Distal Leaflet Research Articles

Predictors of mid-term functional tricuspid regurgitation after device closure of atrial septal defect in adults: Impact of pre-operative tricuspid valve remodeling

BackgroundFunctional TR occurs in adults with congenital atrial septal defect (ASD) due to left-to-right inter-atrial shunting and enlarged right heart. The prevalence and the predictors of TR following ASD closure in adults remain unknown. Moreover, whether preoperative anatomical parameters of tricuspid valve related to TR after ASD closure is understudied. MethodsComprehensive transthoracic echocardiography was performed in 64 consecutive secundum ASD patients before and at 3months after device closure. Tricuspid valve parameters [annulus diameter (TAD), tenting area, tenting height, distal tricuspid septal leaflet angle (TSLA)], right ventricular (RV) function, pulmonary arterial pressures and severity of TR were recorded. Post-closure TR was defined as PISA radius ≥6mm at 3months. ResultsThe TR severity was reduced after ASD closure with post-closure TR in 31 patients (48%) at follow-up. TR patients had larger ventricles, more TR, greater TAD, and larger tenting area/height and TSLA but similar RV long-axis function and pulmonary pressures at baseline. Pre-closure TAD [odds ratio (OR): 6.076, p=0.032] and TSLA (OR: 1.218, p=0.001) were the independent predictors for TR. A TAD >3.5cm and a TSLA >30° had sensitivity, specificity, and area under the curve of 97%, 76%, and 0.82 and 100%, 78%, and 0.84, respectively. Assessment of TSLA showed an incremental value over TAD for predicting TR after closure (χ2=21.3 vs. 9.9, p<0.001). ConclusionNearly half of adults had TR following device closure of ASD. It was related to the excessive pre-operative structural remodeling in tricuspid valve rather than changes in RV.

Lateral suppressor and Goblet act in hierarchical order to regulate ectopic meristem formation at the base of tomato leaflets.

In seed plants, new axes of growth are established by the formation of meristems, groups of pluripotent cells that maintain themselves and initiate the formation of lateral organs. After embryonic development, secondary shoot meristems form in the boundary zones between the shoot apical meristem and leaf primordia, the leaf axils. In addition, many plant species develop ectopic meristems at different positions of the plant body. In the compound tomato leaf, ectopic meristems can initiate at the base of leaflets, which are delimited by two distinct boundary zones, referred to as the proximal (PLB) and distal (DLB) leaflet boundaries. We demonstrate that the two leaflet boundaries differ from each other and that ectopic meristem formation is strictly limited to the DLB. Our data suggest that the DLB harbours a group of pluripotent cells that seems to be the launching pad for meristem formation. Initiation of these meristems is dependent on the activities of the transcriptional regulators Goblet (Gob) and Lateral suppressor (Ls), specifically expressed in the DLB. Gob and Ls act in hierarchical order, because Ls transcript accumulation is dependent on Gob activity, but not vice versa. Ectopic meristem formation at the DLB is also observed in other seed plants, like Cardamine pratensis, indicating that it is part of a widespread developmental program. Ectopic meristem formation leads to an increase in the number of buds, enhances the capacity for survival and opens the route to vegetative propagation.

Channel-forming activity of syringomycin E in two mercury-supported biomimetic membranes

