Aclar discs: a versatile substrate for routine high-pressure freezing of mammalian cell monolayers.

High-pressure freezing avoids the artefacts induced by conventional chemical fixation, and, in combination with freeze-substitution and plastic embedding, is a reliable method for the ultrastructural analysis of mammalian cell monolayers. In order to high-pressure freeze mammalian cell monolayers, cells have to be seeded on a suitable substrate. Unfortunately, electron microscopy analysis is often hampered by poor cell growth, changes in cell morphology induced by the cell substrate or cell loss during processing. We report a method to culture, high-pressure freeze, freeze-substitute and plastic embed mammalian cell monolayers. The method is based on the use of Aclar, a copolymer film with properties very similar to those of tissue culture plastic. We show that Aclar discs support the normal growth and morphology of a wide variety of mammalian cell types, and form an ideal starting point for high-pressure freezing, freeze-substitution and plastic embedding. We present a complete protocol, which, because of its simplicity and reproducibility, provides a method suitable for the routine analysis of mammalian cell monolayers by electron microscopy and tomography.

Exercise echocardiography for the prognostic stratification of patients with low pretest probability of coronary artery disease.

PURPOSE: The aim of this study was to determine whether exercise echocardiography provides incremental data for risk stratification of patients with a low pretest probability of coronary artery disease. PATIENTS AND METHODS: The study included patients referred for exercise echocardiography whose probability of coronary artery disease was 25% or less. We calculated an exercise wall motion score index (on a 1-5 scale), an indicator of the extent and severity of exercise-induced abnormalities. The primary outcomes of the study were subsequent cardiac events (cardiac death and nonfatal myocardial infarction). RESULTS: We studied 571 men and 1047 women; their mean (+/- SD) age was 55 +/- 13 years. During a median follow-up of 3 years, there were 19 cardiac events (6 cardiac deaths and 13 nonfatal myocardial infarctions); an additional 37 patients underwent coronary revascularization. In a multivariate analysis of clinical, exercise electrocardiographic, and echocardiographic parameters, exercise wall motion score index (hazard ratio [HR] = 2.1 per 0.5 units; 95% confidence interval [CI]: 1.3 to 3.4), and age (HR = 2.0 per decade; 95% CI: 1.2-2.8) were independently associated with the risk of cardiac events. Although exercise echocardiographic variables contributed significantly (P = 0.01) to a model of the risk of adverse events, only 9 (47%) of the 19 patients with cardiac events were identified by an abnormal exercise echocardiogram. CONCLUSION: Among patients with low pretest probability of coronary artery disease by clinical criteria, exercise echocardiography identifies some, but not all, patients at risk of future events. Because of the low event rate, routine application of exercise echocardiography in a patient with a low pretest probability does not appear to be cost-effective and therefore cannot be recommended.

Safety of electrical cardioversion in patients with previous embolic events.

OBJECTIVE: To assess thromboembolic complications in cardioversions in patients with atrial fibrillation or flutter and a previous embolic event. PATIENTS AND METHODS: The study population consisted of 104 patients with previous embolic events who underwent 128 electrical cardioversions for termination of atrial fibrillation or flutter. The primary outcome measure was successful cardioversion. RESULTS: Anticoagulants were administered in 118 procedures (92%). Cardioversion was successful in 108 (84%) of the 128 procedures. Only 1 embolic event occurred within 30 days after cardioversion (incidence, 0.9% of successful procedures; 95 % confidence interval, 0.02%-5.3%). The single embolic event was a transient neurologic deficit occurring 22 days after cardioversion in a patient with previous atrial fibrillation. This patient had a sub-therapeutic level of anticoagulation. Transesophageal echocardiography revealed no spontaneous echo contrast or thrombi before the procedure. No thromboembolism was noted in patients who had therapeutic anticoagulation or in those with failed cardioversion. CONCLUSION: Patients with previous embolism are not at additional risk of thromboembolic complications after cardioversion if anticoagulation is adequate.

