Cholesterol In Bilayers Research Articles

<p>CPX-351: a nanoscale liposomal co-formulation of daunorubicin and cytarabine with unique biodistribution and tumor cell uptake properties</p>

Combination regimens are a standard of care for many cancers. However, components of such regimens are typically first developed individually and subsequently combined using strategies to minimize toxicity. Little or no consideration is given to strategies that potentially maximize efficacy. In contrast, CPX-351 (Vyxeos®) is a dual-drug liposomal encapsulation of cytarabine and daunorubicin that was rationally designed to improve efficacy over the traditional 7+3 cytarabine/daunorubicin chemotherapy regimen for patients with acute myeloid leukemia (AML). The notable clinical efficacy of CPX-351 is achieved through maintenance of a synergistic 5:1 molar ratio of cytarabine and daunorubicin within the liposome after intravenous injection. The CPX-351 liposome, which is formulated to contain bilayers of distearoylphosphatidylcholine, distearoylphosphatidylglycerol, and cholesterol at a 7:2:1 molar ratio and remains in a gel phase at body temperature, provides stability without polyethylene glycol, controlled release of cytarabine and daunorubicin, limited systemic drug distribution, and preferential internalization within malignant myeloblasts in the bone marrow via active uptake of liposomes into cytoplasmic vacuoles. Thus, the CPX-351 liposome protects cytarabine and daunorubicin from metabolism and elimination, while overcoming pharmacokinetic differences between the two agents. In clinical studies, these liposome properties markedly increased the elimination half-life of CPX-351 versus free cytarabine and daunorubicin and maintained a synergistic drug ratio for over 24 hrs after administration. Preferential uptake of liposomes by leukemia cells suggests that relatively large amounts of cytarabine and daunorubicin enter malignant cells via liposomes, potentially bypassing P-glycoprotein-based efflux pumps, which are important mediators of chemotherapy resistance, and contribute to the rapid clearance of leukemia cells from the circulation and bone marrow. These pharmacologic advantages, a direct consequence of properties of the encapsulating liposome, may explain the efficacy of CPX-351 in patients with newly diagnosed high-risk/secondary AML and the reduced drug exposure in off-target tissues that contribute to a manageable safety profile.

Atomistic simulations of cholesterol in symmetric lipid bilayers with varying degrees of lipid chain unsaturation

Atomistic simulations of cholesterol in symmetric lipid bilayers with varying degrees of lipid chain unsaturation

Cholesterol in phospholipid bilayers: positions and orientations inside membranes with different unsaturation degrees.

Cholesterol is an essential component of all animal cell membranes and plays an important role in maintaining the membrane structure and physical-chemical properties necessary for correct cell functioning. The presence of cholesterol is believed to be responsible for domain formation (lipid rafts) due to different interactions of cholesterol with saturated and unsaturated lipids. In order to get detailed atomistic insight into the behaviour of cholesterol in bilayers composed of lipids with varying degrees of unsaturation, we have carried out a series of molecular dynamics simulations of saturated and polyunsaturated lipid bilayers with different contents of cholesterol, as well as well-tempered metadynamics simulations with a single cholesterol molecule in these bilayers. From these simulations we have determined distributions of cholesterol across the bilayer, its orientational properties, free energy profiles, and specific interactions of molecular groups able to form hydrogen bonds. Both molecular dynamics and metadynamics simulations showed that the most unsaturated bilayer with 22:6 fatty acid chains shows behaviour which is most different from other lipids. In this bilayer, cholesterol is relatively often found in a "flipped" configuration with the hydroxyl group oriented towards the membrane middle plane. This bilayer has also the highest (least negative) binding free energy among liquid phase bilayers, and the lowest reorientation barrier. Furthermore, cholesterol molecules in this bilayer are often found to form head-to-tail contacts which may lead to specific clustering behaviour. Overall, our simulations support ideas that there can be a subtle interconnection between the contents of highly unsaturated fatty acids and cholesterol, deficiency or excess of each of them is related to many human afflictions and diseases.

