Cholesterol-Dependent Nanomechanical Stability of Phase-Segregated Multicomponent Lipid Bilayers

Cholesterol is involved in endocytosis, exocytosis, and the assembly of sphingolipid/cholesterol-enriched domains, as has been demonstrated in both model membranes and living cells. In this work, we explored the influence of different cholesterol levels (5-40 mol %) on the morphology and nanomechanical stability of phase-segregated lipid bilayers consisting of dioleoylphosphatidylcholine/sphingomyelin/cholesterol (DOPC/SM/Chol) by means of atomic force microscopy (AFM) imaging and force mapping. Breakthrough forces were consistently higher in the SM/Chol-enriched liquid-ordered domains (L_o) than in the DOPC-enriched fluid-disordered phase (L_d) at a series of loading rates. We also report the activation energies (ΔE_a) for the formation of an AFM-tip-induced fracture, calculated by a model for the rupture of molecular thin films. The obtained ΔE_a values agree remarkably well with reported values for fusion-related processes using other techniques. Furthermore, we observed that within the Chol range studied, the lateral organization of bilayers can be categorized into three distinct groups. The results are rationalized by fracture nanomechanics of a ternary phospholipid/sphingolipid/cholesterol mixture using correlated AFM-based imaging and force mapping, which demonstrates the influence of a wide range of cholesterol content on the morphology and nanomechanical stability of model bilayers. This provides fundamental insights into the role of cholesterol in the formation and stability of sphingolipid/cholesterol-enriched domains, as well as in membrane fusion.     

Stability and Liquid-Liquid Phase Separation in Mixed Saturated Lipid Bilayers

The phase stability of a fluid lipid bilayer composed of a mixture of DC₁₈PC, (DSPC), and a shorter DC_ns PC, with n_s from 8 to 17, has been studied using a self-consistent field theory that explicitly includes molecular details and configurational properties of the lipid molecules. Phase separation between two liquid phases was found when there was a sufficient mismatch between the hydrophobic thicknesses of the two bilayers composed entirely of one component or the other. This occurs when n_s ≤ 12 and there is a sufficient concentration of the shorter lipid. The mixture separates into a thin bilayer depleted of DSPC and a thick bilayer enriched in DSPC. Even when there is no phase separation, as in the cases when there is either insufficient concentration of a sufficiently short lipid or any concentration of a lipid with n_s > 12, we observe that the effect of the shorter lipid is to increase the susceptibility of the system to fluctuations in the concentration. This is of interest, given that a common motif for the anchoring of proteins to the plasma membrane is via a myristoyl chain, that is, one with 14 carbons.     

Two-Point Microrheology of Phase-Separated Domains in Lipid Bilayers

Though the importance of membrane fluidity for cellular function has been well established for decades, methods for measuring lipid bilayer viscosity remain challenging to devise and implement. Recently, approaches based on characterizing the Brownian dynamics of individual tracers such as colloidal particles or lipid domains have provided insights into bilayer viscosity. For fluids in general, however, methods based on single-particle trajectories provide a limited view of hydrodynamic response. The technique of two-point microrheology, in which correlations between the Brownian dynamics of pairs of tracers report on the properties of the intervening medium, characterizes viscosity at length-scales that are larger than that of individual tracers and has less sensitivity to tracer-induced distortions, but has never been applied to lipid membranes. We present, to our knowledge, the first two-point microrheological study of lipid bilayers, examining the correlated motion of domains in phase-separated lipid vesicles and comparing one- and two-point results. We measure two-point correlation functions in excellent agreement with the forms predicted by two-dimensional hydrodynamic models, analysis of which reveals a viscosity intermediate between those of the two lipid phases, indicative of global fluid properties rather than the viscosity of the local neighborhood of the tracer.     

Electrophysiological Behavior of the TolC Channel-Tunnel in Planar Lipid Bilayers

Escherichia coli TolC assembles into the unique channel-tunnel structure spanning the outer membrane and periplasmic space. The structure is constricted only at the periplasmic entrance of the tunnel and this must be opened to allow export of substrates bound by cognate inner membrane complexes. We have investigated the electrophysiological behavior of TolC reconstituted into planar lipid bilayers, in particular the influence of the membrane potential, the electrolyte concentration and pH. TolC inserted in one orientation into the membrane. The resultant pores were stable and showed no voltage-dependent opening or closing. Nevertheless, TolC could adopt up to three conductance substates. The pores were cation-selective with a permeability ratio of potassium to chloride ions of 16.5. The single-channel conductance was higher when the protein was inserted from the side with negative potential. It showed a nonlinear dependence on the concentration of the electrolyte in the bulk solution and decreased as the pH was lowered. The calculated pK of the apparent closing was 4.5. The electrophysiological characterization is discussed in relation to the TolC structure, in particular the periplasmic entrance.     

