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PubMed1994

Filipin staining of lipoproteins in polyacrylamide gels: sensitivity and photobleaching of the fluorophore and its use in a double staining method.

We have developed a double staining procedure in which polyacrylamide gels are first stained with filipin to identify lipoproteins, and then with Coomassie Brilliant Blue (CBB) to identify proteins. Filipin staining when performed at 37 degrees C is both more rapid and more sensitive than previously published procedures. After only 5 min, 20 ng of low density lipoprotein (LDL) unesterified cholesterol/mm3 of band volume could be detected, and after 12 h, sensitivity reached 0.8 ng/mm3. A semilogarithmic relationship was found between the amount of LDL unesterified cholesterol applied and filipin fluorescence. Although rapid photobleaching of the fluorophore occurred during UV transillumination of these gels, such photobleaching actually resulted in maximizing of the signal:noise ratio, resulting in better definition of bands. Treatment of gels with filipin had no deleterious effects on the subsequent staining with CBB. This dual staining procedure should prove useful for studies in which both lipoproteins and proteins in plasma need to be documented in the same gel.

Cholesterol↗
PubMed1985

Absence of filipin-sterol complexes from the ciliary necklace of ependymal cells.

The polyene antibiotic filipin reacts specifically with membrane cholesterol and produces membrane perturbations. Prefixed vibratome slices of cat ependymal cells were treated with a glutaraldehyde solution containing 300 microM filipin. Freeze-fracture of these cells revealed numerous 25-30 nm protuberances on fracture faces (filipin-sterol complexes) in the ciliary membrane. In the ciliary necklace, however, these filipin-sterol complexes were absent. These results suggest that the ciliary necklace has a low cholesterol content.

Animals↗
PubMed1981

Freeze-fracture images on filipin-sterol complexes in the thyroid follicle epithelial cell of mice with special regard to absence of cholesterol at the site of micropinocytosis.

In order to clarify the distribution of cholesterol in the plasma- and cyto-membranes of the thyroid follicle cell, freeze-fracture images of the filipin-treated tissues of normal and TSH-treated mice were observed. The filipin-sterol complexes, 25 to 30 nm protuberances or pits are distributed densely and almost homogeneously on the fractured plasma membrane, though the small depressions showing aggregates of intramembrane particles lack the complexes. Each depression corresponds to the coated pit, which might be an initial site for micropinocytosis of the luminal colloid. The limiting membranes of all the large colloid droplets reabsorbed are generally very rich in the complexes, but some small regions on the limiting membrane of the droplet are less in their density. The membranes of the rough endoplasmic reticulum, of the nucleus and of the Golgi apparatus are almost free from the complexes, though small clusters consisting of 2-5 complexes are rarely scattered. In thin sections, the membranes which are rich in the filipin-sterol complexes become obscure in their fine structure after treatment with filipin for 12-24 h.

Animals↗
PubMed1978

Effect of the polyene antibiotic filipin and the calcium ionophore A23187 on sodium transport in isolated frog skin (Rana temporaria).

Addition of filipin (50 micrometer) to the inside bathing solution of the frog skin resulted in a transient increase in the active sodium transport [measured as short-circuit current (SCC)]. The filipin-induced increase in the SCC required the presence of calcium. The calcium ionophore A23187 (4 micrometer) also induced a transient increase in the SCC. After the activation of the SCC by A23187, the SCC could not be activated by filipin. This indicates that the polyene antibiotic filipin acts as a calcium ionophore. Higher concentrations (40 micrometer) of A23187 resulted in a shrinking of the cells in the transporting cell layer. A23187 also increased the potassium-42 exchange in the isolated epithelium. It is suggested that calcium ionophores enhanced the intracellular calcium concentration; this increase in the calcium concentration resulted in an increase in the potassium permeability of the inward-facing membrane. The increase in the potassium permeability might explain the observed increase in the SCC.

Action Potentials↗
PubMed1980

Heterogeneity of toad bladder granular cell luminal membranes. Distribution of filipin-sterol complexes in freeze-fracture.

Glutaraldehyde-fixed toad urinary bladders were incubated with the sterol specific antibiotic, filipin, and the distribution of the resulting filipin-sterol complexes was studied in freeze-fracturing replicas. While the luminal membrane of some cells had a heavy, homogeneous labelling, most had clusters of filipin-sterol complexes scattered on an otherwise poorly labelled membrane. These clusters were seen to overlie cytoplasmic granules, which are found just beneath the luminal plasma membrane of this cell type, and which were always heavily labelled. These results suggest that (a) the granule membranes have a high cholesterol content and (b) sites of interaction between granule and luminal membranes might be enriched in cholesterol. In addition, filipin-sterol complexes were absent from vasopressin-induced particle aggregates on the granular cell luminal membrane.

Animals↗
PubMed1981

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.

