Self-Assembly of Janus Oligomers into Onion-like Vesicles with Layer-by-Layer Water Discharging Capability: A Minimalist Model.

PubMed

Arai, Noriyoshi; Yasuoka, Kenji; Zeng, Xiao Cheng

2016-08-23

A vesicle in a cell is an enclosed structure in which the interior fluid is encompassed by a lipid bilayer. Synthetic vesicles are known as the liposomes. Liposomes with a single phospholipid bilayer are called unilamellar liposomes; otherwise, they are called multilamellar liposomes or onion-like liposomes (vesicles). One prototype synthetic onion-like vesicle, namely, onion-like dendrimersomes, have been recently produced via the self-assembly of amphiphilic Janus dendrimers (Proc. Natl. Acad. Sci. U.S.A. 2016, 113, 1162). Herein, we show computer simulation evidence of another type of onion-like vesicle, namely, onion-like oligomersomes, via the self-assembly of amphiphilic Janus oligomers in water. Specifically, we investigate the minimum-sized oligomers (or minimalist model) that can give rise to the onion-like oligomersomes as well as the composition-dependent phase diagrams. Insights into the formation condition and formation process of the onion-like oligomersomes are obtained. We demonstrate that the discharge of the in-vesicle water is through the remarkable "peeling-one-onion-layer-at-a-time" fashion, a feature that can be utilized for a clinical dosing regimen. The ability to control the formation of onion-like oligomersomes by design can be exploited for applications in drug and gene delivery.

Bubble-induced microstreaming: guiding and destroying lipid vesicles

NASA Astrophysics Data System (ADS)

Marmottant, Philippe; Hilgenfeldt, Sascha

2002-11-01

Micron-sized bubbles respond with strong oscillations when submitted to ultrasound. This has led to their use as echographic contrast enhancers. The large energy and force densities generated by the collapsing bubbles also make them non-invasive mechanical tools: Recently, it has been reported that the interaction of cavitating bubbles with nearby cells can render the latter permeable to large molecules (sonoporation), suggesting prospects for drug delivery and gene transfection. We have developed a laboratory setup that allows for a controlled study of the interaction of single microbubbles with single lipid bilayer vesicles. Substituting vesicles for cell membranes is advantageous because the mechanical properties of vesicles are well-known. Microscopic observations reveal that vesicles near a bubble follow the vivid streaming motion set up by the bubble. The vesicles "bounce" off the bubble, being periodically accelerated towards and away from it, and undergo well-defined shape deformations along their trajectory in accordance with fluid-dynamical theory. Break-up of vesicles could also be observed.

Engineering Globular Protein Vesicles through Tunable Self-Assembly of Recombinant Fusion Proteins

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

Jang, Yeongseon; Choi, Won Tae; Heller, William T.

Vesicles assembled from folded, globular proteins have potential for functions different from traditional lipid or polymeric vesicles. However, they also present challenges in understanding the assembly process and controlling vesicle properties. From detailed investigation of the assembly behavior of recombinant fusion proteins, this work reports a simple strategy to engineer protein vesicles containing functional, globular domains. This is achieved through tunable self-assembly of recombinant globular fusion proteins containing leucine zippers and elastin-like polypeptides. The fusion proteins form complexes in solution via high affinity binding of the zippers, and transition through dynamic coacervates to stable hollow vesicles upon warming. The thermalmore » driving force, which can be tuned by protein concentration or temperature, controls both vesicle size and whether vesicles are single or bi-layered. Lastly, these results provide critical information to engineer globular protein vesicles via self-assembly with desired size and membrane structure.« less

Engineering Globular Protein Vesicles through Tunable Self-Assembly of Recombinant Fusion Proteins

DOE PAGES

Jang, Yeongseon; Choi, Won Tae; Heller, William T.; ...

2017-07-27

Vesicles assembled from folded, globular proteins have potential for functions different from traditional lipid or polymeric vesicles. However, they also present challenges in understanding the assembly process and controlling vesicle properties. From detailed investigation of the assembly behavior of recombinant fusion proteins, this work reports a simple strategy to engineer protein vesicles containing functional, globular domains. This is achieved through tunable self-assembly of recombinant globular fusion proteins containing leucine zippers and elastin-like polypeptides. The fusion proteins form complexes in solution via high affinity binding of the zippers, and transition through dynamic coacervates to stable hollow vesicles upon warming. The thermalmore » driving force, which can be tuned by protein concentration or temperature, controls both vesicle size and whether vesicles are single or bi-layered. Lastly, these results provide critical information to engineer globular protein vesicles via self-assembly with desired size and membrane structure.« less

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

Boreyko, Jonathan B; Mruetusatorn, Prachya; Sarles, Stephen A

Droplet interface bilayers (DIBs) are a robust platform for studying synthetic cellular membranes; however, to date no DIBs have been produced at cellular length scales. Here, we create microscale droplet interface bilayers ( DIBs) at the interface between aqueous femtoliter-volume droplets within an oil-filled microfluidic channel. The uniquely large area-to-volume ratio of the droplets results in strong evaporation effects, causing the system to transition through three distinct regimes. First, the two adjacent droplets shrink into the shape of a single spherical droplet, where an augmented lipid bilayer partitions two hemi-spherical volumes. In the second regime, the combined effects of themore » shrinking monolayers and growing bilayer force the confined bilayer to buckle to conserve its mass. Finally, at a bending moment corresponding to a critical shear stress, the buckling bilayer fissions a vesicle to regulate its shape and stress. The DIBs produced here enable evaporation-induced bilayer dynamics reminiscent of endo- and exocytosis in cells.« less

Probing Biological Processes on Supported Lipid Bilayers with Single-Walled Carbon Nanotube Field-Effect Transistors

NASA Astrophysics Data System (ADS)

Zhou, Xinjian; Moran-Mirabal, Jose Manuel; Craighead, Harold; McEuen, Paul

2006-03-01

We have formed supported lipid bilayers (SLBs) by small unilamellar vesicle fusion on substrates containing single-walled carbon nanotube field-effect transistors (SWNT-FETs). We are able to detect the self-assembly of SLBs electrically with SWNT-FETs since their threshold voltages are shifted by this event. The SLB fully covers the NT surface and lipid molecules can diffuse freely in the bilayer surface across the NT. To study the interactions of important biological entities with receptors imbedded within the membrane, we have also integrated a membrane protein, GT1b ganglioside, in the bilayer. While bare gangliosides can diffuse freely across the NT, interestingly the NT acts as a diffusion barrier for the gangliosides when they are bound with tetanus toxin. This experiment opens the possibility of using SWNT-FETs as biosensors for label-free detection.

Viscoelastic deformation of lipid bilayer vesicles.

PubMed

Wu, Shao-Hua; Sankhagowit, Shalene; Biswas, Roshni; Wu, Shuyang; Povinelli, Michelle L; Malmstadt, Noah

2015-10-07

Lipid bilayers form the boundaries of the cell and its organelles. Many physiological processes, such as cell movement and division, involve bending and folding of the bilayer at high curvatures. Currently, bending of the bilayer is treated as an elastic deformation, such that its stress-strain response is independent of the rate at which bending strain is applied. We present here the first direct measurement of viscoelastic response in a lipid bilayer vesicle. We used a dual-beam optical trap (DBOT) to stretch 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) giant unilamellar vesicles (GUVs). Upon application of a step optical force, the vesicle membrane deforms in two regimes: a fast, instantaneous area increase, followed by a much slower stretching to an eventual plateau deformation. From measurements of dozens of GUVs, the average time constant of the slower stretching response was 0.225 ± 0.033 s (standard deviation, SD). Increasing the fluid viscosity did not affect the observed time constant. We performed a set of experiments to rule out heating by laser absorption as a cause of the transient behavior. Thus, we demonstrate here that the bending deformation of lipid bilayer membranes should be treated as viscoelastic.

Interactive, computer-assisted tracking of speckle trajectories in fluorescence microscopy: application to actin polymerization and membrane fusion.

PubMed

Smith, Matthew B; Karatekin, Erdem; Gohlke, Andrea; Mizuno, Hiroaki; Watanabe, Naoki; Vavylonis, Dimitrios

2011-10-05

Analysis of particle trajectories in images obtained by fluorescence microscopy reveals biophysical properties such as diffusion coefficient or rates of association and dissociation. Particle tracking and lifetime measurement is often limited by noise, large mobilities, image inhomogeneities, and path crossings. We present Speckle TrackerJ, a tool that addresses some of these challenges using computer-assisted techniques for finding positions and tracking particles in different situations. A dynamic user interface assists in the creation, editing, and refining of particle tracks. The following are results from application of this program: 1), Tracking single molecule diffusion in simulated images. The shape of the diffusing marker on the image changes from speckle to cloud, depending on the relationship of the diffusion coefficient to the camera exposure time. We use these images to illustrate the range of diffusion coefficients that can be measured. 2), We used the program to measure the diffusion coefficient of capping proteins in the lamellipodium. We found values ∼0.5 μm(2)/s, suggesting capping protein association with protein complexes or the membrane. 3), We demonstrate efficient measuring of appearance and disappearance of EGFP-actin speckles within the lamellipodium of motile cells that indicate actin monomer incorporation into the actin filament network. 4), We marked appearance and disappearance events of fluorescently labeled vesicles to supported lipid bilayers and tracked single lipids from the fused vesicle on the bilayer. This is the first time, to our knowledge, that vesicle fusion has been detected with single molecule sensitivity and the program allowed us to perform a quantitative analysis. 5), By discriminating between undocking and fusion events, dwell times for vesicle fusion after vesicle docking to membranes can be measured. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Dynamics, Surface Electrostatics and Phase Properties of Nanoscale Curved Lipid Bilayers

NASA Astrophysics Data System (ADS)

Koolivand, Amir

Surface electrostatic potential of a lipid bilayer governs many vital functions of living cells. Several classes of proteins are known of exhibiting strong binding preferences to curved lipid bilayer surfaces. In this project we employed electron paramagnetic resonance (EPR) of a recently introduced phospholipid (IMTSL-PTE) bearing a pH-sensitive nitroxide covalently attached to the lipid head group to measure the surface electrostatics of the lipid membrane and nanopore-confined lipid bilayers as a function of the bilayer curvature. The pKa of the ionizable group of this lipid-based spin probe is reporting on the bilayer surface electrostatics potential by changes in the EPR spectra. Specifically, both rotational dynamics and magnetic parameters of the nitroxide are affected by the probe protonation. Effect of curvature on the surface electrostatic potential and dynamics of lipid bilayer was studied for POPG and DMPG unilamellar vesicles (ULVs). It was found that the magnitude of the negative surface electrostatic potential increased upon decrease in the vesicle diameter for the bilayers in the fluid phase; however, no significant changes were observed for DMPG ULVs in a gel phase. We speculate that biologically relevant fluid bilayer phase allows for a larger variability in the lipid packing density in the lipid polar head group region than a more ordered gel phase and it is likely that the lipid flip-flop is responsible for pH equilibration of IMTSL-PTE. The kinetic EPR study of nitroxide reduction showed that the rate of flip-flop is in the order of 10-5 s-1. The flip-flop rate constant increases when vesicle size deceases. Oxygen permeability measured by X-ban EPR decreases in higher curved vesicles---an observation that is consistent with a tighter packing in smaller vesicles. Partitioning of a small nitroxide molecule TEMPO into ULVs was measured by X-band (9 GHz) and W-band (95 GHz) EPR spectroscopy. The partitioning coefficient of this probe in the lipid phase of the bilayer was higher in smaller vesicles likely due to a larger number of defects in smaller vesicles allowing more water soluble molecules partitioning into lipid bilayers. However, the rotational correlation time for TEMPO slows down in smaller vesicles indicating an increase in the lipid packing. Pulsed EPR techniques, HYSCORE and ESEEM spectroscopy, were used to detect local water concentration and distinguish the hydrogen bonded water to the nitroxide from the bulk one. HYSCORE was then employed to investigate the effect of bilayer curvature on the water penetration into lipid bilayer and it was found that the higher curved lipids allow more water to penetrate into lipid bilayer as a result of more defects in the highly curved lipid vesicles. Nanopore-confined lipid bilayers formed inside ordered nanochannels of anodic aluminum oxide (AAO) have found many practical applications, serving as thermodynamically stable biophysical models of cellular membranes of concave curvature and allowing for stabilization of membrane proteins in functional conformations. It was found that surface potential of POPG lipids inside the AAO pores are higher than that of vesicles---the effect that is attributed to highly ordered and packed lipids inside the AAO nanopores. At pH=7.0 the AAO zeta potential was found to be -29+/-0.64 mV. Cytochrome C and poly glutamic acid as positively and negatively charged macromolecules in physiological pH (7.4) were used to prepare multilayer protein nanotubes and cytochrome c interaction with AAO was studied by CD and UV-Vis spectroscopy. Lipid nanotube arrays containing a transmembrane WALP peptide were also formed and these macroscopically aligned lipid nanotubes were studied by CD spectroscopy. The lipid phase transition of DMPC and binding of melittin, an antibacterial peptide model, were observed from a frequency change for the QCM quartz-AAO-Lipid as a promising "biosensor".

Controlling Two-dimensional Tethered Vesicle Motion Using an Electric Field

PubMed Central

Yoshina-Ishii, Chiaki; Boxer, Steven G.

2008-01-01

We recently introduced methods to tether phospholipid vesicles or proteoliposomes onto a fluid supported lipid bilayer using DNA hybridization. These intact tethered vesicles diffuse in two dimensions parallel to the supporting membrane surface. In this paper, we report the dynamic response of individual tethered vesicles to an electric field applied parallel to the bilayer surface. Vesicles respond to the field by moving in the direction of electro-osmotic flow, and this can be used to reversibly concentrate tethered vesicles against a barrier. By adding increasing amounts of negatively charged phosphatidylserine to the supporting bilayer to increase electro-osmosis, the electrophoretic mobility of the tethered vesicles can be increased. The electro-osmotic contribution can be modeled well by a sphere connected to a cylindrical anchor in a viscous membrane with charged head groups. The electrophoretic force on the negatively charged tethered vesicles opposes the electro-osmotic force. By increasing the amount of negative charge on the tethered vesicle, drift in the direction of electro-osmotic flow can be slowed; at high negative charge on the tethered vesicle, motion can be forced in the direction of electrophoresis. The balance between these forces can be visualized on a patterned supporting bilayer containing negatively charged lipids which themselves reorganize in an externally applied electric field to create a gradient of charge within a corralled region. The charge gradient at the surface creates a gradient of electro-osmotic flow, and vesicles carrying similar amounts of negative charge can be focused to a region perpendicular to the applied field where electrophoresis is balanced by electro-osmosis, away from the corral boundary. Electric fields are effective tools to direct tethered vesicles, concentrate them and to measure the tethered vesicle’s electrostatic properties. PMID:16489833

Kinetics of DNA-mediated docking reactions between vesicles tethered to supported lipid bilayers

PubMed Central

Chan, Yee-Hung M.; Lenz, Peter; Boxer, Steven G.

2007-01-01

Membrane–membrane recognition and binding are crucial in many biological processes. We report an approach to studying the dynamics of such reactions by using DNA-tethered vesicles as a general scaffold for displaying membrane components. This system was used to characterize the docking reaction between two populations of tethered vesicles that display complementary DNA. Deposition of vesicles onto a supported lipid bilayer was performed by using a microfluidic device to prevent mixing of the vesicles in bulk during sample preparation. Once tethered onto the surface, vesicles mixed via two-dimensional diffusion. DNA-mediated docking of two reacting vesicles results in their colocalization after collision and their subsequent tandem motion. Individual docking events and population kinetics were observed via epifluorescence microscopy. A lattice-diffusion simulation was implemented to extract from experimental data the probability, Pdock, that a collision leads to docking. For individual vesicles displaying small numbers of docking DNA, Pdock shows a first-order relationship with copy number as well as a strong dependence on the DNA sequence. Both trends are explained by a model that includes both tethered vesicle diffusion on the supported bilayer and docking DNA diffusion over each vesicle's surface. These results provide the basis for the application of tethered vesicles to study other membrane reactions including protein-mediated docking and fusion. PMID:18025472

Interactions of the GM2 activator protein with phosphatidylcholine bilayers: a site-directed spin-labeling power saturation study.

PubMed

Mathias, Jordan D; Ran, Yong; Carter, Jeffery D; Fanucci, Gail E

2009-09-02

The GM2 activator protein (GM2AP) is an accessory protein that is an essential component in the catabolism of the ganglioside GM2. A function of GM2AP is to bind and extract GM2 from intralysosomal vesicles, forming a soluble protein-lipid complex, which interacts with the hydrolase Hexosaminidase A, the enzyme that cleaves the terminal sugar group of GM2. Here, we used site-directed spin labeling with power saturation electron paramagnetic resonance to determine the surface-bound orientation of GM2AP upon phosphatidylcholine vesicles. Because GM2AP extracts lipid ligands from the vesicle and is undergoing exchange on and off the vesicle surface, we utilized a nickel-chelating lipid to localize the paramagnetic metal collider to the lipid bilayer-aqueous interface. Spin-labeled sites that collide with the lipid-bound metal relaxing agent provide a means for mapping sites of the protein that interact with the lipid bilayer interface. Results show that GM2AP binds to lipid bilayers such that the residues lining the lipid-binding cavity lie on the vesicle surface. This orientation creates a favorable microenvironment that can allow for the lipid tails to flip out of the bilayer directly into the hydrophobic pocket of GM2AP.

Effect of vesicle size on the prodan fluorescence in diheptadecanoylphosphatidylcholine bilayer membrane under atmospheric and high pressures.

PubMed

Goto, Masaki; Sawaguchi, Hiroshi; Tamai, Nobutake; Matsuki, Hitoshi; Kaneshina, Shoji

2010-08-17

The bilayer phase behavior of diheptadecanoylphosphatidylcholine (C17PC) with different vesicle sizes (large multilamellar vesicle (LMV) and giant multilamellar vesicle (GMV)) was investigated by fluorescence spectroscopy using a polarity-sensitive fluorescent probe Prodan under atmospheric and high pressures. The difference in phase transitions and thermodynamic quantities of the transition was hardly observed between LMV and GMV used here. On the contrary, the Prodan fluorescence in the bilayer membranes changed depending on the size of vesicles as well as on the phase states. From the second derivative of fluorescence spectra, the three-dimensional image plots in which we can see the location of Prodan in the bilayer membrane as blue valleys were constructed for LMV and GMV under atmospheric pressure. The following characteristic behavior was found: (1) the Prodan molecules in GMV can be distributed to not only adjacent glycerol backbone region, but also near bulk-water region in the lamellar gel or ripple gel phase; (2) the blue valleys of GMV became deeper than those of LMV because of the greater surface density of the Prodan molecules per unit area of GMV than LMV; (3) the liquid crystalline phase of the bilayer excludes the Prodan molecules to a more hydrophilic region at the membrane surface with an increase in vesicle size; (4) the accurate information as to the phase transitions is gradually lost with increasing vesicle size. Under the high-pressure condition, the difference in Prodan fluorescence between LMV and GMV was essentially the same as the difference under atmospheric pressure except for the existence of the pressure-induced interdigitated gel phase. Further, we found that Prodan fluorescence spectra in the interdigitated gel phase were especially affected by the size of vesicles. This study revealed that the Prodan molecules can move around the headgroup region by responding not only to the phase state but also to the vesicle size, and they become a useful membrane probe, detecting important membrane properties such as the packing stress.

Lipid tail protrusions initiate spontaneous insertion of charged, amphiphilic nanoparticles into lipid bilayers

NASA Astrophysics Data System (ADS)

van Lehn, Reid; Ricci, Maria; Carney, Randy; Voitchovsky, Kislon; Stellacci, Francesco; Alexander-Katz, Alfredo

2014-03-01

Vesicle fusion is a primary mechanism used to mediate the uptake and trafficking of materials both into and between cells. The pathway of vesicle fusion involves the formation of a lipid stalk in which the hydrophobic core regions of two closely associated bilayers merge. The transition state for stalk formation requires the transient protrusion of hydrophobic lipid tails into solvent; favorable contact between these hydrophobic tails then drives stalk creation. In this work, we use unbiased atomistic molecular dynamics simulations to show that lipid tail protrusions can also induce the insertion of charged, amphiphilic nanoparticles (NPs) into lipid bilayers. As in the case of vesicle fusion, the rate-limiting step for NP-bilayer fusion is the stochastic protrusion of aliphatic lipid tails into solvent and into contact with hydrophobic material in the amphiphilic NP monolayer. We confirm our predictions with experiments on supported lipid bilayers. The strong agreement between simulation and experiments indicates that the pre-stalk transition associated with vesicle fusion may be a general mechanism for the insertion of amphiphilic nano-objects that could be prominent in biological systems given the widespread use of NPs in applications ranging from drug delivery to biosensing.

The mechanism of a nuclear pore assembly: a molecular biophysics view.

PubMed

Kuvichkin, Vasily V

2011-06-01

The basic problem of nuclear pore assembly is the big perinuclear space that must be overcome for nuclear membrane fusion and pore creation. Our investigations of ternary complexes: DNA-PC liposomes-Mg²⁺, and modern conceptions of nuclear pore structure allowed us to introduce a new mechanism of nuclear pore assembly. DNA-induced fusion of liposomes (membrane vesicles) with a single-lipid bilayer or two closely located nuclear membranes is considered. After such fusion on the lipid bilayer surface, traces of a complex of ssDNA with lipids were revealed. At fusion of two identical small liposomes (membrane vesicles) < 100 nm in diameter, a "big" liposome (vesicle) with ssDNA on the vesicle equator is formed. ssDNA occurrence on liposome surface gives a biphasic character to the fusion kinetics. The "big" membrane vesicle surrounded by ssDNA is the base of nuclear pore assembly. Its contact with the nuclear envelope leads to fast fusion of half of the vesicles with one nuclear membrane; then ensues a fusion delay when ssDNA reaches the membrane. The next step is to turn inside out the second vesicle half and its fusion to other nuclear membrane. A hole is formed between the two membranes, and nucleoporins begin pore complex assembly around the ssDNA. The surface tension of vesicles and nuclear membranes along with the kinetic energy of a liquid inside a vesicle play the main roles in this process. Special cases of nuclear pore formation are considered: pore formation on both nuclear envelope sides, the difference of pores formed in various cell-cycle phases and linear nuclear pore clusters.

Comparison of Extruded and Sonicated Vesicles for Planar Bilayer Self-Assembly

PubMed Central

Cho, Nam-Joon; Hwang, Lisa Y.; Solandt, Johan J.R.; Frank, Curtis W.

2013-01-01

Lipid vesicles are an important class of biomaterials that have a wide range of applications, including drug delivery, cosmetic formulations and model membrane platforms on solid supports. Depending on the application, properties of a vesicle population such as size distribution, charge and permeability need to be optimized. Preparation methods such as mechanical extrusion and sonication play a key role in controlling these properties, and yet the effects of vesicle preparation method on vesicular properties and integrity (e.g., shape, size, distribution and tension) remain incompletely understood. In this study, we prepared vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid by either extrusion or sonication, and investigated the effects on vesicle size distribution over time as well as the concomitant effects on the self-assembly of solid-supported planar lipid bilayers. Dynamic light scattering (DLS), quartz crystal microbalance with dissipation (QCM-D) monitoring, fluorescence recovery after photobleaching (FRAP) and atomic force microscopy (AFM) experiments were performed to characterize vesicles in solution as well as their interactions with silicon oxide substrates. Collectively, the data support that sonicated vesicles offer more robust control over the self-assembly of homogenous planar lipid bilayers, whereas extruded vesicles are vulnerable to aging and must be used soon after preparation. PMID:28811437

Structure formation in binary mixtures of lipids and detergents: self-assembly and vesicle division.

PubMed

Noguchi, Hiroshi

2013-01-14

Self-assembly dynamics in binary surfactant mixtures and structure changes of lipid vesicles induced by detergent solution are studied using coarse-grained molecular simulations. Disk-shaped micelles, the bicelles, are stabilized by detergents surrounding the rim of a bilayer disk of lipids. The self-assembled bicelles are considerably smaller than bicelles formed from vesicle rupture, and their size is determined by the concentrations of lipids and detergents and the interactions between the two species. The detergent-adsorption induces spontaneous curvature of the vesicle bilayer and results in vesicle division into two vesicles or vesicle rupture into worm-like micelles. The division occurs mainly via the inverse pathway of the modified stalk model. For large spontaneous curvature of the monolayers of the detergents, a pore is often opened, thereby leading to vesicle division or worm-like micelle formation.

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.

Membrane fusion-competent virus-like proteoliposomes and proteinaceous supported bilayers made directly from cell plasma membranes.

PubMed

Costello, Deirdre A; Hsia, Chih-Yun; Millet, Jean K; Porri, Teresa; Daniel, Susan

2013-05-28

Virus-like particles are useful materials for studying virus-host interactions in a safe manner. However, the standard production of pseudovirus based on the vesicular stomatitis virus (VSV) backbone is an intricate procedure that requires trained laboratory personnel. In this work, a new strategy for creating virus-like proteoliposomes (VLPLs) and virus-like supported bilayers (VLSBs) is presented. This strategy uses a cell blebbing technique to induce the formation of nanoscale vesicles from the plasma membrane of BHK cells expressing the hemagglutinin (HA) fusion protein of influenza X-31. These vesicles and supported bilayers contain HA and are used to carry out single particle membrane fusion events, monitored using total internal reflection fluorescence microscopy. The results of these studies show that the VLPLs and VLSBs contain HA proteins that are fully competent to carry out membrane fusion, including the formation of a fusion pore and the release of fluorophores loaded into vesicles. This new strategy for creating spherical and planar geometry virus-like membranes has many potential applications. VLPLs could be used to study fusion proteins of virulent viruses in a safe manner, or they could be used as therapeutic delivery particles to transport beneficial proteins coexpressed in the cells to a target cell. VLSBs could facilitate high throughput screening of antiviral drugs or pathogen-host cell interactions.

Enhanced adsorption of Ca-ATPase containing vesicles on a negatively charged solid-supported-membrane for the investigation of membrane transporters.

PubMed

Sacconi, Alessio; Moncelli, Maria Rosa; Margheri, Giancarlo; Tadini-Buoninsegni, Francesco

2013-11-12

A convenient model system for a biological membrane is a solid-supported membrane (SSM), which consists of a gold-supported alkanethiol|phospholipid bilayer. In combination with a concentration jump method, SSMs have been used for the investigation of several membrane transporters. Vesicles incorporating sarcoplasmic reticulum Ca-ATPase (SERCA) were adsorbed on a negatively charged SSM (octadecanethiol|phosphatidylserine bilayer). The current signal generated by the adsorbed vesicles following an ATP concentration jump was compared to that produced by SERCA-containing vesicles adsorbed on a conventional SSM (octadecanethiol|phosphatidylcholine bilayer). A significantly higher current amplitude was recorded on the serine-based SSM. The adsorption of SERCA-incorporating vesicles on the SSM was then characterized by surface plasmon resonance (SPR). The SPR measurements clearly indicate that in the presence of Ca(2+) and Mg(2+), the amount of adsorbed vesicles on the serine-based SSM is about twice that obtained using the conventional SSM, thereby demonstrating that the higher current amplitude recorded on the negatively charged SSM is correlated with a greater quantity of adsorbed vesicles. The enhanced adsorption of membrane vesicles on the PS-based SSM may be useful to study membrane preparations with a low concentration of transport protein generating small current signals, as in the case of various recombinantly expressed proteins.

Splaying of aliphatic tails plays a central role in barrier crossing during liposome fusion.

PubMed

Mirjanian, Dina; Dickey, Allison N; Hoh, Jan H; Woolf, Thomas B; Stevens, Mark J

2010-09-02

The fusion between two lipid bilayers involves crossing a complicated energy landscape. The limiting barrier in the process appears to be between two closely opposed bilayers and the intermediate state where the outer leaflets are fused. We have performed molecular dynamics simulations to characterize the free energy barrier for the fusion of two liposomes and to examine the molecular details of barrier crossing. To capture the slow dynamics of fusion, a model using coarse-grained representations of lipids was used. The fusion between pairs of liposomes was simulated for four systems: DPPC, DOPC, a 3:1 mixture of DPPC/DPPE, and an asymmetric lipid tail system in which one tail of DPPC was reduced to half the length (ASTail). The weighted histogram method was used to compute the free energy as a function of separation distance. The relative barrier heights for these systems was found to be ASTail > DPPC > DPPC/DPPE > DOPC, in agreement with experimental observations. Further, the free energy curves for all four can be overlaid on a single curve by plotting the free energy versus the surface separation (differing only in the point of fusion). These simulations also confirm that the two main contributions to the free energy barrier are the removal of water between the vesicles and the deformation of the vesicle. The most prominent molecular detail of barrier crossing in all cases examined was the splaying of lipid tails, where initially a single splayed lipid formed a bridge between the two outer leaflets that promotes additional lipid mixing between the vesicles and eventually leads to fusion. The tail splay appears to be closely connected to the energetics of the process. For example, the high barrier for the ASTail is the result of a smaller distance between terminal methyl groups in the splayed molecule. The shortening of this distance requires the liposomes to be closer together, which significantly increases the cost of water removal and bilayer deformation. Before tail splay can initiate fusion, contact must occur between a tail end and the external water. In isolated vesicles, the contact fraction is correlated to the fusogenicity difference between DPPC and DOPC. Moreover, for planar bilayers, the contact fraction is much lower for DPPC, which is consistent with its lack of fusion in giant vesicles. The simulation results show the key roles of lipid tail dynamics in governing the fusion energy landscape.

Understanding crumpling lipid vesicles at the gel phase transition

NASA Astrophysics Data System (ADS)

Hirst, Linda; Ossowski, Adam; Fraser, Matthew

2011-03-01

Wrinkling and crumpling transitions in different membrane types have been studied extensively in recent years both theoretically and computationally. There has also been very interesting recent work on defects in liquid crystalline shells. Lipid bilayer vesicles, widely used in biophysical research can be considered as a single layer smectic shell in the liquid crystalline phase. On cooling the lipid vesicle a transition to the gel phase may take place in which the lipid chains tilt and assume a more ordered packing arrangement. We observe large scale morphological changes in vesicles close to this transition point using fluorescence microscopy and investigate the possible mechanisms for this transition. Confocal microscopy is used to map 3D vesicle shape and crumpling length-scales. We also employ the molecular tilt sensitive dye, Laurdan to investigate the role of tilt domain formation on macroscopic structure. Funded by NSF CAREER award (DMR - BMAT #0852791).

Viscoelastic deformation of lipid bilayer vesicles†

PubMed Central

Wu, Shao-Hua; Sankhagowit, Shalene; Biswas, Roshni; Wu, Shuyang; Povinelli, Michelle L.

2015-01-01

Lipid bilayers form the boundaries of the cell and its organelles. Many physiological processes, such as cell movement and division, involve bending and folding of the bilayer at high curvatures. Currently, bending of the bilayer is treated as an elastic deformation, such that its stress-strain response is independent of the rate at which bending strain is applied. We present here the first direct measurement of viscoelastic response in a lipid bilayer vesicle. We used a dual-beam optical trap (DBOT) to stretch 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) giant unilamellar vesicles (GUVs). Upon application of a step optical force, the vesicle membrane deforms in two regimes: a fast, instantaneous area increase, followed by a much slower stretching to an eventual plateau deformation. From measurements of dozens of GUVs, the average time constant of the slower stretching response was 0.225 ± 0.033 s (standard deviation, SD). Increasing the fluid viscosity did not affect the observed time constant. We performed a set of experiments to rule out heating by laser absorption as a cause of the transient behavior. Thus, we demonstrate here that the bending deformation of lipid bilayer membranes should be treated as viscoelastic. PMID:26268612

Subnanometer structure of an asymmetric model membrane: Interleaflet coupling influences domain properties

DOE PAGES

Heberle, Frederick A.; Marquardt, Drew; Doktorova, Milka; ...

2016-04-29

Cell membranes possess a complex three-dimensional architecture, including nonrandom lipid lateral organization within the plane of a bilayer leaflet, and compositional asymmetry between the two leaflets. As a result, delineating the membrane structure–function relationship has been a highly challenging task. Even in simplified model systems, the interactions between bilayer leaflets are poorly understood, due in part to the difficulty of preparing asymmetric model membranes that are free from the effects of residual organic solvent or osmotic stress. To address these problems, we have modified a technique for preparing asymmetric large unilamellar vesicles (aLUVs) via cyclodextrin-mediated lipid exchange in order tomore » produce tensionless, solvent-free aLUVs suitable for a range of biophysical studies. Leaflet composition and structure were characterized using isotopic labeling strategies, which allowed us to avoid the use of bulky labels. NMR and gas chromatography provided precise quantification of the extent of lipid exchange and bilayer asymmetry, while small-angle neutron scattering (SANS) was used to resolve bilayer structural features with subnanometer resolution. Isotopically asymmetric POPC vesicles were found to have the same bilayer thickness and area per lipid as symmetric POPC vesicles, demonstrating that the modified exchange protocol preserves native bilayer structure. Partial exchange of DPPC into the outer leaflet of POPC vesicles produced chemically asymmetric vesicles with a gel/fluid phase-separated outer leaflet and a uniform, POPC-rich inner leaflet. SANS was able to separately resolve the thicknesses and areas per lipid of coexisting domains, revealing reduced lipid packing density of the outer leaflet DPPC-rich phase compared to typical gel phases. Lastly, our finding that a disordered inner leaflet can partially fluidize ordered outer leaflet domains indicates some degree of interleaflet coupling, and invites speculation on a role for bilayer asymmetry in modulating membrane lateral organization.« less

Experimental study of the bending elasticity of charged lipid bilayers in aqueous solutions with pH5

NASA Astrophysics Data System (ADS)

Mitkova, D.; Stoyanova-Ivanova, A.; Ermakov, Yu A.; Vitkova, V.

2012-12-01

Exposure to high concentrations of contaminations due to air polluting gases, vapours and aerosols and possibly altering the normal pH in the body could lead to undesirable changes in the properties of biological cells. Here, we study experimentally the mechanical properties of synthetic phospholipid bilayers containing increasing molar fractions (up to 0.15) of charged lipid (synthetic phosphatidylserine) in aqueous solutions with controlled ionic strength and at pH 5, which is slightly lower than the physiological values of pH. Our observations in phase contrast and fluorescence testified to the coexistence of two phases in membranes for temperatures below 29°C. Micro-sized inhomogeneities in vesicle membranes were systematically observed at temperatures lower than 29°C and for molar fractions of phosphatidylserine in the bilayer higher than 0.1. For the quantitative determination of the membrane bending rigidity, we applied thermal fluctuation analysis of the shape of quasispherical lipid vesicles. As far as the liquid-crystalline state of the bilayer is a necessary condition for the application of the experimental method, only vesicles satisfying this requirement were processed for determination of their membrane bending rigidity. The value obtained for the bending modulus of bilayers with 0.15 molar content of charged lipid is about two times higher than the bending modulus of uncharged membranes in the same bathing solution. These findings are in qualitative agreement with our previous results for the bending rigidity of charged bilayers, measured by vesicle micromanipulation.

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

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.

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.

Vesicle solubilization by bile salts: comparison of macroscopic theory and simulation.

PubMed

Haustein, M; Wahab, M; Mögel, H-J; Schiller, P

2015-04-14

Lipid metabolism is accompanied by the solubilization of lipid bilayer membranes by bile salts. We use Brownian dynamics simulations to study the solubilization of model membranes and vesicles by sodium cholate. The solubilization pathways of small and large vesicles are found to be different. Both results for small and large vesicles can be compared with predictions of a macroscopic theoretical description. The line tension of bilayer edges is an important parameter in the solubilization process. We propose a simple method to determine the line tension by analyzing the shape fluctuations of planar membrane patches. Macroscopic mechanical models provide a reasonable explanation for processes observed when a spherical vesicle consisting of lipids and adsorbed bile salt molecules is transformed into mixed lipid-bile salt micelles.

Gold nanoparticles covalently assembled onto vesicle structures as possible biosensing platform

PubMed Central

Barroso, M Fátima; Luna, M Alejandra; Tabares, Juan S Flores; Delerue-Matos, Cristina; Correa, N Mariano

2016-01-01

Summary In this contribution a strategy is shown to covalently immobilize gold nanoparticles (AuNPs) onto vesicle bilayers with the aim of using this nanomaterial as platform for the future design of immunosensors. A novel methodology for the self-assembly of AuNPs onto large unilamellar vesicle structures is described. The vesicles were formed with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-undecanethiol (SH). After, the AuNPs photochemically synthesized in pure glycerol were mixed and anchored onto SH–DOPC vesicles. The data provided by voltammetry, spectrometry and microscopy techniques indicated that the AuNPs were successfully covalently anchored onto the vesicle bilayer and decorated vesicles exhibit a spherical shape with a size of 190 ± 10 nm. The developed procedure is easy, rapid and reproducible to start designing a possible immunosensor by using environmentally friendly procedures. PMID:27335755

Associative polymers bridging between layers of multilamellar vesicles.

NASA Astrophysics Data System (ADS)

Choi, Seo; Bhatia, Surita

2006-03-01

Multilamellar vesicles can be found in a variety of pharmaceutical formulations, personal care products, and home care products. Hydrophobically modified associative polymers are often used to stabilize the vesicles or to control the rheological properties of these formulations. The hydrophobic groups are expected to insert themselves into the vesicle bilayers. Recent experimental work shows that hydrophobically modified polymers may from bridges between vesicles or may bridge between layers of a single vesicle. The latter configuration forces an interlayer spacing roughly equal to the radius of gyration of the backbone between associative groups. We have performed simple mean-field calculations on ideal telechelic associative polymers between concentric spherical surfaces. We find that the free energy per chain has an attractive minimum when the layer spacing is approximately N^1/2l, which is consistent with experimental results. The depth of the minimum depends on both chain length and curvature, and as expected when the curvature becomes small, the result for telechelic chains between flat surfaces is recovered.

Rupture and Spreading Dynamics of Lipid Membranes on a Solid Surface

NASA Astrophysics Data System (ADS)

Perazzo, Antonio; Shin, Sangwoo; Colosqui, Carlos; Young, Yuan-Nan; Stone, Howard A.

2017-11-01

The spreading of lipid membranes on solid surfaces is a dynamic phenomenon relevant to drug delivery, endocytosis, biofouling, and the synthesis of supported lipid bilayers. Current technological developments are limited by an incomplete understanding of the spreading and adhesion dynamics of a lipid bilayer under different physicochemical conditions. Here, we present recent experimental and theoretical results for the spreading of giant unilamellar vesicles (GUVs), where the vesicle shell consists of a lipid bilayer. In particular, we study the effect of different background ion concentrations, osmolarity mismatches between the interior and the exterior of the vesicles, and different surface chemistries of the glass substrate. In all of the studied cases, we observe a delay time before a GUV in contact with the solid surface eventually ruptures. The rupture kinetics and subsequent spreading dynamics is controlled by the ionic screening within the thin film of liquid between the vesicle and the surface. Different rupture mechanisms, mobilities of the spreading vesicle, and degrees of substrate coverage are observed by varying the electrolyte concentration, solid surface charge, and osmolarity mismatch.

Changes in lipid bilayer structure caused by the helix-to-sheet transition of an HIV-1 gp41 fusion peptide derivative

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

Heller, William T.; Rai, Durgesh K.

HIV-1, like other enveloped viruses, undergoes fusion with the cell membrane to infect it. Viral coat proteins are thought to bind the virus to the membrane and actively fuse the viral and cellular membranes together. The actual molecular mechanism of fusion is challenging to visualize, resulting in the use of model systems. In this paper, the bilayer curvature modifying properties of a synthetic variant of the HIV-1 gp41 fusion peptide with lipid bilayer vesicles composed of a mixture of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylserine (DMPS) were studied. In 7:3 DMPC:DMPS vesicles made with deuterium-labeled DMPC, the peptide was observedmore » to undergo a concentration-dependent conformational transition between an α-helix and an antiparallel β-sheet. Through the use of small-angle neutron scattering (SANS) and selective deuterium labeling, it was revealed that conformational transition of the peptide is also accompanied by a transition in the structure of the lipid bilayer. In addition to changes in the distribution of the lipid between the leaflets of the vesicle, the SANS data are consistent with two regions having different thicknesses. Finally, of the two different bilayer structures, the one corresponding to the smaller area fraction, being ~8% of the vesicle area, is much thicker than the remainder of the vesicle, which suggests that there are regions of localized negative curvature similar to what takes place at the point of contact between two membranes immediately preceding fusion.« less

Changes in lipid bilayer structure caused by the helix-to-sheet transition of an HIV-1 gp41 fusion peptide derivative

DOE PAGES

Heller, William T.; Rai, Durgesh K.

2017-01-16

HIV-1, like other enveloped viruses, undergoes fusion with the cell membrane to infect it. Viral coat proteins are thought to bind the virus to the membrane and actively fuse the viral and cellular membranes together. The actual molecular mechanism of fusion is challenging to visualize, resulting in the use of model systems. In this paper, the bilayer curvature modifying properties of a synthetic variant of the HIV-1 gp41 fusion peptide with lipid bilayer vesicles composed of a mixture of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylserine (DMPS) were studied. In 7:3 DMPC:DMPS vesicles made with deuterium-labeled DMPC, the peptide was observedmore » to undergo a concentration-dependent conformational transition between an α-helix and an antiparallel β-sheet. Through the use of small-angle neutron scattering (SANS) and selective deuterium labeling, it was revealed that conformational transition of the peptide is also accompanied by a transition in the structure of the lipid bilayer. In addition to changes in the distribution of the lipid between the leaflets of the vesicle, the SANS data are consistent with two regions having different thicknesses. Finally, of the two different bilayer structures, the one corresponding to the smaller area fraction, being ~8% of the vesicle area, is much thicker than the remainder of the vesicle, which suggests that there are regions of localized negative curvature similar to what takes place at the point of contact between two membranes immediately preceding fusion.« less

Gramicidin ion channels in a lipid bilayer supported on polyelectrolyte multilayer films: an electrochemical impedance study.

PubMed

Diamanti, Eleftheria; Gutiérrez-Pineda, Eduart; Politakos, Nikolaos; Andreozzi, Patrizia; Rodriguez-Presa, María José; Knoll, Wolfgang; Azzaroni, Omar; Gervasi, Claudio A; Moya, Sergio E

2017-12-06

Supported membranes on polymer cushions are of fundamental interest as models for cell membranes. The use of polyelectrolyte multilayers (PEMs) assembled by the layer by layer (LbL) technique as supports for a bilayer allows for easy integration of the lipid bilayer on surfaces and devices and for nanoscale tunable spacing of the lipid bilayer. Controlling ionic permeability in lipid bilayers supported on PEMs triggers potential applications in sensing and as models for transport phenomena in cell membranes. Lipid bilayers displaying gramicidin channels are fabricated on top of polyallylamine hydrochloride (PAH) and polystyrene sulfonate (PSS) multilayer films, by the assembly of vesicles of phosphatidylcholine and phosphatidylserine, 50 : 50 M/M, carrying gramicidin (GA). Quartz crystal microbalance with dissipation shows that the vesicles with GA fuse into a bilayer. Atomic force microscopy reveals that the presence of GA alters the bilayer topography resulting in depressions in the bilayer of around 70 nm in diameter. Electrochemical impedance spectroscopy (EIS) studies show that supported bilayers carrying GA have smaller resistances than the bilayers without GA. Lipid layers carrying GA display a higher conductance for K + than for Na + and are blocked in the presence of Ca 2+ .

A modular platform for one-step assembly of multi-component membrane systems by fusion of charged proteoliposomes

NASA Astrophysics Data System (ADS)

Ishmukhametov, Robert R.; Russell, Aidan N.; Berry, Richard M.

2016-10-01

An important goal in synthetic biology is the assembly of biomimetic cell-like structures, which combine multiple biological components in synthetic lipid vesicles. A key limiting assembly step is the incorporation of membrane proteins into the lipid bilayer of the vesicles. Here we present a simple method for delivery of membrane proteins into a lipid bilayer within 5 min. Fusogenic proteoliposomes, containing charged lipids and membrane proteins, fuse with oppositely charged bilayers, with no requirement for detergent or fusion-promoting proteins, and deliver large, fragile membrane protein complexes into the target bilayers. We demonstrate the feasibility of our method by assembling a minimal electron transport chain capable of adenosine triphosphate (ATP) synthesis, combining Escherichia coli F1Fo ATP-synthase and the primary proton pump bo3-oxidase, into synthetic lipid vesicles with sizes ranging from 100 nm to ~10 μm. This provides a platform for the combination of multiple sets of membrane protein complexes into cell-like artificial structures.

Shaped Apertures in Photoresist Films Enhance the Lifetime and Mechanical Stability of Suspended Lipid Bilayers

PubMed Central

Kalsi, Sumit; Powl, Andrew M.; Wallace, B.A.; Morgan, Hywel; de Planque, Maurits R.R.

2014-01-01

Planar lipid bilayers suspended in apertures provide a controlled environment for ion channel studies. However, short lifetimes and poor mechanical stability of suspended bilayers limit the experimental throughput of bilayer electrophysiology experiments. Although bilayers are more stable in smaller apertures, ion channel incorporation through vesicle fusion with the suspended bilayer becomes increasingly difficult. In an alternative bilayer stabilization approach, we have developed shaped apertures in SU8 photoresist that have tapered sidewalls and a minimum diameter between 60 and 100 μm. Bilayers formed at the thin tip of these shaped apertures, either with the painting or the folding method, display drastically increased lifetimes, typically >20 h, and mechanical stability, being able to withstand extensive perturbation of the buffer solution. Single-channel electrical recordings of the peptide alamethicin and of the proteoliposome-delivered potassium channel KcsA demonstrate channel conductance with low noise, made possible by the small capacitance of the 50 μm thick SU8 septum, which is only thinned around the aperture, and unimpeded proteoliposome fusion, enabled by the large aperture diameter. We anticipate that these shaped apertures with micrometer edge thickness can substantially enhance the throughput of channel characterization by bilayer lipid membrane electrophysiology, especially in combination with automated parallel bilayer platforms. PMID:24739164

Vesicular perylene dye nanocapsules as supramolecular fluorescent pH sensor systems.

PubMed

Zhang, Xin; Rehm, Stefanie; Safont-Sempere, Marina M; Würthner, Frank

2009-11-01

Water-soluble, self-assembled nanocapsules composed of a functional bilayer membrane and enclosed guest molecules can provide smart (that is, condition responsive) sensors for a variety of purposes. Owing to their outstanding optical and redox properties, perylene bisimide chromophores are interesting building blocks for a functional bilayer membrane in a water environment. Here, we report water-soluble perylene bisimide vesicles loaded with bispyrene-based energy donors in their aqueous interior. These loaded vesicles are stabilized by in situ photopolymerization to give nanocapsules that are stable over the entire aqueous pH range. On the basis of pH-tunable spectral overlap of donors and acceptors, the donor-loaded polymerized vesicles display pH-dependent fluorescence resonance energy transfer from the encapsulated donors to the bilayer dye membrane, providing ultrasensitive pH information on their aqueous environment with fluorescence colour changes covering the whole visible light range. At pH 9.0, quite exceptional white fluorescence could be observed for such water-soluble donor-loaded perylene vesicles.

Interaction of phospholipid vesicles with cells. Endocytosis and fusion as alternate mechanisms for the uptake of lipid-soluble and water-soluble molecules

PubMed Central

1975-01-01

Depending on their phospholipid composition, liposomes are endocytosed by, or fuse with, the plasma membrane, of Acanthamoeba castellanii. Unilamellar egg lecithin vesicles are endocytosed by amoeba at 28 degrees C with equal uptake of the phospholipid bilayer and the contents of the internal aqueous space of the vesicles. Uptake is inhibited almost completely by incubation at 4 degrees C or in the presence of dinitrophenol. After uptake at 28 degrees C, the vesicle phospholipid can be visualized by electron microscope autoradiography within cytoplasmic vacuoles. In contrast, uptake of unilamellar dipalmitoyl lecithin vesicles and multilamellar dipalmitoyl lecithin liposomes is only partially inhibited at 4 degrees C, by dinitrophenol and by prior fixation of the amoebae with glutaraldehyde, each of which inhibits pinocytosis. Vesicle contents are taken up only about 40% as well as the phospholipid bilayer. Electron micrographs are compatible with the interpretation that dipalmitoyl lecithin vesicles fuse with the amoeba plasma membrane, adding their phospholipid to the cell surface, while their contents enter the cell cytoplasm. Dimyristoyl lecithin vesicles behave like egg lecithin vesicles while distearoyl lecithin vesicles behave like dipalmitoyl lecithin vesicles. PMID:1174130

Dynamics of small unilamellar vesicles

NASA Astrophysics Data System (ADS)

Hoffmann, Ingo; Hoffmann, Claudia; Farago, Bela; Prévost, Sylvain; Gradzielski, Michael

2018-03-01

In this paper, we investigate the dynamics of small unilamellar vesicles with the aid of neutron spin-echo spectroscopy. The purpose of this investigation is twofold. On the one hand, we investigate the influence of solubilised cosurfactant on the dynamics of the vesicle's surfactant bilayer. On the other hand, the small unilamellar vesicles used here have a size between larger vesicles, with dynamics being well described by the Zilman-Granek model and smaller microemulsion droplets which can be described by the Milner-Safran model. Therefore, we want to elucidate the question, which model is more suitable for the description of the membrane dynamics of small vesicles, where the finite curvature of the bilayer is felt by the contained amphiphilic molecules. This question is of substantial relevance for our understanding of membranes and how their dynamics is affected by curvature, a problem that is also of key importance in a number of biological questions. Our results indicate the even down to vesicle radii of 20 nm the Zilman-Granek model appears to be the more suitable one.

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.

1H NMR Shows Slow Phospholipid Flip-Flop in Gel and Fluid Bilayers

DOE PAGES

Marquardt, Drew; Heberle, Frederick A.; Miti, Tatiana; ...

2017-01-20

We measured the transbilayer diffusion of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in large unilamellar vesicles, in both the gel (L β') and fluid (L α) phases. The choline resonance of headgroup-protiated DPPC exchanged into the outer leaflet of headgroup-deuterated DPPC-d13 vesicles was monitored using 1H NMR spectroscopy, coupled with the addition of a paramagnetic shift reagent. This allowed us to distinguish between the inner and outer bilayer leaflet of DPPC, to determine the flip-flop rate as a function of temperature. Flip-flop of fluid-phase DPPC exhibited Arrhenius kinetics, from which we determined an activation energy of 122 kJ mol –1. In gel-phase DPPC vesicles,more » flip-flop was not observed over the course of 250 h. Here, our findings are in contrast to previous studies of solid-supported bilayers, where the reported DPPC translocation rates are at least several orders of magnitude faster than those in vesicles at corresponding temperatures. Finally, we reconcile these differences by proposing a defect-mediated acceleration of lipid translocation in supported bilayers, where long-lived, submicron-sized holes resulting from incomplete surface coverage are the sites of rapid transbilayer movement.« less

Irregular bilayer structure in vesicles prepared from Halobacterium cutirubrum lipids

NASA Technical Reports Server (NTRS)

Lanyi, J. K.

1974-01-01

Fluorescent probes were used to study the structure of the cell envelope of Halobacterium cutirubrum, and, in particular, to explore the effect of the heterogeneity of the lipids in this organism on the structure of the bilayers. The fluorescence polarization of perylene was followed in vesicles of unfractionated lipids and polar lipids as a function of temperature in 3.4 M solutions of NaCl, NaNO3, and KSCN, and it was found that vesicles of unfractionated lipids were more perturbed by chaotropic agents than polar lipids. The dependence of the relaxation times of perylene on temperature was studied in cell envelopes and in vesicles prepared from polar lipids, unfractionated lipids, and mixtures of polar and neutral lipids.

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

PubMed Central

Nikolaus, Joerg; Karatekin, Erdem

2016-01-01

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, "kiss-and-run" fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore "openness", the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo. PMID:27585113

Functionalized Vesicles by Microfluidic Device.

PubMed

Vallejo, Derek; Lee, Shih-Hui; Lee, Abraham

2017-01-01

In recent years, lipid vesicles have become popular vehicles for the creation of biosensors. Vesicles can hold reaction components within a selective permeable membrane that provides an ideal environment for membrane protein biosensing elements. The lipid bilayer allows a protein to retain its native structure and function, and the membrane fluidity can allow for conformational changes and physiological interactions with target analytes. Here, we present two methods for the production of giant unilamellar vesicles (GUVs) within a microfluidic device that can be used as the basis for a biosensor. The vesicles are produced from water-in-oil-in-water (W/O/W) double emulsion templates using a nonvolatile oil phase. To create the GUVs, the oil can be removed via extraction with ethanol, or by altering the interfacial tension between the oil and carrier solution causing the oil to retract into a cap on one side of the structure, leaving behind an exposed lipid bilayer. Methods to integrate sensing elements and membrane protein pores onto the vesicles are also introduced in this work.

Membrane Permeabilization Induced by Sphingosine: Effect of Negatively Charged Lipids

PubMed Central

Jiménez-Rojo, Noemi; Sot, Jesús; Viguera, Ana R.; Collado, M. Isabel; Torrecillas, Alejandro; Gómez-Fernández, J.C.; Goñi, Félix M.; Alonso, Alicia

2014-01-01

Sphingosine [(2S, 3R, 4E)-2-amino-4-octadecen-1, 3-diol] is the most common sphingoid long chain base in sphingolipids. It is the precursor of important cell signaling molecules, such as ceramides. In the last decade it has been shown to act itself as a potent metabolic signaling molecule, by activating a number of protein kinases. Moreover, sphingosine has been found to permeabilize phospholipid bilayers, giving rise to vesicle leakage. The present contribution intends to analyze the mechanism by which this bioactive lipid induces vesicle contents release, and the effect of negatively charged bilayers in the release process. Fluorescence lifetime measurements and confocal fluorescence microscopy have been applied to observe the mechanism of sphingosine efflux from large and giant unilamellar vesicles; a graded-release efflux has been detected. Additionally, stopped-flow measurements have shown that the rate of vesicle permeabilization increases with sphingosine concentration. Because at the physiological pH sphingosine has a net positive charge, its interaction with negatively charged phospholipids (e.g., bilayers containing phosphatidic acid together with sphingomyelins, phosphatidylethanolamine, and cholesterol) gives rise to a release of vesicular contents, faster than with electrically neutral bilayers. Furthermore, phosphorous 31-NMR and x-ray data show the capacity of sphingosine to facilitate the formation of nonbilayer (cubic phase) intermediates in negatively charged membranes. The data might explain the pathogenesis of Niemann-Pick type C1 disease. PMID:24940775

Interactions between antimicrobial polynorbornenes and phospholipid vesicles monitored by light scattering and microcalorimetry.

PubMed

Gabriel, Gregory J; Pool, Joanna G; Som, Abhigyan; Dabkowski, Jeffrey M; Coughlin, E Bryan; Muthukumar, M; Tew, Gregory N

2008-11-04

Antimicrobial polynorbornenes composed of facially amphiphilic monomers have been previously reported to accurately emulate the antimicrobial activity of natural host-defense peptides (HDPs). The lethal mechanism of most HDPs involves binding to the membrane surface of bacteria leading to compromised phospholipid bilayers. In this paper, the interactions between biomimetic vesicle membranes and these cationic antimicrobial polynorbornenes are reported. Vesicle dye-leakage experiments were consistent with previous biological assays and corroborated a mode of action involving membrane disruption. Dynamic light scattering (DLS) showed that these antimicrobial polymers cause extensive aggregation of vesicles without complete bilayer disintegration as observed with surfactants that efficiently solubilize the membrane. Fluorescence microscopy on vesicles and bacterial cells also showed polymer-induced aggregation of both synthetic vesicles and bacterial cells. Isothermal titration calorimetry (ITC) afforded free energy of binding values (Delta G) and polymer to lipid binding ratios, plus revealed that the interaction is entropically favorable (Delta S>0, Delta H>0). It was observed that the strength of vesicle binding was similar between the active polymers while the binding stoichiometries were dramatically different.

Interaction of the Antimicrobial Peptide Aurein 1.2 and Charged Lipid Bilayer

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

Rai, Durgesh K.; Qian, Shuo

Aurein 1.2 is a potent antimicrobial peptide secreted by frog Litoria aurea. As a short membrane-active peptide with only 13 amino acids in sequence, it has been found to be residing on the surface of lipid bilayer and permeabilizing bacterial membranes at high concentration. However, the detail at the molecular level is largely unknown. Here in this study, we investigated the action of Aurein 1.2 in charged lipid bilayers composed of DMPC/DMPG. Oriented Circular Dichroism results showed that the peptide was on the surface of lipid bilayer regardless of the charged lipid ratio. Only at a very high peptide-to-lipid ratiomore » (~1/10), the peptide became perpendicular to the bilayer, however no pore was detected by neutron in-plane scattering. To further understand how it interacted with charged lipid bilayers, we employed Small Angle Neutron Scattering to probe lipid distribution across bilayer leaflets in lipid vesicles. The results showed that Aurein 1.2 interacted strongly with negatively charged DMPG, causing strong asymmetry in lipid bilayer. At high concentration, while the vesicles were intact, we found additional structure feature on the bilayer. Finally, our study provides a glimpse into how Aurein 1.2 disturbs anionic lipid-containing membranes without pore formation.« less

Interaction of the Antimicrobial Peptide Aurein 1.2 and Charged Lipid Bilayer

DOE PAGES

Rai, Durgesh K.; Qian, Shuo

2017-06-16

Aurein 1.2 is a potent antimicrobial peptide secreted by frog Litoria aurea. As a short membrane-active peptide with only 13 amino acids in sequence, it has been found to be residing on the surface of lipid bilayer and permeabilizing bacterial membranes at high concentration. However, the detail at the molecular level is largely unknown. Here in this study, we investigated the action of Aurein 1.2 in charged lipid bilayers composed of DMPC/DMPG. Oriented Circular Dichroism results showed that the peptide was on the surface of lipid bilayer regardless of the charged lipid ratio. Only at a very high peptide-to-lipid ratiomore » (~1/10), the peptide became perpendicular to the bilayer, however no pore was detected by neutron in-plane scattering. To further understand how it interacted with charged lipid bilayers, we employed Small Angle Neutron Scattering to probe lipid distribution across bilayer leaflets in lipid vesicles. The results showed that Aurein 1.2 interacted strongly with negatively charged DMPG, causing strong asymmetry in lipid bilayer. At high concentration, while the vesicles were intact, we found additional structure feature on the bilayer. Finally, our study provides a glimpse into how Aurein 1.2 disturbs anionic lipid-containing membranes without pore formation.« less

Small-Molecule Photostabilizing Agents are Modifiers of Lipid Bilayer Properties

PubMed Central

Alejo, Jose L.; Blanchard, Scott C.; Andersen, Olaf S.

2013-01-01

Small-molecule photostabilizing or protective agents (PAs) provide essential support for the stability demands on fluorescent dyes in single-molecule spectroscopy and fluorescence microscopy. These agents are employed also in studies of cell membranes and model systems mimicking lipid bilayer environments, but there is little information about their possible effects on membrane structure and physical properties. Given the impact of amphipathic small molecules on bilayer properties such as elasticity and intrinsic curvature, we investigated the effects of six commonly used PAs—cyclooctatetraene (COT), para-nitrobenzyl alcohol (NBA), Trolox (TX), 1,4-diazabicyclo[2.2.2]octane (DABCO), para-nitrobenzoic acid (pNBA), and n-propyl gallate (nPG)—on bilayer properties using a gramicidin A (gA)-based fluorescence quench assay to probe for PA-induced changes in the gramicidin monomer↔dimer equilibrium. The experiments were done using fluorophore-loaded large unilamellar vesicles that had been doped with gA, and changes in the gA monomer↔dimer equilibrium were assayed using a gA channel-permeable fluorescence quencher (Tl+). Changes in bilayer properties caused by, e.g., PA adsorption at the bilayer/solution interface that alter the equilibrium constant for gA channel formation, and thus the number of conducting gA channels in the large unilamellar vesicle membrane, will be detectable as changes in the rate of Tl+ influx—the fluorescence quench rate. Over the experimentally relevant millimolar concentration range, TX, NBA, and pNBA, caused comparable increases in gA channel activity. COT, also in the millimolar range, caused a slight decrease in gA channel activity. nPG increased channel activity at submillimolar concentrations. DABCO did not alter gA activity. Five of the six tested PAs thus alter lipid bilayer properties at experimentally relevant concentrations, which becomes important for the design and analysis of fluorescence studies in cells and model membrane systems. We therefore tested combinations of COT, NBA, and TX; the combinations altered the fluorescence quench rate less than would be predicted assuming their effects on bilayer properties were additive. The combination of equimolar concentrations of COT and NBA caused minimal changes in the fluorescence quench rate. PMID:23746513

Transdermal delivery of flurbiprofen from surfactant-based vesicles: particle characterization and the effect of water on in vitro transport.

PubMed

Uchino, Tomonobu; Matsumoto, Yuiko; Murata, Akiko; Oka, Toshihiko; Miyazaki, Yasunori; Kagawa, Yoshiyuki

2014-04-10

Flurbiprofen loaded rigid and elastic vesicles comprising the bilayer-forming surfactant sucrose-ester laurate were prepared by the film rehydration and extrusion method. The charge-inducing agent sodium dodecyl sulfate, and the micelle-forming surfactants, sorbitan monolaurate, polyethylene glycol monolaurate, and polysorbate 20, were used to enhance elasticity. Vesicle formulations were evaluated for size, zeta potential, (1)H and (19)F nuclear magnetic resonance (NMR) spectra, and in vitro skin permeation across Yucatan micropig (YMP) skin. Vesicle formulations were stable for 2 weeks and their mean sizes were 95-135 nm. NMR spectroscopy showed that flurbiprofen molecular mobility was restricted by interaction with vesicle components because of entrapment in vesicle bilayers. Moreover, sorbitan monolaurate-containing vesicles strongly retained flurbiprofen molecules. After non-occlusive application to YMP skin, flurbiprofen transport from all vesicle formulations was superior to that of flurbiprofen alone and remarkably decreased after water vaporization. Polarization microscopy and small-angle X-ray diffraction analysis showed that the vesicle formulation was transferred to liquid crystalline state. Suppression of vesicle transition to the liquid crystalline state was observed with applications of both large quantities and diluted samples. The presence of water in the formulations was associated with maintenance of the vesicle structure and greater flurbiprofen transport across YMP skin. Copyright © 2014 Elsevier B.V. All rights reserved.

Microfluidics based manufacture of liposomes simultaneously entrapping hydrophilic and lipophilic drugs.

PubMed

Joshi, Sameer; Hussain, Maryam T; Roces, Carla B; Anderluzzi, Giulia; Kastner, Elisabeth; Salmaso, Stefano; Kirby, Daniel J; Perrie, Yvonne

2016-11-30

Despite the substantial body of research investigating the use of liposomes, niosomes and other bilayer vesicles for drug delivery, the translation of these systems into licensed products remains limited. Indeed, recent shortages in the supply of liposomal products demonstrate the need for new scalable production methods for liposomes. Therefore, the aim of our research has been to consider the application of microfluidics in the manufacture of liposomes containing either or both a water soluble and a lipid soluble drug to promote co-delivery of drugs. For the first time, we demonstrate the entrapment of a hydrophilic and a lipophilic drug (metformin and glipizide respectively) both individually, and in combination, using a scalable microfluidics manufacturing system. In terms of the operating parameters, the choice of solvents, lipid concentration and aqueous:solvent ratio all impact on liposome size with vesicle diameter ranging from ∼90 to 300nm. In terms of drug loading, microfluidics production promoted high loading within ∼100nm vesicles for both the water soluble drug (20-25% of initial amount added) and the bilayer embedded drug (40-42% of initial amount added) with co-loading of the drugs making no impact on entrapment efficacy. However, co-loading of glipizide and metformin within the same liposome formulation did impact on the drug release profiles; in both instances the presence of both drugs in the one formulation promoted faster (up to 2 fold) release compared to liposomes containing a single drug alone. Overall, these results demonstrate the application of microfluidics to prepare liposomal systems incorporating either or both an aqueous soluble drug and a bilayer loaded drug. Copyright © 2016 Elsevier B.V. All rights reserved.

Vesicle Origami and the Influence of Cholesterol on Lipid Packing.

PubMed

Tanasescu, Radu; Lanz, Martin A; Mueller, Dennis; Tassler, Stephanie; Ishikawa, Takashi; Reiter, Renate; Brezesinski, Gerald; Zumbuehl, Andreas

2016-05-17

The artificial phospholipid Pad-PC-Pad was analyzed in 2D (monolayers at the air/water interface) and 3D (aqueous lipid dispersions) systems. In the gel phase, the two leaflets of a Pad-PC-Pad bilayer interdigitate completely, and the hydrophobic bilayer region has a thickness comparable to the length of a single phospholipid acyl chain. This leads to a stiff membrane with no spontaneous curvature. Forced into a vesicular structure, Pad-PC-Pad has faceted geometry, and in its extreme form, tetrahedral vesicles were found as predicted a decade ago. Above the main transition temperature, a noninterdigitated Lα phase with fluid chains has been observed. The addition of cholesterol leads to a slight decrease of the main transition temperature and a gradual decrease in the transition enthalpy until the transition vanishes at 40 mol % cholesterol in the mixture. Additionally, cholesterol pulls the chains apart, and a noninterdigitated gel phase is observed. In monolayers, cholesterol has an ordering effect on liquid-expanded phases and disorders condensed phases. The wavenumbers of the methylene stretching vibration indicate the formation of a liquid-ordered phase in mixtures with 40 mol % cholesterol.

Response of unilamellar DPPC and DPPC:SM vesicles to hypo and hyper osmotic shocks: A comparison.

PubMed

Ahumada, M; Calderon, C; Alvarez, C; Lanio, M E; Lissi, E A

2015-05-01

DPPC and DPPC:SM large unilamellar vesicles (LUVs), prepared by extrusion, readily respond to osmotic shocks (hypo- and hyper-osmotic) by water influx/efflux (evaluated by changes in turbidity) and by entrapped calcein liberation (measured by an increase in dye fluorescence intensity). On the other hand, small unilamellar vesicles (SUVs) prepared by sonication are almost osmotically insensitive. LUVs water transport, both in hypo- and hyper-osmotic conditions, takes place faster than calcein ejection towards the external solvent. Similarly, response to a hypotonic imbalance is faster than that associated to a hypertonic stress. This difference is particularly noticeable for the increase in calcein fluorescence intensity and can be related to the large reorganization of the bilayer needed to form pores and/or to adsorb the dye to the inner leaflet of the vesicle after water efflux. Conversely, addition of SM to the vesicles barely modify the rate of calcein permeation across the bilayer. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Intrinsic Curvature-Mediated Transbilayer Coupling in Asymmetric Lipid Vesicles

DOE PAGES

Eicher, Barbara; Marquardt, Drew; Heberle, Frederick A.; ...

2018-01-09

We measured the effect of intrinsic lipid curvature, J 0, on structural properties of asymmetric vesicles made of palmitoyl-oleoyl-phosphatidylethanolamine (POPE; J 0 < 0) and palmitoyl-oleoyl-phosphatidylcholine (POPC; J 0 ~ 0). Electron microscopy and dynamic light scattering were used to determine vesicle size and morphology, and x-ray and neutron scattering, combined with calorimetric experiments and solution NMR, yielded insights into leaflet-specific lipid packing and melting processes. Below the lipid melting temperature we observed strong interleaflet coupling in asymmetric vesicles with POPE inner bilayer leaflets and outer leaflets enriched in POPC. This lipid arrangement manifested itself by lipids melting cooperatively inmore » both leaflets, and a rearrangement of lipid packing in both monolayers. On the other hand, no coupling was observed in vesicles with POPC inner bilayer leaflets and outer leaflets enriched in POPE. In this case, the leaflets melted independently and did not affect each other’s acyl chain packing. Furthermore, we found no evidence for transbilayer structural coupling above the melting temperature of either sample preparation. Our results are consistent with the energetically preferred location of POPE residing in the inner leaflet, where it also resides in natural membranes, most likely causing the coupling of both leaflets. The loss of this coupling in the fluid bilayers is most likely the result of entropic contributions.« less

Chromatic response of polydiacetylene vesicle induced by the permeation of methotrexate.

PubMed

Shin, Min Jae; Kim, Ye Jin; Kim, Jong-Duk

2015-07-07

The noble vesicular system of polydiacetylene showed a red shift using two types of detecting systems. One of the systems involves the absorption of target materials from the outer side of the vesicle, and the other system involves the permeation through the vesicular layers from within the vesicle. The chromatic mixed vesicles of N-(2-aminoethyl)pentacosa-10,12-diynamide (AEPCDA) and dimethyldioctadecylammonium chloride (DODAC) were fabricated by sonication, followed by polymerization by UV irradiation. The stability of monomeric vesicles was observed to increase with the polymerization of the vesicles. Methotrexate was used as a target material. The polymerized mixed vesicles having a blue color were exposed to a concentration gradient of methotrexate, and a red shift was observed indicating the adsorption of methotrexate on the polydiacetylene bilayer. In order to check the chromatic change by the permeation of methotrexate, we separated the vesicle portion, which contained methotrexate inside the vesicle, and checked chromatic change during the permeation of methotrexate through the vesicle. The red shift apparently indicates the disturbance in the bilayer induced by the permeation of methotrexate. The maximum contrast of color appeared at the equal molar ratio of AEPCDA and DODAC, indicating that the formation of flexible and deformable vesicular layers is important for red shift. Therefore, it is hypothesized that the system can be applicable for the chromatic detection of the permeation of methotrexate through the polydiacetylene layer.

Transport efficiency of membrane-anchored kinesin-1 motors depends on motor density and diffusivity

PubMed Central

Grover, Rahul; Fischer, Janine; Schwarz, Friedrich W.; Walter, Wilhelm J.; Schwille, Petra; Diez, Stefan

2016-01-01

In eukaryotic cells, membranous vesicles and organelles are transported by ensembles of motor proteins. These motors, such as kinesin-1, have been well characterized in vitro as single molecules or as ensembles rigidly attached to nonbiological substrates. However, the collective transport by membrane-anchored motors, that is, motors attached to a fluid lipid bilayer, is poorly understood. Here, we investigate the influence of motors’ anchorage to a lipid bilayer on the collective transport characteristics. We reconstituted “membrane-anchored” gliding motility assays using truncated kinesin-1 motors with a streptavidin-binding peptide tag that can attach to streptavidin-loaded, supported lipid bilayers. We found that the diffusing kinesin-1 motors propelled the microtubules in the presence of ATP. Notably, we found the gliding velocity of the microtubules to be strongly dependent on the number of motors and their diffusivity in the lipid bilayer. The microtubule gliding velocity increased with increasing motor density and membrane viscosity, reaching up to the stepping velocity of single motors. This finding is in contrast to conventional gliding motility assays where the density of surface-immobilized kinesin-1 motors does not influence the microtubule velocity over a wide range. We reason that the transport efficiency of membrane-anchored motors is reduced because of their slippage in the lipid bilayer, an effect that we directly observed using single-molecule fluorescence microscopy. Our results illustrate the importance of motor–cargo coupling, which potentially provides cells with an additional means of regulating the efficiency of cargo transport. PMID:27803325

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

Thermal and active fluctuations of a compressible bilayer vesicle

NASA Astrophysics Data System (ADS)

Sachin Krishnan, T. V.; Yasuda, Kento; Okamoto, Ryuichi; Komura, Shigeyuki

2018-05-01

We discuss thermal and active fluctuations of a compressible bilayer vesicle by using the results of hydrodynamic theory for vesicles. Coupled Langevin equations for the membrane deformation and the density fields are employed to calculate the power spectral density matrix of membrane fluctuations. Thermal contribution is obtained by means of the fluctuation dissipation theorem, whereas active contribution is calculated from exponentially decaying time correlation functions of active random forces. We obtain the total power spectral density as a sum of thermal and active contributions. An apparent response function is further calculated in order to compare with the recent microrheology experiment on red blood cells. An enhanced response is predicted in the low-frequency regime for non-thermal active fluctuations.

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

Mruetusatorn, Prachya; Boreyko, Jonathan B; Sarles, Stephen A

Droplet interface bilayers (DIBs) are a powerful platform for studying the dynamics of synthetic cellular membranes; however, very little has been done to exploit the unique dynamical features of DIBs. Here, we generate microscale droplet interface bilayers ( DIBs) by bringing together femtoliter-volume water droplets in a microfluidic oil channel, and characterize morphological changes of the DIBs as the droplets shrink due to evaporation. By varying the initial conditions of the system, we identify three distinct classes of dynamic morphology. (1) Buckling and Fission: When forming DIBs using the lipid-out method (lipids in oil phase), lipids in the shrinking monolayersmore » continually pair together and slide into the bilayer to conserve their mass. As the bilayer continues to grow, it becomes confined, buckles, and eventually fissions one or more vesicles. (2) Uniform Shrinking: When using the lipid-in method (lipids in water phase) to form DIBs, lipids uniformly transfer from the monolayers and bilayer into vesicles contained inside the water droplets. (3) Stretching and Unzipping: Finally, when the droplets are pinned to the wall(s) of the microfluidic channel, the droplets become stretched during evaporation, culminating in the unzipping of the bilayer and droplet separation. These findings offer a better understanding of the dynamics of coupled lipid interfaces.« less

Separating attoliter-sized compartments using fluid pore-spanning lipid bilayers.

PubMed

Lazzara, Thomas D; Carnarius, Christian; Kocun, Marta; Janshoff, Andreas; Steinem, Claudia

2011-09-27

Anodic aluminum oxide (AAO) is a porous material having aligned cylindrical compartments with 55-60 nm diameter pores, and being several micrometers deep. A protocol was developed to generate pore-spanning fluid lipid bilayers separating the attoliter-sized compartments of the nanoporous material from the bulk solution, while preserving the optical transparency of the AAO. The AAO was selectively functionalized by silane chemistry to spread giant unilamellar vesicles (GUVs) resulting in large continuous membrane patches covering the pores. Formation of fluid single lipid bilayers through GUV rupture could be readily observed by fluorescence microscopy and further supported by conservation of membrane surface area, before and after GUV rupture. Fluorescence recovery after photobleaching gave low immobile fractions (5-15%) and lipid diffusion coefficients similar to those found for bilayers on silica. The entrapment of molecules within the porous underlying cylindrical compartments, as well as the exclusion of macromolecules from the nanopores, demonstrate the barrier function of the pore-spanning membranes and could be investigated in three-dimensions using confocal laser scanning fluorescence imaging. © 2011 American Chemical Society

Flexible bilayers with spontaneous curvature lead to lamellar gels and spontaneous vesicles

PubMed Central

Coldren, Bret A.; Warriner, Heidi; van Zanten, Ryan; Zasadzinski, Joseph A.; Sirota, Eric B.

2006-01-01

Mixtures of cetyltrimethylammonium tosylate (CTAT) and sodium dodecylbenzene sulfonate (SDBS) in water form a fluid lamellar phase at ≤40 wt % water but surprisingly turn into viscous gels at higher water fractions. The gels are characterized by spherulite and other bilayer defects consistent with a low bending elasticity, κ ∼ kBT, and a nonzero spontaneous curvature. Caillé analysis of the small-angle x-ray line shape confirms that for 7:3 wt:wt CTAT:SDBS bilayers at 50% water, κ = 0.62 ± 0.09 kBT and κ̄ = −0.9 ± 0.2 kBT. For 13:7 wt:wt CTAT:SDBS bilayers, the measured bending elasticity decreases with increasing water dilution in good agreement with predictions based on renormalization theory, giving κo = 0.28 kBT. These results show that surfactant mixing is sufficient to make κ ∼ kBT, which promotes strong, Helfrich-type repulsion between bilayers that can dominate the van der Waals attraction. These are necessary conditions for spontaneous vesicles formed at even higher water fractions to be equilibrium structures. PMID:16467142

Molecular devices: Caroviologens as an approach to molecular wires—synthesis and incorporation into vesicle membranes

PubMed Central

Arrhenius, Thomas S.; Blanchard-Desce, Mireille; Dvolaitzky, Maya; Lehn, Jean-Marie; Malthete, Jacques

1986-01-01

Molecular wires, which would allow electron flow to take place between different components, are important elements in the design of molecular devices. An approach to such species would be molecules possessing an electron-conducting conjugated chain, terminal electroactive polar groups, and a length sufficient to span a lipid membrane. To this end, bispyridinium polyenes of different lengths have been synthesized and their incorporation into the bilayer membrane of sodium dihexadecyl phosphate vesicles has been studied. Since they combine the features of carotenoids and of viologens, they may be termed caroviologens. Vesicles containing the caroviologen whose length approximately corresponds to the thickness of the sodium dihexadecyl phosphate bilayer display temperature-dependent changes of its absorption spectrum reflecting the gel → liquid-crystal phase transition of the membrane. The data agree with a structural model in which the caroviologens of sufficient length span the bilayer membrane, the pyridinium sites being close to the negatively charged outer and inner surfaces of the sodium dihexadecyl phosphate vesicles and the polyene chain crossing the lipidic interior of the membrane. These membranes may now be tested in processes in which the caroviologen would function as a continuous, transmembrane electron channel—i.e., as a molecular wire. Various further developments may be envisaged along these lines. PMID:16593731

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

Eicher, Barbara; Marquardt, Drew; Heberle, Frederick A.

We measured the effect of intrinsic lipid curvature, J 0, on structural properties of asymmetric vesicles made of palmitoyl-oleoyl-phosphatidylethanolamine (POPE; J 0 < 0) and palmitoyl-oleoyl-phosphatidylcholine (POPC; J 0 ~ 0). Electron microscopy and dynamic light scattering were used to determine vesicle size and morphology, and x-ray and neutron scattering, combined with calorimetric experiments and solution NMR, yielded insights into leaflet-specific lipid packing and melting processes. Below the lipid melting temperature we observed strong interleaflet coupling in asymmetric vesicles with POPE inner bilayer leaflets and outer leaflets enriched in POPC. This lipid arrangement manifested itself by lipids melting cooperatively inmore » both leaflets, and a rearrangement of lipid packing in both monolayers. On the other hand, no coupling was observed in vesicles with POPC inner bilayer leaflets and outer leaflets enriched in POPE. In this case, the leaflets melted independently and did not affect each other’s acyl chain packing. Furthermore, we found no evidence for transbilayer structural coupling above the melting temperature of either sample preparation. Our results are consistent with the energetically preferred location of POPE residing in the inner leaflet, where it also resides in natural membranes, most likely causing the coupling of both leaflets. The loss of this coupling in the fluid bilayers is most likely the result of entropic contributions.« less

Characteristics and anti-proliferative activity of azelaic acid and its derivatives entrapped in bilayer vesicles in cancer cell lines.

PubMed

Manosroi, Aranya; Panyosak, Atchara; Rojanasakul, Yon; Manosroi, Jiradej

2007-06-01

The hydrophilicity and lipophilicity of azelaic acid (AA) were modified to diethyl azelate (DA) which was synthesized by Fisher esterification reaction and identified by IR, MS and (1)H NMR and to azelaic acid-beta-cyclodextrin complex (AACD) which was prepared by inclusion complexation and identified by IR, DSC and XRD respectively. AA, DA and AACD were entrapped in liposomes and niosomes comprising of L-alpha-dipalmitoyl phosphatidylcholine (DPPC)/cholesterol at 7:3 molar ratio and Tween61/cholesterol at 1:1 molar ratio, respectively, using a thin-film hydration method with sonication. The size and morphology of these bilayer vesicles were determined by optical and transmission electron microscopy. The particle size was found to be in the range of 90-190 nm. The entrapment efficiency of AA, DA and AACD in all vesicular formulations was more than 80%, as analyzed by HPLC for AA and AACD, and GC for DA. Anti-proliferative activity of AA and its derivatives (DA and AACD) both entrapped and not entrapped in bilayer vesicles, using MTT assay in three cancer cell lines (HeLa, KB and B(16)F(10)) comparing with vincristine, were investigated. AACD showed the highest potency comparing to AA in HeLa, KB and B(16)F(10) of 1.48, 1.6 and 1.5 times, respectively. AA entrapped in liposomes was about 90 times more potent than the free AA, and about 1.5 times less potent than vincristine. When entrapped in bilayer vesicles, DA and AACD were more effective than AA in killing cancer cells. AACD entrapped in liposomes gave the highest anti-proliferation activity in HeLa cell lines with the IC(50) of 2.3 and 327 times more potent than vincristine and AA, respectively. DA in liposomes demonstrated the IC(50) of 0.03 times less potent than vincristine in KB cell lines, while in B(16)F(10) AACD in niosomes showed the IC(50) of 0.05 times less potent than vincristine. This study has suggested that the modification of AA by derivatization and complexation as well as the entrapment in bilayer vesicles can enhance its therapeutic efficacy.

