Membrane Lipid Dynamics Research Articles

Update on lipid membrane microdomains

Lipid membrane microdomains are involved in major types of disease, ranging from vascular and metabolic diseases to neurodegeneration, autoimmunity, infectious and inflammatory diseases, and cancer. This review provides an update of membrane microdomain abnormalities. Lipid membrane microdomains are dynamic assemblies of sphingolipids, cholesterol and proteins that dissociate and associate rapidly and form functional clusters. Membrane microdomain clustering is the key to how membrane microdomains can form lipid-protein platforms in cell membranes, functioning in membrane trafficking, cell polarization and signalling. Clustering of membrane microdomains can be modified, for example by dietary lipids and pharmacological agents. Metabolic overload through a cholesterol-rich and fat-rich diet can trigger metabolic learning, which is associated with membrane microdomain persistence, persistent signalling and disturbed vesicular traffic. Detailed characterization of lipid membrane microdomains and dynamics at the molecular level is necessary and will help to identify new dietary and pharmacological therapeutic targets for the treatment and prevention of lipid membrane microdomain related diseases.

Membrane Insertion and Bilayer Perturbation by Antimicrobial Peptide CM15

Antimicrobial peptides (AMPs) are an important component of innate immunity and have generated considerable interest as a potential new class of antibiotic. The biological activity of AMPs is strongly influenced by peptide-membrane interactions; however, for many of these peptides the molecular details of how they disrupt and/or translocate across target membranes are not known. CM15 is a linear, synthetic hybrid AMP composed of the first seven residues of the cecropin A and residues 2–9 of the bee venom peptide mellitin. Previous studies have shown that upon membrane binding CM15 folds into an α-helix with its helical axis aligned parallel to the bilayer surface and have implicated the formation of 2.2–3.8 nm pores in its bactericidal activity. Here we report site-directed spin labeling electron paramagnetic resonance studies examining the behavior of CM15 analogs labeled with a methanethiosulfonate spin label (MTSL) and a brominated MTSL as a function of increasing peptide concentration and utilize phospholipid-analog spin labels to assess the effects of CM15 binding and accumulation on the physical properties of membrane lipids. We find that as the concentration of membrane-bound CM15 is increased the N-terminal domain of the peptide becomes more deeply immersed in the lipid bilayer. Only minimal changes are observed in the rotational dynamics of membrane lipids, and changes in lipid dynamics are confined primarily to near the membrane surface. However, the accumulation of membrane-bound CM15 dramatically increases accessibility of lipid-analog spin labels to the polar relaxation agent, nickel (II) ethylenediaminediacetate, suggesting an increased permeability of the membrane to polar solutes. These results are discussed in relation to the molecular mechanism of membrane disruption by CM15.

Cytoskeleton dynamics: Fluctuations within the network

Out-of-equilibrium systems, such as the dynamics of a living cytoskeleton (CSK), are inherently noisy with fluctuations arising from the stochastic nature of the underlying biochemical and molecular events. Recently, such fluctuations within the cell were characterized by observing spontaneous nano-scale motions of an RGD-coated microbead bound to the cell surface [Bursac et al., Nat. Mater. 4 (2005) 557–561]. While these reported anomalous bead motions represent a molecular level reorganization (remodeling) of microstructures in contact with the bead, a precise nature of these cytoskeletal constituents and forces that drive their remodeling dynamics are largely unclear. Here, we focused upon spontaneous motions of an RGD-coated bead and, in particular, assessed to what extent these motions are attributable to (i) bulk cell movement (cell crawling), (ii) dynamics of focal adhesions, (iii) dynamics of lipid membrane, and/or (iv) dynamics of the underlying actin CSK driven by myosin motors.

