Membrane-associated proteins affect the formation of filipin-cholesterol complexes in viral membranes

The presence and distribution of cholesterol in biological membranes can be visualized by complex formation with the polyene antibiotic filipin after or during fixation with glutaraldehyde. In the envelopes of budding and immature retroviruses no filipin-cholesterol complexes are formed, but in the plasma membrane of host cells and in the envelopes of mature viruses filipin-cholesterol complexes are easily detected. However, after treatment of glutaraldehyde-fixed cells with pepsin, the presence of cholesterol in the envelopes of budding and immature viral particles could also be demonstrated. This indicates that in these structures the reaction of cholesterol with filipin is inhibited by proteins associated with the cholesterol-containing membrane. Treatment of fixed cells with trypsin, and of unfixed cells with cytochalasin B (CB) had no effect on detectability of cholesterol in these structures. On no occasion were cholesterol-filipin complexes formed in coated pits. The present findings call for caution when interpretating data on absence of filipincholesterol complexes in those membrane domains that are characterized by the presence of closely associated proteins.     

Effect of cholesterol on the functional activity of proteins responsible for the resistance of human lymphocytes to xenobiotics

The influence of agents modifying cholesterol in plasma membranes on the functional activity of transporter proteins (P-glycoprotein (P-gp) and multidrug resistance protein 1 (MRP1)) in human lymphocytes has been studied. It was shown that changes in the lateral distribution of cholesterol using the polyene antibiotic filipin, which disturbs the structure and function of glycolipid microdomains in plasma membranes of lymphocytes lead to a decrease in the transport activity of both P-gp and MRP1. It was found that the treatment of human lymphocytes with the cyclic oligosaccharide methyl-β-cyclodextrin, which leads to cholesterol depletion and reduction of lipid bilayer microviscosity in membranes of these cells, also decreases the functional activity of these proteins. It was concluded that the transport activity of P-gp and MRP1 depends on the modification of cholesterol in the membranes of human lymphocytes, i.e., is closely associated with the level of cholesterol and its lateral distribution.     

Effects of Filipin on the Structure and Biological Activity of Enveloped Viruses

The interaction of the polyene antibiotic filipin with membrane-bound cholesterol in vesicular stomatitis (VS), influenza, and Rauscher leukemia virions was studied. Exposure of virions to filipin resulted in a series of depressions and ridges in the envelope of VS virions, with a periodicity of 15 to 20 nm perpendicular to the long axis of the particle; similar morphological alterations were observed in negatively stained preparations, in thin-sectioned virions, and in protease-treated virions that lack surface glycoproteins. This morphological effect was specific for filipin, since the envelopes of VS virions that had been treated with another polyene antibiotic, amphotericin B, exhibited markedly different morphology. Morphological alterations induced by filipin in influenza and Rauscher leukemia virions differed from those seen in VS virions. The infectivity of filipin-treated VS virions was reduced up to 500-fold, whereas influenza virions were resistant to filipin treatment. Incorporation of filipin into the virions was demonstrated, and no release of either lipids or proteins from virions was detected after filipin treatment. A stoichiometry of approximately 1 mol of bound filipin per mol of cholesterol was found in both intact and protease-treated VS virions. The equilibrium dissociation constant for filipin-cholesterol interaction was approximately 74-fold larger in intact than in protease-treated VS virions. The initial rate of association of filipin with cholesterol in intact virions was slower than that in protease-treated particles. The fluidity of lipids in VS viral membranes, as probed by a stearic acid derivative spin label, was markedly reduced when either intact or protease-treated virions were treated with filipin.     

