Reduce Line Tension Research Articles

Iron-Induced Lipid Oxidation Alters Membrane Mechanics Favoring Permeabilization.

Ferroptosis is a form of regulated necrosis characterized by the iron-dependent accumulation of lipid peroxides in cell membranes. However, how lipid oxidation via iron-mediated Fenton reactions affects the biophysical properties of cellular membranes and how these changes contribute to the opening of plasma membrane pores are major questions in the field. Here, we characterized the dynamics of membrane alterations during lipid oxidation induced onsite by Fenton reactions in chemically defined in vitro model membrane systems. We find that lipid vesicle permeabilization kinetically correlates with the appearance of malondialdehyde (MDA), a product of lipid oxidation. Iron-induced lipid oxidation also alters the lateral organization of supported lipid bilayers (SLBs) with lipid phase coexistence in a time-dependent manner, reducing the lipid phase mismatch and the circularity of liquid ordered domains, which indicates a decrease in line tension at the phase boundaries. Further analysis of oxidized SLBs by force spectroscopy reveals a significant decrease in the average membrane breakthrough force upon oxidation, resulting from changes in lipid bilayer organization that make it more susceptible to permeabilization. Our findings suggest that lipid oxidation via iron-mediated Fenton-like reactions induces strong changes in membrane lipid interactions and mechanical properties leading to reduced line tension in the permeabilized state of the bilayer, which promotes membrane pore formation.

Melatonin: Regulation of Biomolecular Condensates in Neurodegenerative Disorders.

Biomolecular condensates are membraneless organelles (MLOs) that form dynamic, chemically distinct subcellular compartments organizing macromolecules such as proteins, RNA, and DNA in unicellular prokaryotic bacteria and complex eukaryotic cells. Separated from surrounding environments, MLOs in the nucleoplasm, cytoplasm, and mitochondria assemble by liquid–liquid phase separation (LLPS) into transient, non-static, liquid-like droplets that regulate essential molecular functions. LLPS is primarily controlled by post-translational modifications (PTMs) that fine-tune the balance between attractive and repulsive charge states and/or binding motifs of proteins. Aberrant phase separation due to dysregulated membrane lipid rafts and/or PTMs, as well as the absence of adequate hydrotropic small molecules such as ATP, or the presence of specific RNA proteins can cause pathological protein aggregation in neurodegenerative disorders. Melatonin may exert a dominant influence over phase separation in biomolecular condensates by optimizing membrane and MLO interdependent reactions through stabilizing lipid raft domains, reducing line tension, and maintaining negative membrane curvature and fluidity. As a potent antioxidant, melatonin protects cardiolipin and other membrane lipids from peroxidation cascades, supporting protein trafficking, signaling, ion channel activities, and ATPase functionality during condensate coacervation or dissolution. Melatonin may even control condensate LLPS through PTM and balance mRNA- and RNA-binding protein composition by regulating N6-methyladenosine (m6A) modifications. There is currently a lack of pharmaceuticals targeting neurodegenerative disorders via the regulation of phase separation. The potential of melatonin in the modulation of biomolecular condensate in the attenuation of aberrant condensate aggregation in neurodegenerative disorders is discussed in this review.

