Domains In Biological Membranes Research Articles

Phase Behavior and Miscibility in Two-Component Glycolipid Monolayers.

Glycolipids are known to be involved in the formation of ordered functional domains in biological membranes. Since the structural characterization of such domains is difficult, most studies have so far dealt with lipid mixtures containing only one glycolipid component at a time, although biological membranes usually contain several glycolipid species, which can result in more complex structures and phase behavior. Here, we combine classical isotherm measurements with surface-sensitive grazing-incidence X-ray diffraction to investigate the phase behavior and miscibility in Langmuir monolayers of binary glycolipid mixtures. We find that the phase behavior has a subtle dependence on the saccharide headgroup chemistry. For compatible chemistries, molecular superlattice structures formed by one of the glycolipid species are conserved and can host foreign glycolipids up to a defined stoichiometry. In contrast, for sterically incompatible saccharide chemistries, the superlattice is lost even if both species are able to form such structures in their pure forms. Our results suggest that related phenomena may play important roles also in biological contexts.

Effect of cholesterol on the lactosylceramide domains in phospholipid bilayers

Lactosylceramide (LacCer) in the plasma membranes of immune cells is an important lipid for signaling in innate immunity through the formation of LacCer-rich domains together with cholesterol (Cho). However, the properties of the LacCer domains formed in multicomponent membranes remain unclear. In this study, we examined the properties of the LacCer domains formed in Cho-containing 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) membranes by deuterium solid-state NMR and fluorescence lifetimes. The potent affinity of LacCer-LacCer (homophilic interaction) is known to induce a thermally stable gel phase in the unitary LacCer bilayer. In LacCer/Cho binary membranes, Cho gradually destabilized the LacCer gel phase to form the liquid-ordered phase by its potent order effect. In the LacCer/POPC binary systems without Cho, the 2H NMR spectra of 10′,10′-d2-LacCer and 18′,18′,18′-d3-LacCer probes revealed that LacCer was poorly miscible with POPC in the membranes and formed stable gel phases without being distributed in the liquid crystalline domain. The lamellar structure of the LacCer/POPC membrane was gradually disrupted at around 60°C, whereas the addition of Cho increased the thermal stability of the lamellarity. Furthermore, the area of the LacCer gel phase and its chain order were decreased in the LacCer/POPC/Cho ternary membranes, whereas the liquid-ordered domain, which was observed in the LacCer/Cho binary membrane, was not observed. Cho surrounding the LacCer gel domain liberated LacCer and facilitated forming the submicron to nano-scale small domains in the liquid crystalline domain of the LacCer/POPC/Cho membranes, as revealed by the fluorescence lifetimes of trans-parinaric acid and trans-parinaric acid-LacCer. Our findings on the membrane properties of the LacCer domains, particularly in the presence of Cho, would help elucidate the properties of the LacCer domains in biological membranes.

Investigating the role of membrane dipole potential in liquid-liquid phase separation: ether lipids influence domain size

Lateral lipid heterogeneity is thought to play an important role in the functioning of cell membranes. Parameters such as the shape, size, and composition of domains are not well-understood in biological membranes, in part because of their chemical complexity. Factors including line tension, dipole repulsions, and thermal fluctuations contribute to these parameters. To elucidate the impact of these factors, we use simple three- or four- component model membranes. Liquid-liquid phase separation in these systems have provided numerous insights into the behavior of domains in biological membranes.

Computer simulations of a heterogeneous membrane with enhanced sampling techniques.

Computational determination of the equilibrium state of heterogeneous phospholipid membranes is a significant challenge. We wish to explore the rich phase diagram of these multi-component systems. However, the diffusion and mixing times in membranes are long compared to typical time scales of computer simulations. Here, we evaluate the combination of the enhanced sampling techniques molecular dynamics with alchemical steps and Monte Carlo with molecular dynamics with a coarse-grained model of membranes (Martini) to reduce the number of steps and force evaluations that are needed to reach equilibrium. We illustrate a significant gain compared to straightforward molecular dynamics of the Martini model by factors between 3 and 10. The combination is a useful tool to enhance the study of phase separation and the formation of domains in biological membranes.

The Dynamic Behavior Possibilities of Raft-Like Domains in Biological Membranes

A possible scenario of the behavior of a raft-like domain system oscillating near the phase transition point of the Verchulst transition type, when the form of the stationary distribution for the concentration of domains changes stepwise, has been considered. A stationary state of the system is also possible at the indicated phase transition point, as well as fluctuations in the state of the system between the modes of extinction and survival, if the analogy with the Verhulst model is applied. The system behavior is explored in the framework of the stochastic storage model. This model is compared with the Verhulst model of a biological population. Similarities and differences between the models are highlighted. There are no bifurcations and transition to chaos in the domain system. Other features and characteristics of the dynamic behavior and stationary states of the raft-like domain system are considered.