Organization and Dynamics of NBD-Labeled Lipids in Membranes Analyzed by Fluorescence Recovery after Photobleaching

Lateral diffusion of membrane constituents plays an important role in membrane organization and represents a central theme in current models describing the structure and function of biological membranes. Fluorescence recovery after photobleaching (FRAP) is a widely used approach that provides information regarding dynamic properties and spatial distribution of membrane constituents. On the basis of the unique concentration-dependent fluorescence emission properties of a fluorescently labeled cholesterol analogue modified at the tail region, 25-[N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino]-27-norcholesterol (25-NBD-cholesterol), we have previously shown that it exhibits local organization even at very low concentrations in membranes. In this paper, we address aspects regarding the molecular size and dynamics of such an organized assembly of 25-NBD-cholesterol by monitoring its lateral diffusion characteristics using FRAP. To obtain a comprehensive understanding of the organization and dynamics of 25-NBD-cholesterol in the membrane, we compare its diffusion properties to that of a fluorescent phospholipid analogue 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-(1,3-benzoxadiazol-4-yl)) (NBD-PE). Our results indicate significant differences in the membrane dynamics of these NBD-labeled lipids. Importantly, on the basis of a novel wavelength-selective FRAP approach, our results show that the organization of 25-NBD-cholesterol is heterogeneous, with the presence of fast- and slow-diffusing species which could correspond to predominant populations of monomers and dimers of 25-NBD-cholesterol. The potential application of the wavelength-selective FRAP approach to monitor the organization and dynamics of molecules in membranes therefore represents an exciting possibility.

Conformational analysis of lipid molecules by self-organizing maps

The authors have studied the use of the self-organizing map (SOM) in the analysis of lipid conformations produced by atomic-scale molecular dynamics simulations. First, focusing on the methodological aspects, they have systematically studied how the SOM can be employed in the analysis of lipid conformations in a controlled and reliable fashion. For this purpose, they have used a previously reported 50 ns atomistic molecular dynamics simulation of a 1-palmitoyl-2-linoeayl-sn-glycero-3-phosphatidylcholine (PLPC) lipid bilayer and analyzed separately the conformations of the headgroup and the glycerol regions, as well as the diunsaturated fatty acid chain. They have elucidated the effect of training parameters on the quality of the results, as well as the effect of the size of the SOM. It turns out that the main conformational states of each region in the molecule are easily distinguished together with a variety of other typical structural features. As a second topic, the authors applied the SOM to the PLPC data to demonstrate how it can be used in the analysis that goes beyond the standard methods commonly used to study the structure and dynamics of lipid membranes. Overall, the results suggest that the SOM method provides a relatively simple and robust tool for quickly gaining a qualitative understanding of the most important features of the conformations of the system, without a priori knowledge. It seems plausible that the insight given by the SOM could be applied to a variety of biomolecular systems and the design of coarse-grained models for these systems.

Alterations in the Lipid Profile and Membrane Dynamics of Rat Intestinal Brush Border Membrane Induced by Different Classes of Nonsteroidal Anti-Inflammatory Drugs

ABSTRACTNonsteroidal anti-inflammatory drugs (NSAIDs) are known to cause gastrointestinal damage. New anti-inflammatory drugs have been developed in an attempt to improve their gastrointestinal side effect profile. Therefore, the objective of the present study was to compare the effect of three different NSAIDs, aspirin, nimesulide, and celecoxib, on the lipid profile and dynamics of rat intestinal brush border membranes (BBMs). Female Wistar rats were divided into four different groups viz: group I (control), group II (aspirin treated), group III (nimesulide treated), and group IV (celecoxib treated). Groups II, III, and IV received the corresponding drugs orally at a dose of 40 mg/kg body weight dissolved in water, while the control group received the vehicle only. After 28 days, all the treatment groups showed modification in the lipid profile of intestinal BBM as compared to control. Alterations in the intestinal membrane dynamics by fluidity studies showed a considerable increase, which correlated well with the changes in the lipid composition. It was suggested that NSAIDs such as aspirin, nimesulide, and celecoxib pose intestinal side effects due to initial changes in the composition and dynamics of the membranes. It was further concluded that newly discovered NSAIDs such as celecoxib have better safety profiles because of the less intense membrane effects, but studies are still required to comment decisively on the suitability of various NSAIDs depending upon their cyclooxygenase enzyme specificity.