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.

Biologically Complex Planar Cell Plasma Membranes Supported on Polyelectrolyte Cushions Enhance Transmembrane Protein Mobility and Retain Native Orientation.

PubMed

Liu, Han-Yuan; Chen, Wei-Liang; Ober, Christopher K; Daniel, Susan

2018-01-23

Reconstituted supported lipid bilayers (SLB) are widely used as in vitro cell-surface models because they are compatible with a variety of surface-based analytical techniques. However, one of the challenges of using SLBs as a model of the cell surface is the limited complexity in membrane composition, including the incorporation of transmembrane proteins and lipid diversity that may impact the activity of those proteins. Additionally, it is challenging to preserve the transmembrane protein native orientation, function, and mobility in SLBs. Here, we leverage the interaction between cell plasma membrane vesicles and polyelectrolyte brushes to create planar bilayers from cell plasma membrane vesicles that have budded from the cell surface. This approach promotes the direct incorporation of membrane proteins and other species into the planar bilayer without using detergent or reconstitution and preserves membrane constituents. Furthermore, the structure of the polyelectrolyte brush serves as a cushion between the planar bilayer and rigid supporting surface, limiting the interaction of the cytosolic domains of membrane proteins with this surface. Single particle tracking was used to analyze the motion of GPI-linked yellow fluorescent proteins (GPI-YFP) and neon-green fused transmembrane P2X2 receptors (P2X2-neon) and shows that this platform retains over 75% mobility of multipass transmembrane proteins in its native membrane environment. An enzyme accessibility assay confirmed that the protein orientation is preserved and results in the extracellular domain facing toward the bulk phase and the cytosolic side facing the support. Because the platform presented here retains the complexity of the cell plasma membrane and preserves protein orientation and mobility, it is a better representative mimic of native cell surfaces, which may find many applications in biological assays aimed at understanding cell membrane phenomena.

Small-molecule photostabilizing agents are modifiers of lipid bilayer properties.

PubMed

Alejo, Jose L; Blanchard, Scott C; Andersen, Olaf S

2013-06-04

Small-molecule photostabilizing or protective agents (PAs) provide essential support for the stability demands on fluorescent dyes in single-molecule spectroscopy and fluorescence microscopy. These agents are employed also in studies of cell membranes and model systems mimicking lipid bilayer environments, but there is little information about their possible effects on membrane structure and physical properties. Given the impact of amphipathic small molecules on bilayer properties such as elasticity and intrinsic curvature, we investigated the effects of six commonly used PAs--cyclooctatetraene (COT), para-nitrobenzyl alcohol (NBA), Trolox (TX), 1,4-diazabicyclo[2.2.2]octane (DABCO), para-nitrobenzoic acid (pNBA), and n-propyl gallate (nPG)--on bilayer properties using a gramicidin A (gA)-based fluorescence quench assay to probe for PA-induced changes in the gramicidin monomer↔dimer equilibrium. The experiments were done using fluorophore-loaded large unilamellar vesicles that had been doped with gA, and changes in the gA monomer↔dimer equilibrium were assayed using a gA channel-permeable fluorescence quencher (Tl⁺). Changes in bilayer properties caused by, e.g., PA adsorption at the bilayer/solution interface that alter the equilibrium constant for gA channel formation, and thus the number of conducting gA channels in the large unilamellar vesicle membrane, will be detectable as changes in the rate of Tl⁺ influx-the fluorescence quench rate. Over the experimentally relevant millimolar concentration range, TX, NBA, and pNBA, caused comparable increases in gA channel activity. COT, also in the millimolar range, caused a slight decrease in gA channel activity. nPG increased channel activity at submillimolar concentrations. DABCO did not alter gA activity. Five of the six tested PAs thus alter lipid bilayer properties at experimentally relevant concentrations, which becomes important for the design and analysis of fluorescence studies in cells and model membrane systems. We therefore tested combinations of COT, NBA, and TX; the combinations altered the fluorescence quench rate less than would be predicted assuming their effects on bilayer properties were additive. The combination of equimolar concentrations of COT and NBA caused minimal changes in the fluorescence quench rate. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Joint small-angle X-ray and neutron scattering data analysis of asymmetric lipid vesicles

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

Eicher, Barbara; Heberle, Frederick A.; Marquardt, Drew T.

2017-02-28

Low- and high-resolution models describing the internal transbilayer structure of asymmetric lipid vesicles have been developed. These models can be used for the joint analysis of small-angle neutron and X-ray scattering data. The models describe the underlying scattering length density/electron density profiles either in terms of slabs or through the so-called scattering density profile, previously applied to symmetric lipid vesicles. Both models yield structural details of asymmetric membranes, such as the individual area per lipid, and the hydrocarbon thickness of the inner and outer bilayer leaflets. The scattering density profile model, however, comes at a cost of increased computational effortmore » but results in greater structural resolution, showing a slightly lower packing of lipids in the outer bilayer leaflet of ~120 nm diameter palmitoyloleoyl phosphatidylcholine (POPC) vesicles, compared to the inner leaflet. Here, analysis of asymmetric dipalmitoyl phosphatidylcholine/POPC vesicles did not reveal evidence of transbilayer coupling between the inner and outer leaflets at 323 K, i.e.above the melting transition temperature of the two lipids.« less

Functional liposomes and supported lipid bilayers: towards the complexity of biological archetypes.

PubMed

Berti, Debora; Caminati, Gabriella; Baglioni, Piero

2011-05-21

This perspective paper provides some illustrative examples on the interplay between information gathered on planar supported lipid bilayers (SLB) and unilamellar lipid vesicles (ULV) to get an integrated description of phenomena occurring at the nanoscale that involve locally bilayered structures. Similarities and differences are underlined and critically compared in terms of biomimetic fidelity and instrumental accessibility to structural and dynamical parameters, focusing on some recent reports that either explicitly address this comparison or introducing some studies that separately investigate the same process in SLB and lipid vesicles. Despite the structural similarity on the nanoscale, the different topology implies radically different characterization techniques that have evolved in sectorial and separated approaches. The quest for increasing levels of compositional complexity for bilayered systems should not result in a loss of structural and dynamical control: this is the central challenge of future research in this area, where the integrated approach highlighted in this contribution would enable improved levels of understanding. © The Owner Societies 2011

A stepwise mechanism for the permeation of phloretin through a lipid bilayer

PubMed Central

1982-01-01

The thermodynamics of interactions between phloretin and a phosphatidylcholine (PC) vesicle membrane are characterized using equilibrium spectrophotometric titration, stopped-flow, and temperature- jump techniques. Binding of phloretin to a PC vesicle membrane is diffusion limited, with an association rate constant greater than 10(8) M-1s-1, and an interfacial activation free energy of less than 2 kcal/mol. Equilibrium binding of phloretin to a vesicle membrane is characterized by a single class of high-affinity (8 micro M), noninteracting sites. Binding is enthalpy driven (delta H = -4.9 kcal/mol) at 23 degrees C. Analysis of amplitudes of kinetic processes shows that 66 +/- 3% of total phloretin binding sites are exposed at the external vesicle surface. The rate of phloretin movement between binding sites located near the external and internal interfaces is proportional to the concentration of un-ionized phloretin, with a rate constant of 5.7 X 10(4) M-1s-1 at 23 degrees C. The rate of this process is limited by a large enthalpic (9 kcal/mol) and entropic (-31 entropy units) barrier. An analysis of the concentration dependence of the rate of transmembrane movement suggests the presence of multiple intramembrane potential barriers. Permeation of phloretin through a lipid bilayer is modeled quantitatively in terms of discrete steps: binding to a membrane surface, translocation across a series of intramembrane barriers, and dissociation from the opposite membrane surface. The permeability coefficient for phloretin is calculated as 1.9 X 10(-3) cm/s on the basis of the model presented. Structure- function relationships are examined for a number of phloretin analogues. PMID:7142954

Structural transformations in diluted micellar and lamellar systems

NASA Astrophysics Data System (ADS)

Zelaya-Rincon, Blanca

The role of dilution by artificial hard water on nanostructures present in body wash samples provided by Procter and Gamble were investigated using time-resolved cryogenic transmission electron microscopy (cryo-TEM). Samples with and without perfume were examined at 10X, 20X, and 50X dilution. Micellar samples transformed to mostly unilamellar vesicles at 50X dilution, in contrast to the micelle to monomer transition seen in typical samples. At lower dilutions, a change in morphology from spherical to wormlike micelles was observed. For lamellar samples, lower dilution ratios show tightly packed multilamellar vesicles, while higher dilution ratios show more dispersed vesicles with less bilayers. Nanostructural transformations upon dilution were attributed to changes in curvature/packing parameters, which occurred due to dilution with hard water and addition of perfume. The systems experience changes in curvature in order to maintain equilibrium. Also, the addition of perfume in the lamellar samples caused an increase in the number of bilayers present in multilamellar vesicles, because of its role in increasing the packing parameter in the system.

Asymmetric or symmetric bilayer formation during oblique drop impact depends on rheological properties of saturated and unsaturated lipid monolayers.

PubMed

Vranceanu, Marcel; Terinte, Nicoleta; Nirschl, Hermann; Leneweit, Gero

2011-02-01

Bilayer structures are formed by approaching two liquid surfaces with phospholipid monolayers, which are brought into contact by oblique drop impact on a liquid surface. Asymmetric bilayers can be produced by the coupling of drop and target monolayers. In contrast, symmetric bilayers or multilayers are formed by collapse of the compressed target monolayer. We show that under all studied conditions bilayer/multilayer synthesis takes place. The experimental conditions for the synthesis of asymmetric or symmetric bilayers are described quantitatively in terms of the surface rheological (surface elasticity and dilational viscosity) and the hydrodynamical parameters (Weber number and impact angle). The composition and mechanical properties of the phospholipid monolayers strongly influences the patterns of drop impact and the bilayer/multilayer formation. Cholesterol stiffens unsaturated phospholipid monolayers and fluidifies saturated monolayers. All monolayers form asymmetric vesicle-like structures, which are stable in the aqueous medium. Additionally, unsaturated phospholipid monolayers without cholesterol form symmetric vesicles by folding parts of the target monolayer. Sufficient presence of cholesterol in unsaturated phospholipid monolayers inhibits the folding of the target monolayer and the subsequent formation of symmetric bilayers. The rheological properties of saturated and unsaturated phospholipid monolayers and their mixtures with cholesterol are discussed. Based on drop impact results it is shown that the state of a so far undefined region in the DPPC/cholesterol phase diagram is a fluid phase. Copyright © 2010 Elsevier Inc. All rights reserved.

Particle-based simulations of bilayer membranes: self-assembly, structural analysis, and shock-wave damage

NASA Astrophysics Data System (ADS)

Steinhauser, Martin O.; Schindler, Tanja

2017-01-01

We report on the results of particle-based, coarse-grained molecular dynamics simulations of amphiphilic lipid molecules in aqueous environment where the membrane structures at equilibrium are subsequently exposed to strong shock waves, and their damage is analyzed. The lipid molecules self-assemble from unbiased random initial configurations to form stable bilayer membranes, including closed vesicles. During self-assembly of lipid molecules, we observe several stages of clustering, starting with many small clusters of lipids, gradually merging together to finally form one single bilayer membrane. We find that the clustering of lipids sensitively depends on the hydrophobic interaction h_c of the lipid tails in our model and on temperature T of the system. The self-assembled bilayer membranes are quantitatively analyzed at equilibrium with respect to their degree of order and their local structure. We also show that—by analyzing the membrane fluctuations and using a linearized theory— we obtain area compression moduli K_A and bending stiffnesses κ _B for our bilayer membranes which are within the experimental range of in vivo and in vitro measurements of biological membranes. We also discuss the density profile and the pair correlation function of our model membranes at equilibrium which has not been done in previous studies of particle-based membrane models. Furthermore, we present a detailed phase diagram of our lipid model that exhibits a sol-gel transition between quasi-solid and fluid domains, and domains where no self-assembly of lipids occurs. In addition, we present in the phase diagram the conditions for temperature T and hydrophobicity h_c of the lipid tails of our model to form closed vesicles. The stable bilayer membranes obtained at equilibrium are then subjected to strong shock waves in a shock tube setup, and we investigate the damage in the membranes due to their interaction with shock waves. Here, we find a transition from self-repairing membranes (reducing their damage after impact) and permanent (irreversible) damage, depending on the shock front speed. The here presented idea of using coarse-grained (CG) particle models for soft matter systems in combination with the investigation of shock-wave effects in these systems is a quite new approach.

Partitioning of 2,6-Bis(1H-Benzimidazol-2-yl)pyridine Fluorophore into a Phospholipid Bilayer: Complementary Use of Fluorescence Quenching Studies and Molecular Dynamics Simulations

PubMed Central

Kyrychenko, Alexander; Sevriukov, Igor Yu.; Syzova, Zoya A.; Ladokhin, Alexey S.; Doroshenko, Andrey O.

2014-01-01

Successful use of fluorescence sensing in elucidating the biophysical properties of lipid membranes requires knowledge of the distribution and location of an emitting molecule in the bilayer. We report here that 2,6-bis(1H-benzimidazol-2-yl)pyridine (BBP), which is almost non-fluorescent in aqueous solutions, reveals a strong emission enhancement in a hydrophobic environment of a phospholipid bilayer, making it interesting for fluorescence probing of water content in a lipid membrane. Comparing the fluorescence behavior of BBP in a wide variety of solvents with those in phospholipid vesicles, we suggest that the hydrogen bonding interactions between a BBP fluorophore and water molecules play a crucial role in the observed “light switch effect”. Therefore, the loss of water-induced fluorescence quenching inside a membrane are thought to be due to deep penetration of BBP into the hydrophobic, water-free region of a bilayer. Characterized by strong quenching by transition metal ions in solution, BBP also demonstrated significant shielding from the action of the quencher in the presence of phospholipid vesicles. We used the increase in fluorescence intensity, measured upon titration of probe molecules with lipid vesicles, to estimate the partition constant and the Gibbs free energy (ΔG) of transfer of BBP from aqueous buffer into a membrane. Partitioning BBP revealed strongly favorable ΔG, which depends only slightly on the lipid composition of a bilayer, varying in a range from -6.5 to -7.0 kcal/mol. To elucidate the binding interactions of the probe with a membrane on the molecular level, a distribution and favorable location of BBP in a POPC bilayer were modeled via atomistic molecular dynamics (MD) simulations using two different approaches: (i) free, diffusion-driven partitioning of the probe molecules into a bilayer and (ii) constrained umbrella sampling of a penetration profile of the dye molecule across a bilayer. Both of these MD approaches agreed with regard to the preferred location of a BBP fluorophore within the interfacial region of a bilayer, located between the hydrocarbon acyl tails and the initial portion of the lipid headgroups. MD simulations also revealed restricted permeability of water molecules into this region of a POPC bilayer, determining the strong fluorescence enhancement observed experimentally for the membrane-partitioned form of BBP. PMID:21211898

Bursting Bubbles and Bilayers

PubMed Central

Wrenn, Steven P.; Dicker, Stephen M.; Small, Eleanor F.; Dan, Nily R.; Mleczko, Michał; Schmitz, Georg; Lewin, Peter A.

2012-01-01

This paper discusses various interactions between ultrasound, phospholipid monolayer-coated gas bubbles, phospholipid bilayer vesicles, and cells. The paper begins with a review of microbubble physics models, developed to describe microbubble dynamic behavior in the presence of ultrasound, and follows this with a discussion of how such models can be used to predict inertial cavitation profiles. Predicted sensitivities of inertial cavitation to changes in the values of membrane properties, including surface tension, surface dilatational viscosity, and area expansion modulus, indicate that area expansion modulus exerts the greatest relative influence on inertial cavitation. Accordingly, the theoretical dependence of area expansion modulus on chemical composition - in particular, poly (ethylene glyclol) (PEG) - is reviewed, and predictions of inertial cavitation for different PEG molecular weights and compositions are compared with experiment. Noteworthy is the predicted dependence, or lack thereof, of inertial cavitation on PEG molecular weight and mole fraction. Specifically, inertial cavitation is predicted to be independent of PEG molecular weight and mole fraction in the so-called mushroom regime. In the “brush” regime, however, inertial cavitation is predicted to increase with PEG mole fraction but to decrease (to the inverse 3/5 power) with PEG molecular weight. While excellent agreement between experiment and theory can be achieved, it is shown that the calculated inertial cavitation profiles depend strongly on the criterion used to predict inertial cavitation. This is followed by a discussion of nesting microbubbles inside the aqueous core of microcapsules and how this significantly increases the inertial cavitation threshold. Nesting thus offers a means for avoiding unwanted inertial cavitation and cell death during imaging and other applications such as sonoporation. A review of putative sonoporation mechanisms is then presented, including those involving microbubbles to deliver cargo into a cell, and those - not necessarily involving microubbles - to release cargo from a phospholipid vesicle (or reverse sonoporation). It is shown that the rate of (reverse) sonoporation from liposomes correlates with phospholipid bilayer phase behavior, liquid-disordered phases giving appreciably faster release than liquid-ordered phases. Moreover, liquid-disordered phases exhibit evidence of two release mechanisms, which are described well mathematically by enhanced diffusion (possibly via dilation of membrane phospholipids) and irreversible membrane disruption, whereas liquid-ordered phases are described by a single mechanism, which has yet to be positively identified. The ability to tune release kinetics with bilayer composition makes reverse sonoporation of phospholipid vesicles a promising methodology for controlled drug delivery. Moreover, nesting of microbubbles inside vesicles constitutes a truly “theranostic” vehicle, one that can be used for both long-lasting, safe imaging and for controlled drug delivery. PMID:23382772

Bursting bubbles and bilayers.

PubMed

Wrenn, Steven P; Dicker, Stephen M; Small, Eleanor F; Dan, Nily R; Mleczko, Michał; Schmitz, Georg; Lewin, Peter A

2012-01-01

This paper discusses various interactions between ultrasound, phospholipid monolayer-coated gas bubbles, phospholipid bilayer vesicles, and cells. The paper begins with a review of microbubble physics models, developed to describe microbubble dynamic behavior in the presence of ultrasound, and follows this with a discussion of how such models can be used to predict inertial cavitation profiles. Predicted sensitivities of inertial cavitation to changes in the values of membrane properties, including surface tension, surface dilatational viscosity, and area expansion modulus, indicate that area expansion modulus exerts the greatest relative influence on inertial cavitation. Accordingly, the theoretical dependence of area expansion modulus on chemical composition-- in particular, poly (ethylene glyclol) (PEG)--is reviewed, and predictions of inertial cavitation for different PEG molecular weights and compositions are compared with experiment. Noteworthy is the predicted dependence, or lack thereof, of inertial cavitation on PEG molecular weight and mole fraction. Specifically, inertial cavitation is predicted to be independent of PEG molecular weight and mole fraction in the so-called mushroom regime. In the "brush" regime, however, inertial cavitation is predicted to increase with PEG mole fraction but to decrease (to the inverse 3/5 power) with PEG molecular weight. While excellent agreement between experiment and theory can be achieved, it is shown that the calculated inertial cavitation profiles depend strongly on the criterion used to predict inertial cavitation. This is followed by a discussion of nesting microbubbles inside the aqueous core of microcapsules and how this significantly increases the inertial cavitation threshold. Nesting thus offers a means for avoiding unwanted inertial cavitation and cell death during imaging and other applications such as sonoporation. A review of putative sonoporation mechanisms is then presented, including those involving microbubbles to deliver cargo into a cell, and those--not necessarily involving microubbles--to release cargo from a phospholipid vesicle (or reverse sonoporation). It is shown that the rate of (reverse) sonoporation from liposomes correlates with phospholipid bilayer phase behavior, liquid-disordered phases giving appreciably faster release than liquid-ordered phases. Moreover, liquid-disordered phases exhibit evidence of two release mechanisms, which are described well mathematically by enhanced diffusion (possibly via dilation of membrane phospholipids) and irreversible membrane disruption, whereas liquid-ordered phases are described by a single mechanism, which has yet to be positively identified. The ability to tune release kinetics with bilayer composition makes reverse sonoporation of phospholipid vesicles a promising methodology for controlled drug delivery. Moreover, nesting of microbubbles inside vesicles constitutes a truly "theranostic" vehicle, one that can be used for both long-lasting, safe imaging and for controlled drug delivery.

Fatty acyl chain order in lecithin model membranes determined from proton magnetic resonance.

PubMed

Bloom, M; Burnell, E E; MacKay, A L; Nichol, C P; Valic, M I; Weeks, G

1978-12-26

Proton magnetic resonance (1H NMR) has been used to compare the local orientational order of acyl chains in phospholipid bilayers of multilamellar and small sonicated vesicular membranes of dipalmitoyllecithin (DPL) at 50 degrees C and egg yolk lecithin (EYL) at 31 degrees C. The orientational order of the multilamellar systems was characterized using deuterium magnetic resonance order parameters and 1H NMR second moments. 1H NMR line shapes in the vesicle samples were calculated using vesicle size distributions, determined directly using electron microscopy, and a theory of motional narrowing, which takes into account the symmetry properties of the bilayer systems. The predicted non-Lorentzian line shapes and widths were found to be in good agreement with experimental results, indicating that the local orientational order (called "packing" by many workers) in the bilayers of small vesicles and in multilamellar membranes is substantially the same. This results was found to be true not only for the largest 1H NMR line associated with the nonterminal methylene protons but also for the resolved 1H NMR lines due to the alpha-CH2 and the terminal CH3 positions on the acyl chain. Analysis of the vesicle 1H NMR spectra of EYL taken with different medium viscosities yielded a value of approximately 4 X 10(-8) cm2 s-1 for the lateral diffusion constant of the phospholipid molecules at 31 degrees C.

Interactions of the Auxilin-1 PTEN-like Domain with Model Membranes Result in Nanoclustering of Phosphatidyl Inositol Phosphates

PubMed Central

Kalli, Antreas C.; Morgan, Gareth; Sansom, Mark S.P.

2013-01-01

Auxilin-1 is a neuron-specific membrane-binding protein involved in a late stage of clathrin-mediated endocytosis. It recruits Hsc70, thus initiating uncoating of the clathrin-coated vesicles. Interactions of auxilin-1 with the vesicle membrane are crucial for this function and are mediated via an N-terminal PTEN-like domain. We have used multiscale molecular dynamics simulations to probe the interactions of the auxilin-1 PTEN-like domain with lipid bilayers containing differing phospholipid composition, including bilayers containing phosphatidyl inositol phosphates. Our results suggest a novel, to our knowledge, model for the auxilin/membrane encounter and subsequent interactions. Negatively charged lipids (especially PIP2) enhance binding of auxilin to lipid bilayers and facilitate its correct orientation relative to the membrane. Mutations in three basic residues (R301E/R307E/K311E) of the C2 subdomain of the PTEN-like domain perturbed its interaction with the bilayer, changing its orientation. The interaction of membrane-bound auxilin-1 PTEN-like domain with negatively charged lipid headgroups results in nanoclustering of PIP2 molecules in the adjacent bilayer leaflet. PMID:23823232

Molecular organization, localization and orientation of antifungal antibiotic amphotericin B in a single lipid bilayer

PubMed Central

Grudzinski, Wojciech; Sagan, Joanna; Welc, Renata; Luchowski, Rafal; Gruszecki, Wieslaw I.

2016-01-01

Amphotericin B is a popular antifungal antibiotic, a gold standard in treatment of systemic mycotic infections, due to its high effectiveness. On the other hand, applicability of the drug is limited by its considerable toxicity to patients. Biomembranes are a primary target of physiological activity of amphotericin B and both the pharmacologically desired and toxic side effects of the drug relay on its molecular organization in the lipid phase. In the present work, molecular organization, localization and orientation of amphotericin B, in a single lipid bilayer system, was analysed simultaneously, thanks to application of a confocal fluorescence lifetime imaging microscopy of giant unilamellar vesicles. The results show that the presence of sterols, in the lipid phase, promotes formation of supramolecular structures of amphotericin B and their penetration into the membrane hydrophobic core. The fact that such an effect is substantially less pronounced in the case of cholesterol than ergosterol, the sterol of fungal membranes, provides molecular insight into the selectivity of the drug. PMID:27620838

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

PubMed Central

Hormel, Tristan T.; Reyer, Matthew A.; Parthasarathy, Raghuveer

2015-01-01

Though the importance of membrane fluidity for cellular function has been well established for decades, methods for measuring lipid bilayer viscosity remain challenging to devise and implement. Recently, approaches based on characterizing the Brownian dynamics of individual tracers such as colloidal particles or lipid domains have provided insights into bilayer viscosity. For fluids in general, however, methods based on single-particle trajectories provide a limited view of hydrodynamic response. The technique of two-point microrheology, in which correlations between the Brownian dynamics of pairs of tracers report on the properties of the intervening medium, characterizes viscosity at length-scales that are larger than that of individual tracers and has less sensitivity to tracer-induced distortions, but has never been applied to lipid membranes. We present, to our knowledge, the first two-point microrheological study of lipid bilayers, examining the correlated motion of domains in phase-separated lipid vesicles and comparing one- and two-point results. We measure two-point correlation functions in excellent agreement with the forms predicted by two-dimensional hydrodynamic models, analysis of which reveals a viscosity intermediate between those of the two lipid phases, indicative of global fluid properties rather than the viscosity of the local neighborhood of the tracer. PMID:26287625

Gravimetric antigen detection utilizing antibody-modified lipid bilayers.

PubMed

Larsson, Charlotte; Bramfeldt, Hanna; Wingren, Christer; Borrebaeck, Carl; Höök, Fredrik

2005-10-01

Lipid bilayers containing 5% nitrilotriacetic acid (NTA) lipids supported on SiO2 have been used as a template for immobilization of oligohistidine-tagged single-chained antibody fragments (scFvs) directed against cholera toxin. It was demonstrated that histidine-tagged scFvs could be equally efficiently coupled to an NTA-Ni2+-containing lipid bilayer from a purified sample as from an expression supernatant, thereby providing a coupling method that eliminates time-consuming protein prepurification steps. Irrespective of whether the coupling was made from the unpurified or purified antibody preparation, the template proved to be efficient for antigen (cholera toxin) detection, verified using quartz crystal microbalance with dissipation monitoring. In addition, via a secondary amplification step using lipid vesicles containing GM1 (the natural membrane receptor for cholera toxin), the detection limit of cholera toxin was less than 750 pM. To further strengthen the coupling of scFvs to the lipid bilayer, scFvs containing two histidine tags, instead of just one tag, were also evaluated. The increased coupling strength provided via the bivalent anchoring significantly reduced scFv displacement in complex solutions containing large amounts of histidine-containing proteins, verified via cholera toxin detection in serum.

Evidence that electrostatic interactions between vesicle-associated membrane protein 2 and acidic phospholipids may modulate the fusion of transport vesicles with the plasma membrane.

PubMed

Williams, Dumaine; Vicôgne, Jérome; Zaitseva, Irina; McLaughlin, Stuart; Pessin, Jeffrey E

2009-12-01

The juxtamembrane domain of vesicle-associated membrane protein (VAMP) 2 (also known as synaptobrevin2) contains a conserved cluster of basic/hydrophobic residues that may play an important role in membrane fusion. Our measurements on peptides corresponding to this domain determine the electrostatic and hydrophobic energies by which this domain of VAMP2 could bind to the adjacent lipid bilayer in an insulin granule or other transport vesicle. Mutation of residues within the juxtamembrane domain that reduce the VAMP2 net positive charge, and thus its interaction with membranes, inhibits secretion of insulin granules in beta cells. Increasing salt concentration in permeabilized cells, which reduces electrostatic interactions, also results in an inhibition of insulin secretion. Similarly, amphipathic weak bases (e.g., sphingosine) that reverse the negative electrostatic surface potential of a bilayer reverse membrane binding of the positively charged juxtamembrane domain of a reconstituted VAMP2 protein and inhibit membrane fusion. We propose a model in which the positively charged VAMP and syntaxin juxtamembrane regions facilitate fusion by bridging the negatively charged vesicle and plasma membrane leaflets.

Dynamic excitations in membranes induced by optical tweezers.

PubMed Central

Bar-Ziv, R; Moses, E; Nelson, P

1998-01-01

We present the phenomenology of transformations in lipid bilayers that are excited by laser tweezers. A variety of dynamic instabilities and shape transformations are observed, including the pearling instability, expulsion of vesicles, and more exotic ones, such as the formation of passages. Our physical picture of the laser-membrane interaction is based on the generation of tension in the bilayer and loss of surface area. Although tension is the origin of the pearling instability, it does not suffice to explain expulsion of vesicles, where we observe opening of giant pores and creeping motion of bilayers. We present a quantitative theoretical framework to understand most of the observed phenomenology. The main hypothesis is that lipid is pulled into the optical trap by the familiar dielectric effect, is disrupted, and finally is repackaged into an optically unresolvable suspension of colloidal particles. This suspension, in turn, can produce osmotic pressure and depletion forces, driving the observed transformations. PMID:9649388

Investigating structural details of lipid-cholesterol-A β interactions

NASA Astrophysics Data System (ADS)

Rai, Durgesh; Anunciado, Divina; Heller, William; O'Neill, Hugh; Urban, Volker; Qian, Shuo

2015-03-01

Alzheimer's disease (AD) is the most common form of dementia and is predicted to affect 1 in 85 people around the world by 2050. Amyloid beta (A β) -peptide, a peptide composed of 40- 42 amino acids that is the product of cleavage from the amyloid precursor protein (APP), is regarded to play a major role in the development of AD. In addition, accumulating evidence points to a positive association between cholesterol and AD. Here, we present results from our studies about A β-peptide and cholesterol in bilayer by small-angle neutron scattering (SANS) using a combination of dimyristoyl, phosphocholine (DMPC) and partially deuterated cholesterol (cholesterol-d7) with and without A β. We compare the results using grazing incidence and transmission SANS on lipid bilayer films and unilamellar vesicles respectively. The structural details on vesicles and bilayers work in conjunction with the circular dichroism on peptide in solution and oriented circular dichroism in bilayer films. The studies confirm a positive association of A β with the membrane layers. The results from different studies will be compared and contrasted in presentation.

The interactions between ionic surfactants and phosphatidylcholine vesicles: Conductometry

NASA Astrophysics Data System (ADS)

Tsao, Heng-Kwong; Tseng, Wen Liang

2001-11-01

The interaction between ionic surfactants and phosphatidylcholine vesicles, which are prepared without addition of buffer and salt, is investigated by conductivity measurements. On the basis of the vesicle acting as a trap of charge carriers, the bilayer/aqueous phase partition coefficient K and the surfactant/lipid molar ratio Re of nine surfactants are determined. The thermodynamic consistency is satisfied by the measured parameters. The effects of the alkyl chain length (C10-C16) and ionic head group are then studied. The inverse partition coefficient K-1 is linearly related to the critical micelle concentration. The solubilizing ability Reb is a consequence of the competition between the surfactant incorporation into the bilayer and the formation of micelles. Consequently, the K parameter rises whereas the Reb parameter declines as the chain length is increased. The influence due to addition of salt is also discussed.

Engineering plant membranes using droplet interface bilayers.

PubMed

Barlow, N E; Smpokou, E; Friddin, M S; Macey, R; Gould, I R; Turnbull, C; Flemming, A J; Brooks, N J; Ces, O; Barter, L M C

2017-03-01

Droplet interface bilayers (DIBs) have become widely recognised as a robust platform for constructing model membranes and are emerging as a key technology for the bottom-up assembly of synthetic cell-like and tissue-like structures. DIBs are formed when lipid-monolayer coated water droplets are brought together inside a well of oil, which is excluded from the interface as the DIB forms. The unique features of the system, compared to traditional approaches (e.g., supported lipid bilayers, black lipid membranes, and liposomes), is the ability to engineer multi-layered bilayer networks by connecting multiple droplets together in 3D, and the capability to impart bilayer asymmetry freely within these droplet architectures by supplying droplets with different lipids. Yet despite these achievements, one potential limitation of the technology is that DIBs formed from biologically relevant components have not been well studied. This could limit the reach of the platform to biological systems where bilayer composition and asymmetry are understood to play a key role. Herein, we address this issue by reporting the assembly of asymmetric DIBs designed to replicate the plasma membrane compositions of three different plant species; Arabidopsis thaliana , tobacco, and oats, by engineering vesicles with different amounts of plant phospholipids, sterols and cerebrosides for the first time. We show that vesicles made from our plant lipid formulations are stable and can be used to assemble asymmetric plant DIBs. We verify this using a bilayer permeation assay, from which we extract values for absolute effective bilayer permeation and bilayer stability. Our results confirm that stable DIBs can be assembled from our plant membrane mimics and could lead to new approaches for assembling model systems to study membrane translocation and to screen new agrochemicals in plants.

Properties of POPC/POPE supported lipid bilayers modified with hydrophobic quantum dots on polyelectrolyte cushions.

PubMed

Kolasinska-Sojka, Marta; Wlodek, Magdalena; Szuwarzynski, Michal; Kereiche, Sami; Kovacik, Lubomir; Warszynski, Piotr

2017-10-01

The formation and properties of supported lipid bilayers (SLB) containing hydrophobic nanoparticles (NP) was studied in relation to underlying cushion obtained from selected polyelectrolyte multilayers. Lipid vesicles were formed from zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) in phosphate buffer (PBS). As hydrophobic nanoparticles - quantum dots (QD) with size of 3.8nm (emission wavelength of 420nm) were used. Polyelectrolyte multilayers (PEM) were constructed by the sequential, i.e., layer-by-layer (LbL) adsorption of alternately charged polyelectrolytes from their solutions. Liposomes and Liposome-QDs complexes were studied with Transmission Cryo-Electron Microscopy (Cryo-TEM) to verify the quality of vesicles and the position of QD within lipid bilayer. Deposition of liposomes and liposomes with quantum dots on polyelectrolyte films was studied in situ using quartz crystal microbalance with dissipation (QCM-D) technique. The fluorescence emission spectra were analyzed for both: suspension of liposomes with nanoparticles and for supported lipid bilayers containing QD on PEM. It was demonstrated that quantum dots are located in the hydrophobic part of lipid bilayer. Moreover, we proved that such QD-modified liposomes formed supported lipid bilayers and their final structure depended on the type of underlying cushion. Copyright © 2017 Elsevier B.V. All rights reserved.

Visualization of lipids and proteins at high spatial and temporal resolution via interferometric scattering (iSCAT) microscopy

NASA Astrophysics Data System (ADS)

Spindler, Susann; Ehrig, Jens; König, Katharina; Nowak, Tristan; Piliarik, Marek; Stein, Hannah E.; Taylor, Richard W.; Garanger, Elisabeth; Lecommandoux, Sébastien; Alves, Isabel D.; Sandoghdar, Vahid

2016-07-01

Microscopy based on the interferometric detection of light scattered from nanoparticles (iSCAT) was introduced in our laboratory more than a decade ago. In this work, we present various capabilities of iSCAT for biological studies by discussing a selection of our recent results. In particular, we show tracking of lipid molecules in supported lipid bilayers (SLBs), tracking of gold nanoparticles with diameters as small as 5 nm and at frame rates close to 1 MHz, 3D tracking of Tat peptide-coated nanoparticles on giant unilamellar vesicles (GUVs), imaging the formation of lipid bilayers, sensing single unlabelled proteins and tracking their motion under electric fields, as well as challenges of studying live cell membranes. These studies set the ground for future quantitative research on dynamic biophysical processes at the nanometer scale.

Growth and instability of a phospholipid vesicle in a bath of fatty acids

NASA Astrophysics Data System (ADS)

Dervaux, J.; Noireaux, V.; Libchaber, A. J.

2017-06-01

Using a microfluidic trap, we study the behavior of individual phospholipid vesicles in contact with fatty acids. We show that spontaneous fatty acids insertion inside the bilayer is controlled by the vesicle size, osmotic pressure difference across the membrane and fatty acids concentration in the external bath. Depending on these parameters, vesicles can grow spherically or become unstable and fragment into several daughter vesicles. We establish the phase diagram for vesicle growth and we derive a simple thermodynamic model that reproduces the time evolution of the vesicle volume. Finally, we show that stable growth can be achieved on an artificial cell expressing a simple set of bacterial cytoskeletal proteins, paving the way toward artificial cell reproduction.

Interactions between non-steroidal anti-inflammatory drugs and lipid membranes

NASA Astrophysics Data System (ADS)

Boggara, Mohan; Krishnamoorti, Ramanan

2008-03-01

Chronic usage of Non-steroidal anti-inflammatory drugs(NSAIDs) leads to gastrointestinal toxicity and clinical evidences point the cause to direct interactions between NSAIDs and phospholipid membranes. Also, NSAIDs pre-associated with phospholipid vesicles are shown to be safer and therapeutically more effective than unmodified ones. Our initial experiments and simulations on the partitioning of Aspirin and Ibuprofen clearly indicate role played by the drug structure in drug-membrane interactions. Those results motivated systematic molecular dynamics simulations of membranes with NSAIDs of different size, structure and pKa values. Our results suggest high partition coefficients for these NSAIDs in the membrane compared to water and thinning effect on the bilayer. Our small angle neutron scattering and reflectivity studies on DMPC-Ibuprofen systems indicate that the drug affects both ˜5 nm thick bilayer and overall ˜100 nm diameter vesicle, indicating that NSAIDs affect vesicles on various length scales. We will discuss the structural perturbations to membranes due to NSAIDs at clinically relevant molar ratios and their implications on the use of vesicles as delivery vehicles for NSAIDs.

Recruitment of a phospholipase C/sphingomyelinase into non-lamellar lipid droplets during hydrolysis of lipid bilayers.

PubMed

Ibarguren, Maitane; Sot, Jesús; Montes, L Ruth; Vasil, Adriana I; Vasil, Michael L; Goñi, Félix M; Alonso, Alicia

2013-01-01

When giant unilamellar vesicles (GUVs) composed of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, and cholesterol are treated with PlcHR(2), a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa, the initial stages of lipid hydrolysis do not cause large changes in vesicle morphology (Ibarguren et al., 2011). However, when hydrolysis progresses confocal fluorescence microscopy reveals the formation of lipid aggregates, whose morphology is not compatible with that of bilayers. Smaller vesicles or droplets can also be seen inside the GUV. Our studies indicate that these aggregates or droplets are enriched in the non-lamellar lipid ceramide, an end-product of PlcHR(2) reaction. Moreover, the aggregates/droplets appear enriched in the hydrolytic enzyme PlcHR(2). At a final stage GUVs containing the enzyme-enriched droplets disintegrate and vanish from the microscope field. The observed non-lamellar enzyme-rich structures may be related to intermediates in the process of aggregation and fusion although the experimental design prevents vesicle free diffusion in the aqueous medium, thus actual aggregation or fusion cannot be observed. 2012 Elsevier Ireland Ltd. All rights reserved

Interaction of Gramicidin S and its Aromatic Amino-Acid Analog with Phospholipid Membranes

PubMed Central

Jelokhani-Niaraki, Masoud; Hodges, Robert S.; Meissner, Joseph E.; Hassenstein, Una E.; Wheaton, Laura

2008-01-01

To investigate the mechanism of interaction of gramicidin S-like antimicrobial peptides with biological membranes, a series of five decameric cyclic cationic β-sheet-β-turn peptides with all possible combinations of aromatic D-amino acids, Cyclo(Val-Lys-Leu-D-Ar1-Pro-Val-Lys-Leu-D-Ar2-Pro) (Ar ≡ Phe, Tyr, Trp), were synthesized. Conformations of these cyclic peptides were comparable in aqueous solutions and lipid vesicles. Isothermal titration calorimetry measurements revealed entropy-driven binding of cyclic peptides to POPC and POPE/POPG lipid vesicles. Binding of peptides to both vesicle systems was endothermic—exceptions were peptides containing the Trp-Trp and Tyr-Trp pairs with exothermic binding to POPC vesicles. Application of one- and two-site binding (partitioning) models to binding isotherms of exothermic and endothermic binding processes, respectively, resulted in determination of peptide-lipid membrane binding constants (Kb). The Kb1 and Kb2 values for endothermic two-step binding processes corresponded to high and low binding affinities (Kb1 ≥ 100 Kb2). Conformational change of cyclic peptides in transferring from buffer to lipid bilayer surfaces was estimated using fluorescence resonance energy transfer between the Tyr-Trp pair in one of the peptide constructs. The cyclic peptide conformation expands upon adsorption on lipid bilayer surface and interacts more deeply with the outer monolayer causing bilayer deformation, which may lead to formation of nonspecific transient peptide-lipid porelike zones causing membrane lysis. PMID:18621820

Electrochemical and PM-IRRAS Studies of the Effect of Cholesterol on the Structure of a DMPC Bilayer Supported at an Au (111) Electrode Surface, Part 1: Properties of the Acyl Chains

PubMed Central

Bin, Xiaomin; Horswell, Sarah L.; Lipkowski, Jacek

2005-01-01

Charge density measurements and polarization modulation infrared reflection absorption spectroscopy were employed to investigate the spreading of small unilamellar vesicles of a dimyristoylphosphatidylcholine (DMPC)/cholesterol (7:3 molar ratio) mixture onto an Au (111) electrode surface. The electrochemical experiments demonstrated that vesicles fuse and spread onto the Au (111) electrode surface, forming a bilayer, at rational potentials −0.4 V < (E − Epzc) < 0.4 V or field strength <6×107 V m−1. Polarization modulation infrared reflection absorption spectroscopy experiments provided information concerning the conformation and orientation of the acyl chains of DMPC molecules. Deuterated DMPC was used to subtract the contribution of C-H stretching bands of cholesterol and of the polar head region of DMPC from spectra in the C-H stretching region. The absorption spectra of the C-H stretch bands in the acyl chains were determined in this way. The properties of the DMPC/cholesterol bilayer have been compared with the properties of a pure DMPC bilayer. The presence of 30% cholesterol gives a thicker and more fluid bilayer characterized by a lower capacity and lower tilt angle of the acyl chains. PMID:15849259

Adhesive interactions of biologically inspired soft condensed matter

NASA Astrophysics Data System (ADS)

Anderson, Travers Heath

Improving our fundamental understanding of the surface interactions between complex materials is needed to improve existing materials and products as well as develop new ones. The object of this research was to apply the measurements of fundamental surface interactions to real world problems facing chemical engineers and materials scientists. I focus on three systems of biologically inspired soft condensed matter, with an emphasis on the adhesive interactions between them. The formation of phospholipid bilayers of the neutral lipid, dimyristoyl-phosphatidylcholine (DMPC) on silica surfaces from vesicles in aqueous solutions was investigated. The process involves five stages: vesicle adhesion to the substrate surfaces, steric interactions with neighboring vesicles, rupture, spreading via hydrophobic fusion of bilayer edges, and ejection of excess lipid, trapped water and ions into the solution. The forces between DMPC bilayers and silica were measured in the Surface Forces Apparatus (SFA) in phosphate buffered saline. The adhesion energy was found to be much stronger than the expected adhesion predicted by van der Waals interactions, likely due to an attractive electrostatic interaction. The effects of non-adsorbing cationic polyelectrolytes on the interactions between supported cationic surfactant bilayers were studied using the SFA. Addition of polyelectrolyte has a number of effects on the interactions including the induction of a depletion-attraction and screening of the double-layer repulsion. Calculations are made that allow for the conversion of the adhesion energy measured in the SFA to the overall interaction energy between vesicles in solution, which determines the stability behavior of vesicle dispersions. Mussels use a variety of dihydroxyphenyl-alanine (DOPA) rich proteins specifically tailored to adhering to wet surfaces. The SFA was used to study the role of DOPA on the adhesive properties of these proteins to TiO 2 and mica using both real mussel foot proteins (mfp) and a synthetic polypeptide analogue of mfp-3. Adhesion increased with DOPA concentration, although oxidation of DOPA reduces the adhesive capabilities of the proteins. Comparison of the two shows that DOPA is responsible for at least 80% of the adhesion energy of mfp-3 and can be attributed to DOPA groups favorably oriented within or at the interface of these films.

Spontaneous Buckling of Lipid Bilayer and Vesicle Budding Induced by Antimicrobial Peptide Magainin 2: A Coarse-Grained Simulation Study

DTIC Science & Technology

2011-05-30

were added to neutralize each system. The GROMACS software package39 was used for simulations. The molecules in this paper refer to the CGMARTINI...accelerated.19 Most of the peptides on the surfaces ended up in clusters containing transmembrane pores, which appeared to perturb the bilayers significantly

Mechanical properties of lipid bilayers from molecular dynamics simulation

PubMed Central

Venable, Richard M.; Brown, Frank L.H.; Pastor, Richard W.

2015-01-01

Lipid areas (Aℓ), bilayer area compressibilities (KA), bilayer bending constants (KC), and monolayer spontaneous curvatures (c0) from simulations using the CHARMM36 force field are reported for 12 representative homogenous lipid bilayers. Aℓ (or their surrogate, the average deuterium order parameter in the “plateau region” of the chain) agree very well with experiment, as do the KA. Simulated KC are in near quantitative agreement with vesicle flicker experiments, but are somewhat larger than KC from x-ray, pipette aspiration, and neutron spin echo for saturated lipids. Spontaneous curvatures of bilayer leaflets from the simulations are approximately 30% smaller than experimental values of monolayers in the inverse hexagonal phase. PMID:26238099

Choosing the right fluorophore for single-molecule fluorescence studies in a lipid environment.

PubMed

Zhang, Zhenfu; Yomo, Dan; Gradinaru, Claudiu

2017-07-01

Nonspecific interactions between lipids and fluorophores can alter the outcomes of single-molecule spectroscopy of membrane proteins in live cells, liposomes or lipid nanodiscs and of cytosolic proteins encapsulated in liposomes or tethered to supported lipid bilayers. To gain insight into these effects, we examined interactions between 9 dyes that are commonly used as labels for single-molecule fluorescence (SMF) and 6 standard lipids including cationic, zwitterionic and anionic types. The diffusion coefficients of dyes in the absence and presence of set amounts of lipid vesicles were measured by fluorescence correlation spectroscopy (FCS). The partition coefficients and the free energies of partitioning for different fluorophore-lipid pairs were obtained by global fitting of the titration FCS curves. Lipids with different charges, head groups and degrees of chain saturation were investigated, and interactions with dyes are discussed in terms of hydrophobic, electrostatic and steric contributions. Fluorescence imaging of individual fluorophores adsorbed on supported lipid bilayers provides visualization and additional quantification of the strength of dye-lipid interaction in the context of single-molecule measurements. By dissecting fluorophore-lipid interactions, our study provides new insights into setting up single-molecule fluorescence spectroscopy experiments with minimal interference from interactions between fluorescent labels and lipids in the environment. Copyright © 2017 Elsevier B.V. All rights reserved.

Deformed soft matter under constraints

NASA Astrophysics Data System (ADS)

Bertrand, Martin

In the last few decades, an increasing number of physicists specialized in soft matter, including polymers, have turned their attention to biologically relevant materials. The properties of various molecules and fibres, such as DNA, RNA, proteins, and filaments of all sorts, are studied to better understand their behaviours and functions. Self-assembled biological membranes, or lipid bilayers, are also the focus of much attention as many life processes depend on these. Small lipid bilayers vesicles dubbed liposomes are also frequently used in the pharmaceutical and cosmetic industries. In this thesis, work is presented on both the elastic properties of polymers and the response of lipid bilayer vesicles to extrusion in narrow-channels. These two areas of research may seem disconnected but they both concern deformed soft materials. The thesis contains four articles: the first presenting a fundamental study of the entropic elasticity of circular chains; the second, a simple universal description of the effect of sequence on the elasticity of linear polymers such as DNA; the third, a model of the symmetric thermophoretic stretch of a nano-confined polymer; the fourth, a model that predicts the final sizes of vesicles obtained by pressure extrusion. These articles are preceded by an extensive introduction that covers all of the essential concepts and theories necessary to understand the work that has been done.

Scraping and stapling of end-grafted DNA chains by a bioadhesive spreading vesicle to reveal chain internal friction and topological complexity.

PubMed

Nam, Gimoon; Hisette, Marie Laure; Sun, Yuting Liang; Gisler, Thomas; Johner, Albert; Thalmann, Fabrice; Schröder, André Pierre; Marques, Carlos Manuel; Lee, Nam-Kyung

2010-08-20

Stained end-grafted DNA molecules about 20 μm long are scraped away and stretched out by the spreading front of a bioadhesive vesicle. Tethered biotin ligands bind the vesicle bilayer to a streptavidin substrate, stapling the DNAs into frozen confinement paths. Image analysis of the stapled DNA gives access, within optical resolution, to the local stretching values of individual DNA molecules swept by the spreading front, and provides evidence of self-entanglements.

Export of Virulence Genes and Shiga Toxin by Membrane Vesicles of Escherichia coli O157:H7

PubMed Central

Kolling, Glynis L.; Matthews, Karl R.

1999-01-01

Membrane vesicles released by Escherichia coli O157:H7 into culture medium were purified and analyzed for protein and DNA content. Electron micrographs revealed vesicles that are spherical, range in size from 20 to 100 nm, and have a complete bilayer. Analysis of vesicle protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrates vesicles that contain many proteins with molecular sizes similar to outer membrane proteins and a number of cellular proteins. Immunoblot (Western) analysis of vesicles suggests the presence of cell antigens. Treatment of vesicles with exogenous DNase hydrolyzed surface-associated DNA; PCR demonstrated that vesicles contain DNA encoding the virulence genes eae, stx1 and stx2, and uidA, which encodes for β-galactosidase. Immunoblot analysis of intact and lysed, proteinase K-treated vesicles demonstrate that Shiga toxins 1 and 2 are contained within vesicles. These results suggest that vesicles contain toxic material and transfer experiments demonstrate that vesicles can deliver genetic material to other gram-negative organisms. PMID:10223967

Submillisecond Dynamics of Mastoparan X Insertion into Lipid Membranes.

PubMed

Schuler, Erin E; Nagarajan, Sureshbabu; Dyer, R Brian

2016-09-01

The mechanism of protein insertion into a lipid bilayer is poorly understood because the kinetics of this process is difficult to measure. We developed a new approach to study insertion of the antimicrobial peptide Mastoparan X into zwitterionic lipid vesicles, using a laser-induced temperature-jump to initiate insertion on the microsecond time scale and infrared and fluorescence spectroscopies to follow the kinetics. Infrared probes the desolvation of the peptide backbone and yields biphasic kinetics with relaxation lifetimes of 12 and 117 μs, whereas fluorescence probes the intrinsic tryptophan residue located near the N-terminus and yields a single exponential phase with a lifetime of 440 μs. Arrhenius analysis of the temperature-dependent rates yields an activation energy for insertion of 96 kJ/mol. These results demonstrate the complexity of the insertion process and provide mechanistic insight into the interplay between peptides and the lipid bilayer required for peptide transport across cellular membranes.

The effect of cholesterol on the partitioning of 1-octanol into POPC vesicles

NASA Astrophysics Data System (ADS)

Zakariaee Kouchaksaraee, Roja

Microcalorimetry has become a method of choice for sensitive characterization of biomolecular interactions. In this study, isothermal titration calorimetry (ITC) was used to measure the partitioning of 1-octanol into lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), a semi-unsaturated lipid, and cholesterol, a steroid, as a function of cholesterol molar concentration. The ITC instrument measures the heat evolved or absorbed upon titration of a liposome dispersion, at concentrations ranging from 0 to 40% cholesterol, into a suspension of 1-octanol in water. A model function was fit to the data in order to determine the partition coefficient of octanol into POPC bilayers and the enthalpy of interaction. I found that the partition coefficient increases and the heat of interaction becomes less negative with increasing cholesterol content, in contrast to results found by other groups for partitioning of alcohols into lipid-cholesterol bilayers containing saturated lipids. The heat of dilution of vesicles was also measured. Keywords: Partition coefficient; POPC; 1-Octanol; Cholesterol; Isothermal titration calorimetry; Lipid-alcohol interactions. Subject Terms: Calorimetry; Membranes (Biology); Biophysics; Biology -- Technique; Bilayer lipid membranes -- Biotechnology; Lipid membranes -- Biotechnology.

Interaction of triblock co-polymer micelles with phospholipid-bilayer: a spectroscopic investigation using a potential chloride channel blocker.

PubMed

Ganguly, Aniruddha; Ghosh, Soumen; Guchhait, Nikhil

2015-03-07

Interaction of a potential chloride channel blocker, 9-methyl anthroate (9-MA), has been studied with zwitterionic l-α-phosphatidylcholine (egg-PC) lipid vesicles, which ascertains the utility of the drug as an efficient molecular reporter for probing the microheterogeneous environment of lipid-bilayers. The effect of a non-ionic triblock co-polymer P123 on the stability of these drug-bound lipid-bilayers has also been investigated by means of steady state and time-resolved spectroscopic techniques exploiting the fluorescence properties of the drug. Experimental results reveal that the addition of P123 to the drug-bound lipid results in a preferential complexation of the drug with the Pluronic leaving the lipid vesicles aside, which has been attributed to a substantially stronger binding interaction of the drug with P123 than that with egg-PC. The result is of potential interest from a medical perspective owing to the context of excess drug desorption from bio-membranes.

Patterning of supported lipid bilayers and proteins using material selective nitrodopamine-mPEG.

PubMed

Spycher, Philipp R; Hall, Heike; Vogel, Viola; Reimhult, Erik

2015-01-01

We present a generic patterning process by which biomolecules in a passivated background are patterned directly from physiological buffer to microfabricated surfaces without the need for further processing. First, nitrodopamine-mPEG is self-assembled to selectively render TiO2 patterns non-fouling to biomolecule adsorption on hydrophilic and adhesive glass surfaces. After the controlled TiO2 passivation, the biomolecules can be directly adsorbed from solution in a single step creating large scale micropatterned and highly homogeneous arrays of biomolecules with very high pattern definition. We demonstrate the formation of fluid supported lipid bilayers (SLBs) down to the single μm-level limited only by the photolithographic process. Non-specific adsorption of lipid vesicles to the TiO2 background was found to be almost completely suppressed. The SLB patterns can be further selectively functionalized with retained mobility, which we demonstrate through biotin-streptavidin coupling. We envision this single step patterning approach to be very beneficial for membrane-based biosensors and for pattering of cells on a passivated background with complex, sub-cellular geometries; in each application the adherent areas have a tunable mobility of interaction sites controlled by the fluidity of the membrane.

The amphiphilic alkyl ester derivatives of l-ascorbic acid induce reorganization of phospholipid vesicles.

PubMed

Giudice, Francesca; Ambroggio, Ernesto E; Mottola, Milagro; Fanani, Maria Laura

2016-09-01

l-ascorbic acid alkyl esters (ASCn) are lipophilic forms of vitamin C, which maintain some of its antioxidant power. Those properties make this drug family attractive to be used in pharmacological preparations protecting other redox-sensible drugs or designed to reduce possible toxic oxidative processes. In this work, we tested the ability of l-ascorbic acid alkyl esters (ASCn) to modulate the structure, permeability, and rheological properties of phospholipid bilayers. The ASCn studied here (ASC16, ASC14, and ASC12) alter the structural integrity as well as the rheological properties of phospholipid membranes without showing any evident detergent activity. ASC14 appeared as the most efficient drug in destabilize the membrane structure of nano- and micro-size phospholipid liposomes inducing vesicle content leakage and shape elongation on giant unilamellar vesicles. It also was the most potent enhancer of membrane microviscosity and surface water structuring. Only ASC16 induced the formation of drug-enriched condensed domains after its incorporation into the lipid bilayer, while ASC12 appeared as the less membrane-disturbing compound, likely because of its poor, and more superficial, partition into the membrane. We also found that incorporation of ASCn into the lipid bilayers enhanced the reduction of membrane components, compared with soluble vitamin C. Our study shows that ASCn compounds, which vary in the length of the acyl chain, show different effects on phospholipid vesicles used as biomembrane models. Those variances may account for subtly differences in the effectiveness on their pharmacological applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Vesiculation of healthy and defective red blood cells

NASA Astrophysics Data System (ADS)

Li, He; Lykotrafitis, George

2015-07-01

Vesiculation of mature red blood cells (RBCs) contributes to removal of defective patches of the erythrocyte membrane. In blood disorders, which are related to defects in proteins of the RBC membrane, vesiculation of the plasma membrane is intensified. Several hypotheses have been proposed to explain RBC vesiculation but the exact underlying mechanisms and what determines the sizes of the vesicles are still not completely understood. In this work, we apply a two-component coarse-grained molecular dynamics RBC membrane model to study how RBC vesiculation is controlled by the membrane spontaneous curvature and by lateral compression of the membrane. Our simulation results show that the formation of small homogeneous vesicles with a diameter less than 40 nm can be attributed to a large spontaneous curvature of membrane domains. On the other hand, compression on the membrane can cause the formation of vesicles with heterogeneous composition and with sizes comparable with the size of the cytoskeleton corral. When spontaneous curvature and lateral compression are simultaneously considered, the compression on the membrane tends to facilitate formation of vesicles originating from curved membrane domains. We also simulate vesiculation of RBCs with membrane defects connected to hereditary elliptocytosis (HE) and to hereditary spherocytosis (HS). When the vertical connectivity between the lipid bilayer and the membrane skeleton is elevated, as in normal RBCs, multiple vesicles are shed from the compressed membrane with diameters similar to the cytoskeleton corral size. In HS RBCs, where the connectivity between the lipid bilayer and the cytoskeleton is reduced, larger-size vesicles are released under the same compression ratio as in normal RBCs. Lastly, we find that vesicles released from HE RBCs can contain cytoskeletal filaments due to fragmentation of the membrane skeleton while vesicles released from the HS RBCs are depleted of cytoskeletal filaments.

Lipid vesicles and other colloids as drug carriers on the skin.

PubMed

Cevc, Gregor

2004-03-27

Colloids from an aqueous suspension can cross the skin barrier only through hydrophilic pathways. Various colloids have a different ability to do this by penetrating narrow pores of fixed size in the skin, or the relevant nano-pores in barriers modelling the skin. Such ability is governed by colloid adaptability, which must be high enough to allow penetrant deformation to the size of a pore in such barrier: for a 100 nm colloid trespassing the skin this means at least 5-fold deformation/elongation. (Lipid) Bilayer vesicles are normally more adaptable than the comparably large (lipid coated) fluid droplets. One of the reasons for this, and an essential condition for achieving a high bilayer adaptability and pore penetration, is a high bilayer membrane elasticity. The other reason is the relaxation of changing colloid's volume-to-surface constraint during pore penetration; it stands to reason that such relaxation requires a concurrent, but only transient and local, bilayer permeabilisation. Both these phenomena are reflected in bilayer composition sensitivity, which implies non-linear pressure dependency of the apparent barrier penetrability, for example. Amphipats that acceptably weaken a membrane (surfactants, (co)solvents, such as certain alcohols, etc.) consequently facilitate controlled, local bilayer destabilisation and increase lipid bilayer flexibility. When used in the right quantity, such additives thus lower the energetic expense for elastic bilayer deformation, associated with pore penetration. Another prerequisite for aggregate transport through the skin is the colloid-induced opening of the originally very narrow ( approximately 0.4 nm) gaps between cells in the barrier to pores with diameter above 30 nm. Colloids incapable of enforcing such widening-and simultaneously of self-adapting to the size of 20-30 nm without destruction-are confined to the skin surface. All relatively compact colloids seem to fall in this latter category. This includes mixed lipid micelles, solid (nano)particles, nano-droplets, biphasic vesicles, etc. Such colloids, therefore, merely enter the skin through the rare wide gaps between groups of skin cells near the organ surface. Transdermal drug delivery systems based on corresponding drug formulations, therefore, rely on simple drug diffusion through the skin; the colloid then, at best, can modulate drug transport through the barrier. In contrast, the adaptability-and stability-optimised mixed lipid vesicles (Transfersomes, a trademark of IDEA AG) can trespass much narrower pathways between most cells in the skin; such highly adaptable colloids thus mediate drug transport through the skin. Sufficiently stable ultra-adaptable carriers, therefore, can ensure targeted drug delivery deep below the application site. This has already been shown in numerous preclinical tests and several phase I and phase II clinical studies. Drug delivery by means of highly adaptable drug carriers, moreover, allows highly efficient and well-tolerated drug targeting into the skin proper. Sustained drug release through the skin into systemic blood circulation is another field of ultradeformable drug carrier application.

High Cholesterol Obviates a Prolonged Hemifusion Intermediate in Fast SNARE-Mediated Membrane Fusion

PubMed Central

Kreutzberger, Alex J.B.; Kiessling, Volker; Tamm, Lukas K.

2015-01-01

Cholesterol is essential for exocytosis in secretory cells, but the exact molecular mechanism by which it facilitates exocytosis is largely unknown. Distinguishing contributions from the lateral organization and dynamics of membrane proteins to vesicle docking and fusion and the promotion of fusion pores by negative intrinsic spontaneous curvature and other mechanical effects of cholesterol have been elusive. To shed more light on this process, we examined the effect of cholesterol on SNARE-mediated membrane fusion in a single-vesicle assay that is capable of resolving docking and elementary steps of fusion with millisecond time resolution. The effect of cholesterol on fusion pore formation between synaptobrevin-2 (VAMP-2)-containing proteoliposomes and acceptor t-SNARE complex-containing planar supported bilayers was examined using both membrane and content fluorescent markers. This approach revealed that increasing cholesterol in either the t-SNARE or the v-SNARE membrane favors a mechanism of direct fusion pore opening, whereas low cholesterol favors a mechanism leading to a long-lived (>5 s) hemifusion state. The amount of cholesterol in the target membrane had no significant effect on docking of synaptobrevin vesicles. Comparative studies with α-tocopherol (vitamin E) show that the negative intrinsic spontaneous curvature of cholesterol and its presumed promotion of a very short-lived (<50 ms) lipid stalk intermediate is the main factor that favors rapid fusion pore opening at high cholesterol. This study also shows that this single-vesicle fusion assay can distinguish between hemifusion and full fusion with only a single lipid dye, thereby freeing up a fluorescence channel for the simultaneous measurement of another parameter in fast time-resolved fusion assays. PMID:26200867

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 on protein activity and the roles of membrane proteins in disease pathways.

Solvent-assisted lipid bilayer formation on silicon dioxide and gold.

PubMed

Tabaei, Seyed R; Choi, Jae-Hyeok; Haw Zan, Goh; Zhdanov, Vladimir P; Cho, Nam-Joon

2014-09-02

Planar lipid bilayers on solid supports mimic the fundamental structure of biological membranes and can be investigated using a wide range of surface-sensitive techniques. Despite these advantages, planar bilayer fabrication is challenging, and there are no simple universal methods to form such bilayers on diverse material substrates. One of the novel methods recently proposed and proven to form a planar bilayer on silicon dioxide involves lipid deposition in organic solvent and solvent exchange to influence the phase of adsorbed lipids. To scrutinize the specifics of this solvent-assisted lipid bilayer (SALB) formation method and clarify the limits of its applicability, we have developed a simplified, continuous solvent-exchange version to form planar bilayers on silicon dioxide, gold, and alkanethiol-coated gold (in the latter case, a lipid monolayer is formed to yield a hybrid bilayer) and varied the type of organic solvent and rate of solvent exchange. By tracking the SALB formation process with simultaneous quartz crystal microbalance-dissipation (QCM-D) and ellipsometry, it was determined that the acoustic, optical, and hydration masses along with the acoustic and optical thicknesses, measured at the end of the process, are comparable to those observed by employing conventional fabrication methods (e.g., vesicle fusion). As shown by QCM-D measurements, the obtained planar bilayers are highly resistant to protein adsorption, and several, but not all, water-miscible organic solvents could be successfully used in the SALB procedure, with isopropanol yielding particularly high-quality bilayers. In addition, fluorescence recovery after photobleaching (FRAP) measurements demonstrated that the coefficient of lateral lipid diffusion in the fabricated bilayers corresponds to that measured earlier in the planar bilayers formed by vesicle fusion. With increasing rate of solvent exchange, it was also observed that the bilayer became incomplete and a phenomenological model was developed in order to explain this feature. The results obtained allowed us to clarify and discriminate likely steps of the SALB formation process as well as determine the corresponding influence of organic solvent type and flow conditions on these steps. Taken together, the findings demonstrate that the SALB formation method can be adapted to a continuous solvent-exchange procedure that is technically minimal, quick, and efficient to form planar bilayers on solid supports.

Monitoring changes of paramagnetically-shifted 31P signals in phospholipid vesicles

NASA Astrophysics Data System (ADS)

Joyce, Rebecca E.; Williams, Thomas L.; Serpell, Louise C.; Day, Iain J.

2016-03-01

Phospholipid vesicles are commonly used as biomimetics in the investigation of the interaction of various species with cell membranes. In this letter we present a 31P NMR investigation of a simple vesicle system using a paramagnetic shift reagent to probe the inner and outer layers of the lipid bilayer. Time-dependent changes in the 31P NMR signal are observed, which differ whether the paramagnetic species is inside or outside the vesicle, and on the choice of buffer solution used. An interpretation of these results is given in terms of the interaction of the paramagnetic shift reagent with the lipids.

Vesicle electrohydrodynamics.

PubMed

Schwalbe, Jonathan T; Vlahovska, Petia M; Miksis, Michael J

2011-04-01

A small amplitude perturbation analysis is developed to describe the effect of a uniform electric field on the dynamics of a lipid bilayer vesicle in a simple shear flow. All media are treated as leaky dielectrics and fluid motion is described by the Stokes equations. The instantaneous vesicle shape is obtained by balancing electric, hydrodynamic, bending, and tension stresses exerted on the membrane. We find that in the absence of ambient shear flow, it is possible that an applied stepwise uniform dc electric field could cause the vesicle shape to evolve from oblate to prolate over time if the encapsulated fluid is less conducting than the suspending fluid. For a vesicle in ambient shear flow, the electric field damps the tumbling motion, leading to a stable tank-treading state.

Role of lipid phase separations and membrane hydration in phospholipid vesicle fusion.

PubMed

Hoekstra, D

1982-06-08

The relationship between lipid phase separation and fusion of small unilamellar phosphatidylserine-containing vesicles was investigated. The kinetics of phase separation were monitored by following the increase of self-quenching of the fluorescent phospholipid analogue N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine, which occurs when the local concentration of the probe increases upon Ca2+-induced phase separation in phosphatidylserine (PS) bilayers [Hoekstra, D. (1982) Biochemistry 21, 1055-1061]. Fusion was determined by using the resonance energy transfer fusion assay [Struck, D. K., Hoekstra, D., & Pagano, R. E. (1981) Biochemistry 20, 4093-4099], which monitors the mixing of fluorescent lipid donor and acceptor molecules, resulting in an increase in energy transfer efficiency. The results show that in the presence of Ca2+, fusion proceeds much more rapidly (t 1/2 less than 5 s) than the process of phase separation (T 1/2 congruent to 1 min). Mg2+ also induced fusion, albeit at higher concentrations than Ca2+. Mg2+-induced phase separation were not detected, however. Subthreshold concentrations of Ca2+ (0.5 mM) or Mg2+ (2 mM) induced extensive fusion of PS-containing vesicles in poly(ethylene glycol) containing media. This effect did not appear to be a poly(ethylene glycol)-facilitated enhancement of cation binding to the bilayer, and consequently Ca2+-induced phase separation was not observed. The results suggest that macroscopic phase separation may facilitate but does not induced the fusion process and is therefore, not directly involved in the actual fusion mechanism. The fusion experiments performed in the presence of poly(ethylene glycol) suggest that the degree of bilayer dehydration and the creation of "point defects" in the bilayer without rigorous structural rearrangements in the membrane are dominant factors in the initial fusion events.

Effect of Ca2+ on Vesicle Fusion on Solid Surface: An In vitro Model of Protein-Accelerated Vesicle Fusion

NASA Astrophysics Data System (ADS)

Shinozaki, Youichi; Siitonen, Ari M.; Sumitomo, Koji; Furukawa, Kazuaki; Torimitsu, Keiichi

2008-07-01

Lipid vesicle fusion is an important reaction in the cell. Calcium ions (Ca2+) participate in various important biological events including the fusion of vesicles with cell membranes in cells. We studied the effect of Ca2+ on the fusion of egg yolk phosphatidylcholine/brain phosphatidylserine (eggPC/brainPS) lipid vesicles on a mica substrate with fast scanning atomic force microscopy (AFM). When unattached and unfused lipid vesicles on mica were rinsed away, discrete patches of fused vesicles were observed under high Ca2+ concentrations. At 0 mM Ca2+, lipid vesicles were fused on mica and formed continuous supported lipid bilayers (SLBs) covering almost the entire mica surface. The effect of Ca2+ on SLB formation was offset by a Ca2+ chelating agent. When lipid vesicles were added during AFM observation, vesicles fused on mica and covered almost all areas even under high Ca2+ concentrations. These results indicate that force between AFM tip and vesicles overcomes the Ca2+-reduced fusion of lipid vesicles.

Sculpting and fusing biomimetic vesicle networks using optical tweezers.

PubMed

Bolognesi, Guido; Friddin, Mark S; Salehi-Reyhani, Ali; Barlow, Nathan E; Brooks, Nicholas J; Ces, Oscar; Elani, Yuval

2018-05-14

Constructing higher-order vesicle assemblies has discipline-spanning potential from responsive soft-matter materials to artificial cell networks in synthetic biology. This potential is ultimately derived from the ability to compartmentalise and order chemical species in space. To unlock such applications, spatial organisation of vesicles in relation to one another must be controlled, and techniques to deliver cargo to compartments developed. Herein, we use optical tweezers to assemble, reconfigure and dismantle networks of cell-sized vesicles that, in different experimental scenarios, we engineer to exhibit several interesting properties. Vesicles are connected through double-bilayer junctions formed via electrostatically controlled adhesion. Chemically distinct vesicles are linked across length scales, from several nanometres to hundreds of micrometres, by axon-like tethers. In the former regime, patterning membranes with proteins and nanoparticles facilitates material exchange between compartments and enables laser-triggered vesicle merging. This allows us to mix and dilute content, and to initiate protein expression by delivering biomolecular reaction components.

Membrane bending: the power of protein imbalance.

PubMed

Derganc, Jure; Antonny, Bruno; Copič, Alenka

2013-11-01

Many cellular processes require membrane deformation, which is driven by specialized protein machinery and can often be recapitulated using pure lipid bilayers. However, biological membranes contain a large amount of embedded proteins. Recent research suggests that membrane-bound proteins with asymmetric distribution of mass across the bilayer can influence membrane bending in a nonspecific manner due to molecular crowding. This mechanism is physical in nature and arises from collisions between such 'mushroom-shaped' proteins. It can either facilitate or impede the action of protein coats, for example COPII, during vesicle budding. We describe the physics of how molecular crowding can influence membrane bending and discuss the implications for other cellular processes, such as sorting of glycosylphosphatidylinositol-anchored proteins (GPI-APs) and production of intraluminal vesicles. Copyright © 2013 Elsevier Ltd. All rights reserved.

Inverted micellar intermediates and the transitions between lamellar, cubic, and inverted hexagonal lipid phases. II. Implications for membrane-membrane interactions and membrane fusion.

PubMed Central

Siegel, D P

1986-01-01

Results of a kinetic model of thermotropic L alpha----HII phase transitions are used to predict the types and order-of-magnitude rates of interactions between unilamellar vesicles that can occur by intermediates in the L alpha----HII phase transition. These interactions are: outer monolayer lipid exchange between vesicles; vesicle leakage subsequent to aggregation; and (only in systems with ratios of L alpha and HII phase structural dimensions in a certain range or with unusually large bilayer lateral compressibilities) vesicle fusion with retention of contents. It was previously proposed that inverted micellar structures mediate membrane fusion. These inverted micellar structures are thought to form in all systems with such transitions. However, I show that membrane fusion probably occurs via structures that form from these inverted micellar intermediates, and that fusion should occur in only a sub-set of lipid systems that can adopt the HII phase. For single-component phosphatidylethanolamine (PE) systems with thermotropic L alpha----HII transitions, lipid exchange should be observed starting at temperatures several degrees below TH and at all higher temperatures, where TH is the L alpha----HII transition temperature. At temperatures above TH, the HII phase forms between apposed vesicles, and eventually ruptures them (leakage). In most single-component PE systems, fusion via L alpha----HII transition intermediates should not occur. This is the behavior observed by Bentz, Ellens, Lai, Szoka, et al. in PE vesicle systems. Fusion is likely to occur under circumstances in which multilamellar samples of lipid form the so-called "inverted cubic" or "isotropic" phase. This is as observed in the mono-methyl DOPE system (Ellens, H., J. Bentz, and F. C. Szoka. 1986. Fusion of phosphatidylethanolamine containing liposomes and the mechanism of the L alpha-HII phase transition. Biochemistry. In press.) In lipid systems with L alpha----HII transitions driven by cation binding (e.g., Ca2+-cardiolipin), fusion should be more frequent than in thermotropic systems. PMID:3719075

Phospholipid order in gel- and fluid-phase cell-size liposomes measured by digitized video fluorescence polarization microscopy.

PubMed Central

Florine-Casteel, K

1990-01-01

Low-light digitized video fluorescence microscopy has been utilized to measure the steady-state polarized fluorescence from the membrane probe diphenylhexatriene (DPH) and its cationic and phosphatidylcholine derivatives 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 2-[3-(diphenylhexatrienyl)propanoyl]-3-palmitoyl-L-alpha-phosphati dylcholine (DPH-PC), respectively, in cell-size (10-70 microns) unilamellar vesicles composed of gel-or fluid-phase phospholipid. Using an inverted microscope with epi-illumination optics and an intensified silicon intensified target camera interfaced to a minicomputer, fluorescence images of single vesicles were obtained at emission polarizer orientations of 0 degrees, 45 degrees, 90 degrees, and 135 degrees relative to the excitation light polarization direction. Fluorescence intensity ratios F90 degrees/F0 degrees (= F perpendicular/F parallel) and F135 degrees/F45 degrees were calculated on a pixel-by-pixel basis from digitized image pairs. Theoretical expressions were derived for collected polarized fluorescence as a function of position on the membrane surface as well as the degree of lipid order, in terms of the fluorophore's maximum angular motional freedom in the bilayer (identical to theta max), using a modification of the method of D. Axelrod (1979. Biophys. J. 26:557-574) together with the "wobbling-in-a-cone" model of probe rotational diffusion. Comparison of experimental polarization ratios with theoretical ratios yielded the following results. In gel-phase dipalmitoyl-phosphatidylcholine, the data for all three probes correspond to a model in which the cone angle theta max = 17 +/- 2 degrees and there exists a collective tilt of the phospholipid acyl chains of 30 degrees relative to the bilayer normal. In addition, approximately 5% of DPH and TMA-DPH molecules are aligned parallel to the plane of the bilayer. In fluid-phase palmitoyloleoyl-phosphatidylcholine, the data are well fit by models in which theta max = 60 +/- 2 degrees for DPH and DPH-PC and 32 +/- 4 degrees for TMA-DPH, with approximately 20% of DPH molecules and 10% of TMA-DPH molecules aligned parallel to the bilayer plane, and a net phospholipid tilt at or near the headgroup region of approximately 30 degrees. The results demonstrate that lipid order can be measured with a spatial resolution of approximately 1 micron2 in cell-size vesicles even with high aperture observation through a microscope. Images FIGURE 4 FIGURE 7 FIGURE 10 PMID:2393705

Lipid oxidation in bilayer liposomes induced by radicals from the surrounding water phases

NASA Astrophysics Data System (ADS)

Sprinz, H.; Brede, O.

1996-03-01

Some features of the radiation chemistry of organized assemblies were studied in aqueous dispersions of small unilamellar vesicles of egg yolk lecithin. The kinetics for the reaction of OH radicals with the bilayer was determined by pulse radiolysis. The conversion of OH radicals into N 3 radicals results in a remarkable reduction of the radiolysis of the hydrophylic part of the phospholipid and in an enhanced degradation of the most radiosensitive group of polyunsaturated fatty acid residues. The transverse proton relaxation of the choline head group is very sensitive to the radical attack on the bilayer.

Determination of Benzyl-hexadecyldimethylammonium 1,4-Bis(2-ethylhexyl)sulfosuccinate Vesicle Permeability by Using Square Wave Voltammetry and an Enzymatic Reaction.

PubMed

Cobo Solis, Airam K; Correa, N Mariano; Molina, Patricia G

2017-10-31

This report describes the studies performed to determine the permeability coefficient value (P) of 1-naphthyl phosphate (1-NP) through the benzyl-hexadecyldimethylammonium 1,4-bis(2-ethylhexyl)sulfosuccinate (AOT-BHD) vesicle bilayer. 1-NP was added in the external phase and must cross the bilayer of the vesicle to react with the encapsulated enzyme (alkaline phosphatase) to yield 1-naphtholate (NPh - ), the product of the enzymatic hydrolysis. This product is electrochemically detected, at basic pH value, by a square wave voltammetry technique, which can be a good alternative over the spectroscopic one, to measure the vesicle solutions because scattering (due to its turbidity) does not make any influence in the electrochemical signal. The experimental data allow us to propose a mathematical model, and a value of P = (1.00 ± 0.15) × 10 -9 cm s -1 was obtained. Also, a value of P = (2.0 ± 0.5) × 10 -9 cm s -1 was found by using an independent technique, ultraviolet-visible spectroscopy, for comparison. It is evident that the P values obtained from both the techniques are comparable (within the experimental error of both techniques) under the same experimental conditions. This study constitutes the first report of the 1-NP permeability determination in this new vesicle. We want to highlight the importance of the introduction of a new method and the electrochemical response of the product generated through an enzymatic reaction that occurs in the inner aqueous phase of the vesicle, where the enzyme is placed.

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

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

Aqueous self-assembly of poly(ethylene oxide)-block-poly(ε-caprolactone) (PEO-b-PCL) copolymers: disparate diblock copolymer compositions give rise to nano- and meso-scale bilayered vesicles

NASA Astrophysics Data System (ADS)

Qi, Wei; Ghoroghchian, P. Peter; Li, Guizhi; Hammer, Daniel A.; Therien, Michael J.

