Top Leaflet Research Articles

Identification of QTLs and joint QTL segments of leaflet traits at different canopy layers in an interspecific RIL population of soybean.

A leaflet trait on different canopy layers may have different QTLs; leaflet trait QTLs may cluster to form joint QTL segments; all canopy layer QTLs form a complete QTL system for a leaflet trait. As the main part of the plant canopy structure, leaf/leaflet size and shape affect the plant architecture and yield. To explore the leaflet trait QTL system, a population composed of 199 recombinant inbred lines derived from Changling (annual wild, narrow leaflet) and Yiqianli (landrace, broad leaflet) with their parents was tested for leaflet length (LL), width (LW) and length to width (LLW). The population was genotyped with specific-locus amplified fragment sequencing (SLAF-seq) and applied for linkage mapping of the leaflet traits. The results showed that the leaflet traits varied greatly even within a plant, which supported a stratified leaflet sampling strategy to evaluate these traits at top, middle and bottom canopy layers. Altogether, 13 LL, 10 LW and 9 LLW in a total of 32 plus 3 duplicated QTLs were identified, in which, 17 QTLs were new ones, and 48.6%, 28.6% and 22.8% of QTLs were from the top, middle and bottom layers, respectively, indicating the genetic importance of the top layer leaves. Since a leaflet trait may have layer-specific QTLs, all layer QTLs form a complete QTL system. Five QTL clusters each with their QTL supporting intervals overlapped were designated as joint QTL segments (JQSs). In JQS-16, with its linkage map further validated using PCR markers, two QTLs, qLW-16-1 and qLLW-16-1 of the top layer leaflet, were identified six QTL·times. Six candidate genes were predicted, with Glyma.16G127900 as the most potential one for LW and LLW. Three PCR markers, Gm16PAV0653, BARCSOYSSR_16_0796 and YC-16-3, were suggested for marker-assisted selection for LW and LLW in JQS-16.

Self-Uptake Mechanism of Polymyxin-Based Lipopeptide against Gram-Negative Bacterial Membrane: Role of the First Adsorbed Lipopeptide.

Research in the continuously increasing threat of polymyxin-resistant multidrug-resistant Pseudomonas aeruginosa, which causes severe infection in immunocompromised patients, has resulted in the development of several polymyxin-derived cyclic lipopeptides containing l-α-γ- diamino butyric acid-like FADDI-019 (F19). In this work, F19's insertion into a minimal model of the asymmetric outer membrane of the bacterium, which contained only penta-acylated lipid A (LipA) and lacked keto-d-octulosonic acid and O-antigens, in the top leaflet and phospholipids in the bottom leaflet, was studied. F19 exhibited all of the hallmarks of the self-uptake mechanism into the asymmetric bilayer. While a single monomer of the lipopeptide did not get partitioned into the inside of the bilayer, it competitively displaced Ca2+ from the membrane surface, observed as a decrease in Ca2+ coordination number with phosphate groups (1.89 vs 1.718), resulting in membrane destabilization. This resulted in an increment of the average defect size and the probability of interplay between lipid tails and hydrophobic residues of another F19. When more than one monomer was present in the system, the first monomer remained docked on the surface, while other monomers intercalated into the bilayer interior with their hydrophobic moieties "sleeved" by lipid acyl chains. The free energy barrier for partial insertion of the lipopeptide into a bilayer in the presence of surface-docked second F19 was recorded at ∼1.3 kcal/mol using two-dimensional (2D) well-tempered metadynamics, making it a low barrier process at 300 K. This study is an attempt to demonstrate the self-uptake mechanism of F19 during intercalation process into the bilayer interior, which may help in the design of better alternates for polymyxins to work against polymyxin resistance.

