Lipid vesicle-cell interactions. I. Hemagglutination and hemolysis

The interaction of lipid vesicles (liposomes) of several different compositions with erythrocytes has been investigated. Lecithin liposomes, rendered positively charged with stearylamine, exhibit potent hemagglutination activity in media containing low concentrations of electrolytes. The hemagglutination titer is found to be a linear function of the zeta potential of the lipid vesicles. Hemagglutination is reduced when the surface potential of the cells is made more positive by pH adjustment or enzyme treatment. Similarly, hemagglutination is reduced by increasing concentrations of electrolytes. Hemagglutination is examined theoretically and is shown to be consistent with vesicle-cell interactions that are due to only electrostatic forces. Vesicles containing lysolecithin in addition to lecithin and stearylamine cause lysis of erythrocytes, provided the lipids of the vesicles are above the crystal-liquid crystal phase transition temperature. In addition, hemolysis requires close juxtaposition of the vesicle to the cell membrane; vesicles precoated with antibodies exhibit severely diminished hemolytic activities, only a small fraction of which can be attributed to a reduction in hemagglutination titer. Evidence is presented indicating that a single vesicle is sufficient to lyse one cell. With regard to hemagglutination and hemolysis, lipid vesicles of simple composition mimic paramyxoviruses such as Sendai virus.     

Lipid vesicle-cell interactions. III. Introduction of a new antigenic determinant into erythrocyte membranes

The introduction of a new antigenic determinant, 2,4-dinitrophenyl-aminocaproyl-phosphatidylethanolamine (DNP-Cap-PE), into the surface membranes of intact human erythrocytes is described. Fresh cells were incubated in the presence of liposomes composed of 10% DNP-Cap-PE, 5% stearylamine, 20% lysolecithin, and 65% lecithin. Such liposome-treated erythrocytes are shown to be susceptible to immune lysis by anti-DNP serum in the presence of complement. Uptake of DNP-Cap-PE by erythrocyte membranes is also demonstrated by immunofluorescence using indirect staining with rabbit anti-DNP serum followed by fluroescein-conjugated goat anti-rabbit IgG and by electron microscopy using ferritin-conjugated antibody. Antigen uptake did not occur at low temperatures or from vesicles lacking lysolecithin and stearylamine. Fluorescence microscopy shows that the antigen-antibody complexes are free to diffuse over the cell surface, eventually coalescing into a single area on the cell membrane. Electron microscopy suggests that a substantial proportion of the lipid antigen is incorporated by fusion of vesicles with the cell membrane. There are indications that vesicle treatment causes a small proportion of cells to invaginate.     

A membrane-bound fluorescent probe to detect phospholipid vesicle-cell fusion

Summary Pyrenesulfonylphosphatidylethanolamine has been incorporated into sonicated phospholipid vesicles to provide a fluorescent signal from a membrane-bound probe whose spectrum is sensitive to the local concentration of dye molecules. When vesicle material was taken up by viable mouse splenocytes, the disappearance of the pyrene excimer fluorescence emission peak that accompanied dilution of the vesicle membrane lipid could be quantitated. One can thus measure, by a simple and rapid procedure, a new parameter which is related to the extent of vesicle-cell fusion and which is independent of the transfer of aqueous vesicle contents to the cell cytoplasm.Abbreviations used 6-CF 6-carboxyfluorescein - HBSS Hanks Balanced Salt Solution - DMPC dimyristoylphosphatidylcholine - DOPC dioleoyl-phosphatidylcholine - DPPC dipalmitoylphosphatidylcholine - EYL egg yolk lecithin - PE phosphatidylethanolamine - PEG polyethylene glycol - PLV phospholipid vesicle - PSPE pyrenesulfonylphosphatidylethanolamine     