The lipodepsipeptide syringomycin E (SR-E) interacts with two mercury-supported biomimetic membranes, which consist of a self-assembled phospholipid monolayer (SAM) and of a tethered bilayer lipid membrane (tBLM) separated from the mercury surface by a hydrophilic tetraethyleneoxy (TEO) spacer that acts as an ionic reservoir. SR-E interacts more rapidly and effectively with a SAM of dioleoylphosphatidylserine (DOPS) than with one of dioleoylphosphatidylcholine (DOPC). The proximal lipid monolayer of the tBLM has no polar head region, being linked to the TEO spacer via an ether bond, while the distal monolayer consists of either a DOPC or a DOPS leaflet. The ion flow into or out of the spacer through the lipid bilayer moiety of the tBLM was monitored by potential step chronocoulometry and cyclic voltammetry. With the distal monolayer bathed by aqueous 0.1M KCl and 0.8μM SR-E, an ion flow in two stages was monitored with DOPC at pH3 and 5.4 and with DOPS at pH3, while a single stage was observed with DOPS at pH5.4. This behavior was compared with that already described at conventional bilayer lipid membranes. The sigmoidal shape of the chronocoulometric charge transients points to an aggregation of SR-E monomers forming an ion channel via a mechanism of nucleation and growth. The ion flow is mainly determined by potassium ions, and is inhibited by calcium ions. The contribution to the transmembrane potential from the distal leaflet depends more on the nature of the lipid than that of the ion channel.

Synaptotagmin’s Role in Neurotransmitter Release Likely Involves Ca2+-induced Conformational Transition

Neuronal exocytosis is mediated by a Ca2+-triggered membrane fusion event that joins synaptic vesicles and presynaptic membrane. In this event, synaptotagmin I plays a key role as a Ca2+ sensor protein that binds to and bends the presynaptic membrane with its C2B domain, and thereby initiates membrane fusion. We report free energy calculations according to which C2B-induced membrane bending is preceded by a Ca2+- and membrane-dependent conformational transition. In this transition C2B attaches to the membrane, moves its C-terminal helix from the orientation seen in the available (but membrane-free) crystal/NMR structures as pointing away from the membrane (helix-up), to an orientation pointing toward the membrane (helix-down). In the C2B helix-down state, lipid tails in the proximal membrane bilayer leaflet interact with the moved helix and become disordered, whereas tails in the distal leaflet, to keep in contact with the proximal leaflet, become stretched and ordered. The difference in lipid tail packing between the two leaflets results in an imbalance of pressure across the membrane, and thereby causes membrane bending. The lipid-disordering monitored in the simulations is well suited to facilitate Ca2+-triggered membrane fusion.

Gel-to-fluid phase transformations in solid-supported phospholipid bilayers assembled by the Langmuir-Blodgett technique: effect of the Langmuir monolayer phase state and molecular density.

Planar-supported phospholipid bilayers are increasingly used as synthetic membranes for scientific and practical applications. The thermotropic phase properties of supported bilayers are important for recreating biologically relevant situations. Unlike free-standing lipid membranes that undergo one gel-to-fluid or main phase transition, mica-supported single bilayers have been found to undergo two separate leaflet transitions. Although the distinctive nature of the main transition in mica-supported bilayers has been attributed to different effects, determining their relevance has been problematic because vesicle fusion, the technique most widely used to prepare solid-supported bilayer membranes, does not allow one to readily control the lipid surface coverage and molecular density. To circumvent the limitations of the vesicle fusion method and systematically investigate the effects on the individual leaflet transitions of the lipid phase state and molecular density before deposition on the substrate, mica-supported single bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were prepared using the Langmuir-Blodgett technique. The gel-to-fluid transitions of the bilayer leaflets were tracked by controlled-temperature atomic force microscopy to determine the relative fractions of the gel and fluid phases as a function of temperature. The fraction of solid versus temperature data was fit to the van't Hoff equation to determine the leaflet melting temperatures and transition enthalpies. The phase state and molecular density of the Langmuir monolayer precursor at the transfer pressure of 35 mN m(-1) was found to have a greater effect on the main transition temperature and width of the distal (upper) leaflet than that of the proximal (lower) one. The contributions of substrate-mediated condensation, asymmetric lipid densities, and surface area available for thermal expansion of the bilayer are addressed. This work demonstrates the potential of the Langmuir-Blodgett technique as a tool for identifying and manipulating the factors that govern the phase transition properties of surface-confined lipid bilayers.

