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PubMed1991

Intramembranous particle distribution and filipin binding in dysplastic canine retina.

We examined dysplastic canine retina for changes in intramembranous particle (IMP) density and filipin binding to sterols. Differences in IMP density were identified in incipiently dysplastic fetal retina and also in the degree of filipin binding near the onset of the dysplastic process. The data suggest that there are temporal differences in IMPs and filipin-sterol complexes that may be related to the formation of retinal folds and disorganized dysplastic retina proliferation.

Animals↗
PubMed1988

Localization of lipoprotein unesterified cholesterol in nondenaturing gradient gels with filipin.

A method is described for the staining of lipoprotein unesterified cholesterol in nondenaturing polyacrylamide gradient gels with the fluorescent polyene antibiotic, filipin. The sensitivity of the filipin stain was comparable to that of oil red O and Coomassie R250 in terms of the amount of lipoprotein applied. Filipin successfully stained discoidal complexes of apoA-I-phosphatidylcholine-cholesterol, which in turn were stained poorly with oil red O. The potential for the identification of unesterified cholesterol-enriched lipoprotein subclasses was demonstrated.

Cholesterol↗
PubMed1985

Distribution of intramembranous particles and filipin-sterol complexes in the cell membranes of Toxoplasma gondii.

Toxoplasma gondii possesses a plasma membrane covering the whole cell and, below it, two closely apposed unit membranes interrupted at the anterior and posterior tips of the parasite and at the micropore. Density differences of intramembranous particles (IMP) were observed among the various membranes. The polyene antibiotic filipin was used for the detection of sterols in freeze-fractured membranes of the parasite. Protuberances with a mean diameter of 38 nm, indicative of the formation of filipin-sterol complexes, were seen in the P and E faces of the plasma membrane. The density of filipin-sterol complexes on the P and E faces of the plasma membrane was 130 +/- 45 and 101 +/- 50 protuberances/micron2, respectively. Few or no protuberances were seen on both fracture faces of the intermediate and inner membranes. The results obtained are discussed and compared with those obtained in other parasites enclosed with a complex membrane system.

Animals↗
PubMed1979

Activation of particulate guanylate cyclase by nitroprusside and MNNG after filipin treatment.

Particulate guanylate cyclase from rat lung was stimulated less than 2-fold by agents capable of activating the soluble guanylate cyclase, including sodium nitroprusside, MNNG, azide and hydroxylamine. The action of the first two agents was potentiated by 10 mM 2-mercaptoethanol, and that of the last two by catalase. Pretreatment of the particulate enzyme with the polyene antibiotic, filipin, potentiated the stimulatory effects of the activators, activity with 1 mM nitroprusside in the presence of 2-mercaptoethanol being increased 10.4-fold over basal. The enzyme treated with filipin and nitroprusside showed less specificity for Mn2+, as it was able to use Mg2+ as sole cation more efficiently than the untreated enzyme. Since filipin is known to alter membrane fluidity by interacting with membrane cholesterol, it is proposed that the activity of membrane bound guanylate cylase may be regulated in part by the fluid state of the phospholipid matrix.

Animals↗
PubMed1983

Distribution of filipin-sterol complexes in plasma membranes of the kidney. II. The thin limbs of Henle's loop.

Thin limbs of Henle from the rat kidney were investigated by freeze fracture electron microscopy after incubation of the tissue with the polyene antibiotic, filipin, a morphologic probe for membrane cholesterol. Distinct patterns of distribution of filipin-sterol complexes were found in plasma membranes of epithelial cells from each of the four thin limb regions examined. In particular, adjacent cells in the thin ascending limb had either heavily or poorly labeled apical membranes, whereas in the lower part of long descending thin limbs, all apical membranes were poorly labeled. In contrast, both apical and basolateral membranes of short descending limbs were heavily labeled with filipin-sterol complexes. These results demonstrate the existence of plasma membranes with different properties both within specific segments of Henle's loop (ascending thin limb) and between different thin limb segments (short and long descending limbs).

Animals↗
PubMed1982

Distribution of intramembrane particles and filipin-sterol complexes in plasma membranes of kidney. I. Corpuscle of Malpighi.

