Fluid Lipid Bilayer Membranes Research Articles

Detecting asymmetry and lateral heterogeneity caused by antimicrobial peptides in fluid lipid bilayer membranes

Detecting asymmetry and lateral heterogeneity caused by antimicrobial peptides in fluid lipid bilayer membranes

Fixed DNA Molecule Arrays for High-Throughput Single DNA-Protein Interaction Studies.

The DNA Curtains assay is a recently developed experimental platform for protein-DNA interaction studies at the single-molecule level that is based on anchoring and alignment of DNA fragments. The DNA Curtains so far have been made by using chromium barriers and fluid lipid bilayer membranes, which makes such a specialized assay technically challenging and relatively unstable. Herein, we report on an alternative strategy for DNA arraying for analysis of individual DNA-protein interactions. It relies on stable DNA tethering onto nanopatterned protein templates via high affinity molecular recognition. We describe fabrication of streptavidin templates (line features as narrow as 200 nm) onto modified glass coverslips by combining surface chemistry, atomic force microscopy (AFM), and soft lithography techniques with affinity-driven assembly. We have employed such chips for arraying single- and double-tethered DNA strands, and we characterized the obtained molecular architecture: we evaluated the structural characteristics and specific versus nonspecific binding of fluorescence-labeled DNA using AFM and total internal reflection fluorescence microscopy. We demonstrate the feasibility of our DNA molecule arrays for short single-tethered as well as for lambda single- and double-tethered DNA. The latter type of arrays proved very suitable for localization of single DNA-protein interactions employing restriction endonucleases. The presented molecular architecture and facile method of fabrication of our nanoscale platform does not require clean room equipment, and it offers advanced functional studies of DNA machineries and the development of future nanodevices.

Recent advances in liposome surface modification for oral drug delivery.

Oral delivery via the gastrointestinal (GI) tract is the dominant route for drug administration. Orally delivered liposomal carriers can enhance drug solubility and protect the encapsulated theraputic agents from the extreme conditions found in the GI tract. Liposomes, with their fluid lipid bilayer membrane and their nanoscale size, can significantly improve oral absorption. Unfortunately, the clinical applications of conventional liposomes have been hindered due to their poor stability and availability under the harsh conditions typically presented in the GI tract. To overcome this problem, the surface modification of liposomes has been investigated. Although liposome surface modification has been extensively studied for oral drug delivery, no review exists so far that adequately covers this topic. The purpose of this paper is to summarize and critically analyze emerging trends in liposome surface modification for oral drug delivery.

Drag force on a liquid domain moving inside a membrane sheet surrounded by aqueous medium

We compute the drag on a circular and liquid microdomain diffusing in a two-dimensional fluid lipid bilayer membrane surrounded by a fluid above and below. Under the assumptions that the liquids are incompressible and the flow is of low Reynolds number, Stokes’ equations describe the flow in the two-dimensional membrane as well as in the surrounding three-dimensional fluid. The expression for the drag force on the liquid domain involves Fredholm integral equations of the second kind, which we numerically solve using discrete collocation method based on Chebyshev polynomials. We observe that when the domain is more viscous than the surrounding membrane (including the rigid domain case), the drag force is almost independent of the viscosity contrast between the domain and the surrounding membrane, as also observed earlier in experiments by other researchers. The mobility also varies logarithmically with Boussinesq number${\it\beta}$for large${\it\beta}$. On the other hand, for a less viscous domain the dimensionless drag force reduces with increasing viscosity contrast, and a significant change in the drag force, from that when there is no viscosity contrast or when the domain is rigid, has been observed. Further, the logarithmic behaviour of the mobility no longer holds for less viscous domains. Our method of computing the drag force and diffusion coefficient is valid for arbitrary viscosity contrast between the domain and membrane and any domain size (subject to${\it\beta}\geqslant 5$).

