Short-chain alcohols promote accelerated membrane distention in a dynamic liposome model of exocytosis

We have used amperometric measurements in a model system consisting of two liposomes connected with a membrane nanotube to monitor catechol release during artificial exocytosis and thereby to elucidate the effect of small-chain alcohols on this dynamic membrane process. To determine the rate of membrane shape change, catechol release during membrane distention was monitored amperometrically, and the presence of alcohols in the buffer was shown to accelerate the membrane distention process in a concentration-dependent manner. Compression isotherms for the same lipid composition in the absence and presence of ethanol and 1-propanol were measured to determine how these short-chain alcohols affect the lipid packing in monolayers. The isotherms show a marked decrease in lipid packing density that is dependent on the particular alcohol and its concentration. Comparison of the electrochemical and isotherm results suggests a correlation between decreasing lipid packing density and increasing rates of membrane shape change.     

Atomistic simulation of lipid and DiI dynamics in membrane bilayers under tension

Membrane tension modulates cellular processes by initiating changes in the dynamics of its molecular constituents. To quantify the precise relationship between tension, structural properties of the membrane, and the dynamics of lipids and a lipophilic reporter dye, we performed atomistic molecular dynamics (MD) simulations of DiI-labeled dipalmitoylphosphatidylcholine (DPPC) lipid bilayers under physiological lateral tensions ranging from -2.6 mN m(-1) to 15.9 mN m(-1). Simulations showed that the bilayer thickness decreased linearly with tension consistent with volume-incompressibility, and this thinning was facilitated by a significant increase in acyl chain interdigitation at the bilayer midplane and spreading of the acyl chains. Tension caused a significant drop in the bilayer's peak electrostatic potential, which correlated with the strong reordering of water and lipid dipoles. For the low tension regime, the DPPC lateral diffusion coefficient increased with increasing tension in accordance with free-area theory. For larger tensions, free area theory broke down due to tension-induced changes in molecular shape and friction. Simulated DiI rotational and lateral diffusion coefficients were lower than those of DPPC but increased with tension in a manner similar to DPPC. Direct correlation of membrane order and viscosity near the DiI chromophore, which was just under the DPPC headgroup, indicated that measured DiI fluorescence lifetime, which is reported to decrease with decreasing lipid order, is likely to be a good reporter of tension-induced decreases in lipid headgroup viscosity. Together, these results offer new molecular-level insights into membrane tension-related mechanotransduction and into the utility of DiI in characterizing tension-induced changes in lipid packing.     

Effect of chlorogenic acid on the phase transition in phospholipid and phospholipid/cholesterol membranes

Chlorogenic acid (CGA) is present in many plants, especially in green coffee, dry plums, and bilberries. It is an important bioactive polyphenol. Studies showed that CGA has an antioxidative, bacteriostatic, anticancer, antiviral, and anti-inflammatory activity. Despite great interest in this compound, its interaction with the lipid model membrane has not yet been investigated. To better understand the relationship between the biological activity of CGA and its interaction with biological membranes, the thermotropic behavior of model lipid membranes was investigated. The effect of CGA on the model lipid membrane, specifically on the lipid bilayer phase transitions, was examined by the combined methods: differential scanning calorimetry and fluorescence spectroscopy. In particular, the degree of packing order of the hydrophilic phase of the lipid bilayer was determined using the fluorimetric method with Laurdan and Prodan probes, while the fluorescence anisotropy of the hydrophobic phase with the DPH and TMA-DPH probes. The results of the study show that CGA incorporates mainly into the hydrophilic part of membrane, changing the packing order of the polar heads of lipids. No significant changes were recorded in membrane fluidity of the hydrophobic membrane region, for the fluorescence anisotropy practically did not change. One can thus infer that CGA does not penetrate deep into the hydrophobic area of the membrane.     

