Uptake of liposomes containing the photoprotein obelin by rat isolated adipocytes. Adhesion,endocytosis or fusion?

1. The uptake of liposomes containing the photoprotein obelin by rat isolated adipocytes was investigated with the aim of producing liposome–cell fusion, enabling obelin to be introduced into the cytoplasm of intact cells. 2. Incubation of liposomes containing obelin with rat isolated adipocytes resulted in a time-dependent uptake of entrapped obelin by the adipocytes. The uptake by adipocytes (at 2h) of liposomes prepared from phosphatidylcholine, phosphatidylcholine+phosphatidylserine (molar ratio 4:1) and phosphatidylcholine+N-octadecylamine (molar ratio 4:1) was approx. 6, 10 and 10% of original entrapped obelin per g dry wt. of adipocytes respectively. 3. During incubation with adipocytes some of the liposomes became permeable to Ca²⁺ ions, resulting in stimulation of obelin luminescence from within the liposomes. This increase in permeability to Ca²⁺ seemed to be the result of the interaction of liposomes with the cell membrane. 4. Approx. 50% of liposome uptake could be inhibited by cytochalasin B (500μm). This was consistent with this uptake being the result of endocytosis. The remaining uptake was probably the result of adhesion of liposomes to the cell membrane. 5. In an attempt to detect the presence of cytoplasmic obelin, after incubation of adipocytes with liposomes, a method of causing a rapid rise in cell-membrane permeability to Ca²⁺ was developed in which an anti-cell anti-body–complement reaction occurred at the cell membrane. There was no detectable transfer of active obelin into the cell cytoplasm. 6. After incubation of liposomes with adipocytes in the absence of bovine serum albumin, obelin luminescence from a small proportion of liposomes increased (approx. 1.5%) in response to anti-(5′-nucleotidase) antibody plus complement. 7. It was concluded that under the conditions of these experiments, (a) no detectable transfer (<0.1%) of liposome-entrapped obelin to the adipocyte cytoplasm had occurred, (b) an increase in liposome permeability to Ca²⁺ occurred during incubation with adipocytes, (c) at least 50% of liposome uptake by adipocytes was the result of endocytosis, presumably into secondary lysosomes, and the remaining uptake was apparently the result of loose attachment of liposomes to the cell surface, and (d) in the absence of bovine serum albumin, a portion of at least one surface antigen, the ectoenzyme 5′-nucleotidase, was transferred from the adipocyte membrane to the liposome membrane.     

Two distinct adhesion mechanisms in embryonic neural retina cells. I. A kinetic analysis

The reaggregation kinetics of embryonic chick neural retina cells prepared using several different dissociation procedures were monitored through decreases in the small-angle light scattering of aggregating samples. Two distinct modes of aggregation were revealed, one Ca²⁺ independent, the other Ca²⁺ dependent, suggesting the existence of two separate adhesion mechanisms. By varying the concentrations of Ca²⁺ and trypsin in the dissociation medium, we obtained cells which exhibited both, either, or neither mode of aggregation. The Ca²⁺-independent adhesiveness is active in the absence of proteolysis, is resistant to low levels of trypsin (0.001%), but is readily inactivated at higher trypsin concentrations in either the presence or absence of Ca²⁺. It is relatively temperature independent. By contrast, the Ca²⁺-dependent adhesiveness is not detected before exposure of the cells to proteolysis. It is expressed after tryptic proteolysis in the presence of Ca²⁺ and is then highly temperature dependent. It is resistant to further digestion by trypsin in the continued presence of Ca²⁺ but is lost when Ca²⁺ is subsequently removed, apparently through the expression of tryptic cleavage incurred earlier. We suggest that its increased activity may result at least in part from the clustering of surface components into adhesive patches. A provisional model is presented correlating these data.     

