Sterol–Lipid Hybrids: Novel Molecular Tools to Tune Membrane Lateral Phase Separation

Cell membranes contain diverse lipids with distinct architectures and affinities that organize laterally into liquid-ordered (lo) and liquid-disordered (ld) domains. Such lo–ld phase coexistence underlies the lipid raft hypothesis, where proteins preferring one phase can drive site-specific biological activities. This coexistence is well established in ternary mixtures of saturated, unsaturated lipids, and cholesterol— producing micro- and nano-domains. Controlling these domains with molecular additives has major biological and practical applications such as regulation of protein activity and antimicrobial intervention methods. Towards this aim, we studied sterol-modified lipids (SMLs) -engineered hybrids with a sterol molecule substituting one (or both) of the two hydrocarbon chains. To understand the regulatory role of SMLs, we employed a suite of techniques that probe phase separation on multiple spatial scales including fluorescence microscopy, small-angle neutron scattering (SANS), and atomic force microscopy (AFM). Contrary to expectation, full cholesterol replacement with SMLs stabilized highly ordered domains with distinct morphology and fluidity compared to cholesterol-only membranes. However, 50% replacement of cholesterol with SMLs almost completely suppressed domains across both macroscopic and nanoscopic scales. These findings highlight the ability of SMLs to modulate membrane organization with potential applications in therapeutic treatments and spatially patterned artificial cells.