An Asymmetry in Monolayer Tension Regulates Lipid Droplet Budding Direction

Cells store excess energy in the form of neutral lipids that are synthesized and encapsulated within the endoplasmic reticulum intermonolayer space. The lipids next demix to form lipid droplets (LDs), which, surprisingly, bud off mostly toward the cytosol. This directional LD formation is critical to energy metabolism, but its mechanism remains poorly understood. Here, we reconstituted the LD formation topology by embedding artificial LDs into the intermonolayer space of bilayer vesicles. We provide experimental evidence that the droplet behavior in the membrane is recapitulated by the physics of three-phase wetting systems, dictated by the equilibrium of surface tensions. We thereupon determined that slight tension asymmetries between the membrane monolayers regulate the droplet budding side. A differential regulation of lipid or protein composition around a forming LD can generate a monolayer tension asymmetry that will determine the LD budding side. Our results offer, to our knowledge, new insights on how the proteins might regulate LD formation side by generating a monolayer tension asymmetry.     

A nanohybrid membrane with lipid bilayer-like properties utilized as a conductimetric saccharin sensor

Since their introduction, artificial lipid bilayer membranes were used in a wide array of applications, such as sensors, biocompatible materials and study-models of the cell's outer boundary. Here, we present a nanohybrid membrane using an inorganic host and amphiphilic organic molecules with lipid bilayer-like properties. The stability of the presented mimetic membrane is significantly improved when compared to existing methods. The nanohybrid membrane exhibited two thermotropic phases corresponding to the L(alpha) and L(beta) phases that lipid bilayer membranes are known to adopt. Integration of cholesterol molecules into the nanohybrid membrane lead to the same qualitative effects as in lipid bilayers, including expansion of the bilayer spacing and decrease of the L(alpha) to L(beta) transition enthalpy. To further illustrate the similarities of the synthesized membrane with a lipid bilayer, the ability of the nanohybrid membrane to function as saccharin conductimetric sensor was evaluated. The lower limit of detection of the sensor was 6 microM and the linear range of response was from 20 to 400 microM.     

A general mechanism for drug promiscuity: Studies with amiodarone and other antiarrhythmics

Amiodarone is a widely prescribed antiarrhythmic drug used to treat the most prevalent type of arrhythmia, atrial fibrillation (AF). At therapeutic concentrations, amiodarone alters the function of many diverse membrane proteins, which results in complex therapeutic and toxicity profiles. Other antiarrhythmics, such as dronedarone, similarly alter the function of multiple membrane proteins, suggesting that a multipronged mechanism may be beneficial for treating AF, but raising questions about how these antiarrhythmics regulate a diverse range of membrane proteins at similar concentrations. One possible mechanism is that these molecules regulate membrane protein function by altering the common environment provided by the host lipid bilayer. We took advantage of the gramicidin (gA) channels’ sensitivity to changes in bilayer properties to determine whether commonly used antiarrhythmics—amiodarone, dronedarone, propranolol, and pindolol, whose pharmacological modes of action range from multi-target to specific—perturb lipid bilayer properties at therapeutic concentrations. Using a gA-based fluorescence assay, we found that amiodarone and dronedarone are potent bilayer modifiers at therapeutic concentrations; propranolol alters bilayer properties only at supratherapeutic concentration, and pindolol has little effect. Using single-channel electrophysiology, we found that amiodarone and dronedarone, but not propranolol or pindolol, increase bilayer elasticity. The overlap between therapeutic and bilayer-altering concentrations, which is observed also using plasma membrane–like lipid mixtures, underscores the need to explore the role of the bilayer in therapeutic as well as toxic effects of antiarrhythmic agents.     

