Structural Analysis of Gel-Phase-Like Subdomains Formed in Lipid-Raft-Mimetic Bilayers

Lipid rafts are ordered membrane domains composed of sphingomyelin (SM) and cholesterol (chol). They serve as platforms for membrane-based signal transduction. To date, the structural properties of lipid rafts have been investigated using SM/chol/unsaturated-lipid ternary bilayers, which undergo phase separation into SM/chol-rich liquid-ordered (Lo) and unsaturated-lipid-rich liquid-disordered (Ld) phases. Although some previous studies have suggested structural heterogeneity within the Lo phase, the internal organization of lipid-raft-mimetic Lo phases has been largely unexplored owing to the lack of methodologies capable of probing the local membrane structure. Recently, we developed a low-flux electron diffraction (LFED) technique and disclosed the local structures of lipid monolayers. In this study, we examined the local structure within the Lo phase by using LFED. We first tested the applicability of a previously developed rapid-freezing and sublimation protocol to lipid bilayers and successfully prepared dehydrated bilayers with minimal perturbation of lipid chain packing. Next, we optimized the electron beam flux and acquired diffraction patterns from the lipid bilayers in a minimally invasive manner. Finally, we directly examined local chain-packing structures within the Lo phase using LFED for the first time. As a result, sharp-ring and pseudohexagonal-spot diffractions were observed at some locations in the Lo phase. Notably, these diffraction patterns were identical with those observed in pure SM bilayers (gel phase). Hence, these results suggest that subdomains with gel-phase-like lipid packing are formed in the Lo phase and that subdomains consist of almost-pure SM. These results provide experimental evidence for structural heterogeneity within the Lo phase and a new physical basis for understanding the organization and functional versatility of lipid rafts.