Spatially Resolving the Condensing Effect of Cholesterol in Lipid Bilayers

We study the effect of cholesterol on the structure of dipalmitoylphosphatidylcholine phospholipid bilayers. Using extensive molecular dynamics computer simulations at atomistic resolution, we observe and quantify several structural changes upon increasing cholesterol content that are collectively known as the condensing effect: a thickening of the bilayer, an increase in lipid tail order, and a decrease in lateral area. We also observe a change in leaflet interdigitation and a lack thereof in the distributions of dipalmitoylphosphatidylcholine headgroup orientations. These results, obtained over a wide range of cholesterol mole fractions, are then used to calibrate the analysis of phospholipid properties in bilayers containing a single cholesterol molecule per leaflet, which we perform in a spatially resolved way. We find that a single cholesterol molecule affects phospholipids in its first and second solvation shells, which puts the range of this effective interaction to be on the order of 1–2 nm. We also observe a tendency of phospholipids to orient their polar headgroups toward the cholesterol, which provides additional support for the umbrella model of bilayer organization.

Detection of cholesterol bilayer domains in intact biological membranes: Methodology development and its application to studies of eye lens fiber cell plasma membranes

Four purported lipid domains are expected in plasma membranes of the eye lens fiber cells. Three of these domains, namely, bulk, boundary, and trapped lipids, have been detected. The cholesterol bilayer domain (CBD), which has been detected in lens lipid membranes prepared from the total lipids extracted from fiber cell plasma membranes, has not yet been detected in intact fiber cell plasma membranes. Here, a saturation-recovery electron paramagnetic resonance spin-labeling method has been developed that allows identification of CBDs in intact fiber cell plasma membranes of eye lenses. This method is based on saturation-recovery signal measurements of the cholesterol-analog spin label located in the lipid bilayer portion of intact fiber cell membranes as a function of the partial pressure of molecular oxygen with which the samples are equilibrated. The capabilities and limitations of this method are illustrated for intact cortical and nuclear fiber cell plasma membranes from porcine eye lenses where CBDs were detected in porcine nuclear intact membranes for which CBDs were also detected in lens lipid membranes. CBDs were not detected in porcine cortical intact and lens lipid membranes. CBDs were detected in intact membranes isolated from both cortical and nuclear fiber cells of lenses obtained from human donors. The cholesterol content in fiber cell membranes of these donors was always high enough to induce the formation of CBDs in cortical as well as nuclear lens lipid membranes. The results obtained for intact membranes, when combined with those obtained for lens lipid membranes, advance our understanding of the role of high cholesterol content and CBDs in lens biology, aging, and/or cataract formation.

Nucleation and growth of pores in 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) / cholesterol bilayer by antimicrobial peptides melittin, its mutants and cecropin P1.

Antimicrobial peptides are one of the most promising alternatives to antibiotics for targeting pathogens without developing resistance. In this study, pore formation in 1,2-Dimyristoyl-snglycero-3-phosphocholine (DMPC) / cholesterol liposome induced by native melittin, its two mutant variants (G1I and I17 K), and cecropin P1 was investigated by monitoring the dynamics of fluorescence dye leakage. A critical peptide concentration was required for dye leakage with the rate of leakage being dependent on peptide concentration above a critical value. A lag time was required for dye leakage for low peptide concentrations that are above the critical value, which decreased at higher peptide concentrations eventually approaching zero. Lag time was found to be in the order I17 K mutant with lower hydrophobicity and higher net charge > G1I with higher hydrophobicity > melittin > cecropin P1. Cecropin P1 exhibited the highest rate of dye leakage followed by melittin, G1I, and I17 K. Size distribution and transmission electron microscopy (TEM) of liposomes exposed to peptides of different concentrations indicated pore formation with accompanied stretching of liposomes at low peptide concentrations for both melittin and cecropin P1. At much higher concentrations, however, size distribution indicated three peaks for both peptides. In both cases, TEM images show that the middle and small peaks are shown to be due to stretched liposome and broken stretched liposome respectively. For melittin, the large peak is due to peptide aggregates as well as aggregates of liposome. For cecropin P1, however, the large peak indicates cecropin P1 aggregates with solubilized lipids thus suggesting carpet mechanism.

