Sample records for disrupts lipid membranes

  1. Interaction of tau protein with model lipid membranes induces tau structural compaction and membrane disruption

    PubMed Central

    Jones, Emmalee M.; Dubey, Manish; Camp, Phillip J.; Vernon, Briana C.; Biernat, Jacek; Mandelkow, Eckhard; Majewski, Jaroslaw; Chi, Eva Y.

    2012-01-01

    The misfolding and aggregation of the intrinsically disordered, microtubule-associated tau protein into neurofibrillary tangles is implicated in the pathogenesis of Alzheimer's disease. However, the mechanisms of tau aggregation and toxicity remain unknown. Recent work has shown that lipid membrane can induce tau aggregation and that membrane permeabilization may serve as a pathway by which protein aggregates exert toxicity, suggesting that the plasma membrane may play dual roles in tau pathology. This prompted our investigation to assess tau's propensity to interact with membranes and to elucidate the mutually disruptive structural perturbations the interactions induce in both tau and the membrane. We show that although highly charged and soluble, the full-length tau (hTau40) is also highly surface active, selectively inserts into anionic DMPG lipid monolayers and induces membrane morphological changes. To resolve molecular-scale structural details of hTau40 associated with lipid membranes, X-ray and neutron scattering techniques are utilized. X-ray reflectivity indicates hTau40's presence underneath a DMPG monolayer and penetration into the lipid headgroups and tailgroups, whereas grazing incidence X-ray diffraction shows that hTau40 insertion disrupts lipid packing. Moreover, both air/water and DMPG lipid membrane interfaces induce the disordered hTau40 to partially adopt a more compact conformation with density similar to that of a folded protein. Neutron reflectivity shows that tau completely disrupts supported DMPG bilayers while leaving the neutral DPPC bilayer intact. Our results show that hTau40's strong interaction with anionic lipids induces tau structural compaction and membrane disruption, suggesting possible membrane-based mechanisms of tau aggregation and toxicity in neurodegenerative diseases. PMID:22401494

  2. Characterization of 3D Voronoi Tessellation Nearest Neighbor Lipid Shells Provides Atomistic Lipid Disruption Profile of Protein Containing Lipid Membranes

    PubMed Central

    Cheng, Sara Y.; Duong, Hai V.; Compton, Campbell; Vaughn, Mark W.; Nguyen, Hoa; Cheng, Kwan H.

    2015-01-01

    Quantifying protein-induced lipid disruptions at the atomistic level is a challenging problem in membrane biophysics. Here we propose a novel 3D Voronoi tessellation nearest-atom-neighbor shell method to classify and characterize lipid domains into discrete concentric lipid shells surrounding membrane proteins in structurally heterogeneous lipid membranes. This method needs only the coordinates of the system and is independent of force fields and simulation conditions. As a proof-of-principle, we use this multiple lipid shell method to analyze the lipid disruption profiles of three simulated membrane systems: phosphatidylcholine, phosphatidylcholine/cholesterol, and beta-amyloid/phosphatidylcholine/cholesterol. We observed different atomic volume disruption mechanisms due to cholesterol and beta-amyloid Additionally, several lipid fractional groups and lipid-interfacial water did not converge to their control values with increasing distance or shell order from the protein. This volume divergent behavior was confirmed by bilayer thickness and chain orientational order calculations. Our method can also be used to analyze high-resolution structural experimental data. PMID:25637891

  3. How Membrane-Active Peptides Get into Lipid Membranes.

    PubMed

    Sani, Marc-Antoine; Separovic, Frances

    2016-06-21

    The structure-function relationship for a family of antimicrobial peptides (AMPs) from the skin of Australian tree frogs is discussed and compared with that of peptide toxins from bee and Australian scorpion venoms. Although these membrane-active peptides induce a similar cellular fate by disrupting the lipid bilayer integrity, their lytic activity is achieved via different modes of action, which are investigated in relation to amino acid sequence, secondary structure, and membrane lipid composition. In order to better understand what structural features govern the interaction between peptides and lipid membranes, cell-penetrating peptides (CPPs), which translocate through the membrane without compromising its integrity, are also discussed. AMPs possess membrane lytic activities that are naturally designed to target the cellular membrane of pathogens or competitors. They are extremely diverse in amino acid composition and often show specificity against a particular strain of microbe. Since our antibiotic arsenal is declining precariously in the face of the rise in multiantibiotic resistance, AMPs increasingly are seen as a promising alternative. In an effort to understand their molecular mechanism, biophysical studies of a myriad of AMPs have been reported, yet no unifying mechanism has emerged, rendering difficult the rational design of drug leads. Similarly, a wide variety of cytotoxic peptides are found in venoms, the best known being melittin, yet again, predicting their activity based on a particular amino acid composition or secondary structure remains elusive. A common feature of these membrane-active peptides is their preference for the lipid environment. Indeed, they are mainly unstructured in solution and, in the presence of lipid membranes, quickly adsorb onto the surface, change their secondary structure, eventually insert into the hydrophobic core of the membrane bilayer, and finally disrupt the bilayer integrity. These steps define the molecular

  4. DMSO Induces Dehydration near Lipid Membrane Surfaces

    PubMed Central

    Cheng, Chi-Yuan; Song, Jinsuk; Pas, Jolien; Meijer, Lenny H.H.; Han, Songi

    2015-01-01

    Dimethyl sulfoxide (DMSO) has been broadly used in biology as a cosolvent, a cryoprotectant, and an enhancer of membrane permeability, leading to the general assumption that DMSO-induced structural changes in cell membranes and their hydration water play important functional roles. Although the effects of DMSO on the membrane structure and the headgroup dehydration have been extensively studied, the mechanism by which DMSO invokes its effect on lipid membranes and the direct role of water in this process are unresolved. By directly probing the translational water diffusivity near unconfined lipid vesicle surfaces, the lipid headgroup mobility, and the repeat distances in multilamellar vesicles, we found that DMSO exclusively weakens the surface water network near the lipid membrane at a bulk DMSO mole fraction (XDMSO) of <0.1, regardless of the lipid composition and the lipid phase. Specifically, DMSO was found to effectively destabilize the hydration water structure at the lipid membrane surface at XDMSO <0.1, lower the energetic barrier to dehydrate this surface water, whose displacement otherwise requires a higher activation energy, consequently yielding compressed interbilayer distances in multilamellar vesicles at equilibrium with unaltered bilayer thicknesses. At XDMSO >0.1, DMSO enters the lipid interface and restricts the lipid headgroup motion. We postulate that DMSO acts as an efficient cryoprotectant even at low concentrations by exclusively disrupting the water network near the lipid membrane surface, weakening the cohesion between water and adhesion of water to the lipid headgroups, and so mitigating the stress induced by the volume change of water during freeze-thaw. PMID:26200868

  5. Melittin-induced cholesterol reorganization in lipid bilayer membranes

    DOE PAGES

    Qian, Shuo; Heller, William T.

    2015-06-12

    The peptide melittin, a 26 amino acid, cationic peptide from honey bee ( Apis mellifera) venom, disrupts lipid bilayer membranes in a concentration-dependent manner. Rather than interacting with a specific receptor, the peptide interacts directly with the lipid matrix of the membrane in a manner dependent on the lipid composition. Here, a small-angle neutron scattering study of the interaction of melittin with lipid bilayers made of mixtures of dimyristoylphosphatidylcholine (DMPC) and cholesterol (Chol) is presented. Through the use of deuterium-labeled DMPC, changes in the distribution of the lipid and cholesterol in unilamellar vesicles were observed for peptide concentrations below thosemore » that cause pores to form. In addition to disrupting the in-plane organization of Chol, melittin produces vesicles having inner and outer leaflet compositions that depend on the lipid–Chol molar ratio and on the peptide concentration. The changes seen at high cholesterol and low peptide concentration are similar to those produced by alamethicin (Qian, S. et al., J. Phys. Chem. B 2014, 118, 11200–11208), which points to an underlying physical mechanism driving the redistribution of Chol, but melittin displays an additional effect not seen with alamethicin. Furthermore, a model for how the peptide drives the redistribution of Chol is proposed. The results suggest that redistribution of the lipids in a target cell membrane by membrane active peptides takes places as a prelude to the lysis of the cell.« less

  6. Melittin-induced cholesterol reorganization in lipid bilayer membranes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Qian, Shuo; Heller, William T.

    The peptide melittin, a 26 amino acid, cationic peptide from honey bee ( Apis mellifera) venom, disrupts lipid bilayer membranes in a concentration-dependent manner. Rather than interacting with a specific receptor, the peptide interacts directly with the lipid matrix of the membrane in a manner dependent on the lipid composition. Here, a small-angle neutron scattering study of the interaction of melittin with lipid bilayers made of mixtures of dimyristoylphosphatidylcholine (DMPC) and cholesterol (Chol) is presented. Through the use of deuterium-labeled DMPC, changes in the distribution of the lipid and cholesterol in unilamellar vesicles were observed for peptide concentrations below thosemore » that cause pores to form. In addition to disrupting the in-plane organization of Chol, melittin produces vesicles having inner and outer leaflet compositions that depend on the lipid–Chol molar ratio and on the peptide concentration. The changes seen at high cholesterol and low peptide concentration are similar to those produced by alamethicin (Qian, S. et al., J. Phys. Chem. B 2014, 118, 11200–11208), which points to an underlying physical mechanism driving the redistribution of Chol, but melittin displays an additional effect not seen with alamethicin. Furthermore, a model for how the peptide drives the redistribution of Chol is proposed. The results suggest that redistribution of the lipids in a target cell membrane by membrane active peptides takes places as a prelude to the lysis of the cell.« less

  7. Alteration of cellular lipids and lipid metabolism markers in RTL-W1 cells exposed to model endocrine disrupters.

    PubMed

    Dimastrogiovanni, Giorgio; Córdoba, Marlon; Navarro, Isabel; Jáuregui, Olga; Porte, Cinta

    2015-08-01

    This work investigates the suitability of the rainbow trout liver cell line (RTL-W1) as an in-vitro model to study the ability of model endocrine disrupters, namely TBT, TPT, 4-NP, BPA and DEHP, to act as metabolic disrupters by altering cellular lipids and markers of lipid metabolism. Among the tested compounds, BPA and DEHP significantly increased the intracellular accumulation of triacylglycerols (TAGs), while all the compounds -apart from TPT-, altered membrane lipids - phosphatidylcholines (PCs) and plasmalogen PCs - indicating a strong interaction of the toxicants with cell membranes and cell signaling. RTL-W1 expressed a number of genes involved in lipid metabolism that were modulated by exposure to BPA, TBT and TPT (up-regulation of FATP1 and FAS) and 4-NP and DEHP (down-regulation of FAS and LPL). Multiple and complex modes of action of these chemicals were observed in RTL-W1 cells, both in terms of expression of genes related to lipid metabolism and alteration of cellular lipids. Although further characterization is needed, this might be a useful model for the detection of chemicals leading to steatosis or other diseases associated with lipid metabolism in fish. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Plasma membrane lipids and their role in fungal virulence.

    PubMed

    Rella, Antonella; Farnoud, Amir M; Del Poeta, Maurizio

    2016-01-01

    There has been considerable evidence in recent years suggesting that plasma membrane lipids are important regulators of fungal pathogenicity. Various glycolipids have been shown to impart virulent properties in several fungal species, while others have been shown to play a role in host defense. In addition to their role as virulence factors, lipids also contribute to other virulence mechanisms such as drug resistance, biofilm formation, and release of extracellular vesicles. In addition, lipids also affect the mechanical properties of the plasma membrane through the formation of packed microdomains composed mainly of sphingolipids and sterols. Changes in the composition of lipid microdomains have been shown to disrupt the localization of virulence factors and affect fungal pathogenicity. This review gathers evidence on the various roles of plasma membrane lipids in fungal virulence and how lipids might contribute to the different processes that occur during infection and treatment. Insight into the role of lipids in fungal virulence can lead to an improved understanding of the process of fungal pathogenesis and the development of new lipid-mediated therapeutic strategies. Published by Elsevier Ltd.

  9. Lipid partitioning at the nuclear envelope controls membrane biogenesis

    PubMed Central

    Barbosa, Antonio Daniel; Sembongi, Hiroshi; Su, Wen-Min; Abreu, Susana; Reggiori, Fulvio; Carman, George M.; Siniossoglou, Symeon

    2015-01-01

    Partitioning of lipid precursors between membranes and storage is crucial for cell growth, and its disruption underlies pathologies such as cancer, obesity, and type 2 diabetes. However, the mechanisms and signals that regulate this process are largely unknown. In yeast, lipid precursors are mainly used for phospholipid synthesis in nutrient-rich conditions in order to sustain rapid proliferation but are redirected to triacylglycerol (TAG) stored in lipid droplets during starvation. Here we investigate how cells reprogram lipid metabolism in the endoplasmic reticulum. We show that the conserved phosphatidate (PA) phosphatase Pah1, which generates diacylglycerol from PA, targets a nuclear membrane subdomain that is in contact with growing lipid droplets and mediates TAG synthesis. We find that cytosol acidification activates the master regulator of Pah1, the Nem1-Spo7 complex, thus linking Pah1 activity to cellular metabolic status. In the absence of TAG storage capacity, Pah1 still binds the nuclear membrane, but lipid precursors are redirected toward phospholipids, resulting in nuclear deformation and a proliferation of endoplasmic reticulum membrane. We propose that, in response to growth signals, activation of Pah1 at the nuclear envelope acts as a switch to control the balance between membrane biogenesis and lipid storage. PMID:26269581

  10. Lipid rafts are disrupted in mildly inflamed intestinal microenvironments without overt disruption of the epithelial barrier.

    PubMed

    Bowie, Rachel V; Donatello, Simona; Lyes, Clíona; Owens, Mark B; Babina, Irina S; Hudson, Lance; Walsh, Shaun V; O'Donoghue, Diarmuid P; Amu, Sylvie; Barry, Sean P; Fallon, Padraic G; Hopkins, Ann M

    2012-04-15

    Intestinal epithelial barrier disruption is a feature of inflammatory bowel disease (IBD), but whether barrier disruption precedes or merely accompanies inflammation remains controversial. Tight junction (TJ) adhesion complexes control epithelial barrier integrity. Since some TJ proteins reside in cholesterol-enriched regions of the cell membrane termed lipid rafts, we sought to elucidate the relationship between rafts and intestinal epithelial barrier function. Lipid rafts were isolated from Caco-2 intestinal epithelial cells primed with the proinflammatory cytokine interferon-γ (IFN-γ) or treated with methyl-β-cyclodextrin as a positive control for raft disruption. Rafts were also isolated from the ilea of mice in which colitis had been induced in conjunction with in vivo intestinal permeability measurements, and lastly from intestinal biopsies of ulcerative colitis (UC) patients with predominantly mild or quiescent disease. Raft distribution was analyzed by measuring activity of the raft-associated enzyme alkaline phosphatase and by performing Western blot analysis for flotillin-1. Epithelial barrier integrity was estimated by measuring transepithelial resistance in cytokine-treated cells or in vivo permeability to fluorescent dextran in colitic mice. Raft and nonraft fractions were analyzed by Western blotting for the TJ proteins occludin and zonula occludens-1 (ZO-1). Our results revealed that lipid rafts were disrupted in IFN-γ-treated cells, in the ilea of mice with subclinical colitis, and in UC patients with quiescent inflammation. This was not associated with a clear pattern of occludin or ZO-1 relocalization from raft to nonraft fractions. Significantly, a time-course study in colitic mice revealed that disruption of lipid rafts preceded the onset of increased intestinal permeability. Our data suggest for the first time that lipid raft disruption occurs early in the inflammatory cascade in murine and human colitis and, we speculate, may contribute to

  11. Procyanidins can interact with Caco-2 cell membrane lipid rafts: involvement of cholesterol.

    PubMed

    Verstraeten, Sandra V; Jaggers, Grayson K; Fraga, Cesar G; Oteiza, Patricia I

    2013-11-01

    Large procyanidins (more than three subunits) are not absorbed at the gastrointestinal tract but could exert local effects through their interactions with membranes. We previously showed that hexameric procyanidins (Hex), although not entering cells, interact with membranes modulating cell signaling and fate. This paper investigated if Hex, as an example of large procyanidins, can selectively interact with lipid rafts which could in part explain its biological actions. This mechanism was studied in both synthetic membranes (liposomes) and Caco-2 cells. Hex promoted Caco-2 cell membrane rigidification and dehydration, effects that were abolished upon cholesterol depletion with methyl-β-cyclodextrin (MCD). Hex prevented lipid raft structure disruption induced by cholesterol depletion/redistribution by MCD or sodium deoxycholate. Supporting the involvement of cholesterol-Hex bonding in Hex interaction with lipid rafts, the absence of cholesterol markedly decreased the capacity of Hex to prevent deoxycholate- and Triton X-100-mediated disruption of lipid raft-like liposomes. Stressing the functional relevance of this interaction, Hex mitigated lipid raft-associated activation of the extracellular signal-regulated kinases (ERK) 1/2. Results support the capacity of a large procyanidin (Hex) to interact with membrane lipid rafts mainly through Hex-cholesterol bondings. Procyanidin-lipid raft interactions can in part explain the capacity of large procyanidins to modulate cell physiology. © 2013 Elsevier B.V. All rights reserved.

  12. Interactions of Polyethylenimines with Zwitterionic and Anionic Lipid Membranes.

    PubMed

    Kwolek, Urszula; Jamróz, Dorota; Janiczek, Małgorzata; Nowakowska, Maria; Wydro, Paweł; Kepczynski, Mariusz

    2016-05-17

    Interactions between polyethylenimines (PEIs) and phospholipid membranes are of fundamental importance for various biophysical applications of these polymers such as gene delivery. Despite investigations into the nature of these interactions, their molecular basis remains poorly understood. In this article, we combined experimental methods and atomistic molecular dynamics (MD) simulations to obtain comprehensive insight into the effect of linear and branched PEIs on zwitterionic and anionic bilayers used as simple models of mammalian cellular membranes. Our results show that PEIs adsorb only partially on the surface of zwitterionic membranes by forming hydrogen bonds to the lipid headgroups, whereas a large part of the polymer chains dangles freely in the aqueous phase. In contrast, PEIs readily adhere to and insert into the anionic membrane. The attraction of the polymer chains to the membrane is due to electrostatic interactions as well as hydrogen bonding between the amine groups of PEI and the phosphate groups of lipids. These interactions were found to induce a substantial reorganization of the bilayer in the polymer vicinity due to the reorientation of lipid molecules. The lipid headgroups were pulled toward the center of the membrane, which can facilitate transmembrane translocations of anionic lipids. Furthermore, the PEI-lipid interactions affect the stability of liposomal dispersions, but we did not see any evidence of disruption of the vesicular structures into small fragments at polymer concentrations typically used in gene therapy. Our results provide a detailed molecular-level description of the lipid organization in the membrane in the presence of polycations that can be useful in understanding their mechanisms of in vitro and in vivo cytotoxicity.

  13. Disruption of gel phase lipid packing efficiency by sucralose studied with merocyanine 540.

    PubMed

    Barker, Morgan; Kennedy, Anthony

    2017-04-01

    Sucralose, an artificial sweetener, displays very different behavior towards membranes than its synthetic precursor sucrose. The impact of both sugars on model dipalmitoylphosphatidylcholine model membranes was investigated using absorbance and flourescence spectroscopy and the membrane probe merocyanine 540. This probe molecule is highly sensitive to changes in membrane packing, microviscosity and polarity. This work focuses on the impact of sugars on the outer leaflet of unilamellar dipalmitoyl phosphatidylcholine model membranes. The choice of lipid permits access to the gel phase at room temperature and incorporation of the dye after liposome formation allows us to examine the direct impact of the sugar on the outer leaflet while maximizing the response of the dye to changes in the bilayer. The results demonstrate that sucrose has no impact on the packing efficiency of lipids in unilamellar DPPC vesicles in the gel phase. Conversely sucralose decreases the packing efficiency of lipids in the gel phase and results in decreased microviscosity and increased membrane fluidity, which may be as a result of water disruption at the membrane water interface. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Biomembrane disruption by silica-core nanoparticles: effect of surface functional group measured using a tethered bilayer lipid membrane

    PubMed Central

    Liu, Ying; Zhang, Zhen; Zhang, Quanxuan; Baker, Gregory L.; Worden, R. Mark

    2013-01-01

    Engineered nanomaterials (ENM) have desirable properties that make them well suited for many commercial applications. However, a limited understanding of how ENM’s properties influence their molecular interactions with biomembranes hampers efforts to design ENM that are both safe and effective. This paper describes the use of a tethered bilayer lipid membrane (tBLM) to characterize biomembrane disruption by functionalized silica-core nanoparticles. Electrochemical impedance spectroscopy was used to measure the time trajectory of tBLM resistance following nanoparticle exposure. Statistical analysis of parameters from an exponential resistance decay model was then used to quantify and analyze differences between the impedance profiles of nanoparticles that were unfunctionalized, amine-functionalized, or carboxyl-functionalized. All of the nanoparticles triggered a decrease in membrane resistance, indicating nanoparticle-induced disruption of the tBLM. Hierarchical clustering allowed the potency of nanoparticles for reducing tBLM resistance to be ranked in the order amine > carboxyl ~ bare silica. Dynamic light scattering analysis revealed that tBLM exposure triggered minor coalescence for bare and amine-functionalized silica nanoparticles but not for carboxyl-functionalized silica nanoparticles. These results indicate that the tBLM method can reproducibly characterize ENM-induced biomembrane disruption and can distinguish the BLM-disruption patterns of nanoparticles that are identical except for their surface functional groups. The method provides insight into mechanisms of molecular interaction involving biomembranes and is suitable for miniaturization and automation for high-throughput applications to help assess the health risk of nanomaterial exposure or identify ENM having a desired mode of interaction with biomembranes. PMID:24060565

  15. Role of Transbilayer Distribution of Lipid Molecules on the Structure and Protein-Lipid Interaction of an Amyloidogenic Protein on the Membrane Surface

    NASA Astrophysics Data System (ADS)

    Cheng, Kwan; Cheng, Sara

    We used molecular dynamics simulations to examine the effects of transbilayer distribution of lipid molecules, particularly anionic lipids with negatively charged headgroups, on the structure and binding kinetics of an amyloidogenic protein on the membrane surface and subsequent protein-induced structural disruption of the membrane. Our systems consisted of a model beta-sheet rich dimeric protein absorbed on asymmetric bilayers with neutral and anionic lipids and symmetric bilayers with neutral lipids. We observed larger folding, domain aggregation, and tilt angle of the absorbed protein on the asymmetric bilayer surfaces. We also detected more focused bilayer thinning in the asymmetric bilayer due to weak lipid-protein interactions. Our results support the mechanism that the higher lipid packing in the protein-contacting lipid leaflet promotes stronger protein-protein but weaker protein-lipid interactions of an amyloidogenic protein on the membrane surface. We speculate that the observed surface-induced structural and protein-lipid interaction of our model amyloidogenic protein may play a role in the early membrane-associated amyloid cascade pathway that leads to membrane structural damage of neurons in Alzheimer's disease. NSF ACI-1531594.

  16. Cations as Switches of Amyloid-Mediated Membrane Disruption Mechanisms: Calcium and IAPP

    PubMed Central

    Sciacca, Michele F.M.; Milardi, Danilo; Messina, Grazia M.L.; Marletta, Giovanni; Brender, Jeffrey R.; Ramamoorthy, Ayyalusamy; La Rosa, Carmelo

    2013-01-01

    Disruption of the integrity of the plasma membrane by amyloidogenic proteins is linked to the pathogenesis of a number of common age-related diseases. Although accumulating evidence suggests that adverse environmental stressors such as unbalanced levels of metal ions may trigger amyloid-mediated membrane damage, many features of the molecular mechanisms underlying these events are unknown. Using human islet amyloid polypeptide (hIAPP, aka amylin), an amyloidogenic peptide associated with β-cell death in type 2 diabetes, we demonstrate that the presence of Ca2+ ions inhibits membrane damage occurring immediately after the interaction of freshly dissolved hIAPP with the membrane, but significantly enhances fiber-dependent membrane disruption. In particular, dye leakage, quartz crystal microbalance, atomic force microscopy, and NMR experiments show that Ca2+ ions promote a shallow membrane insertion of hIAPP, which leads to the removal of lipids from the bilayer through a detergent-like mechanism triggered by fiber growth. Because both types of membrane-damage mechanisms are common to amyloid toxicity by most amyloidogenic proteins, it is likely that unregulated ion homeostasis, amyloid aggregation, and membrane disruption are all parts of a self-perpetuating cycle that fuels amyloid cytotoxicity. PMID:23332070

  17. Two-Step Mechanism of Membrane Disruption by Aβ through Membrane Fragmentation and Pore Formation

    PubMed Central

    Sciacca, Michele F.M.; Kotler, Samuel A.; Brender, Jeffrey R.; Chen, Jennifer; Lee, Dong-kuk; Ramamoorthy, Ayyalusamy

    2012-01-01

    Disruption of cell membranes by Aβ is believed to be one of the key components of Aβ toxicity. However, the mechanism by which this occurs is not fully understood. Here, we demonstrate that membrane disruption by Aβ occurs by a two-step process, with the initial formation of ion-selective pores followed by nonspecific fragmentation of the lipid membrane during amyloid fiber formation. Immediately after the addition of freshly dissolved Aβ1–40, defects form on the membrane that share many of the properties of Aβ channels originally reported from single-channel electrical recording, such as cation selectivity and the ability to be blockaded by zinc. By contrast, subsequent amyloid fiber formation on the surface of the membrane fragments the membrane in a way that is not cation selective and cannot be stopped by zinc ions. Moreover, we observed that the presence of ganglioside enhances both the initial pore formation and the fiber-dependent membrane fragmentation process. Whereas pore formation by freshly dissolved Aβ1–40 is weakly observed in the absence of gangliosides, fiber-dependent membrane fragmentation can only be observed in their presence. These results provide insights into the toxicity of Aβ and may aid in the design of specific compounds to alleviate the neurodegeneration of Alzheimer’s disease. PMID:22947931

  18. Native and dry-heated lysozyme interactions with membrane lipid monolayers: Lipid packing modifications of a phospholipid mixture, model of the Escherichia coli cytoplasmic membrane.

    PubMed

    Derde, Melanie; Nau, Françoise; Guérin-Dubiard, Catherine; Lechevalier, Valérie; Paboeuf, Gilles; Jan, Sophie; Baron, Florence; Gautier, Michel; Vié, Véronique

    2015-04-01

    Antimicrobial resistance is currently an important public health issue. The need for innovative antimicrobials is therefore growing. The ideal antimicrobial compound should limit antimicrobial resistance. Antimicrobial peptides or proteins such as hen egg white lysozyme are promising molecules that act on bacterial membranes. Hen egg white lysozyme has recently been identified as active on Gram-negative bacteria due to disruption of the outer and cytoplasmic membrane integrity. Furthermore, dry-heating (7 days and 80 °C) improves the membrane activity of lysozyme, resulting in higher antimicrobial activity. These in vivo findings suggest interactions between lysozyme and membrane lipids. This is consistent with the findings of several other authors who have shown lysozyme interaction with bacterial phospholipids such as phosphatidylglycerol and cardiolipin. However, until now, the interaction between lysozyme and bacterial cytoplasmic phospholipids has been in need of clarification. This study proposes the use of monolayer models with a realistic bacterial phospholipid composition in physiological conditions. The lysozyme/phospholipid interactions have been studied by surface pressure measurements, ellipsometry and atomic force microscopy. Native lysozyme has proved able to absorb and insert into a bacterial phospholipid monolayer, resulting in lipid packing reorganization, which in turn has lead to lateral cohesion modifications between phospholipids. Dry-heating of lysozyme has increased insertion capacity and ability to induce lipid packing modifications. These in vitro findings are then consistent with the increased membrane disruption potential of dry heated lysozyme in vivo compared to native lysozyme. Moreover, an eggPC monolayer study suggested that lysozyme/phospholipid interactions are specific to bacterial cytoplasmic membranes. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Interplay of electrostatics and lipid packing determines the binding of charged polymer coated nanoparticles to model membranes.

    PubMed

    Biswas, Nupur; Bhattacharya, Rupak; Saha, Arindam; Jana, Nikhil R; Basu, Jaydeep K

    2015-10-07

    Understanding of nanoparticle-membrane interactions is useful for various applications of nanoparticles like drug delivery and imaging. Here we report on the studies of interaction between hydrophilic charged polymer coated semiconductor quantum dot nanoparticles with model lipid membranes. Atomic force microscopy and X-ray reflectivity measurements suggest that cationic nanoparticles bind and penetrate bilayers of zwitterionic lipids. Penetration and binding depend on the extent of lipid packing and result in the disruption of the lipid bilayer accompanied by enhanced lipid diffusion. On the other hand, anionic nanoparticles show minimal membrane binding although, curiously, their interaction leads to reduction in lipid diffusivity. It is suggested that the enhanced binding of cationic QDs at higher lipid packing can be understood in terms of the effective surface potential of the bilayers which is tunable through membrane lipid packing. Our results bring forth the subtle interplay of membrane lipid packing and electrostatics which determine nanoparticle binding and penetration of model membranes with further implications for real cell membranes.

  20. Structural elucidation of the interaction between neurodegenerative disease-related tau protein with model lipid membranes

    NASA Astrophysics Data System (ADS)

    Jones, Emmalee M.

    A protein's sequence of amino acids determines how it folds. That folded structure is linked to protein function, and misfolding to dysfunction. Protein misfolding and aggregation into beta-sheet rich fibrillar aggregates is connected with over 20 neurodegenerative diseases, including Alzheimer's disease (AD). AD is characterized in part by misfolding, aggregation and deposition of the microtubule associated tau protein into neurofibrillary tangles (NFTs). However, two questions remain: What is tau's fibrillization mechanism, and what is tau's cytotoxicity mechanism? Tau is prone to heterogeneous interactions, including with lipid membranes. Lipids have been found in NFTs, anionic lipid vesicles induced aggregation of the microtubule binding domain of tau, and other protein aggregates induced ion permeability in cells. This evidence prompted our investigation of tau's interaction with model lipid membranes to elucidate the structural perturbations those interactions induced in tau protein and in the membrane. We show that although tau is highly charged and soluble, it is highly surface active and preferentially interacts with anionic membranes. To resolve molecular-scale structural details of tau and model membranes, we utilized X-ray and neutron scattering techniques. X-ray reflectivity indicated tau aggregated at air/water and anionic lipid membrane interfaces and penetrated into membranes. More significantly, membrane interfaces induced tau protein to partially adopt a more compact conformation with density similar to folded protein and ordered structure characteristic of beta-sheet formation. This suggests possible membrane-based mechanisms of tau aggregation. Membrane morphological changes were seen using fluorescence microscopy, and X-ray scattering techniques showed tau completely disrupts anionic membranes, suggesting an aggregate-based cytotoxicity mechanism. Further investigation of protein constructs and a "hyperphosphorylation" disease mimic helped

  1. Increased Degree of Unsaturation in the Lipid of Antifungal Cationic Amphiphiles Facilitates Selective Fungal Cell Disruption.

    PubMed

    Steinbuch, Kfir B; Benhamou, Raphael I; Levin, Lotan; Stein, Reuven; Fridman, Micha

    2018-05-11

    Antimicrobial cationic amphiphiles derived from aminoglycosides act through cell membrane permeabilization but have limited selectivity for microbial cell membranes. Herein, we report that an increased degree of unsaturation in the fatty acid segment of antifungal cationic amphiphiles derived from the aminoglycoside tobramycin significantly reduced toxicity to mammalian cells. A collection of tobramycin-derived cationic amphiphiles substituted with C 18 lipid chains varying in degree of unsaturation and double bond configuration were synthesized. All had potent activity against a panel of important fungal pathogens including strains with resistance to a variety of antifungal drugs. The tobramycin-derived cationic amphiphile substituted with linolenic acid with three cis double bonds (compound 6) was up to an order of magnitude less toxic to mammalian cells than cationic amphiphiles composed of lipids with a lower degree of unsaturation and than the fungal membrane disrupting drug amphotericin B. Compound 6 was 12-fold more selective (red blood cell hemolysis relative to antifungal activity) than compound 1, the derivative with a fully saturated lipid chain. Notably, compound 6 disrupted the membranes of fungal cells without affecting the viability of cocultured mammalian cells. This study demonstrates that the degree of unsaturation and the configuration of the double bond in lipids of cationic amphiphiles are important parameters that, if optimized, result in compounds with broad spectrum and potent antifungal activity as well as reduced toxicity toward mammalian cells.

  2. Cations as switches of amyloid-mediated membrane disruption mechanisms: calcium and IAPP.

    PubMed

    Sciacca, Michele F M; Milardi, Danilo; Messina, Grazia M L; Marletta, Giovanni; Brender, Jeffrey R; Ramamoorthy, Ayyalusamy; La Rosa, Carmelo

    2013-01-08

    Disruption of the integrity of the plasma membrane by amyloidogenic proteins is linked to the pathogenesis of a number of common age-related diseases. Although accumulating evidence suggests that adverse environmental stressors such as unbalanced levels of metal ions may trigger amyloid-mediated membrane damage, many features of the molecular mechanisms underlying these events are unknown. Using human islet amyloid polypeptide (hIAPP, aka amylin), an amyloidogenic peptide associated with β-cell death in type 2 diabetes, we demonstrate that the presence of Ca(2+) ions inhibits membrane damage occurring immediately after the interaction of freshly dissolved hIAPP with the membrane, but significantly enhances fiber-dependent membrane disruption. In particular, dye leakage, quartz crystal microbalance, atomic force microscopy, and NMR experiments show that Ca(2+) ions promote a shallow membrane insertion of hIAPP, which leads to the removal of lipids from the bilayer through a detergent-like mechanism triggered by fiber growth. Because both types of membrane-damage mechanisms are common to amyloid toxicity by most amyloidogenic proteins, it is likely that unregulated ion homeostasis, amyloid aggregation, and membrane disruption are all parts of a self-perpetuating cycle that fuels amyloid cytotoxicity. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  3. Membrane-spanning lipids for an uncompromised monitoring of membrane fusion and intermembrane lipid transfer

    PubMed Central

    Schwarzmann, Günter; Breiden, Bernadette; Sandhoff, Konrad

    2015-01-01

    A Förster resonance energy transfer-based fusion and transfer assay was developed to study, in model membranes, protein-mediated membrane fusion and intermembrane lipid transfer of fluorescent sphingolipid analogs. For this assay, it became necessary to apply labeled reporter molecules that are resistant to spontaneous as well as protein-mediated intermembrane transfer. The novelty of this assay is the use of nonextractable fluorescent membrane-spanning bipolar lipids. Starting from the tetraether lipid caldarchaeol, we synthesized fluorescent analogs with fluorophores at both polar ends. In addition, we synthesized radioactive glycosylated caldarchaeols. These labeled lipids were shown to stretch through bilayer membranes rather than to loop within a single lipid layer of liposomes. More important, the membrane-spanning lipids (MSLs) in contrast to phosphoglycerides proved to be nonextractable by proteins. We could show that the GM2 activator protein (GM2AP) is promiscuous with respect to glycero- and sphingolipid transfer. Saposin (Sap) B also transferred sphingolipids albeit with kinetics different from GM2AP. In addition, we could unambiguously show that the recombinant activator protein Sap C x His6 induced membrane fusion rather than intermembrane lipid transfer. These findings showed that these novel MSLs, in contrast with fluorescent phosphoglycerolipids, are well suited for an uncompromised monitoring of membrane fusion and intermembrane lipid transfer. PMID:26269359

  4. Membrane-spanning lipids for an uncompromised monitoring of membrane fusion and intermembrane lipid transfer.

    PubMed

    Schwarzmann, Günter; Breiden, Bernadette; Sandhoff, Konrad

    2015-10-01

    A Förster resonance energy transfer-based fusion and transfer assay was developed to study, in model membranes, protein-mediated membrane fusion and intermembrane lipid transfer of fluorescent sphingolipid analogs. For this assay, it became necessary to apply labeled reporter molecules that are resistant to spontaneous as well as protein-mediated intermembrane transfer. The novelty of this assay is the use of nonextractable fluorescent membrane-spanning bipolar lipids. Starting from the tetraether lipid caldarchaeol, we synthesized fluorescent analogs with fluorophores at both polar ends. In addition, we synthesized radioactive glycosylated caldarchaeols. These labeled lipids were shown to stretch through bilayer membranes rather than to loop within a single lipid layer of liposomes. More important, the membrane-spanning lipids (MSLs) in contrast to phosphoglycerides proved to be nonextractable by proteins. We could show that the GM2 activator protein (GM2AP) is promiscuous with respect to glycero- and sphingolipid transfer. Saposin (Sap) B also transferred sphingolipids albeit with kinetics different from GM2AP. In addition, we could unambiguously show that the recombinant activator protein Sap C x His6 induced membrane fusion rather than intermembrane lipid transfer. These findings showed that these novel MSLs, in contrast with fluorescent phosphoglycerolipids, are well suited for an uncompromised monitoring of membrane fusion and intermembrane lipid transfer. Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

  5. Probing lipid membrane electrostatics

    NASA Astrophysics Data System (ADS)

    Yang, Yi

    The electrostatic properties of lipid bilayer membranes play a significant role in many biological processes. Atomic force microscopy (AFM) is highly sensitive to membrane surface potential in electrolyte solutions. With fully characterized probe tips, AFM can perform quantitative electrostatic analysis of lipid membranes. Electrostatic interactions between Silicon nitride probes and supported zwitterionic dioleoylphosphatidylcholine (DOPC) bilayer with a variable fraction of anionic dioleoylphosphatidylserine (DOPS) were measured by AFM. Classical Gouy-Chapman theory was used to model the membrane electrostatics. The nonlinear Poisson-Boltzmann equation was numerically solved with finite element method to provide the potential distribution around the AFM tips. Theoretical tip-sample electrostatic interactions were calculated with the surface integral of both Maxwell and osmotic stress tensors on tip surface. The measured forces were interpreted with theoretical forces and the resulting surface charge densities of the membrane surfaces were in quantitative agreement with the Gouy-Chapman-Stern model of membrane charge regulation. It was demonstrated that the AFM can quantitatively detect membrane surface potential at a separation of several screening lengths, and that the AFM probe only perturbs the membrane surface potential by <2%. One important application of this technique is to estimate the dipole density of lipid membrane. Electrostatic analysis of DOPC lipid bilayers with the AFM reveals a repulsive force between the negatively charged probe tips and the zwitterionic lipid bilayers. This unexpected interaction has been analyzed quantitatively to reveal that the repulsion is due to a weak external field created by the internai membrane dipole moment. The analysis yields a dipole moment of 1.5 Debye per lipid with a dipole potential of +275 mV for supported DOPC membranes. This new ability to quantitatively measure the membrane dipole density in a noninvasive

  6. Atmospheric-pressure guided streamers for liposomal membrane disruption

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Svarnas, P.; Aleiferis, Sp.; Matrali, S. H.

    2012-12-24

    The potential to use liposomes (LIPs) as a cellular model in order to study interactions of cold atmospheric-pressure plasma with cells is herein investigated. Cold atmospheric-pressure plasma is formed by a dielectric-barrier discharge reactor. Large multilamellar vesicle liposomes, consisted of phosphatidylcholine and cholesterol, are prepared by the thin film hydration technique, to encapsulate a small hydrophilic dye, i.e., calcein. The plasma-induced release of calcein from liposomes is then used as a measure of liposome membrane integrity and, consequently, interaction between the cold atmospheric plasma and lipid bilayers. Physical mechanisms leading to membrane disruption are suggested, based on the plasma characterizationmore » including gas temperature calculation.« less

  7. Composition-dependent Membrane Disruption by the Proapoptotic Protein PB1F2 from HK97 Influenza A Virus.

    PubMed

    Wang, Yujuan; Yang, Jing; Wang, Jiarong; Zhu, Lei; Wang, Junfeng

    2018-06-22

    PB1F2 is a proapoptotic protein encoded by an alternative reading frame in the influenza A virus. Its accumulation accelerates mitochondrial fragmentation by decreasing the mitochondrial membrane potential following translocation into the mitochondrial inner membrane space, but the mechanistic underpinnings remain unclear. Herein, the PB1F2 from HK97 was expressed and purified in soluble form. The interaction between PB1F2 and the mitochondrial membrane were investigated using three membrane mimics, liposomes, bicelles and nanodiscs. We show that the interactions between PB1F2 and membrane mimics depend on lipid type and are time- and dose-dependent. The primary membrane target of PB1F2 is phosphatidylcholine, the lipid that forms the major component of mitochondrial inner membranes. PB1F2 disrupts the integrity of lipid membranes by forming micelle-like PB1F2-lipid assemblies. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  8. Membrane Interactions of hIAPP Monomer and Oligomer with Lipid Membranes by Molecular Dynamics Simulations.

    PubMed

    Zhang, Mingzhen; Ren, Baiping; Liu, Yonglan; Liang, Guizhao; Sun, Yan; Xu, Lijian; Zheng, Jie

    2017-08-16

    Interaction of human islet amyloid polypeptide (hIAPP) peptides with cell membrane is crucial for the understanding of amyloid toxicity associated with Type II diabetes (T2D). While it is known that the hIAPP-membrane interactions are considered to promote hIAPP aggregation into fibrils and induce membrane disruption, the membrane-induced conformation, orientation, aggregation, and adsorption behaviors of hIAPP peptides have not been well understood at the atomic level. Herein, we perform all-atom explicit-water molecular dynamics (MD) simulations to study the adsorption, orientation, and surface interaction of hIAPP aggregates with different sizes (monomer to tetramer) and conformations (monomer with α-helix and tetramer with β-sheet-rich U-turn) upon adsorption on the lipid bilayers composed of both pure zwitterionic POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and mixed anionic POPC/POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) (3:1) lipids. MD simulation results show that hIAPP monomer with α-helical conformation and hIAPP pentamer with β-sheet conformation can adsorb on both POPC and POPC/POPE bilayers via a preferential orientation of N-terminal residues facing toward the bilayer surface. The hIAPP aggregates show stronger interactions with mixed POPC/POPE lipids than pure POPC lipids, consistent with experimental observation that hIAPP adsorption and fibrililation are enhanced on mixed lipid bilayers. While electrostatic interactions are main attractive forces to drive the hIAPP aggregates to adsorb on both bilayers, the introduction of the more hydrophilic head groups of POPE lipids further promote the formation of the interfacial hydrogen bonds. Complement to our previous studies of hIAPP aggregates in bulk solution, this computational work increases our knowledge about the mechanism of amyloid peptide-membrane interactions that is central to the understanding the progression of all amyloid diseases.

  9. Plasma membrane disruption: repair, prevention, adaptation

    NASA Technical Reports Server (NTRS)

    McNeil, Paul L.; Steinhardt, Richard A.

    2003-01-01

    Many metazoan cells inhabit mechanically stressful environments and, consequently, their plasma membranes are frequently disrupted. Survival requires that the cell rapidly repair or reseal the disruption. Rapid resealing is an active and complex structural modification that employs endomembrane as its primary building block, and cytoskeletal and membrane fusion proteins as its catalysts. Endomembrane is delivered to the damaged plasma membrane through exocytosis, a ubiquitous Ca2+-triggered response to disruption. Tissue and cell level architecture prevent disruptions from occurring, either by shielding cells from damaging levels of force, or, when this is not possible, by promoting safe force transmission through the plasma membrane via protein-based cables and linkages. Prevention of disruption also can be a dynamic cell or tissue level adaptation triggered when a damaging level of mechanical stress is imposed. Disease results from failure of either the preventive or resealing mechanisms.

  10. Structural basis of membrane disruption and cellular toxicity by α-synuclein oligomers.

    PubMed

    Fusco, Giuliana; Chen, Serene W; Williamson, Philip T F; Cascella, Roberta; Perni, Michele; Jarvis, James A; Cecchi, Cristina; Vendruscolo, Michele; Chiti, Fabrizio; Cremades, Nunilo; Ying, Liming; Dobson, Christopher M; De Simone, Alfonso

    2017-12-15

    Oligomeric species populated during the aggregation process of α-synuclein have been linked to neuronal impairment in Parkinson's disease and related neurodegenerative disorders. By using solution and solid-state nuclear magnetic resonance techniques in conjunction with other structural methods, we identified the fundamental characteristics that enable toxic α-synuclein oligomers to perturb biological membranes and disrupt cellular function; these include a highly lipophilic element that promotes strong membrane interactions and a structured region that inserts into lipid bilayers and disrupts their integrity. In support of these conclusions, mutations that target the region that promotes strong membrane interactions by α-synuclein oligomers suppressed their toxicity in neuroblastoma cells and primary cortical neurons. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  11. Electrodiffusion of lipids on membrane surfaces

    NASA Astrophysics Data System (ADS)

    Zhou, Y. C.

    2012-05-01

    Lateral translocation of lipids and proteins is a universal process on membrane surfaces. Local aggregation or organization of lipids and proteins can be induced when the random lateral motion is mediated by the electrostatic interactions and membrane curvature. Although the lateral diffusion rates of lipids on membranes of various compositions are measured and the electrostatic free energies of predetermined protein-membrane-lipid systems can be computed, the process of the aggregation and the evolution to the electrostatically favorable states remain largely undetermined. Here we propose an electrodiffusion model, based on the variational principle of the free energy functional, for the self-consistent lateral drift-diffusion of multiple species of charged lipids on membrane surfaces. Finite sizes of lipids are modeled to enforce the geometrical constraint of the lipid concentration on membrane surfaces. A surface finite element method is developed to appropriate the Laplace-Beltrami operators in the partial differential equations of the model. Our model properly describes the saturation of lipids on membrane surfaces, and correctly predicts that the MARCKS peptide can consistently sequester three multivalent phosphatidylinositol 4,5-bisphosphate lipids through its basic amino acid residues, regardless of a wide range of the percentage of monovalent phosphatidylserine in the membrane.

  12. Electrodiffusion of lipids on membrane surfaces.

    PubMed

    Zhou, Y C

    2012-05-28

    Lateral translocation of lipids and proteins is a universal process on membrane surfaces. Local aggregation or organization of lipids and proteins can be induced when the random lateral motion is mediated by the electrostatic interactions and membrane curvature. Although the lateral diffusion rates of lipids on membranes of various compositions are measured and the electrostatic free energies of predetermined protein-membrane-lipid systems can be computed, the process of the aggregation and the evolution to the electrostatically favorable states remain largely undetermined. Here we propose an electrodiffusion model, based on the variational principle of the free energy functional, for the self-consistent lateral drift-diffusion of multiple species of charged lipids on membrane surfaces. Finite sizes of lipids are modeled to enforce the geometrical constraint of the lipid concentration on membrane surfaces. A surface finite element method is developed to appropriate the Laplace-Beltrami operators in the partial differential equations of the model. Our model properly describes the saturation of lipids on membrane surfaces, and correctly predicts that the MARCKS peptide can consistently sequester three multivalent phosphatidylinositol 4,5-bisphosphate lipids through its basic amino acid residues, regardless of a wide range of the percentage of monovalent phosphatidylserine in the membrane.

  13. Lysosomal degradation of membrane lipids.

    PubMed

    Kolter, Thomas; Sandhoff, Konrad

    2010-05-03

    The constitutive degradation of membrane components takes place in the acidic compartments of a cell, the endosomes and lysosomes. Sites of lipid degradation are intralysosomal membranes that are formed in endosomes, where the lipid composition is adjusted for degradation. Cholesterol is sorted out of the inner membranes, their content in bis(monoacylglycero)phosphate increases, and, most likely, sphingomyelin is degraded to ceramide. Together with endosomal and lysosomal lipid-binding proteins, the Niemann-Pick disease, type C2-protein, the GM2-activator, and the saposins sap-A, -B, -C, and -D, a suitable membrane lipid composition is required for degradation of complex lipids by hydrolytic enzymes. Copyright 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  14. Unraveling the impact of hydroxylation on interactions of bile acid cationic lipids with model membranes by in-depth calorimetry studies.

    PubMed

    Singh, Manish; Bajaj, Avinash

    2014-09-28

    We used eight bile acid cationic lipids differing in the number of hydroxyl groups and performed in-depth differential scanning calorimetry studies on model membranes doped with different percentages of these cationic bile acids. These studies revealed that the number and positioning of free hydroxyl groups on bile acids modulate the phase transition and co-operativity of membranes. Lithocholic acid based cationic lipids having no free hydroxyl groups gel well with dipalmitoylphosphatidylcholine (DPPC) membranes. Chenodeoxycholic acid lipids having one free hydroxyl group at the 7'-carbon position disrupt the membranes and lower their co-operativity. Deoxycholic acid and cholic acid based cationic lipids have free hydroxyl groups at the 12'-carbon position, and at 7'- and 12'-carbon positions respectively. Doping of these lipids at high concentrations increases the co-operativity of membranes suggesting that these lipids might induce self-assembly in DPPC membranes. These different modes of interactions between cationic lipids and model membranes would help in future for exploring their use in DNA/drug delivery.

  15. Membrane Contact Sites: Complex Zones for Membrane Association and Lipid Exchange

    PubMed Central

    Quon, Evan; Beh, Christopher T.

    2015-01-01

    Lipid transport between membranes within cells involves vesicle and protein carriers, but as agents of nonvesicular lipid transfer, the role of membrane contact sites has received increasing attention. As zones for lipid metabolism and exchange, various membrane contact sites mediate direct associations between different organelles. In particular, membrane contact sites linking the plasma membrane (PM) and the endoplasmic reticulum (ER) represent important regulators of lipid and ion transfer. In yeast, cortical ER is stapled to the PM through membrane-tethering proteins, which establish a direct connection between the membranes. In this review, we consider passive and facilitated models for lipid transfer at PM–ER contact sites. Besides the tethering proteins, we examine the roles of an additional repertoire of lipid and protein regulators that prime and propagate PM–ER membrane association. We conclude that instead of being simple mediators of membrane association, regulatory components of membrane contact sites have complex and multilayered functions. PMID:26949334

  16. [Germ cell membrane lipids in spermatogenesis].

    PubMed

    Wang, Ting; Shi, Xiao; Quan, Song

    2016-05-01

    Spermatogenesis is a complex developmental process in which a diploid progenitor germ cell transforms into highly specialized spermatozoa. During spermatogenesis, membrane remodeling takes place, and cell membrane permeability and liquidity undergo phase-specific changes, which are all associated with the alteration of membrane lipids. Lipids are important components of the germ cell membrane, whose volume and ratio fluctuate in different phases of spermatogenesis. Abnormal lipid metabolism can cause spermatogenic dysfunction and consequently male infertility. Germ cell membrane lipids are mainly composed of cholesterol, phospholipids and glycolipids, which play critical roles in cell adhesion and signal transduction during spermatogenesis. An insight into the correlation of membrane lipids with spermatogenesis helps us to better understand the mechanisms of spermatogenesis and provide new approaches to the diagnosis and treatment of male infertility.

  17. Peptide:lipid ratio and membrane surface charge determine the mechanism of action of the antimicrobial peptide BP100. Conformational and functional studies.

    PubMed

    Manzini, Mariana C; Perez, Katia R; Riske, Karin A; Bozelli, José C; Santos, Talita L; da Silva, Marcia A; Saraiva, Greice K V; Politi, Mario J; Valente, Ana P; Almeida, Fábio C L; Chaimovich, Hernan; Rodrigues, Magali A; Bemquerer, Marcelo P; Schreier, Shirley; Cuccovia, Iolanda M

    2014-07-01

    The cecropin-melittin hybrid antimicrobial peptide BP100 (H-KKLFKKILKYL-NH2) is selective for Gram-negative bacteria, negatively charged membranes, and weakly hemolytic. We studied BP100 conformational and functional properties upon interaction with large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs, containing variable proportions of phosphatidylcholine (PC) and negatively charged phosphatidylglycerol (PG). CD and NMR spectra showed that upon binding to PG-containing LUVs BP100 acquires α-helical conformation, the helix spanning residues 3-11. Theoretical analyses indicated that the helix is amphipathic and surface-seeking. CD and dynamic light scattering data evinced peptide and/or vesicle aggregation, modulated by peptide:lipid ratio and PG content. BP100 decreased the absolute value of the zeta potential (ζ) of LUVs with low PG contents; for higher PG, binding was analyzed as an ion-exchange process. At high salt, BP100-induced LUVS leakage requires higher peptide concentration, indicating that both electrostatic and hydrophobic interactions contribute to peptide binding. While a gradual release took place at low peptide:lipid ratios, instantaneous loss occurred at high ratios, suggesting vesicle disruption. Optical microscopy of GUVs confirmed BP100-promoted disruption of negatively charged membranes. The mechanism of action of BP100 is determined by both peptide:lipid ratio and negatively charged lipid content. While gradual release results from membrane perturbation by a small number of peptide molecules giving rise to changes in acyl chain packing, lipid clustering (leading to membrane defects), and/or membrane thinning, membrane disruption results from a sequence of events - large-scale peptide and lipid clustering, giving rise to peptide-lipid patches that eventually would leave the membrane in a carpet-like mechanism. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Mechanics of Lipid Bilayer Membranes

    NASA Astrophysics Data System (ADS)

    Powers, Thomas R.

    All cells have membranes. The plasma membrane encapsulates the cell's interior, acting as a barrier against the outside world. In cells with nuclei (eukaryotic cells), membranes also form internal compartments (organelles) which carry out specialized tasks, such as protein modification and sorting in the case of the Golgi apparatus, and ATP production in the case of mitochondria. The main components of membranes are lipids and proteins. The proteins can be channels, carriers, receptors, catalysts, signaling molecules, or structural elements, and typically contribute a substantial fraction of the total membrane dry weight. The equilibrium properties of pure lipid membranes are relatively well-understood, and will be the main focus of this article. The framework of elasticity theory and statistical mechanics that we will develop will serve as the foundation for understanding biological phenomena such as the nonequilibrium behavior of membranes laden with ion pumps, the role of membrane elasticity in ion channel gating, and the dynamics of vesicle fission and fusion. Understanding the mechanics of lipid membranes is also important for drug encapsulation and delivery.

  19. Lipid-lipid and lipid-drug interactions in biological membranes

    NASA Astrophysics Data System (ADS)

    Martynowycz, Michael W.

    Interactions between lipids and drug molecules in biological membranes help govern proper biological function in organisms. The mechanisms responsible for hydrophobic drug permeation remain elusive. Many small molecule drugs are hydrophobic. These drugs inhibit proteins in the cellular interior. The rise of antibiotic resistance in bacteria is thought to be caused by mutations in protein structure, changing drug kinetics to favor growth. However, small molecule drugs have been shown to have different mechanisms depending in the structure of the lipid membrane of the target cell. Biological membranes are investigated using Langmuir monolayers at the air-liquid interface. These offer the highest level of control in the mimetic system and allow them to be investigated using complementary techniques. Langmuir isotherms and insertion assays are used to determine the area occupied by each lipid in the membrane and the change in area caused by the introduction of a drug molecule, respectively. Specular X-ray reflectivity is used to determine the electron density of the monolayer, and grazing incidence X-ray diffraction is used to determine the in-plane order of the monolayer. These methods determine the affinity of the drug and the mechanism of action. Studies are presented on hydrophobic drugs with mammalian membrane mimics using warfarin along with modified analogues, called superwarfarins. Data shows that toxicity of these modified drugs are modulated by the membrane cholesterol content in cells; explaining several previously unexplained effects of the drugs. Membrane mimics of bacteria are investigated along with their interactions with a hydrophobic antibiotic, novobiocin. Data suggests that permeation of the drug is mediated by modifications to the membrane lipids, and completely ceases translocation under certain circumstances. Circumventing deficiencies in small, hydrophobic drugs is approached by using biologically mimetic oligomers. Peptoids, mimetic of host

  20. Single Lipid Molecule Dynamics on Supported Lipid Bilayers with Membrane Curvature.

    PubMed

    Cheney, Philip P; Weisgerber, Alan W; Feuerbach, Alec M; Knowles, Michelle K

    2017-03-15

    The plasma membrane is a highly compartmentalized, dynamic material and this organization is essential for a wide variety of cellular processes. Nanoscale domains allow proteins to organize for cell signaling, endo- and exocytosis, and other essential processes. Even in the absence of proteins, lipids have the ability to organize into domains as a result of a variety of chemical and physical interactions. One feature of membranes that affects lipid domain formation is membrane curvature. To directly test the role of curvature in lipid sorting, we measured the accumulation of two similar lipids, 1,2-Dihexadecanoyl- sn -glycero-3-phosphoethanolamine (DHPE) and hexadecanoic acid (HDA), using a supported lipid bilayer that was assembled over a nanopatterned surface to obtain regions of membrane curvature. Both lipids studied contain 16 carbon, saturated tails and a head group tag for fluorescence microscopy measurements. The accumulation of lipids at curvatures ranging from 28 nm to 55 nm radii was measured and fluorescein labeled DHPE accumulated more than fluorescein labeled HDA at regions of membrane curvature. We then tested whether single biotinylated DHPE molecules sense curvature using single particle tracking methods. Similar to groups of fluorescein labeled DHPE accumulating at curvature, the dynamics of single molecules of biotinylated DHPE was also affected by membrane curvature and highly confined motion was observed.

  1. Liposome Disruption Assay to Examine Lytic Properties of Biomolecules.

    PubMed

    Jimah, John R; Schlesinger, Paul H; Tolia, Niraj H

    2017-08-05

    Proteins may have three dimensional structural or amino acid features that suggest a role in targeting and disrupting lipids within cell membranes. It is often necessary to experimentally investigate if these proteins and biomolecules are able to disrupt membranes in order to conclusively characterize the function of these biomolecules. Here, we describe an in vitro assay to evaluate the membrane lytic properties of proteins and biomolecules. Large unilamellar vesicles (liposomes) containing carboxyfluorescein at fluorescence-quenching concentrations are treated with the biomolecule of interest. A resulting increase in fluorescence due to leakage of the dye from liposomes and subsequent dilution in the buffer demonstrates that the biomolecule is sufficient for disrupting liposomes and membranes. Additionally, since liposome disruption may occur via pore-formation or via general solubilization of lipids similar to detergents, we provide a method to distinguish between these two mechanisms. Pore-formation can be identified and evaluated by examining the blockade of carboxyfluorescein release with dextran molecules that fit the pore. The methods described here were used to determine that the malaria vaccine candidate CelTOS and proapoptotic Bax disrupt liposomes by pore formation (Saito et al. , 2000; Jimah et al. , 2016). Since membrane lipid binding by a biomolecule precedes membrane disruption, we recommend the companion protocol: Jimah et al. , 2017.

  2. Curvature Forces in Membrane Lipid-Protein Interactions

    PubMed Central

    Brown, Michael F.

    2012-01-01

    Membrane biochemists are becoming increasingly aware of the role of lipid-protein interactions in diverse cellular functions. This review describes how conformational changes of membrane proteins—involving folding, stability, and membrane shape transitions—potentially involve elastic remodeling of the lipid bilayer. Evidence suggests that membrane lipids affect proteins through interactions of a relatively long-range nature, extending beyond a single annulus of next-neighbor boundary lipids. It is assumed the distance scale of the forces is large compared to the molecular range of action. Application of the theory of elasticity to flexible soft surfaces derives from classical physics, and explains the polymorphism of both detergents and membrane phospholipids. A flexible surface model (FSM) describes the balance of curvature and hydrophobic forces in lipid-protein interactions. Chemically nonspecific properties of the lipid bilayer modulate the conformational energetics of membrane proteins. The new biomembrane model challenges the standard model (the fluid mosaic model) found in biochemistry texts. The idea of a curvature force field based on data first introduced for rhodopsin gives a bridge between theory and experiment. Influences of bilayer thickness, nonlamellar-forming lipids, detergents, and osmotic stress are all explained by the FSM. An increased awareness of curvature forces suggests that research will accelerate as structural biology becomes more closely entwined with the physical chemistry of lipids in explaining membrane structure and function. PMID:23163284

  3. Membrane Lipid Oscillation: An Emerging System of Molecular Dynamics in the Plant Membrane.

    PubMed

    Nakamura, Yuki

    2018-03-01

    Biological rhythm represents a major biological process of living organisms. However, rhythmic oscillation of membrane lipid content is poorly described in plants. The development of lipidomic technology has led to the illustration of precise molecular profiles of membrane lipids under various growth conditions. Compared with conventional lipid signaling, which produces unpredictable lipid changes in response to ever-changing environmental conditions, lipid oscillation generates a fairly predictable lipid profile, adding a new layer of biological function to the membrane system and possible cross-talk with the other chronobiological processes. This mini review covers recent studies elucidating membrane lipid oscillation in plants.

  4. Continuity of Monolayer-Bilayer Junctions for Localization of Lipid Raft Microdomains in Model Membranes

    PubMed Central

    Ryu, Yong-Sang; Wittenberg, Nathan J.; Suh, Jeng-Hun; Lee, Sang-Wook; Sohn, Youngjoo; Oh, Sang-Hyun; Parikh, Atul N.; Lee, Sin-Doo

    2016-01-01

    We show that the selective localization of cholesterol-rich domains and associated ganglioside receptors prefer to occur in the monolayer across continuous monolayer-bilayer junctions (MBJs) in supported lipid membranes. For the MBJs, glass substrates were patterned with poly(dimethylsiloxane) (PDMS) oligomers by thermally-assisted contact printing, leaving behind 3 nm-thick PDMS patterns. The hydrophobicity of the transferred PDMS patterns was precisely tuned by the stamping temperature. Lipid monolayers were formed on the PDMS patterned surface while lipid bilayers were on the bare glass surface. Due to the continuity of the lipid membranes over the MBJs, essentially free diffusion of lipids was allowed between the monolayer on the PDMS surface and the upper leaflet of the bilayer on the glass substrate. The preferential localization of sphingomyelin, ganglioside GM1 and cholesterol in the monolayer region enabled to develop raft microdomains through coarsening of nanorafts. Our methodology provides a simple and effective scheme of non-disruptive manipulation of the chemical landscape associated with lipid phase separations, which leads to more sophisticated applications in biosensors and as cell culture substrates. PMID:27230411

  5. Effects of Vanadium-Containing Compounds on Membrane Lipids and on Microdomains Used in Receptor-Mediated Signaling

    PubMed Central

    Roess, Deborah A.; Smith, Steven M. L.; Winter, Peter; Zhou, Jun; Dou, Ping; Baruah, Bharat; Trujillo, Alejandro M.; Levinger, Nancy E.; Yang, Xioda; Barisas, B. George; Crans, Debbie C.

    2011-01-01

    There is increasing evidence for the involvement of plasma membrane microdomains in insulin receptor function. Moreover, disruption of these structures, which are typically enriched in sphingomyelin and cholesterol, results in insulin resistance. Treatment strategies for insulin resistance include the use of vanadium compounds which have been shown in animal models to enhance insulin responsiveness. One possible mechanism for insulin-enhancing effects might involve direct effects of vanadium compounds on membrane lipid organization. These changes in lipid organization promote the partitioning of insulin receptors and other receptors into membrane microdomains where receptors are optimally functional. To explore this possibility, we have used several strategies involving vanadium complexes such as [VO2dipic]− (pyridin-2,6-dicarboxylatodioxovanadium(V)), decavanadate (V10O286−, V10), BMOV (bis(maltolato)oxovanadium(IV)) and [VO(saltris)]2 (2-salicylideniminato-2-(hydroxymethyl)-1,3-dihydroxypropane-oxovanadium(V)). Our strategies include an evaluation of interactions between vanadium-containing compounds and model lipid systems, an evaluation of the effects of vanadium compounds on lipid fluidity in erythrocyte membranes, and studies of the effects of vanadium-containing compounds on signaling events initiated by receptors known to use membrane microdomains as signaling platforms. PMID:18729092

  6. Effects of rutin on the physicochemical properties of skin fibroblasts membrane disruption following UV radiation.

    PubMed

    Dobrzyńska, Izabela; Gęgotek, Agnieszka; Gajko, Ewelina; Skrzydlewska, Elżbieta; Figaszewski, Zbigniew A

    2018-02-25

    Human skin provides the body's first line of defense against physical and environmental assaults. This study sought to determine how rutin affects the membrane electrical properties, sialic acid content, and lipid peroxidation levels of fibroblast membranes after disruption by ultraviolet (UV) radiation. Changes in cell function may affect the basal electrical surface properties of cell membranes, and changes can be detected by electrokinetic measurements. The charge density of the fibroblast membrane surface was measured as a function of pH. A four-component equilibrium model was used to describe the interaction between ions in solution and ions on the membrane surface. Agreement was found between experimental and theoretical charge variation curves of fibroblast cells between pH 2.5 and 8. Sialic acid content was determined by Svennerholm's resorcinol method, and lipid peroxidation was estimated by measuring the malondialdehyde level. Compared to untreated cells, ultraviolet A (UVA)- or ultraviolet B (UVB)-treated skin cell membranes exhibited higher concentrations of acidic functional groups and higher average association constants with hydroxyl ions, but lower average association constants with hydrogen ions. Moreover, our results showed that UVA and UVB radiation is associated with increased levels of sialic acid and lipid peroxidation products in fibroblasts. Rutin protected cells from some deleterious UV-associated membrane changes, including changes in electrical properties, oxidative state, and biological functions. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. HAMLET interacts with lipid membranes and perturbs their structure and integrity.

    PubMed

    Mossberg, Ann-Kristin; Puchades, Maja; Halskau, Øyvind; Baumann, Anne; Lanekoff, Ingela; Chao, Yinxia; Martinez, Aurora; Svanborg, Catharina; Karlsson, Roger

    2010-02-23

    Cell membrane interactions rely on lipid bilayer constituents and molecules inserted within the membrane, including specific receptors. HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded alpha-lactalbumin (HLA) and oleic acid that is internalized by tumor cells, suggesting that interactions with the phospholipid bilayer and/or specific receptors may be essential for the tumoricidal effect. This study examined whether HAMLET interacts with artificial membranes and alters membrane structure. We show by surface plasmon resonance that HAMLET binds with high affinity to surface adherent, unilamellar vesicles of lipids with varying acyl chain composition and net charge. Fluorescence imaging revealed that HAMLET accumulates in membranes of vesicles and perturbs their structure, resulting in increased membrane fluidity. Furthermore, HAMLET disrupted membrane integrity at neutral pH and physiological conditions, as shown by fluorophore leakage experiments. These effects did not occur with either native HLA or a constitutively unfolded Cys-Ala HLA mutant (rHLA(all-Ala)). HAMLET also bound to plasma membrane vesicles formed from intact tumor cells, with accumulation in certain membrane areas, but the complex was not internalized by these vesicles or by the synthetic membrane vesicles. The results illustrate the difference in membrane affinity between the fatty acid bound and fatty acid free forms of partially unfolded HLA and suggest that HAMLET engages membranes by a mechanism requiring both the protein and the fatty acid. Furthermore, HAMLET binding alters the morphology of the membrane and compromises its integrity, suggesting that membrane perturbation could be an initial step in inducing cell death.

  8. Mechanical cell disruption of Parachlorella kessleri microalgae: Impact on lipid fraction composition.

    PubMed

    Clavijo Rivera, E; Montalescot, V; Viau, M; Drouin, D; Bourseau, P; Frappart, M; Monteux, C; Couallier, E

    2018-05-01

    Samples of nitrogen-starved Parachlorella kessleri containing intact cells (IC), cells ground by bead milling (BM), and cells subjected to high-pressure cell disruption (HPD), together with their supernatants after centrifugation, were compared for granulometry and lipid profiles. The effects of disruption on the lipid profile and organisation were evaluated. The quantity of lipids available for extraction increased with disruption, and up to 81% could be recovered in supernatants after centrifugation, but a marked reorganization occurred. The proportion of amphiphilic free fatty acids and lysophosphatidylcholine increased during disruption due to their release or owing to lipid degradation by enzymes or physical conditions. This effect was more marked in HPD than in BM. Lipids contained in the aqueous phase, after disruption and centrifugation, were enriched in unsaturated fatty acids, BM leading to larger droplets than HPD. The larger liquid lipid droplet would be easier to recover in the following downstream processing. Copyright © 2018 Elsevier Ltd. All rights reserved.

  9. How Lipid Membranes Affect Pore Forming Toxin Activity.

    PubMed

    Rojko, Nejc; Anderluh, Gregor

    2015-12-15

    Pore forming toxins (PFTs) evolved to permeate the plasma membrane of target cells. This is achieved in a multistep mechanism that usually involves binding of soluble protein monomer to the lipid membrane, oligomerization at the plane of the membrane, and insertion of part of the polypeptide chain across the lipid membrane to form a conductive channel. Introduced pores allow uncontrolled transport of solutes across the membrane, inflicting damage to the target cell. PFTs are usually studied from the perspective of structure-function relationships, often neglecting the important role of the bulk membrane properties on the PFT mechanism of action. In this Account, we discuss how membrane lateral heterogeneity, thickness, and fluidity influence the pore forming process of PFTs. In general, lipid molecules are more accessible for binding in fluid membranes due to steric reasons. When PFT specifically binds ordered domains, it usually recognizes a specific lipid distribution pattern, like sphingomyelin (SM) clusters or SM/cholesterol complexes, and not individual lipid species. Lipid domains were also suggested to act as an additional concentration platform facilitating PFT oligomerization, but this is yet to be shown. The last stage in PFT action is the insertion of the transmembrane segment across the membranes to build the transmembrane pore walls. Conformational changes are a spontaneous process, and sufficient free energy has to be available for efficient membrane penetration. Therefore, fluid bilayers are permeabilized more readily in comparison to highly ordered and thicker liquid ordered lipid phase (Lo). Energetically more costly insertion into the Lo phase can be driven by the hydrophobic mismatch between the thinner liquid disordered phase (Ld) and large protein complexes, which are unable to tilt like single transmembrane segments. In the case of proteolipid pores, membrane properties can directly modulate pore size, stability, and even selectivity. Finally

  10. Liposomal membrane disruption by means of miniaturized dielectric-barrier discharge in air: liposome characterization

    NASA Astrophysics Data System (ADS)

    Svarnas, P.; Asimakoulas, L.; Katsafadou, M.; Pachis, K.; Kostazos, N.; Antimisiaris, S. G.

    2017-08-01

    The increasing interest of the plasma community in the application of atmospheric-pressure cold plasmas to bio-specimen treatment has led to the creation of the emerging field of plasma biomedicine. Accordingly, plasma setups based on dielectric-barrier discharges have already been widely tested for the inactivation of various cells. Most of these systems refer to the plasma jet concept where noble gases penetrate atmospheric air and are subjected to the influence of high electric fields, thus forming guided streamers. Following the original works of our group where liposomal membranes were proposed as models for studying the interaction between plasma jets and cells, we present herein a study on liposomal membrane disruption by means of miniaturized dielectric-barrier discharge running in atmospheric air. Liposomal membranes of various lipid compositions, lamellarities, and sizes are treated at different times. It is shown that the dielectric-barrier discharge of low mean power leads to efficient liposomal membrane disruption. The latter is achieved in a controllable manner and depends on liposome properties. Additionally, it is clearly demonstrated that liposomal membrane disruption takes place even after plasma extinction, i.e. during post-treatment, resembling thus an ‘apoptosis’ effect, which is well known today mainly for cell membranes. Thus, the adoption of the present concept would be beneficial for tailoring studies on plasma-treated cell-mimics. Finally, the liposome treatment is discussed with respect to possible physicochemical mechanisms and potential discharge modification due to the various compositions of the liquid electrode.

  11. Chemically Stable Lipids for Membrane Protein Crystallization

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ishchenko, Andrii; Peng, Lingling; Zinovev, Egor

    2017-05-01

    The lipidic cubic phase (LCP) has been widely recognized as a promising membrane-mimicking matrix for biophysical studies of membrane proteins and their crystallization in a lipidic environment. Application of this material to a wide variety of membrane proteins, however, is hindered due to a limited number of available host lipids, mostly monoacylglycerols (MAGs). Here, we designed, synthesized, and characterized a series of chemically stable lipids resistant to hydrolysis, with properties complementary to the widely used MAGs. In order to assess their potential to serve as host lipids for crystallization, we characterized the phase properties and lattice parameters of mesophases mademore » of two most promising lipids at a variety of different conditions by polarized light microscopy and small-angle X-ray scattering. Both lipids showed remarkable chemical stability and an extended LCP region in the phase diagram covering a wide range of temperatures down to 4 °C. One of these lipids has been used for crystallization and structure determination of a prototypical membrane protein bacteriorhodopsin at 4 and 20 °C.« less

  12. An enhancer peptide for membrane-disrupting antimicrobial peptides

    PubMed Central

    2010-01-01

    Background NP4P is a synthetic peptide derived from a natural, non-antimicrobial peptide fragment (pro-region of nematode cecropin P4) by substitution of all acidic amino acid residues with amides (i.e., Glu → Gln, and Asp → Asn). Results In the presence of NP4P, some membrane-disrupting antimicrobial peptides (ASABF-α, polymyxin B, and nisin) killed microbes at lower concentration (e.g., 10 times lower minimum bactericidal concentration for ASABF-α against Staphylococcus aureus), whereas NP4P itself was not bactericidal and did not interfere with bacterial growth at ≤ 300 μg/mL. In contrast, the activities of antimicrobial agents with a distinct mode of action (indolicidin, ampicillin, kanamycin, and enrofloxacin) were unaffected. Although the membrane-disrupting activity of NP4P was slight or undetectable, ASABF-α permeabilized S. aureus membranes with enhanced efficacy in the presence of NP4P. Conclusions NP4P selectively enhanced the bactericidal activities of membrane-disrupting antimicrobial peptides by increasing the efficacy of membrane disruption against the cytoplasmic membrane. PMID:20152058

  13. Early and late HIV-1 membrane fusion events are impaired by sphinganine lipidated peptides that target the fusion site.

    PubMed

    Klug, Yoel A; Ashkenazi, Avraham; Viard, Mathias; Porat, Ziv; Blumenthal, Robert; Shai, Yechiel

    2014-07-15

    Lipid-conjugated peptides have advanced the understanding of membrane protein functions and the roles of lipids in the membrane milieu. These lipopeptides modulate various biological systems such as viral fusion. A single function has been suggested for the lipid, binding to the membrane and thus elevating the local concentration of the peptide at the target site. In the present paper, we challenged this argument by exploring in-depth the antiviral mechanism of lipopeptides, which comprise sphinganine, the lipid backbone of DHSM (dihydrosphingomyelin), and an HIV-1 envelope-derived peptide. Surprisingly, we discovered a partnership between the lipid and the peptide that impaired early membrane fusion events by reducing CD4 receptor lateral diffusion and HIV-1 fusion peptide-mediated lipid mixing. Moreover, only the joint function of sphinganine and its conjugate peptide disrupted HIV-1 fusion protein assembly and folding at the later fusion steps. Via imaging techniques we revealed for the first time the direct localization of these lipopeptides to the virus-cell and cell-cell contact sites. Overall, the findings of the present study may suggest lipid-protein interactions in various biological systems and may help uncover a role for elevated DHSM in HIV-1 and its target cell membranes.

  14. The Role of Nanoparticle Surface Functionality in the Disruption of Model Cell Membranes

    PubMed Central

    Moghadam, Babak Y.; Hou, Wen-Che; Corredor, Charlie; Westerhoff, Paul; Posner, Jonathan D.

    2012-01-01

    Lipid bilayers are biomembranes common to cellular life and constitute a continuous barrier between cells and their environment. Understanding the interaction of engineered nanomaterials (ENMs) with lipid bilayers is an important step toward predicting subsequent biological effects. In this study, we assess the effect of varying the surface functionality and concentration of 10 nm-diameter gold (Au) and titanium dioxide (TiO2) ENMs on the disruption of negatively charged lipid bilayer vesicles (liposomes) using a dye leakage assay. Our findings show that Au ENMs having both positive and negative surface charge induce leakage that reaches a steady state after several hours. Positively charged particles with identical surface functionality and different core composition show similar leakage effects and result in faster and greater leakage than negatively charged particles, which suggests that surface functionality, not particle core composition, is a critical factor in determining the interaction between ENMs and lipid bilayers. The results suggest that particles permanently adsorb to bilayers and that only one positively charged particle is required to disrupt a liposome and trigger leakage of its entire contents in contrast to mellitin molecules, the most widely studied membrane lytic peptide, which requires hundred of molecules to generate leakage. PMID:22921268

  15. Influence of membrane phospholipid composition and structural organization on spontaneous lipid transfer between membranes.

    PubMed

    Pankov, R; Markovska, T; Antonov, P; Ivanova, L; Momchilova, A

    2006-09-01

    Investigations were carried out on the influence of phospholipid composition of model membranes on the processes of spontaneous lipid transfer between membranes. Acceptor vesicles were prepared from phospholipids extracted from plasma membranes of control and ras-transformed fibroblasts. Acceptor model membranes with manipulated levels of phosphatidylethanolamine (PE), sphingomyelin and phosphatidic acid were also used in the studies. Donor vesicles were prepared of phosphatidylcholine (PC) and contained two fluorescent lipid analogues, NBD-PC and N-Rh-PE, at a self-quenching concentration. Lipid transfer rate was assessed by measuring the increase of fluorescence in acceptor membranes due to transfer of fluorescent lipid analogues from quenched donor to unquenched acceptor vesicles. The results showed that spontaneous NBD-PC transfer increased upon fluidization of acceptor vesicles. In addition, elevation of PE concentration in model membranes was also accompanied by an increase of lipid transfer to all series of acceptor vesicles. The results are discussed with respect to the role of lipid composition and structural order of cellular plasma membranes in the processes of spontaneous lipid exchange between membrane bilayers.

  16. Tethered bilayer lipid membranes (tBLMs): interest and applications for biological membrane investigations.

    PubMed

    Rebaud, Samuel; Maniti, Ofelia; Girard-Egrot, Agnès P

    2014-12-01

    Biological membranes play a central role in the biology of the cell. They are not only the hydrophobic barrier allowing separation between two water soluble compartments but also a supra-molecular entity that has vital structural functions. Notably, they are involved in many exchange processes between the outside and inside cellular spaces. Accounting for the complexity of cell membranes, reliable models are needed to acquire current knowledge of the molecular processes occurring in membranes. To simplify the investigation of lipid/protein interactions, the use of biomimetic membranes is an approach that allows manipulation of the lipid composition of specific domains and/or the protein composition, and the evaluation of the reciprocal effects. Since the middle of the 80's, lipid bilayer membranes have been constantly developed as models of biological membranes with the ultimate goal to reincorporate membrane proteins for their functional investigation. In this review, after a brief description of the planar lipid bilayers as biomimetic membrane models, we will focus on the construction of the tethered Bilayer Lipid Membranes, the most promising model for efficient membrane protein reconstitution and investigation of molecular processes occurring in cell membranes. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  17. Continuity of monolayer-bilayer junctions for localization of lipid raft microdomains in model membranes

    DOE PAGES

    Ryu, Yong -Sang; Wittenberg, Nathan J.; Suh, Jeng -Hun; ...

    2016-05-27

    We show that the selective localization of cholesterol-rich domains and associated ganglioside receptors prefer to occur in the monolayer across continuous monolayer-bilayer junctions (MBJs) in supported lipid membranes. For the MBJs, glass substrates were patterned with poly(dimethylsiloxane) (PDMS) oligomers by thermally-assisted contact printing, leaving behind 3 nm-thick PDMS patterns. The hydrophobicity of the transferred PDMS patterns was precisely tuned by the stamping temperature. Lipid monolayers were formed on the PDMS patterned surface while lipid bilayers were on the bare glass surface. Due to the continuity of the lipid membranes over the MBJs, essentially free diffusion of lipids was allowed betweenmore » the monolayer on the PDMS surface and the upper leaflet of the bilayer on the glass substrate. The preferential localization of sphingomyelin, ganglioside GM1 and cholesterol in the monolayer region enabled to develop raft microdomains through coarsening of nanorafts. Furthermore, our methodology provides a simple and effective scheme of non-disruptive manipulation of the chemical landscape associated with lipid phase separations, which leads to more sophisticated applications in biosensors and as cell culture substrates.« less

  18. Interaction of Spin-Labeled Lipid Membranes with Transition Metal Ions

    PubMed Central

    2015-01-01

    The large values of spin relaxation enhancement (RE) for PC spin-labels in the phospholipid membrane induced by paramagnetic metal salts dissolved in the aqueous phase can be explained by Heisenberg spin exchange due to conformational fluctuations of the nitroxide group as a result of membrane fluidity, flexibility of lipid chains, and, possibly, amphiphilic nature of the nitroxide label. Whether the magnetic interaction occurs predominantly via Heisenberg spin exchange (Ni) or by the dipole–dipole (Gd) mechanism, it is essential for the paramagnetic ion to get into close proximity to the nitroxide moiety for efficient RE. For different salts of Ni the RE in phosphatidylcholine membranes follows the anionic Hofmeister series and reflects anion adsorption followed by anion-driven attraction of paramagnetic cations on the choline groups. This adsorption is higher for chaotropic ions, e.g., perchlorate. (A chaotropic agent is a molecule in water solution that can disrupt the hydrogen bonding network between water molecules.) However, there is no anionic dependence of RE for model membranes made from negatively charged lipids devoid of choline groups. We used Ni-induced RE to study the thermodynamics and electrostatics of ion/membrane interactions. We also studied the effect of membrane composition and the phase state on the RE values. In membranes with cholesterol a significant difference is observed between PC labels with nitroxide tethers long enough vs not long enough to reach deep into the membrane hydrophobic core behind the area of fused cholesterol rings. This study indicates one must be cautious in interpreting data obtained by PC labels in fluid membranes in terms of probing membrane properties at different immersion depths when it can be affected by paramagnetic species at the membrane surface. PMID:26490692

  19. Imaging of blood plasma coagulation at supported lipid membranes.

    PubMed

    Faxälv, Lars; Hume, Jasmin; Kasemo, Bengt; Svedhem, Sofia

    2011-12-15

    The blood coagulation system relies on lipid membrane constituents to act as regulators of the coagulation process upon vascular trauma, and in particular the 2D configuration of the lipid membranes is known to efficiently catalyze enzymatic activity of blood coagulation factors. This work demonstrates a new application of a recently developed methodology to study blood coagulation at lipid membrane interfaces with the use of imaging technology. Lipid membranes with varied net charges were formed on silica supports by systematically using different combinations of lipids where neutral phosphocholine (PC) lipids were mixed with phospholipids having either positively charged ethylphosphocholine (EPC), or negatively charged phosphatidylserine (PS) headgroups. Coagulation imaging demonstrated that negatively charged SiO(2) and membrane surfaces exposing PS (obtained from liposomes containing 30% of PS) had coagulation times which were significantly shorter than those for plain PC membranes and EPC exposing membrane surfaces (obtained from liposomes containing 30% of EPC). Coagulation times decreased non-linearly with increasing negative surface charge for lipid membranes. A threshold value for shorter coagulation times was observed below a PS content of ∼6%. We conclude that the lipid membranes on solid support studied with the imaging setup as presented in this study offers a flexible and non-expensive solution for coagulation studies at biological membranes. It will be interesting to extend the present study towards examining coagulation on more complex lipid-based model systems. Copyright © 2011 Elsevier Inc. All rights reserved.

  20. DHA-fluorescent probe is sensitive to membrane order and reveals molecular adaptation of DHA in ordered lipid microdomains☆

    PubMed Central

    Teague, Heather; Ross, Ron; Harris, Mitchel; Mitchell, Drake C.; Shaikh, Saame Raza

    2012-01-01

    Docosahexaenoic acid (DHA) disrupts the size and order of plasma membrane lipid microdomains in vitro and in vivo. However, it is unknown how the highly disordered structure of DHA mechanistically adapts to increase the order of tightly packed lipid microdomains. Therefore, we studied a novel DHA-Bodipy fluorescent probe to address this issue. We first determined if the DHA-Bodipy probe localized to the plasma membrane of primary B and immortal EL4 cells. Image analysis revealed that DHA-Bodipy localized into the plasma membrane of primary B cells more efficiently than EL4 cells. We then determined if the probe detected changes in plasma membrane order. Quantitative analysis of time-lapse movies established that DHA-Bodipy was sensitive to membrane molecular order. This allowed us to investigate how DHA-Bodipy physically adapted to ordered lipid microdomains. To accomplish this, we employed steady-state and time-resolved fluorescence anisotropy measurements in lipid vesicles of varying composition. Similar to cell culture studies, the probe was highly sensitive to membrane order in lipid vesicles. Moreover, these experiments revealed, relative to controls, that upon incorporation into highly ordered microdomains, DHA-Bodipy underwent an increase in its fluorescence lifetime and molecular order. In addition, the probe displayed a significant reduction in its rotational diffusion compared to controls. Altogether, DHA-Bodipy was highly sensitive to membrane order and revealed for the first time that DHA, despite its flexibility, could become ordered with less rotational motion inside ordered lipid microdomains. Mechanistically, this explains how DHA acyl chains can increase order upon formation of lipid microdomains in vivo. PMID:22841541

  1. Regulation of lipid droplet and membrane biogenesis by the acidic tail of the phosphatidate phosphatase Pah1p

    PubMed Central

    Karanasios, Eleftherios; Barbosa, Antonio Daniel; Sembongi, Hiroshi; Mari, Muriel; Han, Gil-Soo; Reggiori, Fulvio; Carman, George M.; Siniossoglou, Symeon

    2013-01-01

    Lipins are evolutionarily conserved phosphatidate phosphatases that perform key functions in phospholipid, triglyceride, and membrane biogenesis. Translocation of lipins on membranes requires their dephosphorylation by the Nem1p-Spo7p transmembrane phosphatase complex through a poorly understood mechanism. Here we identify the carboxy-terminal acidic tail of the yeast lipin Pah1p as an important regulator of this step. Deletion or mutations of the tail disrupt binding of Pah1p to the Nem1p-Spo7p complex and Pah1p membrane translocation. Overexpression of Nem1p-Spo7p drives the recruitment of Pah1p in the vicinity of lipid droplets in an acidic tail–dependent manner and induces lipid droplet biogenesis. Genetic analysis shows that the acidic tail is essential for the Nem1p-Spo7p–dependent activation of Pah1p but not for the function of Pah1p itself once it is dephosphorylated. Loss of the tail disrupts nuclear structure, INO1 gene expression, and triglyceride synthesis. Similar acidic sequences are present in the carboxy-terminal ends of all yeast lipin orthologues. We propose that acidic tail–dependent binding and dephosphorylation of Pah1p by the Nem1p-Spo7p complex is an important determinant of its function in lipid and membrane biogenesis. PMID:23657815

  2. A Central Role for Triacylglycerol in Membrane Lipid Breakdown, Fatty Acid β-Oxidation, and Plant Survival under Extended Darkness.

