Adiabatic compressibility of red blood cell membrane: influence of skeleton

Measurements of ultrasound velocity and density were used for determination of the adiabatic compressibility of red blood cells (RBC) during detachment of the membrane skeleton. Skeleton detachment was induced by addition of nystatin into a low ionic strength RBC suspension resulting in an increase (10%) of the ultrasound velocity concentration increment, [u], while the specific volume of cells, phi(v) did not change significantly. Changes of the concentration increment had rather long kinetics and were not completed even after 60 min. Both [u] and phiV values were used for calculation of the specific apparent adiabatic compressibility of RBC, phiK/beta0. The value of the specific apparent compressibility decreases following addition of nystatin. This corresponds to an increase in the volume elastic modulus of RBC membranes during detachment of the membrane skeleton. Control experiments with large unilamellar liposomes at conditions similar to that performed with the RBC did not reveal significant changes of [u] after the addition of nystatin. Our results show that the role of the membrane skeleton probably consists in maintaining higher compressibility of the RBC membranes. This may partly provide conditions for conformational changes of RBC membrane proteins.     

Dimpled vesicles: the interplay between energetics and transient pores

The familiar biconcave shape of the red-blood cell (RBC) deforms as the cell travels through capillaries. Its dimpled configurations are unique cell shapes and display malleability to form echinocytes, discocytes and stomatocytes, in response to external perturbations. Sheetz and Singer introduced intercalating species to the exterior lipid leaflet of the membrane to promote cup-shaped stomatocytes, and observed that additives to the interior had the opposite effect. Shape transformations appear to be controlled via the RBC bilayer and the asymmetric surface areas of the two leaflets [Proc. Natl. Acad. Sci. U.S.A. 1974, 71, 4457]. Our system promotes area-difference between the lipid bilayer leaflets from a fully symmetrical system and has mimicked the RBC discoid. In our analysis, we explore the system energetic and geometric confinements, which points to transient pores as enablers for the vesicles to deflate and thereby to assume lower profiles.     

Spherocyte shape transformation and release of tubular nanovesicles in human erythrocytes

We have studied dodecylmaltoside-induced echinocyte-spheroechincyte-spherocyte shape transformation and membrane vesiculation using transmission electron microscopy (TEM) on freeze-fracture replicas. It is indicated that spherical erythrocyte shape at higher dodecylmaltoside concentration is formed due to loss of membrane in the process where small, mostly tubular nanovesicles are released predominantly from the top of echinocyte and spheroechinocyte spicules.     

Equilibrium of ion-exchange polymeric membrane with aqueous salt solution and its thermodynamic modeling

A thermodynamic model is proposed to describe distribution of the components between a liquid solution and a swollen membrane undergoing structural transformations. Free energy contributions related to formation of solution-filled micro-cavities in the membrane interior are estimated. Formation of the cavities of different shape is accounted for by using the Helfrich expressions for the bending energy of a curved interface. Three adjustable parameters of the model are related to the hydrophobic polymer matrix of the membrane, while the electrostatic contribution is estimated explicitly. Structural changes in the membrane are described as a transition from spherical to cylindrical cavities. Predominance of cavities having definite shape (spheres, cylinders) results in a specific shift of the Donnan equilibrium, which thus, becomes dependent on the structure of the membrane on the mesoscale. The results of model calculations are compared with the experimental data on the distribution of ions (H⁺, Li⁺, Cs⁺, K⁺, Na⁺, Ca²⁺, Mg²⁺) between the aqueous solution and the membrane. Different types of predicted thermodynamic behavior of the membrane in the liquid solution, including the hysteresis of ion-exchange equilibrium curves, are discussed. The model takes into account the effect of micro-inhomogeneties and helps to establish a link between molecular characteristics of the perfluoropolymer membrane and its macroscopic behavior in the liquid solution.     