A quantitative study was performed on glomerular cell types from the rat kidney freeze fractured after conventional fixation or following exposure to the sterol-specific antibiotic, filipin. The results reveal a considerable variation on the size and number of intramembrane particles, as well as in the number of filipin-sterol complexes present on plasma membranes of the different cell types present in the glomerulus. These differences in membrane structure probably reflect specialized functions of the individual plasma membranes within the glomerulus. The most striking observation, however, was the low particle content and poor filipin labeling of podocyte foot process bases (which lie against the basal lamina) compared with the rest of the podocyte plasma membrane. The presence of distinct membrane domains in podocytes may be a factor involved in maintaining their specific architecture that is critical for the normal functioning of the kidney filtration barrier.

Animals↗
PubMed1982

Primary cultures of rat hepatocytes as a model system of canalicular development, biliary secretion, and intrahepatic cholestasis. I. Distribution of filipin-cholesterol complexes during de novo formation of bile canaliculi.

Hepatocytes in primary monolayer culture reconstitute structural intact bile canaliculi sealed by tight junctions. Using filipin as a cytochemical marker for cholesterol-like membrane components in conjunction with the techniques of freeze fracture and thin sectioning, we have studied the distribution of cholesterol during the development of the biliary pole of cultured hepatocytes. It was found that the development of bile canaliculi is characterized in its very early stage by huge accumulations of filipin-cholesterol complexes located at distinct domains of the contiguous membrane. They were surrounded by junction formation zones almost devoid of these complexes, in which the alignment of intramembranous particles takes place. Maturation of the bile canaliculi was accompanied by dispersion of cholesterol within the canalicular membrane and its removal by segregation of cholesterol-rich membrane whorls and vesicles into the lumen. Finally, the luminal membranes, and particularly the areas studied with microvilli, contained only very few filipin-cholesterol complexes. In some cases, these seemed to be still arranged in small clusters. These alterations suggest a crucial role of cholesterol-rich membrane domains during initiation of a biliary polarity. On the other hand, cholesterol-poor (thus probably more fluid) areas might be required for the assembly of tight junctions, and appear to constitute the secretory active apical membrane present in the mature bile canaliculus.

Animals↗
PubMed1996

Filipin III: Configuration Assignment and Confirmation by Synthetic Correlation.

The stereochemical configuration of filipin III (1) was determined using the (13)C acetonide analysis. The relative configurations for the nine stereogenic centers in the top half of filipin were initially identified using just three acetonide derivatives (2, 3, and 4) arising from a two-step protection sequence. The structure was confirmed by synthesis and direct correlation of degradation products 8 (C26-C28) and 10 (C1-C16). Filipin tetraacetonide 2 and triacetonide 4 each contain an anti acetonide in a highly unusual chair conformation. Molecular modeling successfully reproduced the preference for a chair conformation over the normally more stable twist-boat conformation.

Journal Article↗
PubMed1979

Inhomogeneous distribution of filipin-sterol complexes in the ciliary membrane of rat tracheal epithelium.

Pieces of rat trachea fixed in a glutaraldehyde solution containing 300 muM filipin, a sterol-specific polyene antibiotic (Elias et al., '78), were freeze-fractured in order to study the distribution of cholesterol within the ciliary membrane. Filipin-sterol complexes, recognizable as 25-30-nm protrusions on fracture faces, appeared densely and uniformly distributed over most of the ciliary membrane, but were absent from the region of the ciliary necklace. It is possible, therefore, that the ciliary necklace represents a cholesterol-poor area of membrane.

Animals↗
PubMed1983

The Sertoli cell junctional complex: structure and permeability to filipin in the neonatal and adult guinea pig.

The development and maintenance of the Sertoli cell junctional complex were investigated in prepubertal and adult guinea pigs. To correlate the structure of the blood-testis barrier with its permeability, the polyene antibiotic filipin (a cholesterol-binding agent of low molecular weight: 570.70) was added to the fixative as a tracer visible in freeze-fracture replicas. Discontinuous zonules, intermediate junctions (i.e., adhering fasciae) and gap junctions all proved permeable to filipin in the two age groups. Only the continuous occluding zonules characteristic of the adult guinea pig's testis were impermeable to the tracer. In pubertal animals, the establishment of the blood-testis barrier coincided with the completion of the junctional strands in occluding zonules. The formation of occluding zonules was similar in the newborn and the adult. In the adult, the Sertoli cell junctional complexes contained three types of cell junctions: occluding, adhering, and gap junctions. The sequence of occluding and adhering junctions from the base to the apex of the epithelium was the reverse of that demonstrated in most epithelia. The impermeable continuous occluding zonules at the base showed parallel patterns of uninterrupted junctional strands, whereas the permeable discontinuous zonules found higher in the epithelium showed a meandering pattern of broken strands. Our observations indicate that (1) Sertoli cell junctional complexes form near the young germinal cells at the base of the seminiferous epithelium and break down near the older germinal cells toward the apex; (2) the various patterns and orientations of the junctional strands reflect, respectively, the different stages of disintegration of the occluding zonules and the conformation of the mature Sertoli cell to the irregular contours of the germinal cells; (3) there is no relationship between permeability and junctional strand orientation; and (4) the cellular contacts between Sertoli cells and germinal cells situated below the blood-testis barrier may represent the early stages of formation of junctional elements which ultimately become incorporated into the Sertoli cell junctional complex.

