Development of cryogenic electron microscopy for probing phase separation in lipid membranes

在化学系生命过程化学（CLP）项目的支持下，Frederick A.田纳西大学的Heberle和M.休斯顿得克萨斯大学健康科学中心的Neal瓦克瑟姆正在开发用于研究脂质膜的低温电子显微镜（cryo-EM）。这些膜为生物体提供了至关重要的结构，形成了细胞与外部环境之间的边界以及内部细胞区室的边界。长期以来，科学家们一直对在某些细胞膜中发现的异常大量的不同脂质感到困惑;例如，哺乳动物细胞的最外层质膜包含数百种化学上不同的脂质种类。许多组分的混合物通常导致相分离，例如当油与水混合时发生相分离。一个有趣的假设是，类似的相分离发生在一些细胞膜中，其中膜脂质自组织成称为脂筏的簇，其性质不同于周围脂质的性质。大量证据表明，筏对细胞是有用的，并在许多细胞功能中发挥重要作用。然而，它们的小尺寸（不到人类头发宽度的1000倍）使它们不可能用常规显微镜看到，因此我们对筏结构的了解是有限的。通过使用电子束而不是可见光作为照明源，可以对更小的结构进行成像，包括人工膜中的筏状结构域，其模拟细胞膜的脂质组成，如这些实验室的初步研究所示。该项目旨在通过各种增强对比度的方法优化膜的冷冻EM图像的质量，从而优化其信息内容;确定可以检测到的最小筏尺寸;并应用改进的成像方法来获得真正细胞膜中筏的图片。该项目的另一个目标是培训研究生的实验和计算方法，为他们在STEM（科学，技术，工程和数学）领域的职业生涯做好准备。研究人员为其他希望使用cryo-EM研究脂质膜的研究人员创造了有用的免费工具。该项目的一个重要组成部分是一个公共宣传计划，以提高对物理化学研究及其在生物科学中的应用的认识和赞赏。细胞膜具有巨大的自组织能力，这是由其脂质体的结构多样性所赋予的。在最外层的质膜内，不同种类的脂质之间的非理想相互作用导致类似于液相分离的现象，其可以指导膜蛋白的空间组织，从而影响细胞功能。相域或“筏”在正常条件下是纳米级的大小，排除了它们的检测由传统的光学显微镜和激励替代成像技术的发展具有更大的空间分辨率。本项目将开发cryo-EM作为一种这样的技术，用于研究无探针，无支撑膜在与脂筏相关的长度尺度上的相行为。主要目标是优化这种新的专业成像应用的实验和分析工作流程。这项工作的关键是结合使用原子分子模拟和介观囊泡模型来生成合成的地面实况图像数据集，从而能够明确估计测量参数的准确性和精度，并确定该技术的分辨率限制。优化的工作流程将在表征良好的实验系统上进行测试，这些系统的域大小和面积分数已经独立建立。最终的科学目标是将该技术扩展到现在可以使用cryo-EM访问的细胞膜中，从而为更深入地了解复杂脂质和蛋白质混合物的横向异质性铺平道路。PI和co-PI将在脂质相分离和混合物的物理化学研究方面培训研究生，并将开发协议，软件工具，和地面实况数据集，以扩大社区的研究人员使用低温，该奖项反映了NSF的法定使命，并被认为是值得通过使用基金会的知识价值和更广泛的影响审查标准进行评估的支持。

With the support of the Chemistry of Life Processes (CLP) Program in the Division of Chemistry, Frederick A. Heberle of the University of Tennessee and M. Neal Waxham of the University of Texas Health Science Center at Houston are developing cryogenic electron microscopy (cryo-EM) for the study of lipid membranes. These membranes provide crucial structure to living organisms, forming the boundary between a cell and its external environment as well as the boundaries of internal cellular compartments. Scientists have long been puzzled by the unusually large number of different lipids found in some cell membranes; for example, the outermost plasma membrane of mammalian cells contains several hundred chemically distinct lipid species. Mixtures of many components often result in phase separation, such as occurs when oil is mixed with water. An intriguing hypothesis is that an analogous phase separation occurs in some cell membranes, in which membrane lipids self-organize into clusters termed lipid rafts that have properties different from those of the sea of lipids surrounding them. A large body of evidence suggests that rafts are useful to the cell and play an important role in many cell functions. However, their small size (less than 1000 times the width of a human hair) makes them impossible to see with a conventional microscope, and consequently our knowledge of raft structure is limited. By using a beam of electrons rather than visible light as the illumination source, it becomes possible to image much smaller structures, including raft-like domains in artificial membranes that mimic the lipid composition of cell membranes as shown in preliminary studies from these laboratories. This project seeks to optimize the quality of cryo-EM images of membranes and thus, their information content, through various methods of enhancing contrast; to determine the minimum raft size that can be detected; and to apply the improved imaging methodology to obtain pictures of rafts in genuine cell membranes. Another goal of this project is the training of graduate students in experimental and computational methods to prepare them for careers in STEM (science, technology, engineering and mathematics) fields. The researchers create useful, freely available tools for other researchers who wish to use cryo-EM to study lipid membranes. An important part of the project is a public outreach program to enhance awareness of and appreciation for physical chemistry research and its application to the biological sciences. Cell membranes have an enormous capacity for self-organization conferred by the structural diversity of their lipidomes. Within the outermost plasma membrane, non-ideal interactions between different classes of lipids results in a phenomenon akin to liquid phase separation that can direct the spatial organization of membrane proteins and thus influence cell function. The phase domains or “rafts” are nanoscopic in size under normal conditions, precluding their detection by conventional light microscopy and motivating the development of alternative imaging techniques with greater spatial resolution. This project will develop cryo-EM as one such technique for investigating the phase behavior of probe-free, unsupported membranes at length scales relevant to lipid rafts. A primary objective is to optimize the experimental and analysis workflow for this new and specialized imaging application. Key to this effort is the combined use of atomistic molecular simulations and mesoscopic vesicle models to generate synthetic ground-truth image datasets, thereby enabling unambiguous estimates of the accuracy and precision of measured parameters and establishing the resolution limitations of the technique. The optimized workflow will be tested on well-characterized experimental systems for which domain sizes and area fractions have been independently established. A final scientific objective is the expansion of the technique into cellular membranes that are now accessible with cryo-EM, thus paving the way for a deeper understanding of lateral heterogeneity in complex lipid and protein mixtures. The PI and co-PI will train graduate students in the study of lipid phase separation and the physical chemistry of mixtures, and will develop protocols, software tools, and ground-truth data sets to expand the community of researchers using cryo-EM to study lipid membranes.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.