Elucidating the phase behavior of ternary model membrane mixtures containing halogenated cholesterol analogues.

Abstract

Cellular plasma membranes exhibit nanoscale lateral lipid heterogeneity, a feature that is thought to be central to their functionality. However, only a few biophysical methods are capable of detecting domains at sub-micron length scales. We recently showed that cryogenic electron microscopy (cryo-EM) can directly image phase separation in extruded liposomes due to its ability to resolve the intrinsic thickness and electron density differences of ordered and disordered phases. However, the contrast between these phases is poor compared to conventional fluorescence microscopy and is thus both a limiting factor and a focal point for optimization. Because the fundamental source of contrast is the spatial variation in electron density within the bilayer, lipid modifications aimed at selectively increasing the electron density of one phase might enhance the ability to resolve coexisting phases. To this end, we make use of a model membrane mixture of DPPC/DOPC/cholesterol, in which one hydrogen of the methyl group on the steroid ring of cholesterol is replaced by an electron-rich halogen atom. We use a variety of biophysical techniques including confocal fluorescence microscopy, FRET, and cryo-EM to examine phase behavior as a function of composition and temperature. We find that cryo-EM image contrast is increased in liposomes containing halogenated cholesterol as expected. However, the phase behavior of these mixtures is altered compared to mixtures containing native cholesterol providing a cautionary note that halogenated cholesterol has biophysical effects beyond increasing electron density.