An Unexpected Driving Force for Lipid Order Appears in Asymmetric Lipid Bilayers.

An ordered phase in one leaflet of an asymmetric bilayer can induce a precisely superimposed in the apposed leaflet. Order is induced in such simple lipid compositions as dioleoylphosphatidylcholine/cholesterol (DOPC)/chol) which is expected to be a uniform and disordered lipid mixture. Dye partitioning can be used to label and identify coexisting liquid-disordered (Ld), liquid-ordered (Lo), or gel-ordered (Lβ) molecules in a phase-separated leaflet.

Nano-scale domains in the plasma membrane are like macroscopic domains in asymmetric bilayers.

Unfavorable lipid-lipid pairwise interactions between HiTm and LowTm lipids drive liquid-disordered (Ld) + liquid-ordered (Lo) phase separation. Large size of phase domains is opposed by lipid dipole repulsions, which are more significant compared with the pairwise interactions for naturally abundant LowTm lipids such as palmitoyl oleoyl phosphatidylcholine. During the nano-to-macro domain size transition, no lipid phase transition occurs, and measured properties of Ld + Lo nanodomains are found to be essentially the same as those of macrodomains.

Improving our picture of the plasma membrane: Rafts induce ordered domains in a simplified model cytoplasmic leaflet.

By study of asymmetric membranes, models of the cell plasma membrane (PM) have improved, with more realistic properties of the asymmetric lipid composition of the membrane being explored. We used hemifusion of symmetric giant unilamellar vesicles (GUVs) with a supported lipid bilayer (SLB) to engineer bilayer leaflets of different composition. During hemifusion, only the outer leaflets of GUV and SLB are connected, exchanging lipids by simple diffusion.

On the small size of liquid-disordered + liquid-ordered nanodomains.

Four-component phase diagrams reveal that Liquid-disordered + liquid-ordered (Ld + Lo) nanodomains are exclusively found adjacent to a three-phase region, and so cannot be a one-phase microemulsion. Of importance for understanding biological membranes, a small change in lipid bilayer composition can change the size of these coexisting phase domains hundreds of fold, between tens of nanometers and microns. Nanodomain diameter, measured from small angle neutron scattering, is in the range 15-35 nm, consistent with stabilization by repulsive dipole fields.

Dataset of asymmetric giant unilamellar vesicles prepared via hemifusion: Observation of anti-alignment of domains and modulated phases in asymmetric bilayers.

The data provided with this paper are confocal fluorescence images of symmetric giant unilamellar vesicles (GUVs) and asymmetric giant unilamellar vesicles (aGUVs). In this work, aGUVs were prepared using the hemifusion method and are labelled with two different fluorescent dyes, named TFPC and DiD. Both dyes show strong preference for the liquid-disordered (Ld) phase instead of the liquid-ordered (Lo) phase.

Investigation of the domain line tension in asymmetric vesicles prepared via hemifusion.

The plasma membrane (PM) is asymmetric in lipid composition. The distinct and characteristic lipid compositions of the exoplasmic and cytoplasmic leaflets lead to different lipid-lipid interactions and physical-chemical properties in each leaflet. The exoplasmic leaflet possesses an intrinsic ability to form coexisting ordered and disordered fluid domains, whereas the cytoplasmic leaflet seems to form a single fluid phase.

PI(4,5)P Clustering and Its Impact on Biological Functions.

Located at the inner leaflet of the plasma membrane (PM), phosphatidyl-inositol 4,5-bisphosphate [PI(4,5)P] composes only 1-2 mol% of total PM lipids. With its synthesis and turnover both spatially and temporally regulated, PI(4,5)P recruits and interacts with hundreds of cellular proteins to support a broad spectrum of cellular functions. Several factors contribute to the versatile and dynamic distribution of PI(4,5)P in membranes.

Bilayer compositional asymmetry influences the nanoscopic to macroscopic phase domain size transition.

The eukaryotic plasma membrane (PM) exhibits lipid mixing heterogeneities known as lipid rafts. These lipid rafts, the result of liquid-liquid phase separation, can be modeled by coexisting liquid ordered (Lo) and liquid disordered (Ld) domains. Four-lipid component systems with a high-melting lipid, a nanodomain-inducing low-melting lipid, a macrodomain-inducing low-melting lipid, and cholesterol (chol) can give rise to domains of different sizes.

