Filipin as a cholesterol probe. I. Morphology of filipin-cholesterol interaction in lipid model systems

We report some novel morphological observations on the interaction of the polyene antibiotic filipin (crude complex) with cholesterol, studied in non-cellular systems with replication, freeze-fracture, and negative stain techniques. Cholesterol crystals, lecithin liposomes containing 0 to 20 mole% of cholesterol, and liposomes containing 10 mole% of cholesterol and 5 to 40 mole% of sphingomyelin were incubated for varying lengths of time with filipin at different cholesterol: filipin molar ratios. The resulting filipin-induced lesions (FIL) were pleomorphic in all systems studied. In replicas of crystals, FIL appeared as ridges which were either straight, or curved into C- and S-shaped figures or closed circles. Negatively stained preparations showed FIL as white lines of the same configurations and in addition revealed a delicate veil attached to individual FIL. FIL, fused by their veils into clusters or large sheets ("holey sheets"), were shed from crystals. Incubation of liposomes for 1 h at cholesterol:filipin molar ratios of 4:1, 2:1, 1:1, and 1:5, demonstrated that cholesterol detection (i.e. formation of FIL) depend upon the ratio of cholesterol to filipin. At a 1:1 molar ratio FIL formed on liposomes containing 10 mole% cholesterol or more, but detectability increased to 5 mole% at the 1:5 ratio. Increasing the molar ratio of cholesterol:filipin to 2:1 and 4:1 decreased cholesterol detectability to between 10 and 20 mole%. Increasing concentrations of sphingomyelin decreased cholesterol detectability at the 1:1 cholesterol:filipin ratio; further, FIL in sphingomyelin-containing liposomes tended towards larger diameters. Filipin induced aggregation of liposomes and linked them together by holey sheets, providing evidence for filipin-induced extraction of cholesterol from liposomes. Taken together our morphological observations on filipin-cholesterol interaction in non-cellular systems raise pertinent questions as to the feasibility of filipin as a cholesterol probe in cellular systems.     

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.     

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.     

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.     

Increase in membrane cholesterol: A possible trigger for degradation of HMG CoA reductase and crystalloid endoplasmic reticulum in UT-1 cells

The crystalloid endoplasmic reticulum (ER) houses large amounts of HMG CoA reductase, the rate-controlling enzyme in cholesterol synthesis. The crystalloid ER appears in UT-1 cells, a line of Chinese hamster ovary cells that has been chronically starved of cholesterol as a result of growth in the presence of compactin, an inhibitor of reductase. When cholesterol was provided to UT-1 cells in the form of low density lipoprotein (LDL), the reductase and crystalloid ER were destroyed. This destruction was preceded by an increase in the cholesterol content of crystalloid ER membranes, as judged by a 4- to 8-fold increase in their ability to form complexes with filipin, a cholesterol-binding compound that can be visualized in freeze-fracture electron micrographs. Filipin binding to other membranes was unchanged. Thus insertion of cholesterol into the crystalloid ER membrane may trigger the degradation of reductase and the membrane itself.     

Labeling of cholesterol with filipin in cellular membranes of parenchymatous organs

Summary Filipin is used for ultrastructural cytochemical localization of cholesterol in biological membranes. It binds to unesterified 3-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 m thick tissue slices incubated in 400 g/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.     

Localization of Filipin-Sterol Complexes in the Membranes of Beta vulgaris Roots and Spinacia oleracea Chloroplasts

Filipin was used as a cytochemical probe for membrane sterols in the root storage tissue of the red beet Beta vulgaris L. and the chloroplasts of Spinacia oleracea L. In unfixed beet tissue, filipin lysed the cells. Freeze-fracture replicas revealed that the filipin-sterol complexes were tightly aggregated in the plasma membrane, while in thin section the complexes corrugated the plasma membrane. If the cells were fixed with glutaraldehyde prior to the filipin treatment, the cell structure was preserved. Filipin-induced lesions were dispersed or clustered loosely in the plasma membrane. A few filipin-sterol complexes were observed in the tonoplast. In spinach chloroplasts, filipin-sterol complexes were limited to the outer membrane of the envelope and were not found in the inner membrane of the envelope or in the lamellar membranes. If the filipin-sterol complexes accurately mapped the distribution of membrane sterols, then sterol was located predominantly in the plasma membrane of the red beet and in the outer membrane of the chloroplast envelope. Furthermore, the sterol may be heterogenously distributed laterally in both these membranes.     

