Polyene Antibiotic Filipin Research Articles

Sterol dynamics during endocytic trafficking in Arabidopsis.

Sterols are lipids found in membranes of eukaryotic cells. Functions of sterols have been demonstrated for various cellular processes including endocytic trafficking in animal, fungal, and plant cells. The ability to visualize sterols at the subcellular level is crucial to understand sterol distribution and function during endocytic trafficking. In plant cells, the polyene antibiotic filipin is the most extensively used tool for the specific detection of fluorescently labeled 3-β-hydroxysterols in situ. Filipin can to some extent be used to track sterol internalization in live cells, but this application is limited, due to the inhibitory effects filipin exerts on sterol-dependent endocytosis. Nevertheless, filipin-sterol labeling can be performed on aldehyde-fixed cells which allows for sterol detection in endocytic compartments. This approach can combine studies correlating sterol distribution with experimental manipulations of endocytic trafficking pathways. Here, we describe step-by-step protocols and troubleshooting for procedures on live and fixed cells to visualize sterols during endocytic trafficking. We also provide a detailed discussion of advantages and limitations of both methods. Moreover, we illustrate the use of the endocytic recycling inhibitor brefeldin A and a genetically modified version of one of its target molecules for studying endocytic sterol trafficking.

Effect of cholesterol on the functional activity of proteins responsible for the resistance of human lymphocytes to xenobiotics

The influence of agents modifying cholesterol in plasma membranes on the functional activity of transporter proteins (P-glycoprotein (P-gp) and the multidrug resistance protein 1 (MRP1)) in human lymphocytes has been studied. It was shown that changes in lateral distribution of cholesterol using the polyene antibiotic filipin, which disturb the structure and function of glycolipid microdomains in plasma membranes of lymphocytes lead to a decrease in the transport activity of both P-gp and MRP1. It was found that the treatment of human lymphocytes with the cyclic oligosaccharide methyl-beta-cyclodextrine, which leads to cholesterol depletion and reduction of lipid bilayer microviscosity in membranes of these cells, also decreases the functional activity of these proteins. It was concluded that the transport activity of P-gp and MRP1 depends on the modification of cholesterol in the membranes of human lymphocytes, i.e., is closely associated with the level of cholesterol and its lateral distribution.

Spectrophotometric assay of yeast sterols using a polyene antibiotic

Spectral changes resulting from specific interaction between sterols and the polyene antibiotic filipin have been exploited in a simple sterol assay that is not subject to interference. This can be employed to measure the sterol content of brewing yeast prior to use thus permitting improved control of the fermentation process. It may also be applicable to quantification of fungal biomass.

Filipin orientation revealed by linear dichroism. Implication for a model of action.

The organization of the polyene antibiotic filipin in membranes containing cholesterol is a controversial matter of debate. Two contradictory models exist, one suggesting a parallel and the other perpendicular organization of filipin with respect to the plane of the membrane. UV-vis linear dichroism, ATR-FTIR, and fluorescence anisotropy decay techniques were combined to study the orientation of filipin in model systems of membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-palmitoyl-sn-glycero-3-phosphocholine (DPPC) with and without cholesterol. Filipin's orientation is determined by the presence/absence of cholesterol when it is inserted in gel crystalline phase model membranes. When cholesterol (33%) is present in DPPC bilayers, filipin stands perpendicular to the membrane surface as expected in "pore-forming" models. At variance, absence of cholesterol leaves filipin in an essentially random organization in the lipidic matrix. In liquid crystalline phase bilayers (POPC) filipin's orientation is perpendicular to the membrane surface even in absence of cholesterol. Thus filipin's activity/organization depends not only on cholesterol presence but also in the lipid phase domain it is inserted in. These findings were combined with spectroscopy and microscopy data in the literature, solving controversial matters of debate.

