Coupling of lateral and transverse organization in complex biomembranes

复杂生物膜中横向和横向组织的耦合

基本信息

批准号：
10475092
负责人：
Frederick A Heberle
金额：
$ 43.1万
依托单位：
UNIVERSITY OF TENNESSEE KNOXVILLE
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：
2020-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10475092
关键词：
Address Affect Architecture Behavior Biological Biological Models Biological Phenomena Biomimetics Biophysics Cell membrane Cells Cellular Membrane Cellular biology Chemicals Cholesterol Complement Complex Confocal Microscopy Coupled Coupling Cryoelectron Microscopy Data Dependence Electron Microscopy Erythrocytes Eukaryota Evaluation Exhibits Goals Heterogeneity Human Image In Situ Individual Investigation Knowledge Lateral Life Lipids Mammalian Cell Measures Mediating Membrane Membrane Lipids Membrane Proteins Methodology Methods Microscopic Microscopy Modeling Molecular Molecular Profiling Nanostructures Neutrons Pharmacology Phase Phospholipids Physiological Play Process Property Proteins Rest Roentgen Rays Role Structure System Techniques Technology Testing Thick Time Translating Transmission Electron Microscopy base biophysical properties cryogenics density experimental study extracellular fluidity fluorescence imaging insight lipidomics membrane model multidisciplinary nanoscale novel physical property simulation spectroscopic imaging three dimensional structure

项目摘要

Project Summary Membranes play central and fundamental roles in cell biology. In addition to providing the physical and functional interface between cellular life and the extracellular world, membranes enable most intracellular compartmentalization in eukaryotes. Furthermore, close to a third of mammalian proteins are membrane embedded, with their organization and activity intrinsically coupled to the emergent properties resulting from the collective assembly of lipids and proteins into membranes. Despite this central importance, the structure and organization of living plasma membranes (PMs) remain poorly characterized. Most notably, living membranes are largely compositionally asymmetric; however, how those distinct leaflet compositions affect biophysical properties remains almost completely unexplored. This knowledge gap has persisted because robust technologies for exploring asymmetric membranes have not been available. However, recent methodological breakthroughs have enabled the construction and characterization of complex, biomimetic, asymmetric bilayers. In parallel, quantitative approaches have been developed to probe the biophysical asymmetry of living membranes. Here, we propose to extend these studies through an unprecedented integration of lipidomics, biophysical experiments, cryogenic transmission electron microscopy (cryoEM), and advanced molecular simulations, to test our central hypothesis that compositionally asymmetric membranes have unique biophysical properties resulting from robust coupling between lateral and transverse membrane organization. We will approach this goal through three independent yet complementary lines of inquiry. In Aim 1, we will investigate the biophysical coupling between leaflet asymmetry and membrane lateral organization in model membranes. We will use confocal microscopy, cryoEM, and atomistic simulations to probe the dependence of lipid composition on interleaflet coupling, thereby defining the compositional drivers and molecular mechanisms of leaflet coupling in asymmetric bilayers. Aim 2 will extend these studies into more complex systems to define the biophysical disparity between leaflets in compositionally biomimetic, asymmetric bilayers. We will compare symmetric membranes representative of the inner and outer leaflet of mammalian PMs to their asymmetric counterparts to directly identify the novel consequences arising from asymmetric lipid distributions. Finally, in Aim 3 we will extend our studies into membrane asymmetry in live cell membranes. Recently developed techniques to selectively probe individual leaflets of cultured mammalian cell PMs will be combined with manipulations of compositional asymmetry to determine the biophysical asymmetry of the resting PM and its perturbation by lipid scrambling. Finally, we will perform the first detailed cryoEM characterization of PMs in situ to determine membrane thickness and density distributions in asymmetric compared to scrambled living membranes. These studies comprise a comprehensive, integrated approach to characterize for the first time the consequences of leaflet asymmetry on the structure and organization of biological membranes.

项目摘要膜在细胞生物学中扮演着中心和基本角色。除了提供身体和功能细胞寿命与细胞外世界之间的接口，膜可实现最细胞内真核生物中的分室化。此外，接近三分之一的哺乳动物蛋白是膜嵌入的，其组织和活动本质地与由集体组装脂质和蛋白质成膜。尽管这一重要性，但结构和活着的质膜组织（PMS）的特征仍然很差。最值得注意的是，活膜在很大程度上是不对称的；但是，这些不同的小叶组成如何影响生物物理属性几乎完全没有探索。这种知识差距一直存在，因为强大尚未提供用于探索不对称膜的技术。但是，最近的方法论突破使复杂，仿生，不对称双层的结构和表征。同时，已经开发了定量方法来探测生命的生物物理不对称性膜。在这里，我们建议通过前所未有的脂质组学整合，扩展这些研究，生物物理实验，低温透射电子显微镜（冷冻）和晚期分子模拟，以测试我们的中心假设，即成分不对称的膜具有独特的生物物理侧面和横膜组织之间强大的耦合产生的属性。我们将通过三个独立但互补的探究路线实现这一目标。在AIM 1中，我们将调查模型膜中的小叶不对称和膜侧组织之间的生物物理耦合。我们将使用共聚焦显微镜，冷冻和原子模拟来探测脂质的依赖性交流联耦合的组成，从而定义了组成驱动因素和分子机制不对称双层中的小叶耦合。 AIM 2将将这些研究扩展到更复杂的系统以定义在构图生物仿生的，不对称双层中小叶之间的生物物理差异。我们将比较哺乳动物PM的内部和外部小叶的对称膜不对称直接识别不对称脂质分布引起的新型后果的对应物。最后，在 AIM 3我们将将研究扩展到活细胞膜中的膜不对称性。最近开发了选择性探测培养的哺乳动物细胞PM的单个传单的技术将与组成不对称的操纵以确定静息PM的生物物理不对称性脂质扰动的扰动。最后，我们将执行PMS原位的第一个详细的冷冻表征确定与炒生物相比，不对称中的膜厚度和密度分布膜。这些研究包括首次表征全面的综合方法小叶不对称对生物膜结构和组织的后果。