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Conserved signaling cascades monitor protein-folding homeostasis to ensure proper cellular function. One of the evolutionary conserved key players is IRE1, which maintains endoplasmic reticulum (ER) homeostasis through the unfolded protein response (UPR). Upon accumulation of misfolded proteins in the ER, IRE1 forms clusters on the ER membrane to initiate UPR signaling. What regulates IRE1 cluster formation is not fully understood. Here, we show that the ER lumenal domain (LD) of human IRE1α forms biomolecular condensates in vitro. IRE1α LD condensates were stabilized both by binding to unfolded polypeptides as well as by tethering to model membranes, suggesting their role in assembling IRE1α into signaling-competent stable clusters. Molecular dynamics simulations indicated that weak multivalent interactions drive IRE1α LD clustering. Mutagenesis experiments identified disordered regions in IRE1α LD to control its clustering in vitro and in cells. Importantly, dysregulated clustering of IRE1α mutants led to defects in IRE1α signaling. Our results revealed that disordered regions in IRE1α LD control its clustering and suggest their role as a common strategy in regulating protein assembly on membranes.
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Biological membranes are highly complex supramolecular assemblies, which play central roles in biology. However, their complexity makes them challenging to study their nanoscale structures. To overcome this challenge, model membranes assembled using reduced sets of membrane-associated biomolecules have been found to be both excellent and tractable proxies for biological membranes. Due to their relative simplicity, they have been studied using a range of biophysical characterization techniques. In this review article, we will briefly detail the use of fluorescence and electron microscopies, and X-ray and neutron scattering techniques used over the past few decades to study the nanostructure of biological membranes.
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Microscopia , Nêutrons , Biofísica , Membrana Celular , LipídeosRESUMO
Tumor-derived extracellular vesicles (T-EVs) represent valuable markers for tumor diagnosis and treatment guidance. However, nanoscale sizes and the low abundance of marker proteins of T-EVs restrict interfacial affinity reaction, leading to low isolation efficiency and detection sensitivity. Here, we engineer a fluid nanoporous microinterface (FluidporeFace) in a microfluidic chip by decorating supported lipid bilayers (SLBs) on nanoporous herringbone microstructures with a multiscale-enhanced affinity reaction for efficient isolation of T-EVs. At the microscale level, the herringbone micropattern promotes the mass transfer of T-EVs to the surface. At the nanoscale level, nanoporousity can overcome boundary effects for close contact between T-EVs and the interface. At the molecular level, fluid SLBs afford clustering of recognition molecules at the binding site, enabling multivalent binding with an â¼83-fold increase of affinity compared with the nonfluid interface. With the synergetic enhanced mass transfer, interface contact, and binding affinity, FluidporeFace affords ultrasensitive detection of T-EVs with a limit of detection of 10 T-EVs µL-1, whose PD-L1 expression levels successfully distinguish cancer patients from healthy donors. We expect this multiscale enhanced interfacial reaction strategy will inspire the biosensor design and expand liquid biopsy applications, especially for low-abundant targets in clinical samples.
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Técnicas Biossensoriais , Vesículas Extracelulares , Nanoporos , Neoplasias , Humanos , Vesículas Extracelulares/metabolismo , Microfluídica , Neoplasias/diagnóstico , Neoplasias/metabolismoRESUMO
Biological supramolecular assemblies, such as phospholipid bilayer membranes, have been used to demonstrate signal processing via short-term synaptic plasticity (STP) in the form of paired pulse facilitation and depression, emulating the brain's efficiency and flexible cognitive capabilities. However, STP memory in lipid bilayers is volatile and cannot be stored or accessed over relevant periods of time, a key requirement for learning. Using droplet interface bilayers (DIBs) composed of lipids, water and hexadecane, and an electrical stimulation training protocol featuring repetitive sinusoidal voltage cycling, we show that DIBs displaying memcapacitive properties can also exhibit persistent synaptic plasticity in the form of long-term potentiation (LTP) associated with capacitive energy storage in the phospholipid bilayer. The time scales for the physical changes associated with the LTP range between minutes and hours, and are substantially longer than previous STP studies, where stored energy dissipated after only a few seconds. STP behavior is the result of reversible changes in bilayer area and thickness. On the other hand, LTP is the result of additional molecular and structural changes to the zwitterionic lipid headgroups and the dielectric properties of the lipid bilayer that result from the buildup of an increasingly asymmetric charge distribution at the bilayer interfaces.
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Potenciação de Longa Duração , Fosfolipídeos , Potenciação de Longa Duração/fisiologia , Fosfolipídeos/química , Bicamadas Lipídicas/química , Plasticidade Neuronal/fisiologia , Água/químicaRESUMO
The expansion of T cells ex vivo is crucial for effective immunotherapy but currently limited by a lack of expansion approaches that closely mimic in vivo T cell activation. Taking inspiration from bottom-up synthetic biology, a new synthetic cell technology is introduced based on dispersed liquid-liquid phase-separated droplet-supported lipid bilayers (dsLBs) with tunable biochemical and biophysical characteristics, as artificial antigen presenting cells (aAPCs) for ex vivo T cell expansion. These findings obtained with the dsLB technology reveal three key insights: first, introducing laterally mobile stimulatory ligands on soft aAPCs promotes expansion of IL-4/IL-10 secreting regulatory CD8+ T cells, with a PD-1 negative phenotype, less prone to immune suppression. Second, it is demonstrated that lateral ligand mobility can mask differential T cell activation observed on substrates of varying stiffness. Third, dsLBs are applied to reveal a mechanosensitive component in bispecific Her2/CD3 T cell engager-mediated T cell activation. Based on these three insights, lateral ligand mobility, alongside receptor- and mechanosignaling, is proposed to be considered as a third crucial dimension for the design of ex vivo T cell expansion technologies.
