Cholesterol In Bilayers Research Articles

ATNT-03FIRST IN HUMAN STUDY OF RHENIUM NANOLIPOSOMES FOR INTRATUMORAL THERAPY OF GLIOMA

BACKGROUND: Using a special methodology for radiolabeling lipid nanoparticles with therapeutic radionuclides, we have developed 186Re-nanoliposomes (186RNL). The liposomes, not unlike the membranes of human cells, are composed of a bilayer of distearoylphosphatidylcholine and cholesterol, and confer drug delivery control on beta emitting 186Re. This then permits the delivery of 186Re radiation of high specific activity which is retained within the tumor. METHODS: A phase 1 dose escalation study of 186RNL in recurrent glioma utilizing a standard 3 + 3 design was undertaken to determine the maximum tolerated dose of 186RNL by convection enhanced delivery following stereotactic biopsy. 186RNL is administered by the use of the SmartFlow Flex catheter (BrainLab) by convection enhanced delivery intra-tumorally. Catheter placement is guided using iPlan Flow and the Varioguide systems (BrainLab). Following catheter placement, patients are taken to a nuclear medicine suite where direct visualization of 186RNL infusion is followed under whole body planar imaging and SPECT/CT. Repeat imaging is performed immediately following, and at 1, 5, and 8 days after administration to obtain radiation dosimetry and distribution. RESULTS: Three patients have been treated to date with 1mCi to achieve a treatment field volume of 5mL. Catheter placement for all 3 cases has closely matched the stereotactic plan based upon MRI fusion with SPECT/CT. No treatment related adverse effects have been observed to date despite absorbed doses up to 140Gy (37 MBq). For patient 01, loco-regional retention was 76.8% of administered radioactivity on average from time 0 to 72 hours. During the 120 hour monitoring period, there was a trend toward slow loco-regional dispersion of radioactivity, which is indicated by larger extended radioactivity distribution volumes increasing from 19mL at 40 minutes to 31mL at 120 hours. CONCLUSION: Initial results of 186RNL delivery by CED are promising and the study will continue until an MTD is reached.

Peptide Recognition of Cholesterol in Fluid Phospholipid Bilayers.

N-Acetyl-LWYIKC-amide, a cholesterol recognition/interaction amino acid consensus (CRAC) peptide, has been found capable of recognizing an exchangeable form of cholesterol in liquid-disordered (l(d)) bilayers derived from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). In sharp contrast, such recognition is barely detectable in analogous cholesterol-rich, liquid-ordered (l0) bilayers. These findings represent the first evidence for a peptide favoring a sterol as a nearest-neighbor in fluid bilayers. They also reveal that such recognition can be strongly dependent on the degree of compactness of the membrane.

A Parameterization of Cholesterol for Mixed Lipid Bilayer Simulation within the Amber Lipid14 Force Field.

The Amber Lipid14 force field is expanded to include cholesterol parameters for all-atom cholesterol and lipid bilayer molecular dynamics simulations. The General Amber and Lipid14 force fields are used as a basis for assigning atom types and basic parameters. A new RESP charge derivation for cholesterol is presented, and tail parameters are adapted from Lipid14 alkane tails. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers are simulated at a range of cholesterol contents. Experimental bilayer structural properties are compared with bilayer simulations and are found to be in good agreement. With this parameterization, another component of complex membranes is available for molecular dynamics with the Amber Lipid14 force field.

Cis and trans unsaturated phosphatidylcholine bilayers: A molecular dynamics simulation study

Trans unsaturated lipids are uncommon in nature. In the human diet, they occur as natural products of ruminal bacteria or from industrial food processing like hydrogenation of vegetable oils. Consumption of trans unsaturated lipids has been shown to have a negative influence on human health; in particular, the risk of cardiovascular disease is higher when the amount of trans unsaturated lipids in the diet is elevated. In this study, we first performed quantum mechanical calculations to specifically and accurately parameterize cis and trans mono-unsaturated lipids and subsequently validated the newly derived parameter set. Then, we carried out molecular dynamics (MD) simulations of lipid bilayers composed of cis or trans unsaturated lipids with and without cholesterol. Our results show that trans mono-unsaturated chains are more flexible than cis mono-unsaturated chains due to lower barriers for rotation around the single bonds next to the trans double bond than those next to the cis double bond. In effect, interactions between cholesterol and trans unsaturated chains are stronger than cis unsaturated chains, which results in a higher ordering effect of cholesterol in trans unsaturated bilayers.

