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PubMed1996

Association of polyene antibiotics with sterol-free lipid membranes: I. Hydrophobic binding of filipin to dimyristoylphosphatidylcholine bilayers.

The interaction of filipin III with multilamellar vesicles (MLV) of dimyristoylphosphatidylcholine (DMPC ) was studied by four complementary methods leading to the following results: (1) The modifications of the filipin dichroic spectrum, by adding preformed fluid DMPC MLV, provide evidence of a saturable association with the stoichiometry DMPC/filipin = 4.2 +/- 0.5, constant between 24 and 35 degrees Celsius. (2) Thermograms obtained by differential scanning calorimetry (DSC) on mixtures where filipin is incorporated during the formation of MLV exhibit a high-temperature tail the more marked the higher the filipin content and some structures at temperatures which depend on this content. The corresponding evolution with the temperature of the CD spectra reveals that the characteristic bound filipin spectrum appears at the temperature at which a structure emerges. (3) Titration calorimetry measurements reveal that the association process is exothermic in the temperature range of the DSC endotherms in agreement with the filipin-induced ordering of the lipid chains, previously established by 2H-NMR in the same temperature range (Milhaud et al.(1989) Eur. Biophys. J. 17, 151-158). A discussion of the relevancy of this exothermicity to the hydrophobic effect is developed by referring to the paper by Wimley and White ((1993) Biochemistry 32, 6307-6312).

Calorimetry, Differential Scanning↗
PubMed1987

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.

Animals↗
PubMedDate not supplied

Histochemical detection of esterified cholesterol within human atherosclerotic lesions using the fluorescent probe filipin.

The fluorescent cholesterol probe filipin has been used in this study to histochemically examine the morphology and cholesterol composition of intra- and extracellular Sudanophilic lipid deposits which accumulate in human atherosclerotic lesions. Because filipin reacts with unesterified but not esterified cholesterol, detection of cholesteryl esters was carried out by first extracting native unesterified cholesterol with ethanol, and then enzymatically converting esterified to unesterified cholesterol before filipin staining. The size and structure of particles comprising extracellular cholesteryl ester-rich lipid deposits was different in regions of necrosis as detected using filipin compared with the Sudan lipid-soluble dye oil red 0. Whereas oil red 0 staining often indicated that extracellular cholesteryl ester in these regions occurred in amorphous and spherical particles of varying sizes, filipin staining revealed that extracellular cholesteryl ester occurred in spherical particles more uniform in size than indicated by oil red 0 staining. Also, the majority of extracellular cholesteryl ester-rich particles in necrotic regions were smaller than intracellular cholesteryl ester-containing lipid droplets. In addition, the use of filipin to detect intracellular cholesteryl ester allowed distinction of 2 subpopulations of oil red 0-stained cells which did and did not contain detectable cholesteryl ester.

Adolescent↗
PubMed1983

Interaction of filipin with junctional membrane at different stages of the junction's life history.

The relationship of filipin-sterol complexes to tight and gap junctions during their formation, maturation, internalization, and degradation was studied in separate cell lines. Filipin-sterol complexes tended to be excluded from mature junctions in tight junction forming COLO 316 cells and gap junction forming SW-13 cells. Once internalized, unlabeled junctional membrane appeared to fuse with heavily labeled vesicles, presumably lysosomes. Although the absence of filipin-sterol complexes from junctional membrane does not necessarily reflect the absolute sterol content of this membrane, the fact that filipin-sterol complexes are largely excluded from these areas indicates that this membrane is different from surrounding membrane. The absence of filipin-sterol complexes also permits the visualization of 'mixing' of this specialized unlabeled membrane domain with other filipin labeled membrane systems.

Adrenal Gland Neoplasms↗
PubMed1983

Distribution of filipin-sterol complexes on cultured muscle cells: cell-substratum contact areas associated with acetylcholine receptor clusters.

Specialized areas within broad, close, cell-substratum contacts seen with reflection interference contrast microscopy in cultures of Xenopus embryonic muscle cells were studied. These areas usually contained a distinct pattern of light and dark spots suggesting that the closeness of apposition between the membrane and the substratum was irregular. They coincided with areas containing acetylcholine receptor clusters identified by fluorescence labeled alpha-bungarotoxin. Freeze-fracture of the cells confirmed these observations. The membrane in these areas was highly convoluted and contained aggregates of large P-face intramembrane particles (probably representing acetylcholine receptors). If cells were fixed and then treated with the sterol-specific antibiotic filipin before fracturing, the pattern of filipin-sterol complex distribution closely followed the pattern of cell-substratum contact. Filipin-sterol complexes were in low density in the regions where the membrane contained clustered intramembrane particles. These membrane regions were away from the substratum (bright white areas in reflection interference contrast; depressions of the P-face in freeze-fracture). Filipin-sterol complexes were also in reduced density where the membrane was very close to the substratum (dark areas in reflection interference contrast; bulges of the P-face in freeze-fracture). These areas were not associated with clustered acetylcholine receptors (aggregated particles). This result suggests that filipin treatment causes little or no artefact in either acetylcholine receptor distribution or membrane topography of fixed cells and that the distribution of filipin-sterol complexes may closely parallel the microheterogeneity of membranes that exist in living cells.

