Lipid-cell interactions: Liposome adsorption and cell-to-liposome lipid transfer are mediated by the same cell-surface sites

The competitive behavior of solid vs. fluid liposomes in liposome-to-cell adsorption and cell-to-liposome lipid transfer processes was investigated with L cells and FBT epithelial sheets. Binding, transfer and ³¹P-NMR experiments have demonstrated that: (i) solid liposomes adhere to the cell surface as integral vesicles retaining the entrapped substances; (ii) fluid liposomes are partly disintegrated at the cell surface with concomitant entry of entrapped substances into the cytoplasm, while their lipids remain on the cell surface; (iii) fluid liposomes that escape lysis dissociate from the cell, taking away cell lipid molecules. The latter process underlies the mechanism of cell-to-fluid liposome lipid transfer. In contrast, no lipid transfer occurs between the plasma membrane and solid liposomes. Cell-bound solid liposomes interfere with the transfer of cell lipids to fluid liposomes, while these in turn inhibit the binding of solid liposomes to the cell surface. Moreover, cell-induced aggregation of both fluid and solid freshly added liposomes is also inhibited by preincubation of the cells with either solid or fluid liposomes. Thus, different types of interaction of both fluid and solid liposomes with the cell are mediated by the same (or closely related) sites on the cell surface.     

Partitioning of pyrene-labeled phospho- and sphingolipids between ordered and disordered bilayer domains

Here we have studied how the length of the pyrene-labeled acyl chain (n) of a phosphatidylcholine, sphingomyelin, or galactosylceramide affects the partitioning of these lipids between 1), gel and fluid domains coexisting in bovine brain sphingomyelin (BB-SM) or BB-SM/spin-labeled phosphatidylcholine (PC) bilayers or 2), between liquid-disordered and liquid-ordered domains in BB-SM/spin-labeled PC/cholesterol bilayers. The partitioning behavior was deduced either from modeling of pyrene excimer/monomer ratio versus temperature plots, or from quenching of the pyrene monomer fluorescence by spin-labeled PC. New methods were developed to model excimer formation and pyrene lipid quenching in segregated bilayers. The main result is that partition to either gel or liquid-ordered domains increased significantly with increasing length of the labeled acyl chain, probably because the pyrene moiety attached to a long chain perturbs these ordered domains less. Differences in partitioning were also observed between phosphatidylcholine, sphingomyelin, and galactosylceramide, thus indicating that the lipid backbone and headgroup-specific properties are not severely masked by the pyrene moiety. We conclude that pyrene-labeled lipids could be valuable tools when monitoring domain formation in model and biological membranes as well as when assessing the role of membrane domains in lipid trafficking and sorting.     

Melittin induces fusion of unilamellar phospholipid vesicles

Melittin, the soluble lipophilic peptide of bee venom, causes fusion of phospholipid vesicles when vesicle suspensions are heated or cooled through their thermal phase transition. Fusion was detected using a new photochemical method (Morgan, C.G., Hudson, B. and Wolber, P. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 26–30) which monitors lipid mixing. Electron microscopy and gel filtration confirmed that most of the lipid formed large vesicular structures. Fluorescence experiments with a water-soluble, membrane-impermeable complex of terbium (Wilschut, J. and Papahadjopoulos, D. (1979) Nature 281, 690–692) demonstrate that these ionic contents are released during fusion. The large structures formed by melittin-induced fusion are impermeable to these ions and are resistant to further fusion. This is in contrast to the behavior observed for the cationic detergent cetyltrimethylammonium bromide (CETAB). The large size of the vesicles formed, the extreme speed of the fusion event and the appearance of electron microscope images of the vesicles prior to fusion suggest that the mechanism of the fusion process includes a preaggregation step.     

Interaction of biologically active molecules with phospholipid membranes: I. Fluorescence depolarization studies on the effect of polymeric biocide bearing biguanide groups in the main chain

Interaction of poly(hexamethylene biguanide hydrochloride) (PHMB), which is a polymeric biocide bearing biguanide groups in its main chain, with phospholipid bilayers was studied by the fluorescence depolarization method. A strong interaction of PHMB with negatively charged bilayers composed of phosphatidylglycerol(PG) alone or of PG and phosphatidylcholine (PC) was observed, whereas neutral PC bilayers were not affected. On adding PHMB, the fluorescence polarization of diphenylhexatriene embedded in the negatively charged bilayers was reduced to a great extent, especially in the gel phase. This was interpreted in terms of PHMB-induced expansion and fluidization of the bilayer, which enables the probe molecule to undergo less-hindered torsional motion. Similarity between PHMB and polymyxin B in the structure, the mode of action against bacteria and the interaction with lipid membranes is discussed.     

