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-sn-glycero-3-phosphocholine (DSPC), mixed with varying amounts of the unsaturated phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dioleoyl-sn-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.
The spatial organization of lipids and proteins in biological membranes seems to have a functional role in the life of a cell. Diverse evidence supports participation of lipid microdomains (rafts) in membrane processes including protein sorting and signaling. Raft functionality may well involve the reversible coalescence of small and transient domains into larger stable structures that act as platforms for organizing protein machinery. While micrometer-sized domains are observed with some model membrane mixtures, rafts much smaller than 100 nm_beyond the reach of optical micro-scopy_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. These experiments were performed using a four-component model system containing fixed proportions of cholesterol and the saturated phospholipid 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), mixed with varying amounts of the unsaturated phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dioleoyl-sn-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 liquidordered 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.
Membrane raft size measurements are crucial to understanding the stability and functionality of rafts in cells. The challenge of accurately measuring raft size is evidenced by the disparate reports of domain sizes, which range from nanometers to microns for the ternary model membrane system sphingomyelin (SM)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/cholesterol (Chol). Using Förster resonance energy transfer (FRET) and differential scanning calorimetry (DSC), we established phase diagrams for porcine brain SM (bSM)/dioleoyl-sn-glycero-3-phosphocholine (DOPC)/Chol and bSM/POPC/Chol at 15 and 25°C. By combining two techniques with different spatial sensitivities, namely FRET and small-angle neutron scattering (SANS), we have significantly narrowed the uncertainty in domain size estimates for bSM/POPC/Chol mixtures. Compositional trends in FRET data revealed coexisting domains at 15 and 25°C for both mixtures, while SANS measurements detected no domain formation for bSM/POPC/Chol. Together these results indicate that liquid domains in bSM/POPC/Chol are between 2 and 7 nm in radius at 25°C: that is, domains must be on the order of the 2–6 nm Förster distance of the FRET probes, but smaller than the ~7 nm minimum cluster size detectable with SANS. However, for palmitoyl SM (PSM)/POPC/Chol at a similar composition, SANS detected coexisting liquid domains. This increase in domain size upon replacing the natural SM component (which consists of a mixture of chain lengths) with synthetic PSM, suggests a role for SM chain length in modulating raft size in vivo.
Cholesterol and ether lipids are ubiquitous in mammalian cell membranes, and their interactions are crucial in ether lipid mediated cholesterol trafficking. We report on cholesterol’s molecular interactions with ether lipids as determined using a combination of small-angle neutron and X-ray scattering, and all-atom molecular dynamics (MD) simulations. A scattering density profile model for an ether lipid bilayer was developed using MD simulations, which was then used to simultaneously fit the different experimental scattering data. From the analysis of the data the various bilayer structural parameters were obtained. Surface area constrained MD simulations were also performed to reproduce the experimental data. This iterative analysis approach resulted in good agreement between the experimental and simulated form factors. The molecular interactions taking place between cholesterol and ether lipids were then determined from the validated MD simulations. We found that in ether membranes, cholesterol primarily hydrogen bonds with the lipid headgroup phosphate oxygen, while in their ester membrane counterparts, cholesterol hydrogen bonds with the backbone ester carbonyls. This different mode of interaction between ether lipids and cholesterol induces cholesterol to reside closer to the bilayer surface, dehydrating the headgroup’s phosphate moiety. Moreover, the three-dimensional lipid chain spatial density distribution around cholesterol indicates anisotropic chain packing, causing cholesterol to tilt. These insights lend a better understanding of ether lipid mediated cholesterol trafficking and the roles that the different lipid species have in determining the structural and dynamical properties of membrane associated biomolecules.
Some of our recent work has resulted in the detailed structures of fully hydrated, fluid phase phosphatidylcholine (PC) and phosphatidylglycerol (PG) bilayers. These structures were obtained from the joint refinement of small-angle neutron and X-ray data using the scattering density profile (SDP) models developed by Kučerka et al. (Biophys J 95:2356-2367, 2008; J Phys Chem B 116:232-239, 2012). In this review, we first discuss models for the standalone analysis of neutron or X-ray scattering data from bilayers, and assess the strengths and weaknesses inherent to these models. In particular, it is recognized that standalone data do not contain enough information to fully resolve the structure of naturally disordered fluid bilayers, and therefore may not provide a robust determination of bilayer structure parameters, including the much-sought-after area per lipid. We then discuss the development of matter density-based models (including the SDP model) that allow for the joint refinement of different contrast neutron and X-ray data, as well as the implementation of local volume conservation within the unit cell (i.e., ideal packing). Such models provide natural definitions of bilayer thicknesses (most importantly the hydrophobic and Luzzati thicknesses) in terms of Gibbs dividing surfaces, and thus allow for the robust determination of lipid areas through equivalent slab relationships between bilayer thickness and lipid volume. In the final section of this review, we discuss some of the significant findings/features pertaining to structures of PC and PG bilayers as determined from SDP model analyses.
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