2013-10-01

Nanoparticles formed from diblock copolymers of FDA approved PEO and PCL have generated considerable interest as in vivo drug delivery vehicles. Herein, we report the synthesis of the most extensive family PEO-b-PCL copolymers that vary over the largest range of number-average molecular weights (Mn: 3.6-57k), PEO weight fractions (fPEO: 0.08-0.33), and PEO chain lengths (0.75-5.8k) reported to date. These polymers were synthesized in order to establish the full range of aqueous phase behaviours of these diblock copolymers and to specifically identify formulations that were able to generate bilayered vesicles (polymersomes). Cryogenic transmission electron microscopy (cryo-TEM) was utilized in order to visualize the morphology of these structures upon aqueous self-assembly of dry polymer films. Nanoscale polymersomes were formed from PEO-b-PCL copolymers over a wide range of PEO weight fractions (fPEO: 0.14-0.27) and PEO molecular weights (0.75-3.8k) after extrusion of aqueous suspensions. Comparative morphology diagrams, which describe the nature of self-assembled structures as a function of diblock copolymer molecular weight and PEO weight fraction, show that in contrast to micron-scale polymersomes, which form only from a limited range of PEO-b-PCL diblock copolymer compositions, a multiplicity of PEO-b-PCL diblock copolymer compositions are able to give rise to nanoscale vesicles. These data underscore that PEO-b-PCL compositions that spontaneously form micron-sized polymersomes, as well as those that have previously been reported to form polymersomes via a cosolvent fabrication system, provide only limited insights into the distribution of PEO-b-PCL diblocks that give rise to nanoscale vesicles. The broad range of polymersome-forming PEO-b-PCL compositions described herein suggest the ability to construct extensive families of nanoscale vesicles of varied bilayer thickness, providing the ability to tune the timescales of vesicle degradation and encapsulant release based on the intended in vivo application.Nanoparticles formed from diblock copolymers of FDA approved PEO and PCL have generated considerable interest as in vivo drug delivery vehicles. Herein, we report the synthesis of the most extensive family PEO-b-PCL copolymers that vary over the largest range of number-average molecular weights (Mn: 3.6-57k), PEO weight fractions (fPEO: 0.08-0.33), and PEO chain lengths (0.75-5.8k) reported to date. These polymers were synthesized in order to establish the full range of aqueous phase behaviours of these diblock copolymers and to specifically identify formulations that were able to generate bilayered vesicles (polymersomes). Cryogenic transmission electron microscopy (cryo-TEM) was utilized in order to visualize the morphology of these structures upon aqueous self-assembly of dry polymer films. Nanoscale polymersomes were formed from PEO-b-PCL copolymers over a wide range of PEO weight fractions (fPEO: 0.14-0.27) and PEO molecular weights (0.75-3.8k) after extrusion of aqueous suspensions. Comparative morphology diagrams, which describe the nature of self-assembled structures as a function of diblock copolymer molecular weight and PEO weight fraction, show that in contrast to micron-scale polymersomes, which form only from a limited range of PEO-b-PCL diblock copolymer compositions, a multiplicity of PEO-b-PCL diblock copolymer compositions are able to give rise to nanoscale vesicles. These data underscore that PEO-b-PCL compositions that spontaneously form micron-sized polymersomes, as well as those that have previously been reported to form polymersomes via a cosolvent fabrication system, provide only limited insights into the distribution of PEO-b-PCL diblocks that give rise to nanoscale vesicles. The broad range of polymersome-forming PEO-b-PCL compositions described herein suggest the ability to construct extensive families of nanoscale vesicles of varied bilayer thickness, providing the ability to tune the timescales of vesicle degradation and encapsulant release based on the intended in vivo application. Electronic supplementary information (ESI) available: Materials and methods, characterization data. See DOI: 10.1039/c3nr03250g

TRIC-B channels display labile gating: evidence from the TRIC-A knockout mouse model.

PubMed

Venturi, Elisa; Matyjaszkiewicz, Antoni; Pitt, Samantha J; Tsaneva-Atanasova, Krasimira; Nishi, Miyuki; Yamazaki, Daiju; Takeshima, Hiroshi; Sitsapesan, Rebecca

2013-08-01

Sarcoplasmic/endoplasmic reticulum (SR) and nuclear membranes contain two related cation channels named TRIC-A and TRIC-B. In many tissues, both subtypes are co-expressed, making it impossible to distinguish the distinct single-channel properties of each subtype. We therefore incorporated skeletal muscle SR vesicles derived from Tric-a-knockout mice into bilayers in order to characterise the biophysical properties of native TRIC-B without possible misclassification of the channels as TRIC-A, and without potential distortion of functional properties by detergent purification protocols. The native TRIC-B channels were ideally selective for cations. In symmetrical 210 mM K(+), the maximum (full) open channel level (199 pS) was equivalent to that observed when wild-type SR vesicles were incorporated into bilayers. Analysis of TRIC-B gating revealed complex and variable behaviour. Four main sub-conductance levels were observed at approximately 80 % (161 pS), 60 % (123 pS), 46 % (93 pS), and 30 % (60 pS) of the full open state. Seventy-five percent of the channels were voltage sensitive with Po being markedly reduced at negative holding potentials. The frequent, rapid transitions between TRIC-B sub-conductance states prevented development of reliable gating models using conventional single-channel analysis. Instead, we used mean-variance plots to highlight key features of TRIC-B gating in a more accurate and visually useful manner. Our study provides the first biophysical characterisation of native TRIC-B channels and indicates that this channel would be suited to provide counter current in response to Ca(2+) release from the SR. Further experiments are required to distinguish the distinct functional properties of TRIC-A and TRIC-B and understand their individual but complementary physiological roles.

Optical stretching of giant unilamellar vesicles with an integrated dual-beam optical trap.

PubMed

Solmaz, Mehmet E; Biswas, Roshni; Sankhagowit, Shalene; Thompson, James R; Mejia, Camilo A; Malmstadt, Noah; Povinelli, Michelle L

2012-10-01

We have integrated a dual-beam optical trap into a microfluidic platform and used it to study membrane mechanics in giant unilamellar vesicles (GUVs). We demonstrate the trapping and stretching of GUVs and characterize the membrane response to a step stress. We then measure area strain as a function of applied stress to extract the bending modulus of the lipid bilayer in the low-tension regime.

Vesicle dynamics in a confined Poiseuille flow: from steady state to chaos.

PubMed

Aouane, Othmane; Thiébaud, Marine; Benyoussef, Abdelilah; Wagner, Christian; Misbah, Chaouqi

2014-09-01

Red blood cells (RBCs) are the major component of blood, and the flow of blood is dictated by that of RBCs. We employ vesicles, which consist of closed bilayer membranes enclosing a fluid, as a model system to study the behavior of RBCs under a confined Poiseuille flow. We extensively explore two main parameters: (i) the degree of confinement of vesicles within the channel and (ii) the flow strength. Rich and complex dynamics for vesicles are revealed, ranging from steady-state shapes (in the form of parachute and slipper shapes) to chaotic dynamics of shape. Chaos occurs through a cascade of multiple periodic oscillations of the vesicle shape. We summarize our results in a phase diagram in the parameter plane (degree of confinement and flow strength). This finding highlights the level of complexity of a flowing vesicle in the small Reynolds number where the flow is laminar in the absence of vesicles and can be rendered turbulent due to elasticity of vesicles.

Early Stages of Oxidative Stress-Induced Membrane Permeabilization: A Neutron Reflectometry Study

PubMed Central

Smith, Hillary L.; Howland, Michael C.; Szmodis, Alan W.; Li, Qijuan; Daemen, Luke L.; Parikh, Atul N.; Majewski, Jaroslaw

2009-01-01

Neutron reflectometry was used to probe in situ the structure of supported lipid bilayers at the solid–liquid interface during the early stages of UV-induced oxidative degradation. Single-component supported lipid bilayers composed of gel phase, dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and fluid phase, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), phospholipids were exposed to low-dose oxidative stress generated by UV light and their structures were examined by neutron reflectometry. An interrupted illumination mode, involving exposures in 15 min increments with 2 h intervals between subsequent exposures, and a continuous mode involving a single 60 (or 90) min exposure period were employed. In both cases, pronounced differences in the structure of the lipid bilayer after exposure were observed. Interrupted exposure led to a substantial decrease in membrane coverage but preserved its total thickness at reduced scattering length densities. These results indicate that the initial phase during UV-induced membrane degradation involves the formation of hydrophilic channels within the membrane. This is consistent with the loss of some lipid molecules we observe and attendant reorganization of residual lipids forming hemimicellar edges of the hydrophilic channels. In contrast, continuous illumination produced a graded interface of continuously varied scattering length density (and hence hydrocarbon density) extending 100–150 Å into the liquid phase. Exposure of a DPPC bilayer to UV light in the presence of a reservoir of unfused vesicles showed low net membrane disintegration during oxidative stress, presumably because of surface back-filling from the bulk reservoir. Chemical evidence for membrane degradation was obtained by mass spectrometry and Fourier transform infrared spectroscopy. Further evidence for the formation of hydrophilic channels was furnished by fluorescence microscopy and imaging ellipsometry data. PMID:19275260

Zwitterionic lipid assemblies: Molecular dynamics studies of monolayers, bilayers, and vesicles using a new coarse grain force field

PubMed Central

Shinoda, Wataru; DeVane, Russell; Klein, Michael L.

2010-01-01

A new coarse-grained (CG) intermolecular force field is presented for a series of zwitterionic lipids. The model is an extension of our previous work on nonionic surfactants and is designed to reproduce experimental surface/interfacial properties as well as distribution functions from all-atom molecular dynamics (MD) simulations. Using simple functional forms, the force field parameters are optimized for multiple lipid molecules, simultaneously. The resulting CG lipid bilayers have reasonable molecular areas, chain order parameters, and elastic properties. The computed surface pressure vs. area (π-A) curve for a DPPC monolayer demonstrates a significant improvement over the previous CG models. The DPPC monolayer has a longer persistence length than a PEG lipid monolayer, exhibiting a long-lived curved monolayer surface under negative tension. The bud ejected from an oversaturated DPPC monolayer has a large bicelle-like structure, which is different from the micellar bud formed from an oversaturated PEG lipid monolayer. We have successfully observed vesicle formation during CG-MD simulations, starting from an aggregate of DMPC molecules. Depending on the aggregate size, the lipid assembly spontaneously transforms into a closed vesicle or a bicelle. None of the various intermediate structures between these extremes seem to be stable. An attempt to observe fusion of two vesicles through the application of an external adhesion force was not successful. The present CG force field also supports stable multi-lamellar DMPC vesicles. PMID:20438090

A bio-synthetic interface for discovery of viral entry mechanisms.

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

Gutzler, Mike; Maar, Dianna; Negrete, Oscar

2010-09-01

Understanding and defending against pathogenic viruses is an important public health and biodefense challenge. The focus of our LDRD project has been to uncover the mechanisms enveloped viruses use to identify and invade host cells. We have constructed interfaces between viral particles and synthetic lipid bilayers. This approach provides a minimal setting for investigating the initial events of host-virus interaction - (i) recognition of, and (ii) entry into the host via membrane fusion. This understanding could enable rational design of therapeutics that block viral entry as well as future construction of synthetic, non-proliferating sensors that detect live virus in themore » environment. We have observed fusion between synthetic lipid vesicles and Vesicular Stomatitis virus particles, and we have observed interactions between Nipah virus-like particles and supported lipid bilayers and giant unilamellar vesicles.« less

Evidence for dimer formation by an amphiphilic heptapeptide that mediates chloride and carboxyfluorescein release from liposomes

PubMed Central

Pajewski, Robert; Ferdani, Riccardo; Pajewska, Jolanta; Djedovič, Natasha; Schlesinger, Paul H.; Gokel, George W.

2008-01-01

Heptapeptides having dioctadecyl, N-terminal hydrocarbon chains insert in phospholipid bilayer membranes and form pores through which at least chloride ions pass. Although amphiphilic, these compounds do not typically form vesicles themselves. They insert in the bilayers of phospholipid vesicles and mediate the release of carboxyfluorescein. Hill analysis indicates that at least two molecules of the amphiphile are involved in pore formation. In CD2Cl2, dimer formation is detected by NMR chemical shift changes. The anion release activity of individual anion transporters is increased by linking them covalently at the C-terminus or, even more, by linking them at the N-terminus. Evidence is presented that either linked molecule releases chloride from liposomes more effectively and rapidly than the individual transporter molecule at a comparable concentration. PMID:15703797

Assessing the efficacy of vesicle fusion with planar membrane arrays using a mitochondrial porin as reporter

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

Pszon-Bartosz, Kamila; Hansen, Jesper S.; Technical University of Denmark, Department of Micro- and Nanotechnology, DK-2800 Kongens Lyngby

2011-03-04

Research highlights: {yields} We have established a vesicle fusion efficacy assay based on the major non-specific porin of Fusobacterium nucleatum (FomA). {yields} Maximal fusion obtained was almost 150,000 porin insertions during 20 min. {yields} Incorporation can be either first order or exponential kinetics which has implications for establishing protein delivery to biomimetic membranes. -- Abstract: Reconstitution of functionally active membrane protein into artificially made lipid bilayers is a challenge that must be overcome to create a membrane-based biomimetic sensor and separation device. In this study we address the efficacy of proteoliposome fusion with planar membrane arrays. We establish a proteinmore » incorporation efficacy assay using the major non-specific porin of Fusobacterium nucleatum (FomA) as reporter. We use electrical conductance measurements and fluorescence microscopy to characterize proteoliposome fusion with an array of planar membranes. We show that protein reconstitution in biomimetic membrane arrays may be quantified using the developed FomA assay. Specifically, we show that FomA vesicles are inherently fusigenic. Optimal FomA incorporation is obtained with a proteoliposome lipid-to-protein molar ratio (LPR) = 50 more than 10{sup 5} FomA proteins could be incorporated in a bilayer array with a total membrane area of 2 mm{sup 2} within 20 min. This novel assay for quantifying protein delivery into lipid bilayers may be a useful tool in developing biomimetic membrane applications.« less

Gating behavior of endoplasmic reticulum potassium channels of rat hepatocytes in diabetes.

PubMed

Ghasemi, Maedeh; Khodaei, Naser; Salari, Sajjad; Eliassi, Afsaneh; Saghiri, Reza

2014-07-01

Defects in endoplasmic reticulum homeostasis are common occurrences in different diseases, such as diabetes, in which the function of endoplasmic reticulum is disrupted. It is now well established that ion channels of endoplasmic reticulum membrane have a critical role in endoplasmic reticulum luminal homeostasis. Our previous studies showed the presence of an ATP-sensitive cationic channel in endoplasmic reticulum. Therefore, in this study, we examined and compared the activities of this channel in control and diabetic rats using single-channel recording techniques. Male Wistar rats were made diabetic for 2 weeks with a single dose injection of streptozotocin (45 mg/kg). Ion channel incorporation of rough endoplasmic reticulum of diabetic hepatocytes into the bilayer lipid membrane allowed the characterization of K+ channel. Ion channel incorporation of rough endoplasmic reticulum vesicles into the bilayer lipid revealed that the channel current-voltage (I-V) relation with a mean slope conductance of 520 ± 19 pS was unaffected in diabetes. Interestingly, the channel Po-voltage relation was significantly lower in diabetic rats at voltages above +30 mV. We concluded that the endoplasmic reticulum cationic channel is involved in diabetes. Also, this finding could be considered as a goal for further therapeutic plans.

Interactions and Translational Dynamics of Phosphatidylinositol Bisphosphate (PIP2) Lipids in Asymmetric Lipid Bilayers.

PubMed

Shi, Xiaojun; Kohram, Maryam; Zhuang, Xiaodong; Smith, Adam W

2016-02-23

Phosphatidylinositol phosphate (PIP) lipids are critical to many cell signaling pathways, in part by acting as molecular beacons that recruit peripheral membrane proteins to specific locations within the plasma membrane. Understanding the biophysics of PIP-protein interactions is critical to developing a chemically detailed model of cell communication. Resolving such interactions is challenging, even in model membrane systems, because of the difficulty in preparing PIP-containing membranes with high fluidity and integrity. Here we report on a simple, vesicle-based protocol for preparing asymmetric supported lipid bilayers in which fluorescent PIP lipid analogues are found only on the top leaflet of the supported membrane facing the bulk solution. With this asymmetric distribution of lipids between the leaflets, the fluorescent signal from the PIP lipid analogue reports directly on interactions between the peripheral molecules and the top leaflet of the membrane. Asymmetric PIP-containing bilayers are an ideal platform to investigate the interaction of PIP with peripheral membrane proteins using fluorescence-based imaging approaches. We demonstrate their usefulness here with a combined fluorescence correlation spectroscopy and single particle tracking study of the interaction between PIP2 lipids and a polycationic polymer, quaternized polyvinylpyridine (QPVP). With this approach we are able to quantify the microscopic features of the mobility coupling between PIP2 lipids and polybasic QPVP. With single particle tracking we observe individual PIP2 lipids switch from Brownian to intermittent motion as they become transiently trapped by QPVP.

Optical stretching of giant unilamellar vesicles with an integrated dual-beam optical trap

PubMed Central

Solmaz, Mehmet E.; Biswas, Roshni; Sankhagowit, Shalene; Thompson, James R.; Mejia, Camilo A.; Malmstadt, Noah; Povinelli, Michelle L.

2012-01-01

We have integrated a dual-beam optical trap into a microfluidic platform and used it to study membrane mechanics in giant unilamellar vesicles (GUVs). We demonstrate the trapping and stretching of GUVs and characterize the membrane response to a step stress. We then measure area strain as a function of applied stress to extract the bending modulus of the lipid bilayer in the low-tension regime. PMID:23082284

Normal vibrational modes of phospholipid bilayers observed by low-frequency Raman scattering

NASA Astrophysics Data System (ADS)

Surovtsev, N. V.; Dmitriev, A. A.; Dzuba, S. A.

2017-03-01

Low-frequency Raman spectra of multilamellar vesicles made either of 1-palmitoyl-2-oleoyl-s n -glycero-3-phosphocholine (POPC) or 1,2-dipalmitoyl-s n -glycero-3-phosphocholine (DPPC) have been studied in a wide temperature range. Below 0 ∘C two peaks are found at frequencies around 8-9 and 14 -17 c m -1 and attributed to the normal vibrational modes of the phospholipid bilayer, which are determined by the bilayer thickness and stiffness (elastic modulus). The spectral positions of the peaks depend on the temperature and the bilayer composition. It is suggested that the ratio of the intensities of the first and second peaks can serve as a measure of the interleaflet elastic coupling. The addition of cholesterol to the phospholipid bilayer leads to peak shift and broadening, which may be assigned to the composition heterogeneities commonly attributed to the lipid raft formation.

Hybrid continuum-coarse-grained modeling of erythrocytes

NASA Astrophysics Data System (ADS)

Lyu, Jinming; Chen, Paul G.; Boedec, Gwenn; Leonetti, Marc; Jaeger, Marc

2018-06-01

The red blood cell (RBC) membrane is a composite structure, consisting of a phospholipid bilayer and an underlying membrane-associated cytoskeleton. Both continuum and particle-based coarse-grained RBC models make use of a set of vertices connected by edges to represent the RBC membrane, which can be seen as a triangular surface mesh for the former and a spring network for the latter. Here, we present a modeling approach combining an existing continuum vesicle model with a coarse-grained model for the cytoskeleton. Compared to other two-component approaches, our method relies on only one mesh, representing the cytoskeleton, whose velocity in the tangential direction of the membrane may be different from that of the lipid bilayer. The finitely extensible nonlinear elastic (FENE) spring force law in combination with a repulsive force defined as a power function (POW), called FENE-POW, is used to describe the elastic properties of the RBC membrane. The mechanical interaction between the lipid bilayer and the cytoskeleton is explicitly computed and incorporated into the vesicle model. Our model includes the fundamental mechanical properties of the RBC membrane, namely fluidity and bending rigidity of the lipid bilayer, and shear elasticity of the cytoskeleton while maintaining surface-area and volume conservation constraint. We present three simulation examples to demonstrate the effectiveness of this hybrid continuum-coarse-grained model for the study of RBCs in fluid flows.

Inkjet formation of unilamellar lipid vesicles for cell-like encapsulation†

PubMed Central

Stachowiak, Jeanne C.; Richmond, David L.; Li, Thomas H.; Brochard-Wyart, Françoise

2010-01-01

Encapsulation of macromolecules within lipid vesicles has the potential to drive biological discovery and enable development of novel, cell-like therapeutics and sensors. However, rapid and reliable production of large numbers of unilamellar vesicles loaded with unrestricted and precisely-controlled contents requires new technologies that overcome size, uniformity, and throughput limitations of existing approaches. Here we present a high-throughput microfluidic method for vesicle formation and encapsulation using an inkjet printer at rates up to 200 Hz. We show how multiple high-frequency pulses of the inkjet’s piezoelectric actuator create a microfluidic jet that deforms a bilayer lipid membrane, controlling formation of individual vesicles. Variations in pulse number, pulse voltage, and solution viscosity are used to control the vesicle size. As a first step toward cell-like reconstitution using this method, we encapsulate the cytoskeletal protein actin and use co-encapsulated microspheres to track its polymerization into a densely entangled cytoskeletal network upon vesicle formation. PMID:19568667

Interbilayer-crosslinked multilamellar vesicles as synthetic vaccines for potent humoral and cellular immune responses

NASA Astrophysics Data System (ADS)

Moon, James J.; Suh, Heikyung; Bershteyn, Anna; Stephan, Matthias T.; Liu, Haipeng; Huang, Bonnie; Sohail, Mashaal; Luo, Samantha; Ho Um, Soong; Khant, Htet; Goodwin, Jessica T.; Ramos, Jenelyn; Chiu, Wah; Irvine, Darrell J.

2011-03-01

Vaccines based on recombinant proteins avoid the toxicity and antivector immunity associated with live vaccine (for example, viral) vectors, but their immunogenicity is poor, particularly for CD8+ T-cell responses. Synthetic particles carrying antigens and adjuvant molecules have been developed to enhance subunit vaccines, but in general these materials have failed to elicit CD8+ T-cell responses comparable to those for live vectors in preclinical animal models. Here, we describe interbilayer-crosslinked multilamellar vesicles formed by crosslinking headgroups of adjacent lipid bilayers within multilamellar vesicles. Interbilayer-crosslinked vesicles stably entrapped protein antigens in the vesicle core and lipid-based immunostimulatory molecules in the vesicle walls under extracellular conditions, but exhibited rapid release in the presence of endolysosomal lipases. We found that these antigen/adjuvant-carrying vesicles form an extremely potent whole-protein vaccine, eliciting endogenous T-cell and antibody responses comparable to those for the strongest vaccine vectors. These materials should enable a range of subunit vaccines and provide new possibilities for therapeutic protein delivery.

Inhibitory effect of DIDS, NPPB, and phloretin on intracellular chloride channels.

PubMed

Malekova, Lubica; Tomaskova, Jana; Novakova, Marie; Stefanik, Peter; Kopacek, Juraj; Lakatos, Boris; Pastorekova, Silvia; Krizanova, Olga; Breier, Albert; Ondrias, Karol

2007-11-01

We studied the effects of the chloride channel blockers, 5-nitro-2-(phenylpropylamino)-benzoate (NPPB), dihydro-4,4' diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), and phloretin on H2O2-induced primary culture cardiomyocyte apoptosis and activity of intracellular chloride channels obtained from rat heart mitochondrial and lysosomal vesicles. The chloride channel blockers (100 micromol/l) inhibited the H2O2-induced cardiomyocytes apoptosis. We characterized the effect of the blockers on single channel properties of the chloride channels derived from the mitochondrial and lysosomal vesicles incorporated into a bilayer lipid membrane. The single chloride channel currents were measured in 250:50 mmol/l KCl cis/trans solutions. NPPB, DIDS, and phloretin inhibited the chloride channels by decreasing the channel open probability in a concentration-dependent manner with EC50 values of 42, 7, and 20 micromol/l, respectively. NPPB and phloretin inhibited the channel's conductance and open dwell time, indicating that they could affect the chloride selective filter, pore permeability, and gating mechanism of the chloride channels. DIDS and NPPB inhibited the channels from the other side than bongkrekic acid and carboxyatractyloside. The results may contribute to understand a possible involvement of intracellular chloride channels in apoptosis and cardioprotection.

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

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

Metabolic GARD: Replicating Catalytic Network of Lipid-Anchored Metabolites

NASA Astrophysics Data System (ADS)

Lancet, D.; Zidovetzki, R.; Shenhav, B.; Markovitch, O.

2017-07-01

We propose a computer-simulated M-GARD model, with mutually catalytic metabolic network of amphiphiles. It can show compositional reproduction of both bilayer and lumen content of lipid vesicles, thus joining metabolism, compartment and replication.

Polymeric amphiphile branching leads to rare nanodisc shaped planar self-assemblies.

PubMed

Qu, Xiaozhong; Omar, Leila; Le, Thi Bich Hang; Tetley, Laurence; Bolton, Katherine; Chooi, Kar Wai; Wang, Wei; Uchegbu, Ijeoma F

2008-09-16

Self-assembly is fundamental to the biological function of cells and the fabrication of nanomaterials. However, the origin of the shape of various self-assemblies, such as the shape of cells, is not altogether clear. Polymeric, oligomeric, or low molecular weight amphiphiles are a rich source of nanomaterials, and controlling their self-assembly is the route to tailored nanosystems with specific functionalities. Here, we provide direct evidence that a particular molecular architecture, polymeric branching, leads to a rare form of self-assembly, the planar nanodisc. Cholesterol containing self-assemblies formed from amphiphilic linear or branched cetyl poly(ethylenimine) (Mn approximately 1000 Da) or amphiphilic cetyl poly(propylenimine) dendrimer derivatives (Mn approximately 2000 Da) show that branching, by reducing the hydrophilic headgroup area, alters the shape of the self-assemblies transforming closed 60 nm spherical bilayer vesicles to rare 50 nm x 10 nm planar bilayer discs. Increasing the hydrophilic headgroup area, by the inclusion of methoxy poly(ethylene glycol) moieties into the amphiphilic headgroup, transforms the planar discs to 100 nm spherical bilayer vesicles. This study provides insight into the key role played by molecular shape on molecular self-organization into rare nanodiscs.

A Computational Approach for Modeling Neutron Scattering Data from Lipid Bilayers

DOE PAGES

Carrillo, Jan-Michael Y.; Katsaras, John; Sumpter, Bobby G.; ...

2017-01-12

Biological cell membranes are responsible for a range of structural and dynamical phenomena crucial to a cell's well-being and its associated functions. Due to the complexity of cell membranes, lipid bilayer systems are often used as biomimetic models. These systems have led to signficant insights into vital membrane phenomena such as domain formation, passive permeation and protein insertion. Experimental observations of membrane structure and dynamics are, however, limited in resolution, both spatially and temporally. Importantly, computer simulations are starting to play a more prominent role in interpreting experimental results, enabling a molecular under- standing of lipid membranes. Particularly, the synergymore » between scattering experiments and simulations offers opportunities for new discoveries in membrane physics, as the length and time scales probed by molecular dynamics (MD) simulations parallel those of experiments. We also describe a coarse-grained MD simulation approach that mimics neutron scattering data from large unilamellar lipid vesicles over a range of bilayer rigidity. Specfically, we simulate vesicle form factors and membrane thickness fluctuations determined from small angle neutron scattering (SANS) and neutron spin echo (NSE) experiments, respectively. Our simulations accurately reproduce trends from experiments and lay the groundwork for investigations of more complex membrane systems.« less

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.

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.

Controlled membrane translocation provides a mechanism for signal transduction and amplification

NASA Astrophysics Data System (ADS)

Langton, Matthew J.; Keymeulen, Flore; Ciaccia, Maria; Williams, Nicholas H.; Hunter, Christopher A.

2017-05-01

Transmission and amplification of chemical signals across lipid bilayer membranes is of profound significance in many biological processes, from the development of multicellular organisms to information processing in the nervous system. In biology, membrane-spanning proteins are responsible for the transmission of chemical signals across membranes, and signal transduction is often associated with an amplified signalling cascade. The ability to reproduce such processes in artificial systems has potential applications in sensing, controlled drug delivery and communication between compartments in tissue-like constructs of synthetic vesicles. Here we describe a mechanism for transmitting a chemical signal across a membrane based on the controlled translocation of a synthetic molecular transducer from one side of a lipid bilayer membrane to the other. The controlled molecular motion has been coupled to the activation of a catalyst on the inside of a vesicle, which leads to a signal-amplification process analogous to the biological counterpart.

Diffusion behavior of lipid vesicles in entangled polymer solutions.

PubMed Central

Cao, X; Bansil, R; Gantz, D; Moore, E W; Niu, N; Afdhal, N H

1997-01-01

Dynamic light scattering was used to follow the tracer diffusion of phospholipid/cholesterol vesicles in aqueous polyacrylamide solutions and compared with the diffusive behavior of polystyrene (PS) latex spheres of comparable diameters. Over the range of the matrix concentration examined (Cp = 0.1-10 mg/ml), the diffusivities of the PS spheres and the large multilamellar vesicles exhibited the Stokes-Einstein (SE) relation, while the diffusivity of the unilamellar vesicles did not follow the increase of the solution's viscosity caused by the presence of the matrix molecules. The difference between the diffusion behaviors of unilamellar vesicles and hard PS spheres of similar size is possibly due to the flexibility of the lipid bilayer of the vesicles. The unilamellar vesicles are capable of changing their shape to move through the entangled polymer solution so that the hindrance to their diffusion due to the presence of the polymer chains is reduced, while the rigid PS spheres have little flexibility and they encounter greater resistance. The multilamellar vesicles are less flexible, thus their diffusion is similar to the hard PS spheres of similar diameter. Images FIGURE 2 PMID:9336189

Release of the glycosylphosphatidylinositol-anchored enzyme ecto-5'-nucleotidase by phospholipase C: catalytic activation and modulation by the lipid bilayer.

PubMed Central

Lehto, M T; Sharom, F J

1998-01-01

Many hydrolytic enzymes are attached to the extracellular face of the plasma membrane of eukaryotic cells by a glycosylphosphatidylinositol (GPI) anchor. Little is currently known about the consequences for enzyme function of anchor cleavage by phosphatidylinositol-specific phospholipase C. We have examined this question for the GPI-anchored protein 5'-nucleotidase (5'-ribonucleotide phosphohydrolase; EC 3.1.3.5), both in the native lymphocyte plasma membrane, and following purification and reconstitution into defined lipid bilayer vesicles, using Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC). Membrane-bound, detergent-solubilized and cleaved 5'-nucleotidase all obeyed Michaelis-Menten kinetics, with a Km for 5'-AMP in the range 11-16 microM. The GPI anchor was removed from essentially all 5'-nucleotidase molecules, indicating that there is no phospholipase-resistant pool of enzyme. However, the phospholipase was much less efficient at cleaving the GPI anchor when 5'-nucleotidase was present in detergent solution, dimyristoyl phosphatidylcholine, egg phosphatidylethanolamine and sphingomyelin, compared with the native plasma membrane, egg phosphatidylcholine and a sphingolipid/cholesterol-rich mixture. Lipid molecular properties and bilayer packing may affect the ability of PI-PLC to gain access to the GPI anchor. Catalytic activation, characterized by an increase in Vmax, was observed following PI-PLC cleavage of reconstituted 5'-nucleotidase from vesicles of several different lipids. The highest degree of activation was noted for 5'-nucleotidase in egg phosphatidylethanolamine. An increase in Vmax was also noted for a sphingolipid/cholesterol-rich mixture, the native plasma membrane and egg phosphatidylcholine, whereas vesicles of sphingomyelin and dimyristoyl phosphatidylcholine showed little activation. Km generally remained unchanged following cleavage, except in the case of the sphingolipid/cholesterol-rich mixture. Insertion of the GPI anchor into a lipid bilayer appears to reduce the catalytic efficiency of 5'-nucleotidase, possibly via a conformational change in the enzyme, and activity is restored on release from the membrane. PMID:9576857

Colloidosomes formed by nonpolar/polar/nonpolar nanoball amphiphiles

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

Chang, Hung-Yu; Sheng, Yu-Jane, E-mail: yjsheng@ntu.edu.tw, E-mail: hktsao@cc.ncu.edu.tw; Tu, Sheng-Hung

2014-08-07

Fullerene-based amphiphiles are able to form bilayer vesicles in aqueous solution. In this study, the self-assembly behavior of polymer-tethered nanoballs (NBs) with nonpolar/polar/nonpolar (n-p-n{sup ′}) motif in a selective solvent is investigated by dissipative particle dynamics. A model NB bears two hydrophobic polymeric arms (n{sup ′}-part) tethered on an extremely hydrophobic NB (n-part) with hydrophilic patch (p-part) patterned on its surface. Dependent on the hydrophobicity and length of tethered arms, three types of aggregates are exhibited, including NB vesicle, core-shell micelle, and segmented-worm. NB vesicles are developed for a wide range of hydrophobic arm lengths. The presence of tethered armsmore » perturbs the bilayer structure formed by NBs. The structural properties including the order parameter, membrane thickness, and area density of the inner leaflet decrease with increasing the arm length. These results indicate that for NBs with longer arms, the extent of interdigitation in the membrane rises so that the overcrowded arms in the inner corona are relaxed. The transport and mechanical properties are evaluated as well. As the arm length grows, the permeability increases significantly because the steric bulk of tethered arms loosens the packing of NBs. By contrast, the membrane tension decreases owing to the reduction of NB/solvent contacts by the polymer corona. Although fusion can reduce membrane tension, NB vesicles show strong resistance to fusion. Moreover, the size-dependent behavior observed in small liposomes is not significant for NB vesicles due to isotropic geometry of NB. Our simulation results are consistent with the experimental findings.« less

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.

A systematic investigation and insight into the formation mechanism of bilayers of fatty acid/soap mixtures in aqueous solutions.

PubMed

Xu, Wenlong; Song, Aixin; Dong, Shuli; Chen, Jingfei; Hao, Jingcheng

2013-10-08

Vesicles are the most common form of bilayer structures in fatty acid/soap mixtures in aqueous solutions; however, a peculiar bilayer structure called a "planar sheet" was found for the first time in the mixtures. In the past few decades, considerable research has focused on the formation theory of bilayers in fatty acid/soap mixtures. The hydrogen bond theory has been widely accepted by scientists to explain the formation of bilayers. However, except for the hydrogen bond, no other driving forces were proposed systematically. In this work, three kinds of weak interactions were investigated in detail, which could perfectly demonstrate the formation mechanism of bilayer structures in the fatty acid/soap mixtures in aqueous solutions. (i) The influence of hydrophobic interaction was detected by changing the chain length of fatty acid (C(n)H(2n+1)COOH), in which n = 10 to 18, the phase behavior was investigated, and the phase region was presented. With the help of cryogenic transmission electron microscopy (cryo-TEM) observations, deuterium nuclear magnetic resonance ((2)H NMR), and X-ray diffraction (XRD) measurements, the vesicles and planar sheets were determined. The chain length of C(n)H(2n+1)COOH has an important effect on the physical state of the hydrophobic chain, resulting in an obvious difference in the viscoelasticity of the solution samples. (ii) The existence of hydrogen bonds between fatty acids and their soaps in aqueous solutions was demonstrated by Fourier transform infrared (FT-IR) spectroscopy and molecule dynamical simulation. From the pH measurements, the pH ranges of the bilayer formation were at the pKa values of fatty acids, respectively. (iii) Counterions can be embedded in the stern layer of the bilayers and screen the electrostatic repulsion between the COO(-) anionic headgroups. FT-IR characterization demonstrated a bidentate bridging coordination mode between counterions and carboxylates. The conductivity measurements provided the degree of counterion binding (β = 0.854), indicating the importance of the counterions.

Study of the Interaction of the HIV-1 Fusion Peptide with Lipid Bilayer Membranes

NASA Astrophysics Data System (ADS)

Heller, William; Rai, Durgesh

HIV-1 undergoes fusion with the cell membrane through interactions between its coat proteins and the target cell. Visualization of fusion with sufficient detail to determine the molecular mechanism remains elusive. Here, the interaction between a synthetic variant of the HIV-1 gp41 fusion peptide with vesicles composed of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylserine (DMPS) was studied. The peptide was observed to undergo a concentration-dependent conformational transition between an α-helix and an antiparallel β-sheet that is accompanied by a transition in the structure of the lipid bilayer vesicle. The peptide changes the distribution of lipids between the vesicle leaflets. Further, it creates two regions having different thicknesses. The results shed new light on how the peptide modifies the membrane structure to favor fusion. A portion of this research was sponsored by the Laboratory Directed Research and Development Program of Oak Ridge National Laboratory, managed by UT-Battelle, LLC, for the U. S. Department of Energy. Research at Oak Ridge National Laboratory's Spallation Neutron Source was sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, U. S. Department of Energy.

Dynamic nuclear polarization enhanced nuclear magnetic resonance and electron spin resonance studies of hydration and local water dynamics in micelle and vesicle assemblies.

PubMed

McCarney, Evan R; Armstrong, Brandon D; Kausik, Ravinath; Han, Songi

2008-09-16

We present a unique analysis tool for the selective detection of local water inside soft molecular assemblies (hydrophobic cores, vesicular bilayers, and micellar structures) suspended in bulk water. Through the use of dynamic nuclear polarization (DNP), the (1)H NMR signal of water is amplified, as it interacts with stable radicals that possess approximately 658 times higher spin polarization. We utilized stable nitroxide radicals covalently attached along the hydrophobic tail of stearic acid molecules that incorporate themselves into surfactant-based micelle or vesicle structures. Here, we present a study of local water content and fluid viscosity inside oleate micelles and vesicles and Triton X-100 micelles to serve as model systems for soft molecular assemblies. This approach is unique because the amplification of the NMR signal is performed in bulk solution and under ambient conditions with site-specific spin labels that only detect the water that is directly interacting with the localized spin labels. Continuous wave (cw) electron spin resonance (ESR) analysis provides rotational dynamics of the spin-labeled molecular chain segments and local polarity parameters that can be related to hydration properties, whereas we show that DNP-enhanced (1)H NMR analysis of fluid samples directly provides translational water dynamics and permeability of the local environment probed by the spin label. Our technique therefore has the potential to become a powerful analysis tool, complementary to cw ESR, to study hydration characteristics of surfactant assemblies, lipid bilayers, or protein aggregates, where water dynamics is a key parameter of their structure and function. In this study, we find that there is significant penetration of water inside the oleate micelles with a higher average local water viscosity (approximately 1.8 cP) than in bulk water, and Triton X-100 micelles and oleate vesicle bilayers mostly exclude water while allowing for considerable surfactant chain motion and measurable water permeation through the soft structure.

Study of supported phospholipid bilayers by THz-TDS

NASA Astrophysics Data System (ADS)

Ionescu, Alina; Mernea, Maria; Vasile, Ionut; Brandus, Catalina Alice; Barbinta-Patrascu, Marcela Elisabeta; Tugulea, Laura; Mihailescu, Dan; Dascalu, Traian

2012-10-01

Terahertz Time-Domain Spectroscopy (THz-TDS) is a new technique in studying the conformational state of molecules. Cell membranes are important structures in the interaction with extra cellular entities. Their principal building blocks are lipids, amphiphilic molecules that spontaneously self-assemble when in contact with water. In this work we report the use of THz-TDS in transmission mode to examine the behavior of supported phospholipid bilayers (SPBs) within the frequency range of 0.2 THz to 3 THz. SPBs were obtained by vesicle adsorption method involving the spread of a suspension (50-100 μl) of small unilamellar vesicles (SUVs) or multilamellar vesicles (MLVs) dissolved in PBS (phosphate buffer solution) on a support of silicon wafers. Both SUVs and MLVs were obtained from dipalmitoyl phosphatidylcholine (DPPC) and lecithin by using the thin-film hydration method. Broadband THz pulses are generated and detected using photoconductive antennas optically excited by a femtosecond laser pulse emitted from a self-mode locked fiber laser at a wavelength of 780 nm with a pulse widths of 150 fs. THz-TDS was proven to be a useful method in studying SPBs and their hydration states. The absorption coefficient and refractive index of the samples were calculated from THz measurements data. The THz absorption spectra for different lipids in SPBs indicate specific absorption frequency lines. A difference in the magnitude of the refractive index was also observed due to the different structure of supported lipid bilayers. The THz spectrum of DPPC was obtained by using theoretical simulations and then the experimental and theoretical THz spectra were compared.

Statistical Analysis of Bending Rigidity Coefficient Determined Using Fluorescence-Based Flicker-Noise Spectroscopy.

PubMed

Doskocz, Joanna; Drabik, Dominik; Chodaczek, Grzegorz; Przybyło, Magdalena; Langner, Marek

2018-06-01

Bending rigidity coefficient describes propensity of a lipid bilayer to deform. In order to measure the parameter experimentally using flickering noise spectroscopy, the microscopic imaging is required, which necessitates the application of giant unilamellar vesicles (GUV) lipid bilayer model. The major difficulty associated with the application of the model is the statistical character of GUV population with respect to their size and the homogeneity of lipid bilayer composition, if a mixture of lipids is used. In the paper, the bending rigidity coefficient was measured using the fluorescence-enhanced flicker-noise spectroscopy. In the paper, the bending rigidity coefficient was determined for large populations of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphocholine vesicles. The quantity of obtained experimental data allows to perform statistical analysis aiming at the identification of the distribution, which is the most appropriate for the calculation of the value of the membrane bending rigidity coefficient. It has been demonstrated that the bending rigidity coefficient is characterized by an asymmetrical distribution, which is well approximated with the gamma distribution. Since there are no biophysical reasons for that we propose to use the difference between normal and gamma fits as a measure of the homogeneity of vesicle population. In addition, the effect of a fluorescent label and types of instrumental setups on determined values has been tested. Obtained results show that the value of the bending rigidity coefficient does not depend on the type of a fluorescent label nor on the type of microscope used.

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.

Dynamics of vesicles in electric fields

NASA Astrophysics Data System (ADS)

Vlahovska, Petia; Gracia, Ruben

2007-11-01

Electromechanical forces are widely used for cell manipulation. Knowledge of the physical mechanisms underlying the interaction of cells and external fields is essential for practical applications. Vesicles are model cells made of a lipid bilayer membrane. They are examples of ``soft'' particles, i.e., their shape when subjected to flow or electric field is not given a priori but it is governed by the balance of membrane, fluid and electrical stresses. This generic ``softness'' gives rise to a very complex vesicle dynamics in external fields. In an AC electric field, as the frequency is increased, vesicles filled with a fluid less conducting than the surrounding fluid undergo shape transition from prolate to oblate ellipsoids. The opposite effect is observed with drops. We present an electro- hydrodynamic theory based on the leaky dielectric model that quantitatively describes experimental observations. We compare drops and vesicles, and show how their distinct behavior stems from different interfacial properties.

Green Fluorescent Protein Changes the Conductance of Connexin 43 (Cx43) Hemichannels Reconstituted in Planar Lipid Bilayers*

PubMed Central

Carnarius, Christian; Kreir, Mohamed; Krick, Marcel; Methfessel, Christoph; Moehrle, Volker; Valerius, Oliver; Brüggemann, Andrea; Steinem, Claudia; Fertig, Niels

2012-01-01

In mammalian tissues, connexin 43 (Cx43) is the most prominent member of the connexin family. In a single lipid bilayer, six connexin subunits assemble into a hemichannel (connexon). Direct communication of apposing cells is realized by two adjacent hemichannels, which can form gap junction channels. Here, we established an expression system in Pichia pastoris to recombinantly produce and purify Cx43 as well as Cx43 fused to green fluorescent protein (GFP). Proteins were isolated from crude cell membrane fractions via affinity chromatography. Cx43 and Cx43-GFP hemichannels were reconstituted in giant unilamellar vesicles as proven by fluorescence microscopy, and their electrophysiological behavior was analyzed on the single channel level by planar patch clamping. Cx43 and Cx43-GFP both showed an ohmic behavior and a voltage-dependent open probability. Cx43 hemichannels exhibited one major mean conductance of 224 ± 26 picosiemens (pS). In addition, a subconductance state at 124 ± 5 pS was identified. In contrast, the analysis of Cx43-GFP single channels revealed 10 distinct conductance states in the range of 15 to 250 pS, with a larger open probability at 0 mV as compared with Cx43, which suggests that intermolecular interactions between the GFP molecules alter the electrophysiology of the protein. PMID:22139870

Green fluorescent protein changes the conductance of connexin 43 (Cx43) hemichannels reconstituted in planar lipid bilayers.

PubMed

Carnarius, Christian; Kreir, Mohamed; Krick, Marcel; Methfessel, Christoph; Moehrle, Volker; Valerius, Oliver; Brüggemann, Andrea; Steinem, Claudia; Fertig, Niels

2012-01-20

In mammalian tissues, connexin 43 (Cx43) is the most prominent member of the connexin family. In a single lipid bilayer, six connexin subunits assemble into a hemichannel (connexon). Direct communication of apposing cells is realized by two adjacent hemichannels, which can form gap junction channels. Here, we established an expression system in Pichia pastoris to recombinantly produce and purify Cx43 as well as Cx43 fused to green fluorescent protein (GFP). Proteins were isolated from crude cell membrane fractions via affinity chromatography. Cx43 and Cx43-GFP hemichannels were reconstituted in giant unilamellar vesicles as proven by fluorescence microscopy, and their electrophysiological behavior was analyzed on the single channel level by planar patch clamping. Cx43 and Cx43-GFP both showed an ohmic behavior and a voltage-dependent open probability. Cx43 hemichannels exhibited one major mean conductance of 224 ± 26 picosiemens (pS). In addition, a subconductance state at 124 ± 5 pS was identified. In contrast, the analysis of Cx43-GFP single channels revealed 10 distinct conductance states in the range of 15 to 250 pS, with a larger open probability at 0 mV as compared with Cx43, which suggests that intermolecular interactions between the GFP molecules alter the electrophysiology of the protein.

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.

Ca-Mediated Electroformation of Cell-Sized Lipid Vesicles

PubMed Central

Tao, Fei; Yang, Peng

2015-01-01

Cell-sized lipid giant unilamellar vesicles (GUVs) are formed when lipid molecules self-assemble to construct a single bilayer compartment with similar morphology to living cells. The physics of self-assembly process is only generally understood and the size distribution of GUVs tends to be very polydisperse. Herein we report a strategy for the production of controlled size distributions of GUVs by a novel mechanism dissecting the mediation ability of calcium (Ca) on the conventional electroformation of GUVs. We finely construct both of the calcium ion (Ca2+) and calcium carbonate (CaCO3) mineral adsorption layers on a lipid film surface respectively during the electroformation of GUVs. It is found that Ca2+ Slip plane polarized by alternating electric field could induce a pattern of electroosmotic flow across the surface, and thus confine the fusion and growth of GUVs to facilitate the formation of uniform GUVs. The model is further improved by directly using CaCO3 that is in situ formed on a lipid film surface, providing a GUV population with narrow polydispersity. The two models deciphers the new biological function of calcium on the birth of cell-like lipid vesicles, and thus might be potentially relevant to the construction of new model to elucidate the cellular development process. PMID:25950604

Mineral Surface Chemistry and Nanoparticle-aggregation Control Membrane Self-Assembly

NASA Astrophysics Data System (ADS)

Sahai, Nita; Kaddour, Hussein; Dalai, Punam; Wang, Ziqiu; Bass, Garrett; Gao, Min

2017-03-01

The self-assembly of lipid bilayer membranes to enclose functional biomolecules, thus defining a “protocell,” was a seminal moment in the emergence of life on Earth and likely occurred at the micro-environment of the mineral-water interface. Mineral-lipid interactions are also relevant in biomedical, industrial and technological processes. Yet, no structure-activity relationships (SARs) have been identified to predict lipid self-assembly at mineral surfaces. Here we examined the influence of minerals on the self-assembly and survival of vesicles composed of single chain amphiphiles as model protocell membranes. The apparent critical vesicle concentration (CVC) increased in the presence of positively-charged nanoparticulate minerals at high loadings (mg/mL) suggesting unfavorable membrane self-assembly in such situations. Above the CVC, initial vesicle formation rates were faster in the presence of minerals. Rates were correlated with the mineral’s isoelectric point (IEP) and reactive surface area. The IEP depends on the crystal structure, chemical composition and surface hydration. Thus, membrane self-assembly showed rational dependence on fundamental mineral properties. Once formed, membrane permeability (integrity) was unaffected by minerals. Suggesting that, protocells could have survived on rock surfaces. These SARs may help predict the formation and survival of protocell membranes on early Earth and other rocky planets, and amphiphile-mineral interactions in diverse other phenomena.

Formation of protocell-like vesicles in a thermal diffusion column.

PubMed

Budin, Itay; Bruckner, Raphael J; Szostak, Jack W

2009-07-22

Many of the properties of bilayer membranes composed of simple single-chain amphiphiles seem to be well-suited for a potential role as primitive cell membranes. However, the spontaneous formation of membranes from such amphiphiles is a concentration-dependent process in which a significant critical aggregate concentration (cac) must be reached. Since most scenarios for the prebiotic synthesis of fatty acids and related amphiphiles would result in dilute solutions well below the cac, the identification of mechanisms that would lead to increased local amphiphile concentrations is an important aspect of defining reasonable conditions for the origin of cellular life. Narrow, vertically oriented channels within the mineral precipitates of hydrothermal vent towers have previously been proposed to act as natural Clusius-Dickel thermal diffusion columns, in which a strong transverse thermal gradient concentrates dilute molecules through the coupling of thermophoresis and convection. Here we experimentally demonstrate that a microcapillary acting as a thermal diffusion column can concentrate a solution of oleic acid. Upon concentration, self-assembly of large vesicles occurs in regions where the cac is exceeded. We detected vesicle formation by fluorescence microscopy of encapsulated dye cargoes, which simultaneously concentrated in our channels. Our findings suggest a novel means by which simple physical processes could have led to the spontaneous formation of cell-like structures from a dilute prebiotic reservoir.

Ca-mediated electroformation of cell-sized lipid vesicles.

PubMed

Tao, Fei; Yang, Peng

2015-05-07

Cell-sized lipid giant unilamellar vesicles (GUVs) are formed when lipid molecules self-assemble to construct a single bilayer compartment with similar morphology to living cells. The physics of self-assembly process is only generally understood and the size distribution of GUVs tends to be very polydisperse. Herein we report a strategy for the production of controlled size distributions of GUVs by a novel mechanism dissecting the mediation ability of calcium (Ca) on the conventional electroformation of GUVs. We finely construct both of the calcium ion (Ca(2+)) and calcium carbonate (CaCO3) mineral adsorption layers on a lipid film surface respectively during the electroformation of GUVs. It is found that Ca(2+) Slip plane polarized by alternating electric field could induce a pattern of electroosmotic flow across the surface, and thus confine the fusion and growth of GUVs to facilitate the formation of uniform GUVs. The model is further improved by directly using CaCO3 that is in situ formed on a lipid film surface, providing a GUV population with narrow polydispersity. The two models deciphers the new biological function of calcium on the birth of cell-like lipid vesicles, and thus might be potentially relevant to the construction of new model to elucidate the cellular development process.

Mineral Surface Chemistry and Nanoparticle-aggregation Control Membrane Self-Assembly

PubMed Central

Sahai, Nita; Kaddour, Hussein; Dalai, Punam; Wang, Ziqiu; Bass, Garrett; Gao, Min

2017-01-01

The self-assembly of lipid bilayer membranes to enclose functional biomolecules, thus defining a “protocell,” was a seminal moment in the emergence of life on Earth and likely occurred at the micro-environment of the mineral-water interface. Mineral-lipid interactions are also relevant in biomedical, industrial and technological processes. Yet, no structure-activity relationships (SARs) have been identified to predict lipid self-assembly at mineral surfaces. Here we examined the influence of minerals on the self-assembly and survival of vesicles composed of single chain amphiphiles as model protocell membranes. The apparent critical vesicle concentration (CVC) increased in the presence of positively-charged nanoparticulate minerals at high loadings (mg/mL) suggesting unfavorable membrane self-assembly in such situations. Above the CVC, initial vesicle formation rates were faster in the presence of minerals. Rates were correlated with the mineral’s isoelectric point (IEP) and reactive surface area. The IEP depends on the crystal structure, chemical composition and surface hydration. Thus, membrane self-assembly showed rational dependence on fundamental mineral properties. Once formed, membrane permeability (integrity) was unaffected by minerals. Suggesting that, protocells could have survived on rock surfaces. These SARs may help predict the formation and survival of protocell membranes on early Earth and other rocky planets, and amphiphile-mineral interactions in diverse other phenomena. PMID:28266537

Current trends in the use of liposomes for tumor targeting

PubMed Central

Deshpande, Pranali P; Biswas, Swati; Torchilin, Vladimir P

2013-01-01

The use of liposomes for drug delivery began early in the history of pharmaceutical nanocarriers. These nanosized, lipid bilayered vesicles have become popular as drug delivery systems owing to their efficiency, biocompatibility, nonimmunogenicity, enhanced solubility of chemotherapeutic agents and their ability to encapsulate a wide array of drugs. Passive and ligand-mediated active targeting promote tumor specificity with diminished adverse off-target effects. The current field of liposomes focuses on both clinical and diagnostic applications. Recent efforts have concentrated on the development of multifunctional liposomes that target cells and cellular organelles with a single delivery system. This review discusses the recent advances in liposome research in tumor targeting. PMID:23914966

Molecular transport through large-diameter DNA nanopores

NASA Astrophysics Data System (ADS)

Krishnan, Swati; Ziegler, Daniela; Arnaut, Vera; Martin, Thomas G.; Kapsner, Korbinian; Henneberg, Katharina; Bausch, Andreas R.; Dietz, Hendrik; Simmel, Friedrich C.

2016-09-01

DNA-based nanopores are synthetic biomolecular membrane pores, whose geometry and chemical functionality can be tuned using the tools of DNA nanotechnology, making them promising molecular devices for applications in single-molecule biosensing and synthetic biology. Here we introduce a large DNA membrane channel with an ~4 nm diameter pore, which has stable electrical properties and spontaneously inserts into flat lipid bilayer membranes. Membrane incorporation is facilitated by a large number of hydrophobic functionalizations or, alternatively, streptavidin linkages between biotinylated channels and lipids. The channel displays an Ohmic conductance of ~3 nS, consistent with its size, and allows electrically driven translocation of single-stranded and double-stranded DNA analytes. Using confocal microscopy and a dye influx assay, we demonstrate the spontaneous formation of membrane pores in giant unilamellar vesicles. Pores can be created both in an outside-in and an inside-out configuration.

Gating Behavior of Endoplasmic Reticulum Potassium Channels of Rat Hepatocytes in Diabetes

PubMed Central

Ghasemi, Maedeh; Khodaei, Naser; Salari, Sajjad; Eliassi, Afsaneh; Saghiri, Reza

2014-01-01

Background: Defects in endoplasmic reticulum homeostasis are common occurrences in different diseases, such as diabetes, in which the function of endoplasmic reticulum is disrupted. It is now well established that ion channels of endoplasmic reticulum membrane have a critical role in endoplasmic reticulum luminal homeostasis. Our previous studies showed the presence of an ATP-sensitive cationic channel in endoplasmic reticulum. Therefore, in this study, we examined and compared the activities of this channel in control and diabetic rats using single-channel recording techniques. Method: Male Wistar rats were made diabetic for 2 weeks with a single dose injection of streptozotocin (45 mg/kg). Ion channel incorporation of rough endoplasmic reticulum of diabetic hepatocytes into the bilayer lipid membrane allowed the characterization of K+ channel. Results: Ion channel incorporation of rough endoplasmic reticulum vesicles into the bilayer lipid revealed that the channel current-voltage (I-V) relation with a mean slope conductance of 520 ± 19 pS was unaffected in diabetes. Interestingly, the channel Po-voltage relation was significantly lower in diabetic rats at voltages above +30 mV. Conclusion: We concluded that the endoplasmic reticulum cationic channel is involved in diabetes. Also, this finding could be considered as a goal for further therapeutic plans. PMID:24842143

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.

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.

Poly(styrene-co-maleic acid)-based pH-sensitive liposomes mediate cytosolic delivery of drugs for enhanced cancer chemotherapy.

PubMed

Banerjee, Shubhadeep; Sen, Kacoli; Pal, Tapan K; Guha, Sujoy K

2012-10-15

pH-responsive polymers render liposomes pH-sensitive and facilitate the intracellular release of encapsulated payload by fusing with endovascular membranes under mildly acidic conditions found inside cellular endosomes. The present study reports the use of high-molecular weight poly(styrene-co-maleic acid) (SMA), which exhibits conformational transition from a charged extended structure to an uncharged globule below its pK(1) value, to confer pH-sensitive property to liposomes. The changes in the co-polymer chain conformation resulted in destabilization of the liposomes at mildly acidic pH due to vesicle fusion and/or channel formation within the membrane bilayer, and ultimately led to the release of the encapsulated cargo. The vesicles preserved their pH-sensitivity and stability in serum unlike other polymer-based liposomes and exhibited no hemolytic activity at physiological pH. The lysis of RBCs at endosomal pH due to SMA-based liposome-induced alterations in the bilayer organization leading to spherocyte formation indicated the potential of these vesicles to mediate cytosolic delivery of bio-active molecules through endosome destabilization. The SMA-loaded liposomes exhibiting excellent cytocompatibility, efficiently delivered chemotherapeutic agent 5-Fluorouracil (5-FU) within colon cancer cells HT-29 in comparison to neat liposomes. This caused increased cellular-availability of the drug, which resulted in enhanced apoptosis and highlighted the clinical potential of SMA-based vesicles. Copyright © 2012 Elsevier B.V. All rights reserved.

New pH-sensitive liposomes containing phosphatidylethanolamine and a bacterial dirhamnolipid.

PubMed

Sánchez, Marina; Aranda, Francisco J; Teruel, José A; Ortiz, Antonio

2011-01-01

Phosphatidylethanolamine-based pH-sensitive liposomes of various compositions have been described as efficient systems for cytoplasmic delivery of molecules into cells. Incorporation of an amphiphile of appropriate structure is needed for the stabilization and performance of these vesicles. Among the wide variety of interesting activities displayed by Pseudomonas aeruginosa dirhamnolipids (diRL), is their capacity to stabilize bilayer structures in phosphatidylethanolamine systems. In this work, X-ray scattering, dynamic light scattering, fluorescence spectroscopy and fluorescence microscopy have been used to study the structure and pH-dependent behaviour of phosphatidylethanolamine/diRL liposomes. We show that diRL, in combination with dioleoylphosphatidylethanolamine (DOPE), forms stable multilamellar and unilamellar liposomes. Acidification of DOPE/diRL vesicles leads to membrane destabilization, fusion, and release of entrapped aqueous vesicle contents. Finally, DOPE/diRL pH-sensitive liposomes act as efficient vehicles for the cytoplasmic delivery of fluorescent probes into cultured cells. It is concluded that DOPE/diRL form stable pH-sensitive liposomes, and that these liposomes are incorporated into cultured cells through the endocytic pathway, delivering its contents into the cytoplasm, which means a potential use of these liposomes for the delivery of foreign substances into living cells. Our results establish a new application of diRL as a bilayer stabilizer in phospholipid vesicles, and the use of diRL-containing pH-sensitive liposomes as delivery vehicles. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

The motion of a train of vesicles in channel flow

NASA Astrophysics Data System (ADS)

Barakat, Joseph; Shaqfeh, Eric

2017-11-01

The inertialess motion of a train of lipid-bilayer vesicles flowing through a channel is simulated using a 3D boundary integral equation method. Steady-state results are reported for vesicles positioned concentrically inside cylindrical channels of circular, square, and rectangular cross sections. The vesicle translational velocity U and excess channel pressure drop Δp+ depend strongly on the ratio of the vesicle radius to the hydraulic radius λ and the vesicle reduced volume υ. ``Deflated vesicles'' of lower reduced volume υ are more streamlined and translate with greater velocity U relative to the mean flow velocity V. Increasing the vesicle size (λ) increases the wall friction force and extra pressure drop Δp+, which in turn reduces the vesicle velocity U. Hydrodynamic interactions between vesicles in a periodic train are largely screened by the channel walls, in accordance with previous results for spheres and drops. The hydraulic resistance is compared across different cross sections, and a simple correction factor is proposed to unify the results. Nonlinear effects are observed when β - the ratio of membrane bending elasticity to viscous traction - is changed. The simulation results show excellent agreement with available experimental measurements as well as a previously reported ``small-gap theory'' valid for large values of λ. NSF CBET 1066263/1066334.

How To Characterize Individual Nanosize Liposomes with Simple Self-Calibrating Fluorescence Microscopy.

PubMed

Mortensen, Kim I; Tassone, Chiara; Ehrlich, Nicky; Andresen, Thomas L; Flyvbjerg, Henrik

2018-05-09

Nanosize lipid vesicles are used extensively at the interface between nanotechnology and biology, e.g., as containers for chemical reactions at minute concentrations and vehicles for targeted delivery of pharmaceuticals. Typically, vesicle samples are heterogeneous as regards vesicle size and structural properties. Consequently, vesicles must be characterized individually to ensure correct interpretation of experimental results. Here we do that using dual-color fluorescence labeling of vesicles-of their lipid bilayers and lumens, separately. A vesicle then images as two spots, one in each color channel. A simple image analysis determines the total intensity and width of each spot. These four data all depend on the vesicle radius in a simple manner for vesicles that are spherical, unilamellar, and optimal encapsulators of molecular cargo. This permits identification of such ideal vesicles. They in turn enable calibration of the dual-color fluorescence microscopy images they appear in. Since this calibration is not a separate experiment but an analysis of images of vesicles to be characterized, it eliminates the potential source of error that a separate calibration experiment would have been. Nonideal vesicles in the same images were characterized by how their four data violate the calibrated relationship established for ideal vesicles. In this way, our method yields size, shape, lamellarity, and encapsulation efficiency of each imaged vesicle. Applying this procedure to extruded samples of vesicles, we found that, contrary to common assumptions, only a fraction of vesicles are ideal.

Single-molecule fluorescence study of the inhibition of the oncogenic functionality of STAT3

NASA Astrophysics Data System (ADS)

Liu, Baoxu; Badali, Daniel; Fletcher, Steven; Avadisian, Miriam; Gunning, Patrick; Gradinaru, Claudiu

2009-06-01

Signal-Transducer-and-Activator-of-Transcription 3 (STAT3) protein plays an important role in the onset of cancers such as leukemia and lymphoma. In this study, we aim to test the effectiveness of a novel peptide drug designed to tether STAT3 to the phospholipid bilayer of the cell membrane and thus inhibit unwanted transcription. As a first step, STAT3 proteins were successfully labelled with tetramethylrhodamine (TMR), a fluorescent dye with suitable photostability for single molecule studies. The effectiveness of labelling was determined using fluorescence correlation spectroscopy in a custom built confocal microscope, from which diffusion times and hydrodynamic radii of individual proteins were determined. A newly developed fluorescein derivative label (F-NAc) has been designed to be incorporated into the structure of the peptide drug so that peptide-STAT3 interactions can be examined. This dye is spectrally characterized and is found to be well suited for its application to this project, as well as other single-molecule studies. The membrane localization via high-affinity cholesterol-bound small-molecule binding agents can be demonstrated by encapsulating TMR-labeled STAT3 and inhibitors within a vesicle model cell system. To this end, unilaminar lipid vesicles were examined for size and encapsulation ability. Preliminary results of the efficiency and stability of the STAT3 anchoring in lipid membranes obtained via quantitative confocal imaging and single-molecule spectroscopy using a custom-built multiparameter fluorescence microscope are reported here.

Study of vesicle size distribution dependence on pH value based on nanopore resistive pulse method

NASA Astrophysics Data System (ADS)

Lin, Yuqing; Rudzevich, Yauheni; Wearne, Adam; Lumpkin, Daniel; Morales, Joselyn; Nemec, Kathleen; Tatulian, Suren; Lupan, Oleg; Chow, Lee

2013-03-01

Vesicles are low-micron to sub-micron spheres formed by a lipid bilayer shell and serve as potential vehicles for drug delivery. The size of vesicle is proposed to be one of the instrumental variables affecting delivery efficiency since the size is correlated to factors like circulation and residence time in blood, the rate for cell endocytosis, and efficiency in cell targeting. In this work, we demonstrate accessible and reliable detection and size distribution measurement employing a glass nanopore device based on the resistive pulse method. This novel method enables us to investigate the size distribution dependence of pH difference across the membrane of vesicles with very small sample volume and rapid speed. This provides useful information for optimizing the efficiency of drug delivery in a pH sensitive environment.

Translational Diffusion Coefficient and Partition Coefficient of a Spin-Labeled Solute in Lecithin Bilayer Membranes

PubMed Central

Dix, James A.; Diamond, Jared M.; Kivelson, Daniel

1974-01-01

The translational diffusion coefficient and the partition coefficient of a spin-labeled solute, di-t-butyl nitroxide, in an aqueous suspension of dipalmitoyl lecithin vesicles have been studied by electron spin resonance spectroscopy. When the lecithin is cooled through its phase transition temperature near 41°C, some solute is “frozen out” of the bilayer, and the standard partial molar enthalpy and entropy of partition go more positive by a factor of 8 and 6, respectively. However, the apparent diffusion constant in the lecithin phase is only slightly smaller than that in water, both above and below the transition temperature. The fraction of bilayer volume within which solute is distributed may increase with temperature, contributing to the positive enthalpy of partition. Comparison of time constants suggests that there is a permeability barrier to this solute in the periphery of the bilayer. PMID:4360944

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.

The Fusion of Membranes and Vesicles: Pathway and Energy Barriers from Dissipative Particle Dynamics

PubMed Central

Grafmüller, Andrea; Shillcock, Julian; Lipowsky, Reinhard

2009-01-01

The fusion of lipid bilayers is studied with dissipative particle dynamics simulations. First, to achieve control over membrane properties, the effects of individual simulation parameters are studied and optimized. Then, a large number of fusion events for a vesicle and a planar bilayer are simulated using the optimized parameter set. In the observed fusion pathway, configurations of individual lipids play an important role. Fusion starts with individual lipids assuming a splayed tail configuration with one tail inserted in each membrane. To determine the corresponding energy barrier, we measure the average work for interbilayer flips of a lipid tail, i.e., the average work to displace one lipid tail from one bilayer to the other. This energy barrier is found to depend strongly on a certain dissipative particle dynamics parameter, and, thus, can be adjusted in the simulations. Overall, three subprocesses have been identified in the fusion pathway. Their energy barriers are estimated to lie in the range 8–15 kBT. The fusion probability is found to possess a maximum at intermediate tension values. As one decreases the tension, the fusion probability seems to vanish before the tensionless membrane state is attained. This would imply that the tension has to exceed a certain threshold value to induce fusion. PMID:19348749

New Poly(amino acid methacrylate) Brush Supports the Formation of Well-Defined Lipid Membranes

PubMed Central

2015-01-01

A novel poly(amino acid methacrylate) brush comprising zwitterionic cysteine groups (PCysMA) was utilized as a support for lipid bilayers. The polymer brush provides a 12-nm-thick cushion between the underlying hard support and the aqueous phase. At neutral pH, the zeta potential of the PCysMA brush was ∼−10 mV. Cationic vesicles containing >25% DOTAP were found to form a homogeneous lipid bilayer, as determined by a combination of surface analytical techniques. The lipid mobility as measured by FRAP (fluorescence recovery after photobleaching) gave diffusion coefficients of ∼1.5 μm2 s–1, which are comparable to those observed for lipid bilayers on glass substrates. PMID:25746444

Exploring the interactions between peptides and lipid bilayers using coherent anti-Stokes Raman scattering and two-photon fluorescence

NASA Astrophysics Data System (ADS)

Mari, M.; Mouras, R.; Downes, A.; Elfick, A.

2011-06-01

We have used a versatile and powerful microscope[1] for multi-modal biomedical imaging on which we combine Coherent Anti-Stokes Raman Scattering (CARS) with Two Photon Excitation Fluorescence (TPEF) using a Nd: YVO4 pump laser. We acquired 2PEF, CARS, and phase contrast images of Multilamellar Vesicles (MLVs) and Giant Unilamellar Vesicles (GUVs), as well as Raman spectra of the constituent lipids. A wide range of peptides are harmful to cells by altering the structure of the biological membranes. This effect depends on the composition of the membrane and the chemical structure of the peptide. The peptide we studied is the beta amyloid Aβ which is a major component of the amyloid plaques deposited on neuronal membranes of Alzheimer's disease (AD) patients. AD is neurodegenerative disorder in which the hallmark symptoms include cognitive decline and dementia[2] and is characterized by the formation of extracellular amyloid fibrils on the neuronal membranes of the brain. Many questions still remain unanswered concerning the destabilization of cellular ionic homeostasis due to pores formed during the interactions of lipid membranes with peptides. In this project, biomimics of cell membranes are used. The structures that best mimic the plasma membranes are MLVs or GUVs. These vesicles are formed using the gentle hydration technique[3] or the electroformation technique[4] respectively and are composed of phospholipids such as DOPC, DPPC, D62PPC and their binary mixtures. The MLVs and GUVs imaging by CARS and TPEF microscopy not only permits the direct imaging of the leakage phenomenon caused by the toxic peptide (Aβ) on the lipid bilayer, but also records simultaneously the lateral structure of the bilayer and peptide distribution in the plane across the membrane.

Aqueous magnesium as an environmental selection pressure in the evolution of phospholipid membranes on early earth

NASA Astrophysics Data System (ADS)

Dalai, Punam; Ustriyana, Putu; Sahai, Nita

2018-02-01

Early compartmentalization of simple biomolecules by membrane bilayers was, presumably, a critical step in the emergence of the first cell-like entities, protocells. Their membranes were likely composed of single chain amphiphiles (SCAs), but pure SCA membranes especially those with short-chains are highly unstable towards divalent cations, which are ubiquitous in aqueous environments. The prebiotic synthesis of phospholipids (PLs), even in only trace amounts, may also have been possible. PL membranes are much more stable towards divalent cations. Here, we show the transition of fatty acid membranes to mixed fatty acid-PL and, finally, to PL membranes in the presence of Mg2+, which acts as an environmental selection pressure, and we propose different mechanisms for the observed increased Mg2+-immunity. The "fatal" concentration ([Mg2+]fatal) at which vesicles are disrupted increased dramatically by an order of magnitude from OA to mixed to POPC vesicles. Two mechanisms for the increasing immunity were determined. The negative charge density of the vesicles decreased with increasing POPC content, so more Mg2+ was required for disruption. More interestingly, Mg2+ preferentially bound to and abstracted OA from mixed lipid membranes, resulting in relatively POPC-enriched vesicles compared to the initial ratio. The effect was the most dramatic for the largest initial OA-POPC ratio representing the most primitive protocells. Thus, Mg2+ acted to evolve the mixed membrane composition towards PL enrichment. To the best of our knowledge, this is the first report of selective lipid abstraction from mixed SCA-PL vesicles. These results may hold implications for accommodating prebiotic Mg2+-promoted processes such as non-enzymatic RNA polymerization on early Earth.

From faceted vesicles to liquid icoshedra: Where topology and crystallography meet

DOE PAGES

Guttman, Shani; Ocko, Benjamin M.; Deutsch, Moshe; ...

2016-02-17

We study many common amphiphiles that spontaneously self-assemble in aqueous solutions, forming membranes and unilamellar vesicles. While the vesicular membranes are bilayers, with the hydrophilic moieties exposed to the solution, the structure formed by amphiphiles at the oil–water (i.e., alkane–water) interfaces, such as the surface of an oil droplet in water, is typically a monolayer. It has recently been demonstrated that these monolayers and bilayers may crystallize on cooling, with the thermodynamic conditions for this transition set by the geometry of the constituent molecules. While a planar hexagonal packing motif is particularly abundant in these crystals, a hexagonal lattice ismore » incompatible with a closed-surface topology, such as a closed vesicle or the surface of a droplet. Thus, (at least) 12 five-fold defects form, giving rise to a complex interplay between the stretching and the bending energies of these two-dimensional crystals; in addition, a central role is also played by the interfacial tension. This interplay, part of which has been theoretically studied in the past, gives rise to a range of unexpected and counterintuitive phenomena, such as the recently-observed temperature-tunable formation of stable liquid polyhedra, and a tail growing and droplet-splitting akin to the spontaneous emulsification effect.« less

Partitioning of lysolipids, fatty acids and their mixtures in aqueous lipid bilayers: solute concentration/composition effects.

PubMed

Singh, Jasmeet; Lai, Amy Jo; Alaee, Yasmin; Ranganathan, Radha

2014-01-01

Distributions of lysopalmitoylphosphatidylcholine (LPPC), palmitic acid (PA) and their 1:1 mixtures between water and dipalmitoylphosphatidylcholine (DPPC) bilayer were determined using a fluorescence probe that selectively detects only the solutes in water. Water solute concentrations were obtained at each of several lipid concentrations. Dynamic Light Scattering experiments confirmed that the lipid/solute aggregates were vesicles in the concentration range investigated. Lipid concentration dependence of the solute component in water was fit to a thermodynamic model of solute distribution between two coexisting solvents. Water/bilayer partition coefficient and the free energy of transfer, for each of these solutes were determined from the fit. Main findings are: (1) Water/bilayer partition coefficient of solute is greater for 2 to 10% solute mole fraction than for 0 to 2%, signaling solute induced bilayer perturbation that increases bilayer solubility, beginning at 2% solute mole fraction. (2) Partition coefficients are in the order LPPC

Partitioning of Lysolipids, Fatty Acids and Their Mixtures in Aqueous Lipid Bilayers: Solute Concentration / Composition Effects

PubMed Central

Singh, Jasmeet; Lai, Amy Jo; Alaee, Yasmin; Ranganathan, Radha

2013-01-01

Distribution of lysopalmitoylphosphatidylcholine (LPPC), Palmitic acid (PA) and their 1:1 mixtures between water and dipalmitoylphosphatidylcholine (DPPC) bilayer were determined using a fluorescence probe that selectively detects only the solutes in water. Water solute concentrations were obtained at each of several lipid concentrations. Dynamic Light Scattering experiments confirmed that the lipid/solute aggregates were vesicles in the concentration range investigated. Lipid concentration dependence of the solute component in water was fit to a thermodynamic model of solute distribution between two coexisting solvents. Water/bilayer partition coefficient and the free energy of transfer, for each of these solutes were determined from the fit. Main findings are: (1) Water/bilayer partition coefficient of solute is greater for 2 to 10 % solute mole fraction than for 0 to 2 %, signaling solute induced bilayer perturbation that increases bilayer solubility, beginning at 2 % solute mole fraction. (2) Partition coefficients are in the order LPPC

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

High Resistivity Lipid Bilayers Assembled on Polyelectrolyte Multilayer Cushions: An Impedance Study.

PubMed

Diamanti, Eleftheria; Gregurec, Danijela; Rodríguez-Presa, María José; Gervasi, Claudio A; Azzaroni, Omar; Moya, Sergio E

2016-06-28

Supported membranes on top of polymer cushions are interesting models of biomembranes as cell membranes are supported on a polymer network of proteins and sugars. In this work lipid vesicles formed by a mixture of 30% 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 70% 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) are assembled on top of a polyelectrolyte multilayer (PEM) cushion of poly(allylamine hydrochloride) (PAH) and poly(styrene sodium sulfonate) (PSS). The assembly results in the formation of a bilayer on top of the PEM as proven by means of the quartz crystal microbalance with dissipation technique (QCM-D) and by cryo-transmission electron microscopy (cryo-TEM). The electrical properties of the bilayer are studied by electrochemical impedance spectroscopy (EIS). The bilayer supported on the PEMs shows a high resistance, on the order of 10(7) Ω cm(2), which is indicative of a continuous, dense bilayer. Such resistance is comparable with the resistance of black lipid membranes. This is the first time that such values are obtained for lipid bilayers supported on PEMs. The assembly of polyelectrolytes on top of a lipid bilayer decreases the resistance of the bilayer up to 2 orders of magnitude. The assembly of the polyelectrolytes on the lipids induces defects or pores in the bilayer which in turn prompts a decrease in the measured resistance.

Experimental observation of the asymmetric instability of intermediate-reduced-volume vesicles in extensional flow.

PubMed

Dahl, Joanna B; Narsimhan, Vivek; Gouveia, Bernardo; Kumar, Sanjay; Shaqfeh, Eric S G; Muller, Susan J

2016-04-20

Vesicles provide an attractive model system to understand the deformation of living cells in response to mechanical forces. These simple, enclosed lipid bilayer membranes are suitable for complementary theoretical, numerical, and experimental analysis. A recent study [Narsimhan, Spann, Shaqfeh, J. Fluid Mech., 2014, 750, 144] predicted that intermediate-aspect-ratio vesicles extend asymmetrically in extensional flow. Upon infinitesimal perturbation to the vesicle shape, the vesicle stretches into an asymmetric dumbbell with a cylindrical thread separating the two ends. While the symmetric stretching of high-aspect-ratio vesicles in extensional flow has been observed and characterized [Kantsler, Segre, Steinberg, Phys. Rev. Lett., 2008, 101, 048101] as well as recapitulated in numerical simulations by Narsimhan et al., experimental observation of the asymmetric stretching has not been reported. In this work, we present results from microfluidic cross-slot experiments observing this instability, along with careful characterization of the flow field, vesicle shape, and vesicle bending modulus. The onset of this shape transition depends on two non-dimensional parameters: reduced volume (a measure of vesicle asphericity) and capillary number (ratio of viscous to bending forces). We observed that every intermediate-reduced-volume vesicle that extends forms a dumbbell shape that is indeed asymmetric. For the subset of the intermediate-reduced-volume regime we could capture experimentally, we present an experimental phase diagram for asymmetric vesicle stretching that is consistent with the predictions of Narsimhan et al.

Electrostatics of lipid bilayer bending.

PubMed Central

Chou, T; Jarić, M V; Siggia, E D

1997-01-01

The electrostatic contribution to spontaneous membrane curvature is calculated within Poisson-Boltzmann theory under a variety of assumptions and emphasizing parameters in the physiological range. Asymmetrical surface charges can be fixed with respect to bilayer midplane area or with respect to the lipid-water area, but induce curvatures of opposite signs. Unequal screening layers on the two sides of a vesicle (e.g., multivalent cationic proteins on one side and monovalent salt on the other) also induce bending. For reasonable parameters, tubules formed by electrostatically induced bending can have radii in the 50-100-nm range, often seen in many intracellular organelles. Thus membrane associated proteins may induce curvature and subsequent budding, without themselves being intrinsically curved. Furthermore, we derive the previously unexplored effects of respecting the strict conservation of charge within the interior of a vesicle. The electrostatic component of the bending modulus is small under most of our conditions and is left as an experimental parameter. The large parameter space of conditions is surveyed in an array of graphs. Images FIGURE 1 FIGURE 10 PMID:9129807

Chitosan derivatives targeting lipid bilayers: Synthesis, biological activity and interaction with model membranes.

PubMed

Martins, Danubia Batista; Nasário, Fábio Domingues; Silva-Gonçalves, Laiz Costa; de Oliveira Tiera, Vera Aparecida; Arcisio-Miranda, Manoel; Tiera, Marcio José; Dos Santos Cabrera, Marcia Perez

2018-02-01

The antimicrobial activity of chitosan and derivatives to human and plant pathogens represents a high-valued prospective market. Presently, two low molecular weight derivatives, endowed with hydrophobic and cationic character at different ratios were synthesized and characterized. They exhibit antimicrobial activity and increased performance in relation to the intermediate and starting compounds. However, just the derivative with higher cationic character showed cytotoxicity towards human cervical carcinoma cells. Considering cell membranes as targets, the mode of action was investigated through the interaction with model lipid vesicles mimicking bacterial, tumoral and erythrocyte membranes. Intense lytic activity and binding are demonstrated for both derivatives in anionic bilayers. The less charged compound exhibits slightly improved selectivity towards bacterial model membranes, suggesting that balancing its hydrophobic/hydrophilic character may improve efficiency. Observing the aggregation of vesicles, we hypothesize that the "charge cluster mechanism", ascribed to some antimicrobial peptides, could be applied to these chitosan derivatives. Copyright © 2017 Elsevier Ltd. All rights reserved.

Phase transitions in methyl parben doped dipalmitoyl phosphatidylethanolamine vesicles

NASA Astrophysics Data System (ADS)

Panicker, Lata

2013-02-01

Influence of the preservative, methyl paraben (MPB), on the thermal properties of dipalmitoyl phosphatidylethanolamine (DPPE) vesicles was investigated using DSC. DSC measurement of the lipid acyl chain melting transition in DPPE membrane doped with MPB, showed MPB concentration dependant modifications in the membrane thermal properties. The interesting findings are: (1) the presence of parabens increases the membrane fluidity. (2) the MPB molecules seem to be present in the aqueous bilayer interfacial region intercalated between the neighboring lipid polar headgroup (3) high concentration of MPB favored formation of crystalline and glassy phases.

Structural Changes in Lipid Vesicles Generated by the Shock Blast Waves: Coarse-Grained Molecular Dynamics Simulation

DTIC Science & Technology

2013-11-01

duration, or shock-pulse shape. Used in this computational study is a coarse-grained model of the lipid vesicle as a simplified model of a cell...Figures iv List of Tables iv 1. Introduction 1 2. Model and Methods 3 3. Results and Discussion 6 3.1 Simulation of the Blast Waves with Low Peak...realistic detail but to focus on a simple model of the major constituent of a cell membrane, the phospholipid bilayer. In this work, we studied the

Bile salts-containing vesicles: promising pharmaceutical carriers for oral delivery of poorly water-soluble drugs and peptide/protein-based therapeutics or vaccines.

