Sub-ten-nanometer heterogeneity of solid supported lipid membranes determined by solution atomic force microscopy

We review the combined effect of temperature and pressure on the structure, dynamics and phase behavior of lipid bilayers, differing in chain length, headgroup structure and composition as revealed by thermodynamic, spectroscopic and scattering experiments. The effect of additives, such as ions, cholesterol, and anaesthetics is discussed as well. Our data include also reports on the effect of pressure on the lateral organization of heterogeneous lipid membranes and lipid extracts from cellular membranes, as well as the influence of peptide and protein incorporation on the pressure-dependent structure and phase behavior of lipid membranes. Moreover, the effects of pressure on membrane protein function are summarized. Finally, we introduce pressure as a kinetic variable for studying the kinetics of various lipid phase transformations.

Certain fatty acid isomers have been linked to the development of several pathological conditions. For example, while oleic acid – a monounsaturated, cis fatty acid – is considered part of a healthy diet, its trans analogue elaidic acid is associated with an increased risk of developing cardiovascular diseases. Fatty acids can insert into cellular lipid membranes, changing their structural and biophysical features, and these alterations are believed to underpin some of the biomedical effects of these molecules. In this work, we unveil the effect of oleic and elaidic acid on the structure, lateral organization, and microviscosity of model membranes by using a combination of X-ray scattering and viscosity-sensitive dyes (‘molecular rotors’). Our results suggest that the cis isomer, oleic acid, is a more effective lineactant and minimizes the height mismatch between different lipid phases while maintaining the membrane’s lateral heterogeneity. We demonstrate that, as a consequence, the presence of oleic acid successfully dampens changes in membrane microviscosity under tension.

Complex Coacervate Formation on a Heterogeneous Membrane

On the lateral structure of model membranes containing cholesterol

Incorporating membrane proteins, which are major targets of drug discovery, into artificial planar lipid bilayer membranes is important. From the observation at the high spatiotemporal resolution in the microscopic region, the intermolecular interactions between lipids and membrane proteins can be revealed. It leads to elucidation of the reaction mechanisms occurring in biological membranes. However, when a lipid membrane supported on a solid substrate (SLB) is used, the following points are interfering with the research. The first point is the structural change due to the contact between the membrane protein and the underlying substrate. The other is disorder of the membrane protein orientation in the SLB. Research on the incorporation of membrane proteins has been done in the field of both physical and biological chemistry, but few reports have achieved detailed measurements with placing emphasis on the plasticity and diversity of membrane proteins. In this study, the tethered bilayer lipid membrane (tBLM) was formed on the atomically flat self-assembled monolayer modified surface which specifically binds to the membrane proteins in the membrane. The fluidity of artificial lipid bilayer and orientation of membrane proteins are observed by the epi-fluorescence microscopy and atomic force microscopy, respectively. The consequent decreased interaction between the substrate and lipid membrane and limited observation for the specific orientation of membrane proteins suggest that it was possible to reproduce the same environment as the biological membrane even in the artificial lipid bilayer membrane. The present tBLM system should be useful for the creation of basic technologies for structural, functional, and dynamical analysis of membrane proteins. It also leads to the understanding the molecular structure of biological membrane which include the various membrane proteins such as ion channels, transporters, pumps and enzymes with addition of more complex structural properties such as the underlying membrane scaffolds, asymmetric lipid composition and domains.

Towards biomimics of cell membranes: Structural effect of phosphatidylinositol triphosphate (PIP3) on a lipid bilayer.

Lateral organization and dynamics of lipids in plasma membranes are crucial for several cellular processes such as signal transduction across the membrane and still remain elusive. In this paper, using coarse-grained molecular dynamics simulation, we theoretically study the combined effects of headgroup charge and tail unsaturation of lipids on the lateral organization and diffusion of lipids in ternary lipid bilayers. In neutral ternary lipid bilayers composed of saturated lipids, unsaturated lipids, and cholesterols, under the conditions of given temperature and components, the main factor for the phase separation is the unsaturation of unsaturated lipids and the bilayers can be separated into liquid-ordered domains enriched in saturated lipids and cholesterols and liquid-disordered domains enriched in unsaturated lipids. Once the headgroup charge is introduced, the electrostatic repulsion between the negatively charged lipid headgroups will increase the distance between the charged lipids. We find that the lateral organization and diffusion of the lipids in the (partially) charged ternary lipid bilayers are determined by the competition between the headgroup charge and the unsaturation of the unsaturated lipids. In the bilayers containing unsaturated lipids with lower unsaturation, the headgroup charge plays a crucial role in the lateral organization and diffusion of lipids. The headgroup charge may make the lipid domains unstable and even can suppress phase separation of the lipids in some systems. However, in the bilayers containing highly unsaturated lipids, the lateral organization and diffusion of lipids are mainly dominated by the unsaturation of the unsaturated lipids. This work may provide some theoretical insights into understanding the formation of nanosized domains and lateral diffusion of lipids in plasma membranes.