    PubMed

    Fan, Jilian; Yu, Linhui; Xu, Changcheng

    2017-07-01

    Neutral lipid metabolism is a key aspect of intracellular homeostasis and energy balance and plays a vital role in cell survival under adverse conditions, including nutrient deprivation in yeast and mammals, but the role of triacylglycerol (TAG) metabolism in plant stress response remains largely unknown. By thoroughly characterizing mutants defective in SUGAR-DEPENDENT1 (SDP1) triacylglycerol lipase or PEROXISOMAL ABC TRANSPORTER 1 (PXA1), here we show that TAG is a key intermediate in the mobilization of fatty acids from membrane lipids for peroxisomal β-oxidation under prolonged dark treatment. Disruption of SDP1 increased TAG accumulation in cytosolic lipid droplets and markedly enhanced plant tolerance to extended darkness. We demonstrate that blocking TAG hydrolysis enhances plant tolerance to dark treatment via two distinct mechanisms. In pxa1 mutants, in which free fatty acids accumulated rapidly under extended darkness, SDP1 disruption resulted in a marked decrease in levels of cytotoxic lipid intermediates such as free fatty acids and phosphatidic acid, suggesting a buffer function of TAG accumulation against lipotoxicity under fatty acid overload. In the wild type, in which free fatty acids remained low and unchanged under dark treatment, disruption of SDP1 caused a decrease in reactive oxygen species production and hence the level of lipid peroxidation, indicating a role of TAG in protection against oxidative damage. Overall, our findings reveal a crucial role for TAG metabolism in membrane lipid breakdown, fatty acid turnover, and plant survival under extended darkness. © 2017 American Society of Plant Biologists. All Rights Reserved.

  3. Cationic peptide exposure enhances pulsed-electric-field-mediated membrane disruption.

    PubMed

    Kennedy, Stephen M; Aiken, Erik J; Beres, Kaytlyn A; Hahn, Adam R; Kamin, Samantha J; Hagness, Susan C; Booske, John H; Murphy, William L

    2014-01-01

    The use of pulsed electric fields (PEFs) to irreversibly electroporate cells is a promising approach for destroying undesirable cells. This approach may gain enhanced applicability if the intensity of the PEF required to electrically disrupt cell membranes can be reduced via exposure to a molecular deliverable. This will be particularly impactful if that reduced PEF minimally influences cells that are not exposed to the deliverable. We hypothesized that the introduction of charged molecules to the cell surfaces would create regions of enhanced transmembrane electric potential in the vicinity of each charged molecule, thereby lowering the PEF intensity required to disrupt the plasma membranes. This study will therefore examine if exposure to cationic peptides can enhance a PEF's ability to disrupt plasma membranes. We exposed leukemia cells to 40 μs PEFs in media containing varying concentrations of a cationic peptide, polyarginine. We observed the internalization of a membrane integrity indicator, propidium iodide (PI), in real time. Based on an individual cell's PI fluorescence versus time signature, we were able to determine the relative degree of membrane disruption. When using 1-2 kV/cm, exposure to >50 μg/ml of polyarginine resulted in immediate and high levels of PI uptake, indicating severe membrane disruption, whereas in the absence of peptide, cells predominantly exhibited signatures indicative of no membrane disruption. Additionally, PI entered cells through the anode-facing membrane when exposed to cationic peptide, which was theoretically expected. Exposure to cationic peptides reduced the PEF intensity required to induce rapid and irreversible membrane disruption. Critically, peptide exposure reduced the PEF intensities required to elicit irreversible membrane disruption at normally sub-electroporation intensities. We believe that these cationic peptides, when coupled with current advancements in cell targeting techniques will be useful tools in

  4. Artificial Lipid Membranes: Past, Present, and Future

    PubMed Central

    Siontorou, Christina G.; Nikoleli, Georgia-Paraskevi; Nikolelis, Dimitrios P.

    2017-01-01

    The multifaceted role of biological membranes prompted early the development of artificial lipid-based models with a primary view of reconstituting the natural functions in vitro so as to study and exploit chemoreception for sensor engineering. Over the years, a fair amount of knowledge on the artificial lipid membranes, as both, suspended or supported lipid films and liposomes, has been disseminated and has helped to diversify and expand initial scopes. Artificial lipid membranes can be constructed by several methods, stabilized by various means, functionalized in a variety of ways, experimented upon intensively, and broadly utilized in sensor development, drug testing, drug discovery or as molecular tools and research probes for elucidating the mechanics and the mechanisms of biological membranes. This paper reviews the state-of-the-art, discusses the diversity of applications, and presents future perspectives. The newly-introduced field of artificial cells further broadens the applicability of artificial membranes in studying the evolution of life. PMID:28933723

  5. Permeability and electrical properties of planar lipid membranes from thylakoid lipids.

    PubMed Central

    Fuks, B; Homblé, F

    1994-01-01

    Electrical measurements were carried out on planar lipid membranes from thylakoid lipids. The specific capacitance of membranes formed from decane-containing monogalactosyldiacylglycerol (MGDG), which accounts for 57% of the total lipid content of thylakoids, showed that it adopted a bilayer structure. Solvent-free bilayers of MGDG were not formed, with very rare exceptions, indicating that decane is required to stabilize the planar conformation. However, this cone-shaped lipid produces bilayer structures in combination with other cylindrical thylakoid lipids even in the absence of organic solvent. We compared the properties of solvent-free and decane-containing bilayers from MGDG, soybean lecithin, and the quaternary mixture of lipids similar to that found in vivo. The conductance of decane-MGDG was 26 times higher than that of decane-lecithin. The flux through the decane-lecithin bilayer was found to be slightly dependent on pH, whereas the decane-MGDG membrane was not. The specific conductance of bilayers formed from the quaternary mixture of lipids was 5 to 10 times larger than lecithin (with alkane or not). Further experiments with bilayers made in the presence of a KCl gradient showed that decane-MGDG, decane-MGDG/DGDG/SQDG/PG, and solvent-free MGDG/DGDG/SQDG/PG were cation-selective. The permeability coefficient for potassium ranged from 4.9 to 8.3 x 10(-11) cm s-1. The permeability coefficient for protons in galactolipids, however, was determined to be about six orders of magnitude higher than the value for potassium ions. The HCl permeation mechanism through the lipid membranes was determined from diffusion potentials measured in HCl gradients. Our results suggest that HCl was not transported as neutral molecules. The data is discussed with regard to the function of galactolipids in the ion transport through thylakoid membranes. PMID:8061192

  6. Plant lipid environment and membrane enzymes: the case of the plasma membrane H+-ATPase.

    PubMed

    Morales-Cedillo, Francisco; González-Solís, Ariadna; Gutiérrez-Angoa, Lizbeth; Cano-Ramírez, Dora Luz; Gavilanes-Ruiz, Marina

    2015-04-01

    Several lipid classes constitute the universal matrix of the biological membranes. With their amphipathic nature, lipids not only build the continuous barrier that confers identity to every cell and organelle, but they are also active actors that modulate the activity of the proteins immersed in the lipid bilayer. The plasma membrane H(+)-ATPase, an enzyme from plant cells, is an excellent example of a transmembrane protein whose activity is influenced by the hydrophilic compartments at both sides of the membrane and by the hydrophobic domains of the lipid bilayer. As a result, an extensive documentation of the effect of numerous amphiphiles in the enzyme activity can be found. Detergents, membrane glycerolipids, and sterols can produce activation or inhibition of the enzyme activity. In some cases, these effects are associated with the lipids of the membrane bulk, but in others, a direct interaction of the lipid with the protein is involved. This review gives an account of reports related to the action of the membrane lipids on the H(+)-ATPase activity.

  7. Sensing voltage across lipid membranes

    PubMed Central

    Swartz, Kenton J.

    2009-01-01

    The detection of electrical potentials across lipid bilayers by specialized membrane proteins is required for many fundamental cellular processes such as the generation and propagation of nerve impulses. These membrane proteins possess modular voltage-sensing domains, a notable example being the S1-S4 domains of voltage-activated ion channels. Ground-breaking structural studies on these domains explain how voltage sensors are designed and reveal important interactions with the surrounding lipid membrane. Although further structures are needed to fully understand the conformational changes that occur during voltage sensing, the available data help to frame several key concepts that are fundamental to the mechanism of voltage sensing. PMID:19092925

  8. A Central Role for Triacylglycerol in Membrane Lipid Breakdown, Fatty Acid β-Oxidation, and Plant Survival under Extended Darkness1[OPEN

    PubMed Central

    2017-01-01

    Neutral lipid metabolism is a key aspect of intracellular homeostasis and energy balance and plays a vital role in cell survival under adverse conditions, including nutrient deprivation in yeast and mammals, but the role of triacylglycerol (TAG) metabolism in plant stress response remains largely unknown. By thoroughly characterizing mutants defective in SUGAR-DEPENDENT1 (SDP1) triacylglycerol lipase or PEROXISOMAL ABC TRANSPORTER 1 (PXA1), here we show that TAG is a key intermediate in the mobilization of fatty acids from membrane lipids for peroxisomal β-oxidation under prolonged dark treatment. Disruption of SDP1 increased TAG accumulation in cytosolic lipid droplets and markedly enhanced plant tolerance to extended darkness. We demonstrate that blocking TAG hydrolysis enhances plant tolerance to dark treatment via two distinct mechanisms. In pxa1 mutants, in which free fatty acids accumulated rapidly under extended darkness, SDP1 disruption resulted in a marked decrease in levels of cytotoxic lipid intermediates such as free fatty acids and phosphatidic acid, suggesting a buffer function of TAG accumulation against lipotoxicity under fatty acid overload. In the wild type, in which free fatty acids remained low and unchanged under dark treatment, disruption of SDP1 caused a decrease in reactive oxygen species production and hence the level of lipid peroxidation, indicating a role of TAG in protection against oxidative damage. Overall, our findings reveal a crucial role for TAG metabolism in membrane lipid breakdown, fatty acid turnover, and plant survival under extended darkness. PMID:28572457

  9. Fluorescence Imaging of Two-Photon Linear Dichroism: Cholesterol Depletion Disrupts Molecular Orientation in Cell Membranes

    PubMed Central

    Benninger, Richard K. P.; Önfelt, Björn; Neil, Mark A. A.; Davis, Daniel M.; French, Paul M. W.

    2005-01-01

    The plasma membrane of cells is an ordered environment, giving rise to anisotropic orientation and restricted motion of molecules and proteins residing in the membrane. At the same time as being an organized matrix of defined structure, the cell membrane is heterogeneous and dynamic. Here we present a method where we use fluorescence imaging of linear dichroism to measure the orientation of molecules relative to the cell membrane. By detecting linear dichroism as well as fluorescence anisotropy, the orientation parameters are separated from dynamic properties such as rotational diffusion and homo energy transfer (energy migration). The sensitivity of the technique is enhanced by using two-photon excitation for higher photo-selection compared to single photon excitation. We show here that we can accurately image lipid organization in whole cell membranes and in delicate structures such as membrane nanotubes connecting two cells. The speed of our wide-field imaging system makes it possible to image changes in orientation and anisotropy occurring on a subsecond timescale. This is demonstrated by time-lapse studies showing that cholesterol depletion rapidly disrupts the orientation of a fluorophore located within the hydrophobic region of the cell membrane but not of a surface bound probe. This is consistent with cholesterol having an important role in stabilizing and ordering the lipid tails within the plasma membrane. PMID:15520272

  10. Specific Adhesion of Lipid Membranes Can Simultaneously Produce Two Types of Lipid and Protein Heterogeneities

    NASA Astrophysics Data System (ADS)

    Shindell, Orrin; Micah, Natalie; Ritzer, Max; Gordon, Vernita

    2015-03-01

    Living cells adhere to one another and their environment. Adhesion is associated with re-organization of the lipid and protein components of the cell membrane. The resulting heterogeneities are functional structures involved in biological processes. We use artificial lipid membranes that contain a single type of binding protein. Before adhesion, the lipid, protein, and dye components in the membrane are well-mixed and constitute a single disordered-liquid phase (Ld) . After adhesion, two distinct types of heterogeneities coexist in the adhesion zone: a central domain of ordered lipid phase that excludes both binding proteins and membrane dye, and a peripheral domain of disordered lipid phase that is densely packed with adhesion proteins and enriched in membrane dye relative to the non-adhered portion of the vesicle. Thus, we show that adhesion that is mediated by only one type of protein can organize the lipid and protein components of the membranes into heterogeneities that resemble those found in biology, for example the immune synapse.

  11. Anionic lipids and the maintenance of membrane electrostatics in eukaryotes

    PubMed Central

    Platre, Matthieu Pierre

    2017-01-01

    ABSTRACT A wide range of signaling processes occurs at the cell surface through the reversible association of proteins from the cytosol to the plasma membrane. Some low abundant lipids are enriched at the membrane of specific compartments and thereby contribute to the identity of cell organelles by acting as biochemical landmarks. Lipids also influence membrane biophysical properties, which emerge as an important feature in specifying cellular territories. Such parameters are crucial for signal transduction and include lipid packing, membrane curvature and electrostatics. In particular, membrane electrostatics specifies the identity of the plasma membrane inner leaflet. Membrane surface charges are carried by anionic phospholipids, however the exact nature of the lipid(s) that powers the plasma membrane electrostatic field varies among eukaryotes and has been hotly debated during the last decade. Herein, we discuss the role of anionic lipids in setting up plasma membrane electrostatics and we compare similarities and differences that were found in different eukaryotic cells. PMID:28102755

  12. Anionic lipids and the maintenance of membrane electrostatics in eukaryotes.

    PubMed

    Platre, Matthieu Pierre; Jaillais, Yvon

    2017-02-01

    A wide range of signaling processes occurs at the cell surface through the reversible association of proteins from the cytosol to the plasma membrane. Some low abundant lipids are enriched at the membrane of specific compartments and thereby contribute to the identity of cell organelles by acting as biochemical landmarks. Lipids also influence membrane biophysical properties, which emerge as an important feature in specifying cellular territories. Such parameters are crucial for signal transduction and include lipid packing, membrane curvature and electrostatics. In particular, membrane electrostatics specifies the identity of the plasma membrane inner leaflet. Membrane surface charges are carried by anionic phospholipids, however the exact nature of the lipid(s) that powers the plasma membrane electrostatic field varies among eukaryotes and has been hotly debated during the last decade. Herein, we discuss the role of anionic lipids in setting up plasma membrane electrostatics and we compare similarities and differences that were found in different eukaryotic cells.

  13. Single-lipid tracking on nanoscale membrane buds: The effects of curvature on lipid diffusion and sorting.

    PubMed

    Woodward, Xinxin; Stimpson, Eric E; Kelly, Christopher V

    2018-05-29

    Nanoscale membrane curvature in cells is critical for endocytosis/exocytosis and membrane trafficking. However, the biophysical ramifications of nanoscale membrane curvature on the behavior of lipids remain poorly understood. Here, we created an experimental model system of membrane curvature at a physiologically-relevant scale and obtained nanoscopic information on single-lipid distributions and dynamics. Supported lipid bilayers were created over 50 and 70 nm radius nanoparticles to create membrane buds. Single-molecule localization microscopy was performed with diverse mixtures of fluorescent and non-fluorescent lipids. Variations in lipid acyl tales length, saturation, head-group, and fluorescent labeling strategy were tested while maintaining a single fluid lipid phase throughout the membrane. Monte Carlo simulations were used to fit our experimental results and quantify the effects of curvature on the lipid diffusion and sorting. Whereas varying the composition of the non-fluorescent lipids yielded minimal changes to the curvature effects, the labeling strategy of the fluorescent lipids yielded highly varying effects of curvature. Most conditions yield single-population Brownian diffusion throughout the membrane; however, curvature-induced lipid sorting, slowing, and aggregation were observed in some conditions. Head-group labeled lipids such as DPPE-Texas Red and POPE-Rhodamine diffused >2.4× slower on the curved vs. the planar membranes; tail-labeled lipids such as NBD-PPC, TopFluor-PPC, TopFluor-PIP2, DiIC 12 , and DiIC 18 displayed no significant changes in diffusion due to the membrane curvature. This article is part of a Special Issue entitled: Emergence of Complex Behavior in Biomembranes edited by Marjorie Longo. Copyright © 2018. Published by Elsevier B.V.

  14. Cationic Peptide Exposure Enhances Pulsed-Electric-Field-Mediated Membrane Disruption

    PubMed Central

    Kennedy, Stephen M.; Aiken, Erik J.; Beres, Kaytlyn A.; Hahn, Adam R.; Kamin, Samantha J.; Hagness, Susan C.; Booske, John H.; Murphy, William L.

    2014-01-01

    Background The use of pulsed electric fields (PEFs) to irreversibly electroporate cells is a promising approach for destroying undesirable cells. This approach may gain enhanced applicability if the intensity of the PEF required to electrically disrupt cell membranes can be reduced via exposure to a molecular deliverable. This will be particularly impactful if that reduced PEF minimally influences cells that are not exposed to the deliverable. We hypothesized that the introduction of charged molecules to the cell surfaces would create regions of enhanced transmembrane electric potential in the vicinity of each charged molecule, thereby lowering the PEF intensity required to disrupt the plasma membranes. This study will therefore examine if exposure to cationic peptides can enhance a PEF’s ability to disrupt plasma membranes. Methodology/Principal Findings We exposed leukemia cells to 40 μs PEFs in media containing varying concentrations of a cationic peptide, polyarginine. We observed the internalization of a membrane integrity indicator, propidium iodide (PI), in real time. Based on an individual cell’s PI fluorescence versus time signature, we were able to determine the relative degree of membrane disruption. When using 1–2 kV/cm, exposure to >50 μg/ml of polyarginine resulted in immediate and high levels of PI uptake, indicating severe membrane disruption, whereas in the absence of peptide, cells predominantly exhibited signatures indicative of no membrane disruption. Additionally, PI entered cells through the anode-facing membrane when exposed to cationic peptide, which was theoretically expected. Conclusions/Significance Exposure to cationic peptides reduced the PEF intensity required to induce rapid and irreversible membrane disruption. Critically, peptide exposure reduced the PEF intensities required to elicit irreversible membrane disruption at normally sub-electroporation intensities. We believe that these cationic peptides, when coupled with

  15. Lipid Clustering Correlates with Membrane Curvature as Revealed by Molecular Simulations of Complex Lipid Bilayers

    PubMed Central

    Koldsø, Heidi; Shorthouse, David; Hélie, Jean; Sansom, Mark S. P.

    2014-01-01

    Cell membranes are complex multicomponent systems, which are highly heterogeneous in the lipid distribution and composition. To date, most molecular simulations have focussed on relatively simple lipid compositions, helping to inform our understanding of in vitro experimental studies. Here we describe on simulations of complex asymmetric plasma membrane model, which contains seven different lipids species including the glycolipid GM3 in the outer leaflet and the anionic lipid, phosphatidylinositol 4,5-bisphophate (PIP2), in the inner leaflet. Plasma membrane models consisting of 1500 lipids and resembling the in vivo composition were constructed and simulations were run for 5 µs. In these simulations the most striking feature was the formation of nano-clusters of GM3 within the outer leaflet. In simulations of protein interactions within a plasma membrane model, GM3, PIP2, and cholesterol all formed favorable interactions with the model α-helical protein. A larger scale simulation of a model plasma membrane containing 6000 lipid molecules revealed correlations between curvature of the bilayer surface and clustering of lipid molecules. In particular, the concave (when viewed from the extracellular side) regions of the bilayer surface were locally enriched in GM3. In summary, these simulations explore the nanoscale dynamics of model bilayers which mimic the in vivo lipid composition of mammalian plasma membranes, revealing emergent nanoscale membrane organization which may be coupled both to fluctuations in local membrane geometry and to interactions with proteins. PMID:25340788

  16. Lipid clustering correlates with membrane curvature as revealed by molecular simulations of complex lipid bilayers.

    PubMed

    Koldsø, Heidi; Shorthouse, David; Hélie, Jean; Sansom, Mark S P

    2014-10-01

    Cell membranes are complex multicomponent systems, which are highly heterogeneous in the lipid distribution and composition. To date, most molecular simulations have focussed on relatively simple lipid compositions, helping to inform our understanding of in vitro experimental studies. Here we describe on simulations of complex asymmetric plasma membrane model, which contains seven different lipids species including the glycolipid GM3 in the outer leaflet and the anionic lipid, phosphatidylinositol 4,5-bisphophate (PIP2), in the inner leaflet. Plasma membrane models consisting of 1500 lipids and resembling the in vivo composition were constructed and simulations were run for 5 µs. In these simulations the most striking feature was the formation of nano-clusters of GM3 within the outer leaflet. In simulations of protein interactions within a plasma membrane model, GM3, PIP2, and cholesterol all formed favorable interactions with the model α-helical protein. A larger scale simulation of a model plasma membrane containing 6000 lipid molecules revealed correlations between curvature of the bilayer surface and clustering of lipid molecules. In particular, the concave (when viewed from the extracellular side) regions of the bilayer surface were locally enriched in GM3. In summary, these simulations explore the nanoscale dynamics of model bilayers which mimic the in vivo lipid composition of mammalian plasma membranes, revealing emergent nanoscale membrane organization which may be coupled both to fluctuations in local membrane geometry and to interactions with proteins.

  17. Revealing the Effects of Nanoscale Membrane Curvature on Lipid Mobility.

    PubMed

    Kabbani, Abir Maarouf; Woodward, Xinxin; Kelly, Christopher V

    2017-10-18

    Recent advances in nanoengineering and super-resolution microscopy have enabled new capabilities for creating and observing membrane curvature. However, the effects of curvature on single-lipid diffusion have yet to be revealed. The simulations presented here describe the capabilities of varying experimental methods for revealing the effects of nanoscale curvature on single-molecule mobility. Traditionally, lipid mobility is revealed through fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and single particle tracking (SPT). However, these techniques vary greatly in their ability to detect the effects of nanoscale curvature on lipid behavior. Traditionally, FRAP and FCS depend on diffraction-limited illumination and detection. A simulation of FRAP shows minimal effects on lipids diffusion due to a 50 nm radius membrane bud. Throughout the stages of the budding process, FRAP detected minimal changes in lipid recovery time due to the curvature versus flat membrane. Simulated FCS demonstrated small effects due to a 50 nm radius membrane bud that was more apparent with curvature-dependent lipid mobility changes. However, SPT achieves a sub-diffraction-limited resolution of membrane budding and lipid mobility through the identification of the single-lipid positions with ≤15 nm spatial and ≤20 ms temporal resolution. By mapping the single-lipid step lengths to locations on the membrane, the effects of membrane topography and curvature could be correlated to the effective membrane viscosity. Single-fluorophore localization techniques, such SPT, can detect membrane curvature and its effects on lipid behavior. These simulations and discussion provide a guideline for optimizing the experimental procedures in revealing the effects of curvature on lipid mobility and effective local membrane viscosity.

  18. Revealing the Effects of Nanoscale Membrane Curvature on Lipid Mobility

    PubMed Central

    Kabbani, Abir Maarouf; Woodward, Xinxin

    2017-01-01

    Recent advances in nanoengineering and super-resolution microscopy have enabled new capabilities for creating and observing membrane curvature. However, the effects of curvature on single-lipid diffusion have yet to be revealed. The simulations presented here describe the capabilities of varying experimental methods for revealing the effects of nanoscale curvature on single-molecule mobility. Traditionally, lipid mobility is revealed through fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and single particle tracking (SPT). However, these techniques vary greatly in their ability to detect the effects of nanoscale curvature on lipid behavior. Traditionally, FRAP and FCS depend on diffraction-limited illumination and detection. A simulation of FRAP shows minimal effects on lipids diffusion due to a 50 nm radius membrane bud. Throughout the stages of the budding process, FRAP detected minimal changes in lipid recovery time due to the curvature versus flat membrane. Simulated FCS demonstrated small effects due to a 50 nm radius membrane bud that was more apparent with curvature-dependent lipid mobility changes. However, SPT achieves a sub-diffraction-limited resolution of membrane budding and lipid mobility through the identification of the single-lipid positions with ≤15 nm spatial and ≤20 ms temporal resolution. By mapping the single-lipid step lengths to locations on the membrane, the effects of membrane topography and curvature could be correlated to the effective membrane viscosity. Single-fluorophore localization techniques, such SPT, can detect membrane curvature and its effects on lipid behavior. These simulations and discussion provide a guideline for optimizing the experimental procedures in revealing the effects of curvature on lipid mobility and effective local membrane viscosity. PMID:29057801

  19. Cell-sized asymmetric lipid vesicles facilitate the investigation of asymmetric membranes

    NASA Astrophysics Data System (ADS)

    Kamiya, Koki; Kawano, Ryuji; Osaki, Toshihisa; Akiyoshi, Kazunari; Takeuchi, Shoji

    2016-09-01

    Asymmetric lipid giant vesicles have been used to model the biochemical reactions in cell membranes. However, methods for producing asymmetric giant vesicles lead to the inclusion of an organic solvent layer that affects the mechanical and physical characteristics of the membrane. Here we describe the formation of asymmetric giant vesicles that include little organic solvent, and use them to investigate the dynamic responses of lipid molecules in the vesicle membrane. We formed the giant vesicles via the inhomogeneous break-up of a lipid microtube generated by applying a jet flow to an asymmetric planar lipid bilayer. The asymmetric giant vesicles showed a lipid flip-flop behaviour in the membrane, superficially similar to the lipid flip-flop activity observed in apoptotic cells. In vitro synthesis of membrane proteins into the asymmetric giant vesicles revealed that the lipid asymmetry in bilayer membranes improves the reconstitution ratio of membrane proteins. Our asymmetric giant vesicles will be useful in elucidating lipid-lipid and lipid-membrane protein interactions involved in the regulation of cellular functions.

  20. Simulations of simple linoleic acid-containing lipid membranes and models for the soybean plasma membranes.

    PubMed

    Zhuang, Xiaohong; Ou, Anna; Klauda, Jeffery B

    2017-06-07

    The all-atom CHARMM36 lipid force field (C36FF) has been tested with saturated, monounsaturated, and polyunsaturated lipids; however, it has not been validated against the 18:2 linoleoyl lipids with an unsaturated sn-1 chain. The linoleoyl lipids are common in plants and the main component of the soybean membrane. The lipid composition of soybean plasma membranes has been thoroughly characterized with experimental studies. However, there is comparatively less work done with computational modeling. Our molecular dynamics (MD) simulation results show that the pure linoleoyl lipids, 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (18:0/18:2) and 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (di-18:2), agree very well with the experiments, which demonstrates the accuracy of the C36FF for the computational study of soybean membranes. Based on the experimental composition, the soybean hypocotyl and root plasma membrane models are developed with each containing seven or eight types of linoleoyl phospholipids and two types of sterols (sitosterol and stigmasterol). MD simulations are performed to characterize soybean membranes, and the hydrogen bonds and clustering results demonstrate that the lipids prefer to interact with the lipids of the same/similar tail unsaturation. All the results suggest that these two soybean membrane models can be used as a basis for further research in soybean and higher plant membranes involving membrane-associated proteins.

  1. Simulations of simple linoleic acid-containing lipid membranes and models for the soybean plasma membranes

    NASA Astrophysics Data System (ADS)

    Zhuang, Xiaohong; Ou, Anna; Klauda, Jeffery B.

    2017-06-01

    The all-atom CHARMM36 lipid force field (C36FF) has been tested with saturated, monounsaturated, and polyunsaturated lipids; however, it has not been validated against the 18:2 linoleoyl lipids with an unsaturated sn-1 chain. The linoleoyl lipids are common in plants and the main component of the soybean membrane. The lipid composition of soybean plasma membranes has been thoroughly characterized with experimental studies. However, there is comparatively less work done with computational modeling. Our molecular dynamics (MD) simulation results show that the pure linoleoyl lipids, 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (18:0/18:2) and 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (di-18:2), agree very well with the experiments, which demonstrates the accuracy of the C36FF for the computational study of soybean membranes. Based on the experimental composition, the soybean hypocotyl and root plasma membrane models are developed with each containing seven or eight types of linoleoyl phospholipids and two types of sterols (sitosterol and stigmasterol). MD simulations are performed to characterize soybean membranes, and the hydrogen bonds and clustering results demonstrate that the lipids prefer to interact with the lipids of the same/similar tail unsaturation. All the results suggest that these two soybean membrane models can be used as a basis for further research in soybean and higher plant membranes involving membrane-associated proteins.

  2. Differential Effect of Plant Lipids on Membrane Organization

    PubMed Central

    Grosjean, Kevin; Mongrand, Sébastien; Beney, Laurent; Simon-Plas, Françoise; Gerbeau-Pissot, Patricia

    2015-01-01

    The high diversity of the plant lipid mixture raises the question of their respective involvement in the definition of membrane organization. This is particularly the case for plant plasma membrane, which is enriched in specific lipids, such as free and conjugated forms of phytosterols and typical phytosphingolipids, such as glycosylinositolphosphoceramides. This question was here addressed extensively by characterizing the order level of membrane from vesicles prepared using various plant lipid mixtures and labeled with an environment-sensitive probe. Fluorescence spectroscopy experiments showed that among major phytosterols, campesterol exhibits a stronger ability than β-sitosterol and stigmasterol to order model membranes. Multispectral confocal microscopy, allowing spatial analysis of membrane organization, demonstrated accordingly the strong ability of campesterol to promote ordered domain formation and to organize their spatial distribution at the membrane surface. Conjugated sterol forms, alone and in synergy with free sterols, exhibit a striking ability to order membrane. Plant sphingolipids, particularly glycosylinositolphosphoceramides, enhanced the sterol-induced ordering effect, emphasizing the formation and increasing the size of sterol-dependent ordered domains. Altogether, our results support a differential involvement of free and conjugated phytosterols in the formation of ordered domains and suggest that the diversity of plant lipids, allowing various local combinations of lipid species, could be a major contributor to membrane organization in particular through the formation of sphingolipid-sterol interacting domains. PMID:25575593

  3. Interactions of surfactants with lipid membranes.

    PubMed

    Heerklotz, Heiko

    2008-01-01

    Surfactants are surface-active, amphiphilic compounds that are water-soluble in the micro- to millimolar range, and self-assemble to form micelles or other aggregates above a critical concentration. This definition comprises synthetic detergents as well as amphiphilic peptides and lipopeptides, bile salts and many other compounds. This paper reviews the biophysics of the interactions of surfactants with membranes of insoluble, naturally occurring lipids. It discusses structural, thermodynamic and kinetic aspects of membrane-water partitioning, changes in membrane properties induced by surfactants, membrane solubilisation to micelles and other phases formed by lipid-surfactant systems. Each section defines and derives key parameters, mentions experimental methods for their measurement and compiles and discusses published data. Additionally, a brief overview is given of surfactant-like effects in biological systems, technical applications of surfactants that involve membrane interactions, and surfactant-based protocols to study biological membranes.

  4. The potent effect of mycolactone on lipid membranes

    PubMed Central

    Maniti, Ofelia; Marion, Estelle; Marsollier, Laurent; Dufourc, Erick J.; Canaan, Stéphane

    2018-01-01

    Mycolactone is a lipid-like endotoxin synthesized by an environmental human pathogen, Mycobacterium ulcerans, the causal agent of Buruli ulcer disease. Mycolactone has pleiotropic effects on fundamental cellular processes (cell adhesion, cell death and inflammation). Various cellular targets of mycolactone have been identified and a literature survey revealed that most of these targets are membrane receptors residing in ordered plasma membrane nanodomains, within which their functionalities can be modulated. We investigated the capacity of mycolactone to interact with membranes, to evaluate its effects on membrane lipid organization following its diffusion across the cell membrane. We used Langmuir monolayers as a cell membrane model. Experiments were carried out with a lipid composition chosen to be as similar as possible to that of the plasma membrane. Mycolactone, which has surfactant properties, with an apparent saturation concentration of 1 μM, interacted with the membrane at very low concentrations (60 nM). The interaction of mycolactone with the membrane was mediated by the presence of cholesterol and, like detergents, mycolactone reshaped the membrane. In its monomeric form, this toxin modifies lipid segregation in the monolayer, strongly affecting the formation of ordered microdomains. These findings suggest that mycolactone disturbs lipid organization in the biological membranes it crosses, with potential effects on cell functions and signaling pathways. Microdomain remodeling may therefore underlie molecular events, accounting for the ability of mycolactone to attack multiple targets and providing new insight into a single unifying mechanism underlying the pleiotropic effects of this molecule. This membrane remodeling may act in synergy with the other known effects of mycolactone on its intracellular targets, potentiating these effects. PMID:29320578

  5. Modulation of ileal bile acid transporter (ASBT) activity by depletion of plasma membrane cholesterol: association with lipid rafts

    PubMed Central

    Annaba, Fadi; Sarwar, Zaheer; Kumar, Pradeep; Saksena, Seema; Turner, Jerrold R.; Dudeja, Pradeep K.; Gill, Ravinder K.; Alrefai, Waddah A.

    2016-01-01

    Apical sodium-dependent bile acid transporter (ASBT) represents a highly efficient conservation mechanism of bile acids via mediation of their active transport across the luminal membrane of terminal ileum. To gain insight into the cellular regulation of ASBT, we investigated the association of ASBT with cholesterol and sphingolipid-enriched specialized plasma membrane microdomains known as lipid rafts and examined the role of membrane cholesterol in maintaining ASBT function. Human embryonic kidney (HEK)-293 cells stably transfected with human ASBT, human ileal brush-border membrane vesicles, and human intestinal epithelial Caco-2 cells were utilized for these studies. Floatation experiments on Optiprep density gradients demonstrated the association of ASBT protein with lipid rafts. Disruption of lipid rafts by depletion of membrane cholesterol with methyl-β-cyclodextrin (MβCD) significantly reduced the association of ASBT with lipid rafts, which was paralleled by a decrease in ASBT activity in Caco-2 and HEK-293 cells treated with MβCD. The inhibition in ASBT activity by MβCD was blocked in the cells treated with MβCD-cholesterol complexes. Kinetic analysis revealed that MβCD treatment decreased the Vmax of the transporter, which was not associated with alteration in the plasma membrane expression of ASBT. Our study illustrates that cholesterol content of lipid rafts is essential for the optimal activity of ASBT and support the association of ASBT with lipid rafts. These findings suggest a novel mechanism by which ASBT activity may be rapidly modulated by alterations in cholesterol content of plasma membrane and thus have important implications in processes related to maintenance of bile acid and cholesterol homeostasis. PMID:18063707

  6. Understanding carbon nanotube channel formation in the lipid membrane

    NASA Astrophysics Data System (ADS)

    Choi, Moon-ki; Kim, Hyunki; Lee, Byung Ho; Kim, Teayeop; Rho, Junsuk; Kim, Moon Ki; Kim, Kyunghoon

    2018-03-01

    Carbon nanotubes (CNTs) have been considered a prominent nano-channel in cell membranes because of their prominent ion-conductance and ion-selectivity, offering agents for a biomimetic channel platform. Using a coarse-grained molecular dynamics simulation, we clarify a construction mechanism of vertical CNT nano-channels in a lipid membrane for a long period, which has been difficult to observe in previous CNT-lipid interaction simulations. The result shows that both the lipid coating density and length of CNT affect the suitable fabrication condition for a vertical and stable CNT channel. Also, simulation elucidated that a lipid coating on the surface of the CNT prevents the CNT from burrowing into the lipid membrane and the vertical channel is stabilized by the repulsion force between the lipids in the coating and membrane. Our study provides an essential understanding of how CNTs can form stable and vertical channels in the membrane, which is important for designing new types of artificial channels as biosensors for bio-fluidic studies.

  7. Non-Brownian diffusion in lipid membranes: Experiments and simulations.

    PubMed

    Metzler, R; Jeon, J-H; Cherstvy, A G

    2016-10-01

    The dynamics of constituents and the surface response of cellular membranes-also in connection to the binding of various particles and macromolecules to the membrane-are still a matter of controversy in the membrane biophysics community, particularly with respect to crowded membranes of living biological cells. We here put into perspective recent single particle tracking experiments in the plasma membranes of living cells and supercomputing studies of lipid bilayer model membranes with and without protein crowding. Special emphasis is put on the observation of anomalous, non-Brownian diffusion of both lipid molecules and proteins embedded in the lipid bilayer. While single component, pure lipid bilayers in simulations exhibit only transient anomalous diffusion of lipid molecules on nanosecond time scales, the persistence of anomalous diffusion becomes significantly longer ranged on the addition of disorder-through the addition of cholesterol or proteins-and on passing of the membrane lipids to the gel phase. Concurrently, experiments demonstrate the anomalous diffusion of membrane embedded proteins up to macroscopic time scales in the minute time range. Particular emphasis will be put on the physical character of the anomalous diffusion, in particular, the occurrence of ageing observed in the experiments-the effective diffusivity of the measured particles is a decreasing function of time. Moreover, we present results for the time dependent local scaling exponent of the mean squared displacement of the monitored particles. Recent results finding deviations from the commonly assumed Gaussian diffusion patterns in protein crowded membranes are reported. The properties of the displacement autocorrelation function of the lipid molecules are discussed in the light of their appropriate physical anomalous diffusion models, both for non-crowded and crowded membranes. In the last part of this review we address the upcoming field of membrane distortion by elongated membrane

  8. Self-assembled tethered bimolecular lipid membranes.

    PubMed

    Sinner, Eva-Kathrin; Ritz, Sandra; Naumann, Renate; Schiller, Stefan; Knoll, Wolfgang

    2009-01-01

    This chapter describes some of the strategies developed in our group for designing, constructing and structurally and functionally characterizing tethered bimolecular lipid membranes (tBLM). We introduce this platform as a novel model membrane system that complements the existing ones, for example, Langmuir monolayers, vesicular liposomal dispersions and bimolecular ("black") lipid membranes. Moreover, it offers the additional advantage of allowing for studies of the influence of membrane structure and order on the function of integral proteins, for example, on how the composition and organization of lipids in a mixed membrane influence the ion translocation activity of integral channel proteins. The first strategy that we introduce concerns the preparation of tethered monolayers by the self-assembly of telechelics. Their molecular architecture with a headgroup, a spacer unit (the "tether") and the amphiphile that mimics the lipid molecule allows them to bind specifically to the solid support thus forming the proximal layer of the final architecture. After fusion of vesicles that could contain reconstituted proteins from a liposomal dispersion in contact to this monolayer the tethered bimolecular lipid membrane is obtained. This can then be characterized by a broad range of surface analytical techniques, including surface plasmon spectroscopies, the quartz crystal microbalance, fluorescence and IR spectroscopies, and electrochemical techniques, to mention a few. It is shown that this concept allows for the construction of tethered lipid bilayers with outstanding electrical properties including resistivities in excess of 10 MOmega cm2. A modified strategy uses the assembly of peptides as spacers that couple covalently via their engineered sulfhydryl or lipoic acid groups at the N-terminus to the employed gold substrate, while their C-terminus is being activated afterward for the coupling of, for example, dimyristoylphosphatidylethanol amine (DMPE) lipid molecules

  9. Molecular Transport Studies Through Unsupported Lipid Membranes

    NASA Astrophysics Data System (ADS)

    Rock, William; Parekh, Sapun; Bonn, Mischa

    2014-03-01

    Dendrimers, spherical polymeric nanoparticles made from branched monomers around a central core, show great promise as drug delivery vehicles. Dendrimer size, core contents, and surface functionality can be synthetically tuned, providing unprecedented versatility. Polyamidoamine (PAMAM) dendrimers have been shown to enter cells; however, questions remain about their biophysical interactions with the cell membrane, specifically about the presence and size of transient pores. We monitor dendrimer-lipid bilayer interactions using unsupported black lipid membranes (BLMs) as model cell membranes. Custom bilayer slides contain two vertically stacked aqueous chambers separated by a 25 μm Teflon sheet with a 120 μm aperture where the bilayer is formed. We vary the composition of model membranes (cholesterol content and lipid phase) to create biomimetic systems and study the interaction of PAMAM G6 and G3 dendrimers with these bilayers. Dendrimers, dextran cargo, and bilayers are monitored and quantified using time-lapse fluorescence imaging. Electrical capacitance measurements are simultaneously recorded to determine if the membrane is porous, and the pore size is deduced by monitoring transport of fluorescent dextrans of increasing molecular weight. These experiments shed light on the importance of cholesterol content and lipid phase on the interaction of dendrimer nanoparticles with membranes.

  10. A layer model of ethanol partitioning into lipid membranes.

    PubMed

    Nizza, David T; Gawrisch, Klaus

    2009-06-01

    The effect of membrane composition on ethanol partitioning into lipid bilayers was assessed by headspace gas chromatography. A series of model membranes with different compositions have been investigated. Membranes were exposed to a physiological ethanol concentration of 20 mmol/l. The concentration of membranes was 20 wt% which roughly corresponds to values found in tissue. Partitioning depended on the chemical nature of polar groups at the lipid/water interface. Compared to phosphatidylcholine, lipids with headgroups containing phosphatidylglycerol, phosphatidylserine, and sphingomyelin showed enhanced partitioning while headgroups containing phosphatidylethanolamine resulted in a lower partition coefficient. The molar partition coefficient was independent of a membrane's hydrophobic volume. This observation is in agreement with our previously published NMR results which showed that ethanol resides almost exclusively within the membrane/water interface. At an ethanol concentration of 20 mmol/l in water, ethanol concentrations at the lipid/water interface are in the range from 30-15 mmol/l, corresponding to one ethanol molecule per 100-200 lipids.

  11. Influence of chain rigidity on the conformation of model lipid membranes in the presence of cylindrical nanoparticle inclusions

    NASA Astrophysics Data System (ADS)

    Diloreto, Chris; Wickham, Robert

    2012-02-01

    We employ real-space self-consistent field theory to study the conformation of model lipid membranes in the presence of solvent and cylindrical nanoparticle inclusions (''peptides''). Whereas it is common to employ a polymeric Gaussian chain model for the lipids, here we model the lipids as persistent, worm-like chains. Our motivation is to develop a more realistic field theory to describe the action of pore-forming anti-microbial peptides that disrupt the bacterial cell membrane. We employ operator-splitting and a pseudo-spectral algorithm, using SpharmonicKit for the chain tangent degrees of freedom, to solve for the worm-like chain propagator. The peptides, modelled using a mask function, have a surface patterned with hydrophobic and hydrophillic patches, but no charge. We examine the role chain rigidity plays in the hydrophobic mismatch, the membrane-mediated interaction between two peptides, the size and structure of pores formed by peptide aggregates, and the free-energy barrier for peptide insertion into the membrane. Our results suggest that chain rigidity influences both the pore structure and the mechanism of pore formation.

  12. Steric Pressure among Membrane-Bound Polymers Opposes Lipid Phase Separation.

    PubMed

    Imam, Zachary I; Kenyon, Laura E; Carrillo, Adelita; Espinoza, Isai; Nagib, Fatema; Stachowiak, Jeanne C

    2016-04-19

    Lipid rafts are thought to be key organizers of membrane-protein complexes in cells. Many proteins that interact with rafts have bulky polymeric components such as intrinsically disordered protein domains and polysaccharide chains. Therefore, understanding the interaction between membrane domains and membrane-bound polymers provides insights into the roles rafts play in cells. Multiple studies have demonstrated that high concentrations of membrane-bound polymeric domains create significant lateral steric pressure at membrane surfaces. Furthermore, our recent work has shown that lateral steric pressure at membrane surfaces opposes the assembly of membrane domains. Building on these findings, here we report that membrane-bound polymers are potent suppressors of membrane phase separation, which can destabilize lipid domains with substantially greater efficiency than globular domains such as membrane-bound proteins. Specifically, we created giant vesicles with a ternary lipid composition, which separated into coexisting liquid ordered and disordered phases. Lipids with saturated tails and poly(ethylene glycol) (PEG) chains conjugated to their head groups were included at increasing molar concentrations. When these lipids were sparse on the membrane surface they partitioned to the liquid ordered phase. However, as they became more concentrated, the fraction of GUVs that were phase-separated decreased dramatically, ultimately yielding a population of homogeneous membrane vesicles. Experiments and physical modeling using compositions of increasing PEG molecular weight and lipid miscibility phase transition temperature demonstrate that longer polymers are the most efficient suppressors of membrane phase separation when the energetic barrier to lipid mixing is low. In contrast, as the miscibility transition temperature increases, longer polymers are more readily driven out of domains by the increased steric pressure. Therefore, the concentration of shorter polymers required

  13. Membrane-Sculpting BAR Domains Generate Stable Lipid Microdomains

    PubMed Central

    Zhao, Hongxia; Michelot, Alphée; Koskela, Essi V.; Tkach, Vadym; Stamou, Dimitrios; Drubin, David G.; Lappalainen, Pekka

    2014-01-01

    SUMMARY Bin-Amphiphysin-Rvs (BAR) domain proteins are central regulators of many cellular processes involving membrane dynamics. BAR domains sculpt phosphoinositide-rich membranes to generate membrane protrusions or invaginations. Here, we report that, in addition to regulating membrane geometry, BAR domains can generate extremely stable lipid microdomains by “freezing” phosphoinositide dynamics. This is a general feature of BAR domains, because the yeast endocytic BAR and Fes/CIP4 homology BAR (F-BAR) domains, the inverse BAR domain of Pinkbar, and the eisosomal BAR protein Lsp1 induced phosphoinositide clustering and halted lipid diffusion, despite differences in mechanisms of membrane interactions. Lsp1 displays comparable low diffusion rates in vitro and in vivo, suggesting that BAR domain proteins also generate stable phosphoinositide microdomains in cells. These results uncover a conserved role for BAR superfamily proteins in regulating lipid dynamics within membranes. Stable microdomains induced by BAR domain scaffolds and specific lipids can generate phase boundaries and diffusion barriers, which may have profound impacts on diverse cellular processes. PMID:24055060

  14. Membrane-sculpting BAR domains generate stable lipid microdomains.

    PubMed

    Zhao, Hongxia; Michelot, Alphée; Koskela, Essi V; Tkach, Vadym; Stamou, Dimitrios; Drubin, David G; Lappalainen, Pekka

    2013-09-26

    Bin-Amphiphysin-Rvs (BAR) domain proteins are central regulators of many cellular processes involving membrane dynamics. BAR domains sculpt phosphoinositide-rich membranes to generate membrane protrusions or invaginations. Here, we report that, in addition to regulating membrane geometry, BAR domains can generate extremely stable lipid microdomains by "freezing" phosphoinositide dynamics. This is a general feature of BAR domains, because the yeast endocytic BAR and Fes/CIP4 homology BAR (F-BAR) domains, the inverse BAR domain of Pinkbar, and the eisosomal BAR protein Lsp1 induced phosphoinositide clustering and halted lipid diffusion, despite differences in mechanisms of membrane interactions. Lsp1 displays comparable low diffusion rates in vitro and in vivo, suggesting that BAR domain proteins also generate stable phosphoinositide microdomains in cells. These results uncover a conserved role for BAR superfamily proteins in regulating lipid dynamics within membranes. Stable microdomains induced by BAR domain scaffolds and specific lipids can generate phase boundaries and diffusion barriers, which may have profound impacts on diverse cellular processes. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Pushing the lipid envelope: using bio-inspired nanocomposites to understand and exploit lipid membrane limitations

    NASA Astrophysics Data System (ADS)

    Montano, Gabriel

    Lipids serve as the organizing matrix material for biological membranes, the site of interaction of cells with the external environment. . As such, lipids play a critical role in structure/function relationships of an extraordinary number of critical biological processes. In this talk, we will look at bio-inspired membrane assemblies to better understand the roles of lipids in biological systems as well as attempt to generate materials that can mimic and potentially advance upon biological membrane processes. First, we will investigate the response of lipids to adverse conditions. In particular, I will present data that demonstrates the response of lipids to harsh conditions and how such responses can be exploited to generate nanocomposite rearrangements. I will also show the effect of adding the endotoxin lipopolysaccharide (LPS) to lipid bilayer assemblies and describe implications on our understanding of LPS organization in biological systems as well as describe induced lipid modifications that can be exploited to organize membrane composites with precise, two-dimensional geometric control. Lastly, I will describe the use of amphiphilic block copolymers to create membrane nanocomposites capable of mimicking biological systems. In particular, I will describe the use of our polymer-based membranes in creating artificial photosynthetic assemblies that rival biological systems in function in a more flexible, dynamic matrix.

  16. Lipidomics in research on yeast membrane lipid homeostasis.

    PubMed

    de Kroon, Anton I P M

    2017-08-01

    Mass spectrometry is increasingly used in research on membrane lipid homeostasis, both in analyses of the steady state lipidome at the level of molecular lipid species, and in pulse-chase approaches employing stable isotope-labeled lipid precursors addressing the dynamics of lipid metabolism. Here my experience with, and view on mass spectrometry-based lipid analysis is presented, with emphasis on aspects of quantification of membrane lipid composition of the yeast Saccharomyces cerevisiae. This article is part of a Special Issue entitled: BBALIP_Lipidomics Opinion Articles edited by Sepp Kohlwein. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Atomistic-level study of the interactions between hIAPP protofibrils and membranes: Influence of pH and lipid composition.

    PubMed

    Qian, Zhenyu; Zou, Yu; Zhang, Qingwen; Chen, Peijie; Ma, Buyong; Wei, Guanghong; Nussinov, Ruth

    2018-02-09

    The pathology of type 2 diabetes mellitus is associated with the aggregation of human islet amyloid polypeptide (hIAPP) and aggregation-mediated membrane disruption. The interactions of hIAPP aggregates with lipid membrane, as well as the effects of pH and lipid composition at the atomic level, remain elusive. Herein, using molecular dynamics simulations, we investigate the interactions of hIAPP protofibrillar oligomers with lipids, and the membrane perturbation that they induce, when they are partially inserted in an anionic dipalmitoyl-phosphatidylglycerol (DPPG) membrane or a mixed dipalmitoyl-phosphatidylcholine (DPPC)/DPPG (7:3) lipid bilayer under acidic/neutral pH conditions. We observed that the tilt angles and insertion depths of the hIAPP protofibril are strongly correlated with the pH and lipid composition. At neutral pH, the tilt angle and insertion depth of hIAPP protofibrils at a DPPG bilayer reach ~52° and ~1.62 nm with respect to the membrane surface, while they become ~77° and ~1.75 nm at a mixed DPPC/DPPG membrane. The calculated tilt angle of hIAPP at DPPG membrane is consistent with a recent chiral sum frequency generation spectroscopic study. The acidic pH induces a smaller tilt angle of ~40° and a shallower insertion depth (~1.24 nm) of hIAPP at the DPPG membrane surface, mainly due to protonation of His18 near the turn region. These differences mainly result from a combination of distinct electrostatic, van der Waals, hydrogen bonding and salt-bridge interactions between hIAPP and lipid bilayers. The hIAPP-membrane interaction energy analysis reveals that besides charged residues K1, R11 and H18, aromatic residues Phe15 and Phe23 also exhibit strong interactions with lipid bilayers, revealing the crucial role of aromatic residues in stabilizing the membrane-bound hIAPP protofibrils. hIAPP-membrane interactions disturb the lipid ordering and the local bilayer thickness around the peptides. Our results provide atomic-level information

  18. Interaction of cholesterol-conjugated ionizable amino lipids with biomembranes: lipid polymorphism, structure-activity relationship, and implications for siRNA delivery.

    PubMed

    Zhang, Jingtao; Fan, Haihong; Levorse, Dorothy A; Crocker, Louis S

    2011-08-02

    Delivery of siRNA is a major obstacle to the advancement of RNAi as a novel therapeutic modality. Lipid nanoparticles (LNP) consisting of ionizable amino lipids are being developed as an important delivery platform for siRNAs, and significant efforts are being made to understand the structure-activity relationship (SAR) of the lipids. This article uses a combination of small-angle X-ray scattering (SAXS) and differential scanning calorimetry (DSC) to evaluate the interaction between cholesterol-conjugated ionizable amino lipids and biomembranes, focusing on an important area of lipid SAR--the ability of lipids to destabilize membrane bilayer structures and facilitate endosomal escape. In this study, cholesterol-conjugated amino lipids were found to be effective in increasing the order of biomembranes and also highly effective in inducing phase changes in biological membranes in vitro (i.e., the lamellar to inverted hexagonal phase transition). The phase transition temperatures, determined using SAXS and DSC, serve as an indicator for ranking the potency of lipids to destabilize endosomal membranes. It was found that the bilayer disruption ability of amino lipids depends strongly on the amino lipid concentration in membranes. Amino lipids with systematic variations in headgroups, the extent of ionization, tail length, the degree of unsaturation, and tail asymmetry were evaluated for their bilayer disruption ability to establish SAR. Overall, it was found that the impact of these lipid structure changes on their bilayer disruption ability agrees well with the results from a conceptual molecular "shape" analysis. Implications of the findings from this study for siRNA delivery are discussed. The methods reported here can be used to support the SAR screening of cationic lipids for siRNA delivery, and the information revealed through the study of the interaction between cationic lipids and biomembranes will contribute significantly to the design of more efficient si

  19. A Layer Model of Ethanol Partitioning into Lipid Membranes

    PubMed Central

    Nizza, David T.; Gawrisch, Klaus

    2013-01-01

    The effect of membrane composition on ethanol partitioning into lipid bilayers was assessed by headspace gas chromatography. A series of model membranes with different compositions have been investigated. Membranes were exposed to a physiological ethanol concentration of 20 mmol/l. The concentration of membranes was 20 wt% which roughly corresponds to values found in tissue. Partitioning depended on the chemical nature of polar groups at the lipid-water interface. Compared to phosphatidylcholine, lipids with headgroups containing phosphatidylglycerol, phosphatidylserine, and sphingomyelin showed enhanced partitioning while headgroups containing phosphatidylethanolamine resulted in a lower partition coefficient. The molar partition coefficient was independent of a membrane’s hydrophobic volume. This observation is in agreement with our previously published NMR results which showed that ethanol resides almost exclusively within the membrane-water interface. At an ethanol concentration of 20 mmol/l in water, ethanol concentrations at the lipid/water interface are in the range from 30 – 15 mmol/l, corresponding to one ethanol molecule per 100–200 lipids. PMID:19592710

  20. Lipid-protein stoichiometries in a crystalline biological membrane: NMR quantitative analysis of the lipid extract of the purple membrane.

    PubMed

    Corcelli, Angela; Lattanzio, Veronica M T; Mascolo, Giuseppe; Papadia, Paride; Fanizzi, Francesco

    2002-01-01

    The lipid/protein stoichiometries of a naturally crystalline biological membrane, the purple membrane (PM) of Halobacterium salinarum, have been obtained by a combination of (31)P- and (1)H-NMR analyses of the lipid extract. In total, 10 lipid molecules per retinal were found to be present in the PM lipid extract: 2-3 molecules of phosphatidylglycerophosphate methyl ester (PGP-Me), 3 of glycolipid sulfate, 1 of phosphatidylglycerol, 1 of archaeal glycocardiolipin (GlyC), 2 of squalene plus minor amounts of phosphatidylglycerosulfate (PGS) and bisphosphatidylglycerol (archaeal cardiolipin) (BPG) and a negligible amount of vitamin MK8. The novel data of the present study are necessary to identify the lipids in the electron density map, and to shed light on the structural relationships of the lipid and protein components of the PM.

  1. Engineering Lipid Bilayer Membranes for Protein Studies

    PubMed Central

    Khan, Muhammad Shuja; Dosoky, Noura Sayed; Williams, John Dalton

    2013-01-01

    Lipid membranes regulate the flow of nutrients and communication signaling between cells and protect the sub-cellular structures. Recent attempts to fabricate artificial systems using nanostructures that mimic the physiological properties of natural lipid bilayer membranes (LBM) fused with transmembrane proteins have helped demonstrate the importance of temperature, pH, ionic strength, adsorption behavior, conformational reorientation and surface density in cellular membranes which all affect the incorporation of proteins on solid surfaces. Much of this work is performed on artificial templates made of polymer sponges or porous materials based on alumina, mica, and porous silicon (PSi) surfaces. For example, porous silicon materials have high biocompatibility, biodegradability, and photoluminescence, which allow them to be used both as a support structure for lipid bilayers or a template to measure the electrochemical functionality of living cells grown over the surface as in vivo. The variety of these media, coupled with the complex physiological conditions present in living systems, warrant a summary and prospectus detailing which artificial systems provide the most promise for different biological conditions. This study summarizes the use of electrochemical impedance spectroscopy (EIS) data on artificial biological membranes that are closely matched with previously published biological systems using both black lipid membrane and patch clamp techniques. PMID:24185908

  2. Membrane Compartmentalization Reducing the Mobility of Lipids and Proteins within a Model Plasma Membrane.

    PubMed

    Koldsø, Heidi; Reddy, Tyler; Fowler, Philip W; Duncan, Anna L; Sansom, Mark S P

    2016-09-01

    The cytoskeleton underlying cell membranes may influence the dynamic organization of proteins and lipids within the bilayer by immobilizing certain transmembrane (TM) proteins and forming corrals within the membrane. Here, we present coarse-grained resolution simulations of a biologically realistic membrane model of asymmetrically organized lipids and TM proteins. We determine the effects of a model of cytoskeletal immobilization of selected membrane proteins using long time scale coarse-grained molecular dynamics simulations. By introducing compartments with varying degrees of restraints within the membrane models, we are able to reveal how compartmentalization caused by cytoskeletal immobilization leads to reduced and anomalous diffusional mobility of both proteins and lipids. This in turn results in a reduced rate of protein dimerization within the membrane and of hopping of membrane proteins between compartments. These simulations provide a molecular realization of hierarchical models often invoked to explain single-molecule imaging studies of membrane proteins.

  3. Interaction of Aspirin (Acetylsalicylic Acid) with Lipid Membranes

    PubMed Central

    Barrett, Matthew A.; Zheng, Songbo; Roshankar, Golnaz; Alsop, Richard J.; Belanger, Randy K. R.; Huynh, Chris; Kučerka, Norbert; Rheinstädter, Maikel C.

    2012-01-01

    We studied the interaction of Aspirin (acetylsalicylic acid) with lipid membranes using x-ray diffraction for bilayers containing up to 50 mol% of aspirin. From 2D x-ray intensity maps that cover large areas of reciprocal space we determined the position of the ASA molecules in the phospholipid bilayers and the molecular arrangement of the molecules in the plane of the membranes. We present direct experimental evidence that ASA molecules participate in saturated lipid bilayers of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) and preferably reside in the head group region of the membrane. Up to 50 mol% ASA molecules can be dissolved in this type of bilayer before the lateral membrane organization is disturbed and the membranes are found to form an ordered, 2D crystal-like structure. Furthermore, ASA and cholesterol were found to co-exist in saturated lipid bilayers, with the ASA molecules residing in the head group region and the cholesterol molecules participating in the hydrophobic membrane core. PMID:22529913

  4. Maximally asymmetric transbilayer distribution of anionic lipids alters the structure and interaction with lipids of an amyloidogenic protein dimer bound to the membrane surface

    PubMed Central

    Cheng, Sara Y.; Chou, George; Buie, Creighton; Vaughn, Mark W.; Compton, Campbell; Cheng, Kwan H.

    2016-01-01

    We used molecular dynamics simulations to explore the effects of asymmetric transbilayer distribution of anionic phosphatidylserine (PS) lipids on the structure of a protein on the membrane surface and subsequent protein–lipid interactions. Our simulation systems consisted of an amyloidogenic, beta-sheet rich dimeric protein (D42) absorbed to the phosphatidylcholine (PC) leaflet, or protein-contact PC leaflet, of two membrane systems: a single-component PC bilayer and double PC/PS bilayers. The latter comprised of a stable but asymmetric transbilayer distribution of PS in the presence of counterions, with a 1-component PC leaflet coupled to a 1-component PS leaflet in each bilayer. The maximally asymmetric PC/PS bilayer had a non-zero transmembrane potential (TMP) difference and higher lipid order packing, whereas the symmetric PC bilayer had a zero TMP difference and lower lipid order packing under physiologically relevant conditions. Analysis of the adsorbed protein structures revealed weaker protein binding, more folding in the N-terminal domain, more aggregation of the N- and C-terminal domains and larger tilt angle of D42 on the PC leaflet surface of the PC/PS bilayer versus the PC bilayer. Also, analysis of protein-induced membrane structural disruption revealed more localized bilayer thinning in the PC/PS versus PC bilayer. Although the electric field profile in the non-protein-contact PS leaflet of the PC/PS bilayer differed significantly from that in the non-protein-contact PC leaflet of the PC bilayer, no significant difference in the electric field profile in the protein-contact PC leaflet of either bilayer was evident. We speculate that lipid packing has a larger effect on the surface adsorbed protein structure than the electric field for a maximally asymmetric PC/PS bilayer. Our results support the mechanism that the higher lipid packing in a lipid leaflet promotes stronger protein–protein but weaker protein–lipid interactions for a dimeric

  5. Maximally asymmetric transbilayer distribution of anionic lipids alters the structure and interaction with lipids of an amyloidogenic protein dimer bound to the membrane surface.

    PubMed

    Cheng, Sara Y; Chou, George; Buie, Creighton; Vaughn, Mark W; Compton, Campbell; Cheng, Kwan H

    2016-03-01

    We used molecular dynamics simulations to explore the effects of asymmetric transbilayer distribution of anionic phosphatidylserine (PS) lipids on the structure of a protein on the membrane surface and subsequent protein-lipid interactions. Our simulation systems consisted of an amyloidogenic, beta-sheet rich dimeric protein (D42) absorbed to the phosphatidylcholine (PC) leaflet, or protein-contact PC leaflet, of two membrane systems: a single-component PC bilayer and double PC/PS bilayers. The latter comprised of a stable but asymmetric transbilayer distribution of PS in the presence of counterions, with a 1-component PC leaflet coupled to a 1-component PS leaflet in each bilayer. The maximally asymmetric PC/PS bilayer had a non-zero transmembrane potential (TMP) difference and higher lipid order packing, whereas the symmetric PC bilayer had a zero TMP difference and lower lipid order packing under physiologically relevant conditions. Analysis of the adsorbed protein structures revealed weaker protein binding, more folding in the N-terminal domain, more aggregation of the N- and C-terminal domains and larger tilt angle of D42 on the PC leaflet surface of the PC/PS bilayer versus the PC bilayer. Also, analysis of protein-induced membrane structural disruption revealed more localized bilayer thinning in the PC/PS versus PC bilayer. Although the electric field profile in the non-protein-contact PS leaflet of the PC/PS bilayer differed significantly from that in the non-protein-contact PC leaflet of the PC bilayer, no significant difference in the electric field profile in the protein-contact PC leaflet of either bilayer was evident. We speculate that lipid packing has a larger effect on the surface adsorbed protein structure than the electric field for a maximally asymmetric PC/PS bilayer. Our results support the mechanism that the higher lipid packing in a lipid leaflet promotes stronger protein-protein but weaker protein-lipid interactions for a dimeric protein on

  6. Methyl-branched lipids promote the membrane adsorption of α-synuclein by enhancing shallow lipid-packing defects.

    PubMed

    Garten, Matthias; Prévost, Coline; Cadart, Clotilde; Gautier, Romain; Bousset, Luc; Melki, Ronald; Bassereau, Patricia; Vanni, Stefano

    2015-06-28

    Alpha-synuclein (AS) is a synaptic protein that is directly involved in Parkinson's disease due to its tendency to form protein aggregates. Since AS aggregation can be dependent on the interactions between the protein and the cell plasma membrane, elucidating the membrane binding properties of AS is of crucial importance to establish the molecular basis of AS aggregation into toxic fibrils. Using a combination of in vitro reconstitution experiments based on Giant Unilamellar Vesicles (GUVs), confocal microscopy and all-atom molecular dynamics simulations, we have investigated the membrane binding properties of AS, with a focus on the relative contribution of hydrophobic versus electrostatic interactions. In contrast with previous observations, we did not observe any binding of AS to membranes containing the ganglioside GM1, even at relatively high GM1 content. AS, on the other hand, showed a stronger affinity for neutral flat membranes consisting of methyl-branched lipids. To rationalize these results, we used all-atom molecular dynamics simulations to investigate the influence of methyl-branched lipids on interfacial membrane properties. We found that methyl-branched lipids promote the membrane adsorption of AS by creating shallow lipid-packing defects to a larger extent than polyunsaturated and monounsaturated lipids. Our findings suggest that methyl-branched lipids may constitute a remarkably adhesive substrate for peripheral proteins that adsorb on membranes via hydrophobic insertions.

  7. Effect of Membrane Tension on the Electric Field and Dipole Potential of Lipid Bilayer Membrane

    PubMed Central

    Warshaviak, Dora Toledo; Muellner, Michael J.; Chachisvilis, Mirianas

    2011-01-01

    The dipole potential of lipid bilayer membrane controls the difference in permeability of the membrane to oppositely charged ions. We have combined molecular dynamics (MD) simulations and experimental studies to determine changes in electric field and electrostatic potential of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer in response to applied membrane tension. MD simulations based on CHARMM36 force field showed that electrostatic potential of DOPC bilayer decreases by ~45 mV in the physiologically relevant range of membrane tension values (0 to 15 dyn/cm). The electrostatic field exhibits a peak (~0.8×109 V/m) near the water/lipid interface which shifts by 0.9 Å towards the bilayer center at 15 dyn/cm. Maximum membrane tension of 15 dyn/cm caused 6.4% increase in area per lipid, 4.7% decrease in bilayer thickness and 1.4% increase in the volume of the bilayer. Dipole-potential sensitive fluorescent probes were used to detect membrane tension induced changes in DOPC vesicles exposed to osmotic stress. Experiments confirmed that dipole potential of DOPC bilayer decreases at higher membrane tensions. These results are suggestive of a potentially new mechanosensing mechanism by which mechanically induced structural changes in the lipid bilayer membrane could modulate the function of membrane proteins by altering electrostatic interactions and energetics of protein conformational states. PMID:21722624

  8. Synthesis and Characterization of Novel Anchorlipids for Tethered Bilayer Lipid Membranes.

    PubMed

    Andersson, Jakob; Knobloch, Jacqueline J; Perkins, Michael V; Holt, Stephen A; Köper, Ingo

    2017-05-09

    Tethered bilayer lipid membranes are versatile solid-supported model membrane systems. Core to these systems is an anchorlipid that covalently links a lipid bilayer to a support. The molecular structure of these lipids can have a significant impact on the properties of the resulting bilayer. Here, the synthesis of anchorlipids containing ester groups in the tethering part is described. The lipids are used to form bilayer membranes, and the resulting structures are compared with membranes formed using conventional anchorlipids or sparsely tethered membranes. All membranes showed good electrical sealing properties; the disulphide-terminated anchorlipids could be used in a sparsely tethered system without significantly reducing the sealing properties of the lipid bilayers. The sparsely tethered systems also allowed for higher ion transport across the membrane, which is in good correlation with higher hydration of the spacer region as seen by neutron scattering.

  9. Interactions between lipids and proteins are critical for organization of plasma membrane-ordered domains in tobacco BY-2 cells.

    PubMed

    Grosjean, Kevin; Der, Christophe; Robert, Franck; Thomas, Dominique; Mongrand, Sébastien; Simon-Plas, Françoise; Gerbeau-Pissot, Patricia

    2018-06-27

    The laterally heterogeneous plant plasma membrane (PM) is organized into finely controlled specialized areas that include membrane-ordered domains. Recently, the spatial distribution of such domains within the PM has been identified as playing a key role in cell responses to environmental challenges. To examine membrane order at a local level, BY-2 tobacco suspension cell PMs were labelled with an environment-sensitive probe (di-4-ANEPPDHQ). Four experimental models were compared to identify mechanisms and cell components involved in short-term (1 h) maintenance of the ordered domain organization in steady-state cell PMs: modulation of the cytoskeleton or the cell wall integrity of tobacco BY-2 cells; and formation of giant vesicles using either a lipid mixture of tobacco BY-2 cell PMs or the original lipid and protein combinations of the tobacco BY-2 cell PM. Whilst inhibiting phosphorylation or disrupting either the cytoskeleton or the cell wall had no observable effects, we found that lipids and proteins significantly modified both the abundance and spatial distribution of ordered domains. This indicates the involvement of intrinsic membrane components in the local physical state of the plant PM. Our findings support a major role for the 'lipid raft' model, defined as the sterol-dependent ordered assemblies of specific lipids and proteins in plant PM organization.

  10. Membrane proteins bind lipids selectively to modulate their structure and function.

    PubMed

    Laganowsky, Arthur; Reading, Eamonn; Allison, Timothy M; Ulmschneider, Martin B; Degiacomi, Matteo T; Baldwin, Andrew J; Robinson, Carol V

    2014-06-05

    Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments and that lipids can bind to specific sites, for example, in potassium channels. Fundamental questions remain however regarding the extent of membrane protein selectivity towards lipids. Here we report a mass spectrometry approach designed to determine the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis and aquaporin Z (AqpZ) and the ammonia channel (AmtB) from Escherichia coli, using ion mobility mass spectrometry (IM-MS), which reports gas-phase collision cross-sections. We demonstrate that folded conformations of membrane protein complexes can exist in the gas phase. By resolving lipid-bound states, we then rank bound lipids on the basis of their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability; however, the highest-ranking lipid is phosphatidylinositol phosphate, in line with its proposed functional role in mechanosensation. AqpZ is also stabilized by many lipids, with cardiolipin imparting the most significant resistance to unfolding. Subsequently, through functional assays we show that cardiolipin modulates AqpZ function. Similar experiments identify AmtB as being highly selective for phosphatidylglycerol, prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3 Å resolution structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that re-position AmtB residues to interact with the lipid bilayer. Our results demonstrate that resistance to unfolding correlates with specific lipid-binding events, enabling a distinction to be made between lipids that merely bind from those that modulate membrane

  11. All-Atom Molecular Dynamics-Based Analysis of Membrane-Stabilizing Copolymer Interactions with Lipid Bilayers Probed under Constant Surface Tensions.

    PubMed

    Houang, Evelyne M; Bates, Frank S; Sham, Yuk Y; Metzger, Joseph M

    2017-11-30

    An all-atom phospholipid bilayer and triblock copolymer model was developed for molecular dynamics (MD) studies. These were performed to investigate the mechanism of interaction between membrane-stabilizing triblock copolymer P188 and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) lipid bilayers under applied lateral surface tension (γ) to model membrane mechanical stress. Results showed that P188 insertion is driven by the hydrophobic poly(propylene oxide) (PPO) core and dependent on bilayer area per lipid. Moreover, insertion of P188 increased the bilayer's resistance to mechanical rupture, as observed by a significant increase in the absolute lateral pressure required to disrupt the bilayer. To further investigate the specific chemical features of P188 underlying membrane stabilizer function, a series of MD simulations with triblock copolymers of the same class as P188 but of varying chemical composition and sizes were performed. Results showed that triblock copolymer insertion into the lipid bilayer is dependent on overall copolymer hydrophobicity, with higher copolymer hydrophobicity requiring a reduced bilayer area per lipid ratio for insertion. Further analysis revealed that the effect of copolymer insertion on membrane mechanical integrity was also dependent on hydrophobicity. Here, P188 insertion significantly increased the absolute apparent lateral pressure required to rupture the POPC bilayer, thereby protecting the membrane against mechanical stress. In marked contrast, highly hydrophobic copolymers decreased the lateral pressure necessary for membrane rupture and thus rendering the membrane significantly more susceptible to mechanical stress. These new in silico findings align with recent experimental findings using synthetic lipid bilayers and in muscle cells in vitro and mouse models in vivo. Collectively, these data underscore the importance of PEO-PPO-PEO copolymer chemical composition in copolymer-based muscle membrane stabilization

  12. Interactions of the anticancer drug tamoxifen with lipid membranes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Khadka, Nawal K.; Cheng, Xiaolin; Ho, Chian Sing

    Interactions of the hydrophobic anticancer drug tamoxifen (TAM) with lipid model membranes were studied using calcein-encapsulated vesicle leakage, attenuated total reflection Fourier transform infrared (FTIR) spectroscopy, small-angle neutron scattering (SANS), atomic force microscopy (AFM) based force spectroscopy, and all-atom molecular dynamics (MD) simulations. The addition of TAM enhances membrane permeability, inducing calcein to translocate from the interior to the exterior of lipid vesicles. A large decrease in the FTIR absorption band’s magnitude was observed in the hydrocarbon chain region, suggesting suppressed bond vibrational dynamics. Bilayer thickening was determined from SANS data. Force spectroscopy measurements indicate that the lipid bilayer areamore » compressibility modulus KA is increased by a large amount after the incorporation of TAM. MD simulations show that TAM decreases the lipid area and increases chain order parameters. Moreover, orientational and positional analyses show that TAM exhibits a highly dynamic conformation within the lipid bilayer. Lastly, our detailed experimental and computational studies of TAM interacting with model lipid membranes shed new light on membrane modulation by TAM.« less

  13. Interactions of the anticancer drug tamoxifen with lipid membranes

    DOE PAGES

    Khadka, Nawal K.; Cheng, Xiaolin; Ho, Chian Sing; ...

    2015-05-19

    Interactions of the hydrophobic anticancer drug tamoxifen (TAM) with lipid model membranes were studied using calcein-encapsulated vesicle leakage, attenuated total reflection Fourier transform infrared (FTIR) spectroscopy, small-angle neutron scattering (SANS), atomic force microscopy (AFM) based force spectroscopy, and all-atom molecular dynamics (MD) simulations. The addition of TAM enhances membrane permeability, inducing calcein to translocate from the interior to the exterior of lipid vesicles. A large decrease in the FTIR absorption band’s magnitude was observed in the hydrocarbon chain region, suggesting suppressed bond vibrational dynamics. Bilayer thickening was determined from SANS data. Force spectroscopy measurements indicate that the lipid bilayer areamore » compressibility modulus KA is increased by a large amount after the incorporation of TAM. MD simulations show that TAM decreases the lipid area and increases chain order parameters. Moreover, orientational and positional analyses show that TAM exhibits a highly dynamic conformation within the lipid bilayer. Lastly, our detailed experimental and computational studies of TAM interacting with model lipid membranes shed new light on membrane modulation by TAM.« less

  14. Lamellar Biogels: Fluid-Membrane-Based Hydrogels Containing Polymer Lipids

    NASA Astrophysics Data System (ADS)

    Warriner, Heidi E.; Idziak, Stefan H. J.; Slack, Nelle L.; Davidson, Patrick; Safinya, Cyrus R.

    1996-02-01

    A class of lamellar biological hydrogels comprised of fluid membranes of lipids and surfactants with small amounts of low molecular weight poly(ethylene glycol)-derived polymer lipids (PEG-lipids) were studied by x-ray diffraction, polarized light microscopy, and rheometry. In contrast to isotropic hydrogels of polymer networks, these membrane-based birefringent liquid crystalline biogels, labeled Lα,g, form the gel phase when water is added to the liquid-like lamellar L_α phase, which reenters a liquid-like mixed phase upon further dilution. Furthermore, gels with larger water content require less PEG-lipid to remain stable. Although concentrated (~50 weight percent) mixtures of free PEG (molecular weight, 5000) and water do not gel, gelation does occur in mixtures containing as little as 0.5 weight percent PEG-lipid. A defining signature of the Lα,g regime as it sets in from the fluid lamellar L_α phase is the proliferation of layer-dislocation-type defects, which are stabilized by the segregation of PEG-lipids to the defect regions of high membrane curvature that connect the membranes.

  15. Modeling Membrane Deformations and Lipid Demixing upon Protein-Membrane Interaction: The BAR Dimer Adsorption

    PubMed Central

    Khelashvili, George; Harries, Daniel; Weinstein, Harel

    2009-01-01

    We use a self-consistent mean-field theory, designed to investigate membrane reshaping and lipid demixing upon interaction with proteins, to explore BAR domains interacting with large patches of lipid membranes of heterogeneous compositions. The computational model includes contributions to the system free energy from electrostatic interactions and elastic energies of the membrane, as well as salt and lipid mixing entropies. The results from our simulation of a single adsorbing Amphiphysin BAR dimer indicate that it is capable of stabilizing a significantly curved membrane. However, we predict that such deformations will occur only for membrane patches that have the inherent propensity for high curvature, reflected in the tendency to create local distortions that closely match the curvature of the BAR dimer itself. Such favorable preconditioning for BAR-membrane interaction may be the result of perturbations such as local lipid demixing induced by the interaction, or of a prior insertion of the BAR domain's amphiphatic N-helix. From our simulations it appears that local segregation of charged lipids under the influence of the BAR dimer cannot produce high enough asymmetry between bilayer leaflets to induce significant bending. In the absence of additional energy contributions that favor membrane asymmetry, the membrane will remain nearly flat upon single BAR dimer adsorption, relative to the undulation expected from thermal fluctuations. Thus, we conclude that the N-helix insertions have a critical mechanistic role in the local perturbation and curving of the membrane, which is then stabilized by the electrostatic interaction with the BAR dimer. We discuss how these results can be used to estimate the tendency of BARs to bend membranes in terms of a spatially nonisotropic spontaneous curvature. PMID:19751667

  16. Engineering lipid structure for recognition of the liquid ordered membrane phase

    DOE PAGES

    Bordovsky, Stefan S.; Wong, Christopher S.; Bachand, George D.; ...

    2016-08-26

    The selective partitioning of lipid components in phase-separated membranes is essential for domain formation involved in cellular processes. Identifying and tracking the movement of lipids in cellular systems would be improved if we understood how to achieve selective affinity between fluorophore-labeled lipids and membrane assemblies. Furthermore, we investigated the structure and chemistry of membrane lipids to evaluate lipid designs that partition to the liquid ordered (L o) phase. A range of fluorophores at the headgroup position and lengths of PEG spacer between the lipid backbone and fluorophore were examined. On a lipid body with saturated palmityl or palmitoyl tails, wemore » found that although the lipid tails can direct selective partitioning to the L o phase through favorable packing interactions, headgroup hydrophobicity can override the partitioning behavior and direct the lipid to the disordered membrane phase (L d). The PEG spacer can serve as a buffer to mute headgroup–membrane interactions and thus improve L o phase partitioning, but its effect is limited with strongly hydrophobic fluorophore headgroups. We present a series of lipid designs leading to the development of novel fluorescently labeled lipids with selective affinity for the L o phase.« less

  17. Engineering Lipid Structure for Recognition of the Liquid Ordered Membrane Phase.

    PubMed

    Bordovsky, Stefan S; Wong, Christopher S; Bachand, George D; Stachowiak, Jeanne C; Sasaki, Darryl Y

    2016-11-29

    The selective partitioning of lipid components in phase-separated membranes is essential for domain formation involved in cellular processes. Identifying and tracking the movement of lipids in cellular systems would be improved if we understood how to achieve selective affinity between fluorophore-labeled lipids and membrane assemblies. Here, we investigated the structure and chemistry of membrane lipids to evaluate lipid designs that partition to the liquid ordered (L o ) phase. A range of fluorophores at the headgroup position and lengths of PEG spacer between the lipid backbone and fluorophore were examined. On a lipid body with saturated palmityl or palmitoyl tails, we found that although the lipid tails can direct selective partitioning to the L o phase through favorable packing interactions, headgroup hydrophobicity can override the partitioning behavior and direct the lipid to the disordered membrane phase (L d ). The PEG spacer can serve as a buffer to mute headgroup-membrane interactions and thus improve L o phase partitioning, but its effect is limited with strongly hydrophobic fluorophore headgroups. We present a series of lipid designs leading to the development of novel fluorescently labeled lipids with selective affinity for the L o phase.

  18. No Evidence for Spontaneous Lipid Transfer at ER-PM Membrane Contact Sites.

    PubMed

    Merklinger, Elisa; Schloetel, Jan-Gero; Spitta, Luis; Thiele, Christoph; Lang, Thorsten

    2016-04-01

    Non-vesicular lipid transport steps play a crucial role in lipid trafficking and potentially include spontaneous exchange. Since membrane contact facilitates this lipid transfer, it is most likely to occur at membrane contact sites (MCS). However, to date it is unknown whether closely attached biological membranes exchange lipids spontaneously. We have set up a system for studying the exchange of lipids at MCS formed between the endoplasmic reticulum (ER) and the plasma membrane. Contact sites were stably anchored and the lipids cholesterol and phosphatidylcholine (PC) were not capable of transferring spontaneously into the opposed bilayer. We conclude that physical contact between two associated biological membranes is not sufficient for transfer of the lipids PC and cholesterol.

  19. Lipids and ions traverse the membrane by the same physical pathway in the nhTMEM16 scramblase

    PubMed Central

    Jiang, Tao; Yu, Kuai

    2017-01-01

    From bacteria to mammals, different phospholipid species are segregated between the inner and outer leaflets of the plasma membrane by ATP-dependent lipid transporters. Disruption of this asymmetry by ATP-independent phospholipid scrambling is important in cellular signaling, but its mechanism remains incompletely understood. Using MD simulations coupled with experimental assays, we show that the surface hydrophilic transmembrane cavity exposed to the lipid bilayer on the fungal scramblase nhTMEM16 serves as the pathway for both lipid translocation and ion conduction across the membrane. Ca2+ binding stimulates its open conformation by altering the structure of transmembrane helices that line the cavity. We have identified key amino acids necessary for phospholipid scrambling and validated the idea that ions permeate TMEM16 Cl- channels via a structurally homologous pathway by showing that mutation of two residues in the pore region of the TMEM16A Ca2+-activated Cl- channel convert it into a robust scramblase. PMID:28917060

  20. Designing lipids for selective partitioning into liquid ordered membrane domains.

    PubMed

    Momin, Noor; Lee, Stacey; Gadok, Avinash K; Busch, David J; Bachand, George D; Hayden, Carl C; Stachowiak, Jeanne C; Sasaki, Darryl Y

    2015-04-28

    Self-organization of lipid molecules into specific membrane phases is key to the development of hierarchical molecular assemblies that mimic cellular structures. While the packing interaction of the lipid tails should provide the major driving force to direct lipid partitioning to ordered or disordered membrane domains, numerous examples show that the headgroup and spacer play important but undefined roles. We report here the development of several new biotinylated lipids that examine the role of spacer chemistry and structure on membrane phase partitioning. The new lipids were prepared with varying lengths of low molecular weight polyethylene glycol (EGn) spacers to examine how spacer hydrophilicity and length influence their partitioning behavior following binding with FITC-labeled streptavidin in liquid ordered (Lo) and liquid disordered (Ld) phase coexisting membranes. Partitioning coefficients (Kp Lo/Ld) of the biotinylated lipids were determined using fluorescence measurements in studies with giant unilamellar vesicles (GUVs). Compared against DPPE-biotin, DPPE-cap-biotin, and DSPE-PEG2000-biotin lipids, the new dipalmityl-EGn-biotin lipids exhibited markedly enhanced partitioning into liquid ordered domains, achieving Kp of up to 7.3 with a decaethylene glycol spacer (DP-EG10-biotin). We further demonstrated biological relevance of the lipids with selective partitioning to lipid raft-like domains observed in giant plasma membrane vesicles (GPMVs) derived from mammalian cells. Our results found that the spacer group not only plays a pivotal role for designing lipids with phase selectivity but may also influence the structural order of the domain assemblies.

  1. Ballistic impact response of lipid membranes.

    PubMed

    Zhang, Yao; Meng, Zhaoxu; Qin, Xin; Keten, Sinan

    2018-03-08

    Therapeutic agent loaded micro and nanoscale particles as high-velocity projectiles can penetrate cells and tissues, thereby serving as gene and drug delivery vehicles for direct and rapid internalization. Despite recent progress in developing micro/nanoscale ballistic tools, the underlying biophysics of how fast projectiles deform and penetrate cell membranes is still poorly understood. To understand the rate and size-dependent penetration processes, we present coarse-grained molecular dynamics simulations of the ballistic impact of spherical projectiles on lipid membranes. Our simulations reveal that upon impact, the projectile can pursue one of three distinct pathways. At low velocities below the critical penetration velocity, projectiles rebound off the surface. At intermediate velocities, penetration occurs after the projectile deforms the membrane into a tubular thread. At very high velocities, rapid penetration occurs through localized membrane deformation without tubulation. Membrane tension, projectile velocity and size govern which phenomenon occurs, owing to their positive correlation with the reaction force generated between the projectile and the membrane during impact. Two critical membrane tension values dictate the boundaries among the three pathways for a given system, due to the rate dependence of the stress generated in the membrane. Our findings provide broad physical insights into the ballistic impact response of soft viscous membranes and guide design strategies for drug delivery through lipid membranes using micro/nanoscale ballistic tools.

  2. Lipid nanotechnologies for structural studies of membrane-associated proteins.

    PubMed

    Stoilova-McPhie, Svetla; Grushin, Kirill; Dalm, Daniela; Miller, Jaimy

    2014-11-01

    We present a methodology of lipid nanotubes (LNT) and nanodisks technologies optimized in our laboratory for structural studies of membrane-associated proteins at close to physiological conditions. The application of these lipid nanotechnologies for structure determination by cryo-electron microscopy (cryo-EM) is fundamental for understanding and modulating their function. The LNTs in our studies are single bilayer galactosylceramide based nanotubes of ∼20 nm inner diameter and a few microns in length, that self-assemble in aqueous solutions. The lipid nanodisks (NDs) are self-assembled discoid lipid bilayers of ∼10 nm diameter, which are stabilized in aqueous solutions by a belt of amphipathic helical scaffold proteins. By combining LNT and ND technologies, we can examine structurally how the membrane curvature and lipid composition modulates the function of the membrane-associated proteins. As proof of principle, we have engineered these lipid nanotechnologies to mimic the activated platelet's phosphtaidylserine rich membrane and have successfully assembled functional membrane-bound coagulation factor VIII in vitro for structure determination by cryo-EM. The macromolecular organization of the proteins bound to ND and LNT are further defined by fitting the known atomic structures within the calculated three-dimensional maps. The combination of LNT and ND technologies offers a means to control the design and assembly of a wide range of functional membrane-associated proteins and complexes for structural studies by cryo-EM. The presented results confirm the suitability of the developed methodology for studying the functional structure of membrane-associated proteins, such as the coagulation factors, at a close to physiological environment. © 2014 Wiley Periodicals, Inc.