PubMed

Aburahma, Mona Hassan

2016-07-01

Most of the new drugs, biological therapeutics (proteins/peptides) and vaccines have poor performance after oral administration due to poor solubility or degradation in the gastrointestinal tract (GIT). Though, vesicular carriers exemplified by liposomes or niosomes can protect the entrapped agent to a certain extent from degradation. Nevertheless, the harsh GIT environment exemplified by low pH, presence of bile salts and enzymes limits their capabilities by destabilizing them. In response to that, more resistant bile salts-containing vesicles (BS-vesicles) were developed by inclusion of bile salts into lipid bilayers constructs. The effectiveness of orally administrated BS-vesicles in improving the performance of vesicles has been demonstrated in researches. Yet, these attempts did not gain considerable attention. This is the first review that provides a comprehensive overview of utilizing BS-vesicles as a promising pharmaceutical carrier with a special focus on their successful applications in oral delivery of therapeutic macromolecules and vaccines. Insights on the possible mechanisms by which BS-vesicles improve the oral bioavailability of the encapsulated drug or immunological response of entrapped vaccine are explained. In addition, methods adopted to prepare and characterize BS-vesicles are described. Finally, the gap in the scientific researches tackling BS-vesicles that needs to be addressed is highlighted.

Organization and dynamics of pyrene and pyrene lipids in intact lipid bilayers. Photo-induced charge transfer processes.

PubMed Central

Barenholz, Y; Cohen, T; Korenstein, R; Ottolenghi, M

1991-01-01

The dynamics of fluorescence quenching and the organization of a series of pyrene derivatives anchored in various depths in bilayers of phosphatidylcholine small unilamellar vesicles was studied and compared with their behavior in homogeneous solvent systems. The studies include characterization of the environmental polarity of the pyrene fluorophore based on its vibronic peaks, as well as the interaction with three collisional quenchers: the two membrane-soluble quenchers, diethylaniline and bromobenzene, and the water soluble quencher potassium iodide. The system of diethylaniline-pyrene derivatives in the membrane of phosphatidylcholine vesicles was characterized in detail. The diethylaniline partition coefficient between the lipid bilayers and the buffer is approximately 5,800. Up to a diethylaniline/phospholipid mole ratio of 1:3 the perturbation to membrane structure is minimal so that all photophysical studies were performed below this mole ratio. The quenching reaction, in all cases, was shown to take place in the lipid bilayer interior and the relative quenching efficiencies of the various probe molecules was used to provide information on the distribution of both fluorescent probes and quencher molecules in the lipid bilayer. The quenching efficiency by diethylaniline in the lipid bilayer was found to be essentially independent on the length of the methylene chain of the pyrene moiety. These findings suggest that the quenching process, being a diffusion controlled reaction, is determined by the mobility of the diethylaniline quencher (with an effective diffusion coefficient D approximately 10(-7) cm2 s-1) which appears to be homogeneously distributed throughout the lipid bilayer. The pulsed laser photolysis products of the charge-transfer quenching reaction were examined. No exciplex (excited-complex) formation was observed and the yield of the separated radical ions was shown to be tenfold smaller than in homogenous polar solutions. The decay of the radical ions is considerably faster than the corresponding process in homogenous solutions. Relatively high intersystem crossing yields are observed. The results are explained on the basis of the intrinsic properties of a lipid bilayer, primarily, its rigid spatial organization. It is suggested that such properties favor ion-pair formation over exciplex generation. They also enhance primary geminate recombination of initially formed (solvent-shared) ion pairs. Triplet states are generated via secondary geminate recombination of ion pairs in the membrane interior. The results bear on the general mechanism of electron transfer processes in biomembranes. PMID:1883931

Advantages of statistical analysis of giant vesicle flickering for bending elasticity measurements.

PubMed

Méléard, P; Pott, T; Bouvrais, H; Ipsen, J H

2011-10-01

We show how to greatly improve precision when determining bending elasticity of giant unilamellar vesicles. Taking advantage of the well-known quasi-spherical model of liposome flickering, we analyze the full probability distributions of the configurational fluctuations instead of limiting the analysis to the second moment measurements only as usually done in previously published works. This leads to objective criteria to reject vesicles that do not behave according to the model. As a result, the confidence in the bending elasticity determination of individual vesicles that fit the model is improved and, consequently, the reproducibility of this measurement for a given membrane system. This approach uncovers new possibilities for bending elasticity studies like detection of minute influences by solutes in the buffer or into the membrane. In the same way, we are now able to detect the inhomogeneous behavior of giant vesicle systems such as the hazardous production of peroxide in bilayers containing fluorescent dyes. © EDP Sciences / Società Italiana di Fisica / Springer-Verlag 2011

The effect of protein on phase separation in giant unilamellar lipid vesicles.

NASA Astrophysics Data System (ADS)

Hutchison, J. B.; Weis, R. M.; Dinsmore, A. D.

2009-03-01

We explore the coarsening and out of plane curvature (budding) of domains in lipid bilayer vesicles composed of DOPC (unsaturated), PSM (saturated), and cholesterol. Green fluorescent protein (GFP) was added to the membrane in controlled amounts by binding to the Ni-chelating lipid, Ni-DOGS. Vesicles with diameters between 10 and 50 microns were prepared via a standard electroformation procedure. As a sample is lowered through temperature Tmix, a previously homogeneous vesicle phase separates into two fluid phases with distinct compositions. Phase-separated domains have a line tension (energy/length) at the boundary with the major phase which competes with bending energy and lateral tension to determine the overall configuration of the vesicle. Domain budding and coarsening were observed and recorded using both bright field and fluorescence microscopy during temperature scans and with varying concentrations of GFP. The addition of a model protein into our system allows for a broader understanding of the effect of protein, which are ubiquitous in cell membranes, on phase separation, budding, and coarsening.

The effect of spontaneous curvature on a two-phase vesicle

PubMed Central

Cox, Geoffrey; Lowengrub, John

2015-01-01

Vesicles are membrane-bound structures commonly known for their roles in cellular transport and the shape of a vesicle is determined by its surrounding membrane (lipid bilayer). When the membrane is composed of different lipids, it is natural for the lipids of similar molecular structure to migrate towards one another (via spinodal decomposition), creating a multi-phase vesicle. In this article, we consider a two-phase vesicle model which is driven by nature’s propensity to maintain a minimal state of elastic energy. The model assumes a continuum limit, thereby treating the membrane as a closed three-dimensional surface. The main purpose of this study is to reveal the complexity of the Helfrich two-phase vesicle model with non-zero spontaneous curvature and provide further evidence to support the relevance of spontaneous curvature as a modelling parameter. In this paper, we illustrate the complexity of the Helfrich two-phase model by providing multiple examples of undocumented solutions and energy hysteresis. We also investigate the influence of spontaneous curvature on morphological effects and membrane phenomena such as budding and fusion. PMID:26097287

Evidence for phospholipid microdomain formation in liquid crystalline liposomes reconstituted with Escherichia coli lactose permease.

PubMed Central

Lehtonen, J Y; Kinnunen, P K

1997-01-01

The well-characterized integral membrane protein lactose (lac) permease from Escherichia coli was reconstituted together with trace amounts (molar fraction X = 0.005 of the total phospholipid) of different pyrene-labeled phospholipid analogs into 1-palmitoyl-2-oleoyl-sn-glycero-3-sn-glycero-3-phospho-rac'-glycerol (POPG) liposomes. Effects of lac permease on bilayer lipid dynamics were investigated by measuring the excimer-to-monomer fluorescence intensity ratio IE/IM. Compared to control vesicles, the presence of lac permease (at a protein:phospholipid stoichiometry P/L of 1:4.000) increased the rate of excimer formation by 1-palmitoyl-2[6-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC) by approximately fivefold. Decreasing P/L from approximately 1:4.000 to 1:7.600 decreased the IE/IM for PPDPC from 0.16 to 0.05, respectively. An increase in bilayer fluidity due to permease is unlikely, thus implying that the augmented IE/IM should arise from partial lateral segregation of PPDPC in the vesicles. This notion is supported by the further 38% increase in IE/IM observed for the pyrene-labeled Cys-148 lac permease reconstituted into POPG vesicles at P/L 1:4000. The importance of the length of the lipid-protein boundary is implicated by the reduction in IE/IM resulting from the aggregation of the lac permease in vesicles by a monoclonal antibody. Interestingly, excimer formation by 1-palmitoyl-2[6-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphocholine (PPHPC) was enhanced only fourfold in the presence of lac permease. Results obtained with the corresponding pyrenyl phosphatidylglycerols and -methanols were qualitatively similar to those above, thus indicating that lipid headgroup-protein interactions are not involved. Inclusion of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamino-N-(5-fluoresce inthio- carbamoyl) (DPPF, X = 0.005) into reconstituted lactose permease vesicles containing PPDPC caused a nearly 90% decrease in excimer fluorescence, whereas in control vesicles lacking the reconstituted protein only 40% quenching was evident. The addition of 1,2-dipalmitoyl-sn-glycero-3-phospho-rac'-glycerol (DPPG) decreased IE/IM for PPDPC, revealing the driving force for the lateral segregation of this probe to become attenuated. More specifically for protein-free bilayers at XDPPG = 0.10 the rate of lateral diffusion of PPDPC in POPG is diminished, as evidenced by the 24% decrement in IE/IM, under these conditions the increase in IE/IM due to lac permease was strongly reduced, by approximately 84%. The present data are interpreted in terms of the hydrophobic mismatch theory, which predicts that integral membrane proteins will draw lipids of similar hydrophobic thickness into their vicinity. In brief, the approximate lengths of most of the predicted 12 hydrophobic, membrane-spanning alpha-helical segments of lactose permease range between 28.5 and 37.5 A and thus exceed the hydrophobic thickness of POPG of approximately 25.8 A. Therefore, to reduce the free energy of the assembly, longer lipids such as PPDPC and DPPF are accumulated in the immediate vicinity of lactose permease in fluid, liquid crystalline POPG bilayers. PMID:9138570

Trapping, Deformation, and Rotation of Giant Unilamellar Vesicles in Octode Dielectrophoretic Field Cages

PubMed Central

Korlach, J.; Reichle, C.; Müller, T.; Schnelle, T.; Webb, W. W.

2005-01-01

The behavior of freestanding lipid bilayer membranes under the influence of dielectric force potentials was studied by trapping, holding, and rotating individual giant unilamellar vesicles (GUVs) inside dielectrophoretic microfield cages. Using laser scanning confocal microscopy and three-dimensional image reconstructions of GUVs labeled with fluorescent membrane probes, field strength and frequency-dependent vesicle deformations were observed which are explained by calculations of the dielectric force potentials inside the cage. Dynamical membrane properties under the influence of the field cage were studied by fluorescence correlation spectroscopy, circumventing potential artifacts associated with measurements involving GUV immobilization on support surfaces. Lipid transport could be accelerated markedly by the applied fields, aided by hydrodynamic fluid streaming which was also studied by fluorescence correlation spectroscopy. PMID:15863477

A Phase of Liposomes with Entangled Tubular Vesicles

NASA Astrophysics Data System (ADS)

Chiruvolu, Shivkumar; Warriner, Heidi E.; Naranjo, Edward; Idziak, Stefan H. J.; Radler, Joachim O.; Plano, Robert J.; Zasadzinski, Joseph A.; Safinya, Cyrus R.

1994-11-01

An equilibrium phase belonging to the family of bilayer liposomes in ternary mixtures of dimyristoylphosphatidylcholine (DMPC), water, and geraniol (a biological alcohol derived from oil-soluble vitamins that acts as a cosurfactant) has been identified. Electron and optical microscopy reveal the phase, labeled Ltv, to be composed of highly entangled tubular vesicles. In situ x-ray diffraction confirms that the tubule walls are multilamellar with the lipids in the chain-melted state. Macroscopic observations show that the Ltv phase coexists with the well-known L_4 phase of spherical vesicles and a bulk L_α phase. However, the defining characteristic of the Ltv phase is the Weissenberg rod climbing effect under shear, which results from its polymer-like entangled microstructure.

Large Plasma Membrane Disruptions Are Rapidly Resealed by Ca2+-dependent Vesicle–Vesicle Fusion Events

PubMed Central

Terasaki, Mark; Miyake, Katsuya; McNeil, Paul L.

1997-01-01

A microneedle puncture of the fibroblast or sea urchin egg surface rapidly evokes a localized exocytotic reaction that may be required for the rapid resealing that follows this breach in plasma membrane integrity (Steinhardt, R.A,. G. Bi, and J.M. Alderton. 1994. Science (Wash. DC). 263:390–393). How this exocytotic reaction facilitates the resealing process is unknown. We found that starfish oocytes and sea urchin eggs rapidly reseal much larger disruptions than those produced with a microneedle. When an ∼40 by 10 μm surface patch was torn off, entry of fluorescein stachyose (FS; 1,000 mol wt) or fluorescein dextran (FDx; 10,000 mol wt) from extracellular sea water (SW) was not detected by confocal microscopy. Moreover, only a brief (∼5–10 s) rise in cytosolic Ca2+ was detected at the wound site. Several lines of evidence indicate that intracellular membranes are the primary source of the membrane recruited for this massive resealing event. When we injected FS-containing SW deep into the cells, a vesicle formed immediately, entrapping within its confines most of the FS. DiI staining and EM confirmed that the barrier delimiting injected SW was a membrane bilayer. The threshold for vesicle formation was ∼3 mM Ca2+ (SW is ∼10 mM Ca2+). The capacity of intracellular membranes for sealing off SW was further demonstrated by extruding egg cytoplasm from a micropipet into SW. A boundary immediately formed around such cytoplasm, entrapping FDx or FS dissolved in it. This entrapment did not occur in Ca2+-free SW (CFSW). When egg cytoplasm stratified by centrifugation was exposed to SW, only the yolk platelet–rich domain formed a membrane, suggesting that the yolk platelet is a critical element in this response and that the ER is not required. We propose that plasma membrane disruption evokes Ca2+ regulated vesicle–vesicle (including endocytic compartments but possibly excluding ER) fusion reactions. The function in resealing of this cytoplasmic fusion reaction is to form a replacement bilayer patch. This patch is added to the discontinuous surface bilayer by exocytotic fusion events. PMID:9314529

Ordering in bio-inorganic hybrid nanomaterials probed by in situ scanning transmission X-ray microscopy

DOE PAGES

Lee, Jonathan R. I.; Bagge-Hansen, Michael; Tunuguntla, Ramya; ...

2015-04-15

Here, phospholipid bilayer coated Si nanowires are one-dimensional (1D) composites that provide versatile bio-nanoelectronic functionality via incorporation of a wide variety of biomolecules into the phospholipid matrix. The physiochemical behaviour of the phospholipid bilayer is strongly dependent on its structure and, as a consequence, substantial modelling and experimental efforts have been directed at the structural characterization of supported bilayers and unsupported phospholipid vesicles; nonetheless, the experimental studies conducted to date have exclusively involved volume-averaged techniques, which do not allow for the assignment of spatially resolved structural variations that could critically impact the performance of the 1D phospholipid-Si NW composites. Inmore » this manuscript, we use scanning transmission X-ray microscopy (STXM) to probe bond orientation and bilayer thickness as a function of position with a spatial resolution of ~30 nm for Δ9-cis 1,2-dioleoyl-sn-glycero-3-phosphocholine layers prepared Si NWs. When coupled with small angle X-ray scattering measurements, the STXM data reveal structural motifs of the Si NWs that give rise to multi-bilayer formation and enable assignment of the orientation of specific bonds known to affect the order and rigidity of phospholipid bilayers.« less

DSC and EPR investigations on effects of cholesterol component on molecular interactions between paclitaxel and phospholipid within lipid bilayer membrane.

PubMed

Zhao, Lingyun; Feng, Si-Shen; Kocherginsky, Nikolai; Kostetski, Iouri

2007-06-29

Differential scanning calorimetry (DSC) and electron paramagnetic resonance spectroscopy (EPR) were applied to investigate effects of cholesterol component on molecular interactions between paclitaxel, which is one of the best antineoplastic agents found from nature, and dipalmitoylphosphatidylcholine (DPPC) within lipid bilayer vesicles (liposomes), which could also be used as a model cell membrane. DSC analysis showed that incorporation of paclitaxel into the DPPC bilayer causes a reduction in the cooperativity of bilayer phase transition, leading to a looser and more flexible bilayer structure. Including cholesterol component in the DPPC/paclitaxel mixed bilayer can facilitate the molecular interaction between paclitaxel and lipid and make the tertiary system more stable. EPR analysis demonstrated that both of paclitaxel and cholesterol have fluidization effect on the DPPC bilayer membranes although cholesterol has more significant effect than paclitaxel does. The reduction kinetics of nitroxides by ascorbic acid showed that paclitaxel can inhibit the reaction by blocking the diffusion of either the ascorbic acid or nitroxide molecules since the reaction is tested to be a first order one. Cholesterol can remarkably increase the reduction reaction speed. This research may provide useful information for optimizing liposomal formulation of the drug as well as for understanding the pharmacology of paclitaxel.

Enzymatic hydrolysis of short-chain lecithin/long-chain phospholipid unilamellar vesicles: sensitivity of phospholipases to matrix phase state.

PubMed

Gabriel, N E; Agman, N V; Roberts, M F

1987-11-17

Short-chain lecithin/long-chain phospholipid unilamellar vesicles (SLUVs), unlike pure long-chain lecithin vesicles, are excellent substrates for water-soluble phospholipases. Hemolysis assays show that greater than 99.5% of the short-chain lecithin is partitioned in the bilayer. In these binary component vesicles, the short-chain species is the preferred substrate, while the long-chain phospholipid can be treated as an inhibitor (phospholipase C) or poor substrate (phospholipase A2). For phospholipase C Bacillus cereus, apparent Km and Vmax values show that bilayer-solubilized diheptanoylphosphatidylcholine (diheptanoyl-PC) is nearly as good a substrate as pure micellar diheptanoyl-PC, although the extent of short-chain lecithin hydrolysis depends on the phase state of the long-chain lipid. For phospholipase A2 Naja naja naja, both Km and Vmax values show a greater range: in a gel-state matrix, diheptanoyl-PC is hydrolyzed with micellelike kinetic parameters; in a liquid-crystalline matrix, the short-chain lecithin becomes comparable to the long-chain component. Both enzymes also show an anomalous increase in specific activity toward diheptanoyl-PC around the phase transition temperature of the long-chain phospholipid. Since the short-chain lecithin does not exhibit a phase transition, this must reflect fluctuations in head-group area or vertical motions of the short-chain lecithin caused by surrounding long-chain lecithin molecules. These results are discussed in terms of a specific model for SLUV hydrolysis and a general explanation for the "interfacial activation" observed with water-soluble phospholipases.

Phase behavior, rheological property, and transmutation of vesicles in fluorocarbon and hydrocarbon surfactant mixtures.

PubMed

Yuan, Zaiwu; Qin, Menghua; Chen, Xiushan; Liu, Changcheng; Li, Hongguang; Hao, Jingcheng

2012-06-26

We present a detailed study of a salt-free cationic/anionic (catanionic) surfactant system where a strongly alkaline cationic surfactant (tetradecyltrimethylammonium hydroxide, TTAOH) was mixed with a single-chain fluorocarbon acid (nonadecafluorodecanoic acid, NFDA) and a hyperbranched hydrocarbon acid [di-(2-ethylhexyl)phosphoric acid, DEHPA] in water. Typically the concentration of TTAOH is fixed while the total concentration and mixing molar ratio of NFDA and DEHPA is varied. In the absence of DEHPA and at a TTAOH concentration of 80 mmol·L(-1), an isotropic L(1) phase, an L(1)/L(α) two-phase region, and a single L(α) phase were observed successively with increasing mixing molar ratio of NFDA to TTAOH (n(NFDA)/n(TTAOH)). In the NFDA-rich region (n(NFDA)/n(TTAOH) > 1), a small amount of excess NFDA can be solubilized into the L(α) phase while a large excess of NFDA eventually leads to phase separation. When NFDA is replaced gradually by DEHPA, the mixed system of TTAOH/NFDA/DEHPA/H(2)O follows the same phase sequence as that of the TTAOH/NFDA/H(2)O system and the phase boundaries remain almost unchanged. However, the viscoelasticity of the samples in the single L(α) phase region becomes higher at the same total surfactant concentration as characterized by rheological measurements. Cryo-transmission electron microscopic (cryo-TEM) observations revealed a microstructural evolution from unilamellar vesicles to multilamellar ones and finally to gaint onions. The size of the vesicle and number of lamella can be controlled by adjusting the molar ratio of NFDA to DEHPA. The dynamic properties of the vesicular solutions have also been investigated. It is found that the yield stress and the storage modulus are time-dependent after a static mixing process between the two different types of vesicle solutions, indicating the occurrence of a dynamic fusion between the two types of vesicles. The microenvironmental changes induced by aggregate transitions were probed by (19)F NMR as well as (31)P NMR measurements. Upon replacement of NFDA by DEHPA, the signal from the (19)F atoms adjacent to the hydrophilic headgroup disappears and that from the (19)F atoms on the main chain becomes sharper. This could be interpreted as an increase of microfluidity in the mixed vesicle bilayers at higher content of DEHPA, whose alkyl chains are expected to have a lower chain melting point. Our results provide basic knowledge on vesicle formation and their structural evolution in salt-free catanionic surfactant systems containing mixed ion pairs, which may contribute to a deeper understanding of the rules governing the formation and properties of surfactant self-assembly.

Determination of lipid bilayer affinities and solvation characteristics by electrokinetic chromatography using polymer-bound lipid bilayer nanodiscs.

PubMed

Penny, William M; Palmer, Christopher P

2018-03-01

Styrene-maleic acid polymer-bound lipid bilayer nanodiscs have been investigated and characterized by electrokinetic chromatography. Linear solvation energy relationship analysis was employed to characterize the changes in solvation environment of nanodiscs of varied belt to lipid ratio, belt polymer chemistry and molecular weight, and lipid composition. Increases in the lipid to belt polymer ratio resulted in smaller, more cohesive nanodiscs with greater electrophoretic mobility. Nanodisc structures with belt polymers of different chemistry and molecular weight were compared and showed only minor changes in solvent characteristics and selectivity consistent with changes in structure of the lipid bilayer. Seven phospholipid and sphingomyelin nanodiscs of different lipid composition were characterized. Changes in lipid head group structure had a significant effect on bilayer-solute interactions. In most cases, changes in alkyl tail structure had no discernible effect on solvation environment aside from those explained by changes in the gel-liquid transition temperature. Comparison to vesicles of similar lipid composition show only minor differences in solvation environment, likely due to differences in lipid composition and bilayer curvature. Together these results provide evidence that the dominant solute-nanodisc interactions are with the lipid bilayer and that head group chemistry has a greater impact on bilayer-solute interactions than alkyl tail or belt polymer structure. Nanodisc electrokinetic chromatography is demonstrated to allow characterization of solute interactions with lipid bilayers of varied composition. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

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

Interactions of beta-blockers with model lipid membranes: molecular view of the interaction of acebutolol, oxprenolol, and propranolol with phosphatidylcholine vesicles by time-dependent fluorescence shift and molecular dynamics simulations.

PubMed

Först, Gesche; Cwiklik, Lukasz; Jurkiewicz, Piotr; Schubert, Rolf; Hof, Martin

2014-08-01

Since pharmacokinetic and pharmacodynamic activities of drugs are often related to their interactions with biomembranes, it is of high interest to establish an approach for the characterization of these interactions at the molecular level. For the present study, beta-blockers (oxprenolol, propranolol, and acebutolol) were selected due to their well described nonspecific membrane effects (NME). Their interactions with model lipid membranes composed of palmitoyloleoylphosphatidylcholine (POPC) were studied using Time-Dependent Fluorescence Shift (TDFS) and Generalized Polarization (GP) as well as molecular dynamics (MD) simulations. Liposomal vesicles were labeled with fluorescent membrane polarity probes (Laurdan, Prodan, and Dtmac). Increasing beta-blocker concentrations (0-10 mM for acebutolol and oxprenolol, and 0-1.5 mM for propranolol) significantly rigidifies the lipid bilayer at the glycerol and headgroup level, which was detected in the steady-state and in the time-resolved fluorescence data. The effects of propranolol were considerably stronger than those of the two other beta-blockers. The addition of fluorescent probes precisely located at different levels within the lipid bilayer revealed the insertion of the beta-blockers into the POPC bilayer at the glycerol backbone level, which was further confirmed by MD simulations in the case of propranolol. Copyright © 2014 Elsevier B.V. All rights reserved.

Energetics and Kinetics of trans-SNARE Zippering

NASA Astrophysics Data System (ADS)

Rebane, Aleksander A.; Shu, Tong; Krishnakumar, Shyam; Rothman, James E.; Zhang, Yongli

Synaptic exocytosis relies on assembly of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins into a four-helix bundle to drive membrane fusion. Complementary SNAREs anchored to the synaptic vesicle (v-SNARE) and the plasma membrane (t-SNARE) associate from their N-termini, transiting a half-assembled intermediate (trans-SNARE), and ending at their C-termini with a rapid power stroke that leads to membrane fusion. Although cytosolic SNARE assembly has been characterized, it remains unknown how membranes modulate the energetics and kinetics of SNARE assembly. Here, we present optical tweezers measurements on folding of single membrane proteins in phospholipid bilayers. To our knowledge, this is the first such report. We measured the energetics, kinetics, and assembly intermediates of trans-SNAREs formed between a t-SNARE inserted into a bead-supported bilayer and a v-SNARE in a nanodisc. We found that the repulsive force of the apposed membranes increases the lifetime of the half-assembled intermediate. Our findings provide a single-molecule platform to study the regulation of trans-SNARE assembly by proteins that act on the half-assembled state, and thus reveal the mechanistic basis of the speed and high fidelity of synaptic transmission. This work was supported by US National Institutes of Health Grants F31 GM119312-01 (to A.A.R) and R01 GM093341 (to Y.Z.).

A Role for Weak Electrostatic Interactions in Peripheral Membrane Protein Binding

PubMed Central

Khan, Hanif M.; He, Tao; Fuglebakk, Edvin; Grauffel, Cédric; Yang, Boqian; Roberts, Mary F.; Gershenson, Anne; Reuter, Nathalie

2016-01-01

Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (BtPI-PLC) is a secreted virulence factor that binds specifically to phosphatidylcholine (PC) bilayers containing negatively charged phospholipids. BtPI-PLC carries a negative net charge and its interfacial binding site has no obvious cluster of basic residues. Continuum electrostatic calculations show that, as expected, nonspecific electrostatic interactions between BtPI-PLC and membranes vary as a function of the fraction of anionic lipids present in the bilayers. Yet they are strikingly weak, with a calculated ΔGel below 1 kcal/mol, largely due to a single lysine (K44). When K44 is mutated to alanine, the equilibrium dissociation constant for small unilamellar vesicles increases more than 50 times (∼2.4 kcal/mol), suggesting that interactions between K44 and lipids are not merely electrostatic. Comparisons of molecular-dynamics simulations performed using different lipid compositions reveal that the bilayer composition does not affect either hydrogen bonds or hydrophobic contacts between the protein interfacial binding site and bilayers. However, the occupancies of cation-π interactions between PC choline headgroups and protein tyrosines vary as a function of PC content. The overall contribution of basic residues to binding affinity is also context dependent and cannot be approximated by a rule-of-thumb value because these residues can contribute to both nonspecific electrostatic and short-range protein-lipid interactions. Additionally, statistics on the distribution of basic amino acids in a data set of membrane-binding domains reveal that weak electrostatics, as observed for BtPI-PLC, might be a less unusual mechanism for peripheral membrane binding than is generally thought. PMID:27028646

Charge-Triggered Membrane Insertion of Matrix Metalloproteinase-7, Supporter of Innate Immunity and Tumors.

PubMed

Prior, Stephen H; Fulcher, Yan G; Koppisetti, Rama K; Jurkevich, Alexander; Van Doren, Steven R

2015-11-03

Matrix metalloproteinase-7 (MMP-7) sheds signaling proteins from cell surfaces to activate bacterial killing, wound healing, and tumorigenesis. The mechanism targeting soluble MMP-7 to membranes has been investigated. Nuclear magnetic resonance structures of the zymogen, free and bound to membrane mimics without and with anionic lipid, reveal peripheral binding to bilayers through paramagnetic relaxation enhancements. Addition of cholesterol sulfate partially embeds the protease in the bilayer, restricts its diffusion, and tips the active site away from the bilayer. Its insertion of hydrophobic residues organizes the lipids, pushing the head groups and sterol sulfate outward toward the enzyme's positive charge on the periphery of the enlarged interface. Fluorescence probing demonstrates a similar mode of binding to plasma membranes and internalized vesicles of colon cancer cells. Binding of bilayered micelles induces allosteric activation and conformational change in the auto-inhibitory peptide and the adjacent scissile site, illustrating a potential intermediate in the activation of the zymogen. Copyright © 2015 Elsevier Ltd. All rights reserved.

Bilayer membrane interactions with nanofabricated scaffolds

DOE PAGES

Collier, C. Patrick

2015-07-29

Membrane function is facilitated by lateral organization within the lipid bilayer, including phase-separation of lipids into more ordered domains (lipid rafts) and anchoring of the membrane to a cytoskeleton. These features have proven difficult to reproduce in model membrane systems such as black lipid membranes, unilamellar vesicles and supported bilayers. However, advances in micro/nanofabrication have resulted in more realistic synthetic models of membrane-cytoskeleton interactions that can help uncover the design rules responsible for biological membrane formation and organization. This review will focus on describing micro-/nanostructured scaffolds that can emulate the connections of a cellular membrane to an underlying “cytoskeleton”. Thismore » includes molecular-based scaffolds anchored to a solid substrate through surface chemistry, solid-state supports modified by material deposition, lithography and etching, the creation of micro/nanoporous arrays, integration with microfluidics, and droplet-based bilayers at interfaces. Lastly, model systems such as these are increasing our understanding of structure and organization in cell membranes, and how they result in the emergence of functionality at the nanoscale.« less

Centerband-only-detection-of-exchange (31)P nuclear magnetic resonance and phospholipid lateral diffusion: theory, simulation and experiment.

PubMed

Lai, Angel; Saleem, Qasim; Macdonald, Peter M

2015-10-14

Centerband-only-detection-of-exchange (CODEX) (31)P NMR lateral diffusion measurements were performed on dimyristoylphosphatidylcholine (DMPC) assembled into large unilamellar spherical vesicles. Optimization of sample and NMR acquisition conditions provided significant sensitivity enhancements relative to an earlier first report (Q. Saleem, A. Lai, H. Morales, and P. M. Macdonald, Chem. Phys. Lipids, 2012, 165, 721). An analytical description was developed that permitted the extraction of lateral diffusion coefficients from CODEX data, based on a Gaussian-diffusion-on-a-sphere model (A. Ghosh, J. Samuel, and S. Sinha, Europhys. Lett., 2012, 98, 30003-p1) as relevant to CODEX (31)P NMR measurements on a population of spherical unilamellar phospholipid bilayer vesicles displaying a distribution of vesicle radii.

[Regulation of immune responses by exosomes derived from antigen presenting cells].

PubMed

Maravillas-Montero, José Luis; Martínez-Cortés, Ismael

2017-01-01

Cells release several biomolecules to the extracellular environment using them as a communication alternative with neighbor cells. Besides these molecules, cells also release more complex elements, like vesicles; structures composed of a lipidic bilayer with transmembrane proteins that protect a hydrophilic content. Exosomes are a small subtype of vesicles (30-150 nm), produced by many cell types, such as tumor cells, neurons, epithelial cells and immune cells. Included in this last group, antigen presenting cells produce exosomes that contain different types of molecules depending on their activation and/or maturation state. In recent years there has been an exponential interest in exosomes due to the recent evidences that show the immunomodulatory properties of these vesicles and therefore, their great potential in diagnostic approaches and development of therapies for different inflammation-associated pathologies.

Reconstitution of SNARE proteins into solid-supported lipid bilayer stacks and X-ray structure analysis.

PubMed

Xu, Yihui; Kuhlmann, Jan; Brennich, Martha; Komorowski, Karlo; Jahn, Reinhard; Steinem, Claudia; Salditt, Tim

2018-02-01

SNAREs are known as an important family of proteins mediating vesicle fusion. For various biophysical studies, they have been reconstituted into supported single bilayers via proteoliposome adsorption and rupture. In this study we extended this method to the reconstitution of SNAREs into supported multilamellar lipid membranes, i.e. oriented multibilayer stacks, as an ideal model system for X-ray structure analysis (X-ray reflectivity and diffraction). The reconstitution was implemented through a pathway of proteomicelle, proteoliposome and multibilayer. To monitor the structural evolution in each step, we used small-angle X-ray scattering for the proteomicelles and proteoliposomes, followed by X-ray reflectivity and grazing-incidence small-angle scattering for the multibilayers. Results show that SNAREs can be successfully reconstituted into supported multibilayers, with high enough orientational alignment for the application of surface sensitive X-ray characterizations. Based on this protocol, we then investigated the effect of SNAREs on the structure and phase diagram of the lipid membranes. Beyond this application, this reconstitution protocol could also be useful for X-ray analysis of many further membrane proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

Determination of Structural Topology of a Membrane Protein in Lipid -Bilayers using Polarization Optimized Experiments (POE) for Static and MAS Solid State NMR Spectroscopy

PubMed Central

Mote, Kaustubh R.; Gopinath, T.; Veglia, Gianluigi

2013-01-01

The low sensitivity inherent to both the static and magic angle spinning techniques of solid-state NMR (ssNMR) spectroscopy has thus far limited the routine application of multidimensional experiments to determine the structure of membrane proteins in lipid bilayers. Here, we demonstrate the advantage of using a recently developed class of experiments, polarization optimized experiments (POE), for both static and MAS spectroscopy to achieve higher sensitivity and substantial time-savings for 2D and 3D experiments. We used sarcolipin, a single pass membrane protein, reconstituted in oriented bicelles (for oriented ssNMR) and multilamellar vesicles (for MAS ssNMR) as a benchmark. The restraints derived by these experiments are then combined into a hybrid energy function to allow simultaneous determination of structure and topology. The resulting structural ensemble converged to a helical conformation with a backbone RMSD ∼ 0.44 Å, a tilt angle of 24° ± 1°, and an azimuthal angle of 55° ± 6°. This work represents a crucial first step toward obtaining high-resolution structures of large membrane proteins using combined multidimensional O-ssNMR and MAS-ssNMR. PMID:23963722

Examining protein-lipid interactions in model systems with a new squarylium fluorescent dye.

PubMed

Ioffe, Valeriya M; Gorbenko, Galyna P; Tatarets, Anatoliy L; Patsenker, Leonid D; Terpechnig, Ewald A

2006-07-01

The applicability of newly synthesized squarylium dye Sq to probing the changes in physical characteristics of lipid bilayer on the formation of protein-lipid complexes has been evaluated. Lipid vesicles composed of zwitterionic phospholipid phosphatidylcholine (PC) and its mixtures with positively charged detergent cetyltrimethylammonium bromide (CTAB), anionic phospholipid cardiolipin (CL), and cholesterol (Chol) were employed as lipid component of model membrane systems while protein constituent was represented by lysozyme (Lz). Fluorescence intensity of Sq was found to decrease on Lz association with lipid bilayer. This effect was observed in all kinds of model systems suggesting that Sq is sensitive to modification of lipid bilayer physical properties on hydrophobic protein-lipid interactions. It was found that Sq spectral response to variations in Chol content depends on relative contributions of electrostatic and hydrophobic components of Lz-membrane binding.

Micelle-vesicle-micelle transition in aqueous solution of anionic surfactant and cationic imidazolium surfactants: Alteration of the location of different fluorophores.

PubMed

Dutta, Rupam; Ghosh, Surajit; Banerjee, Pavel; Kundu, Sangita; Sarkar, Nilmoni

2017-03-15

The presence of different surfactants can alter the physicochemical behaviors of aqueous organized assemblies. In this article, we have investigated the location of hydrophobic molecule (Coumarin 153, C153) and hydrophilic molecule (Rhodamine 6G perchlorate, R6G) during micelle-vesicle-micelle transition in aqueous medium in presence of anionic surfactant, sodium dodecylbenzenesulfonate (SDBS) and cationic imidazolium-based surfactant, 1-alkyl-3-methylimidazolium chloride (C n mimCl; n=12, 16). Initially, the physicochemical properties of anionic micellar solution of SDBS has been investigated in presence of imidazolium-based surfactant, C n mimCl (n=12, 16) in aqueous medium by visual observation, turbidity measurement, zeta potential (ζ), dynamics light scattering (DLS), and transmission electron microscopy (TEM). Zeta potential (ζ) measurement clearly indicates that the incorporation efficiency of C 16 mimCl in SDBS micelle is better than the other one due to the involvement of strong hydrophobic as well as electrostatic interaction between the two associated molecules. Turbidity and DLS measurements clearly suggest the formation of vesicles over a wide range of concentration. Finally, the rotational motion of C153 and R6G has also been monitored at different mole fractions of C n mimCl in SDBS-C n mimCl (n=12, 16) solution mixtures. The hydrophobic C153 molecules preferentially located in the bilayer region of vesicle, whereas hydrophilic R6G can be solubilized at surface of the bilayer, inner water pool or outer surface of vesicles. It is observed that rotational motion of R6G is altered significantly in SDBS-C n mimCl solution mixtures in presence of different mole fractions of C n mimCl. Additionally, the translational diffusion motion of R6G is monitored using fluorescence correlation spectroscopy (FCS) techniques to get a complete scenario about the location and translational diffusion of R6G. Copyright © 2016 Elsevier Inc. All rights reserved.

Nanodesign of olein vesicles for the topical delivery of the antioxidant resveratrol.

PubMed

Pando, Daniel; Caddeo, Carla; Manconi, Maria; Fadda, Anna Maria; Pazos, Carmen

2013-08-01

The ex-vivo percutaneous absorption of the natural antioxidant resveratrol in liposomes and niosomes was investigated. The influence of vesicle composition on their physicochemical properties and stability was evaluated. Liposomes containing resveratrol were formulated using soy phosphatidylcholine (Phospholipon90G). Innovative niosomes were formulated using mono- or diglycerides: glycerol monooleate (Peceol) and polyglyceryl-3 dioleate (Plurol OleiqueCC), respectively, two suitable skin-compatible oleins used in pharmaceutical formulations as penetration enhancers. Small, negatively charged vesicles with a mean size of approximately 200 nm were prepared. The accelerated stability of vesicles was evaluated using Turbiscan Lab Expert, and the bilayer deformability was also assessed. Ex-vivo transdermal experiments were carried out in Franz diffusion cells, on newborn pig skin, to study the influence of the different vesicle formulations on resveratrol skin delivery. Results indicated a high cutaneous accumulation and a low transdermal delivery of resveratrol, especially when Peceol niosomes were used. Overall, niosomes formulated with Plurol oleique or Peceol showed a better behaviour than liposomes in the cutaneous delivery of resveratrol. © 2013 Royal Pharmaceutical Society.

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.

Extracellular Vesicles Present in Human Ovarian Tumor Microenvironments Induce a Phosphatidylserine Dependent Arrest in the T Cell Signaling Cascade

PubMed Central

Kelleher, Raymond J.; Balu-Iyer, Sathy; Loyall, Jenni; Sacca, Anthony J.; Shenoy, Gautam N.; Peng, Peng; Iyer, Vandana; Fathallah, Anas M.; Berenson, Charles S.; Wallace, Paul K.; Tario, Joseph; Odunsi, Kunle; Bankert, Richard B.

2015-01-01

The identification of immunosuppressive factors within human tumor microenvironments, and the ability to block these factors, would be expected to enhance patients’ anti-tumor immune responses. We previously established that an unidentified factor, or factors, present in ovarian tumor ascites fluids reversibly inhibited the activation of T cells by arresting the T cell signaling cascade. Ultracentrifugation of the tumor ascites fluid has now revealed a pellet that contains small extracellular vesicles (EV) with an average diameter of 80nm. The T cell arrest was determined to be causally linked to phosphatidylserine (PS) that is present on the outer leaflet of the vesicle bilayer, as a depletion of PS expressing EV or a blockade of PS with anti-PS antibody significantly inhibits the vesicle induced signaling arrest. The inhibitory EV were also isolated from solid tumor tissues. The presence of immune suppressive vesicles in the microenvironments of ovarian tumors and our ability to block their inhibition of T cell function represent a potential therapeutic target for patients with ovarian cancer. PMID:26112921

Geometry of phase-separated domains in phospholipid bilayers by diffraction-contrast electron microscopy.

PubMed Central

Hui, S W

1981-01-01

The sizes and shapes of solidus (gel) phase domains in the hydrated molecular bilayers of dilauroylphosphatidylcholine/dipalmitoylphasphatidylcholine (DLPC/DPPC) (1:1) and phosphatidylserine (PS)/DPPC (1:2) are visualized directly by low dose diffraction-contrast electron microscopy. The temperature and humidity of the bilayers are controlled by an environmental chamber set in an electron microscope. The contrast between crystalline domains is enhanced by electron optical filtering of the diffraction patterns of the bilayers. The domains are seen as a patchwork in the plane of the bilayer, with an average width of 0.2-0.5 micrometer. The percentage of solidus area measured from diffraction-contrast micrographs at various temperatures agrees in general with those depicted by known phase diagrams. The shape and size of the domains resemble those seen by freeze-fracture in multilamellar vesicles. Temperature-related changes in domain size and in phase boundary per unit area are more pronounced in the less miscible DLPC/DPPC mixture. No significant change in these geometric parameters with temperature is found in the PS/DPPC mixture. Mapping domains by their molecular diffraction signals not only verifies the existance of areas of different molecular packing during phase separation but also provides a quantitative measurement of structural boundaries and defects in lipid bilayers. Images FIGURE 1 FIGURE 3 FIGURE 6 PMID:6894707

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.

Interplay between alkyl chain asymmetry and cholesterol addition in the rigid ion pair amphiphile bilayer systems

NASA Astrophysics Data System (ADS)

Huang, Fong-yin; Chiu, Chi-cheng

2017-01-01

Ion pair amphiphile (IPA), a molecular complex composed of a pair of cationic and anionic surfactants, has been proposed as a novel phospholipid substitute. Controlling the physical stability of IPA vesicles is important for its application developments such as cosmetic and drug deliveries. To investigate the effects of IPA alkyl chain combinations and the cholesterol additive on the structural and mechanical properties of IPA vesicular bilayers, we conducted a series of molecular dynamics studies on the hexadecyltrimethylammonium-dodecylsulfate (HTMA-DS) and dodecyltrimethylammonium-hexadecylsulfate (DTMA-HS) IPA bilayers with cholesterol. We found that both IPA bilayers are in the gel phase at 298 K, consistent with experimental observations. Compared with the HTMA-DS system, the DTMA-HS bilayer has more disordered alkyl chains in the hydrophobic region. When adding cholesterol, it induces alkyl chain ordering around its rigid sterol ring. Yet, cholesterol increases the molecular areas for all species and disturbs the molecular packing near the hydrophilic region and the bilayer core. Cholesterol also promotes the alkyl chain mismatch between the IPA moieties, especially for the DTMA-HS bilayer. The combined effects lead to non-monotonically enhancement of the membrane mechanical moduli for both IPA-cholesterol systems. Furthermore, cholesterol can form H-bonds with the alkylsulfate and thus enhance the contribution of alkylsulfate to the overall mechanical moduli. Combined results provide valuable molecular insights into the roles of each IPA component and the cholesterol on modulating the IPA bilayer properties.

Effect of intra-membrane C60 fullerenes on the modulus of elasticity and the mechanical resistance of gel and fluid lipid bilayers

NASA Astrophysics Data System (ADS)

Zhou, Jihan; Liang, Dehai; Contera, Sonia

2015-10-01

Penetration and partition of C60 to the lipid bilayer core are both relevant to C60 toxicity, and useful to realise C60 biomedical potential. A key aspect is the effect of C60 on bilayer mechanical properties. Here, we present an experimental study on the mechanical effect of the incorporation of C60 into the hydrophobic core of fluid and gel phase zwitterionic phosphatidylcholine (PC) lipid bilayers. We demonstrate its incorporation inside the hydrophobic lipid core and the effect on the packing of the lipids and the vesicle size using a combination of infrared (IR) spectroscopy, atomic force microscopy (AFM) and laser light scattering. Using AFM we measured the Young's modulus of elasticity (E) of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in the absence (presence) of intra-membranous C60 at 24.5 °C. E of fluid phase supported bilayers is not altered by C60, but E increases with incorporation of C60 in gel phase bilayers. The increase is higher for longer hydrocarbon chains: 1.6 times for DPPC and 2 times for DSPC. However the mechanical resistance of gel phase bilayers of curved bilayered structures decreases with the incorporation of C60. Our combined results indicate that C60 causes a decrease in gel phase lipid mobility, i.e. an increase in membrane viscosity.

Polymer-cushioned bilayers. II. An investigation of interaction forces and fusion using the surface forces apparatus.

PubMed Central

Wong, J Y; Park, C K; Seitz, M; Israelachvili, J

1999-01-01

We have created phospholipid bilayers supported on soft polymer "cushions" which act as deformable substrates (see accompanying paper, Wong, J. Y., J. Majewski, M. Seitz, C. K. Park, J. N. Israelachvili, and G. S. Smith. 1999. Biophys. J. 77:1445-1457). In contrast to "solid-supported" membranes, such "soft-supported" membranes can exhibit more natural (higher) fluidity. Our bilayer system was constructed by adsorption of small unilamellar dimyristoylphosphatidylcholine (DMPC) vesicles onto polyethylenimine (PEI)-supported Langmuir-Blodgett lipid monolayers on mica. We used the surface forces apparatus (SFA) to investigate the long-range forces, adhesion, and fusion of two DMPC bilayers both above and below their main transition temperature (T(m) approximately 24 degrees C). Above T(m), hemi-fusion activation pressures of apposing bilayers were considerably smaller than for solid-supported bilayers, e.g., directly supported on mica. After separation, the bilayers naturally re-formed after short healing times. Also, for the first time, complete fusion of two fluid (liquid crystalline) phospholipid bilayers was observed in the SFA. Below T(m) (gel state), very high pressures were needed for hemi-fusion and the healing process became very slow. The presence of the polymer cushion significantly alters the interaction potential, e.g., long-range forces as well as fusion pressures, when compared to solid-supported systems. These fluid model membranes should allow the future study of integral membrane proteins under more physiological conditions. PMID:10465756

Voltage-dependent blockade of muscle Na+ channels by guanidinium toxins

PubMed Central

1984-01-01

Na+ channels from rat muscle plasma membrane vesicles were inserted into neutral planar phospholipid bilayers and were activated by batrachotoxin. Single channel blocking events induced by the addition of various guanidinium toxins were analyzed to derive the rates of channel-toxin association and dissociation. Blocking by tetrodotoxin, saxitoxin, and six natural saxitoxin derivatives containing sulfate or hydroxyl groups were studied. Although the binding affinities vary over 2,000-fold, all of the toxins exhibit identical voltage dependence of the blocking reactions, regardless of the toxin's net charge. The results suggest that the voltage dependence of toxin binding is due to a voltage-dependent conformational equilibrium of the toxin receptor, rather than to direct entry of the charged toxin molecule into the applied transmembrane electric field. PMID:6096479

AC-electric field dependent electroformation of giant lipid vesicles.

PubMed

Politano, Timothy J; Froude, Victoria E; Jing, Benxin; Zhu, Yingxi

2010-08-01

Giant vesicles of larger than 5 microm, which have been of intense interest for their potential as drug delivery vehicles and as a model system for cell membranes, can be rapidly formed from a spin-coated lipid thin film under an electric field. In this work, we explore the AC-field dependent electroformation of giant lipid vesicles in aqueous media over a wide range of AC-frequency from 1 Hz to 1 MHz and peak-to-peak field strength from 0.212 V/mm to 40 V/mm between two parallel conducting electrode surfaces. By using fluorescence microscopy, we perform in-situ microscopic observations of the structural evolution of giant vesicles formed from spin-coated lipid films under varied uniform AC-electric fields. The real-time observation of bilayer bulging from the lipid film, vesicle growth and fusing further examine the critical role of AC-induced electroosmotic flow of surrounding fluids for giant vesicle formation. A rich AC-frequency and field strength phase diagram is obtained experimentally to predict the AC-electroformation of giant unilamellar vesicles (GUVs) of l-alpha-phosphatidylcholine, where a weak dependence of vesicle size on AC-frequency is observed at low AC-field voltages, showing decreased vesicle size with a narrowed size distribution with increased AC-frequency. Formation of vesicles was shown to be constrained by an upper field strength of 10 V/mm and an upper AC-frequency of 10 kHz. Within these parameters, giant lipid vesicles were formed predominantly unilamellar and prevalent across the entire electrode surfaces. Copyright 2010 Elsevier B.V. All rights reserved.

Structure of the PTEN-like region of auxilin, a detector of clathrin-coated vesicle budding

PubMed Central

Guan, Rong; Han, Dai; Harrison, Stephen C.; Kirchhausen, Tomas

2010-01-01

Auxilin, a J-domain containing protein, recruits the Hsc70 uncoating ATPase to newly budded clathrin-coated vesicles. The timing of auxilin arrival determines that uncoating will commence only after the clathrin lattice has fully assembled and after membrane fission is complete. Auxilin has a region resembling PTEN, a PI3P phosphatase. We have determined the crystal structure of this region of bovine auxilin 1; it indeed resembles PTEN closely. A change in the structure of the P-loop accounts for the lack of phosphatase activity. Inclusion of phosphatidylinositol phosphates substantially enhances liposome binding by wild-type auxilin, but not by various mutants bearing changes in loops of the C2 domain. Nearly all these mutations also prevent recruitment of auxilin to newly budded coated vesicles. We propose a specific geometry for auxilin association with a membrane bilayer and discuss implications of this model for the mechanism by which auxilin detects separation of a vesicle from its parent membrane. PMID:20826345

Recent advances in non-ionic surfactant vesicles (niosomes): self-assembly, fabrication, characterization, drug delivery applications and limitations.

PubMed

Abdelkader, Hamdy; Alani, Adam W G; Alany, Raid G

2014-03-01

Non-ionic surfactant vesicles, simply known as niosomes are synthetic vesicles with potential technological applications. Niosomes have the same potential advantages of phospholipid vesicles (liposomes) of being able to accommodate both water soluble and lipid soluble drug molecules control their release and as such serve as versatile drug delivery devices of numerous applications. Additionally, niosomes can be considered as more economically, chemically, and occasionally physically stable alternatives to liposomes. Niosomes can be fabricated using simple methods of preparations and from widely used surfactants in pharmaceutical technology. Many reports have discussed niosomes in terms of physicochemical properties and their applications as drug delivery systems. In this report, a brief and simplified summary of different theories of self-assembly will be given. Furthermore manufacturing methods, physical characterization techniques, bilayer membrane additives, unconventional niosomes (discomes, proniosomes, elastic and polyhedral niosomes), their recent applications as drug delivery systems, limitations and directions for future research will be discussed.

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

Coupled diffusion in lipid bilayers upon close approach

DOE PAGES

Pronk, Sander; Lindahl, Erik; Kasson, Peter M.

2014-12-23

Biomembrane interfaces create regions of slowed water dynamics in their vicinity. When two lipid bilayers come together, this effect is further accentuated, and the associated slowdown can affect the dynamics of larger-scale processes such as membrane fusion. We have used molecular dynamics simulations to examine how lipid and water dynamics are affected as two lipid bilayers approach each other. These two interacting fluid systems, lipid and water, both slow and become coupled when the lipid membranes are separated by a thin water layer. We show in particular that the water dynamics become glassy, and diffusion of lipids in the apposedmore » leaflets becomes coupled across the water layer, while the “outer” leaflets remain unaffected. This dynamic coupling between bilayers appears mediated by lipid–water–lipid hydrogen bonding, as it occurs at bilayer separations where water–lipid hydrogen bonds become more common than water–water hydrogen bonds. We further show that such coupling occurs in simulations of vesicle–vesicle fusion prior to the fusion event itself. As a result, such altered dynamics at membrane–membrane interfaces may both stabilize the interfacial contact and slow fusion stalk formation within the interface region.« less

A phase of liposomes with entangled tubular vesicles

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

Chiruvolu, S.; Naranjo, E.; Warriner, H.E.

1994-11-18

An equilibrium phase belonging to the family of bilayer liposomes in ternary mixtures of dimyristoylphosphatidylcholine (DMPC), water, and geraniol (a biological alcohol derived from oil-soluble vitamins that acts as a cosurfactant) has been identified. Electron and optical microscopy reveal the phase, labeled L{sub tv}, to be composed of highly entangled tubular vesicles. In situ x-ray diffraction confirms that the tubule walls are multilamellar with the lipids in the chain-melted state. Macroscopic observations show that the L{sub tv} phase coexists with the well-known L{sub 4} phase of spherical vesicles and a bulk L{sub {alpha}} phase. However, the defining characteristic of themore » L{sub tv} phase is the Weissenberg rod climbing effect under shear, which results from its polymer-like entangled microstructure. 26 refs., 5 figs.« less

Partition thermodynamics of ionic surfactants between phosphatidylcholine vesicle and water phases

NASA Astrophysics Data System (ADS)

Chu, Shin-Chi; Hung, Chia-Hui; Wang, Shun-Cheng; Tsao, Heng-Kwong

2003-08-01

The partition of ionic surfactants (sodium alkyl sulfate and alkyl trimethyl ammonium bromide) between phosphatidylcholine vesicles and aqueous phase is investigated by simple conductometry under different temperatures. The experimental results can be well represented by the proposed regular solution theory and the thermodynamic parameters satisfy the thermodynamic consistency. The deviation from ideal partition is manifested through the effective interaction energy between lipid and surfactant wb, which is O(kT) large. It is found that wb rises as the alkyl chain is decreased for a specified head group. This is attributed to significant mismatch of chain lengths between surfactant and lipid molecules. The partition coefficient K declines with increasing temperature. The energy barrier from bilayer to aqueous phase, Δμ/kT∝ln K, is in the range of 16-26 kJ/mol. As the alkyl chain length is decreased for a given head group, Δμ is lowered by 1.3-1.5 kJ/mol per methylene group. Two independent analyses are employed to confirm this result. Using the thermodynamic parameters determined from experiments, the internal energy, entropy, and free energy of the partition process can be derived. Partition is essentially driven by the internal energy gain. The solubilizing ability, which is represented by the maximum surfactant-lipid ratio in the bilayer, Reb also decreases in accord with the K parameter. It is because the change in temperature influences the surfactant incorporation into the bilayer more than the formation of micelles.

Ultrasound, liposomes, and drug delivery: principles for using ultrasound to control the release of drugs from liposomes.

PubMed

Schroeder, Avi; Kost, Joseph; Barenholz, Yechezkel

2009-11-01

Ultrasound is used in many medical applications, such as imaging, blood flow analysis, dentistry, liposuction, tumor and fibroid ablation, and kidney stone disruption. In the past, low frequency ultrasound (LFUS) was the main method to downsize multilamellar (micron range) vesicles into small (nano scale) unilamellar vesicles. Recently, the ability of ultrasound to induce localized and controlled drug release from liposomes, utilizing thermal and/or mechanical effects, has been shown. This review, deals with the interaction of ultrasound with liposomes, focusing mainly on the mechanical mechanism of drug release from liposomes using LFUS. The effects of liposome lipid composition and physicochemical properties, on one hand, and of LFUS parameters, on the other, on liposomal drug release, are addressed. Acoustic cavitation, in which gas bubbles oscillate and collapse in the medium, thereby introducing intense mechanical strains, increases release substantially. We suggest that the mechanism of release may involve formation and collapse of small gas nuclei in the hydrophobic region of the lipid bilayer during exposure to LFUS, thereby inducing the formation of transient pores through which drugs are released. Introducing PEG-lipopolymers to the liposome bilayer enhances responsivity to LFUS, most likely due to absorption of ultrasonic energy by the highly hydrated PEG headgroups. The presence of amphiphiles, such as phospholipids with unsaturated acyl chains, which destabilize the lipid bilayer, also increases liposome susceptibility to LFUS. Application of these principles to design highly LFUS-responsive liposomes is discussed.

In vitro synthesis and stabilization of amorphous calcium carbonate (ACC) nanoparticles within liposomes

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

Tester, Chantel C.; Brock, Ryan E.; Wu, Ching-Hsuan

2012-02-07

We show that amorphous calcium carbonate (ACC) can be synthesized in phospholipid bilayer vesicles (liposomes). Liposome-encapsulated ACC nanoparticles are stable against aggregation, do not crystallize for at least 20 h, and are ideally suited to investigate the influence of lipid chemistry, particle size, and soluble additives on ACC in situ.

Toxins and antimicrobial peptides: interactions with membranes

NASA Astrophysics Data System (ADS)

Schlamadinger, Diana E.; Gable, Jonathan E.; Kim, Judy E.

2009-08-01

The innate immunity to pathogenic invasion of organisms in the plant and animal kingdoms relies upon cationic antimicrobial peptides (AMPs) as the first line of defense. In addition to these natural peptide antibiotics, similar cationic peptides, such as the bee venom toxin melittin, act as nonspecific toxins. Molecular details of AMP and peptide toxin action are not known, but the universal function of these peptides to disrupt cell membranes of pathogenic bacteria (AMPs) or a diverse set of eukaryotes and prokaryotes (melittin) is widely accepted. Here, we have utilized spectroscopic techniques to elucidate peptide-membrane interactions of alpha-helical human and mouse AMPs of the cathelicidin family as well as the peptide toxin melittin. The activity of these natural peptides and their engineered analogs was studied on eukaryotic and prokaryotic membrane mimics consisting of <200-nm bilayer vesicles composed of anionic and neutral lipids as well as cholesterol. Vesicle disruption, or peptide potency, was monitored with a sensitive fluorescence leakage assay. Detailed molecular information on peptidemembrane interactions and peptide structure was further gained through vibrational spectroscopy combined with circular dichroism. Finally, steady-state fluorescence experiments yielded insight into the local environment of native or engineered tryptophan residues in melittin and human cathelicidin embedded in bilayer vesicles. Collectively, our results provide clues to the functional structures of the engineered and toxic peptides and may impact the design of synthetic antibiotic peptides that can be used against the growing number of antibiotic-resistant pathogens.

Numerical computations of the dynamics of fluidic membranes and vesicles

NASA Astrophysics Data System (ADS)

Barrett, John W.; Garcke, Harald; Nürnberg, Robert

2015-11-01

Vesicles and many biological membranes are made of two monolayers of lipid molecules and form closed lipid bilayers. The dynamical behavior of vesicles is very complex and a variety of forms and shapes appear. Lipid bilayers can be considered as a surface fluid and hence the governing equations for the evolution include the surface (Navier-)Stokes equations, which in particular take the membrane viscosity into account. The evolution is driven by forces stemming from the curvature elasticity of the membrane. In addition, the surface fluid equations are coupled to bulk (Navier-)Stokes equations. We introduce a parametric finite-element method to solve this complex free boundary problem and present the first three-dimensional numerical computations based on the full (Navier-)Stokes system for several different scenarios. For example, the effects of the membrane viscosity, spontaneous curvature, and area difference elasticity (ADE) are studied. In particular, it turns out, that even in the case of no viscosity contrast between the bulk fluids, the tank treading to tumbling transition can be obtained by increasing the membrane viscosity. Besides the classical tank treading and tumbling motions, another mode (called the transition mode in this paper, but originally called the vacillating-breathing mode and subsequently also called trembling, transition, and swinging mode) separating these classical modes appears and is studied by us numerically. We also study how features of equilibrium shapes in the ADE and spontaneous curvature models, like budding behavior or starfish forms, behave in a shear flow.

Silica metal-oxide vesicles catalyze comprehensive prebiotic chemistry.

PubMed

Bizzarri, Bruno Mattia; Botta, Lorenzo; Pérez-Valverde, Maritza Iveth; Saladino, Raffaele; Di Mauro, Ernesto; Garcia Ruiz, Juan Manuel

2018-03-30

It has recently been demonstrated that mineral self-assembled structures catalyzing prebiotic chemical reactions may form in natural waters derived from serpentinization, a geological process widespread in the early stages of Earth-like planets. We have synthesized self-assembled membranes by mixing microdrops of metal solutions with alkaline silicate solutions in the presence of formamide (NH2CHO), a single carbon molecule, at 80ºC. We found that these bilayer membranes, made of amorphous silica and metal oxide-hydroxide nanocrystals, catalyze the condensation of formamide, yielding the four nucleobases of RNA, three aminoacids and several carboxylic acids in a single pot experiment. Besides manganese, iron and magnesium, two abundant elements in the earliest Earth crust that are key in serpentinization reactions, are enough to produce all these biochemical compounds. These results suggest that the transition from inorganic geochemistry to prebiotic organic chemistry is common on a universal scale and, most probably, earlier than ever thought for our planet. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Lateral diffusion of proteins on supported lipid bilayers: additive friction of synaptotagmin 7 C2A-C2B tandem domains.

PubMed

Vasquez, Joseph K; Chantranuvatana, Kan; Giardina, Daniel T; Coffman, Matthew D; Knight, Jefferson D

2014-12-23

The synaptotagmin (Syt) family of proteins contains tandem C2 domains, C2A and C2B, which bind membranes in the presence of Ca(2+) to trigger vesicle fusion during exocytosis. Despite recent progress, the role and extent of interdomain interactions between C2A and C2B in membrane binding remain unclear. To test whether the two domains interact on a planar lipid bilayer (i.e., experience thermodynamic interdomain contacts), diffusion of fluorescent-tagged C2A, C2B, and C2AB domains from human Syt7 was measured using total internal reflection fluorescence microscopy with single-particle tracking. The C2AB tandem exhibits a lateral diffusion constant approximately half the value of the isolated single domains and does not change when additional residues are engineered into the C2A-C2B linker. This is the expected result if C2A and C2B are separated when membrane-bound; theory predicts that C2AB diffusion would be faster if the two domains were close enough together to have interdomain contact. Stopped-flow measurements of membrane dissociation kinetics further support an absence of interdomain interactions, as dissociation kinetics of the C2AB tandem remain unchanged when rigid or flexible linker extensions are included. Together, the results suggest that the two C2 domains of Syt7 bind independently to planar membranes, in contrast to reported interdomain cooperativity in Syt1.

Ligand Extraction Properties of the GM2 Activator Protein and Its Interactions with Lipid Vesicles

PubMed Central

Ran, Yong; Fanucci, Gail E.

2009-01-01

Abstract The GM2 activator protein (GM2AP) is an accessory protein required for the enzymatic conversion of GM2 to GM3 by hydrolases in the lysosomal compartments of cells. Here, GM2AP interactions with lipid vesicles are investigated by sucrose-loaded vesicle sedimentation and gel filtration assays, and the effects of pH and lipid composition on membrane binding and lipid extraction are characterized. The sedimentation experiments allow for facile quantification of the percentage of protein in solution and on the bilayer surface, with detailed analysis of the protein:lipid complex that remains in solution. Optimum binding and ligand extraction is found for pH 4.8 where <15% of the protein remains surface associated regardless of the lipid composition. In addition to extracting GM2, we find that GM2AP readily extracts dansyl-headgroup-labeled lipids as well as other phospholipids from vesicles. The ability of GM2AP to extract dansyl-DHPE from vesicles is altered by pH and the specific ligand GM2. Although the unique endosomal lipid, bis(monoacylglycero)phosphate, is not required for ligand extraction, it does enhance the extraction efficiency of GM2 when cholesterol is present in the vesicles. PMID:19580763

Ligand extraction properties of the GM2 activator protein and its interactions with lipid vesicles.

PubMed

Ran, Yong; Fanucci, Gail E

2009-07-08

The GM2 activator protein (GM2AP) is an accessory protein required for the enzymatic conversion of GM2 to GM3 by hydrolases in the lysosomal compartments of cells. Here, GM2AP interactions with lipid vesicles are investigated by sucrose-loaded vesicle sedimentation and gel filtration assays, and the effects of pH and lipid composition on membrane binding and lipid extraction are characterized. The sedimentation experiments allow for facile quantification of the percentage of protein in solution and on the bilayer surface, with detailed analysis of the protein:lipid complex that remains in solution. Optimum binding and ligand extraction is found for pH 4.8 where <15% of the protein remains surface associated regardless of the lipid composition. In addition to extracting GM2, we find that GM2AP readily extracts dansyl-headgroup-labeled lipids as well as other phospholipids from vesicles. The ability of GM2AP to extract dansyl-DHPE from vesicles is altered by pH and the specific ligand GM2. Although the unique endosomal lipid, bis(monoacylglycero)phosphate, is not required for ligand extraction, it does enhance the extraction efficiency of GM2 when cholesterol is present in the vesicles.

Cholesterol affects the interaction between an ionic liquid and phospholipid vesicles. A study by differential scanning calorimetry and nanoplasmonic sensing.

PubMed

Russo, Giacomo; Witos, Joanna; Rantamäki, Antti H; Wiedmer, Susanne K

2017-12-01

The present work aims at studying the interactions between cholesterol-rich phosphatidylcholine-based lipid vesicles and trioctylmethylphosphonium acetate ([P 8881 ][OAc]), a biomass dissolving ionic liquid (IL). The effect of cholesterol was assayed by using differential scanning calorimetry (DSC) and nanoplasmonic sensing (NPS) measurement techniques. Cholesterol-enriched dipalmitoyl-phosphatidylcholine vesicles were exposed to different concentrations of the IL, and the derived membrane perturbation was monitored by DSC. The calorimetric data could suggest that the binding and infiltration of the IL are delayed in the vesicles containing cholesterol. To clarify our findings, NPS was applied to quantitatively follow the resistance of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine incorporating 0, 10, and 50mol% of cholesterol toward the IL exposure over time. The membrane perturbation induced by different concentrations of IL was found to be a concentration dependent process on cholesterol-free lipid vesicles. Moreover, our results showed that lipid depletion in cholesterol-enriched lipid vesicles is inversely proportional to the increasing amount of cholesterol in the vesicles. These findings support that cholesterol-rich lipid bilayers are less susceptible toward membrane disrupting agents as compared to membranes that do not incorporate any sterols. This probably occurs because cholesterol tightens the phospholipid acyl chain packing of the plasma membranes, increasing their resistance and reducing their permeability. Copyright © 2017 Elsevier B.V. All rights reserved.

Polyhedral protein cages encase synaptic vesicles and participate in their attachment to the active zone.

PubMed

Zampighi, G A; Fisher, R S

1997-08-01

In an effort to elucidate the interactions between synaptic vesicles and the membrane of the active zone, we have investigated the structure of interneuronal asymmetric synapses in the neocortex of adult rats using thin-sectioning, freeze-fracture, and negative staining electron microscopy. We identified three subtypes of spherical synaptic vesicles. Type I were agranular vesicles of 47.5 +/- 3.8 nm (mean SD, n = 24) in diameter usually seen aggregated in clusters in the presynaptic bouton. Type II synaptic vesicles were composed of a approximately 45-nm-diameter lipid bilayer sphere encased in a cage 77 +/- 4.6 nm (mean SD, n = 42) in diameter. The cage was composed of open-faced pentamers 20-22 nm/side arranged as a regular polyhedron. Type II caged vesicles were found in clusters at the boutons, adhered to the active zone, and were also present in axons. Type III synaptic vesicles appeared as electron-dense spheres 60-75 nm in diameter abutted to the membrane of the active zone. Clathrin-coated vesicles and pits of 116.6 +/- 9 nm (mean SD, n = 14) in diameter were also present in both the pre- and postsynaptic sides. Freeze-fracture showed that some intrinsic membrane proteins in the active zone were arranged as pentamers exhibiting the same dimension of those forming cages (approximately 22 nm/side). From these data, we concluded that: (a) the presynaptic bouton contains a heterogeneous population of "caged" and "plain" synaptic vesicles and (b) type II synaptic vesicles bind to receptors in the active zone. Therefore, current models of transmitter release should take into account the substantial heterogeneity of the vesicle population and the binding of vesicular cages to the membrane of the active zone.

Synthesis and characterization of tethered lipid assemblies for membrane protein reconstitution (Review).

PubMed

Veneziano, Rémi; Rossi, Claire; Chenal, Alexandre; Brenner, Catherine; Ladant, Daniel; Chopineau, Joël

2017-09-28

Biological membranes and their related molecular mechanisms are essential for all living organisms. Membranes host numerous proteins and are responsible for the exchange of molecules and ions, cell signaling, and cell compartmentation. Indeed, the plasma membrane delimits the intracellular compartment from the extracellular environment and intracellular membranes. Biological membranes also play a major role in metabolism regulation and cellular physiology (e.g., mitochondrial membranes). The elaboration of membrane based biomimetic systems allows us to reconstitute and investigate, in controlled conditions, biological events occurring at the membrane interface. A whole variety of model membrane systems have been developed in the last few decades. Among these models, supported membranes were developed on various hydrophilic supports. The use of solid supports enables the direct use of surface sensitive techniques (e.g., surface plasmon resonance, quartz crystal microbalance, and atomic force microscopy) to monitor and quantify events occurring at the membrane surface. Tethered bilayer membranes (tBLMs) could be considered as an achievement of the first solid supported membranes described by the McConnell group. Tethered bilayers on solid supports were designed to delimit an inside compartment from an outside one. They were used for measuring interactions with ligands or incorporating large membrane proteins or complexes without interference with the support. In this context, the authors developed an easy concept of versatile tBLMs assembled on amino coated substrates that are formed upon the vesicle fusion rupture process applicable to protein-free vesicles as well as proteoliposomes. The phospholipid bilayer (natural or synthetic lipids) incorporated 5% of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly ethylene glycol-N-hydroxy succinimide to ensure the anchorage of the bilayer to the amino coated surface. The conditions for the formation of tBLMs on amino-coated gold and glass were optimized for protein-free vesicles. This biomimetic membrane delimits an inside "trans" compartment separated from an outside reservoir "cis." Using this tBLM construction, the authors were interested in deciphering two complex molecular mechanisms involving membrane-associated proteins. The first one concerns two mitochondrial proteins, i.e., the porin voltage dependent anion channel (VDAC) embedded in the outer membrane and the nucleotide transporter (adenine nucleotide translocase) that interacts dynamically during mitochondrial pathophysiology. The purified VDAC porin was first reconstituted in proteoliposomes that were subsequently assembled on an amino coated support to form a biomimetic membrane. As a major result, VDAC was reconstituted in this tBLM and calcium channeling was demonstrated across the lipid bilayer. The same two-compartment biomimetic membrane design was further engineered to study the translocation mechanism of a bacterial toxin, the adenylate cyclase toxin, CyaA, from Bordetella pertussis. As a result, the authors developed an elegant in vitro translocation toolkit applicable to potentially a large panel of proteins transported across membranes.

Investigating Hydrophilic Pores in Model Lipid Bilayers using Molecular Simulations: Correlating Bilayer Properties with Pore Formation Thermodynamics

PubMed Central

Hu, Yuan; Sinha, Sudipta Kumar

2015-01-01

Cell-penetrating and antimicrobial peptides show remarkable ability to translocate across physiological membranes. Along with factors such as electric potential induced-perturbations of membrane structure and surface tension effects, experiments invoke pore-like membrane configurations during the solute transfer process into vesicles and cells. The initiation and formation of pores are associated with a non-trivial free energy cost, thus necessitating consideration of the factors associated with pore formation and attendant free energetics. Due to experimental and modeling challenges related to the long timescales of the translocation process, we use umbrella-sampling molecular dynamics simulations with a lipid-density based order parameter to investigate membrane pore-formation free energy employing Martini coarse-grained models. We investigate structure and thermodynamic features of the pore in 18 lipids spanning a range of head-groups, charge states, acyl chain lengths and saturation. We probe the dependence of pore-formation barriers on area per lipid, lipid bilayer thickness, membrane bending rigidities in three different lipid classes. The pore formation free energy in pure bilayers and peptide translocating scenarios are significantly coupled with bilayer thickness. Thicker bilayers require more reversible work to create pores. Pore formation free energy is higher in peptide-lipid systems relative to the peptide-free lipid systems due to penalties to maintain solvation of charged hydrophilic solutes within the membrane environment. PMID:25614183

Investigating Hydrophilic Pores in Model Lipid Bilayers Using Molecular Simulations: Correlating Bilayer Properties with Pore-Formation Thermodynamics.

PubMed

Hu, Yuan; Sinha, Sudipta Kumar; Patel, Sandeep

2015-06-23

Cell-penetrating and antimicrobial peptides show a remarkable ability to translocate across physiological membranes. Along with factors such as electric-potential-induced perturbations of membrane structure and surface tension effects, experiments invoke porelike membrane configurations during the solute transfer process into vesicles and cells. The initiation and formation of pores are associated with a nontrivial free-energy cost, thus necessitating a consideration of the factors associated with pore formation and the attendant free energies. Because of experimental and modeling challenges related to the long time scales of the translocation process, we use umbrella sampling molecular dynamics simulations with a lipid-density-based order parameter to investigate membrane-pore-formation free energy employing Martini coarse-grained models. We investigate structure and thermodynamic features of the pore in 18 lipids spanning a range of headgroups, charge states, acyl chain lengths, and saturation. We probe the dependence of pore-formation barriers on the area per lipid, lipid bilayer thickness, and membrane bending rigidities in three different lipid classes. The pore-formation free energy in pure bilayers and peptide translocating scenarios are significantly coupled with bilayer thickness. Thicker bilayers require more reversible work to create pores. The pore-formation free energy is higher in peptide-lipid systems than in peptide-free lipid systems due to penalties to maintain the solvation of charged hydrophilic solutes within the membrane environment.

Competition between Bending and Internal Pressure Governs the Mechanics of Fluid Nanovesicles.

PubMed

Vorselen, Daan; MacKintosh, Fred C; Roos, Wouter H; Wuite, Gijs J L

2017-03-28

Nanovesicles (∼100 nm) are ubiquitous in cell biology and an important vector for drug delivery. Mechanical properties of vesicles are known to influence cellular uptake, but the mechanism by which deformation dynamics affect internalization is poorly understood. This is partly due to the fact that experimental studies of the mechanics of such vesicles remain challenging, particularly at the nanometer scale where appropriate theoretical models have also been lacking. Here, we probe the mechanical properties of nanoscale liposomes using atomic force microscopy (AFM) indentation. The mechanical response of the nanovesicles shows initial linear behavior and subsequent flattening corresponding to inward tether formation. We derive a quantitative model, including the competing effects of internal pressure and membrane bending, that corresponds well to these experimental observations. Our results are consistent with a bending modulus of the lipid bilayer of ∼14k b T. Surprisingly, we find that vesicle stiffness is pressure dominated for adherent vesicles under physiological conditions. Our experimental method and quantitative theory represents a robust approach to study the mechanics of nanoscale vesicles, which are abundant in biology, as well as being of interest for the rational design of liposomal vectors for drug delivery.

Invisible liposomes: refractive index matching with sucrose enables flow dichroism assessment of peptide orientation in lipid vesicle membrane.

PubMed

Ardhammar, Malin; Lincoln, Per; Nordén, Bengt

2002-11-26

Valuable information on protein-membrane organization may in principle be obtained from polarized-light absorption (linear dichroism, LD) measurement on shear-aligned lipid vesicle bilayers as model membranes. However, attempts to probe LD in the UV wavelength region (<250 nm) have so far failed because of strong polarized light scattering from the vesicles. Using sucrose to match the refractive index and suppress the light scattering of phosphatidylcholine vesicles, we have been able to detect LD bands also in the peptide-absorbing region (200-230 nm). The potential of refractive index matching in vesicle LD as a general method for studying membrane protein structure was investigated for the membrane pore-forming oligopeptide gramicidin incorporated into the liposome membranes. In the presence of sucrose, the LD signals arising from oriented tryptophan side chains as well as from n-->pi* and pi-->pi* transitions of the amide chromophore of the polypeptide backbone could be studied. The observation of a strongly negative LD for the first exciton transition ( approximately 204 nm) is consistent with a membrane-spanning orientation of two intertwined parallel gramicidin helices, as predicted by coupled-oscillator theory.

Hemifusion and fusion of giant vesicles induced by reduction of inter-membrane distance

NASA Astrophysics Data System (ADS)

Heuvingh, J.; Pincet, F.; Cribier, S.

2004-07-01

Proteins involved in membrane fusion, such as SNARE or influenza virus hemagglutinin, share the common function of pulling together opposing membranes in closer contact. The reduction of inter-membrane distance can be sufficient to induce a lipid transition phase and thus fusion. We have used functionalized lipids bearing DNA bases as head groups incorporated into giant unilamellar vesicles in order to reproduce the reduction of distance between membranes and to trigger fusion in a model system. In our experiments, two vesicles were isolated and brought into adhesion by the mean of micromanipulation; their evolution was monitored by fluorescence microscopy. Actual fusion only occurred in about 5% of the experiments. In most cases, a state of “hemifusion” is observed and quantified. In this state, the outer leaflets of both vesicles' bilayers merged whereas the inner leaflets and the aqueous inner contents remained independent. The kinetics of the lipid probes redistribution is in good agreement with a diffusion model in which lipids freely diffuse at the circumference of the contact zone between the two vesicles. The minimal density of bridging structures, such as stalks, necessary to explain this redistribution kinetics can be estimated.

Optical stretching as a tool to investigate the mechanical properties of lipid bilayers.

PubMed

Solmaz, Mehmet E; Sankhagowit, Shalene; Biswas, Roshni; Mejia, Camilo A; Povinelli, Michelle L; Malmstadt, Noah

2013-10-07

Measurements of lipid bilayer bending modulus by various techniques produce widely divergent results. We attempt to resolve some of this ambiguity by measuring bending modulus in a system that can rapidly process large numbers of samples, yielding population statistics. This system is based on optical stretching of giant unilamellar vesicles (GUVs) in a microfluidic dual-beam optical trap (DBOT). The microfluidic DBOT system is used here to measure three populations of GUVs with distinct lipid compositions. We find that gel-phase membranes are significantly stiffer than liquid-phase membranes, consistent with previous reports. We also find that the addition of cholesterol does not alter the bending modulus of membranes composed of a monounsaturated phospholipid.

Channels Formed by Botulinum, Tetanus, and Diphtheria Toxins in Planar Lipid Bilayers: Relevance to Translocation of Proteins across Membranes

NASA Astrophysics Data System (ADS)

Hoch, David H.; Romero-Mira, Miryam; Ehrlich, Barbara E.; Finkelstein, Alan; Dasgupta, Bibhuti R.; Simpson, Lance L.

1985-03-01

The heavy chains of both botulinum neurotoxin type B and tetanus toxin form channels in planar bilayer membranes. These channels have pH-dependent and voltage-dependent properties that are remarkably similar to those previously described for diphtheria toxin. Selectivity experiments with anions and cations show that the channels formed by the heavy chains of all three toxins are large; thus, these channels could serve as ``tunnel proteins'' for translocation of active peptide fragments. These findings support the hypothesis that the active fragments of botulinum neurotoxin and tetanus toxin, like that of diphtheria toxin, are translocated across the membranes of acidic vesicles.

Adhesion of liposomes: a quartz crystal microbalance study

NASA Astrophysics Data System (ADS)

Lüthgens, Eike; Herrig, Alexander; Kastl, Katja; Steinem, Claudia; Reiss, Björn; Wegener, Joachim; Pignataro, Bruno; Janshoff, Andreas

2003-11-01

Three different systems are presented, exploring the adhesion of liposomes mediated by electrostatic and lipid-protein interactions as well as molecular recognition of ligand receptor pairs. Liposomes are frequently used to gain insight into the complicated processes involving adhesion and subsequent events such as fusion and fission mainly triggered by specific proteins. We combined liposome technology with the quartz crystal microbalance (QCM) technique as a powerful tool to study the hidden interface between the membrane and functionalized surface. Electrostatic attraction and molecular recognition were employed to bind liposomes to the functionalized quartz crystal. The QCM was used to distinguish between adsorption of vesicles and rupture due to strong adhesive forces. Intact vesicles display viscoelastic behaviour, while planar lipid bilayers as a result of vesicle rupture can be modelled by a thin rigid film. Furthermore, the adhesion of cells was modelled successfully by receptor bearing liposomes. Scanning force microscopy was used to confirm the results obtained by QCM measurements.