Glass transition temperature of water confined in lipid membranes as determined by anelastic spectroscopy

The research of gene delivery vehicles used in gene therapy is focused on nonviral vectors like lipid membranes. Such vectors, nonimmunogenic and biodegradable, are formed by complexation of DNA with a mixture of cationic lipids and a neutral colipid which improve the transfection efficiency. A main topic related to lipid membrane dynamics is their capability to spontaneously confine water. At present the value of the glass transition temperature (Tg) is largely debated and determined only by some indirect methods. Here the authors show that anelastic spectroscopy allows the confined water Tg value to be directly identified in several lipid mixtures.

Exploring the collective dynamics of lipid membranes with inelastic neutron scattering

While most spectroscopic techniques, as e.g., nuclear magnetic resonance or dielectric spectroscopy, probe macroscopic responses, neutron and within some restrictions also x-ray scattering experiments give the unique access to microscopic dynamics at length scales of intermolecular or atomic distances. Only recently, it has become possible to study collective dynamics of planar lipid bilayers using neutron spectroscopy techniques [M. Rheinstädter, C. Ollinger, G. Fragneto, F. Demmel, and T. Salditt, Phys. Rev. Lett. 93, 108107 (2004)]. We determined the dispersion relation of the coherent fast picosecond density fluctuations on nearest-neighbor distances of the phospholipid acyl chains in the gel and in the fluid phases of a dimyristoylphoshatidylcholine bilayer. The experiments shed light on the evolution of structure and dynamics, and the relation between them, in the range of the gel-fluid main phase transition. The scattering volume restriction for inelastic neutron experiments was overcome by stacking several thousands of highly aligned membrane bilayers. By combining different neutron-scattering techniques, namely, three-axis, backscattering, and spin-echo spectroscopies, we present measurements of short- and long-wavelength collective fluctuations in biomimetic and biological membranes in a large range in momentum and energy transfer, covering time scales from about 0.1ps to almost 1μs and length scales from 3Å to about 0.1μm. The neutron-backscattering technique gives information about slow molecular dynamics of lipid acyl chains and the “membrane-water,” i.e., the water molecules in between the stacked bilayers in the nanosecond time range [M. C. Rheinstädter, T. Seydel, F. Demmel, and T. Salditt, Phys. Rev. E 71, 061908 (2005)]. The dispersion relations of the long-wavelength undulation modes in lipid bilayers with nanosecond relaxation times can be determined by quasielastic reflectometry on spin-echo spectrometers and give direct access to the elasticity parameters of the membranes.

Red Cell Membrane Lipid Changes at 3500 m and on Return to Sea Level

Previous studies have shown that acute hypobaric hypoxia, obtained in a hypobaric chamber, and subsequent reoxygenation, give rise to modifications of the erythrocyte membrane lipid dynamics, resulting in an increased lateral diffusivity of the membrane lipids, and this was interpreted as the result of a modified lipid-protein interaction. The aim of the present study was to determine the effect of the reoxygenation condition in individuals after 3 days at an altitude of 3,500 m above sea level. Reoxygenation was a consequence of returning to sea level. Resting blood samples from both conditions were obtained, and erythrocytes were separated and immediately lysed for membrane isolation. We measured the bilayer polarity in membranes with Laurdan, a fluorescent probe. We also measured malondialdehyde in membrane lipids, an indicator of oxidative damage. We found a 12% (p = 0.016, n = 7) increase in the polarity of the membrane bilayer surface, and an increase of 70% (p = 0.005, n = 7) in the formation of malondialdehyde in the membrane after the reoxygenation condition. The membrane bilayer polarity increase is due to an oxidative modification of the phospholipid backbone after reoxygenation. People working and/or recreating at moderate altitude (3,500 m) may be at risk of erythrocyte membrane oxidative damage upon returning to sea level, and therefore a better understanding of the processes occurring upon reoxygenation may lead to proposed strategies to minimize this effect.