  3. Lipid emulsions differentially affect LPS-induced acute monocytes inflammation: in vitro effects on membrane remodeling and cell viability.

    PubMed

    Boisramé-Helms, Julie; Delabranche, Xavier; Klymchenko, Andrey; Drai, Jocelyne; Blond, Emilie; Zobairi, Fatiha; Mely, Yves; Hasselmann, Michel; Toti, Florence; Meziani, Ferhat

    2014-11-01

    The aim of this study was to assess how lipid emulsions for parenteral nutrition affect lipopolysaccharide (LPS)-induced acute monocyte inflammation in vitro. An 18 h long LPS induced human monocyte leukemia cell stimulation was performed and the cell-growth medium was supplemented with three different industrial lipid emulsions: Intralipid(®), containing long-chain triglycerides (LCT--soybean oil); Medialipid(®), containing LCT (soybean oil) and medium-chain triglycerides (MCT--coconut oil); and SMOFlipid(®), containing LCT, MCT, omega-9 and -3 (soybean, coconut, olive and fish oils). Cell viability and apoptosis were assessed by Trypan blue exclusion and flow cytometry respectively. Monocyte composition and membrane remodeling were studied using gas chromatography and NR12S staining. Microparticles released in supernatant were measured by prothrombinase assay. After LPS challenge, both cellular necrosis and apoptosis were increased (threefold and twofold respectively) and microparticle release was enhanced (sevenfold) after supplementation with Medialipid(®) compared to Intralipid(®), SMOFlipid(®) and monocytes in the standard medium. The monocytes differentially incorporated fatty acids after lipid emulsion challenge. Finally, lipid-treated cells displayed microparticles characterized by disrupted membrane lipid order, reflecting lipid remodeling of the parental cell plasma membrane. Our data suggest that lipid emulsions differentially alter cell viability, monocyte composition and thereby microparticle release. While MCT have deleterious effects, we have shown that parenteral nutrition emulsion containing LCT or LCT and MCT associated to n-3 and n-9 fatty acids have no effect on endotoxin-induced cell death and inflammation.

  4. Effect of Amphotericin B antibiotic on the properties of model lipid membrane

    NASA Astrophysics Data System (ADS)

    Kiryakova, S.; Dencheva-Zarkova, M.; Genova, J.

    2014-12-01

    Model membranes formed from natural and synthetic lipids are an interesting object for scientific investigations due to their similarity to biological cell membrane and their simple structure with controlled composition and properties. Amphotericin B is an important polyene antifungal antibiotic, used for treatment of systemic fungal infections. It is known from the literature that the studied antibiotic has a substantial effect on the transmembrane ionic channel structures. When applied to the lipid membranes it has the tendency to create pores and in this way to affect the structure and the properties of the membrane lipid bilayer. In this work the thermally induced shape fluctuations of giant quasi-spherical liposomes have been used to study the influence of polyene antibiotic amphotericin B on the elastic properties of model lipid membranes. It have been shown experimentally that the presence of 3 mol % of AmB in the lipid membrane reduces the bending elasticity of the lipid membrane for both studied cases: pure SOPC membrane and mixed SOPC-Cholesterol membrane. Interaction of the amphotericin B with bilayer lipid membranes containing channels have been studied in this work. Model membranes were self-assembled using the patch-clamp and tip-dip patch clamp technique. We have found that amphotericin B is an ionophore and reduces the resistance of the lipid bilayer.

  5. Membrane Disruption Mechanism of a Prion Peptide (106-126) Investigated by Atomic Force Microscopy, Raman and Electron Paramagnetic Resonance Spectroscopy.

    PubMed

    Pan, Jianjun; Sahoo, Prasana K; Dalzini, Annalisa; Hayati, Zahra; Aryal, Chinta M; Teng, Peng; Cai, Jianfeng; Rodriguez Gutierrez, Humberto; Song, Likai

    2017-05-18

    A fragment of the human prion protein spanning residues 106-126 (PrP106-126) recapitulates many essential properties of the disease-causing protein such as amyloidogenicity and cytotoxicity. PrP106-126 has an amphipathic characteristic that resembles many antimicrobial peptides (AMPs). Therefore, the toxic effect of PrP106-126 could arise from a direct association of monomeric peptides with the membrane matrix. Several experimental approaches are employed to scrutinize the impacts of monomeric PrP106-126 on model lipid membranes. Porous defects in planar bilayers are observed by using solution atomic force microscopy. Adding cholesterol does not impede defect formation. A force spectroscopy experiment shows that PrP106-126 reduces Young's modulus of planar lipid bilayers. We use Raman microspectroscopy to study the effect of PrP106-126 on lipid atomic vibrational dynamics. For phosphatidylcholine lipids, PrP106-126 disorders the intrachain conformation, while the interchain interaction is not altered; for phosphatidylethanolamine lipids, PrP106-126 increases the interchain interaction, while the intrachain conformational order remains similar. We explain the observed differences by considering different modes of peptide insertion. Finally, electron paramagnetic resonance spectroscopy shows that PrP106-126 progressively decreases the orientational order of lipid acyl chains in magnetically aligned bicelles. Together, our experimental data support the proposition that monomeric PrP106-126 can disrupt lipid membranes by using similar mechanisms found in AMPs.

  6. Membrane Disruption Mechanism of a Prion Peptide (106-126) Investigated by Atomic Force Microscopy, Raman and Electron Paramagnetic Resonance Spectroscopy

    PubMed Central

    Pan, Jianjun; Sahoo, Prasana K.; Dalzini, Annalisa; Hayati, Zahra; Aryal, Chinta M.; Teng, Peng; Cai, Jianfeng; Gutierrez, Humberto Rodriguez; Song, Likai

    2018-01-01

    A fragment of the human prion protein spanning residues 106-126 (PrP106-126) recapitulates many essential properties of the disease-causing protein such as amyloidogenicity and cytotoxicity. PrP106-126 has an amphipathic characteristic that resembles many antimicrobial peptides (AMPs). Therefore, the toxic effect of PrP106-126 could arise from a direct association of monomeric peptides with membrane matrix. Several experimental approaches are employed to scrutinize the impacts of monomeric PrP106-126 on model lipid membranes. Porous defects in planar bilayers are observed by using solution atomic force microscopy. Adding cholesterol does not impede defect formation. Force spectroscopy experiment shows that PrP106-126 reduces Young’s modulus of planar lipid bilayers. We use Raman microspectroscopy to study the effect of PrP106-126 on lipid vibrational dynamics. For phosphatidylcholine lipids, PrP106-126 disorders the intra-chain conformation, while the inter-chain interaction is not altered; for phosphatidylethanolamine lipids, PrP106-126 increases the inter-chain interaction, while the intra-chain conformational order remains similar. We explain the observed differences by considering different modes of peptide insertion. Finally, electron paramagnetic resonance spectroscopy shows that PrP106-126 progressively decreases the orientational order of lipid acyl chains in magnetically aligned bicelles. Together, our experimental data support the proposition that monomeric PrP106-126 can disrupt lipid membranes by using similar mechanisms found in AMPs. PMID:28459565

  7. Cell membrane disruption stimulates cAMP and Ca2+ signaling to potentiate cell membrane resealing in neighboring cells.

    PubMed

    Togo, Tatsuru

    2017-12-15

    Disruption of cellular plasma membranes is a common event in many animal tissues, and the membranes are usually rapidly resealed. Moreover, repeated membrane disruptions within a single cell reseal faster than the initial wound in a protein kinase A (PKA)- and protein kinase C (PKC)-dependent manner. In addition to wounded cells, recent studies have demonstrated that wounding of Madin-Darby canine kidney (MDCK) cells potentiates membrane resealing in neighboring cells in the short-term by purinergic signaling, and in the long-term by nitric oxide/protein kinase G signaling. In the present study, real-time imaging showed that cell membrane disruption stimulated cAMP synthesis and Ca 2+ mobilization from intracellular stores by purinergic signaling in neighboring MDCK cells. Furthermore, inhibition of PKA and PKC suppressed the ATP-mediated short-term potentiation of membrane resealing in neighboring cells. These results suggest that cell membrane disruption stimulates PKA and PKC via purinergic signaling to potentiate cell membrane resealing in neighboring MDCK cells. © 2017. Published by The Company of Biologists Ltd.

  8. Raman Imaging in Cell Membranes, Lipid-Rich Organelles, and Lipid Bilayers.

    PubMed

    Syed, Aleem; Smith, Emily A

    2017-06-12

    Raman-based optical imaging is a promising analytical tool for noninvasive, label-free chemical imaging of lipid bilayers and cellular membranes. Imaging using spontaneous Raman scattering suffers from a low intensity that hinders its use in some cellular applications. However, developments in coherent Raman imaging, surface-enhanced Raman imaging, and tip-enhanced Raman imaging have enabled video-rate imaging, excellent detection limits, and nanometer spatial resolution, respectively. After a brief introduction to these commonly used Raman imaging techniques for cell membrane studies, this review discusses selected applications of these modalities for chemical imaging of membrane proteins and lipids. Finally, recent developments in chemical tags for Raman imaging and their applications in the analysis of selected cell membrane components are summarized. Ongoing developments toward improving the temporal and spatial resolution of Raman imaging and small-molecule tags with strong Raman scattering cross sections continue to expand the utility of Raman imaging for diverse cell membrane studies.

  9. Defining the membrane disruption mechanism of kalata B1 via coarse-grained molecular dynamics simulations

    PubMed Central

    Nawae, Wanapinun; Hannongbua, Supa; Ruengjitchatchawalya, Marasri

    2014-01-01

    Kalata B1 has been demonstrated to have bioactivity relating to membrane disruption. In this study, we conducted coarse-grained molecular dynamics simulations to gain further insight into kB1 bioactivity. The simulations were performed at various concentrations of kB1 to capture the overall progression of its activity. Two configurations of kB1 oligomers, termed tower-like and wall-like clusters, were detected. The conjugation between the wall-like oligomers resulted in the formation of a ring-like hollow in the kB1 cluster on the membrane surface. Our results indicated that the molecules of kB1 were trapped at the membrane-water interface. The interfacial membrane binding of kB1 induced a positive membrane curvature, and the lipids were eventually extracted from the membrane through the kB1 ring-like hollow into the space inside the kB1 cluster. These findings provide an alternative view of the mechanism of kB1 bioactivity that corresponds with the concept of an interfacial bioactivity model. PMID:24492660

  10. Mesoscale organization of domains in the plasma membrane - beyond the lipid raft.

    PubMed

    Lu, Stella M; Fairn, Gregory D

    2018-04-01

    The plasma membrane is compartmentalized into several distinct regions or domains, which show a broad diversity in both size and lifetime. The segregation of lipids and membrane proteins is thought to be driven by the lipid composition itself, lipid-protein interactions and diffusional barriers. With regards to the lipid composition, the immiscibility of certain classes of lipids underlies the "lipid raft" concept of plasmalemmal compartmentalization. Historically, lipid rafts have been described as cholesterol and (glyco)sphingolipid-rich regions of the plasma membrane that exist as a liquid-ordered phase that are resistant to extraction with non-ionic detergents. Over the years the interest in lipid rafts grew as did the challenges with studying these nanodomains. The term lipid raft has fallen out of favor with many scientists and instead the terms "membrane raft" or "membrane nanodomain" are preferred as they connote the heterogeneity and dynamic nature of the lipid-protein assemblies. In this article, we will discuss the classical lipid raft hypothesis and its limitations. This review will also discuss alternative models of lipid-protein interactions, annular lipid shells, and larger membrane clusters. We will also discuss the mesoscale organization of plasmalemmal domains including visible structures such as clathrin-coated pits and caveolae.

  11. Monitoring the Orientational Changes of Alamethicin during Incorporation into Bilayer Lipid Membranes.

    PubMed

    Forbrig, Enrico; Staffa, Jana K; Salewski, Johannes; Mroginski, Maria Andrea; Hildebrandt, Peter; Kozuch, Jacek

    2018-02-13

    Antimicrobial peptides (AMPs) are the first line of defense after contact of an infectious invader, for example, bacterium or virus, with a host and an integral part of the innate immune system of humans. Their broad spectrum of biological functions ranges from cell membrane disruption over facilitation of chemotaxis to interaction with membrane-bound or intracellular receptors, thus providing novel strategies to overcome bacterial resistances. Especially, the clarification of the mechanisms and dynamics of AMP incorporation into bacterial membranes is of high interest, and different mechanistic models are still under discussion. In this work, we studied the incorporation of the peptaibol alamethicin (ALM) into tethered bilayer lipid membranes on electrodes in combination with surface-enhanced infrared absorption (SEIRA) spectroscopy. This approach allows monitoring the spontaneous and potential-induced ion channel formation of ALM in situ. The complex incorporation kinetics revealed a multistep mechanism that points to peptide-peptide interactions prior to penetrating the membrane and adopting the transmembrane configuration. On the basis of the anisotropy of the backbone amide I and II infrared absorptions determined by density functional theory calculations, we employed a mathematical model to evaluate ALM reorientations monitored by SEIRA spectroscopy. Accordingly, ALM was found to adopt inclination angles of ca. 69°-78° and 21° in its interfacially adsorbed and transmembrane incorporated states, respectively. These orientations can be stabilized efficiently by the dipolar interaction with lipid head groups or by the application of a potential gradient. The presented potential-controlled mechanistic study suggests an N-terminal integration of ALM into membranes as monomers or parallel oligomers to form ion channels composed of parallel-oriented helices, whereas antiparallel oligomers are barred from intrusion.

  12. Black Lipid Membranes at Bifaces

    PubMed Central

    Ti Tien, H.

    1968-01-01

    Black lipid membranes (BLM) less than 90 A thick have been shown to be the most realistic approach to biological membrane models. This paper describes the formation characteristics, optical properties, and thermodynamics of BLM at water/oil/water bifaces. In particular, the nature of the Plateau-Gibbs border which supports the black membrane is analyzed in some detail. The formation of BLM at the biface involves a spontaneous reduction of the free energy of the system. As long as the integrity of the membrane is maintained, the limiting structure of the BLM represents the lowest free energy configuration. PMID:19873618

  13. Lipid rafts regulate PCB153-induced disruption of occludin and brain endothelial barrier function through protein phosphatase 2A and matrix metalloproteinase-2

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Eum, Sung Yong, E-mail: seum@miami.edu; Jaraki, Dima; András, Ibolya E.

    Occludin is an essential integral transmembrane protein regulating tight junction (TJ) integrity in brain endothelial cells. Phosphorylation of occludin is associated with its localization to TJ sites and incorporation into intact TJ assembly. The present study is focused on the role of lipid rafts in polychlorinated biphenyl (PCB)-induced disruption of occludin and endothelial barrier function. Exposure of human brain endothelial cells to 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB153) induced dephosphorylation of threonine residues of occludin and displacement of occludin from detergent-resistant membrane (DRM)/lipid raft fractions within 1 h. Moreover, lipid rafts modulated the reduction of occludin level through activation of matrix metalloproteinase 2 (MMP-2)more » after 24 h PCB153 treatment. Inhibition of protein phosphatase 2A (PP2A) activity by okadaic acid or fostriecin markedly protected against PCB153-induced displacement of occludin and increased permeability of endothelial cells. The implication of lipid rafts and PP2A signaling in these processes was further defined by co-immunoprecipitation of occludin with PP2A and caveolin-1, a marker protein of lipid rafts. Indeed, a significant MMP-2 activity was observed in lipid rafts and was increased by exposure to PCB153. The pretreatment of MMP-2 inhibitors protected against PCB153-induced loss of occludin and disruption of lipid raft structure prevented the increase of endothelial permeability. Overall, these results indicate that lipid raft-associated processes, such as PP2A and MMP-2 activation, participate in PCB153-induced disruption of occludin function in brain endothelial barrier. This study contributes to a better understanding of the mechanisms leading to brain endothelial barrier dysfunction in response to exposure to environmental pollutants, such as ortho-substituted PCBs. - Highlights: • PCB153 disturbed human brain endothelial barrier through disruption of occludin. • Lipid raft

  14. Immobilization of acetylcholinesterase in lipid membranes deposited on self-assembled monolayers.

    PubMed

    Milkani, Eftim; Khaing, Aung M; Huang, Fei; Gibson, Daniel G; Gridley, Scott; Garceau, Norman; Lambert, Christopher R; McGimpsey, W Grant

    2010-12-21

    Human red blood cell acetylcholinesterase was incorporated into planar lipid membranes deposited on alkanethiol self-assembled monolayers (SAMs) on gold substrates. Activity of the protein in the membrane was detected with a standard photometric assay and was determined to be similar to the protein in detergent solution or incorporated in lipid vesicles. Monolayer and bilayer lipid membranes were generated by fusing liposomes to hydrophobic and hydrophilic SAMs, respectively. Liposomes were formed by the injection method using the lipid dimyristoylphosphatidylcholine (DMPC). The formation of alkanethiol SAMs and lipid monolayers on SAMs was confirmed by sessile drop goniometry, ellipsometry, and electrochemical impedance spectroscopy. In this work, we report acetylcholinesterase immobilization in lipid membranes deposited on SAMs formed on the gold surface and compare its activity to enzyme in solution.

  15. Cell disruption and lipid extraction for microalgal biorefineries: A review.

    PubMed

    Lee, Soo Youn; Cho, Jun Muk; Chang, Yong Keun; Oh, You-Kwan

    2017-11-01

    The microalgae-based biorefinement process has attracted much attention from academic and industrial researchers attracted to its biofuel, food and nutraceutical applications. In this paper, recent developments in cell-disruption and lipid-extraction methods, focusing on four biotechnologically important microalgal species (namely, Chlamydomonas, Haematococcus, Chlorella, and Nannochloropsis spp.), are reviewed. The structural diversity and rigidity of microalgal cell walls complicate the development of efficient downstream processing methods for cell-disruption and subsequent recovery of intracellular lipid and pigment components. Various mechanical, chemical and biological cell-disruption methods are discussed in detail and compared based on microalgal species and status (wet/dried), scale, energy consumption, efficiency, solvent extraction, and synergistic combinations. The challenges and prospects of the downstream processes for the future development of eco-friendly and economical microalgal biorefineries also are outlined herein. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Atomistic Monte Carlo Simulation of Lipid Membranes

    PubMed Central

    Wüstner, Daniel; Sklenar, Heinz

    2014-01-01

    Biological membranes are complex assemblies of many different molecules of which analysis demands a variety of experimental and computational approaches. In this article, we explain challenges and advantages of atomistic Monte Carlo (MC) simulation of lipid membranes. We provide an introduction into the various move sets that are implemented in current MC methods for efficient conformational sampling of lipids and other molecules. In the second part, we demonstrate for a concrete example, how an atomistic local-move set can be implemented for MC simulations of phospholipid monomers and bilayer patches. We use our recently devised chain breakage/closure (CBC) local move set in the bond-/torsion angle space with the constant-bond-length approximation (CBLA) for the phospholipid dipalmitoylphosphatidylcholine (DPPC). We demonstrate rapid conformational equilibration for a single DPPC molecule, as assessed by calculation of molecular energies and entropies. We also show transition from a crystalline-like to a fluid DPPC bilayer by the CBC local-move MC method, as indicated by the electron density profile, head group orientation, area per lipid, and whole-lipid displacements. We discuss the potential of local-move MC methods in combination with molecular dynamics simulations, for example, for studying multi-component lipid membranes containing cholesterol. PMID:24469314

  17. G protein-membrane interactions II: Effect of G protein-linked lipids on membrane structure and G protein-membrane interactions.

    PubMed

    Casas, Jesús; Ibarguren, Maitane; Álvarez, Rafael; Terés, Silvia; Lladó, Victoria; Piotto, Stefano P; Concilio, Simona; Busquets, Xavier; López, David J; Escribá, Pablo V

    2017-09-01

    G proteins often bear myristoyl, palmitoyl and isoprenyl moieties, which favor their association with the membrane and their accumulation in G Protein Coupled Receptor-rich microdomains. These lipids influence the biophysical properties of membranes and thereby modulate G protein binding to bilayers. In this context, we showed here that geranylgeraniol, but neither myristate nor palmitate, increased the inverted hexagonal (H II ) phase propensity of phosphatidylethanolamine-containing membranes. While myristate and palmitate preferentially associated with phosphatidylcholine membranes, geranylgeraniol favored nonlamellar-prone membranes. In addition, Gαi 1 monomers had a higher affinity for lamellar phases, while Gβγ and Gαβγ showed a marked preference for nonlamellar prone membranes. Moreover, geranylgeraniol enhanced the binding of G protein dimers and trimers to phosphatidylethanolamine-containing membranes, yet it decreased that of monomers. By contrast, both myristate and palmitate increased the Gαi 1 preference for lamellar membranes. Palmitoylation reinforced the binding of the monomer to PC membranes and myristoylation decreased its binding to PE-enriched bilayer. Finally, binding of dimers and trimers to lamellar-prone membranes was decreased by palmitate and myristate, but it was increased in nonlamellar-prone bilayers. These results demonstrate that co/post-translational G protein lipid modifications regulate the membrane lipid structure and that they influence the physico-chemical properties of membranes, which in part explains why G protein subunits sort to different plasma membrane domains. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. How sterol tilt regulates properties and organization of lipid membranes and membrane insertions

    PubMed Central

    Khelashvili, George; Harries, Daniel

    2013-01-01

    Serving as a crucial component of mammalian cells, cholesterol critically regulates the functions of biomembranes. This review focuses on a specific property of cholesterol and other sterols: the tilt modulus χ that quantifies the energetic cost of tilting sterol molecules inside the lipid membrane. We show how χ is involved in determining properties of cholesterol-containing membranes, and detail a novel approach to quantify its value from atomistic molecular dynamics (MD) simulations. Specifically, we link χ with other structural, thermodynamic, and mechanical properties of cholesterol-containing lipid membranes, and delineate how this useful parameter can be obtained from the sterol tilt probability distributions derived from relatively small-scale unbiased MD simulations. We demonstrate how the tilt modulus quantitatively describes the aligning field that sterol molecules create inside the phospholipid bilayers, and we relate χ to the bending rigidity of the lipid bilayer through effective tilt and splay energy contributions to the elastic deformations. Moreover, we show how χ can conveniently characterize the “condensing effect” of cholesterol on phospholipids. Finally, we demonstrate the importance of this cholesterol aligning field to the proper folding and interactions of membrane peptides. Given the relative ease of obtaining the tilt modulus from atomistic simulations, we propose that χ can be routinely used to characterize the mechanical properties of sterol/lipid bilayers, and can also serve as a required fitting parameter in multi-scaled simulations of lipid membrane models to relate the different levels of coarse-grained details. PMID:23291283

  19. Membrane raft organization is more sensitive to disruption by (n-3) PUFA than nonraft organization in EL4 and B cells.

    PubMed

    Rockett, Benjamin Drew; Franklin, Andrew; Harris, Mitchel; Teague, Heather; Rockett, Alexis; Shaikh, Saame Raza

    2011-06-01

    Model membrane and cellular detergent extraction studies show (n-3) PUFA predominately incorporate into nonrafts; thus, we hypothesized (n-3) PUFA could disrupt nonraft organization. The first objective of this study was to determine whether (n-3) PUFA disrupted nonrafts of EL4 cells, an extension of our previous work in which we discovered an (n-3) PUFA diminished raft clustering. EPA or DHA treatment of EL4 cells increased plasma membrane accumulation of the nonraft probe 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate by ~50-70% relative to a BSA control. Förster resonance energy transfer imaging showed EPA and DHA also disrupted EL4 nanometer scale nonraft organization by increasing the distance between nonraft molecules by ~25% compared with BSA. However, changes in nonrafts were due to an increase in cell size; under conditions where EPA or DHA did not increase cell size, nonraft organization was unaffected. We next translated findings on EL4 cells by testing if (n-3) PUFA administered to mice disrupted nonrafts and rafts. Imaging of B cells isolated from mice fed low- or high-fat (HF) (n-3) PUFA diets showed no change in nonraft organization compared with a control diet (CD). However, confocal microscopy revealed the HF (n-3) PUFA diet disrupted lipid raft clustering and size by ~40% relative to CD. Taken together, our data from 2 different model systems suggest (n-3) PUFA have limited effects on nonrafts. The ex vivo data, which confirm previous studies with EL4 cells, provide evidence that (n-3) PUFA consumed through the diet disrupt B cell lipid raft clustering.

  20. Enhanced membrane disruption and antibiotic action against pathogenic bacteria by designed histidine-rich peptides at acidic pH.

    PubMed

    Mason, A James; Gasnier, Claire; Kichler, Antoine; Prévost, Gilles; Aunis, Dominique; Metz-Boutigue, Marie-Hélène; Bechinger, Burkhard

    2006-10-01

    The histidine-rich amphipathic cationic peptide LAH4 has antibiotic and DNA delivery capabilities. Here, we explore the interaction of peptides from this family with model membranes as monitored by solid-state (2)H nuclear magnetic resonance and their antibiotic activities against a range of bacteria. At neutral pH, the membrane disruption is weak, but at acidic pH, the peptides strongly disturb the anionic lipid component of bacterial membranes and cause bacterial lysis. The peptides are effective antibiotics at both pH 7.2 and pH 5.5, although the antibacterial activity is strongly affected by the change in pH. At neutral pH, the LAH peptides were active against both methicillin-resistant and -sensitive Staphylococcus aureus strains but ineffective against Pseudomonas aeruginosa. In contrast, the LAH peptides were highly active against P. aeruginosa in an acidic environment, as is found in the epithelial-lining fluid of cystic fibrosis patients. Our results show that modest antibiotic activity of histidine-rich peptides can be dramatically enhanced by inducing membrane disruption, in this case by lowering the pH, and that histidine-rich peptides have potential as future antibiotic agents.

  1. The effect of polyethylene glycol-modified lipids on the interaction of HIV-1 derived peptide-dendrimer complexes with lipid membranes.

    PubMed

    Melikishvili, Sophie; Poturnayova, Alexandra; Ionov, Maksim; Bryszewska, Maria; Vary, Tomáš; Cirak, Julius; Muñoz-Fernández, María Ángeles; Gomez-Ramirez, Rafael; de la Mata, Francisco Javier; Hianik, Tibor

    2016-12-01

    In this study, dendrimers have been purposed as an alternative approach for delivery of HIV peptides to dendritic cells. We have investigated the interaction of dendriplexes formed from polyanionic HIV peptide Nef and cationic carbosilane dendrimer (CBD) with model lipid membranes - large unilamellar vesicles (LUVs), Langmuir monolayers and supported lipid membranes (sBLMs) containing various molar ratio of zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG 2000 ). In our experiments, the lipid membranes represented the model of the plasma membrane of the cell. PEGylated lipids were used in order to model glycocalyx which constitutes the outer leaflet of cellular membranes. The presence of PEGylated lipids resulted in an increase of the phase transition temperature of the lipid bilayer of LUVs, in a decrease of specific volume and adiabatic compressibility. Fluorescence anisotropy study suggests that PEGylated LUVs possessed higher lipid order and decreased fluidity when compared to zwitterionic DMPC vesicles. The interaction of dendriplexes with monolayers was accompanied by the formation of the aggregates as revealed by BAM experiments. This conclusion has been confirmed also by AFM imaging of sBLMs. We have demonstrated that dendriplexes interact with lipid membranes for all types of lipid composition. Moreover, the stronger interaction of cationic dendrimer/peptide complexes with lipid monolayers, vesicles and sBLMs was observed for membranes composed of zwitterionic lipids than for PEGylated lipid membranes. Increased concentration of PEGylated lipids made this interaction weaker. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Antiproliferative effects of γ-tocotrienol are associated with lipid raft disruption in HER2-positive human breast cancer cells.

    PubMed

    Alawin, Osama A; Ahmed, Rayan A; Ibrahim, Baher A; Briski, Karen P; Sylvester, Paul W

    2016-01-01

    A large percentage of human breast cancers are characterized by excessive or aberrant HER2 activity. Lipid rafts are specialized microdomains within the plasma membrane that are required for HER2 activation and signal transduction. Since the anticancer activity of γ-tocotrienol is associated with suppression in HER2 signaling, studies were conducted to examine the effects of γ-tocotrienol on HER2 activation within the lipid raft microdomain in HER2-positive SKBR3 and BT474 human breast cancer cells. Treatment with 0-5μM γ-tocotrienol induced a significant dose-dependent inhibition in cancer cell growth after a 5-day culture period, and these growth inhibitory effects were associated with a reduction in HER2 dimerization and phosphorylation (activation). Phosphorylated HER2 was found to be primarily located in the lipid raft microdomain of the plasma membrane in vehicle-treated control groups, whereas γ-tocotrienol treatment significantly inhibited this effect. Assay of plasma membrane subcellular fractions showed that γ-tocotrienol also accumulates exclusively within the lipid raft microdomain. Hydroxypropyl-β-cyclodextrin (HPβCD) is an agent that disrupts lipid raft integrity. Acute exposure to 3mM HPβCD alone had no effect, whereas an acute 24-h exposure to 20μM γ-tocotrienol alone significantly decreased SKBR3 and BT474 cell viability. However, combined treatment with these agents greatly reduced γ-tocotrienol accumulation in the lipid raft microdomain and cytotoxicity. In summary, these findings demonstrate that the anticancer effects of γ-tocotrienol are associated with its accumulation in the lipid raft microdomain and subsequent interference with HER2 dimerization and activation in SKBR3 and BT474 human breast cancer cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Advances in nanopatterned and nanostructured supported lipid membranes and their applications.

    PubMed

    Reimhult, Erik; Baumann, Martina; Kaufmann, Stefan; Kumar, Karthik; Spycher, Philipp

    2010-01-01

    Lipid membranes are versatile and convenient alternatives to study the properties of natural cell membranes. Self-assembled, artificial, substrate-supported lipid membranes have taken a central role in membrane research due to a combination of factors such as ease of creation, control over complexity, stability and the applicability of a large range of different analytical techniques. While supported lipid bilayers have been investigated for several decades, recent advances in the understanding of the assembly of such membranes from liposomes have spawned a renaissance in the field. Supported lipid bilayers are a highly promising tool to study transmembrane proteins in their native state, an application that could have tremendous impact on, e.g. drug discovery, development of biointerfaces and as platforms for glycomics and probing of multivalent binding which requires ligand mobility. Parallel advances in microfluidics, biosensor design, micro- and nanofabrication have converged to bring self-assembled supported lipid bilayers closer to a versatile and easy to use research tool as well as closer to industrial applications. The field of supported lipid bilayer research and application is thus rapidly expanding and diversifying with new platforms continuously being proposed and developed. In order to use supported lipid bilayers for such applications several advances have to be made: decoupling of the membrane from the support while maintaining it close to the surface, making use of biologically relevant lipid compositions, patterning of lipid membranes into arrays, and application to nanostructured substrates and sensors. This review summarizes recent advances in the field which addresses these challenges.

  4. Solid-Supported Lipid Membranes: Formation, Stability and Applications

    NASA Astrophysics Data System (ADS)

    Goh, Haw Zan

    This thesis presents a comprehensive investigation of the formation of supported lipid membranes with vesicle hemifusion, their stability under detergents and organic solvents and their applications in molecular biology. In Chapter 3, we describe how isolated patches of DOPC bilayers supported on glass surfaces are dissolved by various detergents (decyl maltoside, dodecyl maltoside, CHAPS, CTAB, SDS, TritonX-100 and Tween20) at their CMC, as investigated by fluorescence video microscopy. In general, detergents partition into distal leaflets of bilayers and lead to the expansion of the bilayers through a rolling motion of the distal over the proximal leaflets, in agreement with the first stage of the established 3-stage model of lipid vesicle solubilization by detergents. Subsequently, we study the partitioning of organic solvents (methanol, ethanol, isopropanol, propanol, acetone and chloroform) into isolated bilayer patches on glass in Chapter 4 with fluorescence microscopy. The area expansion of bilayers due to the partitioning of organic solvents is measured. From the titration of organic solvents, we measured the rate of area expansion as a function of the volume fraction of organic solvents, which is proposed to be a measure of strength of interactions between solvents and membranes. From the same experiments, we also measure the maximum expansion of bilayers (or the maximum binding stoichiometry between organic solvents and lipids) before structural breakdown, which depends on the depth of penetration of solvents to the membranes. In Chapter 5, we investigate the formation of sparsely-tethered bilayer lipid membranes (stBLMs) with vesicle hemifusion. In vesicle hemifusion, lipid vesicles in contact with a hydrophobic alkyl-terminated self-assembled monolayer (SAM) deposit a lipid monolayer to the SAM surface, thus completing the bilayer. Electrical Impedance Spectroscopy and Neutron Reflectivity are used to probe the integrity of stBLMs in terms of their

  5. Membrane Curvature and Lipid Composition Synergize To Regulate N-Ras Anchor Recruitment.

    PubMed

    Larsen, Jannik B; Kennard, Celeste; Pedersen, Søren L; Jensen, Knud J; Uline, Mark J; Hatzakis, Nikos S; Stamou, Dimitrios

    2017-09-19

    Proteins anchored to membranes through covalently linked fatty acids and/or isoprenoid groups play crucial roles in all forms of life. Sorting and trafficking of lipidated proteins has traditionally been discussed in the context of partitioning to membrane domains of different lipid composition. We recently showed that membrane shape/curvature can in itself mediate the recruitment of lipidated proteins. However, exactly how membrane curvature and composition synergize remains largely unexplored. Here we investigated how three critical structural parameters of lipids, namely acyl chain saturation, headgroup size, and acyl chain length, modulate the capacity of membrane curvature to recruit lipidated proteins. As a model system we used the lipidated minimal membrane anchor of the GTPase, N-Ras (tN-Ras). Our data revealed complex synergistic effects, whereby tN-Ras binding was higher on planar DOPC than POPC membranes, but inversely higher on curved POPC than DOPC membranes. This variation in the binding to both planar and curved membranes leads to a net increase in the recruitment by membrane curvature of tN-Ras when reducing the acyl chain saturation state. Additionally, we found increased recruitment by membrane curvature of tN-Ras when substituting PC for PE, and when decreasing acyl chain length from 14 to 12 carbons (DMPC versus DLPC). However, these variations in recruitment ability had different origins, with the headgroup size primarily influencing tN-Ras binding to planar membranes whereas the change in acyl chain length primarily affected binding to curved membranes. Molecular field theory calculations recapitulated these findings and revealed lateral pressure as an underlying biophysical mechanism dictating how curvature and composition synergize to modulate recruitment of lipidated proteins. Our findings suggest that the different compositions of cellular compartments could modulate the potency of membrane curvature to recruit lipidated proteins and

  6. Monoolein Lipid Phases as Incorporation and Enrichment Materials for Membrane Protein Crystallization

    PubMed Central

    Wallace, Ellen; Dranow, David; Laible, Philip D.; Christensen, Jeff; Nollert, Peter

    2011-01-01

    The crystallization of membrane proteins in amphiphile-rich materials such as lipidic cubic phases is an established methodology in many structural biology laboratories. The standard procedure employed with this methodology requires the generation of a highly viscous lipidic material by mixing lipid, for instance monoolein, with a solution of the detergent solubilized membrane protein. This preparation is often carried out with specialized mixing tools that allow handling of the highly viscous materials while minimizing dead volume to save precious membrane protein sample. The processes that occur during the initial mixing of the lipid with the membrane protein are not well understood. Here we show that the formation of the lipidic phases and the incorporation of the membrane protein into such materials can be separated experimentally. Specifically, we have investigated the effect of different initial monoolein-based lipid phase states on the crystallization behavior of the colored photosynthetic reaction center from Rhodobacter sphaeroides. We find that the detergent solubilized photosynthetic reaction center spontaneously inserts into and concentrates in the lipid matrix without any mixing, and that the initial lipid material phase state is irrelevant for productive crystallization. A substantial in-situ enrichment of the membrane protein to concentration levels that are otherwise unobtainable occurs in a thin layer on the surface of the lipidic material. These results have important practical applications and hence we suggest a simplified protocol for membrane protein crystallization within amphiphile rich materials, eliminating any specialized mixing tools to prepare crystallization experiments within lipidic cubic phases. Furthermore, by virtue of sampling a membrane protein concentration gradient within a single crystallization experiment, this crystallization technique is more robust and increases the efficiency of identifying productive crystallization

  7. Lipid-protein interactions in plasma membranes of fiber cells isolated from the human eye lens.

    PubMed

    Raguz, Marija; Mainali, Laxman; O'Brien, William J; Subczynski, Witold K

    2014-03-01

    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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Lipid-Protein Interactions in Plasma Membranes of Fiber Cells Isolated from the Human Eye Lens

    PubMed Central

    Raguz, Marija; Mainali, Laxman; O’Brien, William J.; Subczynski, Witold K.

    2014-01-01

    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. PMID:24486794

  9. Active processes make mixed lipid membranes either flat or crumpled

    NASA Astrophysics Data System (ADS)

    Banerjee, Tirthankar; Basu, Abhik

    2018-01-01

    Whether live cell membranes show miscibility phase transitions (MPTs), and if so, how they fluctuate near the transitions remain outstanding unresolved issues in physics and biology alike. Motivated by these questions we construct a generic hydrodynamic theory for lipid membranes that are active, due for instance, to the molecular motors in the surrounding cytoskeleton, or active protein components in the membrane itself. We use this to uncover a direct correspondence between membrane fluctuations and MPTs. Several testable predictions are made: (i) generic active stiffening with orientational long range order (flat membrane) or softening with crumpling of the membrane, controlled by the active tension and (ii) for mixed lipid membranes, capturing the nature of putative MPTs by measuring the membrane conformation fluctuations. Possibilities of both first and second order MPTs in mixed active membranes are argued for. Near second order MPTs, active stiffening (softening) manifests as a super-stiff (super-soft) membrane. Our predictions are testable in a variety of in vitro systems, e.g. live cytoskeletal extracts deposited on liposomes and lipid membranes containing active proteins embedded in a passive fluid.

  10. Cyclohexane Rings Reduce Membrane Permeability to Small Ions in Archaea-Inspired Tetraether Lipids.

    PubMed

    Koyanagi, Takaoki; Leriche, Geoffray; Onofrei, David; Holland, Gregory P; Mayer, Michael; Yang, Jerry

    2016-01-26

    Extremophile archaeal organisms overcome problems of membrane permeability by producing lipids with structural elements that putatively improve membrane integrity compared to lipids from other life forms. Herein, we describe a series of lipids that mimic some key structural features of archaeal lipids, such as: 1) single tethering of lipid tails to create fully transmembrane tetraether lipids and 2) the incorporation of small rings into these tethered segments. We found that membranes formed from pure tetraether lipids leaked small ions at a rate that was about two orders of magnitude slower than common bilayer-forming lipids. Incorporation of cyclopentane rings into the tetraether lipids did not affect membrane leakage, whereas a cyclohexane ring reduced leakage by an additional 40 %. These results show that mimicking certain structural features of natural archaeal lipids results in improved membrane integrity, which may help overcome limitations of many current lipid-based technologies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Determining Membrane Protein-Lipid Binding Thermodynamics Using Native Mass Spectrometry.

    PubMed

    Cong, Xiao; Liu, Yang; Liu, Wen; Liang, Xiaowen; Russell, David H; Laganowsky, Arthur

    2016-04-06

    Membrane proteins are embedded in the biological membrane where the chemically diverse lipid environment can modulate their structure and function. However, the thermodynamics governing the molecular recognition and interaction of lipids with membrane proteins is poorly understood. Here, we report a method using native mass spectrometry (MS), to determine thermodynamics of individual ligand binding events to proteins. Unlike conventional methods, native MS can resolve individual ligand binding events and, coupled with an apparatus to control the temperature, determine binding thermodynamic parameters, such as for protein-lipid interactions. We validated our approach using three soluble protein-ligand systems (maltose binding protein, lysozyme, and nitrogen regulatory protein) and obtained similar results to those using isothermal titration calorimetry and surface plasmon resonance. We also determined for the first time the thermodynamics of individual lipid binding to the ammonia channel (AmtB), an integral membrane protein from Escherichia coli. Remarkably, we observed distinct thermodynamic signatures for the binding of different lipids and entropy-enthalpy compensation for binding lipids of variable chain length. Additionally, using a mutant form of AmtB that abolishes a specific phosphatidylglycerol (PG) binding site, we observed distinct changes in the thermodynamic signatures for binding PG, implying these signatures can identify key residues involved in specific lipid binding and potentially differentiate between specific lipid binding sites.

  12. The role of interfacial lipids in stabilizing membrane protein oligomers.

    PubMed

    Gupta, Kallol; Donlan, Joseph A C; Hopper, Jonathan T S; Uzdavinys, Povilas; Landreh, Michael; Struwe, Weston B; Drew, David; Baldwin, Andrew J; Stansfeld, Phillip J; Robinson, Carol V

    2017-01-19

    Oligomerization of membrane proteins in response to lipid binding has a critical role in many cell-signalling pathways but is often difficult to define or predict. Here we report the development of a mass spectrometry platform to determine simultaneously the presence of interfacial lipids and oligomeric stability and to uncover how lipids act as key regulators of membrane-protein association. Evaluation of oligomeric strength for a dataset of 125 α-helical oligomeric membrane proteins reveals an absence of interfacial lipids in the mass spectra of 12 membrane proteins with high oligomeric stability. For the bacterial homologue of the eukaryotic biogenic transporters (LeuT, one of the proteins with the lowest oligomeric stability), we found a precise cohort of lipids within the dimer interface. Delipidation, mutation of lipid-binding sites or expression in cardiolipin-deficient Escherichia coli abrogated dimer formation. Molecular dynamics simulation revealed that cardiolipin acts as a bidentate ligand, bridging across subunits. Subsequently, we show that for the Vibrio splendidus sugar transporter SemiSWEET, another protein with low oligomeric stability, cardiolipin shifts the equilibrium from monomer to functional dimer. We hypothesized that lipids are essential for dimerization of the Na + /H + antiporter NhaA from E. coli, which has the lowest oligomeric strength, but not for the substantially more stable homologous Thermus thermophilus protein NapA. We found that lipid binding is obligatory for dimerization of NhaA, whereas NapA has adapted to form an interface that is stable without lipids. Overall, by correlating interfacial strength with the presence of interfacial lipids, we provide a rationale for understanding the role of lipids in both transient and stable interactions within a range of α-helical membrane proteins, including G-protein-coupled receptors.

  13. The role of interfacial lipids in stabilising membrane protein oligomers

    PubMed Central

    Uzdavinys, Povilas; Landreh, Michael; Struwe, Weston B.; Drew, David; Baldwin, Andrew J.; Stansfeld, Phillip J.; Robinson, Carol V.

    2017-01-01

    Oligomerisation of membrane proteins in response to lipid binding plays a critical role in many cell-signaling pathways 1 but is often difficult to define 2 or predict 3. Here we develop a mass spectrometry platform to determine simultaneously presence of interfacial lipids and oligomeric stability and discover how lipids act as key regulators of membrane protein association. Evaluation of oligomeric strength for a dataset of 125 α-helical oligomeric membrane proteins revealed an absence of interfacial lipids in the mass spectra of 12 membrane proteins with high oligomeric stability. For the bacterial homologue of the eukaryotic biogenic transporters (LeuT) 4 one of the proteins with the lowest oligomeric stability, we found a precise cohort of lipids within the dimer interface. Delipidation, mutation of lipid binding sites or expression in cardiolipin (CDL) deficient Escherichia coli, abrogated dimer formation. Molecular dynamics simulation revealed that CDL acts as a bidentate ligand bridging across subunits. Subsequently, we show that for the sugar transporter SemiSWEET from Vibrio splendidus 5, another protein with low oligomeric stability, cardiolipin shifts the equilibrium from monomer to functional dimer. We hypothesised that lipids would be essential for dimerisation of the Na+/H+ antiporter NhaA from E. coli, which has the lowest oligomeric strength, but not for substantially more stable, homologous NapA from Thermus thermophilus. We found that lipid binding is obligatory for dimerisation of NhaA, whereas NapA has adapted to form an interface that is stable without lipids. Overall, by correlating interfacial strength with the presence of interfacial lipids we provide a rationale for understanding the role of lipids in both transient and stable interactions within a range of α-helical membrane proteins, including GPCRs. PMID:28077870

  14. Lipid membrane-assisted condensation and assembly of amphiphilic Janus particles

    DOE PAGES

    Chambers, Mariah; Mallory, Stewart Anthony; Malone, Heather; ...

    2016-01-01

    Amphiphilic Janus particles self-assemble into complex metastructures, but little is known about how their assembly might be modified by weak interactions with a nearby biological membrane surface. Here, we report an integrated experimental and molecular dynamics simulation study to investigate the self-assembly of amphiphilic Janus particles on a lipid membrane. We created an experimental system in which Janus particles are allowed to self-assemble in the same medium where zwitterionic lipids form giant unilamellar vesicles (GUVs). Janus particles spontaneously concentrated on the inner leaflet of the GUVs. They exhibited biased orientation and heterogeneous rotational dynamics as revealed by single particle rotationalmore » tracking. The combined experimental and simulation results show that Janus particles concentrate on the lipid membranes due to weak particle–lipid attraction, whereas the biased orientation of particles is driven predominantly by inter-particle interactions. Furthermore, this study demonstrates the potential of using lipid membranes to influence the self-assembly of Janus particles.« less

  15. Isolation and analysis of membrane lipids and lipid rafts in common carp (Cyprinus carpio L.).

    PubMed

    Brogden, Graham; Propsting, Marcus; Adamek, Mikolaj; Naim, Hassan Y; Steinhagen, Dieter

    2014-03-01

    Cell membranes act as an interface between the interior of the cell and the exterior environment and facilitate a range of essential functions including cell signalling, cell structure, nutrient uptake and protection. It is composed of a lipid bilayer with integrated proteins, and the inner leaflet of the lipid bilayer comprises of liquid ordered (Lo) and liquid disordered (Ld) domains. Lo microdomains, also named as lipid rafts are enriched in cholesterol, sphingomyelin and certain types of proteins, which facilitate cell signalling and nutrient uptake. Lipid rafts have been extensively researched in mammals and the presence of functional lipid rafts was recently demonstrated in goldfish, but there is currently very little knowledge about their composition and function in fish. Therefore a protocol was established for the analysis of lipid rafts and membranous lipids in common carp (Cyprinus carpio L.) tissues. Twelve lipids were identified and analysed in the Ld domain of the membrane with the most predominant lipids found in all tissues being; triglycerides, cholesterol, phosphoethanolamine and phosphatidylcholine. Four lipids were identified in lipid rafts in all tissues analysed, triglycerides (33-62%) always found in the highest concentration followed by cholesterol (24-32%), phosphatidylcholine and sphingomyelin. Isolation of lipid rafts was confirmed by identifying the presence of the lipid raft associated protein flotillin, present at higher concentrations in the detergent resistant fraction. The data provided here build a lipid library of important carp tissues as a baseline for further studies into virus entry, protein trafficking or environmental stress analysis. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. Micrometer-Scale Membrane Transition of Supported Lipid Bilayer Membrane Reconstituted with Cytosol of Dictyostelium discoideum.

    PubMed

    Takahashi, Kei; Toyota, Taro

    2017-03-07

    The transformation of the supported lipid bilayer (SLB) membrane by extracted cytosol from living resources, has recently drawn much attention. It enables us to address the question of whether the purified phospholipid SLB membrane, including lipids related to amoeba locomotion, which was discussed in many previous studies, exhibits membrane deformation in the presence of cytosol extracted from amoeba; Methods: In this report, a method for reconstituting a supported lipid bilayer (SLB) membrane, composed of purified phospholipids and cytosol extracted from Dictyostelium discoideum , is described. This technique is a new reconstitution method combining the artificial constitution of membranes with the reconstitution using animate cytosol (without precise purification at a molecular level), contributing to membrane deformation analysis; Results: The morphology transition of a SLB membrane composed of phosphatidylcholines, after the addition of cytosolic extract, was traced using a confocal laser scanning fluorescence microscope. As a result, pore formation in the SLB membrane was observed and phosphatidylinositides incorporated into the SLB membrane tended to suppress pore formation and expansion; Conclusions: The current findings imply that phosphatidylinositides have the potential to control cytoplasm activity and bind to a phosphoinositide-containing SLB membrane.

  17. Liposomes composed of unsaturated lipids for membrane modification of human erythrocytes.

    PubMed

    Stoll, Christoph; Holovati, Jelena L; Acker, Jason P; Wolkers, Willem F

    2011-01-01

    Previous studies have shown that certain saturated lipids protect red blood cells (RBCs) during hypothermic storage but provide little protection during freezing or freeze-drying, whereas various unsaturated lipids destabilize RBCs during hypothermic storage but protect during freezing and freeze-drying. The protective effect of liposomes has been attributed to membrane modifications. We have previously shown that cholesterol exchange and lipid transfer between liposomes composed of saturated lipids and RBCs critically depends on the length of the lipid acyl chains. In this study the effect of unsaturated lipids with differences in their number of unsaturated bonds (18:0/18:1, 18:1/18:1, 18:2/18:2) on RBC membrane properties has been studied. RBCs were incubated in the presence of liposomes and both the liposomal and RBC fraction were analyzed by Fourier transform infrared spectroscopy (FTIR) after incubation. The liposomes caused an increase in RBC membrane conformational disorder at suprazero temperatures. The fluidizing effect of the liposomes on the RBC membranes, however, was found to be similar for the different lipids irrespective of their unsaturation level. The gel to liquid crystalline phase transition temperature of the liposomes increased after incubation with RBCs. RBC membrane fluidity increased linearly during the first 8 hours of incubation in the presence of liposomes. The increase in RBC membrane fluidity was found to be temperature dependent and displayed Arrhenius behaviour between 20 and 40°C, with an activation energy of 88 kJ mol⁻¹. Taken together, liposomes composed of unsaturated lipids increase RBC membrane conformational disorder, which could explain their cryoprotective action.

  18. Membrane Raft Organization Is More Sensitive to Disruption by (n-3) PUFA Than Nonraft Organization in EL4 and B Cells123

    PubMed Central

    Rockett, Benjamin Drew; Franklin, Andrew; Harris, Mitchel; Teague, Heather; Rockett, Alexis; Shaikh, Saame Raza

    2011-01-01

    Model membrane and cellular detergent extraction studies show (n-3) PUFA predominately incorporate into nonrafts; thus, we hypothesized (n-3) PUFA could disrupt nonraft organization. The first objective of this study was to determine whether (n-3) PUFA disrupted nonrafts of EL4 cells, an extension of our previous work in which we discovered an (n-3) PUFA diminished raft clustering. EPA or DHA treatment of EL4 cells increased plasma membrane accumulation of the nonraft probe 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate by ~50–70% relative to a BSA control. Förster resonance energy transfer imaging showed EPA and DHA also disrupted EL4 nanometer scale nonraft organization by increasing the distance between nonraft molecules by ~25% compared with BSA. However, changes in nonrafts were due to an increase in cell size; under conditions where EPA or DHA did not increase cell size, nonraft organization was unaffected. We next translated findings on EL4 cells by testing if (n-3) PUFA administered to mice disrupted nonrafts and rafts. Imaging of B cells isolated from mice fed low- or high-fat (HF) (n-3) PUFA diets showed no change in nonraft organization compared with a control diet (CD). However, confocal microscopy revealed the HF (n-3) PUFA diet disrupted lipid raft clustering and size by ~40% relative to CD. Taken together, our data from 2 different model systems suggest (n-3) PUFA have limited effects on nonrafts. The ex vivo data, which confirm previous studies with EL4 cells, provide evidence that (n-3) PUFA consumed through the diet disrupt B cell lipid raft clustering. PMID:21525263

  19. Interaction pathways between soft lipid nanodiscs and plasma membranes: A molecular modeling study.

    PubMed

    Li, Shixin; Luo, Zhen; Xu, Yan; Ren, Hao; Deng, Li; Zhang, Xianren; Huang, Fang; Yue, Tongtao

    2017-10-01

    Lipid nanodisc, a model membrane platform originally synthesized for study of membrane proteins, has recently been used as the carrier to deliver amphiphilic drugs into target tumor cells. However, the central question of how cells interact with such emerging nanomaterials remains unclear and deserves our research for both improving the delivery efficiency and reducing the side effect. In this work, a binary lipid nanodisc is designed as the minimum model to investigate its interactions with plasma membranes by using the dissipative particle dynamics method. Three typical interaction pathways, including the membrane attachment with lipid domain exchange of nanodiscs, the partial membrane wrapping with nanodisc vesiculation, and the receptor-mediated endocytosis, are discovered. For the first pathway, the boundary normal lipids acting as ligands diffuse along the nanodisc rim to gather at the membrane interface, repelling the central bola lipids to reach a stable membrane attachment. If bola lipids are positioned at the periphery and act as ligands, they diffuse to form a large aggregate being wrapped by the membrane, leaving the normal lipids exposed on the membrane exterior by assembling into a vesicle. Finally, by setting both central normal lipids and boundary bola lipids as ligands, the receptor-mediated endocytosis occurs via both deformation and self-rotation of the nanodiscs. All above pathways for soft lipid nanodiscs are quite different from those for rigid nanoparticles, which may provide useful guidelines for design of soft lipid nanodiscs in widespread biomedical applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Mechanics of water pore formation in lipid membrane under electric field

    NASA Astrophysics Data System (ADS)

    Bu, Bing; Li, Dechang; Diao, Jiajie; Ji, Baohua

    2017-04-01

    Transmembrane water pores are crucial for substance transport through cell membranes via membrane fusion, such as in neural communication. However, the molecular mechanism of water pore formation is not clear. In this study, we apply all-atom molecular dynamics and bias-exchange metadynamics simulations to study the process of water pore formation under an electric field. We show that water molecules can enter a membrane under an electric field and form a water pore of a few nanometers in diameter. These water molecules disturb the interactions between lipid head groups and the ordered arrangement of lipids. Following the movement of water molecules, the lipid head groups are rotated and driven into the hydrophobic region of the membrane. The reorientated lipid head groups inside the membrane form a hydrophilic surface of the water pore. This study reveals the atomic details of how an electric field influences the movement of water molecules and lipid head groups, resulting in water pore formation.

  1. Diphytanoyl lipids as model systems for studying membrane-active peptides.

    PubMed

    Kara, Sezgin; Afonin, Sergii; Babii, Oleg; Tkachenko, Anton N; Komarov, Igor V; Ulrich, Anne S

    2017-10-01

    The branched chains in diphytanoyl lipids provide membranes with unique properties, such as high chemical/physical stability, low water permeability, and no gel-to-fluid phase transition at ambient temperature. Synthetic diphytanoyl phospholipids are often used as model membranes for electrophysiological experiments. To evaluate whether these sturdy lipids are also suitable for solid-state NMR, we have examined their interactions with a typical amphiphilic peptide in comparison with straight-chain lipids. First, their phase properties were monitored using 31 P NMR, and the structural behaviour of the antimicrobial peptide PGLa was studied by 19 F NMR and circular dichroism in oriented membrane samples. Only lipids with choline headgroups (DPhPC) were found to form stable lipid bilayers in oriented samples, while DPhPG, DPhPE and DPhPS display non-lamellar structures. Hence, the experimental temperature and hydration are crucial factors when using supported diphytanoyl lipids, as both parameters must be maintained in an appropriate range to avoid the formation of non-bilayer structures. For the same reason, a high content of other diphytanoyl lipids besides DPhPC in mixed lipid systems is not favourable. Unlike the situation in straight-chain membranes, we found that the α-helical PGLa was not able to insert into the tightly packed fluid bilayer of DPhPC but remained in a surface-bound state even at very high peptide concentration. This behaviour can be explained by the high cohesivity and the negative spontaneous curvature of the diphytanoyl lipids. These characteristic features must therefore be taken into consideration, both, in electrophysiological studies, and when interpreting the structural behaviour of membrane-active peptides in such lipid environment. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Single histidine residue in head-group region is sufficient to impart remarkable gene transfection properties to cationic lipids: evidence for histidine-mediated membrane fusion at acidic pH.

    PubMed

    Kumar, V V; Pichon, C; Refregiers, M; Guerin, B; Midoux, P; Chaudhuri, A

    2003-08-01

    Presence of endosome-disrupting multiple histidine functionalities in the molecular architecture of cationic polymers, such as polylysine, has previously been demonstrated to significantly enhance their in vitro gene delivery efficiencies. Towards harnessing improved transfection property through covalent grafting of endosome-disrupting single histidine functionality in the molecular structure of cationic lipids, herein, we report on the design, the synthesis and the transfection efficiency of two novel nonglycerol-based histidylated cationic amphiphiles. We found that L-histidine-(N,N-di-n-hexadecylamine)ethylamide (lipid 1) and L-histidine-(N,N-di-n-hexadecylamine,-N-methyl)ethylamide (lipid 2) in combination with cholesterol gave efficient transfections into various cell lines. The transfection efficiency of Chol/lipid 1 lipoplexes into HepG2 cells was two order of magnitude higher than that of FuGENE(TM)6 and DC-Chol lipoplexes, whereas it was similar into A549, 293T7 and HeLa cells. A better efficiency was obtained with Chol/lipid 2 lipoplexes when using the cytosolic luciferase expression vector (pT7Luc) under the control of the bacterial T7 promoter. Membrane fusion activity measurements using fluorescence resonance energy transfer (FRET) technique showed that the histidine head-groups of Chol/lipid 1 liposomes mediated membrane fusion in the pH range 5-7. In addition, the transgene expression results using the T7Luc expression vector convincingly support the endosome-disrupting role of the presently described mono-histidylated cationic transfection lipids and the release of DNA into the cytosol. We conclude that covalent grafting of a single histidine amino acid residue to suitable twin-chain hydrophobic compounds is able to impart remarkable transfection properties on the resulting mono-histidylated cationic amphiphile, presumably via the endosome-disrupting characteristics of the histidine functionalities.

  3. Light on fluorescent lipids in rafts: a lesson from model membranes.

    PubMed

    Kahya, Nicoletta

    2010-09-15

    Tracking fluorescent lipids in cellular membranes has been applied for decades to shed light on membrane trafficking, sorting, endocytosis and exocytosis, viral entry, and to understand the functional relevance of membrane heterogeneity, phase separation and lipid rafts. However, fluorescent probes may display different organizing behaviour from their corresponding endogenous lipids. A full characterization of these probes is therefore required for proper interpretation of fluorescence microscopy data in complex membrane systems. Model membrane studies provide essential clues that guide us to design and interpret our experiments, help us to avoid pitfalls and resolve artefacts in complex cellular environments. In the present issue of the Biochemical Journal, Juhasz, Davis and Sharom demonstrate the importance of testing lipid probes systematically in heterogeneous model membranes of specific composition and well-defined thermodynamic properties. The phase-partitioning behaviour of fluorescent probes, alone and/or in combination, cannot simply be assumed, but has to be fully characterized.

  4. Aspirin Increases the Solubility of Cholesterol in Lipid Membranes

    NASA Astrophysics Data System (ADS)

    Alsop, Richard; Barrett, Matthew; Zheng, Sonbo; Dies, Hannah; Rheinstadter, Maikel

    2014-03-01

    Aspirin (ASA) is often prescribed for patients with high levels of cholesterol for the secondary prevention of myocardial events, a regimen known as the Low-Dose Aspirin Therapy. We have recently shown that Aspirin partitions in lipid bilayers. However, a direct interplay between ASA and cholesterol has not been investigated. Cholesterol is known to insert itself into the membrane in a dispersed state at moderate concentrations (under ~37.5%) and decrease fluidity of membranes. We prepared model lipid membranes containing varying amounts of both ASA and cholesterol molecules. The structure of the bilayers as a function of ASA and cholesterol concentration was determined using high-resolution X-ray diffraction. At cholesterol levels of more than 40mol%, immiscible cholesterol plaques formed. Adding ASA to the membranes was found to dissolve the cholesterol plaques, leading to a fluid lipid bilayer structure. We present first direct evidence for an interaction between ASA and cholesterol on the level of the cell membrane.

  5. Methylphenidate disrupts cytoskeletal homeostasis and reduces membrane-associated lipid content in juvenile rat hippocampus.

    PubMed

    Schmitz, Felipe; Pierozan, Paula; Biasibetti-Brendler, Helena; Ferreira, Fernanda Silva; Dos Santos Petry, Fernanda; Trindade, Vera Maria Treis; Pessoa-Pureur, Regina; Wyse, Angela T S

    2017-12-29

    Although methylphenidate (MPH) is ubiquitously prescribed to children and adolescents, the consequences of chronic utilization of this psychostimulant are poorly understood. In this study, we investigated the effects of MPH on cytoskeletal homeostasis and lipid content in rat hippocampus. Wistar rats received intraperitoneal injections of MPH (2.0 mg/kg) or saline solution (controls), once a day, from the 15th to the 44th day of age. Results showed that MPH provoked hypophosphorylation of glial fibrillary acidic protein (GFAP) and reduced its immunocontent. Middle and high molecular weight neurofilament subunits (NF-M, NF-H) were hypophosphorylated by MPH on KSP repeat tail domains, while NFL, NFM and NFH immunocontents were not altered. MPH increased protein phosphatase 1 (PP1) and 2A (PP2A) immunocontents. MPH also decreased the total content of ganglioside and phospholipid, as well as the main brain gangliosides (GM1, GD1a, and GD1b) and the major brain phospholipids (sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine). Total cholesterol content was also reduced in the hippocampi of juvenile rats treated with MPH. These results provide evidence that disruptions of cytoskeletal and lipid homeostasis in hippocampus of juvenile rats are triggers by chronic MPH treatment and present a new basis for understanding the effects and consequences associated with chronic use of this psychostimulant during the development of the central nervous system.

  6. Domain Formation Induced by the Adsorption of Charged Proteins on Mixed Lipid Membranes

    PubMed Central

    Mbamala, Emmanuel C.; Ben-Shaul, Avinoam; May, Sylvio

    2005-01-01

    Peripheral proteins can trigger the formation of domains in mixed fluid-like lipid membranes. We analyze the mechanism underlying this process for proteins that bind electrostatically onto a flat two-component membrane, composed of charged and neutral lipid species. Of particular interest are membranes in which the hydrocarbon lipid tails tend to segregate owing to nonideal chain mixing, but the (protein-free) lipid membrane is nevertheless stable due to the electrostatic repulsion between the charged lipid headgroups. The adsorption of charged, say basic, proteins onto a membrane containing anionic lipids induces local lipid demixing, whereby charged lipids migrate toward (or away from) the adsorption site, so as to minimize the electrostatic binding free energy. Apart from reducing lipid headgroup repulsion, this process creates a gradient in lipid composition around the adsorption zone, and hence a line energy whose magnitude depends on the protein's size and charge and the extent of lipid chain nonideality. Above a certain critical lipid nonideality, the line energy is large enough to induce domain formation, i.e., protein aggregation and, concomitantly, macroscopic lipid phase separation. We quantitatively analyze the thermodynamic stability of the dressed membrane based on nonlinear Poisson-Boltzmann theory, accounting for both the microscopic characteristics of the proteins and lipid composition modulations at and around the adsorption zone. Spinodal surfaces and critical points of the dressed membranes are calculated for several different model proteins of spherical and disk-like shapes. Among the models studied we find the most substantial protein-induced membrane destabilization for disk-like proteins whose charges are concentrated in the membrane-facing surface. If additional charges reside on the side faces of the proteins, direct protein-protein repulsion diminishes considerably the propensity for domain formation. Generally, a highly charged flat face

  7. Incorporation of large guest molecules into liposomes via chemical reactions in lipid membranes.

    PubMed

    Tsuchiya, Yuki; Sugikawa, Kouta; Ueda, Masafumi; Ikeda, Atsushi

    2017-02-22

    The incorporation of hydrophobic guest molecules into lipid membranes by the exchange of the guest molecule from a cyclodextrin (CDx) complex to a liposome is limited to guest molecules that can be included in CDxs. To solve this problem, large guest molecules were incorporated into liposomes by chemical reactions of guest molecules in lipid membranes. Stable lipid-membrane-incorporated fullerene derivatives with large substituent(s) were prepared by Diels-Alder reactions in lipid membranes.

  8. The Formation and Properties of Thin Lipid Membranes from HK and LK Sheep Red Cell Lipids

    PubMed Central

    Andreoli, Thomas E.; Bangham, J. Andrew; Tosteson, Daniel C.

    1967-01-01

    Lipids were obtained from high potassium (HK) and low potassium (LK) sheep red cells by sequential extraction of the erythrocytes with isopropanol-chloroform, chloroform-methanol-0.1 M KCl, and chloroform. The extract contained cholesterol and phospholipid in a molar ratio of 0.8:1.0, and less than 1% protein contaminant. Stable thin lipid membranes separating two aqueous compartments were formed from an erythrocyte lipid-hydrocarbon solution, and had an electrical resistance of ∼108 ohm-cm2 and a capacitance of 0.38–0.4 µf/cm2. From the capacitance values, membrane thickness was estimated to be 46–132 A, depending on the assumed value for the dielectric constant (2.0–4.5). Membrane voltage was recorded in the presence of ionic (NaCl and/or KCl) concentration gradients in the solutions bathing the membrane. The permeability of the membrane to Na+, K+, and Cl- (expressed as the transference number, T ion) was computed from the steady-state membrane voltage and the activity ratio of the ions in the compartments bathing the membrane. T Na and T K were approximately equal (∼0.8) and considerably greater than T Cl (∼0.2). The ionic transference numbers were independent of temperature, the hydrocarbon solvent, the osmolarity of the solutions bathing the membranes, and the cholesterol content of the membranes, over the range 21–38°C. The high degree of membrane cation selectivity was tentatively attributed to the negatively charged phospholipids (phosphatidylethanolamine and phosphatidylserine) present in the lipid extract. PMID:6034765

  9. Exploiting lipopolysaccharide-induced deformation of lipid bilayers to modify membrane composition and generate two-dimensional geometric membrane array patterns

    DOE PAGES

    Adams, Peter G.; Swingle, Kirstie L.; Paxton, Walter F.; ...

    2015-05-27

    Supported lipid bilayers have proven effective as model membranes for investigating biophysical processes and in development of sensor and array technologies. The ability to modify lipid bilayers after their formation and in situ could greatly advance membrane technologies, but is difficult via current state-of-the-art technologies. Here we demonstrate a novel method that allows the controlled post-formation processing and modification of complex supported lipid bilayer arrangements, under aqueous conditions. We exploit the destabilization effect of lipopolysaccharide, an amphiphilic biomolecule, interacting with lipid bilayers to generate voids that can be backfilled to introduce desired membrane components. We further demonstrate that when usedmore » in combination with a single, traditional soft lithography process, it is possible to generate hierarchically-organized membrane domains and microscale 2-D array patterns of domains. Significantly, this technique can be used to repeatedly modify membranes allowing iterative control over membrane composition. This approach expands our toolkit for functional membrane design, with potential applications for enhanced materials templating, biosensing and investigating lipid-membrane processes.« less

  10. Watching individual molecules flex within lipid membranes using SERS

    NASA Astrophysics Data System (ADS)

    Taylor, Richard W.; Benz, Felix; Sigle, Daniel O.; Bowman, Richard W.; Bao, Peng; Roth, Johannes S.; Heath, George R.; Evans, Stephen D.; Baumberg, Jeremy J.

    2014-08-01

    Interrogating individual molecules within bio-membranes is key to deepening our understanding of biological processes essential for life. Using Raman spectroscopy to map molecular vibrations is ideal to non-destructively `fingerprint' biomolecules for dynamic information on their molecular structure, composition and conformation. Such tag-free tracking of molecules within lipid bio-membranes can directly connect structure and function. In this paper, stable co-assembly with gold nano-components in a `nanoparticle-on-mirror' geometry strongly enhances the local optical field and reduces the volume probed to a few nm3, enabling repeated measurements for many tens of minutes on the same molecules. The intense gap plasmons are assembled around model bio-membranes providing molecular identification of the diffusing lipids. Our experiments clearly evidence measurement of individual lipids flexing through telltale rapid correlated vibrational shifts and intensity fluctuations in the Raman spectrum. These track molecules that undergo bending and conformational changes within the probe volume, through their interactions with the environment. This technique allows for in situ high-speed single-molecule investigations of the molecules embedded within lipid bio-membranes. It thus offers a new way to investigate the hidden dynamics of cell membranes important to a myriad of life processes.

  11. Fatty acids from membrane lipids become incorporated into lipid bodies during Myxococcus xanthus differentiation.

    PubMed

    Bhat, Swapna; Boynton, Tye O; Pham, Dan; Shimkets, Lawrence J

    2014-01-01

    Myxococcus xanthus responds to amino acid limitation by producing fruiting bodies containing dormant spores. During development, cells produce triacylglycerides in lipid bodies that become consumed during spore maturation. As the cells are starved to induce development, the production of triglycerides represents a counterintuitive metabolic switch. In this paper, lipid bodies were quantified in wild-type strain DK1622 and 33 developmental mutants at the cellular level by measuring the cross sectional area of the cell stained with the lipophilic dye Nile red. We provide five lines of evidence that triacylglycerides are derived from membrane phospholipids as cells shorten in length and then differentiate into myxospores. First, in wild type cells, lipid bodies appear early in development and their size increases concurrent with an 87% decline in membrane surface area. Second, developmental mutants blocked at different stages of shortening and differentiation accumulated lipid bodies proportionate with their cell length with a Pearson's correlation coefficient of 0.76. Third, peripheral rods, developing cells that do not produce lipid bodies, fail to shorten. Fourth, genes for fatty acid synthesis are down-regulated while genes for fatty acid degradation are up regulated. Finally, direct movement of fatty acids from membrane lipids in growing cells to lipid bodies in developing cells was observed by pulse labeling cells with palmitate. Recycling of lipids released by Programmed Cell Death appears not to be necessary for lipid body production as a fadL mutant was defective in fatty acid uptake but proficient in lipid body production. The lipid body regulon involves many developmental genes that are not specifically involved in fatty acid synthesis or degradation. MazF RNA interferase and its target, enhancer-binding protein Nla6, appear to negatively regulate cell shortening and TAG accumulation whereas most cell-cell signals activate these processes.

  12. Inducing morphological changes in lipid bilayer membranes with microfabricated substrates

    NASA Astrophysics Data System (ADS)

    Liu, Fangjie; Collins, Liam F.; Ashkar, Rana; Heberle, Frederick A.; Srijanto, Bernadeta R.; Collier, C. Patrick

    2016-11-01

    Lateral organization of lipids and proteins into distinct domains and anchoring to a cytoskeleton are two important strategies employed by biological membranes to carry out many cellular functions. However, these interactions are difficult to emulate with model systems. Here we use the physical architecture of substrates consisting of arrays of micropillars to systematically control the behavior of supported lipid bilayers - an important step in engineering model lipid membrane systems with well-defined functionalities. Competition between attractive interactions of supported lipid bilayers with the underlying substrate versus the energy cost associated with membrane bending at pillar edges can be systematically investigated as functions of pillar height and pitch, chemical functionalization of the microstructured substrate, and the type of unilamellar vesicles used for assembling the supported bilayer. Confocal fluorescent imaging and AFM measurements highlight correlations that exist between topological and mechanical properties of lipid bilayers and lateral lipid mobility in these confined environments. This study provides a baseline for future investigations into lipid domain reorganization on structured solid surfaces and scaffolds for cell growth.

  13. Specific membrane lipid composition is important for plasmodesmata function in Arabidopsis.

    PubMed

    Grison, Magali S; Brocard, Lysiane; Fouillen, Laetitia; Nicolas, William; Wewer, Vera; Dörmann, Peter; Nacir, Houda; Benitez-Alfonso, Yoselin; Claverol, Stéphane; Germain, Véronique; Boutté, Yohann; Mongrand, Sébastien; Bayer, Emmanuelle M

    2015-04-01

    Plasmodesmata (PD) are nano-sized membrane-lined channels controlling intercellular communication in plants. Although progress has been made in identifying PD proteins, the role played by major membrane constituents, such as the lipids, in defining specialized membrane domains in PD remains unknown. Through a rigorous isolation of "native" PD membrane fractions and comparative mass spectrometry-based analysis, we demonstrate that lipids are laterally segregated along the plasma membrane (PM) at the PD cell-to-cell junction in Arabidopsis thaliana. Remarkably, our results show that PD membranes display enrichment in sterols and sphingolipids with very long chain saturated fatty acids when compared with the bulk of the PM. Intriguingly, this lipid profile is reminiscent of detergent-insoluble membrane microdomains, although our approach is valuably detergent-free. Modulation of the overall sterol composition of young dividing cells reversibly impaired the PD localization of the glycosylphosphatidylinositol-anchored proteins Plasmodesmata Callose Binding 1 and the β-1,3-glucanase PdBG2 and altered callose-mediated PD permeability. Altogether, this study not only provides a comprehensive analysis of the lipid constituents of PD but also identifies a role for sterols in modulating cell-to-cell connectivity, possibly by establishing and maintaining the positional specificity of callose-modifying glycosylphosphatidylinositol proteins at PD. Our work emphasizes the importance of lipids in defining PD membranes. © 2015 American Society of Plant Biologists. All rights reserved.

  14. Specific Membrane Lipid Composition Is Important for Plasmodesmata Function in Arabidopsis

    PubMed Central

    Grison, Magali S.; Brocard, Lysiane; Fouillen, Laetitia; Nicolas, William; Wewer, Vera; Dörmann, Peter; Nacir, Houda; Benitez-Alfonso, Yoselin; Claverol, Stéphane; Germain, Véronique; Boutté, Yohann; Mongrand, Sébastien; Bayer, Emmanuelle M.

    2015-01-01

    Plasmodesmata (PD) are nano-sized membrane-lined channels controlling intercellular communication in plants. Although progress has been made in identifying PD proteins, the role played by major membrane constituents, such as the lipids, in defining specialized membrane domains in PD remains unknown. Through a rigorous isolation of “native” PD membrane fractions and comparative mass spectrometry-based analysis, we demonstrate that lipids are laterally segregated along the plasma membrane (PM) at the PD cell-to-cell junction in Arabidopsis thaliana. Remarkably, our results show that PD membranes display enrichment in sterols and sphingolipids with very long chain saturated fatty acids when compared with the bulk of the PM. Intriguingly, this lipid profile is reminiscent of detergent-insoluble membrane microdomains, although our approach is valuably detergent-free. Modulation of the overall sterol composition of young dividing cells reversibly impaired the PD localization of the glycosylphosphatidylinositol-anchored proteins Plasmodesmata Callose Binding 1 and the β-1,3-glucanase PdBG2 and altered callose-mediated PD permeability. Altogether, this study not only provides a comprehensive analysis of the lipid constituents of PD but also identifies a role for sterols in modulating cell-to-cell connectivity, possibly by establishing and maintaining the positional specificity of callose-modifying glycosylphosphatidylinositol proteins at PD. Our work emphasizes the importance of lipids in defining PD membranes. PMID:25818623

  15. Lipid self-assembly and lectin-induced reorganization of the plasma membrane.

    PubMed

    Sych, Taras; Mély, Yves; Römer, Winfried

    2018-05-26

    The plasma membrane represents an outstanding example of self-organization in biology. It plays a vital role in protecting the integrity of the cell interior and regulates meticulously the import and export of diverse substances. Its major building blocks are proteins and lipids, which self-assemble to a fluid lipid bilayer driven mainly by hydrophobic forces. Even if the plasma membrane appears-globally speaking-homogeneous at physiological temperatures, the existence of specialized nano- to micrometre-sized domains of raft-type character within cellular and synthetic membrane systems has been reported. It is hypothesized that these domains are the origin of a plethora of cellular processes, such as signalling or vesicular trafficking. This review intends to highlight the driving forces of lipid self-assembly into a bilayer membrane and the formation of small, transient domains within the plasma membrane. The mechanisms of self-assembly depend on several factors, such as the lipid composition of the membrane and the geometry of lipids. Moreover, the dynamics and organization of glycosphingolipids into nanometre-sized clusters will be discussed, also in the context of multivalent lectins, which cluster several glycosphingolipid receptor molecules and thus create an asymmetric stress between the two membrane leaflets, leading to tubular plasma membrane invaginations.This article is part of the theme issue 'Self-organization in cell biology'. © 2018 The Author(s).

  16. The Interaction of Melittin with Dimyristoyl Phosphatidylcholine-Dimyristoyl Phosphatidylserine Lipid Bilayer Membranes.

    PubMed

    Rai, Durgesh K; Qian, Shuo; Heller, William T

    2016-11-01

    Membrane-active peptides (MAPs), which interact directly with the lipid bilayer of a cell and include toxins and host defense peptides, display lipid composition-dependent activity. Phosphatidylserine (PS) lipids are anionic lipids that are found throughout the cellular membranes of most eukaryotic organisms where they serve as both a functional component and as a precursor to phosphatidylethanolamine lipids. The inner leaflet of the plasma membrane contains more PS than the outer one, and the asymmetry is actively maintained. Here, the impact of the MAP melittin on the structure of lipid bilayer vesicles made of a mixture of phosphatidylcholine and phosphatidylserine was studied. Small-angle neutron scattering of the MAP associated with selectively deuterium-labeled lipid bilayer vesicles revealed how the thickness and lipid composition of phosphatidylserine-containing vesicles change in response to melittin. The peptide thickens the lipid bilayer for concentrations up to P/L=1/500, but membrane thinning results when P/L=1/200. The thickness transition is accompanied by a large change in the distribution of DMPS between the leaflets of the bilayer. The change in composition is driven by electrostatic interactions, while the change in bilayer thickness is driven by changes in the interaction of the peptide with the headgroup region of the lipid bilayer. The results provide new information about lipid-specific interactions that take place in mixed composition lipid bilayer membranes. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. The Interaction of Melittin with Dimyristoyl Phosphatidylcholine-Dimyristoyl Phosphatidylserine Lipid Bilayer Membranes

    DOE PAGES

    Rai, Durgesh K.; Qian, Shuo; Heller, William T.

    2016-08-13

    We report that membrane-active peptides (MAPs), which interact directly with the lipid bilayer of a cell and include toxins and host defense peptides, display lipid composition-dependent activity. Phosphatidylserine (PS) lipids are anionic lipids that are found throughout the cellular membranes of most eukaryotic organisms where they serve as both a functional component and as a precursor to phosphatidylethanolamine lipids. The inner leaflet of the plasma membrane contains more PS than the outer one, and the asymmetry is actively maintained. Here, the impact of the MAP melittin on the structure of lipid bilayer vesicles made of a mixture of phosphatidylcholine andmore » phosphatidylserine was studied. Small-angle neutron scattering of the MAP associated with selectively deuterium-labeled lipid bilayer vesicles revealed how the thickness and lipid composition of phosphatidylserine-containing vesicles change in response to melittin. The peptide thickens the lipid bilayer for concentrations up to P/L = 1/500, but membrane thinning results when P/L = 1/200. The thickness transition is accompanied by a large change in the distribution of DMPS between the leaflets of the bilayer. The change in composition is driven by electrostatic interactions, while the change in bilayer thickness is driven by changes in the interaction of the peptide with the headgroup region of the lipid bilayer. Lastly, the results provide new information about lipid-specific interactions that take place in mixed composition lipid bilayer membranes.« less

  18. Brownian Motion at Lipid Membranes: A Comparison of Hydrodynamic Models Describing and Experiments Quantifying Diffusion within Lipid Bilayers.

    PubMed

    Block, Stephan

    2018-05-22

    The capability of lipid bilayers to exhibit fluid-phase behavior is a fascinating property, which enables, for example, membrane-associated components, such as lipids (domains) and transmembrane proteins, to diffuse within the membrane. These diffusion processes are of paramount importance for cells, as they are for example involved in cell signaling processes or the recycling of membrane components, but also for recently developed analytical approaches, which use differences in the mobility for certain analytical purposes, such as in-membrane purification of membrane proteins or the analysis of multivalent interactions. Here, models describing the Brownian motion of membrane inclusions (lipids, peptides, proteins, and complexes thereof) in model bilayers (giant unilamellar vesicles, black lipid membranes, supported lipid bilayers) are summarized and model predictions are compared with the available experimental data, thereby allowing for evaluating the validity of the introduced models. It will be shown that models describing the diffusion in freestanding (Saffman-Delbrück and Hughes-Pailthorpe-White model) and supported bilayers (the Evans-Sackmann model) are well supported by experiments, though only few experimental studies have been published so far for the latter case, calling for additional tests to reach the same level of experimental confirmation that is currently available for the case of freestanding bilayers.

  19. Digital holographic microscopy of phase separation in multicomponent lipid membranes

    NASA Astrophysics Data System (ADS)

    Farzam Rad, Vahideh; Moradi, Ali-Reza; Darudi, Ahmad; Tayebi, Lobat

    2016-12-01

    Lateral in-homogeneities in lipid compositions cause microdomains formation and change in the physical properties of biological membranes. With the presence of cholesterol and mixed species of lipids, phospholipid membranes segregate into lateral domains of liquid-ordered and liquid-disordered phases. Coupling of two-dimensional intralayer phase separations and interlayer liquid-crystalline ordering in multicomponent membranes has been previously demonstrated. By the use of digital holographic microscopy (DHMicroscopy), we quantitatively analyzed the volumetric dynamical behavior of such membranes. The specimens are lipid mixtures composed of sphingomyelin, cholesterol, and unsaturated phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine. DHMicroscopy in a transmission mode is an effective tool for quantitative visualization of phase objects. By deriving the associated phase changes, three-dimensional information on the morphology variation of lipid stacks at arbitrary time scales is obtained. Moreover, the thickness distribution of the object at demanded axial planes can be obtained by numerical focusing. Our results show that the volume evolution of lipid domains follows approximately the same universal growth law of previously reported area evolution. However, the thickness of the domains does not alter significantly by time; therefore, the volume evolution is mostly attributed to the changes in area dynamics. These results might be useful in the field of membrane-based functional materials.

  20. How synthetic membrane systems contribute to the understanding of lipid-driven endocytosis.

    PubMed

    Schubert, Thomas; Römer, Winfried

    2015-11-01

    Synthetic membrane systems, such as giant unilamellar vesicles and solid supported lipid bilayers, have widened our understanding of biological processes occurring at or through membranes. Artificial systems are particularly suited to study the inherent properties of membranes with regard to their components and characteristics. This review critically reflects the emerging molecular mechanism of lipid-driven endocytosis and the impact of model membrane systems in elucidating the complex interplay of biomolecules within this process. Lipid receptor clustering induced by binding of several toxins, viruses and bacteria to the plasma membrane leads to local membrane bending and formation of tubular membrane invaginations. Here, lipid shape, and protein structure and valency are the essential parameters in membrane deformation. Combining observations of complex cellular processes and their reconstitution on minimal systems seems to be a promising future approach to resolve basic underlying mechanisms. This article is part of a Special Issue entitled: Mechanobiology. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Biophysical interactions with model lipid membranes: applications in drug discovery and drug delivery

    PubMed Central

    Peetla, Chiranjeevi; Stine, Andrew; Labhasetwar, Vinod

    2009-01-01

    The transport of drugs or drug delivery systems across the cell membrane is a complex biological process, often difficult to understand because of its dynamic nature. In this regard, model lipid membranes, which mimic many aspects of cell-membrane lipids, have been very useful in helping investigators to discern the roles of lipids in cellular interactions. One can use drug-lipid interactions to predict pharmacokinetic properties of drugs, such as their transport, biodistribution, accumulation, and hence efficacy. These interactions can also be used to study the mechanisms of transport, based on the structure and hydrophilicity/hydrophobicity of drug molecules. In recent years, model lipid membranes have also been explored to understand their mechanisms of interactions with peptides, polymers, and nanocarriers. These interaction studies can be used to design and develop efficient drug delivery systems. Changes in the lipid composition of cells and tissue in certain disease conditions may alter biophysical interactions, which could be explored to develop target-specific drugs and drug delivery systems. In this review, we discuss different model membranes, drug-lipid interactions and their significance, studies of model membrane interactions with nanocarriers, and how biophysical interaction studies with lipid model membranes could play an important role in drug discovery and drug delivery. PMID:19432455

  2. Phospatidylserine or ganglioside--which of anionic lipids determines the effect of cationic dextran on lipid membrane?

    PubMed

    Hąc-Wydro, Katarzyna; Wydro, Paweł; Cetnar, Andrzej; Włodarczyk, Grzegorz

    2015-02-01

    In this work the influence of cationic polymer, namely diethylaminoethyl DEAE-dextran on model lipid membranes was investigated. This polymer is of a wide application as a biomaterial and a drug carrier and its cytotoxicity toward various cancer cells was also confirmed. It was suggested that anticancer effect of cationic dextran is connected with the binding of the polymer to the negatively charged sialic acid residues overexpressed in cancer membrane. This fact encouraged us to perform the studies aimed at verifying whether the effect of cationic DEAE-dextran on membrane is determined only by the presence of the negatively charged lipid in the system or the kind of anionic lipid is also important. To reach this goal systematic investigations on the effect of dextran on various one-component lipid monolayers and multicomponent hepatoma cell model membranes differing in the level and the kind of anionic lipids (phosphatidylserine, sialic acid-containing ganglioside GM3 or their mixture) were done. As evidenced the results the effect of DEAE-dextran on the model system is determined by anionic lipid-polymer electrostatic interactions. However, the magnitude of the effect of cationic polymer is strongly dependent on the kind of anionic lipid in the model system. Namely, the packing and ordering of the mixtures containing ganglioside GM3 were more affected by DEAE-dextran than phosphatidylserine-containing monolayers. Although the experiments were done on model systems and therefore further studies are highly needed, the collected data may indicate that ganglioside may be important in the differentiation of the effect of cationic dextran on membranes. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Hydrostatic pressure decreases membrane fluidity and lipid desaturase expression in chondrocyte progenitor cells.

    PubMed

    Montagne, Kevin; Uchiyama, Hiroki; Furukawa, Katsuko S; Ushida, Takashi

    2014-01-22

    Membrane biomechanical properties are critical in modulating nutrient and metabolite exchange as well as signal transduction. Biological membranes are predominantly composed of lipids, cholesterol and proteins, and their fluidity is tightly regulated by cholesterol and lipid desaturases. To determine whether such membrane fluidity regulation occurred in mammalian cells under pressure, we investigated the effects of pressure on membrane lipid order of mouse chondrogenic ATDC5 cells and desaturase gene expression. Hydrostatic pressure linearly increased membrane lipid packing and simultaneously repressed lipid desaturase gene expression. We also showed that cholesterol mimicked and cholesterol depletion reversed those effects, suggesting that desaturase gene expression was controlled by the membrane physical state itself. This study demonstrates a new effect of hydrostatic pressure on mammalian cells and may help to identify the molecular mechanisms involved in hydrostatic pressure sensing in chondrocytes. © 2013 Elsevier Ltd. All rights reserved.