PI3 kinase enzymology on fluid lipid bilayers

We report the use of fluid lipid bilayer membrane as a model platform to study the influence of the bilayer microenvironment and composition on the enzymology in membrane. As a model system we determined the enzyme kinetics on membranes for the transformation of bilayers containing phosphoinositol(4,5)-bisphosphate (PI(4,5)P2) to phosphoinositol(3,4,5)-trisphosphate (PI(3,4,5)P3) by the enzyme phosphoinositol-3-kinase (PI3K) using radiolabeled ATP. The activity of the enzyme was monitored as a function of the radioactivity incorporated within the bilayer. The transformation of PI(4,5)P2 to PI(3,4,5)P3 was determined using a mass strip assay. The fluidity of the bilayer was confirmed by Fluorescence Recovery After Photobleaching (FRAP) experiments. Kinetic simulations were performed based on Langmuir adsorption and Michaelis-Menton kinetics equations to generate the rate constants for the enzymatic reaction. The effect of cholesterol on the enzyme kinetics was studied by doping the bilayer with 1% cholesterol. This leads to significant reduction in reaction rate due to change in membrane microenvironment. This strategy provides a method to study the enzymology of various kinases and phosphatases occurring at the membrane and also how these reactions are affected by the membrane composition and surface microenvironment.

Lipid Bilayer-Integrated Optoelectronic Tweezers for Nanoparticle Manipulations

Remotely manipulating a large number of microscopic objects is important to soft-condensed matter physics, biophysics, and nanotechnology. Optical tweezers and optoelectronic tweezers have been widely used for this purpose but face critical challenges when applied to nanoscale objects, including severe photoinduced damages, undesired ionic convections, or irreversible particle immobilization on surfaces. We report here the first demonstration of a lipid bilayer-integrated optoelectronic tweezers system for simultaneous manipulation of hundreds of 60 nm gold nanoparticles in an arbitrary pattern. We use a fluid lipid bilayer membrane with a ~5 nm thickness supported by a photoconductive electrode to confine the diffusion of chemically tethered nanoparticles in a two-dimensional space. Application of an external a.c. voltage together with patterned light selectively activates the photoconducting electrode that creates strong electric field localized near the surface. The field strength changes most significantly at the activated electrode surface where the particles tethered to the membrane thus experience the strongest dielectrophoretic forces. This design allows us to efficiently achieve dynamic, reversible, and parallel manipulation of many nanoparticles. Our approach to integrate biomolecular structures with optoelectronic devices offers a new platform enabling the study of thermodynamics in many particle systems and the selective transport of nanoscale objects for broad applications in biosensing and cellular mechanotransductions.

Equilibrium Configurations of Lipid Bilayer Membranes and Carbon Nanostructures

The present article concerns the continuum modelling of the mechanical behaviour and equilibrium shapes of two types of nano-scale objects: fluid lipid bilayer membranes and carbon nanostructures. A unified continuum model is used to handle four different case studies. Two of them consist in representing in analytic form cylindrical and axisymmetric equilibrium configurations of single-wall carbon nanotubes and fluid lipid bilayer membranes subjected to uniform hydrostatic pressure. The third one is concerned with determination of possible shapes of junctions between a single-wall carbon nanotube and a fiat graphene sheet or another single-wall carbon nanotube. The last one deals with the mechanical behaviour of closed fluid lipid bilayer membranes (vesicles) adhering onto a fiat homogeneous rigid substrate subjected to micro-injection and uniform hydrostatic pressure.

Deformation of injected vesicles adhering onto flat rigid substrates

This study is concerned with the determination of the mechanical behaviour of closed fluid lipid bilayer membranes (vesicles) under a uniform hydrostatic pressure, pressed against and adhering onto a flat homogeneous rigid substrate. Assuming that the initial and deformed shapes of the vesicle are axisymmetric, a variational statement of the problem is developed on the ground of the so-called spontaneous curvature model. In this setting, the vesicle is regarded as a closed surface in the three-dimensional Euclidean space and its equilibrium shapes are supposed to provide stationary values of the bending energy functional under the constraint of fixed total area. The corresponding Euler–Lagrange equations and natural boundary conditions are derived, the work done by the pressure being taken into account, and used to determine the forces and moments in the membrane. Several examples of surfaces representing possible equilibrium shapes of so loaded membranes are determined numerically.

Supported membranes embedded with fixed arrays of gold nanoparticles.

We present a supported membrane platform consisting of a fluid lipid bilayer membrane embedded with a fixed array of gold nanoparticles. The system is realized by preforming a hexagonal array of gold nanoparticles (∼5–7 nm) with controlled spacing (∼50–150 nm) fixed to a silica or glass substrate by block copolymer lithography. Subsequently, a supported membrane is assembled over the intervening bare substrate. Proteins or other ligands can be associated with the fluid lipid component, the fixed nanoparticle component, or both, providing a hybrid interface consisting of mobile and immobile components with controlled geometry. We test different biochemical coupling strategies to bind individual proteins to the particles surrounded by a fluid lipid membrane. The coupling efficiency to nanoparticles and the influence of nanoparticle arrays on the surrounding membrane integrity are characterized by fluorescence imaging, correlation spectroscopy, and super-resolution fluorescence microscopy. Finally, the functionality of this system for live cell experiments is tested using the ephrin-A1–EphA2 juxtacrine signaling interaction in human breast epithelial cells.