Photosensitized damage of bilayer lipid membrane in the presence of haematoporphyrin dimethylether

The variations in electrical conductivity and surface tension of planar bilayer lipid membranes (BLMs) sensitized by a haematoporphyrin dimethylether (HpDME) on visible light irradiation are reported. The irradiation of BLMs immediately leads to a decrease in membrane surface tension. On irradiation the conductivity of BLMs remains constant for a certain period of time (induction time), followed by an increase, terminated by membrane breakage. The induction time is not dependent on stirring of the solution, the addition of azide or ferricyanide to the solution, the addition of antioxidant to the lipid or substitution of air for argon in the cell. The induction time decreases for repeated irradiations or for any new BLM formed in the same cell immediately after the previous membrane has been broken. The conductivity shift consists of reversible and irreversible components. These results suggest that the irradiation of BLMs sensitized by HpDME leads to an accumulation of photoproducts in the membrane which induce pore formation and to a decrease in BLM stability when the concentration of the photoproducts exceeds a critical level.     

Fluorescence studies on radiation oxidative damage to membranes with implications to cellular radiosensitivity

Radiation oxidative damage to plasma membrane and its consequences to cellular radiosensitivity have received increasing attention in the past few years. This review gives a brief account of radiation oxidative damage in model and cellular membranes with particular emphasis on results from our laboratory. Fluorescence and ESR spin probes have been employed to investigate the structural and functional alterations in membranes after y-irradiation. Changes in the lipid bilayer in irradiated unilamellar liposomes prepared from egg yolk lecithin (EYL) were measured by using diphenylhexatriene (DPH) as a probe. The observed increase in DPH polarization and decrease in fluorescence intensity after γ-irradiation of liposomes imply radiation-induced decrease in bilayer fluidity. Inclusion of cholesterol in liposome was found to protect lipids against radiation damage, possibly by modulation of bilayer organization e.g. lipid packing. Measurements on dipalmitoyl phosphatidylcholine (DPPC) liposomes loaded with 6-carboxyfluorescein (CF) showed radiation dose-dependent release of the probe indicating radiation-induced increased permeability. Changes in plasma membrane permeability of thymocytes were monitored by fluorescein diacetate (FDA) and induced intracellular reactive oxygen species (ROS) were determined by 2,7-dichlorodihydro fluorescein diacetate (DCH-FDA). Results suggest a correlation between ROS generation and membrane permeability changes induced by radiation within therapeutic doses (0-10 Gy). It is concluded that increase in membrane permeability was the result of ROS-mediated oxidative reactions, which might trigger processes leading to apoptotic cell death after radiation exposure.     

Evaluation of 1-naphthol as a convenient fluorescent probe for monitoring ethanol-induced interdigitation in lipid bilayer membrane.

In this work we have tried to evaluate the usefulness of 1-naphthol as an excited state proton transfer fluorescent probe for studying the ethanol-induced interdigitation in lipid bilayer membranes. When ethanol concentration in lipisome is progressively increased, the neutral form fluorescence of 1-naphthol is found to decrease with corresponding increase in the anionic form intensity. This behavior is in contrast to that observed in the absence of lipid where a reverse effect is noticed. Modification of lipid bilayer is known to occur in the presence of ethanol, which increases the packing density of the membrane. Due to this induction of interdigitated gel phase, redistribution of naphthol between the inner core and interfacial region of the lipid bilayer takes places, accounting for the reduction in neutral form fluorescence intensity. The partition coefficient values and the quenching studies also support the redistribution of 1-naphthol in the liposome membrane. The neutral form fluorescence of 1-naphthol successfully monitors the shift in phase transition temperature due to ethanol-induced interdigitation. It also explains the prevention of interdigitation in lipid bilayer at high cholesterol concentration.     