  4. Manipulating membrane lipid profiles to restore T-cell function in autoimmunity.

    PubMed

    Waddington, Kirsty E; Jury, Elizabeth C

    2015-08-01

    Plasma membrane lipid rafts are heterogeneous cholesterol and glycosphingolipid (GSL)-enriched microdomains, within which the tight packing of cholesterol with the saturated-acyl chains of GSLs creates a region of liquid-order relative to the surrounding disordered membrane. Thus lipid rafts govern the lateral mobility and interaction of membrane proteins and regulate a plethora of signal transduction events, including T-cell antigen receptor (TCR) signalling. The pathways regulating homoeostasis of membrane cholesterol and GSLs are tightly controlled and alteration of these metabolic processes coincides with immune cell dysfunction as is evident in atherosclerosis, cancer and autoimmunity. Indeed, membrane lipid composition is emerging as an important factor influencing the ability of cells to respond appropriately to microenvironmental stimuli. Consequently, there is increasing interest in targeting membrane lipids or their metabolic control as a novel therapeutic approach to modulate immune cell behaviour and our recent work demonstrates that this is a promising strategy in T-cells from patients with the autoimmune disease systemic lupus erythematosus (SLE). © 2015 Authors; published by Portland Press Limited.

  5. Changes in lipid membranes may trigger amyloid toxicity in Alzheimer's disease

    PubMed Central

    Drolle, Elizabeth; Negoda, Alexander; Hammond, Keely; Pavlov, Evgeny

    2017-01-01

    Amyloid-beta peptides (Aβ), implicated in Alzheimer’s disease (AD), interact with the cellular membrane and induce amyloid toxicity. The composition of cellular membranes changes in aging and AD. We designed multi-component lipid models to mimic healthy and diseased states of the neuronal membrane. Using atomic force microscopy (AFM), Kelvin probe force microscopy (KPFM) and black lipid membrane (BLM) techniques, we demonstrated that these model membranes differ in their nanoscale structure and physical properties, and interact differently with Aβ1–42. Based on our data, we propose a new hypothesis that changes in lipid membrane due to aging and AD may trigger amyloid toxicity through electrostatic mechanisms, similar to the accepted mechanism of antimicrobial peptide action. Understanding the role of the membrane changes as a key activating amyloid toxicity may aid in the development of a new avenue for the prevention and treatment of AD. PMID:28767712

  6. Origin of 1/f noise in hydration dynamics on lipid membrane surfaces

    PubMed Central

    Yamamoto, Eiji; Akimoto, Takuma; Yasui, Masato; Yasuoka, Kenji

    2015-01-01

    Water molecules on lipid membrane surfaces are known to contribute to membrane stability by connecting lipid molecules and acting as a water bridge. Although water structures and diffusivities near the membrane surfaces have been extensively studied, hydration dynamics on the surfaces has remained an open question. Here we investigate residence time statistics of water molecules on the surface of lipid membranes using all-atom molecular dynamics simulations. We show that hydration dynamics on the lipid membranes exhibits 1/f noise. Constructing a dichotomous process for the hydration dynamics, we find that residence times in each state follow a power-law with exponential cutoff and that the process can be regarded as a correlated renewal process where interoccurrence times are correlated. The results imply that the origin of the 1/f noise in hydration dynamics on the membrane surfaces is a combination of a power-law distribution with cutoff of interoccurrence times of switching events and a long-term correlation between the interoccurrence times. These results suggest that the 1/f noise attributed to the correlated renewal process may contribute to the stability of the hydration layers and lipid membranes. PMID:25743377

  7. Efficient replacement of plasma membrane outer leaflet phospholipids and sphingolipids in cells with exogenous lipids

    PubMed Central

    Kim, JiHyun; Huang, Zhen; St. Clair, Johnna R.; Brown, Deborah A.; London, Erwin

    2016-01-01

    Our understanding of membranes and membrane lipid function has lagged far behind that of nucleic acids and proteins, largely because it is difficult to manipulate cellular membrane lipid composition. To help solve this problem, we show that methyl-α-cyclodextrin (MαCD)-catalyzed lipid exchange can be used to maximally replace the sphingolipids and phospholipids in the outer leaflet of the plasma membrane of living mammalian cells with exogenous lipids, including unnatural lipids. In addition, lipid exchange experiments revealed that 70–80% of cell sphingomyelin resided in the plasma membrane outer leaflet; the asymmetry of metabolically active cells was similar to that previously defined for erythrocytes, as judged by outer leaflet lipid composition; and plasma membrane outer leaflet phosphatidylcholine had a significantly lower level of unsaturation than phosphatidylcholine in the remainder of the cell. The data also provided a rough estimate for the total cellular lipids residing in the plasma membrane (about half). In addition to such lipidomics applications, the exchange method should have wide potential for investigations of lipid function and modification of cellular behavior by modification of lipids. PMID:27872310

  8. Efficient replacement of plasma membrane outer leaflet phospholipids and sphingolipids in cells with exogenous lipids.

    PubMed

    Li, Guangtao; Kim, JiHyun; Huang, Zhen; St Clair, Johnna R; Brown, Deborah A; London, Erwin

    2016-12-06

    Our understanding of membranes and membrane lipid function has lagged far behind that of nucleic acids and proteins, largely because it is difficult to manipulate cellular membrane lipid composition. To help solve this problem, we show that methyl-α-cyclodextrin (MαCD)-catalyzed lipid exchange can be used to maximally replace the sphingolipids and phospholipids in the outer leaflet of the plasma membrane of living mammalian cells with exogenous lipids, including unnatural lipids. In addition, lipid exchange experiments revealed that 70-80% of cell sphingomyelin resided in the plasma membrane outer leaflet; the asymmetry of metabolically active cells was similar to that previously defined for erythrocytes, as judged by outer leaflet lipid composition; and plasma membrane outer leaflet phosphatidylcholine had a significantly lower level of unsaturation than phosphatidylcholine in the remainder of the cell. The data also provided a rough estimate for the total cellular lipids residing in the plasma membrane (about half). In addition to such lipidomics applications, the exchange method should have wide potential for investigations of lipid function and modification of cellular behavior by modification of lipids.

  9. In Situ Synthesis of Lipid Membranes

    NASA Astrophysics Data System (ADS)

    Devaraj, N. K.

    2017-07-01

    We have a strong interest in applying covalent coupling reactions to the formation and modification of lipid membranes. We have utilized chemoselective reactions, such as copper-catalyzed triazole formation or the native chemical ligation.

  10. Maturation of the Gag core decreases the stability of retroviral lipid membranes.

    PubMed

    Davidoff, Candice; Payne, Riley J; Willis, Sharon H; Doranz, Benjamin J; Rucker, Joseph B

    2012-11-25

    To better understand how detergents disrupt enveloped viruses, we monitored the biophysical stability of murine leukemia virus (MLV) virus-like particles (VLPs) against a panel of commonly used detergents using real-time biosensor measurements. Although exposure to many detergents, such as Triton X-100 and Empigen, results in lysis of VLP membranes, VLPs appeared resistant to complete membrane lysis by a significant number of detergents, including Tween 20, Tween 80, Lubrol, and Saponin. VLPs maintained their structural integrity after exposure to Tween 20 at concentrations up to 500-fold above its CMC. Remarkably, VLPs containing immature cores composed of unprocessed (uncleaved) Gag polyprotein were significantly more resistant to detergent lysis than VLPs with mature cores. Although the maturity of retroviral Gag is known to influence the stability of the protein core structure itself, our studies suggest that the maturity of the Gag core also influences the stability of the lipid bilayer surrounding the core. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Maturation of the Gag core decreases the stability of retroviral lipid membranes

    PubMed Central

    Davidoff, Candice; Payne, Riley; Willis, Sharon H.; Doranz, Benjamin J.; Rucker, Joseph B.

    2012-01-01

    To better understand how detergents disrupt enveloped viruses, we monitored the biophysical stability of murine leukemia virus (MLV) virus-like particles (VLPs) against a panel of commonly used detergents using real-time biosensor measurements. Although exposure to many detergents, such as Triton X-100 and Empigen, results in lysis of VLP membranes, VLPs appeared resistant to complete membrane lysis by a significant number of detergents, including Tween 20, Tween 80, Lubrol, and Saponin. VLPs maintained their structural integrity after exposure to Tween 20 at concentrations up to 500-fold above its CMC. Remarkably, VLPs containing immature cores composed of unprocessed (uncleaved) Gag polyprotein were significantly more resistant to detergent lysis than VLPs with mature cores. Although the maturity of retroviral Gag is known to influence the stability of the protein core structure itself, our studies suggest that the maturity of the Gag core also influences the stability of the lipid bilayer surrounding the core. PMID:22995186

  12. Reconstitution of a Kv channel into lipid membranes for structural and functional studies.

    PubMed

    Lee, Sungsoo; Zheng, Hui; Shi, Liang; Jiang, Qiu-Xing

    2013-07-13

    To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

  13. UV-Visible and Infrared Methods for Investigating Lipid-Rhodopsin Membrane Interactions

    PubMed Central

    Brown, Michael F.

    2017-01-01

    Summary Experimental UV-visible and Fourier transform infrared (FTIR) spectroscopic methods are described for characterizing lipid-protein interactions for the example of rhodopsin in a membrane bilayer environment. The combined use of FTIR and UV-visible difference spectroscopy monitors the structural and functional changes during rhodopsin activation. Such studies investigate how membrane lipids stabilize the various rhodopsin photoproducts, analogous to mutating the protein. Interpretation of the results entails a non-specific flexible surface model for explaining the role of membrane lipid-protein interactions in biological functions. PMID:22976026

  14. Effect of Self-Assembly of Fullerene Nano-Particles on Lipid Membrane

    PubMed Central

    Zhang, Saiqun; Mu, Yuguang; Zhang, John Z. H.; Xu, Weixin

    2013-01-01

    Carbon nanoparticles can penetrate the cell membrane and cause cytotoxicity. The diffusion feature and translocation free energy of fullerene through lipid membranes is well reported. However, the knowledge on self-assembly of fullerenes and resulting effects on lipid membrane is poorly addressed. In this work, the self-assembly of fullerene nanoparticles and the resulting influence on the dioleoylphosphtidylcholine (DOPC) model membrane were studied by using all-atom molecular dynamics simulations with explicit solvents. Our simulation results confirm that gathered small fullerene cluster can invade lipid membrane. Simulations show two pathways: 1) assembly process is completely finished before penetration; 2) assembly process coincides with penetration. Simulation results also demonstrate that in the membrane interior, fullerene clusters tend to stay at the position which is 1.0 nm away from the membrane center. In addition, the diverse microscopic stacking mode (i.e., equilateral triangle, tetrahedral pentahedral, trigonal bipyramid and octahedron) of these small fullerene clusters are well characterized. Thus our simulations provide a detailed high-resolution characterization of the microscopic structures of the small fullerene clusters. Further, we found the gathered small fullerene clusters have significant adverse disturbances to the local structure of the membrane, but no great influence on the global integrity of the lipid membrane, which suggests the prerequisite of high-content fullerene for cytotoxicity. PMID:24204827

  15. Influence of Lipid Membrane Rigidity on Properties of Supporting Polymer

    PubMed Central

    Jablin, Michael S.; Dubey, Manish; Zhernenkov, Mikhail; Toomey, Ryan; Majewski, Jarosław

    2011-01-01

    Temperature-sensitive hydrogel polymers are utilized as responsive layers in various applications. Although the polymer's native characteristics have been studied extensively, details concerning its properties during interaction with biorelated structures are lacking. This work investigates the interaction between a thermoresponsive polymer cushion and different lipid membrane capping layers probed by neutron reflectometry. N-isopropylacrylamide copolymerized with methacroylbenzophenone first supported a lipid bilayer composed of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and subsequently 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). The polymer-membrane systems were investigated above and below the polymer transition temperature (37 and 25°C). Although the same cushion supported each lipid membrane, the polymer hydration profile and thickness were markedly different for DPPE and DPPC systems. Because DPPE and DPPC have different bending rigidities, these results establish that the polymer-membrane interaction is critically mediated by the mechanics of the membrane, providing better insight into cell-hydrogel interactions. PMID:21723822

  16. The Escherichia coli Phospholipase PldA Regulates Outer Membrane Homeostasis via Lipid Signaling.

    PubMed

    May, Kerrie L; Silhavy, Thomas J

    2018-03-20

    The outer membrane (OM) bilayer of Gram-negative bacteria is biologically unique in its asymmetrical organization of lipids, with an inner leaflet composed of glycerophospholipids (PLs) and a surface-exposed outer leaflet composed of lipopolysaccharide (LPS). This lipid organization is integral to the OM's barrier properties. Perturbations of the outer leaflet by antimicrobial peptides or defects in LPS biosynthesis or transport to the OM cause a compensatory flipping of PLs to the outer leaflet. As a result, lipid asymmetry is disrupted and OM integrity is compromised. Recently, we identified an Escherichia coli mutant that exhibits aberrant accumulation of surface PLs accompanied by a cellular increase in LPS production. Remarkably, the observed hyperproduction of LPS is PldA dependent. Here we provide evidence that the fatty acids generated by PldA at the OM are transported into the cytoplasm and simultaneously activated by thioesterification to coenzyme A (CoA) by FadD. The acyl-CoAs produced ultimately inhibit LpxC degradation by FtsH. The increased levels of LpxC, the enzyme that catalyzes the first committed step in LPS biosynthesis, increases the amount of LPS produced. Our data suggest that PldA acts as a sensor for lipid asymmetry in the OM. PldA protects the OM barrier by both degrading mislocalized PLs and generating lipid second messengers that enable long-distance signaling that prompts the cell to restore homeostasis at a distant organelle. IMPORTANCE The outer membrane of Gram-negative bacteria is an effective permeability barrier that protects the cell from toxic agents, including antibiotics. Barrier defects are often manifested by phospholipids present in the outer leaflet of this membrane that take up space normally occupied by lipopolysaccharide. We have discovered a signaling mechanism that operates across the entire cell envelope used by the cell to detect these outer membrane defects. A phospholipase, PldA, that functions to degrade these

  17. Molecular dynamics exploration of poration and leaking caused by Kalata B1 in HIV-infected cell membrane compared to host and HIV membranes.

    PubMed

    Nawae, Wanapinun; Hannongbua, Supa; Ruengjitchatchawalya, Marasri

    2017-06-15

    The membrane disruption activities of kalata B1 (kB1) were investigated using molecular dynamics simulations with membrane models. The models were constructed to mimic the lipid microdomain formation in membranes of HIV particle, HIV-infected cell, and host cell. The differences in the lipid ratios of these membranes caused the formation of liquid ordered (lo) domains of different sizes, which affected the binding and activity of kB1. Stronger kB1 disruptive activity was observed for the membrane with small sized lo domain. Our results show that kB1 causes membrane leaking without bilayer penetration. The membrane poration mechanism involved in the disorganization of the lo domain and in cholesterol inter-leaflet translocation is described. This study enhances our understanding of the membrane activity of kB1, which may be useful for designing novel and potentially therapeutic peptides based on the kB1 framework.

  18. Architectures of Lipid Transport Systems for the Bacterial Outer Membrane.

    PubMed

    Ekiert, Damian C; Bhabha, Gira; Isom, Georgia L; Greenan, Garrett; Ovchinnikov, Sergey; Henderson, Ian R; Cox, Jeffery S; Vale, Ronald D

    2017-04-06

    How phospholipids are trafficked between the bacterial inner and outer membranes through the hydrophilic space of the periplasm is not known. We report that members of the mammalian cell entry (MCE) protein family form hexameric assemblies with a central channel capable of mediating lipid transport. The E. coli MCE protein, MlaD, forms a ring associated with an ABC transporter complex in the inner membrane. A soluble lipid-binding protein, MlaC, ferries lipids between MlaD and an outer membrane protein complex. In contrast, EM structures of two other E. coli MCE proteins show that YebT forms an elongated tube consisting of seven stacked MCE rings, and PqiB adopts a syringe-like architecture. Both YebT and PqiB create channels of sufficient length to span the periplasmic space. This work reveals diverse architectures of highly conserved protein-based channels implicated in the transport of lipids between the membranes of bacteria and some eukaryotic organelles. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Lipophilic oligonucleotides spontaneously insert into lipid membranes, bind complementary DNA strands, and sequester into lipid-disordered domains.

    PubMed

    Bunge, Andreas; Kurz, Anke; Windeck, Anne-Kathrin; Korte, Thomas; Flasche, Wolfgang; Liebscher, Jürgen; Herrmann, Andreas; Huster, Daniel

    2007-04-10

    For the development of surface functionalized bilayers, we have synthesized lipophilic oligonucleotides to combine the molecular recognition mechanism of nucleic acids and the self-assembly characteristics of lipids in planar membranes. A lipophilic oligonucleotide consisting of 21 thymidine units and two lipophilic nucleotides with an alpha-tocopherol moiety as a lipophilic anchor was synthesized using solid-phase methods with a phosphoramadite strategy. The interaction of the water soluble lipophilic oligonucleotide with vesicular lipid membranes and its capability to bind complementary DNA strands was studied using complementary methods such as NMR, EPR, DSC, fluorescence spectroscopy, and fluorescence microscopy. This oligonucleotide inserted stably into preformed membranes from the aqueous phase. Thereby, no significant perturbation of the lipid bilayer and its stability was observed. However, the non-lipidated end of the oligonucleotide is exposed to the aqueous environment, is relatively mobile, and is free to interact with complementary DNA strands. Binding of the complementary single-stranded DNA molecules is fast and accomplished by the formation of Watson-Crick base pairs, which was confirmed by 1H NMR chemical shift analysis and fluorescence resonance energy transfer. The molecular structure of the membrane bound DNA double helix is very similar to the free double-stranded DNA. Further, the membrane bound DNA double strands also undergo regular melting. Finally, in raft-like membrane mixtures, the lipophilic oligonucleotide was shown to preferentially sequester into liquid-disordered membrane domains.

  20. Interactions between HIV-1 Neutralizing Antibodies and Model Lipid Membranes imaged with AFM

    NASA Astrophysics Data System (ADS)

    Zauscher, Stefan; Hardy, Gregory; Alam, Munir; Shapter, Joseph

    2012-02-01

    Lipid membrane interactions with rare, broadly neutralizing antibodies (NAbs), 2F5 and 4E10, play a critical role in HIV-1 neutralization. Our research is motivated by recent immunization studies that have shown that induction of antibodies that avidly bind the gp41-MPER antigen is not sufficient for neutralization. Rather, it is required that antigen designs induce polyreactive antibodies that recognize MPER antigens as well as the viral lipid membrane. However, the mechanistic details of how membrane properties influence NAb-lipid and NAb-antigen interactions remain unknown. Furthermore, it is well established that the native viral membrane is heterogeneous, representing a mosaic of lipid rafts and protein clustering. However, the size, physical properties, and dynamics of these regions are poorly characterized and their potential roles in HIV-1 neutralization are also unknown. To understand how membrane properties contribute to 2F5/4E10 membrane interactions, we have engineered biomimetic supported lipid bilayers (SLBs) and use atomic force microscopy to visualize membrane domains, antigen clustering, and antibody-membrane interactions at sub-nanometer z-resolution. Our results show that localized binding of HIV-1 antigens and NAbs occur preferentially with the most fluid membrane domain. This supports the theory that NAbs may interact with regions of low lateral lipid forces that allow antibody insertion into the bilayer.

  1. Membrane lipid profiles of coral responded to zinc oxide nanoparticle-induced perturbations on the cellular membrane.

    PubMed

    Tang, Chuan-Ho; Lin, Ching-Yu; Lee, Shu-Hui; Wang, Wei-Hsien

    2017-06-01

    Zinc oxide nanoparticles (nZnOs) released from popular sunscreens used during marine recreation apparently endanger corals; however, the known biological effects are very limited. Membrane lipids constitute the basic structural element to create cell a dynamic structure according to the circumstance. Nano-specific effects have been shown to mechanically perturb the physical state of the lipid membrane, and the cells accommodating the actions of nZnOs can be involved in the alteration of the membrane lipid composition. To gain insight into the effects of nanoparticles on coral, glycerophosphocholine (GPC) profiling of the coral Seriatopora caliendrum exposed to nZnOs was performed in this study. Increasing lyso-GPCs, docosapentaenoic acid-possessing GPCs and docosahexaenoic acid-possessing GPCs and decreasing arachidonic acid-possessing GPCs were the predominant changes responded to nZnO exposure in the coral. A backfilling of polyunsaturated plasmanylcholines was observed in the coral exposed to nZnO levels over a threshold. These changes can be logically interpreted as an accommodation to nZnOs-induced mechanical disturbances in the cellular membrane based on the biophysical properties of the lipids. Moreover, the coral demonstrated a difference in the changes in lipid profiles between intra-colonial functionally differentiated polyps, indicating an initial membrane composition-dependent response. Based on the physicochemical properties and physiological functions of these changed lipids, some chronic biological effects can be incubated once the coral receives long-term exposure to nZnOs. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Bioactive Structure of Membrane Lipids and Natural Products Elucidated by a Chemistry-Based Approach.

    PubMed

    Murata, Michio; Sugiyama, Shigeru; Matsuoka, Shigeru; Matsumori, Nobuaki

    2015-08-01

    Determining the bioactive structure of membrane lipids is a new concept, which aims to examine the functions of lipids with respect to their three-dimensional structures. As lipids are dynamic by nature, their "structure" does not refer solely to a static picture but also to the local and global motions of the lipid molecules. We consider that interactions with lipids, which are completely defined by their structures, are controlled by the chemical, functional, and conformational matching between lipids and between lipid and protein. In this review, we describe recent advances in understanding the bioactive structures of membrane lipids bound to proteins and related molecules, including some of our recent results. By examining recent works on lipid-raft-related molecules, lipid-protein interactions, and membrane-active natural products, we discuss current perspectives on membrane structural biology. © 2015 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Lipid composition affects the rate of photosensitized dissipation of cross-membrane diffusion potential on liposomes

    PubMed Central

    Ytzhak, Shany; Wuskell, Joseph P.; Loew, Leslie M.; Ehrenberg, Benjamin

    2010-01-01

    Hydrophobic or amphiphilic tetrapyrrole sensitizers are taken up by cells and are usually located in cellular lipid membranes. Singlet oxygen is photogenerated by the sensitizer and it diffuses in the membrane and causes oxidative damage to membrane components. This damage can occur to membrane lipids and to membrane-localized proteins. Depolarization of the Nernst electric potential on cells’ membranes has been observed in cellular photosensitization, but it was not established whether lipid oxidation is a relevant factor leading to abolishing the resting potential of cells’ membranes and to their death. In this work we studied the effect of liposomes’ lipid composition on the kinetics of hematoporphyrin-photosensitized dissipation of K+-diffusion electric potential that was generated across the membranes. We employed an electrochromic voltage-sensitive spectroscopic probe that possesses a high fluorescence signal response to the potential. We found a correlation between the structure and unsaturation of lipids and the leakage of the membrane, following photosensitization. As the extent of non-conjugated unsaturation of the lipids is increased from 1 to 6 double bonds, the kinetics of depolarization become faster. We also found that the kinetics of depolarization is affected by the percentage of the unsaturated lipids in the liposome: as the fraction of the unsaturated lipids increases the leakage trough the membrane is enhanced. When liposomes are composed of a lipid mixture similar to that of natural membranes and photosensitization is being carried out under usual photodynamic therapy (PDT) conditions, photodamage to the lipids is not likely to cause enhanced permeability of ions through the membrane, which would have been a mechanism that leads to cell death. PMID:20536150

  4. A novel squarylium dye for monitoring oxidative processes in lipid membranes.

    PubMed

    Trusova, Valeriya M; Gorbenko, Galyna P; Deligeorgiev, Todor; Gadjev, Nikolai; Vasilev, Aleksey

    2009-11-01

    A novel squaraine probe SQ-1 has been found to be appropriate for monitoring the peroxidation processes in membrane systems. Formation of free radicals was triggered by methemoglobin (metHb) or cytochrome c (cyt c) binding to the model lipid membranes composed of zwitterionic lipid phosphatidylcholine (PC) and anionic lipid cardiolipin (CL). Protein association with the lipid vesicles was followed by drastic quenching of SQ-1 fluorescence. The observed spectral changes were suppressed in the presence of free radical scavengers, butylated hydroxytoluene (BHT) and thiourea (TM) suggesting that SQ-1 decolorization can be attributed to its reactions with lipid radicals.

  5. Flexible Charged Macromolecules on Mixed Fluid Lipid Membranes: Theory and Monte Carlo Simulations

    PubMed Central

    Tzlil, Shelly; Ben-Shaul, Avinoam

    2005-01-01

    Fluid membranes containing charged lipids enhance binding of oppositely charged proteins by mobilizing these lipids into the interaction zone, overcoming the concomitant entropic losses due to lipid segregation and lower conformational freedom upon macromolecule adsorption. We study this energetic-entropic interplay using Monte Carlo simulations and theory. Our model system consists of a flexible cationic polyelectrolyte, interacting, via Debye-Hückel and short-ranged repulsive potentials, with membranes containing neutral lipids, 1% tetravalent, and 10% (or 1%) monovalent anionic lipids. Adsorption onto a fluid membrane is invariably stronger than to an equally charged frozen or uniform membrane. Although monovalent lipids may suffice for binding rigid macromolecules, polyvalent counter-lipids (e.g., phosphatidylinositol 4,5 bisphosphate), whose entropy loss upon localization is negligible, are crucial for binding flexible macromolecules, which lose conformational entropy upon adsorption. Extending Rosenbluth's Monte Carlo scheme we directly simulate polymer adsorption on fluid membranes. Yet, we argue that similar information could be derived from a biased superposition of quenched membrane simulations. Using a simple cell model we account for surface concentration effects, and show that the average adsorption probabilities on annealed and quenched membranes coincide at vanishing surface concentrations. We discuss the relevance of our model to the electrostatic-switch mechanism of, e.g., the myristoylated alanine-rich C kinase substrate protein. PMID:16126828

  6. Film Balance Studies of Membrane Lipids and Related Molecules

    ERIC Educational Resources Information Center

    Cadenhead, D. A.

    1972-01-01

    Discusses apparatus, techniques, and measurements used to determine cell membrane composition. The use of a film balance to study monolayer membranes of selected lipids is described and results reported. (TS)

  7. Structure formation of lipid membranes: Membrane self-assembly and vesicle opening-up to octopus-like micelles

    NASA Astrophysics Data System (ADS)

    Noguchi, Hiroshi

    2013-02-01

    We briefly review our recent studies on self-assembly and vesicle rupture of lipid membranes using coarse-grained molecular simulations. For single component membranes, lipid molecules self-assemble from random gas states to vesicles via disk-shaped clusters. Clusters aggregate into larger clusters, and subsequently the large disks close into vesicles. The size of vesicles are determined by kinetics than by thermodynamics. When a vesicle composed of lipid and detergent types of molecules is ruptured, a disk-shaped micelle called bicelle can be formed. When both surfactants have negligibly low critical micelle concentration, it is found that bicelles connected with worm-like micelles are also formed depending on the surfactant ratio and spontaneous curvature of the membrane monolayer.

  8. Communication: Alamethicin can capture lipid-like molecules in the membrane

    NASA Astrophysics Data System (ADS)

    Afanasyeva, Ekaterina F.; Syryamina, Victoria N.; Dzuba, Sergei A.

    2017-01-01

    Alamethicin (Alm) is a 19-mer antimicrobial peptide produced by fungus Trichoderma viride. Above a threshold concentration, Alm forms pores across the membrane, providing a mechanism of its antimicrobial action. Here we show that at a small concentration which is below the threshold value, Alm participates in formation of nanoscale lipid-mediated clusters of guest lipid-like molecules in the membrane. These results are obtained by electron spin echo (ESE) technique—a pulsed version of electron paramagnetic resonance—on spin-labeled stearic acid in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer with Alm added at 1/200 peptide-to-lipid ratio. ESE decay measurements are interpreted assuming that stearic acid molecules in the membrane are assembling around the Alm molecule. One may suggest that this Alm capturing effect on the guest lipid-like molecules could be important for the peptide antimicrobial action.

  9. Biomimetic interfaces based on S-layer proteins, lipid membranes and functional biomolecules

    PubMed Central

    Schuster, Bernhard; Sleytr, Uwe B.

    2014-01-01

    Designing and utilization of biomimetic membrane systems generated by bottom-up processes is a rapidly growing scientific and engineering field. Elucidation of the supramolecular construction principle of archaeal cell envelopes composed of S-layer stabilized lipid membranes led to new strategies for generating highly stable functional lipid membranes at meso- and macroscopic scale. In this review, we provide a state-of-the-art survey of how S-layer proteins, lipids and polymers may be used as basic building blocks for the assembly of S-layer-supported lipid membranes. These biomimetic membrane systems are distinguished by a nanopatterned fluidity, enhanced stability and longevity and, thus, provide a dedicated reconstitution matrix for membrane-active peptides and transmembrane proteins. Exciting areas in the (lab-on-a-) biochip technology are combining composite S-layer membrane systems involving specific membrane functions with the silicon world. Thus, it might become possible to create artificial noses or tongues, where many receptor proteins have to be exposed and read out simultaneously. Moreover, S-layer-coated liposomes and emulsomes copying virus envelopes constitute promising nanoformulations for the production of novel targeting, delivery, encapsulation and imaging systems. PMID:24812051

  10. Neutron scattering to study membrane systems: from lipid vesicles to living cells.

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nickels, Jonathan D.; Chatterjee, Sneha; Stanley, Christopher B.

    The existence and role of lateral lipid organization in biological membranes has been studied and contested for more than 30 years. Lipid domains, or rafts, are hypothesized as scalable compartments in biological membranes, providing appropriate physical environments to their resident membrane proteins. This implies that lateral lipid organization is associated with a range of biological functions, such as protein co-localization, membrane trafficking, and cell signaling, to name just a few. Neutron scattering techniques have proven to be an excellent tool to investigate these structural features in model lipids, and more recently, in living cells. I will discuss our recent workmore » using neutrons to probe the structure and mechanical properties in model lipid systems and our current efforts in using neutrons to probe the structure and organization of the bilayer in a living cell. These efforts in living cells have used genetic and biochemical strategies to generate a large neutron scattering contrast, making the membrane visible. I will present our results showing in vivo bilayer structure and discuss the outlook for this approach.« less

  11. Chemotherapy drugs form ion pores in membranes due to physical interactions with lipids.

    PubMed

    Ashrafuzzaman, Mohammad; Tseng, Chih-Yuan; Duszyk, Marek; Tuszynski, Jack A

    2012-12-01

    We demonstrate the effects on membrane of the tubulin-binding chemotherapy drugs: thiocolchicoside and taxol. Electrophysiology recordings across lipid membranes in aqueous phases containing drugs were used to investigate the drug effects on membrane conductance. Molecular dynamics simulation of the chemotherapy drug-lipid complexes was used to elucidate the mechanism at an atomistic level. Both drugs are observed to induce stable ion-flowing pores across membranes. Discrete pore current-time plots exhibit triangular conductance events in contrast to rectangular ones found for ion channels. Molecular dynamics simulations indicate that drugs and lipids experience electrostatic and van der Waals interactions for short periods of time when found within each other's proximity. The energies from these two interactions are found to be similar to the energies derived theoretically using the screened Coulomb and the van der Waals interactions between peptides and lipids due to mainly their charge properties while forming peptide-induced ion channels in lipid bilayers. Experimental and in silico studies together suggest that the chemotherapy drugs induce ion pores inside lipid membranes due to drug-lipid physical interactions. The findings reveal cytotoxic effects of drugs on the cell membrane, which may aid in novel drug development for treatment of cancer and other diseases. © 2012 John Wiley & Sons A/S.

  12. Stabilization of composition fluctuations in mixed membranes by hybrid lipids

    NASA Astrophysics Data System (ADS)

    Safran, Samuel; Palmieri, Benoit

    2013-03-01

    A ternary mixture model is proposed to describe composition fluctuations in mixed membranes composed of saturated, unsaturated and hybrid lipids. The asymmetric hybrid lipid has one saturated and one unsaturated hydrocarbon chain and it can reduce the packing incompatibility between saturated and unsaturated lipids. A methodology to recast the free-energy of the lattice in terms of a continuous isotropic field theory is proposed and used to analyze composition fluctuations above the critical temperature. The effect of hybrid lipids on fluctuations domains rich in saturated/unsaturated lipids is predicted. The correlation length of such fluctuations decreases significantly with increasing amounts of hybrids even if the temperature is maintained close to the critical temperature. This provides an upper bound for the domain sizes expected in rafts stabilized by hybrids, above the critical temperature. When the hybrid composition of the membrane is increased further, a crossover value is found above which ``stripe-like'' fluctuations are observed. The wavelength of these fluctuations decreases with increasing hybrid fraction and tends toward a molecular size in a membrane that contains only hybrids.

  13. Extended synaptotagmins are Ca2+-dependent lipid transfer proteins at membrane contact sites.

    PubMed

    Yu, Haijia; Liu, Yinghui; Gulbranson, Daniel R; Paine, Alex; Rathore, Shailendra S; Shen, Jingshi

    2016-04-19

    Organelles are in constant communication with each other through exchange of proteins (mediated by trafficking vesicles) and lipids [mediated by both trafficking vesicles and lipid transfer proteins (LTPs)]. It has long been known that vesicle trafficking can be tightly regulated by the second messenger Ca(2+), allowing membrane protein transport to be adjusted according to physiological demands. However, it remains unclear whether LTP-mediated lipid transport can also be regulated by Ca(2+) In this work, we show that extended synaptotagmins (E-Syts), poorly understood membrane proteins at endoplasmic reticulum-plasma membrane contact sites, are Ca(2+)-dependent LTPs. Using both recombinant and endogenous mammalian proteins, we discovered that E-Syts transfer glycerophospholipids between membrane bilayers in the presence of Ca(2+) E-Syts use their lipid-accommodating synaptotagmin-like mitochondrial lipid binding protein (SMP) domains to transfer lipids. However, the SMP domains themselves cannot transport lipids unless the two membranes are tightly tethered by Ca(2+)-bound C2 domains. Strikingly, the Ca(2+)-regulated lipid transfer activity of E-Syts was fully recapitulated when the SMP domain was fused to the cytosolic domain of synaptotagmin-1, the Ca(2+)sensor in synaptic vesicle fusion, indicating that a common mechanism of membrane tethering governs the Ca(2+)regulation of lipid transfer and vesicle fusion. Finally, we showed that microsomal vesicles isolated from mammalian cells contained robust Ca(2+)-dependent lipid transfer activities, which were mediated by E-Syts. These findings established E-Syts as a novel class of LTPs and showed that LTP-mediated lipid trafficking, like vesicular transport, can be subject to tight Ca(2+)regulation.

  14. On the interaction between fluoxetine and lipid membranes: Effect of the lipid composition

    NASA Astrophysics Data System (ADS)

    Pham, Vy T.; Nguyen, Trinh Q.; Dao, Uyen P. N.; Nguyen, Trang T.

    2018-02-01

    Molecular interaction between the antidepressant fluoxetine and lipid bilayers was investigated in order to provide insights into the drug's incorporation to lipid membranes. In particular, the effects of lipid's unsaturation degree and cholesterol content on the partitioning of fluoxetine into large unilamellar vesicles (LUVs) comprised of unsaturated 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and saturated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were evaluated using second derivative spectrophotometry and Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR). It was found that fluoxetine partitioned to a greater extent into the liquid-crystalline DOPC LUVs than into the solid-gel DPPC LUVs. The lipid physical state dependence of drug partitioning was verified by increasing the temperature in which the partition coefficient of fluoxetine significantly increased upon the change of the lipid phase from solid-gel to liquid-crystalline. The incorporation of 28 mol% cholesterol into the LUVs exerted a significant influence on the drug partitioning into both DOPC and DPPC LUVs. The ATR-FTIR study revealed that fluoxetine perturbed the conformation of DOPC more strongly than that of DPPC due to the cis-double bonds in the lipid acyl chains. Fluoxetine possibly bound to the carbonyl moiety of the lipids through the hydrogen bonding formation while displaced some water molecules surrounding the PO2- regions of the lipid head groups. Cholesterol, however, could lessen the interaction between fluoxetine and the carbonyl groups of both DOPC and DPPC LUVs. These findings provided a better understanding of the role of lipid structure and cholesterol on the interaction between fluoxetine and lipid membranes, shedding more light into the drug's therapeutic action.

  15. Ion-Induced Defect Permeation of Lipid Membranes

    PubMed Central

    Vorobyov, Igor; Olson, Timothy E.; Kim, Jung H.; Koeppe, Roger E.; Andersen, Olaf S.; Allen, Toby W.

    2014-01-01

    We have explored the mechanisms of uncatalyzed membrane ion permeation using atomistic simulations and electrophysiological recordings. The solubility-diffusion mechanism of membrane charge transport has prevailed since the 1960s, despite inconsistencies in experimental observations and its lack of consideration for the flexible response of lipid bilayers. We show that direct lipid bilayer translocation of alkali metal cations, Cl–, and a charged arginine side chain analog occurs via an ion-induced defect mechanism. Contrary to some previous suggestions, the arginine analog experiences a large free-energy barrier, very similar to those for Na+, K+, and Cl–. Our simulations reveal that membrane perturbations, due to the movement of an ion, are central for explaining the permeation process, leading to both free-energy and diffusion-coefficient profiles that show little dependence on ion chemistry and charge, despite wide-ranging hydration energies and the membrane’s dipole potential. The results yield membrane permeabilities that are in semiquantitative agreement with experiments in terms of both magnitude and selectivity. We conclude that ion-induced defect-mediated permeation may compete with transient pores as the dominant mechanism of uncatalyzed ion permeation, providing new understanding for the actions of a range of membrane-active peptides and proteins. PMID:24507599

  16. Thermal conductivity and rectification in asymmetric archaeal lipid membranes

    NASA Astrophysics Data System (ADS)

    Youssefian, Sina; Rahbar, Nima; Van Dessel, Steven

    2018-05-01

    Nature employs lipids to construct nanostructured membranes that self-assemble in an aqueous environment to separate the cell interior from the exterior environment. Membrane composition changes among species and according to environmental conditions, which allows organisms to occupy a wide variety of different habitats. Lipid bilayers are phase-change materials that exhibit strong thermotropic and lyotropic phase behavior in an aqueous environment, which may also cause thermal rectification. Among different types of lipids, archaeal lipids are of great interest due to their ability to withstand extreme conditions. In this paper, nonequilibrium molecular dynamics simulations were employed to study the nanostructures and thermal properties of different archaeols and to investigate thermal rectification effects in asymmetric archaeal membranes. In particular, we are interested in understanding the role of bridged phytanyl chains and cyclopentane groups in controlling the phase transition temperature and heat flow across the membrane. Our results indicate that the bridged phytanyl chains decrease the molecular packing of lipids, whereas the existence of cyclopentane rings on the tail groups increases the molecular packing by enhancing the interactions between isoprenoid chains. We found that macrocyclic archaeols have the highest thermal conductivity, whereas macrocyclic archaeols with two cyclopentane rings have the lowest. The effect of the temperature on the variation of thermal conductivity was found to be progressive. Our results further indicate that small thermal rectification effects occur in asymmetric archaeol bilayer membranes at around 25 K temperature gradient. The calculated thermal rectification factor was around 0.09 which is in the range of rectification factor obtained experimentally for nanostructures such as carbon nanotubes (0.07). Such phenomena may be of biological significance and could also be optimized for use in various engineering

  17. Probing the Interaction of Dielectric Nanoparticles with Supported Lipid Membrane Coatings on Nanoplasmonic Arrays

    PubMed Central

    Ferhan, Abdul Rahim; Ma, Gamaliel Junren; Jackman, Joshua A.; Sut, Tun Naw; Park, Jae Hyeon; Cho, Nam-Joon

    2017-01-01

    The integration of supported lipid membranes with surface-based nanoplasmonic arrays provides a powerful sensing approach to investigate biointerfacial phenomena at membrane interfaces. While a growing number of lipid vesicles, protein, and nucleic acid systems have been explored with nanoplasmonic sensors, there has been only very limited investigation of the interactions between solution-phase nanomaterials and supported lipid membranes. Herein, we established a surface-based localized surface plasmon resonance (LSPR) sensing platform for probing the interaction of dielectric nanoparticles with supported lipid bilayer (SLB)-coated, plasmonic nanodisk arrays. A key emphasis was placed on controlling membrane functionality by tuning the membrane surface charge vis-à-vis lipid composition. The optical sensing properties of the bare and SLB-coated sensor surfaces were quantitatively compared, and provided an experimental approach to evaluate nanoparticle–membrane interactions across different SLB platforms. While the interaction of negatively-charged silica nanoparticles (SiNPs) with a zwitterionic SLB resulted in monotonic adsorption, a stronger interaction with a positively-charged SLB resulted in adsorption and lipid transfer from the SLB to the SiNP surface, in turn influencing the LSPR measurement responses based on the changing spatial proximity of transferred lipids relative to the sensor surface. Precoating SiNPs with bovine serum albumin (BSA) suppressed lipid transfer, resulting in monotonic adsorption onto both zwitterionic and positively-charged SLBs. Collectively, our findings contribute a quantitative understanding of how supported lipid membrane coatings influence the sensing performance of nanoplasmonic arrays, and demonstrate how the high surface sensitivity of nanoplasmonic sensors is well-suited for detecting the complex interactions between nanoparticles and lipid membranes. PMID:28644423

  18. The incorporation of hydrophobic protein receptors and artificial lipid membranes.

    PubMed

    Reader, T A; Fiszer de Plazas, S; Salas, P J; de Robertis, E

    1976-01-01

    The mechanism of chemical synaptic transmission implies: 1) the existence of a specific protein receptor at the postsynaptic membrane, and 2) the interaction between the transmitter released and the receptor, thus producing a change in ionic permeability. Previous studies from our laboratory have shown that special hydrophobic proteins extracted from postsynpatic membranes of different tissues showed a high affinity binding for the different pharmacological agents. The present paper describes experiments in which different hydrophobic protein binding acetylcholine, noradrenaline, gamma-aminobutyric acid, and glutamate were incorporated into artificial lipid membranes, similar to those first described by Mueller et al. (19). The effect of the different pharmacological agents was tested under experimental conditions of voltage clamp and the d.c. current changes measured. The results were then compared for the different lipid-protein membranes employed during the steady state and during transient conductance changes. The specificity of the responses indicate that artificial lipid membranes containing these hydrophobic proteins from electroplax, myocardium, spleen capsule and shrimp muscle can be used as a model to study pharmacologic receptors.

  19. Asymmetric Hybrid Polymer-Lipid Giant Vesicles as Cell Membrane Mimics.

    PubMed

    Peyret, Ariane; Ibarboure, Emmanuel; Le Meins, Jean-François; Lecommandoux, Sebastien

    2018-01-01

    Lipid membrane asymmetry plays an important role in cell function and activity, being for instance a relevant signal of its integrity. The development of artificial asymmetric membranes thus represents a key challenge. In this context, an emulsion-centrifugation method is developed to prepare giant vesicles with an asymmetric membrane composed of an inner monolayer of poly(butadiene)- b -poly(ethylene oxide) (PBut- b -PEO) and outer monolayer of 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC). The formation of a complete membrane asymmetry is demonstrated and its stability with time is followed by measuring lipid transverse diffusion. From fluorescence spectroscopy measurements, the lipid half-life is estimated to be 7.5 h. Using fluorescence recovery after photobleaching technique, the diffusion coefficient of 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine- N -(lissamine rhodamine B sulfonyl) (DOPE-rhod, inserted into the POPC leaflet) is determined to be about D = 1.8 ± 0.50 μm 2 s -1 at 25 °C and D = 2.3 ± 0.7 μm 2 s -1 at 37 °C, between the characteristic values of pure POPC and pure polymer giant vesicles and in good agreement with the diffusion of lipids in a variety of biological membranes. These results demonstrate the ability to prepare a cell-like model system that displays an asymmetric membrane with transverse and translational diffusion properties similar to that of biological cells.

  20. Interaction of Dendritic Polymers with Synthetic Lipid and Cell Membranes

    NASA Astrophysics Data System (ADS)

    Mecke, Almut; Hong, Seungpyo; Bielinska, Anna U.; Banaszak Holl, Mark M.; Orr, Bradford G.; Baker, James R., Jr.

    2004-03-01

    Polyamidoamine (PAMAM) dendrimers are promising candidates for the development of nanoscale therapeutic transport agents. Here we present studies on dendrimer-membrane interactions leading to a better understanding of possible uptake mechanisms into cells. Using synthetic lipid and natural cell membranes as model systems it is shown that the effect of PAMAM dendrimers on a membrane strongly depends on the dendrimer generation, architecture and chemical properties of the branch end groups. Atomic force microscopy data indicates that generation 7 dendrimers have the ability to form small ( 10-100 nm) holes in a lipid bilayer. When dendrimers with otherwise identical chemical properties are arranged in a covalently linked cluster, no hole formation occurs. Dendrimer-lipid micelle formation is proposed and investigated as a possible mechanism for this behavior. Smaller dendrimers (generation 5) have a greatly reduced ability to remove lipid molecules from a bilayer. In addition to the size of the dendrimer, the charge of the branch end groups plays a significant role for dendrimer-membrane interactions. These results agree well with biological studies using cultured cells and point to a new mechanism of specific targeting and uptake into cells.

  1. Lipid-glass adhesion in giga-sealed patch-clamped membranes.

    PubMed

    Opsahl, L R; Webb, W W

    1994-01-01

    Adhesion between patch-clamped lipid membranes and glass micropipettes is measured by high contrast video imaging of the mechanical response to the application of suction pressure across the patch. The free patch of membrane reversibly alters both its contact angle and radius of curvature on pressure changes. The assumption that an adhesive force between the membrane and the pipette can sustain normal tension up to a maximum Ta at the edge of the free patch accounts for the observed mechanical responses. When the normal component of the pressure-induced membrane tension exceeds Ta membrane at the contact point between the free patch and the lipid-glass interface is pulled away from the pipette wall, resulting in a decreased radius of curvature for the patch and an increased contact angle. Measurements of the membrane radius of curvature as a function of the suction pressure and pipette radius determine line adhesion tensions Ta which range from 0.5 to 4.0 dyn/cm. Similar behavior of patch-clamped cell membranes implies similar adhesion mechanics.

  2. Effects of Lipid Composition on Bilayer Membranes Quantified by All-Atom Molecular Dynamics.

    PubMed

    Ding, Wei; Palaiokostas, Michail; Wang, Wen; Orsi, Mario

    2015-12-10

    Biological bilayer membranes typically contain varying amounts of lamellar and nonlamellar lipids. Lamellar lipids, such as dioleoylphosphatidylcholine (DOPC), are defined by their tendency to form the lamellar phase, ubiquitous in biology. Nonlamellar lipids, such as dioleoylphosphatidylethanolamine (DOPE), prefer instead to form nonlamellar phases, which are mostly nonbiological. However, nonlamellar lipids mix with lamellar lipids in biomembrane structures that remain overall lamellar. Importantly, changes in the lamellar vs nonlamellar lipid composition are believed to affect membrane function and modulate membrane proteins. In this work, we employ atomistic molecular dynamics simulations to quantify how a range of bilayer properties are altered by variations in the lamellar vs nonlamellar lipid composition. Specifically, we simulate five DOPC/DOPE bilayers at mixing ratios of 1/0, 3/1, 1/1, 1/3, and 0/1. We examine properties including lipid area and bilayer thickness, as well as the transmembrane profiles of electron density, lateral pressure, electric field, and dipole potential. While the bilayer structure is only marginally altered by lipid composition changes, dramatic effects are observed for the lateral pressure, electric field, and dipole potential profiles. Possible implications for membrane function are discussed.

  3. Lipid domains control myelin basic protein adsorption and membrane interactions between model myelin lipid bilayers

    PubMed Central

    Lee, Dong Woog; Banquy, Xavier; Kristiansen, Kai; Kaufman, Yair; Boggs, Joan M.; Israelachvili, Jacob N.

    2014-01-01

    The surface forces apparatus and atomic force microscope were used to study the effects of lipid composition and concentrations of myelin basic protein (MBP) on the structure of model lipid bilayers, as well as the interaction forces and adhesion between them. The lipid bilayers had a lipid composition characteristic of the cytoplasmic leaflets of myelin from “normal” (healthy) and “disease-like” [experimental allergic encephalomyelitis (EAE)] animals. They showed significant differences in the adsorption mechanism of MBP. MBP adsorbs on normal bilayers to form a compact film (3–4 nm) with strong intermembrane adhesion (∼0.36 mJ/m2), in contrast to its formation of thicker (7–8 nm) swelled films with weaker intermembrane adhesion (∼0.13 mJ/m2) on EAE bilayers. MBP preferentially adsorbs to liquid-disordered submicron domains within the lipid membranes, attributed to hydrophobic attractions. These results show a direct connection between the lipid composition of membranes and membrane–protein adsorption mechanisms that affects intermembrane spacing and adhesion and has direct implications for demyelinating diseases. PMID:24516125

  4. Electrostatic interactions among hydrophobic ions in lipid bilayer membranes.

    PubMed Central

    Andersen, O S; Feldberg, S; Nakadomari, H; Levy, S; McLaughlin, S

    1978-01-01

    We have shown that the absorption of tetraphenylborate into black lipid membranes formed from either bacterial phosphatidylethanolamine or glycerolmonooleate produces concentration-dependent changes in the electrostatic potential between the membrane interior and the bulk aqueous phases. These potential changes were studied by a variety of techniques: voltage clamp, charge pulse, and "probe" measurements on black lipid membranes; electrophroetic mobility measurements on phospholipid vesicles; and surface potential measurements on phospholipid monolayers. The magnitude of the potential changes indicates that tetraphenylborate absorbs into a region of the membrane with a low dielectric constant, where it produces substantial boundary potentials, as first suggested by Markin et al. (1971). Many features of our data can be explained by a simple three-capacitor model, which we develop in a self-consistent manner. Some discrepancies between our data and the simple model suggest that discrete charge phenomena may be important within these thin membranes. PMID:620077

  5. Combined Enzymatic and Mechanical Cell Disruption and Lipid Extraction of Green Alga Neochloris oleoabundans

    PubMed Central

    Wang, Dongqin; Li, Yanqun; Hu, Xueqiong; Su, Weimin; Zhong, Min

    2015-01-01

    Microalgal biodiesel is one of the most promising renewable fuels. The wet technique for lipids extraction has advantages over the dry method, such as energy-saving and shorter procedure. The cell disruption is a key factor in wet oil extraction to facilitate the intracellular oil release. Ultrasonication, high-pressure homogenization, enzymatic hydrolysis and the combination of enzymatic hydrolysis with high-pressure homogenization and ultrasonication were employed in this study to disrupt the cells of the microalga Neochloris oleoabundans. The cell disruption degree was investigated. The cell morphology before and after disruption was assessed with scanning and transmission electron microscopy. The energy requirements and the operation cost for wet cell disruption were also estimated. The highest disruption degree, up to 95.41%, assessed by accounting method was achieved by the combination of enzymatic hydrolysis and high-pressure homogenization. A lipid recovery of 92.6% was also obtained by the combined process. The combined process was found to be more efficient and economical compared with the individual process. PMID:25853267

  6. Membrane on a Chip: A Functional Tethered Lipid Bilayer Membrane on Silicon Oxide Surfaces

    PubMed Central

    Atanasov, Vladimir; Knorr, Nikolaus; Duran, Randolph S.; Ingebrandt, Sven; Offenhäusser, Andreas; Knoll, Wolfgang; Köper, Ingo

    2005-01-01

    Tethered membranes have been proven during recent years to be a powerful and flexible biomimetic platform. We reported in a previous article on the design of a new architecture based on the self-assembly of a thiolipid on ultrasmooth gold substrates, which shows extremely good electrical sealing properties as well as functionality of a bilayer membrane. Here, we describe the synthesis of lipids for a more modular design and the adaptation of the linker part to silane chemistry. We were able to form a functional tethered bilayer lipid membrane with good electrical sealing properties covering a silicon oxide surface. We demonstrate the functional incorporation of the ion carrier valinomycin and of the ion channel gramicidin. PMID:16127170

  7. Cationic nanoparticles induce nanoscale disruption in living cell plasma membranes.

    PubMed

    Chen, Jiumei; Hessler, Jessica A; Putchakayala, Krishna; Panama, Brian K; Khan, Damian P; Hong, Seungpyo; Mullen, Douglas G; Dimaggio, Stassi C; Som, Abhigyan; Tew, Gregory N; Lopatin, Anatoli N; Baker, James R; Holl, Mark M Banaszak; Orr, Bradford G

    2009-08-13

    It has long been recognized that cationic nanoparticles induce cell membrane permeability. Recently, it has been found that cationic nanoparticles induce the formation and/or growth of nanoscale holes in supported lipid bilayers. In this paper, we show that noncytotoxic concentrations of cationic nanoparticles induce 30-2000 pA currents in 293A (human embryonic kidney) and KB (human epidermoid carcinoma) cells, consistent with a nanoscale defect such as a single hole or group of holes in the cell membrane ranging from 1 to 350 nm(2) in total area. Other forms of nanoscale defects, including the nanoparticle porating agents adsorbing onto or intercalating into the lipid bilayer, are also consistent; although the size of the defect must increase to account for any reduction in ion conduction, as compared to a water channel. An individual defect forming event takes 1-100 ms, while membrane resealing may occur over tens of seconds. Patch-clamp data provide direct evidence for the formation of nanoscale defects in living cell membranes. The cationic polymer data are compared and contrasted with patch-clamp data obtained for an amphiphilic phenylene ethynylene antimicrobial oligomer (AMO-3), a small molecule that is proposed to make well-defined 3.4 nm holes in lipid bilayers. Here, we observe data that are consistent with AMO-3 making approximately 3 nm holes in living cell membranes.

  8. Changes in membrane lipids drive increased endocytosis following Fas ligation.

    PubMed

    Degli Esposti, Mauro; Matarrese, Paola; Tinari, Antonella; Longo, Agostina; Recalchi, Serena; Khosravi-Far, Roya; Malorni, Walter; Misasi, Roberta; Garofalo, Tina; Sorice, Maurizio

    2017-05-01

    Once activated, some surface receptors promote membrane movements that open new portals of endocytosis, in part to facilitate the internalization of their activated complexes. The prototypic death receptor Fas (CD95/Apo1) promotes a wave of enhanced endocytosis that induces a transient intermixing of endosomes with mitochondria in cells that require mitochondria to amplify death signaling. This initiates a global alteration in membrane traffic that originates from changes in key membrane lipids occurring in the endoplasmic reticulum (ER). We have focused the current study on specific lipid changes occurring early after Fas ligation. We analyzed the interaction between endosomes and mitochondria in Jurkat T cells by nanospray-Time-of-flight (ToF) Mass Spectrometry. Immediately after Fas ligation, we found a transient wave of lipid changes that drives a subpopulation of early endosomes to merge with mitochondria. The earliest event appears to be a decrease of phosphatidylcholine (PC), linked to a metabolic switch enhancing phosphatidylinositol (PI) and phosphoinositides, which are crucial for the formation of vacuolar membranes and endocytosis. Lipid changes occur independently of caspase activation and appear to be exacerbated by caspase inhibition. Conversely, inhibition or compensation of PC deficiency attenuates endocytosis, endosome-mitochondria mixing and the induction of cell death. Deficiency of receptor interacting protein, RIP, also limits the specific changes in membrane lipids that are induced by Fas activation, with parallel reduction of endocytosis. Thus, Fas activation rapidly changes the interconversion of PC and PI, which then drives enhanced endocytosis, thus likely propagating death signaling from the cell surface to mitochondria and other organelles.

  9. Membrane lipids and the origin of life

    NASA Technical Reports Server (NTRS)

    Oro, J.; Holzer, G.; Rao, M.; Tornabene, T. G.

    1981-01-01

    The current state of knowledge regarding the development of biological systems is briefly reviewed. At a crucial stage concerning the evolution of such systems, the mechanisms leading to more complex structures must have evolved within the confines of a protected microenvironment, similar to those provided by the contemporary cell membranes. The major components found normally in biomembranes are phospholipids. The structure of the biomembrane is examined, and attention is given to questions concerning the availability of the structural components which are necessary in the formation of primitive lipid membranes. Two approaches regarding the study of protomembranes are discussed. The probability of obtaining ether lipids under prebiotic conditions is considered, taking into account the formation of cyclic and acyclic isoprenoids by the irradiation of isoprene with UV.

  10. Probing protein-lipid interactions by FRET between membrane fluorophores

    NASA Astrophysics Data System (ADS)

    Trusova, Valeriya M.; Gorbenko, Galyna P.; Deligeorgiev, Todor; Gadjev, Nikolai

    2016-09-01

    Förster resonance energy transfer (FRET) is a powerful fluorescence technique that has found numerous applications in medicine and biology. One area where FRET proved to be especially informative involves the intermolecular interactions in biological membranes. The present study was focused on developing and verifying a Monte-Carlo approach to analyzing the results of FRET between the membrane-bound fluorophores. This approach was employed to quantify FRET from benzanthrone dye ABM to squaraine dye SQ-1 in the model protein-lipid system containing a polycationic globular protein lysozyme and negatively charged lipid vesicles composed of phosphatidylcholine and phosphatidylglycerol. It was found that acceptor redistribution between the lipid bilayer and protein binding sites resulted in the decrease of FRET efficiency. Quantification of this effect in terms of the proposed methodology yielded both structural and binding parameters of lysozyme-lipid complexes.

  11. Analysis of Toxic Amyloid Fibril Interactions at Natively Derived Membranes by Ellipsometry

    PubMed Central

    Smith, Rachel A. S.; Nabok, Aleksey; Blakeman, Ben J. F.; Xue, Wei-Feng; Abell, Benjamin; Smith, David P.

    2015-01-01

    There is an ongoing debate regarding the culprits of cytotoxicity associated with amyloid disorders. Although small pre-fibrillar amyloid oligomers have been implicated as the primary toxic species, the fibrillar amyloid material itself can also induce cytotoxicity. To investigate membrane disruption and cytotoxic effects associated with intact and fragmented fibrils, the novel in situ spectroscopic technique of Total Internal Reflection Ellipsometry (TIRE) was used. Fibril lipid interactions were monitored using natively derived whole cell membranes as a model of the in vivo environment. We show that fragmented fibrils have an increased ability to disrupt these natively derived membranes by causing a loss of material from the deposited surface when compared with unfragmented fibrils. This effect was corroborated by observations of membrane disruption in live cells, and by dye release assay using synthetic liposomes. Through these studies we demonstrate the use of TIRE for the analysis of protein-lipid interactions on natively derived lipid surfaces, and provide an explanation on how amyloid fibrils can cause a toxic gain of function, while entangled amyloid plaques exert minimal biological activity. PMID:26172440

  12. Novel tilt-curvature coupling in lipid membranes

    NASA Astrophysics Data System (ADS)

    Terzi, M. Mert; Deserno, Markus

    2017-08-01

    On mesoscopic scales, lipid membranes are well described by continuum theories whose main ingredients are the curvature of a membrane's reference surface and the tilt of its lipid constituents. In particular, Hamm and Kozlov [Eur. Phys. J. E 3, 323 (2000)] have shown how to systematically derive such a tilt-curvature Hamiltonian based on the elementary assumption of a thin fluid elastic sheet experiencing internal lateral pre-stress. Performing a dimensional reduction, they not only derive the basic form of the effective surface Hamiltonian but also express its emergent elastic couplings as trans-membrane moments of lower-level material parameters. In the present paper, we argue, though, that their derivation unfortunately missed a coupling term between curvature and tilt. This term arises because, as one moves along the membrane, the curvature-induced change of transverse distances contributes to the area strain—an effect that was believed to be small but nevertheless ends up contributing at the same (quadratic) order as all other terms in their Hamiltonian. We illustrate the consequences of this amendment by deriving the monolayer and bilayer Euler-Lagrange equations for the tilt, as well as the power spectra of shape, tilt, and director fluctuations. A particularly curious aspect of our new term is that its associated coupling constant is the second moment of the lipid monolayer's lateral stress profile—which within this framework is equal to the monolayer Gaussian curvature modulus, κ¯ m. On the one hand, this implies that many theoretical predictions now contain a parameter that is poorly known (because the Gauss-Bonnet theorem limits access to the integrated Gaussian curvature); on the other hand, the appearance of κ¯ m outside of its Gaussian curvature provenance opens opportunities for measuring it by more conventional means, for instance by monitoring a membrane's undulation spectrum at short scales.

  13. Novel tilt-curvature coupling in lipid membranes.

    PubMed

    Terzi, M Mert; Deserno, Markus

    2017-08-28

    On mesoscopic scales, lipid membranes are well described by continuum theories whose main ingredients are the curvature of a membrane's reference surface and the tilt of its lipid constituents. In particular, Hamm and Kozlov [Eur. Phys. J. E 3, 323 (2000)] have shown how to systematically derive such a tilt-curvature Hamiltonian based on the elementary assumption of a thin fluid elastic sheet experiencing internal lateral pre-stress. Performing a dimensional reduction, they not only derive the basic form of the effective surface Hamiltonian but also express its emergent elastic couplings as trans-membrane moments of lower-level material parameters. In the present paper, we argue, though, that their derivation unfortunately missed a coupling term between curvature and tilt. This term arises because, as one moves along the membrane, the curvature-induced change of transverse distances contributes to the area strain-an effect that was believed to be small but nevertheless ends up contributing at the same (quadratic) order as all other terms in their Hamiltonian. We illustrate the consequences of this amendment by deriving the monolayer and bilayer Euler-Lagrange equations for the tilt, as well as the power spectra of shape, tilt, and director fluctuations. A particularly curious aspect of our new term is that its associated coupling constant is the second moment of the lipid monolayer's lateral stress profile-which within this framework is equal to the monolayer Gaussian curvature modulus, κ¯ m . On the one hand, this implies that many theoretical predictions now contain a parameter that is poorly known (because the Gauss-Bonnet theorem limits access to the integrated Gaussian curvature); on the other hand, the appearance of κ¯ m outside of its Gaussian curvature provenance opens opportunities for measuring it by more conventional means, for instance by monitoring a membrane's undulation spectrum at short scales.

  14. Recent progress on lipid lateral heterogeneity in plasma membranes: from rafts to submicrometric domains

    PubMed Central

    Carquin, Mélanie; D'Auria, Ludovic; Pollet, Hélène; Bongarzone, Ernesto R.; Tyteca, Donatienne

    2016-01-01

    The concept of transient nanometric domains known as lipid rafts has brought interest to reassess the validity of the Singer-Nicholson model of a fluid bilayer for cell membranes. However, this new view is still insufficient to explain the cellular control of surface lipid diversity or membrane deformability. During the past decade, the hypothesis that some lipids form large (submicrometric/mesoscale vs nanometric rafts) and stable (> min vs sec) membrane domains has emerged, largely based on indirect methods. Morphological evidence for stable submicrometric lipid domains, well-accepted for artificial and highly specialized biological membranes, was further reported for a variety of living cells from prokaryotes to yeast and mammalian cells. However, results remained questioned based on limitations of available fluorescent tools, use of poor lipid fixatives, and imaging artifacts due to non-resolved membrane projections. In this review, we will discuss recent evidence generated using powerful and innovative approaches such as lipid-specific toxin fragments that support the existence of submicrometric domains. We will integrate documented mechanisms involved in the formation and maintenance of these domains, and provide a perspective on their relevance on membrane deformability and regulation of membrane protein distribution. PMID:26738447

  15. Lipid Biomembrane in Ionic Liquids

    NASA Astrophysics Data System (ADS)

    Yoo, Brian; Jing, Benxin; Shah, Jindal; Maginn, Ed; Zhu, Y. Elaine; Department of Chemical and Biomolecular Engineering Team

    2014-03-01

    Ionic liquids (ILs) have been recently explored as new ``green'' chemicals in several chemical and biomedical processes. In our pursuit of understanding their toxicities towards aquatic and terrestrial organisms, we have examined the IL interaction with lipid bilayers as model cell membranes. Experimentally by fluorescence microscopy, we have directly observed the disruption of lipid bilayer by added ILs. Depending on the concentration, alkyl chain length, and anion hydrophobicity of ILs, the interaction of ILs with lipid bilayers leads to the formation of micelles, fibrils, and multi-lamellar vesicles for IL-lipid complexes. By MD computer simulations, we have confirmed the insertion of ILs into lipid bilayers to modify the spatial organization of lipids in the membrane. The combined experimental and simulation results correlate well with the bioassay results of IL-induced suppression in bacteria growth, thereby suggesting a possible mechanism behind the IL toxicity. National Science Foundation, Center for Research Computing at Notre Dame.

  16. Entropic elasticity based coarse-grained model of lipid membranes

    NASA Astrophysics Data System (ADS)

    Feng, Shuo; Hu, Yucai; Liang, Haiyi

    2018-04-01

    Various models for lipid bilayer membranes have been presented to investigate their morphologies. Among them, the aggressive coarse-grained models, where the membrane is represented by a single layer of particles, are computationally efficient and of practical importance for simulating membrane dynamics at the microscopic scale. In these models, soft potentials between particle pairs are used to maintain the fluidity of membranes, but the underlying mechanism of the softening requires further clarification. We have analyzed the membrane area decrease due to thermal fluctuations, and the results demonstrate that the intraparticle part of entropic elasticity is responsible for the softening of the potential. Based on the stretching response of the membrane, a bottom-up model is developed with an entropic effect explicitly involved. The model reproduces several essential properties of the lipid membrane, including the fluid state and a plateau in the stretching curve. In addition, the area compressibility modulus, bending rigidity, and spontaneous curvature display linear dependence on model parameters. As a demonstration, we have investigated the closure and morphology evolution of membrane systems driven by spontaneous curvature, and vesicle shapes observed experimentally are faithfully reproduced.

  17. Cholesterol Regulates μ-Opioid Receptor-Induced β-Arrestin 2 Translocation to Membrane Lipid RaftsS⃞

    PubMed Central

    Qiu, Yu; Wang, Yan; Chen, Hong-Zhuan; Loh, Horace H.

    2011-01-01

    μ-Opioid receptor (OPRM1) is mainly localized in lipid raft microdomains but internalizes through clathrin-dependent pathways. Our previous studies demonstrated that disruption of lipid rafts by cholesterol-depletion reagent blocked the agonist-induced internalization of OPRM1 and G protein-dependent signaling. The present study demonstrated that reduction of cholesterol level decreased and culturing cells in excess cholesterol increased the agonist-induced internalization and desensitization of OPRM1, respectively. Further analyses indicated that modulation of cellular cholesterol level did not affect agonist-induced receptor phosphorylation but did affect membrane translocation of β-arrestins. The translocation of β-arrestins was blocked by cholesterol reduction, and the effect could be reversed by incubating with cholesterol. OptiPrep gradient separation of lipid rafts revealed that excess cholesterol retained more receptors in lipid raft domains and facilitated the recruitment of β-arrestins to these microdomains upon agonist activation. Moreover, excess cholesterol could evoke receptor internalization and protein kinase C-independent extracellular signal-regulated kinases activation upon morphine treatment. Therefore, these results suggest that cholesterol not only can influence OPRM1 localization in lipid rafts but also can effectively enhance the recruitment of β-arrestins and thereby affect the agonist-induced trafficking and agonist-dependent signaling of OPRM1. PMID:21518774

  18. Generation of wavy structure on lipid membrane by peripheral proteins: a linear elastic analysis.

    PubMed

    Mahata, Paritosh; Das, Sovan Lal

    2017-05-01

    We carry out a linear elastic analysis to study wavy structure generation on lipid membrane by peripheral membrane proteins. We model the lipid membrane as linearly elastic and anisotropic material. The hydrophobic insertion by proteins into the lipid membrane has been idealized as penetration of rigid rod-like inclusions into the membrane and the electrostatic interaction between protein and membrane has been modeled by a distributed surface traction acting on the membrane surface. With the proposed model we study curvature generation by several binding domains of peripheral membrane proteins containing BAR domains and amphipathic alpha-helices. It is observed that electrostatic interaction is essential for curvature generation by the BAR domains. © 2017 Federation of European Biochemical Societies.

  19. Anomalous Dynamics of a Lipid Recognition Protein on a Membrane Surface

    PubMed Central

    Yamamoto, Eiji; Kalli, Antreas C.; Akimoto, Takuma; Yasuoka, Kenji; Sansom, Mark S. P.

    2015-01-01

    Pleckstrin homology (PH) domains are lipid-binding modules present in peripheral membrane proteins which interact with phosphatidyl-inositol phosphates (PIPs) in cell membranes. We use multiscale molecular dynamics simulations to characterize the localization and anomalous dynamics of the DAPP1 PH domain on the surface of a PIP-containing lipid bilayer. Both translational and rotational diffusion of the PH domain on the lipid membrane surface exhibit transient subdiffusion, with an exponent α ≈ 0.5 for times of less than 10 ns. In addition to a PIP3 molecule at the canonical binding site of the PH domain, we observe additional PIP molecules in contact with the protein. Fluctuations in the number of PIPs associated with the PH domain exhibit 1/f noise. We suggest that the anomalous diffusion and long-term correlated interaction of the PH domain with the membrane may contribute to an enhanced probability of encounter with target complexes on cell membrane surfaces. PMID:26657413

  20. The effect of charged lipids on bacteriorhodopsin membrane reconstitution and its photochemical activities

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang Zhen; Bai Jing; Xu Yuhong

    2008-07-11

    Bacteriorhodopsin (BR) was reconstituted into artificial lipid membrane containing various charged lipid compositions. The proton pumping activity of BR under flash and continuous illumination, proton permeability across membrane, as well as the decay kinetics of the photocycle intermediate M{sub 412} were studied. The results showed that lipid charges would significantly affect the orientation of BR inserted into lipid membranes. In liposomes containing anionic lipids, BRs were more likely to take natural orientation as in living cells. In neutral or positively charged liposomes, most BRs were reversely assembled, assuming an inside out orientation. Moreover, the lipids charges also affect BR's Mmore » intermediate kinetics, especially the slow component in M intermediate decay. The half-life M{sub 412s} increased significantly in BRs in liposomes containing cationic lipids, while decreased in those in anionic liposomes.« less

  1. Proteomic Analysis of Lipid Raft-Like Detergent-Resistant Membranes of Lens Fiber Cells.

    PubMed

    Wang, Zhen; Schey, Kevin L

    2015-12-01

    Plasma membranes of lens fiber cells have high levels of long-chain saturated fatty acids, cholesterol, and sphingolipids-key components of lipid rafts. Thus, lipid rafts are expected to constitute a significant portion of fiber cell membranes and play important roles in lens biology. The purpose of this study was to characterize the lens lipid raft proteome. Quantitative proteomics, both label-free and iTRAQ methods, were used to characterize lens fiber cell lipid raft proteins. Detergent-resistant, lipid raft membrane (DRM) fractions were isolated by sucrose gradient centrifugation. To confirm protein localization to lipid rafts, protein sensitivity to cholesterol removal by methyl-β-cyclodextrin was quantified by iTRAQ analysis. A total of 506 proteins were identified in raft-like detergent-resistant membranes. Proteins identified support important functions of raft domains in fiber cells, including trafficking, signal transduction, and cytoskeletal organization. In cholesterol-sensitivity studies, 200 proteins were quantified and 71 proteins were strongly affected by cholesterol removal. Lipid raft markers flotillin-1 and flotillin-2 and a significant fraction of AQP0, MP20, and AQP5 were found in the DRM fraction and were highly sensitive to cholesterol removal. Connexins 46 and 50 were more abundant in nonraft fractions, but a small fraction of each was found in the DRM fraction and was strongly affected by cholesterol removal. Quantification of modified AQP0 confirmed that fatty acylation targeted this protein to membrane raft domains. These data represent the first comprehensive profile of the lipid raft proteome of lens fiber cells and provide information on membrane protein organization in these cells.

  2. Fluorescent probes for lipid rafts: from model membranes to living cells.

    PubMed

    Klymchenko, Andrey S; Kreder, Rémy

    2014-01-16

    Membrane microdomains (rafts) remain one of the controversial issues in biophysics. Fluorescent molecular probes, which make these lipid nanostructures visible through optical techniques, are one of the tools currently used to study lipid rafts. The most common are lipophilic fluorescent probes that partition specifically into liquid ordered or liquid disordered phase. Their partition depends on the lipid composition of a given phase, which complicates their use in cellular membranes. A second class of probes is based on environment-sensitive dyes, which partition into both phases, but stain them by different fluorescence color, intensity, or lifetime. These probes can directly address the properties of each separate phase, but their cellular applications are still limited. The present review focuses on summarizing the current state in the field of developing and applying fluorescent molecular probes to study lipid rafts. We highlight an urgent need to develop new probes, specifically adapted for cell plasma membranes and compatible with modern fluorescence microscopy techniques to push the understanding of membrane microdomains forward. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Kinetic analysis of the interaction between poly(amidoamine) dendrimers and model lipid membranes.

    PubMed

    Tiriveedhi, Venkataswarup; Kitchens, Kelly M; Nevels, Kerrick J; Ghandehari, Hamidreza; Butko, Peter

    2011-01-01

    We used fluorescence spectroscopy and surface tensiometry to study the interaction between low-generation (G1 and G4) poly(amidoamine) (PAMAM) dendrimers, potential vehicles for intracellular drug delivery, and model lipid bilayers. Membrane association of fluorescently labeled dendrimers, measured by fluorescence anisotropy, increased with increasing size of the dendrimer and with increasing negative charge density in the membrane, indicating the electrostatic nature of the interaction. When the membrane was doped with pyrene-labeled phosphatidyl glycerol (pyrene-PG), pyrene excimer fluorescence demonstrated a dendrimer-induced selective aggregation of negatively charged lipids when the membrane was in the liquid crystalline state. A nonlinear Stern-Volmer quenching of dendrimer fluorescence with cobalt bromide suggested a dendrimer-induced aggregation of lipid vesicles, which increased with the dendrimer's generation number. Surface tensiometry measurements showed that dendrimers penetrated into the lipid monolayer only at subphysiologic surface pressures (<30mN/m). We conclude that the low-generation PAMAM dendrimers associate with lipid membranes predominantly electrostatically, without significantly compromising the bilayer integrity. They bind stronger to membranes with higher fluidity and lower surface pressure, which are characteristic of rapidly dividing cells. Copyright © 2010 Elsevier B.V. All rights reserved.

  4. Xenon and Other Volatile Anesthetics Change Domain Structure in Model Lipid Raft Membranes

    PubMed Central

    Weinrich, Michael; Worcester, David L.

    2014-01-01

    Inhalation anesthetics have been in clinical use for over 160 years, but the molecular mechanisms of action continue to be investigated. Direct interactions with ion channels received much attention after it was found that anesthetics do not change the structure of homogeneous model membranes. However, it was recently found that halothane, a prototypical anesthetic, changes domain structure of a binary lipid membrane. The noble gas xenon is an excellent anesthetic and provides a pivotal test of the generality of this finding, extended to ternary lipid raft mixtures. We report that xenon and conventional anesthetics change the domain equilibrium in two canonical ternary lipid raft mixtures. These findings demonstrate a membrane-mediated mechanism whereby inhalation anesthetics can affect the lipid environment of trans-membrane proteins. PMID:24299622

  5. Lipid Domain Structure of the Plasma Membrane Revealed by Patching of Membrane Components

    PubMed Central

    Harder, Thomas; Scheiffele, Peter; Verkade, Paul; Simons, Kai

    1998-01-01

    Lateral assemblies of glycolipids and cholesterol, “rafts,” have been implicated to play a role in cellular processes like membrane sorting, signal transduction, and cell adhesion. We studied the structure of raft domains in the plasma membrane of non-polarized cells. Overexpressed plasma membrane markers were evenly distributed in the plasma membrane. We compared the patching behavior of pairs of raft markers (defined by insolubility in Triton X-100) with pairs of raft/non-raft markers. For this purpose we cross-linked glycosyl-phosphatidylinositol (GPI)-anchored proteins placental alkaline phosphatase (PLAP), Thy-1, influenza virus hemagglutinin (HA), and the raft lipid ganglioside GM1 using antibodies and/or cholera toxin. The patches of these raft markers overlapped extensively in BHK cells as well as in Jurkat T–lymphoma cells. Importantly, patches of GPI-anchored PLAP accumulated src-like protein tyrosine kinase fyn, which is thought to be anchored in the cytoplasmic leaflet of raft domains. In contrast patched raft components and patches of transferrin receptor as a non-raft marker were sharply separated. Taken together, our data strongly suggest that coalescence of cross-linked raft elements is mediated by their common lipid environments, whereas separation of raft and non-raft patches is caused by the immiscibility of different lipid phases. This view is supported by the finding that cholesterol depletion abrogated segregation. Our results are consistent with the view that raft domains in the plasma membrane of non-polarized cells are normally small and highly dispersed but that raft size can be modulated by oligomerization of raft components. PMID:9585412

  6. Dye-release assay for investigation of antimicrobial peptide activity in a competitive lipid environment.

    PubMed

    Sani, Marc-Antoine; Gagne, Eve; Gehman, John D; Whitwell, Thomas C; Separovic, Frances

    2014-09-01

    A dye-release method for investigating the effect of a competitive lipid environment on the activity of two membrane-disrupting antimicrobial peptides (AMP), maculatin 1.1 and aurein 1.2, is presented. The results support the general conclusion that AMP have greater affinity for negatively charged membranes, for example bacterial membranes, than for the neutral membrane surface found in eukaryotic cells, but only within a competitive lipid environment. Indeed, in a single-model membrane environment, both peptides were more potent against neutral vesicles than against charged vesicles. The approach was also used to investigate the effect of pre-incubating the peptides in a neutral lipid environment then introducing charged lipid vesicles. Maculatin was shown to migrate from the neutral lipid bilayers, where pores had already formed, to the charged membrane bilayers. This result was also observed for charged-to-charged bilayers but, interestingly, not for neutral-to-neutral lipid interfaces. Aurein was able to migrate from either lipid environment, indicating weaker binding to lipid membranes, and a different molecular mechanism for lysis of lipid bilayers. Competitive lipid environments could be used to assess other critical conditions that modulate the activity of membrane peptides or proteins.

  7. Lipid Raft: A Floating Island Of Death or Survival

    PubMed Central

    George, Kimberly S.; Wu, Shiyong

    2012-01-01

    Lipid rafts are microdomains of the plasma membrane enriched in cholesterol and sphingolipids, and play an important role in the initiation of many pharmacological agent-induced signaling pathways and toxicological effects. The structure of lipid rafts is dynamic, resulting in an ever-changing content of both lipids and proteins. Cholesterol, as a major component of lipid rafts, is critical for the formation and configuration of lipid rafts microdomains, which provide signaling platforms capable of activating both pro-apoptotic and anti-apoptotic signaling pathways. A change of cholesterol level can result in lipid rafts disruption and activate or deactivate raft-associated proteins, such as death receptor proteins, protein kinases, and calcium channels. Several anti-cancer drugs are able to suppress growth and induce apoptosis of tumor cells through alteration of lipid raft contents via disrupting lipid raft integrity. PMID:22289360

  8. Probing membrane permeabilization by the antimicrobial peptide distinctin in mercury-supported biomimetic membranes.

    PubMed

    Becucci, Lucia; Papini, Martina; Mullen, Daniel; Scaloni, Andrea; Veglia, Gianluigi; Guidelli, Rolando

    2011-11-01

    The mechanism of membrane permeabilization by the antimicrobial peptide distinctin was investigated by using two different mercury-supported biomimetic membranes, namely a lipid self-assembled monolayer and a lipid bilayer tethered to the mercury surface through a hydrophilic spacer (tethered bilayer lipid membrane: tBLM). Incorporation of distinctin into a lipid monolayer from its aqueous solution yields rapidly ion channels selective toward inorganic cations, such as Tl(+) and Cd(2+). Conversely, its incorporation in a tBLM allows the formation of ion channels permeable to potassium ions only at non-physiological transmembrane potentials, more negative than -340mV. These channels, once formed, are unstable at less negative transmembrane potentials. The kinetics of their formation is consistent with the disruption of distinctin clusters adsorbed on top of the lipid bilayer, incorporation of the resulting monomers and their aggregation into hydrophilic pores by a mechanism of nucleation and growth. Comparing the behavior of distinctin in tBLMs with that in conventional black lipid membranes strongly suggests that distinctin channel formation in lipid bilayer requires the partitioning of distinctin molecules between the two sides of the lipid bilayer. We can tentatively hypothesize that an ion channel is formed when one distinctin cluster on one side of the lipid bilayer matches another one on the opposite side. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. Quantitative analysis of molecular partition towards lipid membranes using surface plasmon resonance

    NASA Astrophysics Data System (ADS)

    Figueira, Tiago N.; Freire, João M.; Cunha-Santos, Catarina; Heras, Montserrat; Gonçalves, João; Moscona, Anne; Porotto, Matteo; Salomé Veiga, Ana; Castanho, Miguel A. R. B.

    2017-03-01

    Understanding the interplay between molecules and lipid membranes is fundamental when studying cellular and biotechnological phenomena. Partition between aqueous media and lipid membranes is key to the mechanism of action of many biomolecules and drugs. Quantifying membrane partition, through adequate and robust parameters, is thus essential. Surface Plasmon Resonance (SPR) is a powerful technique for studying 1:1 stoichiometric interactions but has limited application to lipid membrane partition data. We have developed and applied a novel mathematical model for SPR data treatment that enables determination of kinetic and equilibrium partition constants. The method uses two complementary fitting models for association and dissociation sensorgram data. The SPR partition data obtained for the antibody fragment F63, the HIV fusion inhibitor enfuvirtide, and the endogenous drug kyotorphin towards POPC membranes were compared against data from independent techniques. The comprehensive kinetic and partition models were applied to the membrane interaction data of HRC4, a measles virus entry inhibitor peptide, revealing its increased affinity for, and retention in, cholesterol-rich membranes. Overall, our work extends the application of SPR beyond the realm of 1:1 stoichiometric ligand-receptor binding into a new and immense field of applications: the interaction of solutes such as biomolecules and drugs with lipids.

  10. Acyl transfer from membrane lipids to peptides is a generic process.

    PubMed

    Dods, Robert H; Bechinger, Burkhard; Mosely, Jackie A; Sanderson, John M

    2013-11-15

    The generality of acyl transfer from phospholipids to membrane-active peptides has been probed using liquid chromatography-mass spectrometry analysis of peptide-lipid mixtures. The peptides examined include melittin, magainin II, PGLa, LAK1, LAK3 and penetratin. Peptides were added to liposomes with membrane lipid compositions ranging from pure phosphatidylcholine (PC) to mixtures of PC with phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol. Experiments were typically conducted at pH7.4 at modest salt concentrations (90 mM NaCl). In favorable cases, lipidated peptides were further characterized by tandem mass spectrometry methods to determine the sites of acylation. Melittin and magainin II were the most reactive peptides, with significant acyl transfer detected under all conditions and membrane compositions. Both peptides were lipidated at the N-terminus by transfer from PC, phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol, as well as at internal sites: lysine for melittin; serine and lysine for magainin II. Acyl transfer could be detected within 3h of melittin addition to negatively charged membranes. The other peptides were less reactive, but for each peptide, acylation was found to occur in at least one of the conditions examined. The data demonstrate that acyl transfer is a generic process for peptides bound to membranes composed of diacylglycerophospholipids. Phospholipid membranes cannot therefore be considered as chemically inert toward peptides and by extension proteins. © 2013. Published by Elsevier Ltd. All rights reserved.

  11. Permeability modes in fluctuating lipid membranes with DNA-translocating pores.

    PubMed

    Moleiro, L H; Mell, M; Bocanegra, R; López-Montero, I; Fouquet, P; Hellweg, Th; Carrascosa, J L; Monroy, F

    2017-09-01

    Membrane pores can significantly alter not only the permeation dynamics of biological membranes but also their elasticity. Large membrane pores able to transport macromolecular contents represent an interesting model to test theoretical predictions that assign active-like (non-equilibrium) behavior to the permeability contributions to the enhanced membrane fluctuations existing in permeable membranes [Maneville et al. Phys. Rev. Lett. 82, 4356 (1999)]. Such high-amplitude active contributions arise from the forced transport of solvent and solutes through the open pores, which becomes even dominant at large permeability. In this paper, we present a detailed experimental analysis of the active shape fluctuations that appear in highly permeable lipid vesicles with large macromolecular pores inserted in the lipid membrane, which are a consequence of transport permeability events occurred in an osmotic gradient. The experimental results are found in quantitative agreement with theory, showing a remarkable dependence with the density of membrane pores and giving account of mechanical compliances and permeability rates that are compatible with the large size of the membrane pore considered. The presence of individual permeation events has been detected in the fluctuation time-series, from which a stochastic distribution of the permeation events compatible with a shot-noise has been deduced. The non-equilibrium character of the membrane fluctuations in a permeation field, even if the membrane pores are mere passive transporters, is clearly demonstrated. Finally, a bio-nano-technology outlook of the proposed synthetic concept is given on the context of prospective uses as active membrane DNA-pores exploitable in gen-delivery applications based on lipid vesicles. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Evaluation of the membrane lipid selectivity of the pea defensin Psd1.