Animals↗
PubMed1979

Effects of phospholipase A2 and filipin on the activation of adenylate cyclase.

Rat liver plasma membranes were incubated with phospholipase A2 (purified from snake venom) or with filipin, a polyene antibiotic, followed by analysis of the binding of glucagon to receptors, effects of GTP on the glucagon-receptor complex, and the activity and responses of adenylate cyclase to glucagon + GTP, GTP, Gpp(NH)p, and F-. Phospholipase A2 treatment resulted in concomitant lossess of glucagon binding and of activation of cyclase by glucagon + GTP. Greater than 85% of maximal hydrolysis of membrane phospholipids was required before significant effects of phospholipase A2 on receptor binding and activity response to glucagon were observed. The stimulatory effects of Gpp(NH)p or F- remained essentially unaffected even at maximal hydrolysis of phospholipids, whereas the stimulatory effect of GTP was reduced. Detailed analysis of receptor binding indicates that phospholipase A2 treatment affected the affinity but not the number of glucagon receptors. The receptors remain sensitive to the effects of GTP on hormone binding. Filipin also caused marked reduction in activation by glucagon + GTP. However, in contrast to phospholipase A2 treatment, the binding of glucagon to receptors was unaffected. The effect of GTP on the binding process was also not affected. The most sensitive parameter of activity altered by filipin was stimulation by GTP or Gpp(NH)p; basal and fluoride-stimulated activities were least affected. It is concluded from these findings that phospholipase A2 and filipin, as was previously shown with phospholipase C, are valuable tools for differentially affecting the components involved in hormone, guanyl nucleotide, and fluoride action on hepatic adenylate cyclase.

Adenosine Triphosphate↗
PubMed1982

Freeze-fracture cytochemistry of neuronal membranes: inhomogeneous distribution of filipin-sterol complexes in perikarya, dendrites and axons.

Vibratome slices of cerebellar cortex fixed in a glutaraldehyde solution containing filipin, a sterol-specific probe, were freeze-fractured to study the distribution of filipin-sterol complexes in neuronal plasma membranes. These complexes appear as 25-30 nm protuberances or pits in the fracture face of plasma membranes, and their density was low in dendrites of Purkinje and granule cells. In contrast, the plasma membranes of neuronal perikarya showed an abundant filipin labeling, 4-6 times greater than in dendrites. Parallel fibers, the axons of granule cells also had significantly more filipin-sterol complexes than granule cell dendrites, but fewer than granule cell perikarya. The results reveal difference in the organization of specific regions of neuronal plasma membranes, which are also characterized by a different pattern of synaptic contacts.

Animals↗
PubMed1977

Cholestane spin label study of filipin action on lipid planar multibilayers.

EPR spectra of a cholestane probe dissolved in egg yolk lecithin and lecithin-cholesterol planar multibilayers were observed as a function of the filipin dose. The probe is structurally similar to cholesterol; its normal position when dissolved is with the long axis approximately along the bilayer normal. Both cholesterol-containing and cholesterol-free samples showed spectral components characteristic of bilayer fragmentation (tilted domains) which increased with dose. Furthermore, the cholesterol-free spectra indicated that some of the probe was frozen with the long molecular axis perpendicular to the slide normal. The frozen spectral component increased with dose. Spectra from a fatty acid probe did not have this feature. We interpret this as due to probe complexed with filipin (in place of cholesterol) in accordance with the filipin-cholesterol aggregate model of deKruijff and Demel. An ultraviolet study of filipin-probe interaction indicates that the probe is capable of complexing in just such a manner but has less affinity for the drug than cholesterol. Spectra from the cholesttane probe in liposomes were also observed.

Cholestanes↗
PubMed1999

The pentaene macrolide antibiotic filipin prefers more rigid DPPC bilayers: a fluorescence pressure dependence study.

Filipin is a pentaene macrolide antibiotic which was previously shown to incorporate more extensively into DPPC bilayers below the main phase transition temperature than above this temperature. This result was extremely unusual because drugs tend to be expelled from ordered gel phases. However, such results could not be safely attributed to the phase change of the bilayer itself because the temperature was changing concomitantly. In this work we changed the bilayer phase isothermally (53 degrees C) by hydrostatic pressure variation and discovered that filipin has a slightly more extensive incorporation in the pure DPPC gel phase (P>ca. 54.4 MPa): Kp,lc approximately 3x10(3) vs. Kp,gel approximately 6x10(3). The presence of sterols (45% molar ergosterol or cholesterol) caused an increase in the partition coefficients, regardless of pressure, ergosterol having a more pronounced effect (Kp approximately 2x10(4)-6x10(4)). Kp was pressure dependent in both cases, but mainly with cholesterol (Kp approximately 2x10(3)-2x10(4)). At variance with cholesterol, when ergosterol was used, no phase transition was detected. This difference cannot be due to a more extended uptake of filipin by cholesterol-containing membranes, and so must be due to specific interactions with cholesterol. In agreement with this finding, we discovered that filipin is more tightly packed (lower partial molar volume) in the cholesterol-rich phase than in the ergosterol-rich phase. Our results also point to a 2:1 DPPC:cholesterol stoichiometry in the cholesterol-rich phase (17% molar cholesterol). All partition coefficients were calculated from steady-state fluorescence anisotropy measurements.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1995

Filipin fluorescence quenching by spin-labeled probes: studies in aqueous solution and in a membrane model system.

