Membrane Line Tension Research Articles

Impact of Lipid Peroxidation on the Response of Cell Membranes to High-Speed Equibiaxial Stretching: A Computational Study.

The difference between diseased and healthy cellular membranes in response to mechanical stresses is crucial for biology, as well as in the development of medical devices. However, the biomolecular mechanisms by which mechanical stresses interact with diseased cellular components remain largely unknown. In this work, we focus on the response of diseased cellular membranes with lipid peroxidation to high-speed tensile loadings. We find that the critical areal strain (ξc, when the pore forms) is highly sensitive to lipid peroxidation. For example, ξc of a fully oxidized bilayer is only 64 and 69% of the nonoxidized one at the stretching speed of 0.1 and 0.6 m/s, respectively. ξc decreases with the increase in the oxidized lipid ratio, regardless of the speeds. Also, the critical rupture tension of membranes exhibits a similar change. It is obvious that the oxidized membranes are more easily damaged than normal ones by high-speed stretching, which coincides with experimental findings. The reason is that peroxidation introduces a polar group to the tail of lipids, increases the hydrophilicity of tails, and warps the tails to the membrane-water interface, which causes loose accumulation and disorder of lipid tails. This can be deduced from the variation in the area per lipid and order parameter. In addition, the lowering stretching modulus and line tension of membranes (i.e., softening) after lipid peroxidation is also a significant factor. We reveal the difference between the peroxidized (diseased) and normal membrane in response to high-speed stretching, give the ξc value in the pore formation of membranes and analyze the influence of the stretching speed, peroxidation ratio, and molecular structure of phospholipids. We hope that the molecular-level information will be useful for the development of biological and medical devices in the future.

Electrostatic effects on the electrical tension-induced irreversible pore formation in giant unilamellar vesicles

Irreversible electroporation (IRE) is a new technique in which a series of short pulses with high frequency electrical energy is applied on the targeted regions of cells or vesicles for their destruction or rupture formation. IRE induces lateral tension in the membranes of vesicles. We have investigated the electrostatic interaction effects on the constant electrical tension-induced rate constant of irreversible pore formation in the membranes of giant unilamellar vesicles (GUVs). The electrostatic interaction has been varied by changing the salt concentration in buffer and the surface charge density of membranes. The membranes of GUVs are synthesized by a mixture of negatively charged lipid dioleoylphosphatidylglycerol (DOPG) and neutral lipid dioleoylphosphatidylcholine (DOPC) using the natural swelling method. The rate constant of pore formation increases with the decrease of salt concentration in buffer along with the increase of surface charge density of membranes. The tension dependent probability of pore formation and the rate constant of pore formation are fitted to the theoretical equation, and obtained the line tension of membranes. The decrease in energy barrier of a prepore due to electrostatic interaction is the key factor causing an increase of rate constant of pore formation.

Influence of cholesterol on electroporation in lipid membranes of giant vesicles.

Irreversible electroporation (IRE) is primarily a nonthermal ablative technology that uses a series of high-voltage and ultra-short pulses with high-frequency electrical energy to induce cell death. This paper presents the influence of cholesterol on the IRE-induced probability of pore formation and the rate constant of pore formation in giant unilamellar vesicles (GUVs). The GUVs are prepared by a mixture of dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylcholine (DOPC) and cholesterol using the natural swelling method. An IRE signal of frequency 1.1kHz is applied to the membranes of GUVs. The probability of pore formation and the rate constant of pore formation events are obtained using statistical analysis from several single GUVs. The time-dependent fraction of intact GUVs among all those examined is fitted to a single exponential decay function from where the rate constant of pore formation is calculated. The probability of pore formation and the rate constant of pore formation decreases with an increase in cholesterol content in the membranes of GUVs. Theoretical equations are fitted to the tension-dependent rate constant of pore formation and to the probability of pore formation, which allows us to obtain the line tension of membranes. The obtained line tension increases with an increase in cholesterol in the membranes. The increase in the energy barrier of the prepore state, due to the increase of cholesterol in membranes, is the main factor explaining the decrease in the rate constant of pore formation.

Transendothelial Perforations and the Sphere of Influence of Single-Site Sonoporation

Acoustically driven gas bubble cavitation locally concentrates energy and can result in physical phenomena including sonoluminescence and erosion. In biomedicine, ultrasound-driven microbubbles transiently increase plasma membrane permeability (sonoporation) to promote drug/gene delivery. Despite its potential, little is known about cellular response in the aftermath of sonoporation. In the work described here, using a live-cell approach, we assessed the real-time interplay between transendothelial perforations (∼30–60 s) up to 650 µm2, calcium influx, breaching of the local cytoskeleton and sonoporation resealing upon F-actin recruitment to the perforation site (∼5–10 min). Through biophysical modeling, we established the critical role of membrane line tension in perforation resealing velocity (10–30 nm/s). Membrane budding/shedding post-sonoporation was observed on complete perforation closure, yet successful pore repair does not mark the end of sonoporation: protracted cell mobility from 8 µs of ultrasound is observed up to 4 h post-treatment. Taken holistically, we established the biophysical context of endothelial sonoporation repair with application in drug/gene delivery.

