Intramembrane particles and filipin labelling on the membranes of autophagic vacuoles and lysosomes in mouse liver

Summary Morphologically detectable protein (intramembrane particles) and cholesterol (filipin labelling) in the membranes of autophagic vacuoles and lysosomes were studied in mouse hepatocytes using thin-section and freeze-fracture electron microscopy. Both isolated autophagic vacuoles and lysosomes, and intact tissue blocks were used due to the facts (i) that lysosomes are difficult to recognize in freeze-fracture replicas of intact hepatocytes, and (i) that filipin penetration into the tissue blocks is unsatisfactory. Intramembrane particle density was low in the membranes of early autophagic vacuoles (defined as round-shaped vacuoles in which an inner membrane parallel with the outer limiting membrane was clearly visible). The lysosomal membranes contained considerably more intramembrane particles. Particle-rich lysosomes or other vesicles were observed to fuse with the early autophagic vacuoles. The membranes of nascent autophagic vacuoles with morphologically intact contents were usually not labelled by filipin, whereas the membranes of all other autophagic vacuoles and lysosomes were heavily labelled. The increased cholesterol in the membranes of slightly older autophagic vacuoles is presumably derived from cholesterol-rich lysosomes or other vesicles fusing with the vacuoles and from the degrading organelles inside the autophagic vacuoles.     

Ultrastructural characterization of the delimiting membranes of isolated autophagosomes and amphisomes by freeze-fracture electron microscopy

The delimiting membranes of isolated autophagosomes from rat liver had extremely few transmembrane proteins, as indicated by the paucity of intramembrane particles in freeze-fracture images (about 20 particles/microm2, whereas isolated lysosomes had about 2000 particles/microm2). The autophagosomes also appeared to lack peripheral surface membrane proteins, since attempts to surface-biotinylate intact autophagosomes only yielded biotinylation of proteins from contaminating damaged mitochondria. All the membrane layers of multilamellar autophagosomes were equally particle-poor; the same was true of the autophagosome-forming, sequestering membrane complexes (phagophores). Isolated amphisomes (vacuoles formed by fusion between autophagosomes and endosomes) had more intramembrane particles than the autophagosomes (about 90 particles/microm2), and freeze-fracture images of these organelles frequently showed particle-rich endosomes fusing with particle-poor or particle-free autophagosomes. The appearence of multiple particle clusters suggested that a single autophagic vacuole could undergo multiple fusions with endosomes. Only the outermost membrane of bi- or multilamellar autophagic vacuoles appeared to engage in such fusions.     

Topology of morphologically detectable protein and cholesterol in membranes of polypeptide-secreting cells

The freeze-fracture morphology of intracellular and plasma membranes in endocrine and exocrine polypeptide-secreting cells has been studied to detect changes while these membranes interact during secretion. A qualitative and quantitative evaluation of intramembrane particles and filipin binding as indicators of protein and cholesterol content of the membranes, respectively, reveals the following changes. From the forming of the maturing pole of the Golgi complex, membranes lose morphologically detectable protein and gain morphologically detectable cholesterol. The protein-poor, cholesterol-rich secretory granule membrane then interacts with a richly particulate plasma membrane in endocrine cells and with a moderately particulate luminal membrane in exocrine cells. The site of interaction between secretory granule and plasma membrane is characterized by a local clearing of intramembrane particles; by contrast, filipin-binding sites revealing cholesterol are present in this area. In exocrine cells, the fused secretory granule, which is initially rich in filipin-cholesterol complexes and poor in particles, appears to lose progressively its filipin labelling to resemble the poorly labelled luminal membrane. These findings, although they cannot be interpreted definitely at present, clearly show impressive changes of membrane structure along the secretory pathway and suggest that a corresponding degree of functional specialization is needed for proper interaction to occur.     