Layer-by-layer cell membrane assembly

NASA Astrophysics Data System (ADS)

Matosevic, Sandro; Paegel, Brian M.

2013-11-01

Eukaryotic subcellular membrane systems, such as the nuclear envelope or endoplasmic reticulum, present a rich array of architecturally and compositionally complex supramolecular targets that are as yet inaccessible. Here we describe layer-by-layer phospholipid membrane assembly on microfluidic droplets, a route to structures with defined compositional asymmetry and lamellarity. Starting with phospholipid-stabilized water-in-oil droplets trapped in a static droplet array, lipid monolayer deposition proceeds as oil/water-phase boundaries pass over the droplets. Unilamellar vesicles assembled layer-by-layer support functional insertion both of purified and of in situ expressed membrane proteins. Synthesis and chemical probing of asymmetric unilamellar and double-bilayer vesicles demonstrate the programmability of both membrane lamellarity and lipid-leaflet composition during assembly. The immobilized vesicle arrays are a pragmatic experimental platform for biophysical studies of membranes and their associated proteins, particularly complexes that assemble and function in multilamellar contexts in vivo.

Label-free tracking of single extracellular vesicles in a nano-fluidic optical fiber (Conference Presentation)

NASA Astrophysics Data System (ADS)

van der Pol, Edwin; Weidlich, Stefan; Lahini, Yoav; Coumans, Frank A. W.; Sturk, Auguste; Nieuwland, Rienk; Schmidt, Markus A.; Faez, Sanli; van Leeuwen, Ton G.

2016-03-01

Background: Extracellular vesicles, such as exosomes, are abundantly present in human body fluids. Since the size, concentration and composition of these vesicles change during disease, vesicles have promising clinical applications, including cancer diagnosis. However, since ~70% of the vesicles have a diameter <70 nm, detection of single vesicles remains challenging. Thus far, vesicles <70 nm have only be studied by techniques that require the vesicles to be adhered to a surface. Consequently, the majority of vesicles have never been studied in their physiological environment. We present a novel label-free optical technique to track single vesicles <70 nm in suspension. Method: Urinary vesicles were contained within a single-mode light-guiding silica fiber containing a 600 nm nano-fluidic channel. Light from a diode laser (660 nm wavelength) was coupled to the fiber, resulting in a strongly confined optical mode in the nano-fluidic channel, which continuously illuminated the freely diffusing vesicles inside the channel. The elastic light scattering from the vesicles, in the direction orthogonal to the fiber axis, was collected using a microscope objective (NA=0.95) and imaged with a home-built microscope. Results: We have tracked single urinary vesicles as small as 35 nm by elastic light scattering. Please note that vesicles are low-refractive index (n<1.4) particles, which we confirmed by combining data on thermal diffusion and light scattering cross section. Conclusions: For the first time, we have studied vesicles <70 nm freely diffusing in suspension. The ease-of-use and performance of this technique support its potential for vesicle-based clinical applications.

Morphological transitions of brain sphingomyelin are determined by the hydration protocol: ripples re-arrange in plane, and sponge-like networks disintegrate into small vesicles.

PubMed

Meyer, H W; Bunjes, H; Ulrich, A S

1999-06-01

The phase transition of hydrated brain sphingomyelin occurs at around 35 degrees C, which is close to the physiological temperature. Freeze-fracture electron microscopy is used to characterize different gel state morphologies in terms of solid-ordered and liquid-ordered phase states, according to the occurrence of ripples and other higher-dimensional bilayer deformations. Evidently, the natural mixed-chain sphingomyelin does not assume the flat L beta, phase but instead the rippled P beta, phase, with symmetric and asymmetric ripples as well as macroripples and an egg-carton pattern, depending on the incubation conditions. An unexpected difference was observed between samples that are hydrated above and below the phase transition temperature. When the lipid is hydrated at low temperature, a sponge-like network of bilayers is formed in the gel state, next to some normal lamellae. The network loses its ripples during cold-incubation, which indicates the formation of a liquid-ordered (lo) gel phase. Ripples re-appear upon warming and the sponge-like network disintegrates spontaneously and irreversibly into small vesicles above the phase transition.

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

Characterization of a Ca(2+)-dependent anion channel from sheep tracheal epithelium incorporated into planar bilayers.

PubMed Central

Alton, E W; Manning, S D; Schlatter, P J; Geddes, D M; Williams, A J

1991-01-01

1. Anion-selective channels from the apical membrane of respiratory epithelia are involved in the secretion of chloride into the airway lumen. In cystic fibrosis (CF) there is an abnormality of phosphorylation-regulated chloride transport in this tissue, whilst a calcium-dependent pathway appears to function normally. 2. Using incorporation of apical membrane vesicles into planar phospholipid bilayers, we have characterized the most commonly seen anion-selective channel from sheep tracheal epithelium. 3. In symmetrical 200 mM-NaCl solutions the channel showed rectification, with a chord conductance at negative voltages of 107 pS and at positive voltages of 67 pS. The channel characteristically demonstrated subconductance states at 1/3 and 3/4 of the fully open level. Selectivity for chloride over sodium was approximately 6:1. 4. The channel required a minimum of approximately 100 microM-calcium on the presumed cytoplasmic surface (cis) for opening events to be observed. Open probability (Po) of the fully open state was markedly voltage dependent, but little effect of voltage was seen on the 1/3 subconductance state. 5. The relative permeabilities of monovalent anions monitored under bi-ionic conditions gave the following sequence: NO3- greater than I- greater than Cl- = Br- much much greater than F-. The order of conductances in symmetrical solutions was Cl- = NO3- greater than Br- greater than I- much much greater than F-. 6. The chloride channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) produced a dose-related reduction in Po with a flickering block at 10-50 microM and complete block at higher concentrations. 7. ATP produced a dose-related reduction in Po with effects at 1 microM and complete closing at 1 mM. These effects were only seen with addition to the cis chamber. 8. The catalytic subunit of protein kinase A, either when incubated with vesicles prior to incorporation into bilayers, or when added directly to either chamber, produced no effect. 9. Channels with very similar properties were seen from transfected human tracheo-bronchial cells. 10. Recent whole-cell patch-clamp studies have suggested a distinct calcium-activated chloride current in secretory epithelia. The described channel has properties in common with this current and may be a candidate for its single-channel basis. PMID:1726592

NO and CO binding profiles of hemoglobin vesicles as artificial oxygen carriers.

PubMed

Sakai, Hiromi; Sato, Atsushi; Sobolewski, Peter; Takeoka, Shinji; Frangos, John A; Kobayashi, Koichi; Intaglietta, Marcos; Tsuchida, Eishun

2008-10-01

Hemoglobin vesicles (HbVs) are artificial oxygen carriers encapsulating purified and concentrated Hb solution in phospholipid vesicles (liposomes). We examined in-vitro reaction profiles of a formulation of HbV with NO and CO in anaerobic and aerobic conditions using stopped-flow spectrophotometry and a NO electrode. Reaction rate constants of NO to deoxygenated and oxygenated HbV were considerably smaller than those of cell-free Hb because of the intracellular NO-diffusion barrier. The reaction of CO with deoxygenated HbV was slightly slower than that of cell-free Hb solely because of the co-encapsulated allosteric effector, pyridoxal 5'-phosphate. The NO depletion in an aerobic condition in the presence of empty vesicles was monitored using a NO electrode, showing that the hydrophobic bilayer membrane of HbV, which might have higher gas solubility, does not markedly facilitate the O(2) and NO reaction, and that the intracellular Hb is the major component of NO depletion. In conclusion, HbV shows retarded gas reactions, providing some useful information to explain the absence of vasoconstriction and hypertension when they are intravenously injected.

Vesicle Adhesion and Fusion Studied by Small-Angle X-Ray Scattering.

PubMed

Komorowski, Karlo; Salditt, Annalena; Xu, Yihui; Yavuz, Halenur; Brennich, Martha; Jahn, Reinhard; Salditt, Tim

2018-04-24

We have studied the adhesion state (also denoted by docking state) of lipid vesicles as induced by the divalent ions Ca 2+ or Mg 2+ at well-controlled ion concentration, lipid composition, and charge density. The bilayer structure and the interbilayer distance in the docking state were analyzed by small-angle x-ray scattering. A strong adhesion state was observed for DOPC:DOPS vesicles, indicating like-charge attraction resulting from ion correlations. The observed interbilayer separations of ∼1.6 nm agree quantitatively with the predictions of electrostatics in the strong coupling regime. Although this phenomenon was observed when mixing anionic and zwitterionic (or neutral) lipids, pure anionic membranes (DOPS) with highest charge density σ resulted in a direct phase transition to a multilamellar state, which must be accompanied by rupture and fusion of vesicles. To extend the structural assay toward protein-controlled docking and fusion, we have characterized reconstituted N-ethylmaleimide-sensitive factor attachment protein receptors in controlled proteoliposome suspensions by small-angle x-ray scattering. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Vesicle encapsulation of a nonbiological photochemical system capable of reducing NAD(+) to NADH.

PubMed

Summers, David P; Rodoni, David

2015-10-06

One of the fundamental structures of a cell is the membrane. Self-assembling lipid bilayer vesicles can form the membrane of an artificial cell and could also have plausibly assembled prebiotically for the origin of life. Such cell-like structures, that encapsulate some basic subset of the functions of living cells, are important for research to infer the minimum chemistry necessary for a cell, to help understand the origin of life, and to allow the production of useful species in microscopic containers. We show that the encapsulation of TiO2 particles has the potential to provide the basis for an energy transduction system inside vesicles which can be used to drive subsequent chemistry. TiO2 encapsulated in vesicles can be used to produce biochemical species such as NADH. The NADH is formed from NAD(+) reduction and is produced in a form that is able to drive further enzymatic chemistry. This allows us to link a mineral-based, nonbiological photosystem to biochemical reactions. This is a fundamental step toward being able to use this mineral photosystem in a protocell/artificial cell.

Large divalent cations and electrostatic potentials adjacent to membranes. Experimental results with hexamethonium.

PubMed Central

Alvarez, O; Brodwick, M; Latorre, R; McLaughlin, A; McLaughlin, S; Szabo, G

1983-01-01

A simple extension of the Gouy-Chapman theory predicts that the ability of a divalent cation to screen charges at a membrane-solution interface decreases significantly if the distance between the charges on the cation is comparable with the Debye length. We tested this prediction by investigating the effect of hexamethonium on the electrostatic potential adjacent to negatively charged phospholipid bilayer membranes. The distance between the two charges of an extended hexamethonium molecule is approximately 1 nm, which is the Debye length in the 0.1 M monovalent salt solutions used in these experiments. Six different experimental approaches were utilized. We measured the electrophoretic mobility of multilamellar vesicles to determine the zeta potential, the line width of the 31P nuclear magnetic resonance (NMR) signal from sonicated vesicles to calculate the change in potential at the phosphodiester moiety of the lipid, and the conductance of planar bilayer membranes exposed to either carriers (nonactin) or pore formers (gramicidin) to estimate the change in potential within the membrane. We also measured directly the effect of hexamethonium on the potential above a monolayer formed from negative lipids, and attempted to calculate the change in the surface potential of a bilayer membrane from capacitance measurements. With the exception of the capacitance calculations, each of the techniques gave comparable results: hexamethonium exerts a smaller effect on the potential than that predicted by the classic screening theory. The results are consistent with the predictions of the extended Gouy-Chapman theory and are relevant to the interpretation of physiological and pharmacological experiments that utilize hexamethonium and other large divalent cations. PMID:6198001

Fabrication Procedures and Birefringence Measurements for Designing Magnetically Responsive Lanthanide Ion Chelating Phospholipid Assemblies.

PubMed

Isabettini, Stéphane; Baumgartner, Mirjam E; Fischer, Peter; Windhab, Erich J; Liebi, Marianne; Kuster, Simon

2018-01-03

Bicelles are tunable disk-like polymolecular assemblies formed from a large variety of lipid mixtures. Applications range from membrane protein structural studies by nuclear magnetic resonance (NMR) to nanotechnological developments including the formation of optically active and magnetically switchable gels. Such technologies require high control of the assembly size, magnetic response and thermal resistance. Mixtures of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and its lanthanide ion (Ln 3+ ) chelating phospholipid conjugate, 1,2-dimyristoyl-sn-glycero-3-phospho-ethanolamine-diethylene triaminepentaacetate (DMPE-DTPA), assemble into highly magnetically responsive assemblies such as DMPC/DMPE-DTPA/Ln 3+ (molar ratio 4:1:1) bicelles. Introduction of cholesterol (Chol-OH) and steroid derivatives in the bilayer results in another set of assemblies offering unique physico-chemical properties. For a given lipid composition, the magnetic alignability is proportional to the bicelle size. The complexation of Ln 3+ results in unprecedented magnetic responses in terms of both magnitude and alignment direction. The thermo-reversible collapse of the disk-like structures into vesicles upon heating allows tailoring of the assemblies' dimensions by extrusion through membrane filters with defined pore sizes. The magnetically alignable bicelles are regenerated by cooling to 5 °C, resulting in assembly dimensions defined by the vesicle precursors. Herein, this fabrication procedure is explained and the magnetic alignability of the assemblies is quantified by birefringence measurements under a 5.5 T magnetic field. The birefringence signal, originating from the phospholipid bilayer, further enables monitoring of polymolecular changes occurring in the bilayer. This simple technique is complementary to NMR experiments that are commonly employed to characterize bicelles.

Structure and functions of simple membrane-water interfaces. [Abstract only

NASA Technical Reports Server (NTRS)

Pohorille, A.; Wilson, M. A.

1994-01-01

The structure and functions of the earliest ancestors of contemporary cells are focal points in studies of the origin of life. Probably the first cell-like structures were vesicles - closed, spheroidal structures with aqueous medium trapped inside. The membranous walls of vesicles were most likely bilayers composed of simple amphiphilic material available on early earth. The membrane studied was composed of glycerol 1-monooleate (GMO). Glycerol forms the polar head group and the oily tail contains 18 carbon atoms. All head groups have been found to be located in two narrow regions at the interfaces with water. The membrane interior, formed by the hydrophobic tails, is quite fluid with chain disorder increasing towards the center of the bilayer. These results are in agreement with x-ray and neutron scattering data from related bilayers. The width of the membrane is not constant, but fluctuates in time and space. Occasional thinning defects in the membrane, observed during the course of the simulations, may have a significant influence on rates of passive transport of small molecules across membranes. It has been found that water penetrates the head group region but not the oily interior of the membrane. Water molecules near the interface are oriented by dipoles of the head groups. The resulting electrostatic potential across the interface, determined in our simulations, has been found to be markedly larger than across the water-oil interface. This quantity has been implicated as the source of selectivity, with respect to the sign of the charge, as an ion approaches the interface and during transport of hydrophobic ions across membranes.

Vesicles from pH-regulated reversible gemini amino-acid surfactants as nanocapsules for delivery.

PubMed

Lv, Jing; Qiao, Weihong; Li, Zongshi

2016-10-01

Reversible transition from micelles to vesicles by regulating pH were realized by gemini amino-acid surfactants N,N'-dialkyl-N,N'-diacetate ethylenediamine. Measurement results of ζ-potential at different pH and DLS at varying solvents revealed that the protonation between H(+) and double NCH2COO(-) groups (generating NH(+)CH2COO(-)), expressed as pKa1 and pKa2, is the key driving force to control the aggregation behaviors of gemini surfactant molecule. Effect of pH on the bilayer structure was studied in detail by using steady-state fluorescence spectroscopy of hydrophobic pyrene and Coumarin 153 (C153) respectively and fluorescence resonance energy transfer (FRET) from C153 to Rhodamine 6G (R6G). Various pH-regulated and pH-reversible self-assemblies were obtained in one surfactant system. Vitamin D3 was encapsulated in vesicle bilayers to form nano-VD3-capsules as VD3 supplement agent for health care products. By using the electrostatic attraction between Ca(2+) and double -COO(-) groups, nano-VD3-capsules with Ca(2+) coated outermost layers were prepared as a formulation for VD3 and calcium co-supplement agent. DLS and TEM were performed to check stability and morphology of the nano-capsules. It is concluded that the pH-regulated gemini amino-acid surfactants can be used to construct colloidal systems for delivering hydrophobic drugs or nutritions without lipids at human physiological pH level. Copyright © 2016 Elsevier B.V. All rights reserved.

Membrane fusion between baculovirus budded virus-enveloped particles and giant liposomes generated using a droplet-transfer method for the incorporation of recombinant membrane proteins.

PubMed

Nishigami, Misako; Mori, Takaaki; Tomita, Masahiro; Takiguchi, Kingo; Tsumoto, Kanta

2017-07-01

Giant proteoliposomes are generally useful as artificial cell membranes in biochemical and biophysical studies, and various procedures for their preparation have been reported. We present here a novel preparation technique that involves the combination of i) cell-sized lipid vesicles (giant unilamellar vesicles, GUVs) that are generated using the droplet-transfer method, where lipid monolayer-coated water-in-oil microemulsion droplets interact with oil/water interfaces to form enclosed bilayer vesicles, and ii) budded viruses (BVs) of baculovirus (Autographa californica nucleopolyhedrovirus) that express recombinant transmembrane proteins on their envelopes. GP64, a fusogenic glycoprotein on viral envelopes, is activated by weak acids and is thought to cause membrane fusion with liposomes. Using confocal laser scanning microscopy (CLSM), we observed that the single giant liposomes fused with octadecyl rhodamine B chloride (R18)-labeled wild-type BV envelopes with moderate leakage of entrapped soluble compounds (calcein), and the fusion profile depended on the pH of the exterior solution: membrane fusion occurred at pH ∼4-5. We further demonstrated that recombinant transmembrane proteins, a red fluorescent protein (RFP)-tagged GPCR (corticotropin-releasing hormone receptor 1, CRHR1) and envelope protein GP64 could be partly incorporated into membranes of the individual giant liposomes with a reduction of the pH value, though there were also some immobile fluorescent spots observed on their circumferences. This combination may be useful for preparing giant proteoliposomes containing the desired membranes and inner phases. Copyright © 2017 Elsevier B.V. All rights reserved.

LDRD final report on light-powered nanovehicles.

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

Shelnutt, John Allen; van Swol, Frank B.; Miller, James Edward

2003-11-01

We have investigated the possibility of constructing nanoscale metallic vehicles powered by biological motors or flagella that are activated and powered by visible light. The vehicle's body is to be composed of the surfactant bilayer of a liposome coated with metallic nanoparticles or nanosheets grown together into a porous single crystal. The diameter of the rigid metal vesicles is from about 50 nm to microns. Illumination with visible light activates a photosynthetic system in the bilayer that can generate a pH gradient across the liposomal membrane. The proton gradient can fuel a molecular motor that is incorporated into the membrane.more » Some molecular motors require ATP to fuel active transport. The protein ATP synthase, when embedded in the membrane, will use the pH gradient across the membrane to produce ATP from ADP and inorganic phosphate. The nanoscale vehicle is thus composed of both natural biological components (ATPase, flagellum; actin-myosin, kinesin-microtubules) and biomimetic components (metal vehicle casing, photosynthetic membrane) as functional units. Only light and storable ADP, phosphate, water, and weak electron donor are required fuel components. These nano-vehicles are being constructed by self-assembly and photocatalytic and autocatalytic reactions. The nano-vehicles can potentially respond to chemical gradients and other factors such as light intensity and field gradients, in a manner similar to the way that magnetic bacteria navigate. The delivery package might include decision-making and guidance components, drugs or other biological and chemical agents, explosives, catalytic reactors, and structural materials. We expected in one year to be able only to assess the problems and major issues at each stage of construction of the vehicle and the likely success of fabricating viable nanovehicles with our biomimetic photocatalytic approach. Surprisingly, we have been able to demonstrate that metallized photosynthetic liposomes can indeed be made. We have completed the synthesis of metallized liposomes with photosynthetic function included and studied these structures by electron microscopy. Both platinum and palladium nanosheeting have been used to coat the micelles. The stability of the vehicles to mechanical stress and the solution environment is enhanced by the single-crystalline platinum or palladium coating on the vesicle. With analogous platinized micelles, it is possible to dry the vehicles and re-suspend them with full functionality. However, with the liposomes drying on a TEM grid may cause the platinized liposomes to collapse, although probably stay viable in solution. It remains to be shown whether a proton motive force across the metallized bilayer membrane can be generated and whether we will also be able to incorporate various functional capabilities including ATP synthesis and functional molecular motors. Future tasks to complete the nanovehicles would be the incorporation of ATP synthase into metallized liposomes and the incorporation of a molecular motor into metallized liposomes.« less

Interaction of staphylococcal delta-toxin and synthetic analogues with erythrocytes and phospholipid vesicles. Biological and physical properties of the amphipathic peptides.

PubMed

Alouf, J E; Dufourcq, J; Siffert, O; Thiaudiere, E; Geoffroy, C

1989-08-01

Staphylococcal delta-toxin, a 26-residue amphiphilic peptide is lytic for cells and phospholipid vesicles and is assumed to insert as an amphipathic helix and oligomerize in membranes. For the first time, the relationship between these properties and toxin structure is investigated by means of eight synthetic peptides, one identical in sequence to the natural toxin, five 26-residue analogues and two shorter peptides corresponding to residues 1-11 and 11-26. These peptides were designed by the Edmundson wheel axial projection in order to maintain: (a) the hydrophilic/hydrophobic balance while rationalizing the sequence, (b) the alpha-helical configuration and (c) the common epitopic structure. The fluorescence of the single Trp residue was used to monitor the behaviour of the natural toxin and analogues. All 26-residue analogues were hemolytically active although to a lesser extent than natural toxin. The peptide of residues 11-26 bound lipids weakly and was hemolytic at high concentration. The peptide of residues 1-11 did not bind lipids and was hemolytically inactive. All peptides except the latter cross-reacted in immunoprecipitation tests with the natural toxin. The study of a 26-residue analogue by circular dichroism revealed an alpha-helical configuration in both the free and lipid-bound state. Changes in the fluorescence of the peptides in the presence of lipid micelles and bilayers varied according to the position of the reporter group. When bound to lipids, Trp5, Trp16 and the Fmoc-1 positions of the analogues became buried while Trp15 of the natural toxin and its synthetic replicate remained more exposed. All changes are rationalized by the proposal of an amphipathic helix whose hydrophobic face is embedded within the apolar core of bilayers while the hydrophilic and charged face remains more exposed to solvent.

Metal Sorbing Vesicles: Light Scattering Characterization and Metal Sorbtion Behavior.

NASA Astrophysics Data System (ADS)

van Zanten, John Hollis

1992-01-01

The research described herein consisted of two parts: light scattering characterization of vesicles and kinetic investigations of metal sorbing vesicles. Static light scattering techniques can be used to determine the geometric size, shape and apparent molecular weight of phosphatidylcholine vesicles in aqueous suspension. A Rayleigh-Gans-Debye (RGD) approximation analysis of multiangle scattered light intensity data yields the size and degree of polydispersity of the vesicles in solution, while the Zimm plot technique provides the radius of gyration and apparent weight-average molecular weight. Together the RGD approximation and Zimm plots can be used to confirm the geometric shape of vesicles and can give a good estimate of the vesicle wall thickness in some cases. Vesicles varying from 40 to 115 nm in diameter have been characterized effectively. The static light scattering measurements indicate that, as expected, phosphatidylcholine vesicles in this size range scatter light as isotropic hollow spheres. Additionally, static and dynamic light scattering measurements have been made and compared with one another. The values for geometric radii determined by static light scattering typically agree with those estimated by dynamic light scattering to within a few percent. Interestingly however, dynamic measurements suggest that there is a significant degree of polydispersity present in the vesicle dispersions, while static measurements indicate near size monodisperse dispersions. Metal sorbing vesicles which harbor ionophores, such as antibiotic A23187 and synthetic carriers, in their bilayer membranes have been produced. These vesicles also encapsulate the chelating compound, nitrilotriacetate, to provide the driving force for metal ion uptake. Very dilute dispersions (on the order of 0.03% w/v) of these metal sorbing vesicles were capable of removing Cd ^{2+} and Pb^{2+ } from dilute aqueous solution (5 ppm and less) and concentrating these metal ions several hundred to more than a thousand fold in the vesicle interior in a few minutes time. Synthetic ionophores were found to preferentially transport Pb^{2+} over Cd^{2+}, thus suggesting that engineered vesicle dispersions can be used as selective separations media. The effect of ionophore concentration, solution pH, solution ionic strength, initial metal ion concentration and vesicle concentration have been investigated.

Energy Transduction Inside of Amphiphilic Vesicles: Encapsulation of Photochemically Active Semiconducting Particles

NASA Astrophysics Data System (ADS)

Summers, David P.; Noveron, Juan; Basa, Ranor C. B.

2009-04-01

Amphiphilic bilayer membrane structures (vesicles) have been postulated to have been abiotically formed and spontaneously assemble on the prebiotic Earth, providing compartmentalization for the origin of life. These vesicles are similar to modern cellular membranes and can serve to contain water-soluble species, concentrate species, and have the potential to catalyze reactions. The origin of the use of photochemical energy in metabolism (i.e. energy transduction) is one of the central issues in the origin of life. This includes such questions as how energy transduction may have occurred before complex enzymatic systems, such as required by contemporary photosynthesis, had developed and how simple a photochemical system is possible. It has been postulated that vesicle structures developed the ability to capture and transduce light, providing energy for reactions. It has also been shown that pH gradients across the membrane surface can be photochemically created, but coupling these to drive chemical reactions has been difficult. Colloidal semiconducting mineral particles are known to photochemically drive redox chemistry. We propose that encapsulation of these particles has the potential to provide a source of energy transduction inside vesicles, and thereby drive protocellular chemistry, and represents a model system for early photosynthesis. In our experiments we show that TiO2 particles, in the ~20 nm size range, can be incorporated into vesicles and retain their photoactivity through the dehydration/rehydration cycles that have been shown to concentrate species inside a vesicle.

Energy transduction inside of amphiphilic vesicles: encapsulation of photochemically active semiconducting particles.

PubMed

Summers, David P; Noveron, Juan; Basa, Ranor C B

2009-04-01

Amphiphilic bilayer membrane structures (vesicles) have been postulated to have been abiotically formed and spontaneously assemble on the prebiotic Earth, providing compartmentalization for the origin of life. These vesicles are similar to modern cellular membranes and can serve to contain water-soluble species, concentrate species, and have the potential to catalyze reactions. The origin of the use of photochemical energy in metabolism (i.e. energy transduction) is one of the central issues in the origin of life. This includes such questions as how energy transduction may have occurred before complex enzymatic systems, such as required by contemporary photosynthesis, had developed and how simple a photochemical system is possible. It has been postulated that vesicle structures developed the ability to capture and transduce light, providing energy for reactions. It has also been shown that pH gradients across the membrane surface can be photochemically created, but coupling these to drive chemical reactions has been difficult. Colloidal semiconducting mineral particles are known to photochemically drive redox chemistry. We propose that encapsulation of these particles has the potential to provide a source of energy transduction inside vesicles, and thereby drive protocellular chemistry, and represents a model system for early photosynthesis. In our experiments we show that TiO2 particles, in the approximately 20 nm size range, can be incorporated into vesicles and retain their photoactivity through the dehydration/rehydration cycles that have been shown to concentrate species inside a vesicle.

Structure and energetics of carbon, hexagonal boron nitride, and carbon/hexagonal boron nitride single-layer and bilayer nanoscrolls

NASA Astrophysics Data System (ADS)

Siahlo, Andrei I.; Poklonski, Nikolai A.; Lebedev, Alexander V.; Lebedeva, Irina V.; Popov, Andrey M.; Vyrko, Sergey A.; Knizhnik, Andrey A.; Lozovik, Yurii E.

2018-03-01

Single-layer and bilayer carbon and hexagonal boron nitride nanoscrolls as well as nanoscrolls made of bilayer graphene/hexagonal boron nitride heterostructure are considered. Structures of stable states of the corresponding nanoscrolls prepared by rolling single-layer and bilayer rectangular nanoribbons are obtained based on the analytical model and numerical calculations. The lengths of nanoribbons for which stable and energetically favorable nanoscrolls are possible are determined. Barriers to rolling of single-layer and bilayer nanoribbons into nanoscrolls and barriers to nanoscroll unrolling are calculated. Based on the calculated barriers nanoscroll lifetimes in the stable state are estimated. Elastic constants for bending of graphene and hexagonal boron nitride layers used in the model are found by density functional theory calculations.

Development of novel diolein-niosomes for cutaneous delivery of tretinoin: influence of formulation and in vitro assessment.

PubMed

Manca, Maria Letizia; Manconi, Maria; Nacher, Amparo; Carbone, Claudia; Valenti, Donatella; Maccioni, Anna Maria; Sinico, Chiara; Fadda, Anna Maria

2014-12-30

This work describes innovative niosomes, composed of diolein alone or in association with the hydrophilic penetration enhancer Labrasol(®), as carriers for cutaneous drug delivery. The model drug was tretinoin and conventional, and Labrasol(®) containing liposomes was used as controls to evaluate the influence of vesicle composition and the role of Labrasol(®) on vesicle physico-chemical properties and performance as skin delivery system. Vesicles, prepared by the thin film hydration technique, were characterized in terms of size distribution, morphology, zeta potential, structure, incorporation efficiency, and rheological properties. The influence of carrier composition on tretinoin delivery to human skin was evaluated by in vitro percutaneous experiments, while formulation distribution on human skin and cellular uptake in human keratinocytes were studied using confocal laser scanning microscopy. showed that tretinoin loaded diolein-niosomes formed unilamellar vesicles very similar in physico-chemical properties to liposomes. The role of Labrasol(®) was similar in niosomes and liposomes. Its addition affected vesicle structure and size, by formation of an interdigitate bilayer with higher curvature and larger vesicle size, and rheological properties. Indeed, the presence of Labrasol(®) allowed both niosomes and liposomes to shift from Newtonian to pseudo-plastic behavior. Confocal laser microscopy highlighted an important contemporaneous deposition of hydrophilic and lipophilic vesicle components in stratum corneum and a high vesicle affinity for skin appendages when Labrasol(®) was added to the diolein-niosomes. Moreover, all samples were internalized in human keratinocytes in vitro. Copyright © 2014 Elsevier B.V. All rights reserved.

Roles of head group architecture and side chain length on colorimetric response of polydiacetylene vesicles to temperature, ethanol and pH.

PubMed

Charoenthai, Nipaphat; Pattanatornchai, Thanutpon; Wacharasindhu, Sumrit; Sukwattanasinitt, Mongkol; Traiphol, Rakchart

2011-08-15

In this contribution, we report the relationship between molecular structures of polydiacetylene (PDA) vesicles, fabricated by using three monomers, 10,12-tricosadiynoic acid (TCDA), 10,12-pentacosadiynoic acid (PCDA) and N-(2-aminoethyl)pentacosa-10,12-diynamide (AEPCDA), and their color-transition behaviors. The modification of side chain length and head group of the PDA vesicles strongly affects the colorimetric response to temperature, ethanol and pH. A shorter side chain of poly(TCDA) yields weaker inter- and intra-chain dispersion interactions in the bilayers compared to the system of poly(PCDA), which in turn results in a faster color transition upon exposure to all stimuli. A change of head group in poly(AEPCDA) slightly reduces the transition temperature. Interestingly, the colorimetric response of poly(AEPCDA) vesicles to the addition of ethanol is found to occur in a two-step fashion while the response of poly(PCDA) vesicles takes place in a one-step process. The amount of ethanol required for inducing complete color-transition of poly(AEPCDA) vesicles is also much higher, about 87% v/v. The increase of pH to ~9 and ~10 causes a color-transition of poly(TCDA) and poly(PCDA) vesicles, respectively. The poly(AEPCDA) vesicles, on the other hand, change color upon decreasing pH to ~0. The colorimetric response also occurs in a multi-step fashion. These discrepancies are attributed to the architecture of surface layers of poly(AEPCDA), constituting amine and amide groups separated by ethyl linkers. Copyright © 2011 Elsevier Inc. All rights reserved.

Pressure effects on dipalmitoylphosphatidylcholine bilayers measured by sub 2 H nuclear magnetic resonance

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

Driscoll, D.A.; Samarasinghe, S.; Adamy, S.

1991-04-02

The effects of pressure, up to 5 kbar, on multilamellar vesicles of 1,2-dipalmitoyl-sn-phosphatidylcholine perdeuterated in the acyl chains (DPPC-d{sub 62}) were examined by using high-pressure NMR techniques. A deuterium probe was built, and the quadrupole splitting was measured against pressure at various temperatures. The experiments were performed on pure lipid bilayers in the liquid-crystalline state and on bilayers in the liquid-crystalline state containing the local anesthetic tetracaine. The results show that the order parameter of all segments of the acyl chains increases with pressure in the liquid-crystalline state. The more highly ordered regions of the chains are affected slightly moremore » than the regions near the methyl ends. The addition of tetracaine increases the disorder of the chains, and pressure reverses the effect of anesthetic on the lipid as seen by the reversal of the changes in line shape and the measured order parameter.« less

Role of the array geometry in multi-bilayer hair cell sensors

NASA Astrophysics Data System (ADS)

Tamaddoni, Nima J.; Sarles, Stephen A.

2014-03-01

Recently, a bio-inspired, synthetic membrane-based hair cell sensor was fabricated and characterized. This sensor generates current in response to mechanical stimuli, such as airflow or free vibration, which perturb the sensor's hair. Vibration transferred from the hair to a lipid membrane (lipid bilayer) causes a voltage-dependent time rate of change in electrical capacitance of the membrane, which produces measurable current. Studies to date have been performed on systems containing only two droplets and a single bilayer, even though an array of multiple bilayers can be formed with more than 2 droplets. Thus, it is yet to be determined how multiple lipid bilayers affect the sensing response of a membrane-based hair cell sensor. In this work, we assemble serial droplet arrays with more than 1 bilayer to experimentally study the current generated by each membrane in response to perturbation of a single hair element. Two serial array configurations are studied: The first consists of a serial array of 3 bilayers formed using 4 droplets with the hair positioned in an end droplet. The second configuration consists of 3 droplets and 2 bilayers in series with the hair positioned in the central droplet. In serial arrays of up to four droplets, we observe that mechanotransduction of the hair's motion into a capacitive current occurs at every membrane, with bilayers positioned adjacent to the droplet containing the hair generating the largest sensing current. The measured currents suggest the total current generated by all bilayers in a 4-droplet, 3-bilaye array is greater than the current produced by a single-membrane sensor and similar in magnitude to the sum of currents output by 3, single-bilayer sensors operated independently. Moreover, we learned that bilayers positioned on the same side of the hair produce sensing currents that are in-phase, whereas bilayers positioned on opposite sides of the droplet containing the hair generate out-of-phase responses.

The multilayer nanoparticles for deep penetration of docetaxel into tumor parenchyma to overcome tumor microenvironment.

PubMed

Khaliq, Nisar Ul; Park, Dal Yong; Lee, Jae Young; Joo, Yeonhee; Oh, Keun Sang; Kim, Jung Seok; Kim, Jin-Seok; Kim, In-San; Kwon, Ick Chan; Yuk, Soon Hong

2016-10-01

Deep penetration of the anticancer drug, docetaxel (DTX), into tumor parenchyma was demonstrated to achieve improved chemotherapy. For this purpose, a multistage nanostructure was designed and characterized using the multilayer nanoparticles (NPs). The multilayer NPs had a core/shell structure. The core was composed of the DTX-loaded Pluronic NPs (diameter: 12nm) that were transferred into the inner side of vesicles to form the vesicle NPs. Förster resonance energy transfer (FRET) in the NPs was observed to verify the incorporation of the DTX-loaded Pluronic NPs into the inner side of the vesicles during the formation of the vesicle NPs. Subsequently, the vesicle NPs were stabilized through Pluronic-lipid bilayer interaction to form the multilayer NPs. To examine the morphology and size distribution of the multilayer NPs, transmittance electron microscopy and dynamic light scattering were used. In vitro release behavior and toxicity were observed to verify the functionality of the multilayer NPs as nanocarriers for cancer therapy. Multistage functionality was evaluated by cellular uptake and tissue distribution behaviors of the multilayer NPs. The biodistribution of the multilayer NPs and their antitumor efficacy were also observed to understand the role of multistage functionality for improved chemotherapy. Copyright © 2016 Elsevier B.V. All rights reserved.

The dynamics of giant unilamellar vesicle oxidation probed by morphological transitions.

PubMed

Sankhagowit, Shalene; Wu, Shao-Hua; Biswas, Roshni; Riche, Carson T; Povinelli, Michelle L; Malmstadt, Noah

2014-10-01

We have studied the dynamics of Lissamine Rhodamine B dye sensitization-induced oxidation of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) giant unilamellar vesicles (GUVs), where the progression of the underlying chemical processes was followed via vesicle membrane area changes. The surface-area-to-volume ratio of our spherical GUVs increased after as little as ten seconds of irradiation. The membrane area expansion was coupled with high amplitude fluctuations not typical of GUVs in isoosmotic conditions. To accurately measure the area of deformed and fluctuating membranes, we utilized a dual-beam optical trap (DBOT) to stretch GUV membranes into a geometrically regular shape. Further oxidation led to vesicle contraction, and the GUVs became tense, with micron-scale pores forming in the bilayer. We analyzed the GUV morphological behaviors as two consecutive rate-limiting steps. We also considered the effects of altering DOPC and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (RhDPPE) concentrations. The resulting kinetic model allows us to measure how lipid molecular area changes during oxidation, as well as to determine the rate constants controlling how quickly oxidation products are formed. Controlled membrane oxidation leading to permeabilization is also a potential tool for drug delivery based on engineered photosensitizer-containing lipid vesicles. Copyright © 2014 Elsevier B.V. All rights reserved.

The Bretherton Problem for a Vesicle

NASA Astrophysics Data System (ADS)

Barakat, Joseph; Spann, Andrew; Shaqfeh, Eric

2016-11-01

The motion of a lipid bilayer vesicle through a circular tube is investigated by singular perturbation theory in the limit of vanishing clearance. The vesicle is treated as a sac of fluid enclosed by a thin, elastic sheet that admits a bending stiffness. It is assumed that the vesicle is axisymmetric and swollen to a near-critical volume such that the clearance "e" between the membrane and the tube wall is very small. In this limit, bending resistance is of negligible importance compared to the isotropic tension, allowing the vesicle to be treated as a "no-slip bubble." The effective membrane tension is found to scale inversely with "e" raised to the 3/2 power with a comparatively weak Marangoni gradient. The extra pressure drop is found to have a leading contribution due to the cylindrical midsection, which scales inversely with "e," as well as a correction due to the end caps, which scales inversely with the square root of "e." The apparent viscosity is predicted as a unique function of the geometry. The theory exhibits excellent agreement with a simplified, "quasi-parallel" theory and with direct numerical simulations using the boundary element method. The results of this work are compared to those for bubbles, rigid particles, and red blood cells in confined flows.

Potassium-doped n-type bilayer graphene

NASA Astrophysics Data System (ADS)

Yamada, Takatoshi; Okigawa, Yuki; Hasegawa, Masataka

2018-01-01

Potassium-doped n-type bilayer graphene was obtained. Chemical vapor deposited bilayer and single layer graphene on copper (Cu) foils were used. After etching of Cu foils, graphene was dipped in potassium hydroxide aqueous solutions to dope potassium. Graphene on silicon oxide was characterized by X-ray photoelectron spectroscopy (XPS), energy dispersive X-ray spectroscopy (EDX), and Raman spectroscopy. Both XPS and EDX spectra indicated potassium incorporation into the bilayer graphene via intercalation between the graphene sheets. The downward shift of the 2D peak position of bilayer graphene after the potassium hydroxide (KOH) treatment was confirmed in Raman spectra, indicating that the KOH-treated bilayer graphene was doped with electrons. Electrical properties were measured using Hall bar structures. The Dirac points of bilayer graphene were shifted from positive to negative by the KOH treatment, indicating that the KOH-treated bilayer graphene was n-type conduction. For single layer graphene after the KOH treatment, although electron doping was confirmed from Raman spectra, the peak of potassium in the X-ray photoelectron spectroscopy (XPS) spectrum was not detected. The Dirac points of single layer graphene with and without the KOH treatment showed positive.

Assessment of bilayer silicene to probe as quantum spin and valley Hall effect

NASA Astrophysics Data System (ADS)

Rehman, Majeed Ur; Qiao, Zhenhua

2018-02-01

Silicene takes precedence over graphene due to its buckling type structure and strong spin orbit coupling. Motivated by these properties, we study the silicene bilayer in the presence of applied perpendicular electric field and intrinsic spin orbit coupling to probe as quantum spin/valley Hall effect. Using analytical approach, we calculate the spin Chern-number of bilayer silicene and then compare it with monolayer silicene. We reveal that bilayer silicene hosts double spin Chern-number as compared to single layer silicene and therefore accordingly has twice as many edge states in contrast to single layer silicene. In addition, we investigate the combined effect of intrinsic spin orbit coupling and the external electric field, we find that bilayer silicene, likewise single layer silicene, goes through a phase transitions from a quantum spin Hall state to a quantum valley Hall state when the strength of the applied electric field exceeds the intrinsic spin orbit coupling strength. We believe that the results and outcomes obtained for bilayer silicene are experimentally more accessible as compared to bilayer graphene, because of strong SO coupling in bilayer silicene.

Anion channels in the sea urchin sperm plasma membrane.

PubMed

Morales, E; de la Torre, L; Moy, G W; Vacquier, V D; Darszon, A

1993-10-01

Ionic fluxes in sea urchin sperm plasma membrane regulate cell motility and the acrosome reaction (AR). Although cationic channels mediate some of the ionic movements, little is known about anion channels in these cells. The fusion of sperm plasma membranes into lipid bilayers allowed identification of a 150 pS anion channel. This anion channel was enriched from detergent-solubilized sperm plasma membranes using a wheat germ agglutinin Sepharose column. Vesicles formed from this preparation were fused into black lipid membranes (BLM), yielding single channel anion-selective activity with the properties of those found in the sperm membranes. The following anion selectivity sequence was found: NO3- > CNS- > Br- > Cl-. This anion channel has a high open probability at the holding potentials tested, it is partially blocked by 4,4'-diisothiocyano-2,2'-stilbendisulfonic acid (DIDS), and it often displays substates. The sperm AR was also inhibited by DIDS.

D IR Line Shapes for Determining the Structure of a Peptide in a Bilayer

NASA Astrophysics Data System (ADS)

Woys, Ann Marie; Lin, Y. S.; Skinner, J. S.; Zanni, M. T.; Reddy, A. S.; de Pablo, J. J.

2010-06-01

Structure of the antimicrobial peptide, ovispirin, on a lipid bilayer was determined using 2D IR spectroscopy and spectra calculated from molecular dynamics simulations. Ovispirin is an 18 residue amphipathic peptide that binds parallel to the membrane in a mostly alpha helical conformation. 15 of the 18 residues were ^1^3C^1^8O isotopically labeled on the backbone to isolate the amide I vibration at each position. 2D IR spectra were collected for each labeled peptide in 3:1 POPC/POPG vesicles, and peak width along the diagonal was measured. The diagonal line width is sensitive to the vibrator's electrostatic environment, which varies through the bilayer. We observe an oscillatory line width spanning 10 to 24 cm-1 and with a period of nearly 3.6 residues. To further investigate the position of ovispirin in a bilayer, molecular dynamics simulations determined the peptide depth to be just below the lipid headgroups. The trajectory of ovispirin at this depth was used to calculate 2D IR spectra, from which the diagonal line width is measured. Both experimental and simulated line widths are similar in periodicity and suggest a kink in the peptide backbone and the tilt in the bilayer. A. Woys, Y. S. Lin, A. S. Reddy, W. Xiong, J. J. de Pablo, J. S. Skinner, and M. T. Zanni, JACS 132, 2832-2838 (2010).

Highly selective water channel activity measured by voltage clamp: analysis of planar lipid bilayers reconstituted with purified AqpZ.

PubMed

Pohl, P; Saparov, S M; Borgnia, M J; Agre, P

2001-08-14

Aquaporins are membrane channels selectively permeated by water or water plus glycerol. Conflicting reports have described ion conductance associated with some water channels, raising the question of whether ion conductance is a general property of the aquaporin family. To clarify this question, a defined system was developed to simultaneously measure water permeability and ion conductance. The Escherichia coli water channel aquaporin-Z (AqpZ) was studied, because it is a highly stable tetramer. Planar lipid bilayers were formed from unilamellar vesicles containing purified AqpZ. The hydraulic conductivity of bilayers made from the total extract of E. coli lipids increased 3-fold if reconstituted with AqpZ, but electric conductance was unchanged. No channel activity was detected under voltage-clamp conditions, indicating that less than one in 10(9) transport events is electrogenic. Microelectrode measurements were simultaneously undertaken adjacent to the membrane. Changes in sodium concentration profiles accompanying transmembrane water flow permitted calculation of the activation energies: 14 kcal/mol for protein-free lipid bilayers and 4 kcal/mol for lipid bilayers containing AqpZ. Neither the water permeability nor the electric conductivity exhibited voltage dependence. This sensitive system demonstrated that AqpZ is permeated by water but not charged ions and should permit direct analyses of putative electrogenic properties of other aquaporins.

Super-Sensitive and Robust Biosensors from Supported Polymer Bilayers

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

Paxton, Walter F.

2015-09-01

Biological organisms are potentially the most sensitive and selective biological detection systems known, yet we are currently severely limited in our ability to exploit biological interactions in sensory devices, due in part to the limited stability of biological systems and derived materials. This proposal addresses an important aspect of integrating biological sensory materials in a solid state device. If successful, such technology could enable entirely new classes of robust biosensors that could be miniaturized and deployed in the field. The critical aims of the proposed work were 1) the calibration of a more versatile approach to measuring pH, 2) themore » use of this method to monitor pH changes caused by the light-induced pumping of protons across vesicles with bacteriorhodopsin integrated into the membranes (either polymer or lipid); 3) the preparation of bilayer assemblies on platinum surfaces; 4) the enhanced detection of lightinduced pH changes driven by bR-loaded supported bilayers. I have developed a methodology that may enable that at interfaces and developed a methodology to characterize the functionality of bilayer membranes with reconstituted membrane proteins. The integrity of the supported bilayer films however must be optimized prior to the full realization of the work originally envisioned in the original proposal. Nevertheless, the work performed on this project and the encouraging results it has produced has demonstrated that these goals are challenging yet within reach.« less

Definition of the specific roles of lysolecithin and palmitic acid in altering the susceptibility of dipalmitoylphosphatidylcholine bilayers to phospholipase A2.

PubMed

Henshaw, J B; Olsen, C A; Farnbach, A R; Nielson, K H; Bell, J D

1998-07-28

Bilayers composed of phosphatidylcholine initially resist catalysis by phospholipase A2. However, after a latency period, they become susceptible when sufficient reaction products (lysolecithin and fatty acid) accumulate in the membrane. Temperature near the main bilayer phase transition and calcium concentration modulate the effectiveness of the reaction products. The purpose of this study was to examine the individual contributions of lysolecithin and palmitic acid to the susceptibility of dipalmitoylphosphatidylcholine vesicles and to rationalize the effects of temperature and calcium. Various fluorescent probes (Prodan, Laurdan, pyrene-labeled fatty acid, and dansyl-labeled phospholipid) were used to assess changes in the ability of the reaction products to perturb the bilayer and to affect the interactions with the enzyme. Un-ionized palmitic acid decreased bilayer polarity and perturbed the membrane surface exposing some of the Prodan to bulk water. Lysolecithin increased bilayer polarity and the rate of dipolar relaxation in response to the excited states of Laurdan and Prodan. A combination of the individual contributions of each product was observed when palmitic acid and lysolecithin were present together at low calcium, and the effects of lysolecithin dominated at high calcium. Palmitic acid, but not lysolecithin, promoted the binding of phospholipase A2 to the bilayer surface in the absence of calcium. Lysolecithin reduced the ability of fatty acid to enhance binding apparently by altering the structure of fatty acid domains in the membrane. Furthermore, increased temperature and ionization of the fatty acid tended to cause segregation of bound phospholipase A2 into domains poor in phospholipid content which presumably impeded bilayer hydrolysis. In contrast, un-ionized palmitic acid and lysolecithin promoted hydrolysis by augmenting a step distal to the adsorption of enzyme to the bilayer. This kinetic response to lysolecithin was calcium-dependent. A model accounting for these varied influences of the reaction products is presented.

Role of Water in Proton-Hydroxide Conductance Across Membranes

DTIC Science & Technology

1988-06-28

a~a,:v %~ ’ diffusion , rather than hopping along water wires, and there should be little or no deuterium effect. References Bangham , A.D...CONTRACT TITLE: Role of water in proton-hydroxide conductance across model and biological membranes. START DATE: October 1, 1987 RESEARCH OBJECTIVE: To...hypothesis of Bangham and Mason (1980) who suggested that general anesthetics might introduce defects into bilayers of synaptic vesicle membranes which lead

Charge Inversion in semi-permeable membranes

NASA Astrophysics Data System (ADS)

Das, Siddhartha; Sinha, Shayandev; Jing, Haoyuan

Role of semi-permeable membranes like lipid bilayer is ubiquitous in a myriad of physiological and pathological phenomena. Typically, lipid membranes are impermeable to ions and solutes; however, protein channels embedded in the membrane allow the passage of selective, small ions across the membrane enabling the membrane to adopt a semi-permeable nature. This semi-permeability, in turn, leads to electrostatic potential jump across the membrane, leading to effects such as regulation of intracellular calcium, extracellular-vesicle-membrane interactions, etc. In this study, we theoretically demonstrate that this semi-permeable nature may trigger the most remarkable charge inversion (CI) phenomenon in the cytosol-side of the negatively-charged lipid bilayer membrane that are selectively permeable to only positive ions of a given salt. This CI is manifested as the changing of the sign of the electrostatic potential from negative to positive from the membrane-cytosol interface to deep within the cytosol. We study the impact of the parameters such as the concentration of this salt with selectively permeable ions as well as the concentration of an external salt in the development of this CI phenomenon. We anticipate such CI will profoundly influence the interaction of membrane and intra-cellular moieties (e.g., exosome or multi-cellular vesicles) having implications for a host of biophysical processes.

Structure of Immune Stimulating Complex Matrices and Immune Stimulating Complexes in Suspension Determined by Small-Angle X-Ray Scattering

PubMed Central

Pedersen, Jan Skov; Oliveira, Cristiano L.P.; Hübschmann, Henriette Baun; Arleth, Lise; Manniche, Søren; Kirkby, Nicolai; Nielsen, Hanne Mørck

2012-01-01

Immune stimulating complex (ISCOM) particles consisting of a mixture of Quil-A, cholesterol, and phospholipids were structurally characterized by small-angle x-ray scattering (SAXS). The ISCOM particles are perforated vesicles of very well-defined structures. We developed and implemented a novel (to our knowledge) modeling method based on Monte Carlo simulation integrations to describe the SAXS data. This approach is similar to the traditional modeling of SAXS data, in which a structure is assumed, the scattering intensity is calculated, and structural parameters are optimized by weighted least-squares methods when the model scattering intensity is fitted to the experimental data. SAXS data from plain ISCOM matrix particles in aqueous suspension, as well as those from complete ISCOMs (i.e., with an antigen (tetanus toxoid) incorporated) can be modeled as a polydisperse distribution of perforated bilayer vesicles with icosahedral, football, or tennis ball structures. The dominating structure is the tennis ball structure, with an outer diameter of 40 nm and with 20 holes 5–6 nm in diameter. The lipid bilayer membrane is 4.6 nm thick, with a low-electron-density, 2.0-nm-thick hydrocarbon core. Surprisingly, in the ISCOMs, the tetanus toxoid is located just below the membrane inside the particles. PMID:22677391

Calcium-dependent regulation of SNARE-mediated membrane fusion by calmodulin.

PubMed

Di Giovanni, Jerome; Iborra, Cécile; Maulet, Yves; Lévêque, Christian; El Far, Oussama; Seagar, Michael

2010-07-30

Neuroexocytosis requires SNARE proteins, which assemble into trans complexes at the synaptic vesicle/plasma membrane interface and mediate bilayer fusion. Ca(2+) sensitivity is thought to be conferred by synaptotagmin, although the ubiquitous Ca(2+)-effector calmodulin has also been implicated in SNARE-dependent membrane fusion. To examine the molecular mechanisms involved, we examined the direct action of calmodulin and synaptotagmin in vitro, using fluorescence resonance energy transfer to assay lipid mixing between target- and vesicle-SNARE liposomes. Ca(2+)/calmodulin inhibited SNARE assembly and membrane fusion by binding to two distinct motifs located in the membrane-proximal regions of VAMP2 (K(D) = 500 nm) and syntaxin 1 (K(D) = 2 microm). In contrast, fusion was increased by full-length synaptotagmin 1 anchored in vesicle-SNARE liposomes. When synaptotagmin and calmodulin were combined, synaptotagmin overcame the inhibitory effects of calmodulin. Furthermore, synaptotagmin displaced calmodulin binding to target-SNAREs. These findings suggest that two distinct Ca(2+) sensors act antagonistically in SNARE-mediated fusion.

Calcium-activated potassium channels in basolateral membranes of colon epithelial cells; reconstitution and functional properties.

PubMed

Wiener, H; Turnheim, K

1990-10-26

Using differential sedimentation, isopycnic and Ficoll-400 barrier centrifugation, basolateral membrane vesicles of surface and crypt cells of the rabbit distal colon were enriched 34- and 9-fold, respectively. 86Rb(+)-uptake into these vesicles, driven by an electrical potential difference, was stimulated by submicromolar Ca2+ activities and inhibited by Ba2+. These findings indicate the presence of Ca2(+)-activated K+ channels. The K+ channels in surface and crypt cell membranes differed with respect to inhibition by the bee venom apamin, the scorpion venom charybdotoxin and tetraethylammonium and exhibited a different pH dependence. Fusion of basolateral membrane vesicles with planar phospholipid bilayers revealed the presence of high-conductance Ba2(+)-sensitive K+ channels which were activated by micromolar Ca2+ and inhibited by crude scorpion venom and trifluoperazine. These K+ channels may be involved in the coupling of apical and basolateral membrane conductances during Na+ absorption and Cl- secretion, but they may also play a role in cell volume regulation.

Synaptotagmin-mediated bending of the target membrane is a critical step in Ca2+-regulated fusion

PubMed Central

Hui, Enfu; Johnson, Colin P.; Yao, Jun; Dunning, F. Mark; Chapman, Edwin R.

2009-01-01

Summary Decades ago it was proposed that exocytosis involves invagination of the target membrane, resulting in a highly localized site of contact between the bilayers destined to fuse. The vesicle protein synaptotagmin-I (syt) bends membranes in response to Ca2+, but whether this drives localized invagination of the target membrane to accelerate fusion has not been determined; previous studies relied on reconstituted vesicles that were already highly curved and used mutations in syt that were not selective for membrane-bending activity. Here, we directly address this question by utilizing vesicles with different degrees of curvature. A tubulation-defective syt mutant was able to promote fusion between highly curved SNARE-bearing liposomes, but exhibited a marked loss of activity when the membranes were relatively flat. Moreover, bending of flat membranes by adding an N-BAR domain rescued the function of the tubulation-deficient syt mutant. Hence, syt-mediated membrane bending is a critical step in membrane fusion. PMID:19703397

Extracellular Vesicles in Cardiovascular Theranostics

PubMed Central

Bei, Yihua; Das, Saumya; Rodosthenous, Rodosthenis S.; Holvoet, Paul; Vanhaverbeke, Maarten; Monteiro, Marta Chagas; Monteiro, Valter Vinicius Silva; Radosinska, Jana; Bartekova, Monika; Jansen, Felix; Li, Qian; Rajasingh, Johnson; Xiao, Junjie

2017-01-01

Extracellular vesicles (EVs) are small bilayer lipid membrane vesicles that can be released by most cell types and detected in most body fluids. EVs exert key functions for intercellular communication via transferring their bioactive cargos to recipient cells or activating signaling pathways in target cells. Increasing evidence has shown the important regulatory effects of EVs in cardiovascular diseases (CVDs). EVs secreted by cardiomyocytes, endothelial cells, fibroblasts, and stem cells play essential roles in pathophysiological processes such as cardiac hypertrophy, cardiomyocyte survival and apoptosis, cardiac fibrosis, and angiogenesis in relation to CVDs. In this review, we will first outline the current knowledge about the physical characteristics, biological contents, and isolation methods of EVs. We will then focus on the functional roles of cardiovascular EVs and their pathophysiological effects in CVDs, as well as summarize the potential of EVs as therapeutic agents and biomarkers for CVDs. Finally, we will discuss the specific application of EVs as a novel drug delivery system and the utility of EVs in the field of regenerative medicine. PMID:29158817

Single Molecule Measurements of Interaction Free Energies Between the Proteins Within Binary and Ternary SNARE Complexes

PubMed Central

Liu, W.; Montana, Vedrana; Parpura, Vladimir; Mohideen, U.

2010-01-01

We use an Atomic Force Microscope based single molecule measurements to evaluate the activation free energy in the interaction of SNARE proteins syntaxin 1A, SNAP25B and synaptobrevin 2 which regulate intracellular fusion of vesicles with target membranes. The dissociation rate of the binary syntaxin-synaptobrevin and the ternary syntaxin-SNAP25B-synaptobrevin complex was measured from the rupture force distribution as a function of the rate of applied force. The temperature dependence of the spontaneous dissociation rate was used to obtain the activation energy to the transition state of 19.8 ± 3.5 kcal/mol = 33 ± 6 kBT and 25.7 ± 3.0 kcal/mol = 43 ± 5 kBT for the binary and ternary complex, respectively. They are consistent with those measured previously for the ternary complex in lipid membranes and are of order expected for bilayer fusion and pore formation. The ΔG was 12.4–16.6 kcal/mol = 21–28 kBT and 13.8–18.0 kcal/mol = 23–30 kBT for the binary and ternary complex, respectively. The ternary complex was more stable by 1.4 kcal/mol = 2.3 kBT, consistent with the spontaneous dissociation rates. The higher adhesion energies and smaller molecular extensions measured with SNAP25B point to its possible unique and important physiological role in tethering/docking the vesicle in closer proximity to the plasma membrane and increasing the probability for fusion completion. PMID:20107522

Measurement of the membrane dipole electric field in DMPC vesicles using vibrational shifts of p-cyanophenylalanine and molecular dynamics simulations.

PubMed

Shrestha, Rebika; Cardenas, Alfredo E; Elber, Ron; Webb, Lauren J

2015-02-19

The magnitude of the membrane dipole field was measured using vibrational Stark effect (VSE) shifts of nitrile oscillators placed on the unnatural amino acid p-cyanophenylalanine (p-CN-Phe) added to a peptide sequence at four unique positions. These peptides, which were based on a repeating alanine-leucine motif, intercalated into small unilamellar DMPC vesicles which formed an α-helix as confirmed by circular dichroic (CD) spectroscopy. Molecular dynamics simulations of the membrane-intercalated helix containing two of the nitrile probes, one near the headgroup region of the lipid (αLAX(25)) and one buried in the interior of the bilayer (αLAX(16)), were used to examine the structure of the nitrile with respect to the membrane normal, the assumed direction of the dipole field, by quantifying both a small tilt of the helix in the bilayer and conformational rotation of the p-CN-Phe side chain at steady state. Vibrational absorption energies of the nitrile oscillator at each position showed a systematic blue shift as the nitrile was stepped toward the membrane interior; for several different concentrations of peptide, the absorption energy of the nitrile located in the middle of the bilayer was ∼3 cm(-1) greater than that of the nitrile closest to the surface of the membrane. Taken together, the measured VSE shifts and nitrile orientations within the membrane resulted in an absolute magnitude of 8-11 MV/cm for the dipole field, at the high end of the range of possible values that have been accumulated from a variety of indirect measurements. Implications for this are discussed.

Measurement of the Membrane Dipole Electric Field in DMPC Vesicles Using Vibrational Shifts of p-Cyanophenylalanine and Molecular Dynamics Simulations

PubMed Central

Shrestha, Rebika; Cardenas, Alfredo E.; Elber, Ron; Webb, Lauren J.

2015-01-01

The magnitude of the membrane dipole field was measured using vibrational Stark effect (VSE) shifts of nitrile oscillators placed on the unnatural amino acid p-cyanophenylalanine (p-CN-Phe) added to a peptide sequence at four unique positions. These peptides, which were based on a repeating alanine-leucine motif, intercalated into small unilamellar DMPC vesicles which formed an α-helix as confirmed by circular dichroic (CD) spectroscopy. Molecular dynamics simulations of the membrane-intercalated helix containing two of the nitrile probes, one near the head-group region of the lipid (αLAX(25)) and one buried in the interior of the bilayer (αLAX(16)), were used to examine the structure of the nitrile with respect to the membrane normal, the assumed direction the dipole field, by quantifying both a small tilt of the helix in the bilayer and conformational rotation of the p-CN-Phe side chain at steady-state. Vibrational absorption energies of the nitrile oscillator at each position showed a systematic blue shift as the nitrile was stepped towards the membrane interior; for several different concentrations of peptide, the absorption energy of the nitrile located in the middle of the bilayer was ~3 cm−1 greater than that of the nitrile closest to the surface of the membrane. Taken together, the measured VSE shifts and nitrile orientations within the membrane resulted in a value of 8 – 11 MV/cm for the dipole field, at the high end of the range of possible values that have been accumulated from a variety of indirect measurements. Implications for this are discussed. PMID:25602635

Surface electrostatics of lipid bilayers by EPR of a pH-sensitive spin-labeled lipid.

PubMed

Voinov, Maxim A; Rivera-Rivera, Izarys; Smirnov, Alex I

2013-01-08

Many biophysical processes such as insertion of proteins into membranes and membrane fusion are governed by bilayer electrostatic potential. At the time of this writing, the arsenal of biophysical methods for such measurements is limited to a few techniques. Here we describe a, to our knowledge, new spin-probe electron paramagnetic resonance (EPR) approach for assessing the electrostatic surface potential of lipid bilayers that is based on a recently synthesized EPR probe (IMTSL-PTE) containing a reversibly ionizable nitroxide tag attached to the lipids' polar headgroup. EPR spectra of the probe directly report on its ionization state and, therefore, on electrostatic potential through changes in nitroxide magnetic parameters and the degree of rotational averaging. Further, the lipid nature of the probe provides its full integration into lipid bilayers. Tethering the nitroxide moiety directly to the lipid polar headgroup defines the location of the measured potential with respect to the lipid bilayer interface. Electrostatic surface potentials measured by EPR of IMTSL-PTE show a remarkable (within ±2%) agreement with the Gouy-Chapman theory for anionic DMPG bilayers in fluid (48°C) phase at low electrolyte concentration (50 mM) and in gel (17°C) phase at 150-mM electrolyte concentration. This agreement begins to diminish for DMPG vesicles in gel phase (17°C) upon varying electrolyte concentration and fluid phase bilayers formed from DMPG/DMPC and POPG/POPC mixtures. Possible reasons for such deviations, as well as the proper choice of an electrostatically neutral reference interface, have been discussed. Described EPR method is expected to be fully applicable to more-complex models of cellular membranes. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Surface Electrostatics of Lipid Bilayers by EPR of a pH-Sensitive Spin-Labeled Lipid

PubMed Central

Voinov, Maxim A.; Rivera-Rivera, Izarys; Smirnov, Alex I.

2013-01-01

Many biophysical processes such as insertion of proteins into membranes and membrane fusion are governed by bilayer electrostatic potential. At the time of this writing, the arsenal of biophysical methods for such measurements is limited to a few techniques. Here we describe a, to our knowledge, new spin-probe electron paramagnetic resonance (EPR) approach for assessing the electrostatic surface potential of lipid bilayers that is based on a recently synthesized EPR probe (IMTSL-PTE) containing a reversibly ionizable nitroxide tag attached to the lipids’ polar headgroup. EPR spectra of the probe directly report on its ionization state and, therefore, on electrostatic potential through changes in nitroxide magnetic parameters and the degree of rotational averaging. Further, the lipid nature of the probe provides its full integration into lipid bilayers. Tethering the nitroxide moiety directly to the lipid polar headgroup defines the location of the measured potential with respect to the lipid bilayer interface. Electrostatic surface potentials measured by EPR of IMTSL-PTE show a remarkable (within ±2%) agreement with the Gouy-Chapman theory for anionic DMPG bilayers in fluid (48°C) phase at low electrolyte concentration (50 mM) and in gel (17°C) phase at 150-mM electrolyte concentration. This agreement begins to diminish for DMPG vesicles in gel phase (17°C) upon varying electrolyte concentration and fluid phase bilayers formed from DMPG/DMPC and POPG/POPC mixtures. Possible reasons for such deviations, as well as the proper choice of an electrostatically neutral reference interface, have been discussed. Described EPR method is expected to be fully applicable to more-complex models of cellular membranes. PMID:23332063

Inositol Depletion Restores Vesicle Transport in Yeast Phospholipid Flippase Mutants

PubMed Central

Yamagami, Kanako; Yamamoto, Takaharu; Sakai, Shota; Mioka, Tetsuo; Sano, Takamitsu; Igarashi, Yasuyuki; Tanaka, Kazuma

2015-01-01

In eukaryotic cells, type 4 P-type ATPases function as phospholipid flippases, which translocate phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of the lipid bilayer. Flippases function in the formation of transport vesicles, but the mechanism remains unknown. Here, we isolate an arrestin-related trafficking adaptor, ART5, as a multicopy suppressor of the growth and endocytic recycling defects of flippase mutants in budding yeast. Consistent with a previous report that Art5p downregulates the inositol transporter Itr1p by endocytosis, we found that flippase mutations were also suppressed by the disruption of ITR1, as well as by depletion of inositol from the culture medium. Interestingly, inositol depletion suppressed the defects in all five flippase mutants. Inositol depletion also partially restored the formation of secretory vesicles in a flippase mutant. Inositol depletion caused changes in lipid composition, including a decrease in phosphatidylinositol and an increase in phosphatidylserine. A reduction in phosphatidylinositol levels caused by partially depleting the phosphatidylinositol synthase Pis1p also suppressed a flippase mutation. These results suggest that inositol depletion changes the lipid composition of the endosomal/TGN membranes, which results in vesicle formation from these membranes in the absence of flippases. PMID:25781026

Measuring H(+) Pumping and Membrane Potential Formation in Sealed Membrane Vesicle Systems.

PubMed

Wielandt, Alex Green; Palmgren, Michael G; Fuglsang, Anja Thoe; Günther-Pomorski, Thomas; Justesen, Bo Højen

2016-01-01

The activity of enzymes involved in active transport of matter across lipid bilayers can conveniently be assayed by measuring their consumption of energy, such as ATP hydrolysis, while it is more challenging to directly measure their transport activities as the transported substrate is not converted into a product and only moves a few nanometers in space. Here, we describe two methods for the measurement of active proton pumping across lipid bilayers and the concomitant formation of a membrane potential, applying the dyes 9-amino-6-chloro-2-methoxyacridine (ACMA) and oxonol VI. The methods are exemplified by assaying transport of the Arabidopsis thaliana plasma membrane H(+)-ATPase (proton pump), which after heterologous expression in Saccharomyces cerevisiae and subsequent purification has been reconstituted in proteoliposomes.

Confocal Raman Microscopy for pH-Gradient Preconcentration and Quantitative Analyte Detection in Optically Trapped Phospholipid Vesicles.

PubMed

Hardcastle, Chris D; Harris, Joel M

2015-08-04

The ability of a vesicle membrane to preserve a pH gradient, while allowing for diffusion of neutral molecules across the phospholipid bilayer, can provide the isolation and preconcentration of ionizable compounds within the vesicle interior. In this work, confocal Raman microscopy is used to observe (in situ) the pH-gradient preconcentration of compounds into individual optically trapped vesicles that provide sub-femtoliter collectors for small-volume samples. The concentration of analyte accumulated in the vesicle interior is determined relative to a perchlorate-ion internal standard, preloaded into the vesicle along with a high-concentration buffer. As a guide to the experiments, a model for the transfer of analyte into the vesicle based on acid-base equilibria is developed to predict the concentration enrichment as a function of source-phase pH and analyte concentration. To test the concept, the accumulation of benzyldimethylamine (BDMA) was measured within individual 1 μm phospholipid vesicles having a stable initial pH that is 7 units lower than the source phase. For low analyte concentrations in the source phase (100 nM), a concentration enrichment into the vesicle interior of (5.2 ± 0.4) × 10(5) was observed, in agreement with the model predictions. Detection of BDMA from a 25 nM source-phase sample was demonstrated, a noteworthy result for an unenhanced Raman scattering measurement. The developed model accurately predicts the falloff of enrichment (and measurement sensitivity) at higher analyte concentrations, where the transfer of greater amounts of BDMA into the vesicle titrates the internal buffer and decreases the pH gradient. The predictable calibration response over 4 orders of magnitude in source-phase concentration makes it suitable for quantitative analysis of ionizable compounds from small-volume samples. The kinetics of analyte accumulation are relatively fast (∼15 min) and are consistent with the rate of transfer of a polar aromatic molecule across a gel-phase phospholipid membrane.

Comparative structure-function characterization of the saposin-like domains from potato, barley, cardoon and Arabidopsis aspartic proteases.

PubMed

Bryksa, Brian C; Grahame, Douglas A; Yada, Rickey Y

2017-05-01

The present study characterized the aspartic protease saposin-like domains of four plant species, Solanum tuberosum (potato), Hordeum vulgare L. (barley), Cynara cardunculus L. (cardoon; artichoke thistle) and Arabidopsis thaliana, in terms of bilayer disruption and fusion, and structure pH-dependence. Comparison of the recombinant saposin-like domains revealed that each induced leakage of bilayer vesicles composed of a simple phospholipid mixture with relative rates Arabidopsis>barley>cardoon>potato. When compared for leakage of bilayer composed of a vacuole-like phospholipid mixture, leakage was approximately five times higher for potato saposin-like domain compared to the others. In terms of fusogenic activity, distinctions between particle size profiles were noted among the four proteins, particularly for potato saposin-like domain. Bilayer fusion assays in reducing conditions resulted in altered fusion profiles except in the case of cardoon saposin-like domain which was virtually unchanged. Secondary structure profiles were similar across all four proteins under different pH conditions, although cardoon saposin-like domain appeared to have higher overall helix structure. Furthermore, increases in Trp emission upon protein-bilayer interactions suggested that protein structure rearrangements equilibrated with half-times ranging from 52 to 120s, with cardoon saposin-like domain significantly slower than the other three species. Overall, the present findings serve as a foundation for future studies seeking to delineate protein structural features and motifs in protein-bilayer interactions based upon variability in plant aspartic protease saposin-like domain structures. Copyright © 2017 Elsevier B.V. All rights reserved.

The influence of hyaluronan on the structure of a DPPC-bilayer under high pressures.

PubMed

Zander, Thomas; Wieland, D C Florian; Raj, Akanksha; Wang, Min; Nowak, Benedikt; Krywka, Christina; Dėdinaitė, Andra; Claesson, Per Martin; Garamus, Vasil M; Schreyer, Andreas; Willumeit-Römer, Regine

2016-06-01

The superior lubrication properties of synovial joints have inspired many studies aiming at uncovering the molecular mechanisms which give rise to low friction and wear. However, the mechanisms are not fully understood yet, and, in particular, it has not been elucidated how the biolubricants present at the interface of cartilage respond to high pressures, which arise during high loads of joints. In this study we utilize a simple model system composed of two biomolecules that have been implied as being important for joint lubrication. It consists of a solid supported dipalmitoylphosphatidylcholin (DPPC) bilayer, which was formed via vesicles fusion on a flat Si wafer, and the anionic polysaccharide hyaluronan (HA). We first characterized the structure of the HA layer that adsorbed to the DPPC bilayers at ambient pressure and different temperatures using X-ray reflectivity (XRR) measurements. Next, XRR was utilized to evaluate the response of the system to high hydrostatic pressures, up to 2kbar (200MPa), at three different temperatures. By means of fluorescence microscopy images the distribution of DPPC and HA on the surface was visualized. Our data suggest that HA adsorbs to the headgroup region that is oriented towards the water side of the supported bilayer. Phase transitions of the bilayer in response to temperature and pressure changes were also observed in presence and absence of HA. Our results reveal a higher stability against high hydrostatic pressures for DPPC/HA composite layers compared to that of the DPPC bilayer in absence of HA. Copyright © 2016 Elsevier B.V. All rights reserved.

The vesosome-- a multicompartment drug delivery vehicle.

PubMed

Kisak, E T; Coldren, B; Evans, C A; Boyer, C; Zasadzinski, J A

2004-01-01

Assembling structures to divide space controllably and spontaneously into subunits at the nanometer scale is a significant challenge, although one that biology has solved in two distinct ways: prokaryotes and eukaryotes. Prokaryotes have a single compartment delimited by one or more lipid-protein membranes. Eukaryotes have nested-membrane structures that provide internal compartments--such as the cell nucleus and cell organelles in which specialized functions are carried out. We have developed a simple method of creating nested bilayer compartments in vitro via the "interdigitated" bilayer phase formed by adding ethanol to a variety of saturated phospholipids. At temperatures below the gel-liquid crystalline transition, T(m), the interdigitated lipid-ethanol sheets are rigid and flat; when the temperature is raised above T(m), the sheets become flexible and close on themselves and the surrounding solution to form closed compartments. During this closure, the sheets can entrap other vesicles, biological macromolecules, or colloidal particles. The result is efficient and spontaneous encapsulation without disruption of even fragile materials to form biomimetic nano-environments for possible use in drug delivery, colloidal stabilization, or as microreactors. The vesosome structure can take full advantage of the 40 years of progress in liposome development including steric stabilization, pH loading of drugs, and intrinsic biocompatibility. However, the multiple compartments of the vesosome give better protection to the interior contents in serum, leading to extended release of model compounds in comparison to unilamellar liposomes.

Compartmentalisation Strategies for Hydrocarbon-based Biota on Titan

NASA Astrophysics Data System (ADS)

Norman, Lucy; Skipper, N.; Fortes, A. D.; Crawford, I.

2012-05-01

The goal of our study is to determine the nature of compartimentalisation strategies for any organisms inhabiting the hydrocarbon polar lakes of Titan (the largest moon of Saturn). Titan is the only moon in the solar system with a substantial atmosphere; it has a remarkably earth- like ‘hydrological' cycle, with evidence for storm cloud activity, rainfall and river systems, often with subsequent drainage into lakes and seas at high latitudes (1, 2). However, at the low surface temperature of 94 K, the liquid involved is not water, but a mixture of methane, ethane and propane (3). Due to Titan's plethora of organic chemistries it has long been recognised that it may provide useful insights into the pre-biotic evolution of early Earth (4). Since receiving huge amounts of data via the Cassini-Huygens mission to the Saturnian system astrobiologists have speculated that exotic biota might currently inhabit this environment, consuming acetylene (snowed onto the surface as a result of atmospheric photochemistry) and hydrogen whilst excreting methane (5, 6). This consumption should lead to an anomalous hydrogen depletion near the surface; and evidence to suggest this depletion exists has been published (7). Nevertheless, many questions still remain concerning the possible physiological traits of biota in these environments, including whether cell- like structures can form in low temperature, low molecular weight hydrocarbons. Terrestrial cell membranes are vesicular structures composed primarily of a phospholipid bilayer with the hydrophilic head groups arranged around the periphery. Simplified analogues of these structures, called liposomes, plus vesicles prepared from other surfactant types e.g. polymers, are artificially prepared primarily for pharmaceutical reasons e.g. drug delivery. However, these types of model cell membrane are also thought to be akin to the first proto-cells that terrestrial life utilised (8). Recently reversed aggregate types, such as reverse spherical micelles (a single lipid layer with a polar core) and reverse vesicles (a bilayer with a nonpolar core - see Fig.1), have been studied in nonpolar liquids, such as hydrocarbon solvents, due to pharmaceutical interests (9). However, these reverse vesicles may also be ideal model cell membranes for hydrocarbon-based organisms inhabiting Titan's hydrocarbon lakes (10). A variety of different surfactants have been used to create reverse vesicles in liquid hydrocarbons to date; non-ionic ethers (11) and esters (9, 12); catanionic surfactant mixtures (13); zwitterionic gemini surfactants (14); coblock polymer surfactants (15); and amphiphilic phospholipid surfactants (16). In order to discover whether certain amphiphiles (a compound possessing both hydrophilic and lipophilic properties) will exhibit vesicular behaviour within liquid hydrocarbons, and to analyse their structure, we will carry out experimental studies using environmental conditions that are increasing comparable to those found on the surface of Titan. Experimental studies to determine the presence of vesicles include the use of microscopy, the Tyndall scattering effect, transmission electron microscopy (TEM), nanoparticle tracking anaylsis (NTA), and - if beamtime is awarded - small-angle neutron scattering (SANS) and small-angle x-ray scattering (SAXS). These potential ‘biomarkers' could be searched for in results from proposed missions to the lakes, such as the proposed Titan lake lander - ‘TiME' (17).

Fabrication of patterned surface by soft lithographic technique for confinement of lipid bilayer

NASA Astrophysics Data System (ADS)

Moulick, Ranjita Ghosh; Mayer, Dirk

2018-04-01

In this paper we demonstrated that a 3D pattern can be well transferred from a silicon Master to a gold substrate using µcontact printing. In this process 1-Octadecanthiol served as an ink and printing followed by etching generated the desired pattern on the gold substrate. The prepatterned substrate was also used for lipid vesicle fusion and revealed that lipid molecules selectively bind to the gold layer.

Expression, purification and functional reconstitution of slack sodium-activated potassium channels.

PubMed

Yan, Yangyang; Yang, Youshan; Bian, Shumin; Sigworth, Fred J

2012-11-01

The slack (slo2.2) gene codes for a potassium-channel α-subunit of the 6TM voltage-gated channel family. Expression of slack results in Na(+)-activated potassium channel activity in various cell types. We describe the purification and reconstitution of Slack protein and show that the Slack α-subunit alone is sufficient for potassium channel activity activated by sodium ions as assayed in planar bilayer membranes and in membrane vesicles.

Synergistic effect of temperature and point defect on the mechanical properties of single layer and bi-layer graphene

NASA Astrophysics Data System (ADS)

Debroy, Sanghamitra; Pavan Kumar, V.; Vijaya Sekhar, K.; Acharyya, Swati Ghosh; Acharyya, Amit

2017-10-01

The present study reports a comprehensive molecular dynamics simulation of the effect of a) temperature (300-1073 K at intervals of every 100 K) and b) point defect on the mechanical behaviour of single (armchair and zigzag direction) and bilayer layer graphene (AA and AB stacking). Adaptive intermolecular reactive bond order (AIREBO) potential function was used to describe the many-body short-range interatomic interactions for the single layer graphene sheet. Moreover, Lennard Jones model was considered for bilayer graphene to incorporate the van der Waals interactions among the interlayers of graphene. The effect of temperature on the strain energy of single layer and bilayer graphene was studied in order to understand the difference in mechanical behaviour of the two systems. The strength of the pristine single layer graphene was found to be higher as compared to bilayer AA stacked graphene at all temperatures. It was observed at 1073 K and in the presence of vacancy defect the strength for single layer armchair sheet falls by 30% and for bilayer armchair sheet by 33% as compared to the pristine sheets at 300 K. The AB stacked graphene sheet was found to have a two-step rupture process. The strength of pristine AB sheet was found to decrease by 22% on increase of temperature from 300 K to 1073 K.

P-glycoprotein substrate transport assessed by comparing cellular and vesicular ATPase activity.

PubMed

Nervi, Pierluigi; Li-Blatter, Xiaochun; Aänismaa, Päivi; Seelig, Anna

2010-03-01

We compared the P-glycoprotein ATPase activity in inside-out plasma membrane vesicles and living NIH-MDR1-G185 cells with the aim to detect substrate transport. To this purpose we used six substrates which differ significantly in their passive influx through the plasma membrane. In cells, the cytosolic membrane leaflet harboring the substrate binding site of P-glycoprotein has to be approached by passive diffusion through the lipid membrane, whereas in inside-out plasma membrane vesicles, it is accessible directly from the aqueous phase. Compounds exhibiting fast passive influx compared to active efflux by P-glycoprotein induced similar ATPase activity profiles in cells and inside-out plasma membrane vesicles, because their concentrations in the cytosolic leaflets were similar. Compounds exhibiting similar influx as efflux induced in contrast different ATPase activity profiles in cells and inside-out vesicles. Their concentration was significantly lower in the cytosolic leaflet of cells than in the cytosolic leaflet of inside-out membrane vesicles, indicating that P-glycoprotein could cope with passive influx. P-glycoprotein thus transported all compounds at a rate proportional to ATP hydrolysis (i.e. all compounds were substrates). However, it prevented substrate entry into the cytosol only if passive influx of substrates across the lipid bilayer was in a similar range as active efflux. Copyright 2009 Elsevier B.V. All rights reserved.