Phase separation pattern transition of three-domain vesicles

Based on the Helfrich elastic curvature energy model, the stable shapes for the two patterns of three-domain phase separation are studied in detail for the experimental parameters with direct minimization method in order to explain the interesting experimental results by Yanagisawa et al. (2010 Phys. Rev. E 82 051928). According to their experimental results, there are two transition processes. In the first process, the three-domain vesicles are formed, which are metastable. After several tens of minutes, the three-domain vesicles begin to bud, which is the second process. In the first process, the three-domain vesicles are formed with two patterns. The pattern with the liquid-ordered (Lo) phase in the middle with roughly cylindrical shape and two cap-shape liquid disordered (Ld) domains on each side of the Lo domain is termed pattern I in our paper, and the pattern with Ld domain in the middle with roughly cylindrical shape and two cap-shape Lo domains on each side is referred to as pattern Ⅱ. In the same paper of M. Yanagisawa et al., an approximate calculation is made with the vesicle shapes of the two patterns approximately represented by spheroids. Their calculation shows that the transition point of the two patterns is at o* 0.27 in the case of = 0.02 (or v = 0.942) and = 50, in contrast with the experimental result of o* 0.5. Here o is the area fraction of Lo phase, and is the excess area (which is usually represented by reduced volume v in the previous literatures), is the reduced line tension at the boundary of two adjacent domains. Thus the problem comes down to whether the transition point of the two patterns conforming with the experimental result can be obtained by the Helfrich elastic curvature energy theory if one performs a more precise calculation. Our calculation is performed with the direct minimization method, with the two boundaries of domains constrained in two parallel planes, this is an effective method to guarantee the smoothness of the boundary. To allow the vesicle to have a sufficient freedom to evolve, only constraints of fixed reduced volume and area fraction are imposed (The usual implementation method of constraints with the enclosed volume and the area of each phase fixed is not appropriate in this case. It does not allow the vesicle to have enough freedom to evolve, since the two boundaries are constrained in two preassigned planes). For the experimental parameters of = 50 and = 0.02, the transition point for the two patterns is obtained to be o* = 0.49, which is quite close to the experimental result of o* = 0.5. In order to understand the budding process in the second process, a detailed study is also made with the direct minimization method. It is found that the budding process can occur only for high enough value ( qslant 7.0) and permeable membrane (in other words, no constraint of reduced volume is exerted). One possible mechanism of the permeation is the temporary passage caused by the defect in the bilayer membrane due to large reduced line tension, which needs to be further checked experimentally. The three-domain vesicles found in the experiment have rotational symmetry in the case of small (or large v). What is more, they have a reflective symmetric plane perpendicular to the rotational symmetric axis, thus only vesicles with Dh symmetry are considered in this paper.

Effect of Chain Length of Hybrid Lipids on Line Tension in Phase-Separated Giant Unilamellar Vesicles

Hybrid lipids (HL) are phospholipids with one saturated and one unsaturated chain and they are abundant in cell membranes. HLs are hypothesized to act as linactants and reducing line tension in cell membranes. Here we compare two hybrid lipids of different chain length in their abilities to alter line tension of lipid domains. We found that in both PAPC/DSPC/Cholesterol and SAPC/DSPC/Cholesterol mixtures, the liquid-ordered (lo) and liquid-disordered (ld) lipid domains are micron-sized. Our line tension measurement indicates that hybrid lipids can reduce line tension through reducing physical property differences of domains. Although in both mixtures line tension is reduced compared to a mixture without hybrid lipid, in the SAPC/DSPC/Chol mixture it is reduced more, indicating chain length plays a role in line tension reduction.

Complex Roles of Hybrid Lipids in the Composition, Order, and Size of Lipid Membrane Domains

Hybrid lipids (HL) are phospholipids with one saturated chain and one unsaturated chain. HL are hypothesized to act as linactants (i.e., 2D surfactants) in cell membranes, reducing line tension and creating nanoscopic lipid domains. Here we compare three hybrid lipids of different chain unsaturation (16:0-18:1PC (POPC), 16:0-18:2PC (PLPC), and 16:0-20:4PC (PAPC)) in their abilities to alter the composition, line tension, order, and compactness of lipid domains. We found that the liquid-ordered (Lo) and liquid-disordered (Ld) lipid domains in PAPC/di18:0PC(DSPC)/cholesterol and PLPC/DSPC/cholesterol mixtures are micrometer-sized, and only the POPC/DSPC/cholesterol system has nanoscopic domains. The results indicate that some HLs with polyunsaturated chains are not linactants, and the monounsaturated POPC displays both properties of weak linactants and "Ld-phase" lipids such as di18:1PC (DOPC). The obtained phase boundaries from giant unilamellar vesicles (GUV) show that both POPC and PLPC partition well in the Lo phases. Our MD simulations reveal that these hybrid lipids decrease the order and compactness of Lo domains. Thus, hybrid lipids distinguish themselves from other lipid groups in this combined "partitioning and loosening" ability, which could explain why the Lo domains of GUVs, which often do not contain HL, are more compact than the raft domains in cell membranes. Our line tension measurement and Monte Carlo simulation both show that even the monounsaturated POPC is a weak linactant with only modest ability to occupy domain boundaries and reduce line tension. A more important property of HLs is that they can reduce physical property differences of Lo and Ld bulk domains, which also reduces line tension at domain boundaries.