Drug binding and mobility relating to the thermal fluctuation in fluid lipid membranes

Drug binding and mobility in fluid lipid bilayer membranes are quantified in situ by using the multinuclear solution NMR combined with the pulsed-field-gradient technique. One-dimensional and pulsed-field-gradient (19)F and (1)H NMR signals of an anticancer drug, 5-fluorouracil (5FU) are analyzed at 283-313 K in the presence of large unilamellar vesicles (LUVs) of egg phosphatidylcholine (EPC) as model cell membranes. The simultaneous observation of the membrane-bound and free 5FU signals enables to quantify in what amount of 5FU is bound to the membrane and how fast 5FU is moving within the membrane in relation to the thermal fluctuation of the soft, fluid environment. It is shown that the mobility of membrane-bound 5FU is slowed down by almost two orders of magnitude and similar to the lipid movement in the membrane, the movement closely related to the intramembrane fluidity. The mobility of 5FU and EPC is, however, not similar at 313 K; the 5FU movement is enhanced in the membrane as a result of the loose binding of 5FU in the lipid matrices. The membrane-bound fraction of 5FU is approximately 0.1 and almost unaltered over the temperature range examined. It is also independent of the 5FU concentration from 2 to 30 mM with respect to the 40-50 mM LUV. The free energy of the 5FU binding is estimated at -4 to -2 kJ/mol, the magnitude always close to the thermal fluctuation, 2.4-2.6 kJ/mol.

Cylindrical equilibrium shapes of fluid membranes

Within the framework of the well-known curvature models, a fluid lipid bilayer membrane is regarded as a surface embedded in the three-dimensional Euclidean space whose equilibrium shapes are described in terms of its mean and Gaussian curvatures by the so-called membrane shape equation. In the present paper, all solutions to this equation determining cylindrical membrane shapes are found and presented, together with the expressions for the corresponding position vectors, in explicit analytic form. The necessary and sufficient conditions for such a surface to be closed are derived and several sufficient conditions for its directrix to be simple or self-intersecting are given.

Flicker Spectroscopy of Thermal Lipid Bilayer Domain Boundary Fluctuations

This contribution describes measurements of lipid bilayer domain line tension based on two-dimensional thermal undulations of membranes with liquid ordered/liquid disordered phase coexistence and near-critical composition at room temperature. Lateral inhomogeneity of lipid and protein composition is currently a subject of avid research aimed at determining both fundamental properties and biological relevance of membrane domains. Line tension at fluid lipid bilayer membrane domain boundaries controls the kinetics of domain growth and therefore regulates the size of compositional heterogeneities. High line tension promotes membrane domain budding and fission. Line tension could therefore be an important control parameter regulating functional aspects of biological membranes. Here the established method of fluid domain flicker spectroscopy is applied to examine thermal domain wall fluctuations of phase-separated bilayer membranes. We find a Gaussian probability distribution for the first few excited mode amplitudes, which permits an analysis by means of appropriately specialized capillary wave theory. Time autocorrelation functions are found to decay exponentially, and relaxation times are fitted by means of a hydrodynamic theory relating line tensions and excited mode relaxation kinetics. Line tensions below 1 pN are obtained, with these two approaches yielding similar results. We examine experimental artifacts that perturb the Fourier spectrum of domain traces and discuss ways to identify the number of modes that yield reliable line tension information.