Influence of polymyxins on the structural dynamics of Escherichia coli lipid membranes

Polymyxins are a family of nonribosomic cationic peptide antibiotics highly effective against Gram-negative bacteria. Two members of this family, Polymyxins B and E (PxB, PxE), form molecular vesicle-vesicle contacts and promote a selective exchange of phospholipids at very low concentrations in the membrane, a biophysical phenomenon that can be the basis of their antibiotic mode of action. To get more insight into the interaction of these antibiotics with the lipid membrane, their effect on the structural dynamics of bilayers prepared with lipids extracted from the membrane of Escherichia coli was determined using fluorescently labeled phopholipids. Steady-state anisotropy measurements with probes that localize at different positions in the membrane give information on the effects of polymyxins on the mobility of the phospholipids. Results with PxB, PxE, colymycin M and polymyxin B nonapeptide (PxB-NP), a deacylated derivative with no antibiotic properties, are compared. At low peptide concentrations (<2 mol%) PxB and PxE bind to the membranes superficially, affecting very slightly the ordering of the lipids at the outermost part of the bilayer. Above this concentration, PxB and PxE insert more deeply in the bilayer, increasing lipid order both in the gel and liquid-crystal states and modifying phase transitions. Fluorescence experiments with pyrene labeled phospholipids indicate that the increase in lipid packing is accompanied by an enrichment of phospholipids in the bilayers. In contrast, colymycin M and PxB-NP did not modify lipid packing or phase transition, nor did they induce microdomain formation. The possible significance of these results in the antibiotic mode of action of PxB and PxE is discussed. The combination of spectroscopic techniques described here can be useful as part of a general method of screening for new antibiotics that act on the membrane by the same mechanism as polymyxins.     

Surface tension effect on transmembrane channel stability in a model membrane

The effect of surface tension on the lipid bilayer membrane is a question that has drawn considerable research effort. This interest has been driven both by the desire to determine the surface tension effects on the lipid bilayer and from the suggestion that adding finite surface tension to a small membrane system may provide more realistic lipid properties in molecular dynamics simulations. Here, the effect of surface tension on a palmitololelylphosphatidylcholine (POPC) bilayer membrane containing a four-helix transmembrane alamethicin peptide bundle is investigated. Simulations of 10 ns were undertaken for two different ensembles, NPT and NP(z)gammaT with a surface tension, gamma, of 20 mN m(-1) per interface, which is near the pore-forming region. The significance of differences between the tension-free and surface tension simulations was determined using nonparametric statistical analysis on replicate simulations with different initial conditions. The results suggest that, when the membrane is under surface tension, the peptide helical structure is perturbed from that in the tension-free state but that the bundle conformation is more stable than that in the tension-free state, with hydrogen bonding playing an important stabilizing role. Surface tension counteracts the influence of the transmembrane helix bundle on nearby lipid order, making the lipid order more uniform throughout the membrane in the tension state. Conversely, the lipid mobility was less uniform in the tension state, with lipids far from the bundle being significantly more mobile than those near the bundle. One general implication of the results is that surface tension can affect the membrane nonuniformly, in that the properties of lipids near the peptide are different from those further away.     

Switchable Solvatochromic Probes for Live‐Cell Super‐resolution Imaging of Plasma Membrane Organization

Visualization of the nanoscale organization of cell membranes remains challenging because of the lack of appropriate fluorescent probes. Herein, we introduce a new design concept for super‐resolution microscopy probes that combines specific membrane targeting, on/off switching, and environment sensing functions. A functionalization strategy for solvatochromic dye Nile Red that improves its photostability is presented. The dye is grafted to a newly developed membrane‐targeting moiety composed of a sulfonate group and an alkyl chain of varied lengths. While the long‐chain probe with strong membrane binding, NR12A, is suitable for conventional microscopy, the short‐chain probe NR4A, owing to the reversible binding, enables first nanoscale cartography of the lipid order exclusively at the surface of live cells. The latter probe reveals the presence of nanoscopic protrusions and invaginations of lower lipid order in plasma membranes, suggesting a subtle connection between membrane morphology and lipid organization.     

Spectral Imaging to Measure Heterogeneity in Membrane Lipid Packing

Physicochemical properties of the plasma membrane have been shown to play an important role in cellular functionality. Among those properties, the molecular order of the lipids, or the lipid packing, is of high importance. Changes in lipid packing are believed to compartmentalize cellular signaling by initiating coalescence and conformational changes of proteins. A common way to infer membrane lipid packing is by using membrane‐embedded polarity‐sensitive dyes, whose emission spectrum is dependent on the molecular order of the immediate membrane environment. Here, we report on an improved determination of such spectral shifts in the emission spectrum of the polarity‐sensitive dyes. This improvement is based on the use of spectral imaging on a scanning confocal fluorescence microscope in combination with an improved analysis, which considers the whole emission spectrum instead of just single wavelength ranges. Using this approach and the polarity‐sensitive dyes C‐Laurdan or Di‐4‐ANEPPDHQ, we were able to image—with high accuracy—minute differences in the lipid packing of model and cellular membranes.     