Lipid-polyethylene glycol interactions: I. Induction of fusion between liposomes

Summary Fusion between unilamellar vesicles of both egg phosphatidylcholine and bovine phosphatidylserine was induced by polyethylene glycol. Aggregation and fusion events were monitored by electron microscopy and turbidity measurements. The threshold concentration of polyethylene glycol for aggregation and fusion is found to be independent of lipid concentration. Typically, aggregation of phosphatidylcholine vesicles starts at 2.5% (wt/wt) polyethylene glycol, but fusion is not significant until the polyethylene glycol concentration reaches 35%. Multilamellar vesicles were formed as a result of fusion.Abbreviations PEG Polyethylene glycol - IMP Intramembranous particle - PC Phosphatidylcholine - PS Phosphatidylserine - SUV Small unilamellar vesicles - MLV Multilamellar vesicles - DPPC Dipalmitoyl phosphatidylcholine - DSC Differential scanning calorimetry     

Intercellular interactions preceding muscle cell fusion

Membrane interactions of myogenic cells of the 7-8 old chick embryos were investigated by electron microscopy. Tannic acid was used as specific fixative for biological membranes. Three types of specialized contacts can be revealed in fused myogenic cells: the first is like gap junctions, the second represents a pentalamellar structure, and the third is a so far non-described type of contact showing a membrane complex consisting of two parallel membranes arranged at a distance of approximately 15 nm and connected by electron-dense "bridges". The third type contact is revealed only by tannin-fixation. It is suggested that the "bridge" contact precedes the pentalamellar structure. A transition from the first type of contact to the second one is possible. The pentalamellar structure can be considered as an initial phase of fusion.     

Biochemical studies on cell fusion. I. Lipid composition of fusion- resistant cells

A series of stable cell mutants of mouse fibroblasts were previously isolated (Roos, D. S. and R. L. Davidson, 1980, Somatic Cell Genet., 6:381-390) that exhibit varying degrees of resistance to the fusion-inducing effect of polyethylene glycol (PEG), but are morphologically similar to the parental cells from which they were derived. Biochemical analysis of these mutant cell lines has revealed differences in whole cell lipid composition which are directly correlated with their susceptibility to fusion. Fusion-resistant cells contain elevated levels of neutral lipids, particularly triglycerides and an unusual ether-linked lipid, O-alkyl, diacylglycerol. This ether lipid is increased approximately 35-fold over parental cells in the most highly PEG-resistant cell line. Fusion-resistant cells also contain more highly saturated fatty acyl chains (ratio of saturated to polyunsaturated fatty acids [S/P ratio] approximately 4:1) than the parental line (S/P ratio approximately 1:1). Cells which are intermediate in their resistance to PEG have ether lipid and fatty acid composition which is intermediate between the parental cells and the most fusion-resistant mutants. In a related communication (Roos, D. S. and P. W. Choppin, 1985, J. Cell. Biol., 100:1591-1598) evidence is presented that alteration of lipid content can predictably control the fusion response of these cells.     

Biochemical studies on cell fusion. II. Control of fusion response by lipid alteration

The preceding communication (Roos, D.S. and P.W. Choppin, 1985, J. Cell Biol. 101:1578-1590) described the lipid composition of a series of mouse fibroblast cell lines which vary in susceptibility to the fusogenic effects of polyethylene glycol (PEG). Two alterations in lipid content were found to be directly correlated with resistance to PEG-induced cell fusion: increases in fatty acyl chain saturation, and the elevation of neutral glycerides, including an unusual ether-linked compound. In this study, we have probed the association between lipid composition and cell fusion through the use of fatty acid supplements to the cellular growth medium, and show that the fusibility of cells can be controlled by altering their acyl chain composition. The parental Clone 1D cells contain moderately unsaturated fatty acids with a ratio of saturates to polyunsaturates (S/P) approximately 1 and fuse virtually to completion following a standard PEG treatment. By contrast, the lipids of a highly fusion-resistant mutant cell line, F40, are highly saturated (S/P approximately 4). When the S/P ratio of Clone 1D cells was increased to approximate that normally found in F40 cells by growth in the presence of high concentrations of saturated fatty acids, they became highly resistant to PEG. Reduction of the S/P ratio of F40 cells by growth in cis-polyunsaturated fatty acids rendered them susceptible to fusion. Cell lines F8, F16, etc., which are normally intermediate between Clone 1D and F40 in both lipid composition and fusion response, can be altered in either direction (towards either increased or decreased susceptibility to fusion) by the addition of appropriate fatty acids to the growth medium. Although trans-unsaturated fatty acids have phase-transition temperatures roughly similar to saturated compounds, and might therefore be expected to affect membrane fluidity in a similar manner, trans-unsaturated fatty acids exerted the same effect as cis-unsaturates on the control of PEG-induced cell fusion. This observation suggests that the control of cell fusion by alteration of fatty acid content is not due to changes in membrane fluidity, and thus that the fatty acids are involved in some other way in the modulation of cell fusion.     

