Large-scale co-aggregation of fluorescent lipid probes with cell surface proteins

Large scale aggregation of fluorescein-labeled immunoglobulin E (IgE) receptor complexes on the surface of RBL cells results in the co- aggregation of a large fraction of the lipophilic fluorescent probe 3,3'-dihexadecylindocarbocyanine (diI) that labels the plasma membranes much more uniformly in the absence of receptor aggregation. Most of the diI molecules that are localized in patches of aggregated receptors have lost their lateral mobility as determined by fluorescence photobleaching recovery. The diI outside of patches is mobile, and its mobility is similar to that in control cells without receptor aggregates. It is unlikely that the co-aggregation of diI with IgE receptors is due to specific interactions between these components, as two other lipophilic probes of different structures are also observed to redistribute with aggregated IgE receptors, and aggregation of two other cell surface antigens also results in the coredistribution of diI at the RBL cell surface. Quantitative analysis of CCD images of labeled cells reveals some differences in the spatial distributions of co- aggregated diI and IgE receptors. The results indicate that cross- linking of specific cell surface antigens causes a substantial change in the organization of the plasma membrane by redistributing pre- existing membrane domains or causing their formation.     

Pattern photobleaching of fluorescent lipid vesicles using polarized laser light.

A burst of linearly polarized laser radiation incident on a spherical lipid vesicle, liposome, or biological cell can produce a well-defined nonuniform distribution of membrane-bound fluorescent molecules, provided the absorption transition dipole moment of the fluorescent label has a nonrandom orientation relative to the membrane surface and can be photobleached by the laser radiation. The return (recovery) of fluorescent membrane-bound molecules to a uniform distribution can be monitored using the same polarized radiation source. Under appropriate conditions this recovery is characterized by a single exponential time constant tau. This time constant is related to the radius R of the vesicle and the lateral diffusion coefficient D of the fluorescent membrane-bound molecules by the equation R2 = 6D tau. In the case of vesicle membranes this result is not limited by diffraction and so should be applicable to vesicles whose radii are less than the wavelength of light. The above considerations are illustrated by the polarized light photobleaching-recovery of lipid vesicles containing a fluorescent lipid, N-4-nitro-benzo-2-oxa,1,3-diazole l-alpha-dimyristoylphosphatidylethanolamine (NBD-DMPE).     

Antibody interaction with a membrane-bound fluorescent ligand on synthetic lipid vesicles

The interaction of membrane-bound ligand with bivalent and monovalent fragments of monoclonal antibody was studied by fluorescence and precipitation analysis using synthetic lipid vesicles. The ligand N epsilon-[5-(dimethylamino)-naphthyl-1-sulfonyl]lysine was linked to the hydrophobic anchor dipalmitoylphosphatidylethanolamine and ranged between 0.01 and 1 mol% of the membrane components. The effects of cholesterol on the specific interaction were observed over the range of 0-50 mol%. A precipitation assay was developed to evaluate various factors related to the cross-linking of small unilamellar vesicles by bivalent antibody. The cholesterol content was critical for this process as demonstrated by the increased efficiency of precipitation over the range of 0-40 mol% of this component. Fluorescence analysis yielded the parallel finding of increased accessibility of the ligand to the antibody with greater cholesterol content. Increased surface density of the ligand also was found to enhance the intervesicle interaction. Finally, a comparison of the kinetics by fluorescence analysis of the binding of monovalent and bivalent fragments indicated that the bivalent interaction involved primarily the cross-linking of vesicles in accord with published findings of the interaction of monoclonal antibody with cell membrane antigens.     