Examining the Effects of Cholesterol: Laurdan and Patman see it Differently

The liquid-ordered phase induced by cholesterol in phosphatidylcholine bilayers can be detected with Laurdan fluorescence by an increase in the associated Generalized Polarization (GP) value. This increase in GP is usually interpreted as a reduction in the access of water molecules to the bilayer at depths approaching the phospholipid glycerol backbones. Comparisons of Laurdan fluorescence over a broad range of temperatures with various saturated and unsaturated phosphatidylcholines demonstrated that cholesterol has little effect on GP at temperatures below the melting point of the pure lipid (tm). However, above tm, increasing cholesterol concentrations monotonically raised the value of GP. The resulting GP increments generally did not vary with temperature above tm. In contrast, the observations were more complex with Patman, a charged derivative of Laurdan. First, cholesterol raised the value of Patman GP at temperatures below tm, and its effect as a function of concentration was bimodal. Second, although cholesterol increased Patman GP above tm (similar to Laurdan), the effect size was smaller and was bimodal as a function of cholesterol concentration. Finally, the GP values at high versus low cholesterol concentration converged as temperature was raised well beyond tm. In some cases, such as with unsaturated lipids ≥54°C above tm, this convergence of Patman GP values reached a cross-over point; Patman GP was reduced by the presence of cholesterol beyond this point. These results suggest that the charge associated with Patman, or possibly the slightly deeper location of the probe in the bilayer, provides a means for identifying additional effects of cholesterol on the membrane that are not visible with Laurdan.

Cholesterol Bilayer Domain in Phospholipid Bilayer Membranes can be Detected by Confocal Microscope

The unique feature of the eye lens fiber-cell plasma membrane is its extremely high cholesterol content; cholesterol/phospholipid molar ratio can be as high as 4 in human lens nucleus. Cholesterol saturates bulk phospholipid bilayers and induces formation of immiscible cholesterol bilayer domains (CBDs) within membranes. The presence of CBDs plays crucial role ensuring that surrounding phospholipid bilayers are saturated with cholesterol which keeps the bulk physical properties of lens-lipid membranes consistent and independent of changes in phospholipid composition. Thus, CBDs help to maintain lens-membrane homeostasis when the membrane phospholipid composition changes significantly with age. Our previous experiments were based on electron paramagnetic resonance spin-labeling methods. They allowed to detect CBDs in model and lens lipid membranes at high cholesterol content (1,2). In current experiments the giant unilamellar vesicles made of cholesterol/distearoylphosphatidylcholine (Chol/DSPC) mixtures, with mixing ration from 0.5 to 5 and labeled with phospholipid analog (1,1′-Dioctadecyl-3,3,3’,3′-Tetramethylindocarbocyanine-5,5′-Disulfonic Acid) and cholesterol analog (23-(dipyrrometheneboron difluoride)-24-norcholesterol) fluorescent probes, were prepared using the electroformation method (3). Confocal microscopy experiment allowed us to confirm that membranes with higher Chol/DSPC molar ratio contain two distinct lipid environments, the bulk phospholipid-cholesterol domain (PCD) and the CBD. The amount of CBD was greater in membranes with higher Chol/DSPC ratio. According to these results, CBD is always located on the top of the giant vesicle. Rationale for this discovery is that CBD has smaller mass per unit area of the vesicle compared with PCD. Additionally, it can be concluded that vesicles with higher Chol/DSPC molar ratio are more rigid and show less wobbling motion in water environment.1. Raguz et al. CPL 2011 164(8):819-29.2. Mainali et al. BBA 2013 1828(6):1432-40.3. Veatch SL. Methods Mol Biol. 2007 398:59-72.AcknowledgementsSupported by NIH grants EY015526 and EY001931.