The Unique Roles of Hybrid Lipids in Lipid Membrane Domain Size and Order

Hybrid lipids (HL) are phospholipids with one saturated and one unsaturated chains. Although it is believed that hybrid lipids can act as linactants (i.e., 2D surfactants) in cell membranes to reduce line tension of lipid domains, as a group, their fundamental roles in membrane heterogeneity have not yet been systematically investigated. In this study, three hybrid lipids of different degrees of chain unsaturation (i.e., 16:0-18:1PC (POPC), 16:0-18:2PC (PLPC), and 16:0-20:4PC (PAPC)) are compared in their abilities to alter the composition, order and compactness of lipid domains. The liquid-ordered and liquid-disordered (Lo+Ld) phase boundaries of HL/di18:0PC(DSPC)/cholesterol systems were determined from giant unilamellar vesicles (GUV). We found that the Lo and Ld lipid domains in PAPC/DSPC/CHOL and PLPC/DSPC/CHOL mixtures are micron-sized, and only POPC/DSPC/CHOL system has nanoscopic domains. The results show that some poly-unsaturated HLs essentially behave like the double-unsaturated di18:1PC (DOPC), and the mono-unsaturated POPC clearly displays both properties of unsaturated lipid and linactant. The obtained phase boundaries indicate that both POPC and PLPC have significant partition in the Lo phases. Our MD simulations reveal that these hybrid lipids decrease the order and compactness of Lo domains. Thus, hybrid lipids distinguish themselves from other lipids in this combined “partitioning and loosening” ability in Lo domains, and this ability could facilitate membrane proteins to partition into lipid-raft-like domains in biomembranes. Our line tension measurement and Monte Carlo simulation both show that even in the best cases, HLs are weak linactants with only modest ability in reducing line tension. Furthermore, the reduction is through two separated mechanisms: one through reducing the compositional differences of Lo and Ld domains, and the other through weak linactant action.

The sticholysin family of pore-forming toxins induces the mixing of lipids in membrane domains

Sticholysins (Sts) I and II (StI/II) are pore-forming toxins (PFTs) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin family, a unique class of eukaryotic PFTs exclusively found in sea anemones. The role of lipid phase co-existence in the mechanism of the action of membranolytic proteins and peptides is not clearly understood. As for actinoporins, it has been proposed that phase separation promotes pore forming activity. However little is known about the effect of sticholysins on the phase separation of lipids in membranes. To gain insight into the mechanism of action of sticholysins, we evaluated the effect of these proteins on lipid segregation using differential scanning calorimetry (DSC) and atomic force microscopy (AFM). New evidence was obtained reflecting that these proteins reduce line tension in the membrane by promoting lipid mixing. In terms of the relevance for the mechanism of action of actinoporins, we hypothesize that expanding lipid disordered phases into lipid ordered phases decreases the lipid packing at the borders of the lipid raft, turning it into a more suitable environment for N-terminal insertion and pore formation.

Effect of cholesterol nanodomains on monolayer morphology and dynamics.

At low mole fractions, cholesterol segregates into 10- to 100-nm-diameter nanodomains dispersed throughout primarily dipalmitoylphosphatidylcholine (DPPC) domains in mixed DPPC:cholesterol monolayers. The nanodomains consist of 6:1 DPPC:cholesterol "complexes" that decorate and lengthen DPPC domain boundaries, consistent with a reduced line tension, λ. The surface viscosity of the monolayer, ηs, decreases exponentially with the area fraction of the nanodomains at fixed surface pressure over the 0.1- to 10-Hz range of frequencies common to respiration. At fixed cholesterol fraction, the surface viscosity increases exponentially with surface pressure in similar ways for all cholesterol fractions. This increase can be explained with a free-area model that relates ηs to the pure DPPC monolayer compressibility and collapse pressure. The elastic modulus, G', initially decreases with cholesterol fraction, consistent with the decrease in λ expected from the line-active nanodomains, in analogy to 3D emulsions. However, increasing cholesterol further causes a sharp increase in G' between 4 and 5 mol% cholesterol owing to an evolution in the domain morphology, so that the monolayer is elastic rather than viscous over 0.1-10 Hz. Understanding the effects of small mole fractions of cholesterol should help resolve the controversial role cholesterol plays in human lung surfactants and may give clues as to how cholesterol influences raft formation in cell membranes.