Small cargo proteins and large aggregates can traverse the Golgi by a common mechanism without leaving the lumen of cisternae.

Procollagen (PC)-I aggregates transit through the Golgi complex without leaving the lumen of Golgi cisternae. Based on this evidence, we have proposed that PC-I is transported across the Golgi stacks by the cisternal maturation process. However, most secretory cargoes are small, freely diffusing proteins, thus raising the issue whether they move by a transport mechanism different than that used by PC-I. To address this question we have developed procedures to compare the transport of a small protein, the G protein of the vesicular stomatitis virus (VSVG), with that of the much larger PC-I aggregates in the same cell. Transport was followed using a combination of video and EM, providing high resolution in time and space. Our results reveal that PC-I aggregates and VSVG move synchronously through the Golgi at indistinguishable rapid rates. Additionally, not only PC-I aggregates (as confirmed by ultrarapid cryofixation), but also VSVG, can traverse the stack without leaving the cisternal lumen and without entering Golgi vesicles in functionally relevant amounts. Our findings indicate that a common mechanism independent of anterograde dissociative carriers is responsible for the traffic of small and large secretory cargo across the Golgi stack.

Dynamin is membrane-active: lipid insertion is induced by phosphoinositides and phosphatidic acid.

Dynamin is a large GTPase involved in the regulation of membrane constriction and fission during receptor-mediated endocytosis. Dynamin contains a pleckstrin-homology domain which is essential for endocytosis and which binds to anionic phospholipids. Here, we show for the first time that dynamin is a membrane-active molecule capable of penetrating into the acyl chain region of membrane lipids. Lipid penetration is strongly stimulated by phosphatidic acid (PA), phosphatidylinositol 4-phosphate, and phosphatidylinositol 4, 5-bisphosphate. Though binding is more efficient in the presence of the phosphoinositides, a much larger part of the dynamin molecule penetrates into PA-containing mixed-lipid systems. Thus, local lipid metabolism will dramatically influence dynamin-lipid interactions, and dynamin-lipid interactions are likely to play an important role in dynamin-dependent endocytosis. Our data suggest that dynamin is directly involved in membrane destabilization, a prerequisite to membrane fission.

Greasing membrane fusion and fission machineries.

Biological membrane fusion is a local-point event, extremely fast, and under strict control. Proteins are responsible for the mutual recognition of the fusion partners and for the initiation of biomembrane fusion, and thus determine where and when fusion occurs. However, the central event during membrane fusion is the merger of two membranes, which requires a transient reorganization of membrane lipids into highly curved fusion intermediates. This review focuses on the potential role of lipids in the generation of membrane curvature, and thus in the regulation of membrane fusion and fission.

CtBP/BARS induces fission of Golgi membranes by acylating lysophosphatidic acid.

Membrane fission is essential in intracellular transport. Acyl-coenzyme As (acyl-CoAs) are important in lipid remodelling and are required for fission of COPI-coated vesicles. Here we show that CtBP/BARS, a protein that functions in the dynamics of Golgi tubules, is an essential component of the fission machinery operating at Golgi tubular networks, including Golgi compartments involved in protein transport and sorting. CtBP/BARS-induced fission was preceded by the formation of constricted sites in Golgi tubules, whose extreme curvature is likely to involve local changes in the membrane lipid composition. We find that CtBP/BARS uses acyl-CoA to selectively catalyse the acylation of lysophosphatidic acid to phosphatidic acid both in pure lipidic systems and in Golgi membranes, and that this reaction is essential for fission. Our results indicate a key role for lipid metabolic pathways in membrane fission.

Interaction of the anti-cancer drug cisplatin with phosphatidylserine in intact and semi-intact cells.