Atomistic Simulations of Pore Formation and Closure in Lipid Bilayers

Cellular membranes separate distinct aqueous compartments, but can be breached by transient hydrophilic pores. A large energetic cost prevents pore formation, which is largely dependent on the composition and structure of the lipid bilayer. The softness of bilayers and the disordered structure of pores make their characterization difficult. We use molecular-dynamics simulations with atomistic detail to study the thermodynamics, kinetics, and mechanism of pore formation and closure in DLPC, DMPC, and DPPC bilayers, with pore formation free energies of 17, 45, and 78 kJ/mol, respectively. By using atomistic computer simulations, we are able to determine not only the free energy for pore formation, but also the enthalpy and entropy, which yields what is believed to be significant new insights in the molecular driving forces behind membrane defects. The free energy cost for pore formation is due to a large unfavorable entropic contribution and a favorable change in enthalpy. Changes in hydrogen bonding patterns occur, with increased lipid-water interactions, and fewer water-water hydrogen bonds, but the total number of overall hydrogen bonds is constant. Equilibrium pore formation is directly observed in the thin DLPC lipid bilayer. Multiple long timescale simulations of pore closure are used to predict pore lifetimes. Our results are important for biological applications, including the activity of antimicrobial peptides and a better understanding of membrane protein folding, and improve our understanding of the fundamental physicochemical nature of membranes.     

Nonlamellar-Phase-Promoting Colipids Enhance Segregation of Palmitoyl Ceramide in Fluid Bilayers

Ceramide is an important intermediate in sphingolipid homeostasis. We examined how colipids, with negative intrinsic curvature and which may induce curvature stress in the bilayers, affected the segregation of palmitoyl ceramide (PCer). Such colipids include 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and tetra-linoleoyl cardiolipin (CL). In 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers, PCer formed ordered, gel-like domains at concentrations above 10 mol% at 23°C, as evidenced by the change in the average lifetime of the trans-parinaric acid emission. When POPE or DOPE were included in the DOPC bilayer (at 20:80 or 40:60 POPE or DOPE to DOPC, by mol), the lateral segregation of PCer was facilitated in a concentration-dependent manner, and less PCer was required for the formation of the ordered ceramide-rich domains. Inclusion of CL in the DOPE bilayer (at 10:90 or 20:80 CL to PC, by mol) also caused a similar facilitation of the lateral segregation of PCer. The PCer-rich domains formed in the presence of POPE, DOPE, or CL in DOPC bilayers were slightly more thermostable (by 2–10°C) when compared to PCer-rich domains in DOPC-only bilayers. Nonlamellar phases were not present in bilayers in which the effects of POPE or DOPE on PCer segregation were the largest, as verified by ³¹P NMR. When palmitoyl sphingomyelin was added to the different bilayer compositions at 5 mol%, relative to the phospholipids, PCer segregated into gel domains at lower concentrations (2–3 mol% PCer), and the effect of POPE on PCer segregation was eliminated. We suggest that the effects of POPE, DOPE, and CL on PCer segregation was in part influenced by their effects on membrane curvature stress and in part because of unfavorable interactions with PCer due to their unsaturated acyl chains. These lipids are abundant in mitochondrial membranes and are likely to affect functional properties of saturated ceramides in them.     

Behavior of Doxorubicin Lipophilic Conjugates in Liposomal Lipid Bilayers

Russian Journal of Bioorganic Chemistry - Preparation of liposomal formulations containing water-soluble drugs in the form of lipophilic prodrugs in their lipid bilayer is of considerable interest....     

