原激活会诱导短暂的、高度局部化的、非特异性的脂质扰乱，这与在目标 2 中，我们将鉴定与细胞凋亡相关的典型永久 PS 暴露。使用超分辨率显微镜，我们将监测纳米尺度的机制。各种脂质转运蛋白、翻转脂质和免疫信号级联中关键机制的组织。我们期望观察到这些关键分子在专门的 PM 域中的空间分离和限制。最后，在目标 3 中，我们将通过评估 PM 的影响来检查脂质扰乱的功能含义我们认为激活引起的扰乱会降低蛋白质-膜相互作用的不对称性。内部 PM 小叶的电荷密度，导致电荷依赖性信号蛋白从为了验证这一假设，我们将使用原位荧光显微镜以及生物物理工具。体外和计算机 - 检查含有多碱基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 将成功实现这些目标。对膜组织及其生理调节变化产生重要而新颖的见解此外，这些研究将提供第一个详细的脂质组学和生物物理学研究。未受干扰和功能修饰的 PM 不对称性的表征这些发现可能是可扩展的。细胞激活在各种其他情况下，因此预计将通过揭示产生深远的影响理解和治疗病理学的新的潜在目标。