Cholesterol-rich microdomains in rat and porcine thyroid membranes involved in TSH-induced endocytotic processes

Filipin, a polyene antibiotic, was used to detect cholesterol in thyroid membranes in vivo and in culture during TSH stimulation. We found that apical and basolateral plasma membranes were heterogeneously modified by filipin which induced abundant lesions in apical membranes, whereas Golgi apparatus, endoplasmic reticulum, nuclear membranes were unmodified. Small apical vesicles and colloid droplets were generally highly enriched in these complexes, suggesting a high cholesterol concentration in their membranes. Pseudopod membranes, known to be highly specialized domains in the apical plasma membrane, appeared enriched in cholesterol. Consequently, we suggest that an increased cholesterol content may be involved in the stabilization of thyroid membranes during endocytotic processes.     

Filipin-cholesterol complexes form in uncoated vesicle membrane derived from coated vesicles during receptor-mediated endocytosis of low density lipoprotein

Filipin has been widely used as an electron microscopic probe to detect 3-beta-hydroxysterols, principally cholesterol, in cellular membranes. When it complexes with sterol, it forms globular deposits that disrupt the planar organization of the membrane. Previous studies have shown that coated pits and coated vesicles, specialized membranes involved in receptor-mediated endocytosis, do not appear to bind filipin. This has led to the suggestion that these membranes are low in cholesterol compared with the remainder of the plasma membrane. Since coated endocytic vesicles become uncoated vesicles during the transport of internalized ligands to the lysosome, we have carried out studies to determine whether or not the membranes that surround these transport vesicles are unable to bind filipin and therefore, are also low in cholesterol. Cells were incubated with ferritin-conjugated ligands that bind to low density lipoprotein (LDL) receptors in coated pits. After allowing internalization of the conjugates, we fixed the cells in either the presence or absence of filipin. This permitted us to identify all of the vesicles involved in the transport of LDL to the lysosome and to determine whether the membranes of these vesicles were able to bind filipin. We found that, coordinate with the dissociation of the clathrin coat from the endocytic vesicles, the membranes became sensitive to the formation of filipin-sterol complexes. Furthermore, all of the uncoated endocytic vesicle membranes, as well as the lysosomal membranes, bound filipin. This suggests either that coated membrane contains normal cholesterol levels, which is not easily detected with filipin, or that cholesterol rapidly moves into endocytic vesicles after the clathrin coat dissociates from the membrane.     

Filipin-cholesterol complexes in plasma membranes and cell junctions of Tenebrio molitor epidermis

The polyene antibiotic filipin combines with cholesterol in membranes to form complexes that are readily identifiable in the electron microscope. The distribution of filipin-cholesterol (FC) complexes is most easily studied by freeze-fracture. Larval epidermis of Tenebrio molitor (Insecta, Coleoptera) was maintained in vitro for 48 hr, since the electrophysiological properties of the cells are best characterized under these conditions. The cells were fixed in buffered 3.0% glutaraldehyde at RT for 15 min, transferred to fresh fixative containing 1% DMSO and filipin (final concentration; 0.5 mg/ml) for 3 hr RT. Control cells were treated in fixative containing 1% DMSO only. In freeze fracture replicas, FC complexes appear on the plasma membrane as large circular protrusions measuring 26.5 +/- 6.8 nm (x +/- s.d.) n = 50, in diameter and 17.1 +/- 2.8 nm, n = 50, in height and 11.7 +/- 2.6 nm, n = 25, in depth. Protrusions are about two times more frequent on the E face while pits are several times more frequent on the P face. FC complexes are most abundant (greater than 50/mu m2) on the basal membrane surface of the cells but are excluded from regions of hemidesmosomal plaques that anchor the cells to the basal lamina. FC complexes are also abundant on the apical surfaces of the cells where cuticle secretion occurs. In the lateral regions below the junctional belt, FC complexes are less numerous but often appear to increase in frequency in a graded fashion away from the junctional region. The septate junctions are relatively free of FC complexes except in regions where they open to form islands. These islands often contain gap junctions but the FC complexes rarely invade the particle domains of the gap junctions. Single FC complexes were seen in three out of a total of 97 gap junctions. Exposure of the epidermis to 20-hydroxyecdysone for 24 hr in vitro did not induce the appearance of FC complexes within the cell junctions.     