Membrane mobility agents. II. Active promoters of cell fusion.

The membrane mobility agent, A2C, actively promotes the fusion of hen erythrocytes under conditions similar to those used by Lucy et al. for glyceryl monooleate.     

Lipid-cell interactions. Induction of microvilli on the cell surface by liposomes

Treatment of cells with sonicated egg lecithin (EL) liposomes induced microvilli on the surface of mouse embryo fibroblasts and cells of an epithelioid MPTR strain. Unsonicated liposomes and sonicated dipalmitoyllecithin (DPL) liposomes do not induce microvilli on the surface of these cells. It is supposed that microvilli induction is caused by modification of the cell surface composition by small liquid-crystalline liposomes (e.g. by fusion with plasma membrane or transfer of some components from the cell surface to liposomes and vice versa). Microvilli induction by lipsomes provides a system for investigation of their role in cell life.     

Lipid interactions of the hemagglutinin HA2 NH2-terminal segment during influenza virus-induced membrane fusion.

Fusion of influenza viruses with target membranes is induced by acid and involves complex changes in the viral fusion protein hemagglutinin (HA) and in the contact sites between viruses and target membranes (Stegmann, T., White, J. M., and Helenius, A. (1990) EMBO J. 9, 4231-4241). At 0 degrees C, in a first, kinetically distinct step, target membranes irreversibly adhere to the viruses. Fusion itself starts only after a lag-phase of several minutes (X-31 strain viruses) or after raising the temperature (PR8/34 strain viruses). We now provide evidence that the initial conformational change resulting in virus-target membrane adhesion is restricted to a (minor) subpopulation of the HA molecules. These molecules become susceptible to bromelain digestion, and they could be labeled with the photoactivatable reagent [3H]PTPC/11, a nonexchangeable lipid present in the target lipid bilayer (Harter, C., B?chi, T., Semenza, G., and Brunner, J. (1988) Biochemistry 27, 1856-1864). Only the HA2 subunit was labeled, and analyses of 2-nitro-5-thio-cyanobenzoic acid fragments derived thereof indicate that the HA2 NH2-terminal segment (fusion peptide) inserted into the target membrane bilayer. When the temperature was raised to trigger fusion of PR8/34 viruses, labeling of HA2 increased by a factor of 130. Most (74%) of that label was incorporated into the COOH-terminal membrane anchor region, but there was also a strong increase (about 30-fold) of NH2-terminal fusion peptide labeling. This suggests that fusion is preceded., or accompanied, by further changes in HA which lead to additional extensive lipid insertions of HA2 fusion peptides.     

Studies on membrane fusion. II. Induction of fusion in pure phospholipid membranes by calcium ions and other divalent metals