Molecular Dynamics Investigation of the Ternary Bilayer Formed by Saturated Phosphotidylcholine, Sphingomyelin, and Cholesterol

Using the all-atom CHARMM36 force field, we have performed molecular dynamics simulations of the equimolar ternary dimyristoylphosphatidylcholine (DMPC)/ N-palmitoylsphingomyelin (PSM)/cholesterol bilayer. Analysis of its structural and kinetic properties has led us to the following conclusions. First, the DMPC/PSM/cholesterol bilayer features favorable interactions of cholesterol with DMPC and, particularly, PSM lipids, which are supported by hydrogen bonds. In contrast, the interactions between cholesterol molecules are strongly suppressed. Further analysis shows that about 60% of PSM molecules form hydrogen bonds with other PSM or cholesterol molecules and, to a lesser degree, with DMPC lipids. Second, local lipid packing around PSM molecules favors parallel or antiparallel alignments, resulting in the appearance of large anisotropic chain-like clusters formed by PSM lipids. In contrast, the distribution of DMPC lipids is closer to ideal, whereas the cholesterol distribution is sharply shifted toward small clusters or isolated molecules. Third, intermolecular interactions and mismatch in fatty acid tail lengths significantly slow down PSM rotational relaxation compared to that for DMPC, and PSM, but not DMPC lipids, tend to combine axial rotation with wobbling. Fourth, the analysis of cholesterol hydration indicates that lipid headgroups at most block about 30% of water molecules from reaching cholesterol, hinting at limited applicability of the umbrella model. Finally, we show that PSM and DMPC lipids predominantly exist in two equally probable conformational states, with and without a kink in their fatty acid tails. The appearance of these states is linked to the impact of cholesterol. Comparisons with previous studies allow us to delineate contributions of cholesterol, PSM, and DMPC to bilayer properties.

Presence and Role of Midplane Cholesterol in Lipid Bilayers Containing Registered or Antiregistered Phase Domains

Three-component lipid mixtures can produce coexisting liquid ordered and liquid disordered phases, a model for eukaryotic plasma membrane rafts. In compositionally symmetric bilayers with twophase-separated leaflets, phase domains of the two leaflets may align through registration, where domains are found across from domains of the same phase, or else antiregistration, where domains are found across from domains of the opposite phase. This alignment could serve as a method of information communication across the plasma membrane. We used coarse-grained molecular dynamics simulations to study ternary mixtures of a high-melting-temperature phospholipid, a low-melting-temperature phospholipid, and cholesterol. We found a significant presence of cholesterol molecules at the bilayer midplane rather than in a leaflet in some systems, corresponding to a lack of registration. Increasing the length of the acyl chains from 16 to 24 carbons in high-melting-temperature phospholipids or increasing the concentration of cholesterol from 20 to 35 mol % in the bilayer produced a transition from registration to antiregistration and gave rise to significant populations of midplane cholesterol.

Transverse distribution of plasma membrane bilayer cholesterol: Picking sides.

The transverse asymmetry (sidedness) of phospholipids in plasma membrane bilayers is well characterized, distinctive, actively maintained and functionally important. In contrast, numerous studies using a variety of techniques have concluded that plasma membrane bilayer cholesterol is either mostly in the outer leaflet or the inner leaflet or is fairly evenly distributed. Sterols might simply partition according to their differing affinities for the asymmetrically disposed phospholipids, but some studies have proposed that it is actively transported to the outer leaflet. Other work suggests that the sterol is enriched in the inner leaflet, driven by either positive interactions with the phosphatidylethanolamine on that side or by its exclusion from the outer leaflet by the long chain sphingomyelin molecules therein. This uncertainty raises three questions: is plasma membrane cholesterol sidedness fixed in a given cell or cell type; is it generally the same among mammalian species; and does it serve specific physiological functions? This review grapples with these issues.