We study the stress distribution profiles and the height and thickness fluctuations of lipid membranes in the gel Lβ′ state by Monte Carlo simulations of a generic coarse-grained model for lipid membranes, which reproduces many known properties of dipalmitoylphosphatidyncholine (DPPC) bilayers. The results are related to the corresponding properties of fluid membranes, and to theoretical predictions for crystalline and hexatic membranes. One striking observation is that the spontaneous curvature of the monolayers changes sign from the fluid to the gel phase. In the gel-phase, the long-wavelength height fluctuations are suppressed, and the fluctuation spectrum is highly anisotropic. In the direction of tilt, it carries the signature of soft modes with wavelengths compatible to those of the ripple phase Pβ′, which emerges in the transition region between the fluid and the gel state. In the direction perpendicular to the tilt, the thickness fluctuations are almost entirely suppressed, and the height fluctuations seem to be dominated by an interfacial energy, i.e., by out-of-layer fluctuations, up to length scales of tens of nanometres.

We investigate the hydrodynamic effects on the dynamics of critical concentration fluctuations in multicomponent fluid membranes. Two geometrical cases are considered; (i) confined membrane case and (ii) supported membrane case. We numerically calculate the wavenumber dependence of the effective diffusion coefficient by changing the temperature and/or the thickness of the bulk fluid. For some limiting cases, the result is compared with the previously obtained analytical expression. An analogy of the multicomponent membrane to 2D microemulsions is explored for the confined membrane geometry.

Sphingomyelin Acyl Chains Influence the Formation of Sphingomyelin- and Cholesterol-Enriched Domains

Multiscale simulations of heterogeneous model membranes

Biophysical Characterization of Lipid Membranes: Effect of Lipid Head Groups and Tails on Synthetic and Natural Lipid Membranes

In the central nervous system, lipid-protein interactions are pivotal for myelin maintenance, as these interactions regulate protein transport to the myelin membrane as well as the molecular organization within the sheath. To improve our understanding of the fundamental properties of myelin, we focused here on the lateral membrane organization and dynamics of peripheral membrane protein 18.5-kDa myelin basic protein (MBP) and transmembrane protein proteolipid protein (PLP) as a function of the typical myelin lipids galactosylceramide (GalC), and sulfatide, and exogenous factors such as the extracellular matrix proteins laminin-2 and fibronectin, employing an oligodendrocyte cell line, selectively expressing the desired galactolipids. The dynamics of MBP were monitored by z-scan point fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS), while PLP dynamics in living cells were investigated by circular scanning FCS. The data revealed that on an inert substrate the diffusion rate of 18.5-kDa MBP increased in GalC-expressing cells, while the diffusion coefficient of PLP was decreased in sulfatide-containing cells. Similarly, when cells were grown on myelination-promoting laminin-2, the lateral diffusion coefficient of PLP was decreased in sulfatide-containing cells. In contrast, PLP's diffusion rate increased substantially when these cells were grown on myelination-inhibiting fibronectin. Additional biochemical analyses revealed that the observed differences in lateral diffusion coefficients of both proteins can be explained by differences in their biophysical, i.e., galactolipid environment, specifically with regard to their association with lipid rafts. Given the persistence of pathological fibronectin aggregates in multiple sclerosis lesions, this fundamental insight into the nature and dynamics of lipid-protein interactions will be instrumental in developing myelin regenerative strategies.

Phase-Field Modeling and Simulations of Lipid Membranes Coupling Composition with Membrane Mechanical Properties

Myelin basic protein (MBP) is an intrinsically disordered protein and in the central nervous system (CNS) mainly responsible for connecting the cytoplasmic surfaces of the multilamellar, compact myelin. Increased posttranslational modification of MBP is linked to both, the natural development (from adolescent to adult brains) of myelin, and features of multiple sclerosis. Here, we study how a combination of this intrinsically disordered myelin protein with varying the natural cholesterol content may alter the characteristics of myelin-like membranes and interactions between these membranes. Large unilamellar vesicles (LUVs) with a composition mimicking the cytoplasmic leaflet of myelin were chosen as the model system, in which different parameters contributing to the interactions between the lipid membrane and MBP were investigated. While we use cryo-transmission electron microscopy (TEM) for imaging, dynamic light scattering (DLS) and electrophoretic measurements through continuously-monitored phase-analysis light scattering (cmPALS) were used for a more global overview of particle size and charge, and electron paramagnetic resonance (EPR) spectroscopy was utilized for local behavior of lipids in the vesicles' membranes in aqueous solution. The cholesterol content was varied from 060 % in these LUVs and measurements were performed in the presence and absence of MBP. We find that the composition of the lipid layers is relevant to the interaction with MBP. Not only the size, the shape and the aggregation behavior of the vesicles depend on the cholesterol content, but also within each membrane, cholesterol's freedom of movement, its environmental polarity and its distribution were found to depend on the content using the EPR-active spin-labeled cholesterol (CSOSL). In addition, DLS and EPR measurements probing the transition temperatures of the lipid phases allow a correlation of specific behavior with the human body temperature of 37 °C. Overall, our results aid in understanding the importance of the native cholesterol content in the healthy myelin membrane, which serves as the basis for stable and optimum protein-bilayer interactions. Although studied in this specific myelin-like system, from a more general and materials science-oriented point of view, we could establish how membrane and vesicle properties depend on cholesterol and/or MBP content, which might be useful generally when specific membrane and vesicle characteristics are sought for.