Two distinct adhesion mechanisms in embryonic neural retina cells. II. An immunological analysis

Antibodies were raised against neural retina cells prepared by dissociation in EGTA alone (E cells, Ca²⁺-independent aggregation), in trypsin + Ca²⁺ (TC cells, Ca²⁺-dependent aggregation), or in trypsin + EGTA (TE cells, nonadhesive). Anti-E-cell Fab selectively inhibited Ca²⁺-independent aggregation, anti-TC-cell Fab selectively inhibited Ca²⁺-dependent aggregation, and anti-TE-cell Fab inhibited neither. Fab from a fourth preparation, also raised against E cells, inhibited both Ca²⁺-independent and Ca²⁺-dependent aggregation but was separated by immunoadsorption into two fractions, one specific for each mode of aggregation. In cells which utilize both modes simultaneously (LTC cells), each was inhibited exclusively by the appropriate Fab. The immunological data presented here demonstrate the existence in the same cells of two distinct and functionally independent adhesion mechanisms, each responsible for one of the two modes of aggregation. The differing adhesive properties of retinal cells prepared by different procedures are explained by the presence, absence, or degree of activity of these two mechanisms, qualities regulated by the concentrations of trypsin and Ca²⁺ used in the tissue dissociation.     

Cationic polypeptide-induced fusion of acidic liposomes

Fusion of acidic liposomes was induced by Mg²⁺, Ca²⁺, polylysine and polymyxin B. The extent of fusion and the concomitant change in liposome permeability induced by divalent cations depended on the concentration of liposomes in the suspension as well as on the cation concentration. In contradistinction, the extent of fusion and the change in permeability induced by the polypeptides depended only on the polycation concentration. The difference in the pattern of interaction, between the liposomes and the various cations, is a result of different binding affinities. The binding of the polypeptides to the liposomes, in contrast to divalent cations, is practically irreversible. The potential of polylysine to induce fusion of acidic phosphatidylethanolamine-devoid liposomes was used to demonstrate that in order to obtain fusion, both membranes involved must be susceptible, at least to a certain degree, to fusion by the proper inducer. When lysophosphatidylcholine substituted for phosphatidylcholine in phosphatidylethanolamine-rich acidic liposomes, extensive polylysine-induced fusion was obtained without concomitant spillage of the liposome contents.     

Calcium-dependent proteins in platelets response of calcium-activated protease in normal and thrombasthenic platelets to aggregating agents

Recent studies have suggested a role for Ca²⁺-dependent proteolysis in the regulation of microfilament disassembly by high molecular weight actin-binding protein. A Ca²⁺-activated protease similar to myofibrillar Ca²⁺-activated protease has been described in platelets. To explore the role of Ca²⁺-activated proteolysis of actin-binding protein in platelet function, we have examined the effects of platelet aggregating agents on platelet Ca²⁺-activated protease-like activity. The hydrolysis of actin-binding protein by Ca²⁺-activated protease was determined electrophoretically. The calcium ionophore, A23187, produced a dose-dependent stimulation of Ca²⁺-activated protease-like activity in the presence of exogenous calcium but had no effect in the absence of external calcium. Both normal and thrombasthenic platelets generated Ca²⁺-activated protease-like activity in response to A23187. Ionophore-induced stimulation of Ca²⁺-activated protease-like activity was not affected by prior incubation of platelets with 8-bromo cyclic GMP, 8-bromo cyclic AMP, prostaglandin E₁, prostaglandin I₂, indomethacin or tetracaine, but was inhibited by the sulfhydryl inhibitor N-ethylmaleimide. These results confirm the presence of Ca²⁺-activated protease in platelets and indicate that the source of calcium important in Ca²⁺-activated protease stimulation is in part extracellular. Other aggregating agents, thrombin, epinephrine, and ADP, were not accompanied by hydrolysis of actin-binding protein, indicating that the alteration in ionic calcium that occurs during aggregation by these other agents is insufficient to generate Ca²⁺-activated protease-like activity as measured by the present analytical technique.     