The vesicle-to-micelle transition of phosphatidylcholine vesicles induced by nonionic detergents: effects of sodium chloride, sucrose and urea.

PubMed

Walter, A; Kuehl, G; Barnes, K; VanderWaerdt, G

2000-11-23

The vesicle-to-micelle transition of egg phosphatidylcholine LUVs induced by octylglucoside was studied in buffers with 0-4 M sodium chloride, sucrose or urea. We used both light scattering and fluorescent probes to follow the lipid-detergent complexes in these buffers. The vesicle-to-micelle transition process was fundamentally the same in each solute. However, the detergent-to-lipid ratio required for micelle formation shifted in ways that depended on the aqueous solute. The partitioning of octylglucoside between the vesicles and the aqueous phase was primarily determined by the change in its critical micelle concentration (cmc) induced by each solute. Specifically, the cmc decreased in high salt and sucrose buffers but increased in high concentrations of urea. Cmc for two additional nonionic detergents, decyl- and dodecyl-maltoside, and three zwittergents (3-12, 3-14 and 3-16) were determined as a function of concentration for each of the solutes. In all cases NaCl and sucrose decreased the solubility of the detergents, whereas urea increased their solubilities. The effects clearly depended on acyl chain length in urea-containing solutions, but this dependence was less clear with increasing NaCl and sucrose concentrations. The contributions of these solutes to solubility and to interfacial interactions in the bilayers, pure and mixed micelles are considered.

Poly(aniline) nanowires in sol-gel coated ITO: A pH-responsive substrate for planar supported lipid bilayers

PubMed Central

Ge, Chenhao; Orosz, Kristina S.; Armstrong, Neal R.; Saavedra, S. Scott

2011-01-01

Facilitated ion transport across an artificial lipid bilayer coupled to a solid substrate is a function common to several types of bioelectronic devices based on supported membranes, including biomimetic fuel cells and ion channel biosensors. Described here is fabrication of a pH-sensitive transducer composed of a porous sol-gel layer derivatized with poly(aniline) (PANI) nanowires grown from an underlying planar indium-tin oxide (ITO) electrode. The upper sol-gel surface is hydrophilic, smooth, and compatible with deposition of a planar supported lipid bilayer (PSLB) formed via vesicle fusion. Conducting tip AFM was used to show that the PANI wires are connected to the ITO, which convert this electrode into a potentiometric pH sensor. The response to changes in the pH of the buffer contacting the PANI nanowire/sol-gel/ITO electrode is blocked by the very low ion permeability of the overlying, fluid PSLB. The feasibility of using this assembly to monitor facilitated proton transport across the PSLB was demonstrated by doping the membrane with lipophilic ionophores that respond to a transmembrane pH gradient, which produced an apparent proton permeability several orders of magnitude greater than values measured for undoped lipid bilayers. PMID:21707069

Nano- and microparticles at fluid and biological interfaces.

PubMed

Dasgupta, S; Auth, T; Gompper, G

2017-09-20

Systems with interfaces are abundant in both technological applications and biology. While a fluid interface separates two fluids, membranes separate the inside of vesicles from the outside, the interior of biological cells from the environment, and compartmentalize cells into organelles. The physical properties of interfaces are characterized by interface tension, those of membranes are characterized by bending and stretching elasticity. Amphiphilic molecules like surfactants that are added to a system with two immiscible fluids decrease the interface tension and induce a bending rigidity. Lipid bilayer membranes of vesicles can be stretched or compressed by osmotic pressure; in biological cells, also the presence of a cytoskeleton can induce membrane tension. If the thickness of the interface or the membrane is small compared with its lateral extension, both can be described using two-dimensional mathematical surfaces embedded in three-dimensional space. We review recent work on the interaction of particles with interfaces and membranes. This can be micrometer-sized particles at interfaces that stabilise emulsions or form colloidosomes, as well as typically nanometer-sized particles at membranes, such as viruses, parasites, and engineered drug delivery systems. In both cases, we first discuss the interaction of single particles with interfaces and membranes, e.g. particles in external fields, non-spherical particles, and particles at curved interfaces, followed by interface-mediated interaction between two particles, many-particle interactions, interface and membrane curvature-induced phenomena, and applications.

Nano- and microparticles at fluid and biological interfaces

NASA Astrophysics Data System (ADS)

Dasgupta, S.; Auth, T.; Gompper, G.

2017-09-01

Systems with interfaces are abundant in both technological applications and biology. While a fluid interface separates two fluids, membranes separate the inside of vesicles from the outside, the interior of biological cells from the environment, and compartmentalize cells into organelles. The physical properties of interfaces are characterized by interface tension, those of membranes are characterized by bending and stretching elasticity. Amphiphilic molecules like surfactants that are added to a system with two immiscible fluids decrease the interface tension and induce a bending rigidity. Lipid bilayer membranes of vesicles can be stretched or compressed by osmotic pressure; in biological cells, also the presence of a cytoskeleton can induce membrane tension. If the thickness of the interface or the membrane is small compared with its lateral extension, both can be described using two-dimensional mathematical surfaces embedded in three-dimensional space. We review recent work on the interaction of particles with interfaces and membranes. This can be micrometer-sized particles at interfaces that stabilise emulsions or form colloidosomes, as well as typically nanometer-sized particles at membranes, such as viruses, parasites, and engineered drug delivery systems. In both cases, we first discuss the interaction of single particles with interfaces and membranes, e.g. particles in external fields, non-spherical particles, and particles at curved interfaces, followed by interface-mediated interaction between two particles, many-particle interactions, interface and membrane curvature-induced phenomena, and applications.

Asymmetric distribution of charged lipids between the leaflets of a vesicle bilayer induced by melittin and alamethicin

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

Qian, Shuo; Heller, William T

2011-01-01

Cellular membranes are complex mixtures of lipids, proteins, and other small molecules that provide functional, dynamic barriers between the cell and its environment, as well as between environments within the cell. The lipid composition of the membrane is highly specific and controlled in terms of both content and lipid localization. The membrane structure results from the complex interplay between the wide varieties of molecules present. Here, small-angle neutron scattering and selective deuterium labeling were used to probe the impact of the membrane-active peptides melittin and alamethicin on the structure of lipid bilayers composed of a mixture of the lipids dimyristoylmore » phosphatidylglycerol (DMPG) and chain-perdeuterated dimyristoyl phosphatidylcholine (DMPC). We found that both peptides enriched the outer leaflet of the bilayer with the negatively charged DMPG, creating an asymmetric distribution of lipids. The level of enrichment is peptide concentration-dependent and is stronger for melittin than it is for alamethicin. The enrichment between the inner and outer bilayer leaflets occurs at very low peptide concentrations and increases with peptide concentration, including when the peptide adopts a membrane-spanning, pore-forming state. The results suggest that these membrane-active peptides may have a secondary stressful effect on target cells at low concentrations that results from a disruption of the lipid distribution between the inner and outer leaflets of the bilayer that is independent of the formation of transmembrane pores.« less

Temperature dependence of structure, bending rigidity, and bilayer interactions of dioleoylphosphatidylcholine bilayers.

PubMed

Pan, Jianjun; Tristram-Nagle, Stephanie; Kucerka, Norbert; Nagle, John F

2008-01-01

X-ray diffuse scattering was measured from oriented stacks and unilamellar vesicles of dioleoylphosphatidylcholine lipid bilayers to obtain the temperature dependence of the structure and of the material properties. The area/molecule, A, was 75.5 A(2) at 45 degrees C, 72.4 A(2) at 30 degrees C, and 69.1 A(2) at 15 degrees C, which gives the area expansivity alpha(A) = 0.0029/deg at 30 degrees C, and we show that this value is in excellent agreement with the polymer brush theory. The bilayer becomes thinner with increasing temperature; the contractivity of the hydrocarbon portion was alpha(Dc) = 0.0019/deg; the difference between alpha(A) and alpha(Dc) is consistent with the previously measured volume expansivity alpha(Vc) = 0.0010/deg. The bending modulus K(C) decreased as exp(455/T) with increasing T (K). Our area compressibility modulus K(A) decreased with increasing temperature by 5%, the same as the surface tension of dodecane/water, in agreement again with the polymer brush theory. Regarding interactions between bilayers, the compression modulus B as a function of interbilayer water spacing D'(W) was found to be nearly independent of temperature. The repulsive fluctuation pressure calculated from B and K(C) increased with temperature, and the Hamaker parameter for the van der Waals interaction was nearly independent of temperature; this explains why the fully hydrated water spacing, D'(W), that we obtain from our structural results increases with temperature.

Liposome formation in microgravity.

PubMed

Claassen, D E; Spooner, B S

1996-01-01

Liposomes are artificial vesicles with a phospholipid bilayer membrane. The formation of liposomes is a self-assembly process that is driven by the amphipathic nature of phospholipid molecules and can be observed during the removal of detergent from phospholipids dissolved in detergent micelles. As detergent concentration in the mixed micelles decreases, the non-polar tail regions of phospholipids produce a hydrophobic effect that drives the micelles to fuse and form planar bilayers in which phospholipids orient with tail regions to the center of the bilayer and polar head regions to the external surface. Remaining detergent molecules shield exposed edges of the bilayer sheet from the aqueous environment. Further removal of detergent leads to intramembrane folding and membrane folding and membrane vesiculation, forming liposomes. We have observed that the formation of liposomes is altered in microgravity. Liposomes that were formed at 1-g did not exceed 150 nm in diameter, whereas liposomes that were formed during spaceflight exhibited diameters up to 2000 nm. Using detergent-stabilized planar bilayers, we determined that the stage of liposome formation most influenced by gravity is membrane vesiculation. In addition, we found that small, equipment-induced fluid disturbances increased vesiculation and negated the size-enhancing effects of microgravity. However, these small disturbances had no effect on liposome size at 1-g, likely due to the presence of gravity-induced buoyancy-driven fluid flows (e.g., convection currents). Our results indicate that fluid disturbances, induced by gravity, influence the vesiculation of membranes and limit the diameter of forming liposomes.

Liposome formation in microgravity

NASA Astrophysics Data System (ADS)

Claassen, D. E.; Spooner, B. S.

Liposomes are artificial vesicles with a phospholipid bilayer membrane. The formation of liposomes is a self-assembly process that is driven by the amphipathic nature of phospholipid molecules and can be observed during the removal of detergent from phospholipids dissolved in detergent micelles. As detergent concentration in the mixed micelles decreases, the non-polar tail regions of phospholipids produce a hydrophobic effect that drives the micelles to fuse and form planar bilayers in which phospholipids orient with tail regions to the center of the bilayer and polar head regions to the external surface. Remaining detergent molecules shield exposed edges of the bilayer sheet from the aqueous environment. Further removal of detergent leads to intramembrane folding and membrane vesiculation, forming liposomes. We have observed that the formation of liposomes is altered in microgravity. Liposomes that were formed at 1-g did not exceed 150 nm in diameter, whereas liposomes that were formed during spaceflight exhibited diameters up to 2000 nm. Using detergent-stabilized planar bilayers, we determined that the stage of liposome formation most influenced by gravity is membrane vesiculation. In addition, we found that small, equipment-induced fluid disturbances increased vesiculation and negated the size-enhancing effects of microgravity. However, these small disturbances had no effect on liposome size at 1-g, likely due to the presence of gravity-induced buoyancy-driven fluid flows (e.g., convection currents). Our results indicate that fluid disturbances, induced by gravity, influence the vesiculation of membranes and limit the diameter of forming liposomes.

A novel vesicular carrier, transethosome, for enhanced skin delivery of voriconazole: characterization and in vitro/in vivo evaluation.

PubMed

Song, Chung Kil; Balakrishnan, Prabagar; Shim, Chang-Koo; Chung, Suk-Jae; Chong, Saeho; Kim, Dae-Duk

2012-04-01

This study describes a novel carrier, transethosome, for enhanced skin delivery of voriconazole. Transethosomes (TELs) are composed of phospholipid, ethanol, water and edge activator (surfactants) or permeation enhancer (oleic acid). Characterization of the TELs was based on results from recovery, particle size, transmission electron microscopy (TEM), zeta potential and elasticity studies. In addition, skin permeation profile was obtained using static vertical diffusion Franz cells and hairless mouse skin treated with TELs containing 0.3% (w/w) voriconazole, and compared with those of ethosomes (ELs), deformable liposomes (DLs), conventional liposomes (CLs) and control (polyethylene glycol, PG) solutions. The recovery of the studied vesicles was above 90% in all vesicles, as all of them contained ethanol (7-30%). There was no significant difference in the particles size of all vesicles. The TEM study revealed that the TELs were in irregular spherical shape, implying higher fluidity due to perturbed lipid bilayer compared to that of other vesicles which were of spherical shape. The zeta potential of vesicles containing sodium taurocholate or oleic acid showed higher negative value compared to other vesicles. The elasticities of ELs and TELs were much higher than that of CLs and DLs. Moreover, TELs dramatically enhanced the skin permeation of voriconazole compared to the control and other vesicles (p<0.05). Moreover, the TELs enhanced both in vitro and in vivo skin deposition of voriconazole in the dermis/epidermis region compared to DLs, CLs and control. Therefore, based on the current study, the novel carrier TELs could serve as an effective dermal delivery for voriconazole. Copyright © 2011 Elsevier B.V. All rights reserved.

Phase equilibria and formation of vesicles of dioleoylphosphatidylcholine in glycerol/water mixtures.

PubMed

Johansson, L B; Kalman, B; Wikander, G; Fransson, A; Fontell, K; Bergenståhl, B; Lindblom, G

1993-07-04

The lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) forms a lamellar liquid crystalline phase (L alpha) in arbitrary mixtures of glycerol and water. The phase has been characterized by means of X-ray diffraction, 31P-NMR spectroscopy and differential scanning calorimetry (DSC). In the L alpha state, and for DOPC concentrations greater than 50% (w/w), the thickness of the lipid bilayer decreases, while the area of the polar head group increases with increasing glycerol concentration. The phase transition from gel to L alpha state occurs in the range of 240 to 260 K. Contrary to a previous (McDaniel, R.V., McIntosh, T.J. and Simon, S.A. (1983) Biochim. Biophys. Acta 731, 97) study of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) we find that in the gel state, the thickness of the DOPC lipid bilayer is greater than that in the L alpha state. This suggests that in the gel state, the lipid acyl chains of DOPC are in extended configuration. The lamellar phase reaches its maximum swelling at about 50% (w/w) of DOPC. At lower DOPC concentrations a two-phase system is formed where the lamellar phase exists in equilibrium with excess of solvent. Unilamellar vesicles can be prepared from a diluted suspension of the lamellar phase either by using the sonicator or extruder technique. We show this by means of 31P-NMR, EPR and fluorescence spectroscopy. The mean radius of the vesicles, prepared by a sonicator, has been determined at different glycerol/water mixtures. It is found to decrease continuously from 100 A at 100% water to a minimum of 75 A at about 50% water in the solvent mixture. By further decreasing the water content in the solution, the radius rapidly increases, and a mean radius of 450 A is estimated at a water content of 10%. The rotational relaxation times of a fluorescent probe and two EPR spin probes, solubilized in DOPC vesicles, have been measured at different glycerol/water mixtures. It is found that the rotational rates are always much slower in the systems containing glycerol.

Correlating antimicrobial activity and model membrane leakage induced by nylon-3 polymers and detergents

PubMed Central

Hovakeemian, Sara G.; Liu, Runhui; Gellman, Samuel H.; Heerklotz, Heiko

2015-01-01

Most antimicrobial peptides act upon target microorganisms by permeabilizing their membranes. The mode of action is often assessed by vesicle leakage experiments that use model membranes, with the assumption that biological activity arises from permeabilization of the lipid bilayer. The current work aims to extend the interpretation of vesicle leakage results and examine the correlation between vesicle leakage and antimicrobial activity. To this end, we used a lifetime-based leakage assay with calcein-loaded vesicles to study the membrane permeabilizing properties of a novel antifungal polymer poly-NM, two of its analogs, and a series of detergents. In conjunction, the biological activities of these compounds against Candida albicans were assessed and correlated with data from vesicle leakage. Poly-NM induces all-or-none leakage in polar yeast lipid vesicles at the polymer’s MIC, 3 μg/mL. At this and higher concentrations, complete leakage after an initial lag time was observed. Concerted activity tests imply that this polymer acts independently of the detergent octyl glucoside (OG) for both vesicle leakage and activity against C. albicans spheroplasts. In addition, Poly-NM was found to have negligible activity against zwitterionic vesicles and red blood cells. Our results provide a consistent, detailed picture of the mode of action of Poly-NM: this polymer induces membrane leakage by electrostatic lipid clustering. In contrast, Poly-MM:CO, a nylon-3 polymer comprised of both cationic and hydrophobic segments, seems to act by a different mechanism that involves membrane asymmetry stress. Vesicle leakage for this polymer is transient (limited to <100%) and graded, non-specific among zwitterionic and polar yeast lipid vesicles, additive with detergent action, and correlates poorly with biological activity. Based on these results, we conclude that comprehensive leakage experiments can provide a detailed description of the mode of action of membrane permeabilizing compounds. Without this thorough approach, it would have been logical to assume that the two nylon-3 polymers we examined act via similar mechanisms; it is surprising that their mechanisms are so distinct. Some, but not all mechanisms of vesicle permeabilization allow for antimicrobial activity. PMID:26234884

Correlating antimicrobial activity and model membrane leakage induced by nylon-3 polymers and detergents.

PubMed

Hovakeemian, Sara G; Liu, Runhui; Gellman, Samuel H; Heerklotz, Heiko

2015-09-14

Most antimicrobial peptides act upon target microorganisms by permeabilizing their membranes. The mode of action is often assessed by vesicle leakage experiments that use model membranes, with the assumption that biological activity correlates with the permeabilization of the lipid bilayer. The current work aims to extend the interpretation of vesicle leakage results and examine the correlation between vesicle leakage and antimicrobial activity. To this end, we used a lifetime-based leakage assay with calcein-loaded vesicles to study the membrane permeabilizing properties of a novel antifungal polymer poly-NM, two of its analogs, and a series of detergents. In conjunction, the biological activities of these compounds against Candida albicans were assessed and correlated with data from vesicle leakage. Poly-NM induces all-or-none leakage in polar yeast lipid vesicles at the polymer's MIC, 3 μg mL(-1). At this and higher concentrations, complete leakage after an initial lag time was observed. Concerted activity tests imply that this polymer acts independently of the detergent octyl glucoside (OG) for both vesicle leakage and activity against C. albicans spheroplasts. In addition, poly-NM was found to have negligible activity against zwitterionic vesicles and red blood cells. Our results provide a consistent, detailed picture of the mode of action of poly-NM: this polymer induces membrane leakage by electrostatic lipid clustering. In contrast, poly-MM:CO, a nylon-3 polymer comprised of both cationic and hydrophobic segments, seems to act by a different mechanism that involves membrane asymmetry stress. Vesicle leakage for this polymer is transient (limited to <100%) and graded, non-specific among zwitterionic and polar yeast lipid vesicles, additive with detergent action, and correlates poorly with biological activity. Based on these results, we conclude that comprehensive leakage experiments can provide a detailed description of the mode of action of membrane permeabilizing compounds. Without this thorough approach, it would have been logical to assume that the two nylon-3 polymers we examined act via similar mechanisms; it is surprising that their mechanisms are so distinct. Some, but not all mechanisms of vesicle permeabilization allow for antimicrobial activity.

Phospholipid Bilayers: Stability and Encapsulation of Nanoparticles

NASA Astrophysics Data System (ADS)

Alipour, Elnaz; Halverson, Duncan; McWhirter, Samantha; Walker, Gilbert C.

2017-05-01

Nanoparticles are widely studied for their potential medical uses in diagnostics and therapeutics. The interface between a nanoparticle and its target has been a focus of research, both to guide the nanoparticle and to prevent it from deactivating. Given nature's frequent use of phospholipid vesicles as carriers, much attention has been paid to phospholipids as a vehicle for drug delivery. The physical chemistry of bilayer formation and nanoparticle encapsulation is complex, touching on fundamental properties of hydrophobicity. Understanding the design rules for particle synthesis and encapsulation is an active area of research. The aim of this review is to provide a perspective on what preparative guideposts have been empirically discovered and how these are related to theoretical understanding. In addition, we aim to summarize how modern theory is beginning to help guide the design of functional particles that can effectively cross biological membranes.

Influence of ceramide on the internal structure and hydration of the phospholipid bilayer studied by neutron and X-ray scattering

NASA Astrophysics Data System (ADS)

Kiselev, M. A.; Zemlyanaya, E. V.; Ryabova, N. Y.; Hauss, T.; Almasy, L.; Funari, S. S.; Zbytovska, J.; Lombardo, D.

2014-07-01

Small angle neutron scattering (SANS), neutron diffraction and X-ray powder diffraction were used to investigate influence of N-stearoyl phytosphingosine (CER[NP]) and α-hydroxy- N-stearoyl phytosphingosine (CER[AP]) on the internal structure and hydration of DMPC membrane in fully and partly hydrated states at T = 30 °C. Application of Fourier analysis for diffraction data and model calculations for the SANS data evidence that addition of both CER[NP] and CER[AP] in small concentrations promotes significant changes in the organization of DMPC bilayers, such as the increase of the hydrophobic core region. SANS data evidence a decrease in the average radius and polydispersity of the vesicles that can be ascribed to hydrogen bonds interactions that favor tight lipid packing with a compact, more rigid character.

Small-angle x-ray scattering from lipid bilayers is well described by modified Caillé theory but not by paracrystalline theory.

PubMed Central

Zhang, R; Tristram-Nagle, S; Sun, W; Headrick, R L; Irving, T C; Suter, R M; Nagle, J F

1996-01-01

X-ray scattering data at high instrumental resolution are reported for multilamellar vesicles of L alpha phase lipid bilayers of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine at 50 degrees C under varying osmotic pressure. The data are fitted to two theories that account for noncrystalline disorder, paracrystalline theory (PT) and modified Caillé theory (MCT). The MCT provides good fits to the data, much better than the PT fits. The particularly important characteristic of MCT is the long power law tails in the scattering. PT fits (as well as ordinary integration with no attempt to account for the noncrystalline disorder) increasingly underestimate this scattering intensity as the order h increases, thereby underestimating the form factors used to obtain electron density profiles. Images FIGURE 4 PMID:8770211

Factors influencing uptake and retention of amino-containing drugs in large unilamellar vesicles exhibiting transmembrane pH gradients.

PubMed

Maurer-Spurej, E; Wong, K F; Maurer, N; Fenske, D B; Cullis, P R

1999-01-12

The level of uptake and retention of amino-containing drugs in large unilamellar vesicles (LUVs) following uptake in response to a transmembrane pH gradient (DeltapH) can vary dramatically depending on the drug. For example, the anticancer drugs doxorubicin and epirubicin can be readily retained, whereas the anticancer drug vincristine and the antibiotic ciprofloxacin tend to leak out rapidly. In this investigation, we examine the influence of the hydrophobicity of the entrapped amines (that induce the DeltapH) and the anionic lipid content of the LUV on drug retention. It is shown that entrapment of increasingly hydrophobic monoamines (methylamine to amylamine) all lead to an induced DeltapH of 3 units and essentially complete drug uptake under the conditions employed, but do not lead to improved retention of vincristine and ciprofloxacin. However, significantly improved retention could be achieved by substitution of the anionic lipid distearoylphosphatidylglycerol (DSPG) for distearoylphosphatidylcholine (DSPC) in the LUV bilayer. Further, it is shown that if the induced DeltapH is reduced to 1.4 units (driven by entrapped diamine) nearly 100% accumulation of doxorubicin and epirubicin could be achieved, whereas only 25% loading for vincristine and ciprofloxacin was observed. Taken together these results provide methodology for improving (weak base) drug retention in liposomes and indicate that drugs that can partition into the lipid bilayer exhibit improved uptake and retention characteristics.

Large Femtosecond Two-Photon Absorption Cross-Sections of Fullerosome Vesicle Nanostructures Derived from Highly Photoresponsive Amphiphilic C60-Light-Harvesting Fluorene Dyad

PubMed Central

Wang, Min; Nalla, Venkatram; Jeon, Seaho; Mamidala, Venkatesh; Ji, Wei; Tan, Loon-Seng; Cooper, Thomas; Chiang, Long Y.

2011-01-01

We demonstrated ultrafast femtosecond nonlinear optical (NLO) absorption characteristics of bilayered fullerosome vesicle nanostructures derived from molecular self-assembly of amphiphilic oligo(ethylene glycolated) C60-(light-harvesting diphenylaminofluorene antenna). Fullerene conjugates were designed to enhance photoresponse in a femtosecond time scale by applying an isomerizable periconjugation linker between the C60 cage and diphenylaminofluorene antenna subunit in an intramolecular contact distance of only < 3.0 Å. Morphology of C60(>DPAF-EG12C1)-based fullerosome nanovesicles in H2O was characterized to consist of a bilayered shell with a sphere diameter of 20–70 nm and a chromophore shell-width of 9.0–10 nm, fitting well with a head-to-head packing configuration of the molecular length. At the estimated effective nanovesicle concentration as low as 5.5 × 10−8 MV (molecular molar concentration of 5.0 × 10−4 M) in H2O, two-photon absorption (2PA) phenomena were found to be the dominating photophysical events showing a large molar concentration-insensitive 2PA cross-section value equivalent to 8500 GM in a form of nanovesicles, on average. The observed NLO characteristics led to a sharp trend of efficient light-transmittance intensity reduction at the input laser intensity above 100 GW/cm2. PMID:22022620

Evolution and development of model membranes for physicochemical and functional studies of the membrane lateral heterogeneity.

PubMed

Morigaki, Kenichi; Tanimoto, Yasushi

2018-03-14

One of the main questions in the membrane biology is the functional roles of membrane heterogeneity and molecular localization. Although segregation and local enrichment of protein/lipid components (rafts) have been extensively studied, the presence and functions of such membrane domains still remain elusive. Along with biochemical, cell observation, and simulation studies, model membranes are emerging as an important tool for understanding the biological membrane, providing quantitative information on the physicochemical properties of membrane proteins and lipids. Segregation of fluid lipid bilayer into liquid-ordered (Lo) and liquid-disordered (Ld) phases has been studied as a simplified model of raft in model membranes, including giant unilamellar vesicles (GUVs), giant plasma membrane vesicles (GPMVs), and supported lipid bilayers (SLB). Partition coefficients of membrane proteins between Lo and Ld phases were measured to gauze their affinities to lipid rafts (raftophilicity). One important development in model membrane is patterned SLB based on the microfabrication technology. Patterned Lo/Ld phases have been applied to study the partition and function of membrane-bound molecules. Quantitative information of individual molecular species attained by model membranes is critical for elucidating the molecular functions in the complex web of molecular interactions. The present review gives a short account of the model membranes developed for studying the lateral heterogeneity, especially focusing on patterned model membranes on solid substrates. Copyright © 2018 Elsevier B.V. All rights reserved.

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.

Interaction of two overlapped synthetic peptides from GB virus C with charged mono and bilayers.

PubMed

Alay, M; Haro, I; Alsina, M A; Girona, V; Prat, J; Busquets, M A

2013-05-01

The physical chemistry properties and interactions of E2 (125-139) and E2 (120-139) peptide sequences from GB virus C with model cell membranes were investigated by means of several biophysical techniques in order to gain better understanding of the effect of peptide length and lipid charge on membrane binding. The peptides, having one net negative charge at the pH of the assays, interacted with monolayers of all the phospholipids regardless of the charge but with more extent with the cationic DPTAP thus indicating that the interaction had both a hydrophobic and an electrostatic component as has been observed for other peptides of the same family. The peptides were able to leakage contents of liposomes and showed fluorescence energy transfer in vesicles depending on the vesicles lipid composition. On another hand, circular dichroism has shown that the peptides exist mainly as a mixture of disordered structure and β-type conformations in aqueous solution but diminished its unstructured content, folding preferentially into α-helical conformation upon interaction with hydrophobic solvents or positively charged lipid surfaces. Altogether, results of this work indicate that the peptides interact at a surface level, penetrate into bilayers composed of fluid lipids and that conformational changes could be responsible for this effect. Copyright © 2012 Elsevier B.V. All rights reserved.

Permeability of acetic acid across gel and liquid-crystalline lipid bilayers conforms to free-surface-area theory.

PubMed Central

Xiang, T X; Anderson, B D

1997-01-01

Solubility-diffusion theory, which treats the lipid bilayer membrane as a bulk lipid solvent into which permeants must partition and diffuse across, fails to account for the effects of lipid bilayer chain order on the permeability coefficient of any given permeant. This study addresses the scaling factor that must be applied to predictions from solubility-diffusion theory to correct for chain ordering. The effects of bilayer chemical composition, temperature, and phase structure on the permeability coefficient (Pm) of acetic acid were investigated in large unilamellar vesicles by a combined method of NMR line broadening and dynamic light scattering. Permeability values were obtained in distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, and dilauroylphosphatidylcholine bilayers, and their mixtures with cholesterol, at various temperatures both above and below the gel-->liquid-crystalline phase transition temperatures (Tm). A new scaling factor, the permeability decrement f, is introduced to account for the decrease in permeability coefficient from that predicted by solubility-diffusion theory owing to chain ordering in lipid bilayers. Values of f were obtained by division of the observed Pm by the permeability coefficient predicted from a bulk solubility-diffusion model. In liquid-crystalline phases, a strong correlation (r = 0.94) between f and the normalized surface density sigma was obtained: in f = 5.3 - 10.6 sigma. Activation energies (Ea) for the permeability of acetic acid decreased with decreasing phospholipid chain length and correlated with the sensitivity of chain ordering to temperature, [symbol: see text] sigma/[symbol: see text](1/T), as chain length was varied. Pm values decreased abruptly at temperatures below the main phase transition temperatures in pure dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine bilayers (30-60-fold) and below the pretransition in dipalmitoylphosphatidylcholine bilayers (8-fold), and the linear relationship between in f and sigma established for liquid-crystalline bilayers was no longer followed. However, in both gel and liquid-crystalline phases in f was found to exhibit an inverse correlation with free surface area (in f = -0.31 - 29.1/af, where af is the average free area (in square angstroms) per lipid molecule). Thus, the lipid bilayer permeability of acetic acid can be predicted from the relevant chain-packing properties in the bilayer (free surface area), regardless of whether chain ordering is varied by changes in temperature, lipid chain length, cholesterol concentration, or bilayer phase structure, provided that temperature effects on permeant dehydration and diffusion and the chain-length effects on bilayer barrier thickness are properly taken into account. PMID:8994607

Introducing a fluorescence-based standard to quantify protein partitioning into membranes.

PubMed

Thomas, Franziska A; Visco, Ilaria; Petrášek, Zdeněk; Heinemann, Fabian; Schwille, Petra

2015-11-01

The affinity of peripheral membrane proteins for a lipid bilayer can be described using the partition coefficient (KP). Although several methods to determine KP are known, all possess limitations. To address some of these issues, we developed both: a versatile method based on single molecule detection and fluorescence imaging for determining KP, and a simple measurement standard employing hexahistidine-tagged enhanced green fluorescent protein (eGFP-His6) and free standing membranes of giant unilamellar vesicles (GUVs) functionalized with NTA(Ni) lipids as binding sites. To ensure intrinsic control, our method features two measurement modes. In the single molecule mode, fluorescence correlation spectroscopy (FCS) is applied to quantify free and membrane associated protein concentrations at equilibrium and calculate KP. In the imaging mode, confocal fluorescence images of GUVs are recorded and analyzed with semi-automated software to extract protein mean concentrations used to derive KP. Both modes were compared by determining the affinity of our standard, resulting in equivalent KP values. As observed in other systems, eGFP-His6 affinity for membranes containing increasing amounts of NTA(Ni) lipids rises in a stronger-than-linear fashion. We compared our dual approach with a FCS-based assay that uses large unilamellar vesicles (LUVs), which however fails to capture the stronger-than-linear trend for our NTA(Ni)-His6 standard. Hence, we determined the KP of the MARCKS effector domain with our FCS approach on GUVs, whose results are consistent with previously published data using LUVs. We finally provide a practical manual on how to measure KP and understand it in terms of molecules per lipid surface. Copyright © 2015. Published by Elsevier B.V.

Properties of Plasma Membrane from Pea Root Seedlings under Altered Gravity

NASA Astrophysics Data System (ADS)

Klymchuk, D.; Baranenko, V.; Vorobyova, T. V.; Kurylenko, I.; Chyzhykova, O.; Dubovoy, V.

In this study, the properties of pea (Pisum sativum L.) plasma membrane were examined to determine how the membrane structure and functions are regulated in response to clinorotation (2 rev/min) conditions. Membrane preparations enriched by plasma membrane vesicles were obtained by aqueous two-phase partitioning from 6-day seedling roots. The specific characteristics of H^+-ATPase, lípid composition and peroxidation intensity as well as fluidity of lipid bilayer were analysed. ATP hydrolytic activity was inhibited by ortovanadate and was insensitive to aside and nitrate in sealed plasma membrane vesicles isolated from both clinorotated and control seedlings. Plasma membrane vesicles from clinorotated seedlings in comparison to controls were characterised by increase in the total lipid/protein ratio, ATP hydrolytic activity and intensifying of lipid peroxidation. Sitosterol and campesterol were the predominant free sterol species. Clinorotated seedlings contained a slightly higher level of unsaturated fatty acid than controls. Plasma membrane vesicles were labelled with pyrene and fluorescence originating from monomeric (I_M) molecules and excimeric (I_E) aggregates were measured. The calculated I_E/I_M values were higher in clinorotated seedlings compared with controls reflecting the reduction in membrane microviscosity. The involvement of the changes in plasma membrane lipid content and composition, fluidity and H^+-ATPase activity in response of pea seedlings to altered gravity is discussed.

Janus dendrimersomes coassembled from fluorinated, hydrogenated, and hybrid Janus dendrimers as models for cell fusion and fission.

PubMed

Xiao, Qi; Sherman, Samuel E; Wilner, Samantha E; Zhou, Xuhao; Dazen, Cody; Baumgart, Tobias; Reed, Ellen H; Hammer, Daniel A; Shinoda, Wataru; Klein, Michael L; Percec, Virgil

2017-08-22

A three-component system of Janus dendrimers (JDs) including hydrogenated, fluorinated, and hybrid hydrogenated-fluorinated JDs are reported to coassemble by film hydration at specific ratios into an unprecedented class of supramolecular Janus particles (JPs) denoted Janus dendrimersomes (JDSs). They consist of a dumbbell-shaped structure composed of an onion-like hydrogenated vesicle and an onion-like fluorinated vesicle tethered together. The synthesis of dye-tagged analogs of each JD component enabled characterization of JDS architectures with confocal fluorescence microscopy. Additionally, a simple injection method was used to prepare submicron JDSs, which were imaged with cryogenic transmission electron microscopy (cryo-TEM). As reported previously, different ratios of the same three-component system yielded a variety of structures including homogenous onion-like vesicles, core-shell structures, and completely self-sorted hydrogenated and fluorinated vesicles. Taken together with the JDSs reported herein, a self-sorting pathway is revealed as a function of the relative concentration of the hybrid JD, which may serve to stabilize the interface between hydrogenated and fluorinated bilayers. The fission-like pathway suggests the possibility of fusion and fission processes in biological systems that do not require the assistance of proteins but instead may result from alterations in the ratios of membrane composition.

Interaction of recombinant human epidermal growth factor with phospholipid vesicles. A steady-state and time-resolved fluorescence study of the bis-tryptophan sequence (Trp49-Trp50).

PubMed

Li De La Sierra, I M; Vincent, M; Padron, G; Gallay, J

1992-01-01

The interaction of recombinant human epidermal growth factor with small unilamellar phospholipid vesicles was studied by steady-state and time-resolved fluorescence of the bis-tryptophan sequence (Trp49-Trp50). Steady-state anisotropy measurements demonstrate that strong binding occurred with small unilamellar vesicles made up of acidic phospholipids at acidic pH only (pH < or = 4.7). An apparent stoichiometry for 1,2-dimyristoyl-sn-phosphoglycerol of about 12 phospholipid molecules per molecule of human epidermal growth factor was estimated. The binding appears to be more efficient at temperatures above the gel to liquid-crystalline phase transition. The conformation and the environment of the Trp-Trp sequence are not greatly modified after binding, as judged from the invariance of the excited state lifetime distribution and from that of the fast processes affecting the anisotropy decay. This suggests that the Trp-Trp sequence is not embedded within the bilayer, in contrast to the situation in surfactant micelles (Mayo et al. 1987; Kohda and Inigaki 1992).

pH-sensitive niosomes: Effects on cytotoxicity and on inflammation and pain in murine models.

PubMed

Rinaldi, Federica; Del Favero, Elena; Rondelli, Valeria; Pieretti, Stefano; Bogni, Alessia; Ponti, Jessica; Rossi, François; Di Marzio, Luisa; Paolino, Donatella; Marianecci, Carlotta; Carafa, Maria

2017-12-01

pH-sensitive nonionic surfactant vesicles (niosomes) by polysorbate-20 (Tween-20) or polysorbate-20 derivatized by glycine (added as pH sensitive agent), were developed to deliver Ibuprofen (IBU) and Lidocaine (LID). For the physical-chemical characterization of vesicles (mean size, size distribution, zeta potential, vesicle morphology, bilayer properties and stability) dynamic light scattering (DLS), small angle X-ray scattering and fluorescence studies were performed. Potential cytotoxicity was evaluated on immortalized human keratinocyte cells (HaCaT) and on immortalized mouse fibroblasts Balb/3T3. In vivo antinociceptive activity (formalin test) and anti-inflammatory activity tests (paw edema induced by zymosan) in murine models were performed on drug-loaded niosomes. pH-sensitive niosomes were stable in the presence of 0 and 10% fetal bovine serum, non-cytotoxic and able to modify IBU or LID pharmacological activity in vivo. The synthesis of stimuli responsive surfactant, as an alternative to add pH-sensitive molecules to niosomes, could represent a promising delivery strategy for anesthetic and anti-inflammatory drugs.

Experimental Aspects of Colloidal Interactions in Mixed Systems of Liposome and Inorganic Nanoparticle and Their Applications

PubMed Central

Michel*, Raphael; Gradzielski*, Michael

2012-01-01

In the past few years, growing attention has been devoted to the study of the interactions taking place in mixed systems of phospholipid membranes (for instance in the form of vesicles) and hard nanoparticles (NPs). In this context liposomes (vesicles) may serve as versatile carriers or as a model system for biological membranes. Research on these systems has led to the observation of novel hybrid structures whose morphology strongly depends on the charge, composition and size of the interacting colloidal species as well as on the nature (pH, ionic strength) of their dispersing medium. A central role is played by the phase behaviour of phospholipid bilayers which have a tremendous influence on the liposome properties. Another central aspect is the incorporation of nanoparticles into vesicles, which is intimately linked to the conditions required for transporting a nanoparticle through a membrane. Herein, we review recent progress made on the investigations of the interactions in liposome/nanoparticle systems focusing on the particularly interesting structures that are formed in these hybrid systems as well as their potential applications. PMID:23109874

The glycolipid GM1 reshapes asymmetric biomembranes and giant vesicles by curvature generation.

PubMed

Dasgupta, Raktim; Miettinen, Markus S; Fricke, Nico; Lipowsky, Reinhard; Dimova, Rumiana

2018-05-29

The ganglioside GM1 is present in neuronal membranes at elevated concentrations with an asymmetric spatial distribution. It is known to generate curvature and can be expected to strongly influence the neuron morphology. To elucidate these effects, we prepared giant vesicles with GM1 predominantly present in one leaflet of the membrane, mimicking the asymmetric GM1 distribution in neuronal membranes. Based on pulling inward and outward tubes, we developed a technique that allowed the direct measurement of the membrane spontaneous curvature. Using vesicle electroporation and fluorescence intensity analysis, we were able to quantify the GM1 asymmetry across the membrane and to subsequently estimate the local curvature generated by the molecule in the bilayer. Molecular-dynamics simulations confirm the experimentally determined dependence of the membrane spontaneous curvature as a function of GM1 asymmetry. GM1 plays a crucial role in connection with receptor proteins. Our results on curvature generation of GM1 point to an additional important role of this ganglioside, namely in shaping neuronal membranes. Copyright © 2018 the Author(s). Published by PNAS.

Physical and chemical stability of marine lipid-based liposomes under acid conditions.

PubMed

Nacka, F; Cansell, M; Gouygou, J P.; Gerbeaud, C; Méléard, P; Entressangles, B

2001-03-01

Liposomes made from a marine lipid extract containing a high polyunsaturated fatty lipid ratio were submitted to large pH variations, ranging from 1 to 8. Shape transformations were followed by video microscopy using giant liposomes and micromanipulation experiments. Acidification induced a decrease of the vesicle size simultaneous to the appearance of invaginations. These pH-dependent structural rearrangements were interpreted in terms of osmotic shocks and chemical modifications of the membranes. Liposomes produced by direct filtration were studied using turbidity measurements and optical microscopy observations. A low pH led to an instantaneous vesicle aggregation and to complex supramolecular and/or morphological changes as a function of time. The subsequent buffer neutralization of the liposome suspensions induced a partial reversion of the aggregation phenomenon while the structural membrane rearrangements were persisting. Furthermore, weak chemical degradations (oxidation and hydrolysis) were evidenced when the vesicles were incubated at low pH up to a 24-h incubation time. Thus, although acidification revealed liposome size and shape changes, the bilayer structure was maintained indicating that marine lipid-based liposomes could be used as oral administration vectors.

Electrical and structural characterization of plasma polymerized polyaniline/TiO2 heterostructure diode: a comparative study of single and bilayer TiO2 thin film electrode.

PubMed

Ameen, Sadia; Akhtar, M Shaheer; Kimi, Young Soon; Yang, O-Bong; Shin, Hyung-Shik

2011-04-01

A heterostructure was fabricated using p-type plasma polymerized polyaniline (PANI) and n-type (single and bilayer) titanium dioxide (TiO2) thin film on FTO glass. The deposition of single and bilayer TiO2 thin film on FTO substrate was achieved through doctor blade followed by dip coating technique before subjected to plasma enhanced polymerization. To fabricate p-n heterostructure, a plasma polymerization of aniline was conducted using RF plasma at 13.5 MHz and at the power of 120 W on the single and bilayer TiO2 thin film electrodes. The morphological, optical and the structural characterizations revealed the formation of p-n heterostructures between PANI and TiO2 thin film. The PANI/bilayer TiO2 heterostructure showed the improved current-voltage (I-V) characteristics due to the substantial deposition of PANI molecules into the bilayer TiO2 thin film which provided good conducting pathway and reduced the degree of excitons recombination. The change of linear I-V behavior of PANI/TiO2 heterostructure to non linear behavior with top Pt contact layer confirmed the formation of Schottky contact at the interfaces of Pt layer and PANI/TiO2 thin film layers.

Planar Supported Membranes with Mobile SNARE Proteins and Quantitative Fluorescence Microscopy Assays to Study Synaptic Vesicle Fusion

PubMed Central

Kiessling, Volker; Liang, Binyong; Kreutzberger, Alex J. B.; Tamm, Lukas K.

2017-01-01

Synaptic vesicle membrane fusion, the process by which neurotransmitter gets released at the presynaptic membrane is mediated by a complex interplay between proteins and lipids. The realization that the lipid bilayer is not just a passive environment where other molecular players like SNARE proteins act, but is itself actively involved in the process, makes the development of biochemical and biophysical assays particularly challenging. We summarize in vitro assays that use planar supported membranes and fluorescence microscopy to address some of the open questions regarding the molecular mechanisms of SNARE-mediated membrane fusion. Most of the assays discussed in this mini-review were developed in our lab over the last 15 years. We emphasize the sample requirements that we found are important for the successful application of these methods. PMID:28360838

Raman imaging of lipid bilayer membrane by surface enhanced Raman scattering

NASA Astrophysics Data System (ADS)

Mori, Motoaki; Abe, Shunsuke; Kondo, Takahiro; Saito, Yuika

2018-04-01

We investigated two-dimensional lipid bilayers by spectroscopic imaging with surface enhanced Raman spectroscopy (SERS). A DSPC lipid bilayer incubated on a glass substrate was coated with a thin layer of silver. Due to the strong electromagnetic enhancement of the silver film and the affinity to lipid molecules, the Raman spectrum of a single bilayer was obtained in a 1 s exposure time with 0.1 mW of incident laser power. In the C-H vibrational region of the spectra, which is sensitive to bilayer configurations, a randomly stacked area was dominated by the CH3 asymmetric-stretch mode, whereas flat areas including double bilayers showed typical SERS spectra. The spectral features of the randomly stacked area are explained by the existence of many free lipid molecules, which is supported by DFT calculations of paired DSPC molecules. Our method can be applied to reveal the local crystallinity of single lipid bilayers, which is difficult to assess by conventional Raman imaging.

Interaction of pH-sensitive non-phospholipid liposomes with cellular mimetic membranes.

PubMed

Marianecci, Carlotta; Rinaldi, Federica; Di Marzio, Luisa; Pozzi, Daniela; Caracciolo, Giulio; Manno, Daniela; Dini, Luciana; Paolino, Donatella; Celia, Christian; Carafa, Maria

2013-04-01

Surfactant nanocarriers have received considerable attention in the last several years as interesting alternative to classic liposomes. Different pH-sensitive vesicular colloidal carriers based on Tween 20 derivatives, obtained after functionalization of the head groups of the surfactant with natural, or simply modified, amino acids, were proposed as drug nanocarriers. Dynamic light scattering, Small Angle X-ray Scattering, Trasmission Electron Microscopy and fluorescence studies were used for the physico-chemical characterization of vesicles and mean size, size distribution, zeta potential, vesicle morphology and bilayer properties were evaluated. The pH-sensitivity and the stability of formulations, in absence and in presence of foetal bovine serum, were also evaluated. Moreover, the contact between surfactant vesicles and liposomes designed to model the cellular membrane was investigated by fluorescence studies to preliminary explore the potential interaction between vesicle and cell membranes. Experimental findings showed that physico-chemical and technological features of pH-sensitive vesicles were influenced by the composition of the carriers. Furthermore, proposed carriers are able to interact with mimetic cell membrane and it is reasonable to attribute the observed differences in interaction to the architectural/structural properties of Tween 20 derivatives. The findings reported in this investigation showed that a deep and extensive physico-chemical characterization of the carrier is a fundamental step, according to the evidence that the knowledge of nanocarrier properties is necessary to translate its potentiality to in vitro/in vivo applications.

Mechanistic insights for block copolymer morphologies: how do worms form vesicles?

PubMed

Blanazs, Adam; Madsen, Jeppe; Battaglia, Giuseppe; Ryan, Anthony J; Armes, Steven P

2011-10-19

Amphiphilic diblock copolymers composed of two covalently linked, chemically distinct chains can be considered to be biological mimics of cell membrane-forming lipid molecules, but with typically more than an order of magnitude increase in molecular weight. These macromolecular amphiphiles are known to form a wide range of nanostructures (spheres, worms, vesicles, etc.) in solvents that are selective for one of the blocks. However, such self-assembly is usually limited to dilute copolymer solutions (<1%), which is a significant disadvantage for potential commercial applications such as drug delivery and coatings. In principle, this problem can be circumvented by polymerization-induced block copolymer self-assembly. Here we detail the synthesis and subsequent in situ self-assembly of amphiphilic AB diblock copolymers in a one pot concentrated aqueous dispersion polymerization formulation. We show that spherical micelles, wormlike micelles, and vesicles can be predictably and efficiently obtained (within 2 h of polymerization, >99% monomer conversion) at relatively high solids in purely aqueous solution. Furthermore, careful monitoring of the in situ polymerization by transmission electron microscopy reveals various novel intermediate structures (including branched worms, partially coalesced worms, nascent bilayers, "octopi", "jellyfish", and finally pure vesicles) that provide important mechanistic insights regarding the evolution of the particle morphology during the sphere-to-worm and worm-to-vesicle transitions. This environmentally benign approach (which involves no toxic solvents, is conducted at relatively high solids, and requires no additional processing) is readily amenable to industrial scale-up, since it is based on commercially available starting materials.

Permeability and channel-mediated transport of boric acid across membrane vesicles isolated from squash roots.

PubMed

Dordas, C; Chrispeels, M J; Brown, P H

2000-11-01

Boron is an essential micronutrient for plant growth and the boron content of plants differs greatly, but the mechanism(s) of its uptake into cells is not known. Boron is present in the soil solution as boric acid and it is in this form that it enters the roots. We determined the boron permeability coefficient of purified plasma membrane vesicles obtained from squash (Cucurbita pepo) roots and found it to be 3 x 10(-7) +/-1.4 x 10(-8) cm s(-1), six times higher than the permeability of microsomal vesicles. Boric acid permeation of the plasma membrane vesicles was partially inhibited (30%-39%) by mercuric chloride and phloretin, a non-specific channel blocker. The inhibition by mercuric chloride was readily reversible by 2-mercaptoethanol. The energy of activation for boron transport into the plasma membrane vesicles was 10.2 kcal mol(-1). Together these data indicate that boron enters plant cells in part by passive diffusion through the lipid bilayer of the plasma membrane and in part through proteinaceous channels. Expression of the major intrinsic protein (MIP) PIP1 in Xenopus laevis oocytes resulted in a 30% increase in the boron permeability of the oocytes. Other MIPs tested (PIP3, MLM1, and GlpF) did not have this effect. We postulate that certain MIPs, like those that have recently been shown to transport small neutral solutes, may also be the channels through which boron enters plant cells.

Permeability and Channel-Mediated Transport of Boric Acid across Membrane Vesicles Isolated from Squash Roots1

PubMed Central

Dordas, Christos; Chrispeels, Maarten J.; Brown, Patrick H.

2000-01-01

Boron is an essential micronutrient for plant growth and the boron content of plants differs greatly, but the mechanism(s) of its uptake into cells is not known. Boron is present in the soil solution as boric acid and it is in this form that it enters the roots. We determined the boron permeability coefficient of purified plasma membrane vesicles obtained from squash (Cucurbita pepo) roots and found it to be 3 × 10−7 ±1.4 × 10−8 cm s−1, six times higher than the permeability of microsomal vesicles. Boric acid permeation of the plasma membrane vesicles was partially inhibited (30%–39%) by mercuric chloride and phloretin, a non-specific channel blocker. The inhibition by mercuric chloride was readily reversible by 2-mercaptoethanol. The energy of activation for boron transport into the plasma membrane vesicles was 10.2 kcal mol−1. Together these data indicate that boron enters plant cells in part by passive diffusion through the lipid bilayer of the plasma membrane and in part through proteinaceous channels. Expression of the major intrinsic protein (MIP) PIP1 in Xenopus laevis oocytes resulted in a 30% increase in the boron permeability of the oocytes. Other MIPs tested (PIP3, MLM1, and GlpF) did not have this effect. We postulate that certain MIPs, like those that have recently been shown to transport small neutral solutes, may also be the channels through which boron enters plant cells. PMID:11080310

Small-angle X-ray and neutron scattering demonstrates that cell-free expression produces properly formed disc-shaped nanolipoprotein particles

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

Cleveland, Thomas E.; He, Wei; Evans, Angela C.

Nanolipoprotein particles (NLPs), composed of membrane scaffold proteins and lipids, have been used to support membrane proteins in a native-like bilayer environment for biochemical and structural studies. Traditionally, these NLPs have been prepared by the controlled removal of detergent from a detergent-solubilized protein-lipid mixture. Recently, an alternative method has been developed using direct cell-free expression of the membrane scaffold protein in the presence of preformed lipid vesicles, which spontaneously produces NLPs without the need for detergent at any stage. Using SANS/SAXS, we show here that NLPs produced by this cell-free expression method are structurally indistinguishable from those produced using detergentmore » removal methodologies. This further supports the utility of single step cell-free methods for the production of lipid binding proteins. Lastly, in addition, detailed structural information describing these NLPs can be obtained by fitting a capped core-shell cylinder type model to all SANS/SAXS data simultaneously.« less

Cholesterol suppresses membrane leakage by decreasing water penetrability.

PubMed

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

2018-06-13

Membrane fusion is a fundamental biological process that lies at the heart of enveloped virus infection, synaptic signaling, intracellular vesicle trafficking, gamete fertilization, and cell-cell fusion. Membrane fusion is initiated as two apposed membranes merge to a single bilayer called a hemifusion diaphragm. It is believed that the contents of the two fusing membranes are released through a fusion pore formed at the hemifusion diaphragm, and yet another possible pathway has been proposed in which an undefined pore may form outside the hemifusion diaphragm at the apposed membranes, leading to the so-called leaky fusion. Here, we performed all-atom molecular dynamics simulations to study the evolution of the hemifusion diaphragm structure with various lipid compositions. We found that the lipid cholesterol decreased water penetrability to inhibit leakage pore formation. Biochemical leakage experiments support these simulation results. This study may shed light on the underlying mechanism of the evolution pathways of the hemifusion structure, especially the understanding of content leakage during membrane fusion.

Small-angle X-ray and neutron scattering demonstrates that cell-free expression produces properly formed disc-shaped nanolipoprotein particles

DOE PAGES

Cleveland, Thomas E.; He, Wei; Evans, Angela C.; ...

2018-02-13

Nanolipoprotein particles (NLPs), composed of membrane scaffold proteins and lipids, have been used to support membrane proteins in a native-like bilayer environment for biochemical and structural studies. Traditionally, these NLPs have been prepared by the controlled removal of detergent from a detergent-solubilized protein-lipid mixture. Recently, an alternative method has been developed using direct cell-free expression of the membrane scaffold protein in the presence of preformed lipid vesicles, which spontaneously produces NLPs without the need for detergent at any stage. Using SANS/SAXS, we show here that NLPs produced by this cell-free expression method are structurally indistinguishable from those produced using detergentmore » removal methodologies. This further supports the utility of single step cell-free methods for the production of lipid binding proteins. Lastly, in addition, detailed structural information describing these NLPs can be obtained by fitting a capped core-shell cylinder type model to all SANS/SAXS data simultaneously.« less

Vesicle biomechanics in a time-varying magnetic field.

PubMed

Ye, Hui; Curcuru, Austen

2015-01-01

Cells exhibit distortion when exposed to a strong electric field, suggesting that the field imposes control over cellular biomechanics. Closed pure lipid bilayer membranes (vesicles) have been widely used for the experimental and theoretical studies of cellular biomechanics under this electrodeformation. An alternative method used to generate an electric field is by electromagnetic induction with a time-varying magnetic field. References reporting the magnetic control of cellular mechanics have recently emerged. However, theoretical analysis of the cellular mechanics under a time-varying magnetic field is inadequate. We developed an analytical theory to investigate the biomechanics of a modeled vesicle under a time-varying magnetic field. Following previous publications and to simplify the calculation, this model treated the inner and suspending media as lossy dielectrics, the membrane thickness set at zero, and the electric resistance of the membrane assumed to be negligible. This work provided the first analytical solutions for the surface charges, electric field, radial pressure, overall translational forces, and rotational torques introduced on a vesicle by the time-varying magnetic field. Frequency responses of these measures were analyzed, particularly the frequency used clinically by transcranial magnetic stimulation (TMS). The induced surface charges interacted with the electric field to produce a biomechanical impact upon the vesicle. The distribution of the induced surface charges depended on the orientation of the coil and field frequency. The densities of these charges were trivial at low frequency ranges, but significant at high frequency ranges. The direction of the radial force on the vesicle was dependent on the conductivity ratio between the vesicle and the medium. At relatively low frequencies (<200 KHz), including the frequency used in TMS, the computed radial pressure and translational forces on the vesicle were both negligible. This work provides an analytical framework and insight into factors affecting cellular biomechanics under a time-varying magnetic field. Biological effects of clinical TMS are not likely to occur via alteration of the biomechanics of brain cells.

DNA-mediated self-assembly of artificial vesicles.

PubMed

Hadorn, Maik; Eggenberger Hotz, Peter

2010-03-26

Although multicompartment systems made of single unilamellar vesicles offer the potential to outperform single compartment systems widely used in analytic, synthetic, and medical applications, their use has remained marginal to date. On the one hand, this can be attributed to the binary character of the majority of the current tethering protocols that impedes the implementation of real multicomponent or multifunctional systems. On the other hand, the few tethering protocols theoretically providing multicompartment systems composed of several distinct vesicle populations suffer from the readjustment of the vesicle formation procedure as well as from the loss of specificity of the linking mechanism over time. In previous studies, we presented implementations of multicompartment systems and resolved the readjustment of the vesicle formation procedure as well as the loss of specificity by using linkers consisting of biotinylated DNA single strands that were anchored to phospholipid-grafted biotinylated PEG tethers via streptavidin as a connector. The systematic analysis presented herein provides evidences for the incorporation of phospholipid-grafted biotinylated PEG tethers to the vesicle membrane during vesicle formation, providing specific anchoring sites for the streptavidin loading of the vesicle membrane. Furthermore, DNA-mediated vesicle-vesicle self-assembly was found to be sequence-dependent and to depend on the presence of monovalent salts. This study provides a solid basis for the implementation of multi-vesicle assemblies that may affect at least three distinct domains. (i) Analysis. Starting with a minimal system, the complexity of a bottom-up system is increased gradually facilitating the understanding of the components and their interaction. (ii) Synthesis. Consecutive reactions may be implemented in networks of vesicles that outperform current single compartment bioreactors in versatility and productivity. (iii) Personalized medicine. Transport and targeting of long-lived, pharmacologically inert prodrugs and their conversion to short-lived, active drug molecules directly at the site of action may be accomplished if multi-vesicle assemblies of predefined architecture are used.

Influence of the state of phase of lipid bilayer on the exposure of glucose residues on the surface of liposomes.

PubMed

Villalva, Denise Gradella; Giansanti, Luisa; Mauceri, Alessandro; Ceccacci, Francesca; Mancini, Giovanna

2017-11-01

The presence of carbohydrate-binding proteins (i.e. lectins) on the surface of various bacterial strains and their overexpression in some tumor tissues makes the use of glycosylated liposomes a promising approach for the specific drug delivery in antibacterial and anti-cancer therapies. However, the functionalization of liposome surface with sugar moieties by glycosylated amphiphiles does not ensure the binding of sugar-coated vesicles with lectins. In fact, the composition and properties of lipid bilayer play a pivotal role in the exposure of sugar residues and in the interaction with lectins. The influence of the length of the hydrophilic spacer that links the sugar to liposome surface and of the presence of saturated or unsaturated phospholipids in the lipid bilayer on the ability of glucosylated liposomes to interact with a model lectin, Concanavalin A, was investigated. Our results demonstrate that both the chain length and the prensece of unsaturation, parameters that strongly affect the fluidity of the lipid bilayer, affect agglutination. In particular, agglutination is favored when liposomes are in the gel phase within a defined range of temperature. Moreover, the obtained results confirm that the length of the PEG spacer, that influences both lipid organization and the exposure of sugar moieties to the bulk, plays a crucial role in liposome/lectin interaction. Copyright © 2017 Elsevier B.V. All rights reserved.

Integrative self-assembly of functional hybrid nanoconstructs by inorganic wrapping of single biomolecules, biomolecule arrays and organic supramolecular assemblies

NASA Astrophysics Data System (ADS)

Patil, Avinash J.; Li, Mei; Mann, Stephen

2013-07-01

Synthesis of functional hybrid nanoscale objects has been a core focus of the rapidly progressing field of nanomaterials science. In particular, there has been significant interest in the integration of evolutionally optimized biological systems such as proteins, DNA, virus particles and cells with functional inorganic building blocks to construct mesoscopic architectures and nanostructured materials. However, in many cases the fragile nature of the biomolecules seriously constrains their potential applications. As a consequence, there is an on-going quest for the development of novel strategies to modulate the thermal and chemical stabilities, and performance of biomolecules under adverse conditions. This feature article highlights new methods of ``inorganic molecular wrapping'' of single or multiple protein molecules, individual double-stranded DNA helices, lipid bilayer vesicles and self-assembled organic dye superstructures using inorganic building blocks to produce bio-inorganic nanoconstructs with core-shell type structures. We show that spatial isolation of the functional biological nanostructures as ``armour-plated'' enzyme molecules or polynucleotide strands not only maintains their intact structure and biochemical properties, but also enables the fabrication of novel hybrid nanomaterials for potential applications in diverse areas of bionanotechnology.

Integrative self-assembly of functional hybrid nanoconstructs by inorganic wrapping of single biomolecules, biomolecule arrays and organic supramolecular assemblies.

PubMed

Patil, Avinash J; Li, Mei; Mann, Stephen

2013-08-21

Synthesis of functional hybrid nanoscale objects has been a core focus of the rapidly progressing field of nanomaterials science. In particular, there has been significant interest in the integration of evolutionally optimized biological systems such as proteins, DNA, virus particles and cells with functional inorganic building blocks to construct mesoscopic architectures and nanostructured materials. However, in many cases the fragile nature of the biomolecules seriously constrains their potential applications. As a consequence, there is an on-going quest for the development of novel strategies to modulate the thermal and chemical stabilities, and performance of biomolecules under adverse conditions. This feature article highlights new methods of "inorganic molecular wrapping" of single or multiple protein molecules, individual double-stranded DNA helices, lipid bilayer vesicles and self-assembled organic dye superstructures using inorganic building blocks to produce bio-inorganic nanoconstructs with core-shell type structures. We show that spatial isolation of the functional biological nanostructures as "armour-plated" enzyme molecules or polynucleotide strands not only maintains their intact structure and biochemical properties, but also enables the fabrication of novel hybrid nanomaterials for potential applications in diverse areas of bionanotechnology.

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.

Complex biomembrane mimetics on the sub-nanometer scale

DOE PAGES

Heberle, Frederick A.; Pabst, Georg

2017-07-17

Biomimetic lipid vesicles are indispensable tools for gaining insight into the biophysics of cell physiology on the molecular level. The level of complexity of these model systems has steadily increased, and now spans from domain forming lipid mixtures to asymmetric lipid bilayers. We review recent progress in the development and application of elastic neutron and X-ray scattering techniques for studying these systems in situ and under physiologically relevant conditions on the nanometer to sub-nanometer length scales. Particularly we focus on: (i) structural details of coexisting liquid-ordered and liquid-disordered domains, including their thickness and lipid packing mismatch as a function ofmore » a size transition from nanoscopic to macroscopic domains; (ii) membrane-mediated protein partitioning into lipid domains; (iii) the role of the aqueous medium in tuning interactions between membranes and domains; and (iv) leaflet specific structure in asymmetric bilayers and passive lipid flip-flop.« less

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.

Complex biomembrane mimetics on the sub-nanometer scale

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

Heberle, Frederick A.; Pabst, Georg

Biomimetic lipid vesicles are indispensable tools for gaining insight into the biophysics of cell physiology on the molecular level. The level of complexity of these model systems has steadily increased, and now spans from domain forming lipid mixtures to asymmetric lipid bilayers. We review recent progress in the development and application of elastic neutron and X-ray scattering techniques for studying these systems in situ and under physiologically relevant conditions on the nanometer to sub-nanometer length scales. Particularly we focus on: (i) structural details of coexisting liquid-ordered and liquid-disordered domains, including their thickness and lipid packing mismatch as a function ofmore » a size transition from nanoscopic to macroscopic domains; (ii) membrane-mediated protein partitioning into lipid domains; (iii) the role of the aqueous medium in tuning interactions between membranes and domains; and (iv) leaflet specific structure in asymmetric bilayers and passive lipid flip-flop.« less

Helicity, membrane incorporation, orientation and thermal stability of the large conductance mechanosensitive ion channel from E. coli

NASA Technical Reports Server (NTRS)

Arkin, I. T.; Sukharev, S. I.; Blount, P.; Kung, C.; Brunger, A. T.

1998-01-01

In this report, we present structural studies on the large conductance mechanosensitive ion channel (MscL) from E. coli in detergent micelles and lipid vesicles. Both transmission Fourier transform infrared spectroscopy and circular dichroism (CD) spectra indicate that the protein is highly helical in detergents as well as liposomes. The secondary structure of the proteins was shown to be highly resistant towards denaturation (25-95 degrees C) based on an ellipticity thermal profile. Amide H+/D+ exchange was shown to be extensive (ca. 66%), implying that two thirds of the protein are water accessible. MscL, reconstituted in oriented lipid bilayers, was shown to possess a net bilayer orientation using dichroic ratios measured by attenuated total-reflection Fourier transform infrared spectroscopy. Here, we present and discuss this initial set of structural data on this new family of ion-channel proteins.

Amphiphilic naproxen prodrugs: differential scanning calorimetry study on their interaction with phospholipid bilayers.

PubMed

Giuffrida, Maria Chiara; Pignatello, Rosario; Castelli, Francesco; Sarpietro, Maria Grazia

2017-09-01

Naproxen, a nonsteroid anti-inflammatory drug studied for Alzheimer's disease, was conjugated with lipoamino acids (LAA) directly or through a diethylamine (EDA) spacer to improve the drug lipophilicity and the interaction with phospholipid bilayers. The interaction of naproxen and its prodrugs with biomembrane models consisting of dimyristoylphosphatidylcholine multilamellar vesicles was studied by differential scanning calorimetry. The transfer of prodrugs from a lipophilic carrier to a biomembrane model was also studied. Naproxen conjugation to lipoamino acids improves its interaction with biomembrane models and affects the transfer from a lipophilic carrier to biomembrane model. LAA portion may localize between the phospholipid chains; the entity of the interaction depends not only on the presence of the spacer but also on the LAA chain length. Variation of LAA portion can modulate the naproxen prodrugs affinity towards the biological membrane as well as towards the lipophilic carrier. © 2017 Royal Pharmaceutical Society.

Improved electrical performance and bias stability of solution-processed active bilayer structure of indium zinc oxide based TFT.

PubMed

Seo, Jin-Suk; Bae, Byeong-Soo

2014-09-10

We fabricated active single- and bilayer structure thin film transistors (TFTs) with aluminum or gallium doped (IZO:Al or IZO:Ga) and undoped indium zinc oxide (IZO) thin film layers using an aqueous solution process. The electrical performance and bias stability of these active single- and bilayer structure TFTs were investigated and compared to reveal the effects of Al/Gal doping and bilayer structure. The single-layer structure IZO TFT shows a high mobility of 19 cm(2)/V · s with a poor positive bias stability (PBS) of ΔVT + 3.4 V. However, Al/Ga doped in IZO TFT reduced mobility to 8.5-9.9 cm(2)/V · s but improved PBS to ΔVT + 1.6-1.7 V due to the reduction of oxygen vacancy. Thus, it is found the bilayer structure TFTs with a combination of bottom- and top-layer compositions modify both the mobility and bias stability of the TFTs to be optimized. The bilayer structure TFT with an IZO:X bottom layer possess high mobility and an IZO bottom layer improves the PBS.

pH gradients across phospholipid membranes caused by fast flip-flop of un-ionized fatty acids.

PubMed Central

Kamp, F; Hamilton, J A

1992-01-01

A central, unresolved question in cell physiology is how fatty acids move across cell membranes and whether protein(s) are required to facilitate transbilayer movement. We have developed a method for monitoring movement of fatty acids across protein-free model membranes (phospholipid bilayers). Pyranin, a water-soluble, pH-sensitive fluorescent molecule, was trapped inside well-sealed phosphatidylcholine vesicles (with or without cholesterol) in Hepes buffer (pH 7.4). Upon addition of a long-chain fatty acid (e.g., oleic acid) to the external buffer (also Hepes, pH 7.4), a decrease in fluorescence of pyranin was observed immediately (within 10 sec). This acidification of the internal volume was the result of the "flip" of un-ionized fatty acids to the inner leaflet, followed by a release of protons from approximately 50% of these fatty acid molecules (apparent pKa in the bilayer = 7.6). The proton gradient thus generated dissipated slowly because of slow cyclic proton transfer by fatty acids. Addition of bovine serum albumin to vesicles with fatty acids instantly removed the pH gradient, indicating complete removal of fatty acids, which requires rapid "flop" of fatty acids from the inner to the outer monolayer layer. Using a four-state kinetic diagram of fatty acids in membranes, we conclude that un-ionized fatty acid flip-flops rapidly (t1/2 < or = 2 sec) whereas ionized fatty acid flip-flops slowly (t1/2 of minutes). Since fatty acids move across phosphatidylcholine bilayers spontaneously and rapidly, complex mechanisms (e.g., transport proteins) may not be required for translocation of fatty acids in biological membranes. The proton movement accompanying fatty acid flip-flop is an important consideration for fatty acid metabolism in normal physiology and in disease states such as cardiac ischemia. Images PMID:1454821

Hypoxia directly increases serotonin transport by porcine pulmonary artery endothelial cell (PAEC) plasma membrane vesicles

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

Bhat, G.B.; Block, E.R.

1990-02-26

Alterations in the physical state and composition of membrane lipids have been shown to interfere with a number of critical cellular and membrane functions including transmembrane transport. The authors have reported that hypoxia has profound effects upon the physical state and lipid composition of the PAEC plasma membrane bilayer and have suggested that this is responsible for increased serotonin uptake by these cells. In order to determine whether hypoxia has a direct effect on the plasma membrane transport of serotonin, they measured serotonin transport activity (1) in plasma membrane vesicles isolated from normoxic (20% O{sub 2}-5% CO{sub 2}) and hypoxicmore » (0% O{sub 2}-5% CO{sub 2}) PAEC and (2) in PAEC plasma membrane vesicles that were exposed directly to normoxia or hypoxia. A 24-h exposure of PAEC to hypoxia resulted in a 40% increase in specific serotonin transport by plasma membrane vesicles derived from these cells. When plasma membrane vesicles were isolated and then directly exposed to normoxia or hypoxia for 1 h at 37C, a 31% increase in specific 5-HT transport was observed in hypoxic vesicles. Hypoxia did not alter the Km of serotonin transport (normoxia = 3.47 {mu}M versus hypoxia = 3.76 {mu}M) but markedly increased the maximal rate of transport (V{sup max}) (normoxia = 202.4 pmol/min/mg protein versus hypoxia = 317.9 pmol/min/mg protein). These results indicate that hypoxia increases serotonin transport in PAEC by a direct effect on the plasma membrane leading to an increase in the effective number of transporter molecules without alteration in transporter affinity for serotonin.« less

NMR-NOE and MD simulation study on phospholipid membranes: dependence on membrane diameter and multiple time scale dynamics.

PubMed

Shintani, Megumi; Yoshida, Ken; Sakuraba, Shun; Nakahara, Masaru; Matubayasi, Nobuyuki

2011-07-28

Motional correlation times between the hydrophilic and hydrophobic terminal groups in lipid membranes are studied over a wide range of curvatures using the solution-state (1)H NMR-nuclear Overhauser effect (NOE) and molecular dynamics (MD) simulation. To enable (1)H NMR-NOE measurements for large vesicles, the transient NOE method is combined with the spin-echo method, and is successfully applied to a micelle of 1-palmitoyl-lysophosphatidylcholine (PaLPC) with diameter of 5 nm and to vesicles of dipalmitoylphosphatidylcholine (DPPC) with diameters ranging from 30 to 800 nm. It is found that the NOE intensity increases with the diameter up to ∼100 nm, and the model membrane is considered planar on the molecular level beyond ∼100 nm. While the NOE between the hydrophilic terminal and hydrophobic terminal methyl groups is absent for the micelle, its intensity is comparable to that for the neighboring group for vesicles with larger diameters. The origin of NOE signals between distant sites is analyzed by MD simulations of PaLPC micelles and DPPC planar bilayers. The slow relaxation is shown to yield an observable NOE signal even for the hydrophilic and hydrophobic terminal sites. Since the information on distance and dynamics cannot be separated in the experimental NOE alone, the correlation time in large vesicles is determined by combining the experimental NOE intensity and MD-based distance distribution. For large vesicles, the correlation time is found to vary by 2 orders of magnitude over the proton sites. This study shows that NOE provides dynamic information on large vesicles when combined with MD, which provides structural information. © 2011 American Chemical Society

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.

Buffer-induced swelling and vesicle budding in binary lipid mixtures of dioleoylphosphatidylcholine:dioleoylphosphatidylethanolamine and dioleoylphosphatidylcholine:lysophosphatidylcholine using small-angle X-ray scattering and ³¹P static NMR.

PubMed

Barriga, Hanna M G; Bazin, Richard; Templer, Richard H; Law, Robert V; Ces, Oscar

2015-03-17

A large variety of data exists on lipid phase behavior; however, it is mostly in nonbuffered systems over nonbiological temperature ranges. We present biophysical data on lipid mixtures of dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), and lysophosphatidylcholine (LysoPC) examining their behaviors in excess water and buffer systems over the temperature range 4-34 °C. These mixtures are commonly used to investigate the effects of spontaneous curvature on integral membrane proteins. Using small-angle X-ray scattering (SAXS) and (31)P NMR, we observed lamellar and vesicle phases, with the buffer causing an increase in the layer spacing. Increasing amounts of DOPE in a DOPC bilayer decreased the layer spacing of the mesophase, while the opposite trend was observed for increasing amounts of LysoPC. (31)P static NMR was used to analyze the DOPC:LysoPC samples to investigate the vesicle sizes present, with evidence of vesicle budding observed at LysoPC concentrations above 30 mol %. NMR line shapes were fitted using an adapted program accounting for the distortion of the lipids within the magnetic field. The distortion of the vesicle, because of magnetic susceptibility, varied with LysoPC content, and a discontinuity was found in both the water and buffer samples. Generally, the distortion increased with LysoPC content; however, at a ratio of DOPC:LysoPC 60:40, the sample showed a level of distortion of the vesicle similar to that of pure DOPC. This implies an increased flexibility in the membrane at this point. Commonly, the assumption is that for increasing LysoPC concentration there is a reduction in membrane tension, implying that estimations of membrane tension based on spontaneous curvature assumptions may not be accurate.

Fractal avalanche ruptures in biological membranes

NASA Astrophysics Data System (ADS)

Gözen, Irep; Dommersnes, Paul; Czolkos, Ilja; Jesorka, Aldo; Lobovkina, Tatsiana; Orwar, Owe

2010-11-01

Bilayer membranes envelope cells as well as organelles, and constitute the most ubiquitous biological material found in all branches of the phylogenetic tree. Cell membrane rupture is an important biological process, and substantial rupture rates are found in skeletal and cardiac muscle cells under a mechanical load. Rupture can also be induced by processes such as cell death, and active cell membrane repair mechanisms are essential to preserve cell integrity. Pore formation in cell membranes is also at the heart of many biomedical applications such as in drug, gene and short interfering RNA delivery. Membrane rupture dynamics has been studied in bilayer vesicles under tensile stress, which consistently produce circular pores. We observed very different rupture mechanics in bilayer membranes spreading on solid supports: in one instance fingering instabilities were seen resulting in floral-like pores and in another, the rupture proceeded in a series of rapid avalanches causing fractal membrane fragmentation. The intermittent character of rupture evolution and the broad distribution in avalanche sizes is consistent with crackling-noise dynamics. Such noisy dynamics appear in fracture of solid disordered materials, in dislocation avalanches in plastic deformations and domain wall magnetization avalanches. We also observed similar fractal rupture mechanics in spreading cell membranes.

Physicochemical properties of liposomes as potential anticancer drugs carriers. Interaction of etoposide and cytarabine with the membrane: Spectroscopic studies

NASA Astrophysics Data System (ADS)

Pentak, Danuta

2014-03-01

The interactions between etoposide, cytarabine and 1,2-dihexadecanoyl-sn-glycerol-3-phosphocholine bilayers were studied using differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR). These techniques have proven to be a very powerful tool in studying the structure and dynamics of phospholipid bilayers. In particular, DSC can provide information on the phase transition temperature and cooperativity of the lipid molecules in the absence and presence of the drug. Vibrational spectroscopy is well suited to the study of drug-lipid interactions, since it allows for an investigation of the conformation of phospholipid molecules at different levels in lipid bilayers and follows structural changes that occur during the gel to liquid-crystalline phase transition. NMR supported the determination of the main phase transition temperatures (TC) of 1,2-dihexadecanoyl-sn-glycerol-3-phosphocholine (DPPC). The main phase transition temperature (TC) determined by 1H NMR is comparable with values obtained by DSC for all studied liposomes. The location of cytarabine and etoposide in liposomes was also determined by NMR. Atomic force microscopy (AFM) images, acquired immediately after sample deposition on a mica surface, revealed the spherical shape of lipid vesicles.

Membrane Deformation and Permeabilization Caused by Microplasma Irradiation

NASA Astrophysics Data System (ADS)

Motomura, Hideki; Nagaiwa, Hidenori; Yamamoto, Kenta; Kido, Yugo; Ikeda, Yoshihisa; Satoh, Susumu; Jinno, Masafumi

2016-09-01

The microplasma irradiation achieves high gene taransfection efficiency and high cell survivability simultaneously. For this purpose, we have developed a special plasma source using a microcapillary electrode. However, it is not clear how the stimuli of effective factors generated by plasma, such as current, charge, field, chemical species, cause transfection. In this study, we used artificial cell which is a spherical vesicle consisting of a lipid bilayer to visualize membrane dynamics and permeabilization caused by microplasma irradiation. Dioleoyl phosphatidylcholine (DOPC) was used as phospholipid molecules forming the lipid bilayer. The artificial cells were prepared by natural swelling method. Fluorescent labeled polyethylene glycol (PEG) polymers (Nanocs, MPEG Fluorescein, MW = 1000) were encapsulated in the artificial cells. The artificial cells were exposed to the microplasma for 5 ms and 10-20% of decrease of the dye fluorescence in the artificial cells was observed. This result suggests the outflow of the MPEG polymers through temporary poration or deformation of the lipid bilayer. The membrane deformation dynamics was directly observed with a microscope and the relationship to the polymer outflow will be shown at the conference. This work was partly supported by a Grant-in-Aid (25108509 and 15H00896) from JSPS and a grant from Ehime University.

Membrane-Assisted Growth of DNA Origami Nanostructure Arrays

PubMed Central

2015-01-01

Biological membranes fulfill many important tasks within living organisms. In addition to separating cellular volumes, membranes confine the space available to membrane-associated proteins to two dimensions (2D), which greatly increases their probability to interact with each other and assemble into multiprotein complexes. We here employed two DNA origami structures functionalized with cholesterol moieties as membrane anchors—a three-layered rectangular block and a Y-shaped DNA structure—to mimic membrane-assisted assembly into hierarchical superstructures on supported lipid bilayers and small unilamellar vesicles. As designed, the DNA constructs adhered to the lipid bilayers mediated by the cholesterol anchors and diffused freely in 2D with diffusion coefficients depending on their size and number of cholesterol modifications. Different sets of multimerization oligonucleotides added to bilayer-bound origami block structures induced the growth of either linear polymers or two-dimensional lattices on the membrane. Y-shaped DNA origami structures associated into triskelion homotrimers and further assembled into weakly ordered arrays of hexagons and pentagons, which resembled the geometry of clathrin-coated pits. Our results demonstrate the potential to realize artificial self-assembling systems that mimic the hierarchical formation of polyhedral lattices on cytoplasmic membranes. PMID:25734977

Membrane-assisted growth of DNA origami nanostructure arrays.