Properties of membranes derived from the total lipids extracted from clear and cataractous lenses of 61-70-year-old human donors.

Human lens-lipid membranes prepared from the total lipids extracted from clear and cataractous lens cortexes and nuclei of 61-70-year-old donors by use of a rapid solvent-exchange method were investigated. The measured cholesterol-to-phospholipid (Chol/PL) molar ratio in these membranes was 1.8 and 4.4 for cortex and nucleus of clear lenses, respectively, and 1.14 and 1.45 for cataractous lenses. Properties and organization of the lipid bilayer were investigated by use of electron paramagnetic resonance spin-labeling methods. Formation of Chol crystals was confirmed by use of differential scanning calorimetry. Pure cholesterol bilayer domains (CBDs) were formed in all the membranes investigated. It was shown that in clear lens membranes of the nucleus, Chol exists in three different environments: (1) dispersed in phospholipid bilayers (PCDs), (2) in CBDs, and (3) in Chol crystals. In clear lens membranes of the cortex, and in cortical and nuclear cataractous lens membranes, Chol crystals were not detected, because of the lower Chol content. Profiles of membrane properties (alkyl-chain order, fluidity, oxygen transport, and hydrophobicity) across the PCD were very similar for clear and cataractous membranes. Profiles ofthe oxygen transportparameter across the CBD were, however, different for cortical clear and cataractous membranes-the amount and size of CBDs was less in cataractous membranes. These results suggest that high Chol content, formation of CBDs, and formation of Chol crystals should not be regarded as major predispositions for the development of age-related cataracts.

Structure of Cholesterol in Lipid Rafts.

Rafts, or functional domains, are transient nano-or mesoscopic structures in the plasma membrane and are thought to be essential for many cellular processes such as signal transduction, adhesion, trafficking, and lipid or protein sorting. Observations of these membrane heterogeneities have proven challenging, as they are thought to be both small and short lived. With a combination of coarse-grained molecular dynamics simulations and neutron diffraction using deuterium labeled cholesterol molecules, we observe raftlike structures and determine the ordering of the cholesterol molecules in binary cholesterol-containing lipid membranes. From coarse-grained computer simulations, heterogenous membranes structures were observed and characterized as small, ordered domains. Neutron diffraction was used to study the lateral structure of the cholesterol molecules. We find pairs of strongly bound cholesterol molecules in the liquid-disordered phase, in accordance with the umbrella model. Bragg peaks corresponding to ordering of the cholesterol molecules in the raftlike structures were observed and indexed by two different structures: a monoclinic structure of ordered cholesterol pairs of alternating direction in equilibrium with cholesterol plaques, i.e., triclinic cholesterol bilayers.

Dynamics of lipids, cholesterol, and transmembrane α-helices from microsecond molecular dynamics simulations.

Extensive all-atom molecular dynamics (∼24 μs total) allowed exploration of configurational space and calculation of lateral diffusion coefficients of the components of a protein-embedded, cholesterol-containing model bilayer. The three model membranes are composed of an ∼50/50 (by mole) dipalmitoylphosphatidylcholine (DPPC)/cholesterol bilayer and contained an α-helical transmembrane protein (HIV-1 gp41 TM). Despite the high concentration of cholesterol, normal Brownian motion was observed and the calculated diffusion coefficients (on the order of 10–9 cm2/s) are consistent with experiments. Diffusion is sensitive to a variety of parameters, and a temperature difference of ∼4 K from thermostat artifacts resulted in 2–10-fold differences in diffusion coefficients and significant differences in lipid order, membrane thickness, and unit cell area. Also, the specific peptide sequence likely underlies the consistently observed faster diffusion in one leaflet. Although the simulations here present molecular dynamics (MD) an order of magnitude longer than those from previous studies, the three systems did not approach ergodicity. The distributions of cholesterol and DPPC around the peptides changed on the microsecond time scale, but not significantly enough to thoroughly explore configurational space. These simulations support conclusions of other recent microsecond MD in that even longer time scales are needed for equilibration of model membranes and simulations of more realistic cellular or viral bilayers.