Internalization of pancreatic polypeptide Y4 receptors: correlation of receptor intake and affinity

Unlike neuropeptide Y receptors, the pancreatic polypeptide Y4 receptors display considerable differences in sequence and ligand-binding affinity across mammalian species. This could produce different receptor turnover rates in the same cellular membrane environment. Comparing rat, human and guinea-pig Y4 receptors expressed in Chinese hamster ovary (CHO) cells ( K d with human pancreatic polypeptide 14, 45 and 116 pM, respectively), we indeed found human pancreatic polypeptide internalization in the rank order of receptor affinities. A large fraction of the internalized human pancreatic polypeptide, similar across the Y4 species, was associated with secondary endosomes (density≈1.05 in Percoll gradients) and lysosomes (density≈1.11). For all Y4 receptors examined, this intake was potently and selectively inhibited by cholesterol-complexing polyene antibiotic filipin III and also by clathrin lattice formation inhibitor, phenylarsine oxide. Internalization differences found across Y4 receptor species to a degree compare with those observed for the cloned guinea-pig neuropeptide Y Y1 and human neuropeptide Y Y5 receptors and, generally, support ligand-binding affinities as important determinants of internalization for neuropeptide receptors.

Filipin Prevents Pathological Prion Protein Accumulation by Reducing Endocytosis and Inducing Cellular PrP Release

Conversion of the normal membrane-bound prion protein (PrP-sen) to its pathological isoform (PrP-res) is a key event in the pathogenesis of transmissible spongiform encephalopathies. Although the subcellular sites of conversion are poorly characterized, several lines of evidence have suggested the involvement of membrane lipid rafts in the conversion process. Here we report that copper stimulates the endocytosis of PrP-sen via a caveolin-dependent pathway in both microglia and neuroblastoma cells. We show that the polyene antibiotic filipin both limits endocytosis of PrP-sen and dramatically reduces the amount of membrane-bound PrP-sen. This reduction results from a rapid and massive release of full matured PrP-sen into the culture medium. Finally, we demonstrate that filipin is a potent inhibitor of PrP-res formation into chronically infected neuroblastoma cells. Our results reinforce the role of rafts in PrP trafficking and raise the possibility that the release of PrP-sen from the plasma membrane decreases the amount of available substrate PrP-sen at the conversion sites.

A photophysical study of the polyene antibiotic filipin: Self-aggregation and filipin–ergosterol interaction

Filipin, a macrolide polyene antibiotic, is known to interact selectively with ergosterol, a constituent of fungi membranes. In this work, the fluorescence resonance energy transfer (FRET) between a fluorescent analog of ergosterol, dehydroergosterol (DHE), and filipin was measured in small unilamellar vesicles of dipalmitoylphosphatidylcholine at 25°C. The time-resolved FRET results were rationalized in the framework of the mean concentration model, and were complemented with steady-state fluorescence intensity, anisotropy and absorption measurements. The results point to the formation of both DHE–filipin aggregates (evidence from static quenching of DHE fluorescence by filipin) and filipin–filipin aggregates (evidence from: (i) the FRET acceptor concentration distributions; (ii) spectral changes of filipin absorption in the vesicles, the excitonic interaction suggesting a stack arrangement; (iii) filipin fluorescence self-quenching), even in presence of DHE and low antibiotic mole fractions (<1 mol%). These results point out that apparently contradictory biochemical models for the action of filipin (some based on the presence of sterols, others not) can be equally valid. Moreover, since results (ii) and (iii) are also observed when a sterol is present, both models of action can actually coexist in membranes with a low sterol content.

Heterogeneous distribution of membrane cholesterol at the attachment site of Cryptosporidium muris to host cells.

Distribution of membrane cholesterol at the attachment site of Cryptosporidium muris was investigated by freeze-fracture cytochemistry using a polyene antibiotic filipin. Since the host plasma membrane enveloped C. muris, the inner and outer membranes were continuous with the parasite plasma membrane at the annular ring and with host membrane at the dense band, respectively. Although many filipin-cholesterol complexes were observed on the plasma membrane of host cells and parasites, a line showing no complexes was evident at the above two membrane junctures. These observations indicate that parasitic infection of C. muris altered the organization of membrane cholesterol.

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

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

Three probe flow cytometry of a human foam-cell forming macrophage.