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.     

Morphological detection of filipin-sterol complexes in the cytoplasmic membrane of staphylococcal L-form

Filipin, a sterol-specific antibiotic, and freeze-fracture electron microscopy were used to study the presence and distribution of sterol in the cytoplasmic membrane of stable staphylococcal L-form cells. Fixed cells were treated with filipin, and then observed by freeze-fracture electron microscopy. Freeze-fractured profiles of the L-form cells treated with filipin demonstrated irregular distribution of protuberances or pits of 25-30 nm, representing filipin-sterol complexes, on the proto-plasmic fracture face (PF) and exoplasmic fracture face (EF) of the cytoplasmic membrane. In contrast, no such structure was detected in the filipin-treated parent cells or protoplasts. The results suggest that some sterol molecules, which are usually not found in staphylococcal or other bacterial cells, emerged on the cytoplasmic membrane after the cells were converted to the stable L-form.     

Filipin resistance in intermediate junction membranes of guinea pig ependyma: possible relationship to filamentous underlying

Plasma membranes in intermediate junctions of ependymal cells are found to show considerable resistance to the antibiotic filipin, suggesting low cholesterol in these membranes. Further, ependymal cells were treated with cytochalasin B (CB) infused into the cerebral ventricle in vivo, and then incubated with filipin. When treated with CB, intermediate junctions show a decrease in their underlying density, mainly composed of microfilaments, and their membranes are found to be more affected by filipin. This reduction of resistance to the antibiotic is clearly demonstrated by thin-section and freeze-fracture as well as quantitative analysis. Nonjunctional lateral membranes, however, show no significant difference in the degree of filipin effect whether treated with CB or not. Although biochemical data on lipid composition have not been available for the intermediate junction membranes, we bring forward a possibility that resistance to filipin in these membranes may come not from less cholesterol but from morphological membrane stability brought about by the filamentous underlying.     

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 labelling and intramembrane particles on the membranes of early and later autophagic vacuoles in Ehrlich ascites cells

Cholesterol and intramembrane particle distribution on autophagic vacuole membranes was studied in Ehrlich ascites cells using filipin labelling and freeze-fracture electron microscopy. Unsaturated fatty acids were stained using imidazole-buffered osmium tetroxide. Autophagocytosis was induced with vinblastine, and early autophagic vacuoles were accumulated by lowering the ATP level in the cells with iodoacetate. Filipin labelling was observed in the limiting membranes of later, apparently hydrolase-containing autophagic vacuoles, whereas the most newly-formed, double-membrane limited vacuoles were not labelled. The limiting membranes of late, residual body-type vacuoles either showed patchy filipin-induced deformation or were completely smooth. Imidazole-buffered osmium tetroxide stained the membranes of newly-formed or developing autophagic vacuoles partly or entirely. The membranes of older vacuoles stained more weakly. Intramembrane particle density on the P-face of the outer limiting membranes of newly-formed autophagic vacuoles was similar to that on endoplasmic reticulum, and the density seemed to increase slightly later on. The size of the P-face particles increased when the vacuoles became older. The limiting membranes of late, residual body-type vacuoles were almost smooth. The inner limiting membranes and the membranes inside the autophagic were always almost particle-free. In conclusion, the amount of cholesterol, unsaturated fatty acids and protein in autophagic vacuole membranes changes during vacuole maturation.     

