Fluorimetric detection of phospholipid vesicles bound to planar phospholipid membranes.

The first step in the fusion of two phospholipid membranes culminates in the aggregation of the two lipid bilayers. We have used a custom-built fluorimeter to detect multilamellar vesicles (liposomes) containing the fluorescent dye, 6-carboxyfluorescein (6-CF), bound to a planar lipid bilayer (BLM). Liposomes were added to one side of the BLM, and unbound vesicles were perfused out. This left a residual fluorescence from the BLM, but only when the membranes contained anionic lipids, and then only when millimolar levels of calcium were present. This residual fluorescence was consistently detected only when calcium was included in the buffer during the perfusion. This residual fluorescence originated from liposomes bound to the BLM. Breaking the BLM or lysing the adsorbed vesicles with distilled water abolished it. free 6-CF and/or calcium in the absence of liposomes resulted in no residual fluorescence. No residual fluorescence was detected when both the liposomes and the BLM were composed entirely of zwitterionic lipids. This was found to result from the insensitivity of the fluorimeter to a small number of liposomes adsorbed to the BLM. For this system, we conclude that calcium is necessary for both the initiation and maintenance of the state in which the vesicle membrane is bound to the planar bilayer when the membranes contain negatively charged lipids. This attachment is stronger than the interaction between zwitterionic membranes.     

Formation of supported phospholipid bilayers via co-adsorption with beta-D-dodecyl maltoside

We have investigated the formation of supported model membranes via the adsorption of phospholipid-surfactant mixtures at the Si-water interface by specular neutron reflection. The adsorption of mixed micelles of the nonionic surfactant beta-D-dodecyl maltoside and DOPC or POPC was determined as a function of bulk concentration, and using d25-beta-D-dodecyl maltoside, the composition of DOPC and POPC bilayers was determined. Bilayer thicknesses of 39+/-3 A for DOPC and 41+/-3 A for POPC agree well with data from bulk lamellar phases for both lipids, and the average area per lipid molecule can be varied from 62 to 115 A2 by varying the bulk concentrations used. The amount of surfactant in the bilayer is very sensitive to the bulk volume-to-surface area ratio, but it can be fully eliminated by ensuring a sufficiently large dilution/rinsing volume of the solution.     

Protein-mediated intermembrane contact facilitates fusion of lipid vesicles with planar bilayers

Fusion of phospholipid vesicles with planar bilayer membranes occurs provided there is an intermembrane contact, which can be mediated by phospholipid-binding proteins, even in the absence of calcium. The firm attachment phase is then followed by the osmotically-driven fusion. These results show that hydrophobic proteins (not necessarily Ca²⁺-binding proteins) may enhance fusion by promoting contact of membranes. Such proteins may operate synergistically with Ca²⁺ to reduce the threshold concentration of Ca²⁺ needed for fusion of biological membranes. Protein-mediated intermembrane contact resulting in fusion may play a crucial role in the regulation and catalysis of biological fusion events.     

Natively folded HypF-N and its early amyloid aggregates interact with phospholipid monolayers and destabilize supported phospholipid bilayers

Recent data depict membranes as the main sites where proteins/peptides are recruited and concentrated, misfold, and nucleate amyloids; at the same time, membranes are considered key triggers of amyloid toxicity. The N-terminal domain of the prokaryotic hydrogenase maturation factor HypF (HypF-N) in 30% trifluoroethanol undergoes a complex path of fibrillation starting with initial 2-3-nm oligomers and culminating with the appearance of mature fibrils. Oligomers are highly cytotoxic and permeabilize lipid membranes, both biological and synthetic. In this article, we report an in-depth study aimed at providing information on the surface activity of HypF-N and its interaction with synthetic membranes of different lipid composition, either in the native conformation or as amyloid oligomers or fibrils. Like other amyloidogenic peptides, the natively folded HypF-N forms stable films at the air/water interface and inserts into synthetic phospholipid bilayers with efficiencies depending on the type of phospholipid. In addition, HypF-N prefibrillar aggregates interact with, insert into, and disassemble supported phospholipid bilayers similarly to other amyloidogenic peptides. These results support the idea that, at least in most cases, early amyloid aggregates of different peptides and proteins produce similar effects on the integrity of membrane assembly and hence on cell viability.     

Calcium-induced fusion of sea urchin egg secretory vesicles with planar phospholipid bilayer membranes.