A fluorescent sterol probe study of cholesterol/phospholipid membranes

The behavior of dehydroergosterol in -α-dimyristoylphosphatidylcholine (DMPC) unsonicated multilamellar liposomes was characterized by absorption spectroscopy and fluorescence measurements. Dehydroergosterol exhibited a lowered absorption coefficient in multilamellar liposomes whiel the steady-state fluorescence anisotropy of dehydroergosterol in these membranes decreased significantly with increasing dehydroergosterol concentration, suggesting membrane sterol-sterol interactions. The comparative steady-state anisotropy of 0.9 mole percent dehydroergosterol in multilamellar liposomes was lower than in small unilamellar vesicles suggesting different sterol environments for dehydroergosterol. Dehydroergosterol fluorescence lifetime was relatively independent of membrane sterol content and yielded similar values in sonicated and unsonicated model membranes. In multilamellar liposomes containing 5 mole percent cholesterol, the gel-to-liqui crystalline phase transition of DMPC detected by 0.9 mole percent dehydroergosterol was significantly broadened when compared to the phase transition detected by dehydroergosterol in the absence of membrane cholesterol (Smutzer, G. et al. (1986) Biochim. Biophys. Acta 862, 361–371). In multilamellar liposomes containing 10 mole percent cholesterol, the major fluorescence lifetime of dehydroergosterol did not detect the gel-to-liquid crystalline phase transition of DMPC. Time-correlated fluorescence anisotropy decays of dehydroergosterol in DMPC multilamellar liposomes in the absence and presence of 5 mole percent cholesterol exhibited a single rotational correlation time near one nanosecond that was relatively independent of temperature and low concentrations of membrane cholesterol. The limiting anisotropy of 0.9 mole percent dehydroergosterol decreased above the gel-to-liquid crystalline phase transition in membranes without cholesterol and was not significantly affected by the phase transition in membranes containing 5 mole percent cholesterol. These results suggested hindered rotational diffusion of dehydroergosterol in multilamellar liposomes. Lifetime and time-correlated fluorescence measurements of 0.9 mole percent dehydroergosterol in multilamellar liposomes further suggested this fluorophore was detecting physical properties of the bulk membrane phospholipids in membranes devoid of cholesterol and was detecting sterol-rich regions in membranes of low sterol concentration.     

Membrane events involved in myoblast fusion

Myoblast fusion has been studied in cultures of chick embryonic muscle utilizing ultrastructural techniques. The multinucleated muscle cells (myotubes) are generated by the fusion of two plasma membranes from adjacent cells, apparently by forming a single bilayer that is particle-free in freeze-fracture replicas. This single bilayer subsequently collapses, and cytoplasmic continuity is established between the cells. The fusion between the two plasma membranes appears to take place primarily within particle-free domains (probably phospholipid enriched), and cytoplasmic unilamellar, particle-free vesicles are occasionally associated with these regions. These vesicles structurally resemble phospholipid vesicles (liposomes). They are present in normal myoblasts, but they are absent in certain fusion-arrested myoblast popluations, such as those treated with either 5-bromo-deoxyuridine (BUdR), cycloheximide (CHX), or pospholipase C (PLC). The unilamellar, particle-free vesicles are present in close proximity to the plasma membranes, and physical contact is observed frequently between the vesicle membrane and the plasma membrane. The regions of vesicle membrane-plasma membrane interaction are characteristically free of intramembrane particles. A model for myoblast fusion is presented that is based onan interpretation of these observations. This model suggests that the cytoplasmic vesicles initiate the generation of particle-depleted membrane domains, both being essential components in the fusion process.     

Interactions between digitonin and bilayer membranes

The morphology of interactions between digitonin and cholesterol has been investigated. When precipitated from ethanolic solutions, digitonin-cholesterol complexes form in flat lamellar sheets. In contrast, when the complex is formed in a bilayer membrane, the membrane is deformed into corrugations of hemitubules. The polarity of the deformations formed in bilayer membranes is highly correlated with the direction of entry of digitonin into the membrane. We suggest that the morphology of digitonin/cholesterol hemitubules is dependent upon the complex being formed within a bilayer and, in addition, is not correlated with asymmetry of cholesterol concentration across the membrane.     

Localization of filipin-sterol complexes in cell membranes of eosinophils

Summary The polyene antibiotic filipin was used as a probe for the detection of cholesterol in the cell membranes of eosinophils isolated from the peritoneal exudate of rats. A homogenous distribution of filipin-sterol complexes was observed, both in thin sections and freeze-fracture replicas throughout the whole plasma membrane but not in the membrane of pynocytic vesicles, Golgi complex, endoplasmic reticulum, mitochondria and the nucleus. Few complexes were seen in freeze-fracture replicas showing the membrane of the specific granules. Treatment of living cells with filipin induced aggregation of filipin-sterol complexes at some points of the plasma membrane.     

Localization of filipin-sterol complexes in cell membranes of eosinophils

The polyene antibiotic filipin was used as a probe for the detection of cholesterol in the cell membranes of eosinophils isolated from the peritoneal exudate of rats. A homogenous distribution of filipin-sterol complexes was observed, both in thin sections and freeze-fracture replicas throughout the whole plasma membrane but not in the membrane of pynocytic vesicles, Golgi complex, endoplasmic reticulum, mitochondria and the nucleus. Few complexes were seen in freeze-fracture replicas showing the membrane of the specific granules. Treatment of living cells with filipin induced aggregation of filipin-sterol complexes at some points of the plasma membrane.     