A human cell line THP-1 was differentiated into macrophages expressing the scavenger receptor for uptake of modified lipoproteins. The cells were exposed to native low-density lipoprotein (n-LDL), acetylated-low-density lipoprotein (Ac-LDL), oxidised-LDL, or 25-OH cholesterol, leading to the accumulation of cholesteryl esters within the cells. Harvested macrophages were studied using three separate probes: 1) 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diI)-labelled LDL to study lipoprotein uptake; 2) the lipophilic fluorescent dye Nile Red to study cholesteryl ester accumulation within the cells; and 3) the polyene antibiotic Filipin III to study free cholesterol homeostasis. Cells were analysed using fluorescence flow cytometry and the three signals analysed separately. THP-1 macrophages incubated with diI-labelled modified lipoproteins produced a concentration dependent increase in the fluorescence emissions, consistent with accumulation of the labelled particles. Macrophages exposed to unlabelled modified LDLs were demonstrated, by staining with Nile Red, to accumulate cholesteryl esters within their cytoplasm and to alter their cholesterol content as judged by staining with Filipin. The foam-cell forming macrophage and its response to modified lipoproteins is considered a key step in the development of atherosclerosis. The use of these three probes during the formation of foam-cells in vitro offers a way of studying their behaviour at the single cell level.

Absorption and fluorescence spectra of polyene antibiotics in the presence of cholesterol.

The alterations in the absorption and fluorescence spectra observed for the polyene antibiotics filipin and nystatin in the presence of cholesterol are due to an exciton interaction (polyene aggregates) and cannot be attributed to a specific sterol-antibiotic complex. Filipin and nystatin molecules partition into the sterol aggregates, these structures being very efficient to induce exciton interaction; the observed splitting profile indicates that the chromophores are in a stacked arrangement (parallel transition dipoles). For filipin incorporated in lipid bilayers, the sterol is able to induce the same type of aggregate, at variance with nystatin.

The nature of the conductance increase induced by filipin in cholesterol-containing planar lipid bilayers

The effects of the polyene antibiotic filipin on the conductance and permeability of planar lipid bilayers were investigated under voltage-clamp conditions. The membrane conductance of lipid bilayers containing no cholesterol was not affected by filipin. In the presence of cholesterol containing lipid bilayers, filipin induced a 10 4–10 5-fold increase in transmembrane conductance. This conductance increase was dependent on the ionic species present in solution, decreasing in the following order: G CsCl > G NaAc > G KCl > G NaCl > G CaCl 2 > G Na 2SO 4 > G BaCl 2 > G MgCl 2 . Reversal potential measurements in simple biionic conditions revealed the following relative permeability sequence: P K > P Cl > P Na ≃ P ac ≃ P Ba > P Cs > P Mg ≃ P Ca > P sulphate. The filipin-sterol mediated increase in membrane conductance was independent of the membrane potential. The increase in membrane current following a step alteration in membrane potential occurred instantaneously and had no dependence on the previous value of the holding membrane potential. We propose that the filipin-sterol complex forms ion channels in lipid membranes. These channels are found in a single configuration (open state) and select preferentially monovalent cations or anions over divalent ions. Our experimental results are discussed in relation to the effects of other polyene antibiotics on the membrane permeability, and also in relation to experimental problems previously reported with the use of filipin in planar lipid bilayers.

Sterol effects on phospholipid biosynthesis in the yeast strain GL7.

Cells of the yeast sterol auxotroph GL7 were grown on either ergosterol or cholesterol to mid-logarithmic phase and total membrane fractions prepared. Activities of phospholipid biosynthetic enzymes in the two cell types were determined. The rates of phosphatidyl-ethanolamine-phosphatidyl-choline-N-methyl transferase and acyl-CoA-alpha-glycerol-3-phosphate transcylase were significantly greater in ergosterol-grown than in cholesterol-grown cells. These reactions were also inhibited by the polyene antibiotic filipin. By contrast the activities of long-chain fatty acyl-CoA synthetase, CTP-phosphatidate-cytidyl transferase, phosphatidylserine decarboxylase and of phosphatidylinositol synthetase were identical in the two (ergosterol and cholesterol) cultures and unaffected by filipin. The ergosterol effect on phosphatidyl-ethanolamine N-methyl transferase was greatest in cells harvested in early log phase, intermediate in the mid-log phase cells, and not significant in stationary phase cells.