Atomistic Simulations of Pore Formation and Closure in Lipid Bilayers

Cellular membranes separate distinct aqueous compartments, but can be breached by transient hydrophilic pores. A large energetic cost prevents pore formation, which is largely dependent on the composition and structure of the lipid bilayer. The softness of bilayers and the disordered structure of pores make their characterization difficult. We use molecular-dynamics simulations with atomistic detail to study the thermodynamics, kinetics, and mechanism of pore formation and closure in DLPC, DMPC, and DPPC bilayers, with pore formation free energies of 17, 45, and 78 kJ/mol, respectively. By using atomistic computer simulations, we are able to determine not only the free energy for pore formation, but also the enthalpy and entropy, which yields what is believed to be significant new insights in the molecular driving forces behind membrane defects. The free energy cost for pore formation is due to a large unfavorable entropic contribution and a favorable change in enthalpy. Changes in hydrogen bonding patterns occur, with increased lipid-water interactions, and fewer water-water hydrogen bonds, but the total number of overall hydrogen bonds is constant. Equilibrium pore formation is directly observed in the thin DLPC lipid bilayer. Multiple long timescale simulations of pore closure are used to predict pore lifetimes. Our results are important for biological applications, including the activity of antimicrobial peptides and a better understanding of membrane protein folding, and improve our understanding of the fundamental physicochemical nature of membranes.     

Triglyceride Blisters in Lipid Bilayers: Implications for Lipid Droplet Biogenesis and the Mobile Lipid Signal in Cancer Cell Membranes

Triglycerides have a limited solubility, around 3%, in phosphatidylcholine lipid bilayers. Using millisecond-scale course grained molecular dynamics simulations, we show that the model lipid bilayer can accommodate a higher concentration of triolein (TO) than earlier anticipated, by sequestering triolein molecules to the bilayer center in the form of a disordered, isotropic, mobile neutral lipid aggregate, at least 17 nm in diameter, which forms spontaneously, and remains stable on at least the microsecond time scale. The results give credence to the hotly debated existence of mobile neutral lipid aggregates of unknown function present in malignant cells, and to the early biogenesis of lipid droplets accommodated between the two leaflets of the endoplasmic reticulum membrane. The TO aggregates give the bilayer a blister-like appearance, and will hinder the formation of multi-lamellar phases in model, and possibly living membranes. The blisters will result in anomalous membrane probe partitioning, which should be accounted for in the interpretation of probe-related measurements.     

Differential Scanning Calorimetry (DSC): A Tool to Study the Thermal Behavior of Lipid Bilayers and Liposomal Stability

Thermodynamical techniques are applied for determining the thermal stress of medicinal compounds of the excipients as well as their interactions during the formulation process.

The physicochemical properties and the stability of the medicinal products could be measured as a function of temperature or time using thermal analysis.

Differential Scanning Calorimetry (DSC) is a suitable thermal analysis technique for determining the purity, the polymorphic forms and the melting point of a sample in the Pharmaceutical Industry. It is also considered as a tool to study the thermal behavior of lipid bilayers and of lipidic drug delivery systems, like liposomes by measuring thermodynamic parameters (i.e. ΔH and Tm), which affect the stability of the liposomal suspension under given storage conditions.     

Amyloids of Alpha-Synuclein Affect the Structure and Dynamics of Supported Lipid Bilayers

Interactions of monomeric alpha-synuclein (αS) with lipid membranes have been suggested to play an important role in initiating aggregation of αS. We have systematically analyzed the distribution and self-assembly of monomeric αS on supported lipid bilayers. We observe that at protein/lipid ratios higher than 1:10, αS forms micrometer-sized clusters, leading to observable membrane defects and decrease in lateral diffusion of both lipids and proteins. An αS deletion mutant lacking amino-acid residues 71–82 binds to membranes, but does not observably affect membrane integrity. Although this deletion mutant cannot form amyloid, significant amyloid formation is observed in the wild-type αS clusters. These results suggest that the process of amyloid formation, rather than binding of αS on membranes, is crucial in compromising membrane integrity.     

Amyloids of Alpha-Synuclein Affect the Structure and Dynamics of Supported Lipid Bilayers

Interactions of monomeric alpha-synuclein (αS) with lipid membranes have been suggested to play an important role in initiating aggregation of αS. We have systematically analyzed the distribution and self-assembly of monomeric αS on supported lipid bilayers. We observe that at protein/lipid ratios higher than 1:10, αS forms micrometer-sized clusters, leading to observable membrane defects and decrease in lateral diffusion of both lipids and proteins. An αS deletion mutant lacking amino-acid residues 71–82 binds to membranes, but does not observably affect membrane integrity. Although this deletion mutant cannot form amyloid, significant amyloid formation is observed in the wild-type αS clusters. These results suggest that the process of amyloid formation, rather than binding of αS on membranes, is crucial in compromising membrane integrity.     

