Fission of the bilayer lipid tube

The fusion of two black lipid membranes results in the formation of peculiar bilayer lipid tubes (‘cylindrical’) membranes (Neher, E. (1974) Biochim. Biophys. Acta 373, 328–336 and Melikyan, G.B., Abidor, L.G., Chernomordik, L.V. and Chailakhyan, L.M. (1983) Biochim. Biophys. Acta 730, 395–398). The mechanical stability of such tubes has been investigated experimentally and theoretically. With increasing hydrostatic pressure on the outside of the tube the radius of its middle part decreases. After this radius has reached a critical value, which constitutes 0.55 of the radius of the tube base, there occurs a collapse of the tube and its disintegration into two planar bilayers (fission). Expressions are obtained which relate the transmembrane difference of the hydrostatic pressure, causing the collapse, to the geometrical characteristics of the tube (its length and the radius of its base) and to the tension of the lipid bilayer. A method for measuring the membrane tension is proposed on the basis of the phenomenon considered.     

Detection of motional heterogeneities in lipid bilayer membranes by dual probe fluorescence correlation spectroscopy

We report the detection of heterogeneities in the diffusion of lipid molecules for the three-component mixture dipalmitoyl-PC/dilauroyl-PC/cholesterol, a chemically simple lipid model for the mammalian plasma membrane outer leaflet. Two-color fluorescence correlation spectroscopy (FCS) was performed on giant unilamellar vesicles (GUVs) using fluorescent probes that have differential lipid phase partition behavior—DiO-C18:2 favors disordered fluid lipid phases, whereas DiI-C20:0 prefers spatially ordered lipid phases. Simultaneously-obtained fluorescence autocorrelation functions from the same excitation volume for each dye showed that, depending on the lipid composition of this ternary mixture, the two dyes exhibited different lateral mobilities in regions of the phase diagram with previously proposed submicroscopic two-phase coexistence. In one-phase regions, both dyes reported identical diffusion coefficients. Two-color FCS thus may be detecting local membrane heterogeneities at size scales below the optical resolution limit, either due to short-range order in a single phase or due to submicroscopic phase separation.     

Oxygen permeability of the lipid bilayer membrane made of calf lens lipids

The oxygen permeability coefficient across the membrane made of the total lipid extract from the plasma membrane of calf lens was estimated from the profile of the oxygen transport parameter (local oxygen diffusion-concentration product) and compared with those estimated for membranes made of an equimolar 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol (POPC/Chol) mixture and of pure POPC. Profiles of the oxygen transport parameter were obtained by observing the collision of molecular oxygen with nitroxide radical spin labels placed at different depths in the membrane using the saturation-recovery EPR technique and were published by us earlier (J. Widomska, M. Raguz, J. Dillon, E. R. Gaillard, W. K. Subczynski, Biochim. Biophys. Acta. 1768 (2007) 1454-1465). At 35 °C, the estimated oxygen permeability coefficients were 51.3, 49.7, and 157.4 cm/s for lens lipid, POPC/Chol, and POPC membranes, respectively (compared with 53.3 cm/s for a water layer with the same thickness as a membrane). Membrane permeability significantly decreases at lower temperatures. In the lens lipid membrane, resistance to the oxygen transport is located in and near the polar headgroup region of the membrane to the depth of the ninth carbon, which is approximately where the steroid-ring structure of cholesterol reaches into the membrane. In the central region of the membrane, oxygen transport is enhanced, significantly exceeding that in bulk water. It is concluded that the high level of cholesterol in lens lipids is responsible for these unique membrane properties.     

Effects of membrane composition and lipid structure on the photopolymerization of lipid diacetylenes in bilayer membranes

Molecules analogous to biological and synthetic lipids have been prepared with conjugated diacetylene moieties in the long alkyl chain. These lipid diacetylenes form bilayer structures when suspended in aqueous buffers. Ultraviolet light (254 nm) exposure initiates the polymerization of the diacetylenes in the lipid bilayer to give a fully conjugated, highly colored product. The reaction is topotactic, and its efficiency depends on the correct alignment of the monomeric units. Thus, the lipid diacetylenes are photopolymerizable if the hydrocarbon chains are in a regular lattice found at temperatures below the lipid transition temperature; polymerization is inhibited above this transition. The photopolymerization of a diacetylenic glycerophosphocholine in lipid bilayer membranes was observed in two-component mixtures with a nonpolymerizable lipid, either dioleoylphosphatidylcholine or distearoylphosphatidylcholine. The photochemical and thermochemical characteristics suggest that the diacetylenic glycerophosphocholine exists largely in separate domains in the mixed bilayers. Lipid diacetylenes analogous to a dialkyldimethylammonium salt and to a dialkyl phosphate have a plane of symmetry, which suggests that both chains penetrate equally into the bilayer. The photopolymerization of these symmetrical synthetic species is more than 10³-times more efficient than that of the diacetylenic glycerophosphocholine. These differences are interpretable in terms of the expected conformational preference of the lipid molecules.     

Galectin-3 interacts with membrane lipids and penetrates the lipid bilayer

The precise mechanism by which galectin-3 and other cytosolic proteins that lack signal peptides are secreted is yet to be elucidated. In the present analyses, we determined that galectin-3, a beta-galactoside binding protein, can interact directly with membrane lipids in solid phase binding assays. More interestingly, we determined by spectrophotometric methods that it can spontaneously penetrate the lipid bilayer of liposomes in either direction. These findings suggest that galectin-3 on its own has the capacity to traverse the lipid bilayer. Whereas the situation is rather simplified in liposomes, the interaction of galectin-3 with the plasma membrane may involve cholesterol-rich membrane domains where galectin-3 can be concentrated and form multimers or interact covalently with other proteins.     