Inward growth by nucleation: Multiscale self-assembly of ordered membranes.

Striking morphological similarities found between superstructures of a wide variety of seemingly unrelated crystalline membrane systems hint at the existence of a common formation mechanism. Resembling systems such as multiwalled carbon nanotubes, bacterial protein shells, or peptide nanotubes, the self-assembly of SDS/β-cyclodextrin complexes leads to monodisperse multilamellar microtubes. We uncover the mechanism of this hierarchical self-assembly process by time-resolved small- and ultrasmall-angle x-ray scattering. In particular, we show that symmetric crystalline bilayers bend into hollow cylinders as a consequence of membrane line tension and an anisotropic elastic modulus. Starting from single-walled microtubes, successive nucleation of new cylinders inside preexisting ones drives an inward growth. As both the driving forces that underlie the self-assembly behavior and the resulting morphologies are common to systems of ordered membranes, we believe that this formation mechanism has a similarly general applicability.

Antimicrobial Peptides Share a Common Interaction Driven by Membrane Line Tension Reduction

Antimicrobial peptides (AMPs) are a class of host-defense molecules that neutralize a broad range of pathogens. Their membrane-permeabilizing behavior has been commonly attributed to the formation of pores; however, with the continuing discovery of AMPs, many are uncharacterized and their exact mechanism remains unknown. Using atomic force microscopy, we previously characterized the disruption of model membranes by protegrin-1 (PG-1), a cationic AMP from pig leukocytes. When incubated with zwitterionic membranes of dimyristoylphosphocholine, PG-1 first induced edge instability at low concentrations, then porous defects at intermediate concentrations, and finally worm-like micelle structures at high concentrations. These rich structural changes suggested that pore formation constitutes only an intermediate state along the route of PG-1’s membrane disruption process. The formation of these structures could be best understood by using a mesophase framework of a binary mixture of lipids and peptides, where PG-1 acts as a line-active agent in lowering interfacial bilayer tensions. We have proposed that rather than being static pore formers, AMPs share a common ability to lower interfacial tensions that promote membrane transformations. In a study of 13 different AMPs, we found that peptide line-active behavior was not driven by the overall charge, and instead was correlated with their adoption of imperfect secondary structures. These peptide structures commonly positioned charged residues near the membrane interface to promote deformation favorable for their incorporation into the membrane. Uniquely, the data showed that barrel-stave-forming peptides such as alamethicin are not line-active, and that the seemingly disparate models of toroidal pores and carpet activity are actually related. We speculate that this interplay between peptide structure and the distribution of polar residues in relation to the membrane governs AMP line activity in general and represents a novel, to our knowledge, avenue for the rational design of new drugs.

Theoretical study of n-budding opening-up vesicle based on the spontaneous curvature model

N-budding lipid vesicles with single holes were analyzed theoretically by taking into account the line tension of membrane with a hole and the bending energy of membranes based on the Spontaneous-Curvature (SC) model. We solved numerically the Euler-Lagrange equation and the boundary conditions holding on the membrane edge to obtain axisymmetric vesicle shapes, which minimize the total energy. The numerical results show that the size of the opening-up vesicle is larger when the line tension is lower. In other words, additive protein molecules reduce the line tension of lipid membranes, such as talin dose.

The Copolymer Surfactant P188 Reduces Tension in Permeabilized Cell Membranes

Introduction: Certain biocompatible block copolymer surfactants have been found to have important cytoprotective membrane sealing properties. Resealing of disrupted cell membranes through normal cellular endogenous vesicle- membrane fusion pathways is preceeded by a decrease in membrane line tension. It has been postulated that a similar decrease in membrane line tension preceeds sealing of permeabilized cell membranes by block copolymer surfactants such as Poloxamer 188 (P188). Methods: To test this concept we measured effects of sub-CMC P188 on line-tension in saponin permeabilized plasma membranes of MDCK cells in monolayer cell culture. 10 kDa neutral dextran was used an osmotic control. By recording quasi-static strain laser optical tweezer distraction of membrane-bound antibody coated latex 0.5μm diameter beads, we measured maximum tether length of detergent (0.005% saponin) disrupted MDCK cell membranes before and after treatment with either 0.2mM P188 or 0.2mM 10kDa neutral dextran (ND). Bead selection was determined by position and traction was parallel to cytoskeletal protein orientation. Steady-state tether length for maximum traction force was compared. Results and Discussion: Maximum tether length reflects available membrane reservoir and membrane material properties. Recorded tether lengths were 18.30±2.02 (mean±s.e.m.) μm for normal control cells. 6.46±0.82 μm for cells permeabilized with saponin, 6.18x0.96 μm for permeabilized cells treated with ND, and 11.09±1.09 μm for permeabilized cells treate with P188. Saponin extraction of membrane lipid increases membrane stiffness by reducing reservoir size and creation of water-filled defects.Because it is unlikely that P188 restores membrane reservoir volume, these results suggest P188 reduced membrane tension by resealing water-filled defects.