Competition between cholesterol and phospholipids for binding to filipin

The interaction of the polyene antibiotic filipin with large unilamellar vesicles (LUV) has been studied by circular dichroism (CD). (1) On the one hand, the interaction has been followed with preformed LUV. In the presence of cholesterol-free dimyristoylphosphatidylcholine (DMPC) LUV, monomeric filipin, above a critical concentration of about 1·10 −6 mol/1 and up to a DMPC/filipin molar ratio of approximately 4, bound to LUV and self-associated in the membranes as demonstrated by the presence of an excitonic doublet. For increasing DMPC/filipin molar ratios, filipin remained bound to membranes but became more and more under monomeric form. In the presence of preformed cholesterol-containing DMPC LUV, for increasing concentrations of filipin, the formation of a 1:1 filipin-cholesterol complex was first observed; then, for an antibiotic/cholesterol molar ratio higher than 1 an interaction between filipin and phospholipid, identical to the one observed with cholesterol-free LUV, was observed. (2) On the other hand, the interaction with cholesterol-containing model membranes has been followed in the same experimental conditions as those used in recent studies by 2H-NMR (Dufourc, E.J. and Smith, I.C.P. (1985) Biochemistry 24, 2420–2424) and by ESR (Maurin, L., Bancel, F., Morin, P. and Bienvenue, A. (1988) Biochim. Biophys. Acta 939, 102–110), that is with filipin incorporated into membranes during their preparation. For preparations with filipin/cholesterol molar ratios of about 1:1 we confirm the results obtained by both methods. In the presence of an excess of filipin (filipin/cholesterol molar ratio 10:1), the CD spectrum observed with egg-yolk PC at 6°C (conditions used in the ESR study) is identical to that observed in the presence of pure DMPC above the transition temperature (Milhaud, J., Mazerski, J., Bolard, J. and Dufourc, E.J. (1989) Eur. Biophys. J. 17, 151–158). We conclude that in these conditions a filipin-phospholipid association occurs once the sterolic sites of fixation have been occupied.

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

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

Interaction of the polyene antibiotic filipin with model and natural membranes containing plant sterols

The interaction of the polyene antibiotic, filipin, with individual or mixed plant sterols (stigmasterol, sitosterol, campesterol and 24-methylpollinastanol) incorporated into large unilamellar vesicles (LUV) of soybean phosphatidylcholine (PC) as well as the filipin interaction with purified membrane fractions from maize roots containing these sterols was investigated by ultraviolet (UV) absorption and circular dichroism (CD) spectroscopy. With both types of membrane preparation, dramatic changes in the UV absorption and CD spectra of the antibiotic were evidenced. When LUV containing stigmasterol, sitosterol and/or campesterol were incubated with low filipin concentrations (i.e., for filipin/sterol molar ratios ( r st) lower than 1), CD signal characteristic of the formation of filipin-sterol complexes were observed. At higher r st values, the filipin-sterol interaction was shown to be in competition with a filipin-phospholipid interaction. With 24-methylpollinastanol-containing LUV, the filipin-phospholipid interaction was detected even at r st values lower than 1, which suggests a lower affinity of filipin for this sterol and emphasizes the structural differences between Δ 5-sterols and 9β,19-cyclopropylsterols. With sterol-free soybean PC LUV, a filipin-phospholipid interaction could also be evidenced. With maize root cell membranes containing either Δ 5-sterols or 9β,19-cyclopropylsterols, CD spectra similar to those obtained in the presence of LUV having these sterols as components were observed. Thus, the protein component of the membranes does not appear to be an important feature.

Freeze-fracture cytochemistry of cholesterol content in neuronal plasma membrane following cerebral ischaemia.

Minor changes on the neuronal perikaryon cytoplasm and large alterations in neuronal processes have been demonstrated with transmission electron microscopy after 60 minutes of focal and selective cerebral ischaemia. The distribution of cholesterol in neuronal plasma membrane and perikarya plasmalemma of normal and ischaemic caudate nucleus was investigated with the polyene antibiotic filipin, a morphological probe for membrane cholesterol domains in freeze-fracture replicas. After filipin incubation of prefixed vibratome slices, filipin-cholesterol complexes appeared as 20-30 proturberances and pits on P- and E-faces. Distinct patterns of filipin-cholesterol complexes were found in non-ischaemic and ischaemic neuronal membrane. The filipin-treated specimens showed a 35-40% drop in cholesterol content in the neuronal plasmalemma one hour after cerebral ischaemia.