Animals↗
PubMed1988

Orthogonal arrays of particles in non-pigmented cells of rat ciliary epithelium: relation to distribution of filipin- and digitonin-induced alterations of the basolateral membrane.

It has been suggested that orthogonal arrays of particles may increase the rigidity of plasma membrane, as does cholesterol. Therefore, using freeze-fractured non-pigmented ciliary epithelium, the distribution of such arrays was compared to the distribution of membrane deformations induced by the sterol-probes filipin and digitonin in different domains of the basolateral plasma membrane. The distribution of orthogonal arrays of particles was homogeneous between different regions of the basolateral membrane of the non-pigmented ciliary epithelium, while the number of filipin-induced alterations was nearly 4 times higher in the membrane domains not in contact with the basal lamina than in domains in contact with it. Contrary to the homogeneous distribution of arrays, digitonin-induced deformations also differed markedly in these two basolateral membrane domains. Considering that a marked positive response to sterol probes implies a high sterol content, we conclude that orthogonal arrays of particles can occur in plasma membrane regions well-provided with cholesterol and not in direct contact with the basal lamina. Other possible roles of these arrays are discussed.

Animals↗
PubMed1982

Tissue connections in a transplantable virus-producing sebaceous adenoma of the mouse. II. A freeze-fracture study in conjunction with filipin.

The distribution, size, and configuration of intercellular junctions in the sebaceous tumor of mice were examined using the freeze-fracture technique. Three types of junctions were observed: desmosomes, gap, and tight junctions. Tight junctions in general consisted of short linear unbranched fibrils, and macular or complex tight junctional patterns were present only occasionally. Gap junctions ranged from small sports of 0.9 x 10(-3) micrometer2 to areas of approximately 0.46 micrometers2. Desmosomes were the most frequent junctional specializations, and it is concluded that they are at least partially responsible for this tumor not being metastatic. Filipin in conjunction with freeze-fracture showed filipin-sterol complexes on the plasma membrane, the nuclear envelope, and the membranes of the endoplasmic reticulum and mitochondria. The intercellular junctions were devoid of these complexes indicating that these regions are low in cholesterol.

Adenoma↗
PubMed2004

Decrease of P-glycoprotein activity in K562/ADR cells by MbetaCD and filipin and lack of effect induced by cholesterol oxidase indicate that this transporter is not located in rafts.

The effect of low-density membrane domains on function of the plasma membrane transporter P-glycoprotéine (P-gp), involved in multidrug resistance (MDR) phenotype, has been investigated in K562/ADR cells. To this end we reversibly altered the cholesterol content of K562/ADR cells by using methyl-beta-cyclodextrin as a cholesterol chelator and conversely we repleted them through incubation with cholesterol in culture medium. We also used the cholesterol-binding fluorochrome filipin and cholesterol oxidase. Our data show that either cholesterol depletion or complex formation with filipin resulted in a strong decrease of P-gp activity. However, when cells were incubated with cholesterol oxidase that are known to disrupt rafts, no modification of the P-gp activity was observed. In addition, using a free-detergent methodology to separate by ultracentrifugation, "light," "heavy," and "extra heavy" fractions we show that no P-gp is found in the "light" fraction where rafts are usually detected. Altogether, our data strongly suggest that, in this cell line, P-gp is not localized in rafts.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1986

A freeze-fracture cytochemical study of filipin-sterol complexes in pulp fibroblasts of the rat incisor.