Enzymes inside lipid vesicles: preparation, reactivity and applications

There are a number of methods that can be used for the preparation of enzyme-containing lipid vesicles (liposomes) which are lipid dispersions that contain water-soluble enzymes in the trapped aqueous space. This has been shown by many investigations carried out with a variety of enzymes. A review of these studies is given and some of the main results are summarized. With respect to the vesicle-forming amphiphiles used, most preparations are based on phosphatidylcholine, either the natural mixtures obtained from soybean or egg yolk, or chemically defined compounds, such as DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) or POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine). Charged enzyme-containing lipid vesicles are often prepared by adding a certain amount of a negatively charged amphiphile (typically dicetylphosphate) or a positively charged lipid (usually stearylamine). The presence of charges in the vesicle membrane may lead to an adsorption of the enzyme onto the interior or exterior site of the vesicle bilayers. If (i) the high enzyme encapsulation efficiencies; (ii) avoidance of the use of organic solvents during the entrapment procedure; (iii) relatively monodisperse spherical vesicles of about 100 nm diameter; and (iv) a high degree of unilamellarity are required, then the use of the so-called 'dehydration-rehydration method', followed by the 'extrusion technique' has shown to be superior over other procedures. In addition to many investigations in the field of cheese production--there are several studies on the (potential) medical and biomedical applications of enzyme-containing lipid vesicles (e.g. in the enzyme-replacement therapy or for immunoassays)--including a few in vivo studies. In many cases, the enzyme molecules are expected to be released from the vesicles at the target site, and the vesicles in these cases serve as the carrier system. For (potential) medical applications as enzyme carriers in the blood circulation, the preparation of sterically stabilized lipid vesicles has proven to be advantageous. Regarding the use of enzyme-containing vesicles as submicrometer-sized nanoreactors, substrates are added to the bulk phase. Upon permeation across the vesicle bilayer(s), the trapped enzymes inside the vesicles catalyze the conversion of the substrate molecules into products. Using physical (e.g. microwave irradiation) or chemical methods (e.g. addition of micelle-forming amphiphiles at sublytic concentration), the bilayer permeability can be controlled to a certain extent. A detailed molecular understanding of these (usually) submicrometer-sized bioreactor systems is still not there. There are only a few approaches towards a deeper understanding and modeling of the catalytic activity of the entrapped enzyme molecules upon externally added substrates. Using micrometer-sized vesicles (so-called 'giant vesicles') as simple models for the lipidic matrix of biological cells, enzyme molecules can be microinjected inside individual target vesicles, and the corresponding enzymatic reaction can be monitored by fluorescence microscopy using appropriate fluorogenic substrate molecules.     

Association of a fluorescent amphiphile with lipid bilayer vesicles in regions of solid-liquid-disordered phase coexistence

The effects of solid-fluid phase separations on the kinetics of association of a single-chain fluorescent amphiphile were investigated in two different systems: pure DMPC (dimyristoylphosphatidylcholine) and a 1:1 mixture of DMPC and DSPC (distearoylphosphatidylcholine). In pure DMPC vesicles, solid (s) and fluid (l(d)) phases coexist at the phase transition temperature, T(m), whereas a 1:1 mixture of DMPC and DSPC shows a stable s-l(d) phase separation over a large temperature interval. We found that in single-component bilayers, within the main phase transition, the experimental kinetics of association are clearly not single-exponential, the deviation from that function becoming maximal at the T(m). This observation can be accounted for by a rate of desorption that is slower than desorption from either fluid or solid phases, leaving the rates of insertion unchanged, but a treatment in terms of stable fluid and solid domains may not be adequate for the analysis of the association of an amphiphile with pure DMPC vesicles at the T(m). In DMPC/DSPC mixtures with solid-fluid phase coexistence, association occurs overall faster than expected based on phase composition. The observed kinetics can be described by an increase in the rate of insertion, leaving the desorption rates unchanged. The fast kinetics of insertion of the amphiphile into two-phase bilayers in two-component vesicles is attributed to a more rapid insertion into defect-rich regions, which are most likely phase boundaries between solid and fluid domains. A two-component mixture of lipids that shows a stable phase separation between l(d)-s phases over a large temperature interval thus behaves very differently from a single-component bilayer at the T(m), with respect to insertion of amphiphiles.     