Detection of Pure Cholesterol Bilayer Domains in Biological Membranes Overloaded with Cholesterol: Methodology Development and its Application to Porcine Lens Membrane Studies

Detection of Pure Cholesterol Bilayer Domains in Biological Membranes Overloaded with Cholesterol: Methodology Development and its Application to Porcine Lens Membrane Studies

Effect of Chain Unsaturation and Temperature on Oxygen Diffusion Through Lipid Membranes from Simulations.

Rafts are nanoscale ordered domains in biological membranes that are rich in saturated phospholipids. In this study, the influence of chain unsaturation and temperature on oxygen diffusion through lipid membranes is examined using advanced computational modeling. The studied phospholipids with increasing unsaturation are: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The unsaturation correlates with the area per lipid and the order parameter. Oxygen diffusion is found to be faster at higher temperature, and the solubility of oxygen in the membrane with respect to water decreases. Diffusion varies over a larger range across the membrane at 323 K in DPPC than in DOPC, whereas POPC has intermediate diffusivity. Oxygen diffusion in saturated lipids is faster at the membrane center and slower near the head group region than in unsaturated lipids. Oxygen solubility in DPPC is higher than in unsaturated lipids.

Identification of Sphingolipid-binding Motif in G Protein-coupled Receptors.

Sphingolipids correspond to a major class of lipids which serve as indispensable structural components of membranes and play an important role in various cellular functions. They constitute ~10-20% of total membrane lipids and are known to form segregated domains in biological membranes. Sphingolipids have been shown to play a vital role in the function of various G protein-coupled receptors (GPCRs). We report here the presence of sphingolipid-binding motif (SBM) in representative GPCRs such as cholecystokinin, oxytocin and secretin receptors, and subtypes of human serotonin receptors. We previously reported the importance of sphingolipids in the function of the serotonin1A receptor, a representative member of the GPCR superfamily, involved in behavioral, cognitive, and developmental functions. In this work, we show that the serotonin1A receptor contains a putative SBM, corresponding to amino acids 205 to 213. In addition, our analysis shows that SBM is an intrinsic characteristic feature of the serotonin1A receptor and is conserved throughout the course of natural evolution. Our results represent the first report on the presence of SBM in serotonin1A receptors and provide novel insight on the molecular mechanism of GPCR-sphingolipid interaction.

Impact of external influences on characteristics of domains in biological membranes

Living systems continually interact with the environment. External influences evoke complex processes in the entire system. In this work, the impact of external influences on the behavior of the domains in biological membranes is considered both in general and on special examples. A general approach of nonequilibrium statistical thermodynamics proposed by L.A. Stratonovich along with the same approach adapted to stochastic storage processes are applied. Both the Stratonovich theory and the storage models allow characterizing specific features of the stationary non-equilibrium states as well as the behavior of the highly non-equilibrium processes in open systems, such as live organisms.

Lateral Heterogeneity of Cholesterol on Binary Lipid Mixtures of POPC/Chol Imaged with AFM

Phase diagrams on ternary and quaternary lipid mixtures showing the existence of liquid-ordered (lo), liquid-disordered (ld) and a mixed phase (lo+ld) are now well established. However the existence of such a phase diagram on binary mixtures is still in debate despite reports of a phase diagram on the mixtures POPC/Chol and POPC/Erg and nystatin activity co-related to this purported phase diagram; with a maximal activity appearing in the mixed phase. Recent Molecular Dynamics simulations of POPC/Chol at different temperatures and cholesterol concentrations along this phase diagram, suggest the existence of cholesterol enriched nano-sites in this mixed phase, indicating the presence of cholesterol lateral heterogeneity. Here we present atomic force microscopy images of supported lipid bilayers made up of POPC and different cholesterol concentrations along the phase diagram. We observe structures that are consistent with the Molecular Dynamics predictions of order parameters and membrane thickness, supporting the idea of cholesterol lateral heterogeneity in binary mixtures, which can enrich the discussion on the existence of lipid rafts and domains in biological membranes. Funding: DGAPA-PAPIIT-RG100416.

Dynamics of sphingomyelin- and cholesterol-enriched lipid domains during cytokinesis.