The sterols in microdomains of the cell membrane of pulp fibroblasts react with the polyene antibiotic, filipin, to form filipin-sterol complexes (FSC). The FSC appear in ultra-thin sections as minute corrugations or deformations of the membrane. In freeze-fracture replicas, individual FSC were 20-30 nm elevations and were abundant on filopodial cell processes. They were not found in the membrane of cell-to-matrix attachment plaques or in cell-to-cell adherens junctions. The findings suggest that stabilization of the membrane at these sites may interfere with FSC formation and conversely that the absence of FSC may be an indication of membrane stabilization.

Animals↗
PubMed1976

Freeze-fracture ultrastructural alterations induced by filipin, pimaricin, nystatin and amphotericin B in the plasmia membranes of Epidermophyton, Saccharomyces and red complex-induced membrane lesions.

The effects of chemically different polyenes on fungal membranes (Epidermaphyton floccosum, a human pathogenic fungus, and Saccharomyces cerevisiae) and human red blood cell membranes were studied by freeze-fracture electron microscopy in order to elucidate the interaction of these antibiotics with ergosterol. Each type of neutral, small amphoteric and large amphoteric polyenes produces a distinct morphoneutral, small amphoteric and large amphoteric polyenes produces a distinct morphological effect on the fungal membranes: (1) Pit formation type. Filipin, a neutral polyene, produces 250-300 A diameter "pits" or "invagination" both in ergosterol-containing fungal plasma membranes and cholesterol-containing red blood cell ghost membranes. (2) Network particle aggregation type. The small amphoteric polyene, pimaricin, produces a network of membrane particle aggregation which encloses 1000 A diameter particle-free areas in fungal membranes. These areas are slightly elevated toward the outside of the cell. (3) Random particle aggregation type. The large amphoteric polyenes, amphotericin B and nystatin, cause a random segregation of the fungal plasma membrane and the red blood cell ghost membranes into particle-free and aggregated areas. It is concluded that these morphological differences are due to different mechanisms of polyene-sterol interactions in which the different size of the mocrolide ring in the antibiotic structure may be involved. Since all of these antibiotics, except filipin, cause no alterations on whole red blood cells detectable by negative staining and freeze-fracture electron microscopy, it is possible that they have a higher affinity to ergosterol than cholesterol in membranes.

Amphotericin B↗
PubMed1987

Filipin-cholesterol binding in CNS axons prior to myelination: evidence for microheterogeneity in premyelinated axolemma.

The distribution of cholesterol in axonal membrane of developing rat optic nerves prior to myelination was studied by freeze-fracture cytochemistry. Binding of the cholesterol-specific probe, filipin, to the axolemma of premyelinated axons was heterogeneous; this suggests the presence of microdomains of axolemma with different membrane composition and/or cytoskeletal/extracellular matrix association. Although the reasons for this binding pattern have not yet been determined, heterogeneity occurs prior to association of glia with the axon, and may reflect regional differences in lipid/sterol composition of the axonal membrane bilayer, or distribution of membrane-associated cytoskeleton. The distribution of intramembranous particles was not obviously associated with the pattern of filipin binding in early developing axons, however, as might have been expected from the attending differences in fluidity of the membrane microdomains. Microheterogeneity in axonal membranes of developing axons could have an influence on several membrane properties, and may be associated with processes important for growth and differentiation of axons.

Aging↗
PubMed1987

Distribution of filipin-sterol complexes in the unmyelinated nerve fibre.

The filipin-sterol technique was employed, together with freeze-fracture, to investigate the fine structure of the unmyelinated nerve fibre in the peripheral nerve. No heterogeneity was observed in the distribution of filipin labelling either in the Schwann cell plasma membrane or along the axolemma. The distribution of labelling is contrasted with that in the myelinated nerve fibre and related to the relative morphology and electrophysiology of the two types of fibre.

Animals↗
PubMed1991

Changes in filipin-sterol binding in the rat cingulate cortex after the administration of antidepressant drugs. A freeze-fracture study.

The amount of cholesterol in cell membranes of cingulate cortex nerve cells from rats treated with Imipramine (tricyclic antidepressant drug) or mianserin (non-tricyclic antidepressant) was investigated using filipin-sterol binding and freeze-fracture. The number of filipin-sterol complexes decreased within membranes of neurons from cingulate cortex chronically treated with imipramine or mianserin. These results suggest that the decreased content of cholesterol causes the cell membrane to become more fluid. This increased fluidity of the cell membrane may play an important role in regulating the interaction of transmitters and drugs with their membrane receptors.

Animals↗