Systematic measurements of interleaflet friction in supported bilayers

When lipid membranes curve or are subjected to strong shear forces, the two apposed leaflets of the bilayer slide past each other. The drag that one leaflet creates on the other is quantified by the coefficient of interleaflet friction, b. Existing measurements of this coefficient range over several orders of magnitude, so we used a recently developed microfluidic technique to measure it systematically in supported lipid membranes. Fluid shear stress was used to force the top leaflet of a supported membrane to slide over the stationary lower leaflet. Here, we show that this technique yields a reproducible measurement of the friction coefficient and is sensitive enough to detect differences in friction between membranes made from saturated and unsaturated lipids. Adding cholesterol to saturated and unsaturated membranes increased interleaflet friction significantly. We also discovered that fluid shear stress can reversibly induce gel phase in supported lipid bilayers that are close to the gel-transition temperature.

Inside and Out of the Pore: Comparing Interactions and Molecular Dynamics of Influenza A M2 Viroporin Complexes in Standard Lipid Bilayers.

Ion channels located at viral envelopes (viroporins) have a critical function for the replication of infectious viruses and are important drug targets. Over the last decade, the number and duration of molecular dynamics (MD) simulations of the influenza A M2 ion channel owing to the increased computational efficiency. Here, we aimed to define the system setup and simulation conditions for the correct description of the protein-pore and the protein-lipid interactions for influenza A M2 in comparison with experimental data. We performed numerous MD simulations of the influenza A M2 protein in complex with adamantane blockers in standard lipid bilayers using OPLS2005 and CHARMM36 (C36) force fields. We explored the effect of varying the M2 construct (M2(22-46) and M2(22-62)), the lipid buffer size and type (stiffer DMPC or softer POPC with or without 20% cholesterol), the simulation time, the H37 protonation site (Nδ or Νε), the conformational state of the W41 channel gate, and M2's cholesterol binding sites (BSs). We report that the 200 ns MD with M2(22-62) (having Nε Η37) in the 20 Å lipid buffer with the C36 force field accurately describe: (a) the M2 pore structure and interactions inside the pore, that is, adamantane channel blocker location, water clathrate structure, and water or chloride anion blockage/passage from the M2 pore in the presence of a channel blocker and (b) interactions between M2 and the membrane environment as reflected by the calculation of the M2 bundle tilt, folding of amphipathic helices, and cholesterol BSs. Strikingly, we also observed that the C36 1 μs MD simulations using M2(22-62) embedded in a 20 Å POPC:cholesterol (5:1) scrambled membrane produced frequent interactions with cholesterol, which when combined with computational kinetic analysis, revealed the experimentally observed BSs of cholesterol and suggested three similarly long-interacting positions in the top leaflet that have previously not been observed experimentally. These findings promise to be useful for other viroporin systems.

Immobilization of Phosphatidylinositides Revealed by Bilayer Leaflet Decoupling.

Phosphatidylinositol 4,5-bisphosphate (PIP2) has a significantly lower mobile fraction than most other lipids in supported lipid bilayers (SLBs). Moreover, the fraction of mobile PIP2 continuously decreases with time. To explore this, a bilayer unzipping technique was designed to uncouple the two leaflets of the SLB. The results demonstrate that PIP2 molecules in the top leaflet are fully mobile, while the PIP2 molecules in the lower leaflet are immobilized on the oxide support. Over time, mobile PIP2 species flip from the top leaflet to the bottom leaflet and become trapped. It was found that PIP2 flipped between the leaflets through a defect-mediated process. The flipping could be completely inhibited when holes in the bilayer were backfilled with bovine serum albumin (BSA). Moreover, by switching from H2O to D2O, it was shown that the primary interaction between PIP2 and the underlying substrate was due to hydrogen bond formation, which outcompeted electrostatic repulsion. Using substrates with fewer surface silanol groups, like oxidized polydimethylsiloxane, led to a large increase in the mobile fraction of PIP2. Moreover, PIP2 immobilization also occurred when the bilayer was supported on a protein surface rather than glass. These results may help to explain the behavior of PIP2 on the inner leaflet of the plasma membrane, where it is involved in attaching the membrane to the underlying cytoskeleton.