Energetics of ErbB1 Transmembrane Domain Dimerization in Lipid Bilayers

One of the most extensively studied receptor tyrosine kinases is EGFR/ErbB1. Although our knowledge of the role of the extracellular domains and ligands in ErbB1 activation has increased dramatically based on solved domain structures, the exact mechanism of signal transduction across the membrane remains unknown. The transmembrane domains are expected to play an important role in the dimerization process, but the contribution of ErbB1 TM domain to dimer stability is not known, with published results contradicting one another. We address this controversy by showing that ErbB1 TM domain dimerizes in lipid bilayers and by calculating its contribution to stability as −2.5 kcal/mol. The stability calculations use two different methods based on Förster resonance energy transfer, which give the same result. The ErbB1 TM domain contribution to stability exceeds the change in receptor tyrosine kinases dimerization propensities that can convert normal signaling processes into pathogenic processes, and is thus likely important for biological function.     

Penetration of Cell Membranes and Synthetic Lipid Bilayers by Nanoprobes

Nanoscale devices have been proposed as tools for measuring and controlling intracellular activity by providing electrical and/or chemical access to the cytosol. Unfortunately, nanostructures with diameters of 50–500 nm do not readily penetrate the cell membrane, and rationally optimizing nanoprobes for cell penetration requires real-time characterization methods that are capable of following the process of membrane penetration with nanometer resolution. Although extensive work has examined the rupture of supported synthetic lipid bilayers, little is known about the applicability of these model systems to living cell membranes with complex lipid compositions, cytoskeletal attachment, and membrane proteins. Here, we describe atomic force microscopy (AFM) membrane penetration experiments in two parallel systems: live HEK293 cells and stacks of synthetic lipid bilayers. By using the same probes in both systems, we were able to clearly identify membrane penetration in synthetic bilayers and compare these events with putative membrane penetration events in cells. We examined membrane penetration forces for three tip geometries and 18 chemical modifications of the probe surface, and in all cases the median forces required to penetrate cellular and synthetic lipid bilayers with nanoprobes were greater than 1 nN. The penetration force was sensitive to the probe''s sharpness, but not its surface chemistry, and the force did not depend on cell surface or cytoskeletal properties, with cells and lipid stacks yielding similar forces. This systematic assessment of penetration under various mechanical and chemical conditions provides insights into nanoprobe-cell interactions and informs the design of future intracellular nanoprobes.     

Penetration of Cell Membranes and Synthetic Lipid Bilayers by Nanoprobes

Nanoscale devices have been proposed as tools for measuring and controlling intracellular activity by providing electrical and/or chemical access to the cytosol. Unfortunately, nanostructures with diameters of 50–500 nm do not readily penetrate the cell membrane, and rationally optimizing nanoprobes for cell penetration requires real-time characterization methods that are capable of following the process of membrane penetration with nanometer resolution. Although extensive work has examined the rupture of supported synthetic lipid bilayers, little is known about the applicability of these model systems to living cell membranes with complex lipid compositions, cytoskeletal attachment, and membrane proteins. Here, we describe atomic force microscopy (AFM) membrane penetration experiments in two parallel systems: live HEK293 cells and stacks of synthetic lipid bilayers. By using the same probes in both systems, we were able to clearly identify membrane penetration in synthetic bilayers and compare these events with putative membrane penetration events in cells. We examined membrane penetration forces for three tip geometries and 18 chemical modifications of the probe surface, and in all cases the median forces required to penetrate cellular and synthetic lipid bilayers with nanoprobes were greater than 1 nN. The penetration force was sensitive to the probe's sharpness, but not its surface chemistry, and the force did not depend on cell surface or cytoskeletal properties, with cells and lipid stacks yielding similar forces. This systematic assessment of penetration under various mechanical and chemical conditions provides insights into nanoprobe-cell interactions and informs the design of future intracellular nanoprobes.     