The Structure of Lipid Bilayers Adsorbed on Activated Carboxy-Terminated Monolayers Investigated by Sum Frequency Generation Spectroscopy

The formation and structure of isotopically asymmetric supported bilayer membranes (SBMs) has been investigated using sum frequency generation (SFG) vibrational spectroscopy supplemented by reflection absorption infrared spectroscopy (RAIRS). The bilayers were composed of a proximal and distal leaflet of the phospholipid dipalmitoyl phosphatidylethanolamine (DPPE) supported on a gold surface. The proximal leaflet was chemically tethered to the gold via an 11-mercapto-undecanoic acid (MUA) self-assembled monolayer (SAM) that had been chemically modified to produce an activated succinimidyl ester headgroup using N-hydroxysuccinimide (NHS) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC). The activation of the MUA and the tethering of the DPPE were monitored and confirmed using SFG and RAIRS. The distal leaflet of the bilayer was added using either vesicle fusion (VF) or Langmuir-Blodgett (LB) deposition. To gain insight into the structure of each layer of the SBM perdeuterated DPPE (d-DPPE) and MUA (d-MUA) were used to create SBMs with a layer that was isotopically distinguishable from the rest. The polar orientation and conformational ordering of the lipids was determined using SFG. It was found that the tethering of the proximal lipid leaflet resulted in an increase in the conformational order of the MUA SAM. Furthermore, by careful analysis and comparison of spectra recorded in both the C-H (2800-3000 cm(-1)) and C-D (2000-2300 cm(-1)) stretching regions it was concluded that a better ordered and more biologically relevant lipid bilayer was formed when the distal leaflet was added using LB deposition. On the other hand the SFG spectra of the SBMs in which the distal leaflet was added by VF showed little evidence of conformational ordering on the time scale of minutes, suggesting the presence of an incomplete monolayer or of multilayer formation.

C2B Domain in Synaptotagmin I Induces Membrane Bending Only After Conformational Change

Neuronal exocytosis is mediated by a Ca2+-triggered membrane fusion event that joins synaptic vesicles and presynaptic membrane. In this event, synaptotagmin I plays a key role as a Ca2+ sensor protein that binds to and bends the presynaptic membrane with its C2B domain and, thereby, triggers membrane fusion. We report free energy calculations according to which C2B-induced membrane bending is preceded by a Ca2+- and membrane-dependent conformational transition, in which C2B attaches to the membrane, moves its C-terminal helix from the orientation seen in the membrane-free crystal/NMR structures as pointing away from the membrane (helix up), to an orientation pointing towards the membrane (helix down). In the C2B “helix down” state, lipid tails in the proximal membrane bilayer leaflet interact with the moved helix and become disordered, while tails in the distal leaflet, to keep in contact with the proximal leaflet, become stretched and ordered. The difference in lipid tail packing between the two leaflets results in an imbalance of pressure across the membrane and, thereby, causes membrane bending. The lipid disordering in the proximal membrane leaflet should facilitate Ca2+-triggered membrane fusion.

Asymmetric Supported Lipid Bilayer Formed via Methyl-β-Cyclodextrin Mediated Lipid Exchange: a Membrane Model System to Study Phase Separation and Transbilayer Lipid Movement

Supported lipid bilayers (SLBs) are broadly used as a membrane model system and commonly produced by vesicle fusion (VF). Despite its advantages, VF does not allow the controlled formation of asymmetric bilayers that mimic the asymmetry in lipid composition normally found in biological systems. Here we present a simple, quick and versatile method to produce stable SLBs with a desired asymmetric lipid composition. We apply a methyl-β-cyclodextrin (MβCD) mediated lipid exchange method to SLBs formed by VF to enrich the distal leaflet of the bilayer with sphingomyelin (SM). Bilayer asymmetry is assessed by fluorescence correlation spectroscopy (FCS), measuring lipid mobility separately in each leaflet. To check the compatibility of the method with the most common protein reconstitution approaches, we also report the production of asymmetric SLBs (aSLBs) in presence of a membrane protein, reconstituted both via direct protein insertion (i.e. directional insertion) and proteoliposomes fusion. aSLBs are a suitable membrane model system to study phase separation and transbilayer lipid movement of raft-mimicking lipid mixtures. Although their phase behavior was extensively investigated, the effect of compositional asymmetry on phase separation remains unclear. To address this question, we compared SLBs and aSLBs with the same overall lipid composition. The observed differences in terms of phase separation provide further experimental evidence that the transversal lipid distribution affect the overall lipid miscibility and allow to temporally investigate leaflet mixing (i.e. lipid flip-flop). Additionally, cholesterol distribution between cytosolic and extracellular side of the plasma membrane is of great importance in determining protein-membrane interaction and the formation of raft-like domains in vivo. Therefore, we use a combined FCS and mass spectrometric approach to investigate cholesterol distribution between inner and outer leaflet of SM-containing aSLBs.