Trout Erythrocyte Membranes. Thermoresistivity and thermally induced structural transitions

Differential scanning microcalorimetry and equilibrium thermohemolysis procedure were used to study the effect of acclimation temperature on thermally induced transitions and thermoresistivity of fish (trout) erythrocyte membranes. Strong correlation has been found between the rates and activation energies of erythrocyte thermohemolysis and acclimation temperature. Transition temperatures of five thermodynamically irreversible and one partially reversible transitions at about 87°C as well as the overall shape of microcalorimetric curves of the erythrocyte ghosts do not vary with acclimation temperature. The results suggest an essential conservation of phospholipid microenvironment of membrane skeleton proteins despite the compensatory response in lipid composition of erythrocyte membrane bilayer. This revised version was published online in July 2006 with corrections to the Cover Date.     

Surface tension in bilayer membranes with fixed projected area

We study the elastic response of bilayer membranes with fixed projected area to both the stretching and shape deformations. A surface tension is associated to each of these deformations. By using model amphiphilic membranes and computer simulations, we are able to observe both the types of deformation, and thus, both the surface tensions, related to each type of deformation, are measured for the same system. These surface tensions are found to assume different values in the same bilayer membrane, in particular, they vanish for different values of the projected area. We introduce a simple theory which relates the two quantities and successfully apply it to the data obtained with computer simulations.     

Nonlocal membrane bending: A reflection,the facts and its relevance

About forty years ago it was realized that phospholipid membranes, because they are composed of two layers, exhibit particular, and specific mechanical properties [1–3]. This led to the concept of nonlocal membrane bending, often called area difference elasticity. We present a short history of the development of the concept, followed by arguments for a proper definition of the corresponding elastic constant. The effects of the nonlocal bending energy on vesicle shape are explained. It is demonstrated that lipid vesicles, cells and cellular aggregates exhibit phenomena that can only be described in a complete manner by considering nonlocal bending.     

Statistical mechanics of bilayer membrane with a fixed projected area

The equilibrium and fluctuation methods for determining the surface tension, sigma, and bending modulus, kappa, of a bilayer membrane with a fixed projected area are discussed. In the fluctuation method the elastic coefficients sigma and kappa are measured from the amplitude of thermal fluctuations of the planar membrane, while in the equilibrium method the free energy required to deform the membrane is considered. The latter approach is used to derive new expressions for sigma and kappa (as well as for the saddle-splay modulus), which relate them to the pair-interactions between the amphiphiles forming the membrane. We use linear response theory to argue that the two routes lead to similar values for sigma and kappa. This argument is confirmed by Monte Carlo simulations of a model membrane whose elastic coefficients are calculated using both methods.     

Mechanical stability of micropipet-aspirated giant vesicles with fluid phase coexistence

Micropipet aspiration of phase-separated lipid bilayer vesicles can elucidate physicochemical aspects of membrane fluid phase coexistence. Recently, we investigated the composition dependence of line tension at the boundary between liquid-ordered and liquid-disordered phases of giant unilamellar vesicles obtained from ternary lipid mixtures using this approach. Here we examine mechanical equilibria and stability of dumbbell-shaped vesicles deformed by line tension. We present a relationship between the pipet aspiration pressure and the aspiration length in vesicles with two coexisting phases. Using a strikingly simple mechanical model for the free energy of the vesicle, we predict a relation that is in almost quantitative agreement with experiment. The model considers the vesicle free energy to be proportional to line tension and assumes that the vesicle volume, domain area fraction, and total area are conserved during aspiration. We also examine a mechanical instability encountered when releasing a vesicle from the pipet. We find that this releasing instability is observed within the framework of our model that predicts a change of the compressibility of a pipet-aspirated membrane cylinder from positive (i.e., stable) to negative (unstable) values, at the experimental instability. The model furthermore includes an aspiration instability that has also previously been experimentally described. Our method of studying micropipet-induced shape transitions in giant vesicles with fluid domains could be useful for investigating vesicle shape transitions modulated by bending stiffness and line tension.     