Dynamic fluorescence polarization studies on lipid mobilities in phospholipid vesicles in the presence of calcium mediators

The influence of Ca²⁺ mediators (nifedipine, verapamil and prostaglandin F_2α) on fluorescence polarization of l-anilino-8-napthalene-sulphonate in dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine liposomes was studied at various temperatures to understand the dynamic behaviour of membrane lipids. We also studied the effect of change in calcium concentration on the fluorescence polarization of the dye in the liposomes. Our results show increase in polarization (indicative of stiffening of the membrane) in the presence of Ca²⁺ ions. In the case of dimyristoyl phosphatidylcholine liposomes, all 3 drugs caused decrease in fluorescence polarization (increase in fluidity of the membrane) with or without Ca²⁺ ions in the medium. Contrary to this, in the case of dipalmitoyl phosphatidylcholine liposomes, the fluidization effect is observed for all the 3 drugs in the absence of Ca²⁺ ions; in the presence of Ca²⁺ ions stiffening is observed upon addition of nifedipine and verapamil which are antagonists, and fluidization is observed upon addition of prostaglandin F_2α. The role of drug-induced fluidity changes in membranes in therapy planning is discussed in the paper.     

Catalytic properties of phospholipid exchange protein from bovine heart

A protein which catalyzes the exchange of phosphatidylcholine between membranes has been purified from heart tissue homogenates up to 300-fold by acidic pH precipitation, (NH₄)₂SO₄ precipitation, gel filtration, and ion-exchange chromatography. Binding of the protein to phosphatidylcholine liposomes as measured by Sepharose chromatography was nondetectable. However, isoelectric focusing experiments showed that individual molecules of phosphatidylcholine were transferred from liposomes to the soluble, partially purified protein. Exchange of phospholipid between liposomes and mitochondria was not affected by the presence of moderate amounts of cholesterol in liposomes. A search for competitive inhibitors among moieties similar to phosphatidylcholine failed to show strong binding sites in the hydrophilic part of the substrate. High concentrations of Na⁺, Ca²⁺ and Mg²⁺ impaired the exchange activity.     

Calcium and magnesium induced changes in the relative fluidity of phosphatidylcholine liposomes

The effect of Ca²⁺ and Mg²⁺ on relative fluidity of phosphatidylcholine liposomes was studied by measuring the degree of chlorophyll fluorescence polarization. An increase in the degree of fluorescence polarization was observed on incubation of liposomes with different concentrations of Ca²⁺ or Mg²⁺. The results have been interpreted on the basis of increase in the size of liposomes which could be brought about by calcium or magnesium induced fusion of small unilamellar liposomes to form larger vesicles. Fusion of liposomes has also been confirmed by the experiments on efficiency of energy transfer from chlorophyll b to chlorophyll a, and transmission electron microscopy of liposomes before and after incubation with Ca²⁺ and Mg²⁺.     

Membranotropic effects of ω-hydroxypalmitic acid and Ca<Superscript>2+</Superscript> on rat liver mitochondria and lecithin liposomes. Aggregation and membrane permeabilization

The paper examines membranotropic Ca²⁺-dependent effects of ω-hydroxypalmitic acid (HPA), a product of ω-oxidation of fatty acids, on the isolated rat liver mitochondria and artificial membrane systems (liposomes). It was established that in the presence of Ca²⁺, HPA induced aggregation of liver mitochondria, which was accompanied by the release of cytochrome c from the organelles. It was further demonstrated that the addition of Ca²⁺ to HPA-containing liposomes induced their aggregation and/or fusion. Ca²⁺ also caused the release of the fluorescent dye sulforhodamine B from liposomes, indicating their permeabilization. HPA was shown to induce a high-amplitude swelling of Ca²⁺-loaded mitochondria, to decrease their membrane potential, to induce the release of Ca²⁺ from the organelles and to result in the oxidation of the mitochondrial NAD(P)H pool. Those effects of HPA were not blocked by the MPT pore inhibitor CsA, but were suppressed by the mitochondrial calcium uniporter inhibitor ruthenium red. The effects of HPA were also observed when Ca²⁺ was replaced with Sr²⁺ (but not with Ba²⁺ or Mg²⁺). A supposition is made that HPA can induce a Ca²⁺-dependent aggregation of mitochondria, as well as Ca²⁺dependent CsA-insensitive permeabilization of the inner mitochondrial membrane – with the subsequent lysis of the organelles.     