Not All Hybrid Lipids are Linactants

Hybrid lipids are thought to be able to perform the function of linactants at membrane domain interfaces: They can reduce line tension and stabilize nanoscopic lipid raft domains in biomembranes. Hybrid lipids are lipids with one saturated chain and one unsaturated chain. Here we provide evidences that only certain hybrid lipids behave like linactants. In this study, we compared three hybrid lipids (i.e., 16:0-181PC (POPC), 16:0-18:2PC, and 16:0-22:4PC) in their abilities to reduce lipid domain size and shift phase boundary. The Lo-Ld phase boundaries of hybrid-lipid/di18:0PC(DSPC)/cholesterol systems were determined from giant unilamellar vesicles (GUV) using fluorescence microscopy. We found that 16:0-22:4PC behaves similarly to a fluid-phase lipid: The Lo and Ld lipid domains in 16:0-22:4PC/DSPC/CHOL mixtures are macroscopic and the phase coexisting region is very wide. On the other hand, 16:0-18:2PC/DSPC/CHOL system has a much narrower Lo-Ld phase coexisting region; however, the lipid domains are still macroscopic. Only POPC/DSPC/CHOL system contains nanoscopic lipid domains. These results were compared with Monte Carlo simulations. Based on the magnitudes of interaction energies, it appears that only mono-unsaturated hybrid lipids behave like linactants, while poly-unsaturated hybrid lipids behave more or less like fluid-phase lipids.

Imaging the membrane lytic activity of bioactive peptide latarcin 2a

Latarcin 2a (ltc2a, GLFGKLIKKFGRKAISYAVKKARGKH-COOH) is a short linear antimicrobial and cytolytic peptide extracted from the venom of the Central Asian spider, Lachesana tarabaevi, with lytic activity against Gram-positive and Gram-negative bacteria, erythrocytes, and yeast at micromolar concentrations. Ltc2a adopts a helix–hinge–helix structure in membrane mimicking environment, whereas its derivative latarcin 2aG11A (ltc2aG11A, GLFGKLIKKFARKAISYAVKKARGKH-COOH), likely adopts a more rigid structure, demonstrates stronger nonspecific interaction with the zwitterionic membrane, and is potentially more toxic against eukaryotic cells. In this work, interactions of these two ltc2a derivatives with supported “raft” lipid bilayer (1,2-dioleoyl-sn-glycero-3-phosphocholin/egg sphingomyelin/cholesterol 40/40/20mol%) were studied by in situ atomic force microscopy in order to investigate the potential anticancer activity of the peptides since some breast and prostate cancer cell lines contain higher levels of cholesterol-rich lipid rafts than non-cancer cells. Both peptides induced reorganization of the raft model membrane by reducing line tension of the liquid ordered phase. Ltc2aG11A induced membrane thinning likely due to membrane interdigitation. Formation of large pores by the peptides in the bilayer was observed. Cholesterol was found to attenuate membrane disruption by the peptides. Finally, leakage assay showed that both peptides have similar membrane permeability toward various model membrane vesicles.

Lipid Bilayer Membrane Shape Remodeling by Lipid-Modified Ras Proteins

Ras proteins are small GTP-hydrohydrolyzing enzymes that operate as molecular switches in signal transduction pathways and are present in a mutant, activated state in many human tumors. It has been shown that the lipid-modified C-terminus drive lateral segregation of Ras proteins into membrane sub-structure on the plasma membrane. Such transient and dynamic molecular assembly, so-call nanocluster, is emerging as a crucial mechanism by which cells achieve high-fidelity signal transmission. However, little is known about the underlying force driving the formation of nanoclusters on the plasma membrane and their influence on the membrane structure. To investigate the interaction between nanocluster and the membrane, we carried out extensive semi-atomistic molecular dynamics simulations of the C-terminal membrane-targeting motif of H-ras (tH) in a phase separated lipid bilayer composed of 2000 DPPC, DliPC and cholesterol molecules. We found out that at ambient temperature approximately 30–40% of tH molecules assemble into clusters of 4-9 proteins, and preferentially localize at the interface between the liquid-order and liquid-disorder phases of the membrane. In the nanoclusters, tH backbones preferably exhibits an extended conformation and align linearly at the phase interface. With such molecular-level organization, tH molecule act as a linactant at the interface and reduce line tension of the anchor-containing monolayer and induces shape transition of the overall membrane due to the tendency of the system to minimize the free energy. The resultant geometry of the membrane is determined by the different elastic bending modulus of different phases as well as the phase boundary line tension, which is strongly affected by the localization of the nanoclusters. Our findings were explained based on the domain-induced budding model and shed light on the interaction between membrane-bounded proteins and membrane domains, such as lipid rafts.