Pigment containing lipid vesicles

Summary It was shown previously (Walz, 1976) that chlorophylla incorporated into the membrane of lecithin vesicles is a probe which detects the aggregational state of the lipids. This phenomenon is interpreted in terms of a solvatochromism, i.e., the effect of various solvents on the absorption spectrum of the solute. The sensor characteristics can be expressed by a set of solvatochromic coefficients, which are pertinent to the electronic transitions occuring in chlorophylla on excitation with light, and by means of the absorption bands associated with these transitions. An unambiguous resolution of spectra into absorption bands is not yet practicable, but at least part of the bands can be approximated by, Gaussian components which then allows us to estimate the solvatochromic coefficients From these data and based on the currently available theoretical and experimental information about, solvatochromism, it is concluded that the chromophore, i.e., the porphyrin ring of chlorophylla, is located adjacent to the glycerol-ester moieties of the lecithin molecules in the membrane, and that the sensor ability relies on different orientations of the chromophore for lecithin in different states of aggregation.     

Pigment containing lipid vesicles

Summary Vesicles obtained by sonication of chlorophylla-lecithin mixtures dispersed in an aqueous medium closely resemble the well-characterized vesicles similarly prepared from pure lipids. They are bounded by one spherical lipid bilayer which contains the chlorophylla. Appropriate conditions for sonication prevent substantial degradation of the membrane constituents. Up to one chlorophylla molecule per 55 lecithins can be incorporated into the membranes. The average Stokes' radius of the vesicles determined by analytical sieve chromatography is 102±5 Å and independent of the chlorophylla content. The membrane is visible in the electron-microscope when the vesicles are treated with osmium tetroxide prior to negative staining. The osmium fixation is, however, not strong enough to allow for a preparation of the vesicles for thin sectioning (dehydration, embedding in epoxide).     

Structural characterization of labeled clathrin and coated vesicles

Clathrin (8 S) and coated vesicles have been covalently labeled by using the sulfhydryl-labeling fluorescent probe N-(1-anilinonaphthalene)maleimide. A large increase in energy transfer from Trp to anilinonaphthalene (AN) residues was observed in clathrin in the pH range approximately 6.5-6.0, where the rate of clathrin self-association increased rapidly. The change in energy transfer was indicative of a conformational rearrangement, which could be responsible for the initiation of the clathrin self-association reaction to form coat structure. The AN label was found in both the coat and membrane proteins after dissociation of coated vesicles at pH 8.5. The labeled coat and membrane proteins readily recombined to form coated vesicles after reducing the pH to 6.5, indicating that the labeling did not interfere with the ability of clathrin to self-associate and interact with uncoated vesicles to form coat structure. A comparison of the AN fluorescence with the Coomassie blue pattern after electrophoresis in sodium dodecyl sulfate-gels revealed that a 180,000-Da protein (clathrin) was mainly labeled in coated vesicles, while a 110,000-Da protein was also strongly labeled in uncoated vesicles. AN-labeled baskets and coated vesicles have been prepared. Trypsin digestion reduced the sedimentation rate of baskets from 150 S to 120 S and of coated vesicles from 200 S to 150 S. Gel electrophoresis of baskets and coated vesicles showed extensive conversion of clathrin (Mr 180,000) to a product of Mr approximately equal to 110,000, suggesting equivalent structural organization of the coat in coated vesicles as in baskets. In both cases, the peptide(s) released from the vesicles by digestion were essentially free of fluorescent label. In the case of the uncoated vesicles, tryptic digestion released most of the proteins remaining after coat removal.     