Sum Frequency Generation Vibrational Spectroscopy of Cholesterol in Hybrid Bilayer Membranes

The assignment of the vibrational spectrum of cholesterol is surprisingly incomplete for such a fundamental molecule. To improve our understanding, a new investigation of the spectra of cholesterol in the C-H stretching region has been undertaken using the surface specific technique of Sum Frequency Generation (SFG) vibrational spectroscopy and the complementary technique of Reflection Absorption Infrared Spectroscopy (RAIRS). They were used to record the spectra of monolayers of cholesterol in hybrid bilayer membranes (HBMs). In addition to cholesterol, spectra were recorded of HBMs comprising monolayers of the partially deuterated cholesterol isotopologues d6-cholesterol and d7-cholesterol, and the cholesterol analogues cholestanol and androstanol to aid assignment of the spectra. Monolayers of each of the five molecules were used to form the distal leaflet of HBMs with the proximal leaflet consisting of a monolayer of deuterated mercaptoundecanoic acid (d-MUA) self-assembled on a gold substrate. Although cholesterol has five methyl groups and eleven methylene groups, by using molecules in which certain groups were either deuterated or entirely absent, it was possible to assign vibrational bands to specific sets of methyl or methylene groups either in the alkyl chain or sterol ring system of the molecule. Analysis of the spectra showed that the alkyl chains of cholesterol are orientated away from the substrate, which is opposite to their orientation in HBMs when the proximal leaflet is a hydrophobic self-assembled monolayer of octadecane thiol (ODT) adsorbed on gold. Additionally it was shown that in the d-MUA HBM, the α-face of the cholesterol ring is inclined toward the layer of air above the film, and the β-face is inclined toward the gold substrate.

Electrochemical and PM-IRRAS Characterization of Cholera Toxin Binding at a Model Biological Membrane

A mixed phospholipid-cholestrol bilayer, with cholera toxin B (CTB) units attached to the monosialotetrahexosylganglioside (GM1) binding sites in the distal leaflet, was deposited on a Au(111) electrode surface. Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) measurements were used to characterize structural and orientational changes in this model biological membrane upon binding CTB and the application of the electrode potential. The data presented in this article show that binding cholera toxin to the membrane leads to an overall increase in the tilt angle of the fatty acid chains; however, the conformation of the bilayer remains relatively constant as indicated by the small decrease in the total number of gauche conformers of acyl tails. In addition, the bound toxin caused a significant decrease in the hydration of the ester group contained within the lipid bilayer. Furthermore, changes in the applied potential had a minimal effect on the overall structure of the membrane. In contrast, our results showed significant voltage-dependent changes in the average orientation of the protein α-helices that may correspond to the voltage-gated opening and closing of the central pore that resides within the B subunit of cholera toxin.

Diffusion of complex objects embedded in free and supported lipid bilayer membranes: role of shape anisotropy and leaflet structure

We present a versatile numerical scheme to predict diffusion coefficients for arbitrarily shaped objects embedded in lipid bilayer membranes. Diffusion coefficients for micron-scale diamond-shaped solid domains are calculated for direct comparison to recent experiments. In supported membranes, identical objects in the distal and proximal leaflets may diffuse differently from one another; quantitative predictions for this asymmetry are provided, both for experimental systems and coarse-grained molecular simulations. We show that though recent experiments comparing the diffusion of monomeric, dimeric and trimeric protein assemblies moving over the surface of supported bilayers are inconsistent with the simplest Saffman–Delbrück model, they may be explained by a hydrodynamic model appropriate for supported membranes.