Elastic deformation of membrane bilayers probed by deuterium NMR relaxation

In deuterium ((2)H) NMR spectroscopy of fluid lipid bilayers, the average structure is manifested in the segmental order parameters (S(CD)) of the flexible molecules. The corresponding spin-lattice relaxation rates (R(1Z) depend on both the amplitudes and the rates of the segmental fluctuations, and indicate the types of lipid motions. By combining (2)H NMR order parameter measurements with relaxation studies, we have obtained a more comprehensive picture of lipids in the liquid-crystalline (L(alpha)) state than formerly possible. Our data suggest that a lipid bilayer constitutes an ordered fluid, in which the phospholipids are grafted to the aqueous interface via their polar headgroups, whereas the fatty acyl chains are in effect liquid hydrocarbon. Studies of (2)H-labeled saturated lipids indicate their R(1Z) rates and S(CD) order parameters are correlated by a model-free, square-law functional dependence, signifying the presence of relatively slow bilayer fluctuations. A new composite membrane deformation model explains simultaneously the frequency (magnetic field) dependence and the angular anisotropy of the relaxation. The results imply the R(1Z) rates are due to a broad spectrum of 3-D collective bilayer excitations, together with effective axial rotations of the lipids. For the first time, NMR relaxation studies show that the viscoelastic properties of membrane lipids at megahertz frequencies are modulated by the lipid acyl length (bilayer thickness), polar headgroups (bilayer interfacial area), inclusion of a nonionic detergent (C(12)E(8)), and the presence of cholesterol, leading to a range of bilayer softness. Our findings imply the concept of elastic deformation is relevant on lengths approaching the bilayer thickness and less (the mesoscopic scale), and suggest that application of combined R(1Z) and S(CD) studies of phospholipids can be used as a simple membrane elastometer. Heuristic estimates of the bilayer bending rigidity kappa and the area elastic modulus K(a) enable comparison to other biophysical studies, involving macroscopic deformation of thin membrane lipid films. Finally, the bilayer softness may be correlated with the lipid diversity of biomembranes, for example, with regard to membrane curvature, repulsive interactions between bilayers, and lipid-protein interactions.     

Vesicle shapes from molecular dynamics simulations

Lipid bilayer membranes are known to form various structures such as large sheets or vesicles. When the two leaflets of the bilayer have an equal composition, the membrane preferentially forms a flat sheet or a spherical vesicle. However, a difference in the composition of the two leaflets may result in a curved bilayer or in a wide variety of vesicle shapes. Vesicles with different shapes have already been shown in experiments and diverse vesicle shapes have been predicted theoretically from energy minimization of continuous curves. Here we present a molecular dynamics study of the effect of small changes in the phospholipid headgroups on the spontaneous curvature of the bilayer and on the resulting vesicle shape transformations. Small asymmetries in the bilayers already result in high spontaneous curvature and large vesicle deformations. Vesicle shapes that are formed include ellipsoids, discoids, pear-shaped vesicles, cup-shaped vesicles, as well as budded vesicles. Comparison of these vesicles with theoretically derived vesicle shapes shows both resemblances and differences.     

Mechanism of unassisted ion transport across membrane bilayers

To establish how charged species move from water to the nonpolar membrane interior and to determine the energetic and structural effects accompanying this process, we performed molecular dynamics simulations of the transport of Na+ and Cl- across a lipid bilayer located between two water lamellae. The total length of molecular dynamics trajectories generated for each ion was 10 ns. Our simulations demonstrate that permeation of ions into the membrane is accompanied by the formation of deep, asymmetric thinning defects in the bilayer, whereby polar lipid head groups and water penetrate the nonpolar membrane interior. Once the ion crosses the midplane of the bilayer the deformation "switches sides"; the initial defect slowly relaxes, and a defect forms in the outgoing side of the bilayer. As a result, the ion remains well solvated during the process; the total number of oxygen atoms from water and lipid head groups in the first solvation shell remains constant. A similar membrane deformation is formed when the ion is instantaneously inserted into the interior of the bilayer. The formation of defects considerably lowers the free energy barrier to transfer of the ion across the bilayer and, consequently, increases the permeabilities of the membrane to ions, compared to the rigid, planar structure, by approximately 14 orders of magnitude. Our results have implications for drug delivery using liposomes and peptide insertion into membranes.     