One amino acid drives the lipid droplet targeting sequence of a new noncoding RNA-encoded protein to mitochondrion

Over the past decade, the majority of the mammalian genome considered to be noncoding has been revealed to be able to produce proteins. Many RNA molecules, mis-annotated as noncoding, actually are predicted to code for proteins. Some of those proteins have been identified and verified to play critical roles in multiple biological processes. The lipid droplet (LD) is a unique cellular organelle bound with a phospholipid monolayer membrane, and is closely associated with cellular lipid metabolism and metabolic disorders. However, it is still unclear how a protein targets to LDs. Here we identified a new protein on LDs, LDANP2, which is encoded by noncoding RNA, through a proteomics-based strategy. The key sequence for its localization on LDs, Truncation 3, is predicted to form an amphipathic helix. Surprisingly, the deletion of the first amino acid in Truncation 3 resulted in mitochondrial localization. How the types of amino acids would determine the LD or mitochondrial localizations of the protein was studied. The findings introduce a useful strategy to mine for new proteins and would provide clues to the understanding of how a protein would find its right organelle, with phospholipid monolayer or bilayer membrane.     

Concentration effects of volatile anesthetics on the properties of model membranes: a coarse-grain approach

To gain insights into the molecular level mechanism of drug action at the membrane site, we have carried out extensive molecular dynamics simulations of a model membrane in the presence of a volatile anesthetic using a coarse-grain model. Six different anesthetic (halothane)/lipid (dimyristoylphosphatidylcholine) ratios have been investigated, going beyond the low doses typical of medical applications. The volatile anesthetics were introduced into a preassembled fully hydrated 512-molecule lipid bilayer and each of the molecular dynamics simulations were carried out at ambient conditions, using the NPT ensemble. The area per lipid increases monotonically with the halothane concentration and the lamellar spacing decreases, whereas the lipid bilayer thickness shows no appreciable differences and only a slight increase upon addition of halothane. The density profiles of the anesthetic molecules display a bimodal distribution along the membrane normal with maxima located close to the lipid-water interface region. We have studied how halothane molecules fluctuate between the two maxima of the bimodal distribution and we observed a different mechanism at low and high anesthetic concentrations. Through the investigation of the reorientational motions of the lipid tails, we found that the anesthetic molecules increase the segmental order of the lipids close to the membrane surface.     

Membrane-protein integration and the role of the translocation channel

Most eukaryotic membrane proteins are integrated into the lipid bilayer during their synthesis at the endoplasmic reticulum (ER). Their integration occurs with the help of a protein-conducting channel formed by the heterotrimeric Sec61 membrane-protein complex. The crystal structure of an archaeal homolog of the complex suggests mechanisms that enable the channel to open across the membrane and to release laterally hydrophobic transmembrane segments of nascent membrane proteins into lipid. Many aspects of membrane-protein integration remain controversial and poorly understood, but new structural data provide testable hypotheses. We propose a model of how the channel recognizes transmembrane segments, orients them properly with respect to the plane of the membrane and releases them into lipid. We also discuss how the channel would prevent small molecules from crossing the lipid bilayer while it is integrating proteins.     

Lipid-protein interactions

Intrinsic membrane proteins are solvated by a shell of lipid molecules interacting with the membrane-penetrating surface of the protein; these lipid molecules are referred to as annular lipids. Lipid molecules are also found bound between transmembrane α-helices; these are referred to as non-annular lipids. Annular lipid binding constants depend on fatty acyl chain length, but the dependence is less than expected from models based on distortion of the lipid bilayer alone. This suggests that hydrophobic matching between a membrane protein and the surrounding lipid bilayer involves some distortion of the transmembrane α-helical bundle found in most membrane proteins, explaining the importance of bilayer thickness for membrane protein function. Annular lipid binding constants also depend on the structure of the polar headgroup region of the lipid, and hotspots for binding anionic lipids have been detected on some membrane proteins; binding of anionic lipid molecules to these hotspots can be functionally important. Binding of anionic lipids to non-annular sites on membrane proteins such as the potassium channel KcsA can also be important for function. It is argued that the packing preferences of the membrane-spanning α-helices in a membrane protein result in a structure that matches nicely with that of the surrounding lipid bilayer, so that lipid and protein can meet without either having to change very much.     