Differential activation of valvulogenic, chondrogenic, and osteogenic pathways in mouse models of myxomatous and calcific aortic valve disease

Studies of human diseased aortic valves have demonstrated increased expression of genetic markers of valve progenitors and osteogenic differentiation associated with pathogenesis. Three potential mouse models of valve disease were examined for cellular pathology, morphology, and induction of valvulogenic, chondrogenic, and osteogenic markers. Osteogenesis imperfecta murine (Oim) mice, with a mutation in Col1a2, have distal leaflet thickening and increased proteoglycan composition characteristic of myxomatous valve disease. Periostin null mice also exhibit dysregulation of the ECM with thickening in the aortic midvalve region, but do not have an overall increase in valve leaflet surface area. Klotho null mice are a model for premature aging and exhibit calcific nodules in the aortic valve hinge-region, but do not exhibit leaflet thickening, ECM disorganization, or inflammation. Oim/oim mice have increased expression of valve progenitor markers Twist1, Col2a1, Mmp13, Sox9 and Hapln1, in addition to increased Col10a1 and Asporin expression, consistent with increased proteoglycan composition. Periostin null aortic valves exhibit relatively normal gene expression with slightly increased expression of Mmp13 and Hapln1. In contrast, Klotho null aortic valves have increased expression of Runx2, consistent with the calcified phenotype, in addition to increased expression of Sox9, Col10a1, and osteopontin. Together these studies demonstrate that oim/oim mice exhibit histological and molecular characteristics of myxomatous valve disease and Klotho null mice are a new model for calcific aortic valve disease.

The Mitral Valve in Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) has evoked both fascination and controversy since its first modern description >50 years ago.1 Originally described as “functional aortic subvalvar stenosis” by Brock, the majority of articles about HCM in the ensuing first decade emanated from observations made in the operating room or in the cardiac catheterization laboratory.1 The actual cause of left ventricular outflow tract (LVOT) obstruction in patients with this condition had not yet been clarified. Ironically, the mechanisms of LVOT obstruction in HCM were only elucidated several years after surgery was first performed in these patients. In fact, an early report by Morrow described the septal myotomy procedure as surgical treatment for HCM and proposed that the myotomy interfered with the sphincter-like muscular contraction ring in the LVOT.1 Article see p 40 Noninvasive cardiovascular imaging studies have greatly advanced our knowledge about HCM.2 Hypertrophic cardiomyopathy is now felt to have tremendous genetic,3 clinical, morphological, and hemodynamic heterogeneity.1 Nevertheless, the presence of LVOT obstruction in patients with HCM has continued to inspire interest. The often contentious debates surrounding LVOT obstruction have centered on its very existence, its causes, its clinical impact and prognosis, and its optimal management.1 These issues have inextricably included the mitral valve, which has long been felt to play a pivotal role in the pathophysiology of LVOT obstruction in patients with HCM. In the present issue of Circulation , Maron et al use cardiovascular magnetic resonance (CMR) imaging to characterize mitral valve leaflets in patients with HCM.4 This study assessed the lengths of the anterior mitral leaflet (AML) and posterior mitral leaflet in 172 consecutive patients with HCM. Their findings were compared with 172 normal control subjects matched for age, sex, and body surface area. In addition, the study included a small cohort of …