Filipin as a cholesterol probe. II. Filipin-cholesterol interaction in red blood cell membranes

Filipin, a mixture of polyene antibiotics which form complexes with cholesterol, perturbs membrane lipid organization, and causes hemolysis of erythrocytes, is increasingly used as a cytochemical probe for the distribution of cholesterol in cell membranes. We used light (phase-contrast, dark-field and fluorescence) and electron microscopical techniques (whole-mount shadowing, negative staining, and freeze-fracture) to study the interaction of filipin with unfixed and glutaraldehyde-fixed human red blood cell (RBC) membranes. Lysis time and extent depended upon the cholesterol:filipin (C:F) ratio. Lysis was prevented by osmotic protection with high MW dextran. Filipin treated cells fluoresced, but variation in fluorescence intensity among unfixed as well as among fixed cells was evident both at low and high C:F ratios. Negatively stained preparations of unfixed cells lysed on grids or in suspension revealed ring- or C-shaped filipin-induced lesions (FIL) equipped with a veil-like appendage; single FIL, and FIL fused by their veils into aggregates, were shed from membranes. FIL at the surface proper of shadowed whole-mounts and of freeze-etched preparations of prefixed cells appeared as single, dispersed or aggregated cylinders protruding to variable heights above the membrane's plane; aggregated FIL were shed from cells. The freeze-fracture appearance of FIL differed in membranes fixed before or after filipin treatment. E- and P-faces of post-fixed membranes exhibited cylindrical protrusions and depressions, respectively; in essence, the reverse was found in pre-fixed RBC. Both pre- and post-fixed membranes showed considerable variation in the number of FIL on individual cells whether incubated at high (1:1) or low (1:5) C:F ratios, or for a short (10 min) or a long (80-180 min) time. Aggregation and shedding of FIL was evident in all preparations. Thin layer chromatography of the incubation fluid after sedimentation of cells showed that membrane cholesterol was shed from incubated cells. The presented data question the feasibility of filipin as a probe for the topographical distribution of cholesterol in cell membranes.     

Absence of filipin-sterol complexes from the membranes of active zones and acetylcholine receptor aggregates at frog neuromuscular junctions

The polyene antibiotic filipin reacts specifically with membrane cholesterol and produces distinctive membrane lesions. We treated frog cutaneous and sartorius muscles with 0.04% filipin in a glutaraldehyde solution with or without prefixation with glutaraldehyde. Freeze- fracture of these muscles revealed numerous 19 to 38-nm protuberances and depressions (filipin-sterol complexes) in most areas of muscle, axon, and Schwann cell membranes. In the presynaptic membrane, however, these filipin-sterol complexes were absent from active zones consisting of ridges bordered with double rows of particles. In the postsynaptic membrane, filipin-sterol complexes were also virtually absent from the areas occupied by aggregates of large particles representing acetylcholine receptors. These results suggest that the membrane regions of active zones and acetylcholine receptor aggregates have a low cholesterol content.     

Stimulation of ion release from liposomes by the polyene antibiotic, filipin.

The effect of the polyene antibiotic, filipin, upon release of the ions Ca²⁺, Sr²⁺, SO₄^2? and phosphate out of phospholipid and phospholipid-cholesterol liposomal vesicles was studied. The addition of filipin at concentrations stoichiometrically comparable to the cholesterol concentration in the liposomes, resulted in 2–10 × stimulation of the rate of release of all of these ions. The filipin mediated stimulation of release of ions from liposomes was dependent upon the presence of cholesterol. The relative effectiveness of filipin increased when the mole percent of cholesterol incorporated into the liposomes increased from 10 to 50% and when the molar filipin:cholesterol ratio increased from 0.2 to 1.0. It has been previously shown that there is a 1:1 stoichiometry of interaction between filipin and cholesterol [Biochem. Biophys. Acta339, 57 (1974)]. The present studies suggest that this 1:1 stoichiometric interaction may also be responsible for the increased release of entrapped ions.A possible mechanism of action of polyene antibiotics is discussed which suggests that the rearrangement of membrane constituents occurring upon interaction of filipin with cholesterol is the basis for the enhancement of ion release. This would imply that the ion specificity observed upon interaction of polyene antibiotics with membranes would not only be determined by the polyene antibiotic itself, but also by the intrinsic properties of the membrane.     