The superdiamagnetic effect of magnetic fields on one and two component multilamellar liposomes

For multilamella vesicles of DMPC, DPPC, DSPC, binary mixtures of DMPC-DPPC, DMPC-DSPC, DMPC-DPPE, DOPC and egg lecithin, the optical turbidity decreases significantly on the application of a magnetic field in excess of about 0.2 T, provided that the temperature is above the pretransition value. The turbidity reaches a limiting value for magnetic fields of about 2 T. The effect is attributed to augmentation of the diamagnetic anisotropy of the lipid molecules by clustering within the bilayer, with consequent orientation of either the individual ‘superdiamagnetic’ clusters or the whole liposome. It is suggested that, since most animal cell membranes are largely in the liquid crystalline phase, it is possible that homogeneous magnetic fields as low as 0.2 T may cause biologically significant changes within the membrane.     

Thermodynamics of lipid interactions in complex bilayers

The mutual interactions between lipids in bilayers are reviewed, including mixtures of phospholipids, and mixtures of phospholipids and cholesterol (Chol). Binary mixtures and ternary mixtures are considered, with special emphasis on membranes containing Chol, an ordered phospholipid, and a disordered phospholipid. Typically the ordered phospholipid is a sphingomyelin (SM) or a long-chain saturated phosphatidylcholine (PC), both of which have high phase transitions temperatures; the disordered phospholipid is 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) or dioleoylphosphatidylcholine (DOPC). The unlike nearest-neighbor interaction free energies (ω_AB) between lipids (including Chol), obtained by an variety of unrelated methods, are typically in the range of 0-400 cal/mol in absolute value. Most are positive, meaning that the interaction is unfavorable, but some are negative, meaning it is favorable. It is of special interest that favorable interactions occur mainly between ordered phospholipids and Chol. The interpretation of domain formation in complex mixtures of Chol and phospholipids in terms of phase separation or condensed complexes is discussed in the light of the values of lipid mutual interactions.     

A restatement of melittin-induced effects on the thermotropism of zwitterionic phospholipids

Perturbations induced by melittin on the thermotropism of dimyristoyl-, dipalmitoyl-, distearoylphosphatidylcholine and natural sphingomyelin are investigated and rationalized from data obtained by fluorescence polarization, differential scanning calorimetry and Raman spectroscopy. Depending on the technique and / or experimental conditions used, the observed effects differ at the same lipid to protein molar ratio, due to partial binding of melittin. The binding is more efficient for tetrameric than for monomeric melittin, but in both cases its affinity is weaker for phosphatidylcholine dispersions in the gel phase than for sonicated vesicles. For temperatures $\text{T ? T}{}_{\mathrm{<mtext>m</mtext>}}$ efficient binding occurs whatever the initial state of the lipids is. One can summarize the effects induced by melittin on the transition temperature as follows: (i) No upward shift is observed on synthetic phosphatidylcholines when lipid degradation is avoided. This is achieved by using highly purified melittin, phospholipase inhibitors, and / or non-hydrolysable lipids. (ii) Melittin monomer does not change $\text{T}{}_{\mathrm{<mtext>m</mtext>}}$. (iii) When melittin tetramer is stabilized, it decreases $\text{T}{}_{\mathrm{<mtext>m</mtext>}}$ by 10–15 deg. C. The transition broadens, and is finally abolished for $\text{R}{}_{\mathrm{<mtext>i</mtext>}}\text{? 2}$. Very similar results are found for natural sphingomyelin. Fluorescence polarization indicates similar changes in order and dynamics of the acyl chains for all lipid studied. For $\text{T ? T}{}_{\mathrm{<mtext>m</mtext>}}$, fluorescence and Raman show that melittin decreases the amount of CH₂ groups in ‘trans’ conformation and the intermolecular order of the chains. According to fluorescence data, there is an increase of the rigid-body orientational order at $\text{T ? T}{}_{\mathrm{<mtext>m</mtext>}}$, while from Raman the positional intermolecular order decreases without significant change in the CH₂ groups ‘trans’/‘gauche’ ratio.     