The Pathway to Fusion in Synthetic Membrane Systems

Considerable evidence indicates the pathway to fusion of synthetic and biological membranes consists of two stages. The first is a transition to a hemifused intermediate where only contacting leaflets are fused and distal leaflets engaged in a hemifusion diaphragm (HD). The second consists in rupture of the growing or fully developed HD to give fusion. Here we present a quantitative model for this pathway in protein-free systems. The first stage is described by our model of hemifusion (presented elsewhere) which outputs time-evolutions of HD tension and area. We find that complete fusion results only if the HD tension, initially twice that outside the HD, excedes the lysis threshold for sufficiently long. Fusion does not occur instantaneously after hemifusion nucleation since membranes transiently tolerate tensions above lysis as previously demonstrated by micropipet aspiration of vesicles [Evans et al, Biophys J, 2003]. Tension relaxes rapidly with HD size such that a growing HD surviving the initial high tension episode matures into a dead-end hemifused state. Thus fusion and dead-end hemifusion are alternative outcomes; the favored outcome depends critically on cation concentration, lipid composition and other physical factors. We find higher salt levels create higher tensions which favor complete fusion. Predicted fusion times are in quantitative agreement with a study of giant vesicles where HD rupture yielded fusion after ∼1-2 s in 6 mM Ca2+ [Nikolaus et al, Biophys J, 2010]. We predict dead-end hemifusion at lower salt, consistent with stable HDs reported in 2 mM Mg2+. In suspended bilayer-vesicle systems we find cation-induced vesicle tensions are dissipated upon hemifusion and thus Ca2+ drives dead-end hemifusion only. This is in agreement with the consistently reported experimental finding that calcium alone cannot fuse vesicles to bilayers, and fusion requires additional osmotic swelling.

The Mechanism of Calcium-Mediated Hemifusion on the Pathway to Fusion

In many synthetic and biological membrane systems the pathway to fusion passes through a hemifused intermediate where only proximal leaflets are fused. Evidence suggests the unfused distal leaflets engage one another in a hemifusion diaphragm (HD) but the nature of this intermediate is poorly understood even in protein-free systems where divalent cations drive fusion of lipid vesicles. Recently lipid vesicle hemifusion was directly visualized and quantitatively characterized for the first time [Nikolaus et al, Biophys J, 2010]: nucleated HDs grew at ∼20 υm2/s and fully developed equilibrium HDs were microns in diameter and occupied ∼9% of the surface area of the hemifusing vesicles. We have developed a general model of hemifusion which mechanistically explains these findings and predicts HD properties in many other published fusion experiments. We show membrane tension and cationic electrostatic forces, resisted by interleaflet tension, peel back proximal leaflets and grow the HD until the forces balance in equilibrium. Model predictions are in excellent agreement with dynamic and equilibrium HD measurements of Nikolaus et al. Our model predicts HDs occupied 3-7% of vesicle areas in other studies depending on the lipid composition and calcium concentration. The predictions for time-dependent HD area and HD tension constitute the necessary information for a quantitative model of the entire hemifusion-fusion pathway via HD rupture. In vesicle-planar bilayer systems [Chanturiaya et al, PNAS, 1997], a common model of vesicle-plasma membrane fusion, we show the equilibrated HD constitutes half the area of the vesicle which straddles the planar bilayer. In cellular systems we argue that calcium levels that trigger intracellular fusion are often sufficient to induce strong local tensions which may drive HD growth in preparation for SNARE-dependent fusion.

Predictors of Mitral Regurgitation Recurrence in Patients With Heart Failure Undergoing Mitral Valve Annuloplasty