Lipid rafts and membrane dynamics

Cell membranes contain a variety of lipid species that differ in their physico-chemical properties. Lipid-lipid immiscibility gives rise to lateral heterogeneities in the membrane plane, a subset of which are termed lipid rafts ([Simons and Vaz, 2004][1]). Originally defined biochemically as

Distribution, Lateral Mobility and Function of Membrane Proteins Incorporated into Giant Unilamellar Vesicles

GUVs have been widely used for studies on lipid mobility, membrane dynamics and lipid domain (raft) formation, using single molecule techniques like fluorescence correlation spectroscopy. Reports on membrane protein dynamics in these types of model membranes are by far less advanced due to the difficulty of incorporating proteins into GUVs in a functional state. We have used sucrose to prevent four distinct membrane protein(s) (complexes) from inactivating during the dehydration step of the GUV-formation process. The amount of sucrose was optimized such that the proteins retained 100% biological activity, and many proteo-GUVs were obtained. Although GUVs could be formed by hydration of lipid mixtures composed of neutral and anionic lipids, an alternate current electric field was required for GUV formation from neutral lipids. Distribution, lateral mobility, and function of an ATP-binding cassette transport system, an ion-linked transporter, and a mechanosensitive channel in GUVs were determined by confocal imaging, fluorescence correlation spectroscopy, patch-clamp measurements, and biochemical techniques. In addition, we show that sucrose slows down the lateral mobility of fluorescent lipid analogs, possibly due to hydrogen-bonding with the lipid headgroups, leading to larger complexes with reduced mobility.

1SC54 膜リン脂質のダイナミクスと細胞の形態形成(機能場としての生体膜 : タンパク質と脂質による動的超分子構造と機能の制御)

1SC54 膜リン脂質のダイナミクスと細胞の形態形成(機能場としての生体膜 : タンパク質と脂質による動的超分子構造と機能の制御)

Human Platelet Membrane Lipid Dynamics: Agonist Stimulation Results in the Aggregation of Lipid Rafts and the Lateral Phase Separation of like Lipids in Intact Human Platelets.

Sphingomyelin and cholesterol rich platelet membrane domains are organized into lipid rafts, which are present in the liquid ordered state, and which have been suggested to be key membrane components both during cold-induced human platelet activation (Gousset et al. 2002 Evidence for a physiological role for membrane rafts in human platelets. J. Cellular Physiol. 190:117–128) as well as during agonist induced activation. We have previous demonstrated that platelets have two membrane phase transitions, a raft transition at 34–40°C (Gousset et al., 2002 ) and a phospholipid phase transition at 10–20°C (Tablin et al. 1996 The membrane phase transition of intact human platelets: correlation with cold-induced activation. J. Cellular Physiol. 168:305–313). Using Fourier transform infrared spectroscopy (FTIR) we are able to sample all the key phases adopted by intact platelet phospholipids and sphingolipids (gel, liquid crystalline, liquid ordered (raft<INS cite=mailto:John%20H%20Crowe dateTime=2004-04-08T16:45>)</INS>, hexagonal). This method offers unique advantages for the study of phospholipid acyl chain structures and interactions, as it is non-invasive and can detect molecular vibrations produced by dipole moment oscillations at infrared frequencies. Equally important<INS cite=mailto:John%20H%20Crowe dateTime=2004-04-08T16:45>,</INS> these vibrational frequencies are well characterized for individual phospholipids, permitting unambiguous assignment of the transitions to membranes in intact cells (Crowe et al. 1999. Are lipid phase transitions responsible for chilling damage in human platelets? Cryobiology 38:180-101). Most recently, using FTIR we have examined platelet lipid membrane dynamics associated with agonist induced human platelet activation. FTIR analysis of thrombin ( 1U/ml and 0.5U/ml) activated human platelets demonstrates that agonist stimulation results in the development of multiple (four or more) phase transitions, strongly suggestive of phase separation of lipids into like-lipid domains, also referred to as lateral phase separation. These like-lipid domains have phase transition temperatures very similar to that of isolated single lipid species, which, we have previously demonstrated combined to form the main phospholipids phase transition at between 10–20°C (Crowe et al. 1999). Furthermore, when human platelets are repeatedly scanned by FTIR - despite the temperature excursions which might normally result in the remixing of pure lipid populations, the thrombin treated platelets maintained their multiple phase transitions, suggesting that the lateral phase separation is an irreversible event during agonist induced activation. In addition to FTIR studies of human platelet populations, we have also examined the distribution of diIC18 in washed resting, and agonist stimulated human platelets. This dye, which preferentially partitions into liquid ordered or gel phases was uniformly distributed in resting platelets, but became aggregated when platelets were treated by either low concentrations of thrombin (0.01U/ml) or collagen (3.0ug/ml). In addition, platelets treated with higher concentrations of either agonist, demonstrated lipid rafts which further aggregated into larger liquid ordered domains. These results strongly suggest that platelet membrane lipid organization plays a key role in membrane function and further downstream signaling. Funded by NHLBI and DARPA.