An Atomistic Molecular Dynamics Study of Titanium Dioxide Adhesion to Lipid Bilayers

Titanium dioxide (TiO2) nanoparticles are found in an array of consumer and industrial products, and human exposure to these nanoparticles involves interaction with biological membranes. To understand the effect of the membrane lipid composition on bilayer perturbation by TiO2 we performed all-atom molecular dynamics simulations of nanosized TiO2 interacting with three model single component bilayers differing only in their head group composition: the zwitterionic DOPC, which is overall neutral containing negatively charged phosphate and positively charged choline in its head, DOPG, which is overall anionic containing negatively charged phosphate and neutral glycerol and the anionic DOPS, containing negatively charged phosphate attached to the hydroxyl side-chain of the amino acid, serine containing negatively charged carboxyl and positively charged ammonium. The nanoparticle adheres to all three bilayers causing a negative curvature on their top leaflet. However, the local deformation of DOPG was more pronounced than DOPC and DOPS. The anionic DOPG, which is the thinnest of the three bilayers, interacted most strongly with the TiO2. DOPS has the next strongest interaction, however this bilayer's high bending modulus enables it to resist deformation by the nanoparticle. DOPC has the weakest interaction with the nanoparticle of the three as it has the highest bending modulus and its zwitterionic head groups have strong cohesive interactions amongst themselves. We also observed a non-uniform response of the bilayers: the orientational order of the lipids near the nanoparticle decreases, while that of the lipids away from the nanoparticle increases. The overall thickness and bending modulus of DOPG increased upon contact with the nanoparticle owing to overall stiffening of the bilayer despite local softening, while the average structural and mechanical properties of DOPC and DOPS remain unchanged, which can be explained in part by the greater bilayer bending elasticicty of DOPC and DOPS. The above findings suggest regions of biological membranes populated by anionic lipids with weaker bending elasticity will be more susceptible to perturbation by TiO2 nanoparticles than zwitterionic-rich regions.

Structural behavior of amphiphilic polyion complexes interacting with saturated lipid membranes investigated by coarse-grained molecular dynamic simulations.

Neutral polyelectrolyte complexes (PECs) made from an amphiphilic multiblock copolymer of type (AnBn)m and an oppositely charged polyion and interacting with a dipalmitoylphosphatidylcholine (DPPC) lipid membrane have been examined employing a coarse-grained model with implicit solvent and molecular dynamics simulations. One systematically explored the influence of the size of the hydrophobic block B and of the number of these blocks per chain on the PEC tendency to adhere to the membrane surface and to intercalate into the membrane core. Simulation results showed that PECs bound irreversibly to the lipid bilayer without polyion unwinding from the complex and the adsorbed conformation was strongly affected by the size of the hydrophobic block B. The adsorption kinetics at low B size were characterized by a relaxation phase dominated by the spreading of PEC constituents along the outer leaflet of the membrane. Upon increasing the size of the hydrophobic block B to reach core–shell organization of the free PEC, the relaxation pathway of the complex corona in close contact with the headgroup lipids facilitated the transient exposure of the PEC hydrophobic core to the lipids and its subsequent cooperative internalization and solubilization in the membrane inner part associated with an internal reorganization of the lipid bilayer. In the generated snorkeling-type conformation, the charged blocks A and the oppositely charged polyion were confined to the headgroup region of the top leaflet, without spontaneous flipping to the headgroup region of the distal leaflet.