Sphingomyelin (SM) and cholesterol (Chol) are the major lipids in the mammalian cells, which are mainly localized to the plasma membrane. Multiple lines of evidence suggest that these lipids form local lipid domains in the plasma membrane, playing functional roles in the cell. Several observations have suggested that these lipid domains are required for cytokinesis. In this chapter, we show the methods for visualizing SM-rich and/or Chol-rich membrane domains at cytokinesis by using specific lipid-binding proteins. Lysenin, equinatoxin II, perfringolysin O, and pleurotolysin A2 bind specifically to clustered SM-rich domain, dispersed SM-rich domain, Chol-rich domain, and SM/Chol mixtures, respectively. Nontoxic forms of these lipid-binding proteins fused to fluorescent proteins are used for imaging lipid domains in biological membranes at cytokinesis. The image analysis reveals the structures and functions of SM-rich and/or Chol-rich domains at the time of cytokinesis.

Stochastic model of domain kinetics in biological membranes

A stochastic storage model based on the behavior of macroscopic variables of the system is used to describe the kinetics of raft-like domains in biological membranes. For a simple output model, we examine the features of the system behavior corresponding to the noise-induced nonequilibrium phase transitions. Characteristics of the behavior of the statistical system are obtained: an explicit form of the stationary distribution of the number of domains; ratios for the phase transition points; the expression for the first two moments of the random domain concentration, and the expression for the lifetime of membrane domains in the stationary state.

The Biophysics of Living Membranes: Protein Partitioning and Functional Differentiation in Ordered Plasma Membrane Domains

The lipid raft hypothesis posits that ordered, lateral membrane domains are important for the regulation of plasma membrane (PM) structure and function in eukaryotes. Giant Plasma Membrane Vesicles (GPMVs) are isolated plasma membranes that microscopically separate into coexisting ordered and disordered phases, facilitating experimental analysis of the composition and physical properties of ordered raft domains in biological membranes. In these studies, we analyze the biophysical and lipidomic differentiation of the plasma membrane in Mesenchymal Stem Cells (MSC) as the cells undergo differentiation into adipocytes and osteoblasts. During differentiation, dramatic remodeling the PM lipidome, including lipid acyl chain length and unsaturation, results inchanges to membrane order and phase separation. These observations elucidate the compositional determinants of biophysical properties in biological membranes, as well as identify lineage-specific PM features. These results facilitate rational remodeling of membrane phenotypes to direct differentiation, as supplementation with ꙍ-3 docosahexaneoic acid (DHA) promoted the osteoblastic PM phenotype and potentiated osteogenic differentiation. The differences in the physical properties of the coexisting domains lead to preferential protein partitioning between them. We evaluate the structural determinants of raft partitioning of a model transmembrane protein, and find that raft phase partitioning is related to features of the protein's transmembrane domain (TMD), namely the hydrophobic length and surface area of the TMD. Longer TMDs impart greater raft association while TMDs with larger, bulkier amino acid side chains prefer the non-raft phase. We present a simple physical model wherein raft partitioning is driven by phase-dependent differences in interfacial energy between the TMD and its surrounding lipid matrix, and find excellent quantitative agreement with observations that provide the first predictions of protein-lipid surface tension. These results point the way to a general rule for raft partitioning of transmembrane proteins.

Combination of MD Simulations with Two-State Kinetic Rate Modeling Elucidates the Chain Melting Transition of Phospholipid Bilayers for Different Hydration Levels.

The phase behavior of membrane lipids plays an important role in the formation of functional domains in biological membranes and crucially affects molecular transport through lipid layers, for instance, in the skin. We investigate the thermotropic chain melting transition from the ordered Lβ phase to the disordered Lα phase in membranes composed of dipalmitoylphosphatidylcholine (DPPC) by atomistic molecular dynamics simulations in which the membranes are subject to variable heating rates. We find that the transition is initiated by a localized nucleus and followed by the propagation of the phase boundary. A two-state kinetic rate model allows characterizing the transition state in terms of thermodynamic quantities such as transition state enthalpy and entropy. The extrapolated equilibrium melting temperature increases with reduced membrane hydration and thus in tendency reproduces the experimentally observed dependence on dehydrating osmotic stress.