The fusion of sea urchin egg secretory vesicles to planar phospholipid bilayer membranes was studied by differential interference contrast (DIC) and fluorescent microscopy, in combination with electrical recordings of membrane conductance. A strong binding of vesicles to protein-free planar membranes was observed in the absence of calcium. Calcium-induced fusion of vesicles was detected using two independent assays: loss of the contents of individual vesicles visible by DIC microscopy: and vesicle content discharge across the planar membrane detected by an increase in the fluorescence of a dye. In both cases, no increase in the membrane conductance was observed unless vesicles were incubated with either Amphotericin B or digitonin prior to applying them to the planar membrane, an indication that native vesicles are devoid of open channels. Pre-incubation of vesicles with n-ethylmaleimide (NEM) abolished calcium-induced fusion. Fusion was also detected when vesicles were osmotically swollen to the point of lysis. In contrast, no fusion of vesicles to planar bilayers was seen when vesicles on plasma membrane (native cortices) were applied to a phospholipid membrane, despite good binding of vesicles to the planar membrane and fusion of vesicles to plasma membrane. It is suggested that cortical vesicles (CVs) have sufficient calcium-sensitive proteins for fusion to lipid membranes, but in native cortices granular fusion sites are oriented toward the plasma membrane. Removal of vesicles from the plasma membrane may allow fusion sites on vesicles access to new membranes.     

Parameters affecting the fusion of unilamellar phospholipid vesicles with planar bilayer membranes

It was previously shown (Cohen, F. S., J. Zimmerberg, and A. Finkelstein, 1980, J. Gen. Physiol., 75:251-270) that multilamellar phospholipid vesicles can fuse with decane-containing phospholipid bilayer membranes. An essential requirement for fusion was an osmotic gradient across the planar membrane, with the vesicle-containing (cis) side hyperosmotic with respect to the opposite (trans) side. We now report that unilamellar vesicles will fuse with "hydrocarbon-free" membranes subject to these same osmotic conditions. Thus the same conditions that apply to fusion of multilamellar vesicles with planar bilayer membranes also apply to fusion of unilamellar vesicles with these membranes, and hydrocarbon is not required for the fusion process. If the vesicles and/or planar membrane contain negatively charged lipids, divalent cation (approximately 15 mM Ca++) is required in the cis compartment (in addition to the osmotic gradient across the membrane) to obtain substantial fusion rates. On the other hand, vesicles made from uncharged lipids readily fuse with planar phosphatidylethanolamine planar membranes in the near absence of divalent cation with just an osmotic gradient. Vesicles fuse much more readily with phosphatidylethanolamine-containing than with phosphatidylcholine-containing planar membranes. Although hydrocarbon (decane) is not required in the planar membrane for fusion, it does affect the rate of fusion and causes the fusion process to be dependent on stirring in the cis compartment.     

Atomic force microscopy of supported planar membrane bilayers.

Membrane bilayers of dipalmitoyl phosphatidylcholine (DPPC) and dipalmitoyl phosphatidylethanolamine (DPPE) adsorbed to a freshly cleaved mica substrate have been imaged by Atomic Force Microscopy (AFM). The membranes were mounted for imaging by two methods: (a) by dialysis of a detergent solution of the lipid in the presence of the substrate material, and (b) by adsorption of lipid vesicles onto the substrate surface from a vesicle suspension. The images were taken in air, and show lipid bilayers adhering to the surface either in isolated patches or in continuous sheets, depending on the deposition conditions. Epifluorescence light-microscopy shows that the lipid is distributed on the substrate surfaces as seen in the AFM images. In some instances, when DPPE was used, whole, unfused vesicles, which were bound to the substrate, could be imaged by the AFM. Such membranes should be capable of acting as natural anchors for imaging membrane proteins by AFM.     

Filipin-induced lesions in planar phospholipid bilayers imaged by atomic force microscopy.