DNA-Induced Aggregation and Fusion of Phosphatidylcholine Liposomes in the Presence of Multivalent Cations Observed by the Cryo-TEM Technique

By means of cryoelectron transmission microscopy (cryo-TEM), we were able to demonstrate the formation of ternary complexes (TC): DNA–phosphatidylcholine liposome–divalent metal cations. Addition of Ba²⁺ to TC led to visualization of DNA compacting on the liposome surface. Staining the TC by Tb³⁺ cations revealed the changed secondary structure of DNA located between fused liposomes. Cryo-TEM and liposome turbidity data were analyzed during TC formation. Liposome aggregation and the liposome fusion induced by DNA in TC were observed. Because TC displayed the property of DNA cationic liposome complexes as well as their own unique properties, we were able to consider cationic lipoplexes as a particular case of TC. The involvement of TC and direct DNA–lipid interactions in the formation nuclear pore complexes were assumed.     

In vitro reaggregation of dissociated mouse cerebellar cells : I. Demonstration of different aggregation mechanisms

Reaggregation of mechanically dissociated mouse cerebellar cells (M cells) was compared with cells that received an additional trypsinization either before (T cells) or after (MT cells) the dissociation step. Reaggregation behaviour was followed by measuring the number and size distribution of particles with a Coulter counter. Aggregation rates which were calculated as percentage of decrease of particles could be measured reproducibly. Since the percentage of very large particles (> 100 cells) formed during aggregation varied considerably from one experiment to the next, size distribution curves of particles were used more to distinguish qualitative differences in a less quantitative way.Whereas aggregation rates and size distribution of particles with M cells were almost identical when aggregation occurred in medium of high (1.1 mM) or low (0.1 mM) Ca²⁺ concentrations, T and MT cells aggregated better at high Ca²⁺ concentration. Their aggregation rates were reduced by approx. 50% at low Ca²⁺ concentrations and larger aggregates were hardly formed under these conditions. The aggregation rates of T and MT cells showed a clear dependence on Ca²⁺ concentration, being half maximal at approx. 0.1 mM Ca²⁺.The ability of M cells to aggregate at low or high Ca²⁺ concentrations was influenced by subsequent trypsinization to produce MT cells. When the trypsin concentration was changed from 0.001 to 0.1% during this procedure the aggregation rates at high Ca²⁺ concentration were reduced to approx. 80% of the maximal value, whereas those at low Ca²⁺ concentrations were reduced to 35%. Variation of the Ca²⁺ concentration between 1.1 and 0.1 mM during the trypsinization step (0.015% trypsin) revealed no difference on the aggregation rates.We propose that M cells aggregate mainly or exclusively by a Ca²⁺-independent binding mechanism, whereas T or MT cells aggregate using a Ca²⁺-dependent one which may be functionally silent in M cells.     

Effect of Ca2+ on thermotropic properties of saturated phosphatidylcholine liposomes

The effect of various concentrations of calcium ion (Ca²⁺) on dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) and mixed DPPC/DSPC (1:1) liposomes was studied by differential scanning calorimetry. Ca²⁺ concentrations of 10 mM and above split the main transition peak of DPPC into two distinguishable components, and, at 30 mM and above, also resulted in the disappearance of a pre-transition peak. The effect of Ca²⁺ on DSPC liposomes was even more dramatic in that it induced a more definitive split in the main transition peak and caused the loss of the pretransition peak at only 10 mM concentration. The thermograms of the DPPC/DSPC mixed liposomes were unaltered in the presence of Ca²⁺, even at a concentration of 50 mM. Whether or not Ca²⁺ is able to alter thermograms of phosphatidylcholine liposomes appears to be dependent on the degree of molecular order of the bilayer prior to interaction with Ca²⁺.     