Fluorescent Flipper Probes: Comprehensive Twist Coverage

To image the membrane tension in living cells, planarizable push–pull probes have been introduced. The first operational probe is built around two dithieno[3,2-b:2′,3′-d]thiophenes (DTTs) that are twisted out of co-planarity and polarized with donors and acceptors at either end. In this report, the chemical space available for the twisting of “flipper probes” is assessed comprehensively. The result is, not surprisingly, that every atom matters: Removal of one methyl group in the twist region yields probes that planarize already in solution and are thus less sensitive to membrane tension. Addition of one or more carbons in the same region hinders non-interfering probe alignment along lipid tails and thus partitioning into lipid bilayer membranes as well as mechanosensitivity. However, substitution of one methyl by an isosteric trifluoromethyl group in the twist region, achieved by quite substantial multistep organic synthesis, yields excitation maxima that shift over +100 nm to the red in response to increasing order of the surrounding membrane. This record redshift comes with record changes in fluorescence intensity and lifetime, high push–pull transition dipoles and higher rotational barriers. Supported by distinct dependence on viscosity and twist of the push–pull probes, kinetic competition between dark, fully twisted and bright, fully planarized relaxed excited states emerges as unifying origin of fluorescence quantum yields.     

Spin probe study on the interaction of chitosan-derived polymer surfactants with lipid membrane

Chitosan-derived polymer surfactants, sulfated N-acyl-chitosan (S-Cn-Chitosan), were synthesized and compared with commonly used low-molecular-weight surfactants, sodium dodecyl sulfate (SDS), dodecyltrimethyl ammonium chloride (DTAC), and octaethyleneglycol mono n-dodecyl ether (BL8SY), in their interaction with a lipid membrane using a spin probe method. A suspension of dipalmitoylphosphatidyl-choline (DPPC) spin-labeled strongly (10%) with a spin probe, 1-palmitoyl-2-(12-doxyl) stearoyl-phosphatidylcholine, was mixed with the surfactant solutions. The dissolution time of the DPPC membrane was estimated from the peak height change vs time, which was caused by the decrease in spin-exchange interaction. The times were 2, 4, and 70 s for BL8SY, SDS, and DTAC and 1.2 and 8.8 h for S-C10-Chitosan and S-C14-Chitosan, respectively, showing that the dissolution of the lipid membrane with polymer surfactants was far slower than that with low-molecular-weight surfactants. In addition, the time depended on the length of the alkyl chains of the polymer surfactants. Simulations of the ESR spectra of the DPPC-surfactant systems containing small amounts of surfactants were carried out in order to examine how the membrane structure was changed by the incorporation of surfactant molecules. By this analysis, it was revealed that the rigidity of the membrane was decreased by the addition of low-molecular-weight surfactants in the order of DTAC>SDS>BL8SY, inverse to the order of dissolution times. S-Cn-Chitosan, in contrast, increased the rigidity of the membrane, suggesting that polymer surfactants adhered to the lipid membrane and tightly enfolded the riposome anchoring their alkyl chains.     

Loosening of Lipid Packing Promotes Oligoarginine Entry into Cells

Despite extensive use of arginine‐rich cell‐penetrating peptides (CPPs)—including octaarginine (R8)—as intracellular delivery vectors, mechanisms for their internalization are still under debate. Lipid packing in live cell membranes was characterized using a polarity‐sensitive dye (di‐4‐ANEPPDHQ), and evaluated in terms of generalized polarization. Treatment with membrane curvature‐inducing peptides led to significant loosening of the lipid packing, resulting in an enhanced R8 penetration. Pyrenebutyrate (PyB) is known to facilitate R8 membrane translocation by working as a hydrophobic counteranion. Interestingly, PyB also actively induced membrane curvature and perturbed lipid packing. R8 is known to directly cross cell membranes at elevated concentrations. The sites of R8 influx were found to have looser lipid packing than surrounding areas. Lipid packing loosening is proposed as a key factor that governs the membrane translocation of CPPs.     