Restoration of gap junction-like structure after detergent solubilization of the proteins from liver gap junctions

Gap junctions isolated from rat liver were partially solubilized with a mixture of digitonin and octyl glucoside. After supplementation with lecithin and cholesterol, the octyl glucoside was removed from the soluble fraction by dialysis. The membranes of the reconstituted vesicles, observed in freeze-fracture, contained particles ranging from 7 to 12 nm diameter, more or less aggregated depending on the protein-to-lipid ratio. At every protein concentration, the arrangement of particles in contact areas between adjacent membranes closely resembles the organization of intact gap junctions. We conclude that the mixture of digitonin and octyl glucoside is able to solubilize the proteins of the liver gap junctions while preserving their property of restoring a gap junction-like structure.     

Electron microscopic observations of reconstituted proteoliposomes with the purified major intrinsic membrane protein of eye lens fibers

The purified major intrinsic protein of the lens fiber plasma membrane (MP26) reconstituted into liposomes favored membrane-to-membrane close contacts as visualized by freeze fracture and immunoelectron microscopy. Reconstituted apposed unilamellar vesicles formed pentalaminar profiles, and multilamellar liposomes showed regions of stacked bilayers. Immunogold labeling, using antibody directed against MP26, demonstrated that this polypeptide is present in regions of membrane-to-membrane close interaction. Fracture faces displayed both randomly distributed clusters of 8-nm polygonal intramembrane particles and membrane domains where a bidimensional lattice of repeating subunits was present. The structural pleomorphism which characterized the MP26-reconstituted proteoliposomes seems quite comparable to that visualized in natural fiber plasma membrane domains.     

Effects of cholesterol on lipid organization in human erythrocyte membrane

The molar ratio of cholesterol to phospholipid (C/P) in human erythrocyte membrane is modified by incubating the cells with liposomes of various C/P ratios. The observed increase in cell surface area may be accounted for by the addition of cholesterol molecules. Fusion between liposomes and cells or attachment of liposomes to cells is not a significant factor in the alteration of C/P ratio. Onset temperatures for lipid phase separation in modified membranes are measured by electron diffraction. The onset temperature increases with decreasing C/P ration from 2 degrees C at C/P = 0.95 to 20 degrees C at C/P = 0.5. Redistribution of intramembrane particles is observed in membranes freeze-quenched from temperatures below the onset temperature. The heterogeneous distribution of intramembrane particles below the onset temperature suggests phase separation of lipid, with concomitant segregation of intramembrane protein into domains, even in the presence of an intact spectrin network.     

Ultrastructural detection of cholesterol in the liver of rats with alcoholic intoxication

The deposits of cholesterol in hepatocytes in alcoholic rats were studied by means of cytochemical method. It was shown the localization of cholesterol in intracellular as well as extracellular compartment. In numerous hepatocytes there was observed a destruction of mitochondria, rough endoplasmic reticulum and plasma membranes. It was observed two structural forms of digitonin cholesterol complexes: structures like "crinkles" and small cylinders and multilamellar ones.     

Enveloped viruses as model membrane systems: microviscosity of vesicular stomatitis virus and host cell membranes.

The fluorescence probe 1,6-diphenyl-1,3,5-hexatriene was used to study and compare the dynamic properties of the hydrophobic region of vesicular stomatitis virus grown on L-929 cells, plasma membrane of L-929 cells prepared by two different methods, liposomes prepared from virus lipids and plasma membrane lipids, and intact L-929 cells. The rate of penetration of the probe into the hydrophobic region of the lipid bilayer was found to be much faster in the lipid vesicle bilayer as compared with the intact membrane, but in all cases the fluorescence anisotropy was constant with time. The L-cell plasma membranes, the vesicles prepared from the lipids derived from the plasma membranes, and intact cells are found to have much lower microviscosity values than the virus or virus lipid vesicles throughout a wide range of temperatures. The microviscosity of plasma membrane and plasma membrane lipid vesicles was found to depend on the procedure for plasma membrane preparation as the membranes prepared by different methods had different microviscosities. The intact virus and liposomes prepared from the virus lipids were found to have very similar microviscosity values. Plasma membrane and liposomes prepared from plasma membrane lipids also had similar microviscosity values. Factors affecting microviscosity in natural membranes and artificially mixed lipid membranes are discussed.     