A Glycoprotein from Rat Liver Endoplasmic Reticulum Promotes Both Aggregation and Fusion of Liposomes at Acidic pH

Low-pH-induced fusion of liposomes with rat liver endoplasmic reticulum was evidenced. Fusion was inactivated by treatment of microsomes with trypsin or EEDQ (N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline), indicating the involvement of a protein. The protein was purified 555-fold by chromatographic steps. The identification and purification to homogeneity was obtained by electroelution from a slab gel, which gave a still active protein of about 50 kDa. The protein promoted the fusion of liposomes; laser light scattering showed an increase of mean radius of vesicles from 60 up to about 340 nm. Fusion was studied as mass action kinetics, describing the overall fusion as a two-step sequence of a second order aggregation followed by a first order fusion of liposomes. For phosphatidylcholine containing liposomes aggregation was not rate-limiting at pH 5.0 and fusion followed first order kinetics with a rate constant of 13 · 10⁻³ sec⁻¹. For phosphatidylethanolamine/phosphatidic acid liposomes aggregation was rate-limiting; however, the overall fusion was first order process, suggesting that fusogenic protein influences both aggregation and fusion of liposomes. The protein binds to the lipid bilayer of liposomes, independently of pH, probably by a hydrophobic segment. Exposed carboxylic groups might be able to trigger pH-dependent aggregation and fusion. It is proposed that the protein inserted in the lipid bilayer bridges with an adjacent liposome forming a fused doublet. Since at endoplasmic reticulum level proton pumps are operating to generate a low-pH environment, the membrane bound fusogenic protein may be responsible for both aggregation and fusion of neighboring membranes and therefore could operate in the exchange of lipidic material between intracellular membranes. Received: 25 August 1997/Revised: 28 April 1998     

CALCIUM-DEPENDENT BINDING OF BRAIN GLUTAMATE DECARBOXYLASE TO PHOSPHOLIPID VESICLES

The binding of glutamate decarboxylase (GAD), to phospholipid vesicles (liposomes) in the absence and in the presence of several Ca²⁺ and Mg²⁺ concentrations was studied. Phosphatidylcho-line-phosphatidylserine (4:1) liposomes are capable of binding GAD in a Ca²⁺-dependent manner. The per cent of GAD bound increased from 5 to 65°., in a sigmoid shape with Ca²⁺ concentrations in the 0.2-4 mm range. Mg²⁺ also induces GAD binding but is less effective than Ca²⁺ The Ca²⁺ -dependent binding of GAD is not the result of unspecific association of protein, since Ca²⁺ did not promote any binding of choline acetyltransferase or lactate dehydrogenase. Furthermore, the relative specific activity (^o_o enzyme activity/% protein) of GAD associated to liposomes increases 4-fold from 0 to 2 mm Ca²⁺. The per cent of GAD bound attains a plateau at a ratio phospholipid/protein of about 1.5. and decreases when the pH increases from 6.5 or 6.8 to 7 or 7.25. Na⁺ or K⁺ at a 100mm concentration also induce binding of GAD to liposomes. Phosphatidylcholine liposomes (without phosphatidylserine) practically did not bind GAD at any Ca²⁺ concentration. The Ca²⁺-dependent association of GAD to phosphatidylcholine-phosphatidylserine liposomes is very similar to that previously reported using brain membranes, and it correlates also well with the reported Ca²⁺-dependent aggregation of phosphatidylserine molecules in phospholipid membranes of similar composition. It is concluded that phosphatidylserine is probably involved in the Ca²⁺-dependent binding of GAD to brain membranes. Phospholipid vesicles seem to be a useful experimental model for studying the mechanisms of this GAD association to membranes and the possible physiological implications of the GAD-Ca²⁺-membrane interaction regarding the release of newly synthesized GABA from nerve endings.     

