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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：
10319193
负责人：
Milka Doktorova
金额：
$ 6.98万
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 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10319193
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 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.

项目摘要/摘要