Restrictive mitral annuloplasty is a surgical treatment option for patients with heart failure (HF) and functional mitral regurgitation (MR). However, recurrent MR has been reported at mid-term follow-up. The aim of the present study was to identify the echocardiographic predictors of recurrent MR in patients with HF undergoing mitral annuloplasty. During a mean follow-up of 2.6 +/- 1.6 years, 109 patients with HF (49% ischemic and 51% idiopathic dilated cardiomyopathy) who had undergone mitral valve repair were followed up (of 122 total patients). The severity of MR was quantified, and the following parameters were measured before intervention and at the mid-term follow-up examination: left ventricular (LV) and left atrial volumes and dimensions, LV sphericity index, mitral annular area, and mitral valve geometry parameters. At mid-term follow-up, 21 patients presented with significant MR (grade 2 to 4), and 88 patients had only MR grade 0 to 1. Both groups of patients had had a similar preoperative MR grade, mitral annular area, and LV volume and dimension. In contrast, patients with recurrent MR had had increased preoperative posterior and anterior leaflet angles, tenting height, tenting area, and LV sphericity index compared to the patients without recurrent MR. Of the different parameters of mitral and LV geometry, the distal mitral anterior leaflet angle (hazard ratio 1.48, 95% confidence interval 1.32 to 1.66, p <0.001) and posterior leaflet angle (hazard ratio 1.13, 95% confidence interval 1.07 to 1.19, p <0.001) were independent determinants of MR at mid-term follow-up. In conclusion, in patients with HF of ischemic or idiopathic etiology and functional MR, distal mitral leaflet tethering and posterior mitral leaflet tethering were associated with recurrent MR after restrictive mitral annuloplasty.

Force Spectroscopy Study of Langmuir−Blodgett Asymmetric Bilayers of Phosphatidylethanolamine and Phosphatidylglycerol

Phosphatidylethanolamine (PE) and phosphatidylgycerol (PG) are the main components of the inner membrane of Escherichia coli. Mixtures of PE and PG mimicking the proportions found in E. coli have been extensively used to reconstitute transmembrane proteins as lactose permease (LacY) in proteoliposomes because in this environment the protein shows maximal activity. Hence, the study of the physicochemical properties of this phospholipid matrix becomes of potential interest. In previous studies, we used atomic force microscopy (AFM) and force spectroscopy (FS) to study the topographic and nanomechanical properties of supported lipid bilayers (SLBs) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and of POPE and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (3:1, mol/mol). The study reported here was extended for completeness to asymmetric SLBs obtained by the Langmuir-Blodgett (LB) method. Thus, we prepared SLBs with the proximal leaflet extracted at 30 mN x m(-1) and the distal leaflet extracted at 25 mN x m(-1). We prepared SLBs with both leaflets with same composition (POPG/POPG), and also with the proximal leaflet of POPE and the distal leaflet of POPG or POPE:POPG (3:1, mol/mol). The topography of the SLBs acquired in liquid was compared with the topography of the monolayers acquired in air. Breakthrough (F(y)) and adhesion forces (F(adh)) of SLBs were extracted from force curves. The values obtained are discussed in terms of the possible involvement of the nanomechanical properties of the SLBs in membrane protein insertion. The results provide means for the observation that insertion of LacY in POPE:POPG (3:1, mol/mol) occurs preferentially in the fluid phase, which is the phase with the lower F(y) and the higher F(adh).

In-plane homogeneity and lipid dynamics in tethered bilayer lipid membranes (tBLMs).

Tethered bilayer lipid membranes (tBLMs) were prepared by the self-assembly of thiolated lipidic anchor molecules on gold, followed by phospholipid precipitation via rapid solvent exchange. They were characterized by their in-plane structure, dynamics and dielectric properties. We find that the in-plane homogeneity and resistivity of the tBLMs depend critically on a well-controlled sample environment during the rapid solvent-exchange procedure. The in-plane dynamics of the systems, assessed by fluorescence correlation spectroscopy (FCS) as the diffusivity of free, labeled phospholipid dissolved in the membrane, depend on the density of the lipidic anchors in the bilayer leaflet proximal to the substrate as well as on details of the molecular structure of the anchor lipid. In DOPC tBLMs in which tethers are laterally dilute (sparsely tethered bilayer lipid membranes, stBLMs), measured diffusivities, D ≈ 4 μm(2) s(-1), are only slightly greater than those reported in physisorbed bilayers (M. Przybylo, J. Sykora, J. Humpolíckova, A. Benda, A. Zan and M. Hof, Langmuir, 2006, 22, 9096-9099). However, when we distinguish label diffusion in the proximal and in the distal bilayer leaflets, we observe distinct diffusivities, D ≈ 2 μm(2) s(-1) and 7 μm(2) s(-1), respectively. The value observed in the distal leaflet is identical to that in free membranes. stBLMs completed with phytanoyl lipids (DPhyPC) show consistently lower label diffusivity than those completed with unsaturated chains (DOPC). As the length of the tether chain increases, a reduction in the apparent diffusivity is observed, which we interpret as an increased propensity of the proximal bilayer leaflet to host free lipid. We also investigated preparation conditions that control whether the tBLMs are laterally homogeneous, as assessed by optical microscopy. In laterally heterogeneous bilayers, the label diffusivity varies only by a factor of ~2 to 4, indicating that the regions in the bilayers with different label solubilities do not correspond to distinct phases, such as a fluid phase coexisting with a gel phase.