A detailed photophysical study of the fluorescence quenching (transient and steady state) of the macrolide antibiotic filipin by nitroxide-substituted fatty acids and a cholesterol derivative was carried out, aimed at determining its transverse position in a model system of membranes (multilamellar vesicles of dipalmitoylphosphatidylcholine). Filipin partitions efficiently into membranes (Kp = (5.0 +/- 1.0).10(3), 20 degrees C) and it was concluded that the antibiotic is buried in the membrane, away from the lipid-water interface. In addition, information on the organization of the quenchers was also obtained. The 5-nitroxide derivative of the fatty acid is essentially randomly distributed, while the 16-nitroxide is aggregated at concentrations higher than approximately 5% molar. For the cholesterol compound the results point to a phase separation at concentrations higher than 3% molar (below this limit concentration filipin associates with the derivatized sterol with KA = 20 M-1, assuming a 1:1 interaction). We propose that this phase separation and the aggregation state of filipin in the aqueous solution may be key processes in the antibiotic mode of action. A systematic and general approach to fluorescence quenching data analysis in complex (e.g., biochemical) systems is also presented.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1987

Quantitative freeze-fracture and filipin-binding study of retinal pigment epithelial-cell basal membranes in diabetic rats.

Breakdown of the blood-retinal barrier in diabetes may be related to alterations in the retinal pigment epithelial (RPE) cell layer. Morphological studies suggest increased permeability of diabetic RPE plasma membranes, and proliferation and flattening of the RPE basal infoldings have been observed in diabetic animals. In order to determine whether these phenomena are associated with changes in membrane protein or sterol composition, we have used quantitative electron-microscope freeze-fracture and filipin-binding techniques to study the RPE basal membrane in streptozotocin diabetic and 3-O-methyl glucose control rats. Perfusion-fixed retinas were processed for freeze-fracture and filipin-binding analysis. Filipin, a polyene antibiotic, binds specifically to 3-beta-hydroxy-sterols to produce membrane deformations recognizable by freeze-fracture. These analyses revealed an 11% increase in the density of intramembrane particles within the cytoplasmic (P-face) leaflet in diabetic rats as compared with the controls (P less than 0.01, t test). The increase occurred primarily in 6-9-nm particles, while smaller particles were decreased (P less than 0.001, chi-square test). Filipin binding was the same in both groups. These results suggest that alterations in intrinsic membrane proteins may contribute to permeability and surface area changes in the diabetic RPE but that RPE membrane sterols are not affected by diabetes.

Animals↗
PubMed2000

Solution NMR structures of the polyene macrolide antibiotic filipin III.

The solution structure of filipin III, an antifungal polyene macrolide biosynthesized by Streptomyces filipinensis and widely used for the detection and the quantitation of cholesterol in biomembranes, has been calculated with a set of geometrical restraints derived from 1H NMR in DMSO-d(6) at 25 degrees C. Filipin III appears as a rod-shaped molecule of 18 A length. Its amphiphilic structure is made of an all-syn 1,3-polyol motif, stabilized by intramolecular hydrogen bonds on one side, and a conjugated pentaene moiety on the other side of the molecule. The overall shape is comparable to cholesterol, and the molecular structure of filipin III affords a first molecular basis to the comprehensive understanding of the interactions possible in the filipin III-cholesterol complex which is still unknown at the atomic resolution.

Antifungal Agents↗
PubMedDate not supplied

Failure of filipin to detect cholesterol-rich domains in smooth muscle plasma membrane.

Using the cholesterol probe filipin and freeze-fracture electron microscopy, Montesano reported an apparent difference in cholesterol content between contiguous zones of the smooth muscle plasma membrane. In this membrane, microinvaginations termed caveolae are organized in band-like domains, which were highly sensitive to filipin (and therefore cholesterol-rich) compared with the surrounding non-caveolar inter-band regions (assumed to be cholesterol-poor). Using another cholesterol probe, the saponin tomatin, applied alone and after filipin treatment, we now present evidence that both the band and inter-band domains of the smooth muscle plasma membrane are cholesterol-rich. This leads to the conclusion that the lack of inter-band response to filipin is a false-negative cytochemical result.

Animals↗
PubMed1981

Heterogeneous distribution of filipin--cholesterol complexes across the cisternae of the Golgi apparatus.