Animals↗
PubMed1985

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.

Alanine Transaminase↗
PubMed1983

Freeze-fracture localization of filipin-cholesterol complexes in the plasma membrane of Trypanosoma cruzi.

The polyene antibiotic filipin was used as a probe for the detection of cholesterol on the P and E faces of the cell body and the flagellar membranes of epimastigotes of Trypanosoma cruzi. When glutaraldehyde-fixed cells were treated with filipin the filipin-cholesterol complexes were homogenously distributed throughout the parasite's plasma membrane. In some cells, mainly on their P faces, such complexes were arranged linearly forming parallel bands with a distance between the bands similar to that existent between the subpellicular microtubules that are attached to the plasma membrane. Filipin-cholesterol complexes were more abundant in the flagellar membrane than in the membrane that enclosed the cell body. No filipin-cholesterol complexes were seen in the membrane of intracellular organelles. Treatment of living epimastigotes with filipin-induced aggregation of cholesterol and intramembranous particles.

Animals↗
PubMed1972

Fluorescence studies of the binding of the polyene antibiotics filipin 3, amphotericin B, nystatin, and lagosin to cholesterol.

The interactions of filipin III, amphotericin B, nystatin, and lagosin with sterols in aqueous suspension and in vesicles were followed by fluorescence excitation spectra and by measurement of polarized fluorescence intensities. The equilibrium constants for association of the polyene antibiotics with aqueous suspensions of cholesterol follow the order filipin III > amphotericin B > nystatin > lagosin, in agreement with the order reported for the extent of damage these antibiotics cause in natural and model membranes. Fluorescence polarization measurements show that hydrophobic forces are primarily responsible for the formation of the complexes. Filipin III undergoes a large enhancement in fluorescence polarization on binding to aqueous suspensions of cholesterol and epi-cholesterol, and to vesicles of lecithin-cholesterol, lecithin-beta-cholestanol, and lecithinergosterol. Small increases in polarization occur on interaction of filipin III with vesicles derived from lecithin and epi-cholesterol, thiocholesterol, and androstan-3beta-ol. Amphotericin B undergoes a relatively constant enhancement in fluorescence polarization on interaction with the various lecithin-sterol vesicles used and does not display the selectivity exhibited by filipin III. It is suggested that filipin III serves as a probe of lecithin-sterol interaction.

Amphotericin B↗
PubMed1973

Effects of Filipin and Cholesterol on K Movement in Etiolated Stem Cells of Pisum sativum L.

Filipin, a polyene antibiotic known to induce leakage of materials from various cells, depresses K(+) and NO(3) (-) uptake in etiolated pea epicotyl segments. Filipin concentrations which strongly reduce K(+) influx have little effect on efflux; however, high concentrations enhance K(+) efflux. Filipin has no effect on respiration rates or cell electropotentials; its action is presumed to be on the cell membranes. Cholesterol, but not a thiol-protecting agent (dithiothreitol), enhances K(+) influx and counteracts the inhibition by filipin. Although this effect of cholesterol may be due to an interaction with filipin in the outer solution, there is reason to believe that its major effect is to impart stability to the membrane; filipin is believed to act by interfering with sterol stabilization of phospholipid layers. The predominant native sterols of etiolated pea stem (Pisum sativum L. var. Alaska), which cholesterol probably mimics, are beta-sitosterol, campesterol, and stigmasterol.

Journal Article↗
PubMed1995

Changes in free cholesterol content, measured by filipin fluorescence and flow cytometry, correlate with changes in cholesterol biosynthesis in THP-1 macrophages.

The free cholesterol content of cells can be monitored by the intensity of fluorescence emissions from the polyene antibiotic filipin. In a previous study (Hassall: Cytometry 13:381-388, 1992) using THP-1 macrophages, a decrease in filipin fluorescence in response to increasing concentrations of modified lipoprotein was observed, suggesting a reduction in the free cholesterol content of the cells. In this study, THP-1 macrophages were treated with a number of agents known to modulate cholesterol biosynthesis and cholesterol esterification. Changes in filipin fluorescence emissions were measured by flow cytometry, and correlated with changes in cholesterol biosynthesis measured by incorporation of [14C]acetate into cholesterol. A correlation between decreases in filipin fluorescence and reductions in cholesterol biosynthesis was apparent, even when cholesterol esterification was inhibited. These results suggest that the decreases in filipin fluorescence observed may be due, at least in part, to reduction in cholesterol biosynthesis.