The anti-cancer drug cisplatin (cis-diamminedichloroplatinum(II)) forms a stable coordination complex with phosphatidylserine (PS) in model membrane systems (Speelmans et al., Biochemistry 36 (1997) 10545-10550). Because a similar interaction in vivo would be expected to have important physiological implications we studied cisplatin-PS interaction in human erythrocytes and tumor cell lines. Although cisplatin was efficiently taken up by intact erythrocytes, a cisplatin-PS complex was only detected in cells which had lysed as a result of prolonged storage or hypotonic shock. Despite the use of highly sensitive detection methods, and despite efficient cellular uptake of cisplatin, a complex could also not be detected in four human tumor cell lines, unless cells were permeabilized. In experiments in which cisplatin was incubated with PS-containing liposomes in the presence of an alternative cellular substrate, such as reduced glutathione, the relative affinity of cisplatin for PS was found to be low. Moreover, loading erythrocyte ghosts with physiological concentrations of glutathione strongly reduced cisplatin-PS complexation. Thus, in intact (tumor) cells a complex is not detected, most likely, because of the presence of higher affinity substrates. Though a transient complexation of cisplatin to PS cannot be excluded, our data suggest that cisplatin-PS does not play a direct role in the cellular (cyto)toxicity of cisplatin.

Topology of sphingolipid galactosyltransferases in ER and Golgi: transbilayer movement of monohexosyl sphingolipids is required for higher glycosphingolipid biosynthesis.

Glucosylceramide (GlcCer) is synthesized at the cytosolic surface of the Golgi complex while enzymes acting in late steps of glycosphingolipid biosynthesis have their active centers in the Golgi lumen. However, the topology of the "early" galactose-transferring enzymes is largely unknown. We used short-chain ceramides with either an 2-hydroxy fatty acid (HFA) or a normal fatty acid (NFA) to determine the topology of the galactosyltransferases involved in the formation of HFA- and NFA-galactosylceramide (GalCer), lactosylceramide (LacCer), and galabiosylceramide (Ga2Cer). Although the HFA-GalCer synthesizing activity colocalized with an ER marker, the other enzyme activities fractionated at the Golgi density of a sucrose gradient. In cell homogenates and permeabilized cells, newly synthesized short-chain GlcCer and GalCer were accessible to serum albumin, whereas LacCer and Ga2Cer were protected. From this and from the results obtained after protease treatment, and after interfering with UDP-Gal import into the Golgi, we conclude that (a) GlcCer and NFA-GalCer are synthesized in the cytosolic leaflet, while LacCer and Ga2Cer are synthesized in the lumenal leaflet of the Golgi. (b) HFA-GalCer is synthesized in the lumenal leaflet of the ER, but has rapid access to the cytosolic leaflet. (c) GlcCer, NFA-GalCer, and HFA-GalCer translocate from the cytosolic to the lumenal leaflet of the Golgi membrane. The transbilayer movement of GlcCer and NFA-GalCer in the Golgi complex is an absolute requirement for higher glycosphingolipid biosynthesis and for the cell surface expression of these monohexosyl sphingolipids.

Sphingolipid trafficking--sorted out?

Studies of intracellular membrane traffic have traditionally focused on the protein components of membranes, but what about lipids? Recent findings have drawn attention to the transport of one type of lipid, the sphingolipids. Their unique physical properties may allow them to aggregate into microdomains in membranes that concentrate sphingolipids into specific transport pathways. Gerrit van Meer and Koert Burger consider here the routes of sphingolipid biosynthesis and transport, and the role of proteins in their targeting. The following article by Deborah Brown turns the tables to review the evidence suggesting that sphingolipid domains are important in specific targeting of GPI-anchored proteins to the plasma membrane.

Glucosylceramide is synthesized at the cytosolic surface of various Golgi subfractions.