Caveolin-2 is targeted to lipid droplets, a new "membrane domain" in the cell

Caveolin-1 and -2 constitute a framework of caveolae in nonmuscle cells. In the present study, we showed that caveolin-2, especially its beta isoform, is targeted to the surface of lipid droplets (LD) by immunofluorescence and immunoelectron microscopy, and by subcellular fractionation. Brefeldin A treatment induced further accumulation of caveolin-2 along with caveolin-1 in LD. Analysis of mouse caveolin-2 deletion mutants revealed that the central hydrophobic domain (residues 87-119) and the NH(2)-terminal (residues 70-86) and COOH-terminal (residues 120-150) hydrophilic domains are all necessary for the localization in LD. The NH(2)- and COOH-terminal domains appeared to be related to membrane binding and exit from ER, respectively, implying that caveolin-2 is synthesized and transported to LD as a membrane protein. In conjunction with recent findings that LD contain unesterified cholesterol and raft proteins, the result implies that the LD surface may function as a membrane domain. It also suggests that LD is related to trafficking of lipid molecules mediated by caveolins.     

Setting up and optimization of membrane protein simulations

In this paper we describe a method for setting up an atomistic simulation of a membrane protein in a hydrated lipid bilayer and report the effect of differing electrostatic parameters on the drift in the protein structure during the subsequent simulation. The method aims to generate a suitable cavity in the interior of a lipid bilayer, using the solvent-accessible surface of the protein as a template, during the course of a short steered molecular dynamics simulation of a solvated lipid membrane. This is achieved by a two-stage process: firstly, lipid molecules whose headgroups are inside a cylindrical volume equivalent to that defined by the protein surface are removed; then the protein-lipid interface is optimized by applying repulsive forces perpendicular to the protein surface, and of gradually increased magnitude, to the remaining lipid atoms inside the volume occupied by the protein surface until it is emptied. The protein itself may then be inserted. Using the bacterial membrane proteins KcsA and FhuA as test cases, we show how the method achieves the formation of a suitable cavity in the interior of a dimyristoylphosphatidylcholine lipid bilayer without perturbing the configuration of the non-interfacial regions of the previously equilibrated lipid bilayer, even in cases of membrane proteins with irregular geometrical shapes. In addition, we compare subsequent simulations in which the long-range electrostatic interactions are treated via either a cut-off or a particle-mesh Ewald method. The results show that the drift from the initial structure is less in the latter case, especially for KcsA and for the non-core secondary structural elements (i.e. surface loops) of both proteins.     

The organization of melatonin in lipid membranes

Melatonin is a hormone that has been shown to have protective effects in several diseases that are associated with cholesterol dysregulation, including cardiovascular disease, Alzheimer's disease, and certain types of cancers. We studied the interaction of melatonin with model membranes made of dimyristoylphosphatidylcholine (DMPC) at melatonin concentrations ranging from 0.5 mol% to 30 mol%. From 2-dimensional X-ray diffraction measurements, we find that melatonin induces a re-ordering of the lipid membrane that is strongly dependent on the melatonin concentration. At low melatonin concentrations, we observe the presence of melatonin-enriched patches in the membrane, which are significantly thinner than the lipid bilayer. The melatonin molecules were found to align parallel to the lipid tails in these patches. At high melatonin concentrations of 30 mol%, we observe a highly ordered melatonin structure that is uniform throughout the membrane, where the melatonin molecules align parallel to the bilayers and one melatonin molecule associates with 2 lipid molecules. Understanding the organization and interactions of melatonin in membranes, and how these are dependent on the concentration, may shed light into its anti-amyloidogenic, antioxidative and photoprotective properties and help develop a structural basis for these properties.     

Molecular study of the diffusional process of DMSO in double lipid bilayers

As a way to quantify the diffusion process of molecular compounds through biological membranes, we investigated in this study the dynamics of DMSO through an 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC) bilayer system. To properly account for the diffusion of DMSO due to a concentration gradient, a double DPPC bilayer was setup for our simulations. In such configuration, the aqueous phases can be explicitly associated with the extra and intracellular domains of the membrane, which is seldom the case in studies of single lipid bilayer due to the periodicity imposed by the simulations. DMSO molecules were initially contained in one of the aqueous phases (extracellular region) at a concentration of 5 wt.%. Molecular dynamics simulation was performed in this system for 95 ns at 350 K and 1 bar. The simulations showed that although many DMSO molecules penetrated the lipid bilayer, only about 10% of them crossed the bilayer to reach the other aqueous phase corresponding to the intracellular region of the membrane. The simulation time considered was insufficient to reach equilibrium of the DMSO concentration between the aqueous phases. However, the simulations provided sufficient information to estimate parameters to apply Fick's Law to model the diffusion process of the system. Using this model, we predicted that for the time considered in our simulation, the concentration of DMSO in the intracellular domain should have been about half of the actual value obtained. The model also predicted that equilibrium of the DMSO concentration in the system would be reached after about 2000 ns, approximately 20 times longer than the performed simulation.     