Membrane Disk and Sphere: Controllable Mesoscopic Structures for the Capture and Release of a Targeted Object

Design of molecules for self-assembled mesoscopic structures with specific functions is an important and interesting challenge that spans across disciplines such as nanosciences. A closed lipid membrane is a good example of a self-assembled mesostructure. In this study, we developed controllable membrane formation by making a subtle change at the molecular level. We utilized a synthetic photosensitive amphiphile (KAON12) to achieve the photobased molecular manipulation of the opening and closing of membranes through reversible transitions between sphere and disk structures. We found that the mechanism is based on the photoswitching of the membrane line tension, as deduced from the fluctuation of the membrane edge, through the action of KAON12. Furthermore, we demonstrated the controllable capture and release of colloidal particles into and from a membrane sphere. The observation of Brownian motion of the particle confirmed colloidal encapsulation. This successful photomanipulation of mesoscopic membrane structures in a noncontact and reversible manner should lead to a better understanding of the mechanism of membrane self-organization and may see wider application, such as in microreactors and drug-delivery systems.

Effects of temperature, acyl chain length, and flow-rate ratio on liposome formation and size in a microfluidic hydrodynamic focusing device

Microfluidic hydrodynamic focusing of an alcohol–lipid mixture into a narrow fluid stream by two oblique buffer streams provides a controlled and reproducible method of forming phospholipid bilayer vesicles (i.e., liposomes) with relatively monodisperse and specific size ranges. Previous work has established that liposome size can be controlled by changing the relative and absolute flow rates of the fluids. In other previous work, a kinetic (non-equilibrium) theoretical description of the detergent dilution liposome formation method was developed, in which planar lipid bilayer discs aggregate until they become sufficiently large to close into spherical liposomes. In this work, we show that an approximation of the kinetic theory can help explain liposome formation for our microfluidic method. This approximation predicts that the liposome radius should be approximately proportional to the ratio of the membrane bending elasticity modulus to the line tension of the hydrophobic edges of the lipid bilayer disc. In combination with very fast microfluidic mixing, this theory enables a new method to measure the ratio of the elasticity modulus to the line tension of membranes. The theory predicts that the temperature should change the liposome size primarily as a result of its effect on the ratio of the membrane bending elasticity modulus to the line tension, in contrast to previous work on microdroplet and microbubble formation, which showed that the effect of temperature on droplet/bubble size was primarily due to viscosity changes. In agreement with theory, most membrane compositions form larger liposomes close to or below the gel-to-liquid crystalline phase transition temperature, where the membrane elasticity modulus is much larger, and they have a much smaller dependence of size on temperature far above the transition temperature, where the membrane elasticity modulus is relatively constant. Other parameters modulated by the temperature (e.g., viscosity, free energy, and diffusion coefficients) appear to have little or no effect on liposome size, because they have counteracting effects on the lipid aggregation rate and the liposome closure time. Experiments are performed using phospholipids with varying hydrophobic acyl chain lengths that have phase transition temperatures ranging from −1 °C to 55 °C, so that the temperature dependence is examined below, above, and around the transition temperature. In addition, the effect of IPA stabilizing the edges of the bilayer discs can be examined by comparing the liposome sizes obtained at different flow-rate ratios. Finally, polydispersity is shown to increase as the median liposome size increases, regardless of whether the change in size is due to changing temperature or flow-rate ratio.

Polyethylene Glycol Enhances Axolemmal Resealing following Transection in Cultured Cells and inEx VivoSpinal Cord

The integrity of the neuronal membrane is critical for its function as well as survival, and ineffective repair of damaged membranes may be one of the key factors underlying the neuronal degeneration and overall functional loss that occurs after spinal cord injury and traumatic brain injury. Previously, we showed that polyethylene glycol (PEG) can reseal axonal membranes following compression in isolated guinea pig spinal cord white matter. We now report that 10 mM PEG can also significantly enhance membrane resealing following transection in the clinically relevant conditions of low extracellular Ca(2+) and low temperature. Such beneficial effects were demonstrated both functionally, through membrane potential measured by double sucrose gap apparatus, and anatomically, through horseradish peroxidase and tetramethyl rhodamine dextran dye exclusion assays. We further noted that axons with small diameters preferentially benefited from PEG-mediated axolemmal resealing. Using atomic force microscopy, we further showed that PEG can effectively reduce neuronal membrane surface tension. We hypothesize that PEG may promote axolemmal resealing by increasing membrane line tension and reducing membrane tension, thus creating conditions more favorable to membrane resealing. In summary, these studies suggest that PEG is effective under the clinically relevant conditions of low Ca(2+) and temperature, and thus has the potential to be used in combination with other more established interventions in spinal cord and traumatic brain injury.

