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 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.     

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.     

Observations of hamster sperm-egg fusion in freeze-fracture replicas including the use of filipin as a sterol marker

We have extended the observations of previous transmission electron microscopy studies of sperm-egg fusion to include those of freeze-fracture replicas showing sperm-egg interactions before, during, and following sperm head fusion with the egg membrane. Hamster eggs were incubated with hamster sperm under polyspermic conditions and were observed after a period of 5-30 minutes. After fixation, the eggs and sperm were exposed to filipin, which binds beta-OH-sterols to form visible complexes in freeze-fracture replicas. Filipin can act as a marker for egg plasma membrane wherein it is abundant, while filipin is relatively scarce in the acrosome-reacted hamster sperm membrane, found only in the plasma membrane of the equatorial segment. The earliest sperm-egg interactions are observed between the egg microvilli and the perforatorium and the equatorial segment of the sperm, and the initial fusion between egg and sperm occurs in the vicinity of the equatorial segment. At later stages of fusion involving the postacrosomal segment, a clear line of demarcation is observed between the filipin-rich egg membrane and the filipin-poor sperm postacrosomal segment, suggesting that filipin binding lipids from the egg intercalate into the sperm membrane following membrane fusion. The anterior segment of the sperm does not fuse with the egg but is instead incorporated into a cytoplasmic vesicle derived from both sperm and egg membranes. In this latter step, filipin-sterol complexes are not found in sperm-derived membranes suggesting that there may be barriers to the movement of filipin binding lipids from the egg into these sperm membranes.     

Filipin-cholesterol complexes (FCC) in the goblet cell granule membrane of the distal colon are arranged in a rhombic pattern

After fixation in the presence of filipin, the membrane of the goblet cell granules of the distal colonic mucosa (guinea pig) displays, in freeze-fracture replicas, a rhombic pattern composed of intersecting "lines" of unknown nature. The filipin-cholesterol complexes of the granule membrane are ordered according to the same geometry. This arrangement--but not the "lines"--has been found for the filipin-cholesterol complexes of other membranes of the same goblet cells as well as of other colonic cells. These observations indicate that in some biological membranes cholesterol has an ordered distribution, comparable to that suggested by investigations on artificial membranes.     

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-sterol complexes in disrupted myelin in the rat

Using filipin as a cytochemical probe for cholesterol we have compared the distribution of filipin labelling in mildly disrupted myelin and normal myelin. The myelin lamellae in rat sciatic nerve were separated either by hypotonic saline (0.035-0.07 M) or nerve section (24-32 h) before aldehyde fixation and filipin treatment. Myelin separation was assessed in ultrathin sections and filipin distribution in freeze-fracture replicas. In separated myelin lamellae filipin labelling was similar throughout the myelin sheath while in normal control myelin filipin occurred most in the outer (abaxonal), least in the inner (adaxonal) and intermediate in the middle lamellae. It is concluded that this heterogeneous filipin labelling in normal myelin is a result of diffusion gradients to filipin within the myelin sheath and that in vivo cholesterol is uniformly distributed throughout all the lamellae of the myelin sheath. The site of the diffusion barrier to filipin within normal myelin is considered.     

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.     

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.     

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.     

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.     

Cholesterol localization in the membranes of granular cells in the bladder epithelium of the frog during stimulated water transport

The polyene antibiotic filipin has been used to characterize the cholesterol distribution in the membranes of resting and ADH-stimulated frog urinary bladder in freeze-fracture replicas. In general, the intracellular membranes takes up filipin only insignificantly. An exception is the cholesterol rich granule membrane. Both density and polarity of filipin-induced deformations were evaluated, and the asymmetry in membrane cholesterol was analysed. Upon ADH-stimulation of water flow both density and polarity of filipin-induced deformations altered differently in apical and basolateral regions of the plasma membrane. This difference is presumably due to the stretching of the basolateral membrane as a result of swelling, on the one hand, and to incorporation of aggregate containing membranes into the apical membrane, on the other one. The results obtained may suggest that the appearance of ADH-induced intramembranous particle aggregates in the apical membrane be accompanied with a relative cholesterol decrease in this apical membrane.     

SDS-digested freeze-fracture replica labeling electron microscopy to study the two-dimensional distribution of integral membrane proteins and phospholipids in biomembranes: practical procedure, interpretation and application

 Recently, we have developed a quick-freezing/freeze-fracture replica labeling technique, sodium dodecyl sulfate (SDS)-digested freeze-fracture replica labeling (SDS-FRL), to study the two-dimensional distribution of cytochemical labeling on the membrane surface and the relationship of this distribution to images of freeze-fracture replicas created by platinum shadowing. In SDS-FRL, unfixed, quick-frozen cells, after freeze-fracture and platinum/carbon shadowing, are treated with SDS. The detergent dissolves unfractured areas of the cell membranes, with the release of the cytoplasmic contents. The cytoplasmic and exoplasmic membrane surfaces can be then labeled cytochemically. Integral membrane proteins, revealed as intramembrane particles by freeze-fracture replication, which are indistinguishable on a purely morphological basis, can be selectively labeled by SDS-FRL with specific antibody. In addition, this approach can be applied to examine the transmembrane phospholipid distribution in various cell and intracellular membranes. In this review, we describe the practical procedure for SDS-FRL in detail, present its application to labeling of various membrane components, and briefly discuss the possibility of a combination of SDS-FRL with atomic force microscopy. Accepted: 1 November 1996     