Cholesterol modulates curcumin partitioning and membrane effects

Curcumin, a polyphenol molecule, presents a wide range of biological activities as antioxidant, anticancer, anti-inflammatory, antimicrobial and wound healing. Although some strengths attributed to curcumin derive from promiscuous biological activity, possibly because curcumin can interfere on many membrane located processes, knowledge of underlying interactions are lacking. Mammalian cell membranes characteristically contain 25 to 50% cholesterol/phospholipid ratio; however, most studies involving lipid bilayers and curcumin consider pure phosphatidylcholine and compare effects of curcumin on membranes with those of cholesterol. We investigated the interaction of curcumin with lipid bilayers containing cholesterol mimicking mammalian cells, and used spectroscopy techniques to determine partition coefficients, rigidity parameters and lytic activity. We found that curcumin partitions into different lipid bilayers (104 order coefficients that vary by less than a factor of two), containing cholesterol or not, and in the presence of sphingomyelin or phosphatidylserine. Curcumin decreases rigidity in all tested compositions, except that containing 40% cholesterol in which it increases the lipid packing order. In addition, curcumin induces leakage from giant unilamellar vesicles on a cholesterol concentration dependent way. Our results are compatible with the hypothesis of curcumin interaction with membranes being modulated by the liquid disordered phase and by the coexistence of liquid-ordered/liquid disordered phases. In bilayers containing cholesterol, curcumin assumes a more superficial location, drastically stiffens the 40% cholesterol bilayer and decreases the lytic effect. Our study may help researchers in the analysis of the biological effects of curcumin and curcumin-derived formulations by calling the attention to the discriminating role of the cholesterol content.

Link between Membrane Composition and Permeability to Drugs.

Prediction of membrane permeability to small molecules represents an important aspect of drug discovery. First-principles calculations of this quantity require an accurate description of both the thermodynamics and kinetics that underlie translocation of the permeant across the lipid bilayer. In this contribution, the membrane permeability to three drugs, or drug-like molecules, namely, 9-anthroic acid (ANA), 2',3'-dideoxyadenosine (DDA), and hydrocortisone (HYL), are estimated in a pure 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and in a POPC:cholesterol (2:1) mixture. On the basis of independent 2-5-μs free-energy calculations combined with a time-fractional Smoluchowski determination of the diffusivity, the estimated membrane permeabilities to these chemically diverse permeants fall within an order of magnitude from the experimental values obtained in egg-lecithin bilayers, with the exception of HYL in pure POPC. This exception is particularly interesting because the calculated permeability of the sterol-rich bilayer to HYL, in close agreement with the experimental value, is about 600 times lower than that of the pure POPC bilayer to HYL. In contrast, the permeabilities to ANA and DDA differ by less than a factor of 10 between the pure POPC and POPC:cholesterol bilayers. The unusual behavior of HYL, a large, amphiphilic compound, may be linked with the longer range perturbation of the lipid bilayer it induces, compared to ANA and DDA, suggestive of a possibly different translocation mechanism. We find that the tendency of lower permeabilities of the POPC:cholesterol bilayer relative to those of the pure POPC one is a consequence of increased free-energy barriers. Beyond reporting accurate estimates of the membrane permeability, the present contribution also demonstrates that rigorous free-energy calculations and a fractional-diffusion model are key in revealing the molecular phenomena linking the composition of a membrane to its permeability to drugs.

Electroporation of Skin Stratum Corneum Lipid Bilayer and Molecular Mechanism of Drug Transport: A Molecular Dynamics Study.

The electroporation technique has been used significantly to increase drug permeation through the skin. This technique relies on the application of short-timed (microseconds to millisecond) electric fields (generally, order of 50--300 V) on the skin to create microscopic pores. However, the molecular mechanism of pore formation, resulting in an enhanced flux of active molecules through the skin, remains poorly understood. In this study, extensive atomistic molecular dynamics simulation of skin lipids [made up of ceramide (CER), cholesterol (CHOL), and free fatty acid (FFA)] has been performed at various external electric fields. We show for the first time the pore formation in the skin lipid bilayer during electroporation. We show the effect of the applied external electrical field (0.6-1.0 V/nm) on the pore formation dynamics in the lipid bilayer of different sizes (154, 616, and 2464 lipids) and compositions (CER/CHOL/FFA, 1:0:0, 1:0:1, 1:1:0, 1:1:1). The pore formation and resealing kinetics were different and were found to be highly dependent on the composition of the skin lipid bilayer. The pore formation time decreased with increase in the bilayer size. The pore sustaining electric field was found to be in the range of 0.20-0.25 V/nm for equimolar CER, CHOL, and FFA lipid bilayers. The skin lipid bilayer (1:1:1) sealed itself within 20 ns after the removal of the external electric field. We also present the molecular mechanism of enhancement of drug permeation in the presence of external field as compared to the passive diffusion. The molecular-level understanding obtained here could help in optimizing/designing the electroporation experiments for effective drug delivery. For a given skin composition and size of the drug molecule, the combination of pore formation time and pore growth model can be used to know a priori the desired electric field and time for the application of the electric field.