Mechanisms of PI(4,5)P2 Enrichment in HIV-1 Viral Membranes.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is critical for HIV-1 virus assembly. The viral membrane is enriched in PIP2, suggesting that the virus assembles at PIP2-rich microdomains. We showed previously that in model membranes PIP2 can form nanoscopic clusters bridged by multivalent cations.

Asymmetric Bilayers by Hemifusion: Method and Leaflet Behaviors.

We describe a new method to prepare asymmetric giant unilamellar vesicles (aGUVs) via hemifusion. Hemifusion of giant unilamellar vesicles and a supported lipid bilayer, triggered by calcium, promotes the lipid exchange of the fused outer leaflets mediated by lipid diffusion. We used different fluorescent dyes to monitor the inner and the outer leaflets of the unsupported aGUVs.

Physical Principles of Membrane Shape Regulation by the Glycocalyx.

Cells bend their plasma membranes into highly curved forms to interact with the local environment, but how shape generation is regulated is not fully resolved. Here, we report a synergy between shape-generating processes in the cell interior and the external organization and composition of the cell-surface glycocalyx. Mucin biopolymers and long-chain polysaccharides within the glycocalyx can generate entropic forces that favor or disfavor the projection of spherical and finger-like extensions from the cell surface.

Molecular Dynamics Simulations Reveal Leaflet Coupling in Compositionally Asymmetric Phase-Separated Lipid Membranes.

The eukaryotic plasma membrane has an asymmetric distribution of its component lipids. Rafts that result from liquid-liquid phase separation are a feature of its exoplasmic leaflet, but how these exoplasmic leaflet domains are coupled to the cytoplasmic leaflet is not understood. These rafts can be studied in model membranes of three-component mixtures that produce coexisting liquid ordered (Lo) and liquid disordered (Ld) domains.

Gramicidin Increases Lipid Flip-Flop in Symmetric and Asymmetric Lipid Vesicles.

Unlike most transmembrane proteins, phospholipids can migrate from one leaflet of the membrane to the other. Because this spontaneous lipid translocation (flip-flop) tends to be very slow, cells facilitate the process with enzymes that catalyze the transmembrane movement and thereby regulate the transbilayer lipid distribution. Nonenzymatic membrane-spanning proteins with unrelated primary functions have also been found to accelerate lipid flip-flop in a nonspecific manner and by various hypothesized mechanisms.

Lowering line tension with high cholesterol content induces a transition from macroscopic to nanoscopic phase domains in model biomembranes.

Chemically simplified lipid mixtures are used here as models of the cell plasma membrane exoplasmic leaflet. In such models, phase separation and morphology transitions controlled by line tension in the liquid-disordered (Ld) + liquid-ordered (Lo) coexistence regime have been described [1]. Here, we study two four-component lipid mixtures at different cholesterol fractions: brain sphingomyelin (BSM) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/cholesterol (Chol).

Presence and Role of Midplane Cholesterol in Lipid Bilayers Containing Registered or Antiregistered Phase Domains.

Three-component lipid mixtures can produce coexisting liquid ordered and liquid disordered phases, a model for eukaryotic plasma membrane rafts. In compositionally symmetric bilayers with two phase-separated leaflets, phase domains of the two leaflets may align through registration, where domains are found across from domains of the same phase, or else antiregistration, where domains are found across from domains of the opposite phase. This alignment could serve as a method of information communication across the plasma membrane.

Multivalent Cation-Bridged PI(4,5)P Clusters Form at Very Low Concentrations.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P or PIP2), is a key component of the inner leaflet of the plasma membrane in eukaryotic cells. In model membranes, PIP2 has been reported to form clusters, but whether these locally different conditions could give rise to distinct pools of unclustered and clustered PIP2 is unclear. By use of both fluorescence self-quenching and Förster resonance energy transfer assays, we have discovered that PIP2 self-associates at remarkably low concentrations starting below 0.

Membrane Bending Moduli of Coexisting Liquid Phases Containing Transmembrane Peptide.

