Coupling of lateral and transverse organization in complex biomembranes

项目摘要 膜在细胞生物学中起着核心和基础的作用。除了提供物理和功能上的 细胞生命和细胞外世界之间的界面，膜使大多数细胞内 真核生物中的区隔。此外，近三分之一的哺乳动物蛋白质是膜蛋白。 它们的组织和活动本质上耦合到由 将脂类和蛋白质集体组装成膜。尽管这一点至关重要，但该结构和 活的质膜(PM)的组织结构仍然没有得到很好的描述。最值得注意的是，活的膜 在很大程度上是不对称的；然而，这些不同的小叶成分如何影响生物物理 房地产几乎完全没有被开发过。这种知识鸿沟之所以持续存在，是因为 目前还没有探索不对称膜的技术。然而，最近的方法论 这些突破使得复杂的、仿生的、不对称的双层膜的构建和表征成为可能。 与此同时，已经开发出定量方法来探索生命的生物物理不对称性。 膜。在这里，我们建议通过史无前例的脂质组学整合来扩展这些研究， 生物物理实验、低温透射式电子显微镜和先进分子 模拟，以验证我们的中心假设，即成分不对称的膜具有独特的生物物理 由于横向和横向膜组织之间的强健耦合而产生的特性。我们会 通过三条独立但相辅相成的调查路线来实现这一目标。在目标1中，我们将调查 模型膜中小叶不对称性与膜侧向组织之间的生物物理耦合。 我们将使用共聚焦显微镜、低温电子显微镜和原子模拟来探索脂质的依赖性 小叶间联结的组成，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。目标2将把这些研究扩展到更复杂的系统，以定义 在成分仿生的不对称双层中，小叶之间的生物物理差异。我们会比较一下 代表哺乳动物PM的内外叶的对称膜对其不对称性 直接确定不对称类脂分布引起的新后果。最后，在 目的3我们将把我们的研究扩展到活细胞膜中的膜不对称。最近开发的 选择性探测培养的哺乳动物细胞PM的个别小叶的技术将与 成分不对称性的处理以确定静息PM及其生物物理不对称性 由脂质扰乱引起的扰动。最后，我们将在原位对PM进行第一次详细的低温EM表征 测定非对称生物与杂乱生物的膜厚度和密度分布 膜。这些研究包括一种全面、综合的方法，首次表征了 小叶不对称对生物膜结构和组织的影响。

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.