Electric breakdown of lipid vesicles

Theoretical expression for free energy F of spherical lipid vesicle containing through pore in the presence of diffusional potential difference is derived. It is assumed that the pore radius is small in comparison with vesicle size. According to estimation the variation of elastic energy of vesicle membrane with pore radius is small. Therefore electrical breakdown becomes reversible for reasonable region of r values. Conditions of equilibrium and dynamic modes of breakdown are analyzed. Random oscillation mode of intravesicular label discharge is shown for some region of vesicle parameters.     

Affinity purification of lipid vesicles

We present a novel column chromatography technique for recovery and purification of lipid vesicles, which can be extended to other macromolecular assemblies. This technique is based on reversible binding of biotinylated lipids to monomeric avidin. Unlike the very strong binding of biotin and biotin-functionalized molecules to streptavidin, the interaction between biotin-functionalized molecules and monomeric avidin can be disrupted effectively by ligand competition from free biotin. In this work, biotin-functionalized lipids (biotin-PEG-PE) were incorporated into synthetic lipid vesicles (DOPC), resulting in unilamellar biotinylated lipid vesicles. The vesicles were bound to immobilized monomeric avidin, washed extensively with buffer, and eluted with a buffer supplemented with free biotin. Increasing the biotinyl lipid molar ratio beyond 0.53% of all lipids did not increase the efficiency of vesicle recovery. A simple adsorption model suggests 1.1 x 10(13) active binding sites/mL of resin with an equilibrium binding constant of K = 1.0 x 10(8) M(-1). We also show that this method is very robust and reproducible and can accommodate vesicles of varying sizes with diverse contents. This method can be scaled up to larger columns and/or high throughput analysis, such as a 96-well plate format.     

Auto-oxidation-induced fusion of lipid vesicles

Spontaneous size changes of small unilamellar vesicles with initial mean diameters of 25 nm measured by quasi-elastic light scattering (QELS) and electron microscopy are reported. After the size conversion the vesicles have mean diameters of about 70 nm and are of the unilamellar and multilamellar type. The fact that auto-oxidation initiates this process is established by the comparison of the results for vesicles which differ only in the degree of auto-oxidation. The role of phosphatidylcholine hydroperoxides as fusogens is discussed.     

Biosynthetic preparation of labeled 4,4-dimethylzymosterol

Labeled 4,4-dimethyl-5 alpha -cholesta-8,24-dien-3 beta-ol (4,4-dimethylzymosterol) was prepared by incubating labeled mevalonate with rat liver extracts in the presence of arsenite and lanosterol.     

Large-scale preparation of plasma membrane vesicles from PC-12 Pheochromocytoma cells and their use in noradrenaline transport studies

Plasma membranes were isolated from rat pheochromocytoma cells (PC-12) grown in spinner culture. The rapid and simple isolation procedure consisted of a differential and isopycnic centrifugation (in a linear sucrose gradient) with the aid of a high capacity fixed angle rotor equipped with siliconized centrifuge tubes. The isolated membranes were closed and osmotically active vesicles (about 0.3 μm in diameter) with a mean intravesicular water space of 1.84 μl / mg protein. In the presence of an inward gradient of sodium chloride and an outward gradient of potassium, [³H]noradrenaline (50 nM) was taken up and accumulated 550-fold (at 31°C). The uptake and accumulation of [³H]noradrenaline was temperature-sensitive and inhibited by the tricyclic antidepressant desipramine. Membrane vesicles isolated from PC-12 cells represent a useful model for the investigation of the molecular mechanism of the neuronal noradrenaline transport system.     