Single-particle fusion of influenza viruses reveals complex interactions with target membranes

The first step in infection of influenza A virus is contact with the host cell membrane, with which it later fuses. The composition of the target bilayer exerts a complex influence on both fusion efficiency and time. Here, an in vitro, single-particle approach is used to study this effect. Using total internal reflection fluorescence (TIRF) microscopy and a microfluidic flow cell, the hemifusion of single virions is visualized. Hemifusion efficiency and kinetics are studied while altering target bilayer cholesterol content and sialic-acid donor. Cholesterol ratios tested were 0%, 10%, 20%, and 40%. Sialic-acid donors GD1a and GYPA were used. Both cholesterol ratio and sialic-acid donors proved to have a significant effect on hemifusion efficiency. Furthermore, comparison between GD1a and GYPA conditions shows that the cholesterol dependence of the hemifusion time is severely affected by the sialic-acid donor. Only GD1a shows a clear increasing trend in hemifusion efficiency and time with increasing cholesterol concentration of the target bilayer with maximum rates for GD1A and 40% cholesterol. Overall our results show that sialic acid donor and target bilayer composition should be carefully chosen, depending on the desired hemifusion time and efficiency in the experiment.

Solid-State NMR of highly 13C-enriched cholesterol in lipid bilayers.

Cholesterol (Chol) is vital for cell function as it is essential to a myriad of biochemical and biophysical processes. The atomistic details of Chol's interactions with phospholipids and proteins is therefore of fundamental interest, and NMR offers unique opportunities to interrogate these properties at high resolution. Towards this end, here we describe approaches for examining the structure and dynamics of Chol in lipid bilayers using high levels of 13C enrichment in combination with magic-angle spinning (MAS) methods. We quantify the incorporation levels and demonstrate high sensitivity and resolution in 2D 13C-13C and 1H-13C spectra, enabling de novo assignments and site-resolved order parameter measurements obtained in a fraction of the time required for experiments with natural abundance sterols. We envision many potential future applications of these methods to study sterol interactions with drugs, lipids and proteins.

Stretch-Induced Interdigitation of a Phospholipid/Cholesterol Bilayer.

The interdigitated gel (LβI) phase is one of the membrane phases of phospholipid molecules in which the hydrophobic tails of the phospholipid molecules penetrate the opposite leaflet of the bilayer. Recent molecular dynamics (MD) simulations have shown that interdigitation can take place as a phase transition from the liquid-ordered (Lo) phase to the LβI phase under stretching. However, there is still no conclusive experimental evidence for this process, so its existence remains controversial. In this study, to explain the transition from energy balance, we propose a free-energy model. The model consists of three energy components: the elastic deformation energy, surface energy at the bilayer-water interface, and interphase boundary energy. To determine the parameters of the model, we perform MD simulations of a stretched 1,2-dipalmitoyl- sn-glycero-3-phosphocholine/cholesterol bilayer. The phase diagrams from our model are in good agreement with those obtained from MD simulations. The energy balance among the components in the stretched bilayer quantitatively explains the stretch-induced transition. In the model, increasing the system size to that used in experiments shows that interdigitation is favorable for rigid bilayers under stretching or in alcohol solutions. These results suggest that the stretch-induced interdigitation might be observed in microscopic experiments.

Molecular Dynamics Simulations Reveal the Role of Membrane Cholesterol during Pore Forming Pathway of Cytolysin A

Cytolysin A (ClyA), an α pore-forming toxin expressed by E. Coli as a water-soluble monomer, undergoes a large conformational change during pore formation. Although cholesterol is known to enhance the lytic activity of ClyA, molecular aspects of the interactions between cholesterol and ClyA during pore formation are not well understood. Using all-atom molecular dynamics simulations ranging from 0.5 - 0.9 µs, we study a single membrane-inserted protomer, a dimer (two protomers) and the dodecameric ClyA pore embedded in a DOPC+30% cholesterol bilayer. In the single membrane-inserted protomer, high cholesterol occupancy was observed around the transmembrane residues of N-terminus which form part of a CRAC motif and also around residues of the β-tongue. Although high cholesterol occupancy sites were not observed near the N-terminus in the dimer simulations, a cholesterol molecule was preferentially located in the pocket formed between two adjacent β-tongues of the dimer. Cholesterol spent 97% of the simulation time (600 ns) inside this pocket sampling two major orientations. Energies of two conformations were reported from docking simulations. Formation of transmembrane water channels were observed in both single membrane inserted and dimer ClyA simulations. From the dodecameric pore simulations, density map showed regions of high cholesterol population between the β-tongue pockets and mobility map indicated slower cholesterol in the vicinity of the pore as compared to bulk. Our simulations elucidate specific interactions with cholesterol that could stabilize both the single membrane inserted protomeric state as well as the dodecameric pore.

