Cholestatrienol Research Articles

Ergosterol promotes aggregation of natamycin in the yeast plasma membrane

Polyene macrolides are antifungal substances, which interact with cells in a sterol-dependent manner. While being widely used, their mode of action is poorly understood. Here, we employ ultraviolet-sensitive (UV) microscopy to show that the antifungal polyene natamycin binds to the yeast plasma membrane (PM) and causes permeation of propidium iodide into cells. Right before membrane permeability became compromised, we observed clustering of natamycin in the PM that was independent of PM protein domains. Aggregation of natamycin was paralleled by cell deformation and membrane blebbing as revealed by soft X-ray microscopy. Substituting ergosterol for cholesterol decreased natamycin binding and caused a reduced clustering of natamycin in the PM. Blocking of ergosterol synthesis necessitates sterol import via the ABC transporters Aus1/Pdr11 to ensure natamycin binding. Quantitative imaging of dehydroergosterol (DHE) and cholestatrienol (CTL), two analogues of ergosterol and cholesterol, respectively, revealed a largely homogeneous lateral sterol distribution in the PM, ruling out that natamycin binds to pre-assembled sterol domains. Depletion of sphingolipids using myriocin increased natamycin binding to yeast cells, likely by increasing the ergosterol fraction in the outer PM leaflet. Importantly, binding and membrane aggregation of natamycin was paralleled by a decrease of the dipole potential in the PM, and this effect was enhanced in the presence of myriocin. We conclude that ergosterol promotes binding and aggregation of natamycin in the yeast PM, which can be synergistically enhanced by inhibitors of sphingolipid synthesis.

Formation of a Tight Complex between Amphidinol 3 and Sterols in Lipid Bilayers Revealed by Short-Range Energy Transfer

Abstract The exploration of molecular recognition in lipid bilayers is still extremely difficult. In this report, we leveraged short-range energy transfer (ET) that enabled detection of close contacts within 1 nm distance, and applied it to the interaction of natural products with sterols in lipid bilayers. Amphidinol 3 (AM3), a polyhydroxy–polyene metabolite from a marine dinoflagellate, possesses potent antifungal activity by forming membrane pores in a sterol-dependent manner. Although AM3 was shown to interact directly with membrane sterols, the mode of interaction is yet to be fully elucidated. Herein, we found that AM3 and cholestatrienol (CTL), a fluorescent sterol, can be an ET pair because the emission spectrum of the former overlaps with the excitation of the latter. We further confirmed that CTL exerts the sterol-dependent pore formation of AM3 as in the case of cholesterol. Then, titration using intermolecular ET in bilayers revealed that AM3 and CTL form a 1:1 complex with a dissociation constant of 1.4 × 10−5 M. The distance between the AM3 polyene and CTL triene was estimated to be less than 1 nm. Based on this information, we proposed a tight binding model between AM3 and the sterol in lipid bilayers.

Computational analysis of altered one- and two-photon CD of sterols inside a protein binding pocket

Niemann Pick C2 (NPC2) is a small glycoprotein involved in cellular trafficking of cholesterol. Its dysfunction causes accumulation of cholesterol in the lysosomal organelles, a hallmark of Niemann Pick type C disease. Therefore, understanding cholesterol transport and the accumulation mechanism that may result is essential to understand the occurrence of this neurodegenerative disease. Cholestatrienol (CTL) is a fluorescent sterol widely used as an alternative to cholesterol because of its improved optical properties in spectroscopy and live-cell imaging. In this paper, the electronic circular dichroism (ECD) and the two-photon circular dichroism (TPCD) of CTL within the NPC2 binding pockets are simulated using response theory employing time-dependent density functional theory (TD-DFT). Moreover, an explicit embedding method has been used to include the effect of the protein residues and the water solvent in calculating the spectroscopic properties. In both cases, an alteration of the CD signal is observed.