The effect of linoleic acid and benzyl alcohol on the activity of glycosyltransferases of rat liver golgi membranes and some soluble glycosyltransferases

The effects of the membrane perturbing reagents linoleic acid and benzyl alcohol on the activities of four rat liver Golgi membrane enzymes, N-acetylglucosaminyl-, N-acetylgalactosaminyl-, galactosyl-, and sialytransferases and several soluble glycosyltransferases, bovine milk galactosyl- and N-acetylglucosaminyltransferases and porcine submaxillary N-acetylgalactosaminyltransferases have been studied. In rat liver Golgi membranes, linoleic acid inhibited the activities of N-acetylgalactosaminyl- and galactosyltransferases by 50% or greater, sialyltransferase by 10–15%, and N-acetylglucosaminyltransferase not at all. The isolated bovine milk N-acetylglucosaminyltransferase and porcine submaxillary N-acetylgalactosylaminyltranferase were not inhibited but bovine milk galactosyltransferase was inhibited by 95% or greater. The inhibition by linoleic acid on Golgi membrane galactosyltransferase appears to be a direct effect of the reagent on the enzyme. Incorporation of bovine milk galactosyltransferase into liposomes formed from saturated phospholipids, DMPC, DPPC, and DSPC (dimyristoyl-, dipalmitoyl-, and distearoylphosphatidylcholine) prevented inhibition of the enzyme activity suggesting that the lipid formed a barrier which did not allow linoleic acid access to the enzyme. The water soluble benzyl alcohol was more effective in inhibiting enzymes of the isolated rat liver Golgi complex. All four glycosyltransferases were inhibited, the N-acetylglucosaminyl- and N-acetylgalactosaminyltransferases by more than 95%. A higher concentration of benzyl alcohol was necessary to inhibit the galactosyltransferases than was required for the other Golgi enzymes. Benzyl alcohol also inhibited the isolated bovine milk N-acetylglucosaminyl- and galactosyltransferases 90% to 95%, respectively, but did not affect the isolated porcine submaxillary gland N-acetylgalactosaminyltransferase. Benzyl alcohol did not inhibit the milk galactosyltransferase incorporated into DMPC or DPPC liposomes but showed a complex effect on the activity of the enzyme incorporated into DSPC vesicles, a stimulation of activity at low concentrations followed by an inhibition. A lipid environment consisting of saturated lipids appears to present a barrier to inhibiting substances such as linoleic acid and benzyl alcohol, or lipid may stabilize the active conformation of the enzyme. The different effects of these reagents on four transferases of the Golgi complex suggest that the lipid environment around these enzymes may be different for each transferase.     

Kinetic and thermodynamic studies of the fusion of small unilamellar phospholipid vesicles

Small phospholipid vesicles, prepared so as to minimize impurities, fuse relatively slowly resulting in the time-dependent development of a characteristic endotherm in differential scanning calorimetry and corresponding changes in the Raman spectrum. The stability of small vesicles towards fusion increases with increasing acyl chain length for the series C-14 through 18. Within the protocols of these experiments, the fusion rate remains unchanged whether the vesicles are held at 10°C below T_m or at T_m itself. We have determined enthalpies of transition for small vesicles and fusion product for C-14 through C-18. In each case ΔH for small vesicles is lower than that of the corresponding multilamellar vesicles, while the fusion product ΔH is intermediate between small and multilamellar vesicles. The apparent lack of concensus in the literature as to the nature of the fusion process is ascribed to the variety of protocols used as well as the presence or absence of fusion-inducing impurities.     

Separation of liquid phases in giant vesicles of ternary mixtures of phospholipids and cholesterol

We use fluorescence microscopy to directly observe liquid phases in giant unilamellar vesicles. We find that a long list of ternary mixtures of high melting temperature (saturated) lipids, low melting temperature (usually unsaturated) lipids, and cholesterol produce liquid domains. For one model mixture in particular, DPPC/DOPC/Chol, we have mapped phase boundaries for the full ternary system. For this mixture we observe two coexisting liquid phases over a wide range of lipid composition and temperature, with one phase rich in the unsaturated lipid and the other rich in the saturated lipid and cholesterol. We find a simple relationship between chain melting temperature and miscibility transition temperature that holds for both phosphatidylcholine and sphingomyelin lipids. We experimentally cross miscibility boundaries both by changing temperature and by the depletion of cholesterol with beta-cyclodextrin. Liquid domains in vesicles exhibit interesting behavior: they collide and coalesce, can finger into stripes, and can bulge out of the vesicle. To date, we have not observed macroscopic separation of liquid phases in only binary lipid mixtures.