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In two-component phosphatidylcholine bilayers with coexisting liquid and P beta' gel phases, the distribution between phases of low concentrations of glycosphingolipids can be determined by freeze-etch electron microscopy after labeling the glycolipid with a suitable protein. We have found that the distribution depends upon the glycosphingolipid species (Rock, P. et al., (1991) Biochemistry 30, 19-25). Using this technique with cholera toxin as the protein label and bilayers formed from dipalmitoyl- and dielaidoylphosphatidylcholine (1:1) containing < 1 mol% GM1, we have studied the distribution of a family of GM1 homologues differing in the acyl chain and sphingoid base moieties. The GM1 preference for the P beta' ripple phase decreases with decreasing acyl chain length and increasing unsaturation. GM1 with either a C18:1 or C20:1 sphingoid base shows similar distributions in liquid and gel phases. When the molecules are preferentially found in the P beta' phase, they are positioned along unique loci in both A and A/2 forms of the ripple structure. This localization and acyl chain dependence reflect the volume, shape and localization of molecular packing defects in the P beta' phase. The ganglioside inclusions stabilize the P beta' phase and form compositional domains of unique topography.

We demonstrate that supported synthetic phospholipid bilayers, which are stabilized by lateral cross-linking in both leaflets, can be used for specimen preparation for atomic force microscopy of purified membrane proteins with high stability and excellent reproducibility under water or low-salt buffer. A bilayer containing 1,2-dipentacosa-10,12-diynoyl-phosphatidylcholine and 20 mol% ganglioside (GMI) was transferred onto the surface of mica from a Langmuir trough. Cholera toxin, both the B-subunit and the complete molecule randomly bound to the gangliosides, were imaged by atomic force microscopy in solution with a resolution of better than 2 nm. The pentameric structure of the B-subunit oligomers was well resolved. This result indicates that, with this preparation procedure, other membrane proteins may be studied at intermediate to high resolution under physiologically relevant conditions without the need for crystallization.

Multilamellar liposomes composed of 1:1 dielaidoylphosphatidylcholine: dipalmitoylphosphatidylcholine at 20 degrees C contain laterally separated gel and liquid-crystalline phases that can be identified by electron microscopy in freeze-etch replicas on the basis of their distinctive morphology. Visualization of marker proteins that specifically bind to glycosphingolipids included in these liposomes has revealed that, at 1 mol % or less, the ganglioside GM1 and the neutral asialo-GM1 derived from it are localized within the gel-phase regions exclusively. Increasing the mole fraction of the glycosphingolipids results in the appearance of marker in the fluid-phase regions. Another neutral glycosphingolipid, Forssman, does not display a phase preference and is found in both phases at a low mole percent. The phase preference of these three glycosphingolipids depends primarily upon interactions between the hydrophobic moieties of these molecules and the matrix phosphatidylcholines.

The techniques of ultrafast freezing and freeze-etch electron microscopy have been successfully employed to visualize IgG molecules and Fab fragments specifically bound to the neutral glycosphingolipids Forssman and asialo-GM1 incorporated into phosphatidylcholine liposomes. Monovalent Fab is the superior marker because of its small size and because it does not cause liposomal aggregation with concomitant glycolipid reorganization. Analysis of Fab labeling of liposomes containing these neutral glycosphingolipids leads to the conclusion that the Forssman glycosphingolipid is dispersed in clusters of not more than several molecules when present at low mole fraction in fluid-phase 1-palmitoyl-2-oleoylphosphatidylcholine liposomes. In contrast to this, asialo-GM1 under the same conditions is present in clusters of about 15 molecules in this phospholipid matrix.

A 37-year-old female with thrombocytopenia since childhood developed diffuse osteosclerosis followed by bone marrow aplasia 23 years after Thorotrast administration. Tissue deposition of Thorotrast was documented by radiospectroscopy (liver, bone) and autoradiography (bone). Bone marrow failure in this patient was clinically ascribed to diffuse myelofibrosis, but at autopsy marrow fibrosis was only focal. An unsuspected bone mineralization defect was manifested by thick osteoid seams. It is suggested that osteosclerosis, osteomalacia, and bone marrow aplasia represent a combined experession of Thorotrast cytotoxicity.