Membrane organization and intracellular transport of a fluorescent analogue of 27-hydroxycholesterol

Oxysterols are cholesterol metabolites with multiple functions in controlling cellular homeostasis. In particular, 27-hydroxycholesterol (27-OH-Chol) has been shown to regulate a variety of physiological functions, but little is known about its uptake, intracellular trafficking, and efflux from cells. This is largely due to a lack of suitable analogs of 27-OH-Chol, which mimic this oxysterol closely. Here, we present the intrinsically fluorescent 27-hydroxy-cholestatrienol (27-OH-CTL), which differs from 27-OH-Chol only by having two additional double bonds in the steroid ring system. Based on molecular dynamics (MD) simulations, we show that 27-OH-CTL possesses almost identical membrane properties compared to 27-OH-Chol. By comparative imaging of 27-OH-CTL and of the cholesterol analogue cholestatrienol (CTL) in living cells, we assess the impact of a single hydroxy group on sterol trafficking. We find that human fibroblasts take up more CTL than 27-OH-CTL, but efflux the oxysterol analogue more efficiently. For both sterols, efflux includes shedding of vesicles from the plasma membrane. Intracellular, 27-OH-CTL accumulates primarily in lipid droplets (LDs), while CTL is mostly found in endosomes and lysosomes. Using fluorescence recovery after photobleaching (FRAP), we find for both sterols a rapidly exchanging pool, which moves orders of magnitude faster than sterol containing vesicles and LDs. In summary, by applying a new fluorescent derivative of 27-OH-Chol we demonstrate that human cells can distinguish sterols based on a single hydroxy group in the side chain, resulting in different transport itineraries, dynamics, and efflux kinetics. Both intrinsically fluorescent cholesterol and oxysterol analogues show rapid non-vesicular transport in human fibroblasts.

Binding and intracellular transport of 25-hydroxycholesterol by Niemann-Pick C2 protein

Side-chain oxidized cholesterol derivatives, like 25-hydroxycholesterol (25-OH-Chol) are important regulators of cellular cholesterol homeostasis. How transport of oxysterols through the endo-lysosomal pathway contributes to their biological function is not clear. The Niemann-Pick C2 protein (NPC2) is a small lysosomal sterol transfer protein required for export of cholesterol from late endosomes and lysosomes (LE/LYSs). Here, we show that 25-hydroxy-cholestatrienol, (25-OH-CTL), an intrinsically fluorescent analogue of 25-OH-Chol, becomes trapped in LE/LYSs of NPC2-deficient fibroblasts, but can efflux from the cells even in the absence of NPC2 upon removal of the sterol source. Fluorescence recovery after photobleaching (FRAP) of 25-OH-CTL in endo-lysosomes was rapid and extensive and only partially dependent on NPC2 function. Using quenching of NPC2's intrinsic fluorescence, we show that 25-OH-Chol and 25-OH-CTL can bind to NPC2 though with lower affinity compared to cholesterol and its fluorescent analogues, cholestatrienol (CTL) and dehydroergosterol (DHE). This is confirmed by calculations of binding energies which additionally show that 25-OH-CTL can bind in two orientations to NPC2, in stark contrast to cholesterol and its analogues. We conclude that NPC2's affinity for all sterols is energetically favored over their self-aggregation in the lysosomal lumen. Lysosomal export of 25-OH-Chol is not strictly dependent on the NPC2 protein.

Live-cell imaging of new polyene sterols for improved analysis of intracellular cholesterol transport.

Analysis of intracellular cholesterol transport by fluorescence microscopy requires suitable fluorescent analogues of cholesterol. Most existing cholesterol analogues contain lipophilic dyes which can compromise the sterol properties in membranes. An alternative strategy is to introduce additional double bonds into the sterol ring system resulting in intrinsic fluorescence, while at the same time keeping the cholesterol-like properties of the analogues. Existing polyene sterols, such as dehydroergosterol (DHE) or cholestatrienol (CTL), however, contain only three double bonds and suffer from low brightness, significant photobleaching and excitation/emission in the ultraviolet region. Thus, special equipment is required to image such sterols. Here, we describe synthesis, characterization and intracellular imaging of new polyene sterols containing four conjugated double bonds in the sterol ring system. We show that such analogues have red-shifted excitation and emission by ∼20 nm compared to DHE or CTL. The red shift was even more pronounced when preventing keto-enol tautomer equilibration by protecting the 3'-hydroxy group with acetate. We show that the latter analogue can be imaged on a conventional wide field microscope with a DAPI/filipin filter cube. The new polyene sterols show reduced photobleaching compared to DHE or CTL allowing for improved deconvolution microscopy of sterol containing cellular membranes.

Synthesis and Live-Cell Imaging of Fluorescent Sterols for Analysis of Intracellular Cholesterol Transport.