Filipin is a macrolide polyene with antifungal activity belonging to the same family of antibiotics as amphotericin B and nystatin. Despite the spectroscopy and electron microscopy studies of its interaction with natural membranes and membrane model systems, several aspects of its biochemical action, such as the role of membrane sterols, remain to be completely understood. We have used atomic force microscopy (AFM) to study the effect of filipin on dipalmitoylphosphatidylethanolamine bilayers in the presence and absence of cholesterol. The bilayers were prepared by Langmuir-Blodgett deposition over mica and imaged under water. It was shown that filipin-induced lesions could only be found in membranes with cholesterol. In close agreement with electron microscopy results, we have reported the presence of densely packed circular protrusions in the membrane with a mean diameter of 19 nm (corrected for convolution with AFM tip) and 0.4 nm height. Larger circular protrusions (90 nm diameter and 2.5 nm height) and doughnut-shaped lesions were also detected. These results demonstrate that filipin-induced lesions in membranes previously observed by electron microscopy are not biased by artifacts resulting from sample preparation. Filipin aggregates in aqueous solution could also be imaged for the first time. These polydisperse spherical structures were observed in samples with and without cholesterol.     

Electric field-induced concentration gradients in planar supported bilayers.

A simple method of generating electric field-induced concentration gradients in planar supported bilayers has been developed. Gradients of charged, fluorescently labeled probes were visualized by epifluorescence microscopy and could be observed at field strengths as low as 1 V/cm. Steady-state concentration gradients can be described by a simple competition between random diffusion and electric field-induced drift. A model based on this principle has been used to determine the diffusion coefficient of the fluorescent probes. This technique achieves a degree of electrical manipulation of supported bilayers that offers a variety of possibilities for the development of new molecular architectures and the study of biological membranes.     

Calcium-induced fusion of sea urchin egg secretory vesicles with planar phospholipid bilayer membranes

The fusion of sea urchin egg secretory vesicles to planar phospholipid bilayer membranes was studied by differential interference contrast (DIC) and fluorescent microscopy, in combination with electrical recordings of membrane conductance. A strong binding of vesicles to protein-free planar membranes was observed in the absence of calcium. Calciuminduced fusion of vesicles was detected using two independent assays: loss of the contents of individual vesicles visible by DIC microscopy; and vesicle content discharge across the planar membrane detected by an increase in the fluorescence of a dye. In both cases, no increase in the membrane conductance was observed unless vesicles were incubated with either Amphotericin B or digitonin prior to applying them to the planar membrane, an indication that native vesicles are devoid of open channels. Pre-incubation of vesicles with n-ethylmaleimide (NEM) abolished calcium-induced fusion. Fusion was also detected when vesicles were osmotically swollen to the point of lysis. In contrast, no fusion of vesicles to planar bilayers was seen when vesicles on plasma membrane (native cortices) were applied to a phospholipid membrane, despite good binding of vesicles to the planar membrane and fusion of vesicles to plasma membrane. It is suggested that cortical vesicles (CVs) have sufficient calcium-sensitive proteins for fusion to lipid membranes, but in native cortices granular fusion sites are oriented toward the plasma membrane. Removal of vesicles from the plasma membrane may allow fusion sites on vesicles access to new membranes.     

Raman spectra of planar supported lipid bilayers

Raman scattering has been used to obtain high quality vibrational spectra of planar supported lipid bilayers (pslb's) at the silica/water interface without the use of resonance or surface enhancement. A total internal reflection geometry was used both to increase the bilayer signal and to suppress the water background. Polarization control permits the determination of four components of the Raman tensor, of which three are independent for a uniaxial film. Spectra are reported of the phospholipids DMPC, DPPC, and POPC, in the C-H stretching region and the fingerprint region. The temperature-dependent polarized spectra of POPC show only small changes over the range 14-41 °C. The corresponding spectra of DMPC and DPPC bilayers show large thermal changes consistent with a decreasing tilt angle from the surface normal and increasing chain ordering at lower temperatures. The thermal behavior of DMPC pslb's is similar to that of vesicles of the same lipid in bulk suspension. In contrast to calorimetry, which shows a sharp phase transition (L_α-L_β') with decreasing temperature, the changes in the Raman spectra occur over a temperature range of ca. 10 °C commencing at the calorimetric phase transition temperature.     