A radioactively-labelled glycosphingolipid, asialo-GM1, has been incorporated into phosphatidylcholine multilamellar vesicles. After incubation with ferritin-Ricinus communis agglutinin 60 (RCA 60) conjugate at different temperatures, the vesicles were separated from the conjugate by discontinuous density gradient ultracentrifugation. Measurement of the distribution of the radioactively-labelled asialo-GM1 in the pelleted conjugate fraction and freeze-etch electron microscopy of the vesicle fraction indicate that the decrease in labelling of asialo-G M 1 -containing vesicles by ferritin-RCA 60 conjugate with increasing temperatures (Tillack, T.W., Wong, M., Allietta, M. and Thompson, T.E. (1982) Biochim. Biophys. Acta 691, 261–273) reflects a decrease in apparent binding affinity rather than an ability of the conjugate to extract glycolipid from the phospholipid bilayer after binding.

Small sonicated dipalmitoylphosphatidylcholine vesicles when incubated at 4 degrees C and high concentrations are shown to fuse completely to vesicles about 700-A diameter in 7 days, and these further fuse to about 950 A diameter vesicles after 3-4 weeks. The 950 A diameter vesicles are spherical, homogeneous, mostly unilamellar, have an internal aqueous space about 10 times that of small vesicles, and are stable for at least 6 months. The 950-A vesicles are characterized by agarose gel chromatography, freeze-fracturing electron microscopy, trapped volume measurements, differential scanning calorimetry, and diphenylhexatriene fluorescence polarization.

Molecules of the ganglioside GM1 are randomly distributed in liquid-crystalline 1-palmitoyl-2-oleoyl phosphatidylcholine bilayers. This conclusion is based on a freeze-etch electron microscopic study using ferritin-conjugated cholera toxin and cholera toxin alone as ganglioside labels. The average number of GM1 molecules under a label is calculated by a novel method from the dependence of the fraction of bilayer area covered by the label on the mole fraction of GM1 in the bilayer.

During the initial 5 minutes of T2 bacteriophage infection of Escherichia coli, there is substantial incorporation of labeled guanine into the transfer RNA (tRNA) fraction, strongly suggesting that new tRNA is synthesized early in infection. In this 5-minute period the amount of labeled guanine incorporated into the tRNA fraction of T2-infected bacteria is 23% of that in uninfected bacteria during an equivalent period. The tRNA fractions from T2-infected bacteria and uninfected bacteria were compared by pulse-labeling cultures of each for 5 minutes with 3H and 14C isotopes of guanine and partially resolving a mixture of the doubly labeled tRNA species by reversed phase chromatography. The chromatographic pattern of the labeled tRNA from uninfected cultures of E. coli was very similar to that of nonlabeled carrier tRNA from uninfected cultures. tRNA prepared from pulse-labeled T2-infected cultures of E. coli had a chromatographic pattern that was radically different from that of tRNA prepared from uninfected cultures. These results indicate either that the tRNA synthesized after T2 infection of E. coli represents species of tRNA entirely different from those made in uninfected bacteria, or that relative proportions of the different species made are greatly altered after phage infection, some being made preferentially and others in reduced amounts. Evidence is presented that a substantial amount of the material in the tRNA fraction made during T2 infection can accept amino acids.

The disappearance and reappearance of the P beta' ripple in multilamellar liposomes of dipalmitoylphosphatidylcholine (DPPC) has been examined by freeze-etch electron microscopy. The presence of less than 10 mol% of various glycosphingolipids or cholesterol in the liposomes markedly increases the time required for ripple disappearance when the vesicles are cooled from 38 degrees C to 30 degrees C, as compared to the pure phospholipid. Once the ripples have begun to disappear in the two-component vesicles, they do not uniformly reappear until the system is heated above the main transition of DPPC and allowed to cool into the pretransition region. These results suggest that the long time for ripple disappearance in the two-component systems reflects a slow molecular reorganization process which occurs when the systems are forced to change from the P beta' gel to the L beta' gel by a temperature downshift.

An affinity purified monovalent ferritin conjugate of Ricinus communis agglutinin (RCA 60) is used with freeze-etch electron microscopy to study the ultrastructural localization of the glycosphingolipid asialo-G M1 in multilamellar phosphatidylcholine liposomes. Dimyristoylphosphatidylcholine (DMPC) liposomes containing up to 20 mol% asialo-G M1 and quenched below the main transition temperature show a striking linear localization of ferritin-RCA 60 between phospholipid ridges. The glycosphingolipid localization is similar to that postulated for up to 20 mol% cholesterol in pure phosphatidylcholine bilayers by Copeland, B.R. and McConnell, H.M. (Biochim. Biophys. Acta, 599, 95–109 (1980)). Above the main phase transition temperature, asialo-G M1 appears to be organized into clusters, especially in palmitoyloleoylphosphatidylcholine (POPC) liposomes. This clustered distribution of glycosphingolipids seen above the phase transition temperature suggests that this type of lipid may exhibit compositional domain structure in biological membranes.