Organization and dynamics of NBD-labeled lipids in lipid bilayer analyzed by FRET using the small membrane fluorescent probe AHBA as donor

Fluorescent probes are employed to investigate natural and model membranes. It is important to know probe location and extent of perturbations they cause into the lipid bilayer. Förster Resonance Energy Transfer (FRET) is a useful tool to investigate phenomena involving plasma membranes, and reports in literature used relatively large fluorophores like 1,6-diphenylhexatriene, located at the center of the hydrophobic region, 4-aminophthalimide-based molecules located at lipid/water interfaces and BODIPY-labeled phosphatidylcholine. In this work we explored FRET process in 1,2-dimyristoyl-L-α-GPC large unilamellar vesicles, in gel and fluid phase, using as donor the very small group o-Abz bound to hexadecyl chain (2-amino-N-hexadecyl-benzamide - AHBA) and 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD) labeled lipids as acceptor. From the intensity decay of donor in presence of acceptors, the FRET efficiency was calculated, and used to fit the model proposed by Fung and Stryer to that efficiency. Using lipid bilayer structural data, the procedure allowed the determination of Förster distance for each donor-acceptor pair in vesicles, without imposing any value for the orientational factor κ². From distance distributions between o-Abz in AHBA and NBD in lipid bilayer obtained using the program CONTIN, we obtained donor-acceptor populations having different separation distances. The populations reflect the occurrence of FRET involving probes in the same or in opposite leaflet. A dynamic picture emerged showing how relative position of the probes is dependent on the structural thermal phase of the DMPC bilayer. The results emphasize the need of careful analysis in order to understand processes involving fluorescent probes in model membranes.     

Pore forming activity of the major outer membrane protein of Rhodobacter capsulatus in lipid bilayer membranes

Porin of the outer membrane of Rhodobacter capsulatus St. Louis (ATCC 23782) was isolated and reconstituted into lipid bilayer membranes. The porin was obtained either by the sodium dodecyl sulfate treatment of cell envelopes (SDS-porin) or by saline extraction of whole cells (NaCl-porin). Nanomolar concentrations of both porin preparations resulted in a strong conductance increase of the lipid bilayer membranes by many orders of magnitude. At small protein concentrations the conductance increased in a stepwise fashion, the average single channel conductance being about 0.35 nS in 0.1 M KCl for SDS-porin and NaCl-porin as well. The single channel conductance was a linear function of the specific conductance of the aqueous phase. The results were consistent with the assumption that the porin formed large water-filled transmembrane channels in the membrane. From the average value of the single channel conductance in 0.1 M KCl an effective channel diameter of about 1.5 nm was estimated for both types of porins.Abbreviations EDTA ethylenediamine tetraacetic acid - SDS sodium dodecyl sulfate     

Propofol, a general anesthetic, promotes the formation of fluid phase domains in model membranes

The molecular site of anesthetic action remains an area of intense research interest. It is not clear whether general anesthetics act through direct binding to proteins or by perturbing the membrane properties of excitable tissues. Several studies indicate that anesthetics affect the properties of either membrane lipids or proteins. However, gaps remain in our understanding of the molecular mechanism of anesthetic action. Recent developments in membrane biology have led to the concept of small-scale domain structures in lipid and lipid--protein coupled systems. The role of such domain structures in anesthetic action has not been studied in detail. In the present study, we investigated the effect of anesthetics on lipid domain structures in model membranes using the fluorescent spectral properties of Laurdan (6-dodecanoyl-2-dimethylamino naphthalene). Propofol, a general anesthetic, promoted the formation of fluid domains in model membranes of dipalmitoyl phosphatidyl choline (DPPC) or mixtures of lipids of varying acyl chains (DPPC:DMPC dimyristoyl phosphatidyl choline 1:1). The estimated size of these domains is 20--50 A. Based on these studies, we speculate that the mechanism of anesthetic action may involve effects on protein--lipid coupled systems through alterations in small-scale lipid domain structures.     

Differences in membrane lipid composition and fluidity of transplanted grsl lymphoma cells, depending on their site of growth in the mouse

GRSL lymphoma cells were isolated from various growth sites in the host. The relative membrane lipid fluidities of these cells and of normal lymphoid cells were estimated by fluorescence polarization, using the probe diphenylhexatriene and by measuring the (free) cholesterol/phospholipid molar ratio in whole cells. The results indicate that the membrane fluidity (reciprocal of the lipid structural order) of the lymphoma cells increases in the order of their location: peripheral blood < spleen < mesenterial lymph node < ascites fluid. The membrane fluidities of normal lymphocytes from thymus, mesenterial lymph node and spleen were about the same, but higher than of peripheral blood lymphocytes, and between those of the lymphoma cells from lymph node and spleen. These results are confirmed by more extensive analysis on purified plasma membranes from the splenic and ascitic GRSL lymphoma cells and from normal splenocytes and thymocytes. The significantly higher lipid order parameter found in the GRSL plasma membrane isolated from the spleen as compared to those from the ascites cells could be fully explained by the differences measured in the major chemical determinants of the fluidity, i.e., the cholesterol/phospholipid ratio, the sphingomyelin content and the degree of saturation of the fatty acyl groups of the phospholipids. It was also found that the cholesterol/phospholipid ratio in erythrocyte membranes isolated from the peripheral blood of the tumor bearers was higher than in those from normal control mice. The observed differences in membrane fluidity between distinct subsets of tumor cells may be relevant to the sensitivity of these cells to immune attack or to drugs.