Design and Characterization of a Membrane Protein Unfolding Platform in Lipid Bilayers

Accurate measurement of membrane protein stability—and particularly how it may vary as a result of disease-phenotypic mutations—ideally requires a denaturant that can unfold a membrane-embedded structure while leaving the solubilizing environment unaffected. The steric trap method fulfills this requirement by using monovalent streptavidin (mSA) molecules to unfold membrane proteins engineered with two spatially close biotin tags. Here we adapted this method to an 87-residue helix-loop-helix (hairpin) construct derived from helices 3 and 4 in the transmembrane domain of the human cystic fibrosis transmembrane conductance regulator (CFTR), wherein helix-helix tertiary interactions are anticipated to confer a portion of construct stability. The wild type CFTR TM3/4 hairpin construct was modified with two accessible biotin tags for mSA-induced unfolding, along with two helix-terminal pyrene labels to monitor loss of inter-helical contacts by pyrene excimer fluorescence. A series of eight constructs with biotin tags at varying distances from the helix-terminal pyrene labels were expressed, purified and labeled appropriately; all constructs exhibited largely helical circular dichroism spectra. We found that addition of mSA to an optimized construct in lipid vesicles led to a complete and reversible loss in pyrene excimer fluorescence and mSA binding, and hence hairpin unfolding—results further supported by SDS-PAGE visualization of mSA bound and unbound species. While some dimeric/oligomeric populations persist that may affect quantitation of the unfolding step, our characterization of the design yields a promising prototype of a future platform for the systematic study of membrane protein folding in a lipid bilayer environment.     

Permeability of Boric Acid Across Lipid Bilayers and Factors Affecting It

Boron enters plant roots as undissociated boric acid (H₃BO₃). Significant differences in B uptake are frequently observed even when plants are grown under identical conditions. It has been theorized that these differences reflect species differences in permeability coefficient of H₃BO₃ across plasma membrane. The permeability coefficient of boric acid however, has not been experimentally determined across any artificial or plant membrane. In the experiments described here the permeability coefficient of boric acid in liposomes made of phosphatidylcholine was 4.9 × 10⁻⁶ cm sec⁻¹, which is in good agreement with the theoretical value. The permeability coefficient varied from 7 × 10⁻⁶ to 9.5 × 10⁻⁹ cm sec⁻¹ with changes in sterols (cholesterol), the type of phospholipid head group, the length of the fatty acyl chain, and the pH of the medium. In this study we also used Arabidopsis thaliana mutants which differ in lipid composition to study the effect of lipid composition on B uptake. The chs1-1 mutant which has lower proportion of sterols shows 30% higher B uptake compared with the wild type, while the act1-1 mutant which has an increased percentage of longer fatty acids, exhibited 35% lower uptake than the wild type. Lipid composition changes in each of the remaining mutants influenced B uptake to various extents. These data suggest that lipid composition of the plasma membrane can affect total B uptake. Received: 15 October 1999/Revised: 11 February 2000     

Interaction of Adriamycin and N-Trifluoroacetyladriamycin-14-Valerate with Cardiolipin-Containing Lipid Bilayers

Abstract

The interaction of adriamycin (ADR) and N-Trifluoroacetyladriamycin-14-valerate (AD 32) with cardiolipin (CL)-containing multilamellar vesicles was studied by high-sensitivity differential scanning calorimetry, using liposomes formed from either dipalmitoylphosphatidylcholine (DPPC) or dipalmitoylphosphatidylglycerol (DPPG) containing small amounts of CL. the drugs partitioned into cardiolipin-containing neutral and acidic bilayers in a manner similar to that observed earlier with CL-free bilayers. the partition coefficient of adriamycin in bilayers of vesicles prepared from DPPG or DPPG in admixture with CL was much higher than that obtained with neutral DPPC vesicles or the DPPC together with CL. Under all conditions, AD 32 was essentially completely partitioned into the lipid phase of the investigated phospholipid membranes. As expected, addition of adriamycin to CL-containing vesicles did not significantly change the thermotropic behavior of these bilayers, whereas the fluidizing effect of AD 32 was directly related to the CL content of the vesicles. the multipeak transitions produced by the anthracyclines in pure DPPG bilayers were preserved in the presence of CL, but the endotherms were broader and slightly shifted to lower temperatures, a finding indicative of stronger interactions. Chlorpromazine (CPZ), which was used as a reference compound to compare the effects produced by the anthracyclines, was found to behave similarly to AD 32 and to be more effective than quinidine (QND), in agreement with their behavior in CL-free liposomes.