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

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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.