The use of iodinated cholesterol to enhance contrast in cryo-EM images of lipid membranes.

Abstract

The lipid raft hypothesis posits nanoscopic domains in the plasma membrane (PM) that arise from unfavorable interactions between low-melting phospholipids and high-melting sphingomyelins in the PM outer leaflet. Cholesterol modulates the properties of these domains (for example, ensuring their fluidity) and can influence the coupling of phase behavior between the outer and inner leaflets. However, the interleaflet distribution of cholesterol has proven difficult to measure. Cryogenic electron microscopy (cryo-EM) is among the few techniques capable of directly visualizing the cholesterol distribution, but this would require an electron-dense cholesterol analog that faithfully mimics the properties of native cholesterol. Here, we investigated a variant of cholesterol in which one hydrogen on the C19 methyl group was replaced with iodine. As expected, cryo-EM images of model membranes containing cholesterol-I19 revealed enhanced contrast compared to vesicles with native cholesterol. However, fluorescence microscopy of GUVs showed that cholesterol-I19 does not support macroscopic liquid-ordered+liquid-disordered phase separation in ternary mixtures with DPPC and DOPC, although FRET and cryo-EM measurements were consistent with nanoscopic heterogeneity in compositions that appeared uniform in GUVs.