Formation of Flat Lamellar Intramembrane Lipid Structures in Microorganisms

Analysis of freeze-fracture replicas and thin sections of cells of the bacteria Sulfobacillus thermosulfidooxidans and Anaerobacter polyendosporus showed that their cytoplasmic membranes contain some regions in the form of flat lamellar inverted lipid membranes a few tenths of nanometers to a few microns in size. The specific features of these membrane structures are as follows: (i) they contain no familiar intramembrane particles commonly present on freeze-fracture replicas; (ii) in cross thin sections, intramembrane structures are bifurcate on the periphery and look like thylakoids; and (iii) the leaflets of intramembrane structures in S. thermosulfidooxidans cells are corrugated. These structures were revealed in bacterial cells cultivated under normal growth conditions. The data obtained suggest the occurrence of a complex type of compartmentalization in biological membranes. Received: 17 July 2000/Revised: 22 November 2000     

Localization of cholesterol in the colonic epithelium of the guinea pig: regional differences and functional implications

Summary It is generally accepted that variations in membrane cholesterol content affect the fluidity of the bilayer, thus altering its permeability. In the biological membranes, in physiological conditions, a high cholesterol content rigidifies the bilayer decreasing its permeability, a lower cholesterol content induces the opposite effect by increasing the permeability. Since differences in the epithelial permeability for short chain fatty acids have previously been demonstrated in the proximal and distal colon of the guinea pig, these two regions were investigated to establish whether differences in membrane cholesterol content of the absorbing cells can be demonstrated. Freeze-fracture replicas of filipin-treated colonic tissue were used. The results show that in the proximal colon the density of filipin cholesterol complexes located on the luminal plasma membrane of the columnar absorbing cells was significantly higher (about twice) than in the distal colon. Therefore the lower amount of cholesterol present in the membrane of the absorbing cells in the distal colon indicates a greater fluidity of the membranes of the epithelial cells in this region. Such fluidity could be correlated to the higher absorption rates of shortchain fatty acids characteristic of this region.     

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.     

Introduction of a high-resolution cytochemical method for studying the distribution of phospholipids in biological tissues

A novel cytochemical method for the in situ, ultrastructural localization of phospholipids in biological tissues is reported. The method is based on the enzyme-gold approach (M. Bendayan: J. Histochem. Cytochem. 29, 531, 1981). Phospholipase A2 from bee venom was adsorbed on colloidal gold particles (PLA2-gold) and applied for the specific labeling of its substrate, sn3-glycerophospholipids. The binding and enzymic competence of the PLA2-gold complex were confirmed by in vitro, preembedding experiments with erythrocytes and a crude lung surfactant preparation. The substrate specificity of the probe was assessed by labeling Epon thin sections of pure phospholipids. To test the potential applications of the PLA2-gold complex, lung and pancreatic tissues were fixed with glutaraldehyde-osmium and embedded in Epon for transmission electron microscopy (TEM). They were also prepared for critical-point-drying fracture-label (CPD-FL) replicas and thin-section fracture-label (TS-FL) specimens. On TEM thin sections incubated with PLA2-gold, all cellular membranes were labeled. The labeling density over each membrane compartment, as quantitated in lung type II pneumocytes, was classified in order of magnitude as follows: a) nuclear membranes; b) outer mitochondrial membrane and rough endoplasmic reticulm (RER); and c) Golgi complex, mitochondrial cristae and plasma membranes. In lung alveoli, the phospholipid-rich surfactant material was intensely labeled. Labeling of lung thin sections from chlorphentermine-treated rats (phospholipidosis-inducing drug) further demonstrates the reliability of PLA2-gold to label phospholipids. CPD-FL replicas and TS-FL specimens further extended the TEM observations: nuclear membranes and RER were more intensely labeled than plasma membranes. In exocrine pancreatic cells, two distinct labeling patterns were found for secretory granule membranes: sparse and dense. The specificity and reliability of the labeling were confirmed through several control experiments. The studies performed thus demonstrate the great potential of the PLA2-gold technique as a new approach to the high-resolution study of phospholipid distribution and density among biological structures.     

Distribution of filipin-sterol complexes in the myelinated nerve fiber

Using filipin as a cytochemical probe to reveal the distribution of cholesterol, myelinated peripheral nerve fibers were examined in freeze-fracture replicas. Filipin-sterol complexes were most abundant in the Schwann cell and axonal plasma membranes. In the Schwann cell plasma membrane there was no heterogeneity in complex distribution in relation to the subjacent cytoplasmic network. In myelin lamellae there was a decrease in complexes from outer to inner lamellae and some aggregation of complexes in individual lamellae. The density of complexes in cytoplasmic organelles varied from absent in mitochondria to high in lysosome-like bodies. The results are interpreted in terms of the related biochemical composition and biophysical properties of cell membranes, with particular reference to the myelinated nerve fiber. The influence of diffusion barriers and gradients on the formation of complexes by filipin is considered.     

Effects of lipid-phase separation on the filipin action on membranes of ergosterol-replaced Tetrahymena cells, as studied by freeze-fracture electron microscopy.

The effects of lipid-phase separation on the filipin action on pellicle membranes of ergosterol-replaced Tetrahymena pyriformis cells were studied by freeze-fracture electron microscopy. The pellicle membranes with phase separations induced by chilling from 34 degrees C (growth temperature) to lower temperatures (30, 22 and 15 degrees C) were treated with filipin. This produced filipin-induced lesions ("pits") only in the particulated (liquid) regions along the margin between solid and liquid domains, while they were produced in the particle-free (solid) areas when membranes were chilled to 15 degrees C. The pellicle membranes with lesions induced by filipin at 34 degrees C were chilled to 22 degrees C. This chilling raised larger particle-free areas and more condensed particle-aggregations on the membranes than on the membranes without the filipin treatment. These results suggest that the membrane fluidity affects induction and development of the ergosterol-filipin complex in the membrane.