Screening Winged Bean (Psophocarpus tetragonolobus (L) DC) Accessions Using Agronomic Characters

Aim:This research aimed at screening winged bean accessions using morphological characters.
 Study Design: The experiment was laid out in Randomized Complete Block Design and replicated three times.
 Place and Duration of Study: The experiment was carried out at the Teaching and Research Farm, Ladoke Akintola University of Technology, Ogbomoso situated on longitude 8°7' N, latitude 4°14' E, and at altitude of 323.5 m above sea level. 
 Methodology: Seeds of thirty-eight accessions of winged bean were obtained from the International Institute of Tropical Agriculture, Ibadan and were evaluated. Data were collected on germination count at 2, 3 and 4 weeks after planting (WAP), days to first and 50% flowering, days to first and 50% podding, top leaflet length, petiole length, secondary vine lengths at 8 and 9 WAP, pod length, and number of seeds per pod and were subjected to analyses variance.
 Results: Accessions exhibited significant (P≤0.05) variation for flowering and podding dates, petiole length and top leaflet length. Days to first flowering varied from 68 to 114 days after planting (DAP) with an average of 76 days while days to 50% flowering ranged between 78 and 121 days with a mean of 83 days. Number of seeds per pod varied from 7 to 15 seeds per pod among the accessions with a mean of 12.5 seeds per pod while pod length varied from 12.3 cm to 25.6 cm with a mean pod length of 22.2 cm. Accessions TPT 26 and TPT 32 were consistent for early flowering and high seed yield and are therefore recommended for further adaptation and nutritional trials.

The molecular behavior of a single β-amyloid inside a dipalmitoylphosphatidylcholine bilayer at three different temperatures: An atomistic simulation study: Aβ interaction with DPPC: Atomistic simulation.

The behavior of a single Aβ40 molecule within a dipalmitoylphosphatidylcholine (DPPC) bilayer was studied by all-atom molecular dynamics simulations. The effect of membrane structure was investigated on Aβ40 behavior, secondary structure, and insertion depth. Simulations were performed at three temperatures (323, 310, and 300 K) to probe three different bilayer fluidities. Results show that at all above temperatures, the peptide contains two short helices, coil, bend, and turn structures. At 300 K, the peptide contains a region with β structure in C-terminal region. Our results also show that Aβ decreases the bilayer thickness and the order of lipids in its vicinity which leads to water insertion into the bilayer and concomitant increase in the local fluidity. The peptide remains embedded in the bilayer at all temperatures, and become inserted into the bilayer up to several residues at 323 and 310 K. At 310 and 300 K, the dominant interaction energy between Aβ and bilayer changes from electrostatic to van der Waals. It can be proposed that at higher temperatures (e.g., 323 K), Lys28 and the C-terminal region of the peptide play the role of two anchors that keep Aβ inside the top leaflet. This study demonstrates that Aβ molecule can perturb the integrity of cellular membranes. Proteins 2017; 85:1298-1310. © 2017 Wiley Periodicals, Inc.

Molecular dynamics simulations reveal how characteristics of surface and permeant affect permeation events at the surface of soft matter

Molecular dynamics simulations are particularly useful in providing details that permit understanding of phenomena occurring at surfaces, phenomena characterised by surface-sensitive experimental methods yielding average properties. The methods and examples that we consider here reveal how the characteristics of the surface and the permeant affect permeation events at the surface of soft matter, in particular, lipid bilayers. We choose permeation of lipid membranes as our example of the ability of molecular dynamics simulations to provide molecular-level mechanisms that are otherwise not available via other means. Molecular permeation through lipid membranes is a fundamental biological process that is important for small molecules such as therapeutics as well as nanoparticles that have become important modes of drug delivery. We describe methods applicable to such large systems and use examples where the permeants are uncharged particles: small molecules (Xe, O2, CO2), bare gold nanocrystals, gold-core nanoparticles with hydrophobic ligands (alkane thiols of various lengths) and gold-core nanoparticles with hydrophilic ligands (methyl-terminated polyethylene glycol of various lengths). In each example, we have previously validated our findings by comparisons with experimental data, using such information as is available from X-ray diffraction, electron paramagnetic resonance, nuclear magnetic resonance, atomic force microscopy, various imaging methods, diffusion measurements, dynamic light scattering. In addition to spherical core nanoparticles, we also examine the characteristic permeation mechanisms of gold nanorods with polyethylene glycol ligands, where the aspect ratio different from 1 makes the permeation event dependent on the angle of the rod axis relative to the membrane surface. This review examines the phenomena associated with the interaction of various permeants with the lipid bilayer that serves as our model membrane. We consider adsorption at the interface, the permeant within the top lipid leaflet, in the middle of the membrane within the lipid tail region, within the bottom lipid leaflet and finally exiting the membrane on the way to recovery, for various permeants: gas molecules, bare gold nanocrystal, gold nanoparticles with alkane thiol ligands, PEGylated gold nanoparticles and PEGylated gold nanorods. We observe formation of a water pore, occasional transport of ions, lipid flip-flops, lipid displacement from the membrane, and rotational behaviour of PEGylated nanorods during the permeation process. These events differ depending on the chemical nature (hydrophobic or hydrophilic) of the ligands, their length, the coverage density on the gold surface and the aspect ratio of the gold core. Direct comparisons across the board are possible by using identical interaction models (MARTINI coarse grain), molecular dynamics methods, simulation set-ups and analyses of MD results, thereby permitting generalisations to be made about mechanisms for the various events and how they are affected by these factors.