Cholesterol induced asymmetry in DOPC bilayers probed by AFM force spectroscopy

Cholesterol induced mechanical effects on artificial lipid bilayers are well known and have been thoroughly investigated by AFM force spectroscopy. However, dynamics of cholesterol impingement into bilayers at various cholesterol concentrations and their effects have not been clearly understood. In this paper we present, the effect of cholesterol as a function of its concentration in a simple single component dioleoylphosphatidylcholine (DOPC) bilayer. The nature of measured breakthrough forces on a bilayer with the addition of cholesterol, suggested that it is not just responsible to increase the mechanical stability but also introduces irregularities across the leaflets of the bilayer. This cholesterol induced asymmetry across the (in the inner and outer leaflets) bilayer is related to the phenomena of interleaflet coupling and is a function of cholesterol concentration probed by AFM can provide an unprecedented direction on mechanical properties of lipid membrane as it can be directly correlated to biophysical properties of a cell membrane.     

Hepatitis C virus core protein uses triacylglycerols to fold onto the endoplasmic reticulum membrane

Lipid droplets (LDs) are involved in viral infections, but exactly how remains unclear. Here, we study the hepatitis C virus (HCV) whose core capsid protein binds to LDs but is also involved in the assembly of virions at the endoplasmic reticulum (ER) bilayer. We found that the amphipathic helix-containing domain of core, D2, senses triglycerides (TGs) rather than LDs per se. In the absence of LDs, D2 can bind to the ER membrane but only if TG molecules are present in the bilayer. Accordingly, the pharmacological inhibition of the diacylglycerol O-acyltransferase enzymes, mediating TG synthesis in the ER, inhibits D2 association with the bilayer. We found that TG molecules enable D2 to fold into alpha helices. Sequence analysis reveals that D2 resembles the apoE lipid-binding region. Our data support that TG in LDs promotes the folding of core, which subsequently relocalizes to contiguous ER regions. During this motion, core may carry TG molecules to these regions where HCV lipoviroparticles likely assemble. Consistent with this model, the inhibition of Arf1/COPI, which decreases LD surface accessibility to proteins and ER-LD material exchange, severely impedes the assembly of virions. Altogether, our data uncover a critical function of TG in the folding of core and HCV replication and reveals, more broadly, how TG accumulation in the ER may provoke the binding of soluble amphipathic helix-containing proteins to the ER bilayer.     

Modeling of full-length Piezo1 suggests importance of the proximal N-terminus for dome structure

Piezo1 forms a mechanically activated calcium-permeable nonselective cation channel that is functionally important in many cell types. Structural data exist for C-terminal regions, but we lack information about N-terminal regions and how the entire channel interacts with the lipid bilayer. Here, we use computational approaches to predict the three-dimensional structure of the full-length Piezo1 and simulate it in an asymmetric membrane. A number of novel insights are suggested by the model: 1) Piezo1 creates a trilobed dome in the membrane that extends beyond the radius of the protein, 2) Piezo1 changes the lipid environment in its vicinity via preferential interactions with cholesterol and phosphatidylinositol 4,5-bisphosphate (PIP₂) molecules, and 3) cholesterol changes the depth of the dome and PIP₂ binding preference. In vitro alteration of cholesterol concentration inhibits Piezo1 activity in a manner complementing some of our computational findings. The data suggest the importance of N-terminal regions of Piezo1 for dome structure and membrane cholesterol and PIP₂ interactions.     