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 NO₃⁻ 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 β-sitosterol, campesterol, and stigmasterol.     

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.     

beta-Hydroxysterol distribution as determined by freeze-fracture cytochemistry

Summary Filipin a polyene antibiotic, fluoresces and forms 15–25 nm aggregates when combined with -hydroxysterols, rendering sterols detectable by fluorescence microscopy and by electron microscopy of thin sections and freeze-fracture replicas. We applied filipin in a glutaraldehyde fixative to tissue-cultured cells ofDrosophila melanogaster larvae, in which sterol concentration can be regulated. Since the number of filipin-sterol aggregates observed in membranes was found to be preportional to the amount of sterol experimentally inserted, utilizing filipin is a valid method for quantifying, as well as for mapping, sterol distribution in biological membranes. Other antibiotics may be similarly used for localizing some species of negatively charged phospholipids.In addition to cytochemical identification of specific lipids, rapid freezing and deep etching of unfixed, non-cryoprotected cells may permit us to examine membrane lipids in different physical states liquid-crystalline and gel. Combining these several techniques has resulted in new data concerning the disposition of lipids during the intimate juxtaposition of membranes preceding fusion. For example, in guinea-pig sperm, foci of closely apposed membranes are bereft of -hydroxysterols and intramembranous particles. Such regions of membrane sometimes exist in a crystalline state and may be rimmed by negatively charged phospholipids. As previously noted in other areas of cytochemistry, thein situ localization of specific substances provides information unobtainable by morphological or biochemical techniques alone.     

Different patterns of filipin-sterol labeling in prostate nuclear membranes from normal and castrated rats

Filipin was used as cytochemical probe for sterol detection in freeze-fractured prostate nuclear membranes from rats under different hormonal conditions. Isolated prostate acini and nuclei were fixed in glutaraldehyde and post-treated with filipin, according to Robinson and Karnovsky (1980). In general, most plasma and intracellular cytoplasmic membranes displayed a marked response to filipin in either epithelial and stromal cells from normal and castrated animals. Nuclear membranes from epithelial secretory cells were systematically negative to filipin labeling in normal animals, although after castration a positive response was detected. Stromal nuclear membranes were labeled both in normal and castrated animals. Filipin-treated isolated nuclei displayed the same overall labeling pattern but there was a different distribution of induced deformations relative to intact cell nuclei. These observations indicate that: a) nuclear membranes from different cell types have different responses to filipin; b) a change in the molecular organization of nuclear membranes from prostate secretory cells follow castration; c) nuclei isolation affects the distribution of filipin induced deformations on the membranes.     

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.     

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.     

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.     

Filipin-induced lesions in planar phospholipid bilayers imaged by atomic force microscopy.

Filipin is a macrolide polyene with antifungal activity belonging to the same family of antibiotics as amphotericin B and nystatin. Despite the spectroscopy and electron microscopy studies of its interaction with natural membranes and membrane model systems, several aspects of its biochemical action, such as the role of membrane sterols, remain to be completely understood. We have used atomic force microscopy (AFM) to study the effect of filipin on dipalmitoylphosphatidylethanolamine bilayers in the presence and absence of cholesterol. The bilayers were prepared by Langmuir-Blodgett deposition over mica and imaged under water. It was shown that filipin-induced lesions could only be found in membranes with cholesterol. In close agreement with electron microscopy results, we have reported the presence of densely packed circular protrusions in the membrane with a mean diameter of 19 nm (corrected for convolution with AFM tip) and 0.4 nm height. Larger circular protrusions (90 nm diameter and 2.5 nm height) and doughnut-shaped lesions were also detected. These results demonstrate that filipin-induced lesions in membranes previously observed by electron microscopy are not biased by artifacts resulting from sample preparation. Filipin aggregates in aqueous solution could also be imaged for the first time. These polydisperse spherical structures were observed in samples with and without cholesterol.