Studies on the mechanism of membrane fusion. Role of head-group composition in calcium- and magnesium-induced fusion of mixed phospholipid vesicles

We have investigated the contribution of various phospholipids to membrane fusion induced by divalent cations. Fusion was followed by means of a new fluorescence assay monitoring the mixing of internal aqueous contents of large (0.1 μm diameter) unilamellar liposomes. The rate and extent of fusion induced by Ca²⁺ in mixed phosphatidylserine/phosphatidylcholine vesicles were lower compared to those in pure phosphatidylserine vesicles. The presence of 50% phosphatidylcholine completely inhibited fusion, although the vesicles aggregated upon Ca²⁺ addition. When phosphatidylserine was mixed with phosphatidylethanolamine, however, rapid fusion could be induced by Ca²⁺ even in mixtures that contained only 25% phosphatidylserine. Phosphatidylethanolamine also facilitated fusion by Mg²⁺ which could not fuse pure phosphatidylserine vesicles. In phosphatidylserine/phosphatidylethanolamine/phosphatidylcholine mixtures, in which the phosphatidylcholine content was kept at 25%, phosphatidylethanolamine could not substitute for phosphatidylserine, and the fusogenic capacity of Mg²⁺ was abolished by the presence of merely 10% phosphatidylcholine. The initial rate of release of vesicle contents was slower than the rate of fusion in all the mixtures used. The presence of phosphate effected a considerable decrease in the threshold concentration of Ca²⁺ and also enhanced     

Changes in surface charge density on liposomes induced by Escherichia coli endotoxin

Changes in surface charge density of liposomes induced by E. coli endotoxin were studied by microelectrophoresis. Endotoxin altered the surface charge of phosphatidylcholine liposomes from neutral to negative. The negative charge of the endotoxin-phosphatidylcholine complex was neutralized electrostatically by binding with Ca²⁺ (2 mM). Phosphatidylcholine liposomes were made positive by addition of the positively charged detergent, hexadecyltrimethylammonium chloride. Endotoxin made the positively charged liposomes less charged. On the other hand, phosphatidylserine liposomes which were negatively charged became less charged in the presence of high concentration of endotoxin (8 mg/ml). The endotoxin effect on phosphatidylserine liposomes was abolished by EDTA (1 mM) but potentiated by CaCl₂ (0.1–2 mM). These results indicate that endotoxin interacts with liposomes both hydrophobically and electrostatically.     

Transbilayer diffusion of divalent cations into liposomes mediated by lipidic particles of phosphatidate

Liposomes formed from egg-yolk phosphatidylcholine:egg-yolk phosphatidate (molar ratio 2:1) containing pBR322 DNA and DNase I were induced to form, with divalent cations, bilayer/nonbilayer phase transitions of phosphatidate which allowed cation diffusion into liposomes; then cation diffusion was measured by the activation of the hydrolysis of DNase I on DNA. The formation of phosphatidate transitions on liposomes was demonstrated by freeze-fracture and ³¹P NMR, and a direct correlation between the formation of phosphatidate transitions and the transbilayer diffusion of cations was found: only Ca²⁺ and Mn²⁺, which induce phase transitions, were able to penetrate liposomes and triggered the DNase I activity; in addition, Ca²⁺ at higher concentrations (10 mM) caused fusion of liposomes, whereas Mn²⁺ did not, suggesting that transitions induced by Mn²⁺ participated only in the diffusion of this ion; furthermore, Mg²⁺ neither formed phase transitions nor triggered the enzymatic activity. The liposomes studied represent more dynamic structures that can form phosphatidate structures involved in both (1) the interchange of divalent cations with the surroundings, thereby modulating encapsulated enzymes, and (2) the fusion of lipid vesicles probably implicated in the enrichment of liposomal content in the early Precambian Earth.Correspondence to: C. Argüello     

Immunological detection of cell surface components related with aggregation of Chinese hamster and chick embryonic cells.