Line tension at lipid phase boundaries regulates formation of membrane vesicles in living cells

Ternary lipid compositions in model membranes segregate into large-scale liquid-ordered (L o) and liquid-disordered (L d) phases. Here, we show μm-sized lipid domain separation leading to vesicle formation in unperturbed human HaCaT keratinocytes. Budding vesicles in the apical portion of the plasma membrane were predominantly labelled with L d markers 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, 1,1′-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate and weakly stained by L o marker fluorescein-labeled cholera toxin B subunit which labels ganglioside GM 1 enriched plasma membrane rafts. Cholesterol depletion with methyl-β-cyclodextrin enhanced DiI vesiculation, GM 1/DiI domain separation and was accompanied by a detachment of the subcortical cytoskeleton from the plasma membrane. Based on these observations we describe the energetic requirements for plasma membrane vesiculation. We propose that the decrease in total ‘L o/L d’ boundary line tension arising from the coalescence of smaller L d-like domains makes it energetically favourable for L d-like domains to bend from flat μm-sized surfaces to cap-like budding vesicles. Thus living cells may utilize membrane line tension energies as a control mechanism of exocytic events.

Theoretical analysis of opening-up vesicles with single and two holes

Cuplike lipid vesicles with a single hole and tubelike vesicles with two holes were theoretically analyzed by taking into account the line tension of membrane holes and the bending energy of membranes, using the area difference elasticity model. We numerically solved the Euler-Lagrange equation and the boundary conditions holding on the membrane edge to obtain axisymmetric vesicle shapes that minimize the total energy. The numerical results showed that when the line tension is very low, and for appropriate values of the relaxed area difference between the two monolayers of bilayer membranes, the model yields cup-, tube-, and funnel-shaped vesicles that closely resemble previously observed shapes of opening-up vesicles with additive guest molecules such as the protein talin and some detergents. This strongly suggests that these additive molecules greatly reduce the line tension of lipid membranes. The effect of the Gaussian bending modulus on the shape of the opening-up vesicles was also evaluated and the effect is greatest when the size of hole is small.

Cholesterol and the activity of bacterial toxins

Cholesterol may affect the activity of microbial toxins in a direct, specific way, or it may exert indirect effects because of its role in membrane fluidity, membrane line tension, and in the stabilization of rafts in the cytoplasmic membrane. The thiol-activated toxins of gram-positive bacteria, and the cytolysin of Vibrio cholerae are presented as examples of specific toxin-cholesterol interaction. Several mechanisms of indirect effects of cholesterol are discussed using examples such as Staphylococcus aureusα-hemolysin, aerolysin, and diphtheria toxin.

Cholesterol and the activity of bacterial toxins

Cholesterol may affect the activity of microbial toxins in a direct, specific way, or it may exert indirect effects because of its role in membrane fluidity, membrane line tension, and in the stabilization of rafts in the cytoplasmic membrane. The thiol-activated toxins of gram-positive bacteria, and the cytolysin of Vibrio cholerae are presented as examples of specific toxin–cholesterol interaction. Several mechanisms of indirect effects of cholesterol are discussed using examples such as Staphylococcus aureus α-hemolysin, aerolysin, and diphtheria toxin.

Interaction of hagfish cathelicidin antimicrobial peptides with model lipid membranes

Hagfish intestinal antimicrobial peptides (HFIAPs) are a family of polycationic peptides exhibiting potent, broad-spectrum bactericidal activity. In an attempt to unravel the mechanism of action of HFIAPs, we have studied their interaction with model membranes. Synthetic HFIAPs selectively bound to liposomes mimicking bacterial membranes, and caused the release of vesicle-encapsulated fluorescent markers in a size-dependent manner. In planar lipid bilayer membranes, HFIAPs induced erratic current fluctuations and reduced membrane line tension according to a general theory for lipidic pores, suggesting that HFIAP pores contain lipid molecules. Consistent with this notion, lipid transbilayer redistribution accompanied HFIAP pore formation, and membrane monolayer curvature regulated HFIAP pore formation. Based on these studies, we propose that HFIAPs kill target cells, at least in part, by interacting with their plasma membrane to induce formation of lipid-containing pores. Such a membrane-permeabilizing function appears to be an evolutionarily conserved host-defense mechanism of antimicrobial peptides.