Cellular cholesterol homeostasis relies on precise control of the sterol content of organelle membranes. Obtaining insight into cholesterol trafficking pathways and kinetics by live-cell imaging relies on two conditions. First, one needs to develop suitable analogs that resemble cholesterol as closely as possible with respect to their biophysical and biochemical properties. Second, the cholesterol analogs should have good fluorescence properties. This interferes, however, often with the first requirement, such that the imaging instrumentation must be optimized to collect photons from suboptimal fluorophores, but good cholesterol mimics, such as the intrinsically fluorescent sterols, cholestatrienol (CTL) or dehydroergosterol (DHE). CTL differs from cholesterol only in having two additional double bonds in the ring system, which is why it is slightly fluorescent in the ultraviolet (UV). In the first part of this protocol, we describe how to synthesize and image CTL in living cells relative to caveolin, a structural component of caveolae. In the second part, we explain in detail how to perform time-lapse experiments of commercially available BODIPY-tagged cholesterol (TopFluor-cholesterol®; TF-Chol) in comparison to DHE. Finally, using two-photon time-lapse imaging data of TF-Chol, we demonstrate how to use our imaging toolbox SpatTrack for tracking sterol rich vesicles in living cells over time.

A Combined Effect of Proteins SP-B and SP-C and Membrane Curvature on Cholesterol Partition in Lung Surfactant Membranes: Answers from Fluorescence

Cholesterol is the major neutral lipid present in lung surfactant, a lipid-protein complex responsible for the stabilization of the air-liquid interface in mammalian lungs, which is essential for the breathing function. It has been proposed that cholesterol content is tightly regulated in surfactant and that protein SP-C could have a role in this respect. To further analyse the affinity of surfactant membranes for cholesterol in the absence or presence of proteins, we have used cyclodextrin as a cholesterol solubilising agent and a fluorescent analog of cholesterol, cholestatrienol (CTL), whose partition can be followed in membrane vesicles with different lipid/protein compositions and sizes. Our results suggest that there is a direct effect of vesicle size/curvature on the affinity of membranes for CTL. This is translated into lower partition coefficients (Kx) for membranes with higher curvatures, possibly caused by an increased exposure of CTL in more curved bilayers. Surfactant hydrophobic proteins SP-B and SP-C were found to have a curvature-independent effect on CTL mobility, as well as in the mobility of other probes such as transparinaric acid-sphingomyelin (tPA-SM) or diphenylhexatriene-phosphatidylcholine (DPH-PC) in membranes of the same composition. Furthermore, in the absence of SP-B, SP-C-containing liposomes were found to spontaneously split into 30 nm vesicles increasing membrane curvature regardless lipid composition, which correlated with a reduction of Kx compared to pure lipid systems. Taken all together, our results indicate that curvature alters cholesterol accessibility in surfactant lipid bilayers and that lung surfactant proteins disturb membranes in cholesterol containing domains, which may have important physiological consequences for cholesterol mobilization in lung surfactant membranes and films.

Characterization of the sterol and phosphatidylinositol 4-phosphate binding properties of Golgi-associated OSBP-related protein 9 (ORP9).

Oxysterol binding protein (OSBP) and OSBP-related proteins (ORPS) have a conserved lipid-binding fold that accommodates cholesterol, oxysterols and/or phospholipids. The diversity of OSBP/ORPs and their potential ligands has complicated the analysis of transfer and signalling properties of this mammalian gene family. In this study we explored the use of the fluorescent sterol cholestatrienol (CTL) to measure sterol binding by ORP9 and competition by other putative ligands. Relative to cholesterol, CTL and dehydroergosterol (DHE) were poor ligands for OSBP. In contrast, both long (ORP9L) and short (ORP9S) variants of ORP9 rapidly extracted CTL, and to a lesser extent DHE, from liposomes. ORP9L and ORP9S also extracted [32P]phosphatidylinositol 4-phosphate (PI-4P) from liposomes, which was inhibited by mutating two conserved histidine residues (HH488,489AA) at the entrance to the binding pocket but not by a mutation in the lid region that inhibited cholesterol binding. Results of direct binding and competition assays showed that phosphatidylserine was poorly extracted from liposomes by ORP9 compared to CTL and PI-4P. ORP9L and PI-4P did not co-localize in the trans-Golgi/TGN of HeLa cells, and siRNA silencing of ORP9L expression did not affect PI-4P distribution in the Golgi apparatus. However, transient overexpression of ORP9L or ORP9S in CHO cells, but not the corresponding PI-4P binding mutants, prevented immunostaining of Golgi-associated PI-4P. The apparent sequestration of Golgi PI-4P by ORP9S was identified as a possible mechanism for its growth inhibitory effects. These studies identify ORP9 as a dual sterol/PI-4P binding protein that could regulate PI-4P in the Golgi apparatus.

