Nanometer-Scale Lipid Clusters in Model Membranes Revealed by Atomic Recombination in Nanosims

Abstract

Much of what is known about the lateral organization of biological membranes is inferred from the analogy between phase diagrams of ternary mixtures of lipids and the plasma membrane of mammalian cells. However, the relevance of phase diagrams of simple lipid mixtures to the compositionally complex and dynamic plasma membrane has not yet been established. Additionally, the fluorescence and spin labels used to build phase diagrams often interact with lipid bilayers, and detecting nanometer-scale phases remains challenging. We have previously introduced atomic recombination in dynamic SIMS as a technique for detecting lateral heterogeneities that occur on the nanometer length scale, below the resolution of both fluorescence microscopy and conventional NanoSIMS imaging. In this method, the formation of 13C15N- secondary ions from 13C and 15N atoms installed on different lipids only occurs if the isotopically-labeled lipids are within approximately 3 nm of each other. Isotope labels are desirable because they avoid problems caused by fluorescent dyes and because they directly report on the organization of the lipids of interest. Facile isotope-labeling chemistry allows the labels to be placed on any lipid of interest, allowing us to determine which lipids are clustering in complex mixtures. Here, we expand this technique to study the formation and composition of nanodomains of lipids in more complex mixtures for which phase diagrams have not been determined and would be too laborious to fully solve. Furthermore, we explore the formation of lipid clusters outside of phase boundaries, where lipids may still be inhomogenously distributed but display no first order phase transition. These more complex model membranes may be better models for lipid rafts given that lipids in the plasma membrane show no micrometer scale phase separation and no first order phase transition.