Spontaneous lipid vesicle fusion with electropermeabilized cells

Fusion is obtained between electropermeabilized mammalian cells and intact large unilamellar lipid vesicles. This is monitored by a fluorescence assay. Prepulse contact is obtained by Ca2+ when negatively charged lipids are present in the liposomes. The mixing of the liposome content in the cell cytoplasm is observed under conditions preserving cell viability. Electric conditions are such that free liposomes are not affected by the external field. Therefore destabilization of only one of the two membranes of the partners is sufficient for fusion. The comparison between the efficiency of dye delivery for different liposome preparations (multilamellar vesicles, large unilamellar vesicles, small unilamellar vesicles) is indicative that more metastable liposomes are more fusable with electropulsated cells. This observation is discussed within the framework of the recent hypothesis that occurrence of a contact induced electrostatic destabilization of the plasma membrane is a key step in the exocytosis process.     

Ultrastructural and cytochemical characterization of adrenal medullary plasma membrane vesicles and their interaction with chromaffin granules

Plasma membrane vesicles obtained by density gradient centrifugation of bovine adrenal medullary homogenates were analyzed by electron microscopic methods, including negative staining, ultrathin sections and freeze-fracture replicas. Rapid freezing showed the intramembrane structure of plasma membrane vesicles to be distinct from that of other organelle membranes, such as chromaffin granules. Cytochemical demonstration of acetylcholinesterase (EC 3.1.1.7) activity on most membrane profiles confirmed that plasma membrane vesicles are derived predominantly from plasma membranes. About half of the plasma membrane vesicles were smaller than 0.15 micron and almost none larger than 0.55 micron. Practically all were composed of single shells. Most vesicles were impermeable to cytochemical markers of the size of Ruthenium red (Mr 800) and none were permeable to markers larger than 40 kDa. Surface charge probes, concanavalin A binding and endogenous actin decoration with heavy meromyosin indicated that the major fraction of plasma membrane vesicles is oriented right-side-out. A minor population with opposite orientation could also be detected. Isotonic ionic media caused vesicle aggregation in suspensions of plasma membrane vesicles and chromaffin granules. Freeze-fracturing always revealed clusters of membrane-intercalated particles at the sites of contact between aggregated membranes.     

Translocation of histone proteins across lipid bilayers and Mycoplasma membranes

We show that the three core histones H2A, H3 and H4 can transverse lipid bilayers of large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs). In contrast, the histone H2B, although able to bind to the liposomes, fails to penetrate the unilamellar and the multilamellar vesicles. Translocation across the lipid bilayer was determined using biotin-labeled histones and an ELISA-based system. Following incubation with the liposomes, external membrane-bound biotin molecules were neutralized by the addition of avidin. Penetrating biotin-histone conjugates were exposed by Triton treatment of the neutralized liposomes. The intraliposomal biotin-histone conjugates, in contrast to those attached only to the external surface, were attached to the detergent lysed lipid molecules. Thus, biotinylated histone molecules that were exposed only following detergent treatment of the liposomes were considered to be located at the inner leaflet of the lipid bilayers. The penetrating histone molecules failed to mediate translocation of BSA molecules covalently attached to them. Translocation of the core histones, including H2B, was also observed across mycoplasma cell membranes. The extent of this translocation was inversely related to the degree of membrane cholesterol. The addition of cholesterol also reduced the extent of histone penetration into the MLVs. Although able to bind biotinylated histones, human erythrocytes, erythrocyte ghosts and Escherichia coli cells were impermeable to them. Based on the present and previous data histones appear to be characterized by the same features that characterize cell penetrating peptides and proteins (CPPs).     

Membrane-damaging action of staphylococcal alpha-toxin on phospholipid-cholesterol liposomes

The mechanism of membrane damage by staphylococcal alpha-toxin was studied using carboxyfluorescein (internal marker)-loaded multilamellar liposomes prepared from various phospholipids and cholesterol. Liposomes composed of phosphatidylcholine or sphingomyelin and cholesterol bound alpha-toxin and released carboxyfluorescein in a dose dependent manner, when they were exposed to alpha-toxin of concentrations higher than 1 or 8 micrograms/ml, respectively. In contrast, the other liposomes composed of phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol or phosphatidylinositol plus cholesterol were not susceptible to the toxin even at high concentrations up to 870 micrograms/ml. The insensitive liposomes containing either phosphatidylserine or phosphatidylglycerol were made sensitive to alpha-toxin by inserting phosphatidylcholine into the liposomal membranes. In addition, phosphorylcholine inhibited the toxin-induced marker release from liposomes. These results indicated that the choline-containing phospholipids are required for the interaction between alpha-toxin and liposomal membranes. Susceptibility of liposomes containing phosphatidylcholine or sphingomyelin increased with the increase in cholesterol contents of the liposomes. Based on these results, we propose that the choline-containing phospholipids are possible membrane components or structures responsible for the toxin-membrane interaction, which leads to damage of membranes. Furthermore, cholesterol may facilitate the interaction between alpha-toxin and membrane as a structural component of the membrane.     

