Phase Diagram of Ternary Cholesterol/Palmitoylsphingomyelin/ Palmitoyloleoyl-Phosphatidylcholine Mixtures - Spin-Label EPR Study of Lipid-Raft Formation

Oct 26, 2026

Upload by noah

Electron paramagnetic resonance (EPR) of spin-labeled phospholipids is used to determine the ternary phase diagram for canonical lipid-raft mixtures of cholesterol (chol), N-palmitoylsphingomyelin (PSM), and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) at 23°C. No phase boundaries are detected for binary POPC/chol mixtures or for ternary mixtures with PSM below 24 mol%. Continuous La-to-Lo conversion is indicated. Gel–fluid coexistence appears at low cholesterol or low POPC, and La–Lb coexistence occurs for PSM/POPC. Fluid–fluid La–Lo separations are mapped and linked to raft-like Lo compositions.

LanguageEnglish

CategoryHealthcare

FormatPDF

Pages10

Reading Time25 min

AccessFree to download

Abstract

Introduction

Lipid raft background and phase behavior

Experimental rationale for domain detection methods

Materials and Methods

Spin-label EPR approach for ternary mixtures

Results

Ternary phase diagram at 23°C

Binary and ternary coexistence regions

Gel–fluid and fluid–fluid separations

Discussion

Tie lines and phase-rule consistency

Raft-like Lo compositions for biological relevance

Phase Diagram of Ternary Cholesterol/Palmitoylsphingomyelin/ Palmitoyloleoyl-Phosphatidylcholine Mixtures: Spin-Label EPR Study of Lipid-Raft Formation Irina V. Ionova, Vsevolod A. Livshits, and Derek Marsh* Centre of Photochemistry, Russian Academy of Sciences, Moscow, Russia; and Max-Planck-Institut f¨ur biophysikalische Chemie, G¨ottingen, Germany ABSTRACT For canonical lipid raft mixtures of cholesterol (chol), N-palmitoylsphingomyelin (PSM), and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), electron paramagnetic resonance (EPR) of spin-labeled phospholipids—which is insensitive to domain size—is used to determine the ternary phase diagram at 23􀀁 C. No phase boundaries are found for binary POPC/chol mixtures, nor for ternary mixtures with PSM content <24 mol % . EPR lineshapes indicate that conversion from the liquid-disordered (La) to liquid-ordered (Lo) phase occurs continuously in this region. Two-component EPR spectra and several tie lines attributable to coexistence of gel (Lb) and ﬂuid phases are found for ternary mixtures with low cholesterol or low POPC content. For PSM/POPC alone, coexistence of La and Lb phases occurs over the range 50􀀃95.5 mol % PSM. A further tie line is found at 3 mol % chol with endpoints at 50 and R77 mol % PSM. For PSM/chol, Lb 􀀃Lo coexistence occurs over the range 10􀀃38 mol % chol and further tie lines are found at 4 .5 and 7 mol % POPC. Two-component EPR spectra indicative of ﬂuid-ﬂuid (La􀀃Lo) phase separation are found for lipid compositions: 25% POPC>10%, and conﬁrmed by nonlinear EPR. Tie lines are identiﬁed in the La􀀃Lo coexistence region, indicating that the ﬂuid domains are of sufﬁcient size to obey the phase rule. The three-phase triangle is bounded approximately by the compositions 40 and 75 mol % PSM with 10 mol % chol, and 60 mol % PSM with 25 mol % chol. These studies deﬁne the compositions of raft-like Lo phases for a minimal realistic biological lipid mixture. INTRODUCTION There is now accumulating evidence that the in-plane organization of plasma membranes is heterogeneous and contains lateral nanodomains enriched in cholesterol and sphingolipids (lipid rafts) that supposedly function as platforms for self-assembly and transport of speciﬁc protein complexes involved in signal transduction, and in membrane trafﬁc within the cell (1–3) . To gain insight into the formation and properties of such domains it is useful to study well-deﬁned bilayer membranes that have simpliﬁed lipid compositions similar to those of cell membranes. Mixtures of a lipid with high chain-melting temperature (of which sphingolipids are representative) and cholesterol are of central interest because these are known to form the so-called liquid-ordered phase, Lo (4,5) . The latter is distinguished from the usual liquid-disordered (or La) phase by having high local orientational order, but still retaining the long-range translational disorder that is characteristic of ﬂuid lipid membranes. A recent critical survey of phospholipid/cholesterol binary phase diagrams has revealed that direct experimental evidence for the existence of Lo-phases is, however, not so extensive as is generally assumed (6) . This is especially the case for coexistence with the ﬂuid La-phase, which is presumed to correspond with the formation of lipid raftsin biological membranes. It has long been recognized that Submitted January 13, 2012, and accepted for publication March 20, 2012. *Correspondence: [dmarsh@gwdg.de](dmarsh@gwdg.de)[ ](dmarsh@gwdg.de)Editor: Paulo Almeida. 􀀁 2012 by the Biophysical Society 0006-3495/12/04/1856/10 $2.00 coexistence of immiscible ﬂuid phases (as opposed to gelﬂuid coexistence) is most likely to be of biological signiﬁcance (7) . Direct evidence for coexistence of ﬂuid La-and Lo-phase domains is somewhat more abundant in ternary systems, as found in a compendium of phase diagrams for mixtures of a high-melting lipid and a low-melting lipid with choles

FAQs

What technique is used to map the ternary lipid phase diagram?

Spin-label electron paramagnetic resonance (EPR) spectroscopy is used to determine the ternary phase diagram for cholesterol, PSM, and POPC mixtures at 23°C.

Is phase separation observed in POPC/chol binary mixtures?

No phase boundaries are found for binary POPC/chol mixtures, indicating no detectable phase separation under the reported conditions.

What does the EPR analysis suggest about the La-to-Lo transition?

EPR lineshapes indicate that conversion from the liquid-disordered (La) to liquid-ordered (Lo) phase occurs continuously in the studied ternary region.

Oct 26, 2026

Upload by noah