Glycosphingolipids, in contrast to glycerol-based lipids, are relatively minor components of mammalian cell membranes. They are, however, confined to the external surface of the plasma membrane and in this surface may collectively be a major component (1–4). In some cell types very small amounts of these lipids have been found associated with Golgi membranes, their probable site of biosynthesis (1). Their location on the trans-cytoplasmic side of the plasma membrane causes them, together with glycosylated membrane proteins, to be the primary components of the cell to interact with the molecules and other cells of the immediate environment. Thus specific glycosphingolipids have been shown to serve as receptors for toxins, viruses and some hormones (2,5–11). They have long been known to act as antigenic determinants and to mediate immune responses (11–13). There is much evidence to suggest that glycosphingolipids play a role in cell-cell interaction and recognition (7). Alterations in the amounts and types of these lipids on the cell surface are very often associated with growth, differentiation, development, aging (7,14,15), and with oncogenic transformation (11,16). Although there is considerable information about the molecular structure of many glycosphingolipids, relatively little is known about the organization of molecules of this class in phospholipid bilayers and in the bilayers of biological membranes. It seems certain that their molecular organization is a critical parameter underlying many of the functions of glycosphingolipids (17).

To determine whether epithelial plasma membrane glycolipids are polarized in a manner analogous to membrane proteins, MDCK cells grown on permeable filters were analyzed for the expression of Forssman ceramide pentasaccharide, the major neutral glycolipid in these cells. In contrast to a recent report which described exclusive apical localization of the Forssman glycolipid (Hansson, G.C., Simons, K. and Van Meer, G. (1986) EMBO J. 5, 483-489), immunofluorescence and immunoelectron microscopic staining revealed the Forssman glycolipid on both the apical and basolateral surfaces of polarized cells. Immunoblots indicated that the Forssman antigen was detectable only on glycolipids and not on proteins. Analysis of metabolically labeled glycolipids released into the apical and basal culture medium, either as shed membrane vesicles or in budding viruses, also demonstrated the presence of the Forssman glycolipid on both apical and basolateral membranes of polarized cells. Quantitation of the released glycolipid indicated that the Forssman glycolipid was concentrated in the apical membrane. These results are consistent with previous reports which described quantitative enrichment of glycolipids in the apical domain of several epithelia.

Developmental changes in the protein, phospholipid, cholesterol, and fatty acid composition of skeletal and cardiac microsomes were correlated with Ca2+ transport activity in chicken embryos and during the early postnatal period. In skeletal muscle microsomes isolated from leg, superficial pectoralis, or deep pectoralis muscles, the rapid increase in the rate and extent of calcium accumulation around the time of hatching was accompanied by increase in total and Ca2+-sensitive ATPase activity, the concentration of phosphoprotein intermediate and the amount of transport ATPase enzyme determined by gel electrophoresis. The increased enzyme content of the membrane is reflected in its increased density and a marked decrease in phospholipid to protein ratio with development. The concentration of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, and phosphatidylinositol changed relatively little during the first 50 days of development but the appearance of calcium transport activity was accompanied by a marked decrease in the palmitate and increase in the linoleate content of membrane phospholipids and a decrease in cholesterol concentration. These changes were clearly apparent in purified sarcoplasmic reticulum membranes isolated by calcium loading or in less pure membrane fractions obtained by differential or sucrose gradient centrifugation. Corresponding but much less pronounced changes in the enzymatic activity of cardiac microsomes were observed during development. Although the observations are consistent with nearly all possible mechanisms of membrane assembly they may suggest that the phospholipid-rich membranes of embryonic muscle are gradually converted during development into calcium-transporting structures by stepwise insertion of the ATPase enzyme and other components of the transport system.

Previous investigations have demonstrated the presence of circular negatively stained lesions in phospholipid-cholesterol liposomes and in the cell membranes of erythrocytes that have been treated with the polyene antibiotic, filipin; these lesions are morphologically similar to those found in erythrocyte cell membranes after immune lysis. Filipin has been shown also to cause release of both glucose and macromolecular enzyme markers from liposomes that contain cholesterol. In the present study, freeze-etching was employed to determine if these lesions are holes through the membrane. Structural alterations (pits, doughnut-shaped craters and protrusions), whose formation was dependent on the presence of cholesterol, were demonstrable in liposomes after incubation with the antibiotic; analogous changes were also produced by filipin in erythrocyte membranes. However, examination of outer and inner surfaces, and outer and inner fracture faces, did not indicate that these structural alterations could be equated with transverse holes. It is, therefore, unlikely that the negatively stained lesions represent stable open channels through liposomal bilayers and erythrocyte membranes which are responsible for the escape of marker compounds and cytoplasmic constituents.