In our attempt to assess the topology of glucosylceramide biosynthesis, we have employed a truncated ceramide analogue that permeates cell membranes and is converted into water soluble sphingolipid analogues both in living and in fractionated cells. Truncated sphingomyelin is synthesized in the lumen of the Golgi, whereas glucosylceramide is synthesized at the cytosolic surface of the Golgi as shown by (a) the insensitivity of truncated sphingomyelin synthesis and the sensitivity of truncated glucosylceramide synthesis in intact Golgi membranes from rabbit liver to treatment with protease or the chemical reagent DIDS; and (b) sensitivity of truncated sphingomyelin export and insensitivity of truncated glucosylceramide export to decreased temperature and the presence of GTP-gamma-S in semiintact CHO cells. Moreover, subfractionation of rat liver Golgi demonstrated that the sphingomyelin synthase activity was restricted to fractions containing marker enzymes for the proximal Golgi, whereas the capacity to synthesize truncated glucosylceramide was also found in fractions containing distal Golgi markers. A similar distribution of glucosylceramide synthesizing activity was observed in the Golgi of the human liver derived HepG2 cells. The cytosolic orientation of the reaction in HepG2 cells was confirmed by complete extractability of newly formed NBD-glucosylceramide from isolated Golgi membranes or semiintact cells by serum albumin, whereas NBD-sphingomyelin remained protected against such extraction.

Interaction of influenza virus hemagglutinin with a lipid monolayer. A comparison of the surface activities of intact virions, isolated hemagglutinins, and a synthetic fusion peptide.

In the infectious entry pathway of influenza virus, the low pH of the endosomal compartment induces an irreversible conformational change in influenza virus hemagglutinin, leading to fusion of viral and endosomal membranes. In the current report, we characterized the low-pH-induced activation of hemagglutinin of influenza strain X31 by studying its interaction with a lipid monolayer. The surface activities of virions, of isolated hemagglutinins and its proteolytic fragments, and of a synthetic peptide mimicking the amino terminus of subunit 2 of hemagglutinin are compared. The data indicate that the surface activity of both virions and isolated hemagglutinin develop as a result of the low-pH-induced conformational change in hemagglutinin. The surface activity of isolated hemagglutinin is mainly caused by penetration into the lipid monolayer of protein domains other than the amino terminus of subunit 2 of hemagglutinin; domains in subunit 1 may be involved. The surface activity of virions appears to be a secondary effect of the conformational change and is explained by assuming a net transfer of viral lipids to the lipid monolayer.

Phospholipase C activity-induced fusion of pure lipid model membranes. A freeze fracture study.

The structural effects of in situ production of diacylglycerol by phospholipase C in pure lipid model membranes have been examined by freeze fracture electron microscopy. Phospholipase C-activity induces massive aggregation and fusion of large unilamellar lipid vesicles and leads to the formation of a 'sealed' lipid aggregate; the outer membrane of this aggregate appears to be continuous. In some areas lipid arranges into a honeycomb structure; this structure is probably a precursor of a discontinuous inverted (type II) cubic phase. Similarly, enzyme treatment of multilamellar vesicles leads to extensive membrane fusion and vesiculation. Thus morphological evidence is obtained showing the ability of phospholipase C to induce bilayer destabilization and fusion. It is speculated that phospholipase C-induced membrane fusion involves a type II fusion intermediate induced by diacylglycerol produced locally.

The interaction of synthetic analogs of the N-terminal fusion sequence of influenza virus with a lipid monolayer. Comparison of fusion-active and fusion-defective analogs.

The amino terminus of subunit-2 of influenza virus hemagglutinin (NHA2) plays a crucial role in the induction of fusion between viral and endosomal membranes leading to the infection of a cell. Three synthetic analogs with an amino acid sequence corresponding to NHA2 of variant hemagglutinins were studied in a monolayer set up. Comparison of the interaction of a fusion-active and two fusion-defective analogs with a lipid monolayer revealed a greater surface activity of the fusion-active analog. Pronounced differences were found if the pure peptides were spread at the air/water interface; the fusion-active analog showed a higher collapse pressure and a greater limiting molecular area. Circular dichroism measurements on collected lipid monolayers indicated a high content of alpha-helical structure for the fusion-active and one of the fusion-defective analogs. A simple relation between alpha-helical content and fusogenicity does not seem to exist. Instead, the extent of penetration, a defined tertiary structure or orientation of the alpha-helical peptide may be essential for its membrane perturbing activity.