Formation of cholesterol-rich supported membranes using solvent-assisted lipid self-assembly.

This paper describes the application of a solvent-exchange method to prepare supported membranes containing high fractions of cholesterol (up to ∼57 mol %) in an apparent equilibrium. The method exploits the phenomenon of reverse-phase evaporation, in which the deposition of lipids in alcohol (e.g., isopropanol) is followed by the slow removal of the organic solvent from the water-alcohol mixture. This in turn induces a series of lyotropic phase transitions successively producing inverse-micelles, monomers, micelles, and vesicles in equilibrium with supported bilayers at the contacting solid surface. By using the standard cholesterol depletion by methyl-β-cyclodextrin treatment, a quartz crystal microbalance with dissipation monitoring assay confirms that the cholesterol concentration in the supported membranes is comparable to that in the surrounding bulk phase. A quantitative characterization of the biophysical properties of the resultant bilayer, including lateral diffusion constants and phase separation, using epifluorescence microscopy and atomic force microscopy establishes the formation of laterally contiguous supported lipid bilayers, which break into a characteristic domain-pattern of coexisting phases in a cholesterol concentration-dependent manner. With increasing cholesterol fraction in the supported bilayer, the size of the domains increases, ultimately yielding two-dimensional cholesterol bilayer domains near the solubility limit. A unique feature of the approach is that it enables preparation of supported membranes containing limiting concentrations of cholesterol near the solubility limit under equilibrium conditions, which cannot be obtained using conventional techniques (i.e., vesicle fusion).

Spin-label W-band EPR with seven-loop-six-gap resonator: Application to lens membranes derived from eyes of a single donor.

Spin-label W-band (94 GHz) EPR with a five-loop-four-gap resonator (LGR) was successfully applied to study membrane properties (L. Mainali, J.S. Hyde, W.K. Subczynski, Using spin-label W-band EPR to study membrane fluidity in samples of small volume, J. Magn. Reson. 226 (2013) 35-44). In that study, samples were equilibrated with the selected gas mixture outside the resonator in a sample volume ~100 times larger than the sensitive volume of the LGR and transferred to the resonator in a quartz capillary. A seven-loop-six-gap W-band resonator has been developed. This resonator permits measurements on aqueous samples of 150 nL volume positioned in a polytetrafluoroethylene (PTFE) gas permeable sample tube. Samples can be promptly deoxygenated or equilibrated with an air/nitrogen mixture inside the resonator, which is significant in saturation-recovery measurements and in spin-label oximetry. This approach was tested for lens lipid membranes derived from lipids extracted from two porcine lenses (single donor). Profiles of membrane fluidity and the oxygen transport parameter were obtained from saturation-recovery EPR using phospholipid analog spin-labels. Cholesterol analog spin-labels allowed discrimination of the cholesterol bilayer domain and acquisition of oxygen transport parameter profiles across this domain. Results were compared with those obtained previously for membranes derived from a pool of 100 lenses. Results demonstrate that EPR at W-band can be successfully used to study aqueous biological samples of small volume under controlled oxygen concentration.