PubMed

Kocabey, Samet; Kempter, Susanne; List, Jonathan; Xing, Yongzheng; Bae, Wooli; Schiffels, Daniel; Shih, William M; Simmel, Friedrich C; Liedl, Tim

2015-01-01

Biological membranes fulfill many important tasks within living organisms. In addition to separating cellular volumes, membranes confine the space available to membrane-associated proteins to two dimensions (2D), which greatly increases their probability to interact with each other and assemble into multiprotein complexes. We here employed two DNA origami structures functionalized with cholesterol moieties as membrane anchors--a three-layered rectangular block and a Y-shaped DNA structure--to mimic membrane-assisted assembly into hierarchical superstructures on supported lipid bilayers and small unilamellar vesicles. As designed, the DNA constructs adhered to the lipid bilayers mediated by the cholesterol anchors and diffused freely in 2D with diffusion coefficients depending on their size and number of cholesterol modifications. Different sets of multimerization oligonucleotides added to bilayer-bound origami block structures induced the growth of either linear polymers or two-dimensional lattices on the membrane. Y-shaped DNA origami structures associated into triskelion homotrimers and further assembled into weakly ordered arrays of hexagons and pentagons, which resembled the geometry of clathrin-coated pits. Our results demonstrate the potential to realize artificial self-assembling systems that mimic the hierarchical formation of polyhedral lattices on cytoplasmic membranes.

Interaction of phloretin and 6-ketocholestanol with DPPC-liposomes as phospholipid model membranes.

PubMed

Auner, Barbara G; O'Neill, Michael A A; Valenta, Claudia; Hadgraft, Jonathan

2005-04-27

Phloretin and 6-ketocholestanol are penetration enhancers for percutaneous delivery of certain topically applied drugs. In the present study some physicochemical experiments have been performed to elucidate the mechanism of action of phloretin and 6-ketocholestanol. The penetration enhancing effect of phloretin and 6-ketocholestanol is believed to be due to their increase of the fluidity of the intercellular lipid bilayers of the stratum corneum. Phospholipid vesicles were chosen as a simple model to represent these bilayers. The effect of phloretin and 6-ketocholestanol on phase transition temperature and enthalpy was studied using differential scanning calorimetry. Beside of that the size of liposomes was monitored when the amount of penetration enhancer in the liposome preparation was changed. Addition of increasing amounts of phloretin and 6-ketocholestanol to the bilayer resulted in lowering of phase transition temperatures and increasing the enthalpy. Additionally the size of the liposomes was increased when penetration enhancer was added. The results suggest that phloretin as well as 6-ketocholestanol would interact with stratum corneum lipids in a similar manner, both reduce the diffusional resistance of the stratum corneum to drugs with balanced hydrophilic-lipophilic characteristics.

Strain and deformations engineered germanene bilayer double gate-field effect transistor by first principles

NASA Astrophysics Data System (ADS)

Meher Abhinav, E.; Chandrasekaran, Gopalakrishnan; Kasmir Raja, S. V.

2017-10-01

Germanene, silicene, stanene, phosphorene and graphene are some of single atomic materials with novel properties. In this paper, we explored bilayer germanene-based Double Gate-Field Effect Transistor (DG-FET) with various strains and deformations using Density Functional Theory (DFT) and Green's approach by first-principle calculations. The DG-FET of 1.6 nm width, 6 nm channel length (Lch) and HfO2 as gate dielectric has been modeled. For intrinsic deformation of germanene bilayer, we have enforced minute mechanical deformation of wrap and twist (5°) and ripple (0.5 Å) on germanene bilayer channel material. By using NEGF formalism, I-V Characteristics of various strains and deformation tailored DG-FET was calculated. Our results show that rough edge and single vacancy (5-9) in bilayer germanene diminishes the current around 47% and 58% respectively as compared with pristine bilayer germanene. In case of strain tailored bilayer DG-FET, multiple NDR regions were observed which can be utilized in building stable multiple logic states in digital circuits and high frequency oscillators using negative resistive techniques.

Synthesis of Large-grain, Single-crystalline Monolayer and AB-stacking Bilayer Graphene

NASA Astrophysics Data System (ADS)

Zhang, Luyao; Lin, Yung-Chen; Zhang, Yi; Chang, Han-Wen; Yeh, Wen-Cheng; Zhou, Chongwu; USC Nanotechnology Research Laboratory Team

2013-03-01

We report the growth of large-grain, single-crystalline monolayer and AB-stacking bilayer graphene by the combination of ambient pressure chemical vapor deposition and low pressure chemical vapor deposition. The shape of the monolayer graphene was modified to be either hexagons or flowers under different growth conditions. The size of the bilayer graphene region was enlarged under ambient pressure growth conditions with low methane concentration. Raman spectra and selected area electron diffraction of individual graphene grain indicated that the each graphene grain is single-crystalline. With electron beam lithography patterned PMMA seeds, graphene nucleation can be controlled and graphene monolayer and bilayer arrays were synthesized on copper foil. Electron backscatter diffraction study revealed that the graphene morphology had little correlation with the crystalline orientation of underlying copper substrate. Mork Family Department of Chemical Engineering and Materials Science

Ca2+-independent Binding of Anionic Phospholipids by Phospholipase C δ1 EF-hand Domain*

PubMed Central

Cai, Jingfei; Guo, Su; Lomasney, Jon W.; Roberts, Mary F.

2013-01-01

Recombinant EF-hand domain of phospholipase C δ1 has a moderate affinity for anionic phospholipids in the absence of Ca2+ that is driven by interactions of cationic and hydrophobic residues in the first EF-hand sequence. This region of PLC δ1 is missing in the crystal structure. The relative orientation of recombinant EF with respect to the bilayer, established with NMR methods, shows that the N-terminal helix of EF-1 is close to the membrane interface. Specific mutations of EF-1 residues in full-length PLC δ1 reduce enzyme activity but not because of disturbing partitioning of the protein onto vesicles. The reduction in enzymatic activity coupled with vesicle binding studies are consistent with a role for this domain in aiding substrate binding in the active site once the protein is transiently anchored at its target membrane. PMID:24235144

Microspectroscopic Study of Liposome-to-cell Interaction Revealed by Förster Resonance Energy Transfer.

PubMed

Yefimova, Svetlana L; Kurilchenko, Irina Yu; Tkacheva, Tatyana N; Kavok, Nataliya S; Todor, Igor N; Lukianova, Nataliya Yu; Chekhun, Vasyl F; Malyukin, Yuriy V

2014-03-01

We report the Förster resonance energy transfer (FRET)-labeling of liposomal vesicles as an effective approach to study in dynamics the interaction of liposomes with living cells of different types (rat hepatocytes, rat bone marrow, mouse fibroblast-like cells and human breast cancer cells) and cell organelles (hepatocyte nuclei). The in vitro experiments were performed using fluorescent microspectroscopic technique. Two fluorescent dyes (DiO as the energy donor and DiI as an acceptor) were preloaded in lipid bilayers of phosphatidylcholine liposomes that ensures the necessary distance between the dyes for effective FRET. The change in time of the donor and acceptor relative fluorescence intensities was used to visualize and trace the liposome-to-cell interaction. We show that FRET-labeling of liposome vesicles allows one to reveal the differences in efficiency and dynamics of these interactions, which are associated with composition, fluidity, and metabolic activity of cell plasma membranes.

Targeted Approach for Proteomic Analysis of a Hidden Membrane Protein.

PubMed

Martins-Marques, Tania; Anjo, Sandra I; Ribeiro-Rodrigues, Teresa; Manadas, Bruno; Girao, Henrique

2017-01-01

Given the properties of plasma membrane proteins, namely, their hydrophobicity, low solubility, and high resistance to digestion and extraction, their identification by traditional mass spectrometry (MS) has been a challenging task. Hence, proteomic studies involving the transmembrane protein connexin43 (Cx43) are scarce. Additionally, studies demonstrating the presence of proteins embedded in the lipid bilayer of extracellular vesicles (EVs) are difficult to perform and require specific changes and fine adjustments in the experimental and technical procedure to allow their detection by MS. In this review, we provide a detailed description of the protocol we have used to detect Cx43 in EVs of human peripheral blood. This includes some of the modifications that we have introduced in order to improve the detection of Cx43 in EVs, including an optimization of vesicle isolation, Cx43 purification, MS acquisition data, and further analysis.

Placing and shaping liposomes with reconfigurable DNA nanocages

NASA Astrophysics Data System (ADS)

Zhang, Zhao; Yang, Yang; Pincet, Frederic; C. Llaguno, Marc; Lin, Chenxiang

2017-07-01

The diverse structure and regulated deformation of lipid bilayer membranes are among a cell's most fascinating features. Artificial membrane-bound vesicles, known as liposomes, are versatile tools for modelling biological membranes and delivering foreign objects to cells. To fully mimic the complexity of cell membranes and optimize the efficiency of delivery vesicles, controlling liposome shape (both statically and dynamically) is of utmost importance. Here we report the assembly, arrangement and remodelling of liposomes with designer geometry: all of which are exquisitely controlled by a set of modular, reconfigurable DNA nanocages. Tubular and toroid shapes, among others, are transcribed from DNA cages to liposomes with high fidelity, giving rise to membrane curvatures present in cells yet previously difficult to construct in vitro. Moreover, the conformational changes of DNA cages drive membrane fusion and bending with predictable outcomes, opening up opportunities for the systematic study of membrane mechanics.

Solvent relaxation of fluorescent labels as a new tool for the detection of polarity and rigidity changes in membranes

NASA Astrophysics Data System (ADS)

Hof, Martin; Hutterer, Rudi

1998-04-01

Since solvent relaxation (SR) exclusively depends on the physical properties of the dye environment, SR spectroscopy of defined located labels in amphiphilic assemblies accomplishes the characterisation of specific domains. The most accurate way to characterise SR is the determination of the time-dependent Stokes shift. The time course of the Stokes shift, expressed as a solvent relaxation time, gives information about both the rigidity and polarity of the dye environment. The absolute value of the Stokes shift following the excitation is correlated with the polarity of the probed region. The validity of this approach for the investigation of phospholipid bilayers is illustrated by listing the parameters influencing the SR kinetics of appropriate membrane labels: membrane curvature, percentage of phosphatidylserine (PS) in small unilamell vesicles (SUV), addition of Ca2+ ions, binding of vitamin-K dependent proteins, percentage of diether-lipids in phosphatidylcholine (PC)-vesicles, and temperature.

Placing and shaping liposomes with reconfigurable DNA nanocages.

PubMed

Zhang, Zhao; Yang, Yang; Pincet, Frederic; Llaguno, Marc C; Lin, Chenxiang

2017-06-23

The diverse structure and regulated deformation of lipid bilayer membranes are among a cell's most fascinating features. Artificial membrane-bound vesicles, known as liposomes, are versatile tools for modelling biological membranes and delivering foreign objects to cells. To fully mimic the complexity of cell membranes and optimize the efficiency of delivery vesicles, controlling liposome shape (both statically and dynamically) is of utmost importance. Here we report the assembly, arrangement and remodelling of liposomes with designer geometry: all of which are exquisitely controlled by a set of modular, reconfigurable DNA nanocages. Tubular and toroid shapes, among others, are transcribed from DNA cages to liposomes with high fidelity, giving rise to membrane curvatures present in cells yet previously difficult to construct in vitro. Moreover, the conformational changes of DNA cages drive membrane fusion and bending with predictable outcomes, opening up opportunities for the systematic study of membrane mechanics.

Regulation of PLCβ2 by the electrostatic and mechanical properties of lipid bilayers

PubMed Central

Arduin, Alessia; Gaffney, Piers R. J.; Ces, Oscar

2015-01-01

Phosphoinositide-specific phospholipase C (PLC) is an important family of enzymes constituting a junction between phosphoinositide lipid signaling and the trans-membrane signal transduction processes that are crucial to many living cells. However, the regulatory mechanism of PLC is not yet understood in detail. To address this issue, activity studies were carried out using lipid vesicles in a model system that was specifically designed to study protein-protein and lipid-protein interactions in concert. Evidence was found for a direct interaction between PLC and the GTPases that mediate phospholipase activation. Furthermore, for the first time, the relationships between PLC activity and substrate presentation in lipid vesicles of various sizes, as well as lipid composition and membrane mechanical properties, were analyzed. PLC activity was found to depend upon the electrostatic potential and the stored curvature elastic stress of the lipid membranes. PMID:26243281

Behavior of atypical amphiphilic molecules

NASA Astrophysics Data System (ADS)

Ko, John

1997-08-01

The physical behavior of several atypical amphiphilic molecules was studied in various environments including micelles, model bilayer membranes, and emulsions. The molecules under investigation were nor-chenodeoxycholic acid (nor-CDCA), ursodeoxycholic acid (UDCA), sphingosine (Sp), sphingosine hydrochloride (SpċHCl), and tetrahydrolipstatin (THL). The bile acids, nor-CDCA and UDCA, were studied using 13C-Nuclear Magnetic Resonance ([13C) -NMR) in micelles of taurocholate and in bilayers of phosphatidylcholine. The pK a values of the bile acids in each environment were determined by [13C) -NMR and are as follows: 6.08 ±.03 for nor-CDCA and 6.27 ±.01 for UDCA in micelles, and 7.04 ± 12 for nor-CDCA and 6.89 ±.05 for UDCA in vesicles. Using line shape analysis, the transbilayer movement rate at 36oC for nor-CDCA and UDCA was calculated to be 580 sec--1 and 409 sec-1, respectively. [13C) -NMR titration of Sp gave pK a values of 9.09 ±.02 in micelles and 9.69 ±.21 in bilayers. Differential scanning calorimetry (DSC) and X-ray diffraction were used to establish the Spċwater and SpċHClċwater phase diagrams. Anhydrous and hydrated samples ranging from 5- 90% water were analyzed. The DSC thermograms traced out the transition temperatures of each molecule while the X- ray diffraction patterns revealed their chain and crystalline lattice packing structures. In general, sphingosine exists as a hydrated crystal with β packing phase below 43oC and melts into an Lα phase. Sphingosine hydrochloride, however, exists as a gel phase (L_beta or /beta/sp') below 42oC that swells to 61% hydration. At low water concentrations (0-64%), a lamellar liquid crystal phase (L_alpha) is formed above the chain melting transition of 42oC. At medium concentration (65%), a Hexagonal I phase is present, and at high water concentrations (66-90%), a micellar phase is present. THL, a specific inhibitor of lipases, was analyzed with [ 13C) -NMR to study its behavior in various environments, ranging from carbon tetrachloride to water to pure triolein. THL was also incorporated into phosphatidylcholine bilayers and into microemulsions of triolein and phosphatidylcholine. [ 13C) -NMR analysis revealed that THL gets incorporated into the surface of vesicles, and into both the surface and core of microemulsion particles.

Characterization of Phospholipids in Insulin Secretory Granules and Mitochondria in Pancreatic Beta Cells and Their Changes with Glucose Stimulation*

PubMed Central

MacDonald, Michael J.; Ade, Lacmbouh; Ntambi, James M.; Ansari, Israr-Ul H.; Stoker, Scott W.

2015-01-01

The lipid composition of insulin secretory granules (ISG) has never previously been thoroughly characterized. We characterized the phospholipid composition of ISG and mitochondria in pancreatic beta cells without and with glucose stimulation. The phospholipid/protein ratios of most phospholipids containing unsaturated fatty acids were higher in ISG than in whole cells and in mitochondria. The concentrations of negatively charged phospholipids, phosphatidylserine, and phosphatidylinositol in ISG were 5-fold higher than in the whole cell. In ISG phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin, fatty acids 12:0 and 14:0 were high, as were phosphatidylserine and phosphatidylinositol containing 18-carbon unsaturated FA. With glucose stimulation, the concentration of many ISG phosphatidylserines and phosphatidylinositols increased; unsaturated fatty acids in phosphatidylserine increased; and most phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophosphatidylcholines were unchanged. Unsaturation and shorter fatty acid length in phospholipids facilitate curvature and fluidity of membranes, which favors fusion of membranes. Recent evidence suggests that negatively charged phospholipids, such as phosphatidylserine, act as coupling factors enhancing the interaction of positively charged regions in SNARE proteins in synaptic or secretory vesicle membrane lipid bilayers with positively charged regions in SNARE proteins in the plasma membrane lipid bilayer to facilitate docking of vesicles to the plasma membrane during exocytosis. The results indicate that ISG phospholipids are in a dynamic state and are consistent with the idea that changes in ISG phospholipids facilitate fusion of ISG with the plasma membrane-enhancing glucose-stimulated insulin exocytosis. PMID:25762724

Hemoglobin-vesicle, a cellular artificial oxygen carrier that fulfils the physiological roles of the red blood cell structure.

PubMed

Sakai, Hiromi; Sou, Keitaro; Horinouchi, Hirohisa; Kobayashi, Koichi; Tsuchida, Eishun

2010-01-01

Hb-vesicles (HbV) are artificial O(2) carriers encapsulating concentrated Hb solution (35 g/dL) with a phospholipid bilayer membrane (liposome). The concentration of the HbV suspension is extremely high ([Hb] = 10 g/dL) and it has an O(2) carrying capacity that is comparable to that of blood. HbV is much smaller than RBC (250 vs. 8000 nm), but it recreates the functions of RBCs; (i) the slower rate of O(2) unloading than Hb solution; (ii) colloid osmotic pressure is zero; (iii) the viscosity of a HbV suspension is adjustable to that of blood; (iv) HbV is finally captured by and degraded in RES; (v) co-encapsulation of an allosteric effector to regulate O(2) affinity; (vi) the lipid bilayer membrane prevents direct contact of Hb and vasculature; (vii) NO-binding is retarded to some extent by an intracellular diffusion barrier, and HbV does not induce vasoconstriction. (viii) Both RBC and HbV can be a carrier of not only O(2) but also exogenous CO. However, HbV has limitations such as a shorter functional half-life when compared with RBCs. On the other hand, the advantages of HbV are that it is pathogen-free and blood-type-antigen-free; moreover, it can withstand long-term storage of a few years, none of which can be achieved by the RBC transfusion systems.

Diffusion in Single Supported Lipid Bilayers

NASA Astrophysics Data System (ADS)

Armstrong, C. L.; Trapp, M.; Rheinstädter, M. C.

2011-03-01

Despite their potential relevance for the development of functionalized surfaces and biosensors, the study of single supported membranes using neutron scattering has been limited by the challenge of obtaining relevant dynamic information from a sample with minimal material. Using state of the art neutron instrumentation we have, for the first time, modeled lipid diffusion in single supported lipid bilayers. While we find that the diffusion coefficient for the single bilayer system is comparable to a multi-lamellar lipid system, the molecular mechanism for lipid motion in the single bilayer is a continuous diffusion process with no sign of the flow-like ballistic motion reported in the stacked membrane system. In the future, these membranes will be used to hold and align proteins, mimicking physiological conditions enabling the study of protein structure, function and interactions in relevant and highly topical membrane/protein systems with minimal sample material. C.L. Armstrong, M.D. Kaye, M. Zamponi, E. Mamontov, M. Tyagi, T. Jenkins and M.C. Rheinstädter, Soft Matter Communication, 2010, Advance Article, DOI: 10.1039/C0SM00637H

The in vitro kinetics of the interactions between PEG-ylated magnetic-fluid-loaded liposomes and macrophages.

PubMed

Martina, Marie-Sophie; Nicolas, Valerie; Wilhelm, Claire; Ménager, Christine; Barratt, Gillian; Lesieur, Sylviane

2007-10-01

Binding and uptake kinetics of magnetic-fluid-loaded liposomes (MFL) by endocytotic cells were investigated in vitro on the model cell-line J774. MFL consisted of unilamellar phosphatidylcholine vesicles (mean hydrodynamic diameter close to 200nm) encapsulating 8-nm nanocrystals of maghemite (gamma-Fe(2)O(3)) and sterically stabilized by introducing 5mol% of distearylphosphatidylcholine poly(ethylene glycol)(2,000) (DSPE-PEG(2,000)) in the vesicle bilayer. The association processes with living macrophages were followed at two levels. On one hand, the lipid vesicles were imaged by confocal fluorescence microscopy. For this purpose 1mol% of rhodamine-marked phosphatidylethanolamine was added to the liposome composition. On the other hand, the iron oxide particles associated with cells were independently quantified by magnetophoresis. All the experiments were similarly performed with PEG-ylated or conventional MFL to point out the role of polymer coating. The results showed cell association with both types of liposomes resulting from binding followed by endocytosis. Steric stabilization by PEG chains reduced binding efficiency limiting the amount of MFL internalized by the macrophages. In contrast, PEG coating did not change the kinetics of endocytosis which exhibited the same first-order rate constant for both conventional and PEG-ylated liposomes. Moreover, lipids and iron oxide particle uptakes were perfectly correlated, indicating that MFL vesicle structure and encapsulation rate were preserved upon cell penetration.

Bovine binder-of-sperm protein BSP1 promotes protrusion and nanotube formation from liposomes

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

Lafleur, Michel, E-mail: michel.lafleur@umontreal.ca; Courtemanche, Lesley; Karlsson, Goeran

Research highlights: {yields} Binder-of-sperm protein 1 (BSP1) modifies the morphology of lipidic vesicles inducing bead necklace-like and thread-like structures. {yields} In the presence of multilamellar liposomes, BSP1 leads to the formation of long nanotubes. {yields} The insertion of BSP1 in the external lipid leaflet of membranes induces local changes in bilayer curvature. -- Abstract: Binder-of-sperm (BSP) proteins interact with sperm membranes and are proposed to extract selectively phosphatidylcholine and cholesterol from these. This change in lipid composition is a key step in sperm capacitation. The present work demonstrates that the interactions between the protein BSP1 and model membranes composed withmore » phosphatidylcholine lead to drastic changes in the morphology of the lipidic self-assemblies. Using cryo-electron microscopy and fluorescence microscopy, we show that, in the presence of the protein, the lipid vesicles elongate, and form bead necklace-like structures that evolve toward small vesicles or thread-like structures. In the presence of multilamellar vesicles, where a large reservoir of lipid is available, the presence of BSP proteins lead to the formation of long nanotubes. Long spiral-like threads, associated with lipid/protein complexes, are also observed. The local curvature of lipid membranes induced by the BSP proteins may be involved in lipid domain formation and the extraction of some lipids during the sperm maturation process.« less

Energy transduction inside vesicles, photocatalysis by titanium dioxide and formation of NADH

NASA Astrophysics Data System (ADS)

Summers, David; Noveron, Juan; Rodoni, David; Basa, Ranor

A number of theories on the origin and early evolution of life have focused on the role of lipid bilayer membrane structures (vesicles). These vesicles are similar to modern cellular membranes , and have been postulated to have been abiotically formed and spontaneously assemble on the prebiotic Earth to provide compartments for early cellular life. They can contain water-soluble species, concentrate species, and have the potential to catalyze reactions. The origin of the use of photochemical energy to drive metabolism (ie. energy transduction) is also one of the central issues in our attempts to understand the origin and evolution of life. When did energy transduction and photosynthesis begin? What was the original system for capturing photochemical energy? How simple can such a system be? It has been postulated that vesicle structures developed the ability to capture and transduce light, providing energy for reactions. It has been shown that pH gradients can be photo-chemically created, but it has been found difficult to couple these to drive chemical reactions. Minerals can introduce a number of properties to a vesicle system. The incorporation of clay particles into vesicles can provide catalytic activity that mediates both vesicle assembly and RNA oligomerization. It is known that colloidal semiconducting mineral particles can act as photocatalysts and drive redox chemistry. We show that encapsulation of these particles has the potential to provide a source of energy transduction inside vesicles, and thereby drive protocellular chemistry and represent a model system for early photosynthesis. TiO2 particles can be incorporated into vesicles and retain their photoactivity through the dehydration/rehydration cycles that have been shown to be able concentrate species inside a vesicle. It is shown that these can be used to produce biochemical species such as enzymatically active NADH in such structures. This system demonstrates a simple energy source inside vesicles/protocells suitable either for simple prebiotic systems and/or for more complex "protobiochemical" systems. It could act as a precursor to metabolic systems and provide a model of how metabolism could have developed prebiotically in a vesicle based "protocell origin of life". It can provide a source of prebiotic compounds inside vesicles, an environment considered to be of great importance for the origin of life. An energy transduction system that is simple enough to have formed at an early stage of the origin of life (even before the formation of enzymatic or ribozymal activity) makes an autotrophic origin of life easier to envision.

Synthesis, characterization, and application of monosized mesoporous silica nanoparticle-supported lipid bilayers for targeted therapeutic delivery to individual cells

NASA Astrophysics Data System (ADS)

Durfee, Paul Nicholas

Mesoporous silica nanoparticle (MSNP) supported-lipid bilayers, termed 'protocells,' represent a potentially transformative class of therapeutic and theranostic delivery vehicles. The field of targeted drug delivery poses considerable challenges that cannot be addressed with a single 'magic bullet'. Consequently, the protocell has been designed as a modular platform composed of interchangeable biocompatible components. The mesoporous silica core can have variable size and shape to direct biodistribution and controlled pore size and surface chemistry to accommodate diverse cargos. The encapsulating supported lipid bilayer can be modified with targeting and trafficking ligands as well as polyethylene glycol (PEG) to effect selective binding, endosomal escape of cargo, drug efflux prevention, and potent therapeutic delivery, while maintaining in vivo colloidal stability. Many nanocarrier cancer therapeutics currently under development, as well as those used in the clinical setting, rely upon the enhanced permeability and retention (EPR) effect to passively accumulate in the tumor microenvironment and kill cancer cells. In leukemia, where leukemogenic stem cells and their progeny circulate within the peripheral blood or bone marrow, the EPR effect may not be operative. Thus, for leukemia therapeutics, it is essential to target and bind individual circulating cells. Here, we investigate protocells, an emerging class of nanocarriers, and establish the synthesis conditions and lipid bilayer composition needed to achieve highly monodisperse protocells that remain stable in complex media as assessed in vitro by dynamic light scattering and cryo-electron microscopy and ex ovo by direct imaging within a chick chorioallantoic membrane (CAM) model. We show that for vesicle fusion conditions where the lipid surface area exceeds the external surface area of the MSNP and the ionic strength exceeds 20 mM, we form monosized protocells (polydispersity index < 0.1) on MSNP cores with varying size, shape, and pore size, whose conformal zwitterionic supported lipid bilayer confers excellent stability as judged by circulation in the CAM and minimal opsonization in vivo in a mouse model. Having established protocell formulations that are stable colloids, we further modified them with anti-EGFR antibodies as targeting agents and re-verified their monodispersity and stability. Then using intravital imaging in the CAM we directly observed in real time the progression of selective targeting of individual leukemia cells (using the established REH leukemia cell line transduced with EGFR) and delivery of a model cargo. Overall we have established the effectiveness of the protocell platform for individual cell targeting and delivery needed for leukemia and other disseminated disease.

Single lipid bilayer deposition on polymer surfaces using bicelles.

PubMed

Saleem, Qasim; Zhang, Zhenfu; Petretic, Amy; Gradinaru, Claudiu C; Macdonald, Peter M

2015-03-09

A lipid bilayer was deposited on a 3 μm diameter polystyrene (PS) bead via hydrophobic anchoring of bicelles containing oxyamine-bearing cholesteric moieties reacting with the aldehyde functionalized bead surface. Discoidal bicelles were formed by mixing dimyristoylphosphatidylcholine (DMPC), dihexanoylphosphatidylcholine (DHPC), dimyristoyltrimethylammonium propane (DMTAP), and the oxyamine-terminated cholesterol derivative, cholest-5-en-3β-oxy-oct-3,6-oxa-an-8-oxyamine (CHOLOA), in the molar ratio DMPC/DHCP/DMTAP/CHOLOA (1/0.5/0.01/0.05) in water. Upon exposure to aldehyde-bearing PS beads, a stable single lipid bilayer coating rapidly formed at the bead surface. Fluorescence recovery after photobleaching demonstrated that the deposited lipids fused into an encapsulating lipid bilayer. Electrospray ionization mass spectrometry showed that the short chain lipid DHPC was entirely absent from the PS adherent lipid coating. Fluorescence quenching measurements proved that the coating was a single lipid bilayer. The bicelle coating method is thus simple and robust, can be modified to include membrane-associated species, and can be adapted to coat any number of different surfaces.

Cl- channels of the gastric parietal cell that are active at low pH.

PubMed

Cuppoletti, J; Baker, A M; Malinowska, D H

1993-06-01

HCl secretion across mammalian gastric parietal cell apical membrane may involve Cl- channels. H(+)-K(+)-ATPase-containing membranes isolated from gastric mucosa of histamine-stimulated rabbits were fused to planar lipid bilayers. Channels were recorded with symmetric 800 mM CsCl solutions, pH 7.4. A linear current-voltage (I-V) relationship was obtained, and conductance was 28 +/- 1 pS at 800 mM CsCl. Conductance was 6.9 +/- 2 pS at 150 mM CsCl. Reversal potential was +22 mV with a fivefold cis-trans CsCl concentration gradient, indicating that the channel was anion selective with a discrimination ratio of 6:1 for Cl- over Cs+. Anion selectivity of the channel was I- > Cl- > or = Br- > NO3-, and gluconate was impermeant. Channels obtained at pH 7.4 persisted when pH of medium bathing the trans side of the bilayer (pHtrans) was reduced to pH 3, without a change in conductance, linearity of I-V relationship, or ion selectivity. In contrast, asymmetric reduction of pH of medium bathing the cis side of the bilayer from 7.4 to 3 always resulted in loss of channel activity. At pH 7.4, open probability (Po) of the channel was voltage dependent, i.e., predominantly open at +80 mV but mainly closed at -80 mV. In contrast, with low pHtrans, channel Po at -80 mV was increased 3.5-fold. The Cl- channel was Ca2+ indifferent. In absence of ionophores, ion selectivity for support of H(+)-K(+)-ATPase activity and H+ transport was consistent with that exhibited by the channel and could be limited by substitution with NO3-, whereas maximal H(+)-K(+)-ATPase activity was indifferent to anion present, demonstrating that anion transport can be rate limiting. Cl- channels with similar characteristics (conductance, linear I-V relationship, and ion selectivity) were also present in H(+)-K(+)-ATPase-containing vesicles isolated from resting (cimetidine-treated) gastric mucosa, exhibiting at -80 mV a pH-independent approximately 3.5-fold lower Po than stimulated vesicle channels. At -80 mV, reduction of pHtrans increased Po of both resting and stimulated Cl- channels by five- to sixfold. Changing membrane potential from 0 to -80 mV across stimulated vesicles increased Cl- channel activity an additional 10-fold.(ABSTRACT TRUNCATED AT 400 WORDS)

The role of charge in the surfactant-assisted stabilization of the natural product curcumin.

PubMed

Wang, Zifan; Leung, Mandy H M; Kee, Tak W; English, Douglas S

2010-04-20

Colloidal solutions of surfactants that form micelles or vesicles are useful for solubilizing and stabilizing hydrophobic molecules that are otherwise sparingly soluble in aqueous solutions. In this paper we investigate the use of micelles and vesicles prepared from ionic surfactants for solubilizing and stabilizing curcumin, a medicinal natural product that undergoes alkaline hydrolysis in water. We identify spectroscopic signatures to evaluate curcumin partitioning and deprotonation in surfactant mixtures containing micelles or vesicles. These spectroscopic signatures allow us to monitor the interaction of curcumin with charged surfactants over a wide range of pH values. Titration data are presented to show the pH dependence of curcumin interactions with negatively and positively charged micelles and vesicles. In solutions of cationic micelles or positively charged vesicles, strong interaction between the Cur(-1) phenoxide ion and the positively charged surfactants results in a change in the acidity of the phenolic hydrogen and a lowering of the apparent lowest pK(a) value for curcumin. In the microenvironments formed by anionic micelles or negatively charged bilayers, our data indicates that curcumin partitions as the Cur(0) species, which is stabilized by interactions with the respective surfactant aggregates, and this leads to an increase in the apparent pK(a) values. Our results may explain some of the discrepancies within the literature with respect to reported pK(a) values and the acidity of the enolic versus phenolic protons. Hydrolysis rates, quantum yields, and molar absorption coefficients are reported for curcumin in a variety of solutions.

He, Ne and Ar systematics in single vesicles: Mantle isotopic ratios and origin of the air component in basaltic glasses

NASA Astrophysics Data System (ADS)

Raquin, Aude; Moreira, Manuel Alexis; Guillon, Fabien

2008-09-01

An outstanding problem in understanding the origin of the gaseous phase, particularly the rare gas compositions in magmatic rocks, is the ubiquitous atmospheric component in bulk rock samples, and whether this atmospheric component is a late stage contamination of the sample, or a recycled component though sediments or altered oceanic crust. In the present study we address this problem by analyzing single vesicles from the "popping rock 2∏D43" sample from the Mid-Atlantic Ridge using a UV laser ablation system. We have determined both elemental and isotopic compositions of He, Ne and Ar in single vesicles as well as Kr and Xe abundances. All vesicles analyzed have an isotopic composition identical to the referred degassed mantle value estimated from this same sample, despite analyzing vesicles from a wide size distribution. The atmospheric component, which is always detected in bulk samples by crushing or heating, was not detected in the single vesicles. This implies that the recycling of atmospheric noble gases in the mantle cannot explain the air-like component of this sample. The addition of the atmospheric component must occur either during the eruption, or after sample recovery.

Single-Molecule Resolution of Antimicrobial Peptide Interactions with Supported Lipid A Bilayers.

PubMed

Nelson, Nathaniel; Schwartz, Daniel K

2018-06-05

The molecular interactions between antimicrobial peptides (AMPs) and lipid A-containing supported lipid bilayers were probed using single-molecule total internal reflection fluorescence microscopy. Hybrid supported lipid bilayers with lipid A outer leaflets and phospholipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)) inner leaflets were prepared and characterized, and the spatiotemporal trajectories of individual fluorescently labeled LL37 and Melittin AMPs were determined as they interacted with the bilayer surfaces comprising either monophosphoryl or diphosphoryl lipid A (from Escherichia coli) to determine the impact of electrostatic interactions. Large numbers of trajectories were obtained and analyzed to obtain the distributions of surface residence times and the statistics of the spatial trajectories. Interestingly, the AMP species were sensitive to subtle differences in the charge of the lipid, with both peptides diffusing more slowly and residing longer on the diphosphoryl lipid A. Furthermore, the single-molecule dynamics indicated a qualitative difference between the behavior of AMPs on hybrid Lipid A bilayers and on those composed entirely of DOPE. Whereas AMPs interacting with a DOPE bilayer exhibited two-dimensional Brownian diffusion with a diffusion coefficient of ∼1.7 μm 2 /s, AMPs adsorbed to the lipid A surface exhibited much slower apparent diffusion (on the order of ∼0.1 μm 2 /s) and executed intermittent trajectories that alternated between two-dimensional Brownian diffusion and desorption-mediated three-dimensional flights. Overall, these findings suggested that bilayers with lipid A in the outer leaflet, as it is in bacterial outer membranes, are valuable model systems for the study of the initial stage of AMP-bacterium interactions. Furthermore, single-molecule dynamics was sensitive to subtle differences in electrostatic interactions between cationic AMPs and monovalent or divalent anionic lipid A moieties. Copyright © 2018 Biophysical Society. All rights reserved.

Large effect of membrane tension on the fluid-solid phase transitions of two-component phosphatidylcholine vesicles.

PubMed

Chen, Dong; Santore, Maria M

2014-01-07

Model phospholipid membranes and vesicles have long provided insight into the nature of confined materials and membranes while also providing a platform for drug delivery. The rich thermodynamic behavior and interesting domain shapes in these membranes have previously been mapped in extensive studies that vary temperature and composition; however, the thermodynamic impact of tension on bilayers has been restricted to recent reports of subtly reduced fluid-fluid transition temperatures. In two-component phosphatidylcholine unilamellar vesicles [1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)], we report a dramatic influence of tension on the fluid-solid transition and resulting phases: At fixed composition, systematic variations in tension produce differently shaped solid domains (striped or irregular hexagons), shift fluid-solid transition temperatures, and produce a triple-point-like intersection of coexistence curves at elevated tensions, about 3 mN/m for 30% DOPC/70% DPPC. Tension therefore represents a potential switch of microstructure in responsive engineered materials; it is an important morphology-determining variable in confined systems, and, in biological membranes, it may provide a means to regulate dynamic structure.

Measuring the change in hydration of a polypeptide-based block polymer vesicle as a function of pH

NASA Astrophysics Data System (ADS)

Smith, Ian; Charlier, Alban; Shishlov, Alexander; Savin, Daniel

Amphiphilic AB2 star polymers undergo directed self-assembly into vesicles in aqueous solution. The overall structure of the assembly is responsive to a change in solution pH by incorporating an ionizable polypeptide as the A-block and two lipid-like tails for the B-blocks. Herein, we present some recent results in the solution characterization of polyglutamate-octadecanethiol2 (PE-DDT2) star polymers using static and dynamic light scattering, as well as transmission electron microscopy. An increase in pH will induce a transition in secondary structure of the PE block from an α-helix to an extended coil, thereby perturbing the morphological structure and resulting in an expansion of the vesicle. The magnitude of this response is much larger than what is expected based on the conformational transition of the peptide. The mechanism of this process can be probed by measuring the change in hydration at the surface of the hydrophobic bilayer. Towards this end, we utilize 2,4,6-trichloro-1,3,5-triazine (TCT) as a modular linker to install spin labels into the assembly as a mechanism to directly interrogate local hydrophobicity using electron paramagnetic resonance (EPR). NSF 1539347.

Fusion activity of influenza virus PR8/34 correlates with a temperature-induced conformational change within the hemagglutinin ectodomain detected by photochemical labeling

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

Brunner, J.; Zugliani, C.; Mischler, R.

1991-03-05

Fusion of influenza viruses with membranes is catalyzed by the viral spike protein hemagglutinin (HA). Under mildly acidic conditions ({approximately}pH 5) this protein undergoes a conformational change that triggers the exposure of the fusion peptide, the hydrophobic N-terminal segment of the HA2 polypeptide chain. Insertion of this segment into the target membrane (or viral membrane ) is likely to represent a key step along the fusion pathway, but the details are far from being clear. The photoreactive phospholipid 1-palmitoyl-2-(11-(4-(3-(trifluoromethyl)diazirinyl)phenyl)(2-{sup 3}H)undecanoyl)-sn-glycero-3-phosphocholine (({sup 3}H)PTPC/11), inserted into the bilayer of large unilamellar vesicles (LUVs), allowed the authors to investigate both the interaction ofmore » viruses with the vesicles under perfusion conditions and the fusion process itself occurring at elevated temperatures only. Despite the observed binding of viruses to LUVs at pH 5 and 0C, labeling of HA2 was very weak. They have studied also the effect of temperature on the acid-induced (pH 5) interaction of bromelain-solubilized HA (BHA) with vesicles.« less

Sustained Release of an Anti-Glaucoma Drug: Demonstration of Efficacy of a Liposomal Formulation in the Rabbit Eye

PubMed Central

Ang, Marcus; Darwitan, Anastasia; Foo, Selin; Zhen, Ma; Koo, Magdalene; Wong, Tina T.; Venkatraman, Subbu S.

2011-01-01

Topical medication remains the first line treatment of glaucoma; however, sustained ocular drug delivery via topical administration is difficult to achieve. Most drugs have poor penetration due to the multiple physiological barriers of the eye and are rapidly cleared if applied topically. Currently, daily topical administration for lowering the intra-ocular pressure (IOP), has many limitations, such as poor patient compliance and ocular allergy from repeated drug administration. Poor compliance leads to suboptimal control of IOP and disease progression with eventual blindness. The delivery of drugs in a sustained manner could provide the patient with a more attractive alternative by providing optimal therapeutic dosing, with minimal local toxicity and inconvenience. To investigate this, we incorporated latanoprost into LUVs (large unilamellar vesicles) derived from the liposome of DPPC (di-palmitoyl-phosphatidyl-choline) by the film hydration technique. Relatively high amounts of drug could be incorporated into this vesicle, and the drug resides predominantly in the bilayer. Vesicle stability monitored by size measurement and DSC (differential scanning calorimetry) analysis showed that formulations with a drug/lipid mole ratio of about 10% have good physical stability during storage and release. This formulation demonstrated sustained release of latanoprost in vitro, and then tested for efficacy in 23 rabbits. Subconjunctival injection and topical eye drop administration of the latanoprost/liposomal formulation were compared with conventional daily administration of latanoprost eye drops. The IOP lowering effect with a single subconjunctival injection was shown to be sustained for up to 50 days, and the extent of IOP lowering was comparable to daily eye drop administration. Toxicity and localized inflammation were not observed in any treatment groups. We believe that this is the first demonstration, in vivo, of sustained delivery to the anterior segment of the eye that is safe and efficacious for 50 days. PMID:21931735

Mechanical properties of electrospun bilayer fibrous membranes as potential scaffolds for tissue engineering.

PubMed

Pu, Juan; Komvopoulos, Kyriakos

2014-06-01

Bilayer fibrous membranes of poly(l-lactic acid) (PLLA) were fabricated by electrospinning, using a parallel-disk mandrel configuration that resulted in the sequential deposition of a layer with fibers aligned across the two parallel disks and a layer with randomly oriented fibers, both layers deposited in a single process step. Membrane structure and fiber alignment were characterized by scanning electron microscopy and two-dimensional fast Fourier transform. Because of the intricacies of the generated electric field, bilayer membranes exhibited higher porosity than single-layer membranes consisting of randomly oriented fibers fabricated with a solid-drum collector. However, despite their higher porosity, bilayer membranes demonstrated generally higher elastic modulus, yield strength and toughness than single-layer membranes with random fibers. Bilayer membrane deformation at relatively high strain rates comprised multiple abrupt microfracture events characterized by discontinuous fiber breakage. Bilayer membrane elongation yielded excessive necking of the layer with random fibers and remarkable fiber stretching (on the order of 400%) in the layer with fibers aligned in the stress direction. In addition, fibers in both layers exhibited multiple localized necking, attributed to the nonuniform distribution of crystalline phases in the fibrillar structure. The high membrane porosity, good mechanical properties, and good biocompatibility and biodegradability of PLLA (demonstrated in previous studies) make the present bilayer membranes good scaffold candidates for a wide range of tissue engineering applications. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Modulation of Plasma Membrane Ca2+-ATPase by Neutral Phospholipids

PubMed Central

Pignataro, María Florencia; Dodes-Traian, Martín M.; González-Flecha, F. Luis; Sica, Mauricio; Mangialavori, Irene C.; Rossi, Juan Pablo F. C.

2015-01-01

The effects of lipids on membrane proteins are likely to be complex and unique for each membrane protein. Here we studied different detergent/phosphatidylcholine reconstitution media and tested their effects on plasma membrane Ca2+ pump (PMCA). We found that Ca2+-ATPase activity shows a biphasic behavior with respect to the detergent/phosphatidylcholine ratio. Moreover, the maximal Ca2+-ATPase activity largely depends on the length and the unsaturation degree of the hydrocarbon chain. Using static light scattering and fluorescence correlation spectroscopy, we monitored the changes in hydrodynamic radius of detergent/phosphatidylcholine particles during the micelle-vesicle transition. We found that, when PMCA is reconstituted in mixed micelles, neutral phospholipids increase the enzyme turnover. The biophysical changes associated with the transition from mixed micelles to bicelles increase the time of residence of the phosphorylated intermediate (EP), decreasing the enzyme turnover. Molecular dynamics simulations analysis of the interactions between PMCA and the phospholipid bilayer in which it is embedded show that in the 1,2-dioleoyl-sn-glycero-3-phosphocholine bilayer, charged residues of the protein are trapped in the hydrophobic core. Conversely, in the 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayer, the overall hydrophobic-hydrophilic requirements of the protein surface are fulfilled the best, reducing the thermodynamic cost of exposing charged residues to the hydrophobic core. The apparent mismatch produced by a 1,2-dioleoyl-sn-glycero-3-phosphocholine thicker bilayer could be a structural foundation to explain its functional effect on PMCA. PMID:25605721

Self-Assembly of Phosphate Amphiphiles in Mixtures of Prebiotically Plausible Surfactants

PubMed Central

Albertsen, A.N.; Duffy, C.D.; Sutherland, J.D.

2014-01-01

Abstract The spontaneous formation of closed bilayer structures from prebiotically plausible amphiphiles is an essential requirement for the emergence of early cells on prebiotic Earth. The sources of amphiphiles could have been both endo- and exogenous (accretion of meteorite carbonaceous material or interstellar dust particles). Among all prebiotic possible amphiphile candidates, those containing phosphate are the least investigated species because their self-assembly occurs in a seemingly too narrow range of conditions. The self-assembly of simple phosphate amphiphiles should, however, be of great interest, as contemporary membranes predominantly contain phospholipids. In contrast to common expectations, we show that these amphiphiles can be easily synthesized under prebiotically plausible environmental conditions and can efficiently form bilayer structures in the presence of various co-surfactants across a large range of pH values. Vesiculation was even observed in crude reaction mixtures that contained 1-decanol as the amphiphile precursor. The two best co-surfactants promoted vesicle formation over the entire pH range in aqueous solutions. Expanding the pH range where bilayer membranes self-assemble and remain intact is a prerequisite for the emergence of early cell-like compartments and their preservation under fluctuating environmental conditions. These mixed bilayers also retained small charged solutes, such as dyes. These results demonstrate that alkyl phosphate amphiphiles might have played a significant role as early compartment building blocks. Key Words: Vesicles—Alkyl phosphate—Prebiotic synthesis—Amphiphile mixtures. Astrobiology 14, 462–472. PMID:24885934

Compression-triggered instabilities of multi-layer systems: From thin elastic membranes to lipid bilayers on flexible substrates

NASA Astrophysics Data System (ADS)

Stone, Howard A.

2013-03-01

Instabilities are triggered when elastic materials are subjected to compression. We explore new features of two distinct systems of this type. First, we describe a two-layer polymeric system under biaxial compressive stress, which exhibits a repetitive wrinkle-to-fold transition that subsequently generates a hierarchical network of folds during reorganization of the stress field. The folds delineate individual domains, and each domain subdivides into smaller ones over multiple generations. By modifying the boundary conditions and geometry, we demonstrate control over the final network morphology. Some analogies to the venation pattern of leaves are indicated. Second, motivated by the confined configurations common to cells, which are wrapped in lipid bilayer membranes, we study a lipid bilayer, coupled to an elastic sheet, and demonstrate that, upon straining, the confined lipid membrane is able to passively regulate its area. In particular, by stretching the elastic support, the bilayer laterally expands without rupture by fusing adhered lipid vesicles; upon compression, lipid tubes grow out of the membrane plane, thus reducing its area. These transformations are reversible, as we show using cycles of expansion and compression, and closely reproduce membrane processes found in cells during area regulation. The two distinct systems illustrate the influence of the substrate on finite amplitude shape changes, for which we describe the time-dependent shape evolution as the stress relaxes. This talk describes joint research with Manouk Abkarian, Marino Arroyo, Pilnam Kim, Mohammad Rahimi and Margarita Staykova.

Acetylsalicylic acid (aspirin) and salicylic acid interaction with the human erythrocyte membrane bilayer induce in vitro changes in the morphology of erythrocytes.

PubMed

Suwalsky, Mario; Belmar, Jessica; Villena, Fernando; Gallardo, María José; Jemiola-Rzeminska, Malgorzata; Strzalka, Kazimierz

2013-11-01

Despite the well-documented information, there are insufficient reports concerning the effects of salicylate compounds on the structure and functions of cell membranes, particularly those of human erythrocytes. With the aim to better understand the molecular mechanisms of the interaction of acetylsalicylic acid (ASA) and salicylic acid (SA) with cell membranes, human erythrocyte membranes and molecular models were utilized. These consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. The capacity of ASA and SA to perturb the multibilayer structures of DMPC and DMPE was evaluated by X-ray diffraction while DMPC unilamellar vesicles (LUV) were studied by fluorescence spectroscopy. Moreover, we took advantage of the capability of differential scanning calorimetry (DSC) to detect the changes in the thermotropic phase behavior of lipid bilayers resulting from ASA and SA interaction with PC and PE molecules. In an attempt to further elucidate their effects on cell membranes, the present work also examined their influence on the morphology of intact human erythrocytes by means of defocusing and scanning electron microscopy, while isolated unsealed human erythrocyte membranes (IUM) were studied by fluorescence spectroscopy. Results indicated that both salicylates interact with human erythrocytes and their molecular models in a concentration-dependent manner perturbing their bilayer structures. Copyright © 2013 Elsevier Inc. All rights reserved.

Behavior of 2,6-bis(decyloxy)naphthalene inside lipid bilayer.

PubMed

Kepczynski, Mariusz; Kumorek, Marta; Stepniewski, Michał; Róg, Tomasz; Kozik, Bartłomiej; Jamróz, Dorota; Bednar, Jan; Nowakowska, Maria

2010-12-02

Interactions between small organic molecules and lipid or cell membranes are important because of their role in the distribution of biologically active substances inside the membrane and their permeation through the cell membranes. In the current paper, we have explored the effect of the attachment of long hydrocarbon tails on the behavior of small organic molecule inside the lipid membrane. Naphthalene with two decyloxy groups attached at the opposite sites of the ring (2,6-bis(decyloxy)naphthalene, 3) was synthesized and incorporated into phosphatidylcholine (PC) vesicles. Fluorescence methods as well as molecular dynamic (MD) simulations were used to estimate the position, orientation, and migration of compound 3 in PC bilayer. It was found that the naphthalene ring of compound 3 resides in the upper acyl chain region of the bilayer and the hydrocarbon tails are directed to the center of the bilayer. As was shown with cryotransmission electron microscopy (cryo-TEM), such lipidlike conformation enables compound 3 to be incorporated into liposomes at a very high content without their disintegration. Moreover, compound 3 can migrate from one leaflet to other. The mechanism of this process is, however, different from that characteristic of the flip-flop event of lipid molecules in the membrane. Finally, the possible application of compound 3 as a rotational molecular probe for monitoring fluidity of liposomal membrane in the acyl side chain region was checked by studies of the effect of cholesterol on the fluorescence anisotropy of 3.

Biomimetic particles for isolation and reconstitution of receptor function.

PubMed

Moura, Sérgio P; Carmona-Ribeiro, Ana M

2006-01-01

Biomimetic particles supporting lipid bilayers are becoming increasingly important to isolate and reconstitute protein function. Cholera toxin (CT) from Vibrio cholerae, an 87-kDa AB5 hexameric protein, and its receptor, the monosialoganglioside GM1, a cell membrane glycolipid, self-assembled on phosphatidylcholine (PC) bilayer-covered silica particles at 1 CT/5 GM1 molar ratio in perfect agreement with literature. This receptor-ligand recognition represented a proof-of-concept that receptors in general can be isolated and their function reconstituted using biomimetic particles, i.e., bilayer-covered silica. After incubation of colloidal silica with small unilamellar PC vesicles in saline solution, pH 7.4, PC adsorption isotherms on silica from inorganic phosphorus analysis showed a high PC affinity for silica with maximal PC adsorption at bilayer deposition. At 0.3 mM PC, fluorescence of pyrene-labeled GM(1) showed that GM(1) incorporation in biomimetic particles increased as a function of particles concentration. At 1 mg/mL silica, receptor incorporation increased to a maximum of 40% at 0.2-0.3 mM PC and then decreased as a function of PC concentration. At 5 microM GM(1), 0.3 mM PC, and 1 mg/mL silica, CT binding increased as a function of CT concentration with a plateau at 2 mg bound CT/m2 silica, which corresponded to the 5 GM(1)/1 CT molar proportion and showed successful reconstitution of receptor-ligand interaction.

Fluid-membrane tethers: minimal surfaces and elastic boundary layers.

PubMed

Powers, Thomas R; Huber, Greg; Goldstein, Raymond E

2002-04-01

Thin cylindrical tethers are common lipid bilayer membrane structures, arising in situations ranging from micromanipulation experiments on artificial vesicles to the dynamic structure of the Golgi apparatus. We study the shape and formation of a tether in terms of the classical soap-film problem, which is applied to the case of a membrane disk under tension subject to a point force. A tether forms from the elastic boundary layer near the point of application of the force, for sufficiently large displacement. Analytic results for various aspects of the membrane shape are given.

Single-vesicle imaging reveals different transport mechanisms between glutamatergic and GABAergic vesicles.

PubMed

Farsi, Zohreh; Preobraschenski, Julia; van den Bogaart, Geert; Riedel, Dietmar; Jahn, Reinhard; Woehler, Andrew

2016-02-26

Synaptic transmission is mediated by the release of neurotransmitters, which involves exo-endocytotic cycling of synaptic vesicles. To maintain synaptic function, synaptic vesicles are refilled with thousands of neurotransmitter molecules within seconds after endocytosis, using the energy provided by an electrochemical proton gradient. However, it is unclear how transmitter molecules carrying different net charges can be efficiently sequestered while maintaining charge neutrality and osmotic balance. We used single-vesicle imaging to monitor pH and electrical gradients and directly showed different uptake mechanisms for glutamate and γ-aminobutyric acid (GABA) operating in parallel. In contrast to glutamate, GABA was exchanged for protons, with no other ions participating in the transport cycle. Thus, only a few components are needed to guarantee reliable vesicle filling with different neurotransmitters. Copyright © 2016, American Association for the Advancement of Science.

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.

Modeling the effect of glutamate diffusion and uptake on NMDA and non-NMDA receptor saturation.

PubMed Central

Holmes, W R

1995-01-01

One- and two-dimensional models of glutamate diffusion, uptake, and binding in the synaptic cleft were developed to determine if the release of single vesicles of glutamate would saturate NMDA and non-NMDA receptors. Ranges of parameter values were used in the simulations to determine the conditions when saturation could occur. Single vesicles of glutamate did not saturate NMDA receptors unless diffusion was very slow and the number of glutamate molecules in a vesicle was large. However, the release of eight vesicles at 400 Hz caused NMDA receptor saturation for all parameter values tested. Glutamate uptake was found to reduce NMDA receptor saturation, but the effect was smaller than that of changes in the diffusion coefficient or in the number of glutamate molecules in a vesicle. Non-NMDA receptors were not saturated unless diffusion was very slow and the number of glutamate molecules in a vesicle was large. The release of eight vesicles at 400 Hz caused significant non-NMDA receptor desensitization. The results suggest that NMDA and non-NMDA receptors are not saturated by single vesicles of glutamate under usual conditions, and that tetanic input, of the type typically used to induce long-term potentiation, will increase calcium influx by increasing receptor binding as well as by reducing voltage-dependent block of NMDA receptors. Images FIGURE 1 PMID:8580317

Ca2+ Dependence of Synaptic Vesicle Endocytosis.

PubMed

Leitz, Jeremy; Kavalali, Ege T

2016-10-01

Ca(2+)-dependent synaptic vesicle recycling is essential for structural homeostasis of synapses and maintenance of neurotransmission. Although, the executive role of intrasynaptic Ca(2+) transients in synaptic vesicle exocytosis is well established, identifying the exact role of Ca(2+) in endocytosis has been difficult. In some studies, Ca(2+) has been suggested as an essential trigger required to initiate synaptic vesicle retrieval, whereas others manipulating synaptic Ca(2+) concentrations reported a modulatory role for Ca(2+) leading to inhibition or acceleration of endocytosis. Molecular studies of synaptic vesicle endocytosis, on the other hand, have consistently focused on the roles of Ca(2+)-calmodulin dependent phosphatase calcineurin and synaptic vesicle protein synaptotagmin as potential Ca(2+) sensors for endocytosis. Most studies probing the role of Ca(2+) in endocytosis have relied on measurements of synaptic vesicle retrieval after strong stimulation. Strong stimulation paradigms elicit fusion and retrieval of multiple synaptic vesicles and therefore can be affected by several factors besides the kinetics and duration of Ca(2+) signals that include the number of exocytosed vesicles and accumulation of released neurotransmitters thus altering fusion and retrieval processes indirectly via retrograde signaling. Studies monitoring single synaptic vesicle endocytosis may help resolve this conundrum as in these settings the impact of Ca(2+) on synaptic fusion probability can be uncoupled from its putative role on synaptic vesicle retrieval. Future experiments using these single vesicle approaches will help dissect the specific role(s) of Ca(2+) and its sensors in synaptic vesicle endocytosis. © The Author(s) 2015.

ER-associated SNAREs and Sey1p mediate nuclear fusion at two distinct steps during yeast mating.

PubMed

Rogers, Jason V; Arlow, Tim; Inkellis, Elizabeth R; Koo, Timothy S; Rose, Mark D

2013-12-01

During yeast mating, two haploid nuclei fuse membranes to form a single diploid nucleus. However, the known proteins required for nuclear fusion are unlikely to function as direct fusogens (i.e., they are unlikely to directly catalyze lipid bilayer fusion) based on their predicted structure and localization. Therefore we screened known fusogens from vesicle trafficking (soluble N-ethylmaleimide-sensitive factor attachment protein receptors [SNAREs]) and homotypic endoplasmic reticulum (ER) fusion (Sey1p) for additional roles in nuclear fusion. Here we demonstrate that the ER-localized SNAREs Sec20p, Ufe1p, Use1p, and Bos1p are required for efficient nuclear fusion. In contrast, Sey1p is required indirectly for nuclear fusion; sey1Δ zygotes accumulate ER at the zone of cell fusion, causing a block in nuclear congression. However, double mutants of Sey1p and Sec20p, Ufe1p, or Use1p, but not Bos1p, display extreme ER morphology defects, worse than either single mutant, suggesting that retrograde SNAREs fuse ER in the absence of Sey1p. Together these data demonstrate that SNAREs mediate nuclear fusion, ER fusion after cell fusion is necessary to complete nuclear congression, and there exists a SNARE-mediated, Sey1p-independent ER fusion pathway.

Charging the quantum capacitance of graphene with a single biological ion channel.

PubMed

Wang, Yung Yu; Pham, Ted D; Zand, Katayoun; Li, Jinfeng; Burke, Peter J

2014-05-27

The interaction of cell and organelle membranes (lipid bilayers) with nanoelectronics can enable new technologies to sense and measure electrophysiology in qualitatively new ways. To date, a variety of sensing devices have been demonstrated to measure membrane currents through macroscopic numbers of ion channels. However, nanoelectronic based sensing of single ion channel currents has been a challenge. Here, we report graphene-based field-effect transistors combined with supported lipid bilayers as a platform for measuring, for the first time, individual ion channel activity. We show that the supported lipid bilayers uniformly coat the single layer graphene surface, acting as a biomimetic barrier that insulates (both electrically and chemically) the graphene from the electrolyte environment. Upon introduction of pore-forming membrane proteins such as alamethicin and gramicidin A, current pulses are observed through the lipid bilayers from the graphene to the electrolyte, which charge the quantum capacitance of the graphene. This approach combines nanotechnology with electrophysiology to demonstrate qualitatively new ways of measuring ion channel currents.

Charging the Quantum Capacitance of Graphene with a Single Biological Ion Channel

PubMed Central

2015-01-01

The interaction of cell and organelle membranes (lipid bilayers) with nanoelectronics can enable new technologies to sense and measure electrophysiology in qualitatively new ways. To date, a variety of sensing devices have been demonstrated to measure membrane currents through macroscopic numbers of ion channels. However, nanoelectronic based sensing of single ion channel currents has been a challenge. Here, we report graphene-based field-effect transistors combined with supported lipid bilayers as a platform for measuring, for the first time, individual ion channel activity. We show that the supported lipid bilayers uniformly coat the single layer graphene surface, acting as a biomimetic barrier that insulates (both electrically and chemically) the graphene from the electrolyte environment. Upon introduction of pore-forming membrane proteins such as alamethicin and gramicidin A, current pulses are observed through the lipid bilayers from the graphene to the electrolyte, which charge the quantum capacitance of the graphene. This approach combines nanotechnology with electrophysiology to demonstrate qualitatively new ways of measuring ion channel currents. PMID:24754625

Formulation of Nanoliposomal Vitamin D3 for Potential Application in Beverage Fortification

PubMed Central

Mohammadi, Maryam; Ghanbarzadeh, Babak; Hamishehkar, Hamed

2014-01-01

Purpose: Vitamin D, a liposoluble vitamin has many benefits on health. Encapsulation of bioactives in lipid-based carrier systems like nanoliposomes preserves their native properties against oxidation over time along with providing its stable aqueous dispersion. Methods: In the current study, vitamin D3 nanoliposomes were prepared using thin-film hydration-sonication method and fully characterized by different instrumental techniques. Results: According to FTIR and DSC results, no interaction was observed between encapsulated nutraceutical and liposome constituents. The particle size and size distribution (Span value) were calculated 82–90 nm and 0.70–0.85, respectively. TEM analysis showed nano sized globular and bilayer vesicles. In all formations, the encapsulation efficiency of vitamin D3 was calculated more than 93%. Addition of cholesterol to lecithin bilayer increased the negative zeta potential from -29 to -43mV. Conclusion: The results of this study concluded that the liposomal nanoparticles may be introduced as a suitable carrier for fortification of beverages with vitamin D3. PMID:25671191

Magnetic liposomes based on nickel ferrite nanoparticles for biomedical applications.

PubMed

Rodrigues, Ana Rita O; Gomes, I T; Almeida, Bernardo G; Araújo, J P; Castanheira, Elisabete M S; Coutinho, Paulo J G

2015-07-21

Nickel ferrite nanoparticles with superparamagnetic behavior at room temperature were synthesized using a coprecipitation method. These magnetic nanoparticles were either covered with a lipid bilayer, forming dry magnetic liposomes (DMLs), or entrapped in liposomes, originating aqueous magnetoliposomes (AMLs). A new and promising method for the synthesis of DMLs is described. The presence of the lipid bilayer in DMLs was confirmed by FRET (Förster Resonance Energy Transfer) measurements between the fluorescent-labeled lipids NBD-C12-HPC (NBD acting as a donor) included in the second lipid layer and rhodamine B-DOPE (acceptor) in the first lipid layer. An average donor-acceptor distance of 3 nm was estimated. Assays of the non-specific interactions of magnetoliposomes with biological membranes (modeled using giant unilamellar vesicles, GUVs) were performed. Membrane fusion between both aqueous and dry magnetoliposomes and GUVs was confirmed by FRET, which is an important result regarding applications of these systems both as hyperthermia agents and antitumor drug nanocarriers.

The Structure of the Mouse Serotonin 5-HT3 Receptor in Lipid Vesicles.

PubMed

Kudryashev, Mikhail; Castaño-Díez, Daniel; Deluz, Cédric; Hassaine, Gherici; Grasso, Luigino; Graf-Meyer, Alexandra; Vogel, Horst; Stahlberg, Henning

2016-01-05

The function of membrane proteins is best understood if their structure in the lipid membrane is known. Here, we determined the structure of the mouse serotonin 5-HT3 receptor inserted in lipid bilayers to a resolution of 12 Å without stabilizing antibodies by cryo electron tomography and subtomogram averaging. The reconstruction reveals protein secondary structure elements in the transmembrane region, the extracellular pore, and the transmembrane channel pathway, showing an overall similarity to the available X-ray model of the truncated 5-HT3 receptor determined in the presence of a stabilizing nanobody. Structural analysis of the 5-HT3 receptor embedded in a lipid bilayer allowed the position of the membrane to be determined. Interactions between the densely packed receptors in lipids were visualized, revealing that the interactions were maintained by the short horizontal helices. In combination with methodological improvements, our approach enables the structural analysis of membrane proteins in response to voltage and ligand gating. Copyright © 2016 Elsevier Ltd. All rights reserved.

Precise detection of pH inside large unilamellar vesicles using membrane-impermeable dendritic porphyrin-based nanoprobes.

PubMed

Leiding, Thom; Górecki, Kamil; Kjellman, Tomas; Vinogradov, Sergei A; Hägerhäll, Cecilia; Arsköld, Sindra Peterson

2009-05-15

Accurate real-time measurements of proton concentration gradients are pivotal to mechanistic studies of proton translocation by membrane-bound enzymes. Here we report a detailed characterization of the pH-sensitive fluorescent nanoprobe Glu(3), which is well suited for pH measurements in microcompartmentalized biological systems. The probe is a polyglutamic porphyrin dendrimer in which multiple carboxylate termini ensure its high water solubility and prevent its diffusion across phospholipid membranes. The probe's pK is in the physiological pH range, and its protonation can be followed ratiometrically by absorbance or fluorescence in the ultraviolet-visible spectral region. The usefulness of the probe was enhanced by using a semiautomatic titration system coupled to a charge-coupled device (CCD) spectrometer, enabling fast and accurate titrations and full spectral coverage of the system at millisecond time resolution. The probe's pK was measured in bulk solutions as well as inside large unilamellar vesicles in the presence of physiologically relevant ions. Glu(3) was found to be completely membrane impermeable, and its distinct spectroscopic features permit pH measurements inside closed membrane vesicles, enabling quantitative mechanistic studies of membrane-spanning proteins. Performance of the probe was demonstrated by monitoring the rate of proton leakage through the phospholipid bilayer in large vesicles with and without the uncoupler gramicidin present. Overall, as a probe for biological proton translocation measurements, Glu(3) was found to be superior to the commercially available pH indicators.

The dissimilar effect of diacylglycerols on Ca(2+)-induced phosphatidylserine vesicle fusion.

PubMed Central

Sánchez-Migallón, M P; Aranda, F J; Gómez-Fernández, J C

1995-01-01

We have studied the effect of physiological concentrations of different diacylglycerols on Ca(2+)-induced fusion between phosphatidylserine vesicles. We monitored vesicle fusion as mixing of membrane lipids under conditions where the limiting factor was the aggregation and also in conditions where this aggregation was not the limiting factor. We found that diacylglycerols have a different modulating effect on the Ca(2+)-induced fusion: i) depending on their interfacial conformation, so that 1,2-isomers of diacylglycerols containing unsaturated or short saturated acyl chains stimulated fusion and their 1,3-isomers did not, and ii) depending on their specific type of bilayer interior perturbation, so that diacylglycerols containing unsaturated or short chain saturated acyl chains stimulated fusion but those containing long-chain saturated acyl chains did not. These requirements resembled those required for the diacylglycerol activation of protein kinase C, suggesting that diacylglycerol acts in both the specific activation of this enzyme and the induction of membrane fusion through the same perturbation of lipid structure. We found that polylysine affected the stimulatory role of 1,2-dioleoylglycerol differently, depending on whether aggregation was the limiting factor of fusion. When we studied the effect of very low concentrations of diacylglycerols on the bulk structural properties of phosphatidylserine, we found that they neither significantly perturbed the thermotropic transitions of phosphatidylserine nor affected the interaction of Ca2+ with the phosphate group of phosphatidylserine. The underlying mechanism of fusion between phosphatidylserine vesicles is discussed. PMID:7696508

Characterization of phospholipids in insulin secretory granules and mitochondria in pancreatic beta cells and their changes with glucose stimulation.

PubMed

MacDonald, Michael J; Ade, Lacmbouh; Ntambi, James M; Ansari, Israr-Ul H; Stoker, Scott W

2015-04-24

The lipid composition of insulin secretory granules (ISG) has never previously been thoroughly characterized. We characterized the phospholipid composition of ISG and mitochondria in pancreatic beta cells without and with glucose stimulation. The phospholipid/protein ratios of most phospholipids containing unsaturated fatty acids were higher in ISG than in whole cells and in mitochondria. The concentrations of negatively charged phospholipids, phosphatidylserine, and phosphatidylinositol in ISG were 5-fold higher than in the whole cell. In ISG phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin, fatty acids 12:0 and 14:0 were high, as were phosphatidylserine and phosphatidylinositol containing 18-carbon unsaturated FA. With glucose stimulation, the concentration of many ISG phosphatidylserines and phosphatidylinositols increased; unsaturated fatty acids in phosphatidylserine increased; and most phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophosphatidylcholines were unchanged. Unsaturation and shorter fatty acid length in phospholipids facilitate curvature and fluidity of membranes, which favors fusion of membranes. Recent evidence suggests that negatively charged phospholipids, such as phosphatidylserine, act as coupling factors enhancing the interaction of positively charged regions in SNARE proteins in synaptic or secretory vesicle membrane lipid bilayers with positively charged regions in SNARE proteins in the plasma membrane lipid bilayer to facilitate docking of vesicles to the plasma membrane during exocytosis. The results indicate that ISG phospholipids are in a dynamic state and are consistent with the idea that changes in ISG phospholipids facilitate fusion of ISG with the plasma membrane-enhancing glucose-stimulated insulin exocytosis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Sodium selective ion channel formation in living cell membranes by polyamidoamine dendrimer.

PubMed

Nyitrai, Gabriella; Keszthelyi, Tamás; Bóta, Attila; Simon, Agnes; Tőke, Orsolya; Horváth, Gergő; Pál, Ildikó; Kardos, Julianna; Héja, László

2013-08-01

Polyamidoamine (PAMAM) dendrimers are highly charged hyperbranched protein-like polymers that are known to interact with cell membranes. In order to disclose the mechanisms of dendrimer-membrane interaction, we monitored the effect of PAMAM generation five (G5) dendrimer on the membrane permeability of living neuronal cells followed by exploring the underlying structural changes with infrared-visible sum frequency vibrational spectroscopy (SVFS), small angle X-ray scattering (SAXS) and transmission electron microscopy (TEM). G5 dendrimers were demonstrated to irreversibly increase the membrane permeability of neurons that could be blocked in low-[Na(+)], but not in low-[Ca(2+)] media suggesting the formation of specific Na(+) permeable channels. SFVS measurements on silica supported DPPG-DPPC bilayers suggested G5-specific trans-polarization of the membrane. SAXS data and freeze-fracture TEM imaging of self-organized DPPC vesicle systems demonstrated disruption of DPPC vesicle layers by G5 through polar interactions between G5 terminal amino groups and the anionic head groups of DPPC. We propose a nanoscale mechanism by which G5 incorporates into the membrane through multiple polar interactions that disrupt proximate membrane bilayer and shape a unique hydrophilic Na(+) ion permeable channel around the dendrimer. In addition, we tested whether these artificial Na(+) channels can be exploited as antibiotic tools. We showed that G5 quickly arrest the growth of resistant bacterial strains below 10μg/ml concentration, while they show no detrimental effect on red blood cell viability, offering the chance for the development of new generation anti-resistant antibiotics. Copyright © 2013 Elsevier B.V. All rights reserved.

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.

Structure and Membrane Interactions of the Antibiotic Peptide Dermadistinctin K by Multidimensional Solution and Oriented 15N and 31P Solid-State NMR Spectroscopy

PubMed Central

Verly, Rodrigo M.; Moraes, Cléria Mendonça de; Resende, Jarbas M.; Aisenbrey, Christopher; Bemquerer, Marcelo Porto; Piló-Veloso, Dorila; Valente, Ana Paula; Almeida, Fábio C.L.; Bechinger, Burkhard

2009-01-01

DD K, a peptide first isolated from the skin secretion of the Phyllomedusa distincta frog, has been prepared by solid-phase chemical peptide synthesis and its conformation was studied in trifluoroethanol/water as well as in the presence of sodium dodecyl sulfate and dodecylphosphocholine micelles or small unilamellar vesicles. Multidimensional solution NMR spectroscopy indicates an α-helical conformation in membrane environments starting at residue 7 and extending to the C-terminal carboxyamide. Furthermore, DD K has been labeled with 15N at a single alanine position that is located within the helical core region of the sequence. When reconstituted into oriented phosphatidylcholine membranes the resulting 15N solid-state NMR spectrum shows a well-defined helix alignment parallel to the membrane surface in excellent agreement with the amphipathic character of DD K. Proton-decoupled 31P solid-state NMR spectroscopy indicates that the peptide creates a high level of disorder at the level of the phospholipid headgroup suggesting that DD K partitions into the bilayer where it severely disrupts membrane packing. PMID:19289046

Spontaneous formation of nanometer scale tubular vesicles in aqueous mixtures of lipid and block copolymer amphiphiles.

PubMed

Lim, Seng Koon; Wong, Andrew S W; de Hoog, Hans-Peter M; Rangamani, Padmini; Parikh, Atul N; Nallani, Madhavan; Sandin, Sara; Liedberg, Bo

2017-02-08

Many common amphiphiles self-assemble in water to produce heterogeneous populations of discrete and symmetric but polydisperse and multilamellar vesicles isolating the encapsulated aqueous core from the surrounding bulk. But when mixtures of amphiphiles of vastly different elastic properties co-assemble, their non-uniform molecular organization can stabilize lower symmetries and produce novel shapes. Here, using high resolution electron cryomicroscopy and tomography, we identify the spontaneous formation of a membrane morphology consisting of unilamellar tubular vesicles in dilute aqueous solutions of binary mixtures of two different amphiphiles of vastly different origins. Our results show that aqueous phase mixtures of a fluid-phase phospholipid and an amphiphilic block copolymer spontaneously assume a bimodal polymorphic character in a composition dependent manner: over a broad range of compositions (15-85 mol% polymer component), a tubular morphology co-exists with spherical vesicles. Strikingly, in the vicinity of equimolar compositions, an exclusively tubular morphology (L t ; diameter, ∼15 nm; length, >1 μm; core, ∼2.0 nm; wall, ∼5-6 nm) emerges in an apparent steady state. Theory suggests that the spontaneous stabilization of cylindrical vesicles, unaided by extraneous forces, requires a significant spontaneous bilayer curvature, which in turn necessitates a strongly asymmetric membrane composition. We confirm that such dramatic compositional asymmetry is indeed produced spontaneously in aqueous mixtures of a lipid and polymer through two independent biochemical assays - (1) reduction in the quenching of fluorophore-labeled lipids and (2) inhibition in the activity of externally added lipid-hydrolyzing phospholipase A2, resulting in a significant enrichment of the polymer component in the outer leaflet. Taken together, these results illustrate the coupling of the membrane shape with local composition through spontaneous curvature generation under conditions of asymmetric distribution of mixtures of disparate amphiphiles.

Enhancing the Therapeutic Efficacy of Tamoxifen Citrate Loaded Span-Based Nano-Vesicles on Human Breast Adenocarcinoma Cells.

PubMed

Kassem, Mohammed A; Megahed, Mohamed A; Abu Elyazid, Sherif K; Abd-Allah, Fathy I; Abdelghany, Tamer M; Al-Abd, Ahmed M; El-Say, Khalid M

2018-05-01

Serious adverse effects and low selectivity to cancer cells are the main obstacles of long term therapy with Tamoxifen (Tmx). This study aimed to develop Tmx-loaded span-based nano-vesicles for delivery to malignant tissues with maximum efficacy. The effect of three variables on vesicle size (Y 1 ), zeta potential (Y 2 ), entrapment efficiency (Y 3 ) and the cumulative percent release after 24 h (Y 4 ) were optimized using Box-Behnken design. The optimized formula was prepared and tested for its stability in different storage conditions. The observed values for the optimized formula were 310.2 nm, - 42.09 mV, 75.45 and 71.70% for Y 1 , Y 2 , Y 3 , and Y 4 , respectively. The examination using electron microscopy confirmed the formation of rounded vesicles with distinctive bilayer structure. Moreover, the cytotoxic activity of the optimized formula on both breast cancer cells (MCF-7) and normal cells (BHK) showed enhanced selectivity (9.4 folds) on cancerous cells with IC 50 values 4.7 ± 1.5 and 44.3 ± 1.3 μg/ml on cancer and normal cells, respectively. While, free Tmx exhibited lower selectivity (2.5 folds) than optimized nano-vesicles on cancer cells with IC 50 values of 9.0 ± 1.1 μg/ml and 22.5 ± 5.3 μg/ml on MCF-7 and BHK cells, respectively. The promising prepared vesicular system, with greater efficacy and selectivity, provides a marvelous tool to overcome breast cancer treatment challenges.

Influence of bilayer resist processing on p-i-n OLEDs: towards multicolor photolithographic structuring of organic displays

NASA Astrophysics Data System (ADS)

Krotkus, Simonas; Nehm, Frederik; Janneck, Robby; Kalkura, Shrujan; Zakhidov, Alex A.; Schober, Matthias; Hild, Olaf R.; Kasemann, Daniel; Hofmann, Simone; Leo, Karl; Reineke, Sebastian

2015-03-01

Recently, bilayer resist processing combined with development in hydrofluoroether (HFE) solvents has been shown to enable single color structuring of vacuum-deposited state-of-the-art organic light-emitting diodes (OLED). In this work, we focus on further steps required to achieve multicolor structuring of p-i-n OLEDs using a bilayer resist approach. We show that the green phosphorescent OLED stack is undamaged after lift-off in HFEs, which is a necessary step in order to achieve RGB pixel array structured by means of photolithography. Furthermore, we investigate the influence of both, double resist processing on red OLEDs and exposure of the devices to ambient conditions, on the basis of the electrical, optical and lifetime parameters of the devices. Additionally, water vapor transmission rates of single and bilayer system are evaluated with thin Ca film conductance test. We conclude that diffusion of propylene glycol methyl ether acetate (PGMEA) through the fluoropolymer film is the main mechanism behind OLED degradation observed after bilayer processing.

Cell-mediated lysis of lipid vesicles containing eye muscle protein: Implications regarding pathogenesis of Graves ophthalmopathy*

PubMed Central

Kriss, Joseph P.; Mehdi, S. Qasim

1979-01-01

We prepared artificial vesicles that are lysed upon cell-mediated immunological attack by human lymphocytes. These vesicles are made from a mixture of dimyristoyl lecithin, dipalmitoyl lecithin, and cholesterol, have eye muscle membrane protein (EMP) inserted into the bilayer wall, and contain intravesicular 99mTc marker. Injury to the vesicular membrane was assessed by measurement of 99mTc release. Thyroglobulin (Tg) and Tg-anti-Tg complex (TgA) bind to EMP-vesicles to an extent equal to or greater than to native eye muscle membranes in vitro; this binding requires the presence of normal human IgG. The role of Tg, TgA, IgG, and peripheral blood lymphocytes in altering membrane permeability was analyzed. Incubation of vesicles for up to 3 hr alone, with added IgG alone, or with further addition of Tg or TgA did not result in 99mTc release. Addition of lymphocytes from normal donors to the above four preparations showed release in the presence of TgA. Lymphocytes from each of eight patients with Graves ophthalmopathy caused release not only in the presence of TgA, but also in the presence of Tg. Separation of a patient's lymphocytes into high- and low-affinity rosette-formers (T and K cells, respectively) showed that cell-mediated vesicle lysis in the presence of TgA was greater with K cells than with T cells, while vesicle lysis in the presence of Tg was greater with T cells than with K cells. Vesicles made with inserted Tg but lacking EMP were not lysed by such T cells. Lymphocytes failed to induce permeability changes in vesicles containing other inserted proteins obtained from human nonextraocular muscle, liver, spleen, or adrenal, even if Tg or TgA were present. The results support the concept that muscle cell damage in Graves ophthalmopathy is immunological, cell-mediated, and of two types: (i) K lymphocytes reacting to immune complex, TgA, on the eye muscle cell surface (i.e., antibody-dependent cytotoxicity) and (ii) sensitized T lymphocytes reacting to Tg on the eye muscle cell surface. An antigenic role for EMP is possible, but has not been unequivocally proven. PMID:88053

Transparent Nanopore Cavity Arrays Enable Highly Parallelized Optical Studies of Single Membrane Proteins on Chip.

PubMed

Diederichs, Tim; Nguyen, Quoc Hung; Urban, Michael; Tampé, Robert; Tornow, Marc

2018-06-13

Membrane proteins involved in transport processes are key targets for pharmaceutical research and industry. Despite continuous improvements and new developments in the field of electrical readouts for the analysis of transport kinetics, a well-suited methodology for high-throughput characterization of single transporters with nonionic substrates and slow turnover rates is still lacking. Here, we report on a novel architecture of silicon chips with embedded nanopore microcavities, based on a silicon-on-insulator technology for high-throughput optical readouts. Arrays containing more than 14 000 inverted-pyramidal cavities of 50 femtoliter volumes and 80 nm circular pore openings were constructed via high-resolution electron-beam lithography in combination with reactive ion etching and anisotropic wet etching. These cavities feature both, an optically transparent bottom and top cap. Atomic force microscopy analysis reveals an overall extremely smooth chip surface, particularly in the vicinity of the nanopores, which exhibits well-defined edges. Our unprecedented transparent chip design provides parallel and independent fluorescent readout of both cavities and buffer reservoir for unbiased single-transporter recordings. Spreading of large unilamellar vesicles with efficiencies up to 96% created nanopore-supported lipid bilayers, which are stable for more than 1 day. A high lipid mobility in the supported membrane was determined by fluorescent recovery after photobleaching. Flux kinetics of α-hemolysin were characterized at single-pore resolution with a rate constant of 0.96 ± 0.06 × 10 -3 s -1 . Here, we deliver an ideal chip platform for pharmaceutical research, which features high parallelism and throughput, synergistically combined with single-transporter resolution.