Ultrastructure of reconstituted rat liver microsomal membranes and cytochrome b5- or P-450-containing proteoliposomes

Thin sectioning and freeze-fracture electron microscopy have been used to show that it is possible to obtain topologically closed vesicles by means of reconstitution of rat liver microsomal membrane "ghosts." The reconstitution by 15 hr dialysis resulted in the formation of vesicles with intramembrane particles (IMP) while after 40 hr dialysis no IMP were observed in the membranes. The protein/lipid ratio and functional activity of NADPH- and NADH-linked enzyme systems were similar in both cases. Cytochrome P-450 (LM2) was incorporated into liposomes of different composition (protein: lipid ratio--1:200). IMP were observed only when the incorporation of cytochrome P-450 was performed in the presence of detergent Emulgen 913 as specific additive to the initial protein-lipid-sodium cholate mixture or in the course of incubation of proteoliposomal suspensions at 37 degrees C. After the incorporation of cytochrome b5 into azolectin liposomes vesicular membranes contain IMP if the incorporated membrane protein: lipid ratio is at least 1:50. Pronase-induced splitting off of a 11 kDa heme-containing fragment of cytochrome b5 did not affect IMP content. The conditions of IMP formation in reconstituted membranes and in microsomal ghosts are discussed.     

Heterogeneous distribution of filipin-sterol complexes in nuclear membranes

Filipin, a sterol-specific polyene antibiotic, has been shown by electron microscopy to form complexes in membranes of mouse urinary bladder cells. Following instillation of a glutaraldehyde-filipin-dimethylsulfoxide solution into the bladder lumen, filipin-cholesterol complexes appear as membrane corrugations in thin sections and as 20–25 nm protuberances and depressions on PF and EF faces in freeze-fracture replicas. The complexes are observed in plasmalemma, Golgi membrane, rough endoplasmic reticulum and nuclear membrane of five different cell types (urothelial, endothelial, mesothelial, smooth muscle and fibroblasts). In the present report, we direct particular attention to the localization of numerous filipin-cholesterol complexes present in the nuclear envelopes of these cells. Our results suggest that enrichment of cell membranes with cholesterol occurs at an earlier stage in the flow-differentiation process than previously suspected. In addition, the unequal distribution of complexes in favor of the outer nuclear membrane suggests that it has a higher cholesterol content than the inner membrane.     

The inhibition of erythrocyte glyceraldehyde-3-phosphate dehydrogenase. In situ PMR studies

In order to investigate the role of the plasma membrane in determining the kinetics of removal of cholesterol from cells, the efflux of [3H]cholesterol from intact cells and plasma membrane vesicles has been compared. The release of cholesterol from cultures of Fu5AH rat hepatoma and WIRL-3C rat liver cells to complexes of egg phosphatidylcholine (1 mg/ml) and human high-density apolipoprotein is first order with respect to concentration of cholesterol in the cells, with half-times (t 1/2) for at least one-third of the cell cholesterol of 3.2 +/- 0.6 and 14.3 +/- 1.5 h, respectively. Plasma membrane vesicles (0.5-5.0 micron diameter) were produced from both cell lines by incubating the cells with 50 mM formaldehyde and 2 mM dithiothreitol for 90 min. The efflux of cholesterol from the isolated vesicles follows the same kinetics as the intact, parent cells: the t 1/2 values for plasma membrane vesicles of Fu5AH and WIRL cells are 3.9 +/- 0.5 and 11.2 +/- 0.7 h, respectively. These t 1/2 values reflect the rate-limiting step in the cholesterol efflux process, which is the desorption of cholesterol molecules from the plasma membrane into the extracellular aqueous phase. The fact that intact cells and isolated plasma membranes release cholesterol at the same rates indicates that variations in the plasma membrane structure account for differences in the kinetics of cholesterol release from different cell types. In order to investigate the role of plasma membrane lipids, the kinetics of cholesterol desorption from small unilamellar vesicles prepared from the total lipid isolated from plasma membrane vesicles of Fu5AH and WIRL cells were measured. Half-times of cholesterol release from plasma membrane lipid vesicles of Fu5AH and WIRL cells were the same, with values of 3.1 +/- 0.1 and 2.9 +/- 0.2 h, respectively. Since bilayers formed from isolated plasma membrane lipids do not reproduce the kinetics of cholesterol efflux observed with the intact plasma membranes, it is likely that the local domain structure, as influenced by membrane proteins, is responsible for the differences in t 1/2 values for cholesterol efflux from these cell lines.