Perspective and limitations of cryo-electron microscopy. From model systems to biological specimens.

We investigated the possibility of vitrifying temperature-sensitive lipid phases as well as (small) biological specimens. From a suspension of unilamellar vesicles, prepared from dipalmitoyl-phosphatidylcholine (DPPC), thin aqueous films were formed at various temperatures. With cryo-electron microscopy vesicles were found to be smooth, rippled and faceted or faceted only, depending on the temperature of thin-film formation (318, 312 and 296 K respectively). The morphology and the electron diffraction patterns indicate that membranes can by physically fixed by vitrification in their high-temperature configuration and studied at low temperature by cryo-electron microscopy. This finding suggests that it may also be possible to preserve, in their original state, the more complex membrane systems found in living organisms by initiating rapid-cooling at a physiological temperature. This was explored by vitrification of thin films formed on specimen grids with (human) blood platelets adhering to collagen fibres. Low-temperature observation with an acceleration voltage of 120 kV revealed subcellular details, More details were observed when using higher accelerating voltages (200 and 300 kV) of the electron beam. The results presented in this paper illustrate the great potential of cryo-electron microscopy in the study of membrane dynamics, both in relatively simple model membrane systems and in more complex biological membrane systems.

Lipid polymorphism as observed by cryo-electron microscopy.

Lipid polymorphism was studied with the aim to gain more insight in bilayer to non-bilayer phase transitions, with particular emphasis on the development of cubic structures on one hand and inverted hexagonal structures on the other hand. Thin vitrified films prepared from aqueous lipid suspensions were used in this study. The entire hydrated contents of these films can be visualized in their two-dimensional projection by cryo-electron microscopy. As the starting material, unilamellar vesicles were prepared from mixtures of dioleoylphosphatidylethanolamine, dioleoylphosphatidylcholine and cholesterol. By heating of the suspension, vesicle fusion (Frederik et al. (1989) Biochim. Biophys. Acta 979, 275-278) and lipid polymorphism was induced. From these suspensions thin films were prepared at various temperatures, and vitrified for low temperature observation. In a parallel series of experiments samples were fast-frozen for freeze-fracture analysis. In vitrified thin films bilayer structures were often observed in coexistence with an inverted hexagonal structure. The bilayer areas were frequently of a complex structure because multiple contacts between stacked membranes were found. These contact points were variable in size and shape and usually had the form of a diabolo (when viewed side-on) giving the impression of a bilayer contact with an aqueous channel. This structure is compatible with the interlamellar attachment site (ILA) proposed by Siegel ((1986) Biophys. J. 49, 1155-1170). In some specimens ILA's seemed to merge into arrays. After thermal cycling of the suspension, arrays of packed globules were observed, which are likely the result of close packing of ILA's. The arrays probably represent a cubic structure. A comparison of freeze-fracture replicas and vitrified thin films indicated that both techniques may provide valuable structural information on lipid polymorphism. Most of the lipidic particles observed by freeze-fracturing probably correspond to the ILA's (fractured around their waist region) as observed in vitrified thin films. The results obtained with vitrified thin films were interpreted in relation to the principles of thin-film formation. Finally, we speculate that lipid structures occurring close to each other in space may represent a developmental series of structures occurring successively in time.

Gramicidin A induced fusion of large unilamellar dioleoylphosphatidylcholine vesicles and its relation to the induction of type II nonbilayer structures.