Behavior of Fluorescent Cholesterol Analogues Dehydroergosterol and Cholestatrienol in Lipid Bilayers: A Molecular Dynamics Study

Molecular dynamics simulations of bilayer systems consisting of varying proportions of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol (Chol), and intrinsically fluorescent Chol analogues dehydroergosterol (DHE) or cholestatrienol (CTL) were carried out to study in detail the extent to which these fluorescent probes mimic Chol's behavior (location, orientation, dynamics) in membranes as well as their effect on host bilayer structure and dynamics (namely their ability to induce membrane ordering in comparison with Chol). Control properties of POPC and POPC/Chol bilayers agree well with published experimental and simulation work. Both probes and Chol share similar structural and dynamical properties within the bilayers. Additionally, the fluorescent sterols induce membrane ordering to a similar (slightly lower) extent to that of Chol. These findings combined demonstrate that the two studied fluorescent sterols are adequate analogues of Chol, and may be used with advantage over side-chain labeled sterols. The small structural differences between the three studied sterols are responsible for the slight variations in the calculated properties, with CTL presenting a more similar behavior to Chol (correlating with its larger structural similarity to Chol) compared to DHE.

Cholesterol's interactions with serine phospholipids — A comparison of N-palmitoyl ceramide phosphoserine with dipalmitoyl phosphatidylserine

In this study we have prepared ceramide phosphoserine (CerPS) and examined its sterol-interacting properties. CerPS is a hydrogen-bonding sphingolipid, but its head group differs from that found in sphingomyelin (SM). Based on diphenylhexatriene steady-state anisotropy measurements, we observed that fully hydrated N-palmitoyl CerPS had a gel-to-liquid crystalline phase transition temperature of about 51°C in 50mM sodium phosphate buffer (pH 7.4). This was close to the Tm measured for 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS) bilayers (Tm 50.5°C). Based on cholestatrienol (CTL) quenching experiments in liquid disordered ternary bilayers (containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphcholine; POPC), cholesterol/CTL formed sterol-enriched ordered domains with CerPS. These had similar thermostability as the sterol domains formed with N-palmitoyl SM. Cholesterol failed to form sterol-enriched ordered domains with DPPS under comparable conditions. Based on the equilibrium partitioning of CTL, we observed that the affinity of sterol for bilayers containing POPC/CerPS/cholesterol (6:3:1 by mol) was much higher than the affinity measured for control fluid POPC/cholesterol (9:1 by mol) bilayers, but slightly less than seen for comparable PSM-containing bilayers. We conclude that the phosphoserine head group was less efficient than the phosphocholine head group in stabilizing sterol/sphingolipid interaction. However, hydrogen bonding apparently can overcome some of the negative effects of the phosphoserine head group, since CerPS interacted more favorably with cholesterol compared to DPPS.

Hydrophobic Matching Between Phospholipid Bilayers and Helical Peptides Affects the Memrbane Affinity of Sterols

The lateral organization of cell membranes is important for several cellular functions including signaling and membrane trafficking. In addition pathogen uptake and influenza virus budding is associated with sphingolipid and cholesterol enriched lateral membrane domains. The lateral structure of membranes is in part controlled by lipid self-organization, but it is becoming increasingly clear that also membrane proteins can play an active role in the maintenance of the lateral structure. Cholesterol is thought to have an important role in lateral organization of eukaryotic cell membranes. As cholesterol also has been implicated to take part in the sorting of cellular transmembrane proteins it is a good starting point to determine how transmembrane proteins influence the lateral sorting of cholesterol in phospholipid bilayers. Insight into this can be obtained by studying how cholesterol interacts with bilayer membranes of different composition in the presence of different transmembrane peptides, mimicking the transmembrane helices of proteins. By measuring the equilibrium partitioning of the fluorescent cholesterol analogue cholestatrienol (CTL) between large unilamellar vesicles and methyl-beta-cyclodextrin the effect of hydrophobic matching on the affinity of sterols for phospholipid bilayers was determined. The results showed that increasing positive mismatch led to higher affinity of the sterol for the bilayers. This suggests that hydrophobic matching could affect the lateral organization of cholesterol in cell membranes and have an important role especially in membrane trafficking.