Effects of cholesterol concentration on the interaction of cytarabine with lipid membranes: a molecular dynamics simulation study

Liposomal cytarabine, DepoCyt, is a chemotherapy agent which is used in cancer treatment. This form of cytarabine has more efficacy and fewer side effects relative to the other forms. Since DepoCyt contains the cytarabine encapsulated within phosphatidylcholine and the sterol molecules, we modeled dioleoylphosphatidylcholine (DOPC)/cholesterol bilayer membrane as a carrier for cytarabine to study drug–bilayer interactions. For this purpose, we performed a series of united-atom molecular dynamics (MD) simulations for 25?ns to investigate the interactions between cytarabine and cholesterol-containing DOPC lipid bilayers. Only the uncharged form of cytarabine molecule was investigated. In this study, different levels of the cholesterol content (0, 20, and 40%) were used. MD simulations allowed us to determine dynamical and structural properties of the bilayer membrane and to estimate the preferred location and orientation of the cytarabine molecule inside the bilayer membrane. Properties such as membrane thickness, area per lipid, diffusion coefficient, mass density, bilayer packing, order parameters, and intermolecular interactions were examined. The results show that by increasing the cholesterol concentration in the lipid bilayers, the bilayer thickness increases and area per lipid decreases. Moreover, in accordance with the experiments, our calculations show that cholesterol molecules have ordering effect on the hydrocarbon acyl chains. Furthermore, the cytarabine molecule preferentially occupies the polar region of the lipid head groups to form specific interactions (hydrogen bonds). Our results fully support the experimental data. Our finding about drug–bilayer interaction is crucial for the liposomal drug design.     

Insertion of amphiphilic molecules into membranes is catalyzed by a high molecular weight non-ionic surfactant

We have employed an amphiphilic fluorescent probe to elucidate the mechanism by which a class of oxyethylene-oxypropylene copolymers catalyzes the insertion of hydrophobic or amphiphilic molecules into membranes. The rate of binding can be accelerated by over two orders of magnitude in the presence of the catalyst which does not itself disrupt the lipid bilayer. The rate of probe binding to lipid vesicles does not depend on the lipid concentration in the presence or absence of catalyst but is linearly related to the concentration of the catalyst. Probe binding to the polyol surfactant appears to be a component of the catalytic mechanism and equilibrium binding parameters can be determined; these are used to indirectly establish quantitative binding parameters for the probe to the vesicle membrane. The polyol surfactant is also shown to catalyze insertion of the probe into the outer leaflet of a hemispherical lipid bilayer and the plasma membrane of HeLa cells. The latter were also stained by catalyzed transfer of a fluorescent lipid from lipid vesicles. The permeability of the cell membrane is not significantly altered under any of the catalytic conditions. These data, taken together, suggest that the polyol surfactant extracts a monomeric substrate molecule from its aggregate or microcrystal and passes it to the membrane via a loose and transient contact.     

Interaction of salicylate and a terpenoid plant extract with model membranes: reconciling experiments and simulations

We investigate the effects of two structurally similar small cyclic molecules: salicylic acid and perillic acid on a zwitterionic model lipid bilayer, and show that both molecules might have biological activity related to membrane thinning. Salicylic acid is a nonsteroidal antiinflammatory drug, some of the pharmacological properties of which arise from its interaction with the lipid bilayer component of the plasma membrane. Prior simulations show that salicylate orders zwitterionic lipid membranes. However, this is in conflict with Raman scattering and vesicle fluctuation analysis data, which suggest the opposite. We show using extensive molecular dynamics simulations, cumulatively >2.5 μs, that salicylic acid indeed disorders membranes with concomitant membrane thinning and that the conflict arose because prior simulations suffered from artifacts related to the sodium-ion induced condensation of zwitterionic lipids modeled by the Berger force field. Perillic acid is a terpenoid plant extract that has antiinfective and anticancer properties, and is extensively used in eastern medicine. We found that perillic acid causes large-scale membrane thinning and could therefore exert its antimicrobial properties via a membrane-lytic mechanism reminiscent of antimicrobial peptides. Being more amphipathic, perillic acid is more potent in disrupting lipid headgroup packing, and significantly modifies headgroup dipole orientation. Like salicylate, the membrane thinning effect of perillic acid is masked by the presence of sodium ions. As an alternative to sodium cations, we advocate the straightforward solution of using larger countercations like potassium or tetra-methyl-ammonium that will maintain electroneutrality but not interact strongly with, and thus not condense, the lipid bilayer.     