TOAC Spin Labels in the Backbone of Alamethicin: EPR Studies in Lipid Membranes

Alamethicin is a 19-amino-acid residue hydrophobic peptide that produces voltage-dependent ion channels in membranes. Analogues of the Glu(OMe) 7,18,19 variant of alamethicin F50/5 that are rigidly spin-labeled in the peptide backbone have been synthesized by replacing residue 1, 8, or 16 with 2,2,6,6-tetramethyl-piperidine-1-oxyl-4-amino-4-carboxyl (TOAC), a helicogenic nitroxyl amino acid. Conventional electron paramagnetic resonance spectra are used to determine the insertion and orientation of the TOAC n alamethicins in fluid lipid bilayer membranes of dimyristoyl phosphatidylcholine. Isotropic 14N-hyperfine couplings indicate that TOAC 8 and TOAC 16 are situated in the hydrophobic core of the membrane, whereas the TOAC 1 label resides closer to the membrane surface. Anisotropic hyperfine splittings show that alamethicin is highly ordered in the fluid membranes. Experiments with aligned membranes demonstrate that the principal diffusion axis lies close to the membrane normal, corresponding to a transmembrane orientation. Combination of data from the three spin-labeled positions yields both the dynamic order parameter of the peptide backbone and the intramolecular orientations of the TOAC groups. The latter are compared with x-ray diffraction results from alamethicin crystals. Saturation transfer electron paramagnetic resonance, which is sensitive to microsecond rotational motion, reveals that overall rotation of alamethicin is fast in fluid membranes, with effective correlation times <30 ns. Thus, alamethicin does not form large stable aggregates in fluid membranes, and ionic conductance must arise from transient or voltage-induced associations.

Biosensing Using Lipid Bilayers Suspended on Porous Silicon

We demonstrate for the first time the formation of a fluid lipid bilayer membrane on mesoporous silicon substrates for bioapplications. Using fluorescence recovery after photobleaching, the diffusion coefficients for the bilayers supported on oxidized, amino-, and biotin-functionalized mesoporous silicon were determined. The biodetection of a single human umbilical vein endothelial cell was accomplished using confocal microscopy and exploiting Foerster resonance energy transfer effects after the incorporation of RGD covalently linked lipid soluble dyes, with fluorescence donor and acceptor components, within the fluid membrane. A signal response of greater than 100% was achieved via the clustering of RGD peptides binding with areas of high integrin density on the surface of a single cell. These results are a testament to the usefulness of such functional molecular assemblies, based on mobile receptors, mimicking the cell membrane in the development of a new generation of biosensors.

Constant-pressure and constant-surface tension simulations in dissipative particle dynamics

We present a method for constant-pressure and constant-surface tension simulations in dissipative particle dynamics using a Langevin piston approach. We demonstrate that the corresponding equations of motion lead to the relevant ensembles and propose an appropriate scheme of integration. After having identified a suitable set of parameters for the approach, we demonstrate the feasibility of the approach by applying it to two different systems, a simple isotropic fluid and an anisotropic fluid lipid-bilayer membrane in water. Results are presented for, respectively, isothermal bulk compressibility, tracer diffusion coefficient, lipid head-group area, and isothermal area compressibility. We find that our Langevin piston approach leads to improvements over other approaches in terms of faster equilibration and shorter correlation times of various system variables.

Dynamics of shape fluctuations of quasi-spherical vesicles revisited.

In this paper, the dynamics of spontaneous shape fluctuations of a single, giant quasi-spherical vesicle formed from a single lipid species is revisited theoretically. A coherent physical theory for the dynamics is developed based on a number of fundamental principles and considerations, and a systematic formulation of the theory is also established. From the systematic theoretical formulation, an analytical description of the dynamics of shape fluctuations of quasi-spherical vesicles is derived. In particular, in developing the theory we have made a new interpretation of some of the phenomenological constants in a canonical continuum description of fluid lipid-bilayer membranes and shown the consequences of this new interpretation in terms of the characteristics of the dynamics of vesicle shape fluctuations. Moreover, we have used the systematic formulation of our theory as a framework against which we have discussed the previously existing theories and their discrepancies. Finally, we have made a systematic prediction about the system-dependent characteristics of the relaxation dynamics of shape fluctuations of quasi-spherical vesicles with a view of experimental studies of the phenomenon and also discussed, based on our theory, a recently published experimental work on the topic.

Patterning Barriers to Lateral Diffusion in Supported Lipid Bilayer Membranes by Blotting and Stamping

Two new methods are introduced for patterning fluid lipid bilayer membranes on solid supports. These methods are based on the observation that supported membranes undergo self-limiting lateral expansion when bilayer material is removed from the surface or when it is deposited in a pattern on a surface. Spatially selective (patterned) removal of bilayer material can be achieved by using a poly(dimethylsiloxane) (PDMS) stamp. Following slight lateral expansion into the bilayer-free region created by this blotting process, stable barriers to lateral diffusion are formed. Inspection of the barrier regions indicates that nearly all of the bilayer material is removed, implying that it has been transferred to the stamp. As a consequence, it also proves possible to transfer the lifted material from the stamp onto a fresh surface. The transferred material retains the original pattern from the stamp and is also laterally mobile, and the mobility is confined to the printed region. Alternatively, bilayers assembled o...