Sterol effects on phospholipid biosynthesis in the yeast strain GL7

Cells of the yeast sterol auxotroph GL7 were grown on either ergosterol or cholesterol to mid-logarithmic phase and total membrane fractions prepared. Activities of phospholipid biosynthetic enzymes in the two cell types were determined. The rates of phosphatidyl-ethanolamine-phosphatidyl-choline-N-methyl transferase and acyl-CoA-alpha-glycerol-3-phosphate transcylase were significantly greater in ergosterol-grown than in cholesterol-grown cells. These reactions were also inhibited by the polyene antibiotic filipin. By contrast the activities of long-chain fatty acyl-CoA synthetase, CTP-phosphatidate-cytidyl transferase, phosphatidylserine decarboxylase and of phosphatidylinositol synthetase were identical in the two (ergosterol and cholesterol) cultures and unaffected by filipin. The ergosterol effect on phosphatidyl-ethanolamine N-methyl transferase was greatest in cells harvested in early log phase, intermediate in the mid-log phase cells, and not significant in stationary phase cells.     

The tight junction as a barrier to cholesterol in canine epithelial cells

Filipin has been used to test several models of continuity or flow of lipid components through the tight junction. Cultured canine kidney cells (MDCK) were fixed and incubated in the presence of filipin. Freeze-fracture replicas were analyzed and densities of filipin-cholesterol complexes measured. Fractures of membranes linked with tight junctions were compared statistically to determine whether filipin-cholesterol complexes (protrusions and pits, independently) were randomly distributed between the two membranes of cells separated by the tight junction. The results indicate that filipin-cholesterol complexes are not randomly distributed across the tight junction. If the density of filipin-cholesterol complexes is an accurate indication of membrane cholesterol concentration, then there is a difference in the cholesterol concentration between leaflets of membranes joined by tight junctions and models of the tight junction which suggest leaflet continuity across the junction are in error.     

Influence of the membrane undercoat on filipin perturbation of the red blood cell membrane

Filipin, a polyene antibiotic, interacts with beta-hydroxy sterols such as cholesterol in most cell membranes, forming bumps and pits that are visible by electron microscopy of freeze-fracture replicas. The markedly reduced perturbability of the red blood cell (RBC) membrane, compared to other cells, has been attributed to the constraining influence of the red cell membrane skeleton, the undercoat composed of spectrin, actin, and protein 4.1. To test the influence of the membrane skeleton on filipin-induced perturbation of the RBC membrane, we studied the interaction of filipin with red cells that were inherently devoid of spectrin and RBC in which spectrin had been crosslinked or denatured. These spectrin-deficient, crosslinked, and denatured cells have a fivefold increase in the number of filipin-induced perturbations as compared to control cells, despite equivalent membrane cholesterol content. These findings confirm that the spectrin-based membrane skeleton strongly influences the organization of the membrane so as to limit perturbation by filipin:cholesterol interaction and that for membranes in which the cholesterol content is known, filipin is a useful probe for testing the avidity of spectrin-based cytoskeletal attachment.     