Sterols Have Higher Affinity for Sphingomyelin than for Phosphatidylcholine Bilayers even at Equal Acyl-Chain Order

The interaction between cholesterol and phospholipids in bilayer membranes is important for the formation and maintenance of membrane structure and function. However, cholesterol does not interact favorably with all types of phospholipids and, for example, prefers more ordered sphingomyelins (SMs) over phosphatidylcholines (PCs). The reason for this preference is not clear. Here we have studied whether acyl-chain order could be responsible for the preferred sterol interaction with SMs. Acyl-chain order was deduced from diphenylhexatriene anisotropy and from the deuterium order parameter obtained by 2H-NMR on bilayers made from either 14:0/14:0(d27)-PC, or 14:0(d27)-SM. Sterol/phospholipid interaction was determined from sterol bilayer partitioning. Cholestatrienol (CTL) was used as a fluorescence probe for cholesterol, because its relative membrane partitioning is similar to cholesterol. When CTL was allowed to reach equilibrium partitioning between cyclodextrins and unilamellar vesicles made from either 14:0/14:0-PC or 14:0-SM, the molar-fraction partitioning coefficient (Kx) was approximately twofold higher for SM bilayers than for PC bilayers. This was even the case when the temperature in the SM samples was raised to achieve equal acyl-chain order, as determined from 1,6-diphenyl-1,3,5-hexatriene (DPH) anisotropy and the deuterium order parameter. Although the Kx did increase with acyl-chain order, the higher Kx for SM bilayers was always evident. At equal acyl-chain order parameter (DPH anisotropy), the Kx was also higher for 14:0-SM bilayers than for bilayers made from either 14:0/15:0-PC or 15:0-/14:0-PC, suggesting that minor differences in chain length or molecular asymmetry are not responsible for the difference in Kx. We conclude that acyl-chain order affects the bilayer affinity of CTL (and thus cholesterol), but that it is not the cause for the preferred affinity of sterols for SMs over matched PCs. Instead, it is likely that the interfacial properties of SMs influence and stabilize interactions with sterols in bilayer membranes.

Effect of hydrophobic mismatch and interdigitation on sterol/sphingomyelin interaction in ternary bilayer membranes

Sphingomyelin (SM) is a major phospholipid in most cell membranes. SMs are composed of a long-chain base (often sphingosine, 18:1Δ4t), and N-linked acyl chains (often 16:0, 18:0 or 24:1Δ15c). Cholesterol interacts with SM in cell membranes, but the acyl chain preference of this interaction is not fully elucidated. In this study we have examined the effects of hydrophobic mismatch and interdigitation on cholesterol/sphingomyelin interaction in complex bilayer membranes. We measured the capacity of cholestatrienol (CTL) and cholesterol to form sterol-enriched ordered domains with saturated SM species having different chain lengths (14 to 24 carbons) in ternary bilayer membranes. We also determined the equilibrium bilayer partitioning coefficient of CTL with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes containing 20mol% of saturated SM analogs. Ours results show that while CTL and cholesterol formed sterol-enriched domains with both short and long-chain SM species, the sterols preferred interaction with 16:0-SM over any other saturated chain length SM analog. When CTL membrane partitioning was determined with fluid POPC bilayers containing 20mol% of a saturated chain length SM analog, the highest affinity was seen with 16:0-SM (both at 23 and 37°C). These results indicate that hydrophobic mismatch and/or interdigitation attenuate sterol/SM association and thus affect lateral distribution of sterols in the bilayer membrane.

Influence of α-helical Transmembrane Peptides on the Affinity of Sterols for Phospholipid Bilayers

It is well known that lipids can segregate laterally into nanoscopic domains or different phases. Yet very little is known about proteins can influence the lateral organization in cellular membranes. As most biomembranes contain relatively high concentrations of transmembrane proteins it is important to learn more about how lipid-protein interplay affects the lateral organization in membranes. Cholesterol is thought to have an important role in lateral organization of eukaryotic cell membranes. As cholesterol also has been implicated to take part in the sorting of cellular transmembrane proteins it is a good starting point to determine how transmembrane proteins influence the lateral sorting of cholesterol in phospholipid bilayers. Insight into this can be obtained by studying how cholesterol interacts with bilayer membranes of different composition in the presence of different transmembrane peptides, mimicking the transmembrane helices of proteins. For this purpose an assay, in which the partitioning of the fluorescent cholesterol analogue cholestatrienol (CTL) between large unilamellar vesicles (LUVs) and methyl-β-cyclodextrin (CD) can be measured, has been developed. The partition assay showed that CTL partition preferentially into fluid phospholipid bilayers with a more ordered acyl chain region, as has been observed previously with cholesterol. It is known that proteins can decrease or increase the order in lipid bilayers and that the nature of this effect is dependent on both the structure of the protein and the composition of the bilayer. In order to assess how such protein induced order changes in the lipid bilayer affects cholesterol partitioning we have measured CTL's affinity for bilayers with varying lipid composition and containing various transmembrane peptides.