Calcium/phosphate-induced immobilization of fluorescent phosphatidylserine in synthetic bilayer membranes: inhibition of lipid transfer between vesicles

Resonance energy transfer between 4-nitro-2,1,3-benzoxadiazole (NBD) acyl chain labeled phospholipid analogues and (lissamine) rhodamine B labeled phosphatidylethanolamine was used to monitor the rate of NBD-labeled lipid transfer between a variety of small unilamellar donor vesicles and dioleoylphosphatidylcholine (DOPC) acceptor vesicles. In the presence of appropriate concentrations of Ca2+ and phosphate, the transfer rate of NBD-phosphatidylserine (NBD-PS) from vesicles composed of lipid extracts from human red blood cells was reduced by approximately 10-fold, while the transfer rates of NBD-phosphatidylcholine, -ethanolamine, -glycerol, -N-succinylethanolamine, and -phosphatidic acid were essentially unaffected. A systematic evaluation of the lipid composition needed to facilitate the Ca2+/phosphate-induced inhibition of NBD-PS transfer revealed that the process was dependent upon the inclusion of both cholesterol and phosphatidylethanolamine (PE) in the donor vesicle population. Inhibition of NBD-PS transfer required the sequential addition of phosphate and Ca2+ to the vesicles, indicating that the combined interaction of Ca2+ and phosphate at the membrane surface was a prerequisite for inhibition to occur. Parallel experiments designed to determine the possible mechanism of this phenomenon showed that inhibition of NBD-PS transfer was not due to Ca2+-mediated phase separations or vesicle-vesicle fusion. However, the addition of Ca2+ and phosphate to vesicles composed of total red blood cell lipids or cholesterol/PE did result in their aggregation. On the other hand, aggregation per se did not seem to be responsible for the inhibition of transfer since NBD-PS-containing vesicles composed of DOPC or DOPC/DOPE also aggregated, although NBD-PS transfer was unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preparation and characteristics of lipid vesicles

Summary As determined by electron microscopy, lipid sonicated in buffer initially forms large vesicles which may be multilamellar. Prolonged sonication results in a population of vesicles of smaller, but not uniform diameters. These vesicles are bounded by only one bilayer. The lipid suspension can be partially fractionated according to size by column chromatography. A fraction of the eluate has been selected for further study. The weight-average vesicle weight and average radius of gyration are obtained by lightscattering measurements. The volume of buffer enclosed by the vesicles is determined using¹⁴C- or³H-labelled sugars as a marker. These values are in reasonable agreement with the corresponding values calculated from the size distribution of the vesicle fraction obtained by electron microscopy.     

Location of valinomycin in lipid vesicles

The location of the cyclododecadepsipeptide, valinomycin in vesicles formed from two synthetic lipids is studied by differential scanning calorimetry, spin-label partitioning electron paramagnetic resonance and [¹H]-nuclear magnetic resonance. The results show that valinomycin is located near the head group region of dipalmitoyl phosphatidyl choline vesicles and in the hydrophobic core of the dimyristoyl phosphatidyl choline vesicles in the liquid crystalline phase.     

Further studies of nerve membranes labeled with fluorescent probes

Summary By using the technique of intracellular perfusion combined with fluorescence measurements, the mode of binding of 6-p-toluidinylnaphthalene-2-sulfonate (2–6 TNS) in a squid giant axon was examined. The apparent dissociation constant for the binding sites in axons was found to be roughly 0.22mm. Out of approximately 5×10¹⁴ molecules/cm² of 2–6 TNS bound to the sites in and near the axonal membrane, roughly 2×10¹⁰ molecules/cm² are shown to contribute to a transient decrease in fluorescence during nerve excitation. By recording fluorescence signals with a polarizer and analyzer inserted in four different combinations of orientations, studies were made of the directions of the transition moments of various probe molecules relative to the longitudinal axis of the axon. Among hydrophobic probes examined, the polarization characteristics of the fluorescence signals obtained with 1–8 derivatives of aminonaphthalenesulfonate (1-8 ANS, 1-8 TNS and 1-8 AmNS) were found to be very different from those obtained with 2–6 derivatives (2-6 ANS, 2-6 TNS and 2-6 MANS). A tentative interpretation is proposed to account for this difference in physiological behavior between 1–8 and 2–6 derivatives. It is emphasized that measurements of fluorescence polarization yield significant information concerning the structure of the axonal membrane.