Arbuscular Mycorrhizal Fungi Regulate the Growth and Phyto-Active Compound of Salvia miltiorrhiza Seedlings

Roots and rhizomes of Salvia miltiorrhiza (S. miltiorrhiza) are widely used for the treatment of cardiovascular diseases. Arbuscular mycorrhizal fungi (AMFs) have been shown to enhance plant growth and increase secondary metabolites concentration in many plant species. However, effects of AMFs on S. miltiorrhiza have not been explored. A pot culture was designed as one control (non-AMF) treatment and four AMFs (G.m, Glomus mosseae; G.a, Glomus aggregatum; G.v, Glomus versiforme; G.i, Glomus intraradices) treatments were performed in order to evaluate the effects of AMFs on plant growth, as well as phyto-active compounds’ concentration of S. miltiorrhiza seedlings. Plants were harvested after 90 days: agronomic traits and concentration; and an accumulation of mineral elements, as well as phyto-active compounds were detected. All AMFs inoculated plants formed mycorrhizal structures, and an infection ratio; also, the intensity of inoculated roots was higher than 84.61% and 23.86%, respectively. Mycorrhizal dependency was above 144.62%. Seedlings with AMFs inoculation had significantly higher plant height, leather leaf length, top leaflet size, base leaflet length, taproot length, taproot diameter and biomass than those with non-AMF inoculation. In addition, inoculation with AMFs increased N, P, and K accumulation significantly, but barely had any effect on mineral elements’ concentrations. AMFs inoculation also significantly improved tanshinones concentrations and stimulation in order to accumulate salvianolic acid B. G.v and G.i were effective for seedlings growth; G.m and G.i were also effective for phyto-active compounds. In total, S. miltiorrhiza inoculation with AMFs had positive effects on growth and active components, especially inoculation with G.v.

Interactions and Translational Dynamics of Phosphatidylinositol Bisphosphate (PIP2) Lipids in Asymmetric Lipid Bilayers.

Phosphatidylinositol phosphate (PIP) lipids are critical to many cell signaling pathways, in part by acting as molecular beacons that recruit peripheral membrane proteins to specific locations within the plasma membrane. Understanding the biophysics of PIP-protein interactions is critical to developing a chemically detailed model of cell communication. Resolving such interactions is challenging, even in model membrane systems, because of the difficulty in preparing PIP-containing membranes with high fluidity and integrity. Here we report on a simple, vesicle-based protocol for preparing asymmetric supported lipid bilayers in which fluorescent PIP lipid analogues are found only on the top leaflet of the supported membrane facing the bulk solution. With this asymmetric distribution of lipids between the leaflets, the fluorescent signal from the PIP lipid analogue reports directly on interactions between the peripheral molecules and the top leaflet of the membrane. Asymmetric PIP-containing bilayers are an ideal platform to investigate the interaction of PIP with peripheral membrane proteins using fluorescence-based imaging approaches. We demonstrate their usefulness here with a combined fluorescence correlation spectroscopy and single particle tracking study of the interaction between PIP2 lipids and a polycationic polymer, quaternized polyvinylpyridine (QPVP). With this approach we are able to quantify the microscopic features of the mobility coupling between PIP2 lipids and polybasic QPVP. With single particle tracking we observe individual PIP2 lipids switch from Brownian to intermittent motion as they become transiently trapped by QPVP.