Electric quadrupole model on the formation of molecular chirality dependent domain shapes of lipid monolayers at the air-water interface

Shapes and orientational deformation of a lipid monolayer domain have been analyzed taking into account the surface pressure, line tension, and electrostatic energy due to the spontaneous polarization and electric quadrupole density generated from the domain. The electrostatic energy due to the generation of spontaneous polarization and electric quadrupole density contributes to the formation of orientational deformation as the Frank elastic energy and spontaneous splay, respectively. Since the orientational configuration of the electric quadrupole density and in-plane spontaneous polarization is dependent on the molecular chirality, and the positive splay deformation of electric quadrupole density is induced by the spontaneous splay, the bending direction of in-plane spontaneous polarization depends on the chirality of constituent lipids. The electrostatic energy due to the in-plane spontaneous polarization is dependent on the orientational deformation of in-plane spontaneous polarization, and bends the domain shape towards the bending direction of the in-plane spontaneous polarization. It has been demonstrated that the chiral dependence of the domain shapes of lipid monolayers originated from the chiral dependence of orientational structure due to the electric quadrupole density.     

Thermally induced transformation of mammalian red blood cells during hyperthermia

The structural and transport characteristics of membranes are mainly determined by the state of the cytoskeleton. The characteristic changes in morphology of human (adult donor and cord) and rat Red Blood Cells (RBC) and of their membrane, induced by hyperthermia (46-51 degrees C) have been analyzed. Two different types of morphological changes have been observed to take place during hyperthermia in all studied RBC groups. We have observed either formation and exfoliation of spiculas from membrane, resulting in the formation of large (4-5 mum) sphere-like cell body and small (0.5-1.5 mum) vesicles or cell fragmentation with formation of large (3-3.5 mum) vesicles. The two distinct phenomena are likely to be determined by the heterogeneity of the RBC population in terms of cell age. There was noted the difference of cord RBC from the donor ones in temperature value of transformation beginning, as well as the character of deformation and vesicle formation, that may testify to their less thermoresistance. The ultrastructure of the membrane, studied with the freeze-fracturing technique, testifies to an irreversible character of membrane changes. The aggregation of intramembrane particles (IMPs) as a continuous network testifies to the strengthening of the interactions between denatured spectrin and bilayer integral components.     

Enhanced hydrophobicity of fluorinated lipid bilayer: a molecular dynamics study

A molecular dynamics simulation of a partially fluorinated phospholipid bilayer has been carried out to understand the effects of fluorination of the hydrophobic chains on the structure and water permeability across the membrane. Fluorocarbon chains typically have an all-trans conformation, showing a highly ordered structure in the membrane core compared to ordinary hydrocarbon chains. The free energy profiles of water across the bilayers were successfully estimated by a revised cavity insertion Widom method. The fluorinated bilayer showed a higher free energy barrier than an ordinary nonfluorinated lipid bilayer by about 1.2 kcal/mol, suggesting a lower water permeability of the fluorinated bilayer membrane. A cavity distribution analysis elucidated the reduced free volume in the fluorinated membrane due to the neatly packed chains, which should account for the higher free energy barrier.     