The E5 protein of the human papillomavirus type 16 modulates composition and dynamics of membrane lipids in keratinocytes

The E5 protein of the human papillomavirus type 16 is a small protein found associated to membranes, mainly in the Golgi apparatus, and expressed in the early stages of viral infection. Its expression modifies the cell response towards growth factors and stress exposures, and also blocks the surface expression of MHC molecules. A global explanation for these multiple effects is hitherto not available. Here we present data showing that the expression of HPV16-E5 increases the amount of free cholesterol readily extractable from the plasma membrane, without altering the total cholesterol content. In addition, HPV16-E5 modifies the composition of the cell membranes, increasing the synthesis rate of phosphatidylcholine and phosphatidylserine, while diminishing that of phosphatidylglycerol. We propose that these changes in the lipid composition of the membrane are the central effect of HPV16-E5 on the cell. The multiple and apparently disconnected effects of HPV16-E5 on tyrosine-kinase receptors, induction of the apoptosis and impairment of MHC trafficking could follow the initial alteration on the membrane composition.

Lipid Dynamics and Domain Formation in Model Membranes Composed of Ternary Mixtures of Unsaturated and Saturated Phosphatidylcholines and Cholesterol

In recent years, the implication of sphingomyelin in lipid raft formation has intensified the long sustained interest in this membrane lipid. Accumulating evidences show that cholesterol preferentially interacts with sphingomyelin, conferring specific physicochemical properties to the bilayer membrane. The molecular packing created by cholesterol and sphingomyelin, which presumably is one of the driving forces for lipid raft formation, is known in general to differ from that of cholesterol and phosphatidylcholine membranes. However, in many studies, saturated phosphatidylcholines are still considered as a model for sphingolipids. Here, we investigate the effect of cholesterol on mixtures of dioleoyl-phosphatidylcholine (DOPC) and dipalmitoyl-phosphatidylcholine (DPPC) or distearoyl-phosphatidylcholine (DSPC) and compare it to that on mixtures of DOPC and sphingomyelin analyzed in previous studies. Giant unilamellar vesicles prepared from ternary mixtures of various lipid compositions were imaged by confocal fluorescence microscopy and, within a certain range of sterol content, domain formation was observed. The assignment of distinct lipid phases and the molecular mobility in the membrane bilayer was investigated by fluorescence correlation spectroscopy. Cholesterol was shown to affect lipid dynamics in a similar way for DPPC and DSPC when the two phospholipids were combined with cholesterol in binary mixtures. However, the corresponding ternary mixtures exhibited different spatial lipid organization and dynamics. Finally, evidences of a weaker interaction of cholesterol with saturated phosphatidylcholines than with sphingomyelin (with matched chain length) are discussed.

Copper Excess Reduces the Fluidity of Plasma Membrane Lipids of Wheat Roots: a Spin Probe EPR Study

Electron paramagnetic resonance spectroscopy was applied to investigate changes in the organization and dynamics of plasma membrane (PM) lipids of roots caused by growing wheat (Triticum durum Desf. cv. Creso) seedlings under copper excess (50 μM Cu2+). To this purpose, unilamellar vesicles were made from total lipid extracts of PMs isolated either from copper-stressed or control roots, and probed at different bilayer depth using stearic acids bearing a doxyl group at two different positions of the alkyl chain, namely at the fifth (5-DSA) and 16th (16-DSA) carbon. EPR spectra were recorded as a function of temperature in the range between 273 and 323 K and interpreted in terms of a microscopically ordered and macroscopically disordered model, in which orientational ordering and dynamics of the spin probes were described by means of the order parameter S and the rotational diffusion coefficient R, respectively. It was found that growth in Cu excess resulted in a decreased root PM fluidity. The results were...