Modulation of the spontaneous curvature and bending rigidity of lipid membranes by interfacially adsorbed amphipathic peptides

Amphipathic alpha-helical peptides are often ascribed an ability to induce curvature stress in lipid membranes. This may lead directly to a bending deformation of the host membrane, or it may promote the formation of defects that involve highly curved lipid layers present in membrane pores, fusion intermediates, and solubilized peptide-micelle complexes. The driving force is the same in all cases: peptides induce a spontaneous curvature in the host lipid layer, the sign of which depends sensitively on the peptide's structural properties. We provide a quantitative account for this observation on the basis of a molecular-level method. To this end, we consider a lipid membrane with peptides interfacially adsorbed onto one leaflet at high peptide-to-lipid ratio. The peptides are modeled generically as rigid cylinders that interact with the host membrane through a perturbation of the conformational properties of the lipid chains. Through the use of a molecular-level chain packing theory, we calculate the elastic properties, that is, the spontaneous curvature and bending stiffness, of the peptide-decorated lipid membrane as a function of the peptide's insertion depth. We find a positive spontaneous curvature (preferred bending of the membrane away from the peptide) for small penetration depths of the peptide. At a penetration depth roughly equal to half-insertion into the hydrocarbon core, the spontaneous curvature changes sign, implying negative spontaneous curvature (preferred bending of the membrane toward the peptide) for large penetration depths. Despite thinning of the membrane upon peptide insertion, we find an increase in the bending stiffness. We discuss these findings in terms of how the peptide induces elastic stress.     

Electrostriction of biotin-streptavidin-modified bilayer lipid membranes on a metal support

Using the electrostriction method we have studied the elasticity modulus perpendicular to the membrane plane, E⊥, electrical capacitance, C, coefficient of dynamic viscosity, η, and membrane potential difference δф_m of supported bilayer lipid membranes (s-BLM) modified by biotin-streptavidin, as a function of d.c. voltage applied to the membrane. Binding of streptavidin to biotin-modified s-BLM resulted in a slight decrease of membrane capacitance, increase of E_⊥ and increase of η, while δф_m did not change. The val of E_⊥ of unmodified membranes was found to change considerably with increasing d.c. voltage and the rate of voltage change. Modification of s-BLM by streptavidin leads to reduced changes of E_⊥ with the rate of d.c. voltage change, and it made s-BLM extremely stable even at an external d.c. voltage of 2 V. Our results indicate that streptavidin considerably stabilized s-BLM by means of the formation of a complex with biotin-modified phospholipids.     

Rationalizing the membrane interactions of cationic amphipathic antimicrobial peptides by their molecular shape

Biophysical and structural studies of cationic amphipathic antimicrobial peptides have revealed new mechanistic details concerning their membrane interactions. In interfacial environments the peptides adopt amphipathic conformations and the resulting distribution of polar, charged and hydrophobic residues allows them to partition into the bilayer interface. For several helical peptides it was found that their long axis is oriented parallel to the membrane surface, an arrangement which results in considerable perturbations in the packing of the lipid bilayer. Within the molecular shape concept the peptides act as wedge-like structures which impose positive curvature strain on the membrane. As a consequence a wide variety of morphologies are observed of peptide–lipid mixtures which strongly depend on the detailed peptide sequence, the membrane lipid composition, buffer, temperature and other environmental parameters. Therefore, the peptide–lipid systems are best described by phase diagrams, similar to the ones of detergent–lipid mixtures, encompassing on the one extreme regions where the peptide stabilizes the bilayer and on the other extreme regions where membrane lysis occurs. The effects of peptide sequence, membrane penetration depth, lipid composition and membrane surface charge density on membrane-association, -morphology and the resulting phase boundaries are discussed.     