A coarse-grained molecular dynamics investigation of the phase behavior of DPPC/cholesterol mixtures

We report a systematic molecular dynamics computer simulation investigation of the phase behavior of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/cholesterol (CHOL) bilayers for a wide range of temperatures and cholesterol compositions (0–50%). Our simulations consist of several thousand phospholipids modeled with the coarse grained MARTINI forcefield covering timescales of tens of microseconds. We employ Voronoi and Delaunay constructions to quantify local compositions in an attempt to identify coexistence regions with the liquid ordered (Lo) phase. We combine this analysis with the chain order parameter and in plane pair correlation functions and propose a phase coexistence diagram for the DPPC/CHOL mixture. We find stable liquid ordered phases Lo for high cholesterol content (40–50%). At these concentrations the cholesterol molecules arrange themselves in linear clusters. Our simulations provide evidence for coexistence between the Lo and gel phases at low temperatures, while at high temperatures the transition from the liquid disordered phase to the Lo phase is continuous and coexistence is not observed. The lipid molecules dynamics depends strongly on temperature and cholesterol concentration. At 323K and short times, t∼0.1μs the lipid molecules’ feature an anomalous sub-diffusive behavior with the mean square displacement (MSD) scaling as ∼t1/2. The characteristic time needed to reach the diffusive regime MSD ∼t, increases with the cholesterol content, reaching 0.2μs for 40% cholesterol content. The lipid diffusion activation energy in the Lo phase is 57kJ/mol, in good agreement with the available experimental estimates.

Effects of cholesterol concentration on the interaction of cytarabine with lipid membranes: a molecular dynamics simulation study

Liposomal cytarabine, DepoCyt, is a chemotherapy agent which is used in cancer treatment. This form of cytarabine has more efficacy and fewer side effects relative to the other forms. Since DepoCyt contains the cytarabine encapsulated within phosphatidylcholine and the sterol molecules, we modeled dioleoylphosphatidylcholine (DOPC)/cholesterol bilayer membrane as a carrier for cytarabine to study drug–bilayer interactions. For this purpose, we performed a series of united-atom molecular dynamics (MD) simulations for 25 ns to investigate the interactions between cytarabine and cholesterol-containing DOPC lipid bilayers. Only the uncharged form of cytarabine molecule was investigated. In this study, different levels of the cholesterol content (0, 20, and 40%) were used. MD simulations allowed us to determine dynamical and structural properties of the bilayer membrane and to estimate the preferred location and orientation of the cytarabine molecule inside the bilayer membrane. Properties such as membrane thickness, area per lipid, diffusion coefficient, mass density, bilayer packing, order parameters, and intermolecular interactions were examined. The results show that by increasing the cholesterol concentration in the lipid bilayers, the bilayer thickness increases and area per lipid decreases. Moreover, in accordance with the experiments, our calculations show that cholesterol molecules have ordering effect on the hydrocarbon acyl chains. Furthermore, the cytarabine molecule preferentially occupies the polar region of the lipid head groups to form specific interactions (hydrogen bonds). Our results fully support the experimental data. Our finding about drug–bilayer interaction is crucial for the liposomal drug design.

Effect of biocompatible gluconamide-type cationic surfactants on thermotropic phase behavior of phosphatidylcholine/cholesterol bilayers

• Interaction of new sugar-derived cationic surfactants with artificial membranes by DSC. • Toxicity with respect to L929 mouse fibroblast and A549 human lung cancer cells. • The effect of the gluconamide moiety on cytotoxicity. • Computations of molecular properties important in interactions with membrane. A series of soft cationic surfactants, containing a saccharide-derived moiety in their hydrophilic grouping – 2-(alkyldimethylammonio) ethylgluconamide bromides, denoted as C n GAB, n = 10, 12, 14 and 16 – were analyzed with respect to their influence on the thermotropic phase behavior of phosphatidylcholine (DPPC)/cholesterol bilayers. The compounds were found to be uniformly distributed in the studied lipid bilayer. As usual, the longer the chain of C n GAB, the stronger was the effect on T m and Δ H m , except C 16 GAB. Having the longest chain, C 16 GAB reduces T m to the smallest extent and does not affect Δ H m until the molar ratio of surfactant/DPPC is 0.15. The interactions with model cells in vitro were determined by cytotoxicity evaluation with respect to two cell lines – mouse fibroblast cell line L929 and human lung cancer cell line A549. The toxicity of C n GABs is much lower than a classical DTAB.