What is the Difference Between a Supported and a Free Lipid Bilayer?

Supported Lipid Bilayers are an abundant research platform for understanding the behavior of real cell membranes as they allow for additional mechanical stability and enable characterization techniques not reachable otherwise. However, in computer simulations these systems have been studied only rarely up to now. Here we present a systematic study of the changes that a support inflicts on a phospholipid bilayer using coarse-grained molecular modeling.We characterize the density and pressure profiles as well as the density imbalance induced by the support. It turns out that the changes in pressure profile are strong enough that protein function should be impacted leading to a previously neglected mechanism of transmembrane protein malfunction in supported bilayers. We also determine the diffusion coefficients and characterize the influence of different corrugations of the support. We then determine the free energy of transfer of phospholipids between the proximal (close to the surface) and distal leaflet of a supported membrane using the coarse-grained Martini model. It turns out that there is at equilibrium about a 2-3% higher density in the proximal leaflet. These results are in favorable agreement with recent data obtained by very large scale modeling using a water free model where flip-flop can be observed directly. We compare results of the free energy of transfer obtained by pulling the lipid across the membrane in different ways. There are small quantitative differences but the overall picture is consistent. We are additionally characterizing the intermediate states which determine the barrier height and therefore the rate of translocation. Simulations in atomistic detail are performed for selected systems in order to confirm the findings.

Lipid Diffusion in Tethered Bilayer Lipid Membranes (tBLMs)

The complexity of cells makes molecular-scale characterizations of structure and interactions of biomembranes in vivo extremely difficult, thus driving the development of synthetic membrane models. tBLMs are resilient biomimetic systems stabilized by the proximity of an inorganic interface.1,2 We characterize their in-plane structure, dynamics and dielectric properties using fluorescence microscopy, fluorescence correlation spectroscopy (FCS) and electrochemical impedance spectroscopy (EIS). The in-plane dynamics of tBLMs depend on structural details of the anchor lipid and its lateral density in the bilayer leaflet proximal to the substrate.3 In tBLMs with homogeneous lateral label distributions, the fluidity of the distal leaflet is comparable to that in vesicle membranes (2D diffusion constant, D ∼ 7 μm2/s) while that in the proximal leaflet is moderately reduced (D ∼ 2-3 μm2/s). tBLMs completed with phytanoyl lipids (DPhyPC) show lower label diffusivity than those completed with unsaturated chains (DOPC). In laterally heterogeneous bilayers, the label diffusivity varies only slightly, indicating that distinct regions in the bilayers do not correspond to distinct phases. Concurrently, we investigate the effect of charged lipids (DOPS) and cholesterol on lipid diffusivity. This aims at a characterization of changes in membrane dynamics as amyloid-β (Aβ) oligomers associate with tBLMs where they have been shown to affect the resistance of the bilayer to ion conduction.4,5Supported by the NIH (1P01AG032131) and the AHAF (A2008-307).1McGillivray, D.J., et al. 2007. Biointerphases 2:21-33.2Vockenroth, I.K., et al. 2008. Biointerphases 3:FA68-73.3Shenoy, S., et al., 2009. Soft Matter, submitted.4Arispe, N., et al., 1993. Proc. Natl. Acad. Sci. U.S.A. 90:567-571.5Valincius, G., et al., 2008. Biophys. J. 95:4845-4861.