Modulation of a Small Two-Domain Lipid Vesicle by Linactants

Linactants, molecules that preferentially localize at the boundary of lipid membrane domains, are attracting considerable attention in recent years due to the recognition that they might regulate lipid-phase separation and thereby modulate membrane morphology. Recent studies have also shown that clustering of some line active agents enhances their ability to modulate membrane curvature. However, the molecular origin of this phenomenon, and the degree to which it impacts biological membranes, remains poorly understood. In this work, we have investigated how linactants induce shape change in multidomain small unilamallar vesicles (SUVs) using extensive dissipative particle dynamics simulations. The linactant was modeled as a two-tailed hybrid lipid with the two tails differing in preference for different lipid domains. We found that addition of a small amount of linactants (∼1%) to a two-domain vesicle leads to substantial reduction in the line tension and neck curvature at the domain boundary. Using cross-linking as a surrogate for clustering, we further show that linactant clusters substantially enhance the boundary preference and therefore the reduction in neck curvature. Moreover, on the basis of analyses of the corresponding changes in the membrane energetics, we highlight how linactants might stabilize nanoscale domains. These results have important implications for the potential existence and physical explanations of nanosized domains in biological membranes.

Identifying Key Juxtamembrane Interactions in Cell Membranes Using AraC-based Transcriptional Reporter Assay (AraTM)

Dimerization is a key regulatory mechanism in activation of transmembrane (TM) receptors during signal transduction. This process involves a coordinated interplay between extracellular (EX), TM, and cytoplasmic (CYTO) regions to form a specific interface required for both ligand binding and intracellular signaling to occur. While several transcriptional activator-based methods exist for investigating TM interactions in bacterial membranes, expression of TM chimera in these methods occurs in a reverse orientation, and are limited to only TM domains for proper membrane trafficking and integration. We therefore developed a new, AraC-based transcriptional reporter assay (AraTM) that expresses EX-TM-CYTO chimera in their native orientation, thereby enabling membrane trafficking to occur independent of the TM chimera used as well as permitting analysis of EX-TM-CYTO interactions in biological membranes. Using integrin α(IIb) TM-CYTO as a model, we observe a large increase in homodimerization for the constitutively active TM mutant L980A relative to wild-type in the TM-CYTO construct (A963-E1008). We also characterized the receptor for advanced glycation endproducts (RAGE), whose homooligomeric state is critical in ligand recognition, and find the specific juxtamembrane region within the CYTO (A375-P394) mediates homodimerization, and is dominant over effects observed when the extracellular C2 domain is included. Furthermore, we find good agreement between our AraTM measurements in bacterial membranes and BRET measurements made on corresponding RAGE constructs expressed in transfected HEK293 cells. Overall, the AraTM assay provides a new approach to identify specific interactions between receptor EX-TM-CYTO domains in biological membranes that are important in regulation of signal transduction.

First steps to rafts?

Single-molecule observations reveal that lipid- and protein-based interactions jointly contribute to the interactions among glycosylphosphatidylinositol-anchored proteins in membranes. Understanding these interactions will help to refine long-evolving (and still debated) models of 'raft' domains in biological membranes.

Concentration fluctuations and phase transitions in coupled modulated bilayers

We consider the formation of finite-size domains in lipid bilayers consisting of saturated and hybrid lipids. First, we describe a monolayer model that includes a coupling between a compositional scalar field and a two-dimensional vectorial order parameter. Such a coupling yields an effective two-dimensional microemulsion free energy for the lipid monolayer, and its characteristic length of compositional modulations can be considered as the origin of finite-size domains in biological membranes. Next, we consider a coupled bilayer composed of two modulated monolayers and discuss the static and dynamic properties of concentration fluctuations above the transition temperature. We also investigate the micro-phase separation below the transition temperature and compare the micro-phase separated structures with statics and dynamics of concentration fluctuations above the transition.

ChemInform Abstract: Crystalline Lipid Domains: Characterization by X‐Ray Diffraction and Their Relation to Biology

AbstractReview: ca. 80 refs.

Water Meniscus-Directed Organization of Liquid-Ordered Domains in Lipid Monolayer

We report on a spatial organization process of liquid-ordered (l(o)) domains in a lipid monolayer into a two dimensional array on structurally patterned substrates. The curvature of a lipid monolayer which strongly obeys the water meniscus at an air-water interface is manipulated to create two different, curved and flat, zones during the water evaporation in the structural patterns. Due to the high bending rigidity, l(o) domains are diffused into the flat region to avoid the elastic deformation. The Helfrich-type thermodynamic criterion for coarsening of l(o) domains was first presented and confirmed through experimental results obtained in various patterns. Our topographic system will be very useful for understanding a fundamental concept as well as a quantitative criterion for curvature-driven organization of l(o) domains in biological membranes.