Occluding Junctions in MDCK Cells: Modulation of Transepithelial Permeability by the Cytoskeleton

In MDCK cell monolayers the opening and resealing of occluding junctions can be induced by removal and restoration of calcium to the external medium. The overall changes in permeability of the occluding junctions in the monolayer can be monitored by the drop and recovery of the total transepithelial electrical resistance. We have investigated the effects of cytochalasin B (CB) on this process. When CB is added to sealed monolayers there is a gradual drop in the electrical resistance across the monolayer. This drop is accompanied by a slow disorganization of the microfilament pattern of these cells, including a disturbance of a ring of cortical microfilaments that is normally associated with the junctions. Cells in open monolayers treated with CB will not reseal and have an altered filament distribution. These cells do not have a continuous cortical ring. We have used a voltage scanning technique that uses a microelectrode to measure the resistance at selected points along the junction which surrounds a single cell. In untreated, closed monolayers, the junction is heterogeneous with alternating points of high and low conductance. In closed monolayers treated with CB, although there are low conductance points, we have observed an increased frequency of high conductance points that correlates with the change in the overall conductance. The frequency of high conductance points along the junction and the overall conductance both increase with time of exposure to CB. In an effort to understand the molecular basis for the permeability changes induced by EGTA and CB, we have looked for differences in the protein components of the cell membranes of open, closed, and CB-treated MDCK monolayers. This was done by radioiodinating the surface membrane proteins under control and experimental conditions that bring about permeability changes. No significant differences in the labeled protein patterns were found under these conditions. These results suggest that the permeability changes involve only a structural rearrangement of membrane components. In addition we have observed that about 36% of the surface label remains bound to the insoluble cytoskeletons obtained from cells in control and experimental conditions that alter the permeability of the tight junctions. The iodinated proteins attached to the CS include polypeptides with M_r of ≥ 120K daltons as well as peptides with M_r = 56K, 50K, 36K, and 18K daltons.     

Cholesterol distribution and structural differentiation in the sarcoplasmic reticulum of rat cardiac muscle cells

Summary The polyene compound, filipin, was used as a probe to localize cholesterol in the membranes of the rat cardiac muscle cell, with particular reference to the sarcoplasmic reticulum (SR). Filipin binds specifically to cholesterol (and related 3--hydroxysterols) in membranes, producing distinct deformations which can be viewed by freeze-fracture and used as markers for the presence of cholesterol-rich regions in the membrane plane. In freeze-fracture replicas of filipin-treated rat myocardium, the muscle cells revealed abundant deformations in their plasma membranes, no deformations in mitochondrial membranes, and an intermediate response in the SR. These results are in agreement with the levels of cholesterol reported in isolated fractions of the different membrane types, and confirm the specificity of filipin action. Within the SR, the filipin-induced deformations were not randomly distributed but occurred more commonly in free SR at or near the Z-region of the sarcomere than in other parts of the free SR or the junctional SR. This finding is interpreted as evidence for a non-homogeneous distribution of cholesterol in cardiac muscle cell SR. The possible significance of cholesterol in relation to structural differentiation and function of the SR is discussed.     

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.     

Depletion of cellular cholesterol interferes with intracellular trafficking of liposome-encapsulated ovalbumin

Cholesterol is a major constituent of plasma cell membranes and influences the functions of proteins residing in the membrane. To assess the role of cholesterol in phagocytosis and intracellular trafficking of liposomal antigen, macrophages were treated with inhibitors of cholesterol biosynthesis for various time periods and levels of cholesterol depletion were assessed by thin layer chromatography. In control macrophages, cholesterol was present in the plasma membrane and in intracellular stores, as visualised by staining with the cholesterol-binding compound filipin, whereas macrophages treated with cholesterol inhibitors failed to stain with filipin. However, these macrophages were still capable of phagocytosis as evidenced by their internalisation of fluorescent-labelled bacteria and liposome-encapsulated Texas red labelled-ovalbumin, L(TR-OVA). While fluorescent ovalbumin (OVA) was consistently transported to the Golgi in macrophages incubated with L(TR-OVA), in cells treated with cholesterol inhibitors, OVA remained spread diffusely throughout the cytoplasm. Even though the mean fluorescence intensity of MHC class I molecules on cholesterol inhibitor-treated macrophages was equivalent to that of the control macrophages, the amount of MHC class I-liposomal OVA-peptide complex detected on the cell surface of cholesterol inhibitor-treated macrophages, was only 45.6 +/- 7.4% (n = 4, mean +/- SEM) of control levels after intracellular processing of L(OVA). We conclude that cholesterol depletion does not eliminate phagocytosis or MHC class I surface expression, but does affect the trafficking and consequently the MHC class I antigen-processing pathway.     