Nitric oxide enhances the capacitance of self-assembled,supported bilayer lipid membranes

The effect of nitric oxide (NO) at biologically relevant concentrations on the electrochemical features of the membrane was investigated by cyclic voltammetry (CV) at self-assembled, stainless steel supported lipid bilayer membranes (s-BLMs) using a three-electrode system. The results showed that the membrane capacitance (C_m) of s-BLMs was dramatically enhanced by the presence of increasing NO concentration from 0 to 70 μM. For comparison, fullerene C₆₀ doped s-BLMs (C₆₀@s-BLMs) was also studied. The C_m of C₆₀@s-BLMs increased with NO concentration from 0 to 16 μM and gradually reached a plateau value when NO concentration was over 16 μM. We concluded that (i) NO accumulated inside lipid bilayer increases the C_m of s-BLMs, and (ii) C₆₀ inside s-BLMs changes the dielectric constant of lipid bilayer, thus reducing the effect of NO on the C_m of C₆₀@s-BLMs. This novel self-assembled lipid modified probe provides a simple yet interesting model to study the effect of NO on the electrical conductance of the membrane.     

Size-dependent properties of small unilamellar vesicles formed by model lipids

The size-dependent behavior of small unilamellar vesicles is explored by dissipative particle dynamics, including the membrane characteristics and mechanical properties. The spontaneously formed vesicles are in the metastable state and the vesicle size is controlled by the concentration of model lipids. As the vesicle size decreases, the bilayer gets thinner and the area density of heads declines. Nonetheless, the area density in the inner leaflet is higher than that in the outer. The packing parameters are calculated for both leaflets. The result indicates that the shape of lipid in the outer leaflet is like a truncated cone but that in the inner leaflet resembles an inverted truncated cone. Based on a local order parameter, our simulations indication that the orientation order of lipid molecules decreases as the size of the vesicle reduces and this fact reveals that the bilayer becoming thinner for smaller vesicle is mainly attributed to the orientation disorder of the lipids. The membrane tension can be obtained through the Young-Laplace equation. The tension is found to grow with reducing vesicle size. Therefore, small vesicles are less stable against fusion. Using the inflation method, the area stretching and bending moduli can be determined and those moduli are found to grow with reducing size. Nonetheless, a general equation with a single numerical constant can relate bending modulus, area stretching modulus, and bilayer thickness irrespective of the vesicle size. Finally, a simple metastable model is proposed to explain the size-dependent behavior of bilayer thickness, orientation, and tension.     

Further reflections on the one-dimensional packing problem

The one-dimensional packing problem may be stated as follows: When objects of lengthL are randomly placed on a line of lengthN until no more placement is possible, how much space remains unoccupied? In a previous paper, the authors showed that, forL = 2, the fraction of unoccupied space is dependent on the model governing the placement mechanism. In this paper, these results are extended from the discrete to the continuous case by allowing bothN andL to increase, while keeping their ratio constant. The methodology was validated by reproducing the analytical results for limiting cases.     

Influence of pyrene-labeling on fluid lipid membranes

We elucidate the influence of pyrene-labeled phospholipids on the structural properties of a fluid dipalmitoylphosphatidylcholine lipid membrane. To this end, we employ extensive atomic-scale molecular dynamics simulations with varying concentrations of pyrene-linked lipids. We find pyrene labeling to perturb the membrane structure significantly in the vicinity of the probe, the correlation length in the bilayer plane being about 1.0-1.5 nm. The local perturbations lead to enhanced ordering and packing of lipid acyl chains located in the vicinity of the probe. Surprisingly, this holds true not only for lipids that reside in the same leaflet as the pyrene-labeled probe but also for lipids in the opposite monolayer. The latter is due to substantial interdigitation of the pyrene moiety into the opposite leaflet, suggesting that occasional excimer formation may take place for probes in different leaflets. As a related issue, we also discuss the location and conformational orientation of the pyrene moieties. In particular, the orientational distribution of pyrene turns out to be more broad and diverse than the distribution of the corresponding acyl tails of nonlabeled lipids.