Penetration and fusion of phospholipid vesicles by lysozyme

The lysozyme-induced fusion of phosphatidylserine/phosphatidylethanolamine vesicles as studied at a wide range of pH is found to correlate well with the binding of this protein to the vesicles. An identical 6000 molecular weight segment of lysozyme at the N-terminal region is found to be protected from tryptic digestion when initially incubated with vesicles at several pH values. Only this segment is labeled by dansyl chloride, which is partitioned into the bilayer. These results suggest the penetration of one segment of lysozyme into the bilayer. Photoactivated labeling of the membrane-penetrating segment of lysozyme with 3-(trifluoromethyl)-3-([125I]iodophenyl)diazirine ([125I]TID) and subsequent identification of the labeled residues by Edman degradation and gamma-ray counting indicate that four amino acids from the N-terminal are located outside the hydrophobic core of the bilayer. Although treatment of the membrane-embedded segment with aminopeptidase failed to cleave any amino acids from the N-terminal, it appears that a loop of lysozyme segment near the N-terminal penetrates into the bilayer at acidic pH. A helical wheel diagram shows that the labeling is done mainly on one surface of the alpha-helix. The penetration kinetics as studied by time-dependent [125I]TID labeling coincide with the fusion kinetics, strongly suggesting that the penetration of the lysozyme segment into the vesicles is the cause of the fusion.     

Raman spectra of planar supported lipid bilayers

Raman scattering has been used to obtain high quality vibrational spectra of planar supported lipid bilayers (pslb's) at the silica/water interface without the use of resonance or surface enhancement. A total internal reflection geometry was used both to increase the bilayer signal and to suppress the water background. Polarization control permits the determination of four components of the Raman tensor, of which three are independent for a uniaxial film. Spectra are reported of the phospholipids DMPC, DPPC, and POPC, in the C-H stretching region and the fingerprint region. The temperature-dependent polarized spectra of POPC show only small changes over the range 14-41 degrees C. The corresponding spectra of DMPC and DPPC bilayers show large thermal changes consistent with a decreasing tilt angle from the surface normal and increasing chain ordering at lower temperatures. The thermal behavior of DMPC pslb's is similar to that of vesicles of the same lipid in bulk suspension. In contrast to calorimetry, which shows a sharp phase transition (L alpha-L beta') with decreasing temperature, the changes in the Raman spectra occur over a temperature range of ca. 10 degrees C commencing at the calorimetric phase transition temperature.     

The insertion of D-beta-hydroxybutyrate apodehydrogenase into phospholipid monolayers and phospholipid vesicles

The strong interaction of D-beta-hydroxybutyrate dehydrogenase with phospholipid monomolecular films is demonstrated by the surface pressure increase of a film compressed up to 33 mN/m. Although the D-beta-hydroxybutyrate apodehydrogenase was able to penetrate many phospholipid monolayers, it interacted preferentially with negatively charged monolayers such as those made from diphosphatidylglycerol. The weakest interaction was found with phosphatidylcholine, which is the reactivating phospholipid for the enzyme. These interactions were dependent on the phospholipid chain length, ionic strength, and pH. At basic pH the apoenzyme lost its specificity for negatively charged phospholipids, suggesting the deprotonation of a cationic amino acid residue of the enzyme polypeptide chain. The charge effects are in agreement with results obtained using phospholipid vesicles. Beside the electrostatic interactions, the influence of phospholipid chain length and the ionic strength indicate that D-beta-hydroxybutyrate apodehydrogenase penetrates into the hydrophobic part of the lipid interface.     

Investigation of AC-DC magnetic field effects in planar phospholipid bilayers.

Observations recently reported by others indicate that a combination of a weak dc magnetic field and extremely-low-frequency ac magnetic field can produce resonant effects in biological systems. We report measurements of the effects of combined dc and ac magnetic fields on the dc current through channel-free planar phospholipid membranes. The combined dc-ac magnetic fields did affect the dc current through planar phospholipid membranes, but not in every membrane, and not consistently at the same values of magnetic flux density and frequency. None of our measurements showed resonant response akin to the cyclotron-like resonance reported in diatoms [Smith et al., 1987] and lymphocytes [Liboff et al., 1987].     

Protein-induced fusion of phospholipid vesicles of heterogeneous sizes.

We have investigated the fusion of phospholipid vesicles induced by lysozyme and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Vesicles were composed of dimyristoylphosphatidylcholine/dioleoylphosphatidylethanolamine/ cholesterol (DMPC:DOPE:Chol, 2:1:1). Small unilamellar vesicles (SUV, diameter ca. 30 nm) obtained by extensive sonication or large unilamellar vesicles (LUV, diameters ranged from 100 to 400 nm) obtained by extrusion methods were used. Fusion of LUV induced by lysozyme and GAPDH was drastically decreased when the diameter of the vesicles increased over a value of 100 nm. Lysozyme effect was stopped at the aggregation step while GAPDH effect was stopped at the fusion (lipid mixing) step. Fusion of heterogeneous vesicle populations (SUV with LUV) was observed only with GAPDH and this happened only when the lipids were in the liquid-crystalline state.     

