Compositional Heterogeneity In Biologically Relevant Membrane Models

Lateral organization of the cellular plasma membrane promotes biological function by permitting regulation of cellular processes. Evidence now supports the hypothesis that the coexistence of a variety of lipid molecules, with different melting temperatures and acyl chain lengths, contribute to this lateral organization by forming lipid rafts, nanoscale domains with distinct biophysical properties. Phase separation into immiscible micron-sized domains is readily observed in model membranes, chemically simplified lipid mixtures that have been studied under equilibrium conditions to understand how composition and temperature affect domain properties. As lipid domains have yet to be imaged directly in live resting cells, the relevance of model membranes is uncertain. We have focused on characterizing the biologically relevant outer leaflet membrane model brain sphingomyelin (bSM)/1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC)/cholesterol (Chol) in which nanoscale domains challenge conventional imaging techniques. We have determined the temperature-dependent ternary phase diagrams for bSM/POPC/Chol and bSM/1,2-dioleoyl-sn-glycero-3phosphocholine (DOPC)/Chol using Förster resonance energy transfer (FRET) and differential scanning calorimetry, and we have confirmed the biologically relevant liquid-disordered (Ld) and liquid-ordered (Lo) coexistence region using electron spin resonance spectroscopy. We have determined that the size of coexisting Ld+Lo domains in bSM/POPC/Chol is 2-6 nm radius using FRET and small-angle neutron scattering. Ultimately, this careful characterization of model membrane will serve as a starting point for investigating the influence of peptides on domain size and other biophysical properties. Rafts can be stabilized to form larger platforms through protein-protein and protein-lipid interactions. Understanding how these domains form, grow, and stabilize in model systems is a first step toward elucidating their roles in important membrane-mediated processes

Comparison of Three Ternary Lipid Bilayer Mixtures: FRET and ESR Reveal Nanodomains

Phase diagrams of ternary lipid mixtures containing cholesterol have provided valuable insight into cell membrane behaviors, especially by describing regions of coexisting liquid-disordered (Ld) and liquid-ordered (Lo) phases. Fluorescence microscopy imaging of giant unilamellar vesicles has greatly assisted the determination of phase behavior in these systems. However, the requirement for optically resolved Ld + Lo domains can lead to the incorrect inference that in lipid-only mixtures, Ld + Lo domain coexistence generally shows macroscopic domains. Here we show this inference is incorrect for the low melting temperature phosphatidylcholines abundant in mammalian plasma membranes. By use of high compositional resolution Förster resonance energy transfer measurements, together with electron spin resonance data and spectral simulation, we find that ternary mixtures of DSPC and cholesterol together with either POPC or SOPC, do indeed have regions of Ld + Lo coexistence. However, phase domains are much smaller than the optical resolution limit, likely on the order of the Förster distance for energy transfer (R0, ∼2–8 nm)

Bilayer Thickness Mismatch Controls Domain Size in Model Membranes

The observation of lateral phase separation in lipid bilayers has received considerable attention, especially in connection to lipid raft phenomena in cells. It is widely accepted that rafts play a central role in cellular processes, notably signal transduction. While micrometer-sized domains are observed with some model membrane mixtures, rafts much smaller than 100 nmbeyond the reach of optical microscopyare now thought to exist, both in vitro and in vivo. We have used small-angle neutron scattering, a probe free technique, to measure the size of nanoscopic membrane domains in unilamellar vesicles with unprecedented accuracy. These experiments were performed using a four-component model system containing fixed proportions of cholesterol and the saturated phospholipid 1,2-distearoyl-<i>sn</i>-glycero-3-phosphocholine (DSPC), mixed with varying amounts of the unsaturated phospholipids 1-palmitoyl-2-oleoyl-<i>sn</i>-glycero-3-phosphocholine (POPC) and 1,2-dioleoyl-<i>sn</i>-glycero-3-phosphocholine (DOPC). We find that liquid domain size increases with the extent of acyl chain unsaturation (DOPC:POPC ratio). Furthermore, we find a direct correlation between domain size and the mismatch in bilayer thickness of the coexisting liquid-ordered and liquid-disordered phases, suggesting a dominant role for line tension in controlling domain size. While this result is expected from line tension theories, we provide the first experimental verification in free-floating bilayers. Importantly, we also find that changes in bilayer thickness, which accompany changes in the degree of lipid chain unsaturation, are entirely confined to the disordered phase. Together, these results suggest how the size of functional domains in homeothermic cells may be regulated through changes in lipid composition