Interactions of caveolin-1 scaffolding and intramembrane regions containing a CRAC motif with cholesterol in lipid bilayers

Caveolin-1 is a major structural protein of caveolae and specifically binds cholesterol (Chol). The caveolin scaffolding domain is thought to be involved in caveolin–Chol interaction through the sequence V94-T-K-Y-W-F-Y-R101, a motif that matches a cholesterol recognition amino-acid consensus (CRAC). In the present work, three CRAC-containing peptides, corresponding to caveolin-1 94–101, 82–101 and 93–126, were tested to study the role of the CRAC motif in the caveolin–Chol interaction in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers using differential scanning calorimetry (DSC), fluorescence and circular dichroism (CD). The Y97I substituents of the three peptides and one peptide segment corresponding to caveolin-1 101–126 that excludes the CRAC motif were also tested for comparison. Our results showed the potency of these CRAC-containing peptides in sequestering Chol into domains and the enhanced role of the intramembrane domain and scaffolding domain for the potency. Of the three CRAC-containing peptides, the peptide 93–126 was particularly effective in promoting Chol segregation, while the peptide 82–101 was less potent in promoting the formation of domains than the peptide 93–126, but was more potent than the peptide 94–101. The domain partition of DPPC/Chol bilayers was not observed in the presence of the peptide 101–126, in contrast to the case in the presence of the peptide 93–126 at the same concentrations of peptide and Chol. The potency of the CRAC motif in Chol segregation was lowered by the Y97I mutation. The difference in structure may be a factor that contributes to different effects of these peptides on the distribution of Chol in the lipid membrane.

Enhancing physical stability of positively charged catanionic vesicles in the presence of calcium chloride via cholesterol-induced fluidic bilayer characteristic

An ion pair amphiphile (IPA), hexadecyltrimethylammonium-dodecylsulfate (HTMA-DS), and a double-chained cationic surfactant, dimethyldimyristylammonium bromide (DTDAB), could form positively charged catanionic vesicles with a potential application in gene delivery. To improve the gene delivery efficiency, the addition of CaCl2 into cationic liposomal systems has been proposed in the literature. In this study, detrimental effect of calcium chloride on the physical stability of the positively charged HTMA-DS/DTDAB catanionic vesicles was demonstrated by the size and zeta potential analyses of the vesicles. It was noted that the reduced electrostatic interaction between the catanionic vesicles could not fully explain the lowered physical stability of the vesicles in the presence of CaCl2. Apparently, the molecular packing/interaction in the vesicular bilayers played an important role in the vesicle physical stability. To modify the molecular packing/interaction in the vesicular bilayers, cholesterol was adopted as an additive to form catanionic vesicles with HTMA-DS/DTDAB. It was found that the physical stability of the catanionic vesicles was significantly improved with the presence of cholesterol in the vesicular bilayers even in the presence of 50 mM CaCl2. An infrared analysis suggested that with the incorporation of cholesterol into HTMA-DS/DTDAB vesicular bilayers, the alkyl chain motion was enhanced, and the molecular packing became less ordered. The cholesterol-induced fluidic bilayer characteristic allowed the vesicular bilayers to be adjusted to a stable status, resulting in improved physical stability of the catanionic vesicles even in the presence of CaCl2 with a high concentration.

DNA-cholesterol barges as programmable membrane-exploring agents.

DNA nanotechnology enables the precise construction of nanoscale devices that mimic aspects of natural biomolecular systems yet exhibit robustly programmable behavior. While many important biological processes involve dynamic interactions between components associated with phospholipid membranes, little progress has been made toward creating synthetic mimics of such interfacial systems. We report the assembly and characterization of cholesterol-labeled DNA origami "barges" capable of reversible association with and lateral diffusion on supported lipid bilayers. Using single-particle fluorescence microscopy, we show that these DNA barges rapidly and stably embed in lipid bilayers and exhibit Brownian diffusion in a manner dependent on both cholesterol labeling and bilayer composition. Tracking of individual barges rapidly generates super-resolution maps of the contiguous regions of a membrane. Addition of appropriate command oligonucleotides enables membrane-associated barges to reversibly exchange fluorescent cargo with bulk solution, dissociate from the membrane, or form oligomers within the membrane, opening up new possibilities for programmable membrane-bound molecular devices.