Phase transition behaviors of the supported DPPC bilayer investigated by sum frequency generation (SFG) vibrational spectroscopy and atomic force microscopy (AFM).

The phase transition behaviors of a supported bilayer of dipalmitoylphosphatidyl-choline (DPPC) have been systematically evaluated by in situ sum frequency generation (SFG) vibrational spectroscopy and atomic force microscopy (AFM). By using an asymmetric bilayer composed of per-deuterated and per-protonated monolayers, i.e., DPPC-d75/DPPC and a symmetric bilayer of DPPC/DPPC, we were able to probe the molecular structural changes during the phase transition process of the lipid bilayer by SFG spectroscopy. It was found that the DPPC bilayer is sequentially melted from the top (adjacent to the solution) to bottom leaflet (adjacent to the substrate) over a wide temperature range. The conformational ordering of the supported bilayer does not decrease (even slightly increases) during the phase transition process. The conformational defects in the bilayer can be removed after the complete melting process. The phase transition enthalpy for the bottom leaflet was found to be approximately three times greater than that for the top leaflet, indicating a strong interaction of the lipids with the substrate. The present SFG and AFM observations revealed similar temperature dependent profiles. Based on these results, the temperature-induced structural changes in the supported lipid bilayer during its phase transition process are discussed in comparison with previous studies.

Effect of Asymmetry on Bilayer Membrane Systems

Many biological processes, such as membrane fusion and the action of microbial proteins, involve asymmetric uptake of proteins. In addition, it has been demonstrated that large mechaosensitive channels open by the asymmetric inclusion of lyso-lipids in the bilayer in the absence of external pressure. These phenomena can be interpreted in terms of the asymmetric local pressure profile in the bilayer, which lacks the quantitative understanding. By noting that such asymmetric pressure profile can be induced in bilayer membranes with asymmetric number of lipids between leaflets, to provide quantitative understanding, we performed molecular dynamics simulations of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayer membranes for a set of systems with different level of asymmetry with and without gramicidin A (gA) or WALP23. We observed that even highly asymmetric lipid bilayers with and without gA or WALP23 were stable during the whole simulation time, which is consistent with the previous study of dipalmitoyl-phosphatidylcholine (DPPC) bilayers [J. Am. Chem. Soc. 131, 15194 (2009)]. As the number of lipids in the lower leaflet becomes smaller, the lipids are more tightly packed in the top leaflet and less ordered in the bottom leaflet, which are quantified in terms the lateral pressure profile, the order parameter, the area per lipid, etc. However, the hydrophobic thickness for pure lipid bilayers does not show meaningful dependence on the asymmetry, which results from the stretched and less ordered lipids in the upper and lower leaflets, respectively. We will also discuss the influence of the asymmetry on the properties of protein-lipid interactions, such as the distance dependence of the lipid properties and the interaction patterns around gA or WALP23.