Dynamics and instabilities of lipid bilayer membrane shapes

Biological membranes undergo constant shape remodeling involving the formation of highly curved structures. The lipid bilayer represents the fundamental architecture of the cellular membrane with its shapes determined by the Helfrich curvature bending energy. However, the dynamics of bilayer shape transitions, especially their modulation by membrane proteins, and the resulting shape instabilities, are still not well understood. Here, we review in a unifying manner several theories that describe the fluctuations (i.e. undulations) of bilayer shapes as well as their local coupling with lipid or protein density variation. The coupling between local membrane curvature and lipid density gives rise to a ‘slipping mode’ in addition to the conventional ‘bending mode’ for damping the membrane fluctuation. This leads to a number of interesting experimental phenomena regarding bilayer shape dynamics. More importantly, curvature-inducing proteins can couple with membrane shape and eventually render the membrane unstable. A criterion for membrane shape instability is derived from a linear stability analysis. The instability criterion reemphasizes the importance of membrane tension in regulating the stability and dynamics of membrane geometry. Recent progresses in understanding the role of membrane tension in regulating dynamical cellular processes are also reviewed. Protein density is emphasized as a key factor in regulating membrane shape transitions: a threshold density of curvature coupling proteins is required for inducing membrane morphology transitions.     

Reduction of lapachones and their reaction with L-cysteine and mercaptoethanol on glassy carbon electrodes

We estimated in vitro membrane fluidity gradient in erythrocytes (RBC) from diabetic patients, using a fluorescent dye 1,6-diphenyl-1,3,5-hexatriene (DPH). The rate constant of DPH incorporation (k) into the membranes was determined by fitting experimental data to an exponential equation. Four important findings were made. First, membrane fluidity in the hydrocarbon region of RBC from diabetic patients is decreased compared with control cells (P<0.01). Second, the rate constant k of DPH incorporation into the membranes of RBC from diabetic patients was lower (P<0.01), which indicates an altered fluidity gradient in the membranes. Third, resorcylidene aminoguanidine (RAG) decreased significantly (P<0.001) the anisotropy values in RBC membranes from diabetic patients, which means that it apparently acted as a fluidizing agent. Lastly, no significant differences in the rate constants k were found between the control membranes (from RAG untreated RBC) and the membranes isolated from RAG pretreated blood from diabetic patients, as well as between the control membranes and those from RAG pretreated control blood. In conclusion, RAG affects lipid-protein interactions in RBC membranes, which results in membrane lipid bilayer fluidization and leads to the restoration of natural physiological membrane dynamic parameters in RBC from diabetic patients.     

Reconfigurable Photonic Capsules Containing Cholesteric Liquid Crystals with Planar Alignment

Cholesteric liquid crystals (CLCs) reflect selected wavelengths of light owing to their periodic helical structures. The encapsulation of CLCs leads to photonic devices that can be easily processed and might be used as stand‐alone microsensors. However, when CLCs are enclosed by polymeric membranes, they usually lose their planar alignment, leading to a deterioration of the optical performance. A microfluidics approach was employed to integrate an ultrathin alignment layer into microcapsules to separate the CLC core and the elastomeric solid membrane using triple‐emulsion drops as the templates. The thinness of the alignment layer provides high lubrication resistance, preserving the layer integrity during elastic deformation of the membrane. The CLCs in the microcapsules can thus maintain their planar alignment, rendering the shape and optical properties highly reconfigurable.     

Free Energy of Bare and Capped Gold Nanoparticles Permeating through a Lipid Bilayer

Herein, we study the permeation free energy of bare and octane‐thiol‐capped gold nanoparticles (AuNPs) translocating through a lipid membrane. To investigate this, we have pulled the bare and capped AuNPs from bulk water to the membrane interior and estimated the free energy cost. The adsorption of the bare AuNP on the bilayer surface is energetically favorable but further loading inside it requires energy. However, the estimated free‐energy barrier for loading the capped AuNP into the lipid membrane is much higher compared to bare AuNP. We also demonstrate the details of the permeation process of bare and capped AuNPs. Bare AuNP induces the curvature in the lipid membrane whereas capped AuNP creates an opening in the interacting monolayer and get inserted into the membrane. The insertion of capped AuNP induces a partial unzipping of the lipid bilayer, which results in the ordering of the local lipids interacting with the nanoparticle. However, bare AuNP disrupts the lipid membrane by pushing the lipid molecules inside the membrane. We also analyze pore formation due to the insertion of capped AuNP into the membrane, which results in water molecules penetrating the hydrophobic region.