Interrelationships between motility, c-AMP, respiration and calcium uptake of ram and boar sperm

Uptake of radioactive calcium by washed ejaculated ram and epididymal boar spermatozoa was inhibited by theophylline and dibutyryl cyclic AMP (diBc-AMP) which increases cyclic AMP (c-AMP) levels in cells, by the uncouplers 2,4-dinitrophenol (2,4-DNP) and carbonylcyanide m-chlorophenylhydrazone (CCP), by sodium azide, rotenone and antimycin A and by ruthenium red and La 3+. Nicotine, eserine and ouabain stimulated the calcium uptake of caput boar sperm and decamethonium inhibited it. Inhibition of calcium uptake was accompanied by a decrease in oxygen uptake in the case of the metabolic inhibitors (rotenone, antimycin A and sodium azide) and substances that interfere with cell calcium transport (ruthenium red and La 3+). Respiration was increased in the presence of 2,4-DNP and CCP, and theophylline and diBc-AMP. The phosphodiesterase inhibitors (caffeine and theophylline), diBc-AMP, 2,4-DNP, bicarbonate ion and ouabain increased the motility of caput boar sperm. Most other substances depressed motility. The ionophore A23187 severely inhibited the motility of ram and boar sperm with an accompanying increase in their calcium and oxygen uptake which was largely unaffected by addition of inhibitors, activators and surfactants. This may be explained by the operation of competing mitochondrial and plasma membrane pumps. However, a selective increase in the permeability of the sperm plasma membrane on addition of the polyene antibiotic filipin released calcium accumulated in the presence of A23187. This provides further evidence for the lack of mitochondrial involvement in the influx of calcium produced by the ionophore.

The surface membrane of Leishmania mexicana mexicana: comparison of amastigote and promastigote using freeze-fracture cytochemistry.

The freeze fracture replica technique has been used to compare the plasma membranes of amastigote and promastigote stages of Leishmania mexicana mexicana with respect to intramembranous particle (integral protein) distribution and to beta-hydroxysterols content as revealed by the distribution of lesions induced by the polyene antibiotic filipin. Intramembranous particle (IMP) density was greater in promastigote than in amastigote plasma membranes. Intramembranous particles were more abundant in the protoplasmic face (PF) than in the exoplasmic face (EF) of promastigotes, but this situation was found to be reversed in amastigotes. Filipin-induced lesions in glutaraldehyde-fixed parasites indicated higher levels of beta-hydroxysterols in the amastigote than in the promastigote plasma membrane, and in the promastigote flagellar membrane than in the body membrane. Amphotericin B (a related polyene antibiotic used in chemotherapy of leishmaniasis) induced IMP aggregation in the PF of unfixed amastigotes but did not appear to influence sterol distribution as demonstrated by freeze-fracture of subsequently-fixed and filipin-treated organisms.

Filipin-sterol complexes in the plasma membrane of zebrafish spermatozoa.

The presence and distribution of filipin-sterol complexes in the plasma membrane of zebrafish (Brachydanio rerio) sperm was investigated. The zebrafish sperm plasma membrane, treated with freeze-fracture techniques, is seen to contain a multitude of intramembranous particles that, in a specific region of the posterior part of the sperm head, are organized into unusual particle arrays that appear as simple hexagons or parallelograms. The polyene antibiotic filipin forms complexes with 3-beta-OH sterols to produce characteristic protrusions and pits in membranes that are readily observable in freeze-fracture replicas. Numerous filipin-sterol complexes were found to populate the sperm plasma membrane, and the complexes exhibited variability in their distribution in different sperm. This appears to be the first illustrated example of an acrosomeless sperm that exhibits a high concentration of filipin-sterol complexes. In contrast, the unique grating formed by the intramembranous particles as well as variable amounts of membrane surrounding the unusual particle arrays were always free of the filipin-sterol complexes. Thus, while cholesterol appears to be present in the plasma membrane of the zebrafish sperm, it is not apparent in the highly differentiated region of the membrane based on the observed distribution of the filipin-sterol complexes.