Low-flux scanning electron diffraction reveals substructures inside the ordered membrane domain

Abstract

Ordered/disordered phase separation occurring in bio-membranes has piqued researchers’ interest because these ordered domains, called lipid rafts, regulate important biological functions. The structure of the ordered domain has been examined with artificial membranes, which undergo macroscopic ordered/disordered phase separation. However, owing to technical difficulties, the local structure inside ordered domains remains unknown. In this study, we employed electron diffraction to examine the packing structure of the lipid carbon chains in the ordered domain. First, we prepared dehydrated monolayer samples using a rapid-freezing and sublimation protocol, which attenuates the shrinkage of the chain-packing lattice in the dehydration process. Then, we optimised the electron flux to minimise beam damage to the monolayer sample. Finally, we developed low-flux scanning electron diffraction and assessed the chain packing structure inside the ordered domain formed in a distearoylphosphatidylcholine/dioleoylphosphatidylcholine binary monolayer. Consequently, we discovered that the ordered domain contains multiple subdomains with different crystallographic axes. Moreover, the size of the subdomain is larger in the domain centre than that near the phase boundary. To our knowledge, this is the first study to reveal the chain packing structures inside an ordered domain.

Double Electron–Electron Resonance of Spin-Labeled Cholestane in Model Membranes: Evidence for Substructures inside the Lipid Rafts

“…Then, these clusters (substructures or subdomains) may serve as the platform for the DChl superlattice observed in the present study. Note in this respect that, recently, Kinoshita and co-workers, using the electron diffraction technique, found that the ordered domain in the membrane contains multiple subdomains with different crystallographic axes.…”

“…17−19 However, despite the extensive literature about the cholesterol−lipid interactions in lipid membranes, direct experimental information on the molecular structure of lipid and cholesterol assemblies remains scarce. 15,20,21 Moreover, controversies about the properties, composition, and even the very existence of lipid rafts remain unresolved. 20 Double electron−electron resonance spectroscopy (DEER, also known as PELDOR) allows studying nanoscale spatial structures of spin-labeled molecules and their assemblies.…”

“…Models of cholesterol spatial distributions and models of phase separation in the lipid membranes are highly discussed in the literature, see, e.g., refs − . Chol dimers and higher oligomers were found in lipid bilayers using NMR and MD simulations. , Also, Chol is known to form a superlattice in model membranes. − However, despite the extensive literature about the cholesterol–lipid interactions in lipid membranes, direct experimental information on the molecular structure of lipid and cholesterol assemblies remains scarce. ,, Moreover, controversies about the properties, composition, and even the very existence of lipid rafts remain unresolved …”

Behavior of Triterpenoid Saponin Ginsenoside Rh2 in Ordered and Disordered Phases in Model Membranes Consisting of Sphingomyelin, Phosphatidylcholine, and Cholesterol

“…The subphase temperature and ambient temperature were kept at 25.0 ± 0.1 and 25 ± 1 °C, respectively. The measurements were repeated thrice under the same conditions to obtain reliable data …”

“…The measurements were repeated thrice under the same conditions to obtain reliable data. 34 Molecular Dynamics Simulation. The ginsenoside Rh2 molecule was built based on the reported X-ray structure of ginsenoside compound K (20-O-β-glucopyranosyl-20(S)-protopanaxadiol) 35 and parametrized using the CHARMM General FF 36 based on the CHARMM36 lipid force field 37 and carbohydrate force fields and the previously reported cholesteryl-β-glucopyranoside (ChoGlc).…”