Phase Transition-Controlled Flip-Flop in Asymmetric Lipid Membranes

Lipid membrane asymmetry is of fundamental importance for biological systems and also provides an attractive means for molecular control over biomaterial surface properties (including drug carriers). In particular, temperature-dependent changes of surface properties can be achieved by taking advantage of distinct phase transitions in lipid membrane coatings where lipids exchange (flip-flop) between leaflets. In this study, temperature is used to control flip-flop of lipids in asymmetric lipid membranes on planar solid supports, where the two leaflets of the lipid membrane are in different phase states. More specifically, the lower leaflet is prepared from a supported lipid membrane composed of a high Tm lipid mixture of phosphocholine (PC), phosphatidylserine (PS), and a bioactive lipid on TiO2, followed by selective removal of the top leaflet by detergent. Next, at a lower temperature, where the remaining leaflet is in the gel state, a top leaflet of a different lipid composition and in the fluid phase is formed. Phase transition-induced changes in membrane surface properties following upon temperature-activation of the prepared asymmetric membrane are demonstrated by the detection of biotinylated lipids, which were initially located (thus "hidden") in the lower-gel phase leaflet, at the surface of the top leaflet. These processes were monitored in real-time by the quartz crystal microbalance with dissipation (QCM-D) and the dual polarization interferometry (DPI) techniques, allowing modeling of the mass and the anisotropic property of the lipid structures in different phase states.

Electrochemical SERS study of a biomimetic membrane supported at a nanocavity patterned Ag electrode

Lipid bilayers in which two leaflets were made of dimyristoyl-phosphatidylcholine (DMPC) and hybrid bilayers with one leaflet composed of hydrogenated lipid and another leaflet of deuterium substituted molecules (d63-DMPC) were deposited on highly ordered nanocavity patterned Ag electrodes using LB–LS and vesicle fusion techniques. In situ electrochemical surface enhanced Raman scattering (EC-SERS) was then used to study potential driven changes in these model biological membranes. The nanocavity structures provided a SERS active substrate with uniformly distributed surface enhancement. To ensure that the bilayer was separated from the metal surface by a hydrophilic space layer, the electrodes were chemically modified with a self-assembled monolayer (SAM) of β-thioglucose (TG) molecules. The monolayer of TG ensured that the solid substrate surface was hydrophilic. The electrochemical properties of the bilayer were monitored by recording differential capacitance curves. EC-SERS indicated that at the silver surface modified by the monolayer of TG the lower leaflet (in contact with the support) is more ordered than the top leaflet that is contact with solution. However, both leaflets remained in the liquid crystalline (LC) state for the entire range of investigated potentials. The results of this study show that the DMPC bilayer at the nanocavity patterned Ag surface may be used as a good biomimetic membrane model in future SERS studies of membrane proteins. The information concerning the effect of the electrode potential on membrane stability may be useful for the development of biosensors.

Bilayer Asymmetry Influences Integrin Sequestering in Raft-Mimicking Lipid Mixtures

There is growing recognition that lipid heterogeneities in cellular membranes play an important role in the distribution and functionality of membrane proteins. However, the detection and characterization of such heterogeneities at the cellular level remains challenging. Here we report on the poorly understood relationship between lipid bilayer asymmetry and membrane protein sequestering in raft-mimicking model membrane mixtures using a powerful experimental platform comprised of confocal spectroscopy XY-scan and photon-counting histogram analyses. This experimental approach is utilized to probe the domain-specific sequestering and oligomerization state of αvβ3 and α5β1 integrins in bilayers, which contain coexisting liquid-disordered/liquid-ordered (ld/lo) phase regions exclusively in the top leaflet of the bilayer (bottom leaflet contains ld phase). Comparison with previously reported integrin sequestering data in bilayer-spanning lo-ld phase separations demonstrates that bilayer asymmetry has a profound influence on αvβ3 and α5β1 sequestering behavior. For example, both integrins sequester preferentially to the lo phase in asymmetric bilayers, but to the ld phase in their symmetric counterparts. Furthermore, our data show that bilayer asymmetry significantly influences the role of native ligands in integrin sequestering.

Molecular dynamics simulations of DPPC bilayers using "LIME", a new coarse-grained model.

