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 作为细胞生命和细胞外世界之间的界面，膜使大多数细胞内 真核生物中的区室化。此外，近三分之一的哺乳动物蛋白质是膜蛋白。 嵌入式的，其组织和活动本质上耦合到由 脂质和蛋白质的集体组装成膜。尽管具有核心重要性， 活质膜（PMs）的组织仍然很难表征。最值得注意的是， 在很大程度上是组成上不对称的;然而，这些不同的小叶组成如何影响生物物理 几乎完全未被开发。这种知识差距一直存在，因为 用于探索不对称膜的技术还没有得到。然而，最近的方法 这些突破使得能够构建和表征复杂的、仿生的、不对称的双层。 与此同时，定量的方法已经被开发出来，以探测生物物理的不对称性， 膜。在这里，我们建议通过前所未有的脂质组学整合来扩展这些研究， 生物物理实验，低温透射电子显微镜（cryoEM），和先进的分子生物学技术。 模拟，以测试我们的中心假设，即组成不对称的膜具有独特的生物物理特性， 由于横向和横向膜组织之间的强大耦合而产生的特性。我们将 通过三条独立但互补的调查路线来实现这一目标。在目标1中，我们将研究 模型膜中瓣叶不对称性和膜横向组织之间的生物物理耦合。 我们将使用共聚焦显微镜、冷冻电镜和原子模拟来探测脂质的依赖性。 组合物对小叶间偶联的影响，从而定义了组合物的驱动因素和分子机制， 不对称双层中的小叶偶联。目标2将把这些研究扩展到更复杂的系统， 在组成仿生、不对称双层中小叶之间的生物物理差异。我们将比较 代表哺乳动物PM的内小叶和外小叶的对称膜 直接识别由不对称脂质分布引起的新后果。最后在 目的3我们将把我们的研究扩展到活细胞膜的膜不对称性。最近开发 选择性探测培养的哺乳动物细胞PM的单个小叶的技术将与 操纵成分不对称性，以确定静息PM及其 通过脂质扰乱的扰动。最后，我们将进行第一次详细的冷冻电镜表征PM原位 以确定与乱序生活相比不对称的膜厚度和密度分布， 膜。这些研究包括一个全面的，综合的方法来表征首次 小叶不对称性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.