    PubMed

    Gonçalves, Sónia; Teixeira, Alexandre; Abade, João; de Medeiros, Luciano Neves; Kurtenbach, Eleonora; Santos, Nuno C

    2012-05-01

    Psd1, a 46 amino acid residues defensin isolated from the pea Pisum sativum seeds, exhibits anti-fungal activity by a poorly understood mechanism of action. In this work, the interaction of Psd1 with biomembrane model systems of different lipid compositions was assessed by fluorescence spectroscopy. Partition studies showed a marked lipid selectivity of this antimicrobial peptide (AMP) toward lipid membranes containing ergosterol (the main sterol in fungal membranes) or specific glycosphingolipid components, with partition coefficients (K(p)) reaching uncommonly high values of 10(6). By the opposite, Psd1 does not partition to cholesterol-enriched lipid bilayers, such as mammalian cell membranes. The Psd1 mutants His36Lys and Gly12Glu present a membrane affinity loss relative to the wild type. Fluorescence quenching data obtained using acrylamide and membrane probes further clarify the mechanism of action of this peptide at the molecular level, pointing out the potential therapeutic use of Psd1 as a natural antimycotic agent. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. An ER protein functionally couples neutral lipid metabolism on lipid droplets to membrane lipid synthesis in the ER.

    PubMed

    Markgraf, Daniel F; Klemm, Robin W; Junker, Mirco; Hannibal-Bach, Hans K; Ejsing, Christer S; Rapoport, Tom A

    2014-01-16

    Eukaryotic cells store neutral lipids such as triacylglycerol (TAG) in lipid droplets (LDs). Here, we have addressed how LDs are functionally linked to the endoplasmic reticulum (ER). We show that, in S. cerevisiae, LD growth is sustained by LD-localized enzymes. When LDs grow in early stationary phase, the diacylglycerol acyl-transferase Dga1p moves from the ER to LDs and is responsible for all TAG synthesis from diacylglycerol (DAG). During LD breakdown in early exponential phase, an ER membrane protein (Ice2p) facilitates TAG utilization for membrane-lipid synthesis. Ice2p has a cytosolic domain with affinity for LDs and is required for the efficient utilization of LD-derived DAG in the ER. Ice2p breaks a futile cycle on LDs between TAG degradation and synthesis, promoting the rapid relocalization of Dga1p to the ER. Our results show that Ice2p functionally links LDs with the ER and explain how cells switch neutral lipid metabolism from storage to consumption. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  14. Reorganization of lipid domain distribution in giant unilamellar vesicles upon immobilization with different membrane tethers.

    PubMed

    Sarmento, M J; Prieto, M; Fernandes, Fábio

    2012-11-01

    Characterization of phase coexistence in biologically relevant lipid mixtures is often carried out through confocal microscopy of giant unilamellar lipid vesicles (GUVs), loaded with fluorescent membrane probes. This last analysis is generally limited to the vesicle hemisphere further away from the coverslip, in order to avoid artifacts induced by the interaction with the solid surface, and immobilization of vesicles is in many cases required in order to carry out intensity, lifetime or single-molecule based microscopy. This is generally achieved through the use of membrane tethers adhering to a coverslip surface. Here, we aimed to determine whether GUV immobilization through membrane tethers induces changes in lipid domain distribution within liposomes displaying coexistence of lipid lamellar phases. Confocal imaging and a Förster resonance energy transfer (FRET) methodology showed that biotinylated phospholipids present significantly different membrane phase partition behavior upon protein binding, depending on the presence or absence of a linker between the lipid headgroup and the biotinyl moiety. Membrane phases enriched in a membrane tether displayed in some cases a dramatically increased affinity for the immobilization surface, effectively driving sorting of lipid domains to the adherent membrane area, and in some cases complete sequestering of a lipid phase to the interaction surface was observed. On the light of these results, we conclude that tethering of lipid membranes to protein surfaces has the potential to drastically reorganize the distribution of lipid domains, and this reorganization is solely dictated by the partition properties of the protein-tether complex. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. The structure of ions and zwitterionic lipids regulates the charge of dipolar membranes.

    PubMed

    Szekely, Or; Steiner, Ariel; Szekely, Pablo; Amit, Einav; Asor, Roi; Tamburu, Carmen; Raviv, Uri

    2011-06-21

    In pure water, zwitterionic lipids form lamellar phases with an equilibrium water gap on the order of 2 to 3 nm as a result of the dominating van der Waals attraction between dipolar bilayers. Monovalent ions can swell those neutral lamellae by a small amount. Divalent ions can adsorb onto dipolar membranes and charge them. Using solution X-ray scattering, we studied how the structure of ions and zwitterionic lipids regulates the charge of dipolar membranes. We found that unlike monovalent ions that weakly interact with all of the examined dipolar membranes, divalent and trivalent ions adsorb onto membranes containing lipids with saturated tails, with an association constant on the order of ∼10 M(-1). One double bond in the lipid tail is sufficient to prevent divalent ion adsorption. We suggest that this behavior is due to the relatively loose packing of lipids with unsaturated tails that increases the area per lipid headgroup, enabling their free rotation. Divalent ion adsorption links two lipids and limits their free rotation. The ion-dipole interaction gained by the adsorption of the ions onto unsaturated membranes is insufficient to compensate for the loss of headgroup free-rotational entropy. The ion-dipole interaction is stronger for cations with a higher valence. Nevertheless, polyamines behave as monovalent ions near dipolar interfaces in the sense that they interact weakly with the membrane surface, whereas in the bulk their behavior is similar to that of multivalent cations. Advanced data analysis and comparison with theory provide insight into the structure and interactions between ion-induced regulated charged interfaces. This study models biologically relevant interactions between cell membranes and various ions and the manner in which the lipid structure governs those interactions. The ability to monitor these interactions creates a tool for probing systems that are more complex and forms the basis for controlling the interactions between dipolar

  16. Lipid peroxidation in sarcoplasmic reticulum membranes: effect on functional and biophysical properties.

    PubMed

    Dinis, T C; Almeida, L M; Madeira, V M

    1993-03-01

    The fluorescent polyunsaturated parinaric acid (PnA) incorporated in sarcoplasmic reticulum membranes (SR) was used to probe the initial stages of membrane lipid peroxidation. The experimental set up of the PnA assay was investigated by means of several peroxidation initiators to ascertain peroxidation conditions. This assay in SR is particularly useful to evaluate the membrane susceptibility to peroxidation and to ascertain suitable conditions (concentration of initiators and cofactors) to challenge peroxidation in each preparation under study. On the basis of the PnA assay, Fe2+/ascorbate was selected among the different initiator systems to assess the effect of lipid peroxidation upon biochemical and biophysical parameters of SR membranes. Under mildly controlled conditions at 25 degrees C, the lipid degradative process, as detected by fatty acid analysis, decreases the Ca2+ uptake (up to about 50% of control) and reduces the Ca2+ pump efficiency (Ca2+/ATP ratio) up to about 58% of control, without inactivation the ATPase enzyme turnover. The effect of lipid peroxidation on the SR bilayer organization is dependent either on the extent of lipid peroxidation or on the depth of the bilayer as probed by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene and by intramolecular excimerization of 1,3-di(1-pyrenyl)propane. It is concluded that the effect of mild lipid peroxidation on Ca2+ pump activity is partially exerted through the alteration of physical properties in the lipid phase or lipid-protein interfaces.

  17. Proteomic Analysis of Lipid Raft-Like Detergent-Resistant Membranes of Lens Fiber Cells

    PubMed Central

    Wang, Zhen; Schey, Kevin L.

    2015-01-01

    Purpose Plasma membranes of lens fiber cells have high levels of long-chain saturated fatty acids, cholesterol, and sphingolipids—key components of lipid rafts. Thus, lipid rafts are expected to constitute a significant portion of fiber cell membranes and play important roles in lens biology. The purpose of this study was to characterize the lens lipid raft proteome. Methods Quantitative proteomics, both label-free and iTRAQ methods, were used to characterize lens fiber cell lipid raft proteins. Detergent-resistant, lipid raft membrane (DRM) fractions were isolated by sucrose gradient centrifugation. To confirm protein localization to lipid rafts, protein sensitivity to cholesterol removal by methyl-β-cyclodextrin was quantified by iTRAQ analysis. Results A total of 506 proteins were identified in raft-like detergent-resistant membranes. Proteins identified support important functions of raft domains in fiber cells, including trafficking, signal transduction, and cytoskeletal organization. In cholesterol-sensitivity studies, 200 proteins were quantified and 71 proteins were strongly affected by cholesterol removal. Lipid raft markers flotillin-1 and flotillin-2 and a significant fraction of AQP0, MP20, and AQP5 were found in the DRM fraction and were highly sensitive to cholesterol removal. Connexins 46 and 50 were more abundant in nonraft fractions, but a small fraction of each was found in the DRM fraction and was strongly affected by cholesterol removal. Quantification of modified AQP0 confirmed that fatty acylation targeted this protein to membrane raft domains. Conclusions These data represent the first comprehensive profile of the lipid raft proteome of lens fiber cells and provide information on membrane protein organization in these cells. PMID:26747763

  18. Interaction of nanoparticles with lipid membranes: a multiscale perspective

    NASA Astrophysics Data System (ADS)

    Montis, Costanza; Maiolo, Daniele; Alessandri, Ivano; Bergese, Paolo; Berti, Debora

    2014-05-01

    Freestanding lipid bilayers were challenged with 15 nm Au nanospheres either coated by a citrate layer or passivated by a protein corona. The effect of Au nanospheres on the bilayer morphology, permeability and fluidity presents strong differences or similarities, depending on the observation length scale, from the colloidal to the molecular domains. These findings suggest that the interaction between nanoparticles and lipid membranes should be conveniently treated as a multiscale phenomenon.Freestanding lipid bilayers were challenged with 15 nm Au nanospheres either coated by a citrate layer or passivated by a protein corona. The effect of Au nanospheres on the bilayer morphology, permeability and fluidity presents strong differences or similarities, depending on the observation length scale, from the colloidal to the molecular domains. These findings suggest that the interaction between nanoparticles and lipid membranes should be conveniently treated as a multiscale phenomenon. Electronic supplementary information (ESI) available: All the experimental details, figures and tables. See DOI: 10.1039/c4nr00838c

  19. DNA release from lipoplexes by anionic lipids: correlation with lipid mesomorphism, interfacial curvature, and membrane fusion

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tarahovsky, Yury S.; Koynova, Rumiana; MacDonald, Robert C.

    2010-01-18

    DNA release from lipoplexes is an essential step during lipofection and is probably a result of charge neutralization by cellular anionic lipids. As a model system to test this possibility, fluorescence resonance energy transfer between DNA and lipid covalently labeled with Cy3 and BODIPY, respectively, was used to monitor the release of DNA from lipid surfaces induced by anionic liposomes. The separation of DNA from lipid measured this way was considerably slower and less complete than that estimated with noncovalently labeled DNA, and depends on the lipid composition of both lipoplexes and anionic liposomes. This result was confirmed by centrifugalmore » separation of released DNA and lipid. X-ray diffraction revealed a clear correlation of the DNA release capacity of the anionic lipids with the interfacial curvature of the mesomorphic structures developed when the anionic and cationic liposomes were mixed. DNA release also correlated with the rate of fusion of anionic liposomes with lipoplexes. It is concluded that the tendency to fuse and the phase preference of the mixed lipid membranes are key factors for the rate and extent of DNA release. The approach presented emphasizes the importance of the lipid composition of both lipoplexes and target membranes and suggests optimal transfection may be obtained by tailoring lipoplex composition to the lipid composition of target cells.« less

  20. Breathable NIPAAm Network with Controllable Hydration Supports Model Lipid Membrane

    NASA Astrophysics Data System (ADS)

    Jablin, Michael; Smith, Hillary; Zhernenkov, Mikhail; Vidyasagar, Ajay; Toomey, Ryan; Saiz, Jessica; Toperverg, Boris; Watkins, Erik; Kuhl, Tonya; Hurd, Alan; Majewski, Jaroslaw

    2009-03-01

    The interaction of a model lipid bilayer composed of DPPC with a surface-tethered poly(N-isopropylacrylamide) (NIPAAm) was explored with neutron reflectometry (NR). The Langmuir-Blodgett / Langmuir-Schaeffer method was used to deposit a lipid bilayer onto the polymer. NR measurements were used to probe the in- and out-of-plane structure of the system as a function of temperature. NR with fluorescence microscopy show that the polymer supports a lipid bilayer, and hydration of the support can be controlled. At low temp. the membrane develops out-of-plane undulations visible in off-specular scattering. Analysis of the off-specular reveals in-plane correlation of the bilayer fluctuations. The separation of the lipid bilayer from the solid support of a substrate constitutes a significant step towards a more realistic model of biological membranes.

  1. Ordering of lipid membranes altered by boron nitride nanosheets.

    PubMed

    Zhang, Yonghui; Li, Zhen; Chan, Chun; Ma, Jiale; Zhi, Chunyi; Cheng, Xiaolin; Fan, Jun

    2018-02-07

    Boron nitride nanosheets are novel promising nanomaterials with a lower cytotoxicity than graphene making them a better candidate for biomedical applications. However, there is no systematic study on how they interact with cell membranes. Here we employed large scale all-atom molecular dynamics simulations to provide molecular details of the structure and properties of membranes after the insertion of boron nitride nanosheets. Our results reveal that the boron nitride nanosheet can extract phospholipids from the lipid bilayers and is enveloped by the membrane. Afterwards, the acyl chains of lipid molecules re-orient and become more ordered. As a result, a fluid to gel phase transition occurs in the 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayer. Consequently, the bending moduli of the bilayers increase, and the diffusivity of the individual lipid molecule decreases. These changes will affect relevant cellular activities, such as endocytosis and signal transduction. Our study provides novel insights into the biocompatibility and cytotoxicity of boron nitride nanosheets, which may facilitate the design of safer nanocarriers, antibiotics and other bio-nanotechnology applications.

  2. Membrane Interaction of Antimicrobial Peptides Using E. coli Lipid Extract as Model Bacterial Cell Membranes and SFG Spectroscopy

    PubMed Central

    Soblosky, Lauren; Ramamoorthy, Ayyalusamy; Chen, Zhan

    2015-01-01

    Supported lipid bilayers are used as a convenient model cell membrane system to study biologically important molecule-lipid interactions in situ. However, the lipid bilayer models are often simple and the acquired results with these models may not provide all pertinent information related to a real cell membrane. In this work, we use sum frequency generation (SFG) vibrational spectroscopy to study molecular-level interactions between the antimicrobial peptides (AMPs) MSI-594, ovispirin-1 G18, magainin 2 and a simple 1,2-dipalmitoyl-d62-sn-glycero-3-phosphoglycerol (dDPPG)-1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) bilayer. We compared such interactions to those between the AMPs and a more complex dDPPG/E. coli polar lipid extract bilayer. We show that to fully understand more complex aspects of peptide-bilayer interaction, such as interaction kinetics, a heterogeneous lipid composition is required, such as the E. coli polar lipid extract. The discrepancy in peptide-bilayer interaction is likely due in part to the difference in bilayer charge between the two systems since highly negative charged lipids can promote more favorable electrostatic interactions between the peptide and lipid bilayer. Results presented in this paper indicate that more complex model bilayers are needed to accurately analyze peptide-cell membrane interactions and demonstrates the importance of using an appropriate lipid composition to study AMP interaction properties. PMID:25707312

  3. Differential effect of plant lipids on membrane organization: specificities of phytosphingolipids and phytosterols.

    PubMed

    Grosjean, Kevin; Mongrand, Sébastien; Beney, Laurent; Simon-Plas, Françoise; Gerbeau-Pissot, Patricia

    2015-02-27

    The high diversity of the plant lipid mixture raises the question of their respective involvement in the definition of membrane organization. This is particularly the case for plant plasma membrane, which is enriched in specific lipids, such as free and conjugated forms of phytosterols and typical phytosphingolipids, such as glycosylinositolphosphoceramides. This question was here addressed extensively by characterizing the order level of membrane from vesicles prepared using various plant lipid mixtures and labeled with an environment-sensitive probe. Fluorescence spectroscopy experiments showed that among major phytosterols, campesterol exhibits a stronger ability than β-sitosterol and stigmasterol to order model membranes. Multispectral confocal microscopy, allowing spatial analysis of membrane organization, demonstrated accordingly the strong ability of campesterol to promote ordered domain formation and to organize their spatial distribution at the membrane surface. Conjugated sterol forms, alone and in synergy with free sterols, exhibit a striking ability to order membrane. Plant sphingolipids, particularly glycosylinositolphosphoceramides, enhanced the sterol-induced ordering effect, emphasizing the formation and increasing the size of sterol-dependent ordered domains. Altogether, our results support a differential involvement of free and conjugated phytosterols in the formation of ordered domains and suggest that the diversity of plant lipids, allowing various local combinations of lipid species, could be a major contributor to membrane organization in particular through the formation of sphingolipid-sterol interacting domains. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Computational and theoretical approaches for studies of a lipid recognition protein on biological membranes

    PubMed Central

    Yamamoto, Eiji

    2017-01-01

    Many cellular functions, including cell signaling and related events, are regulated by the association of peripheral membrane proteins (PMPs) with biological membranes containing anionic lipids, e.g., phosphatidylinositol phosphate (PIP). This association is often mediated by lipid recognition modules present in many PMPs. Here, I summarize computational and theoretical approaches to investigate the molecular details of the interactions and dynamics of a lipid recognition module, the pleckstrin homology (PH) domain, on biological membranes. Multiscale molecular dynamics simulations using combinations of atomistic and coarse-grained models yielded results comparable to those of actual experiments and could be used to elucidate the molecular mechanisms of the formation of protein/lipid complexes on membrane surfaces, which are often difficult to obtain using experimental techniques. Simulations revealed some modes of membrane localization and interactions of PH domains with membranes in addition to the canonical binding mode. In the last part of this review, I address the dynamics of PH domains on the membrane surface. Local PIP clusters formed around the proteins exhibit anomalous fluctuations. This dynamic change in protein-lipid interactions cause temporally fluctuating diffusivity of proteins, i.e., the short-term diffusivity of the bound protein changes substantially with time, and may in turn contribute to the formation/dissolution of protein complexes in membranes. PMID:29159013

  5. Ionic protein-lipid interaction at the plasma membrane: what can the charge do?

    PubMed

    Li, Lunyi; Shi, Xiaoshan; Guo, Xingdong; Li, Hua; Xu, Chenqi

    2014-03-01

    Phospholipids are the major components of cell membranes, but they have functional roles beyond forming lipid bilayers. In particular, acidic phospholipids form microdomains in the plasma membrane and can ionically interact with proteins via polybasic sequences, which can have functional consequences for the protein. The list of proteins regulated by ionic protein-lipid interaction has been quickly expanding, and now includes membrane proteins, cytoplasmic soluble proteins, and viral proteins. Here we review how acidic phospholipids in the plasma membrane regulate protein structure and function via ionic interactions, and how Ca(2+) regulates ionic protein-lipid interactions via direct and indirect mechanisms. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Protein Diffusion on Charged Membranes: A Dynamic Mean-Field Model Describes Time Evolution and Lipid Reorganization

    PubMed Central

    Khelashvili, George; Weinstein, Harel; Harries, Daniel

    2008-01-01

    As charged macromolecules adsorb and diffuse on cell membranes in a large variety of cell signaling processes, they can attract or repel oppositely charged lipids. This results in lateral membrane rearrangement and affects the dynamics of protein function. To address such processes quantitatively we introduce a dynamic mean-field scheme that allows self-consistent calculations of the equilibrium state of membrane-protein complexes after such lateral reorganization of the membrane components, and serves to probe kinetic details of the process. Applicable to membranes with heterogeneous compositions containing several types of lipids, this comprehensive method accounts for mobile salt ions and charged macromolecules in three dimensions, as well as for lateral demixing of charged and net-neutral lipids in the membrane plane. In our model, the mobility of membrane components is governed by the diffusion-like Cahn-Hilliard equation, while the local electrochemical potential is based on nonlinear Poisson-Boltzmann theory. We illustrate the method by applying it to the adsorption of the anionic polypeptide poly-Lysine on negatively charged lipid membranes composed of binary mixtures of neutral and monovalent lipids, or onto ternary mixtures of neutral, monovalent, and multivalent lipids. Consistent with previous calculations and experiments, our results show that at steady-state multivalent lipids (such as PIP2), but not monovalent lipid (such as phosphatidylserine), will segregate near the adsorbing macromolecules. To address the corresponding diffusion of the adsorbing protein in the membrane plane, we couple lipid mobility with the propagation of the adsorbing protein through a dynamic Monte Carlo scheme. We find that due to their higher mobility dictated by the electrochemical potential, multivalent lipids such as PIP2 more quickly segregate near oppositely charged proteins than do monovalent lipids, even though their diffusion constants may be similar. The

  7. Photopolymerization of Dienoyl Lipids Creates Planar Supported Poly(lipid) Membranes with Retained Fluidity.

    PubMed

    Orosz, Kristina S; Jones, Ian W; Keogh, John P; Smith, Christopher M; Griffin, Kaitlyn R; Xu, Juhua; Comi, Troy J; Hall, H K; Saavedra, S Scott

    2016-02-16

    Polymerization of substrate-supported bilayers composed of dienoylphosphatidylcholine (PC) lipids is known to greatly enhance their chemical and mechanical stability; however, the effects of polymerization on membrane fluidity have not been investigated. Here planar supported lipid bilayers (PSLBs) composed of dienoyl PCs on glass substrates were examined to assess the degree to which UV-initiated polymerization affects lateral lipid mobility. Fluorescence recovery after photobleaching (FRAP) was used to measure the diffusion coefficients (D) and mobile fractions of rhodamine-DOPE in unpolymerized and polymerized PSLBs composed of bis-sorbyl phosphatidylcholine (bis-SorbPC), mono-sorbyl-phosphatidylcholine (mono-SorbPC), bis-dienoyl-phosphatidylcholine (bis-DenPC), and mono-dienoyl phosphatidylcholine (mono-DenPC). Polymerization was performed in both the Lα and Lβ phase for each lipid. In all cases, polymerization reduced membrane fluidity; however, measurable lateral diffusion was retained which is attributed to a low degree of polymerization. The D values for sorbyl lipids were less than those of the denoyl lipids; this may be a consequence of the distal location of polymerizable group in the sorbyl lipids which may facilitate interleaflet bonding. The D values measured after polymerization were 0.1-0.8 of those measured before polymerization, a range that corresponds to fluidity intermediate between that of a Lα phase and a Lβ phase. This D range is comparable to ratios of D values reported for liquid-disordered (Ld) and liquid-ordered (Lo) lipid phases and indicates that the effect of UV polymerization on lateral diffusion in a dienoyl PSLB is similar to the transition from a Ld phase to a Lo phase. The partial retention of fluidity in UV-polymerized PSLBs, their enhanced stability, and the activity of incorporated transmembrane proteins and peptides is discussed.

  8. Photopolymerization of dienoyl lipids creates planar supported poly(lipid) membranes with retained fluidity

    PubMed Central

    Orosz, Kristina S.; Jones, Ian W.; Keogh, John P.; Smith, Christopher M.; Griffin, Kaitlyn R.; Xu, Juhua; Comi, Troy J.; Hall, H. K.

    2016-01-01

    Polymerization of substrate-supported bilayers composed of dienoyl phosphatidylcholine (PC) lipids is known to greatly enhance their chemical and mechanical stability, however the effects of polymerization on membrane fluidity have not been investigated. Here planar supported lipid bilayers (PSLBs) composed of dienoyl PCs on glass substrates were examined to assess the degree to which UV-initiated polymerization affects lateral lipid mobility. Fluorescence recovery after photobleaching (FRAP) was used to measure the diffusion coefficients (D) and mobile fractions of rhodamine-DOPE in unpolymerized and polymerized PSLBs composed of bis-sorbyl phosphatidylcholine (bis-SorbPC), mono-sorbyl phosphatidylcholine (mono-SorbPC), bis-dienoyl phosphatidylcholine (bis-DenPC) and mono-dienoyl phosphatidylcholine (mono-DenPC). Polymerization was performed in both the Lα and Lβ phase for each lipid. In all cases, polymerization reduced membrane fluidity, however measurable lateral diffusion was retained which is attributed to a low degree of polymerization. The D values for sorbyl lipids were less than those of the denoyl lipids; this may be a consequence of the distal location of polymerizable group in the sorbyl lipids which may facilitate inter-leaflet bonding. The D values measured after polymerization were 0.1 to 0.8 of those measured before polymerization, a range that corresponds to fluidity intermediate between that of a Lα phase and a Lβ phase. This D range is comparable to ratios of D values reported for liquid-disordered (Ld) and liquid-ordered (Lo) lipid phases, and indicates that the effect of UV polymerization on lateral diffusion in a dienoyl PSLB is similar to the transition from a Ld phase to a Lo phase. The partial retention of fluidity in UV polymerized PSLBs, their enhanced stability, and the activity of incorporated transmembrane proteins and peptides is discussed. PMID:26794208

  9. Membrane Lipid Replacement for chronic illnesses, aging and cancer using oral glycerolphospholipid formulations with fructooligosaccharides to restore phospholipid function in cellular membranes, organelles, cells and tissues.

    PubMed

    Nicolson, Garth L; Ash, Michael E

    2017-09-01

    Membrane Lipid Replacement is the use of functional, oral supplements containing mixtures of cell membrane glycerolphospholipids, plus fructooligosaccharides (for protection against oxidative, bile acid and enzymatic damage) and antioxidants, in order to safely replace damaged, oxidized, membrane phospholipids and restore membrane, organelle, cellular and organ function. Defects in cellular and intracellular membranes are characteristic of all chronic medical conditions, including cancer, and normal processes, such as aging. Once the replacement glycerolphospholipids have been ingested, dispersed, complexed and transported, while being protected by fructooligosaccharides and several natural mechanisms, they can be inserted into cell membranes, lipoproteins, lipid globules, lipid droplets, liposomes and other carriers. They are conveyed by the lymphatics and blood circulation to cellular sites where they are endocytosed or incorporated into or transported by cell membranes. Inside cells the glycerolphospholipids can be transferred to various intracellular membranes by lipid globules, liposomes, membrane-membrane contact or by lipid carrier transfer. Eventually they arrive at their membrane destinations due to 'bulk flow' principles, and there they can stimulate the natural removal and replacement of damaged membrane lipids while undergoing further enzymatic alterations. Clinical trials have shown the benefits of Membrane Lipid Replacement in restoring mitochondrial function and reducing fatigue in aged subjects and chronically ill patients. Recently Membrane Lipid Replacement has been used to reduce pain and other symptoms as well as removing hydrophobic chemical contaminants, suggesting that there are additional new uses for this safe, natural medicine supplement. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights

  10. Spontaneous vesicle formation at lipid bilayer membranes.

    PubMed

    Edwards, D A; Schneck, F; Zhang, I; Davis, A M; Chen, H; Langer, R

    1996-09-01

    Unilamellar vesicles are observed to form spontaneously at planar lipid bilayers agitated by exothermic chemical reactions. The membrane-binding reaction between biotin and streptavidin, two strong transmembrane neutralization reactions, and a weak neutralization reaction involving an "antacid" buffer, all lead to spontaneous vesicle formation. This formation is most dramatic when a viscosity differential exists between the two phases bounding the membrane, in which case vesicles appear exclusively in the more viscous phase. A hydrodynamic analysis explains the phenomenon in terms of a membrane flow driven by liberated reaction energy, leading to vesicle formation. These results suggest that energy liberated by intra- and extracellular chemical reactions near or at cell and internal organelle membranes can play an important role in vesicle formation, membrane agitation, or enhanced transmembrane mass transfer.

  11. Insights into the complex association of bovine factor Va with acidic-lipid-containing synthetic membranes.

    PubMed Central

    Cutsforth, G A; Koppaka, V; Krishnaswamy, S; Wu, J R; Mann, K G; Lentz, B R

    1996-01-01

    The mechanism of binding of blood coagulation cofactor factor Va to acidic-lipid-containing membranes has been addressed. Binding isotherms were generated at room temperature using the change in fluorescence anisotropy of pyrene-labeled bovine factor Va to detect binding to sonicated membrane vesicles containing either bovine brain phosphatidylserine (PS) or 1,2-dioleoyl-3-sn-phosphatidylglycerol (DOPG) in combination with 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC). The composition of the membranes was varied from 0 to 40 mol% for PS/POPC and from 0 to 65 mol % for DOPG/POPC membranes. Fitting the data to a classical Langmuir adsorption model yielded estimates of the dissociation constant (Kd) and the stoichiometry of binding. The values of Kd defined in this way displayed a maximum at low acidic lipid content but were nearly constant at intermediate to high fractions of acidic lipid. Fitting the binding isotherms to a two-process binding model (nonspecific adsorption in addition to binding of acidic lipids to sites on the protein) suggested a significant acidic-lipid-independent binding affinity in addition to occupancy of three protein sites that bind PS in preference to DOPG. Both analyses indicated that interaction of factor Va with an acidic-lipid-containing membrane is much more complex than those of factor Xa or prothrombin. Furthermore, a change in the conformation of bound pyrene-labeled factor Va with surface concentration of acidic lipid was implied by variation of both the saturating fluorescence anisotropy and the binding parameters with the acidic lipid content of the membrane. Finally, the results cannot support the contention that binding occurs through nonspecific adsorption to a patch or domain of acidic lipids in the membrane. Factor Va is suggested to associate with membranes by a complex process that includes both acidic-lipid-specific and acidic-lipid-independent sites and a protein structure change induced by occupancy of acidic-lipid

  12. The antifungal activity and membrane-disruptive action of dioscin extracted from Dioscorea nipponica.

    PubMed

    Cho, Jaeyong; Choi, Hyemin; Lee, Juneyoung; Kim, Mi-Sun; Sohn, Ho-Yong; Lee, Dong Gun

    2013-03-01

    Dioscin is a kind of steroidal saponin isolated from the root bark of wild yam Dioscorea nipponica. We investigated the antifungal effect of dioscin against different fungal strains and its antifungal mechanism(s) in Candida albicans cells. Using the propidium iodide assay and calcein-leakage measurement, we confirmed that dioscin caused fungal membrane damage. Furthermore, we evaluated the ability of dioscin to disrupt the plasma membrane potential, using 3,3'-dipropylthiadicarbocyanine iodide [DiSC(3)(5)] and bis-(1,3-dibarbituric acid)-trimethine oxanol [DiBAC(4)(3)]. Cells stained with the dyes had a significant increase in fluorescent intensity after exposure to dioscin, indicating that dioscin has an effect on the membrane potential. To visualize the effect of dioscin on the cell membrane, we synthesized rhodamine-labeled giant unilamellar vesicles (GUVs) mimicking the outer leaflet of the plasma membrane of C. albicans. As seen in the result, the membrane disruptive action of dioscin caused morphological change and rhodamine leakage of the GUVs. In three-dimensional contour-plot analysis using flow cytometry, we observed a decrease in cell size, which is in agreement with our result from the GUV assay. These results suggest that dioscin exerts a considerable antifungal activity by disrupting the structure in membrane after invading into the fungal membrane, resulting in fungal cell death. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. The Inner Nuclear Membrane Is a Metabolically Active Territory that Generates Nuclear Lipid Droplets.

    PubMed

    Romanauska, Anete; Köhler, Alwin

    2018-06-13

    The inner nuclear membrane (INM) encases the genome and is fused with the outer nuclear membrane (ONM) to form the nuclear envelope. The ONM is contiguous with the endoplasmic reticulum (ER), the main site of phospholipid synthesis. In contrast to the ER and ONM, evidence for a metabolic activity of the INM has been lacking. Here, we show that the INM is an adaptable membrane territory capable of lipid metabolism. S. cerevisiae cells target enzymes to the INM that can promote lipid storage. Lipid storage involves the synthesis of nuclear lipid droplets from the INM and is characterized by lipid exchange through Seipin-dependent membrane bridges. We identify the genetic circuit for nuclear lipid droplet synthesis and a role of these organelles in regulating this circuit by sequestration of a transcription factor. Our findings suggest a link between INM metabolism and genome regulation and have potential relevance for human lipodystrophy. Copyright © 2018 Elsevier Inc. All rights reserved.

  14. Brownian dynamics simulations of lipid bilayer membrane with hydrodynamic interactions in LAMMPS

    NASA Astrophysics Data System (ADS)

    Fu, Szu-Pei; Young, Yuan-Nan; Peng, Zhangli; Yuan, Hongyan

    2016-11-01

    Lipid bilayer membranes have been extensively studied by coarse-grained molecular dynamics simulations. Numerical efficiencies have been reported in the cases of aggressive coarse-graining, where several lipids are coarse-grained into a particle of size 4 6 nm so that there is only one particle in the thickness direction. Yuan et al. proposed a pair-potential between these one-particle-thick coarse-grained lipid particles to capture the mechanical properties of a lipid bilayer membrane (such as gel-fluid-gas phase transitions of lipids, diffusion, and bending rigidity). In this work we implement such interaction potential in LAMMPS to simulate large-scale lipid systems such as vesicles and red blood cells (RBCs). We also consider the effect of cytoskeleton on the lipid membrane dynamics as a model for red blood cell (RBC) dynamics, and incorporate coarse-grained water molecules to account for hydrodynamic interactions. The interaction between the coarse-grained water molecules (explicit solvent molecules) is modeled as a Lennard-Jones (L-J) potential. We focus on two sets of LAMMPS simulations: 1. Vesicle shape transitions with varying enclosed volume; 2. RBC shape transitions with different enclosed volume. This work is funded by NSF under Grant DMS-1222550.

  15. Brownian dynamics simulations of lipid bilayer membrane with hydrodynamic interactions in LAMMPS

    NASA Astrophysics Data System (ADS)

    Fu, Szu-Pei; Young, Yuan-Nan; Peng, Zhangli; Yuan, Hongyan

    Lipid bilayer membranes have been extensively studied by coarse-grained molecular dynamics simulations. Numerical efficiency has been reported in the cases of aggressive coarse-graining, where several lipids are coarse-grained into a particle of size 4 6 nm so that there is only one particle in the thickness direction. Yuan et al. proposed a pair-potential between these one-particle-thick coarse-grained lipid particles to capture the mechanical properties of a lipid bilayer membrane (such as gel-fluid-gas phase transitions of lipids, diffusion, and bending rigidity). In this work we implement such interaction potential in LAMMPS to simulate large-scale lipid systems such as vesicles and red blood cells (RBCs). We also consider the effect of cytoskeleton on the lipid membrane dynamics as a model for red blood cell (RBC) dynamics, and incorporate coarse-grained water molecules to account for hydrodynamic interactions. The interaction between the coarse-grained water molecules (explicit solvent molecules) is modeled as a Lennard-Jones (L-J) potential. We focus on two sets of LAMMPS simulations: 1. Vesicle shape transitions with varying enclosed volume; 2. RBC shape transitions with different enclosed volume.

  16. Lateral mobility of plasma membrane lipids in dividing Xenopus eggs.

    PubMed

    Tetteroo, P A; Bluemink, J G; Dictus, W J; van Zoelen, E J; de Laat, S W

    1984-07-01

    The lateral mobility of plasma membrane lipids was analyzed during first cleavage of Xenopus laevis eggs by fluorescence photobleaching recovery (FPR) measurements, using the lipid analogs 5-(N-hexadecanoyl)aminofluorescein ("HEDAF") and 5-(N-tetradecanoyl)aminofluorescein ("TEDAF") as probes. The preexisting plasma membrane of the animal side showed an inhomogeneous, dotted fluorescence pattern after labeling and the lateral mobility of both probes used was below the detection limits of the FPR method (D much less than 10(-10) cm2/sec). In contrast, the preexisting plasma membrane of the vegetal side exhibited homogeneous fluorescence and the lateral diffusion coefficient of both probes used was relatively high (HEDAF, D = 2.8 X 10(-8) cm2/sec; TEDAF, D = 2.4 X 10(-8) cm2/sec). In the cleaving egg visible transfer of HEDAF or TEDAF from prelabeled plasma membrane to the new membrane in the furrow did not occur, even on the vegetal side. Upon labeling during cleavage, however, the new membrane was uniformly labeled and both probes were mobile, as in the vegetal preexisting plasma membrane. These data show that the membrane of the dividing Xenopus egg comprises three macrodomains: (i) the animal preexisting plasma membrane; (ii) the vegetal preexisting plasma membrane; (iii) the new furrow membrane.

  17. Lipid composition and sensitivity of Prototheca wickerhamii to membrane-active antimicrobial agents.

    PubMed Central

    Sud, I J; Feingold, D S

    1979-01-01

    The lipid composition of Prototheca wickerhamii ATCC 16529 is presented and discussed in relation to the unique susceptibility of the organism to drugs of three membrane-active antimicrobial classes: the polyenes, the polymyxins, and the imidazoles. The presence of ergosterol in the neutral lipid fraction of the membrane is likely responsible for the exquisite susceptibility to amphotericin B. The presence of a large quantity of free fatty acids in the membrane appears responsible for imidazole susceptibility. The membrane determinants of polymyxin B susceptibility are less well defined. PMID:518077

  18. Nonanesthetic alcohols dissolve in synaptic membranes without perturbing their lipids.

    PubMed Central

    Miller, K W; Firestone, L L; Alifimoff, J K; Streicher, P

    1989-01-01

    While many theories of general anesthesia postulate a lipid site of action, there has been no adequate explanation for the lack of anesthetic potency of the highly hydrophobic primary alkanols with more than 12 carbons (the cut-off). Some work suggests that these nonanesthetic alcohols do not dissolve in membranes. Other work contradicts this and suggests that an anesthetic site on a protein provides a better explanation. Here we show that both the anesthetic dodecanol and the nonanesthetic tetradecanol are taken up equally well into the tissues of animals and into isolated postsynaptic membranes. When a group of Rana pipiens tadpoles were treated with dodecanol, half were anesthetized by 4.7 microM (free aqueos concentration), and the corresponding concentration in the tissues was found to be 0.4 mmol per kg wet weight. Prolonged exposure (92 hr) to tetradecanol produced even higher tissue concentrations (0.7 mmol per kg wet weight), yet no anesthetic effects were observed. Furthermore, general anesthetics are thought to act on postsynaptic membranes but both alkanols partitioned into postsynaptic membranes from Torpedo electroplaques. The spin label, 12-doxyl stearate, was incorporated into these membranes. The lipid order parameter it reported was decreased by the anesthetic alcohols (octanol, decanol, and dodecanol), whereas the nonanesthetic alcohols either did not change it significantly (tetradecanol) or actually increased it (hexadecanol and octadecanol). Thus, although lipid solubility is unable to account for the pharmacology of the cut-off in potency of the long-chain alcohols, lipid perturbations provide an accurate description. PMID:2783782

  19. Aromaticity/Bulkiness of Surface Ligands to Promote the Interaction of Anionic Amphiphilic Gold Nanoparticles with Lipid Bilayers.

    PubMed

    Gao, Jinhong; Zhang, Ouyang; Ren, Jing; Wu, Chuanliu; Zhao, Yibing

    2016-02-16

    The presence of large hydrophobic aromatic residues in cell-penetrating peptides or proteins has been demonstrated to be advantageous for their cell penetration. This phenomenon has also been observed when AuNPs were modified with peptides containing aromatic amino acids. However, it is still not clear how the presence of hydrophobic and aromatic groups on the surface of anionic AuNPs affects their interaction with lipid bilayers. Here, we studied the interaction of a range of anionic amphiphilic AuNPs coated by different combinations of hydrophobic and anionic ligands with four different types of synthetic lipid vesicles. Our results demonstrated the important role of the surface aromatic or bulky groups, relative to the hydrocarbon chains, in the interaction of anionic AuNPs with lipid bilayers. Hydrophobic interaction itself arising from the insertion of aromatic/bulky ligands on the surface of AuNPs into lipid bilayers is sufficiently strong to cause overt disruption of lipid vesicles and cell membranes. Moreover, by comparing the results obtained from AuNPs coated with aromatic ligands and cyclohexyl ligands lacking aromaticity respectively, we demonstrated that the bulkiness of the terminal groups in hydrophobic ligands instead of the aromatic character might be more important to the interaction of AuNPs with lipid bilayers. Finally, we further correlated the observation on model liposomes with that on cell membranes, demonstrating that AuNPs that are more disruptive to the more negatively charged liposomes are also substantially more disruptive to cell membranes. In addition, our results revealed that certain cellular membrane domains that are more susceptible to disruption caused by hydrophobic interactions with nanoparticle surfaces might determine the threshold of AuNP-mediated cytotoxicity.

  20. Indentation of Graphene-Covered Atomic Force Microscopy Probe Across a Lipid Bilayer Membrane: Effect of Tip Shape, Size, and Surface Hydrophobicity.

    PubMed

    Lv, Kang; Li, Yinfeng

    2018-06-21

    Understanding the interaction of graphene with cell membranes is crucial to the development of graphene-based biological applications and the management of graphene safety issues. To help reveal the key factors controlling the interaction between graphene and cell membranes, here we adopt the dissipative particle dynamics method to analyze the evolution of interaction force and free energy as the graphene-covered atomic force microscopy (AFM) probe indents across a lipid bilayer. The simulation results show that the graphene-covered AFM probe can cause severe deformation of the cell membrane which drives the lipid molecule to adsorb and diffuse at the surface of graphene. The breakthrough force and free energy are calculated to study the effects of the tip shape, size, and surface hydrophobicity on the piercing behaviors of graphene-covered AFM. In addition, the deformation of cell membrane can decrease the dependency of the breakthrough force on the tip shape. The analysis of surface functionalization suggests that the horizontal patterns on graphene can change the preferred orientation in the penetration process, but the vertical patterns on graphene may disrupt the cell membrane. What's more, the bending stiffness of graphene has little influence on the penetration process as graphene pierces into the cell membrane. These results provide useful guidelines for the molecular design of graphene materials with controllable cell penetrability.

  1. Reconstituted high-density lipoprotein suppresses leukocyte NADPH oxidase activation by disrupting lipid rafts.

    PubMed

    Peshavariya, Hitesh; Dusting, Gregory J; Di Bartolo, Belinda; Rye, Kerry-Anne; Barter, Philip J; Jiang, Fan

    2009-08-01

    Reconstituted discoidal high-density lipoprotein (rHDL) has potent vascular protective actions. Native HDL suppresses cellular generation of reactive oxygen species, whereas this antioxidant effect of rHDL is less clear. This study examined the effects of rHDL on NADPH oxidase, a major source of cellular superoxide generation, in both leukocytes and human umbilical vein endothelial cells. Superoxide was measured with lucigenin-enhanced chemiluminescence. Expression of NADPH oxidase sub-units was determined by real-time PCR. Pre-treatment of HL-60 cells with rHDL (10 and 25 microM) for 1 h significantly reduced phorbol 12-myristate 13-acetate-stimulated superoxide production. Treatment with rHDL for up to 24 h did not change the mRNA expression of NADPH oxidase sub-units. In HL-60 cells, depletion of cholesterol from the plasma membrane by methyl-beta-cyclodextrin mimicked the effect of rHDL, whereas cholesterol repletion blunted the effects of rHDL. Treatment with rHDL induced disruption of the lipid raft structures and blunted PMA-induced redistribution of p47phox into lipid rafts. In contrast, treatment of endothelial cells with rHDL for up to 18 h had no effect on either basal or tumour necrosis factor-alpha-stimulated NADPH oxidase activity, but markedly suppressed the cytokine-induced expression of proinflammatory adhesion molecules. The results suggest that rHDL inhibits NADPH oxidase activation in leukocytes, probably by interrupting the assembly of NADPH oxidase sub-units at the lipid rafts. This effect may contribute to the vascular protective actions of rHDL against inflammation-mediated oxidative damage.

  2. Autonomous buckling of micrometer-sized lipid-protein membrane patches constructed by Dictyostelium discoideum.

    PubMed

    Takahashi, Kei; Toyota, Taro

    2015-01-01

    The cytosol of amoeba cells controls the membrane deformation during their motion in vivo. To investigate such ability of the cytosol of amoeba cell, Dictyostelium discoideum (Dictyostelium), in vitro, we used lipids extracted from Dictyostelium and commercially available phospholipids, and prepared substrate-supported lipid membrane patches on the micrometer scale by spin coating. We found that the spin coater holder, which has pores (pore size = 3.1 mm) of negative pressure to hold the cover glass induced the concave surface of the cover glass. The membrane lipid patches were formed at each position in the vicinity of the holder pores and their sizes were in the range of 2.7 to 3.2 × 10(4) μm(2). After addition of the cytosol extracted from Dictyostelium to the lipid membrane patches, through time-lapse observation with a confocal laser scanning fluorescence microscope, we observed an autonomous buckling of the Dictyostelium lipid patches and localized behaviours of proteins found within. The current method serves as the novel technique for the preparation of film patches in which the positions of patches are controlled by the holder pores without fabricating, modifying, and arranging the chemical properties of the solution components of lipids. The findings imply that lipid-binding proteins in the cytosol were adsorbed and accumulated within the Dictyostelium lipid patches, inducing the transformation of the cell-sized patch.

  3. Membrane solubilisation and reconstitution by octylglucoside: comparison of synthetic lipid and natural lipid extract by isothermal titration calorimetry.

    PubMed

    Krylova, Oxana O; Jahnke, Nadin; Keller, Sandro

    2010-08-01

    We have studied the solubilisation and reconstitution of lipid membranes composed of either synthetic phosphatidylcholine or Escherichia. coli polar lipid extract by the non-ionic detergent octylglucoside. For both lipid systems, composition-dependent transformations of unilamellar vesicles into micelles or vice versa were followed by high-sensitivity isothermal titration calorimetry. Data obtained over a range of detergent and lipid concentrations could be rationalised in terms of a three-stage phase separation model involving bilayer, bilayer/micelle coexistence, and micellar ranges, yielding the detergent/lipid phase diagrams and the bilayer-to-micelle partition coefficients of both detergent and lipid. The most notable difference between the lipids investigated was a substantial widening of the bilayer/micelle coexistence range for E. coli lipid, which was due to an increased preference of the detergent and a decreased affinity of the lipid for the micellar phase as compared with the bilayer phase. These effects on the bilayer-to-micelle partition coefficients could be explained by the high proportion in E. coli membranes of lipids possessing negative spontaneous curvature, which hampers both their transfer into strongly curved micellar structures as well as the insertion of detergent into condensed bilayers.

  4. [The effects of electromagnetic pulse on fluidity and lipid peroxidation of mitochondrial membrane].

    PubMed

    Wang, Changzhen; Cong, Jianbo; Xian, Hong; Cao, Xiaozhe; Sun, Cunpu; Wu, Ke

    2002-08-01

    To study the effects of intense electromagnetic pulse(EMP) on the biological effects of mitochondrial membrane. Rat liver mitochondrial suspension was exposed to EMP at 60 kV/m level. The changes of membrane lipid fluidity and membrane protein mobility were detected by ESR and spin label technique. Malondialdehyde(MDA) was detected by spectrophotometer. The mobility of membrane protein decreased significantly(P < 0.05). Correlation time (tau c) of control group was (0.501 +/- 0.077) x 10(-9)s, and tau c of EMP group was (0.594 +/- 0.049) x 10(-9)s, indicating that the mobility of protein was restricted. The fluidity of mitochondrial membrane increased significantly(P < 0.05) at the same time. Order parameter(S) of mitochondrial membrane lipid in control group was 0.63 +/- 0.01, while S of EMP group was 0.61 +/- 0.01(P < 0.05). MDA decreased significantly. The mobility and lipid peroxidation of mitochondrial membrane may be disturbed after EMP exposure.

  5. Membrane interaction of antimicrobial peptides using E. coli lipid extract as model bacterial cell membranes and SFG spectroscopy.

    PubMed

    Soblosky, Lauren; Ramamoorthy, Ayyalusamy; Chen, Zhan

    2015-04-01

    Supported lipid bilayers are used as a convenient model cell membrane system to study biologically important molecule-lipid interactions in situ. However, the lipid bilayer models are often simple and the acquired results with these models may not provide all pertinent information related to a real cell membrane. In this work, we use sum frequency generation (SFG) vibrational spectroscopy to study molecular-level interactions between the antimicrobial peptides (AMPs) MSI-594, ovispirin-1 G18, magainin 2 and a simple 1,2-dipalmitoyl-d62-sn-glycero-3-phosphoglycerol (dDPPG)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) bilayer. We compared such interactions to those between the AMPs and a more complex dDPPG/Escherichia coli (E. coli) polar lipid extract bilayer. We show that to fully understand more complex aspects of peptide-bilayer interaction, such as interaction kinetics, a heterogeneous lipid composition is required, such as the E. coli polar lipid extract. The discrepancy in peptide-bilayer interaction is likely due in part to the difference in bilayer charge between the two systems since highly negative charged lipids can promote more favorable electrostatic interactions between the peptide and lipid bilayer. Results presented in this paper indicate that more complex model bilayers are needed to accurately analyze peptide-cell membrane interactions and demonstrates the importance of using an appropriate lipid composition to study AMP interaction properties. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  6. Golgi Membrane Dynamics Viewed Through a Lens of Lipids

    PubMed Central

    Bankaitis, Vytas A.; Garcia-Mata, Rafael; Mousley, Carl J.

    2012-01-01

    Summary The striking morphology of the Golgi complex has fascinated cell biologists since its discovery over 100 years ago. Yet, despite intense efforts to understand how membrane flow relates to Golgi form and function, this organelle continues to baffle cell biologists and biochemists alike. Fundamental questions regarding Golgi function, while hotly debated, remain unresolved. While Golgi function is historically described from a protein-centric point of view, we now appreciate that conceptual frameworks for how lipid metabolism is integrated with Golgi biogenesis and function are essential for a mechanistic understanding of this fascinating organelle. It is from a lipid-centric perspective that we discuss the larger question of Golgi dynamics and membrane trafficking. We review the growing body of evidence for how lipid metabolism is integrally written into the engineering of the Golgi system, and highlight questions for future study. PMID:22625862

  7. Labeling viral envelope lipids with quantum dots by harnessing the biotinylated lipid-self-inserted cellular membrane.

    PubMed

    Lv, Cheng; Lin, Yi; Liu, An-An; Hong, Zheng-Yuan; Wen, Li; Zhang, Zhenfeng; Zhang, Zhi-Ling; Wang, Hanzhong; Pang, Dai-Wen

    2016-11-01

    Highly efficient labeling of viruses with quantum dots (QDs) is the prerequisite for the long-term tracking of virus invasion at the single virus level to reveal mechanisms of virus infection. As one of the structural components of viruses, viral envelope lipids are hard to be labeled with QDs due to the lack of efficient methods to modify viral envelope lipids. Moreover, it is still a challenge to maintain the intactness and infectivity of labeled viruses. Herein, a mild method has been developed to label viral envelope lipids with QDs by harnessing the biotinylated lipid-self-inserted cellular membrane. Biotinylated lipids can spontaneously insert in cellular membranes of host cells during culture and then be naturally assembled on progeny Pseudorabies virus (PrV) via propagation. The biotinylated PrV can be labeled with streptavidin-conjugated QDs, with a labeling efficiency of ∼90%. Such a strategy to label lipids with QDs can retain the intactness and infectivity of labeled viruses to the largest extent, facilitating the study of mechanisms of virus infection at the single virus level. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Capacitive Detection of Low-Enthalpy, Higher-Order Phase Transitions in Synthetic and Natural Composition Lipid Membranes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Taylor, Graham J.; Heberle, Frederick A.; Seinfeld, Jason S.

    In-plane lipid organization and phase separation in natural membranes play key roles in regulating many cellular processes. Highly cooperative, first-order phase transitions in model membranes consisting of few lipid components are well understood and readily detectable via calorimetry, densitometry, and fluorescence. However, far less is known about natural membranes containing numerous lipid species and high concentrations of cholesterol, for which thermotropic transitions are undetectable by the above-mentioned techniques. We demonstrate that membrane capacitance is highly sensitive to low-enthalpy thermotropic transitions taking place in complex lipid membranes. Specifically, we measured the electrical capacitance as a function of temperature for droplet interfacemore » bilayer model membranes of increasing compositional complexity, namely, (a) a single lipid species, (b) domain-forming ternary mixtures, and (c) natural brain total lipid extract (bTLE). We observed that, for single-species lipid bilayers and some ternary compositions, capacitance exhibited an abrupt, temperature-dependent change that coincided with the transition detected by other techniques. In addition, capacitance measurements revealed transitions in mixed-lipid membranes that were not detected by the other techniques. Most notably, capacitance measurements of bTLE bilayers indicated a transition at ~38 °C not seen with any other method. Likewise, capacitance measurements detected transitions in some well-studied ternary mixtures that, while known to yield coexisting lipid phases, are not detected with calorimetry or densitometry. These results indicate that capacitance is exquisitely sensitive to low-enthalpy membrane transitions because of its sensitivity to changes in bilayer thickness that occur when lipids and excess solvent undergo subtle rearrangements near a phase transition. Our findings also suggest that heterogeneity confers stability to natural membranes that function near

  9. Capacitive Detection of Low-Enthalpy, Higher-Order Phase Transitions in Synthetic and Natural Composition Lipid Membranes

    DOE PAGES

    Taylor, Graham J.; Heberle, Frederick A.; Seinfeld, Jason S.; ...

    2017-08-15

    In-plane lipid organization and phase separation in natural membranes play key roles in regulating many cellular processes. Highly cooperative, first-order phase transitions in model membranes consisting of few lipid components are well understood and readily detectable via calorimetry, densitometry, and fluorescence. However, far less is known about natural membranes containing numerous lipid species and high concentrations of cholesterol, for which thermotropic transitions are undetectable by the above-mentioned techniques. We demonstrate that membrane capacitance is highly sensitive to low-enthalpy thermotropic transitions taking place in complex lipid membranes. Specifically, we measured the electrical capacitance as a function of temperature for droplet interfacemore » bilayer model membranes of increasing compositional complexity, namely, (a) a single lipid species, (b) domain-forming ternary mixtures, and (c) natural brain total lipid extract (bTLE). We observed that, for single-species lipid bilayers and some ternary compositions, capacitance exhibited an abrupt, temperature-dependent change that coincided with the transition detected by other techniques. In addition, capacitance measurements revealed transitions in mixed-lipid membranes that were not detected by the other techniques. Most notably, capacitance measurements of bTLE bilayers indicated a transition at ~38 °C not seen with any other method. Likewise, capacitance measurements detected transitions in some well-studied ternary mixtures that, while known to yield coexisting lipid phases, are not detected with calorimetry or densitometry. These results indicate that capacitance is exquisitely sensitive to low-enthalpy membrane transitions because of its sensitivity to changes in bilayer thickness that occur when lipids and excess solvent undergo subtle rearrangements near a phase transition. Our findings also suggest that heterogeneity confers stability to natural membranes that function near

  10. Depletion with Cyclodextrin Reveals Two Populations of Cholesterol in Model Lipid Membranes

    PubMed Central

    Litz, Jonathan P.; Thakkar, Niket; Portet, Thomas; Keller, Sarah L.

    2016-01-01

    Recent results provide evidence that cholesterol is highly accessible for removal from both cell and model membranes above a threshold concentration that varies with membrane composition. Here we measured the rate at which methyl-β-cyclodextrin depletes cholesterol from a supported lipid bilayer as a function of cholesterol mole fraction. We formed supported bilayers from two-component mixtures of cholesterol and a PC (phosphatidylcholine) lipid, and we directly visualized the rate of decrease in area of the bilayers with fluorescence microscopy. Our technique yields the accessibility of cholesterol over a wide range of concentrations (30–66 mol %) for many individual bilayers, enabling fast acquisition of replicate data. We found that the bilayers contain two populations of cholesterol, one with low surface accessibility and the other with high accessibility. A larger fraction of the total membrane cholesterol appears in the more accessible population when the acyl chains of the PC-lipid tails are more unsaturated. Our findings are most consistent with the predictions of the condensed-complex and cholesterol bilayer domain models of cholesterol-phospholipid interactions in lipid membranes. PMID:26840728

  11. Membrane contact sites, ancient and central hubs of cellular lipid logistics.

    PubMed

    Jain, Amrita; Holthuis, Joost C M

    2017-09-01

    Membrane contact sites (MCSs) are regions where two organelles are closely apposed to facilitate molecular communication and promote a functional integration of compartmentalized cellular processes. There is growing evidence that MCSs play key roles in controlling intracellular lipid flows and distributions. Strikingly, even organelles connected by vesicular trafficking exchange lipids en bulk via lipid transfer proteins that operate at MCSs. Herein, we describe how MCSs developed into central hubs of lipid logistics during the evolution of eukaryotic cells. We then focus on how modern eukaryotes exploit MCSs to help solve a major logistical problem, namely to preserve the unique lipid mixtures of their early and late secretory organelles in the face of extensive vesicular trafficking. This article is part of a Special Issue entitled: Membrane Contact Sites edited by Christian Ungermann and Benoit Kornmann. Copyright © 2017. Published by Elsevier B.V.

  12. Lipid rafts generate digital-like signal transduction in cell plasma membranes.

    PubMed

    Suzuki, Kenichi G N

    2012-06-01

    Lipid rafts are meso-scale (5-200 nm) cell membrane domains where signaling molecules assemble and function. However, due to their dynamic nature, it has been difficult to unravel the mechanism of signal transduction in lipid rafts. Recent advanced imaging techniques have revealed that signaling molecules are frequently, but transiently, recruited to rafts with the aid of protein-protein, protein-lipid, and/or lipid-lipid interactions. Individual signaling molecules within the raft are activated only for a short period of time. Immobilization of signaling molecules by cytoskeletal actin filaments and scaffold proteins may facilitate more efficient signal transmission from rafts. In this review, current opinions of how the transient nature of molecular interactions in rafts generates digital-like signal transduction in cell membranes, and the benefits this phenomenon provides, are discussed. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation

    PubMed Central

    Barros, Marilia; Nanda, Hirsh

    2016-01-01

    ABSTRACT By assembling in a protein lattice on the host's plasma membrane, the retroviral Gag polyprotein triggers formation of the viral protein/membrane shell. The MA domain of Gag employs multiple signals—electrostatic, hydrophobic, and lipid-specific—to bring the protein to the plasma membrane, thereby complementing protein-protein interactions, located in full-length Gag, in lattice formation. We report the interaction of myristoylated and unmyristoylated HIV-1 Gag MA domains with bilayers composed of purified lipid components to dissect these complex membrane signals and quantify their contributions to the overall interaction. Surface plasmon resonance on well-defined planar membrane models is used to quantify binding affinities and amounts of protein and yields free binding energy contributions, ΔG, of the various signals. Charge-charge interactions in the absence of the phosphatidylinositide PI(4,5)P2 attract the protein to acidic membrane surfaces, and myristoylation increases the affinity by a factor of 10; thus, our data do not provide evidence for a PI(4,5)P2 trigger of myristate exposure. Lipid-specific interactions with PI(4,5)P2, the major signal lipid in the inner plasma membrane, increase membrane attraction at a level similar to that of protein lipidation. While cholesterol does not directly engage in interactions, it augments protein affinity strongly by facilitating efficient myristate insertion and PI(4,5)P2 binding. We thus observe that the isolated MA protein, in the absence of protein-protein interaction conferred by the full-length Gag, binds the membrane with submicromolar affinities. IMPORTANCE Like other retroviral species, the Gag polyprotein of HIV-1 contains three major domains: the N-terminal, myristoylated MA domain that targets the protein to the plasma membrane of the host; a central capsid-forming domain; and the C-terminal, genome-binding nucleocapsid domain. These domains act in concert to condense Gag into a membrane

  14. Mutual adaptation of a membrane protein and its lipid bilayer during conformational changes.

    PubMed

    Sonntag, Yonathan; Musgaard, Maria; Olesen, Claus; Schiøtt, Birgit; Møller, Jesper Vuust; Nissen, Poul; Thøgersen, Lea

    2011-01-01

    The structural elucidation of membrane proteins continues to gather pace, but we know little about their molecular interactions with the lipid environment or how they interact with the surrounding bilayer. Here, with the aid of low-resolution X-ray crystallography, we present direct structural information on membrane interfaces as delineated by lipid phosphate groups surrounding the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) in its phosphorylated and dephosphorylated Ca(2+)-free forms. The protein-lipid interactions are further analysed using molecular dynamics simulations. We find that SERCA adapts to membranes of different hydrophobic thicknesses by inducing local deformations in the lipid bilayers and by undergoing small rearrangements of the amino-acid side chains and helix tilts. These mutually adaptive interactions allow smooth transitions through large conformational changes associated with the transport cycle of SERCA, a strategy that may be of general nature for many membrane proteins.

  15. Dynamic interactions between a membrane binding protein and lipids induce fluctuating diffusivity

    PubMed Central

    Yamamoto, Eiji; Akimoto, Takuma; Kalli, Antreas C.; Yasuoka, Kenji; Sansom, Mark S. P.

    2017-01-01

    Pleckstrin homology (PH) domains are membrane-binding lipid recognition proteins that interact with phosphatidylinositol phosphate (PIP) molecules in eukaryotic cell membranes. Diffusion of PH domains plays a critical role in biological reactions on membrane surfaces. Although diffusivity can be estimated by long-time measurements, it lacks information on the short-time diffusive nature. We reveal two diffusive properties of a PH domain bound to the surface of a PIP-containing membrane using molecular dynamics simulations. One is fractional Brownian motion, attributed to the motion of the lipids with which the PH domain interacts. The other is temporally fluctuating diffusivity; that is, the short-time diffusivity of the bound protein changes substantially with time. Moreover, the diffusivity for short-time measurements is intrinsically different from that for long-time measurements. This fluctuating diffusivity results from dynamic changes in interactions between the PH domain and PIP molecules. Our results provide evidence that the complexity of protein-lipid interactions plays a crucial role in the diffusion of proteins on biological membrane surfaces. Changes in the diffusivity of PH domains and related membrane-bound proteins may in turn contribute to the formation/dissolution of protein complexes in membranes. PMID:28116358

  16. Quantifying lipid changes in various membrane compartments using lipid binding protein domains.

    PubMed

    Várnai, Péter; Gulyás, Gergő; Tóth, Dániel J; Sohn, Mira; Sengupta, Nivedita; Balla, Tamas

    2017-06-01

    One of the largest challenges in cell biology is to map the lipid composition of the membranes of various organelles and define the exact location of processes that control the synthesis and distribution of lipids between cellular compartments. The critical role of phosphoinositides, low-abundant lipids with rapid metabolism and exceptional regulatory importance in the control of almost all aspects of cellular functions created the need for tools to visualize their localizations and dynamics at the single cell level. However, there is also an increasing need for methods to determine the cellular distribution of other lipids regulatory or structural, such as diacylglycerol, phosphatidic acid, or other phospholipids and cholesterol. This review will summarize recent advances in this research field focusing on the means by which changes can be described in more quantitative terms. Published by Elsevier Ltd.

  17. Manipulating lipid membrane architecture by liquid crystal-analog curvature elasticity (Presentation Recording)

    NASA Astrophysics Data System (ADS)

    Lee, Sin-Doo

    2015-10-01

    Soft matters such as liquid crystals and biological molecules exhibit a variety of interesting physical phenomena as well as new applications. Recently, in mimicking biological systems that have the ability to sense, regulate, grow, react, and regenerate in a highly responsive and self-adaptive manner, the significance of the liquid crystal order in living organisms, for example, a biological membrane possessing the lamellar order, is widely recognized from the viewpoints of physics and chemistry of interfaces and membrane biophysics. Lipid bilayers, resembling cell membranes, provide primary functions for the transport of biological components of ions and molecules in various cellular activities, including vesicle budding and membrane fusion, through lateral organization of the membrane components such as proteins. In this lecture, I will describe how the liquid crystal-analog curvature elasticity of a lipid bilayer plays a critical role in developing a new platform for understanding diverse biological functions at a cellular level. The key concept is to manipulate the local curvature at an interface between a solid substrate and a model membrane. Two representative examples will be demonstrated: one of them is the topographic control of lipid rafts in a combinatorial array where the ligand-receptor binding event occurs and the other concerns the reconstitution of a ring-type lipid raft in bud-mimicking architecture within the framework of the curvature elasticity.

  18. Lipid diffusion in the distal and proximal leaflets of supported lipid bilayer membranes studied by single particle tracking

    NASA Astrophysics Data System (ADS)

    Schoch, Rafael L.; Barel, Itay; Brown, Frank L. H.; Haran, Gilad

    2018-03-01

    Supported lipid bilayers (SLBs) have been studied extensively as simple but powerful models for cellular membranes. Yet, potential differences in the dynamics of the two leaflets of a SLB remain poorly understood. Here, using single particle tracking, we obtain a detailed picture of bilayer dynamics. We observe two clearly separate diffusing populations, fast and slow, that we associate with motion in the distal and proximal leaflets of the SLB, respectively, based on fluorescence quenching experiments. We estimate diffusion coefficients using standard techniques as well as a new method based on the blur of images due to motion. Fitting the observed diffusion coefficients to a two-leaflet membrane hydrodynamic model allows for the simultaneous determination of the intermonolayer friction coefficient and the substrate-membrane friction coefficient, without any prior assumptions on the strengths of the relevant interactions. Remarkably, our calculations suggest that the viscosity of the interfacial water confined between the membrane and the substrate is elevated by ˜104 as compared to bulk water. Using hidden Markov model analysis, we then obtain insight into the transbilayer movement of lipids. We find that lipid flip-flop dynamics are very fast, with half times in the range of seconds. Importantly, we find little evidence for membrane defect mediated lipid flip-flop for SLBs at temperatures well above the solid-to-liquid transition, though defects seem to be involved when the SLBs are cooled down. Our work thus shows that the combination of single particle tracking and advanced hydrodynamic modeling provides a powerful means to obtain insight into membrane dynamics.

  19. Changes in membrane lipid composition during saline growth of the fresh water cyanobacterium Synechococcus 6311

    NASA Technical Reports Server (NTRS)

    Huflejt, M. E.; Tremolieres, A.; Pineau, B.; Lang, J. K.; Hatheway, J.; Packer, L.

    1990-01-01

    Growth of Synechococcus 6311 in the presence of 0.5 molar NaCl is accompanied by significant changes in membrane lipid composition. Upon transfer of the cells from a low salt' (0.015 molar NaCl) to high salt' (0.5 molar NaCl) growth medium at different stages of growth, a rapid decrease in palmitoleic acid (C16:1 delta 9) content was accompanied by a concomitant increase in the amount of the two C18:1 acids (C18:1 delta 9, C18:1 delta 11), with the higher increase in oleic acid C18:1 delta 9 content. These changes began to occur within the first hour after the sudden elevation of NaCl and progressed for about 72 hours. The percentage of palmitic acid (C16:0) and stearic acid (C18:0) remained almost unchanged in the same conditions. High salt-dependent changes within ratios of polar lipid classes also occurred within the first 72 hours of growth. The amount of monogalactosyl diacylglycerol (bilayer-destabilizing lipid) decreased and that of the digalactosyl diacylglycerol (bilayer-stabilizing lipid) increased. Consequently, in the three day old cells, the ratio of monogalactosyl diacylglycerol to digalactosyl diacylglycerol in the membranes of high salt-grown cells was about half of that in the membranes of low salt-grown cells. The total content of anionic lipids (phosphatidylglycerol and sulfoquinovosyl diacylglycerol) was always higher in the isolated membranes and the whole cells from high salt-grown cultures compared to that in the cells and membranes from low salt-grown cultures. All the observed rearrangements in the lipid environment occurred in both thylakoid and cytoplasmic membranes. Similar lipid composition changes, however, to a much lesser extent, were also observed in the aging, low salt-grown cultures. The observed changes in membrane fatty acids and lipids composition correlate with the alterations in electron and ion transport activities, and it is concluded that the rearrangement of the membrane lipid environment is an essential part of the

  20. Nonlinear adhesion dynamics of confined lipid membranes

    NASA Astrophysics Data System (ADS)

    To, Tung; Le Goff, Thomas; Pierre-Louis, Olivier

    Lipid membranes, which are ubiquitous objects in biological environments are often confined. For example, they can be sandwiched between a substrate and the cytoskeleton between cell adhesion, or between other membranes in stacks, or in the Golgi apparatus. We present a study of the nonlinear dynamics of membranes in a model system, where the membrane is confined between two flat walls. The dynamics derived from the lubrication approximation is highly nonlinear and nonlocal. The solution of this model in one dimension exhibits frozen states due to oscillatory interactions between membranes caused by the bending rigidity. We develope a kink model for these phenomena based on the historical work of Kawasaki and Otha. In two dimensions, the dynamics is more complex, and depends strongly on the amount of excess area in the system. We discuss the relevance of our findings for experiments on model membranes, and for biological systems. Supported by the grand ANR Biolub.

  1. Calculating the Bending Modulus for Multicomponent Lipid Membranes in Different Thermodynamic Phases

    PubMed Central

    2013-01-01

    We establish a computational approach to extract the bending modulus, KC, for lipid membranes from relatively small-scale molecular simulations. Fluctuations in the splay of individual pairs of lipids faithfully inform on KC in multicomponent membranes over a large range of rigidities in different thermodynamic phases. Predictions are validated by experiments even where the standard spectral analysis-based methods fail. The local nature of this method potentially allows its extension to calculations of KC in protein-laden membranes. PMID:24039553

  2. Neuronal sphingolipidoses: Membrane lipids and sphingolipid activator proteins regulate lysosomal sphingolipid catabolism.

    PubMed

    Sandhoff, Konrad

    2016-11-01

    Glycosphingolipids and sphingolipids of cellular plasma membranes (PMs) reach luminal intra-lysosomal vesicles (LVs) for degradation mainly by pathways of endocytosis. After a sorting and maturation process (e.g. degradation of sphingomyelin (SM) and secretion of cholesterol), sphingolipids of the LVs are digested by soluble enzymes with the help of activator (lipid binding and transfer) proteins. Inherited defects of lipid-cleaving enzymes and lipid binding and transfer proteins cause manifold and fatal, often neurodegenerative diseases. The review summarizes recent findings on the regulation of sphingolipid catabolism and cholesterol secretion from the endosomal compartment by lipid modifiers, an essential stimulation by anionic membrane lipids and an inhibition of crucial steps by cholesterol and SM. Reconstitution experiments in the presence of all proteins needed, hydrolase and activator proteins, reveal an up to 10-fold increase of ganglioside catabolism just by the incorporation of anionic lipids into the ganglioside carrying membranes, whereas an additional incorporation of cholesterol inhibits GM2 catabolism substantially. It is suggested that lipid and other low molecular modifiers affect the genotype-phenotype relationship observed in patients with lysosomal diseases. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  3. Alteration of interleaflet coupling due to compounds displaying rapid translocation in lipid membranes

    PubMed Central

    Reigada, Ramon

    2016-01-01

    The spatial coincidence of lipid domains at both layers of the cell membrane is expected to play an important role in many cellular functions. Competition between the surface interleaflet tension and a line hydrophobic mismatch penalty are conjectured to determine the transversal behavior of laterally heterogeneous lipid membranes. Here, by a combination of molecular dynamics simulations, a continuum field theory and kinetic equations, I demonstrate that the presence of small, rapidly translocating molecules residing in the lipid bilayer may alter its transversal behavior by favoring the spatial coincidence of similar lipid phases. PMID:27596355

  4. Role of Pterocarpus santalinus against mitochondrial dysfunction and membrane lipid changes induced by ulcerogens in rat gastric mucosa.

    PubMed

    Narayan, Shoba; Devi, R S; Devi, C S Shyamala

    2007-11-20

    Free radicals produced by ulcerogenic agents affect the TCA cycle enzymes located in the outer membrane of the mitochondria. Upon induction with ulcerogens, peroxidation of membrane lipids bring about alterations in the mitochondrial enzyme activity. This indicates an increase in the permeability levels of the mitochondrial membrane. The ability of PSE to scavenge the reactive oxygen species results in restoration of activities of TCA cycle enzymes. NSAIDs interfere with the mitochondrial beta-oxidation of fatty acids in vitro and in vivo, resulting in uncoupling of mitochondrial oxidative phosphorylation process. This usually results in diminished cellular ATP production. The recovery of gastric mucosal barrier function through maintenance of energy metabolism results in maintenance of ATP levels, as observed in this study upon treatment with PSE. Membrane integrity altered by peroxidation is known to have a modified fatty acid composition, a disruption of permeability, a decrease in electrical resistance, and increase in flip-flopping between monolayers and inactivated cross-linked proteins. The severe depletion of arachidonic acid in ulcer induced groups was prevented upon treatment with PSE. The acid inhibitory property of the herbal extract enables the maintenance of GL activity upon treatment with PSE. The ability to prevent membrane peroxidation has been traced to the presence of active constituents in the PSE. In essence, PSE has been found to prevent mitochondrial dysfunction, provide mitochondrial cell integrity, through the maintenance of lipid bilayer by its ability to provide a hydrophobic character to the gastric mucosa, further indicating its ability to reverse the action of NSAIDs and mast cell degranulators in gastric mucosa.

  5. Silicon-on-insulator based nanopore cavity arrays for lipid membrane investigation.

    PubMed

    Buchholz, K; Tinazli, A; Kleefen, A; Dorfner, D; Pedone, D; Rant, U; Tampé, R; Abstreiter, G; Tornow, M

    2008-11-05

    We present the fabrication and characterization of nanopore microcavities for the investigation of transport processes in suspended lipid membranes. The cavities are situated below the surface of silicon-on-insulator (SOI) substrates. Single cavities and large area arrays were prepared using high resolution electron-beam lithography in combination with reactive ion etching (RIE) and wet chemical sacrificial underetching. The locally separated compartments have a circular shape and allow the enclosure of picoliter volume aqueous solutions. They are sealed at their top by a 250 nm thin Si membrane featuring pores with diameters from 2 µm down to 220 nm. The Si surface exhibits excellent smoothness and homogeneity as verified by AFM analysis. As biophysical test system we deposited lipid membranes by vesicle fusion, and demonstrated their fluid-like properties by fluorescence recovery after photobleaching. As clearly indicated by AFM measurements in aqueous buffer solution, intact lipid membranes successfully spanned the pores. The nanopore cavity arrays have potential applications in diagnostics and pharmaceutical research on transmembrane proteins.

  6. A method for detergent-free isolation of membrane proteins in their local lipid environment.

    PubMed

    Lee, Sarah C; Knowles, Tim J; Postis, Vincent L G; Jamshad, Mohammed; Parslow, Rosemary A; Lin, Yu-Pin; Goldman, Adrian; Sridhar, Pooja; Overduin, Michael; Muench, Stephen P; Dafforn, Timothy R

    2016-07-01

    Despite the great importance of membrane proteins, structural and functional studies of these proteins present major challenges. A significant hurdle is the extraction of the functional protein from its natural lipid membrane. Traditionally achieved with detergents, purification procedures can be costly and time consuming. A critical flaw with detergent approaches is the removal of the protein from the native lipid environment required to maintain functionally stable protein. This protocol describes the preparation of styrene maleic acid (SMA) co-polymer to extract membrane proteins from prokaryotic and eukaryotic expression systems. Successful isolation of membrane proteins into SMA lipid particles (SMALPs) allows the proteins to remain with native lipid, surrounded by SMA. We detail procedures for obtaining 25 g of SMA (4 d); explain the preparation of protein-containing SMALPs using membranes isolated from Escherichia coli (2 d) and control protein-free SMALPS using E. coli polar lipid extract (1-2 h); investigate SMALP protein purity by SDS-PAGE analysis and estimate protein concentration (4 h); and detail biophysical methods such as circular dichroism (CD) spectroscopy and sedimentation velocity analytical ultracentrifugation (svAUC) to undertake initial structural studies to characterize SMALPs (∼2 d). Together, these methods provide a practical tool kit for those wanting to use SMALPs to study membrane proteins.

  7. Organization of Lipids in Fiber-Cell Plasma Membranes of the Eye Lens

    PubMed Central

    Subczynski, Witold K.; Mainali, Laxman; Raguz, Marija; O’Brien, William J.

    2016-01-01

    The plasma membrane together with the cytoskeleton forms the only supramolecular structure of the matured fiber cell which accounts for mostly all fiber cell lipids. The purpose of this review is to inform researchers about the importance of the lipid bilayer portion of the lens fiber cell plasma membranes in the maintaining lens homeostasis, and thus protecting against cataract development. PMID:26988627

  8. Effect of elevated temperature on membrane lipid saturation in Antarctic notothenioid fish.

    PubMed

    Malekar, Vanita C; Morton, James D; Hider, Richard N; Cruickshank, Robert H; Hodge, Simon; Metcalf, Victoria J

    2018-01-01

    Homeoviscous adaptation (HVA) is a key cellular response by which fish protect their membranes against thermal stress. We investigated evolutionary HVA (long time scale) in Antarctic and non-Antarctic fish. Membrane lipid composition was determined for four Perciformes fish: two closely related Antarctic notothenioid species ( Trematomus bernacchii and Pagothenia borchgrevinki ); a diversified related notothenioid Antarctic icefish ( Chionodraco hamatus ); and a New Zealand species ( Notolabrus celidotus ). The membrane lipid compositions were consistent across the three Antarctic species and these were significantly different from that of the New Zealand species. Furthermore, acclimatory HVA (short time periods with seasonal changes) was investigated to determine whether stenothermal Antarctic fish, which evolved in the cold, stable environment of the Southern Ocean, have lost the acclimatory capacity to modulate their membrane saturation states, making them vulnerable to anthropogenic global warming. We compared liver membrane lipid composition in two closely related Antarctic fish species acclimated at 0 °C (control temperature), 4 °C for a period of 14 days in T. bernacchii and 28 days for P. borchgrevinki, and 6 °C for 7 days in both species. Thermal acclimation at 4 °C did not result in changed membrane saturation states in either Antarctic species. Despite this, membrane functions were not compromised, as indicated by declining serum osmolality, implying positive compensation by enhanced hypo-osmoregulation. Increasing the temperature to 6 °C did not change the membrane lipids of P. borchgrevinki. However, in T. bernacchii, thermal acclimation at 6 °C resulted in an increase of membrane saturated fatty acids and a decline in unsaturated fatty acids. This is the first study to show a homeoviscous response to higher temperatures in an Antarctic fish, although for only one of the two species examined.

  9. Hybrid and Nonhybrid Lipids Exert Common Effects on Membrane Raft Size and Morphology

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Heberle, Frederick A; Doktorova, Milka; Goh, Shih Lin

    2013-01-01

    Nanometer-scale domains in cholesterolrich model membranes emulate lipid rafts in cell plasma membranes (PMs). The physicochemical mechanisms that maintain a finite, small domain size are, however, not well understood. A special role has been postulated for chainasymmetric or hybrid lipids having a saturated sn-1 chain and an unsaturated sn-2 chain. Hybrid lipids generate nanodomains in some model membranes and are also abundant in the PM. It was proposed that they align in a preferred orientation at the boundary of ordered and disordered phases, lowering the interfacial energy and thus reducing domain size. We used small-angle neutron scattering and fluorescence techniquesmore » to detect nanoscopic and modulated liquid phase domains in a mixture composed entirely of nonhybrid lipids and cholesterol. Our results are indistinguishable from those obtained previously for mixtures containing hybrid lipids, conclusively showing that hybrid lipids are not required for the formation of nanoscopic liquid domains and strongly implying a common mechanism for the overall control of raft size and morphology. We discuss implications of these findings for theoretical descriptions of nanodomains.« less

  10. Lipid packing determines protein-membrane interactions: challenges for apolipoprotein A–I and High Density Lipoproteins

    PubMed Central

    Sánchez, Susana A.; Tricerri, M. Alejandra; Ossato, Giulia; Gratton, Enrico

    2010-01-01

    Summary Protein and protein-lipid interactions, with and within specific areas in the cell membrane, are critical in order to modulate the cell signaling events required to maintain cell functions and viability. Biological bilayers are complex, dynamic platforms, and thus in vivo observations usually need to be preceded by studies on model systems that simplify and discriminate the different factors involved in lipid-protein interactions. Fluorescence microscopy studies using giant unilamellar vesicles (GUVs) as membrane model systems provide a unique methodology to quantify protein binding, interaction and lipid solubilization in artificial bilayers. The large size of lipid domains obtainable on GUVs, together with fluorescence microscopy techniques, provides the possibility to localize and quantify molecular interactions. FCS (Fluorescence Correlation Spectroscopy) can be performed using the GUV model to extract information on mobility and concentration. Two-photon Laurdan GP (Generalized Polarization) reports on local changes in membrane water content (related to membrane fluidity) due to protein binding or lipid removal from a given lipid domain. In this review, we summarize the experimental microscopy methods used to study the interaction of human apolipoprotein A–I (apoA-I) in lipid-free and lipid-bound conformations with bilayers and natural membranes. Results described here help us to understand cholesterol homeostasis, and offer a methodological design suited to different biological systems. PMID:20347719

  11. Bacillus subtilis Lipid Extract, A Branched-Chain Fatty Acid Model Membrane

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nickels, Jonathan D.; Chatterjee, Sneha; Mostofian, Barmak

    Lipid extracts are an excellent choice of model biomembrane; however at present, there are no commercially available lipid extracts or computational models that mimic microbial membranes containing the branched-chain fatty acids found in many pathogenic and industrially relevant bacteria. Here, we advance the extract of Bacillus subtilis as a standard model for these diverse systems, providing a detailed experimental description and equilibrated atomistic bilayer model included as Supporting Information to this Letter and at (http://cmb.ornl.gov/members/cheng). The development and validation of this model represents an advance that enables more realistic simulations and experiments on bacterial membranes and reconstituted bacterial membrane proteins.

  12. Structure and dynamics of water and lipid molecules in charged anionic DMPG lipid bilayer membranes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Rønnest, A. K.; Peters, G. H.; Hansen, F. Y., E-mail: flemming@kemi.dtu.dk

    2016-04-14

    Molecular dynamics simulations have been used to investigate the influence of the valency of counter-ions on the structure of freestanding bilayer membranes of the anionic 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) lipid at 310 K and 1 atm. At this temperature, the membrane is in the fluid phase with a monovalent counter-ion and in the gel phase with a divalent counter-ion. The diffusion constant of water as a function of its depth in the membrane has been determined from mean-square-displacement calculations. Also, calculated incoherent quasielastic neutron scattering functions have been compared to experimental results and used to determine an average diffusion constant for allmore » water molecules in the system. On extrapolating the diffusion constants inferred experimentally to a temperature of 310 K, reasonable agreement with the simulations is obtained. However, the experiments do not have the sensitivity to confirm the diffusion of a small component of water bound to the lipids as found in the simulations. In addition, the orientation of the dipole moment of the water molecules has been determined as a function of their depth in the membrane. Previous indirect estimates of the electrostatic potential within phospholipid membranes imply an enormous electric field of 10{sup 8}–10{sup 9} V m{sup −1}, which is likely to have great significance in controlling the conformation of translocating membrane proteins and in the transfer of ions and molecules across the membrane. We have calculated the membrane potential for DMPG bilayers and found ∼1 V (∼2 ⋅ 10{sup 8} V m{sup −1}) when in the fluid phase with a monovalent counter-ion and ∼1.4 V (∼2.8 ⋅ 10{sup 8} V m{sup −1}) when in the gel phase with a divalent counter-ion. The number of water molecules for a fully hydrated DMPG membrane has been estimated to be 9.7 molecules per lipid in the gel phase and 17.5 molecules in the fluid phase, considerably smaller than inferred

  13. Lipids and topological rules of membrane protein assembly: balance between long and short range lipid-protein interactions.

    PubMed

    Vitrac, Heidi; Bogdanov, Mikhail; Heacock, Phil; Dowhan, William

    2011-04-29

    The N-terminal six-transmembrane domain (TM) bundle of lactose permease of Escherichia coli is uniformly inverted when assembled in membranes lacking phosphatidylethanolamine (PE). Inversion is dependent on the net charge of cytoplasmically exposed protein domains containing positive and negative residues, net charge of the membrane surface, and low hydrophobicity of TM VII acting as a molecular hinge between the two halves of lactose permease (Bogdanov, M., Xie, J., Heacock, P., and Dowhan, W. (2008) J. Cell Biol. 182, 925-935). Net neutral lipids suppress the membrane translocation potential of negatively charged amino acids, thus increasing the cytoplasmic retention potential of positively charged amino acids. Herein, TM organization of sucrose permease (CscB) and phenylalanine permease (PheP) as a function of membrane lipid composition was investigated to extend these principles to other proteins. For CscB, topological dependence on PE only becomes evident after a significant increase in the net negative charge of the cytoplasmic surface of the N-terminal TM bundle. High negative charge is required to overcome the thermodynamic block to inversion due to the high hydrophobicity of TM VII. Increasing the positive charge of the cytoplasmic surface of the N-terminal TM hairpin of PheP, which is misoriented in PE-lacking cells, favors native orientation in the absence of PE. PheP and CscB also display co-existing dual topologies dependent on changes in the charge balance between protein domains and the membrane lipids. Therefore, the topology of both permeases is dependent on PE. However, CscB topology is governed by thermodynamic balance between opposing lipid-dependent electrostatic and hydrophobic interactions.

  14. Role of Alcohols in Growth, Lipid Composition, and Membrane Fluidity of Yeasts, Bacteria, and Archaea ▿

    PubMed Central

    Huffer, Sarah; Clark, Melinda E.; Ning, Jonathan C.; Blanch, Harvey W.; Clark, Douglas S.

    2011-01-01

    Increased membrane fluidity, which causes cofactor leakage and loss of membrane potential, has long been documented as a cause for decreased cell growth during exposure to ethanol, butanol, and other alcohols. Reinforcement of the membrane with more complex lipid components is thus thought to be beneficial for the generation of more tolerant organisms. In this study, organisms with more complex membranes, namely, archaea, did not maintain high growth rates upon exposure to alcohols, indicating that more complex lipids do not necessarily fortify the membrane against the fluidizing effects of alcohols. In the presence of alcohols, shifts in lipid composition to more saturated and unbranched lipids were observed in most of the organisms tested, including archaea, yeasts, and bacteria. However, these shifts did not always result in a decrease in membrane fluidity or in greater tolerance of the organism to alcohol exposure. In general, organisms tolerating the highest concentrations of alcohols maintained membrane fluidity after alcohol exposure, whereas organisms that increased membrane rigidity were less tolerant. Altered lipid composition was a common response to alcohol exposure, with the most tolerant organisms maintaining a modestly fluid membrane. Our results demonstrate that increased membrane fluidity is not the sole cause of growth inhibition and that alcohols may also denature proteins within the membrane and cytosol, adversely affecting metabolism and decreasing cell growth. PMID:21784917

  15. Crystallizing Membrane Proteins Using Lipidic Mesophases

    PubMed Central

    Caffrey, Martin; Cherezov, Vadim

    2009-01-01

    A detailed protocol for crystallizing membrane proteins that makes use of lipidic mesophases is described. This has variously been referred to as the lipid cubic phase or in meso method. The method has been shown to be quite general in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and α-helical and β-barrel proteins. Its most recent successes are the human engineered β2-adrenergic and adenosine A2A G protein-coupled receptors. Protocols are provided for preparing and characterizing the lipidic mesophase, for reconstituting the protein into the monoolein-based mesophase, for functional assay of the protein in the mesophase, and for setting up crystallizations in manual mode. Methods for harvesting micro-crystals are also described. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour. PMID:19390528

  16. Cyclotides Insert into Lipid Bilayers to Form Membrane Pores and Destabilize the Membrane through Hydrophobic and Phosphoethanolamine-specific Interactions*

    PubMed Central

    Wang, Conan K.; Wacklin, Hanna P.; Craik, David J.

