Direct Imaging of Nanoscale Lipid Organization in Probe-Free Biomimetic Membranes

Abstract

The three-dimensional architecture of biological membranes has functional consequences for living cells. In the outer leaflet of the plasma membrane (PM), lipids are thought to organize into ordered yet fluid domains, with diverse evidence supporting participation of these “rafts” in membrane processes including protein sorting and signaling. However, many details of PM domain structure remain elusive due to a lack of methods to directly probe membrane features at the nanoscale. We report direct imaging of coexisting nanoscopic domains in synthetic and bio-derived membranes without extrinsic probes using cryoEM. Analysis of images from vesicles composed of lipids ranging from 14 to 22 carbons in length show that cryoEM can resolve sub-angstrom differences in average bilayer thickness. Features in experimental images were reproduced in simulated images constructed from atomistic molecular dynamics simulations, revealing relationships between bilayer thickness measured with small-angle X-ray scattering and calculated from cryoEM images. We further used simulations to predict two sources of contrast between coexisting phases within the same vesicle, namely 1) differences in membrane thickness, and 2) electron density arising from different lipid packing in the two phases. We observed both sources of contrast in vesicles composed of saturated lipids, unsaturated lipids, and cholesterol. We conclude that cryoEM imaging enables quantitative analysis of membrane parameters such as thickness, domain size, and lipid packing, opening new avenues for direct investigation of nanoscopic membrane organization.