Spatiotemporal analysis of endocytosis and membrane distribution of fluorescent sterols in living cells

Distribution and dynamics of cholesterol in the plasma membrane as well as internalization pathways for sterol from the cell surface are of great cell biological interest. Here, UV-sensitive wide field microscopy of the intrinsically fluorescent sterols, dehydroergosterol (DHE) and cholestatrienol (CTL) combined with advanced image analysis were used to study spatiotemporal sterol distribution in living macrophages, adipocytes and fibroblasts. Sterol endocytosis was directly visualized by time-lapse imaging and noise-robust tracking revealing confined motion of DHE containing vesicles in close proximity to the cell membrane. Spatial surface intensity patterns of DHE as well as that of the lipid marker DiIC12 being assessed by statistical image analysis persisted over several minutes in cells having a constant overall curvature. Sites of sterol endocytosis appeared indistinguishable from other regions of the cell surface, and endocytosis contributed by 62% to total sterol uptake in J774 cells. DHE co-localized with fluorescent transferrin (Tf) in vesicles right after onset of endocytosis and in deepened surface patches of energy depleted cells. Surface caveolae labeled with GFP-tagged caveolin were not particularly enriched in DHE or CTL. Some sterol co-localized with internalized caveolin suggesting that caveolar endocytosis contributes to vesicular sterol uptake. These findings demonstrate that plasma membrane sterol is internalized by several endocytic pathways. Sterol endocytosis does not require formation of microscopically resolvable sterol clusters or enrichment of sterol in surface caveolae.

Acyl chain length affects ceramide action on sterol/sphingomyelin-rich domains

The effects of ceramides with varying saturated N-linked acyl chains (C2–C14) on cholesterol displacement from sphingomyelin-rich domains and on the stability of ordered domains were studied. The bilayers examined were made from 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC), d- erythro-N-palmitoyl-sphingomyelin (PSM), d- erythro-N- acyl-sphingosine, and cholesterol (60:15:15:10 mol%, respectively). Cholestatrienol (CTL) or d- erythro-N- trans-parinoyl-sphingomyelin (tParSM) were used as reporter molecules (at 1 mol%) for the ordered domains, and 1-palmitoyl-2-stearoyl-(7-doxyl)- sn-glycero-3-phosphocholine (7SLPC) as a fluorescence quencher (30 mol%, replacing POPC) in the liquid-disordered phase. The results indicate that the ceramide had to have an N-linked acyl chain with at least 8 methylene units in order for it to displace cholesterol from the sphingomyelin-rich domains at the concentration used. The melting of the sphingomyelin-rich domain shifted to higher temperatures (compared to the ceramide-free control) with C2, C12 and longer chain ceramides, whereas C4–C10 ceramides led to domain melting at lower temperatures than control. This study shows that short-chain ceramides do not have the same effects on sterol- and sphingomyelin-rich domains as naturally occurring longer-chain ceramides have.

Domain Formation and Stability in Complex Lipid Bilayers as Reported by Cholestatrienol

In this study, we used cholestatrienol (CTL) as a fluorescent reporter molecule to study sterol-rich L(o) domains in complex lipid bilayers. CTL is a fluorescent cholesterol analog that mimics the behavior of cholesterol well. The ability of 12SLPC to quench the fluorescence of cholestatrienol gives a measure of the amount of sterol included in L(o) domains in mixed lipid membranes. The stability of sterol-rich domains formed in complex lipid mixtures containing saturated sphingomyelins, phosphatidylcholines, or galactosylceramide as potential domain-forming lipids were studied. The amount of sterol associated with sterol-rich domains seemed to always increase with increasing temperature. The quenching efficiency was highly dependent on the domain-forming lipid present in complex lipid mixtures. Sphingomyelins formed stable sterol-enriched domains and were able to shield CTL from quenching better than the other lipids included in this study. The saturated phosphatidylcholines also formed sterol-rich domains, but the quenching efficiency in membranes with these was higher than with sphingomyelins and the domains melted at lower temperatures. PGalCer was not able to form sterol-enriched domains. However, we found that PGalCer stabilized sterol-rich domains formed in PSM-containing bilayers. Using a fluorescent ceramide analog, we also demonstrated that N-palmitoyl-ceramide displaced the sterol from sphingolipid-rich domains in mixed bilayer membranes.