Acetates↗
PubMed1991

Distribution of filipin-sterol complexes in the plasma membrane of stallion spermatozoa during the epididymal maturation process.

The presence and distribution of cholesterol in mature and immature epididymal spermatozoa was analyzed using filipin as a cytochemical tool in freeze-fracture replicas and thin section preparations. The polyenic-antibiotic filipin formed complexes with 3, beta -OH sterols, producing characteristic protrusions, or pits, that were heterogeneously distributed in the plasma membrane of stallion spermatozoa, revealing a specific organization in a functionally specialized area of the gamete. The acrosomal region of the sperm head presented a significantly higher density of filipin sterol complexes than the postacrosomal region, which was usually free of these complexes. The plasma membrane of the flagellum also showed filipin sterol complexes randomly distributed in freeze-fracture replicas. The strong filipin labeling observed in the membrane of spermatozoa obtained from the caput region of the epididymis decreased significantly during epididymal passage. The significance of these changes is not completely understood, but they might contribute to establishing the molecular organization necessary for sperm transit and storage in the epididymis as well as to development of motile spermatozoa that are able to fertilize the oocyte and induce normal embryonic development.

Animals↗
PubMed1988

Filipin-labelled complexes are polarized in their distribution in the cytoplasm of meiotically mature mouse eggs.

Unfertilized (germinal vesicle [GV] stage, superovulated and naturally ovulated) and fertilized mouse eggs were treated with the polyene antibiotic filipin, which complexes with unesterified sterols; specimens were observed by fluorescence microscopy and scanning electron microscopy (SEM). In all oocytes examined, filipin fluorescence was localized to the plasma membrane and to subcellular structures of various sizes. In the unfertilized oocyte, polarity was observed both in the plasma membrane stain and in the pattern formed by the subcellular structures. SEM of filipin-treated oocytes had several characteristic features including a specific distribution of heterogeneous microvilli that appears to have a spatial relationship with the fluorescent pattern of the filipin-positive subcellular structures. In GV stage and fertilized eggs the filipin-positive subcellular structures were associated with the germinal vesicle and in fertilized eggs they were associated with the site of polar body abstriction.

Animals↗
PubMed2001

Differential effects of filipin and methyl-beta-cyclodextrin on B cell receptor signaling.

Methyl-beta-cyclodextrin and filipin are cholesterol-binding reagents often used interchangeably to investigate functional requirements for lipid rafts in receptor-mediated signal transduction. Recently, contradictory results were reported by two groups using these reagents in different model systems to investigate the role of lipid rafts in BCR signaling. We confirm here that BCR-mediated calcium release is inhibited by filipin and enhanced by cyclodextrin. The inhibitory effect of filipin could not be attributed to raft disruption, however, because its ability to release raft-associated proteins into the detergent-soluble phase of cell lysates was less than that of cyclodextrin. In contrast, we found that filipin profoundly inhibited phosphorylation of the raft-associated adaptor protein Cbp/PAG, whereas the effect of cyclodextrin was minor. Thus, filipin and cyclodextrin modify cholesterol-rich microdomains through different mechanisms with different consequences on receptor signaling. In addition, the enhanced calcium release observed under conditions of maximum raft disruption suggests that rafts have a role in negatively regulating BCR signals.

Anti-Bacterial Agents↗
PubMed1987

Filipin-sterol complexes in golden hamster sperm membranes with special reference to epididymal maturation.

The distribution of membrane filipin-sterol complexes (FSCs) was qualitatively surveyed on freeze-fracture replicas of spermatozoa from the male reproductive tract and ejaculates of golden hamster. In the head, the acrosomal plasma membrane showed the strongest filipin labeling on the principal segment, but it was absent in the "quilt-like pattern" areas. These latter were observed in both caput and corpus epididymal spermatozoa, but were absent in mature spermatozoa. The postacrosomal plasma membrane had few FSCs and both the outer and inner acrosomal membranes were always negative to filipin. The nuclear membrane of the principal segment was constantly filipin-positive. The nuclear membrane of the postacrosomal region had more FSCs than that of the principal segment, particularly in mature spermatozoa. Many linear, "rod-like FSCs" were observed on the postacrosomal nuclear membrane of mature spermatozoa, especially in the uterine spermatozoan samples. In the neck, the plasma membrane had only a few FSCs. The redundant nuclear membrane was slightly filipin-positive, while the membrane scroll of mature spermatozoa was heavily labeled. In the tail, the plasma membrane of both the middle and principal piece was moderately labeled.