    2012-01-01

    Cyclotides are a family of plant-derived circular proteins with potential therapeutic applications arising from their remarkable stability, broad sequence diversity, and range of bioactivities. Their membrane-binding activity is believed to be a critical component of their mechanism of action. Using isothermal titration calorimetry, we studied the binding of the prototypical cyclotides kalata B1 and kalata B2 (and various mutants) to dodecylphosphocholine micelles and phosphoethanolamine-containing lipid bilayers. Although binding is predominantly an entropy-driven process, suggesting that hydrophobic forces contribute significantly to cyclotide-lipid complex formation, specific binding to the phosphoethanolamine-lipid headgroup is also required, which is evident from the enthalpic changes in the free energy of binding. In addition, using a combination of dissipative quartz crystal microbalance measurements and neutron reflectometry, we elucidated the process by which cyclotides interact with bilayer membranes. Initially, a small number of cyclotides bind to the membrane surface and then insert first into the outer membrane leaflet followed by penetration through the membrane and pore formation. At higher concentrations of cyclotides, destabilization of membranes occurs. Our results provide significant mechanistic insight into how cyclotides exert their bioactivities. PMID:23129773

  17. Microscopic Description of Thermodynamics of Lipid Membrane at Liquid-Gel Phase Transition

    NASA Astrophysics Data System (ADS)

    Kheyfets, B.; Galimzyanov, T.; Mukhin, S.

    2018-05-01

    A microscopic model of the lipid membrane is constructed that provides analytically tractable description of the physical mechanism of the first order liquid-gel phase transition. We demonstrate that liquid-gel phase transition is cooperative effect of the three major interactions: inter-lipid van der Waals attraction, steric repulsion and hydrophobic tension. The model explicitly shows that temperature-dependent inter-lipid steric repulsion switches the system from liquid to gel phase when the temperature decreases. The switching manifests itself in the increase of lateral compressibility of the lipids as the temperature decreases, making phase with smaller area more preferable below the transition temperature. The model gives qualitatively correct picture of abrupt change at transition temperature of the area per lipid, membrane thickness and volume per hydrocarbon group in the lipid chains. The calculated dependence of phase transition temperature on lipid chain length is in quantitative agreement with experimental data. Steric repulsion between the lipid molecules is shown to be the only driver of the phase transition, as van der Waals attraction and hydrophobic tension are weakly temperature dependent.

  18. Hydrodynamic coupling of particle inclusions embedded in curved lipid bilayer membranes

    DOE PAGES

    Sigurdsson, Jon Karl; Atzberger, Paul J.

    2016-06-27

    Here, we develop theory and computational methods to investigate particle inclusions embedded within curved lipid bilayer membranes. We consider the case of spherical lipid vesicles where inclusion particles are coupled through (i) intramembrane hydrodynamics, (ii) traction stresses with the external and trapped solvent fluid, and (iii) intermonolayer slip between the two leaflets of the bilayer. We investigate relative to flat membranes how the membrane curvature and topology augment hydrodynamic responses. We show how both the translational and rotational mobility of protein inclusions are effected by the membrane curvature, ratio of intramembrane viscosity to solvent viscosity, and intermonolayer slip. For generalmore » investigations of many-particle dynamics, we also discuss how our approaches can be used to treat the collective diffusion and hydrodynamic coupling within spherical bilayers.« less

  19. Hydrodynamic coupling of particle inclusions embedded in curved lipid bilayer membranes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sigurdsson, Jon Karl; Atzberger, Paul J.

    Here, we develop theory and computational methods to investigate particle inclusions embedded within curved lipid bilayer membranes. We consider the case of spherical lipid vesicles where inclusion particles are coupled through (i) intramembrane hydrodynamics, (ii) traction stresses with the external and trapped solvent fluid, and (iii) intermonolayer slip between the two leaflets of the bilayer. We investigate relative to flat membranes how the membrane curvature and topology augment hydrodynamic responses. We show how both the translational and rotational mobility of protein inclusions are effected by the membrane curvature, ratio of intramembrane viscosity to solvent viscosity, and intermonolayer slip. For generalmore » investigations of many-particle dynamics, we also discuss how our approaches can be used to treat the collective diffusion and hydrodynamic coupling within spherical bilayers.« less

  20. Complex roles of hybrid lipids in the composition, order, and size of lipid membrane domains.

    PubMed

    Hassan-Zadeh, Ebrahim; Baykal-Caglar, Eda; Alwarawrah, Mohammad; Huang, Juyang

    2014-02-11

    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.

  1. Lipid transfer proteins do their thing anchored at membrane contact sites… but what is their thing?

    PubMed

    Wong, Louise H; Levine, Tim P

    2016-04-15

    Membrane contact sites are structures where two organelles come close together to regulate flow of material and information between them. One type of inter-organelle communication is lipid exchange, which must occur for membrane maintenance and in response to environmental and cellular stimuli. Soluble lipid transfer proteins have been extensively studied, but additional families of transfer proteins have been identified that are anchored into membranes by transmembrane helices so that they cannot diffuse through the cytosol to deliver lipids. If such proteins target membrane contact sites they may be major players in lipid metabolism. The eukaryotic family of so-called Lipid transfer proteins Anchored at Membrane contact sites (LAMs) all contain both a sterol-specific lipid transfer domain in the StARkin superfamily (related to StART/Bet_v1), and one or more transmembrane helices anchoring them in the endoplasmic reticulum (ER), making them interesting subjects for study in relation to sterol metabolism. They target a variety of membrane contact sites, including newly described contacts between organelles that were already known to make contact by other means. Lam1-4p target punctate ER-plasma membrane contacts. Lam5p and Lam6p target multiple contacts including a new category: vacuolar non-NVJ cytoplasmic ER (VancE) contacts. These developments confirm previous observations on tubular lipid-binding proteins (TULIPs) that established the importance of membrane anchored proteins for lipid traffic. However, the question remaining to be solved is the most difficult of all: are LAMs transporters, or alternately are they regulators that affect traffic more indirectly? © 2016 Authors; published by Portland Press Limited.

  2. Membrane perturbation activity of cationic phenylene ethynylene oligomers and polymers: selectivity against model bacterial and mammalian membranes.

    PubMed

    Wang, Ying; Tang, Yanli; Zhou, Zhijun; Ji, Eunkyung; Lopez, Gabriel P; Chi, Eva Y; Schanze, Kirk S; Whitten, David G

    2010-08-03

    Poly(phenylene ethyneylene) (PPE)-based cationic conjugated polyelectrolytes (CPEs) and cationic phenylene ethynylene oligomers (OPEs) exhibit broad-spectrum antimicrobial activity, and their main target is believed to be the cell membrane. To understand better how these antimicrobial molecules interact with membranes, a series of PPE-based CPEs and OPEs with different side chains were studied. Large unilamellar vesicles with lipid compositions mimicking those of mammalian or bacterial membranes were used as model membranes. Among the CPEs and OPEs tested, the anionic CPE, PPE-SO(3)(2-) and the smallest cationic OPE-1 are inactive against all vesicles. Other cationic CPEs and OPEs show significant membrane perturbation ability against bacterial membrane mimics but are inactive against a mammalian cell membrane mimic with the exception of PPE-DABCO and two end-only-functionalized OPEs, which also disrupted a mammalian cell membrane mimic. The results suggest that the phospholipid composition of vesicles dominates the interaction of CPE and OPE with lipid membranes.

  3. Anesthetics mechanism on a DMPC lipid membrane model: Insights from molecular dynamics simulations.

    PubMed

    Saeedi, Marzieh; Lyubartsev, Alexander P; Jalili, Seifollah

    2017-07-01

    To provide insight into the molecular mechanisms of local anesthetic action, we have carried out an extensive investigation of two amide type local anesthetics, lidocaine and articaine in both charged and uncharged forms, interacting with DMPC lipid membrane. We have applied both standard molecular dynamics simulations and metadynamics simulations to provide a detailed description of the free energy landscape of anesthetics embedded in the lipid bilayer. The global minimum of the free energy surface (equilibrium position of anesthetics in the lipid membrane) occurred around 1nm of the bilayer center. The uncharged anesthetics show more affinity to bind to this region compared to the charged drugs. The binding free energy of uncharged lidocaine in the membrane (-30.3kJ/mol) is higher than uncharged articaine (-24.0kJ/mol), which is in good agreement with higher lipid solubility of lidocaine relative to the articaine. The octanol/water partition coefficient of uncharged drugs was also investigated using expanded ensemble simulations. In addition, complementary standard MD simulations were carried out to study the partitioning behavior of multiple anesthetics inside the lipid bilayer. The results obtained here are in line with previously reported simulations and suggest that the different forms of anesthetics induce different structural modifications in the lipid bilayer, which can provide new insights into their complex membrane translocation phenomena. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. The structural role of cholesterol in cell membranes: from condensed bilayers to lipid rafts.

    PubMed

    Krause, Martin R; Regen, Steven L

    2014-12-16

    CONSPECTUS: Defining the two-dimensional structure of cell membranes represents one of the most daunting challenges currently facing chemists, biochemists, and biophysicists. In particular, the time-averaged lateral organization of the lipids and proteins that make up these natural enclosures has yet to be established. As the classic Singer-Nicolson model of cell membranes has evolved over the past 40 years, special attention has focused on the structural role played by cholesterol, a key component that represents ca. 30% of the total lipids that are present. Despite extensive studies with model membranes, two fundamental issues have remained a mystery: (i) the mechanism by which cholesterol condenses low-melting lipids by uncoiling their acyl chains and (ii) the thermodynamics of the interaction between cholesterol and high- and low-melting lipids. The latter bears directly on one of the most popular notions in modern cell biology, that is, the lipid raft hypothesis, whereby cholesterol is thought to combine with high-melting lipids to form "lipid rafts" that float in a "sea" of low-melting lipids. In this Account, we first describe a chemical approach that we have developed in our laboratories that has allowed us to quantify the interactions between exchangeable mimics of cholesterol and low- and high-melting lipids in model membranes. In essence, this "nearest-neighbor recognition" (NNR) method involves the synthesis of dimeric forms of these lipids that contain a disulfide moiety as a linker. By means of thiolate-disulfide interchange reactions, equilibrium mixtures of dimers are then formed. These exchange reactions are initiated either by adding dithiothreitol to a liposomal dispersion to generate a small amount of thiol monomer or by including a small amount of thiol monomer in the liposomes at pH 5.0 and then raising the pH to 7.4. We then show how such NNR measurements have allowed us to distinguish between two very different mechanisms that have been

  5. ATP-induced lipid membrane reordering in the myelinated nerve fiber identified using Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Kutuzov, N. P.; Brazhe, A. R.; Yusipovich, A. I.; Maksimov, G. V.; Dracheva, O. E.; Lyaskovskiy, V. L.; Bulygin, F. V.; Rubin, A. B.

    2013-07-01

    We demonstrate a successful application of Raman spectroscopy to the problem of lipid ordering with microscopic resolution in different regions of the myelinated nerve fiber. Simultaneous collection of Raman spectra of lipids and carotenoids has enabled us to characterize membrane fluidity and the degree of lipid ordering based on intensity ratios for the 1527/1160 and 2940/2885 cm-1 bands. We show that the intensity profiles of the major Raman bands vary significantly between the three major regions of myelinated nerve fiber: internode, paranode and the node of Ranvier. Mapping Raman peak intensities over these areas suggested that the carotenoid molecules are localized in the myelin membranes of nerve cells. Paranodal membranes were sensitive to extracellular ATP. ATP solutions (7 mM) influenced the 1527/1160 and 2940/2885 cm-1 intensity ratios. Changes in both carotenoid and lipid Raman spectra were in accord and indicated an increase in lipid ordering degree and decrease in membrane fluidity under ATP administration. The collected data provide evidence for the existence of a regulatory purinergic signaling pathway in the peripheral nervous system.

  6. Randomly organized lipids and marginally stable proteins: a coupling of weak interactions to optimize membrane signaling.

    PubMed

    Rice, Anne M; Mahling, Ryan; Fealey, Michael E; Rannikko, Anika; Dunleavy, Katie; Hendrickson, Troy; Lohese, K Jean; Kruggel, Spencer; Heiling, Hillary; Harren, Daniel; Sutton, R Bryan; Pastor, John; Hinderliter, Anne

    2014-09-01

    Eukaryotic lipids in a bilayer are dominated by weak cooperative interactions. These interactions impart highly dynamic and pliable properties to the membrane. C2 domain-containing proteins in the membrane also interact weakly and cooperatively giving rise to a high degree of conformational plasticity. We propose that this feature of weak energetics and plasticity shared by lipids and C2 domain-containing proteins enhance a cell's ability to transduce information across the membrane. We explored this hypothesis using information theory to assess the information storage capacity of model and mast cell membranes, as well as differential scanning calorimetry, carboxyfluorescein release assays, and tryptophan fluorescence to assess protein and membrane stability. The distribution of lipids in mast cell membranes encoded 5.6-5.8bits of information. More information resided in the acyl chains than the head groups and in the inner leaflet of the plasma membrane than the outer leaflet. When the lipid composition and information content of model membranes were varied, the associated C2 domains underwent large changes in stability and denaturation profile. The C2 domain-containing proteins are therefore acutely sensitive to the composition and information content of their associated lipids. Together, these findings suggest that the maximum flow of signaling information through the membrane and into the cell is optimized by the cooperation of near-random distributions of membrane lipids and proteins. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Highly stable and self-repairing membrane-mimetic 2D nanomaterials assembled from lipid-like peptoids

    PubMed Central

    Jin, Haibao; Jiao, Fang; Daily, Michael D.; Chen, Yulin; Yan, Feng; Ding, Yan-Huai; Zhang, Xin; Robertson, Ellen J.; Baer, Marcel D.; Chen, Chun-Long

    2016-01-01

    An ability to develop sequence-defined synthetic polymers that both mimic lipid amphiphilicity for self-assembly of highly stable membrane-mimetic 2D nanomaterials and exhibit protein-like functionality would revolutionize the development of biomimetic membranes. Here we report the assembly of lipid-like peptoids into highly stable, crystalline, free-standing and self-repairing membrane-mimetic 2D nanomaterials through a facile crystallization process. Both experimental and molecular dynamics simulation results show that peptoids assemble into membranes through an anisotropic formation process. We further demonstrated the use of peptoid membranes as a robust platform to incorporate and pattern functional objects through large side-chain diversity and/or co-crystallization approaches. Similar to lipid membranes, peptoid membranes exhibit changes in thickness upon exposure to external stimuli; they can coat surfaces in single layers and self-repair. We anticipate that this new class of membrane-mimetic 2D nanomaterials will provide a robust matrix for development of biomimetic membranes tailored to specific applications. PMID:27402325

  8. Amphiphilic gold nanoparticles as modulators of lipid membrane fusion

    NASA Astrophysics Data System (ADS)

    Tahir, Mukarram; Alexander-Katz, Alfredo

    The fusion of lipid membranes is central to biological functions like inter-cellular transport and signaling and is coordinated by proteins of the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) superfamily. We utilize molecular dynamics simulations to demonstrate that gold nanoparticles functionalized with a mixed-monolayer of hydrophobic and hydrophilic alkanethiol ligands can act as synthetic analogues of these fusion proteins and mediate lipid membrane fusion by catalyzing the formation of a toroidal stalk between adjacent membranes and enabling the formation of a fusion pore upon influx of Ca2+ into the exterior solvent. The fusion pathway enabled by these synthetic nanostructures is analogous to the regulated fast fusion pathway observed during synaptic vesicle fusion; it therefore provides novel physical insights into this important biological process while also being relevant in a number of single-cell therapeutic applications. Computational resources from NSF XSEDE contract TG-DMR130042. Financial support from DOE CSGF fellowship DE-FG02-97ER25308.

  9. Anthrax toxin-induced rupture of artificial lipid bilayer membranes

    PubMed Central

    Nablo, Brian J.; Panchal, Rekha G.; Bavari, Sina; Nguyen, Tam L.; Gussio, Rick; Ribot, Wil; Friedlander, Art; Chabot, Donald; Reiner, Joseph E.; Robertson, Joseph W. F.; Balijepalli, Arvind; Halverson, Kelly M.; Kasianowicz, John J.

    2013-01-01

    We demonstrate experimentally that anthrax toxin complexes rupture artificial lipid bilayer membranes when isolated from the blood of infected animals. When the solution pH is temporally acidified to mimic that process in endosomes, recombinant anthrax toxin forms an irreversibly bound complex, which also destabilizes membranes. The results suggest an alternative mechanism for the translocation of anthrax toxin into the cytoplasm. PMID:23947891

  10. Anthrax toxin-induced rupture of artificial lipid bilayer membranes

    NASA Astrophysics Data System (ADS)

    Nablo, Brian J.; Panchal, Rekha G.; Bavari, Sina; Nguyen, Tam L.; Gussio, Rick; Ribot, Wil; Friedlander, Art; Chabot, Donald; Reiner, Joseph E.; Robertson, Joseph W. F.; Balijepalli, Arvind; Halverson, Kelly M.; Kasianowicz, John J.

    2013-08-01

    We demonstrate experimentally that anthrax toxin complexes rupture artificial lipid bilayer membranes when isolated from the blood of infected animals. When the solution pH is temporally acidified to mimic that process in endosomes, recombinant anthrax toxin forms an irreversibly bound complex, which also destabilizes membranes. The results suggest an alternative mechanism for the translocation of anthrax toxin into the cytoplasm.

  11. Organization of lipids in fiber-cell plasma membranes of the eye lens.

    PubMed

    Subczynski, Witold K; Mainali, Laxman; Raguz, Marija; O'Brien, William J

    2017-03-01

    The plasma membrane together with the cytoskeleton forms the only supramolecular structure of the matured fiber cell which accounts for mostly all fiber cell lipids. The purpose of this review is to inform researchers about the importance of the lipid bilayer portion of the lens fiber cell plasma membranes in the maintaining lens homeostasis, and thus protecting against cataract development. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Lipid Bilayer Vesicles with Numbers of Membrane-Linking Pores

    NASA Astrophysics Data System (ADS)

    Ken-ichirou Akashi,; Hidetake Miyata,

    2010-06-01

    We report that phospholipid membranes spontaneously formed in aqueous medium giant unilamellar vesicles (GUVs) possessing many membranous wormhole-like structures (membrane-linking pores, MLPs). By phase contract microscopy and confocal fluorescence microscopy, the structures of the MLPs, consisting of lipid bilayer, were resolvable, and a variety of vesicular shapes having many MLPs (a high genus topology) were found. These vesicles were stable but easily deformed by micromanipulation with a microneedle. We also observed the size reduction of the MLPs with the increase in membrane tension, which was qualitatively consistent with a prediction from a simple dynamical model.

  13. Distinct lipid rafts in subdomains from human placental apical syncytiotrophoblast membranes.

    PubMed

    Godoy, Valeria; Riquelme, Gloria

    2008-01-01

    We report on the characteristics of raft domains in the apical membrane from human placental syncytiotrophoblast (hSTB), an epithelium responsible for maternal-fetal exchange. Previously, we described two isolated fractions of the hSTB apical membrane: a classical microvillous membrane (MVM) and a light microvillous membrane (LMVM). Detergent-resistant microdomains (DRMs) from MVM and LMVM were prepared with Triton X-100 followed by flotation in a sucrose gradient and tested by Western and dot blot with raft markers (placental alkaline phosphatase, lipid ganglioside, annexin 2) and transferrin receptor as a nonraft marker. DRMs from both fractions showed a consistent peak for these markers, except that the DRMs from MVM had no annexin 2 mark. Cholesterol depletion modified the segregation in both groups of DRMs. Our results show two distinguishable lipid raft subsets from MVM and LMVM. Additionally, we found significant differences between MVM and LMVM in cholesterol content and in expression of cytoskeletal proteins. MVM is enriched in ezrin and beta-actin; in contrast, cholesterol and cytokeratin-7 are more abundant in LMVM. These differences may explain the distinct properties of the lipid raft subtypes.

  14. Anesthetic diffusion through lipid membranes depends on the protonation rate.

    PubMed

    Pérez-Isidoro, Rosendo; Sierra-Valdez, F J; Ruiz-Suárez, J C

    2014-12-18

    Hundreds of substances possess anesthetic action. However, despite decades of research and tests, a golden rule is required to reconcile the diverse hypothesis behind anesthesia. What makes an anesthetic to be local or general in the first place? The specific targets on proteins, the solubility in lipids, the diffusivity, potency, action time? Here we show that there could be a new player equally or even more important to disentangle the riddle: the protonation rate. Indeed, such rate modulates the diffusion speed of anesthetics into lipid membranes; low protonation rates enhance the diffusion for local anesthetics while high ones reduce it. We show also that there is a pH and membrane phase dependence on the local anesthetic diffusion across multiple lipid bilayers. Based on our findings we incorporate a new clue that may advance our understanding of the anesthetic phenomenon.

  15. Anesthetic Diffusion Through Lipid Membranes Depends on the Protonation Rate

    PubMed Central

    Pérez-Isidoro, Rosendo; Sierra-Valdez, F. J.; Ruiz-Suárez, J. C.

    2014-01-01

    Hundreds of substances possess anesthetic action. However, despite decades of research and tests, a golden rule is required to reconcile the diverse hypothesis behind anesthesia. What makes an anesthetic to be local or general in the first place? The specific targets on proteins, the solubility in lipids, the diffusivity, potency, action time? Here we show that there could be a new player equally or even more important to disentangle the riddle: the protonation rate. Indeed, such rate modulates the diffusion speed of anesthetics into lipid membranes; low protonation rates enhance the diffusion for local anesthetics while high ones reduce it. We show also that there is a pH and membrane phase dependence on the local anesthetic diffusion across multiple lipid bilayers. Based on our findings we incorporate a new clue that may advance our understanding of the anesthetic phenomenon. PMID:25520016

  16. Interaction of Aβ(1-42) amyloids with lipids promotes "off-pathway" oligomerization and membrane damage.

    PubMed

    Henry, Sarah; Vignaud, Hélène; Bobo, Claude; Decossas, Marion; Lambert, Oliver; Harte, Etienne; Alves, Isabel D; Cullin, Christophe; Lecomte, Sophie

    2015-03-09

    The toxicity of amyloids, as Aβ(1-42) involved in Alzheimer disease, is a subject under intense scrutiny. Many studies link their toxicity to the existence of various intermediate structures prior to fiber formation and/or their specific interaction with membranes. In this study we focused on the interaction between membrane models and Aβ(1-42) peptides and variants (L34T, mG37C) produced in E. coli and purified in monomeric form. We evaluated the interaction of a toxic stable oligomeric form (oG37C) with membranes as comparison. Using various biophysical techniques as fluorescence and plasmon waveguide resonance, we clearly established that the oG37C interacts strongly with membranes leading to its disruption. All the studied peptides destabilized liposomes and accumulated slowly on the membrane (rate constant 0.02 min(-1)). Only the oG37C exhibited a particular pattern of interaction, comprising two steps: the initial binding followed by membrane reorganization. Cryo-TEM was used to visualize the peptide effect on liposome morphologies. Both oG37C and mG37C lead to PG membrane fragmentation. The PG membrane promotes peptide oligomerization, implicated in membrane disruption. WT (Aβ(1-42)) also perturbs liposome organization with membrane deformation rather than disruption. For all the peptides studied, their interaction with the membranes changes their fibrillization process, with less fibers and more small aggregates being formed. These studies allowed to establish, a correlation between toxicity, fiber formation, and membrane disruption.

  17. Membrane protein crystallization in meso: lipid type-tailoring of the cubic phase.

    PubMed Central

    Cherezov, Vadim; Clogston, Jeffrey; Misquitta, Yohann; Abdel-Gawad, Wissam; Caffrey, Martin

    2002-01-01

    Hydrated monoolein forms the cubic-Pn3m mesophase that has been used for in meso crystallization of membrane proteins. The crystals have subsequently provided high-resolution structures by crystallographic means. It is possible that the hosting cubic phase created by monoolein alone, which itself is not a common membrane component, will limit the range of membrane proteins crystallizable by the in meso method. With a view to expanding the range of applicability of the method, we investigated by x-ray diffraction the degree to which the reference cubic-Pn3m phase formed by hydrated monoolein could be modified by other lipid types. These included phosphatidylcholine (PC), phosphatidylethanolamine, phosphatidylserine, cardiolipin, lyso-PC, a polyethylene glycol-lipid, 2-monoolein, oleamide, and cholesterol. The results show that all nine lipids were accommodated in the cubic phase to some extent without altering phase identity. The positional isomer, 2-monoolein, was tolerated to the highest level. The least well tolerated were the anionic lipids, followed by lyso-PC. The others were accommodated to the extent of 20-25 mol %. Beyond a certain concentration limit, the lipid additives either triggered one or a series of phase transitions or saturated the phase and separated out as crystals, as seen with oleamide and cholesterol. The series of phases observed and their order of appearance were consistent with expectations in terms of interfacial curvature changes. The changes in phase type and microstructure have been rationalized on the basis of lipid molecular shape, interfacial curvature, and chain packing energy. The data should prove useful in the rational design of cubic phase crystallization matrices with different lipid profiles that match the needs of a greater range of membrane proteins. PMID:12496106

  18. Salt Bridge Formation between the I-BAR Domain and Lipids Increases Lipid Density and Membrane Curvature.

    PubMed

    Takemura, Kazuhiro; Hanawa-Suetsugu, Kyoko; Suetsugu, Shiro; Kitao, Akio

    2017-07-28

    The BAR domain superfamily proteins sense or induce curvature in membranes. The inverse-BAR domain (I-BAR) is a BAR domain that forms a straight "zeppelin-shaped" dimer. The mechanisms by which IRSp53 I-BAR binds to and deforms a lipid membrane are investigated here by all-atom molecular dynamics simulation (MD), binding energy analysis, and the effects of mutation experiments on filopodia on HeLa cells. I-BAR adopts a curved structure when crystallized, but adopts a flatter shape in MD. The binding of I-BAR to membrane was stabilized by ~30 salt bridges, consistent with experiments showing that point mutations of the interface residues have little effect on the binding affinity whereas multiple mutations have considerable effect. Salt bridge formation increases the local density of lipids and deforms the membrane into a concave shape. In addition, the point mutations that break key intra-molecular salt bridges within I-BAR reduce the binding affinity; this was confirmed by expressing these mutants in HeLa cells and observing their effects. The results indicate that the stiffness of I-BAR is important for membrane deformation, although I-BAR does not act as a completely rigid template.

  19. AFM of the ultrastructural and mechanical properties of lipid-raft-disrupted and/or cold-treated endothelial cells.

    PubMed

    Wu, Li; Huang, Jie; Yu, Xiaoxue; Zhou, Xiaoqing; Gan, Chaoye; Li, Ming; Chen, Yong

    2014-02-01

    The nonionic detergent extraction at 4 °C and the cholesterol-depletion-induced lipid raft disruption are the two widely used experimental strategies for lipid raft research. However, the effects of raft disruption and/or cold treatment on the ultrastructural and mechanical properties of cells are still unclear. Here, we evaluated the effects of raft disruption and/or cold (4 °C) treatment on these properties of living human umbilical vein endothelial cells (HUVECs). At first, the cholesterol-depletion-induced raft disruption was visualized by confocal microscopy and atomic force microscopy (AFM) in combination with fluorescent quantum dots. Next, the cold-induced cell contraction and the formation of end-branched filopodia were observed by confocal microscopy and AFM. Then, the cell-surface ultrastructures were imaged by AFM, and the data showed that raft disruption and cold treatment induced opposite effects on cell-surface roughness (a significant decrease and a significant increase, respectively). Moreover, the cell-surface mechanical properties (stiffness and adhesion force) of raft-disrupted- and/or cold-treated HUVECs were measured by the force measurement function of AFM. We found that raft disruption and cold treatment induced parallel effects on cell stiffness (increase) or adhesion force (decrease) and that the combination of the two treatments caused dramatically strengthened effects. Finally, raft disruption was found to significantly impair cell migration as previously reported, whereas temporary cold treatment only caused a slight but nonsignificant decrease in cell migration performed at physiological temperature. Although the mechanisms for causing these results might be complicated and more in-depth studies will be needed, our data may provide important information for better understanding the effects of raft disruption or cold treatment on cells and the two strategies for lipid raft research.

  20. Changes Caused by Fruit Extracts in the Lipid Phase of Biological and Model Membranes

    PubMed Central

    Pruchnik, Hanna; Oszmiański, Jan; Sarapuk, Janusz; Kleszczyńska, Halina

    2010-01-01

    The aim of the study was to determine changes incurred by polyphenolic compounds from selected fruits in the lipid phase of the erythrocyte membrane, in liposomes formed of erythrocyte lipids and phosphatidylcholine liposomes. In particular, the effect of extracts from apple, chokeberry, and strawberry on the red blood cell morphology, on packing order in the lipid hydrophilic phase, on fluidity of the hydrophobic phase, as well as on the temperature of phase transition in DPPC liposomes was studied. In the erythrocyte population, the proportions of echinocytes increased due to incorporation of polyphenolic compounds. Fluorimetry with a laurdan probe indicated increased packing density in the hydrophilic phase of the membrane in presence of polyphenolic extracts, the highest effect being observed for the apple extract. Using the fluorescence probes DPH and TMA-DPH, no effect was noted inside the hydrophobic phase of the membrane, as the lipid bilayer fluidity was not modified. The polyphenolic extracts slightly lowered the phase transition temperature of phosphatidylcholine liposomes. The studies have shown that the phenolic compounds contained in the extracts incorporate into the outer region of the erythrocyte membrane, affecting its shape and lipid packing order, which is reflected in the increasing number of echinocytes. The compounds also penetrate the outer part of the external lipid layer of liposomes formed of natural and DPPC lipids, changing its packing order. PMID:21423329

  1. Compositional changes in lipid microdomains of air-blood barrier plasma membranes in pulmonary interstitial edema.

    PubMed

    Palestini, Paola; Calvi, Chiara; Conforti, Elena; Daffara, Rossella; Botto, Laura; Miserocchi, Giuseppe

    2003-10-01

    We evaluated in anesthetized rabbits the compositional changes of plasmalemmal lipid microdomains from lung tissue samples after inducing pulmonary interstitial edema (0.5 ml/kg for 3 h, leading to approximately 5% increase in extravascular water). Lipid microdomains (lipid rafts and caveolae) were present in the detergent-resistant fraction (DRF) obtained after discontinuous sucrose density gradient. DRF was enriched in caveolin-1, flotillin, aquaporin-1, GM1, cholesterol, sphingomyelin, and phosphatidylserine, and their contents significantly increased in interstitial edema. The higher DRF content in caveolin, flotillin, and aquaporin-1 and of the ganglioside GM1 suggests an increase both in caveolar domains and in lipid rafts, respectively. Compositional changes could be ascribed to endothelial and epithelial cells that provide most of plasma membrane surface area in the air-blood barrier. Alterations in lipid components in the plasma membrane may reflect rearrangement of floating lipid platforms within the membrane and/or lipid translocation from intracellular stores. Lipid traffic could be stimulated by the marked increase in hydraulic interstitial pressure after initial water accumulation, from approximately -10 to 5 cmH2O, due to the low compliance of the pulmonary tissue, in particular in the basement membranes and in the interfibrillar substance. Compositional changes in lipid microdomains represent a sign of cellular activation and suggest the potential role of mechanotransduction in response to developing interstitial edema.

  2. A novel biotinylated lipid raft reporter for electron microscopic imaging of plasma membrane microdomains[S

    PubMed Central

    Krager, Kimberly J.; Sarkar, Mitul; Twait, Erik C.; Lill, Nancy L.; Koland, John G.

    2012-01-01

    The submicroscopic spatial organization of cell surface receptors and plasma membrane signaling molecules is readily characterized by electron microscopy (EM) via immunogold labeling of plasma membrane sheets. Although various signaling molecules have been seen to segregate within plasma membrane microdomains, the biochemical identity of these microdomains and the factors affecting their formation are largely unknown. Lipid rafts are envisioned as submicron membrane subdomains of liquid ordered structure with differing lipid and protein constituents that define their specific varieties. To facilitate EM investigation of inner leaflet lipid rafts and the localization of membrane proteins therein, a unique genetically encoded reporter with the dually acylated raft-targeting motif of the Lck kinase was developed. This reporter, designated Lck-BAP-GFP, incorporates green fluorescent protein (GFP) and biotin acceptor peptide (BAP) modules, with the latter allowing its single-step labeling with streptavidin-gold. Lck-BAP-GFP was metabolically biotinylated in mammalian cells, distributed into low-density detergent-resistant membrane fractions, and was readily detected with avidin-based reagents. In EM images of plasma membrane sheets, the streptavidin-gold-labeled reporter was clustered in 20–50 nm microdomains, presumably representative of inner leaflet lipid rafts. The utility of the reporter was demonstrated in an investigation of the potential lipid raft localization of the epidermal growth factor receptor. PMID:22822037

  3. Menthol-induced action potentials in Conocephalum conicum as a result of unspecific interactions between menthol and the lipid phase of the plasma membrane.

    PubMed

    Kupisz, Kamila; Trebacz, Kazimierz; Gruszecki, Wiesław I

    2015-07-01

    Our previous study has shown that the liverwort Conocephalum conicum generates action potentials (APs) in response to both temperature drop and menthol, which are also activators of the TRPM8 (transient receptor potential melastatin 8) receptor in animals. Not only similarities but also differences between electrical reactions to menthol and cooling observed in the liverwort aroused our interest in the action of menthol at the molecular level. Patch-clamp investigations have shown that menthol causes a reduction of current flowing through slow vacuolar (SV) channels to 29 ± 10% of the initial value (n = 9); simultaneously, it does not influence magnitudes of currents passing through a single SV channel. This may point to an unspecific interaction between menthol and the lipid phase of the membrane. An influence of menthol on lipid organization in membranes was investigated in two-component monomolecular layers formed with menthol and dipalmitoylphosphatidylcholine (DPPC) at the argon-water interface. Analyses of the mean molecular area parameters vs the molar fraction of the menthol component have shown over-additivity (approximately 20 Å(2) ) in the region of high molar fractions of menthol. Infrared absorption spectroscopy studies have shown that menthol, most probably, induces breaking of a hydrogen bond network formed by ester carbonyl groups and water bridges in the lipid membrane and binds to the polar head group region of DPPC. We conclude that the disruption in the lipid phase of the membrane influences ion channels and/or pumps and subsequently causes generation of APs in excitable plants such as C. conicum. © 2014 Scandinavian Plant Physiology Society.

  4. Mitochondrial cardiolipin/phospholipid trafficking: the role of membrane contact site complexes and lipid transfer proteins.

    PubMed

    Schlattner, Uwe; Tokarska-Schlattner, Malgorzata; Rousseau, Denis; Boissan, Mathieu; Mannella, Carmen; Epand, Richard; Lacombe, Marie-Lise

    2014-04-01

    Historically, cellular trafficking of lipids has received much less attention than protein trafficking, mostly because its biological importance was underestimated, involved sorting and translocation mechanisms were not known, and analytical tools were limiting. This has changed during the last decade, and we discuss here some progress made in respect to mitochondria and the trafficking of phospholipids, in particular cardiolipin. Different membrane contact site or junction complexes and putative lipid transfer proteins for intra- and intermembrane lipid translocation have been described, involving mitochondrial inner and outer membrane, and the adjacent membranes of the endoplasmic reticulum. An image emerges how cardiolipin precursors, remodeling intermediates, mature cardiolipin and its oxidation products could migrate between membranes, and how this trafficking is involved in cardiolipin biosynthesis and cell signaling events. Particular emphasis in this review is given to mitochondrial nucleoside diphosphate kinase D and mitochondrial creatine kinases, which emerge to have roles in both, membrane junction formation and lipid transfer. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  5. Anomalous Surface Diffusion of Protons on Lipid Membranes

    PubMed Central

    Wolf, Maarten G.; Grubmüller, Helmut; Groenhof, Gerrit

    2014-01-01

    The cellular energy machinery depends on the presence and properties of protons at or in the vicinity of lipid membranes. To asses the energetics and mobility of a proton near a membrane, we simulated an excess proton near a solvated DMPC bilayer at 323 K, using a recently developed method to include the Grotthuss proton shuttling mechanism in classical molecular dynamics simulations. We obtained a proton surface affinity of −13.0 ± 0.5 kJ mol−1. The proton interacted strongly with both lipid headgroup and linker carbonyl oxygens. Furthermore, the surface diffusion of the proton was anomalous, with a subdiffusive regime over the first few nanoseconds, followed by a superdiffusive regime. The time- and distance dependence of the proton surface diffusion coefficient within these regimes may also resolve discrepancies between previously reported diffusion coefficients. Our simulations show that the proton anomalous surface diffusion originates from restricted diffusion in two different surface-bound states, interrupted by the occasional bulk-mediated long-range surface diffusion. Although only a DMPC membrane was considered in this work, we speculate that the restrictive character of the on-surface diffusion is highly sensitive to the specific membrane conditions, which can alter the relative contributions of the surface and bulk pathways to the overall diffusion process. Finally, we discuss the implications of our findings for the energy machinery. PMID:24988343

  6. Association of gamma-secretase with lipid rafts in post-Golgi and endosome membranes.

    PubMed

    Vetrivel, Kulandaivelu S; Cheng, Haipeng; Lin, William; Sakurai, Takashi; Li, Tong; Nukina, Nobuyuki; Wong, Philip C; Xu, Huaxi; Thinakaran, Gopal

    2004-10-22

    Alzheimer's disease-associated beta-amyloid peptides (Abeta) are generated by the sequential proteolytic processing of amyloid precursor protein (APP) by beta- and gamma-secretases. There is growing evidence that cholesterol- and sphingolipid-rich membrane microdomains are involved in regulating trafficking and processing of APP. BACE1, the major beta-secretase in neurons is a palmitoylated transmembrane protein that resides in lipid rafts. A subset of APP is subject to amyloidogenic processing by BACE1 in lipid rafts, and this process depends on the integrity of lipid rafts. Here we describe the association of all four components of the gamma-secretase complex, namely presenilin 1 (PS1)-derived fragments, mature nicastrin, APH-1, and PEN-2, with cholesterol-rich detergent insoluble membrane (DIM) domains of non-neuronal cells and neurons that fulfill the criteria of lipid rafts. In PS1(-/-)/PS2(-/-) and NCT(-/-) fibroblasts, gamma-secretase components that still remain fail to become detergent-resistant, suggesting that raft association requires gamma-secretase complex assembly. Biochemical evidence shows that subunits of the gamma-secretase complex and three TGN/endosome-resident SNAREs cofractionate in sucrose density gradients, and show similar solubility or insolubility characteristics in distinct non-ionic and zwitterionic detergents, indicative of their co-residence in membrane microdomains with similar protein-lipid composition. This notion is confirmed using magnetic immunoisolation of PS1- or syntaxin 6-positive membrane patches from a mixture of membranes with similar buoyant densities following Lubrol WX extraction or sonication, and gradient centrifugation. These findings are consistent with the localization of gamma-secretase in lipid raft microdomains of post-Golgi and endosomes, organelles previously implicated in amyloidogenic processing of APP.

  7. Interaction of nanoparticles with lipid membranes: a multiscale perspective.

    PubMed

    Montis, Costanza; Maiolo, Daniele; Alessandri, Ivano; Bergese, Paolo; Berti, Debora

    2014-06-21

    Freestanding lipid bilayers were challenged with 15 nm Au nanospheres either coated by a citrate layer or passivated by a protein corona. The effect of Au nanospheres on the bilayer morphology, permeability and fluidity presents strong differences or similarities, depending on the observation length scale, from the colloidal to the molecular domains. These findings suggest that the interaction between nanoparticles and lipid membranes should be conveniently treated as a multiscale phenomenon.

  8. Membrane Protein Mobility and Orientation Preserved in Supported Bilayers Created Directly from Cell Plasma Membrane Blebs.

    PubMed

    Richards, Mark J; Hsia, Chih-Yun; Singh, Rohit R; Haider, Huma; Kumpf, Julia; Kawate, Toshimitsu; Daniel, Susan

    2016-03-29

    Membrane protein interactions with lipids are crucial for their native biological behavior, yet traditional characterization methods are often carried out on purified protein in the absence of lipids. We present a simple method to transfer membrane proteins expressed in mammalian cells to an assay-friendly, cushioned, supported lipid bilayer platform using cell blebs as an intermediate. Cell blebs, expressing either GPI-linked yellow fluorescent proteins or neon-green fused transmembrane P2X2 receptors, were induced to rupture on glass surfaces using PEGylated lipid vesicles, which resulted in planar supported membranes with over 50% mobility for multipass transmembrane proteins and over 90% for GPI-linked proteins. Fluorescent proteins were tracked, and their diffusion in supported bilayers characterized, using single molecule tracking and moment scaling spectrum (MSS) analysis. Diffusion was characterized for individual proteins as either free or confined, revealing details of the local lipid membrane heterogeneity surrounding the protein. A particularly useful result of our bilayer formation process is the protein orientation in the supported planar bilayer. For both the GPI-linked and transmembrane proteins used here, an enzymatic assay revealed that protein orientation in the planar bilayer results in the extracellular domains facing toward the bulk, and that the dominant mode of bleb rupture is via the "parachute" mechanism. Mobility, orientation, and preservation of the native lipid environment of the proteins using cell blebs offers advantages over proteoliposome reconstitution or disrupted cell membrane preparations, which necessarily result in significant scrambling of protein orientation and typically immobilized membrane proteins in SLBs. The bleb-based bilayer platform presented here is an important step toward integrating membrane proteomic studies on chip, especially for future studies aimed at understanding fundamental effects of lipid interactions

  9. Detergent effects on enzyme activity and solubilization of lipid bilayer membranes.

    PubMed

    Womack, M D; Kendall, D A; MacDonald, R C

    1983-09-07

    Over 50 detergents were tested to establish which would be most effective in releasing proteins from membrane-bounded compartments without denaturing them. Various concentrations each of detergent were tested for two activities: (1) solubilization of egg phospholipid liposomes as measured by reduction of turbidity and (2) effect of detergent concentration on the activities of soluble, hydrolytic enzymes. Those detergents most effective in solubilizing 0.2% lipid and least detrimental to enzymes were five pure, synthetic compounds recently introduced: CHAPS, CHAPSO, Zwittergents 310 and 312, and octylglucoside. Industrial detergents were generally much inferior, insofar as they solubilized membranes inefficiently and/or inactivated certain hydrolytic enzymes readily. The five detergents were characterized by (a) an unusually high critical micelle concentration and (b) a preference for forming mixed micelles with lipids instead of forming pure micelles, as indicated by an ability to solubilize lipid at concentrations of detergent significantly below the critical micelle concentration. This characteristic permits solubilization of high concentrations of membrane below the critical micelle concentration of the detergent so that protein denaturation is minimized. A generally applicable guideline that emerged from this study is that detergents should be used at approximately their critical micelle concentration which should not be exceeded by the concentration of membrane. Similar considerations should apply to the use of detergents in purifying and reconstituting intrinsic membrane proteins.

  10. Interaction of hematoporphyrin with lipid membranes.

    PubMed

    Stępniewski, Michał; Kepczynski, Mariusz; Jamróz, Dorota; Nowakowska, Maria; Rissanen, Sami; Vattulainen, Ilpo; Róg, Tomasz

    2012-04-26

    Natural or synthetic porphyrins are being used as photosensitizers in photodiagnosis (PD) and photodynamic therapy (PDT) of malignancies and some other diseases. Understanding the interactions between porphyrins and cell membranes is therefore important to rationalize the uptake of photosensitizers and their passive transport through cell membranes. In this study, we consider the properties of hematoporphyrin (Hp), a well-known photosensitizer for PD and PDT, in the presence of a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer that we use as a model system for protein-free cell membranes. For this purpose, we employed 200 ns atomic-scale molecular dynamics (MD) simulations for five systems containing the neutral (Hp(0)) or the dianionic form (Hp(2-)) of Hp and the POPC bilayer. MD simulations allowed one to estimate the position, orientation, and dynamics of Hp molecules inside the membrane. The dye molecules were found to reside in the phospholipid headgroup area close to the carbonyl groups of the POPC acyl chains. Their orientations were dependent on the protonation state of two propionic groups. Hp(2-) was found to have a lower affinity to enter the membrane than the neutral form. The dianions, being in the aqueous phase, formed stable dimers with a strictly determined geometry. Our results fully supported the experimental data and provide a more detailed molecular-level description of the interactions of photosensitizers with lipid membranes.

  11. Mixing, diffusion, and percolation in binary supported membranes containing mixtures of lipids and amphiphilic block copolymers.

    PubMed

    Gettel, Douglas L; Sanborn, Jeremy; Patel, Mira A; de Hoog, Hans-Peter; Liedberg, Bo; Nallani, Madhavan; Parikh, Atul N

    2014-07-23

    Substrate-mediated fusion of small polymersomes, derived from mixtures of lipids and amphiphilic block copolymers, produces hybrid, supported planar bilayers at hydrophilic surfaces, monolayers at hydrophobic surfaces, and binary monolayer/bilayer patterns at amphiphilic surfaces, directly responding to local measures of (and variations in) surface free energy. Despite the large thickness mismatch in their hydrophobic cores, the hybrid membranes do not exhibit microscopic phase separation, reflecting irreversible adsorption and limited lateral reorganization of the polymer component. With increasing fluid-phase lipid fraction, these hybrid, supported membranes undergo a fluidity transition, producing a fully percolating fluid lipid phase beyond a critical area fraction, which matches the percolation threshold for the immobile point obstacles. This then suggests that polymer-lipid hybrid membranes might be useful models for studying obstructed diffusion, such as occurs in lipid membranes containing proteins.

  12. A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli*

    PubMed Central

    Urfer, Matthias; Bogdanovic, Jasmina; Lo Monte, Fabio; Moehle, Kerstin; Zerbe, Katja; Omasits, Ulrich; Ahrens, Christian H.; Pessi, Gabriella; Eberl, Leo; Robinson, John A.

    2016-01-01

    Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel β-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected β-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many β-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of β-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target β-barrel proteins and the integrity of the Gram-negative OM. PMID:26627837

  13. Oral mucosal lipids are antibacterial against Porphyromonas gingivalis, induce ultrastructural damage, and alter bacterial lipid and protein compositions.

    PubMed

    Fischer, Carol L; Walters, Katherine S; Drake, David R; Dawson, Deborah V; Blanchette, Derek R; Brogden, Kim A; Wertz, Philip W

    2013-09-01

    Oral mucosal and salivary lipids exhibit potent antimicrobial activity for a variety of Gram-positive and Gram-negative bacteria; however, little is known about their spectrum of antimicrobial activity or mechanisms of action against oral bacteria. In this study, we examine the activity of two fatty acids and three sphingoid bases against Porphyromonas gingivalis, an important colonizer of the oral cavity implicated in periodontitis. Minimal inhibitory concentrations, minimal bactericidal concentrations, and kill kinetics revealed variable, but potent, activity of oral mucosal and salivary lipids against P. gingivalis, indicating that lipid structure may be an important determinant in lipid mechanisms of activity against bacteria, although specific components of bacterial membranes are also likely important. Electron micrographs showed ultrastructural damage induced by sapienic acid and phytosphingosine and confirmed disruption of the bacterial plasma membrane. This information, coupled with the association of treatment lipids with P. gingivalis lipids revealed via thin layer chromatography, suggests that the plasma membrane is a likely target of lipid antibacterial activity. Utilizing a combination of two-dimensional in-gel electrophoresis and Western blot followed by mass spectroscopy and N-terminus degradation sequencing we also show that treatment with sapienic acid induces upregulation of a set of proteins comprising a unique P. gingivalis stress response, including proteins important in fatty acid biosynthesis, metabolism and energy production, protein processing, cell adhesion and virulence. Prophylactic or therapeutic lipid treatments may be beneficial for intervention of infection by supplementing the natural immune function of endogenous lipids on mucosal surfaces.

  14. Lipid membranes and single ion channel recording for the advanced physics laboratory

    NASA Astrophysics Data System (ADS)

    Klapper, Yvonne; Nienhaus, Karin; Röcker, Carlheinz; Ulrich Nienhaus, G.

    2014-05-01

    We present an easy-to-handle, low-cost, and reliable setup to study various physical phenomena on a nanometer-thin lipid bilayer using the so-called black lipid membrane technique. The apparatus allows us to precisely measure optical and electrical properties of free-standing lipid membranes, to study the formation of single ion channels, and to gain detailed information on the ion conduction properties of these channels using statistical physics and autocorrelation analysis. The experiments are well suited as part of an advanced physics or biophysics laboratory course; they interconnect physics, chemistry, and biology and will be appealing to students of the natural sciences who are interested in quantitative experimentation.

  15. Clomipramine counteracts lipid raft disturbance due to short-term muscle disuse.

    PubMed

    Bryndina, Irina G; Shalagina, Maria N; Sekunov, Alexey V; Zefirov, Andrei L; Petrov, Alexey M

    2018-01-18

    Disuse-induced skeletal muscle dysfunction is a serious consequence of long-term spaceflight, numerous diseases and conditions for which treatment possibilities are still strictly limited. We have previously shown that acute hindlimb suspension (HS)-mediated disuse disrupts membrane lipid rafts in the unloaded muscle. Here, we investigated whether pretreatment of rats with the inhibitor of acid sphingomyelinase, clomipramine (1.25mg/g/day, intramuscularly, for 5days before HS), is able to hinder the loss in lipid raft integrity in response to 12h of HS. Clomipramine pretreatment significantly counteracted the decrease in labeling of the plasma membranes with lipid raft markers (fluorescent cholera toxin B subunit and bodipy-GM1-ganglioside) specifically in the junctional regions of the suspended soleus muscle. This was associated with: a) enhancing raft disrupting potential of exogenous sphingomyelinase in the junctional membranes; b) prevention of both ceramide accumulation and cholesterol loss; c) prevention of decline in nicotinic acetylcholine receptor labeling in the unloaded muscle. Our data suggest that sphingomyelinase-mediated raft disturbance serves as one of the earlier events in HS effects. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Erythrocyte membrane model with explicit description of the lipid bilayer and the spectrin network.

    PubMed

    Li, He; Lykotrafitis, George

    2014-08-05

    The membrane of the red blood cell (RBC) consists of spectrin tetramers connected at actin junctional complexes, forming a two-dimensional (2D) sixfold triangular network anchored to the lipid bilayer. Better understanding of the erythrocyte mechanics in hereditary blood disorders such as spherocytosis, elliptocytosis, and especially, sickle cell disease requires the development of a detailed membrane model. In this study, we introduce a mesoscale implicit-solvent coarse-grained molecular dynamics (CGMD) model of the erythrocyte membrane that explicitly describes the phospholipid bilayer and the cytoskeleton, by extending a previously developed two-component RBC membrane model. We show that the proposed model represents RBC membrane with the appropriate bending stiffness and shear modulus. The timescale and self-consistency of the model are established by comparing our results with experimentally measured viscosity and thermal fluctuations of the RBC membrane. Furthermore, we measure the pressure exerted by the cytoskeleton on the lipid bilayer. We find that defects at the anchoring points of the cytoskeleton to the lipid bilayer (as in spherocytes) cause a reduction in the pressure compared with an intact membrane, whereas defects in the dimer-dimer association of a spectrin filament (as in elliptocytes) cause an even larger decrease in the pressure. We conjecture that this finding may explain why the experimentally measured diffusion coefficients of band-3 proteins are higher in elliptocytes than in spherocytes, and higher than in normal RBCs. Finally, we study the effects that possible attractive forces between the spectrin filaments and the lipid bilayer have on the pressure applied on the lipid bilayer by the filaments. We discover that the attractive forces cause an increase in the pressure as they diminish the effect of membrane protein defects. As this finding contradicts with experimental results, we conclude that the attractive forces are moderate and do

  17. Erythrocyte Membrane Model with Explicit Description of the Lipid Bilayer and the Spectrin Network

    PubMed Central

    Li, He; Lykotrafitis, George

    2014-01-01

    The membrane of the red blood cell (RBC) consists of spectrin tetramers connected at actin junctional complexes, forming a two-dimensional (2D) sixfold triangular network anchored to the lipid bilayer. Better understanding of the erythrocyte mechanics in hereditary blood disorders such as spherocytosis, elliptocytosis, and especially, sickle cell disease requires the development of a detailed membrane model. In this study, we introduce a mesoscale implicit-solvent coarse-grained molecular dynamics (CGMD) model of the erythrocyte membrane that explicitly describes the phospholipid bilayer and the cytoskeleton, by extending a previously developed two-component RBC membrane model. We show that the proposed model represents RBC membrane with the appropriate bending stiffness and shear modulus. The timescale and self-consistency of the model are established by comparing our results with experimentally measured viscosity and thermal fluctuations of the RBC membrane. Furthermore, we measure the pressure exerted by the cytoskeleton on the lipid bilayer. We find that defects at the anchoring points of the cytoskeleton to the lipid bilayer (as in spherocytes) cause a reduction in the pressure compared with an intact membrane, whereas defects in the dimer-dimer association of a spectrin filament (as in elliptocytes) cause an even larger decrease in the pressure. We conjecture that this finding may explain why the experimentally measured diffusion coefficients of band-3 proteins are higher in elliptocytes than in spherocytes, and higher than in normal RBCs. Finally, we study the effects that possible attractive forces between the spectrin filaments and the lipid bilayer have on the pressure applied on the lipid bilayer by the filaments. We discover that the attractive forces cause an increase in the pressure as they diminish the effect of membrane protein defects. As this finding contradicts with experimental results, we conclude that the attractive forces are moderate and do

  18. Structure and electrical properties of DNA nanotubes embedded in lipid bilayer membranes

    PubMed Central

    Maiti, Prabal K

    2018-01-01

    Abstract Engineering the synthetic nanopores through lipid bilayer membrane to access the interior of a cell is a long persisting challenge in biotechnology. Here, we demonstrate the stability and dynamics of a tile-based 6-helix DNA nanotube (DNT) embedded in POPC lipid bilayer using the analysis of 0.2 μs long equilibrium MD simulation trajectories. We observe that the head groups of the lipid molecules close to the lumen cooperatively tilt towards the hydrophilic sugar-phosphate backbone of DNA and form a toroidal structure around the patch of DNT protruding in the membrane. Further, we explore the effect of ionic concentrations to the in-solution structure and stability of the lipid-DNT complex. Transmembrane ionic current measurements for the constant electric field MD simulation provide the I-V characteristics of the water filled DNT lumen in lipid membrane. With increasing salt concentrations, the measured values of transmembrane ionic conductance of the porous DNT lumen vary from 4.3 to 20.6 nS. Simulations of the DNTs with ssDNA and dsDNA overhangs at the mouth of the pore show gating effect with remarkable difference in the transmembrane ionic conductivities for open and close state nanopores. PMID:29136243

  19. The mystery of membrane organization: composition, regulation and physiological relevance of lipid rafts

    PubMed Central

    Sezgin, Erdinc; Levental, Ilya; Mayor, Satyajit; Eggeling, Christian

    2017-01-01

    Cellular plasma membranes are laterally heterogeneous, featuring a variety of distinct subcompartments that differ in their biophysical properties and composition. A large body of research has focused on understanding the basis for this heterogeneity and its physiological relevance. The membrane raft hypothesis formalized a physicochemical principle for a subtype of such lateral membrane heterogeneity, wherein the preferential associations of cholesterol and saturated lipids drives the formation of relatively packed (ordered) membrane domains that selectively recruit certain lipids and proteins. Recent years have yielded new insights into this concept and its in vivo relevance, primarily owing to the development of biochemical and biophysical technologies. PMID:28356571

  20. Recent Developments in Fluorescence Correlation Spectroscopy for Diffusion Measurements in Planar Lipid Membranes

    PubMed Central

    Macháň, Radek; Hof, Martin

    2010-01-01

    Fluorescence correlation spectroscopy (FCS) is a single molecule technique used mainly for determination of mobility and local concentration of molecules. This review describes the specific problems of FCS in planar systems and reviews the state of the art experimental approaches such as 2-focus, Z-scan or scanning FCS, which overcome most of the artefacts and limitations of standard FCS. We focus on diffusion measurements of lipids and proteins in planar lipid membranes and review the contributions of FCS to elucidating membrane dynamics and the factors influencing it, such as membrane composition, ionic strength, presence of membrane proteins or frictional coupling with solid support. PMID:20386647

  1. Lipid Gymnastics: Tethers and Fingers in membrane

    NASA Astrophysics Data System (ADS)

    Tayebi, Lobat; Miller, Gregory; Parikh, Atul

    2009-03-01

    A significant body of evidence now links local mesoscopic structure (e.g., shape and composition) of the cell membrane with its function; the mechanisms by which cellular membranes adopt the specific shapes remain poorly understood. Among all the different structures adopted by cellular membranes, the tubular shape is one of the most surprising one. While their formation is typically attributed to the reorganization of membrane cytoskeleton, many exceptions exist. We report the instantaneous formation of tubular membrane mesophases following the hydration under specific thermal conditions. The shapes emerge in a bimodal way where we have two distinct diameter ranges for tubes, ˜20μm and ˜1μm, namely fat fingers and narrow tethers. We study the roughening of hydrated drops of 3 lipids in 3 different spontaneous curvatures at various temp. and ionic strength to figure out the dominant effect in selection of tethers and fingers. Dynamics of the tubes are of particular interest where we observe four distinct steps of birth, coiling, uncoiling and retraction with different lifetime on different thermal condition. These dynamics appear to reflect interplay between membrane elasticity, surface adhesion, and thermal or hydrodynamic gradient.

  2. Two-Phase Contiguous Supported Lipid Bilayer Model for Membrane Rafts via Polymer Blotting and Stenciling.

    PubMed

    Richards, Mark J; Daniel, Susan

    2017-02-07

    The supported lipid bilayer has been portrayed as a useful model of the cell membrane compatible with many biophysical tools and techniques that demonstrate its appeal in learning about the basic features of the plasma membrane. However, some of its potential has yet to be realized, particularly in the area of bilayer patterning and phase/composition heterogeneity. In this work, we generate contiguous bilayer patterns as a model system that captures the general features of membrane domains and lipid rafts. Micropatterned polymer templates of two types are investigated for generating patterned bilayer formation: polymer blotting and polymer lift-off stenciling. While these approaches have been used previously to create bilayer arrays by corralling bilayers patches with various types of boundaries impenetrable to bilayer diffusion, unique to the methods presented here, there are no physical barriers to diffusion. In this work, interfaces between contiguous lipid phases define the pattern shapes, with continuity between them allowing transfer of membrane-bound biomolecules between the phases. We examine effectors of membrane domain stability including temperature and cholesterol content to investigate domain dynamics. Contiguous patterning of supported bilayers as a model of lipid rafts expands the application of the SLB to an area with current appeal and brings with it a useful toolset for characterization and analysis. These combined tools should be helpful to researchers investigating lipid raft dynamics and function and biomolecule partitioning studies. Additionally, this patterning technique may be useful for applications such as bioseparations that exploit differences in lipid phase partitioning or creation of membranes that bind species like viruses preferentially at lipid phase boundaries, to name a few.

  3. Highly stabilized, polymer-lipid membranes prepared on silica microparticles as stationary phases for capillary chromatography

    PubMed Central

    Gallagher, Elyssia S.; Adem, Seid M.; Baker, Christopher A.; Ratnayaka, Saliya N.; Jones, Ian W.; Hall, Henry K.; Saavedra, S. Scott; Aspinwall, Craig A.

    2015-01-01

    The ability to rapidly screen complex libraries of pharmacological modulators is paramount to modern drug discovery efforts. This task is particularly challenging for agents that interact with lipid bilayers or membrane proteins due to the limited chemical, physical, and temporal stability of conventional lipid-based chromatographic stationary phases. Here, we describe the preparation and characterization of a novel stationary phase material composed of highly stable, polymeric-phospholipid bilayers self-assembled onto silica microparticles. Polymer lipid membranes were prepared by photochemical or redox initiated polymerization of 1,2-bis[10-(2′,4′-hexadieoyloxy)decanoyl]-sn-glycero-2-phosphocholine (bis-SorbPC), a synthetic, polymerizable lipid. The resulting polymerized bis-SorbPC (poly(bis-SorbPC)) stationary phases exhibited enhanced stability compared to particles coated with 1,2-dioleoyl-sn-glycero-phosphocholine (unpolymerized) phospholipid bilayers when exposed to chemical (50mM triton X-100 or 50% acetonitrile) and physical (15 min sonication) insults after 30 days of storage. Further, poly(bis-SorbPC)-coated particles survived slurry packing into fused silica capillaries, compared to unpolymerized lipid membranes, where the lipid bilayer was destroyed during packing. Frontal chromatographic analyses of the lipophilic small molecules acetylsalicylic acid, benzoic acid, and salicylic acid showed > 44% increase in retention times (P < 0.0001) for all analytes on poly(bis-SorbPC)-functionalized stationary phase compared to bare silica microspheres, suggesting a lipophilic retention mechanism. Phospholipid membrane-functionalized stationary phases that withstand the chemical and physical rigors of capillary LC conditions can substantially increase the efficacy of lipid membrane affinity chromatography, and represents a key advance towards the development of robust membrane protein-functionalized chromatographic stationary phases. PMID:25670414

  4. Protein Solvation in Membranes and at Water-Membrane Interfaces

    NASA Technical Reports Server (NTRS)

    Pohorille, Andrew; Chipot, Christophe; Wilson, Michael A.

    2002-01-01

    Different salvation properties of water and membranes mediate a host of biologically important processes, such as folding, insertion into a lipid bilayer, associations and functions of membrane proteins. These processes will be discussed in several examples involving synthetic and natural peptides. In particular, a mechanism by which a helical peptide becomes inserted into a model membrane will be described. Further, the molecular mechanism of recognition and association of protein helical segments in membranes will be discussed. These processes are crucial for proper functioning of a cell. A membrane-spanning domain of glycophorin A, which exists as a helical dimer, serves as the model system. For this system, the free energy of dissociation of the helices is being determined for both the wild type and a mutant, in which dimerization is disrupted.

  5. Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Alters Mitochondrial Membrane Lipids

    PubMed Central

    Sandra, Ferry; Esposti, Mauro Degli; Ndebele, Kenneth; Gona, Philimon; Knight, David; Rosenquist, Magnus; Khosravi-Far, Roya

    2010-01-01

    Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) has been shown to have selective antitumor activity. TRAIL induces ubiquitous pathways of cell death in which caspase activation is mediated either directly or via the release of apoptogenic factors from mitochondria; however, the precise components of the mitochondrial signaling pathway have not been well defined. Notably, mitochondria constitute an important target in overcoming resistance to TRAIL in many types of tumors. Bid is considered to be fundamental in engaging mitochondria during death receptor–mediated apoptosis, but this action is dependent on mitochondrial lipids. Here, we report that TRAIL signaling induces an alteration in mitochondrial membrane lipids, particularly cardiolipin. This occurs independently of caspase activation and primes mitochondrial membranes to the proapoptotic action of Bid. We unveil a link between TRAIL signaling and alteration of membrane lipid homeostasis that occurs in parallel to apical caspase activation but does not take over the mode of cell death because of the concurrent activation of caspase-8. In particular, TRAIL-induced alteration of mitochondrial lipids follows an imbalance in the cellular homeostasis of phosphatidylcholine, which results in an elevation in diacylglycerol (DAG). Elevated DAG in turn activates the δ isoform of phospholipid-dependent serine/threonine protein kinase C, which then accelerates the cleavage of caspase-8. We also show that preservation of phosphatidylcholine homeostasis by inhibition of lipid-degrading enzymes almost completely impedes the activation of pro-caspase-9 while scarcely changing the activation of caspase-8. PMID:16166305

  6. Interaction of anions with lipid cubic phase membranes, an electrochemical impedance study.

    PubMed

    Meynaq, Mohammad Yaser Khani; Lindholm-Sethson, Britta; Tesfalidet, Solomon

    2018-05-29

    Electrochemical impedance spectroscopy is useful to monitor anionic interactions with a Lipid Cubic Phase, as previously demonstrated for cationic interaction (Khani Meynaq et al., 2016). It was expected that the smaller hydrophilic anions, acetate and chloride, would interact differently than the large tryptophan anion with its hydrophobic tail. The impedance measurements enabled estimation of resistances and capacitances of a freestanding lipid cubic phase membrane at exposure to 4 and 40 mM solutions of NaCl, NaOAc and NaTrp. Small-angle X-ray scattering was used for cubic phase identification and to track structural changes within the cubic phase when exposed to the different electrolytes. The membrane resistance increases at exposure to the electrolytes in the order Cl -  < OAc -  < Trp - . The membrane resistance decreases with time at exposure to the hydrophilic anions and increases with time at Trp - exposure. The membrane capacitances were lower for NaTrp compared to NaCl and NaOAc at the corresponding concentrations which is consistent with the results from SAXRD. It is concluded that Trp - ions do not enter the aqueous channels of the cubic phase but are strongly adsorbed to the membrane/electrolyte interface leading to large alteration of the lipid phase structure and a high membrane resistance. Copyright © 2018 Elsevier Inc. All rights reserved.

  7. Impact of two different saponins on the organization of model lipid membranes.

    PubMed

    Korchowiec, Beata; Gorczyca, Marcelina; Wojszko, Kamila; Janikowska, Maria; Henry, Max; Rogalska, Ewa

    2015-10-01

    Saponins, naturally occurring plant compounds are known for their biological and pharmacological activity. This activity is strongly related to the amphiphilic character of saponins that allows them to aggregate in aqueous solution and interact with membrane components. In this work, Langmuir monolayer techniques combined with polarization modulation infrared reflection-absorption spectroscopy (PM-IRRAS) and Brewster angle microscopy were used to study the interaction of selected saponins with lipid model membranes. Two structurally different saponins were used: digitonin and a commercial Merck Saponin. Membranes of different composition, namely, cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine or 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) were formed at the air/water and air/saponin solution interfaces. The saponin-lipid interaction was characterized by changes in surface pressure, surface potential, surface morphology and PM-IRRAS signal. Both saponins interact with model membranes and change the physical state of membranes by perturbing the lipid acyl chain orientation. The changes in membrane fluidity were more significant upon the interaction with Merck Saponin. A higher affinity of saponins for cholesterol than phosphatidylglycerols was observed. Moreover, our results indicate that digitonin interacts strongly with cholesterol and solubilize the cholesterol monolayer at higher surface pressures. It was shown, that digitonin easily penetrate to the cholesterol monolayer and forms a hydrogen bond with the hydroxyl groups. These findings might be useful in further understanding of the saponin action at the membrane interface and of the mechanism of membrane lysis. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Association of γ-Secretase with Lipid Rafts in Post-Golgi and Endosome Membranes*

    PubMed Central

    Vetrivel, Kulandaivelu S.; Cheng, Haipeng; Lin, William; Sakurai, Takashi; Li, Tong; Nukina, Nobuyuki; Wong, Philip C.; Xu, Huaxi; Thinakaran, Gopal

    2005-01-01

    Alzheimer’s disease-associated β-amyloid peptides (Aβ) are generated by the sequential proteolytic processing of amyloid precursor protein (APP) by β- and γ-secretases. There is growing evidence that cholesterol- and sphingolipid-rich membrane microdomains are involved in regulating trafficking and processing of APP. BACE1, the major γ-secretase in neurons is a palmi-toylated transmembrane protein that resides in lipid rafts. A subset of APP is subject to amyloidogenic processing by BACE1 in lipid rafts, and this process depends on the integrity of lipid rafts. Here we describe the association of all four components of the γ-secretase complex, namely presenilin 1 (PS1)-derived fragments, mature nicastrin, APH-1, and PEN-2, with cholesterol-rich detergent insoluble membrane (DIM) domains of non-neuronal cells and neurons that fulfill the criteria of lipid rafts. In PS1−/−/PS2−/− and NCT−/− fibroblasts, γ-secretase components that still remain fail to become detergent-resistant, suggesting that raft association requires γ-secretase complex assembly. Biochemical evidence shows that subunits of the γ-secretase complex and three TGN/endosome-resident SNAREs cofractionate in sucrose density gradients, and show similar solubility or insolubility characteristics in distinct non-ionic and zwitterionic detergents, indicative of their co-residence in membrane microdomains with similar protein-lipid composition. This notion is confirmed using magnetic immunoisolation of PS1- or syntaxin 6-positive membrane patches from a mixture of membranes with similar buoyant densities following Lubrol WX extraction or sonication, and gradient centrifugation. These findings are consistent with the localization of γ-secretase in lipid raft microdomains of post-Golgi and endosomes, organelles previously implicated in amyloidogenic processing of APP. PMID:15322084

  9. Saturation recovery EPR spin-labeling method for quantification of lipids in biological membrane domains.

    PubMed

    Mainali, Laxman; Camenisch, Theodore G; Hyde, James S; Subczynski, Witold K

    2017-12-01

    The presence of integral membrane proteins induces the formation of distinct domains in the lipid bilayer portion of biological membranes. Qualitative application of both continuous wave (CW) and saturation recovery (SR) electron paramagnetic resonance (EPR) spin-labeling methods allowed discrimination of the bulk, boundary, and trapped lipid domains. A recently developed method, which is based on the CW EPR spectra of phospholipid (PL) and cholesterol (Chol) analog spin labels, allows evaluation of the relative amount of PLs (% of total PLs) in the boundary plus trapped lipid domain and the relative amount of Chol (% of total Chol) in the trapped lipid domain [ M. Raguz, L. Mainali, W. J. O'Brien, and W. K. Subczynski (2015), Exp. Eye Res., 140:179-186 ]. Here, a new method is presented that, based on SR EPR spin-labeling, allows quantitative evaluation of the relative amounts of PLs and Chol in the trapped lipid domain of intact membranes. This new method complements the existing one, allowing acquisition of more detailed information about the distribution of lipids between domains in intact membranes. The methodological transition of the SR EPR spin-labeling approach from qualitative to quantitative is demonstrated. The abilities of this method are illustrated for intact cortical and nuclear fiber cell plasma membranes from porcine eye lenses. Statistical analysis (Student's t -test) of the data allowed determination of the separations of mean values above which differences can be treated as statistically significant ( P ≤ 0.05) and can be attributed to sources other than preparation/technique.

  10. Phytosphingosine, sphingosine and dihydrosphingosine ceramides in model skin lipid membranes: permeability and biophysics.

    PubMed

    Školová, Barbora; Kováčik, Andrej; Tesař, Ondřej; Opálka, Lukáš; Vávrová, Kateřina

    2017-05-01

    Ceramides based on phytosphingosine, sphingosine and dihydrosphingosine are essential constituents of the skin lipid barrier that protects the body from excessive water loss. The roles of the individual ceramide subclasses in regulating skin permeability and the reasons for C4-hydroxylation of these sphingolipids are not completely understood. We investigated the chain length-dependent effects of dihydroceramides, sphingosine ceramides (with C4-unsaturation) and phytoceramides (with C4-hydroxyl) on the permeability, lipid organization and thermotropic behavior of model stratum corneum lipid membranes composed of ceramide/lignoceric acid/cholesterol/cholesteryl sulfate. Phytoceramides with very long C24 acyl chains increased the permeability of the model lipid membranes compared to dihydroceramides or sphingosine ceramides with the same chain lengths. Either unsaturation or C4-hydroxylation of dihydroceramides induced chain length-dependent increases in membrane permeability. Infrared spectroscopy showed that C4-hydroxylation of the sphingoid base decreased the relative ratio of orthorhombic chain packing in the membrane and lowered the miscibility of C24 phytoceramide with lignoceric acid. The phase separation in phytoceramide membranes was confirmed by X-ray diffraction. In contrast, phytoceramides formed strong hydrogen bonds and highly thermostable domains. Thus, the large heterogeneity in ceramide structures and in their aggregation mechanisms may confer resistance towards the heterogeneous external stressors that are constantly faced by the skin barrier. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Lipid membrane-mediated attraction between curvature inducing objects

    NASA Astrophysics Data System (ADS)

    van der Wel, Casper; Vahid, Afshin; Šarić, Anđela; Idema, Timon; Heinrich, Doris; Kraft, Daniela J.

    2016-09-01

    The interplay of membrane proteins is vital for many biological processes, such as cellular transport, cell division, and signal transduction between nerve cells. Theoretical considerations have led to the idea that the membrane itself mediates protein self-organization in these processes through minimization of membrane curvature energy. Here, we present a combined experimental and numerical study in which we quantify these interactions directly for the first time. In our experimental model system we control the deformation of a lipid membrane by adhering colloidal particles. Using confocal microscopy, we establish that these membrane deformations cause an attractive interaction force leading to reversible binding. The attraction extends over 2.5 times the particle diameter and has a strength of three times the thermal energy (-3.3 kBT). Coarse-grained Monte-Carlo simulations of the system are in excellent agreement with the experimental results and prove that the measured interaction is independent of length scale. Our combined experimental and numerical results reveal membrane curvature as a common physical origin for interactions between any membrane-deforming objects, from nanometre-sized proteins to micrometre-sized particles.

  12. Lamellar Biogels: Fluid-Membrane Based Hydrogels Containing Polymer-Lipids

    NASA Astrophysics Data System (ADS)

    Warriner, Heidi E.; Davidson, P.; Slack, N. L.; Idziak, S. H. J.; Schmidt, H. W.; Safinya, C. R.

    1996-03-01

    A new class of lamellar biogels containing low molecular weight (MW 5181, 2053 and 576 g/mole) polyethylene glycol-surfactants is described (H. Warriner et. al., Science, (in press)). The gels were formed in 7 different systems using two types of polymer-surfactants: (i) polymer-lipids based on the lipid DMPE covalently attached to the different MW of PEG (ii) polymer-surfactants of the two largest PEG MW covalently attached to double-tailed phenyl surfactants with 14 or 18 carbon tails. Unlike isotropic hydrogels of polymer networks, these membrane-based liquid crystalline biogels, labeled L_α,g, form through the addition of water to a liquid-like L_α phase. The signature of the L_α,g regime in these systems is a dramatic increase in layer-dislocation defects, stabilized by aggregation of the PEG-surfactants to the high curvature defect regions. These regions connect and "entangle" the membranes, causing gelation. A simple model describing these phenomena is that the inclusion of the polymer-surfactants in lamellar membranes softens the free energy of high curvature line-defects, leading to proliferation and gelation.

  13. GRIFFIN: A versatile methodology for optimization of protein-lipid interfaces for membrane protein simulations

    PubMed Central

    Staritzbichler, René; Anselmi, Claudio; Forrest, Lucy R.; Faraldo-Gómez, José D.

    2014-01-01

    As new atomic structures of membrane proteins are resolved, they reveal increasingly complex transmembrane topologies, and highly irregular surfaces with crevices and pores. In many cases, specific interactions formed with the lipid membrane are functionally crucial, as is the overall lipid composition. Compounded with increasing protein size, these characteristics pose a challenge for the construction of simulation models of membrane proteins in lipid environments; clearly, that these models are sufficiently realistic bears upon the reliability of simulation-based studies of these systems. Here, we introduce GRIFFIN, which uses a versatile framework to automate and improve a widely-used membrane-embedding protocol. Initially, GRIFFIN carves out lipid and water molecules from a volume equivalent to that of the protein, so as to conserve the system density. In the subsequent optimization phase GRIFFIN adds an implicit grid-based protein force-field to a molecular dynamics simulation of the pre-carved membrane. In this force-field, atoms inside the implicit protein volume experience an outward force that will expel them from that volume, whereas those outside are subject to electrostatic and van-der-Waals interactions with the implicit protein. At each step of the simulation, these forces are updated by GRIFFIN and combined with the intermolecular forces of the explicit lipid-water system. This procedure enables the construction of realistic and reproducible starting configurations of the protein-membrane interface within a reasonable timeframe and with minimal intervention. GRIFFIN is a standalone tool designed to work alongside any existing molecular dynamics package, such as NAMD or GROMACS. PMID:24707227

  14. Tubular lipid membranes pulled from vesicles: Dependence of system equilibrium on lipid bilayer curvature

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Golushko, I. Yu., E-mail: vaniagolushko@yandex.ru; Rochal, S. B.

    2016-01-15

    Conditions of joint equilibrium and stability are derived for a spherical lipid vesicle and a tubular lipid membrane (TLM) pulled from this vesicle. The obtained equations establish relationships between the geometric and physical characteristics of the system and the external parameters, which have been found to be controllable in recent experiments. In particular, the proposed theory shows that, in addition to the pressure difference between internal and external regions of the system, the variable spontaneous average curvature of the lipid bilayer (forming the TLM) also influences the stability of the lipid tube. The conditions for stability of the cylindrical phasemore » of TLMs after switching off the external force that initially formed the TLM from a vesicle are discussed. The loss of system stability under the action of a small axial force compressing the TLM is considered.« less

  15. Clostridium Perfringens Epsilon Toxin Binds to Membrane Lipids and Its Cytotoxic Action Depends on Sulfatide.

    PubMed

    Gil, Carles; Dorca-Arévalo, Jonatan; Blasi, Juan

    2015-01-01

    Epsilon toxin (Etx) is one of the major lethal toxins produced by Clostridium perfringens types B and D, being the causal agent of fatal enterotoxemia in animals, mainly sheep and goats. Etx is synthesized as a non-active prototoxin form (proEtx) that becomes active upon proteolytic activation. Etx exhibits a cytotoxic effect through the formation of a pore in the plasma membrane of selected cell targets where Etx specifically binds due to the presence of specific receptors. However, the identity and nature of host receptors of Etx remain a matter of controversy. In the present study, the interactions between Etx and membrane lipids from the synaptosome-enriched fraction from rat brain (P2 fraction) and MDCK cell plasma membrane preparations were analyzed. Our findings show that both Etx and proEtx bind to lipids extracted from lipid rafts from the two different models as assessed by protein-lipid overlay assay. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. Binding of proEtx to sulfatide, phosphatidylserine, phosphatidylinositol (3)-phosphate and phosphatidylinositol (5)-phosphate was detected. Removal of the sulphate groups via sulfatase treatment led to a dramatic decrease in Etx-induced cytotoxicity, but not in proEtx-GFP binding to MDCK cells or a significant shift in oligomer formation, pointing to a role of sulfatide in pore formation in rafts but not in toxin binding to the target cell membrane. These results show for the first time the interaction between Etx and membrane lipids from host tissue and point to a major role for sulfatides in C. perfringens epsilon toxin pathophysiology.

  16. High fat diet-induced modifications in membrane lipid and mitochondrial-membrane protein signatures precede the development of hepatic insulin resistance in mice

    PubMed Central

    Kahle, M.; Schäfer, A.; Seelig, A.; Schultheiß, J.; Wu, M.; Aichler, M.; Leonhardt, J.; Rathkolb, B.; Rozman, J.; Sarioglu, H.; Hauck, S.M.; Ueffing, M.; Wolf, E.; Kastenmueller, G.; Adamski, J.; Walch, A.; Hrabé de Angelis, M.; Neschen, S.

    2014-01-01

    Objective Excess lipid intake has been implicated in the pathophysiology of hepatosteatosis and hepatic insulin resistance. Lipids constitute approximately 50% of the cell membrane mass, define membrane properties, and create microenvironments for membrane-proteins. In this study we aimed to resolve temporal alterations in membrane metabolite and protein signatures during high-fat diet (HF)-mediated development of hepatic insulin resistance. Methods We induced hepatosteatosis by feeding C3HeB/FeJ male mice an HF enriched with long-chain polyunsaturated C18:2n6 fatty acids for 7, 14, or 21 days. Longitudinal changes in hepatic insulin sensitivity were assessed via the euglycemic-hyperinsulinemic clamp, in membrane lipids via t-metabolomics- and membrane proteins via quantitative proteomics-analyses, and in hepatocyte morphology via electron microscopy. Data were compared to those of age- and litter-matched controls maintained on a low-fat diet. Results Excess long-chain polyunsaturated C18:2n6 intake for 7 days did not compromise hepatic insulin sensitivity, however, induced hepatosteatosis and modified major membrane lipid constituent signatures in liver, e.g. increased total unsaturated, long-chain fatty acid-containing acyl-carnitine or membrane-associated diacylglycerol moieties and decreased total short-chain acyl-carnitines, glycerophosphocholines, lysophosphatidylcholines, or sphingolipids. Hepatic insulin sensitivity tended to decrease within 14 days HF-exposure. Overt hepatic insulin resistance developed until day 21 of HF-intervention and was accompanied by morphological mitochondrial abnormalities and indications for oxidative stress in liver. HF-feeding progressively decreased the abundance of protein-components of all mitochondrial respiratory chain complexes, inner and outer mitochondrial membrane substrate transporters independent from the hepatocellular mitochondrial volume in liver. Conclusions We assume HF-induced modifications in membrane lipid

  17. Critical Comparison of Biomembrane Force Fields: Protein-Lipid Interactions at the Membrane Interface.

    PubMed

    Sandoval-Perez, Angelica; Pluhackova, Kristyna; Böckmann, Rainer A

    2017-05-09

    Molecular dynamics (MD) simulations offer the possibility to study biological processes at high spatial and temporal resolution often not reachable by experiments. Corresponding biomolecular force field parameters have been developed for a wide variety of molecules ranging from inorganic ligands and small organic molecules over proteins and lipids to nucleic acids. Force fields have typically been parametrized and validated on thermodynamic observables and structural characteristics of individual compounds, e.g. of soluble proteins or lipid bilayers. Less strictly, due to the added complexity and missing experimental data to compare to, force fields have hardly been tested on the properties of mixed systems, e.g. on protein-lipid systems. Their selection and combination for mixed systems is further complicated by the partially differing parametrization strategies. Additionally, the presence of other compounds in the system may shift the subtle balance of force field parameters. Here, we assessed the protein-lipid interactions as described in the four atomistic force fields GROMOS54a7, CHARMM36 and the two force field combinations Amber14sb/Slipids and Amber14sb/Lipid14. Four observables were compared, focusing on the membrane-water interface: the conservation of the secondary structure of transmembrane proteins, the positioning of transmembrane peptides relative to the lipid bilayer, the insertion depth of side chains of unfolded peptides absorbed at the membrane interface, and the ability to reproduce experimental insertion energies of Wimley-White peptides at the membrane interface. Significant differences between the force fields were observed that affect e.g. membrane insertion depths and tilting of transmembrane peptides.

  18. The role of polyglutamine expansion and protein context in disease-related huntingtin/lipid interactions

    NASA Astrophysics Data System (ADS)

    Burke, Kathleen Anne

    Huntington's Disease (HD) is a neurodegenerative disorder that is defined by the accumulation of nanoscale aggregates comprised of the huntingtin (htt) protein. Aggregation is directly caused by an expanded polyglutamine (polyQ) domain in htt, leading to a diverse population of aggregate species, such as oligomers, fibrils, and annular aggregates. Furthermore, the length of this polyQ domain is directly related to onset and severity of disease. The first 17 amino acids on the N-terminus (N17) and the polyproline domain on the C-terminal side of the polyQ domain have been shown to further modulate the aggregation process. Additionally, N17 appears to have lipid binding properties as htt interacts with a variety of membrane-containing structures present in cells, such as organelles, and interactions with these membrane surfaces may further modulate htt aggregation. To investigate the interaction between htt exon1 and lipid bilayers, in situ atomic force microscopy (AFM) was used to directly monitor the aggregation of htt exon1 constructs with varying Q-length (35Q, 46Q, 51Q, and myc- 53Q) or synthetic peptides with different polyQ domain flanking sequences (KK-Q35-KK, KK-Q 35-P10-KK, N17-Q35-KK, and N 17-Q35-P10-KK) on supported lipid membranes comprised of total brain lipid extract. The exon1 fragments accumulated on the lipid membranes, causing disruption of the membrane, in a polyQ dependent manner. By adding N-terminal tags to the htt exon1 fragments, the interaction with the lipid bilayer was impeded. The KK-Q35-KK and KK-Q 35-P10-KK peptides had no appreciable interaction with lipid bilayers. Interestingly, polyQ peptides with the N17 flanking sequence interacted with the bilayer. N17-Q35-KK formed discrete aggregates on the bilayer, but there was minimal membrane disruption. The N17-Q35-P10-KK peptide interacted more aggressively with the lipid bilayer in a manner reminiscent of the htt exon1 proteins.

  19. Anomalous surface diffusion of protons on lipid membranes.

    PubMed

    Wolf, Maarten G; Grubmüller, Helmut; Groenhof, Gerrit

    2014-07-01

    The cellular energy machinery depends on the presence and properties of protons at or in the vicinity of lipid membranes. To asses the energetics and mobility of a proton near a membrane, we simulated an excess proton near a solvated DMPC bilayer at 323 K, using a recently developed method to include the Grotthuss proton shuttling mechanism in classical molecular dynamics simulations. We obtained a proton surface affinity of -13.0 ± 0.5 kJ mol(-1). The proton interacted strongly with both lipid headgroup and linker carbonyl oxygens. Furthermore, the surface diffusion of the proton was anomalous, with a subdiffusive regime over the first few nanoseconds, followed by a superdiffusive regime. The time- and distance dependence of the proton surface diffusion coefficient within these regimes may also resolve discrepancies between previously reported diffusion coefficients. Our simulations show that the proton anomalous surface diffusion originates from restricted diffusion in two different surface-bound states, interrupted by the occasional bulk-mediated long-range surface diffusion. Although only a DMPC membrane was considered in this work, we speculate that the restrictive character of the on-surface diffusion is highly sensitive to the specific membrane conditions, which can alter the relative contributions of the surface and bulk pathways to the overall diffusion process. Finally, we discuss the implications of our findings for the energy machinery. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  20. Reorganization of plasma membrane lipid domains during conidial germination.