The Golgi apparatus is a key element in the ordered movement of secretory polypeptides from the rough endoplasmic reticulum to the plasma membrane during secretion. It has been shown that cisternae that receive membranes from the reticulum are morphologically similar to the latter and that cisternae liberating secretory granules resemble that plasma membrane. By using an ultrastructural probe for membrane cholesterol, filipin, on freeze-fractured and thin-sectioned exocrine and endocrine pancreatic cells, we have shown that an enrichment in filipin-cholesterol complexes takes places across the stacked cisternae of the Golgi apparatus; the reticulum-related (forming) cisternae are poor in such complexes, but the secretory granule-related (maturing) cisternae contain numerous complexes. Secretory granule membrane is also richly labeled with filipin-cholesterol complexes. The heterogeneous cholesterol distribution in the membranes of the Golgi apparatus, as shown by filipin, emphasizes the polarity of this organelle, in agreement with its role in organizing the traffic of the secretory polypeptides from the rough endoplasmic reticulum to the plasma membrane.

Animals↗
PubMed2004

Brefeldin A and filipin distinguish two globotriaosyl ceramide/verotoxin-1 intracellular trafficking pathways involved in Vero cell cytotoxicity.

In verotoxin 1 (VT1)-sensitive cells, globotriaosyl ceramide (Gb3) bound VT1 is endocytosed and transported retrogradely to the Golgi/endoplasmic reticulum (ER). The importance of the Golgi-dependent retrograde transport of VT1 is now shown to vary as a function of both VT1 exposure time and concentration. Following 3 h exposure to < 50 ng/ml VT1, Vero cell cytotoxicity and protein synthesis inhibition is absolutely dependent on intact Golgi structure. However, after 24 h incubation with concentrations of VT1 above 50 ng/ml, a filipin-sensitive (caveolae-dependent) route for cytotoxicity becomes significant. Brefeldin A (BFA), which prevents Golgi-dependent retrograde traffic, protects cells from low VT1 concentrations but not following prolonged toxin exposure at higher VT1 concentrations. Under these conditions, only a combination of BFA and filipin is sufficient to fully protect cells. Intracellular VT1 trafficking monitored using the nontoxic B subunit showed accumulation within BFA-collapsed TGN/endosomes. Considerable VT1 B was retained at the surface of filipin-treated cells, but Golgi targeting was still apparent. Filipin-sensitive VT1 cytotoxicity does not require Golgi access and may involve direct transmembrane signaling. Although cell surface VT1 does not colocalize with caveolin 1, a small fraction of endocytosed VT1 is found within caveolin 1-containing vesicles. These studies indicate both a caveolae-dependent and independent pathway for VT1 access to the TGN/Golgi from the cell surface and two noninterconverting pools of membrane Gb3.

Animals↗
PubMed1985

A correlation between mode of growth and regional ultrastructure of the plasma membrane of Schizosaccharomyces pombe as revealed by freeze-fracturing before and after filipin treatment.

The ultrastructure of the plasma membrane of Schizosaccharomyces pombe was studied by freeze-fracture using invaginations of the plasma membrane as natural markers and filipin-induced deformations as artificial markers. In accord with the mode of growth of this organism, ultrastructural aspects of the plasma membrane were related to the following ring zones: the growing pole, adjacent regions, proximal regions, the new cell pole, and the middle in dividing cells. The growing pole and adjacent regions had no or only a few invaginations. Filipin induced numerous deformations in these regions. By contrast, the proximal regions of the plasma membrane had several invaginations and resisted filipin-induced deformation. Concomitantly with commitment to cytokinesis, both the invaginations and the resistance to filipin-induced deformation disappeared in the middle. The results presented here strongly suggest the existence of two states of the plasma membrane of S. pombe, a fact which correlates well with the mode of growth of this organism.

Ascomycota↗
PubMed1981

Characterization of isolated rat-liver cells made permeable with filipin.

When isolated rat-liver cells were incubated for 1 min at 37 degrees C with filipin at a concentration of 50 microM, the plasma membrane became permeable to sucrose, inulin, glycerol 3-phosphate and other low-molecular-weight compounds. Upon removal of the filipin and subsequent incubation of the cells at 37 degrees C there was a gradual leakage of lactate dehydrogenase from the cells. However, the leakage of lactate dehydrogenase could be prevented for about 10 min by including glutathione and ATP in the incubation medium. The filipin-treated cells were able to metabolize phosphorylated sugars. The conversion of fructose 1,6-bisphosphate to fructose 6-phosphate, glucose-6-phosphate and glucose was inhibited by AMP but not by high concentrations of fructose 1,6-bisphosphate. The results indicate that filipin-treated cells can be used to study the kinetic parameters of enzymes in their macromolecular environment in situ.

Adenine Nucleotides↗