Previous studies suggested that Chinese hamster V79 cells possess two mechanisms for their mutual adhesion, Ca²⁺-dependent and Ca²⁺-independent ones. We could prepare cells with only the Ca²⁺-dependent mechanism intact by dispersing cell monolayers with trypsin (0.01%) containing Ca²⁺. In the present study, we found that cells dispersed with a very low concentration of trypsin (0.0001%) in the absence of Ca²⁺ retain only the Ca²⁺-independent mechanism intact. Fab fragments of antibodies directed against surface antigens of V79 cells inhibited the aggregation of V79 cells by the Ca²⁺-independent mechanism, but did not inhibit the aggregation of these cells by the Ca²⁺-dependent mechanism. These results suggest that the two mechanisms of cell adhesion are based on different cellular components. Molecules responsible for the Ca²⁺-independent adhesion mechanism are probably cell surface components, because they were released from cells by the treatment with 0.01% trypsin without losing their specific antigenicity. The presence of adhesion mechanisms similar to those in V79 cells was shown in neural retinal cells of chick embryos. It was assumed, therefore, that these mechanisms of cell adhesion are generally present among a variety of cell types.     

Cleavage stage mouse embryos share a common cell adhesion system with teratocarcinoma cells

We examined similarities in adhesive properties of mouse cleaving embryos at one- to eight-cell stages and of teratocarcinoma cells by aggregation studies. Teratocarcinoma cells and fibroblastic cells have a Ca²⁺-dependent cell-cell adhesion site (CDS), which is resistant to trypsin in the presence of Ca²⁺ but sensitive in the absence of Ca²⁺. When several embryos treated with trypsin in the presence of Ca²⁺ (TC) were kept in contact with each other, they fused into a single aggregate in the medium with Ca²⁺ but not without Ca²⁺. Embryos treated with trypsin in the absence of Ca²⁺ (TE) did not show such Ca²⁺-dependent aggregation. Aggregation of TC-treated embryos was inhibited by Fab fragments of antibody raised against TC-treated teratocarcinoma F9 cells. The aggregation-inhibitory effect of the Fab was removed by absorption with TC-treated teratocarcinoma cells, but not with TE-treated teratocarcinoma cells. This effect was not removed by absorption with fibroblasts and some other tissue cells. TC-treated embryos adhered to TC-treated teratocarcinoma cells, but not to TC-treated fibroblastic cells. These results suggest that early mouse embryos share a common CDS molecule with teratocarcinoma cells but not with fibroblastic cells.     

Fusion of small unilamellar liposomes with phospholipid planar bilayer membranes and large single-bilayer vesicles

Small unilamellar phosphatidylserine/phosphatidylcholine liposomes incubated on one side of planar phosphatidylserine bilayer membranes induced fluctuations and a sharp increase in the membrane conductance when the Ca²⁺ concentration was increased to a threshold of 3–5 mM in 100 mM NaCl, pH 7.4. Under the same ionic conditions, these liposomes fused with large (0.2 μm diameter) single-bilayer phosphatidylserine vesicles, as shown by a fluorescence assay for the mixing of internal aqueous contents of the two vesicle populations. The conductance behavior of the planar membranes was interpreted to be a consequence of the structural rearrangement of phospholipids during individual fusion events and the incorporation of domains of phosphatidylcholine into the Ca²⁺-complexed phosphatidylserine membrane. The small vesicles did not aggregate or fuse with one another at these Ca²⁺ concentrations, but fused preferentially with the phosphatidylserine membrane, analogous to simple exocytosis in biological membranes. Phosphatidylserine vesicles containing gramicidin A as a probe interacted with the planar membranes upon raising the Ca²⁺ concentration from 0.9 to 1.2 mM, as detected by an abrupt increase in the membrane conductance. In parallel experiments, these vesicles were shown to fuse with the large phosphatidylserine liposomes at the same Ca²⁺ concentration.