Cell penetrating peptide modulation of membrane biomechanics by Molecular dynamics

The efficacy of a pharmaceutical treatment is often countered by the inadequate membrane permeability, that prevents drugs from reaching their specific intracellular targets. Cell penetrating peptides (CPPs) are able to route across cells’ membrane various types of cargo, including drugs and nanoparticles. However, CPPs internalization mechanisms are not yet fully understood and depend on a wide variety of aspects. In this contest, the entry of a CPP into the lipid bilayer might induce molecular conformational changes, including marked variations on membrane’s mechanical properties. Understanding how the CPP does influence the mechanical properties of cells membrane is crucial to design, engineer and improve new and existing penetrating peptides. Here, all atom Molecular Dynamics (MD) simulations were used to investigate the interaction between different types of CPPs embedded in a lipid bilayer of dioleoyl phosphatidylcholine (DOPC). In a greater detail, we systematically highlighted how CPP properties are responsible for modulating the membrane bending modulus. Our findings highlighted the CPP hydropathy strongly correlated with penetration of water molecules in the lipid bilayer, thus supporting the hypothesis that the amount of water each CPP can route inside the membrane is modulated by the hydrophobic and hydrophilic character of the peptide. Water penetration promoted by CPPs leads to a local decrease of the lipid order, which emerges macroscopically as a reduction of the membrane bending modulus.     

Models of protein lateral arrangements in lipid bilayer membranes. Application to electron spin resonance studies of cytochrome c oxidase

We consider the situation of integral membrane proteins in a lipid bilayer matrix where the size of the polar group of the protein is important in determining the lateral packing of the proteins. We represent the cross-section of the protein hydrophobic core as a hexagon moving on a lattice, and represent the projection of the polar group onto the plane of the bilayer as a shape, parts of which overlap the hexagon. Lattice sites represent lipid molecules. We calculate the fraction of lipid molecules which are adjacent to the hydrophobic core of at least one protein. We use this data to consider the "motion restricted" spectrum observed in electron spin resonance (ESR) probe studies, and compute the dependence of the "motion restricted" fraction upon protein concentration. The resulting curves can be used to analyse ESR data in order to deduce the size and shape of the proteins' polar segment. We have used the range of models examined to study the dependence upon protein concentration of the particular case of the "motion restricted" spectrum of a spin-labelled lipid freely diffusing or, alternatively, covalently bound to cytochrome c oxidase. We find that our calculations are in accord with a model where approximately 60 lipid molecules can fit around an isolated such protein in both halves of the bilayer, and where the polar segment is substantially anisotropic and extends laterally beyond the limits of the hydrophobic core. The latter is in accord with what is known about the structure of cytochrome c oxidase. We indicate further measurements that should be performed in order to establish more definitively the dependence of the "motion restricted" component upon protein concentration, giving the lipid protein ratios at which they should be performed, and we make predictions concerning the results. Finally we argue for a particular unified way of plotting experimental data.     

ESR and monolayer study of the localization of coenzyme Q10 in artificial membranes

The data obtained from the ESR experiments show a complex, depth dependent effect of CoQ10 on the lipid molecules mobility in the bilayer. These effects depend both on its concentration and the temperature. CoQ10 disturbs not only the hydrophobic core of the membrane but also the region close to the hydrophilic headgroups of phospholipids. Both these effects could be explained by the fact that the high hydrophobicity of CoQ10 causes the molecules to position itself in the interior of the bilayer, but at the same time its water seeking headgroup is located close to the region of the polar headgrops of membrane lipids. The presence of CoQ10 in the hydrophobic core has further implications on the properties of membrane intrinsic domain. Results of monolayer experiments indicate that CoQ10 may form aggregates when mixed with PC molecules in the lipid hydrocarbon chain-length dependent manner. CoQ10 is not fully miscible with DMPC or DPPC but it is well miscible with the long-chain DSPC molecules. Our suggestion is that CoQ10 when present in long-chain phospholipid bilayer, interacts with saturated fatty acyl-chains and adapt the structure which allows such interactions: either parallel to the saturated acyl chains or "pseudo-ring" conformation resembling sterol structure.