Primary vesicles, vesicle-rich segregation structures and recognition of primary and secondary porosities in lava flows from the Paraná igneous province, southern Brazil

NASA Astrophysics Data System (ADS)

Barreto, Carla Joana S.; de Lima, Evandro F.; Goldberg, Karin

2017-04-01

This study focuses on a volcanic succession of pāhoehoe to rubbly lavas of the Paraná-Etendeka Province exposed in a single road profile in southernmost Brazil. This work provides an integrated approach for examining primary vesicles and vesicle-rich segregation structures at the mesoscopic scale. In addition, this study provides a quantitative analysis of pore types in thin section. We documented distinct distribution patterns of vesicle and vesicle-rich segregation structures according to lava thickness. In compound pāhoehoe lavas, the cooling allows only vesicles (<1 cm size) and pipe vesicles to be frozen into place. In inflated pāhoehoe lavas, vesicles of different sizes are common, including pipe vesicles, and also segregation structures such as proto-cylinders, cylinders, cylinder sheets, vesicle sheets, and pods. In rubbly lavas, only vesicles of varying sizes occur. Gas release from melt caused the formation of primary porosity, while hydrothermal alteration and tectonic fracturing are the main processes that generated secondary porosity. Although several forms of porosity were created in the basaltic lava flows, the precipitation of secondary minerals within the pores has tended to reduce the original porosities. Late-stage fractures could create efficient channel networks for possible hydrocarbon/groundwater migration and entrapment owing to their ability to connect single pores. Quantitative permeability data should be gathered in future studies to confirm the potential of these lavas for store hydrocarbons or groundwater.

Hydrodynamic mobility of confined polymeric particles, vesicles, and cancer cells in a square microchannel.

PubMed

Ahmmed, Shamim M; Suteria, Naureen S; Garbin, Valeria; Vanapalli, Siva A

2018-01-01

The transport of deformable objects, including polymer particles, vesicles, and cells, has been a subject of interest for several decades where the majority of experimental and theoretical studies have been focused on circular tubes. Due to advances in microfluidics, there is a need to study the transport of individual deformable particles in rectangular microchannels where corner flows can be important. In this study, we report measurements of hydrodynamic mobility of confined polymeric particles, vesicles, and cancer cells in a linear microchannel with a square cross-section. Our operating conditions are such that the mobility is measured as a function of geometric confinement over the range 0.3 <  λ  < 1.5 and at specified particle Reynolds numbers that are within 0.1 < Re p  < 2.5. The experimental mobility data of each of these systems is compared with the circular-tube theory of Hestroni, Haber, and Wacholder [J. Fluid Mech. 41 , 689-705 (1970)] with modifications made for a square cross-section. For polymeric particles, we find that the mobility data agrees well over a large confinement range with the theory but under predicts for vesicles. The mobility of vesicles is higher in a square channel than in a circular tube, and does not depend significantly on membrane mechanical properties. The mobility of cancer cells is in good agreement with the theory up to λ ≈ 0.8, after which it deviates. Comparison of the mobility data of the three systems reveals that cancer cells have higher mobility than rigid particles but lower than vesicles, suggesting that the cell membrane frictional properties are in between a solid-like interface and a fluid bilayer. We explain further the differences in the mobility of the three systems by considering their shape deformation and surface flow on the interface. The results of this study may find potential applications in drug delivery and biomedical diagnostics.

Substrate Efflux Propensity Is the Key Determinant of Ca2+-independent Phospholipase A-β (iPLAβ)-mediated Glycerophospholipid Hydrolysis*

PubMed Central

Batchu, Krishna Chaithanya; Hokynar, Kati; Jeltsch, Michael; Mattonet, Kenny; Somerharju, Pentti

2015-01-01

The A-type phospholipases (PLAs) are key players in glycerophospholipid (GPL) homeostasis and in mammalian cells; Ca2+-independent PLA-β (iPLAβ) in particular has been implicated in this essential process. However, the regulation of this enzyme, which is necessary to avoid futile competition between synthesis and degradation, is not understood. Recently, we provided evidence that the efflux of the substrate molecules from the bilayer is the rate-limiting step in the hydrolysis of GPLs by some secretory (nonhomeostatic) PLAs. To study whether this is the case with iPLAβ as well, a mass spectrometric assay was employed to determine the rate of hydrolysis of multiple saturated and unsaturated GPL species in parallel using micelles or vesicle bilayers as the macrosubstrate. With micelles, the hydrolysis decreased with increasing acyl chain length independent of unsaturation, and modest discrimination between acyl positional isomers was observed, presumably due to the differences in the structure of the sn-1 and sn-2 acyl-binding sites of the protein. In striking contrast, no significant discrimination between positional isomers was observed with bilayers, and the rate of hydrolysis decreased with the acyl chain length logarithmically and far more than with micelles. These data provide compelling evidence that efflux of the substrate molecule from the bilayer, which also decreases monotonously with acyl chain length, is the rate-determining step in iPLAβ-mediated hydrolysis of GPLs in membranes. This finding is intriguing as it may help to understand how homeostatic PLAs are regulated and how degradation and biosynthesis are coordinated. PMID:25713085

The complex nature of calcium cation interactions with phospholipid bilayers

PubMed Central

Melcrová, Adéla; Pokorna, Sarka; Pullanchery, Saranya; Kohagen, Miriam; Jurkiewicz, Piotr; Hof, Martin; Jungwirth, Pavel; Cremer, Paul S.; Cwiklik, Lukasz

2016-01-01

Understanding interactions of calcium with lipid membranes at the molecular level is of great importance in light of their involvement in calcium signaling, association of proteins with cellular membranes, and membrane fusion. We quantify these interactions in detail by employing a combination of spectroscopic methods with atomistic molecular dynamics simulations. Namely, time-resolved fluorescent spectroscopy of lipid vesicles and vibrational sum frequency spectroscopy of lipid monolayers are used to characterize local binding sites of calcium in zwitterionic and anionic model lipid assemblies, while dynamic light scattering and zeta potential measurements are employed for macroscopic characterization of lipid vesicles in calcium-containing environments. To gain additional atomic-level information, the experiments are complemented by molecular simulations that utilize an accurate force field for calcium ions with scaled charges effectively accounting for electronic polarization effects. We demonstrate that lipid membranes have substantial calcium-binding capacity, with several types of binding sites present. Significantly, the binding mode depends on calcium concentration with important implications for calcium buffering, synaptic plasticity, and protein-membrane association. PMID:27905555

Stochastic simulations of fatty-acid proto-cell models

NASA Astrophysics Data System (ADS)

Mavelli, F.; Ruiz-Mirazo, K.

2007-06-01

In this contribution we tackle the problem of simulating the time behavior of self-assembling fatty acid vesicles in different experimental conditions. These systems have been (and are being) explored by various labs as possible precursor models of cellular compartments. By means of our recently developed stochastic simulation platform ('ENVIRONMENT') we are able to reproduce quite satisfactorily experimental data that have been reported on the different growth behavior of this type of proto-cellular systems, depending on the level of osmotic pressure they are under. The work here presented is part of a more general attempt to gain insight into the problem of how self-assembling vesicles (closed bilayer structures) could progressively turn into minimal self-producing and self-reproducing cells: i.e., into interesting candidates for (proto-)biological systems. This involves crossing the traditional gap between in silico and in vitro approaches, as we try to do here, convinced that major adavances in the field require the correct integration of both theoretical and experimental endeavors.

Rupturing Giant Plasma Membrane Vesicles to Form Micron-sized Supported Cell Plasma Membranes with Native Transmembrane Proteins.

PubMed

Chiang, Po-Chieh; Tanady, Kevin; Huang, Ling-Ting; Chao, Ling

2017-11-09

Being able to directly obtain micron-sized cell blebs, giant plasma membrane vesicles (GPMVs), with native membrane proteins and deposit them on a planar support to form supported plasma membranes could allow the membrane proteins to be studied by various surface analytical tools in native-like bilayer environments. However, GPMVs do not easily rupture on conventional supports because of their high protein and cholesterol contents. Here, we demonstrate the possibility of using compression generated by the air-water interface to efficiently rupture GPMVs to form micron-sized supported membranes with native plasma membrane proteins. We demonstrated that not only lipid but also a native transmembrane protein in HeLa cells, Aquaporin 3 (AQP3), is mobile in the supported membrane platform. This convenient method for generating micron-sized supported membrane patches with mobile native transmembrane proteins could not only facilitate the study of membrane proteins by surface analytical tools, but could also enable us to use native membrane proteins for bio-sensing applications.

Interactions between charged nanoparticles and giant vesicles fabricated from inverted-headgroup lipids

NASA Astrophysics Data System (ADS)

Wang, Lu; Malmstadt, Noah

2017-10-01

The surface chemistry of the cell membrane plays an important role in how cells interact with particulate species. These interactions are dictated in large part by lipid headgroup charge. To investigate the nature of electrostatic interactions between lipid bilayers and nanoparticles in solution, we studied nanoparticles interacting with the zwitterionic lipid 1,2-dioleoyl-glycero-3-phosphocholine (DOPC), and its inverted-headgroup analog DOCP. These interactions were investigated by fabricating giant unilamellar vesicles (GUVs) with DOPC lipids and DOCP lipids respectively, and introducing nanoparticles to suspensions of both. GUVs displayed various deformational modes depending on the charge and size of the nanoparticles as well as the compositions of the GUVs. The differences in the responses of the two lipid species illuminate how the phosphate and choline groups on the lipid interact with charged nanoparticles. This study suggests that the phosphate group dominates the lipid-nanoparticle electrostatic interaction. We speculate that the formation of water clathrate structures around the choline group inhibits interactions between negatively charged nanoparticles and the positively charged choline.

A Model for Membrane Fusion

NASA Astrophysics Data System (ADS)

Ngatchou, Annita

2010-01-01

Pheochromocytoma is a tumor of the adrenal gland which originates from chromaffin cells and is characterized by the secretion of excessive amounts of neurotransmitter which lead to high blood pressure and palpitations. Pheochromocytoma contain membrane bound granules that store neurotransmitter. The release of these stored molecules into the extracellular space occurs by fusion of the granule membrane with the cell plasma membrane, a process called exocytosis. The molecular mechanism of this membrane fusion is not well understood. It is proposed that the so called SNARE proteins [1] are the pillar of vesicle fusion as their cleavage by clostridial toxin notably, Botulinum neurotoxin and Tetanus toxin abrogate the secretion of neurotransmitter [2]. Here, I describe how physical principles are applied to a biological cell to explore the role of the vesicle SNARE protein synaptobrevin-2 in easing granule fusion. The data presented here suggest a paradigm according to which the movement of the C-terminal of synaptobrevin-2 disrupts the lipid bilayer to form a fusion pore through which molecules can exit.

EVpedia: a community web portal for extracellular vesicles research.

PubMed

Kim, Dae-Kyum; Lee, Jaewook; Kim, Sae Rom; Choi, Dong-Sic; Yoon, Yae Jin; Kim, Ji Hyun; Go, Gyeongyun; Nhung, Dinh; Hong, Kahye; Jang, Su Chul; Kim, Si-Hyun; Park, Kyong-Su; Kim, Oh Youn; Park, Hyun Taek; Seo, Ji Hye; Aikawa, Elena; Baj-Krzyworzeka, Monika; van Balkom, Bas W M; Belting, Mattias; Blanc, Lionel; Bond, Vincent; Bongiovanni, Antonella; Borràs, Francesc E; Buée, Luc; Buzás, Edit I; Cheng, Lesley; Clayton, Aled; Cocucci, Emanuele; Dela Cruz, Charles S; Desiderio, Dominic M; Di Vizio, Dolores; Ekström, Karin; Falcon-Perez, Juan M; Gardiner, Chris; Giebel, Bernd; Greening, David W; Gross, Julia Christina; Gupta, Dwijendra; Hendrix, An; Hill, Andrew F; Hill, Michelle M; Nolte-'t Hoen, Esther; Hwang, Do Won; Inal, Jameel; Jagannadham, Medicharla V; Jayachandran, Muthuvel; Jee, Young-Koo; Jørgensen, Malene; Kim, Kwang Pyo; Kim, Yoon-Keun; Kislinger, Thomas; Lässer, Cecilia; Lee, Dong Soo; Lee, Hakmo; van Leeuwen, Johannes; Lener, Thomas; Liu, Ming-Lin; Lötvall, Jan; Marcilla, Antonio; Mathivanan, Suresh; Möller, Andreas; Morhayim, Jess; Mullier, François; Nazarenko, Irina; Nieuwland, Rienk; Nunes, Diana N; Pang, Ken; Park, Jaesung; Patel, Tushar; Pocsfalvi, Gabriella; Del Portillo, Hernando; Putz, Ulrich; Ramirez, Marcel I; Rodrigues, Marcio L; Roh, Tae-Young; Royo, Felix; Sahoo, Susmita; Schiffelers, Raymond; Sharma, Shivani; Siljander, Pia; Simpson, Richard J; Soekmadji, Carolina; Stahl, Philip; Stensballe, Allan; Stępień, Ewa; Tahara, Hidetoshi; Trummer, Arne; Valadi, Hadi; Vella, Laura J; Wai, Sun Nyunt; Witwer, Kenneth; Yáñez-Mó, María; Youn, Hyewon; Zeidler, Reinhard; Gho, Yong Song

2015-03-15

Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging. We present an improved version of EVpedia, a public database for EVs research. This community web portal contains a database of publications and vesicular components, identification of orthologous vesicular components, bioinformatic tools and a personalized function. EVpedia includes 6879 publications, 172 080 vesicular components from 263 high-throughput datasets, and has been accessed more than 65 000 times from more than 750 cities. In addition, about 350 members from 73 international research groups have participated in developing EVpedia. This free web-based database might serve as a useful resource to stimulate the emerging field of EV research. The web site was implemented in PHP, Java, MySQL and Apache, and is freely available at http://evpedia.info. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

The complex nature of calcium cation interactions with phospholipid bilayers

NASA Astrophysics Data System (ADS)

Melcrová, Adéla; Pokorna, Sarka; Pullanchery, Saranya; Kohagen, Miriam; Jurkiewicz, Piotr; Hof, Martin; Jungwirth, Pavel; Cremer, Paul S.; Cwiklik, Lukasz

2016-12-01

Understanding interactions of calcium with lipid membranes at the molecular level is of great importance in light of their involvement in calcium signaling, association of proteins with cellular membranes, and membrane fusion. We quantify these interactions in detail by employing a combination of spectroscopic methods with atomistic molecular dynamics simulations. Namely, time-resolved fluorescent spectroscopy of lipid vesicles and vibrational sum frequency spectroscopy of lipid monolayers are used to characterize local binding sites of calcium in zwitterionic and anionic model lipid assemblies, while dynamic light scattering and zeta potential measurements are employed for macroscopic characterization of lipid vesicles in calcium-containing environments. To gain additional atomic-level information, the experiments are complemented by molecular simulations that utilize an accurate force field for calcium ions with scaled charges effectively accounting for electronic polarization effects. We demonstrate that lipid membranes have substantial calcium-binding capacity, with several types of binding sites present. Significantly, the binding mode depends on calcium concentration with important implications for calcium buffering, synaptic plasticity, and protein-membrane association.

Liposomes entrapping β-cyclodextrin/ibuprofen inclusion complex: Role of the host and the guest on the bilayer integrity and microviscosity.

PubMed

Angelini, Guido; Campestre, Cristina; Boncompagni, Simona; Gasbarri, Carla

2017-12-01

Multilamellar vesicles (MLVs) from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were prepared by using the dehydration-rehydration method. The β-cyclodextrin/Ibuprofen inclusion complex (β-CD/Ibu) was formed and solubilised into the aqueous compartments of the investigated vesicles. The resulting POPC MLVs entrapping β-CD/Ibu complex were essentially homogeneous in shape as demonstrated by Transmission Electron Microscopy (TEM). The liposomal stability was determined at 37.0±0.1°C by following the outflux rate of 5(6)-carboxyfluorescein (CF) at pH 7.40, while the membrane microviscosity was estimated by the ratio of the fluorescence intensities of pyrene in excimer and monomer state. The results presented herein confirm that interactions between POPC and β-CD occur and suggest that associations between POPC and Ibuprofen are also involved in the properties of the investigated liposomes. Copyright © 2017 Elsevier B.V. All rights reserved.

Environmental Scanning Electron Microscope Imaging of Vesicle Systems.

PubMed

Perrie, Yvonne; Ali, Habib; Kirby, Daniel J; Mohammed, Afzal U R; McNeil, Sarah E; Vangala, Anil

2017-01-01

The structural characteristics of liposomes have been widely investigated and there is certainly a strong understanding of their morphological characteristics. Imaging of these systems, using techniques such as freeze-fracturing methods, transmission electron microscopy, and cryo-electron imaging, has allowed us to appreciate their bilayer structures and factors which can influence this. However, there are few methods which all us to study these systems in their natural hydrated state; commonly the liposomes are visualized after drying, staining, and/or fixation of the vesicles. Environmental Scanning Electron Microscopy (ESEM) offers the ability to image a liposome in its hydrated state without the need for prior sample preparation. Within our studies we were the first to use ESEM to study liposomes and niosomes and we have been able to dynamically follow the hydration of lipid films and changes in liposome suspensions as water condenses on to, or evaporates from, the sample in real time. This provides insight into the resistance of liposomes to coalescence during dehydration, thereby providing an alternative assay of liposome formulation and stability.

Direct simulation of amphiphilic nanoparticle mediated membrane interactions

NASA Astrophysics Data System (ADS)

Tahir, Mukarram; Alexander-Katz, Alfredo

Membrane fusion is a critical step in the transport of biological cargo through membrane-bound compartments like vesicles. Membrane proteins that alleviate energy barriers for initial stalk formation and eventual rupture of the hemifusion intermediate during fusion generally assist this process. Gold nanoparticles functionalized with a combination of hydrophobic and hydrophilic alkanethiol ligands have recently been shown to induce membrane re-arrangements that are similar to those associated with these fusion proteins. In this work, we utilize molecular dynamics simulation to systematically design nanoparticles that exhibit targeted interactions with membranes. We introduce a method for rapidly parameterizing nanoparticle topology for the MARTINI biomolecular force field to permit long timescale simulation of their interactions with lipid bilayers. We leverage this model to investigate how ligand chemistry governs the nanoparticle's insertion efficacy and the perturbations it generates in the membrane environment. We further demonstrate through unbiased simulations that these nanoparticles can direct the fusion of lipid assemblies such as micelles and vesicles in a manner that mimics the function of biological fusion peptides and SNARE proteins.

Insights into the Diagnostic Potential of Extracellular Vesicles and Their miRNA Signature from Liquid Biopsy as Early Biomarkers of Diabetic Micro/Macrovascular Complications.

PubMed

La Marca, Valeria; Fierabracci, Alessandra

2017-09-14

Extracellular vesicles (EVs) represent a heterogeneous population of small vesicles, consisting of a phospholipidic bilayer surrounding a soluble interior cargo. Almost all cell types release EVs, thus they are naturally present in all body fluids. Among the several potential applications, EVs could be used as drug delivery vehicles in disease treatment, in immune therapy because of their immunomodulatory properties and in regenerative medicine. In addition to general markers, EVs are characterized by the presence of specific biomarkers (proteins and miRNAs) that allow the identification of their cell or tissue origin. For these features, they represent a potential powerful diagnostic tool to monitor state and progression of specific diseases. A large body of studies supports the idea that endothelial derived (EMPs) together with platelet-derived microparticles (PMPs) are deeply involved in the pathogenesis of diseases characterized by micro- and macrovascular damages, including diabetes. Existing literature suggests that the detection of circulating EMPs and PMPs and their specific miRNA profile may represent a very useful non-invasive signature to achieve information on the onset of peculiar disease manifestations. In this review, we discuss the possible utility of EVs in the early diagnosis of diabetes-associated microvascular complications, specifically related to kidney.

Lowering Low-Density Lipoprotein Particles in Plasma Using Dextran Sulphate Co-Precipitates Procoagulant Extracellular Vesicles.

PubMed

Wang, Jiong-Wei; Zhang, Ya-Nan; Sze, Siu Kwan; van de Weg, Sander M; Vernooij, Flora; Schoneveld, Arjan H; Tan, Sock-Hwee; Versteeg, Henri H; Timmers, Leo; Lam, Carolyn S P; de Kleijn, Dominique P V

2017-12-29

Plasma extracellular vesicles (EVs) are lipid membrane vesicles involved in several biological processes including coagulation. Both coagulation and lipid metabolism are strongly associated with cardiovascular events. Lowering very-low- and low-density lipoprotein ((V)LDL) particles via dextran sulphate LDL apheresis also removes coagulation proteins. It remains unknown, however, how coagulation proteins are removed in apheresis. We hypothesize that plasma EVs that contain high levels of coagulation proteins are concomitantly removed with (V)LDL particles by dextran sulphate apheresis. For this, we precipitated (V)LDL particles from human plasma with dextran sulphate and analyzed the abundance of coagulation proteins and EVs in the precipitate. Coagulation pathway proteins, as demonstrated by proteomics and a bead-based immunoassay, were over-represented in the (V)LDL precipitate. In this precipitate, both bilayer EVs and monolayer (V)LDL particles were observed by electron microscopy. Separation of EVs from (V)LDL particles using density gradient centrifugation revealed that almost all coagulation proteins were present in the EVs and not in the (V)LDL particles. These EVs also showed a strong procoagulant activity. Our study suggests that dextran sulphate used in LDL apheresis may remove procoagulant EVs concomitantly with (V)LDL particles, leading to a loss of coagulation proteins from the blood.

Presence of aquaporin and V-ATPase on the contractile vacuole of Amoeba proteus.

PubMed

Nishihara, Eri; Yokota, Etsuo; Tazaki, Akira; Orii, Hidefumi; Katsuhara, Maki; Kataoka, Kensuke; Igarashi, Hisako; Moriyama, Yoshinori; Shimmen, Teruo; Sonobe, Seiji

2008-03-01

The results of water permeability measurements suggest the presence of an AQP (aquaporin) in the membrane of the CV (contractile vacuole) in Amoeba proteus [Nishihara, Shimmen and Sonobe (2004) Cell Struct. Funct. 29, 85-90]. In the present study, we cloned an AQP gene from A. proteus [ApAQP (A. proteus AQP)] that encodes a 295-amino-acid protein. The protein has six putative TMs (transmembrane domains) and two NPA (Asn-Pro-Ala) motifs, which are conserved among various AQPs and are thought to be involved in the formation of water channels that span the lipid bilayer. Using Xenopus oocytes, we have demonstrated that the ApAQP protein product can function as a water channel. Immunofluorescence microscopy with anti-ApAQP antibody revealed that ApAQP is detected on the CV membrane and on the vesicles around the CV. The presence of V-ATPase (vacuolar H+-ATPase) on the vesicle membrane around the CV was also detected. Our data on ApAQP allow us to provide the first informed explanation of the high water permeability of the CV membrane in amoeba. Moreover, the results suggest that vesicles possessing V-ATPase are involved in generating an osmotic gradient. Based on our findings, we propose a new hypothesis for the mechanism of CV function.

Dark-field-based observation of single-nanoparticle dynamics on a supported lipid bilayer for in situ analysis of interacting molecules and nanoparticles.

PubMed

Lee, Young Kwang; Kim, Sungi; Nam, Jwa-Min

2015-01-12

Observation of single plasmonic nanoparticles in reconstituted biological systems allows us to obtain snapshots of dynamic processes between molecules and nanoparticles with unprecedented spatiotemporal resolution and single-molecule/single-particle-level data acquisition. This Concept is intended to introduce nanoparticle-tethered supported lipid bilayer platforms that allow for the dynamic confinement of nanoparticles on a two-dimensional fluidic surface. The dark-field-based long-term, stable, real-time observation of freely diffusing plasmonic nanoparticles on a lipid bilayer enables one to extract a broad range of information about interparticle and molecular interactions throughout the entire reaction period. Herein, we highlight important developments in this context to provide ideas on how molecular interactions can be interpreted by monitoring dynamic behaviors and optical signals of laterally mobile nanoparticles. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Advancing the vesosome, a multifunctional drug delivery platform, toward applied in vivo testing

NASA Astrophysics Data System (ADS)

Wong, Benjamin J.

An optimal drug delivery vehicle should circulate long enough to reach the site of illness or disease, possess a large drug loading capacity, retain its contents over the course of treatment, and be able deliver its contents at a rate appropriate for maximum therapeutic benefit at the site of interest. The vesosome, a large lipid bilayer enclosing multiple, smaller liposomes, is our solution to addressing these needs. The external lipid bilayer offers a second barrier of protection for interior components and can also serve as the anchor for active targeting components. Furthermore, internal compartmentalization permits customization of separate environments for multiple therapeutics and release triggers. Previous work established the ability of the vesosome to retain its contents in vitro an order of magnitude longer than liposomes. To be viable in vivo, the vesosome must be functionalized for biocompatibility and tracking, and its synthetic procedure must be repeatable, reliable and result in a purified product. The vesosome was functionalized by introducing biocompatible polymers, such as poly(ethylene glycol) (PEG), and fluorescent dyes in their lipid-bound forms into the external membrane of the vesosome. The external vesosomal membrane is formed from large, flat lipid sheets in the interdigitated (L betaI) phase which, when heated, are used to encapsulate smaller drug-containing vesicles. Through X-Ray diffraction (XRD) and freeze-fracture transmission electron microscopy (FF-TEM), we established that the molar amounts of functionalized lipid required to label the vesosome for tracking and biocompatibility (˜5--7mol% total) did not prevent the formation of the interdigitated phase. Thus, functionalization of the external vesosome membrane can be achieved through functionalization of interdigitated sheets. For in vivo testing, functionalized vesosomes must be separated from unencapsulated vesicles and purification was performed using size exclusion chromatography (SEC) and centrifugation. Having functionalized vesosomes for biocompatibility, PEGylated vesosomes were examined in vitro and in vivo. The presence of surface-grafted PEG was shown to reduce vesosome-vesosome aggregation when exposed to human blood and the circulation half-life was determined to be approximately 2 hours. The evolution of biodistribution was examined by functionalizing the vesosome with a near-infrared dye for in vivo fluorescence imaging and preliminary active targeting experiments show increased vesosome presence at the targeted sites. Ex vivo organ analysis showed the ability of the vesosome to maintain structural integrity for at least 24 hours post-injection. By functionalizing the vesosome for biocompatibility and tracking through a repeatable and reliable synthesis, we have obtained a biocompatible vesosome. Through proof-of-concept live animal testing, we have demonstrated the feasibility of the vesosome as a single site, single dose, multi-therapeutic drug delivery vehicle.

Effect of antimicrobial peptide on dynamics of phosphocholine membrane: role of cholesterol and physical state of bilayer

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

Sharma, Veerendra K.; Mamontov, Eugene; Anunciado, Divina B.

Antimicrobial peptides are universal in all forms of life and are well known for their strong interaction with the cell membrane. This makes them a popular target for investigation of peptide-lipid interactions. Here we report the effect of melittin, an important antimicrobial peptide, on the dynamics of membranes based on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid in both the solid gel and fluid phases. To probe the phase transition, elastic neutron intensity temperature scans have been carried out on DMPC-based unilamellar vesicles (ULV) with and without melittin. We have found that addition of a small amount (0.2 mol%) melittin eliminates the steep fallmore » in the elastic intensity at 296 K associated with the solid gel to fluid phase transition, which is observed for pure DMPC vesicles. Quasielastic neutron scattering (QENS) experiments have been carried out on DMPC ULV in the solid gel and fluid phases with and without 0.2 mol % melittin. The data analysis invariably shows the presence of lateral and internal motions of the DMPC molecule. We found that melittin does have a profound effect on the dynamics of lipid molecules, especially on the lateral motion, and affects it in a different way, depending on the phase of the bilayers. In the solid gel phase, it acts as a plasticizer, enhancing the lateral motion of DMPC. However, in the fluid phase it acts as a stiffening agent, restricting the lateral motion of the lipid molecules. These observations are consistent with the mean squared displacements extracted from the elastic intensity temperature scans. Cholesterol is a vital component of eukaryotic membrane, which is a natural target for melittin. To investigate the effect of melittin on vesicles supplemented with cholesterol, QENS experiments have also been carried out on DMPC ULV with 20 mol% cholesterol in the presence and absence of 0.2 mol% melittin. Remarkably, the effects of melittin on the membrane dynamics disappear in the presence of 20 mol % cholesterol. Thus, our measurements indicate that the destabilizing effect of the peptide melittin on membranes can be mitigated by the presence of cholesterol.« less

Effect of antimicrobial peptide on dynamics of phosphocholine membrane: role of cholesterol and physical state of bilayer

DOE PAGES

Sharma, Veerendra K.; Mamontov, Eugene; Anunciado, Divina B.; ...

2015-06-24

Antimicrobial peptides are universal in all forms of life and are well known for their strong interaction with the cell membrane. This makes them a popular target for investigation of peptide-lipid interactions. Here we report the effect of melittin, an important antimicrobial peptide, on the dynamics of membranes based on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid in both the solid gel and fluid phases. To probe the phase transition, elastic neutron intensity temperature scans have been carried out on DMPC-based unilamellar vesicles (ULV) with and without melittin. We have found that addition of a small amount (0.2 mol%) melittin eliminates the steep fallmore » in the elastic intensity at 296 K associated with the solid gel to fluid phase transition, which is observed for pure DMPC vesicles. Quasielastic neutron scattering (QENS) experiments have been carried out on DMPC ULV in the solid gel and fluid phases with and without 0.2 mol % melittin. The data analysis invariably shows the presence of lateral and internal motions of the DMPC molecule. We found that melittin does have a profound effect on the dynamics of lipid molecules, especially on the lateral motion, and affects it in a different way, depending on the phase of the bilayers. In the solid gel phase, it acts as a plasticizer, enhancing the lateral motion of DMPC. However, in the fluid phase it acts as a stiffening agent, restricting the lateral motion of the lipid molecules. These observations are consistent with the mean squared displacements extracted from the elastic intensity temperature scans. Cholesterol is a vital component of eukaryotic membrane, which is a natural target for melittin. To investigate the effect of melittin on vesicles supplemented with cholesterol, QENS experiments have also been carried out on DMPC ULV with 20 mol% cholesterol in the presence and absence of 0.2 mol% melittin. Remarkably, the effects of melittin on the membrane dynamics disappear in the presence of 20 mol % cholesterol. Thus, our measurements indicate that the destabilizing effect of the peptide melittin on membranes can be mitigated by the presence of cholesterol.« less

Label-free visualization of ultrastructural features of artificial synapses via cryo-EM.

PubMed

Gopalakrishnan, Gopakumar; Yam, Patricia T; Madwar, Carolin; Bostina, Mihnea; Rouiller, Isabelle; Colman, David R; Lennox, R Bruce

2011-12-21

The ultrastructural details of presynapses formed between artificial substrates of submicrometer silica beads and hippocampal neurons are visualized via cryo-electron microscopy (cryo-EM). The silica beads are derivatized by poly-d-lysine or lipid bilayers. Molecular features known to exist at presynapses are clearly present at these artificial synapses, as visualized by cryo-EM. Key synaptic features such as the membrane contact area at synaptic junctions, the presynaptic bouton containing presynaptic vesicles, as well as microtubular structures can be identified. This is the first report of the direct, label-free observation of ultrastructural details of artificial synapses.

Feruloyl Dioleoyglycerol Antioxidant Capacity in Phospholipid Vesicles

USDA-ARS?s Scientific Manuscript database

Ferulic acid and its esters are known to be effective antioxidants. Feruloyl dioleoylglycerol was assessed for its ability to serve as an antioxidant in model membrane phospholipid vesicles. The molecule was incorporated into single-lamellar vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine at ...

SFG studies on interactions between antimicrobial peptides and supported lipid bilayers.

PubMed

Chen, Xiaoyun; Chen, Zhan

2006-09-01

The mode of action of antimicrobial peptides (AMPs) in disrupting cell membrane bilayers is of fundamental importance in understanding the efficiency of different AMPs, which is crucial to design antibiotics with improved properties. Recent developments in the field of sum frequency generation (SFG) vibrational spectroscopy have made it a powerful and unique biophysical technique in investigating the interactions between AMPs and a single substrate supported planar lipid bilayer. We will review some of the recent progress in applying SFG to study membrane lipid bilayers and discuss how SFG can provide novel information such as real-time bilayer structure change and AMP orientation during AMP-lipid bilayer interactions in a very biologically relevant manner. Several examples of applying SFG to monitor such interactions between AMPs and a dipalmitoyl phosphatidylglycerol (DPPG) bilayer are presented. Different modes of actions are observed for melittin, tachyplesin I, d-magainin 2, MSI-843, and a synthetic antibacterial oligomer, demonstrating that SFG is very effective in the study of AMPs and AMP-lipid bilayer interactions.

Supported lipid bilayer/carbon nanotube hybrids

NASA Astrophysics Data System (ADS)

Zhou, Xinjian; Moran-Mirabal, Jose M.; Craighead, Harold G.; McEuen, Paul L.

2007-03-01

Carbon nanotube transistors combine molecular-scale dimensions with excellent electronic properties, offering unique opportunities for chemical and biological sensing. Here, we form supported lipid bilayers over single-walled carbon nanotube transistors. We first study the physical properties of the nanotube/supported lipid bilayer structure using fluorescence techniques. Whereas lipid molecules can diffuse freely across the nanotube, a membrane-bound protein (tetanus toxin) sees the nanotube as a barrier. Moreover, the size of the barrier depends on the diameter of the nanotube-with larger nanotubes presenting bigger obstacles to diffusion. We then demonstrate detection of protein binding (streptavidin) to the supported lipid bilayer using the nanotube transistor as a charge sensor. This system can be used as a platform to examine the interactions of single molecules with carbon nanotubes and has many potential applications for the study of molecular recognition and other biological processes occurring at cell membranes.

A study of carbobenzoxy-D-phenylalanine-L-phenylalanine-glycine, an inhibitor of membrane fusion, in phospholipid bilayers with multinuclear magnetic resonance.

PubMed

Dentino, A R; Westerman, P W; Yeagle, P L

1995-05-04

The anti-viral and membrane fusion inhibitor, carbobenzoxy-D-phenylalanine-L-phenylalanine-glycine (ZfFG), was studied in phospholipid bilayers, where earlier studies had indicated this peptide functioned. Multinuclear magnetic resonance (NMR) studies were performed with isotopically labeled peptide. A peptide labeled in the glycine carboxyl with 13C was synthesized, and the isotropic 13C-NMR chemical shift of that carbon was measured as a function of pH. A pKa of 3.6 for the carboxyl was determined from the peptide bound to a phosphatidylcholine bilayer. ZfFG inhibits the formation by sonication of highly curved, small unilamellar vesicles. Experiments as a function of pH revealed that this ability of ZfFG was governed by a pKa of 3.7. Therefore the protonation state of the carboxyl of ZfFG appeared to regulate the effectiveness of this anti-viral peptide at destabilizing highly curved phospholipid assemblies. Such destabilization had previously been discovered to be related to the mechanism of the anti-fusion and anti-viral activity of this peptide. The location of the carboxyl of ZfFG in the membrane was probed with paramagnetic relaxation enhancement of the 13C spin lattice relaxation of the carboxyl carbon in the glycine of ZfFG (enriched in 13C). Results suggested that this carboxyl is at or above the surface of the phospholipid bilayer. The dynamics of the molecule in the membrane were examined with 2H-NMR studies of ZfFG, deuterated in the alpha-carbon protons of the glycine. When ZfFG was bound to membranes of phosphatidylcholine, a sharp 2H-NMR spectral component was observed, consistent with a disordering of the glycine methylene segment of the peptide. When ZfFG was bound to N-methyl dioleoylphosphatidylethanolamine (N-methyl DOPE) bilayers at temperatures below 30 degrees C, a large quadrupole splitting was observed. These results suggest that ZfFG likely inhibits membrane fusion from the surface of the lipid bilayer, but not by forming a tight, stoichiometric complex with the phospholipids.

Effect of hydrostatic pressure on water penetration and rotational dynamics in phospholipid-cholesterol bilayers.

PubMed Central

Bernsdorff, C; Wolf, A; Winter, R; Gratton, E

1997-01-01

The effect of high hydrostatic pressure on the lipid bilayer hydration, the mean order parameter, and rotational dynamics of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) cholesterol vesicles has been studied by time-resolved fluorescence spectroscopy up to 1500 bar. Whereas the degree of hydration in the lipid headgroup and interfacial region was assessed from fluorescence lifetime data using the probe 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), the corresponding information in the upper acyl chain region was estimated from its effect on the fluorescence lifetime of and 3-(diphenylhexatrienyl)propyl-trimethylammonium (TMAP-DPH). The lifetime data indicate a greater level of interfacial hydration for DPPC bilayers than for POPC bilayers, but there is no marked difference in interchain hydration of the two bilayer systems. The addition of cholesterol at levels from 30 to 50 mol% to DPPC has a greater effect on the increase of hydrophobicity in the interfacial region of the bilayer than the application of hydrostatic pressure of several hundred to 1000 bar. Although the same trend is observed in the corresponding system, POPC/30 mol% cholesterol, the observed effects are markedly less pronounced. Whereas the rotational correlation times of the fluorophores decrease in passing the pressure-induced liquid-crystalline to gel phase transition of DPPC, the wobbling diffusion coefficient remains essentially unchanged. The wobbling diffusion constant of the two fluorophores changes markedly upon incorporation of 30 mol% cholesterol, and increases at higher pressures, also in the case of POPC/30 mol% cholesterol. The observed effects are discussed in terms of changes in the rotational characteristics of the fluorophores and the phase-state of the lipid mixture. The results demonstrate the ability of cholesterol to adjust the structural and dynamic properties of membranes composed of different phospholipid components, and to efficiently regulate the motional freedom and hydrophobicity of membranes, so that they can withstand even drastic changes in environmental conditions, such as high external hydrostatic pressure. PMID:9138572

Visualization of Synchronous or Asynchronous Release of Single Synaptic Vesicle in Active-Zone-Like Membrane Formed on Neuroligin-Coated Glass Surface.

PubMed

Funahashi, Junichiro; Tanaka, Hiromitsu; Hirano, Tomoo

2018-01-01

Fast repetitive synaptic transmission depends on efficient exocytosis and retrieval of synaptic vesicles around a presynaptic active zone. However, the functional organization of an active zone and regulatory mechanisms of exocytosis, endocytosis and reconstruction of release-competent synaptic vesicles have not been fully elucidated. By developing a novel visualization method, we attempted to identify the location of exocytosis of a single synaptic vesicle within an active zone and examined movement of synaptic vesicle protein synaptophysin (Syp) after exocytosis. Using cultured hippocampal neurons, we induced formation of active-zone-like membranes (AZLMs) directly adjacent and parallel to a glass surface coated with neuroligin, and imaged Syp fused to super-ecliptic pHluorin (Syp-SEP) after its translocation to the plasma membrane from a synaptic vesicle using total internal reflection fluorescence microscopy (TIRFM). An AZLM showed characteristic molecular and functional properties of a presynaptic active zone. It contained active zone proteins, cytomatrix at the active zone-associated structural protein (CAST), Bassoon, Piccolo, Munc13 and RIM, and showed an increase in intracellular Ca 2+ concentration upon electrical stimulation. In addition, single-pulse stimulation sometimes induced a transient increase of Syp-SEP signal followed by lateral spread in an AZLM, which was considered to reflect an exocytosis event of a single synaptic vesicle. The diffusion coefficient of Syp-SEP on the presynaptic plasma membrane after the membrane fusion was estimated to be 0.17-0.19 μm 2 /s, suggesting that Syp-SEP diffused without significant obstruction. Synchronous exocytosis just after the electrical stimulation tended to occur at multiple restricted sites within an AZLM, whereas locations of asynchronous release occurring later after the stimulation tended to be more scattered.

Lipid Microarray Biosensor for Biotoxin Detection.

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

Singh, Anup K.; Throckmorton, Daniel J.; Moran-Mirabal, Jose C.

2006-05-01

We present the use of micron-sized lipid domains, patterned onto planar substrates and within microfluidic channels, to assay the binding of bacterial toxins via total internal reflection fluorescence microscopy (TIRFM). The lipid domains were patterned using a polymer lift-off technique and consisted of ganglioside-populated DSPC:cholesterol supported lipid bilayers (SLBs). Lipid patterns were formed on the substrates by vesicle fusion followed by polymer lift-off, which revealed micron-sized SLBs containing either ganglioside GT1b or GM1. The ganglioside-populated SLB arrays were then exposed to either Cholera toxin subunit B (CTB) or Tetanus toxin fragment C (TTC). Binding was assayed on planar substrates bymore » TIRFM down to 1 nM concentration for CTB and 100 nM for TTC. Apparent binding constants extracted from three different models applied to the binding curves suggest that binding of a protein to a lipid-based receptor is strongly affected by the lipid composition of the SLB and by the substrate on which the bilayer is formed. Patterning of SLBs inside microfluidic channels also allowed the preparation of lipid domains with different compositions on a single device. Arrays within microfluidic channels were used to achieve segregation and selective binding from a binary mixture of the toxin fragments in one device. The binding and segregation within the microfluidic channels was assayed with epifluorescence as proof of concept. We propose that the method used for patterning the lipid microarrays on planar substrates and within microfluidic channels can be easily adapted to proteins or nucleic acids and can be used for biosensor applications and cell stimulation assays under different flow conditions. KEYWORDS. Microarray, ganglioside, polymer lift-off, cholera toxin, tetanus toxin, TIRFM, binding constant.4« less

Crowding-Induced Mixing Behavior of Lipid Bilayers: Examination of Mixing Energy, Phase, Packing Geometry, and Reversibility.

PubMed

Zeno, Wade F; Rystov, Alice; Sasaki, Darryl Y; Risbud, Subhash H; Longo, Marjorie L

2016-05-10

In an effort to develop a general thermodynamic model from first-principles to describe the mixing behavior of lipid membranes, we examined lipid mixing induced by targeted binding of small (Green Fluorescent Protein (GFP)) and large (nanolipoprotein particles (NLPs)) structures to specific phases of phase-separated lipid bilayers. Phases were targeted by incorporation of phase-partitioning iminodiacetic acid (IDA)-functionalized lipids into ternary lipid mixtures consisting of DPPC, DOPC, and cholesterol. GFP and NLPs, containing histidine tags, bound the IDA portion of these lipids via a metal, Cu(2+), chelating mechanism. In giant unilamellar vesicles (GUVs), GFP and NLPs bound to the Lo domains of bilayers containing DPIDA, and bound to the Ld region of bilayers containing DOIDA. At sufficiently large concentrations of DPIDA or DOIDA, lipid mixing was induced by bound GFP and NLPs. The validity of the thermodynamic model was confirmed when it was found that the statistical mixing distribution as a function of crowding energy for smaller GFP and larger NLPs collapsed to the same trend line for each GUV composition. Moreover, results of this analysis show that the free energy of mixing for a ternary lipid bilayer consisting of DOPC, DPPC, and cholesterol varied from 7.9 × 10(-22) to 1.5 × 10(-20) J/lipid at the compositions observed, decreasing as the relative cholesterol concentration was increased. It was discovered that there appears to be a maximum packing density, and associated maximum crowding pressure, of the NLPs, suggestive of circular packing. A similarity in mixing induced by NLP1 and NLP3 despite large difference in projected areas was analytically consistent with monovalent (one histidine tag) versus divalent (two histidine tags) surface interactions, respectively. In addition to GUVs, binding and induced mixing behavior of NLPs was also observed on planar, supported lipid multibilayers. The mixing process was reversible, with Lo domains reappearing after addition of EDTA for NLP removal.

Phospholamban and its Phosphorylated Form Interact Differently with Lipid Bilayers: A 31P, 2H and 13C Solid-State NMR Spectroscopic Study

PubMed Central

Abu-Baker, Shadi; Lorigan, Gary A.

2008-01-01

Phospholamban (PLB) is a 52-amino acid integral membrane protein that helps to regulate the flow of Ca2+ ions in cardiac muscle cells. Recent structural studies on the PLB pentamer and the functionally active monomer (AFA-PLB) debate whether its cytoplasmic domain, in either the phosphorylated or dephosphorylated states, is α-helical in structure as well as whether it associates with the lipid head groups [Oxenoid, K. (2005) Proc Natl. Acad. Sci. USA 102, 10870–10875, Karim, C. B. (2004) Proc. Natl. Acad. Sci. USA 101, 14437–14442, Andronesi, C.A. (2005) J. Am. Chem. Soc. 127, 12965–12974, Li, J. (2003) Biochemistry 42, 10674–10682, Metcalfe, E. E. (2005) Biochemistry 44, 4386–4396, Clayton, J. C. (2005) Biochemistry 44, 17016–17026]. Comparing the secondary structure of the PLB pentamer and its phosphorylated form (P-PLB) as well as their interaction with the lipid bilayer is crucial in order to understand its regulatory function. Therefore, in this study, the full-length wild-type (WT)-PLB and P-PLB were incorporated into 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) phospholipid bilayers and studied utilizing solid-state NMR spectroscopy. The analysis of the 2H and 31P solid-state NMR data of PLB and P-PLB in POPC multilamellar vesicles (MLVs) indicates that a direct interaction takes place between both proteins and the phospholipid head groups. However, the interaction of P-PLB with POPC bilayers was less significant when compared to PLB. Moreover, the secondary structure using 13C=O site-specific isotopically labeled Ala15-PLB and Ala15-P-PLB in POPC bilayers suggests that this residue, located in the cytoplasmic domain, is a part of an α-helical structure for both PLB and P-PLB. PMID:17073452

Crowding-induced mixing behavior of lipid bilayers: Examination of mixing energy, phase, packing geometry, and reversibility

DOE PAGES

Zeno, Wade F.; Rystov, Alice; Sasaki, Darryl Y.; ...

2016-04-20

In an effort to develop a general thermodynamic model from first-principles to describe the mixing behavior of lipid membranes, we examined lipid mixing induced by targeted binding of small (Green Fluorescent Protein (GFP)) and large (nanolipoprotein particles (NLPs)) structures to specific phases of phase-separated lipid bilayers. Phases were targeted by incorporation of phase-partitioning iminodiacetic acid (IDA)-functionalized lipids into ternary lipid mixtures consisting of DPPC, DOPC, and cholesterol. GFP and NLPs, containing histidine tags, bound the IDA portion of these lipids via a metal, Cu 2+, chelating mechanism. In giant unilamellar vesicles (GUVs), GFP and NLPs bound to the Lo domainsmore » of bilayers containing DPIDA, and bound to the Ld region of bilayers containing DOIDA. At sufficiently large concentrations of DPIDA or DOIDA, lipid mixing was induced by bound GFP and NLPs. The validity of the thermodynamic model was confirmed when it was found that the statistical mixing distribution as a function of crowding energy for smaller GFP and larger NLPs collapsed to the same trend line for each GUV composition. Moreover, results of this analysis show that the free energy of mixing for a ternary lipid bilayer consisting of DOPC, DPPC, and cholesterol varied from 7.9 × 10 –22 to 1.5 × 10 –20 J/lipid at the compositions observed, decreasing as the relative cholesterol concentration was increased. It was discovered that there appears to be a maximum packing density, and associated maximum crowding pressure, of the NLPs, suggestive of circular packing. A similarity in mixing induced by NLP1 and NLP3 despite large difference in projected areas was analytically consistent with monovalent (one histidine tag) versus divalent (two histidine tags) surface interactions, respectively. In addition to GUVs, binding and induced mixing behavior of NLPs was also observed on planar, supported lipid multibilayers. Furthermore, the mixing process was reversible, with Lo domains reappearing after addition of EDTA for NLP removal.« less

Effect of electrostatic interaction between fluoxetine and lipid membranes on the partitioning of fluoxetine investigated using second derivative spectrophotometry and FTIR.

PubMed

Do, Tien T T; Dao, Uyen P N; Bui, Huong T; Nguyen, Trang T

2017-10-01

The interaction between a drug molecule and lipid bilayers is highly important regarding the pharmaceutical activity of the drug. In this study, the interaction of fluoxetine, a well-known selective serotonin reuptake inhibitor antidepressant and lipid bilayers composed of 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was studied from the aspect of electrostatics using second derivative spectrophotometry and Fourier transform infrared spectroscopy (FTIR) in order to provide insights into the drug behavior. Changing pH from 7.4 to 9.5 to increases the neutral state of fluoxetine, the partitioning of fluoxetine into the zwitterionic DPPC large unilamellar vesicles (LUVs) was increased whereas it was reduced into the negatively charged DPPG LUVs. Fluoxetine was found to exhibit a disordering effect on the acyl chains of DPPC and DPPG bilayers upon its partitioning. In addition, increasing concentration of NaCl lessened the binding of fluoxetine into DPPG bilayers due to the reduction in electrostatic attraction between positively charged fluoxetine and negatively charged DPPG LUVs. In addition, the FTIR study revealed that increasing the NaCl concentration could trigger the shift to higher frequency of the CH 2 stretching as well as the notable blue shift in the PO 2 - regions of DPPG, indicating that fluoxetine had deeper penetration into DPPG LUVs. The differences in the NaCl concentration showed a negligible effect on the incorporation of fluoxetine into the zwitterionic DPPC LUVs. In summary, the electrostatic interaction plays an important role on the partitioning of a cationic amphiphilic SSIR drug into the lipid bilayers and the drug partitioning induces the lipids' conformational change. These imply a possible influence on the drug pharmacology. Copyright © 2017 Elsevier B.V. All rights reserved.

Crowding-induced mixing behavior of lipid bilayers: Examination of mixing energy, phase, packing geometry, and reversibility

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

Zeno, Wade F.; Rystov, Alice; Sasaki, Darryl Y.

In an effort to develop a general thermodynamic model from first-principles to describe the mixing behavior of lipid membranes, we examined lipid mixing induced by targeted binding of small (Green Fluorescent Protein (GFP)) and large (nanolipoprotein particles (NLPs)) structures to specific phases of phase-separated lipid bilayers. Phases were targeted by incorporation of phase-partitioning iminodiacetic acid (IDA)-functionalized lipids into ternary lipid mixtures consisting of DPPC, DOPC, and cholesterol. GFP and NLPs, containing histidine tags, bound the IDA portion of these lipids via a metal, Cu 2+, chelating mechanism. In giant unilamellar vesicles (GUVs), GFP and NLPs bound to the Lo domainsmore » of bilayers containing DPIDA, and bound to the Ld region of bilayers containing DOIDA. At sufficiently large concentrations of DPIDA or DOIDA, lipid mixing was induced by bound GFP and NLPs. The validity of the thermodynamic model was confirmed when it was found that the statistical mixing distribution as a function of crowding energy for smaller GFP and larger NLPs collapsed to the same trend line for each GUV composition. Moreover, results of this analysis show that the free energy of mixing for a ternary lipid bilayer consisting of DOPC, DPPC, and cholesterol varied from 7.9 × 10 –22 to 1.5 × 10 –20 J/lipid at the compositions observed, decreasing as the relative cholesterol concentration was increased. It was discovered that there appears to be a maximum packing density, and associated maximum crowding pressure, of the NLPs, suggestive of circular packing. A similarity in mixing induced by NLP1 and NLP3 despite large difference in projected areas was analytically consistent with monovalent (one histidine tag) versus divalent (two histidine tags) surface interactions, respectively. In addition to GUVs, binding and induced mixing behavior of NLPs was also observed on planar, supported lipid multibilayers. Furthermore, the mixing process was reversible, with Lo domains reappearing after addition of EDTA for NLP removal.« less

SU-G-TeP3-03: Dose Enhancement of Gold Nanoparticle in Proton Therapy: A Monte Carlo Study Based On the Transmission Electron Microscopy Imaging

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

Ma, Y; Beaulieu, L; Laprise-Pelletier, M

2016-06-15

Purpose: Gold nanoparticle (GNP) is a promising radiosensitizer that selectively boosts tumor dose in radiotherapy. Transmission electron microscopy (TEM) imaging observations recently revealed for the first time that GNP exists in vivo in the form of highly localized vesicles, instead of hypothetical uniform distribution. This work investigates the corresponding difference of energy deposition in proton therapy. Methods: First, single vesicles of various radii were constructed by packing GNPs (as Φ50 nm gold spheres) in spheres and were simulated, as well as a single GNP. The radial energy depositions (REDs) were scored using 100 concentric spherical shells from 0.1µm to 10µm,more » 0.1µm thickness each, for both vesicles and GNP, and compared. TEM images, 8 days after injection in a PC3 prostate cancer murine model, were used to extract position/dimension of vesicles, as well as contours of cytoplasmic and nucleus membranes. Vesicles were then constructed based on the TEM images. A 100 MeV proton beam was studied by using the Geant4-DNA code, which simulates all energy deposition events. Results: The vesicle REDs, normalized to the same proton energy loss as in a single GNP, are larger (smaller) than that of a single GNP when radius >2µm (<2µm). The peak increase (at about 3µm radius) is about 10% and 18% for Φ1µm and Φ1.6µm vesicles respectively, relative to a single GNP. The TEM-based simulation resulted in a larger energy deposition (by about one order of magnitude) that follows completely different pattern from that of hypothetical GNP distributions (regular dotted pattern in extracellular and/or extranucleus regions). Conclusion: The in vivo energy deposition, both in pattern and magnitude, of proton therapy is greatly affected by the true distribution of the GNP, as illustrated by the presence of GNP vesicles compared to hypothetical scenarios. Work supported by NSERC Discovery Grant #435510, Canada.« less

Superconducting order parameter fluctuations in NbN/NiCu and NbTiN/NiCu bilayer nanostripes for photon detection

NASA Astrophysics Data System (ADS)

Aichner, Bernd; Jausner, Florian; Zechner, Georg; Mühlgassner, Rita; Lang, Wolfgang; Klimov, Andrii; Puźniak, Roman; Słysz, Wojciech; Guziewicz, Marek; Kruszka, Renata; Wegrzecki, Maciej; Sobolewski, Roman

2017-05-01

Thermodynamic fluctuations of the superconducting order parameter in NbN/NiCu and NbTiN/NiCu superconductor/ferromagnet (S/F) thin bilayers patterned to microbridges are investigated. Plain NbN and NbTiN films served as reference materials for the analyses. The samples were grown using dc-magnetron sputtering on chemically cleaned sapphire single-crystal substrates. After rapid thermal annealing at high temperatures, the superconducting films were coated with NiCu overlays, using co-sputtering. The positive magnetoresistance of the superconducting single layers is very small in the normal state but with a sharp upturn close to the superconducting transition, a familiar signature of superconducting fluctuations. The fluctuation-enhanced conductivity (paraconductivity) of the NbN and NbTiN single layer films is slightly larger than the prediction of the parameter-free Aslamazov-Larkin theory for order-parameter fluctuations in two-dimensional superconductors. The addition of a ferromagnetic top layer, however, changes the magnetotransport properties significantly. The S/F bilayers show a negative magnetoresistance up to almost room temperature, while the signature of fluctuations is similar to that in the plain films, demonstrating the relevance of both ferromagnetic and superconducting effects in the S/F bilayers. The paraconductivity is reduced below theoretical predictions, in particular in the NbTiN/NiCu bilayers. Such suppression of the fluctuation amplitude in S/F bilayers could be favorable to reduce dark counts in superconducting photon detectors and lead the way to enhance their performance.

Molecular machines open cell membranes

NASA Astrophysics Data System (ADS)

García-López, Víctor; Chen, Fang; Nilewski, Lizanne G.; Duret, Guillaume; Aliyan, Amir; Kolomeisky, Anatoly B.; Robinson, Jacob T.; Wang, Gufeng; Pal, Robert; Tour, James M.

2017-08-01

Beyond the more common chemical delivery strategies, several physical techniques are used to open the lipid bilayers of cellular membranes. These include using electric and magnetic fields, temperature, ultrasound or light to introduce compounds into cells, to release molecular species from cells or to selectively induce programmed cell death (apoptosis) or uncontrolled cell death (necrosis). More recently, molecular motors and switches that can change their conformation in a controlled manner in response to external stimuli have been used to produce mechanical actions on tissue for biomedical applications. Here we show that molecular machines can drill through cellular bilayers using their molecular-scale actuation, specifically nanomechanical action. Upon physical adsorption of the molecular motors onto lipid bilayers and subsequent activation of the motors using ultraviolet light, holes are drilled in the cell membranes. We designed molecular motors and complementary experimental protocols that use nanomechanical action to induce the diffusion of chemical species out of synthetic vesicles, to enhance the diffusion of traceable molecular machines into and within live cells, to induce necrosis and to introduce chemical species into live cells. We also show that, by using molecular machines that bear short peptide addends, nanomechanical action can selectively target specific cell-surface recognition sites. Beyond the in vitro applications demonstrated here, we expect that molecular machines could also be used in vivo, especially as their design progresses to allow two-photon, near-infrared and radio-frequency activation.

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

Fast Collisional Lipid Transfer Among Polymer-Bounded Nanodiscs

NASA Astrophysics Data System (ADS)

Cuevas Arenas, Rodrigo; Danielczak, Bartholomäus; Martel, Anne; Porcar, Lionel; Breyton, Cécile; Ebel, Christine; Keller, Sandro

2017-04-01

Some styrene/maleic acid (SMA) copolymers solubilise membrane lipids and proteins to form polymer-bounded nanodiscs termed SMA/lipid particles (SMALPs). Although SMALPs preserve a lipid-bilayer core, they appear to be more dynamic than other membrane mimics. We used time-resolved Förster resonance energy transfer and small-angle neutron scattering to determine the kinetics and the mechanisms of phospholipid transfer among SMALPs. In contrast with vesicles or protein-bounded nanodiscs, SMALPs exchange lipids not only by monomer diffusion but also by fast collisional transfer. Under typical experimental conditions, lipid exchange occurs within seconds in the case of SMALPs but takes minutes to days in the other bilayer particles. The diffusional and second-order collisional exchange rate constants for SMALPs at 30 °C are kdif = 0.287 s-1 and kcol = 222 M-1s-1, respectively. Together with the fast kinetics, the observed invariability of the rate constants with probe hydrophobicity and the moderate activation enthalpy of ~70 kJ mol-1 imply that lipids exchange through a “hydrocarbon continuum” enabled by the flexible nature of the SMA belt surrounding the lipid-bilayer core. Owing to their fast lipid-exchange kinetics, SMALPs represent highly dynamic equilibrium rather than kinetically trapped membrane mimics, which has important implications for studying protein/lipid interactions in polymer-bounded nanodiscs.

Molecular machines open cell membranes.

PubMed

García-López, Víctor; Chen, Fang; Nilewski, Lizanne G; Duret, Guillaume; Aliyan, Amir; Kolomeisky, Anatoly B; Robinson, Jacob T; Wang, Gufeng; Pal, Robert; Tour, James M

2017-08-30

Beyond the more common chemical delivery strategies, several physical techniques are used to open the lipid bilayers of cellular membranes. These include using electric and magnetic fields, temperature, ultrasound or light to introduce compounds into cells, to release molecular species from cells or to selectively induce programmed cell death (apoptosis) or uncontrolled cell death (necrosis). More recently, molecular motors and switches that can change their conformation in a controlled manner in response to external stimuli have been used to produce mechanical actions on tissue for biomedical applications. Here we show that molecular machines can drill through cellular bilayers using their molecular-scale actuation, specifically nanomechanical action. Upon physical adsorption of the molecular motors onto lipid bilayers and subsequent activation of the motors using ultraviolet light, holes are drilled in the cell membranes. We designed molecular motors and complementary experimental protocols that use nanomechanical action to induce the diffusion of chemical species out of synthetic vesicles, to enhance the diffusion of traceable molecular machines into and within live cells, to induce necrosis and to introduce chemical species into live cells. We also show that, by using molecular machines that bear short peptide addends, nanomechanical action can selectively target specific cell-surface recognition sites. Beyond the in vitro applications demonstrated here, we expect that molecular machines could also be used in vivo, especially as their design progresses to allow two-photon, near-infrared and radio-frequency activation.

β-CD-dextran polymer for efficient sequestration of cholesterol from phospholipid bilayers: Mechanistic and safe-toxicity investigations.

PubMed

Stelzl, Dominik; Nielsen, Thorbjørn Terndrup; Hansen, Terkel; di Cagno, Massimiliano

2015-12-30

The aim of this work was to investigate the suitability of β-cyclodextrin-dextran (BCD-dextran) polymer as cholesterol sequestering agent in vitro. For this purpose, BCD-dextran-cholesterol complexation was studied by phase solubility studies as well as with a specifically designed in vitro model based on giant unilamellar vesicles (GUVs) to evaluate the ability of this polymer to sequestrate cholesterol from phospholipid bilayers. Cholesterol-sequestering ability of BCD-dextran was also investigated on different cell lines relevant for the hematopoietic system and results were correlated to cells toxicity. BCD-dextran polymer was capable of extracting significant amount of cholesterol from phospholipid bilayers and to a higher extent in comparison to available β-cyclodextrins (BCDs). The ability of BCD-dextran in sequestering cholesterol resulted also very high on cell lines relevant for the hematopoietic system. Moreover, BCD-dextran resulted less toxic on cell cultures due to higher selectivity in sequestering cholesterol in comparison to MBCD (that sequestrated also significant amounts of cholesteryl esters). In conclusion, BCD-dextran resulted an extremely efficient cholesterol-sequestering agent and BCD-dextran resulted more selective to cholesterol extraction in comparison to other BCDs (therefore of lower cytotoxicity). This phenomenon might play a key role to develop an efficient treatment for hypercholesterolemia based on cholesterol segregation. Copyright © 2015 Elsevier B.V. All rights reserved.

Effect of low levels of lipid oxidation on the curvature, dynamics, and permeability of lipid bilayers and their interactions with cationic nanoparticles

NASA Astrophysics Data System (ADS)

Lee, Hwankyu; Malmstadt, Noah

2018-04-01

Lipid bilayers composed of saturated and unsaturated lipids, oxidized lipids, and cholesterol at concentrations of 0–18 mol% oxidized lipid were simulated, showing that the presence of oxidized lipid increases bilayer disorder, curvature, and lateral dynamics at low oxidized-lipid concentrations of 18 mol% or less. The aldehyde terminal of a shortened oxidized-lipid tail tends to interact with water and thus bends toward the bilayer-water interface, in agreement with previous experiments and simulations. In particular, water molecules pass through the oxidized bilayer without pore formation, implying passive permeability. A single nanoparticle, which consists of 300 polystyrene (PS) chains with cationic terminals, added to this bilayer simulation induces negative bilayer curvature and inserts to the bilayer, regardless of the oxidized-lipid concentration. Hydrophobic monomers and cationic terminals of the PS particle interact respectively with lipid tails and headgroups, leading to the wrapping of either lipid monolayer or bilayer along the particle surface. These results indicate that lipid oxidation increases membrane curvature and permeability even at such a low concentration of oxidized lipid, which supports the experimental observations regarding the passive permeability of oxidized bilayer, and also that oxidized lipids of low concentration do not significantly influence the insertion of a cationic PS particle to the bilayer.

Diffusion and intermembrane distance: case study of avidin and E-cadherin mediated adhesion.

PubMed

Fenz, Susanne F; Merkel, Rudolf; Sengupta, Kheya

2009-01-20

We present a biomimetic model system for cell-cell adhesion consisting of a giant unilamellar vesicle (GUV) adhering via specific ligand-receptor interactions to a supported lipid bilayer (SLB). The modification of in-plane diffusion of tracer lipids and receptors in the SLB membrane due to adhesion to the GUV is reported. Adhesion was mediated by either biotin-neutravidin (an avidin analogue) or the extracellular domains of the cell adhesion molecule E-cadherin (Ecad). In the strong interaction (biotin-avidin) case, binding of soluble receptors to the SLB alone led to reduced diffusion of tracer lipids. From theoretical considerations, this could be attributed partially to introduction of obstacles and partially to viscous effects. Further specific binding of a GUV membrane caused additional slowing down of tracers (up to 15%) and immobilization of receptors, and led to accumulation of receptors in the adhesion zone until full coverage was achieved. The intermembrane distance was measured to be 7 nm from microinterferometry (RICM). We show that a crowding effect due to the accumulated receptors alone is not sufficient to account for the slowing downan additional friction from the membrane also plays a role. In the weak binding case (Ecad), the intermembrane distance was about 50 nm, corresponding to partial overlap of the Ecad domains. No significant change in diffusion of tracer lipids was observed upon either protein binding or subsequent vesicle binding. The former was probably due to very small effective size of the obstacles introduced into the bilayer by Ecad binding, whereas the latter was due to the fact that, with such high intermembrane distance, the resulting friction is negligible. We conclude that the effect of intermembrane adhesion on diffusion depends strongly on the choice of the receptors.

The Antimicrobial Mechanism of Action of Epsilon-Poly-l-Lysine

PubMed Central

Hyldgaard, Morten; Mygind, Tina; Vad, Brian S.; Stenvang, Marcel; Otzen, Daniel E.

2014-01-01

Epsilon-poly-l-lysine (ε-PL) is a natural antimicrobial cationic peptide which is generally regarded as safe (GRAS) as a food preservative. Although its antimicrobial activity is well documented, its mechanism of action is only vaguely described. The aim of this study was to clarify ε-PL's mechanism of action using Escherichia coli and Listeria innocua as model organisms. We examined ε-PL's effect on cell morphology and membrane integrity and used an array of E. coli deletion mutants to study how specific outer membrane components affected the action of ε-PL. We furthermore studied its interaction with lipid bilayers using membrane models. In vitro cell studies indicated that divalent cations and the heptose I and II phosphate groups in the lipopolysaccharide layer of E. coli are critical for ε-PL's binding efficiency. ε-PL removed the lipopolysaccharide layer and affected cell morphology of E. coli, while L. innocua underwent minor morphological changes. Propidium iodide staining showed that ε-PL permeabilized the cytoplasmic membrane in both species, indicating the membrane as the site of attack. We compared the interaction with neutral or negatively charged membrane systems and showed that the interaction with ε-PL relied on negative charges on the membrane. Suspended membrane vesicles were disrupted by ε-PL, and a detergent-like disruption of E. coli membrane was confirmed by atomic force microscopy imaging of supported lipid bilayers. We hypothesize that ε-PL destabilizes membranes in a carpet-like mechanism by interacting with negatively charged phospholipid head groups, which displace divalent cations and enforce a negative curvature folding on membranes that leads to formation of vesicles/micelles. PMID:25304506

The antimicrobial mechanism of action of epsilon-poly-l-lysine.

PubMed

Hyldgaard, Morten; Mygind, Tina; Vad, Brian S; Stenvang, Marcel; Otzen, Daniel E; Meyer, Rikke L

2014-12-01

Epsilon-poly-l-lysine (ε-PL) is a natural antimicrobial cationic peptide which is generally regarded as safe (GRAS) as a food preservative. Although its antimicrobial activity is well documented, its mechanism of action is only vaguely described. The aim of this study was to clarify ε-PL's mechanism of action using Escherichia coli and Listeria innocua as model organisms. We examined ε-PL's effect on cell morphology and membrane integrity and used an array of E. coli deletion mutants to study how specific outer membrane components affected the action of ε-PL. We furthermore studied its interaction with lipid bilayers using membrane models. In vitro cell studies indicated that divalent cations and the heptose I and II phosphate groups in the lipopolysaccharide layer of E. coli are critical for ε-PL's binding efficiency. ε-PL removed the lipopolysaccharide layer and affected cell morphology of E. coli, while L. innocua underwent minor morphological changes. Propidium iodide staining showed that ε-PL permeabilized the cytoplasmic membrane in both species, indicating the membrane as the site of attack. We compared the interaction with neutral or negatively charged membrane systems and showed that the interaction with ε-PL relied on negative charges on the membrane. Suspended membrane vesicles were disrupted by ε-PL, and a detergent-like disruption of E. coli membrane was confirmed by atomic force microscopy imaging of supported lipid bilayers. We hypothesize that ε-PL destabilizes membranes in a carpet-like mechanism by interacting with negatively charged phospholipid head groups, which displace divalent cations and enforce a negative curvature folding on membranes that leads to formation of vesicles/micelles. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Dynamic Structure of Bombolitin II Bound to Lipid Bilayers as Revealed by Solid-state NMR and Molecular-Dynamics Simulation

PubMed Central

Toraya, Shuichi; Javkhlantugs, Namsrai; Mishima, Daisuke; Nishimura, Katsuyuki; Ueda, Kazuyoshi; Naito, Akira

2010-01-01

Bombolitin II (BLT2) is one of the hemolytic heptadecapeptides originally isolated from the venom of a bumblebee. Structure and orientation of BLT2 bound to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) membranes were determined by solid-state 31P and 13C NMR spectroscopy. 31P NMR spectra showed that BLT2-DPPC membranes were disrupted into small particles below the gel-to-liquid crystalline phase transition temperature (Tc) and fused to form a magnetically oriented vesicle system where the membrane surface is parallel to the magnetic fields above the Tc. 13C NMR spectra of site-specifically 13C-labeled BLT2 at the carbonyl carbons were observed and the chemical shift anisotropies were analyzed to determine the dynamic structure of BLT2 bound to the magnetically oriented vesicle system. It was revealed that the membrane-bound BLT2 adopted an α-helical structure, rotating around the membrane normal with the tilt angle of the helical axis at 33°. Interatomic distances obtained from rotational-echo double-resonance experiments further showed that BLT2 adopted a straight α-helical structure. Molecular dynamics simulation performed in the BLT2-DPPC membrane system showed that the BLT2 formed a straight α-helix and that the C-terminus was inserted into the membrane. The α-helical axis is tilted 30° to the membrane normal, which is almost the same as the value obtained from solid-state NMR. These results suggest that the membrane disruption induced by BLT2 is attributed to insertion of BLT2 into the lipid bilayers. PMID:21081076

Site-specific epsilon-NH2 monoacylation of pancreatic phospholipase A2. 2. Transformation of soluble phospholipase A2 into a highly penetrating "membrane-bound" form.

PubMed

Van der Wiele, F C; Atsma, W; Roelofsen, B; van Linde, M; Van Binsbergen, J; Radvanyi, F; Raykova, D; Slotboom, A J; De Haas, G H

1988-03-08

Long-chain lecithins present in bilayer structures like vesicles or membranes are only very poor substrates for pancreatic phospholipases A2. This is probably due to the fact that pancreatic phospholipases A2 cannot penetrate into the densely packed bilayer structures. To improve the weak penetrating properties of pancreatic phospholipases A2, we prepared and characterized a number of pancreatic phospholipase A2 mutants that have various long acyl chains linked covalently to Lys116 in porcine and to Lys10 in bovine phospholipase A2 [Van der Wiele, F.C., Atsma, W., Dijkman, R., Schreurs, A.M.M., Slotboom, A.J., & De Haas, G.H. (1988) Biochemistry (preceding paper in this issue)]. When monomolecular surface layers of L- and D-didecanoyllecithin were used, it was found that the introduction of caprinic, lauric, palmitic, and oleic acid at Lys116 in the porcine enzyme increases its penetrating power from 13 to about 17, 20, 32, and 22 dyn/cm, respectively, before long lag periods were obtained. Incorporation of a palmitoyl moiety at Lys10 in the bovine enzyme shifted the penetrating power from 11 to about 25 dyn/cm. Only the best penetrating mutant, viz., porcine phospholipase A2 having a palmitoyl moiety at Lys116, was able to cause complete leakage of 6-carboxyfluorescein entrapped in small unilamellar vesicles of egg lecithin under nonhydrolytic conditions. Similarly, only this latter palmitoylphospholipase A2 completely hydrolyzed all lecithin in the outer monolayer of the human erythrocyte at a rate much faster than Naja naja phospholipase A2, the most powerful penetrating snake venom enzyme presently known.

Lipid Bilayer Membrane in a Silicon Based Micron Sized Cavity Accessed by Atomic Force Microscopy and Electrochemical Impedance Spectroscopy

PubMed Central

Dosoky, Noura Sayed; Patel, Darayas; Weimer, Jeffrey; Williams, John Dalton

2017-01-01

Supported lipid bilayers (SLBs) are widely used in biophysical research to probe the functionality of biological membranes and to provide diagnoses in high throughput drug screening. Formation of SLBs at below phase transition temperature (Tm) has applications in nano-medicine research where low temperature profiles are required. Herein, we report the successful production of SLBs at above—as well as below—the Tm of the lipids in an anisotropically etched, silicon-based micro-cavity. The Si-based cavity walls exhibit controlled temperature which assist in the quick and stable formation of lipid bilayer membranes. Fusion of large unilamellar vesicles was monitored in real time in an aqueous environment inside the Si cavity using atomic force microscopy (AFM), and the lateral organization of the lipid molecules was characterized until the formation of the SLBs. The stability of SLBs produced was also characterized by recording the electrical resistance and the capacitance using electrochemical impedance spectroscopy (EIS). Analysis was done in the frequency regime of 10−2–105 Hz at a signal voltage of 100 mV and giga-ohm sealed impedance was obtained continuously over four days. Finally, the cantilever tip in AFM was utilized to estimate the bilayer thickness and to calculate the rupture force at the interface of the tip and the SLB. We anticipate that a silicon-based, micron-sized cavity has the potential to produce highly-stable SLBs below their Tm. The membranes inside the Si cavity could last for several days and allow robust characterization using AFM or EIS. This could be an excellent platform for nanomedicine experiments that require low operating temperatures. PMID:28678160

Lipid Bilayer Membrane in a Silicon Based Micron Sized Cavity Accessed by Atomic Force Microscopy and Electrochemical Impedance Spectroscopy.

PubMed

Khan, Muhammad Shuja; Dosoky, Noura Sayed; Patel, Darayas; Weimer, Jeffrey; Williams, John Dalton

2017-07-05

Supported lipid bilayers (SLBs) are widely used in biophysical research to probe the functionality of biological membranes and to provide diagnoses in high throughput drug screening. Formation of SLBs at below phase transition temperature ( Tm ) has applications in nano-medicine research where low temperature profiles are required. Herein, we report the successful production of SLBs at above-as well as below-the Tm of the lipids in an anisotropically etched, silicon-based micro-cavity. The Si-based cavity walls exhibit controlled temperature which assist in the quick and stable formation of lipid bilayer membranes. Fusion of large unilamellar vesicles was monitored in real time in an aqueous environment inside the Si cavity using atomic force microscopy (AFM), and the lateral organization of the lipid molecules was characterized until the formation of the SLBs. The stability of SLBs produced was also characterized by recording the electrical resistance and the capacitance using electrochemical impedance spectroscopy (EIS). Analysis was done in the frequency regime of 10 -2 -10⁵ Hz at a signal voltage of 100 mV and giga-ohm sealed impedance was obtained continuously over four days. Finally, the cantilever tip in AFM was utilized to estimate the bilayer thickness and to calculate the rupture force at the interface of the tip and the SLB. We anticipate that a silicon-based, micron-sized cavity has the potential to produce highly-stable SLBs below their Tm . The membranes inside the Si cavity could last for several days and allow robust characterization using AFM or EIS. This could be an excellent platform for nanomedicine experiments that require low operating temperatures.

In vitro effects of the anti-Alzheimer drug memantine on the human erythrocyte membrane and molecular models

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

Zambrano, Pablo; Suwalsky, Mario; Villena, Fernando

Memantine is a NMDA antagonist receptor clinically used for treating Alzheimer's disease. NMDA receptors are present in the human neurons and erythrocyte membranes. The aim of the present study was to investigate the effects of memantine on human erythrocytes. With this purpose, the drug was developed to in vitro interact with human red cells and bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE). The latter represent lipids respectively present in both outer and inner monolayers of the red cell membrane. Results obtained by scanning electron microscopy (SEM) showed that memantine changed the normal biconcave shape of red cells to cup-shaped stomatocytes.more » According to the bilayer-couple hypothesis the drug intercalated into the inner monolayer of the erythrocyte membrane. Experimental results obtained by X-ray diffraction on multibilayers of DMPC and DMPE, and by differential scanning calorimetry on multilamellar vesicles indicated that memantine preferentially interacted with DMPC in a concentration-dependent manner. Thus, it can be concluded that in the low therapeutic plasma concentration of circa 1 μM memantine is located in NMDA receptor channel without affecting the erythrocyte shape. However, at higher concentrations, once the receptors became saturated excess of memantine molecules (20 μM) would interact with phosphoinositide lipids present in the inner monolayer of the erythrocyte membrane inducing the formation of stomatocytes. However, 40–50 μM memantine was required to interact with isolated phosphatidylcholine bilayers. - Highlights: • The interaction of memantine with human erythrocytes and lipid bilayers were assessed. • Memantine induced morphological changes to human erythrocytes. • Memantine interacted with classes of phospholipids present in the erythrocyte membrane. • Results support the hypothesis that memantine interacts with NMDA receptors.« less

The singular behavior of a β-type semi-synthetic two branched polypeptide: three-dimensional structure and mode of action.

PubMed

Manzo, Giorgia; Serra, Ilaria; Pira, Alessandro; Pintus, Manuela; Ceccarelli, Matteo; Casu, Mariano; Rinaldi, Andrea C; Scorciapino, Mariano Andrea

2016-11-16

Dendrimeric peptides make a versatile group of bioactive peptidomimetics and a potential new class of antimicrobial agents to tackle the pressing threat of multi-drug resistant pathogens. These are branched supramolecular assemblies where multiple copies of the bioactive unit are linked to a central core. Beyond their antimicrobial activity, dendrimeric peptides could also be designed to functionalize the surface of nanoparticles or materials for other medical uses. Despite these properties, however, little is known about the structure-function relationship of such compounds, which is key to unveil the fundamental physico-chemical parameters and design analogues with desired attributes. To close this gap, we focused on a semi-synthetic, two-branched peptide, SB056, endowed with remarkable activity against both Gram-positive and Gram-negative bacteria and limited cytotoxicity. SB056 can be considered the smallest prototypical dendrimeric peptide, with the core restricted to a single lysine residue and only two copies of the same highly cationic 10-mer polypeptide; an octanamide tail is present at the C-terminus. Combining NMR and Molecular Dynamics simulations, we have determined the 3D structure of two analogues. Fluorescence spectroscopy was applied to investigate the water-bilayer partition in the presence of vesicles of variable charge. Vesicle leakage assays were also performed and the experimental data were analyzed by applying an iterative Monte Carlo scheme to estimate the minimum number of bound peptides needed to achieve the release. We unveiled a singular beta hairpin-type structure determined by the peptide chains only, with the octanamide tail available for further functionalization to add new potential properties without affecting the structure.

Apolipoprotein C-III Nanodiscs Studied by Site-Specific Tryptophan Fluorescence.

PubMed

Brisbois, Chase A; Lee, Jennifer C

2016-09-06

Apolipoprotein C-III (ApoC-III) is found on high-density lipoproteins (HDLs) and remodels 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles into HDL-like particles known as nanodiscs. Using single-Trp-containing ApoC-III mutants, we have studied local side chain environments and interactions in nanodiscs at positions W42, W54, and W65. Using transmission electron microscopy and circular dichroism spectroscopy, nanodiscs were characterized at the ultrastructural and secondary conformational levels, respectively. Nearly identical particles (15 ± 2 nm) were produced from all proteins containing approximately 25 ± 4 proteins per particle with an average helicity of 45-51% per protein. Distinct residue-specific fluorescence properties were observed with W54 residing in the most hydrophobic environment followed by W42 and W65. Interestingly, time-resolved anisotropy measurements revealed that Trp side chain mobility is uncorrelated to the polarity of its surroundings. W54 is the most mobile compared to W65 and W42, which are more immobile in a nanodisc-bound state. On the basis of Trp spectral comparisons of ApoC-III in micellar and vesicle environments, ApoC-III binding within nanodiscs more closely resembles a bilayer-bound state. Despite the nanodiscs being structurally similar, we found marked differences during nanodisc formation by the Trp variants as a function of temperature, with W42 behaving the most like the wild-type protein. Our data suggest that despite the modest mutations of Trp to Phe at two of the three native sites, the interfacial location of W42 is important for lipid binding and nanodisc assembly, which may be biologically meaningful as of the three Trp residues, only W42 is invariant among mammals.