    PubMed

    Santos, Filipa C; Fernandes, Andreia S; Antunes, Catarina A C; Moreira, Filipe P; Videira, Arnaldo; Marinho, H Susana; de Almeida, Rodrigo F M

    2017-02-01

    Neurospora crassa, a filamentous fungus, in the unicellular conidial stage has ideal features to study sphingolipid (SL)-enriched domains, which are implicated in fundamental cellular processes ranging from antifungal resistance to apoptosis. Several changes in lipid metabolism and in the membrane composition of N. crassa occur during spore germination. However, the biophysical impact of those changes is unknown. Thus, a biophysical study of N. crassa plasma membrane, particularly SL-enriched domains, and their dynamics along conidial germination is prompted. Two N. crassa strains, wild-type (WT) and slime, which is devoid of cell wall, were studied. Conidial growth of N. crassa WT from a dormancy state to an exponential phase was accompanied by membrane reorganization, namely an increase of membrane fluidity, occurring faster in a supplemented medium than in Vogel's minimal medium. Gel-like domains, likely enriched in SLs, were found in both N. crassa strains, but were particularly compact, rigid and abundant in the case of slime cells, even more than in budding yeast Saccharomyces cerevisiae. In N. crassa, our results suggest that the melting of SL-enriched domains occurs near growth temperature (30°C) for WT, but at higher temperatures for slime. Regarding biophysical properties strongly affected by ergosterol, the plasma membrane of slime conidia lays in between those of N. crassa WT and S. cerevisiae cells. The differences in biophysical properties found in this work, and the relationships established between membrane lipid composition and dynamics, give new insights about the plasma membrane organization and structure of N. crassa strains during conidial growth. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Lipid-Mediated Clusters of Guest Molecules in Model Membranes and Their Dissolving in the Presence of Lipid Rafts.

    PubMed

    Kardash, Maria E; Dzuba, Sergei A

    2017-05-25

    The clustering of molecules is an important feature of plasma membrane organization. It is challenging to develop methods for quantifying membrane heterogeneities because of their transient nature and small size. Here, we obtained evidence that transient membrane heterogeneities can be frozen at cryogenic temperatures which allows the application of solid-state experimental techniques sensitive to the nanoscale distance range. We employed the pulsed version of electron paramagnetic resonance (EPR) spectroscopy, the electron spin echo (ESE) technique, for spin-labeled molecules in multilamellar lipid bilayers. ESE decays were refined for pure contribution of spin-spin magnetic dipole-dipolar interaction between the labels; these interactions manifest themselves at a nanometer distance range. The bilayers were prepared from different types of saturated and unsaturated lipids and cholesterol (Chol); in all cases, a small amount of guest spin-labeled substances 5-doxyl-stearic-acid (5-DSA) or 3β-doxyl-5α-cholestane (DChl) was added. The local concentration found of 5-DSA and DChl molecules was remarkably higher than the mean concentration in the bilayer, evidencing the formation of lipid-mediated clusters of these molecules. To our knowledge, formation of nanoscale clusters of guest amphiphilic molecules in biological membranes is a new phenomenon suggested only recently. Two-dimensional 5-DSA molecular clusters were found, whereas flat DChl molecules were found to be clustered into stacked one-dimensional structures. These clusters disappear when the Chol content is varied between the boundaries known for lipid raft formation at room temperatures. The room temperature EPR evidenced entrapping of DChl molecules in the rafts.

  2. In-Plane Correlations in a Polymer-Supported Lipid Membrane Measured by Off-Specular Neutron Scattering

    NASA Astrophysics Data System (ADS)

    Jablin, Michael S.; Zhernenkov, Mikhail; Toperverg, Boris P.; Dubey, Manish; Smith, Hillary L.; Vidyasagar, Ajay; Toomey, Ryan; Hurd, Alan J.; Majewski, Jaroslaw

    2011-04-01

    Polymer-supported single lipid bilayers are models to study configurations of cell membranes. We used off-specular neutron scattering to quantify in-plane height-height correlations of interfacial fluctuations of such a lipid bilayer. As temperature decreased from 37°C to 25°C, the polymer swells and the polymer-supported lipid membrane deviates from its initially nearly planar structure. A correlation length characteristic of capillary waves changes from 30μm at 37°C to 11μm at 25°C, while the membrane bending rigidity remains roughly constant in this temperature range.

  3. Effects of bile salts on glucosylceramide containing membranes.

    PubMed

    Halin, Josefin; Mattjus, Peter

    2011-12-01

    The glycolipid transfer protein (GLTP) is capable of transporting glycolipids from a donor membrane, through the aqueous environment, to an acceptor membrane. The GLTP mediated glycolipid transfer from sphingomyelin membranes is very slow. In contrast, the transfer is fast from membranes composed of phosphatidylcholine. The lateral glycolipid membrane organization is known to be driven by their tendency to mix non-randomly with different membrane lipids. Consequently, the properties of the membrane lipids surrounding the glycolipids play an important role in the ability of GLTP to bind and transfer its substrates. Since GLTP transfer of glycolipids is almost nonexistent from sphingomyelin membranes, we have used this exceptionality to investigate if membrane intercalators can alter the membrane packing and induce glycolipid transfer. We found that the bile salts cholate, deoxycholate, taurocholate and taurodeoxycholate, cause glucosylceramide to become transferrable by GLTP. Other compounds, such as single chain lipids, ceramide and nonionic surfactants, that have membrane-perturbing effects, did not affect the transfer capability of GLTP. We speculate that the strong hydrogen bonding network formed in the interfacial region of glycosphingolipid-sphingomyelin membranes is disrupted by the membrane partition of the bile salts causing the glycosphingolipid to become transferrable. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. The interaction of antibodies with lipid membranes unraveled by fluorescence methodologies

    NASA Astrophysics Data System (ADS)

    Figueira, Tiago N.; Veiga, Ana Salomé; Castanho, Miguel A. R. B.

    2014-12-01

    The interest and investment in antibody therapies has reached an overwhelming scale in the last decade. Yet, little concern has been noticed among the scientific community to unravel important interactions of antibodies with biological structures other than their respective epitopes. Lipid membranes are particularly relevant in this regard as they set the stage for protein-protein recognition, a concept potentially inclusive of antibody-antigen recognition. Fluorescence techniques allow experimental monitoring of protein partition between aqueous and lipid phases, deciphering events of adsorption, insertion and diffusion. This review focuses on the available fluorescence spectroscopy methodologies directed to the study of antibody-membrane interactions.

  5. Effects of temperature and PEG grafting density on the translocation of PEGylated nanoparticles across asymmetric lipid membrane.

    PubMed

    Zhang, Zuoheng; Lin, Xubo; Gu, Ning

    2017-12-01

    Plasma membrane internalization of nanoparticles (NPs) is important for their biomedical applications such as drug-delivery carriers. On one hand, in order to improve their half-life in circulation, PEGylation has been widely used. However, it may hinder the NPs' membrane internalization ability. On the other hand, higher temperature could enhance the membrane permeability and may affect the NPs' ability to enter into or exit from cells. To make full use of their advantages, we systematically investigated the effects of temperature and PEG density on the translocation of PEGylated nanoparticles across the plasma asymmetric membrane of eukaryotic cells, using near-atom level coarse-grained molecular dynamics simulations. Our results showed that higher temperature could accelerate the translocation of NPs across membranes by making lipids more disorder and faster diffusion. On the contrary, steric hindrance effects of PEG would inhibit NPs' translocation process and promote lipids flip-flops. The PEG chains could rearrange themselves to minimize the contacts between PEG and lipid tails during the translocation, which was similar to 'snorkeling effect'. Moreover, lipid flip-flops were affected by PEGylated density as well as NPs' translocation direction. Higher PEG grafting density could promote lipid flip-flops, but inhibit lipid extraction from bilayers. The consequence of lipid flip-flop and extraction was that the membranes got more symmetric. Copyright © 2017. Published by Elsevier B.V.

  6. Interaction of the Spo20 membrane-sensor motif with phosphatidic acid and other anionic lipids, and influence of the membrane environment.

    PubMed

    Horchani, Habib; de Saint-Jean, Maud; Barelli, Hélène; Antonny, Bruno

    2014-01-01

    The yeast protein Spo20 contains a regulatory amphipathic motif that has been suggested to recognize phosphatidic acid, a lipid involved in signal transduction, lipid metabolism and membrane fusion. We have investigated the interaction of the Spo20 amphipathic motif with lipid membranes using a bioprobe strategy that consists in appending this motif to the end of a long coiled-coil, which can be coupled to a GFP reporter for visualization in cells. The resulting construct is amenable to in vitro and in vivo experiments and allows unbiased comparison between amphipathic helices of different chemistry. In vitro, the Spo20 bioprobe responded to small variations in the amount of phosphatidic acid. However, this response was not specific. The membrane binding of the probe depended on the presence of phosphatidylethanolamine and also integrated the contribution of other anionic lipids, including phosphatidylserine and phosphatidyl-inositol-(4,5)bisphosphate. Inverting the sequence of the Spo20 motif neither affected the ability of the probe to interact with anionic liposomes nor did it modify its cellular localization, making a stereo-specific mode of phosphatidic acid recognition unlikely. Nevertheless, the lipid binding properties and the cellular localization of the Spo20 alpha-helix differed markedly from that of another amphipathic motif, Amphipathic Lipid Packing Sensor (ALPS), suggesting that even in the absence of stereo specific interactions, amphipathic helices can act as subcellular membrane targeting determinants in a cellular context.

  7. The lipid composition modulates the influence of the bovine seminal plasma protein PDC-109 on membrane stability.

    PubMed

    Tannert, Astrid; Töpfer-Petersen, Edda; Herrmann, Andreas; Müller, Karin; Müller, Peter

    2007-10-16

    The bovine seminal plasma protein PDC-109 exerts an essential influence on the sperm cell plasma membrane during capacitation. However, by any mechanism, it has to be ensured that this function of the protein on sperm cells is not initiated too early, that is, upon ejaculation when PDC-109 and sperm cells come into first contact, but rather at later stages of sperm genesis in the female genital tract. To answer the question of whether changes of the bovine sperm lipid composition can modulate the effect of PDC-109 on sperm membranes, we have investigated the influence of PDC-109 on the integrity of (i) differently composed lipid vesicles and of (ii) membranes from human red blood cells and bovine spermatozoa. PDC-109 most effectively disturbed lipid membranes composed of choline-containing phospholipids and in the absence of cholesterol. The impact of the protein on lipid vesicles was attenuated in the presence of cholesterol or of noncholine-containing phospholipids, such as phosphatidylethanolamine or phosphatidylserine. An extraction of cholesterol from lipid or biological membranes using methyl-beta-cyclodextrin caused an increased membrane perturbation by PDC-109. Our results argue for a oppositional effect of PDC-109 during sperm cell genesis. We hypothesize that the lipid composition of ejaculated bull sperm cells allows a binding of PDC-109 without leading to an impairment of the plasma membrane. At later stages of sperm cell genesis upon release of cholesterol from sperm membranes, PDC-109 triggers a destabilization of the cells.

  8. Intermonolayer Friction and Surface Shear Viscosity of Lipid Bilayer Membranes

    PubMed Central

    den Otter, W. K.; Shkulipa, S. A.

    2007-01-01

    The flow behavior of lipid bilayer membranes is characterized by a surface viscosity for in-plane shear deformations, and an intermonolayer friction coefficient for slip between the two leaflets of the bilayer. Both properties have been studied for a variety of coarse-grained double-tailed model lipids, using equilibrium and nonequilibrium molecular dynamics simulations. For lipids with two identical tails, the surface shear viscosity rises rapidly with tail length, while the intermonolayer friction coefficient is less sensitive to the tail length. Interdigitation of lipid tails across the bilayer midsurface, as observed for lipids with two distinct tails, strongly enhances the intermonolayer friction coefficient, but hardly affects the surface shear viscosity. The simulation results are compared against the available experimental data. PMID:17468168

  9. Studies of the molecular effects of a solid support upon lipid membranes and membrane bound proteins

    NASA Astrophysics Data System (ADS)

    Hartshorn, Christopher M.

    Often, membrane/protein systems are studied and/or utilized on solid supports. The underlying substrate in solid supported lipid bilayer assemblies causes large perturbations to the membrane, but the nature of these effects are not well understood. To gain an understanding, these effects were studied on two fronts: the effect upon the membrane by itself, and then the effects upon a membrane/protein system. First, all-atom molecular dynamics (MD) simulations of DLPC, DMPC, POPC, and DEPC on a hydroxylated nanocrystalline alpha-quartz (011) slab revealed a pronounced thinning effect in the lipid bilayers. It was shown that this thinning effect proceeded by one of two mechanisms: the first through a curling of the terminal methyl groups at the interface of the opposing leaflets, and the second through increased interdigitation of the alkyl chains. Also, with the introduction of the solid support, marked asymmetries in a number of structural properties were reported. These asymmetries included (a) the surface area per lipid, (b) the electron densities of the polar head groups, (c) the radial distributions of the choline groups, and (d) the average orientation of water surrounding the membranes. Next, the free energy perturbation method was used to begin calculating the change in free energy (DeltaGbinding) from a Gramicidin monomer to its dimeric state, which were simulated via MD of supported DLPC, DMPC, and DEPC bilayers. The most notable effect was an asymmetry of the calculated free energies relative to the bilayer side closest to the solid support. In all three systems, there was a large difference in free energy between the Gramicidin monomers that were close to the support and the monomers further from the support.

  10. Time-dependent effect of rutin on skin fibroblasts membrane disruption following UV radiation.

    PubMed

    Gęgotek, Agnieszka; Bielawska, Katarzyna; Biernacki, Michał; Dobrzyńska, Izabela; Skrzydlewska, Elżbieta

    2017-08-01

    Chronic exposure of the skin to solar UV radiation induces a number of biological alterations, including a redox imbalance; therefore, there is an urgent need for skin cells protective compounds. The aim of this study was to determine the effects of natural, previously extensively examined, polyphenol with antioxidant properties - rutin, on UV-induced skin fibroblasts membrane disruption. Accordingly, fibroblasts exposed to UVA and UVB irradiation were incubated with rutin (12h before and/or up to 24h after irradiation), and the structural and metabolic changes were examined. Rutin penetration through the fibroblast phospholipid bilayer was aided by UVA-induced bilitranslocase activity 2-4h after irradiation, while UVB irradiation led to enhanced phospholipid peroxidation and higher membrane permeability to facilitate the interaction of rutin with phospholipids. Lipidomic analysis revealed that 4h of rutin treatment also partially prevented UVA/B-induced increase in phosphatidylethanolamine and phosphatidylcholine level, as well as their membrane localization, which resulted in an enhanced zeta potential in the cells and liposomes. Moreover, rutin 2h following irradiation, in a various degree, prevented the increased in phospholipase A2 activity and ROS generation, and partially protected against the reduction of arachidonic and linoleic acids level and the lipid peroxidation product 4-hydroxynonenal level increase. Rutin effectively prevented against decrease in glutathione peroxidase, glutathione and vitamins E and C activities/levels, particularly 2h following UVA irradiation. In conclusion, highest skin fibroblasts membrane level of rutin occurred in 2-4h following UVA/B-radiation results in its strongest effect on biomembrane structure and functions and cellular antioxidant system irrespective of the radiation type. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  11. Modeling of interactions between nanoparticles and cell membranes

    NASA Astrophysics Data System (ADS)

    Ban, Young-Min

    Rapid development of nanotechnology and ability to manufacture materials and devices with nanometer feature size leads to exciting innovations in many areas including the medical and electronic fields. However, the possible health and environmental impacts of manufactured nanomaterials are not fully known. Recent experimental reports suggest that some of the manufactured nanomaterials, such as fullerenes and carbon nanotubes, are highly toxic even in small concentrations. The goal of the current work is to understand the mechanisms responsible for the toxicity of nanomaterials. In the current study coarse-grained molecular dynamics simulations are employed to investigate the interactions between NPs and cellular membranes at a molecular level. One of the possible toxicity mechanisms of the nanomaterials is membrane disruption. Possibility of membrane disruption exposed to the manufactured nanomaterials are examined by considering chemical reactions and non-reactive physical interactions as chemical as well as physical mechanisms. Mechanisms of transport of carbon-based nanoparticles (fullerene and its derivative) across a phospholipid bilayer are investigated. The free energy profile is obtained using constrained simulations. It is shown that the considered nanoparticles are hydrophobic and therefore they tend to reside in the interior of the lipid bilayer. In addition, the dynamics of the membrane fluctuations is significantly affected by the nanoparticles at the bilayer-water interface. The hydrophobic interaction between the particles and membrane core induces the strong coupling between the nanoparticle motion and membrane deformation. It is observed that the considered nanoparticles affect several physical properties of the membrane. The nanoparticles embedded into the membrane interior lead to the membrane softening, which becomes more significant with increase in CNT length and concentration. The lateral pressure profile and membrane energy in the membrane

  12. Molecular dynamics study of lipid bilayers modeling the plasma membranes of mouse hepatocytes and hepatomas.

    PubMed

    Andoh, Yoshimichi; Aoki, Noriyuki; Okazaki, Susumu

    2016-02-28

    Molecular dynamics (MD) calculations of lipid bilayers modeling the plasma membranes of normal mouse hepatocytes and hepatomas in water have been performed under physiological isothermal-isobaric conditions (310.15 K and 1 atm). The changes in the membrane properties induced by hepatic canceration were investigated and were compared with previous MD calculations included in our previous study of the changes in membrane properties induced by murine thymic canceration. The calculated model membranes for normal hepatocytes and hepatomas comprised 23 and 24 kinds of lipids, respectively. These included phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. We referred to previously published experimental values for the mole fraction of the lipids adopted in the present calculations. The calculated structural and dynamic properties of the membranes such as lateral structure, order parameters, lateral self-diffusion constants, and rotational correlation times all showed that hepatic canceration causes plasma membranes to become more ordered laterally and less fluid. Interestingly, this finding contrasts with the less ordered structure and increased fluidity of plasma membranes induced by thymic canceration observed in our previous MD study.

  13. Molecular dynamics study of lipid bilayers modeling the plasma membranes of mouse hepatocytes and hepatomas

    NASA Astrophysics Data System (ADS)

    Andoh, Yoshimichi; Aoki, Noriyuki; Okazaki, Susumu

    2016-02-01

    Molecular dynamics (MD) calculations of lipid bilayers modeling the plasma membranes of normal mouse hepatocytes and hepatomas in water have been performed under physiological isothermal-isobaric conditions (310.15 K and 1 atm). The changes in the membrane properties induced by hepatic canceration were investigated and were compared with previous MD calculations included in our previous study of the changes in membrane properties induced by murine thymic canceration. The calculated model membranes for normal hepatocytes and hepatomas comprised 23 and 24 kinds of lipids, respectively. These included phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. We referred to previously published experimental values for the mole fraction of the lipids adopted in the present calculations. The calculated structural and dynamic properties of the membranes such as lateral structure, order parameters, lateral self-diffusion constants, and rotational correlation times all showed that hepatic canceration causes plasma membranes to become more ordered laterally and less fluid. Interestingly, this finding contrasts with the less ordered structure and increased fluidity of plasma membranes induced by thymic canceration observed in our previous MD study.

  14. Bright and photostable push-pull pyrene dye visualizes lipid order variation between plasma and intracellular membranes.

    PubMed

    Niko, Yosuke; Didier, Pascal; Mely, Yves; Konishi, Gen-ichi; Klymchenko, Andrey S

    2016-01-11

    Imaging lipid organization in cell membranes requires advanced fluorescent probes. Here, we show that a recently synthesized push-pull pyrene (PA), similarly to popular probe Laurdan, changes the emission maximum as a function of lipid order, but outperforms it by spectroscopic properties. In addition to red-shifted absorption compatible with common 405 nm diode laser, PA shows higher brightness and much higher photostability than Laurdan in apolar membrane environments. Moreover, PA is compatible with two-photon excitation at wavelengths >800 nm, which was successfully used for ratiometric imaging of coexisting liquid ordered and disordered phases in giant unilamellar vesicles. Fluorescence confocal microscopy in Hela cells revealed that PA efficiently stains the plasma membrane and the intracellular membranes at >20-fold lower concentrations, as compared to Laurdan. Finally, ratiometric imaging using PA reveals variation of lipid order within different cellular compartments: plasma membranes are close to liquid ordered phase of model membranes composed of sphingomyelin and cholesterol, while intracellular membranes are much less ordered, matching well membranes composed of unsaturated phospholipids without cholesterol. These differences in the lipid order were confirmed by fluorescence lifetime imaging (FLIM) at the blue edge of PA emission band. PA probe constitutes thus a new powerful tool for biomembrane research.

  15. Atomic force microscopy of model lipid membranes.

    PubMed

    Morandat, Sandrine; Azouzi, Slim; Beauvais, Estelle; Mastouri, Amira; El Kirat, Karim

    2013-02-01

    Supported lipid bilayers (SLBs) are biomimetic model systems that are now widely used to address the biophysical and biochemical properties of biological membranes. Two main methods are usually employed to form SLBs: the transfer of two successive monolayers by Langmuir-Blodgett or Langmuir-Schaefer techniques, and the fusion of preformed lipid vesicles. The transfer of lipid films on flat solid substrates offers the possibility to apply a wide range of surface analytical techniques that are very sensitive. Among them, atomic force microscopy (AFM) has opened new opportunities for determining the nanoscale organization of SLBs under physiological conditions. In this review, we first focus on the different protocols generally employed to prepare SLBs. Then, we describe AFM studies on the nanoscale lateral organization and mechanical properties of SLBs. Lastly, we survey recent developments in the AFM monitoring of bilayer alteration, remodeling, or digestion, by incubation with exogenous agents such as drugs, proteins, peptides, and nanoparticles.

  16. Differential Interaction of Synthetic Glycolipids with Biomimetic Plasma Membrane Lipids Correlates with the Plant Biological Response.

    PubMed

    Nasir, Mehmet Nail; Lins, Laurence; Crowet, Jean-Marc; Ongena, Marc; Dorey, Stephan; Dhondt-Cordelier, Sandrine; Clément, Christophe; Bouquillon, Sandrine; Haudrechy, Arnaud; Sarazin, Catherine; Fauconnier, Marie-Laure; Nott, Katherine; Deleu, Magali

    2017-09-26

    Natural and synthetic amphiphilic molecules including lipopeptides, lipopolysaccharides, and glycolipids are able to induce defense mechanisms in plants. In the present work, the perception of two synthetic C14 rhamnolipids, namely, Alk-RL and Ac-RL, differing only at the level of the lipid tail terminal group have been investigated using biological and biophysical approaches. We showed that Alk-RL induces a stronger early signaling response in tobacco cell suspensions than does Ac-RL. The interactions of both synthetic RLs with simplified biomimetic membranes were further analyzed using experimental and in silico approaches. Our results indicate that the interactions of Alk-RL and Ac-RL with lipids were different in terms of insertion and molecular responses and were dependent on the lipid composition of model membranes. A more favorable insertion of Alk-RL than Ac-RL into lipid membranes is observed. Alk-RL forms more stable molecular assemblies than Ac-RL with phospholipids and sterols. At the molecular level, the presence of sterols tends to increase the RLs' interaction with lipid bilayers, with a fluidizing effect on the alkyl chains. Taken together, our findings suggest that the perception of these synthetic RLs at the membrane level could be related to a lipid-driven process depending on the organization of the membrane and the orientation of the RLs within the membrane and is correlated with the induction of early signaling responses in tobacco cells.

  17. Length and sequence dependence in the association of Huntingtin protein with lipid membranes

    NASA Astrophysics Data System (ADS)

    Jawahery, Sudi; Nagarajan, Anu; Matysiak, Silvina

    2013-03-01

    There is a fundamental gap in our understanding of how aggregates of mutant Huntingtin protein (htt) with overextended polyglutamine (polyQ) sequences gain the toxic properties that cause Huntington's disease (HD). Experimental studies have shown that the most important step associated with toxicity is the binding of mutant htt aggregates to lipid membranes. Studies have also shown that flanking amino acid sequences around the polyQ sequence directly affect interactions with the lipid bilayer, and that polyQ sequences of greater than 35 glutamine repeats in htt are a characteristic of HD. The key steps that determine how flanking sequences and polyQ length affect the structure of lipid bilayers remain unknown. In this study, we use atomistic molecular dynamics simulations to study the interactions between lipid membranes of varying compositions and polyQ peptides of varying lengths and flanking sequences. We find that overextended polyQ interactions do cause deformation in model membranes, and that the flanking sequences do play a role in intensifying this deformation by altering the shape of the affected regions.

  18. Phosphatidylserine lipids and membrane order precisely regulate the activity of Polybia-MP1 peptide.

    PubMed

    Alvares, Dayane S; Ruggiero Neto, João; Ambroggio, Ernesto E

    2017-06-01

    Polybia-MP1 (IDWKKLLDAAKQIL-NH 2 ) is a lytic peptide from the Brazilian wasp venom with known anti-cancer properties. Previous evidence indicates that phosphatidylserine (PS) lipids are relevant for the lytic activity of MP1. In agreement with this requirement, phosphatidylserine lipids are translocated to the outer leaflet of cells, and are available for MP1 binding, depending on the presence of liquid-ordered domains. Here, we investigated the effect of PS on MP1 activity when this lipid is reconstituted in membranes of giant or large liposomes with different lipid-phase states. By monitoring the membrane and soluble luminal content of giant unilamellar vesicles (GUVs), using fluorescence confocal microscopy, we were able to determine that MP1 has a pore-forming activity at the membrane level. Liquid-ordered domains, which were phase-separated within the membrane of GUVs, influenced the pore-forming activity of MP1. Experiments evaluating the membrane-binding and lytic activity of MP1 on large unilamellar vesicles (LUVs), with the same lipid composition as GUVs, demonstrated that there was synergy between liquid-ordered domains and PS, which enhanced both activities. Based on our findings, we propose that the physicochemical properties of cancer cell membranes, which possess a much higher concentration of PS than normal cells, renders them susceptible to MP1 binding and lytic pore formation. These results can be correlated with MP1's potent and selective anti-cancer activity and pave the way for future research to develop cancer therapies that harness and exploit the properties of MP1. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Using the Localized Surface Plasmon Resonance of Gold Nanoparticles to Monitor Lipid Membrane Assembly and Protein Binding.

    PubMed

    Messersmith, Reid E; Nusz, Greg J; Reed, Scott M

    2013-12-19

    Gold nanoparticles provide a template for preparing supported lipid layers with well-defined curvature. Here, we utilize the localized surface plasmon resonance (LSPR) of gold nanoparticles as a sensor for monitoring the preparation of lipid layers on nanoparticles. The LSPR is very sensitive to the immediate surroundings of the nanoparticle surface and it is used to monitor the coating of lipids and subsequent conversion of a supported bilayer to a hybrid membrane with an outer lipid leaflet and an inner leaflet containing hydrophobic alkanethiol. We demonstrate that both decanethiol and propanethiol are able to form hybrid membranes and that the membrane created over the shorter thiol can be stripped from the gold along with the lipid leaflet using β-mercaptoethanol. The sensitivity of the nanoparticle LSPR to the refractive index (RI) of its surroundings is greater when the shorter thiol is used (37.8 ± 1.5 nm per RI unit) than when the longer thiol is used (27.5 ± 0.5 nm per RI unit). Finally, C-reactive protein binding to the membrane is measured using this sensor allowing observation of both protein-membrane and nanoparticle-nanoparticle interactions without chemical labeling of protein or lipids.

  20. The role of lipid composition for insertion and stabilization of amino acids in membranes

    NASA Astrophysics Data System (ADS)

    Johansson, Anna C. V.; Lindahl, Erik

    2009-05-01

    While most membrane protein helices are clearly hydrophobic, recent experiments have indicated that it is possible to insert marginally hydrophobic helices into bilayers and have suggested apparent in vivo free energies of insertion for charged residues that are low, e.g., a few kcals for arginine. In contrast, a number of biophysical simulation studies have predicted that the bilayer interior is close to a pure hydrophobic environment with large penalties for hydrophilic amino acids—and yet the experimental scales do significantly better at predicting actual membrane proteins from sequence. Here, we have systematically studied the dependence of the free energy profiles on lipid properties, including tail length, saturation, headgroup hydrogen bond strength, and charge, both to see to whether the in vivo insertion can be explained in whole or part from lipid composition of the endoplasmic reticulum (ER) membranes, and if the solvation properties can help interpret how protein function depends on the lipids. We find that lipid charge is important to stabilize charged amino acids inside the bilayer (with implications, e.g., for ion channels), that thicker bilayers have higher solvation costs for hydrophilic side chains, and that headgroup hydrogen bond strength determines how adaptive the lipids are as a hydrophobic/hydrophilic solvent. None of the different free energy profiles are even close to the low apparent in vivo insertion cost, which suggests that regardless of the specific ER membrane composition the current experimental results cannot be explained by normal lipid-type variation.

  1. Biophysical Investigation of the Membrane-Disrupting Mechanism of the Antimicrobial and Amyloid-Like Peptide Dermaseptin S9

    PubMed Central

    Caillon, Lucie; Killian, J. Antoinette; Lequin, Olivier; Khemtémourian, Lucie

    2013-01-01

    Dermaseptin S9 (Drs S9) is an atypical cationic antimicrobial peptide with a long hydrophobic core and with a propensity to form amyloid-like fibrils. Here we investigated its membrane interaction using a variety of biophysical techniques. Rather surprisingly, we found that Drs S9 induces efficient permeabilisation in zwitterionic phosphatidylcholine (PC) vesicles, but not in anionic phosphatidylglycerol (PG) vesicles. We also found that the peptide inserts more efficiently in PC than in PG monolayers. Therefore, electrostatic interactions between the cationic Drs S9 and anionic membranes cannot explain the selectivity of the peptide towards bacterial membranes. CD spectroscopy, electron microscopy and ThT fluorescence experiments showed that the peptide adopts slightly more β-sheet and has a higher tendency to form amyloid-like fibrils in the presence of PC membranes as compared to PG membranes. Thus, induction of leakage may be related to peptide aggregation. The use of a pre-incorporation protocol to reduce peptide/peptide interactions characteristic of aggregates in solution resulted in more α-helix formation and a more pronounced effect on the cooperativity of the gel-fluid lipid phase transition in all lipid systems tested. Calorimetric data together with 2H- and 31P-NMR experiments indicated that the peptide has a significant impact on the dynamic organization of lipid bilayers, albeit slightly less for zwitterionic than for anionic membranes. Taken together, our data suggest that in particular in membranes of zwitterionic lipids the peptide binds in an aggregated state resulting in membrane leakage. We propose that also the antimicrobial activity of Drs S9 may be a result of binding of the peptide in an aggregated state, but that specific binding and aggregation to bacterial membranes is regulated not by anionic lipids but by as yet unknown factors. PMID:24146759

  2. Effects of magnetic cobalt ferrite nanoparticles on biological and artificial lipid membranes

    PubMed Central

    Drašler, Barbara; Drobne, Damjana; Novak, Sara; Valant, Janez; Boljte, Sabina; Otrin, Lado; Rappolt, Michael; Sartori, Barbara; Iglič, Aleš; Kralj-Iglič, Veronika; Šuštar, Vid; Makovec, Darko; Gyergyek, Sašo; Hočevar, Matej; Godec, Matjaž; Zupanc, Jernej

    2014-01-01

    Background The purpose of this work is to provide experimental evidence on the interactions of suspended nanoparticles with artificial or biological membranes and to assess the possibility of suspended nanoparticles interacting with the lipid component of biological membranes. Methods 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid vesicles and human red blood cells were incubated in suspensions of magnetic bare cobalt ferrite (CoFe2O4) or citric acid (CA)-adsorbed CoFe2O4 nanoparticles dispersed in phosphate-buffered saline and glucose solution. The stability of POPC giant unilamellar vesicles after incubation in the tested nanoparticle suspensions was assessed by phase-contrast light microscopy and analyzed with computer-aided imaging. Structural changes in the POPC multilamellar vesicles were assessed by small angle X-ray scattering, and the shape transformation of red blood cells after incubation in tested suspensions of nanoparticles was observed using scanning electron microscopy and sedimentation, agglutination, and hemolysis assays. Results Artificial lipid membranes were disturbed more by CA-adsorbed CoFe2O4 nanoparticle suspensions than by bare CoFe2O4 nanoparticle suspensions. CA-adsorbed CoFe2O4-CA nanoparticles caused more significant shape transformation in red blood cells than bare CoFe2O4 nanoparticles. Conclusion Consistent with their smaller sized agglomerates, CA-adsorbed CoFe2O4 nanoparticles demonstrate more pronounced effects on artificial and biological membranes. Larger agglomerates of nanoparticles were confirmed to be reactive against lipid membranes and thus not acceptable for use with red blood cells. This finding is significant with respect to the efficient and safe application of nanoparticles as medicinal agents. PMID:24741305

  3. Chloride Transport in Porous Lipid Bilayer Membranes

    PubMed Central

    Andreoli, Thomas E.; Watkins, Mary L.

    1973-01-01

    This paper describes dissipative Cl- transport in "porous" lipid bilayer membranes, i.e., cholesterol-containing membranes exposed to 1–3 x 10-7 M amphotericin B. P DCl (cm·s-1), the diffusional permeability coefficient for Cl-, estimated from unidirectional 36Cl- fluxes at zero volume flow, varied linearly with the membrane conductance (Gm, Ω-1·cm-2) when the contributions of unstirred layers to the resistance to tracer diffusion were relatively small with respect to the membranes; in 0.05 M NaCl, P DCl was 1.36 x 10-4 cm·s-1 when Gm was 0.02 Ω-1·cm-2. Net chloride fluxes were measured either in the presence of imposed concentration gradients or electrical potential differences. Under both sets of conditions: the values of P DCl computed from zero volume flow experiments described net chloride fluxes; the net chloride fluxes accounted for ∼90–95% of the membrane current density; and, the chloride flux ratio conformed to the Ussing independence relationship. Thus, it is likely that Cl- traversed aqueous pores in these anion-permselective membranes via a simple diffusion process. The zero current membrane potentials measured when the aqueous phases contained asymmetrical NaCl solutions could be expressed in terms of the Goldman-Hodgkin-Katz constant field equation, assuming that the P DNa/P DCl ratio was 0.05. In symmetrical salt solutions, the current-voltage properties of these membranes were linear; in asymmetrical NaCl solutions, the membranes exhibited electrical rectification consistent with constant-field theory. It seems likely that the space charge density in these porous membranes is sufficiently low that the potential gradient within the membranes is approximately linear; and, that the pores are not electrically neutral, presumably because the Debye length within the membrane phase approximates the membrane thickness. PMID:4708408

  4. Analysis of Cd44-Containing Lipid Rafts

    PubMed Central

    Oliferenko, Snezhana; Paiha, Karin; Harder, Thomas; Gerke, Volker; Schwärzler, Christoph; Schwarz, Heinz; Beug, Hartmut; Günthert, Ursula; Huber, Lukas A.

    1999-01-01

    CD44, the major cell surface receptor for hyaluronic acid (HA), was shown to localize to detergent-resistant cholesterol-rich microdomains, called lipid rafts, in fibroblasts and blood cells. Here, we have investigated the molecular environment of CD44 within the plane of the basolateral membrane of polarized mammary epithelial cells. We show that CD44 partitions into lipid rafts that contain annexin II at their cytoplasmic face. Both CD44 and annexin II were released from these lipid rafts by sequestration of plasma membrane cholesterol. Partition of annexin II and CD44 to the same type of lipid rafts was demonstrated by cross-linking experiments in living cells. First, when CD44 was clustered at the cell surface by anti-CD44 antibodies, annexin II was recruited into the cytoplasmic leaflet of CD44 clusters. Second, the formation of intracellular, submembranous annexin II–p11 aggregates caused by expression of a trans-dominant mutant of annexin II resulted in coclustering of CD44. Moreover, a frequent redirection of actin bundles to these clusters was observed. These basolateral CD44/annexin II–lipid raft complexes were stabilized by addition of GTPγS or phalloidin in a semipermeabilized and cholesterol-depleted cell system. The low lateral mobility of CD44 in the plasma membrane, as assessed with fluorescent recovery after photobleaching (FRAP), was dependent on the presence of plasma membrane cholesterol and an intact actin cytoskeleton. Disruption of the actin cytoskeleton dramatically increased the fraction of CD44 which could be recovered from the light detergent-insoluble membrane fraction. Taken together, our data indicate that in mammary epithelial cells the vast majority of CD44 interacts with annexin II in lipid rafts in a cholesterol-dependent manner. These CD44-containing lipid microdomains interact with the underlying actin cytoskeleton. PMID:10459018

  5. Xanthine oxidoreductase mediates membrane docking of milk-fat droplets but is not essential for apocrine lipid secretion.

    PubMed

    Monks, Jenifer; Dzieciatkowska, Monika; Bales, Elise S; Orlicky, David J; Wright, Richard M; McManaman, James L

    2016-10-15

    Xanthine oxidoreductase (XOR) modulates milk lipid secretion and lactation initiation. XOR is required for butyrophilin1a1 clustering in the membrane during milk lipid secretion. XOR mediates apical membrane reorganization during milk lipid secretion. Loss of XOR delays milk fat globule secretion. XOR loss alters the proteome of milk fat globules. Apocrine secretion is utilized by epithelial cells of exocrine glands. These cells bud off membrane-bound particles into the lumen of the gland, losing a portion of the cytoplasm in the secretion product. The lactating mammary gland secretes milk lipid by this mechanism, and xanthine oxidoreductase (XOR) has long been thought to be functionally important. We generated mammary-specific XOR knockout (MGKO) mice, expecting lactation to fail. Histology of the knockout glands showed very large lipid droplets enclosed in the mammary alveolar cells, but milk analysis showed that these large globules were secreted. Butyrophilin, a membrane protein known to bind to XOR, was clustered at the point of contact of the cytoplasmic lipid droplet with the apical plasma membrane, in the wild-type gland but not in the knockout, suggesting that XOR mediates 'docking' to this membrane. Secreted milk fat globules were isolated from mouse milk of wild-type and XOR MGKO dams, and subjected to LC-MS/MS for analysis of protein component. Proteomic results showed that loss of XOR leads to an increase in cytoplasmic, cytoskeletal, Golgi apparatus and lipid metabolism proteins associated with the secreted milk fat globule. Association of XOR with the lipid droplet results in membrane docking and more efficient retention of cytoplasmic components by the secretory cell. Loss of XOR then results in a reversion to a more rudimentary, less efficient, apocrine secretion mechanism, but does not prevent milk fat globule secretion. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

  6. Research on the Changes to the Lipid/Polymer Membrane Used in the Acidic Bitterness Sensor Caused by Preconditioning

    PubMed Central

    Harada, Yuhei; Noda, Junpei; Yatabe, Rui; Ikezaki, Hidekazu; Toko, Kiyoshi

    2016-01-01

    A taste sensor that uses lipid/polymer membranes can evaluate aftertastes felt by humans using Change in membrane Potential caused by Adsorption (CPA) measurements. The sensor membrane for evaluating bitterness, which is caused by acidic bitter substances such as iso-alpha acid contained in beer, needs an immersion process in monosodium glutamate (MSG) solution, called “MSG preconditioning”. However, what happens to the lipid/polymer membrane during MSG preconditioning is not clear. Therefore, we carried out three experiments to investigate the changes in the lipid/polymer membrane caused by the MSG preconditioning, i.e., measurements of the taste sensor, measurements of the amount of the bitterness substance adsorbed onto the membrane and measurements of the contact angle of the membrane surface. The CPA values increased as the preconditioning process progressed, and became stable after 3 d of preconditioning. The response potentials to the reference solution showed the same tendency of the CPA value change during the preconditioning period. The contact angle of the lipid/polymer membrane surface decreased after 7 d of MSG preconditioning; in short, the surface of the lipid/polymer membrane became hydrophilic during MSG preconditioning. The amount of adsorbed iso-alpha acid was increased until 5 d preconditioning, and then it decreased. In this study, we revealed that the CPA values increased with the progress of MSG preconditioning in spite of the decrease of the amount of iso-alpha acid adsorbed onto the lipid/polymer membrane, and it was indicated that the CPA values increase because the sensor sensitivity was improved by the MSG preconditioning. PMID:26891299

  7. Bacterial membranes: the effects of chill storage and food processing. An overview.

    PubMed

    Russell, Nicholas J

    2002-11-15

    The shelf life of food is extended by refrigeration because the metabolic processes of food-associated microorganisms are slowed by the lowered temperature. Nonetheless, cold-adapted psychrotrophic food-poisoning and food-spoilage bacteria remain a concern because they possess cold-adapted proteins and membrane lipids that facilitate growth at low temperatures. The use of membrane-disrupting novel preservation techniques, such as ultrasound, high hydrostatic pressure or pulsed electric field, offer the potential for an extension of shelf life. This review considers the interacting and potentially synergistic effects of chill storage or mild heat treatment on membrane properties, with the disruptive effects of membrane-targeted physical treatments.

  8. Quantification of Protein-Induced Membrane Remodeling Kinetics In Vitro with Lipid Multilayer Gratings

    PubMed Central

    Lowry, Troy W.; Hariri, Hanaa; Prommapan, Plengchart; Kusi-Appiah, Aubrey; Vafai, Nicholas; Bienkiewicz, Ewa A.; Van Winkle, David H.; Stagg, Scott M.

    2016-01-01

    The dynamic self-organization of lipids in biological systems is a highly regulated process that enables the compartmentalization of living systems at micro- and nanoscopic scales. Consequently, quantitative methods for assaying the kinetics of supramolecular remodeling such as vesicle formation from planar lipid bilayers or multilayers are needed to understand cellular self-organization. Here, a new nanotechnology-based method for quantitative measurements of lipid–protein interactions is presented and its suitability for quantifying the membrane binding, inflation, and budding activity of the membrane-remodeling protein Sar1 is demonstrated. Lipid multilayer gratings are printed onto surfaces using nanointaglio and exposed to Sar1, resulting in the inflation of lipid multilayers into unilamellar structures, which can be observed in a label-free manner by monitoring the diffracted light. Local variations in lipid multilayer volume on the surface is used to vary substrate availability in a microarray format. A quantitative model is developed that allows quantification of binding affinity (KD) and kinetics (kon and koff). Importantly, this assay is uniquely capable of quantifying membrane remodeling. Upon Sar1-induced inflation of single bilayers from surface supported multilayers, the semicylindrical grating lines are observed to remodel into semispherical buds when a critical radius of curvature is reached. PMID:26649649

  9. Phospholipid Chain Interactions with Cholesterol Drive Domain Formation in Lipid Membranes.

    PubMed

    Bennett, W F Drew; Shea, Joan-Emma; Tieleman, D Peter

    2018-06-05

    Cholesterol is a key component of eukaryotic membranes, but its role in cellular biology in general and in lipid rafts in particular remains controversial. Model membranes are used extensively to determine the phase behavior of ternary mixtures of cholesterol, a saturated lipid, and an unsaturated lipid with liquid-ordered and liquid-disordered phase coexistence. Despite many different experiments that determine lipid-phase diagrams, we lack an understanding of the molecular-level driving forces for liquid phase coexistence in bilayers with cholesterol. Here, we use atomistic molecular dynamics computer simulations to address the driving forces for phase coexistence in ternary lipid mixtures. Domain formation is directly observed in a long-timescale simulation of a mixture of 1,2-distearoyl-sn-glycero-3-phosphocholine, unsaturated 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, and cholesterol. Free-energy calculations for the exchange of the saturated and unsaturated lipids between the ordered and disordered phases give insight into the mixing behavior. We show that a large energetic contribution to domain formation is favorable enthalpic interactions of the saturated lipid in the ordered phase. This favorable energy for forming an ordered, cholesterol-rich phase is opposed by a large unfavorable entropy. Martini coarse-grained simulations capture the unfavorable free energy of mixing but do not reproduce the entropic contribution because of the reduced representation of the phospholipid tails. Phospholipid tails and their degree of unsaturation are key energetic contributors to lipid phase separation. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  10. Lipid-regulated sterol transfer between closely apposed membranes by oxysterol-binding protein homologues.

    PubMed

    Schulz, Timothy A; Choi, Mal-Gi; Raychaudhuri, Sumana; Mears, Jason A; Ghirlando, Rodolfo; Hinshaw, Jenny E; Prinz, William A

    2009-12-14

    Sterols are transferred between cellular membranes by vesicular and poorly understood nonvesicular pathways. Oxysterol-binding protein-related proteins (ORPs) have been implicated in sterol sensing and nonvesicular transport. In this study, we show that yeast ORPs use a novel mechanism that allows regulated sterol transfer between closely apposed membranes, such as organelle contact sites. We find that the core lipid-binding domain found in all ORPs can simultaneously bind two membranes. Using Osh4p/Kes1p as a representative ORP, we show that ORPs have at least two membrane-binding surfaces; one near the mouth of the sterol-binding pocket and a distal site that can bind a second membrane. The distal site is required for the protein to function in cells and, remarkably, regulates the rate at which Osh4p extracts and delivers sterols in a phosphoinositide-dependent manner. Together, these findings suggest a new model of how ORPs could sense and regulate the lipid composition of adjacent membranes.

  11. ToF-SIMS observation for evaluating the interaction between amyloid β and lipid membranes.

    PubMed

    Aoyagi, Satoka; Shimanouchi, Toshinori; Kawashima, Tomoko; Iwai, Hideo

    2015-04-01

    The adsorption behaviour of amyloid beta (Aβ), thought to be a key peptide for understanding Alzheimer's disease, was investigated by means of time-of-flight secondary ion mass spectrometry (ToF-SIMS). Aβ aggregates depending on the lipid membrane condition though it has not been fully understood yet. In this study, Aβ samples on different lipid membranes, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), were observed with ToF-SIMS and the complex ToF-SIMS data of the Aβ samples was interpreted using data analysis techniques such as principal component analysis (PCA), gentle-SIMS (G-SIMS) and g-ogram. DOPC and DMPC are liquid crystal at room temperature, while DPPC is gel at room temperature. As primary ion beams, Bi3(+) and Ar cluster ion beams were used and the effect of an Ar cluster ion for evaluating biomolecules was also studied. The secondary ion images of the peptide fragment ions indicated by G-SIMS and g-ogram were consistent with the PCA results. It is suggested that Aβ is adsorbed homogeneously on the liquid-crystalline-phase lipid membranes, while it aggregates along the lipid on the gel-phase lipid membrane. Moreover, in the results using the Ar cluster, the influence of contamination was reduced.

  12. Molecular Dynamics Simulations of the Permeation of Bisphenol A and Pore Formation in a Lipid Membrane

    NASA Astrophysics Data System (ADS)

    Chen, Liang; Chen, Junlang; Zhou, Guoquan; Wang, Yu; Xu, Can; Wang, Xiaogang

    2016-09-01

    Bisphenol A (BPA) is particularly considered as one of the most suspicious endocrine disruptors. Exposure to BPA may bring about possible human toxicities, such as cancerous tumors, birth defects and neoteny. One of the key issues to understand its toxicities is how BPA enters cells. In this paper, we perform molecular dynamics simulations to explore the interactions between BPA and a phospholipid membrane (dipalmitoylphosphatidylcholine, DPPC bilayer). The simulation results show that BPA can easily enter the membrane from the aqueous phase. With the increasing concentrations of BPA in the membrane, BPA tends to aggregate and form into cluster. Meanwhile, several DPPC lipids are pulled out from each leaflet and adsorbed on the cluster surface, leading to pore formation. Detailed observations indicate that the lipid extraction results mainly from the dispersion interactions between BPA cluster and lipid tails, as well as weak electrostatic attractions between lipid headgroups and the two hydroxyl groups on BPA. The lipid extraction and pore formation may cause cell membrane damage and are of great importance to uncover BPA’s cytotoxicity.

  13. Poloxamer 188 decreases susceptibility of artificial lipid membranes to electroporation.

    PubMed Central

    Sharma, V; Stebe, K; Murphy, J C; Tung, L

    1996-01-01

    The effect of a nontoxic, nonionic block co-polymeric surface active agent, poloxamer 188, on electroporation of artificial lipid membranes made of azolectin, was investigated. Two different experimental protocols were used in our study: charge pulse and voltage clamp. For the charge pulse protocol, membranes were pulsed with a 10-micronsecond rectangular voltage waveform, after which membrane voltage decay was observed through an external 1-M omega resistance. For the voltage clamp protocol the membranes were pulsed with a waveform that consisted of an initial 10-microsecond rectangular phase, followed by a negative sloped ramp that decayed to zero in the subsequent 500 microseconds. Several parameters characterizing the electroporation process were measured and compared for the control membranes and membranes treated with 1.0 mM poloxamer 188. For both the charge pulse and voltage clamp experiments, the threshold voltage (amplitude of initial rectangular phase) and latency time (time elapsed between the end of rectangular phase and the onset of membrane electroporation) were measured. Membrane conductance (measured 200 microseconds after the initial rectangular phase) and rise time (tr; the time required for the porated membrane to reach a certain conductance value) were also determined for the voltage clamp experiments, and postelectroporation time constant (PE tau; the time constant for transmembrane voltage decay after onset of electroporation) for the charge pulse experiments. The charge pulse experiments were performed on 23 membranes with 10 control and 13 poloxamer-treated membranes, and voltage pulse experiments on 49 membranes with 26 control and 23 poloxamer-treated membranes. For both charge pulse and voltage clamp experiments, poloxamer 188-treated membranes exhibited a statistically higher threshold voltage (p = 0.1 and p = 0.06, respectively), and longer latency time (p = 0.04 and p = 0.05, respectively). Also, poloxamer 188-treated membranes were

  14. Dietary lipids differentially affect membranes from different areas of rooster sperm.

    PubMed

    Bongalhardo, D C; Leeson, S; Buhr, M M

    2009-05-01

    The present work aimed to compare the effect of dietary flax with other oil sources on rooster sperm membranes and on semen characteristics. White Leghorn roosters (16 per diet) were fed 1 of 4 treatments: control diet (CON), or a diet containing corn oil (CORN), fish oil (FISH), or flax seed (FLAX) as the lipid source. Semen from 4 birds (30 wk old) of each treatment was pooled, the sperm head (HM) and body membranes (BM) were isolated, and lipids were extracted and analyzed. Aspects of lipid composition tested were as follows: percentage of individual fatty acids (C14:0 to C24:1) in total fatty acids, percentage of fatty acid categories [saturated, monounsaturated, polyunsaturated (PUFA), n-3 and n-6 PUFA, and n-6:n-3 ratio] within total fatty acids, and percentage of phospholipids [phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol, phosphatidylserine, and sphingomyelin] in total phospholipids. Sperm characteristics evaluated were as follows: volume, concentration, viability, percentage of motile cells, average path velocity, track speed, progressive velocity, lateral head displacement, straightness, and linearity. Diet did not affect membrane phospholipid ratios in either membrane but modified major fatty acids within certain phospholipids. Birds fed FISH and CORN showed, respectively, the highest and the lowest n-3 in sperm, causing reciprocal significant changes in n-6:n-3 ratio. Feeding FLAX caused intermediate effects in n-3, with values significantly lower than FISH but higher than CORN in HM (PC, PE, and phosphatidylinositol) and PC in BM (P < 0.05). In the PE phospholipids, FISH, followed by FLAX, increased n-3 in BM and decreased n-6 PUFA in HM. Sperm concentration was specifically correlated with the amount of 20:4n-6 in FLAX and 22:4n-6 in CON. In FLAX diets, straightness correlated with C18:0, n-3, and n-6:n-3 ratio. Diets containing distinct lipid sources differentially modify the lipid contents of HM and BM, with minor

  15. The role of counterions in the membrane-disruptive properties of pH-sensitive lysine-based surfactants.

    PubMed

    Nogueira, D R; Mitjans, M; Infante, M R; Vinardell, M P

    2011-07-01

    Surfactants are among the most versatile and widely used excipients in pharmaceuticals. This versatility, together with their pH-responsive membrane-disruptive activity and low toxicity, could also enable their potential application in drug delivery systems. Five anionic lysine-based surfactants which differ in the nature of their counterion were studied. Their capacity to disrupt the cell membrane was examined under a range of pH values, concentrations and incubation times, using a standard hemolysis assay as a model for endosomal membranes. The surfactants showed pH-sensitive hemolytic activity and improved kinetics at the endosomal pH range. Low concentrations resulted in negligible hemolysis at physiological pH and high membrane lytic activity at pH 5.4, which is in the range characteristic of late endosomes. With increasing concentration, the surfactants showed an enhanced capacity to lyse cell membranes, and also caused significant membrane disruption at physiological pH. This observation indicates that, at high concentrations, surfactant behavior is independent of pH. The mechanism of surfactant-mediated membrane destabilization was addressed, and scanning electron microscopy studies were also performed to evaluate the effects of the compounds on erythrocyte morphology as a function of pH. The in vitro cytotoxicity of the surfactants was assessed by MTT and NRU assays with the 3T3 cell line. The influence of different types of counterion on hemolytic activity and the potential applications of these surfactants in drug delivery are discussed. The possibility of using pH-sensitive surfactants for endosome disruption could hold great promise for intracellular drug delivery systems in future therapeutic applications. Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  16. Probing the interplay between amyloidogenic proteins and membranes using lipid monolayers and bilayers.

    PubMed

    Relini, Annalisa; Marano, Nadia; Gliozzi, Alessandra

    2014-05-01

    Many degenerative diseases such as Alzheimer's and Parkinson's involve proteins that have a tendency to misfold and aggregate eventually forming amyloid fibers. This review describes the use of monolayers, bilayers, supported membranes, and vesicles as model systems that have helped elucidate the mechanisms and consequences of the interactions between amyloidogenic proteins and membranes. These are twofold: membranes favor the formation of amyloid structures and these induce damage in those membranes. We describe studies that show how interfaces, especially charged ones, favor amyloidogenic protein aggregation by several means. First, surfaces increase the effective protein concentration reducing a three-dimensional system to a two-dimensional one. Second, charged surfaces allow electrostatic interactions with the protein. Anionic lipids as well as rafts, rich in cholesterol and gangliosides, prove to play an especially important role. Finally, these amphipathic systems also offer a hydrophobic environment favoring conformational changes, oligomerization, and eventual formation of mature fibers. In addition, we examine several models for membrane permeabilization: protein pores, leakage induced by extraction of lipids, chaotic pores, and membrane tension, presenting illustrative examples of experimental evidence in support of these models. The picture that emerges from recent work is one where more than one mechanism is in play. Which mechanism prevails depends on the protein, its aggregation state, and the lipid environment in which the interactions occur. © 2013.

  17. A molecular theory for nonohmicity of the ion leak across the lipid-bilayer membrane.

    PubMed

    Fujitani, Y; Bedeaux, D

    1997-10-01

    The current-voltage relationship of ion leak (i.e., ion transport involving neither special channels nor carriers) across the lipid-bilayer membrane has been observed to be log-linear above the ohmic regime. The coefficient of the linear term has been found to be universal for membranes and penetrants examined. This universality has been explained in terms of diffusion in an external field, where the ion position is described as a Markovian process. Such a diffusion picture can be questioned, however. It is also probable that a leaking ion gets over the potential barrier before experiencing sufficient random collision in the membrane, considering that each ion is surrounded with long lipid molecules aligned almost unidirectionally. As an alternative, we discuss this ion leak in terms of velocity distribution of the ions entering the membrane and density fluctuation of the lipids. We conclude that we can explain the universality without resorting to the diffusion picture.

  18. Diffusion and spectroscopy of water and lipids in fully hydrated dimyristoylphosphatidylcholine bilayer membranes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yang, J.; Martí, J., E-mail: jordi.marti@upc.edu; Calero, C.

    2014-03-14

    Microscopic structure and dynamics of water and lipids in a fully hydrated dimyristoylphosphatidylcholine phospholipid lipid bilayer membrane in the liquid-crystalline phase have been analyzed with all-atom molecular dynamics simulations based on the recently parameterized CHARMM36 force field. The diffusive dynamics of the membrane lipids and of its hydration water, their reorientational motions as well as their corresponding spectral densities, related to the absorption of radiation, have been considered for the first time using the present force field. In addition, structural properties such as density and pressure profiles, a deuterium-order parameter, surface tension, and the extent of water penetration in themore » membrane have been analyzed. Molecular self-diffusion, reorientational motions, and spectral densities of atomic species reveal a variety of time scales playing a role in membrane dynamics. The mechanisms of lipid motion strongly depend on the time scale considered, from fast ballistic translation at the scale of picoseconds (effective diffusion coefficients of the order of 10{sup −5} cm{sup 2}/s) to diffusive flow of a few lipids forming nanodomains at the scale of hundreds of nanoseconds (diffusion coefficients of the order of 10{sup −8} cm{sup 2}/s). In the intermediate regime of sub-diffusion, collisions with nearest neighbors prevent the lipids to achieve full diffusion. Lipid reorientations along selected directions agree well with reported nuclear magnetic resonance data and indicate two different time scales, one about 1 ns and a second one in the range of 2–8 ns. We associated the two time scales of reorientational motions with angular distributions of selected vectors. Calculated spectral densities corresponding to lipid and water reveal an overall good qualitative agreement with Fourier transform infrared spectroscopy experiments. Our simulations indicate a blue-shift of the low frequency spectral bands of hydration water as a

  19. Model Lipid Membranes on a Tunable Polymer Cushion

    NASA Astrophysics Data System (ADS)

    Smith, Hillary L.; Jablin, Michael S.; Vidyasagar, Ajay; Saiz, Jessica; Watkins, Erik; Toomey, Ryan; Hurd, Alan J.; Majewski, Jaroslaw

    2009-06-01

    A hydrated, surface-tethered polymer network capable of fivefold change in thickness over a 25-37°C temperature range has been demonstrated via neutron reflectivity and fluorescence microscopy to be a novel support for single lipid bilayers in a liquid environment. As the polymer swells from 170 to 900 Å, it promotes both in- and out-of-plane fluctuations of the supported membrane. The cushioned bilayer proved to be very robust, remaining structurally intact for 16 days and many temperature cycles. The promotion of membrane fluctuations offers far-reaching applications for this system as a surrogate biomembrane.

  20. Coupling of lipid membrane elasticity and in-plane dynamics

    NASA Astrophysics Data System (ADS)

    Tsang, Kuan-Yu; Lai, Yei-Chen; Chiang, Yun-Wei; Chen, Yi-Fan

    2017-07-01

    Biomembranes exhibit liquid and solid features concomitantly with their in-plane fluidity and elasticity tightly regulated by cells. Here, we present experimental evidence supporting the existence of the dynamics-elasticity correlations for lipid membranes and propose a mechanism involving molecular packing densities to explain them. This paper thereby unifies, at the molecular level, the aspects of the continuum mechanics long used to model the two membrane features. This ultimately may elucidate the universal physical principles governing the cellular phenomena involving biomembranes.

  1. Fractional hereditariness of lipid membranes: Instabilities and linearized evolution.

    PubMed

    Deseri, L; Pollaci, P; Zingales, M; Dayal, K

    2016-05-01

    In this work lipid ordering phase changes arising in planar membrane bilayers is investigated both accounting for elasticity alone and for effective viscoelastic response of such assemblies. The mechanical response of such membranes is studied by minimizing the Gibbs free energy which penalizes perturbations of the changes of areal stretch and their gradients only (Deseri and Zurlo, 2013). As material instabilities arise whenever areal stretches characterizing homogeneous configurations lie inside the spinoidal zone of the free energy density, bifurcations from such configurations are shown to occur as oscillatory perturbations of the in-plane displacement. Experimental observations (Espinosa et al., 2011) show a power-law in-plane viscous behavior of lipid structures allowing for an effective viscoelastic behavior of lipid membranes, which falls in the framework of Fractional Hereditariness. A suitable generalization of the variational principle invoked for the elasticity is applied in this case, and the corresponding Euler-Lagrange equation is found together with a set of boundary and initial conditions. Separation of variables allows for showing how Fractional Hereditariness owes bifurcated modes with a larger number of spatial oscillations than the corresponding elastic analog. Indeed, the available range of areal stresses for material instabilities is found to increase with respect to the purely elastic case. Nevertheless, the time evolution of the perturbations solving the Euler-Lagrange equation above exhibits time-decay and the large number of spatial oscillation slowly relaxes, thereby keeping the features of a long-tail type time-response. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Acyl chain length and charge effect on Tamoxifen-lipid model membrane interactions

    NASA Astrophysics Data System (ADS)

    Bilge, Duygu; Kazanci, Nadide; Severcan, Feride

    2013-05-01

    Tamoxifen (TAM), which is an antiestrogenic agent, is widely used during chemotherapy of breast, pancreas, brain and liver cancers. In this study, TAM and model membrane interactions in the form of multilamellar vesicles (MLVs) were studied for lipids containing different acyl chain length and different charge status as a function of different TAM (1, 6, 9 and 15 mol%) concentrations. Zwitterionic lipids namely dipalmitoyl phosphatidylcholine (DPPC), and dimyristoylphosphatidylcholine (DMPC) lipids were used to see the acyl chain length effect and anionic dipalmitoyl phosphtidylglycerol (DPPG) lipid was used to see the charge effect. For this purpose Fourier transform-infrared (FTIR) spectroscopic and differential scanning calorimetric (DSC) techniques have been conducted. For zwitterionic lipid, concentration dependent different action of TAM was observed both in the gel and liquid crystalline phases by significantly increasing the lipid order and decreasing the dynamics for 1 mol% TAM, while decreasing the lipid order and increasing the dynamics of the lipids for higher concentrations (6, 9 and 15 mol%). However, different than neutral lipids, the dynamics and disorder of DPPG liposome increased for all TAM concentrations. The interactions between TAM and head group of multilamellar liposomes was monitored by analyzing the Cdbnd O stretching and PO2- antisymmetric double bond stretching bands. Increasing Tamoxifen concentrations led to a dehydration around these functional groups in the polar part of the lipids. DSC studies showed that for all types of lipids, TAM eliminates the pre-transition, shifts the main phase transition to lower temperatures and broadened the phase transition curve. The results indicate that not the acyl chain length but the charge status of the polar head group induces different effects on lipid membranes order and dynamics.

  3. Suppression of IAPP fibrillation at anionic lipid membranes via IAPP-derived amyloid inhibitors and insulin.

    PubMed

    Sellin, Daniel; Yan, Li-Mei; Kapurniotu, Aphrodite; Winter, Roland

    2010-08-01

    Aggregation of human islet amyloid polypeptide (hIAPP) into cytotoxic beta-sheet oligomers and amyloid plaques is considered a key event in pancreatic beta-cell degeneration in type 2 diabetes (T2D). hIAPP is synthesized in the pancreatic beta-cells and it is stored, co-processed in the secretory granules, and co-secreted to the extracellular matrix together with insulin. In vivo, hIAPP aggregation may start and proceed at the water-cell membrane interface and anionic lipid membranes strongly enhance the process of hIAPP fibrillization which is causally linked to membrane disintegration and cell degeneration. In this study we explored the amyloidogenic propensity and conformational properties of hIAPP in the presence of negatively charged membrane (DOPC/DOPG phospholipid bilayers) surfaces upon addition of two recently designed potent hIAPP-derived inhibitors of hIAPP amyloidogenesis, the hexapeptide NF(N-Me)GA(N-Me)IL (NFGAIL-GI) and the 37-residue non-amyloidogenic hIAPP analog [(N-Me)G24, (N-Me)I26]-IAPP (IAPP-GI). For comparison, the effects of insulin, which is a natively occurring hIAPP aggregation inhibitor, rat IAPP (rIAPP), which is a natively non-amyloidogenic hIAPP analog, and the hIAPP amyloid core peptide hIAPP(22-27) or NFGAIL were also studied. The aim of our study was to test whether and how the above peptides which have been shown to completely block or suppress hIAPP amyloidogenesis in bulk solution in vitro would also affect these processes in the presence of lipid membranes. To this end, attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) was applied. We find that IAPP-GI, NFGAIL-GI, insulin, and rIAPP are potent inhibitors of hIAPP fibrillization. Importantly, our data also suggest that the hetero-complexes of IAPP-GI, rIAPP, and insulin with hIAPP although non-amyloidogenic per se are still able to adsorb at the lipid membrane. By contrast, in the presence of NFGAIL-GI, interaction of hIAPP with the lipid membrane is

  4. Effects of cholesterol on plasma membrane lipid order in MCF-7 cells by two-photon microscopy

    NASA Astrophysics Data System (ADS)

    Zeng, Yixiu; Chen, Jianling; Yang, Hongqin; Wang, Yuhua; Li, Hui; Xie, Shusen

    2014-09-01

    Lipid rafts are cholesterol- and glycosphingolipids- enriched microdomains on plasma membrane surface of mammal cells, involved in a variety of cellular processes. Depleting cholesterol from the plasma membrane by drugs influences the trafficking of lipid raft markers. Optical imaging techniques are powerful tools to study lipid rafts in live cells due to its noninvasive feature. In this study, breast cancer cells MCF-7 were treated with different concentrations of MβCD to deplete cholesterol and an environmentally sensitive fluorescence probe, Laurdan was loaded to image lipid order by two-photon microscopy. The generalized polarization (GP) values were calculated to distinguish the lipid order and disorder phase. GP images and GP distributions of native and cholesterol-depleted MCF-7 cells were obtained. Our results suggest that even at low concentration (0.5 mM) of MβCD, the morphology of the MCF-7 cells changes. Small high GP areas (lipid order phase) decrease more rapidly than low GP areas (lipid disorder phase), indicating that lipid raft structure was altered more severely than nonraft domains. The data demonstrates that cholesterol dramatically affect raft coverage and plasma membrane fluidity in living cells.

  5. Defense related decadienal elicits membrane lipid remodeling in the diatom Phaeodactylum tricornutum

    PubMed Central

    Sabharwal, Tanya; Sathasivan, Kanagasabapathi

    2017-01-01

    Diatoms rapidly release extracellular oxylipins (oxygenated lipids) including polyunsaturated aldehydes in response to herbivory and other stresses. Oxylipins have several defense-related activities including inhibition of reproduction in herbivores and signaling to distant diatoms. Physiological changes in diatoms exposed to varying levels of oxylipins are only beginning to be understood. In this study, Phaeodactylum tricornutum cultures were treated with sublethal concentrations of the polyunsaturated aldehyde trans,trans-2,4-decadienal (DD) to assess effects on lipid composition and membrane permeability. In cells treated with DD for 3 hr, all measured saturated and unsaturated fatty acids significantly decreased (0.46–0.69 fold of levels in solvent control cells) except for 18:2 (decreased but not significantly). The decrease was greater in the polyunsaturated fatty acid pool than the saturated and monounsaturated fatty acid pool. Analysis of lipid classes revealed increased abundances of phosphatidylethanolamine and phosphatidylcholine at 3 and 6 hr. Concomitantly, these and other membrane lipids exhibited increased saturated and monounsaturated acyl chains content relative to polyunsaturated acyl chains compared to control cells. Evidence of decreased plasma membrane permeability in DD treated cells was obtained, based on reduced uptake of two of three dyes relative to control cells. Additionally, cells pre-conditioned with a sublethal DD dose for 3 hr then treated with a lethal DD dose for 2 hr exhibited greater membrane integrity than solvent pre-conditioned control cells that were similarly treated. Taken together, the data are supportive of the hypothesis that membrane remodeling induced by sublethal DD is a key element in the development of cellular resistance in diatoms to varying and potentially toxic levels of polyunsaturated aldehydes in environments impacted by herbivory or other stresses. PMID:28582415

  6. Sensing small molecule interactions with lipid membranes by local pH modulation.

    PubMed

    Huang, Da; Zhao, Tao; Xu, Wei; Yang, Tinglu; Cremer, Paul S

    2013-11-05

    Herein, we utilized a label-free sensing platform based on pH modulation to detect the interactions between tetracaine, a positively charged small molecule used as a local anesthetic, and planar supported lipid bilayers (SLBs). The SLBs were patterned inside a flow cell, allowing for various concentrations of tetracaine to be introduced over the surface in a buffer solution. Studies with membranes containing POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) yielded an equilibrium dissociation constant value of Kd = 180 ± 47 μm for this small molecule-membrane interaction. Adding cholesterol to the SLBs decreased the affinity between tetracaine and the bilayers, while this interaction tightened when POPE (1-hexadecanoyl-2-(9-Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine) was added. Studies were also conducted with three negatively charged membrane lipids, POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt)), POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (sodium salt)), and ganglioside GM1. All three measurements gave rise to a similar tightening of the apparent Kd value compared with pure POPC membranes. The lack of chemical specificity with the identity of the negatively charged lipid indicated that the tightening was largely electrostatic. Through a direct comparison with ITC measurements, it was found that the pH modulation sensor platform offers a facile, inexpensive, highly sensitive, and rapid method for the detection of interactions between putative drug candidates and lipid bilayers. As such, this technique may potentially be exploited as a screen for drug development and analysis.

  7. Molecular dynamics study of lipid bilayers modeling the plasma membranes of normal murine thymocytes and leukemic GRSL cells.

    PubMed

    Andoh, Yoshimichi; Okazaki, Susumu; Ueoka, Ryuichi

    2013-04-01

    Molecular dynamics (MD) calculations for the plasma membranes of normal murine thymocytes and thymus-derived leukemic GRSL cells in water have been performed under physiological isothermal-isobaric conditions (310.15K and 1 atm) to investigate changes in membrane properties induced by canceration. The model membranes used in our calculations for normal and leukemic thymocytes comprised 23 and 25 kinds of lipids, respectively, including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. The mole fractions of the lipids adopted here were based on previously published experimental values. Our calculations clearly showed that the membrane area was increased in leukemic cells, and that the isothermal area compressibility of the leukemic plasma membranes was double that of normal cells. The calculated membranes of leukemic cells were thus considerably bulkier and softer in the lateral direction compared with those of normal cells. The tilt angle of the cholesterol and the conformation of the phospholipid fatty acid tails both showed a lower level of order in leukemic cell membranes compared with normal cell membranes. The lateral radial distribution function of the lipids also showed a more disordered structure in leukemic cell membranes than in normal cell membranes. These observations all show that, for the present thymocytes, the lateral structure of the membrane is considerably disordered by canceration. Furthermore, the calculated lateral self-diffusion coefficient of the lipid molecules in leukemic cell membranes was almost double that in normal cell membranes. The calculated rotational and wobbling autocorrelation functions also indicated that the molecular motion of the lipids was enhanced in leukemic cell membranes. Thus, here we have demonstrated that the membranes of thymocyte leukemic cells are more disordered and more fluid than normal cell membranes. Copyright © 2013

  8. S4(13)-PV cell-penetrating peptide induces physical and morphological changes in membrane-mimetic lipid systems and cell membranes: implications for cell internalization.

    PubMed

    Cardoso, Ana M S; Trabulo, Sara; Cardoso, Ana L; Lorents, Annely; Morais, Catarina M; Gomes, Paula; Nunes, Cláudia; Lúcio, Marlene; Reis, Salette; Padari, Kärt; Pooga, Margus; Pedroso de Lima, Maria C; Jurado, Amália S

    2012-03-01

    The present work aims to gain insights into the role of peptide-lipid interactions in the mechanisms of cellular internalization and endosomal escape of the S4(13)-PV cell-penetrating peptide, which has been successfully used in our laboratory as a nucleic acid delivery system. A S4(13)-PV analogue, S4(13)-PVscr, displaying a scrambled amino acid sequence, deficient cell internalization and drug delivery inability, was used in this study for comparative purposes. Differential scanning calorimetry, fluorescence polarization and X-ray diffraction at small and wide angles techniques showed that both peptides interacted with anionic membranes composed of phosphatidylglycerol or a mixture of this lipid with phosphatidylethanolamine, increasing the lipid order, shifting the phase transition to higher temperatures and raising the correlation length between the bilayers. However, S4(13)-PVscr, in contrast to the wild-type peptide, did not promote lipid domain segregation and induced the formation of an inverted hexagonal lipid phase instead of a cubic phase in the lipid systems assayed. Electron microscopy showed that, as opposed to S4(13)-PVscr, the wild-type peptide induced the formation of a non-lamellar organization in membranes of HeLa cells. We concluded that lateral phase separation and destabilization of membrane lamellar structure without compromising membrane integrity are on the basis of the lipid-driven and receptor-independent mechanism of cell entry of S4(13)-PV peptide. Overall, our results can contribute to a better understanding of the role of peptide-lipid interactions in the mechanisms of cell-penetrating peptide membrane translocation, helping in the future design of more efficient cell-penetrating peptide-based drug delivery systems. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. Transcriptional Regulation of T-Cell Lipid Metabolism: Implications for Plasma Membrane Lipid Rafts and T-Cell Function.

    PubMed

    Robinson, George A; Waddington, Kirsty E; Pineda-Torra, Ines; Jury, Elizabeth C

    2017-01-01

    It is well established that cholesterol and glycosphingolipids are enriched in the plasma membrane (PM) and form signaling platforms called lipid rafts, essential for T-cell activation and function. Moreover, changes in PM lipid composition affect the biophysical properties of lipid rafts and have a role in defining functional T-cell phenotypes. Here, we review the role of transcriptional regulators of lipid metabolism including liver X receptors α/β, peroxisome proliferator-activated receptor γ, estrogen receptors α/β (ERα/β), and sterol regulatory element-binding proteins in T-cells. These receptors lie at the interface between lipid metabolism and immune cell function and are endogenously activated by lipids and/or hormones. Importantly, they regulate cellular cholesterol, fatty acid, glycosphingolipid, and phospholipid levels but are also known to modulate a broad spectrum of immune responses. The current evidence supporting a role for lipid metabolism pathways in controlling immune cell activation by influencing PM lipid raft composition in health and disease, and the potential for targeting lipid biosynthesis pathways to control unwanted T-cell activation in autoimmunity is reviewed.

  10. Transcriptional Regulation of T-Cell Lipid Metabolism: Implications for Plasma Membrane Lipid Rafts and T-Cell Function

    PubMed Central

    Robinson, George A.; Waddington, Kirsty E.; Pineda-Torra, Ines; Jury, Elizabeth C.

    2017-01-01

    It is well established that cholesterol and glycosphingolipids are enriched in the plasma membrane (PM) and form signaling platforms called lipid rafts, essential for T-cell activation and function. Moreover, changes in PM lipid composition affect the biophysical properties of lipid rafts and have a role in defining functional T-cell phenotypes. Here, we review the role of transcriptional regulators of lipid metabolism including liver X receptors α/β, peroxisome proliferator-activated receptor γ, estrogen receptors α/β (ERα/β), and sterol regulatory element-binding proteins in T-cells. These receptors lie at the interface between lipid metabolism and immune cell function and are endogenously activated by lipids and/or hormones. Importantly, they regulate cellular cholesterol, fatty acid, glycosphingolipid, and phospholipid levels but are also known to modulate a broad spectrum of immune responses. The current evidence supporting a role for lipid metabolism pathways in controlling immune cell activation by influencing PM lipid raft composition in health and disease, and the potential for targeting lipid biosynthesis pathways to control unwanted T-cell activation in autoimmunity is reviewed. PMID:29225604

  11. Electron paramagnetic resonance study of lipid and protein membrane components of erythrocytes oxidized with hydrogen peroxide

    PubMed Central

    Mendanha, S.A.; Anjos, J.L.V.; Silva, A.H.M.; Alonso, A.

    2012-01-01

    Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to monitor membrane dynamic changes in erythrocytes subjected to oxidative stress with hydrogen peroxide (H2O2). The lipid spin label, 5-doxyl stearic acid, responded to dramatic reductions in membrane fluidity, which was correlated with increases in the protein content of the membrane. Membrane rigidity, associated with the binding of hemoglobin (Hb) to the erythrocyte membrane, was also indicated by a spin-labeled maleimide, 5-MSL, covalently bound to the sulfhydryl groups of membrane proteins. At 2% hematocrit, these alterations in membrane occurred at very low concentrations of H2O2 (50 µM) after only 5 min of incubation at 37°C in azide phosphate buffer, pH 7.4. Lipid peroxidation, suggested by oxidative hemolysis and malondialdehyde formation, started at 300 µM H2O2 (for incubation of 3 h), which is a concentration about six times higher than those detected with the probes. Ascorbic acid and α-tocopherol protected the membrane against lipoperoxidation, but did not prevent the binding of proteins to the erythrocyte membrane. Moreover, the antioxidant (+)-catechin, which also failed to prevent the cross-linking of cytoskeletal proteins with Hb, was very effective in protecting erythrocyte ghosts from lipid peroxidation induced by the Fenton reaction. This study also showed that EPR spectroscopy can be useful to assess the molecular dynamics of red blood cell membranes in both the lipid and protein domains and examine oxidation processes in a system that is so vulnerable to oxidation. PMID:22473321

  12. Interaction of saponin 1688 with phase separated lipid bilayers.

    PubMed

    Chen, Maohui; Balhara, Vinod; Jaimes Castillo, Ana Maria; Balsevich, John; Johnston, Linda J

    2017-07-01

    Saponins are a diverse family of naturally occurring plant triterpene or steroid glycosides that have a wide range of biological activities. They have been shown to permeabilize membranes and in some cases membrane disruption has been hypothesized to involve saponin/cholesterol complexes. We have examined the interaction of steroidal saponin 1688-1 with lipid membranes that contain cholesterol and have a mixture of liquid-ordered (L o ) and liquid-disordered (L d ) phases as a model for lipid rafts in cellular membranes. A combination of atomic force microscopy (AFM) and fluorescence was used to probe the effect of saponin on the bilayer. The results demonstrate that saponin forms defects in the membrane and also leads to formation of small aggregates on the membrane surface. Although most of the membrane damage occurs in the liquid-disordered phase, fluorescence results demonstrate that saponin localizes in both ordered and disordered membrane phases, with a modest preference for the disordered regions. Similar effects are observed for both direct incorporation of saponin in the lipid mixture used to make vesicles/bilayers and for incubation of saponin with preformed bilayers. The results suggest that the initial sites of interaction are at the interface between the domains and surrounding disordered phase. The preference for saponin localization in the disordered phase may reflect the ease of penetration of saponin into a less ordered membrane, rather than the actual cholesterol concentration in the membrane. Dye leakage assays indicate that a high concentration of saponin is required for membrane permeabilization consistent with the supported lipid bilayer experiments. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  13. Lipid engineering reveals regulatory roles for membrane fluidity in yeast flocculation and oxygen-limited growth

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Degreif, Daniel; de Rond, Tristan; Bertl, Adam

    Cells modulate lipid metabolism in order to maintain membrane homeostasis. In this paper, we use a metabolic engineering approach to manipulate the stoichiometry of fatty acid unsaturation, a regulator of cell membrane fluidity, in Saccharomyces cerevisiae. Unexpectedly, reduced lipid unsaturation triggered cell-cell adhesion (flocculation), a phenomenon characteristic of industrial yeast but uncommon in laboratory strains. We find that ER lipid saturation sensors induce expression of FLO1 – encoding a cell wall polysaccharide binding protein – independently of its canonical regulator. In wild-type cells, Flo1p-dependent flocculation occurs under oxygen-limited growth, which reduces unsaturated lipid synthesis and thus serves as the environmentalmore » trigger for flocculation. Transcriptional analysis shows that FLO1 is one of the most highly induced genes in response to changes in lipid unsaturation, and that the set of membrane fluidity-sensitive genes is globally activated as part of the cell's long-term response to hypoxia during fermentation. Finally, our results show how the lipid homeostasis machinery of budding yeast is adapted to carry out a broad response to an environmental stimulus important in biotechnology.« less

  14. Lipid engineering reveals regulatory roles for membrane fluidity in yeast flocculation and oxygen-limited growth.

    PubMed

    Degreif, Daniel; de Rond, Tristan; Bertl, Adam; Keasling, Jay D; Budin, Itay

    2017-05-01

    Cells modulate lipid metabolism in order to maintain membrane homeostasis. Here we use a metabolic engineering approach to manipulate the stoichiometry of fatty acid unsaturation, a regulator of cell membrane fluidity, in Saccharomyces cerevisiae. Unexpectedly, reduced lipid unsaturation triggered cell-cell adhesion (flocculation), a phenomenon characteristic of industrial yeast but uncommon in laboratory strains. We find that ER lipid saturation sensors induce expression of FLO1 - encoding a cell wall polysaccharide binding protein - independently of its canonical regulator. In wild-type cells, Flo1p-dependent flocculation occurs under oxygen-limited growth, which reduces unsaturated lipid synthesis and thus serves as the environmental trigger for flocculation. Transcriptional analysis shows that FLO1 is one of the most highly induced genes in response to changes in lipid unsaturation, and that the set of membrane fluidity-sensitive genes is globally activated as part of the cell's long-term response to hypoxia during fermentation. Our results show how the lipid homeostasis machinery of budding yeast is adapted to carry out a broad response to an environmental stimulus important in biotechnology. Copyright © 2017 International Metabolic Engineering Society. All rights reserved.

  15. Fourier transform infrared spectroscopic study of the interactions of a strongly antimicrobial but weakly hemolytic analogue of gramicidin S with lipid micelles and lipid bilayer membranes.

    PubMed

    Lewis, Ruthven N A H; Kiricsi, Monika; Prenner, Elmar J; Hodges, Robert S; McElhaney, Ronald N

    2003-01-21

    Cyclo[VKLdKVdYPLKVKLdYP] (GS14dK(4)), a synthetic tetradecameric ring-size analogue of the naturally occurring antimicrobial peptide gramicidin S (GS), retains the strong antimicrobial activity of GS but is 15-20 times less hemolytic. To characterize its interaction with lipid membranes and to understand the molecular basis of its capacity to lyse bacterial cells, in preference to erythrocytes, we have investigated the interactions of GS14dK(4) with detergent micelles and with lipid bilayer model membranes by Fourier transform infrared spectroscopy and compared our results with those of a similar study of GS [Lewis, R. N. A. H., et al. (1999) Biochemistry 38, 15193-15203]. In both aqueous and organic solvent solutions, GS14dK(4) adopts a beta-sheet conformation that is somewhat distorted and more sensitive to the polarity of its environment than GS. Like GS, GS14dK(4) is completely or partially excluded from gel-state lipid bilayers but interacts strongly with liquid-crystalline lipid bilayers and detergent micelle, and interacts more strongly with more fluid liquid-crystalline lipid systems. However, its interactions are more strongly influenced by membrane lipid order and fluidity, and unlike GS, it is essentially excluded from cholesterol-containing phospholipid bilayers. Also, GS14dK(4) is excluded from cationic lipid bilayers, but partitions more strongly and/or penetrates more deeply into anionic lipid bilayers than into those composed of either zwitterionic or nonionic lipids. Anionic lipids also facilitate GS14dK(4) interactions with multicomponent lipid bilayers which are predominantly zwitterionic or nonionic. Although GS14dK(4) generally penetrates and/or partitions into zwitterionic or uncharged lipid bilayers less strongly than does GS, its greater size and altered distribution of positive charges make it intrinsically more perturbing with regard to membrane organization once associated with lipid bilayers. This fact, combined with its relatively

  16. Examining the Role of Membrane Lipid Composition in Determining the Ethanol Tolerance of Saccharomyces cerevisiae

    PubMed Central

    Henderson, Clark M.

    2014-01-01

    Yeast (Saccharomyces cerevisiae) has an innate ability to withstand high levels of ethanol that would prove lethal to or severely impair the physiology of other organisms. Significant efforts have been undertaken to elucidate the biochemical and biophysical mechanisms of how ethanol interacts with lipid bilayers and cellular membranes. This research has implicated the yeast cellular membrane as the primary target of the toxic effects of ethanol. Analysis of model membrane systems exposed to ethanol has demonstrated ethanol's perturbing effect on lipid bilayers, and altering the lipid composition of these model bilayers can mitigate the effect of ethanol. In addition, cell membrane composition has been correlated with the ethanol tolerance of yeast cells. However, the physical phenomena behind this correlation are likely to be complex. Previous work based on often divergent experimental conditions and time-consuming low-resolution methodologies that limit large-scale analysis of yeast fermentations has fallen short of revealing shared mechanisms of alcohol tolerance in Saccharomyces cerevisiae. Lipidomics, a modern mass spectrometry-based approach to analyze the complex physiological regulation of lipid composition in yeast and other organisms, has helped to uncover potential mechanisms for alcohol tolerance in yeast. Recent experimental work utilizing lipidomics methodologies has provided a more detailed molecular picture of the relationship between lipid composition and ethanol tolerance. While it has become clear that the yeast cell membrane composition affects its ability to tolerate ethanol, the molecular mechanisms of yeast alcohol tolerance remain to be elucidated. PMID:24610851

  17. Nanosecond pulsed electric field (nsPEF) enhance cytotoxicity of cisplatin to hepatocellular cells by microdomain disruption on plasma membrane.

    PubMed

    Yin, Shengyong; Chen, Xinhua; Xie, Haiyang; Zhou, Lin; Guo, Danjing; Xu, Yuning; Wu, Liming; Zheng, Shusen

    2016-08-15

    Previous studies showed nanosecond pulsed electric field (nsPEF) can ablate solid tumors including hepatocellular carcinoma (HCC) but its effect on cell membrane is not fully understood. We hypothesized nsPEF disrupt the microdomains on outer-cellular membrane with direct mechanical force and as a result the plasma membrane permeability increases to facilitate the small molecule intake. Three HCC cells were pulsed one pulse per minute, an interval longer than nanopore resealing time. The cationized ferritin was used to mark up the electronegative microdomains, propidium iodide (PI) for membrane permeabilization, energy dispersive X-ray spectroscopy (EDS) for the negative cell surface charge and cisplatin for inner-cellular cytotoxicity. We demonstrated that the ferritin marked-microdomain and negative cell surface charge were disrupted by nsPEF caused-mechanical force. The cell uptake of propidium and cytotoxicity of DNA-targeted cisplatin increased with a dose effect. Cisplatin gains its maximum inner-cellular cytotoxicity when combining with nsPEF stimulation. We conclude that nsPEF disrupt the microdomains on the outer cellular membrane directly and increase the membrane permeabilization for PI and cisplatin. The microdomain disruption and membrane infiltration changes are caused by the mechanical force from the changes of negative cell surface charge. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Lennard-Jones type pair-potential method for coarse-grained lipid bilayer membrane simulations in LAMMPS

    NASA Astrophysics Data System (ADS)

    Fu, S.-P.; Peng, Z.; Yuan, H.; Kfoury, R.; Young, Y.-N.