The fusogenic properties of gramicidin were investigated by using large unilamellar dioleoylphosphatidylcholine vesicles. It is shown that gramicidin induces aggregation and fusion of these vesicles at peptide to lipid molar ratios exceeding 1/100. Both intervesicle lipid mixing and mixing of aqueous contents were demonstrated. Furthermore, increased static and dynamic light scattering and a broadening of 31P NMR signals occurred concomitant with lipid mixing. Freeze-fracture electron microscopy revealed a moderate vesicle size increase. Lipid mixing is paralleled by changes in membrane permeability: small solutes like carboxyfluorescein and smaller dextrans, FD-4(Mr approximately 4000), rapidly (1-2 min) leak out of the vesicles. However, larger molecules like FD-10 and FD-17 (Mr approximately 9400 and 17,200) are retained in the vesicles for greater than 10 min after addition of gramicidin, thereby making detection of contents mixing during lipid mixing possible. At low lipid concentrations (5 microM), lipid mixing and leakage are time resolved: leakage of CF shows a lag phase of 1-3 min, whereas lipid mixing is immediate and almost reaches completion during this lag phase. It is therefore concluded that leakage, just as contents mixing, occurs subsequent to aggregation and lipid mixing. Although addition of gramicidin at a peptide/lipid molar ratio exceeding 1/50 eventually leads to hexagonal HII phase formation and a loss of vesicle contents, it is concluded that leakage during fusion (1-2 min) is not the result of HII phase formation but is due to local changes in lipid structure caused by precursors of this phase. By making use of gramicidin derivatives and different solvent conformations, it is shown that there is a close parallel between the ability of the peptide to induce the HII phase and its ability to induce intervesicle lipid mixing and leakage. It is suggested that gramicidin-induced fusion and HII phase formation share common intermediates.

Membrane fusion.

The factors involved in the regulation of biological membrane fusion and models proposed for the molecular mechanism of biomembrane fusion are reviewed. The results obtained in model systems are critically discussed in the light of the known properties of biomembranes and characteristics of biomembrane fusion. Biological membrane fusion is a local-point event; extremely fast, non-leaky, and under strict control. Fusion follows on a local and most probably protein-modulated destabilization, and a transition of the interacting membranes from a bilayer to a non-bilayer lipid structure. The potential role of type II non-bilayer preferring lipids and of proteins in the local destabilization of the membranes is evaluated. Proteins are not only responsible for the mutual recognition of the fusion partners, but are most likely also to be involved in the initiation of biomembrane fusion, by locally producing or activating fusogens, or by acting as fusogens.

Formation of multilamellar vesicles by addition of tannic acid to phosphatidylcholine-containing small unilamellar vesicles.

Tannic acid induces aggregation and formation of multilamellar vesicles when added to preparations of small unilamellar vesicles, specifically those containing phosphatidylcholine. Aggregation and clustering of vesicles was demonstrated by cryo-electron microscopy of thin films and by freeze-fracture technique. Turbidity measurements revealed an approximately one-to-one molar ratio between tannic acid and phosphatidylcholine necessary for a fast and massive aggregation of the small unilamellar vesicles. When tannic acid-induced aggregates were dehydrated and embedded for conventional thin-section electron microscopy, multilamellar vesicles were retrieved in thin sections. It is concluded from morphological studies, as well as previous tracer studies, that tannic acid, at least to a great extent, prevents the extraction of phosphatidylcholine. Multilamellar vesicles were also observed in tannic acid-treated vesicles prepared from total lipid extracts from either rabbit or rat hearts. Substantially more multilamellar vesicles were retrieved in the rabbit vesicle preparation. This difference can probably be explained by the difference in the proportion of the plasmalogen phosphatidylcholine, and possibly the content of sphingomyelin, in lipid extracts of rabbit and rat hearts. It is concluded that the dual effect (reduced extraction and aggregation) of tannic acid on phosphatidylcholines should be taken into consideration when tannic acid is used in tissue preparation.