Formation of cation channels in planar lipid bilayers by brefeldin A.

Brefeldin A (BFA) is a novel agent with the unique property of effecting a rapid increase of Golgi cisternae volume and subsequent loss of a recognizable Golgi apparatus in treated cells. Although a receptor-mediated mechanism has been proposed, the molecular basis of BFA action remains unknown (Lippincott-Schwartz, J., Glickman, J., Donaldson, J. G., Robbins, J., Kreis, T. E., Seamon, K. B., Sheetz, M. P., and Klausner, R. D. (1991) J. Cell Biol. 112, 567-577). Since a variety of ionophores distort Golgi architecture by initially causing osmotic swelling of the cisternae (Mollenhauer, H. H., Morre, D. J., and Rowe, L. D. (1990) Biochim. Biophys. Acta 1031, 225-246), Golgi membrane permeabilization by BFA seemed possible. We examined the effects of BFA on the conductance of planar lipid bilayers bathed in several aqueous salt solutions. Addition of BFA (1 microgram/ml) quickly augmented alkali cation conductance (K+ greater than Na+ much greater than Li+) but not anion conductance of the bilayer. Lower concentrations (1 ng/ml) indicated that BFA formed discrete, cation-selective channels in these bilayers. Given that Golgi cisternae volume increases immediately upon treatment with BFA, these findings suggest that alteration of ion gradients or Golgi membrane potential followed by an influx of water may be the mechanism by which BFA initiates disruption of Golgi structural integrity. Subsequent functional perturbations may then ensue either as a consequence of these initial structural changes or by a combination of several distinct mechanisms.     

Electrical manipulation of glycan-phosphatidyl inositol-tethered proteins in planar supported bilayers.

Electric fields have been used to manipulate and concentrate glycan-phosphatidyl inositol (GPI)-tethered proteins in planar supported bilayers. Naturally GPI-linked CD48, along with engineered forms of I-Ek and B7-2, in which their transmembrane domains have been genetically replaced with the GPI linkage, were studied. The proteins were labeled with fluorescently tagged antibodies, allowing the electric field-induced behavior to be followed by epifluorescence microscopy. All three protein complexes were observed to migrate toward the cathode with the B7-2 and CD48, each tethered to the membrane by a single GPI linker, moving significantly faster than the I-Ek, which has two GPI linkers. Patterns scratched into the membrane function as barriers to lateral diffusion and were used to isolate the proteins into highly concentrated corrals. All field-induced concentration profiles were completely reversible, indicating that the supported bilayer provides a stable, fluid environment in which GPI-tethered proteins can be manipulated. The ability to electrically control the spatial distribution of membrane-tethered proteins provides new opportunities for the study of biological membranes and the development of membrane-based devices.     

Peroxidase immobilized on phospholipid bilayers supported on au (111) by DTT self‐assembled monolayers: Application to dopamine determination

In this work, horseradish peroxidase (HRP) was immobilized on dimyristoylphosphatidylcholine (DMPC) bilayers supported on Au (111) by dithiotreitol (DTT) self‐assembled monolayers and used as a nanostructured electrochemical biosensor to dopamine determination. The morphology of the phospholipid bilayers and the immobilization of HRP to these layers were characterized by atomic force microscopy (AFM). Square‐wave voltammetry (SWV) experiments were done to investigate the performance of the HRP‐modified electrode. The AFM images indicate that the enzyme is adsorbed at the external layer of the lipid bilayer and, although the electrical charges on the surface were not measured, the enzyme and phospholipids surface interaction occurs probably by electrostatic forces due to the pH used in the experiments. Interestingly, the present system can be used as one‐shot sensor for the rapid detection of dopamine. The analytical performance of this system was linear for dopamine concentrations from 3.3 × 10^?5 to 1.3 × 10^?3 mol L^?1 (r = 0.9997) with a detection limit of 2.0 × 10^?6 mol L^?1. Our results indicate that the use of HRP‐DMPC bilayer system may be useful not only in developing new nanostructured materials for technological purposes, but could be very useful in fundamental studies to investigate the interactions between different micro‐and macromolecules, even with soluble proteins, and lipid membranes. Biotechnol. Bioeng. 2013; 110: 374–382. © 2012 Wiley Periodicals, Inc.