A new intermediate resolution model for phospholipids, LIME, designed for use with discontinuous molecular dynamics (DMD) simulations is presented. The implicit-solvent model was developed using a multiscale modeling approach in which the geometric and energetic parameters are obtained by collecting data from atomistic simulations of a system composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) molecules and explicit water. In the model, 14 coarse-grained sites that are classified as 1 of 6 types represent DPPC. DMD simulations performed on a random solution of DPPC resulted in the formation of a defect-free bilayer in less than 4 h. The bilayer formed quantitatively reproduces the main structural properties (e.g., area per lipid, bilayer thickness, bond order parameters) that are observed experimentally. In addition, the bilayer transitions from a liquid-crystalline phase to a tilted gel phase when the temperature is reduced. Transbilayer movement of a lipid from the bottom leaflet to the top leaflet is observed when the temperature is increased.

Integrin Sequestering in Raft-Mimicking Lipid Mixtures: The Role of Bilayer Asymmetry and Cholesterol Content

It is widely accepted that lipid microdomains play an important functional role in plasma membranes. However, small size and transient nature of lipid/membrane heterogeneities in the plasma membrane make a characterization of microdomains and microdomain-related membrane processes in cellular systems quite challenging. To address this important problem, we recently introduced a powerful model membrane system that allows the investigation of membrane protein sequestering and oligomerization in raft-mimicking lipid mixtures using combined confocal fluorescence spectroscopy, photon counting histogram (PCH), and epifluorescence microscopy (1). Our experiments on bilayer-spanning domains showed that αvβ3 and α5β1 integrins predominantly exist as monomers and sequester preferentially to the liquid-disordered (ld) phase in the absence of ligands. Notably, addition of vitronectin (αvβ3) and fibronectin (α5β1) caused substantial translocations of integrins into the liquid-ordered (lo) phase without altering receptor oligomerization state. Here we expand our previous studies and report on the sequestering and oligomerization state of αvβ3 and α5β1 in asymmetric bilayer compositions containing coexisting lo and ld phases located exclusively in the top leaflet of the bilayer (bottom leaflet shows only ld phase). Remarkably, in such a membrane environment, both integrins show a higher affinity for the top leaflet-restricted lo domains in the absence of their respective ligands. This sequestering behavior of integrins was only slightly modified after addition of their respective native ligands. Our findings show that cholesterol content has a substantial influence on integrin sequestering and oligomerization in raft-mimicking lipid mixtures. The described experimental results highlight the potential importance of membrane asymmetry and lipid composition in the sequestering of membrane proteins in biological membranes.(1) Siegel, A. P. et al. (2011) Biophys J 101(7): 1642-1650.

Bilayer Asymmetry, Cholesterol Content, and Ligand Binding Influence Membrane Protein Sequestering in Raft-Mimicking Lipid Mixtures

Lipid heterogeneities and membrane proteins are widely considered to show an important functional relationship in plasma membranes. However, the analysis of this intriguing relationship is challenging, due to the small size and transient nature of lipid heterogeneities in the plasma membrane environment. Recently, we presented a powerful model membrane platform that allows the thorough analysis of membrane protein sequestering and oligomerization in well-defined heterogeneous lipid environments using confocal fluorescence intensity analysis paired with a photon counting histogram (PCH) method (1). By applying this experimental approach, here we show that bilayer asymmetry has a significant influence on the sequestering of αvβ3 and α5β1 integrins. Our experiments demonstrate a higher affinity of integrins for the liquid-disordered (ld) phase in the presence of bilayer-spanning liquid-ordered (lo)-ld phase separations. In contrast, a preference for the lo phase is observed in an asymmetric bilayer with lo-ld phase separations, which are exclusively located in the top leaflet of the bilayer. Importantly, PCH analysis shows that the observed changes in integrin sequestering are not caused by altered receptor oligomerization states. In another set of experiments, we also demonstrate that changes in cholesterol may have a profound impact on integrin sequestering without altering receptor oligomerization state. The obtained results are discussed in terms of potential changes in lipid packing density and hydrophobic thickness within the model membrane. The experimental model membrane approach is also applied to explore the functionally important relationship between the sequestering, level of dimerization, and ligand binding of the GPI-anchored urokinase receptor.(1) Siegel, A. P. et al. (2011) Biophys J101(7): 1642-1650.