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.     

Acute effects of filipin on the plasmic, hepatic, and biliary cholesterol of the rat

Twenty-one male Wistar rats, 13 weeks old, were fed ad libitum hyperlipidic diets (28% fats) loaded with cholesterol (1.2%) for 5 weeks. One group of 11 rats was fed saturated fats (diet group "S") and another group of 10 rats was fed polyunsaturated fats (diet group "PU"). On the day they were sacrificed 10 of the rats were injected intravenously with 1 mg of filipin. Contrary to the rats in diet group "PU," the rats in diet group "S" treated with filipin presented certain characteristics that were not found in the nontreated group: They provided evidence of biliary cholestasis accompanied by a decline in the level of secretion of bile salts and phospholipids into bile. The concentrations of both free and esterified cholesterol in plasma fell and the amount of (esterified) hepatic cholesterol rose, although there was no change due to the filipin in the amounts of hepatic phospholipids. Explanatory hypotheses for these phenomena were considered, first, at the site of plasma membranes where filipin binds selectively to the cholesterol in the membrane, causing a disruption which probably disturbs the absorbance of circulating lipoproteins, especially that of hepatocyte cells, particularly in diet group "PU." Second, the effects of filipin on subcellular membranes seem to disturb the secretion of lipids and lipoproteins into bile and plasma, especially in diet group "S." Last, at the intracellular level, filipin appears to have a blocking effect on the organelles involved in biliary lipid secretion. The activity of certain enzymes such as cholesterol esterase may also be blocked, particularly in diet group "S," which would explain the accumulation of esterified cholesterol in liver.     

Filipin-sterol complexes in molluscan gill ciliated epithelial cell membranes: intercalation into ciliary necklaces and induction of gap junctional particle arrays

Summary Freeze-fracture electron microscopy has been used in conjunction with the antibiotic filipin to investigate possible differences in the distribution of sterols in ciliary and somatic cell membranes of scallop and mussel gill epithelial cells. Contrary to previous reports, we find that filipin-sterol lesions can occur among the strands of the ciliary necklace but they are partially excluded from the smooth neck region above the necklace where the membrane is tightly apposed to the axonemal microtubules. No obvious differences in filipin-sterol lesions occur in the membranes of mussel gill cilia of varying mechanical sensitivity. Although abundant in the apical plasma membrane, filipin-sterol complexes are rare within the membranes of microvilli. Filipin-sterol lesions form outside the loosely parallel particle strands of septate junctions, sometimes increasing their relative orderliness. At sufficiently high density, filipin-sterol protrusions within the plasma membrane result in mass aggregation of gap junctions, possibly through recruitment of unorganized connexons.     

Rates of amphotericin B and filipin association with sterols. A study of changes in sterol structure and phospholipid composition of vesicles

The influence of structural modifications in sterols and phospholipids on the rate of polyene antibiotic-sterol interaction was studied. For filipin and amphotericin B association with sterols in vesicles, a preferential interaction was found with sterols whose side chain length is close to that of cholesterol. Introduction of trans double bonds into the sterol side chain did not alter the rate of interaction in vesicles. The delta 7-bond of the sterol appears to be of critical importance in amphotericin B-sterol interaction, whereas the delta 5-bond is not essential. These observations are relevant to the well-known effects of amphotericin B on cell membranes containing ergosterol compared with those containing cholesterol. The dependence of the rates of sterol-polyene antibiotic interaction on the phospholipid composition of the vesicles indicates that phospholipid vesicles may be an inadequate model for reaching a comprehensive understanding of the effects exerted on biological membranes by these agents.