Animals↗
PubMed1987

Labeling of cholesterol with filipin in cellular membranes of parenchymatous organs. Standardization of incubation conditions.

Filipin is used for ultrastructural cytochemical localization of cholesterol in biological membranes. It binds to unesterified 3 beta-hydroxy-sterols forming 25 nm complexes which are readily recognized in freeze-fracture replicas. Since most investigations with filipin have been performed in isolated cells (tissue culture, cell suspensions etc.) we have investigated the conditions for reproducible labeling of cholesterol in membranes of parenchymatous organs. Vibratome sections of rat kidney fixed by glutaraldehyde perfusion were incubated in filipin and freeze-fracture replicas were prepared using standard techniques. The concentration of filipin, the thickness of vibratome sections and the incubation time and temperature were varied over a wide range. Optimal results were obtained with 50 micron thick tissue slices incubated in 400 micrograms/ml of filipin for 46 h at room temperature. Under these conditions lysosomes were consistently labeled while mitochondria and the endoplasmatic reticulum were negative. Peroxisomes showed a little or no labeling at all while the nuclear envelope was heavily labeled in some cells being negative in others. The method described here should be useful in investigation of the role of cholesterol in function of biological membranes in parenchymatous organs and compact tissues.

Animals↗
PubMed1985

Filipin-sterol complexes at nodes of Ranvier.

Using the filipin-sterol technique, regional heterogeneity in the axonal and Schwann cell plasma membranes was investigated at the node of Ranvier and paranodes. Filipin-sterol complexes were abundant at the nodal axolemma but infrequent throughout the paranodal axolemma. The paranodal Schwann cell plasma membrane was rich in complexes which extended over the nodal Schwann cell microvilli. There were no regional differences in filipin labelling of the nodal-paranodal Schwann cell plasma membrane in relation to features such as paranodal cytoplasmic columns or mesaxonal furrows. However, the paranodes of adjacent Schwann cells were sometimes markedly different from each other in the amount of filipin labelling. The extent to which filipin labelling is indicative of cholesterol membrane content is discussed and the findings are related to current concepts of distribution, mobility and interaction of protein and lipid in biomembranes, with particular reference to the nodal axolemma.

Animals↗
PubMed1983

Ultrastructural characterization of cholesterol distribution in toad bladder using filipin.

The polyene antibiotic filipin was used to characterize the cholesterol distribution in the membranes of the toad bladder epithelium in freeze-fracture replicas. The apical membranes of granular and mitochondria-rich cells incorporate moderate amounts of filipin while the basolateral membranes of both cell types incorporate substantially greater amounts. Intracellular membranes, in general, take up very little filipin. The major exception to this is the granule membrane, which appears to be rich in cholesterol. An inverse correlation was found between the density of filipin-sterol complexes in the apical membrane and the incidence of granules in the cytoplasm. This suggests that fusion of granules with the apical membrane may be responsible for variation in the concentration of cholesterol in the apical membrane. Thirty minutes following vasopressin exposure, there is no consistent change in the cholesterol content of the apical membrane of granular cells as measured by the incidence of filipin-sterol complexes. The lack of change in the amount of membrane cholesterol indicates that the vasopressin-induced increase in transepithelial water permeability is not mediated by a change in cholesterol content of the apical membrane.

Animals↗
PubMed1988

Interactions between a paramagnetic analogue of cholesterol and filipin.

A paramagnetic analogue of cholesterol (called 25-doxyl-27-norcholesterol (CNO)), labeled near the w-end of the hydrophobic tail, was used to study interactions of cholesterol with filipin. We observed by electron microscopy that CNO- and cholesterol-filipin complexes are structurally equivalent. Two kinds of complexes were seen by ESR spectroscopy and electron microscopy, depending on the stoichiometric R ratio between the antibiotic and sterol. When R was high, an immobilized ESR spectrum appeared, showing strong imbrication between CNO and filipin. When R was nearer to unity, an exchange-broadened spectrum emerged, corresponding to a new phase that was very rich in CNO (a fast exchange between spins could occur by nearest contacts). CNO was easily displaced from its complex (i) by gradual addition of genuine cholesterol; and (ii) by an excess of phospholipids, owing to the very poor affinity of CNO (and cholesterol, by extension) for filipin in the lipidic phase. Almost no difference appeared between the ESR spectra of oriented samples, i.e. the probe showed no long-range order in any complex of CNO with filipin.

Cholesterol↗