    2017-01-01

    Lipid bilayer membranes have been extensively studied by coarse-grained molecular dynamics simulations. Numerical efficiencies have been reported in the cases of aggressive coarse-graining, where several lipids are coarse-grained into a particle of size 4 ∼ 6 nm so that there is only one particle in the thickness direction. Yuan et al. proposed a pair-potential between these one-particle-thick coarse-grained lipid particles to capture the mechanical properties of a lipid bilayer membrane, such as gel-fluid-gas phase transitions of lipids, diffusion, and bending rigidity Yuan et al. (2010). In this work we implement such an interaction potential in LAMMPS to simulate large-scale lipid systems such as a giant unilamellar vesicle (GUV) and red blood cells (RBCs). We also consider the effect of cytoskeleton on the lipid membrane dynamics as a model for RBC dynamics, and incorporate coarse-grained water molecules to account for hydrodynamic interactions. The interaction between the coarse-grained water molecules (explicit solvent molecules) is modeled as a Lennard-Jones (L-J) potential. To demonstrate that the proposed methods do capture the observed dynamics of vesicles and RBCs, we focus on two sets of LAMMPS simulations: 1. Vesicle shape transitions with enclosed volume; 2. RBC shape transitions with different enclosed volume. Finally utilizing the parallel computing capability in LAMMPS, we provide some timing results for parallel coarse-grained simulations to illustrate that it is possible to use LAMMPS to simulate large-scale realistic complex biological membranes for more than 1 ms.

  19. Snake Cytotoxins Bind to Membranes via Interactions with Phosphatidylserine Head Groups of Lipids

    PubMed Central

    Konshina, Anastasia G.; Boldyrev, Ivan A.; Utkin, Yuri N.; Omel'kov, Anton V.; Efremov, Roman G.

    2011-01-01

    The major representatives of Elapidae snake venom, cytotoxins (CTs), share similar three-fingered fold and exert diverse range of biological activities against various cell types. CT-induced cell death starts from the membrane recognition process, whose molecular details remain unclear. It is known, however, that the presence of anionic lipids in cell membranes is one of the important factors determining CT-membrane binding. In this work, we therefore investigated specific interactions between one of the most abundant of such lipids, phosphatidylserine (PS), and CT 4 of Naja kaouthia using a combined, experimental and modeling, approach. It was shown that incorporation of PS into zwitterionic liposomes greatly increased the membrane-damaging activity of CT 4 measured by the release of the liposome-entrapped calcein fluorescent dye. The CT-induced leakage rate depends on the PS concentration with a maximum at approximately 20% PS. Interestingly, the effects observed for PS were much more pronounced than those measured for another anionic lipid, sulfatide. To delineate the potential PS binding sites on CT 4 and estimate their relative affinities, a series of computer simulations was performed for the systems containing the head group of PS and different spatial models of CT 4 in aqueous solution and in an implicit membrane. This was done using an original hybrid computational protocol implementing docking, Monte Carlo and molecular dynamics simulations. As a result, at least three putative PS-binding sites with different affinities to PS molecule were delineated. Being located in different parts of the CT molecule, these anion-binding sites can potentially facilitate and modulate the multi-step process of the toxin insertion into lipid bilayers. This feature together with the diverse binding affinities of the sites to a wide variety of anionic targets on the membrane surface appears to be functionally meaningful and may adjust CT action against different types of

  20. Spatial orientation and electric-field-driven transport of hypericin inside of bilayer lipid membranes.

    PubMed

    Strejčková, Alena; Staničová, Jana; Jancura, Daniel; Miškovský, Pavol; Bánó, Gregor

    2013-02-07

    Fluorescence experiments were carried out to investigate the interaction of hypericin (Hyp), a natural hydrophobic photosensitizer, with artificial bilayer lipid membranes. The spatial orientation of Hyp monomers incorporated in diphytanoyl phosphatidylcholine (DPhPC) membranes was determined by measuring the dependence of the Hyp fluorescence intensity on the angle of incidence of p- and s-polarized excitation laser beams. Inside of the membrane, Hyp monomers are preferentially located in the layers near the membrane/water interface and are oriented with the S(1) ← S(0) transition dipole moments perpendicular to the membrane surface. Transport of Hyp anions between the two opposite sides of the lipid bilayer was induced by applying rectangular electric field pulses to the membrane. The characteristic time for Hyp transport through the membrane center was evaluated by the analysis of the Hyp fluorescence signal during the voltage pulses. In the zero-voltage limit, the transport time approached 70 ms and gradually decreased with higher voltage applied to the membrane. In addition, our measurements indicated an apparent pK(a) constant of 8 for Hyp deprotonation in the membrane.

  1. Gemfibrozil disrupts the metabolism of circulating lipids in bobwhite quails.

    PubMed

    Bussière-Côté, Sophie; Omlin, Teye; de Càssia Pinheiro, Eliana; Weber, Jean-Michel

    2016-01-01

    The circulating lipids of birds play essential roles for egg production and as an energy source for flight and thermogenesis. How lipid-lowering pharmaceuticals geared to prevent heart disease in humans and that are routinely released in the environment affect their metabolism is unknown. This study assesses the impact of the popular drug gemfibrozil (GEM) on the plasma phospholipids (PL), neutral lipids (NL), and nonesterified fatty acids (NEFA) of bobwhite quails (Colinus virginianus). Results show that bird lipoproteins are rapidly altered by GEM, even at environmentally-relevant doses. After 4 days of exposure, pharmacological amounts cause an 83% increase in circulating PL levels, a major decrease in average lipoprotein size measured as a 56% drop in the NL/PL ratio, and important changes in the fatty acid composition of PL and NEFA (increases in fatty acid unsaturation). The levels of PL carrying all individual fatty acids except arachidonate are strongly stimulated. The large decrease in bird lipoprotein size may reflect the effects seen in humans: lowering of LDL that can cause atherosclerosis and stimulation of HDL that promote cholesterol disposal. Lower (environmental) doses of GEM cause a reduction of %palmitate in all the plasma lipid fractions of quails, but particularly in the core triacylglycerol of lipoproteins (NL). No changes in mRNA levels of bird peroxisome proliferator-activated receptor (PPAR) could be demonstrated. The disrupting effects of GEM on circulating lipids reported here suggest that the pervasive presence of this drug in the environment could jeopardize reproduction and migratory behaviours in wild birds. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Aspirin suppresses the abnormal lipid metabolism in liver cancer cells via disrupting an NFκB-ACSL1 signaling

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yang, Guang; Wang, Yuan; Feng, Jinyan

    Abnormal lipid metabolism is a hallmark of tumorigenesis. Hence, the alterations of metabolism enhance the development of hepatocellular carcinoma (HCC). Aspirin is able to inhibit the growth of cancers through targeting nuclear factor κB (NF-κB). However, the role of aspirin in disrupting abnormal lipid metabolism in HCC remains poorly understood. In this study, we report that aspirin can suppress the abnormal lipid metabolism of HCC cells through inhibiting acyl-CoA synthetase long-chain family member 1 (ACSL1), a lipid metabolism-related enzyme. Interestingly, oil red O staining showed that aspirin suppressed lipogenesis in HepG2 cells and Huh7 cells in a dose-dependent manner. Inmore » addition, aspirin attenuated the levels of triglyceride and cholesterol in the cells, respectively. Strikingly, we identified that aspirin was able to down-regulate ACSL1 at the levels of mRNA and protein. Moreover, we validated that aspirin decreased the nuclear levels of NF-κB in HepG2 cells. Mechanically, PDTC, an inhibitor of NF-κB, could down-regulate ACSL1 at the levels of mRNA and protein in the cells. Functionally, PDTC reduced the levels of lipid droplets, triglyceride and cholesterol in HepG2 cells. Thus, we conclude that aspirin suppresses the abnormal lipid metabolism in HCC cells via disrupting an NFκB-ACSL1 signaling. Our finding provides new insights into the mechanism by which aspirin inhibits abnormal lipid metabolism of HCC. Therapeutically, aspirin is potentially available for HCC through controlling abnormal lipid metabolism. - Highlights: • Aspirin inhibits the levels of liquid droplets, triglyceride and cholesterol in HCC cells. • Aspirin is able to down-regulate ACSL1 in HCC cells. • NF-κB inhibitor PDTC can down-regulate ACSL1 and reduces lipogenesis in HCC cells. • Aspirin suppresses the abnormal lipid metabolism in HCC cells via disrupting an NFκB-ACSL1 signaling.« less

  3. Simultaneous treatment (cell disruption and lipid extraction) of wet microalgae using hydrodynamic cavitation for enhancing the lipid yield.

    PubMed

    Lee, Ilgyu; Han, Jong-In

    2015-06-01

    Simultaneous treatment (combining with cell disruption and lipid extraction) using hydrodynamic cavitation (HC) was applied to Nannochloropsis salina to demonstrate a simple and integrated way to produce oil from wet microalgae. A high lipid yield from the HC (25.9-99.0%) was observed compared with autoclave (16.2-66.5%) and ultrasonication (5.4-26.9%) in terms of the specific energy input (500-10,000 kJ/kg). The optimal conditions for the simultaneous treatment were established using a statistical approach. The efficiency of the simultaneous method was also demonstrated by comparing each separate treatment. The maximum lipid yield (predicted: 45.9% and experimental: 45.5%) was obtained using 0.89% sulfuric acid with a cavitation number of 1.17 for a reaction time of 25.05 min via response surface methodology. Considering its comparable extractability, energy-efficiency, and potential for scale-up, HC may be a promising method to achieve industrial-scale microalgae operation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. On scattered waves and lipid domains: detecting membrane rafts with X-rays and neutrons

    DOE PAGES

    Marquardt, Drew; Heberle, Frederick A.; Nickels, Jonathan D.; ...

    2015-09-21

    In order to understand the biological role of lipids in cell membranes, it is necessary to determine the mesoscopic structure of well-defined model membrane systems. Neutron and X-ray scattering are non-invasive, probe-free techniques that have been used extensively in such systems to probe length scales ranging from angstroms to microns, and dynamics occurring over picosecond to millisecond time scales. Finally, recent developments in the area of phase separated lipid systems mimicking membrane rafts will be presented, and the underlying concepts of the different scattering techniques used to study them will be discussed in detail.

  5. Glycosphingolipid-facilitated membrane insertion and internalization of cobra cardiotoxin. The sulfatide.cardiotoxin complex structure in a membrane-like environment suggests a lipid-dependent cell-penetrating mechanism for membrane binding polypeptides.

    PubMed

    Wang, Chia-Hui; Liu, Jyung-Hurng; Lee, Shao-Chen; Hsiao, Chwan-Deng; Wu, Wen-Guey

    2006-01-06

    Cobra cardiotoxins, a family of basic polypeptides having lipid- and heparin-binding capacities similar to the cell-penetrating peptides, induce severe tissue necrosis and systolic heart arrest in snakebite victims. Whereas cardiotoxins are specifically retained on the cell surface via heparan sulfate-mediated processes, their lipid binding ability appears to be responsible, at least in part, for cardiotoxin-induced membrane leakage and cell death. Although the exact role of lipids involved in toxin-mediated cytotoxicity remains largely unknown, monoclonal anti-sulfatide antibody O4 has recently been shown to inhibit the action of CTX A3, the major cardiotoxin from Taiwan cobra venom, on cardiomyocytes by preventing cardiotoxin-induced membrane leakage and CTX A3 internalization into mitochondria. Here, we show that anti-sulfatide acts by blocking the binding of CTX A3 to the sulfatides in the plasma membrane to prevent sulfatide-dependent CTX A3 membrane pore formation and internalization. We also describe the crystal structure of a CTX A3-sulfatide complex in a membrane-like environment at 2.3 angstroms resolution. The unexpected orientation of the sulfatide fatty chains in the structure allows prediction of the mode of toxin insertion into the plasma membrane. CTX A3 recognizes both the headgroup and the ceramide interfacial region of sulfatide to induce a lipid conformational change that may play a key role in CTX A3 oligomerization and cellular internalization. This proposed lipid-mediated toxin translocation mechanism may also shed light on the cellular uptake mechanism of the amphiphilic cell-penetrating peptides known to involve multiple internalization pathways.

  6. Regulation of adhesion behavior of murine macrophage using supported lipid membranes displaying tunable mannose domains

    NASA Astrophysics Data System (ADS)

    Kaindl, T.; Oelke, J.; Pasc, A.; Kaufmann, S.; Konovalov, O. V.; Funari, S. S.; Engel, U.; Wixforth, A.; Tanaka, M.

    2010-07-01

    Highly uniform, strongly correlated domains of synthetically designed lipids can be incorporated into supported lipid membranes. The systematic characterization of membranes displaying a variety of domains revealed that the equilibrium size of domains significantly depends on the length of fluorocarbon chains, which can be quantitatively interpreted within the framework of an equivalent dipole model. A mono-dispersive, narrow size distribution of the domains enables us to treat the inter-domain correlations as two-dimensional colloidal crystallization and calculate the potentials of mean force. The obtained results demonstrated that both size and inter-domain correlation can precisely be controlled by the molecular structures. By coupling α-D-mannose to lipid head groups, we studied the adhesion behavior of the murine macrophage (J774A.1) on supported membranes. Specific adhesion and spreading of macrophages showed a clear dependence on the density of functional lipids. The obtained results suggest that such synthetic lipid domains can be used as a defined platform to study how cells sense the size and distribution of functional molecules during adhesion and spreading.

  7. Thermodynamic evidence of non-muscle myosin II-lipid-membrane interaction

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Schewkunow, Vitali; Sharma, Karan P.; Diez, Gerold

    2008-02-08

    A unique feature of protein networks in living cells is that they can generate their own force. Proteins such as non-muscle myosin II are an integral part of the cytoskeleton and have the capacity to convert the energy of ATP hydrolysis into directional movement. Non-muscle myosin II can move actin filaments against each other, and depending on the orientation of the filaments and the way in which they are linked together, it can produce contraction, bending, extension, and stiffening. Our measurements with differential scanning calorimetry showed that non-muscle myosin II inserts into negatively charged phospholipid membranes. Using lipid vesicles mademore » of DMPG/DMPC at a molar ratio of 1:1 at 10 mg/ml in the presence of different non-muscle myosin II concentrations showed a variation of the main phase transition of the lipid vesicle at around 23 deg. C. With increasing concentrations of non-muscle myosin II the thermotropic properties of the lipid vesicle changed, which is indicative of protein-lipid interaction/insertion. We hypothesize that myosin tail binds to acidic phospholipids through an electrostatic interaction using the basic side groups of positive residues; the flexible, amphipathic helix then may partially penetrate into the bilayer to form an anchor. Using the stopped-flow method, we determined the binding affinity of non-muscle myosin II when anchored to lipid vesicles with actin, which was similar to a pure actin-non-muscle myosin II system. Insertion of myosin tail into the hydrophobic region of lipid membranes, a model known as the lever arm mechanism, might explain how its interaction with actin generates cellular movement.« less

  8. Quantifying pulsed electric field-induced membrane nanoporation in single cells.

    PubMed

    Moen, Erick K; Ibey, Bennett L; Beier, Hope T; Armani, Andrea M

    2016-11-01

    Plasma membrane disruption can trigger a host of cellular activities. One commonly observed type of disruption is pore formation. Molecular dynamic (MD) simulations of simplified lipid membrane structures predict that controllably disrupting the membrane via nano-scale poration may be possible with nanosecond pulsed electric fields (nsPEF). Until recently, researchers hoping to verify this hypothesis experimentally have been limited to measuring the relatively slow process of fluorescent markers diffusing across the membrane, which is indirect evidence of nanoporation that could be channel-mediated. Leveraging recent advances in nonlinear optical microscopy, we elucidate the role of pulse parameters in nsPEF-induced membrane permeabilization in live cells. Unlike previous techniques, it is able to directly observe loss of membrane order at the onset of the pulse. We also develop a complementary theoretical model that relates increasing membrane permeabilization to membrane pore density. Due to the significantly improved spatial and temporal resolution possible with our imaging method, we are able to directly compare our experimental and theoretical results. Their agreement provides substantial evidence that nanoporation does occur and that its development is dictated by the electric field distribution. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Impact of membrane lipid composition on the structure and stability of the transmembrane domain of amyloid precursor protein

    PubMed Central

    Dominguez, Laura; Foster, Leigh; Straub, John E.; Thirumalai, D.

    2016-01-01

    Cleavage of the amyloid precursor protein (APP) by γ-secretase is a crucial first step in the evolution of Alzheimer’s disease. To discover the cleavage mechanism, it is urgent to predict the structures of APP monomers and dimers in varying membrane environments. We determined the structures of the C9923−55 monomer and homodimer as a function of membrane lipid composition using a multiscale simulation approach that blends atomistic and coarse-grained models. We demonstrate that the C9923−55 homodimer structures form a heterogeneous ensemble with multiple conformational states, each stabilized by characteristic interpeptide interactions. The relative probabilities of each conformational state are sensitive to the membrane environment, leading to substantial variation in homodimer peptide structure as a function of membrane lipid composition or the presence of an anionic lipid environment. In contrast, the helicity of the transmembrane domain of monomeric C991−55 is relatively insensitive to the membrane lipid composition, in agreement with experimental observations. The dimer structures of human EphA2 receptor depend on the lipid environment, which we show is linked to the location of the structural motifs in the dimer interface, thereby establishing that both sequence and membrane composition modulate the complete energy landscape of membrane-bound proteins. As a by-product of our work, we explain the discrepancy in structures predicted for C99 congener homodimers in membrane and micelle environments. Our study provides insight into the observed dependence of C99 protein cleavage by γ-secretase, critical to the formation of amyloid-β protein, on membrane thickness and lipid composition. PMID:27559086

  10. Measuring Membrane Protein Dimerization Equilibrium in Lipid Bilayers by Single-Molecule Fluorescence Microscopy.

    PubMed

    Chadda, R; Robertson, J L

    2016-01-01

    Dimerization of membrane protein interfaces occurs during membrane protein folding and cell receptor signaling. Here, we summarize a method that allows for measurement of equilibrium dimerization reactions of membrane proteins in lipid bilayers, by measuring the Poisson distribution of subunit capture into liposomes by single-molecule photobleaching analysis. This strategy is grounded in the fact that given a comparable labeling efficiency, monomeric or dimeric forms of a membrane protein will give rise to distinctly different photobleaching probability distributions. These methods have been used to verify the dimer stoichiometry of the Fluc F - ion channel and the dimerization equilibrium constant of the ClC-ec1 Cl - /H + antiporter in lipid bilayers. This approach can be applied to any membrane protein system provided it can be purified, fluorescently labeled in a quantitative manner, and verified to be correctly folded by functional assays, even if the structure is not yet known. © 2016 Elsevier Inc. All rights reserved.

  11. Structure-function insights into direct lipid transfer between membranes by Mmm1-Mdm12 of ERMES.

    PubMed

    Kawano, Shin; Tamura, Yasushi; Kojima, Rieko; Bala, Siqin; Asai, Eri; Michel, Agnès H; Kornmann, Benoît; Riezman, Isabelle; Riezman, Howard; Sakae, Yoshitake; Okamoto, Yuko; Endo, Toshiya

    2018-03-05

    The endoplasmic reticulum (ER)-mitochondrial encounter structure (ERMES) physically links the membranes of the ER and mitochondria in yeast. Although the ER and mitochondria cooperate to synthesize glycerophospholipids, whether ERMES directly facilitates the lipid exchange between the two organelles remains controversial. Here, we compared the x-ray structures of an ERMES subunit Mdm12 from Kluyveromyces lactis with that of Mdm12 from Saccharomyces cerevisiae and found that both Mdm12 proteins possess a hydrophobic pocket for phospholipid binding. However in vitro lipid transfer assays showed that Mdm12 alone or an Mmm1 (another ERMES subunit) fusion protein exhibited only a weak lipid transfer activity between liposomes. In contrast, Mdm12 in a complex with Mmm1 mediated efficient lipid transfer between liposomes. Mutations in Mmm1 or Mdm12 impaired the lipid transfer activities of the Mdm12-Mmm1 complex and furthermore caused defective phosphatidylserine transport from the ER to mitochondrial membranes via ERMES in vitro. Therefore, the Mmm1-Mdm12 complex functions as a minimal unit that mediates lipid transfer between membranes. © 2018 Kawano et al.

  12. Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography.

    PubMed

    Weierstall, Uwe; James, Daniel; Wang, Chong; White, Thomas A; Wang, Dingjie; Liu, Wei; Spence, John C H; Bruce Doak, R; Nelson, Garrett; Fromme, Petra; Fromme, Raimund; Grotjohann, Ingo; Kupitz, Christopher; Zatsepin, Nadia A; Liu, Haiguang; Basu, Shibom; Wacker, Daniel; Han, Gye Won; Katritch, Vsevolod; Boutet, Sébastien; Messerschmidt, Marc; Williams, Garth J; Koglin, Jason E; Marvin Seibert, M; Klinker, Markus; Gati, Cornelius; Shoeman, Robert L; Barty, Anton; Chapman, Henry N; Kirian, Richard A; Beyerlein, Kenneth R; Stevens, Raymond C; Li, Dianfan; Shah, Syed T A; Howe, Nicole; Caffrey, Martin; Cherezov, Vadim

    2014-01-01

    Lipidic cubic phase (LCP) crystallization has proven successful for high-resolution structure determination of challenging membrane proteins. Here we present a technique for extruding gel-like LCP with embedded membrane protein microcrystals, providing a continuously renewed source of material for serial femtosecond crystallography. Data collected from sub-10-μm-sized crystals produced with less than 0.5 mg of purified protein yield structural insights regarding cyclopamine binding to the Smoothened receptor.

  13. A Simple, Cost-Efficient Method to Separate Microalgal Lipids from Wet Biomass Using Surface Energy-Modified Membranes.

    PubMed

    Kwak, Moo Jin; Yoo, Youngmin; Lee, Han Sol; Kim, Jiyeon; Yang, Ji-Won; Han, Jong-In; Im, Sung Gap; Kwon, Jong-Hee

    2016-01-13

    For the efficient separation of lipid extracted from microalgae cells, a novel membrane was devised by introducing a functional polymer coating onto a membrane surface by means of an initiated chemical vapor deposition (iCVD) process. To this end, a steel-use-stainless (SUS) membrane was modified in a way that its surface energy was systemically modified. The surface modification by conformal coating of functional polymer film allowed for selective separation of oil-water mixture, by harnessing the tuned interfacial energy between each liquid phase and the membrane surface. The surface-modified membrane, when used with chloroform-based solvent, exhibited superb permeate flux, breakthrough pressure, and also separation yield: it allowed separation of 95.5 ± 1.2% of converted lipid (FAME) in the chloroform phase from the water/MeOH phase with microalgal debris. This result clearly supported that the membrane-based lipid separation is indeed facilitated by way of membrane being functionalized, enabling us to simplify the whole downstream process of microalgae-derived biodiesel production.

  14. BamA POTRA Domain Interacts with a Native Lipid Membrane Surface.

    PubMed

    Fleming, Patrick J; Patel, Dhilon S; Wu, Emilia L; Qi, Yifei; Yeom, Min Sun; Sousa, Marcelo Carlos; Fleming, Karen G; Im, Wonpil

    2016-06-21

    The outer membrane of Gram-negative bacteria is an asymmetric membrane with lipopolysaccharides on the external leaflet and phospholipids on the periplasmic leaflet. This outer membrane contains mainly β-barrel transmembrane proteins and lipidated periplasmic proteins (lipoproteins). The multisubunit protein β-barrel assembly machine (BAM) catalyzes the insertion and folding of the β-barrel proteins into this membrane. In Escherichia coli, the BAM complex consists of five subunits, a core transmembrane β-barrel with a long periplasmic domain (BamA) and four lipoproteins (BamB/C/D/E). The BamA periplasmic domain is composed of five globular subdomains in tandem called POTRA motifs that are key to BAM complex formation and interaction with the substrate β-barrel proteins. The BAM complex is believed to undergo conformational cycling while facilitating insertion of client proteins into the outer membrane. Reports describing variable conformations and dynamics of the periplasmic POTRA domain have been published. Therefore, elucidation of the conformational dynamics of the POTRA domain in full-length BamA is important to understand the function of this molecular complex. Using molecular dynamics simulations, we present evidence that the conformational flexibility of the POTRA domain is modulated by binding to the periplasmic surface of a native lipid membrane. Furthermore, membrane binding of the POTRA domain is compatible with both BamB and BamD binding, suggesting that conformational selection of different POTRA domain conformations may be involved in the mechanism of BAM-facilitated insertion of outer membrane β-barrel proteins. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  15. [Role of membrane lipids in myocardial cytoprotection

    NASA Technical Reports Server (NTRS)

    Grynberg, A.

    2000-01-01

    The cardiomyocyte capacity to regulate ATP production to face any change in energy demand is a major determinant of cardiac function. This process is based on a balanced fatty acid (FA) metabolism, because FA is the main fuel of the heart, although the most expensive one in oxygen. The pathway is, however, weakly controlled by the cardiac myocyte which can well regulate FA mitochondrial entry but not cell FA uptake. For this reason, several pathological situations often result from either harmful accumulation of FA and derivatives or excess FA-oxidation. Control of the FA/glucose balance by decreased energy production from FA would thus offer an alternative strategy in the treatment of ischaemia, providing the cardiomyocytes weak ability in handling the non-metabolised FA is controlled. The initiation and the regulation of cardiac contraction both result from membrane activity; the other major role of lipids in the heart is their contribution to membrane homeostasis through phospholipid synthesis pathways and phospholipases. The anti-anginal activity of Trimetazidine, reported as a cytoprotective effect without a haemo-dynamic component; is associated with reduced use of FA for energy. However, accumulation of FA and derivatives has never been observed. Trimetazidine is reported to increase significantly the synthesis of phospholipids without influencing the other lipid classes, thus increasing the incorporation of FA in membrane structures. This cytoprotection appears to be based on the redirection of the use of FA to phospholipid synthesis, which would decrease their availability for energy production. This class of compounds, with the same properties as Trimetazidine, offers a metabolic approach to the treatment of ischaemia.

  16. The role of blood cell membrane lipids on the mode of action of HIV-1 fusion inhibitor sifuvirtide

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Matos, Pedro M.; Freitas, Teresa; Castanho, Miguel A.R.B.

    2010-12-17

    Research highlights: {yields} Sifuvirtide interacts with erythrocyte and lymphocyte membrane in a concentration dependent manner by decreasing its dipole potential. {yields} Dipole potential variations in lipid vesicles show sifuvirtide's lipid selectivity towards saturated phosphatidylcholines. {yields} This peptide-membrane interaction may direct the drug towards raft-like membrane domains where the receptors used by HIV are located, facilitating its inhibitory action. -- Abstract: Sifuvirtide is a gp41 based peptide that inhibits HIV-1 fusion with the host cells and is currently under clinical trials. Previous studies showed that sifuvirtide partitions preferably to saturated phosphatidylcholine lipid membranes, instead of fluid-phase lipid vesicles. We extended themore » study to the interaction of the peptide with circulating blood cells, by using the dipole potential sensitive probe di-8-ANEPPS. Sifuvirtide decreased the dipole potential of erythrocyte and lymphocyte membranes in a concentration dependent manner, demonstrating its interaction. Also, the lipid selectivity of the peptide towards more rigid phosphatidylcholines was confirmed based on the dipole potential variations. Overall, the interaction of the peptide with the cell membranes is a contribution of different lipid preferences that presumably directs the peptide towards raft-like domains where the receptors are located, facilitating the reach of the peptide to its molecular target, the gp41 in its pre-fusion conformation.« less

  17. Cadmium sulfide mediated photoelectric effects in bilayer lipid membranes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Baral, S.; Fendler, J.H.

    1989-03-01

    Development of semiconductor- and magnetic-particle-containing bilayer lipid membranes (BLMs) has been prompted by the mimetic relevance of these systems. In particular, the authors have been interested for some times in artificial photosynthesis. Optimization of a given system requires an understanding of the mechanisms of photoelectron transfers in the environments of, and across, mimetic membranes. They have undertaken, therefore, a systematic study of semiconductor-mediated photoelectric effects in BLMs. The present article reports results on steady-state and time-resolved, cadmium sulfide mediated photovoltage and photocurrent measurements in glyceryl monooleate BLMs.

  18. Effect of Polycation Structure on Interaction with Lipid Membranes.

    PubMed

    Wilkosz, Natalia; Jamróz, Dorota; Kopeć, Wojciech; Nakai, Keita; Yusa, Shin-Ichi; Wytrwal-Sarna, Magdalena; Bednar, Jan; Nowakowska, Maria; Kepczynski, Mariusz

    2017-08-03

    Interaction of polycations with lipid membranes is a very important issue in many biological and medical applications such as gene delivery or antibacterial usage. In this work, we address the influence of hydrophobic substitution of strong polycations containing quaternary ammonium groups on the polymer-zwitterionic membrane interactions. In particular, we focus on the polymer tendency to adsorb on or/and incorporate into the membrane. We used complementary experimental and computational methods to enhance our understanding of the mechanism of the polycation-membrane interactions. Polycation adsorption on liposomes was assessed using dynamic light scattering (DLS) and zeta potential measurements. The ability of the polymers to form hydrophilic pores in the membrane was evaluated using a calcein-release method. The polymer-membrane interaction at the molecular scale was explored by performing atomistic molecular dynamics (MD) simulations. Our results show that the length of the alkyl side groups plays an essential role in the polycation adhesion on the zwitterionic surface, while the degree of substitution affects the polycation ability to incorporate into the membrane. Both the experimental and computational results show that the membrane permeability can be dramatically affected by the amount of alkyl side groups attached to the polycation main chain.

  19. MICOS and phospholipid transfer by Ups2-Mdm35 organize membrane lipid synthesis in mitochondria.

    PubMed

    Aaltonen, Mari J; Friedman, Jonathan R; Osman, Christof; Salin, Bénédicte; di Rago, Jean-Paul; Nunnari, Jodi; Langer, Thomas; Tatsuta, Takashi

    2016-06-06

    Mitochondria exert critical functions in cellular lipid metabolism and promote the synthesis of major constituents of cellular membranes, such as phosphatidylethanolamine (PE) and phosphatidylcholine. Here, we demonstrate that the phosphatidylserine decarboxylase Psd1, located in the inner mitochondrial membrane, promotes mitochondrial PE synthesis via two pathways. First, Ups2-Mdm35 complexes (SLMO2-TRIAP1 in humans) serve as phosphatidylserine (PS)-specific lipid transfer proteins in the mitochondrial intermembrane space, allowing formation of PE by Psd1 in the inner membrane. Second, Psd1 decarboxylates PS in the outer membrane in trans, independently of PS transfer by Ups2-Mdm35. This latter pathway requires close apposition between both mitochondrial membranes and the mitochondrial contact site and cristae organizing system (MICOS). In MICOS-deficient cells, limiting PS transfer by Ups2-Mdm35 and reducing mitochondrial PE accumulation preserves mitochondrial respiration and cristae formation. These results link mitochondrial PE metabolism to MICOS, combining functions in protein and lipid homeostasis to preserve mitochondrial structure and function. © 2016 Aaltonen et al.

  20. Membrane lipid alterations in the metabolic syndrome and the role of dietary oils.

    PubMed

    Perona, Javier S

    2017-09-01

    The metabolic syndrome is a cluster of pathological conditions, including hypertension, hyperglycemia, hypertriglyceridemia, obesity and low HDL levels that is of great concern worldwide, as individuals with metabolic syndrome have an increased risk of type-2 diabetes and cardiovascular disease. Insulin resistance, the key feature of the metabolic syndrome, might be at the same time cause and consequence of impaired lipid composition in plasma membranes of insulin-sensitive tissues like liver, muscle and adipose tissue. Diet intervention has been proposed as a powerful tool to prevent the development of the metabolic syndrome, since healthy diets have been shown to have a protective role against the components of the metabolic syndrome. Particularly, dietary fatty acids are capable of modulating the deleterious effects of these conditions, among other mechanisms, by modifications of the lipid composition of the membranes in insulin-sensitive tissues. However, there is still scarce data based of high-level evidence on the effects of dietary oils on the effects of the metabolic syndrome and its components. This review summarizes the current knowledge on the effects of dietary oils on improving alterations of the components of the metabolic syndrome. It also examines their influence in the modulation of plasma membrane lipid composition and in the functionality of membrane proteins involved in insulin activity, like the insulin receptor, GLUT-4, CD36/FAT and ABCA-1, and their effect in the metabolism of glucose, fatty acids and cholesterol, and, in turn, the key features of the metabolic syndrome. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Modeling the dynamics of shape generation and sensing by proteins on lipid membranes

    NASA Astrophysics Data System (ADS)

    Walani, Nikhil; Arroyo, Marino

    Lipid membranes are fluid surfaces with flexural resistance that interact with proteins to perform their function in a biological context. A set of these proteins are responsible for shaping the lipid membranes, or of sensing curvature. A large body of work has examined the curvature sensing and generation properties of these proteins. Even though such processes are fundamentally dynamical in cells and in in vitro reconstituted systems, theoretical and computational studies have largely focussed on equilibrium thermodynamics. In this work, we propose a theoretical framework based on Onsager's variational principle of irreversible thermodynamics that captures the dynamics of adsorption, diffusion, and shape generation or sensing of proteins on lipid surfaces. We acknowledge the funds from European Research Council CoG- 681434 to support this research.

  2. Optimization of fluorimetric lipid membrane biosensor sensitivity through manipulation of membrane structure and nitrobenzoxadiazole dipalmitoylphosphatidylethanolamine concentration

    NASA Astrophysics Data System (ADS)

    Shrive, Jason D. A.; Krull, Ulrich J.

    1995-01-01

    In the work reported here, surface concentrations of 0.027 and 0.073 molecules nm-2 of the fluorescent membrane probe molecule nitrobenzoxadiazole dipalmitoylphosphatidylethanolamine (NBD-PE) were shown to yield optimum sensitivity for fluorimetric transduction of membrane structural perturbations for lipid membrane-based biosensor development. These optima were obtained through correlation of experimental data with theoretical predictions of optimum surface concentrations based on a model for NBD-PE self quenching previously published by our group. It was also determined that membrane structural heterogeneity improves the sensitivity of NBD-PE labeled membrane transducers. Together with fluorescence microscopy, observations of surface potential change upon compression or expansion of phosphatidylcholine (PC)/phosphatidic acid (PA) monolayers were used to qualitatively indicate the degree of structural heterogeneity in these membranes. It was determined that sub-microscopic domains must exist in microscopically homogeneous egg PC/egg PA membranes in order to facilitate the observed NBD-PE self-quenching responses upon alteration of bulk pH and therefore, membrane surface electrostatics and structure.

  3. Fluorescent molecular probes based on excited state prototropism in lipid bilayer membrane

    NASA Astrophysics Data System (ADS)

    Mohapatra, Monalisa; Mishra, Ashok K.

    2012-03-01

    Excited state prototropism (ESPT) is observed in molecules having one or more ionizable protons, whose proton transfer efficiency is different in ground and excited states. The interaction of various ESPT molecules like naphthols and intramolecular ESPT (ESIPT) molecules like hydroxyflavones etc. with different microheterogeneous media have been studied in detail and excited state prototropism as a probe concept has been gaining ground. The fluorescence of different prototropic forms of such molecules, on partitioning to an organized medium like lipid bilayer membrane, often show sensitive response to the local environment with respect to the local structure, physical properties and dynamics. Our recent work using 1-naphthol as an ESPT fluorescent molecular probe has shown that the incorporation of monomeric bile salt molecules into lipid bilayer membranes composed from dipalmitoylphosphatidylcholine (DPPC, a lung surfactant) and dimyristoylphosphatidylcholine (DMPC), in solid gel and liquid crystalline phases, induce appreciable wetting of the bilayer up to the hydrocarbon core region, even at very low (<= 1 mM) concentrations of the bile salts. The incorporation and location of fisetin, an ESIPT molecule having antioxidant properties, in lipid bilayer membrane has been sensitively monitored from its intrinsic fluorescence behaviour.

  4. Microfluidic array platform for simultaneous lipid bilayer membrane formation.

    PubMed

    Zagnoni, M; Sandison, M E; Morgan, H

    2009-01-01

    In recent years, protein array technologies have found widespread applications in proteomics. However, new methods for high-throughput analysis of protein-protein and protein-compound interactions are still required. In this paper, an array of lipid bilayer membranes formed within a microfluidic system with integrated electrodes is presented. The system is comprised of three layers that are clamped together, thus rendering the device cleanable and reusable. The device microfluidics enable the simultaneous formation of an array of lipid bilayers using a previously developed air-exposure technique, thereby avoiding the need to manually form individual bilayers. The Ag/AgCl electrodes allow for ion channel measurements, each of the sites being independently addressable. Typically, a 50% yield in simultaneous lipid bilayer formation over 12 sites was obtained and ion channel recordings have been acquired over multiple sites. This system has great potential for the development of an automatable platform of suspended lipid bilayer arrays.

  5. Modulation of physiological and pathological activities of lysozyme by biological membranes.

    PubMed

    Trusova, Valeriya

    2012-09-01

    The molecular details of interactions between lipid membranes and lysozyme (Lz), a small polycationic protein with a wide range of biological activities, have long been the focus of numerous studies. The biological consequences of this process are considered to embrace at least two aspects: i) correlation between antimicrobial and membranotropic properties of this protein, and ii) lipid-mediated Lz amyloidogenesis. The mechanisms underlying the lipid-assisted protein fibrillogenesis and membrane disruption exerted by Lz in bacterial cells are believed to be similar. The present investigation was undertaken to gain further insight into Lz-lipid interactions and explore the routes by which Lz exerts its antimicrobial and amyloidogenic actions. Binding and Förster resonance energy transfer studies revealed that upon increasing the content of anionic lipids in lipid vesicles, Lz forms aggregates in a membrane environment. Total internal reflection fluorescence microscopy and pyrene excimerization reaction were employed to study the effect of Lz on the structural and dynamic properties of lipid bilayers. It was found that Lz induces lipid demixing and reduction of bilayer free volume, the magnitude of this effect being much more pronounced for oligomeric protein.

  6. Membrane preparation and solubilization.

    PubMed

    Roy, Ankita

    2015-01-01

    Membrane proteins play an essential role in several biological processes like ion transport, signal transduction, and electron transfer to name a few. For structural and functional studies of integral membrane proteins, it is critically important to isolate proteins from the membrane using biological detergents. Detergents disrupt the native lipid components of the native membrane and encase the membrane protein in an unnatural environment in aqueous solution. However, a particular membrane protein is best solubilized in a specific detergent; therefore, screening for the optimal detergent is essential. Apart from keeping the membrane protein monodispered in solution, the detergent has to be compatible with downstream processes to isolate and characterize a membrane protein. Over the past several years, a number of membrane proteins have been successfully isolated for structural and functional studies that allowed an outline of general strategies for isolating a novel membrane protein of interest. © 2015 Elsevier Inc. All rights reserved.

  7. Prolonged Intake of Dietary Lipids Alters Membrane Structure and T Cell Responses in LDLr-/- Mice.

    PubMed

    Pollock, Abigail H; Tedla, Nicodemus; Hancock, Sarah E; Cornely, Rhea; Mitchell, Todd W; Yang, Zhengmin; Kockx, Maaike; Parton, Robert G; Rossy, Jérémie; Gaus, Katharina

    2016-05-15

    Although it is recognized that lipids and membrane organization in T cells affect signaling and T cell activation, to what extent dietary lipids alter T cell responsiveness in the absence of obesity and inflammation is not known. In this study, we fed low-density lipoprotein receptor knockout mice a Western high-fat diet for 1 or 9 wk and examined T cell responses in vivo along with T cell lipid composition, membrane order, and activation ex vivo. Our data showed that high levels of circulating lipids for a prolonged period elevated CD4(+) and CD8(+) T cell proliferation and resulted in an increased proportion of CD4(+) central-memory T cells within the draining lymph nodes following induction of contact hypersensitivity. In addition, the 9-wk Western high-fat diet elevated the total phospholipid content and monounsaturated fatty acid level, but decreased saturated phosphatidylcholine and sphingomyelin within the T cells. The altered lipid composition in the circulation, and of T cells, was also reflected by enhanced membrane order at the activation site of ex vivo activated T cells that corresponded to increased IL-2 mRNA levels. In conclusion, dietary lipids can modulate T cell lipid composition and responses in lipoprotein receptor knockout mice even in the absence of excess weight gain and a proinflammatory environment. Copyright © 2016 by The American Association of Immunologists, Inc.

  8. DNA probes for monitoring dynamic and transient molecular encounters on live cell membranes

    NASA Astrophysics Data System (ADS)

    You, Mingxu; Lyu, Yifan; Han, Da; Qiu, Liping; Liu, Qiaoling; Chen, Tao; Sam Wu, Cuichen; Peng, Lu; Zhang, Liqin; Bao, Gang; Tan, Weihong

    2017-05-01

    Cells interact with the extracellular environment through molecules expressed on the membrane. Disruption of these membrane-bound interactions (or encounters) can result in disease progression. Advances in super-resolution microscopy have allowed membrane encounters to be examined, however, these methods cannot image entire membranes and cannot provide information on the dynamic interactions between membrane-bound molecules. Here, we show a novel DNA probe that can transduce transient membrane encounter events into readable cumulative fluorescence signals. The probe, which translocates from one anchor site to another, mimicking motor proteins, is realized through a toehold-mediated DNA strand displacement reaction. Using this probe, we successfully monitored rapid encounter events of membrane lipid domains using flow cytometry and fluorescence microscopy. Our results show a preference for encounters within the same lipid domains.

  9. DNA probe for monitoring dynamic and transient molecular encounters on live cell membranes

    PubMed Central

    You, Mingxu; Lyu, Yifan; Han, Da; Qiu, Liping; Liu, Qiaoling; Chen, Tao; Wu, Cuichen Sam; Peng, Lu; Zhang, Liqin; Bao, Gang; Tan, Weihong

    2017-01-01

    Cells interact with the extracellular environment through molecules expressed on the membrane. Disruption of these membrane-bound interactions (or encounters) can result in disease progression. Advances in super-resolution microscopy have allowed membrane encounters to be examined, however, these methods cannot image entire membranes and cannot provide information on the dynamic interactions between membrane-bound molecules. Here, we show a novel DNA probe that can transduce transient membrane encounter events into readable cumulative fluorescence signals. The probe, which translocates from one anchor site to another, such as motor proteins, is realized through a toehold-mediated DNA strand displacement reaction. Using this probe, we successfully monitored rapid encounter events of membrane lipid domains using flow cytometry and fluorescence microscopy. Our results show a preference for encounters within different lipid domains. PMID:28319616

  10. Cell-wall disruption and lipid/astaxanthin extraction from microalgae: Chlorella and Haematococcus.

    PubMed

    Kim, Dong-Yeon; Vijayan, Durairaj; Praveenkumar, Ramasamy; Han, Jong-In; Lee, Kyubock; Park, Ji-Yeon; Chang, Won-Seok; Lee, Jin-Suk; Oh, You-Kwan

    2016-01-01

    Recently, biofuels and nutraceuticals produced from microalgae have emerged as major interests, resulting in intensive research of the microalgal biorefinery process. In this paper, recent developments in cell-wall disruption and extraction methods are reviewed, focusing on lipid and astaxanthin production from the biotechnologically important microalgae Chlorella and Haematococcus, respectively. As a common, critical bottleneck for recovery of intracellular components such as lipid and astaxanthin from these microalgae, the composition and structure of rigid, thick cell-walls were analyzed. Various chemical, physical, physico-chemical, and biological methods applied for cell-wall breakage and lipid/astaxanthin extraction from Chlorella and Haematococcus are discussed in detail and compared based on efficiency, energy consumption, type and dosage of solvent, biomass concentration and status (wet/dried), toxicity, scalability, and synergistic combinations. This report could serve as a useful guide to the implementation of practical downstream processes for recovery of valuable products from microalgae including Chlorella and Haematococcus. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. High-Melting Lipid Mixtures and the Origin of Detergent-Resistant Membranes Studied with Temperature-Solubilization Diagrams

    PubMed Central

    Sot, Jesús; Manni, Marco M.; Viguera, Ana R.; Castañeda, Verónica; Cano, Ainara; Alonso, Cristina; Gil, David; Valle, Mikel; Alonso, Alicia; Goñi, Félix M.

    2014-01-01

    The origin of resistance to detergent solubilization in certain membranes, or membrane components, is not clearly understood. We have studied the solubilization by Triton X-100 of binary mixtures composed of egg sphingomyelin (SM) and either ceramide, diacylglycerol, or cholesterol. Solubilization has been assayed in the 4–50°C range, and the results are summarized in a novel, to our knowledge, form of plots, that we have called temperature-solubilization diagrams. Despite using a large detergent excess (lipid/detergent 1:20 mol ratio) and extended solubilization times (24–48 h) certain mixtures were not amenable to Triton X-100 solubilization at one or more temperatures. DSC of all the lipid mixtures, and of all the lipid + detergent mixtures revealed that detergent resistance was associated with the presence of gel domains at the assay temperature. Once the system melted down, solubilization could occur. In general adding high-melting lipids limited the solubilization, whereas the addition of low-melting lipids promoted it. Lipidomic analysis of Madin-Darby canine kidney cell membranes and of the corresponding detergent-resistant fraction indicated a large enrichment of the nonsolubilized components in saturated diacylglycerol and ceramide. SM-cholesterol mixtures were special in that detergent solubilization was accompanied, for certain temperatures and compositions, by an independent phenomenon of reassembly of the partially solubilized lipid bilayers. The temperature at which lysis and reassembly prevailed was ∼25°C, thus for some SM-cholesterol mixtures solubilization occurred both above and below 25°C, but not at that temperature. These observations can be at the origin of the detergent resistance effects observed with cell membranes, and they also mean that cholesterol-containing detergent-resistant membrane remnants cannot correspond to structures existing in the native membrane before detergent addition. PMID:25517149

  12. Rapid Mobilization of Membrane Lipids in Wheat Leaf-Sheaths during Incompatible Interactions with Hessian Fly*

    PubMed Central

    Zhu, Lieceng; Liu, Xuming; Wang, Haiyan; Khajuria, Chitvan; Reese, John C.; Whitworth, R. Jeff; Welti, Ruth; Chen, Ming-Shun

    2013-01-01

    Hessian fly (HF) is a biotrophic insect that interacts with wheat on a gene-for-gene basis. We profiled changes in membrane lipids in two isogenic wheat lines: a susceptible line and its backcrossed offspring containing the resistance gene H13. Our results revealed a 32 to 45% reduction in total concentrations of 129 lipid species in resistant plants during incompatible interactions within 24 h after HF attack. A smaller and delayed response was observed in susceptible plants during compatible interactions. Microarray and real-time PCR analyses of 168 lipid-metabolism related transcripts revealed that the abundance of many of these transcripts increased rapidly in resistant plants after HF attack, but did not change in susceptible plants. In association with the rapid mobilization of membrane lipids, the concentrations of some fatty acids and 12-oxo-phytodienoic acid (OPDA) increased specifically in resistant plants. Exogenous application of OPDA increased mortality of HF larvae significantly. Collectively, our data, along with previously published results, indicate that the lipids were mobilized through lipolysis, producing free fatty acids, which were likely further converted into oxylipins and other defense molecules. Our results suggest that rapid mobilization of membrane lipids constitutes an important step for wheat to defend against HF attack. PMID:22668001

  13. Lipid Domains in Intact Fiber-Cell Plasma Membranes Isolated from Cortical and Nuclear Regions of Human Eye Lenses of Donors from Different Age Groups

    PubMed Central

    Raguz, Marija; Mainali, Laxman; O’Brien, William J.; Subczynski, Witold K.

    2015-01-01

    The results reported here clearly document changes in the properties and the organization of fiber-cell membrane lipids that occur with age, based on electron paramagnetic resonance (EPR) analysis of lens membranes of clear lenses from donors of age groups from 0 to 20, 21 to 40, and 61 to 80 years. The physical properties, including profiles of the alkyl chain order, fluidity, hydrophobicity, and oxygen transport parameter, were investigated using EPR spin-labeling methods, which also provide an opportunity to discriminate coexisting lipid domains and to evaluate the relative amounts of lipids in these domains. Fiber-cell membranes were found to contain three distinct lipid environments: bulk lipid domain, which appears minimally affected by membrane proteins, and two domains that appear due to the presence of membrane proteins, namely boundary and trapped lipid domains. In nuclear membranes the amount of boundary and trapped phospholipids as well as the amount of cholesterol in trapped lipid domains increased with the donors’ age and was greater than that in cortical membranes. The difference between the amounts of lipids in domains uniquely formed due to the presence of membrane proteins in nuclear and cortical membranes increased with the donors’ age. It was also shown that cholesterol was to a large degree excluded from trapped lipid domains in cortical membranes. It is evident that the rigidity of nuclear membranes was greater than that of cortical membranes for all age groups. The amount of lipids in domains of low oxygen permeability, mainly in trapped lipid domains, were greater in nuclear than cortical membranes and increased with the age of donors. These results indicate that the nuclear fiber cell plasma membranes were less permeable to oxygen than cortical membranes and become less permeable to oxygen with age. In clear lenses, age-related changes in the lens lipid and protein composition and organization appear to occur in ways that increase fiber

  14. Lipid domains in intact fiber-cell plasma membranes isolated from cortical and nuclear regions of human eye lenses of donors from different age groups.

    PubMed

    Raguz, Marija; Mainali, Laxman; O'Brien, William J; Subczynski, Witold K

    2015-03-01

    The results reported here clearly document changes in the properties and the organization of fiber-cell membrane lipids that occur with age, based on electron paramagnetic resonance (EPR) analysis of lens membranes of clear lenses from donors of age groups from 0 to 20, 21 to 40, and 61 to 80 years. The physical properties, including profiles of the alkyl chain order, fluidity, hydrophobicity, and oxygen transport parameter, were investigated using EPR spin-labeling methods, which also provide an opportunity to discriminate coexisting lipid domains and to evaluate the relative amounts of lipids in these domains. Fiber-cell membranes were found to contain three distinct lipid environments: bulk lipid domain, which appears minimally affected by membrane proteins, and two domains that appear due to the presence of membrane proteins, namely boundary and trapped lipid domains. In nuclear membranes the amount of boundary and trapped phospholipids as well as the amount of cholesterol in trapped lipid domains increased with the donors' age and was greater than that in cortical membranes. The difference between the amounts of lipids in domains uniquely formed due to the presence of membrane proteins in nuclear and cortical membranes increased with the donors' age. It was also shown that cholesterol was to a large degree excluded from trapped lipid domains in cortical membranes. It is evident that the rigidity of nuclear membranes was greater than that of cortical membranes for all age groups. The amount of lipids in domains of low oxygen permeability, mainly in trapped lipid domains, were greater in nuclear than cortical membranes and increased with the age of donors. These results indicate that the nuclear fiber cell plasma membranes were less permeable to oxygen than cortical membranes and become less permeable to oxygen with age. In clear lenses, age-related changes in the lens lipid and protein composition and organization appear to occur in ways that increase fiber

  15. Regulation of Lipid Droplet Size in Mammary Epithelial Cells by Remodeling of Membrane Lipid Composition—A Potential Mechanism

    PubMed Central

    Cohen, Bat-Chen; Shamay, Avi; Argov-Argaman, Nurit

    2015-01-01

    Milk fat globule size is determined by the size of its precursors—intracellular lipid droplets—and is tightly associated with its composition. We examined the relationship between phospholipid composition of mammary epithelial cells and the size of both intracellular and secreted milk fat globules. Primary culture of mammary epithelial cells was cultured in medium without free fatty acids (control) or with 0.1 mM free capric, palmitic or oleic acid for 24 h. The amount and composition of the cellular lipids and the size of the lipid droplets were determined in the cells and medium. Mitochondrial quantity and expression levels of genes associated with mitochondrial biogenesis and polar lipid composition were determined. Cells cultured with oleic and palmitic acids contained similar quantities of triglycerides, 3.1- and 3.8-fold higher than in controls, respectively (P < 0.0001). When cultured with oleic acid, 22% of the cells contained large lipid droplets (>3 μm) and phosphatidylethanolamine concentration was higher by 23 and 63% compared with that in the control and palmitic acid treatments, respectively (P < 0.0001). In the presence of palmitic acid, only 4% of the cells contained large lipid droplets and the membrane phosphatidylcholine concentration was 22% and 16% higher than that in the control and oleic acid treatments, respectively (P < 0.0001). In the oleic acid treatment, approximately 40% of the lipid droplets were larger than 5 μm whereas in that of the palmitic acid treatment, only 16% of the droplets were in this size range. Triglyceride secretion in the oleic acid treatment was 2- and 12-fold higher compared with that in the palmitic acid and control treatments, respectively. Results imply that membrane composition of bovine mammary epithelial cells plays a role in controlling intracellular and secreted lipid droplets size, and that this process is not associated with cellular triglyceride content. PMID:25756421

  16. Characterization of Interactions between Curcumin and Different Types of Lipid Bilayers by Molecular Dynamics Simulation.

    PubMed

    Lyu, Yuan; Xiang, Ning; Mondal, Jagannath; Zhu, Xiao; Narsimhan, Ganesan

    2018-03-01

    Curcumin (CUR) is a natural food ingredient with known ability to target microbial cell membrane. In this study, the interactions of CUR with different types of model lipid bilayers (POPE, POPG, POPC, DOPC, and DPPE), mixtures of model lipid bilayers (POPE/POPG), and biological membrane mimics (Escherichia coli and yeast) were investigated by all-atom explicit solvent molecular dynamics (MD) simulation. CUR readily inserts into different types of model lipid bilayer systems in the liquid crystalline state, staying in the lipid tails region near the interface of lipid head and lipid tail. Parallel orientation to the membrane surface is found to be more probable than perpendicular for CUR, as indicated by the tilt angle distribution. This orientation preference is less significant as the fraction of POPE is increased in the system, likely due to the better water solvation of perpendicular orientation in the POPE bilayer. In E. coli and yeast bilayers, tilt angle distributions were similar to that for POPE/POPG mixed bilayer, with water hydration number around CUR for the former being higher. Insertion of CUR resulted in membrane thinning. The results from these simulations provide insights into the possible differences in membrane disrupting activity of CUR against different types of microorganisms.

  17. Effect of anionic and cationic polyamidoamine (PAMAM) dendrimers on a model lipid membrane.

    PubMed

    Lombardo, Domenico; Calandra, Pietro; Bellocco, Ersilia; Laganà, Giuseppina; Barreca, Davide; Magazù, Salvatore; Wanderlingh, Ulderico; Kiselev, Mikhail A

    2016-11-01

    In spite of the growing variety of biological applications of dendrimer-based nanocarriers, a major problem of their potential applications in bio-medicine is related to the disruption of lipid bilayers and the cytotoxicity caused by the aggregation processes involved onto cellular membranes. With the aim to study model dendrimer-biomembrane interaction, the self-assembly processes of a mixture of charged polyamidoamine (PAMAM) dendrimers and dipalmitoylphosphatidylcholine (DPPC) lipids were investigated by means of Zeta potential analysis, Raman and x-ray scattering. Zwitterionic DPPC liposomes showed substantially different behaviors during their interaction with negatively charged (generation G=2.5) sodium carboxylate terminated (COO - Na + ) dendrimers or positively charged (generation G=3.0) amino terminated (-NH 2 ) dendrimers. More specifically the obtained results evidence the sensitive interactions between dendrimer terminals and lipid molecules at the surface of the liposome, with an enhancement of the liposome surface zeta potential, as well as in the hydrophobic region of the bilayers, where dendrimer penetration produce a perturbation of the hydrophobic alkyl chains of the bilayers. Analysis of the SAXS structure factor with a suitable model for the inter-dendrimers electrostatic potential allows an estimation of an effective charge of 15 ǀeǀ for G=2.5 and 7.6 ǀeǀ for G=3.0 PAMAM dendrimers. Only a fraction (about 1/7) of this charge contributes to the linear increase of liposome zeta-potential with increasing PAMAM/DPPC molar fraction. The findings of our investigation may be applied to rationalize the effect of the nanoparticles electrostatic interaction in solution environments for the design of new drug carriers combining dendrimeric and liposomal technology. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. The interaction of polyglutamine peptides with lipid membranes is regulated by flanking sequences associated with huntingtin.

    PubMed

    Burke, Kathleen A; Kauffman, Karlina J; Umbaugh, C Samuel; Frey, Shelli L; Legleiter, Justin

    2013-05-24

    Huntington disease (HD) is caused by an expanded polyglutamine (poly(Q)) repeat near the N terminus of the huntingtin (htt) protein. Expanded poly(Q) facilitates formation of htt aggregates, eventually leading to deposition of cytoplasmic and intranuclear inclusion bodies containing htt. Flanking sequences directly adjacent to the poly(Q) domain, such as the first 17 amino acids on the N terminus (Nt17) and the polyproline (poly(P)) domain on the C-terminal side of the poly(Q) domain, heavily influence aggregation. Additionally, htt interacts with a variety of membraneous structures within the cell, and Nt17 is implicated in lipid binding. To investigate the interaction between htt exon1 and lipid membranes, a combination of in situ atomic force microscopy, Langmuir trough techniques, and vesicle permeability assays were used to directly monitor the interaction of a variety of synthetic poly(Q) peptides with different combinations of flanking sequences (KK-Q35-KK, KK-Q35-P10-KK, Nt17-Q35-KK, and Nt17-Q35-P10-KK) on model membranes and surfaces. Each peptide aggregated on mica, predominately forming extended, fibrillar aggregates. In contrast, poly(Q) peptides that lacked the Nt17 domain did not appreciably aggregate on or insert into lipid membranes. Nt17 facilitated the interaction of peptides with lipid surfaces, whereas the poly(P) region enhanced this interaction. The aggregation of Nt17-Q35-P10-KK on the lipid bilayer closely resembled that of a htt exon1 construct containing 35 repeat glutamines. Collectively, this data suggests that the Nt17 domain plays a critical role in htt binding and aggregation on lipid membranes, and this lipid/htt interaction can be further modulated by the presence of the poly(P) domain.

  19. Proton Gradients as a Key Physical Factor in the Evolution of the Forced Transport Mechanism Across the Lipid Membrane.

    PubMed

    Strbak, Oliver; Kanuchova, Zuzana; Krafcik, Andrej

    2016-11-01

    A critical phase in the transition from prebiotic chemistry to biological evolution was apparently an asymmetric ion flow across the lipid membrane. Due to imbalance in the ion flow, the early lipid vesicles could selectively take the necessary molecules from the environment, and release the side-products from the vesicle. Natural proton gradients played a definitively crucial role in this process, since they remain the basis of energy transfer in the present-day cells. On the basis of this supposition, and the premise of the early vesicle membrane's impermeability to protons, we have shown that the emergence of the proton gradient in the lipid vesicle could be a key physical factor in the evolution of the forced transport mechanism (pore formation and active transport) across the lipid bilayer. This driven flow of protons across the membrane is the result of the electrochemical proton gradient and osmotic pressures on the integrity of the lipid vesicle. At a critical number of new lipid molecules incorporated into the vesicle, the energies associated with the creation of the proton gradient exceed the bending stiffness of the lipid membrane, and overlap the free energy of the lipid bilayer pore formation.

  20. Free energies of stable and metastable pores in lipid membranes under tension.

    PubMed

    den Otter, Wouter K

    2009-11-28

    The free energy profile of pore formation in a lipid membrane, covering the entire range from a density fluctuation in an intact bilayer to a large tension-stabilized pore, has been calculated by molecular dynamics simulations with a coarse-grained lipid model. Several fixed elongations are used to obtain the Helmholtz free energy as a function of pore size for thermodynamically stable, metastable, and unstable pores, and the system-size dependence of these elongations is discussed. A link to the Gibbs free energy at constant tension, commonly known as the Litster model, is established by a Legendre transformation. The change of genus upon pore formation is exploited to estimate the saddle-splay modulus or Gaussian curvature modulus of the membrane leaflets. Details are provided of the simulation approach, which combines the potential of mean constraint force method with a reaction coordinate based on the local lipid density.

  1. Neutron Reflectometry Study of the Conformation of HIV Nef Bound to Lipid Membranes

    PubMed Central

    Kent, Michael S.; Murton, Jaclyn K.; Sasaki, Darryl Y.; Satija, Sushil; Akgun, Bulent; Nanda, Hirsh; Curtis, Joseph E.; Majewski, Jaroslaw; Morgan, Christopher R.; Engen, John R.

    2010-01-01

    Nef is an HIV-1 accessory protein that directly contributes to AIDS progression. Nef is myristoylated on the N-terminus, associates with membranes, and may undergo a transition from a solution conformation to a membrane-associated conformation. It has been hypothesized that conformational rearrangement enables membrane-associated Nef to interact with cellular proteins. Despite its medical relevance, to our knowledge there is no direct information about the conformation of membrane-bound Nef. In this work, we used neutron reflection to reveal what we believe are the first details of the conformation of membrane-bound Nef. The conformation of Nef was probed upon binding to Langmuir monolayers through the interaction of an N-terminal His tag with a synthetic metal-chelating lipid, which models one of the possible limiting cases for myr-Nef. The data indicate that residues are inserted into the lipid headgroups during interaction, and that the core domain lies directly against the lipid headgroups, with a thickness of ∼40 Å. Binding of Nef through the N-terminal His tag apparently facilitates insertion of residues, as no insertion occurred upon binding of Nef through weak electrostatic interactions in the absence of the specific interaction through the His tag. PMID:20858440

  2. Studies of Water Diffusion on Single-Supported Bilayer Lipid Membranes by Quasielastic Neutron Scattering

    NASA Astrophysics Data System (ADS)

    Bai, M.; Miskowiec, A.; Wang, S.-K.; Taub, H.; Jenkins, T.; Tyagi, M.; Neumann, D. A.; Hansen, F. Y.

    2010-03-01

    Bilayer lipid membranes supported on a solid surface are attractive model systems for understanding the structure and dynamics of more complex biological membranes that form the outer boundary of living cells. We have recently demonstrated the feasibility of using quasielastic neutron scattering to study on a ˜1 ns time scale the diffusion of water bound to single-supported bilayer lipid membranes. Two different membrane samples characterized by AFM were investigated: protonated DMPC + D2O and tail-deuterated DMPC + H2O. Both fully hydrated membranes were deposited onto SiO2-coated Si(100) substrates. Measurements of elastic neutron intensity as a function of temperature on the High Flux Backscattering Spectrometer at NIST reveal features in the diffusive motion of water that have not been observed previously using multilayer membrane stacks. On slow cooling, the elastic intensity shows sharp step-like increases in the temperature range 265 to 272 K that we tentatively interpret as successive mobile-to-immobile transitions of water bound to the membrane.

  3. Mechanisms Underlying the Essential Role of Mitochondrial Membrane Lipids in Yeast Chronological Aging

    PubMed Central

    Medkour, Younes; Dakik, Paméla; McAuley, Mélissa; Mohammad, Karamat; Mitrofanova, Darya

    2017-01-01

    The functional state of mitochondria is vital to cellular and organismal aging in eukaryotes across phyla. Studies in the yeast Saccharomyces cerevisiae have provided evidence that age-related changes in some aspects of mitochondrial functionality can create certain molecular signals. These signals can then define the rate of cellular aging by altering unidirectional and bidirectional communications between mitochondria and other organelles. Several aspects of mitochondrial functionality are known to impact the replicative and/or chronological modes of yeast aging. They include mitochondrial electron transport, membrane potential, reactive oxygen species, and protein synthesis and proteostasis, as well as mitochondrial synthesis of iron-sulfur clusters, amino acids, and NADPH. Our recent findings have revealed that the composition of mitochondrial membrane lipids is one of the key aspects of mitochondrial functionality affecting yeast chronological aging. We demonstrated that exogenously added lithocholic bile acid can delay chronological aging in yeast because it elicits specific changes in mitochondrial membrane lipids. These changes allow mitochondria to operate as signaling platforms that delay yeast chronological aging by orchestrating an institution and maintenance of a distinct cellular pattern. In this review, we discuss molecular and cellular mechanisms underlying the essential role of mitochondrial membrane lipids in yeast chronological aging. PMID:28593023

  4. Interaction of miltefosine with the lipid and protein components of the erythrocyte membrane.

    PubMed

    Moreira, Rodrigo Alves; Mendanha, Sebastião Antonio; Hansen, Daiane; Alonso, Antonio

    2013-05-01

    Miltefosine (MT) is an alkylphospholipid that has been approved for the treatment of breast cancer metastasis and visceral leishmaniasis, although its mechanism of action remains poorly understood. Electron paramagnetic resonance spectroscopy of a spin-labeled lipid and a thiol-specific spin label showed that MT causes an increase in the molecular dynamics of erythrocyte ghost membranes and detergent-resistant membranes (DRMs) prepared from erythrocyte ghosts. In the vesicles of lipid raft constituents, it was shown that 20 mol % sphingomyelin could be replaced by 20 mol % MT with no change in the molecular dynamics. The effect of MT in DRMs was more pronounced than in erythrocyte ghosts, supporting the hypothesis that MT is a lipid raft modulator. At the reported MT-plasma concentrations found during the treatment of leishmaniasis (31-90 µg/mL), our measurements in the blood plasma indicated a hemolytic level of 2%-5%. The experiments indicated that MT acts predominantly on the protein component of the membrane. MT aggregates may wrap around the hydrophobic polypeptide chains, forming micelle-like structures that stabilize protein conformations more exposed to the solvent. Proteins with higher hydrophobicity may induce the penetration of the hydrophilic groups of MT into the membrane and cause it to rupture. Copyright © 2013 Wiley Periodicals, Inc.

  5. Permeabilization assay for antimicrobial peptides based on pore-spanning lipid membranes on nanoporous alumina.

    PubMed

    Neubacher, Henrik; Mey, Ingo; Carnarius, Christian; Lazzara, Thomas D; Steinem, Claudia

    2014-04-29

    Screening tools to study antimicrobial peptides (AMPs) with the aim to optimize therapeutic delivery vectors require automated and parallelized sampling based on chip technology. Here, we present the development of a chip-based assay that allows for the investigation of the action of AMPs on planar lipid membranes in a time-resolved manner by fluorescence readout. Anodic aluminum oxide (AAO) composed of cylindrical pores with a diameter of 70 nm and a thickness of up to 10 μm was used as a support to generate pore-spanning lipid bilayers from giant unilamellar vesicle spreading, which resulted in large continuous membrane patches sealing the pores. Because AAO is optically transparent, fluid single lipid bilayers and the underlying pore cavities can be readily observed by three-dimensional confocal laser scanning microscopy (CLSM). To assay the membrane permeabilizing activity of the AMPs, the translocation of the water-soluble dyes into the AAO cavities and the fluorescence of the sulforhodamine 101 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanol-l-amine triethylammonium salt (Texas Red DHPE)-labeled lipid membrane were observed by CLSM in a time-resolved manner as a function of the AMP concentration. The effect of two different AMPs, magainin-2 and melittin, was investigated, showing that the concentrations required for membrane permeabilization and the kinetics of the dye entrance differ significantly. Our results are discussed in light of the proposed permeabilization models of the two AMPs. The presented data demonstrate the potential of this setup for the development of an on-chip screening platform for AMPs.

  6. On the adsorption of phloretin onto a black lipid membrane.

    PubMed Central

    de Levie, R; Rangarajan, S K; Seelig, P F; Andersen, O S

    1979-01-01

    The effect of uncharged, dipolar phloretin on anion and cation conductance through a black lipid membrane can be used to study its adsorption behavior. The adsorption of phloretin can be described by a Langmuir isotherm with weak dipole-dipole interaction. PMID:262390

  7. Accurate potentiometric determination of lipid membrane-water partition coefficients and apparent dissociation constants of ionizable drugs: electrostatic corrections.

    PubMed

    Elsayed, Mustafa M A; Vierl, Ulrich; Cevc, Gregor

    2009-06-01

    Potentiometric lipid membrane-water partition coefficient studies neglect electrostatic interactions to date; this leads to incorrect results. We herein show how to account properly for such interactions in potentiometric data analysis. We conducted potentiometric titration experiments to determine lipid membrane-water partition coefficients of four illustrative drugs, bupivacaine, diclofenac, ketoprofen and terbinafine. We then analyzed the results conventionally and with an improved analytical approach that considers Coulombic electrostatic interactions. The new analytical approach delivers robust partition coefficient values. In contrast, the conventional data analysis yields apparent partition coefficients of the ionized drug forms that depend on experimental conditions (mainly the lipid-drug ratio and the bulk ionic strength). This is due to changing electrostatic effects originating either from bound drug and/or lipid charges. A membrane comprising 10 mol-% mono-charged molecules in a 150 mM (monovalent) electrolyte solution yields results that differ by a factor of 4 from uncharged membranes results. Allowance for the Coulombic electrostatic interactions is a prerequisite for accurate and reliable determination of lipid membrane-water partition coefficients of ionizable drugs from potentiometric titration data. The same conclusion applies to all analytical methods involving drug binding to a surface.

  8. Lipid raft regulates the initial spreading of melanoma A375 cells by modulating β1 integrin clustering.

    PubMed

    Wang, Ruifei; Bi, Jiajia; Ampah, Khamal Kwesi; Zhang, Chunmei; Li, Ziyi; Jiao, Yang; Wang, Xiaoru; Ba, Xueqing; Zeng, Xianlu

    2013-08-01

    Cell adhesion and spreading require integrins-mediated cell-extracellular matrix interaction. Integrins function through binding to extracellular matrix and subsequent clustering to initiate focal adhesion formation and actin cytoskeleton rearrangement. Lipid raft, a liquid ordered plasma membrane microdomain, has been reported to play major roles in membrane motility by regulating cell surface receptor function. Here, we identified that lipid raft integrity was required for β1 integrin-mediated initial spreading of melanoma A375 cells on fibronectin. We found that lipid raft disruption with methyl-β-cyclodextrin led to the inability of focal adhesion formation and actin cytoskeleton rearrangement by preventing β1 integrin clustering. Furthermore, we explored the possible mechanism by which lipid raft regulates β1 integrin clustering and demonstrated that intact lipid raft could recruit and modify some adaptor proteins, such as talin, α-actinin, vinculin, paxillin and FAK. Lipid raft could regulate the location of these proteins in lipid raft fractions and facilitate their binding to β1 integrin, which may be crucial for β1 integrin clustering. We also showed that lipid raft disruption impaired A375 cell migration in both transwell and wound healing models. Together, these findings provide a new insight for the relationship between lipid raft and the regulation of integrins. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Analysis of Lipids and Lipid Rafts in Borrelia.

    PubMed

    Toledo, Alvaro; Huang, Zhen; Benach, Jorge L; London, Erwin

    2018-01-01

    Lipid rafts are membrane microdomains that are involved in cellular processes such as protein trafficking and signaling processes, and which play a fundamental role in membrane fluidity and budding. The lipid composition of the membrane and the biochemical characteristics of the lipids found within rafts define the ability of cells to form microdomains and compartmentalize the membrane. In this chapter, we describe the biophysical, biochemical, and molecular approaches used to define and characterize lipid rafts in the Lyme disease agent, Borrelia burgdorferi.

  10. Design of a homogeneous multifunctional supported lipid membrane on layer-by-layer coated microcarriers.

    PubMed

    Göse, Martin; Pescador, Paula; Reibetanz, Uta

    2015-03-09

    Key challenges in the development of drug delivery systems are the prevention of serum compartment interaction and the targeted delivery of the cargo. Layer-by-Layer microcarriers offer many advantages due to various options in drug assembly and multifunctional design. Surface modification with a supported lipid membrane enhances biocompatibility, drug protection ability, and specific functionality. However, the integration of functionalized lipids strongly influences the membrane formation and is often accompanied by submicrometer irregularities: The accessibility of underlying polymers to serum components may change the carrier's properties and enhances the susceptibility to opsonization. Therefore, the formation of a tightly assembled multifunctional lipid membrane has been emphasized. A phosphatidylserine/phosphatidylcholine (POPS/POPC) bilayer equipped with phosphatidylethanolamine-polyethylene glycol-biotin (PE-PEG-Biotin) was used to facilitate a biotin/streptavidin binding site for a variable attachment of an additional function, such as antibodies for specific targeting. Thus, a prefunctionalized carrier where only the outer functionality needs to be replaced without disturbing the underlying structure could be created.

  11. Lipid Droplets and Peroxisomes: Key Players in Cellular Lipid Homeostasis or A Matter of Fat—Store ’em Up or Burn ’em Down

    PubMed Central

    Kohlwein, Sepp D.; Veenhuis, Marten; van der Klei, Ida J.

    2013-01-01

    Lipid droplets (LDs) and peroxisomes are central players in cellular lipid homeostasis: some of their main functions are to control the metabolic flux and availability of fatty acids (LDs and peroxisomes) as well as of sterols (LDs). Both fatty acids and sterols serve multiple functions in the cell—as membrane stabilizers affecting membrane fluidity, as crucial structural elements of membrane-forming phospholipids and sphingolipids, as protein modifiers and signaling molecules, and last but not least, as a rich carbon and energy source. In addition, peroxisomes harbor enzymes of the malic acid shunt, which is indispensable to regenerate oxaloacetate for gluconeogenesis, thus allowing yeast cells to generate sugars from fatty acids or nonfermentable carbon sources. Therefore, failure of LD and peroxisome biogenesis and function are likely to lead to deregulated lipid fluxes and disrupted energy homeostasis with detrimental consequences for the cell. These pathological consequences of LD and peroxisome failure have indeed sparked great biomedical interest in understanding the biogenesis of these organelles, their functional roles in lipid homeostasis, interaction with cellular metabolism and other organelles, as well as their regulation, turnover, and inheritance. These questions are particularly burning in view of the pandemic development of lipid-associated disorders worldwide. PMID:23275493

  12. N-3 fatty acids and membrane microdomains: from model membranes to lymphocyte function.

    PubMed

    Shaikh, Saame Raza; Teague, Heather

    2012-12-01

    This article summarizes the author's research on fish oil derived n-3 fatty acids, plasma membrane organization and B cell function. We first cover basic model membrane studies that investigated how docosahexaenoic acid (DHA) targeted the organization of sphingolipid-cholesterol enriched lipid microdomains. A key finding here was that DHA had a relatively poor affinity for cholesterol. This work led to a model that predicted DHA acyl chains in cells would manipulate lipid-protein microdomain organization and thereby function. We then review how the predictions of the model were tested with B cells in vitro followed by experiments using mice fed fish oil. These studies reveal a highly complex picture on how n-3 fatty acids target lipid-protein organization and B cell function. Key findings are as follows: (1) n-3 fatty acids target not just the plasma membrane but also endomembrane organization; (2) DHA, but not eicosapentaenoic acid (EPA), disrupts microdomain spatial distribution (i.e. clustering), (3) DHA alters protein lateral organization and (4) changes in membrane organization are accompanied by functional effects on both innate and adaptive B cell function. Altogether, the research over the past 10 years has led to an evolution of the original model on how DHA reorganizes membrane microdomains. The work raises the intriguing possibility of testing the model at the human level to target health and disease. Copyright © 2012 Elsevier Ltd. All rights reserved.

  13. Synthetic ion channels via self-assembly: a route for embedding porous polyoxometalate nanocapsules in lipid bilayer membranes.

    PubMed

    Carr, Rogan; Weinstock, Ira A; Sivaprasadarao, Asipu; Müller, Achim; Aksimentiev, Aleksei

    2008-11-01

    Porous polyoxometalate nanocapsules of Keplerate type are known to exhibit the functionality of biological ion channels; however, their use as an artificial ion channel is tempered by the high negative charge of the capsules, which renders their spontaneous incorporation into a lipid bilayer membrane unlikely. In this Letter we report coarse-grained molecular dynamics simulations that demonstrate a route for embedding negatively charged nanocapsules into lipid bilayer membranes via self-assembly. A homogeneous mixture of water, cationic detergent, and phospholipid was observed to spontaneously self-assemble around the nanocapsule into a layered, liposome-like structure, where the nanocapsule was enveloped by a layer of cationic detergent followed by a layer of phospholipid. Fusion of such a layered liposome with a lipid bilayer membrane was observed to embed the nanocapsule into the lipid bilayer. The resulting assembly was found to remain stable even after the surface of the capsule was exposed to electrolyte. In the latter conformation, water was observed to flow into and out of the capsule as Na(+) cations entered, suggesting that a polyoxometalate nanocapsule can form a functional synthetic ion channel in a lipid bilayer membrane.

  14. Synthetic Ion Channels via Self-Assembly: a Route for Embedding Porous Polyoxometalate Nanocapsules in Lipid Bilayer Membranes

    PubMed Central

    Carr, Rogan; Weinstock, Ira A.; Sivaprasadarao, Asipu; Müller, Achim; Aksimentiev, Aleksei

    2010-01-01

    Porous polyoxometalate nanocapsules of Keplerate type are known to exhibit the functionality of biological ion channels, however, their use as artificial ion channel is tempered by the high negative charge of the capsules, which renders their spontaneous incorporation into a lipid bilayer membrane unlikely. In this letter we report coarse-grained molecular dynamics simulations that demonstrate a route for embedding negatively charged nanocapsules into lipid bilayer membranes via self-assembly. A homogeneous mixture of water, cationic detergent, and phospholipid was observed to spontaneously self-assemble around the nanocapsule into a layered, liposome-like structure, where the nanocapsule was enveloped by a layer of cationic detergent followed by a layer of phospholipid. Fusion of such a layered liposome with a lipid bilayer membrane was observed to embed the nanocapsule into the lipid bilayer. The resulting assembly was found to remain stable even after the surface of the capsule was exposed to electrolyte. In the latter conformation, water was observed to flow into and out of the capsule as Na+ cations entered, suggesting that a polyoxometalate nanocapsule can form a functional synthetic ion channel in a lipid bilayer membrane. PMID:18844424

  15. Phloretin-induced changes in ion transport across lipid bilayer membranes

    PubMed Central

    1977-01-01

    Phloretin, the aglucone derivative of phlorizin, increases cation conductance and decreases anion conductance in lipid bilayer membranes. In this paper we present evidence that phloretin acts almost exclusively by altering the permeability of the membrane interior and not by modifying the partition of the permanent species between the membrane and the bulk aqueous phases. We base our conclusion on an analysis of the current responses to a senylborate, and the cation complex, peptide PV-K+. These results are consistent with the hypothesis that phloretin decreases the intrinsic positive internal membrane potential but does not modify to a great extent the potential energy minima at the membrane interfaces. Phloretin increases the conductance for the nonactin-K+ complex, but above 10(-5) M the steady- state nonactin-K+ voltage-current curve changes from superlinear to sublinear. These results imply that, above 10(-5) M phloretin, the nonactin-5+ transport across the membrane becomes interfacially limited. PMID:576427

  16. Formation of Bimolecular Membranes from Lipid Monolayers and a Study of Their Electrical Properties

    PubMed Central

    Montal, M.; Mueller, P.

    1972-01-01

    Bimolecular membranes are formed from two lipid monolayers at an air-water interface by the apposition of their hydrocarbon chains when an aperture in a Teflon partition separating two aqueous phases is lowered through the interface. Formation of the membrane is monitored by an increase of the electrical capacity, as measured with a voltage clamp. Electrical resistance of the unmodified membrane is analogous to that of conventional planar bilayers (black lipid membranes) prepared in the presence of a hydrocarbon solvent, i.e., 106-108 ohm cm2; the resistance can be lowered to values of 103 ohm cm2 by gramicidin, an antibiotic that modifies the conductance only when the membranes are of biomolecular thickness. In contrast to the resistance, there is a significant difference between the capacity of bilayers made from mono-layers and that of hydrocarbon-containing bilayers made by phase transition; the average values are 0.9 and 0.45 μF cm-2, respectively. The value of 0.9 μF cm-2 approximates that of biological membranes. Assuming a dielectric constant of 2.1 for the hydrocarbon region, the dielectric thickness, as calculated from a capacity of 0.9 μF cm-2, is 22 Å. This value is 6-10 Å smaller than the actual thickness of the hydrocarbon region of bilayers and cell membranes, as determined by x-ray diffraction. The difference may be due to a limited penetration of water into the hydrocarbon region near the ester groups that would lower the electrical resistance of this region and reduce the dielectric thickness. Asymmetric membranes have been formed by adjoining two lipid monolayers of different chemical composition. Images PMID:4509315

  17. Plasma lipids, erythrocyte membrane lipids and blood pressure of hypertensive women after ingestion of dietary oleic acid from two different sources.

    PubMed

    Ruíz-Gutiérrez, V; Muriana, F J; Guerrero, A; Cert, A M; Villar, J

    1996-12-01

    To study the effect of a diet rich in mono-unsaturated fatty acids (MUFA), from high-oleic sunflower oil (HOSO) and olive oil, on plasma lipids, erythrocyte membrane lipids (including fatty acid composition) and blood pressure of hypertensive (normocholesterolaemic or hypercholesterolaemic) women. There were 16 participants who were hypertensive women aged 56.2 +/- 5.4 years. The participants ate a diet enriched with HOSO or olive oil for two 4-week periods with a 4-week washout period before starting the second type of MUFA diet. At entry and during study of each diet, plasma lipids and apolipoproteins were measured by conventional enzymatic methods. Erythrocyte membrane lipid and fatty acid compositions were analysed by means of the latroscan thin-layer chromatography/flame ionization detection technique and by gas chromatography, respectively. Blood pressure was also measured. The statistical analysis was conducted by using Student's two-tailed paired t-test. In both groups of hypertensive patients, there was a significant increase in plasma high-density lipoprotein (HDL) cholesterol concentration after the HOSO or olive oil diets, with regard to baseline. Additionally, a significant decrease in plasma HDL2 cholesterol concentration and an increase in plasma HDL3 cholesterol concentration were evident. The membrane free-cholesterol concentration increased significantly and the phospholipid concentration decreased significantly in erythrocytes after the olive oil diet, though both MUFA diets produced a significant decrease in the concentration of membrane esterified cholesterol. Therefore, the molar ratio of cholesterol to phospholipids was raised significantly in the erythrocyte membrane of hypertensive women after the dietary olive oil, but not after the HOSO diet. In the hypertensive and normo-cholesterolaemic group the HOSO diet significantly increased the content in the erythrocyte membrane of oleic, eicosenoic, arachidonic and docosapentaenoic acids

  18. Protonation Dynamics on Lipid Nanodiscs: Influence of the Membrane Surface Area and External Buffers.

    PubMed

    Xu, Lei; Öjemyr, Linda Näsvik; Bergstrand, Jan; Brzezinski, Peter; Widengren, Jerker

    2016-05-10

    Lipid membrane surfaces can act as proton-collecting antennae, accelerating proton uptake by membrane-bound proton transporters. We investigated this phenomenon in lipid nanodiscs (NDs) at equilibrium on a local scale, analyzing fluorescence fluctuations of individual pH-sensitive fluorophores at the membrane surface by fluorescence correlation spectroscopy (FCS). The protonation rate of the fluorophores was ∼100-fold higher when located at 9- and 12-nm diameter NDs, compared to when in solution, indicating that the proton-collecting antenna effect is maximal already for a membrane area of ∼60 nm(2). Fluorophore-labeled cytochrome c oxidase displayed a similar increase when reconstituted in 12 nm NDs, but not in 9 nm NDs, i.e., an acceleration of the protonation rate at the surface of cytochrome c oxidase is found when the lipid area surrounding the protein is larger than 80 nm(2), but not when below 30 nm(2). We also investigated the effect of external buffers on the fluorophore proton exchange rates at the ND membrane-water interfaces. With increasing buffer concentrations, the proton exchange rates were found to first decrease and then, at millimolar buffer concentrations, to increase. Monte Carlo simulations, based on a simple kinetic model of the proton exchange at the membrane-water interface, and using rate parameter values determined in our FCS experiments, could reconstruct both the observed membrane-size and the external buffer dependence. The FCS data in combination with the simulations indicate that the local proton diffusion coefficient along a membrane is ∼100 times slower than that observed over submillimeter distances by proton-pulse experiments (Ds ∼ 10(-5)cm(2)/s), and support recent theoretical studies showing that proton diffusion along membrane surfaces is time- and length-scale dependent. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  19. Interaction measurement of particles bound to a lipid membrane

    NASA Astrophysics Data System (ADS)

    Sarfati, Raphael; Dufresne, Eric

    2015-03-01

    The local shape and dynamics of the plasma membrane play important roles in many cellular processes. Local membrane deformations are often mediated by the adsorption of proteins (notably from the BAR family), and their subsequent self-assembly. The emerging hypothesis is that self-assembly arises from long-range interactions of individual proteins through the membrane's deformation field. We study these interactions in a model system of micron-sized colloidal particles adsorbed onto a lipid bilayer. We use fluorescent microscopy, optical tweezers and particle tracking to measure dissipative and conservative forces as a function of the separation between the particles. We find that particles are driven together with forces of order 100 fN and remain bound in a potential well with a stiffness of order 100 fN/micron.

  20. The bovine seminal plasma protein PDC-109 extracts phosphorylcholine-containing lipids from the outer membrane leaflet.

    PubMed

    Tannert, Astrid; Kurz, Anke; Erlemann, Karl-Rudolf; Müller, Karin; Herrmann, Andreas; Schiller, Jürgen; Töpfer-Petersen, Edda; Manjunath, Puttaswamy; Müller, Peter

    2007-04-01

    The bovine seminal plasma protein PDC-109 modulates the maturation of bull sperm cells by removing lipids, mainly phosphatidylcholine and cholesterol, from their cellular membrane. Here, we have characterized the process of extraction of endogenous phospholipids and of their respective analogues. By measuring the PDC-109-mediated release of fluorescent phospholipid analogues from lipid vesicles and from biological membranes (human erythrocytes, bovine epididymal sperm cells), we showed that PDC-109 extracts phospholipids with a phosphorylcholine headgroup mainly from the outer leaflet of these membranes. The ability of PDC-109 to extract endogenous phospholipids from epididymal sperm cells was followed by mass spectrometry, which allowed us to characterize the fatty acid pattern of the released lipids. From these cells, PDC-109 extracted phosphatidylcholine and sphingomyelin that contained an enrichment of mono- and di-unsaturated fatty acids as well as short-chain and lyso-phosphatidylcholine species. Based on the results, a model explaining the phospholipid specificity of PDC-109-mediated lipid release is presented.