The effect of divalent metals on the interaction and mixing of membrane components in vesicles prepared from acidic phospholipids has been examined using freeze-fracture electron microscopy and differential scanning calorimetry. Ca²⁺, and to a certain extent Mg²⁺, induce extensive mixing of vesicle membrane components and drastic structural rearrangements to form new membranous structures. In contrast to the mixing of vesicle membrane components in the absence of Ca²⁺ described in the accompanying paper which occurs via diffusion of lipid molecules between vesicles, mixing of membrane components induced by Ca²⁺ or Mg²⁺ results from true fusion of entire vesicles. There appears to be a “threshold” concentration at which Ca²⁺ and Mg²⁺ become effective in inducing vesicle fusion and the threshold concentration varies for different acidic phospholipid species. Different phospholipids also vary markedly in their relative responsiveness to Ca²⁺ and Mg²⁺, with certain phospholipids being much more susceptible to fusion by Ca²⁺ than Mg²⁺. Vesicle fusion induced by divalent cations also requires that the lipids of the interacting membranes be in a “fluid” state ($\text{T > T}{}_{\mathrm{c}}$). Fusion of vesicle membranes by Ca²⁺ and Mg²⁺ does not appear to be due to simple electrostatic charge neutralization. Rather the action of these cations in inducing fusion is related to their ability to induce isothermal phase transitions and phase separations in phospholipid membranes. It is suggested that under these conditions membranes become transiently susceptible to fusion as a result of changes in molecular packing and creation of new phase boundaries induced by Ca²⁺ (or Mg²⁺).     

Induction of cell fusion of plant protoplasts by electrical stimulation

When an electric impulse of a few milliseconds was applied topoint-adherence protoplasts isolated from cultured cells ofRauwolfia serpentina through glass capillary microelectrodes,fusion of the protoplasts was immediately induced. This phenomenonseems to be related to transient changes in the membrane state,such as membrane excitation, induced by electrical stimulation. (Received February 22, 1979; )     

Mechanism of antigenic competition. II. Induction by specific T cell products

Since previous work had indicated that the mechanism of antigenic competition induced by the injection of red cells was due to a reversible block of cooperation at the surface of the macrophage, the possibility that the inhibitor was complexes of IgT and antigen resulting from the previous response (to red cells) was investigated. Macrophages were incubated with supernatants from activated T cells (ATC), containing, or deprived of, IgT by incubation with anti-mouse Ig-coated sepharose beads. Macrophages incubated with ATC supernatants were immunogenic for the corresponding antigen, but were not capable of binding a second ATC supernatant, of different antigenic specificity, in an immunogenic way. In contrast, if IgT was previously removed, the macrophages were not immunogenic and were not blocked. These results support the concept that this form of antigenic competition is due to blockage of macrophage receptor sites for IgT.     

Toxoplasma gondii-vertebrate cell interactions. II. The intracellular reproductive phase.

The reproduction of Toxoplasma gondii RH-strain in vertebrate cells was studied in a controlled-environment culture system. The lag period before reproduction and the doubling time of individual parasites were determined using a least-squares linear regression method of analysis which does not artificially constrain the data. In the majority of cases, the time intercept of the linear regression line was either zero, implying the lack of a lag phase before reproduction, or negative, implying the parasite had completed part of its reproductive cycle before entering the host cell. The mean doubling time of T. gondii is 10.9 h in bovine embryo skeletal muscle cells and 8.3 h in HeLa cells. This difference is not significant at the 5% level. The population doubling times of mouse-derived parasites is best described by a gamma distribution.     

Induction of HLA expression in Daudi cells after cell fusion

The Daudi cell line, established from a Burkitt lymphoma, has recently been found to be HLA- andΒ ₂-microglobulin-negative, although it expresses B lymphocyte alloantigens. This report is concerned with the reexpression of HLA-A10, B38, and B17 on the Daudi cell, after cell fusion with another human cell line (Raji) or with mouse fibroblasts. In the latter fusion, the same HLA specificities are re-expressed, but not humanΒ ₂-microglobulin while mouseΒ ₂-microglobulin andH-2 could be detected. No such reexpression was observed when Daudi was fused with the F9 mouse teratocarcinoma, which lacks mouseΒ ₂-m andH-2. No HLA activity (alloantigenic and xenogenic activity) was detected in the membrane or cytoplasm of Daudi, using salt extraction and sonication. Therefore we postulate thatΒ ₂-microglobulin could be necessary for the expression and possible synthesis of the HLA antigen.