Probing Lipid Mobility of Raft-exhibiting Model Membranes by Fluorescence Correlation Spectroscopy

Confocal fluorescence microscopy and fluorescence correlation spectroscopy (FCS) have been employed to investigate the lipid spatial and dynamic organization in giant unilamellar vesicles (GUVs) prepared from ternary mixtures of dioleoyl-phosphatidylcholine/sphingomyelin/cholesterol. For a certain range of cholesterol concentration, formation of domains with raft-like properties was observed. Strikingly, the lipophilic probe 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-C18) was excluded from sphingomyelin-enriched regions, where the raft marker ganglioside GM1 was localized. Cholesterol was shown to promote lipid segregation in dioleoyl-phosphatidylcholine-enriched, liquid-disordered, and sphingomyelin-enriched, liquid-ordered phases. Most importantly, the lipid mobility in sphingomyelin-enriched regions significantly increased by increasing the cholesterol concentration. These results pinpoint the key role, played by cholesterol in tuning lipid dynamics in membranes. At cholesterol concentrations >50 mol%, domains vanished and the lipid diffusion slowed down upon further addition of cholesterol. By taking the molecular diffusion coefficients as a fingerprint of membrane phase compositions, FCS is proven to evaluate domain lipid compositions. Moreover, FCS data from ternary and binary mixtures have been used to build a ternary phase diagram, which shows areas of phase coexistence, transition points, and, importantly, how lipid dynamics varies between and within phase regions.

The Effect of Fructan on Membrane Lipid Organization and Dynamics in the Dry State

Fructans are a group of fructose-based oligo- and polysaccharides, which appear to be involved in membrane preservation during dehydration by interacting with the membrane lipids. To get further understanding of the protective mechanism, the consequences of the fructan-membrane lipid interaction for the molecular organization and dynamics in the dry state were studied. POPC and DMPC were investigated in the dry state by 2H, 31P NMR, and Fourier transform infrared spectroscopy using two types of fructan and dextran. The order-disorder transition temperature of dry POPC was reduced by 70°C in the presence of fructan. Fructan increased the mobility of the acyl chains, but immobilized the lipid headgroup region. Most likely, fructans insert between the headgroups of lipids, thereby spacing the acyl chains. This results in a much lower phase transition temperature. The headgroup is immobilized by the interaction with fructan. The location of the interaction with the lipid headgroup is different for the inulin-type fructan compared to the levan-type fructan, since inulin shows interaction with the lipid phosphate group, whereas levan does not. Dextran did not influence the phase transition temperature of dry POPC showing that reduction of this temperature is not a general property of polysaccharides.

Curvature effects in vesicle-particle interactions

In this paper I propose a continuum model to describe the dynamics of a lipid membrane and a bead that are interacting. The bead could represent either a colloidal particle or a peripheral protein. The evolution is governed by a system of nonlinear differential equations. Focusing attention on the case where the membrane is fixed gives information about the forces exerted on the bead by the membrane. It turns out that the curvature and the curvature gradient of the membrane play a prominent role in the evolution of the bead, as illustrated by suitable examples.

Evaluation of hydralazine and procainamide effects on fibroblast membrane fluidity

In this study the membrane fluidity of fibroblasts under different pharmacological treatment was investigated. Two drugs, hydralazine and procainamide, were used to treat the immortalized mouse NIH 3T3 and hamster B14 fibroblasts. Membrane lipid dynamics was measured by fluorescence spectroscopy and electron spin resonance techniques. Two kinds of fluorescent probes (TMA-DPH and 12-(9-anthroyloxy)-stearic acid (12-AS)) and two spin labels (5-doxylstearic acid (5-DS) and 12-doxylstearic acid (12-DS)) were used to monitor fluidity in the upper polar and in the hydrophobic core regions of the lipid bilayer. The drugs influenced the membrane hydrophobic core, of which hydralazine induced fluidization and procainamide increased the rigidity. The membrane fluidity at the surface of the lipid bilayer was not modified by the drugs which indicates that both drugs intercalated mainly into the inner core of the cell membrane.