Electroporation of cells and tissues

Electrical pulses that cause the transmembrane voltage of fluid lipid bilayer membranes to reach at least U/sub m//spl ap/0.2 V, usually 0.5-1 V, are hypothesized to create primary membrane "pores" with a minimum radius of -1 nm. Transport of small ions such as Na/sup +/ and Cl/sup -/ through a dynamic pore population discharges the membrane even while an external pulse tends to increase U/sub m/, leading to dramatic electrical behavior. Molecular transport through primary pores and pores enlarged by secondary processes provides the basis for transporting molecules into and out of biological cells. Cell electroporation in vitro is used mainly for transfection by DNA introduction, but many other interventions are possible, including microbial killing. Ex vivo electroporation provides manipulation of cells that are reintroduced into the body to provide therapy. In vivo electroporation of tissues enhances molecular transport through tissues and into their constitutive cells. Tissue electroporation, by longer, large pulses, is involved in electrocution injury. Tissue electroporation by shorter, smaller pulses is under investigation for biomedical engineering applications of medical therapy aimed at cancer treatment, gene therapy, and transdermal drug delivery. The latter involves a complex barrier containing both high electrical resistance, multilamellar lipid bilayer membranes and a tough, electrically invisible protein matrix.

Sequence-related behaviour of transmembrane domains from class I receptor tyrosine kinases

2 H NMR spectroscopy and freeze-fracture electron microscopy were used to compare the transmembrane domains of two Class I protein receptor tyrosine kinases (the EGF receptor and Neu/erbB-2) regarding overall behaviour in fluid lipid bilayer membranes. The 34-residue peptide, EGFR tm, was synthesised to contain the 23 amino acid hydrophobic stretch (Ile 622 to Met 644) thought to span the membrane of the human EGF receptor, plus the first 10 amino acids (Arg 645 to Thr 654) of the cytoplasmic domain. Deuterium probes replaced selected 1 H nuclei at sites corresponding to Ala 623, Met 644, and Val 650. The 38-residue peptide, Neu tm, was synthesised having the 21 residue hydrophobic stretch (Ile 660 to Ile 680) calculated to span the membrane in rat Neu/erbB-2, plus residues Lys 681 to Thr 691 of the contiguous cytoplasmic domain. Deuterium probes replaced selected 1 H nuclei at Ala 661, Leu 667, and Val 676. A third peptide, Neu tm*, was also prepared, corresponding to the transmembrane domain of a constitutively-activating Neu/erbB-2 transformant in which Val 664 is replaced by Glu: it was deuterated in a manner identical to Neu tm. Peptides were studied by 2 H NMR spectroscopy at 1 mol% and 6 mol% in unsonicated fluid bilayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and in POPC containing 33 mol% cholesterol, over the range 12° to 65°C. Overall motion was found to be different for each of the three peptides under a given set of conditions. EGFR tm spectra were characteristic of axially symmetric motion in membranes of POPC alone, and in POPC/cholesterol at 35°C and above. In contrast, spectra of the transmembrane peptides, Neu tm and Neu tm*, were characteristic of significantly axially asymmetric motion under all conditions studied (and regardless of sample preparation method). Addition of 33% cholesterol to membranes was accompanied by spectral changes consistent with increased formation of peptide dimers/oligomers in all cases. The transformant peptide, Neu tm*, showed greater spectral evidence of immobilisation than did the wild type - probably reflecting a greater tendency to form large oligomers. Sequence-related details within the transmembrane domains of Class I receptor tyrosine kinases appear to exert important control over their associations within membranes. Freeze-fracture electron microscopy of the NMR samples demonstrated their liposomal nature. Peptide-related intramembranous particles (IMPs) were present which likely represent oligomers of the transmembrane peptide. IMP size and distribution were similar under a given set of conditions for all three peptides, suggesting that the differences seen by NMR spectroscopy reflect structures smaller than the 2 nm resolution limit of freeze-fracture EM and peptide relationships within its 20 nm accuracy of identifying lateral position.