How well does cholesteryl hemisuccinate mimic cholesterol in saturated phospholipid bilayers?

Cholesteryl hemisuccinate is a detergent that is often used to replace cholesterol in crystallization of membrane proteins. Here we employ atomistic molecular dynamics simulations to characterize how well the properties of cholesteryl hemisuccinate actually match those of cholesterol in saturated protein-free lipid membranes. We show that the protonated form of cholesteryl hemisuccinate mimics many of the membrane properties of cholesterol quite well, while the deprotonated form of cholesteryl hemisuccinate is less convincing in this respect. Based on the results, we suggest that cholesteryl hemisuccinate in its protonated form is a quite faithful mimic of cholesterol for membrane protein crystallization, if specific cholesterol-protein interactions (not investigated here) are not playing a crucial role.

Lipid–protein interactions in plasma membranes of fiber cells isolated from the human eye lens

The protein content in human lens membranes is extremely high, increases with age, and is higher in the nucleus as compared with the cortex, which should strongly affect the organization and properties of the lipid bilayer portion of intact membranes. To assess these effects, the intact cortical and nuclear fiber cell plasma membranes isolated from human lenses from 41- to 60-year-old donors were studied using electron paramagnetic resonance spin-labeling methods. Results were compared with those obtained for lens lipid membranes prepared from total lipid extracts from human eyes of the same age group [Mainali, L., Raguz, M., O’Brien, W. J., and Subczynski, W. K. (2013) Biochim. Biophys. Acta]. Differences were considered to be mainly due to the effect of membrane proteins. The lipid-bilayer portions of intact membranes were significantly less fluid than lipid bilayers of lens lipid membranes, prepared without proteins. The intact membranes were found to contain three distinct lipid environments termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain. However, the cholesterol bilayer domain, which was detected in cortical and nuclear lens lipid membranes, was not detected in intact membranes. The relative amounts of bulk and trapped lipids were evaluated. The amount of lipids in domains uniquely formed due to the presence of membrane proteins was greater in nuclear membranes than in cortical membranes. Thus, it is evident that the rigidity of nuclear membranes is greater than that of cortical membranes. Also the permeability coefficients for oxygen measured in domains of nuclear membranes were significantly lower than appropriate coefficients measured in cortical membranes. Relationships between the organization of lipids into lipid domains in fiber cells plasma membranes and the organization of membrane proteins are discussed.

Structural and nanomechanical effects of cholesterol in binary and ternary spin-coated single lipid bilayers in dry conditions

We investigate the effects of Cholesterol (Chol) in the structural and nanomechanical properties of binary and ternary spin-coated single lipid bilayers made of Dioleoylphosphatidylcholine (DOPC) and Sphingomyelin (SM) in dry conditions. We show that for the DOPC/Chol bilayers, Chol induces an initial increase of the bilayer thickness, followed by decrease for concentrations above 30% Chol. The mechanical properties, instead, appear practically insensitive to the Chol content. For the SM/Chol bilayers we have observed both the thinning of the bilayer and the decrease of the force necessary to break it for Chol content above 40mol%. In both binary mixtures phase separation is not observed. For ternary single bilayers of DOPC/SM/Chol, Chol induces phase segregation and the formation of domains resembling lipid rafts. The domains show a thickness and mechanical response clearly distinct from the surrounding phase and dependent on the relative Chol content. Based on the results obtained for the binary mixtures, DOPC- and SM-enriched domains can be identified. We highlight that many of the effects of Chol reported here for the dry multicomponent single lipid bilayers resemble closely those observed in hydrated bilayers, thus offering an additional insight into their properties.