Interaction Between Lytic Peptide Latarcin 2A and Supported Lipid Bilayers Studied by in Situ Atomic Force Microscopy

Latarcin 2a (ltc2a, GLFGKLIKKFGRKAISYAVKKARGKH-COOH) is one of the seven short linear antimicrobial and cytolytic peptides extracted from the venom of the Central Asian spider, Lachesana tarabaevi, with lytic activity against Gram-positive and Gram-negative bacteria, erythrocytes, and yeast at micromolar concentrations. Ltc2a is known to adopt helix-hinge-helix conformation in membrane mimicking environment, whereas its derivative latarcin 2aG11A (ltc2aG11A, GLFGKLIKKFARKAISYAVKKARGKH-COOH), likely adopts a more rigid helical structure, has higher nonspecific interaction with zwitterionic membrane and is potentially more toxic against eukaryotic cells. In this work, interactions of two ltc2a derivatives with supported lipid bilayers (DOPC/egg SPM/Chol 40/40/20 mol%) were studied by in situ atomic force microscopy. Interactions are affected by membrane fluidity and peptide concentration. Both peptides induced reorganization of raft model membrane by reducing line tension of the liquid ordered phase. Ltc2aG11A-induced raft membrane thinning may be due to membrane interdigitation. Presence of cholesterol was determined to be important to attenuate peptide induced membrane disruption. Finally, leakage assay showed both peptides have similar membrane permeability toward various model membrane vesicles.

Mechanism of structural transformations induced by antimicrobial peptides in lipid membranes

It has long been suggested that pore formation is responsible for the increase in membrane permeability by antimicrobial peptides (AMPs). To better understand the mechanism of AMP activity, the disruption of model membrane by protegrin-1 (PG-1), a cationic antimicrobial peptide, was studied using atomic force microscopy. We present here the direct visualization of the full range of structural transformations in supported lipid bilayer patches induced by PG-1 on zwitterionic 1,2-dimyristoyl-snglycero-phospho-choline (DMPC) membranes. When PG-1 is added to DMPC, the peptide first induces edge instability at low concentrations, then pore-like surface defects at intermediate concentrations, and finally wormlike structures with a specific length scale at high concentrations. The formation of these structures can be understood using a mesophase framework of a binary mixture of lipids and peptides, where PG-1 acts as a line-active agent. Atomistic molecular dynamics simulations on lipid bilayer ribbons with PG-1 molecules placed at the edge or interior positions are carried out to calculate the effect of PG-1 in reducing line tension. Further investigation of the placement of PG-1 and its association with defects in the bilayer is carried out using unbiased assembly of a PG-1 containing bilayer from a random mixture of PG-1, DMPC, and water. A generalized model of AMP induced structural transformations is also presented in this work. This article is part of a Special Issue entitled: Membrane protein structure and function.

Flow-induced melting of condensed domains within a dispersed Langmuir film

During phase transition from the liquid-expanded to the liquid-condensed state, a dispersed Langmuir film of pentadecanoic acid is submitted to an annular shear flow of moderate Reynolds number (Re=10–100). The mesoscopic morphology of this two-phase Langmuir film is investigated based on area fraction distribution of the condensed phase after a permanent regime is established. The distribution demonstrates radially inwards packing along the liquid surface induced by centripetal flow originating from centrifugation of the subphase along the rotating floor. For a growing level of centrifugation, a circular Reynolds ridge arises along the liquid surface. The Langmuir film experiences a strong morphological transition driven by a balance between surface shear and reduced line tension. As a result, a shear-induced melting of the condensed domains generates a new patterning which can be described as a regular and monodispersed matrix of tiny condensed droplets.

Structure and function of surfactant protein B and C in lipid monolayers: a scanning force microscopy study

This study focuses on the impact of surfactant protein C and B on lipid monolayers at various surface pressures. The artificial system is composed of the saturated phospholipids dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) in a molar ratio of 4:1 with 0.2 mol% SP-B and/or 0.4 mol% SP-C. A dominating influence of SP-C on the morphology of lipid monolayers at high surface pressure was found. Even in the presence of both proteins structural peculiarities typical for SP-C were found at elevated pressure employing tapping mode scanning force microscopy of LB-films. Stacked bilayer-protrusions known to be induced by SP-C are visible in films containing SP-C together with SP-B. The findings were corroborated by fluorescence microscopy at the air/water interface and are consistent with the appearance of the corresponding isotherms. In the low pressure regime, however, disc-like protrusions characteristic of SP-B containing films are discernible. Filamentous LE domains with large boundaries arise due to the reduced line tension in the presence of surface active proteins, particularly SP-B. Remarkably, SP-B fluidizes the monolayer to a larger extent than SP-C as revealed by scanning force microscopy images. These findings show that SP-B and SP-C interact independently of each other. Therefore we conclude that SP-C may be responsible for the fast respreading process during the breathing cycle while SP-B removes material from the monolayer in more discrete portions.