A number of highly curved membranes in vivo, such as epithelial cell microvilli, have the relatively high sphingolipid content associated with "raft-like" composition. Given the much lower bending energy measured for bilayers with "nonraft" low sphingomyelin and low cholesterol content, observing high curvature for presumably more rigid compositions seems counterintuitive. To understand this behavior, we measured membrane rigidity by fluctuation analysis of giant unilamellar vesicles.

FRET Detects the Size of Nanodomains for Coexisting Liquid-Disordered and Liquid-Ordered Phases.

Biomembranes with as few as three lipid components can form coexisting liquid-disordered (Ld) and liquid-ordered (Lo) phases. In the coexistence region of Ld and Lo phases, the lipid mixtures 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/chol or brain sphingomyelin (bSM)/DOPC/chol form micron-scale domains that are easily visualized with light microscopy. Although large domains are not observed in the mixtures DSPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/chol and bSM/POPC/chol, lateral heterogeneity is nevertheless detected using techniques with nanometer-scale spatial resolution.

Cholesterol Promotes Protein Binding by Affecting Membrane Electrostatics and Solvation Properties.

Binding of the retroviral structural protein Gag to the cellular plasma membrane is mediated by the protein's matrix (MA) domain. Prominent among MA-PM interactions is electrostatic attraction between the positively charged MA domain and the negatively charged plasma membrane inner leaflet. Previously, we reported that membrane association of HIV-1 Gag, as well as purified Rous sarcoma virus (RSV) MA and Gag, depends strongly on the presence of acidic lipids and is enhanced by cholesterol (Chol).

Line Tension Controls Liquid-Disordered + Liquid-Ordered Domain Size Transition in Lipid Bilayers.

To better understand animal cell plasma membranes, we studied simplified models, namely four-component lipid bilayer mixtures. Here we describe the domain size transition in the region of coexisting liquid-disordered (Ld) + liquid-ordered (Lo) phases. This transition occurs abruptly in composition space with domains increasing in size by two orders of magnitude, from tens of nanometers to microns.

Effects of Membrane Charge and Order on Membrane Binding of the Retroviral Structural Protein Gag.

Unlabelled: The retroviral structural protein Gag binds to the inner leaflet of the plasma membrane (PM), and many cellular proteins do so as well. We used Rous sarcoma virus (RSV) Gag together with membrane sensors to study the principles governing peripheral protein membrane binding, including electrostatics, specific recognition of phospholipid headgroups, sensitivity to phospholipid acyl chain compositions, preference for membrane order, and protein multimerization. We used an in vitro liposome-pelleting assay to test protein membrane binding properties of Gag, the well-characterized MARCKS peptide, a series of fluorescent electrostatic sensor proteins (mNG-KRn), and the specific phosphatidylserine (PS) binding protein Evectin2.

Subnanometer Structure of an Asymmetric Model Membrane: Interleaflet Coupling Influences Domain Properties.

Cell membranes possess a complex three-dimensional architecture, including nonrandom lipid lateral organization within the plane of a bilayer leaflet, and compositional asymmetry between the two leaflets. As a result, delineating the membrane structure-function relationship has been a highly challenging task. Even in simplified model systems, the interactions between bilayer leaflets are poorly understood, due in part to the difficulty of preparing asymmetric model membranes that are free from the effects of residual organic solvent or osmotic stress.

Effects of Transmembrane α-Helix Length and Concentration on Phase Behavior in Four-Component Lipid Mixtures: A Molecular Dynamics Study.

We used coarse-grained molecular dynamics simulations to examine the effects of transmembrane α-helical WALP peptides on the behavior of four-component lipid mixtures. These mixtures contain a high-melting temperature (high-Tm) lipid, a nanodomain-inducing low-Tm lipid, a macrodomain-inducing low-Tm lipid and cholesterol to model the outer leaflet of cell plasma membranes. In a series of simulations, we incrementally replace the nanodomain-inducing low-Tm lipid by the macrodomain-inducing low-Tm lipid and measure how lipid and phase properties are altered by the addition of WALPs of different length.

Controlling the taste receptor accessible structure of rebaudioside A via binding to bovine serum albumin.

We illustrate a method that uses bovine serum albumin (BSA) to control the receptor-accessible part of rebaudioside A (Reb A). The critical micelle concentration (CMC) of Reb A was found to be 4.5 mM and 5 mM at pH 3 and 6.