Cubic Phases in Phosphatidylcholine-Cholesterol Mixtures: Cholesterol as Membrane “Fusogen”

Abstract

X-ray diffraction reveals that mixtures of some unsaturated phosphatidylcholines (PCs) with cholesterol (Chol) readily form inverted bicontinuous cubic phases that are stable under physiological conditions. This effect was studied in most detail for dioleoyl PC/Chol mixtures with molar ratios of 1:1 and 3:7. Facile formation of Im3m and Pn3m phases with lattice constants of 30-50 nm and 25-30 nm, respectively, took place in phosphate-buffered saline, in sucrose solution, and in water near the temperature of the Lalpha-HII transition of the mixtures, as well as during cooling of the HII phase. Once formed, the cubic phases displayed an ability to supercool and replace the initial Lalpha phase over a broad range of physiological temperatures. Conversion into stable cubic phases was also observed for mixtures of Chol with dilinoleoyl PC but not for mixtures with palmitoyl-linoleoyl PC or palmitoyl-oleoyl PC, for which only transient cubic traces were recorded at elevated temperatures. A saturated, branched-chain PC, diphytanoyl PC, also displayed a cubic phase in mixture with Chol. Unlike the PEs, the membrane PCs are intrinsically nonfusogenic lipids: in excess water they only form lamellar phases and not any of the inverted phases on their own. Thus, the finding that Chol induces cubic phases in mixtures with unsaturated PCs may have important implications for its role in fusion. In ternary mixtures, saturated PCs and sphingomyelin are known to separate into liquid-ordered domains along with Chol. Our results thus suggest that unsaturated PCs, which are excluded from these domains, could form fusogenic domains with Chol. Such a dual role of Chol may explain the seemingly paradoxical ability of cell membranes to simultaneously form rigid, low-curvature raft-like patches while still being able to undergo facile membrane fusion.

Caught in the Act: Visualization of SNARE‐Mediated Fusion Events in Molecular Detail

“…[36,37] However, the small circular contact interface between the vesicles enforces bending of the elongated stalk along its perimeter and restricts its topology to that of an inverted micelle. Due to the inherent relation with inverse lipid phases, the formation of IMIs is expected at, for example, a high PE and/or cholesterol content [38,39] or for membranes that are moderately enriched with anionic lipids in the presence of calcium. [40,41] This has readily been observed in simulations for experimentally feasible 1:1 PC/PE mixtures.…”

Development of Dioctadecyldimethylammonium Bromide/Monoolein Liposomes for Gene Delivery

“…Cholesterol is one of such molecules due to its ability to modify bilayer fluidity [83]. Inclusion of cholesterol results in the formation of complexes that are more stable but less efficient in vitro compared to DOPEcontaining lipoplexes.…”

Stalk structures in lipid bilayer fusion studied by x-ray diffraction

“…However, in our case, other space groups can not be ruled out with certainty since only few of the allowed reflections could be observed. Metastability and difficulties in reproducibility of bicontinuous phases, where kinetic factors seem to play an important role, have been frequently noted in the literature [88,146,147,148]. This could explain why the appearance of pattern C depended on the protocol for thermal equilibration prior to the measurements.…”

“…All observed peaks are accounted for if a body-centered cubic lattice (symmetry element I for innenzentriert, reflection condition h +k +ℓ = 2n, n ∈ Z) is assumed. TENCHOV et al observed cubic phases in aqueous dispersions of DOPC/Chol mixtures which were subject to heating/cooling cycles by powder diffraction [146]. In their analysis, the strong {211} and {220} reflections corresponding to a ratio of spacings 6 : 8 indicate space group I a3d containing gyroid structures ( Fig.…”