Kinetic Details of the Interplay between Sodium Binding and Opioid Receptor Signaling

Sodium cations have been known to control ligand binding to and signaling of various rhodopsin-like G Protein-Coupled Receptors (GPCRs) for more than four decades, but the molecular details of the sodium binding site have been revealed only very recently by means of high-resolution crystal structures of receptors. Notwithstanding this important information, a complete understanding of the role of sodium on ligand binding and efficacy, requires additional thermodynamic and kinetic details. Here, we analyze micro-second scale all-atom molecular dynamics simulations of sodium binding to the active or inactive crystallographic conformations of the µ-opioid receptor (MOR) embedded in an explicit 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/cholesterol bilayer. Using information from time-independent component analysis (tICA) of unbiased simulations and multi-ensemble Markov State modeling combining unbiased and umbrella sampling simulations, we estimate the thermodynamics and kinetics parameters of the binding of sodium cations to MOR. While an energetically favorable ion pathway is identified across the active conformation of MOR based on calculations of time-averaged ion spatial distribution, this pathway is occluded in the inactive receptor. Key residues involved in ion binding kinetics emerge from this analysis, and the mutual effects of the ion on both the conformation and solvation of the protein are also characterized. These results generate testable hypotheses of the molecular determinants of ion-receptor binding kinetics, and their role in receptor function.

Detection of Pure Cholesterol Bilayer Domains in Biological Membranes Overloaded with Cholesterol: Methodology Development and its Application to Porcine Lens Membrane Studies

Detection of Pure Cholesterol Bilayer Domains in Biological Membranes Overloaded with Cholesterol: Methodology Development and its Application to Porcine Lens Membrane Studies

Pure Cholesterol Bilayer Domains are Formed at Cholesterol Contents Significantly Lower than Cholesterol Solubility Thresholds in Phospholipid Membranes: EPR and DSC Studies

Pure Cholesterol Bilayer Domains are Formed at Cholesterol Contents Significantly Lower than Cholesterol Solubility Thresholds in Phospholipid Membranes: EPR and DSC Studies

Cholesterol-Induced Membrane Organization Promotes Influenza Virus Binding

The influenza virus attaches to a cell surface by binding sialic-acid containing receptors with the viral ligand hemagglutinin. Stable viral binding requires more than one viral ligand- host receptor interaction to simultaneously occur. Thus, binding can be modulated by the spatial distribution of target receptors in the host membrane. We show that membrane sterols critically control viral attachment and propose that sterols alter the nanoscale clustering of viral receptors to facilitate binding. Using single virus fluorescence microscopy, we demonstrated that viral binding is dependent on the cholesterol content of the target membrane. Fluorescently labeled viral particles preferentially bound synthetic membranes supplemented with increasing amounts of cholesterol (0-40 mol%). Other sterols exhibited a similar effect on binding, independent of their ability to support liquid-liquid phase separation. To develop a molecular explanation for cholesterol's effect, we ran a series of course grained molecular dynamics simulations of lipid bilayers containing the viral receptor, disialoganglioside GD1a. While simulated GD1a molecules self-associated independent of cholesterol, the dissociation rate between pairs of GD1a molecules was a function of bilayer cholesterol concentration. Additionally, cholesterol increased the order parameter of simulated GD1a lipid tails. We suggest that, by preordering the viral receptor in its monomeric state, cholesterol lowers the entropic penalty of receptor association. This in turn promotes the formation of GD1a multimers and increases the influenza virus binding avidity of the lipid bilayer. Our findings assign a critical role to the host cellular membrane in viral infectivity and reveal sterol-dependent membrane organization not associated with phase separation.