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Cushioned lipid bilayers are structures consisting of a lipid bilayer supported on a solid substrate with an intervening layer of soft material. They offer possibilities for studying the behavior and interactions of biological membranes more accurately under physiological conditions. In this work, we continue our studies of cushion formation induced by histatin 5 (24Hst5), focusing on the effect of the length of the peptide chain. 24Hst5 is a short, positively charged, intrinsically disordered saliva peptide, and here, both a shorter (14Hst5) and a longer (48Hst5) peptide variant were evaluated. Experimental surface active techniques were combined with coarse-grained Monte Carlo simulations to obtain information about these peptides. Results show that at 10 mM NaCl, both the shorter and the longer peptide variants behave like 24Hst5 and a cushion below the bilayer is formed. At 150 mM NaCl, however, no interaction is observed for 24Hst5. On the contrary, a cushion is formed both in the case of 14Hst5 and 48Hst5, and in the latter, an additional thick, diffuse, and highly hydrated layer of peptide and lipid molecules is formed, on top of the bilayer. Similar trends were observed from the simulations, which allowed us to hypothesize that positively charged patches of the amino acids lysine and arginine in all three peptides are essential for them to interact with and translocate over the bilayer. We therefore hypothesize that electrostatic interactions are important for the interaction between the solid-supported lipid bilayers and the peptide depending on the linear charge density through the primary sequence and the positively charged patches in the sequence. The understanding of how, why, and when the cushion is formed opens up the possibility for this system to be used in the research and development of new drugs and pharmaceuticals.
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Histatinas , Bicamadas Lipídicas , Método de Monte Carlo , Bicamadas Lipídicas/química , Histatinas/química , Peptídeos Antimicrobianos/químicaRESUMO
Vital biological processes, such as trafficking, sensing, and motility, are facilitated by cellular lipid membranes, which interact mechanically with surrounding fluids. Such lipid membranes are only a few nanometers thick and composed of a liquid crystalline structure known as the lipid bilayer. Here, we introduce an active, noncontact, two-point microrheology technique combining multiple optical tweezers probes with planar freestanding lipid bilayers accessible on both sides. We use the method to quantify both fluid slip close to the bilayer surface and transmission of fluid flow across the structure, and we use numerical simulations to determine the monolayer viscosity and the intermonolayer friction. We find that these physical properties are highly dependent on the molecular structure of the lipids in the bilayer. We compare ordered-phase with liquid disordered-phase lipid bilayers, and we find the ordered-phase bilayers to be 10 to 100 times more viscous but with 100 times less intermonolayer friction. When a local shear is applied by the optical tweezers, the ultralow intermonolayer friction results in full slip of the two leaflets relative to each other and as a consequence, no shear transmission across the membrane. Our study sheds light on the physical principles governing the transfer of shear forces by and through lipid membranes, which underpin cell behavior and homeostasis.
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1,2-Dipalmitoilfosfatidilcolina/química , Membrana Celular/química , Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Fosfatidilcolinas/química , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Fenômenos Biomecânicos , Membrana Celular/metabolismo , Fricção , Hidrodinâmica , Dispositivos Lab-On-A-Chip , Bicamadas Lipídicas/metabolismo , Lipídeos de Membrana/metabolismo , Pinças Ópticas , Fosfatidilcolinas/metabolismo , Reologia , Propriedades de Superfície , ViscosidadeRESUMO
The plasma membrane forms the boundary between a living entity and its environment and acts as a barrier to permeation and flow of substances. Several computational means of calculating permeability have been implemented for molecular dynamics (MD) simulations-based approaches. Except for double bilayer systems, most permeability studies have been performed under equilibrium conditions, in large part due to the challenges associated with creating concentration gradients in simulations utilizing periodic boundary conditions. To enhance the scientific understanding of permeation and complement the existing computational means of characterizing membrane permeability, we developed a non-equilibrium method that enables the generation and maintenance of steady-state gradients in MD simulations. We utilize PBCs advantageously by imposing a directional bias to the motion of permeants so that their crossing of the boundary replenishes the gradient, like a previous study on ions. Under these conditions, a net flow of permeants across membranes may be observed to determine bulk permeability by a direct application of J=PΔc. In the present study, we explore the results of its application to an exemplary O2 and POPC bilayer system, demonstrating accurate and precise permeability measurements. In addition, we illustrate the impact of permeant concentration and the choice of thermostat on the permeability. Moreover, we demonstrate that energetics of permeation can be closely examined by the dissipation of the gradient across the membrane to gain nuanced insights into the thermodynamics of permeability.
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Febre Familiar do Mediterrâneo , Simulação de Dinâmica Molecular , Humanos , Membranas , Membrana Celular , Permeabilidade da Membrana CelularRESUMO
Current understanding of the structure and functioning of biomembranes is impossible without determining the mechanism of formation of membrane lipid rafts. The formation of liquid-ordered and disordered phases (Lo and Ld) and lipid rafts in membranes and their simplified models is discussed. A new consideration of the processes of formation of lipid phases Lo and Ld and lipid rafts is proposed, taking into account the division of each of the glycerophospholipids into several groups. Generally accepted three-component schemes for modeling the membrane structure are critically considered. A four-component scheme is proposed, which is designed to more accurately assume the composition of lipids in the resulting Lo and Ld phases. The role of the polar head groups of phospholipids and, in particular, phosphatidylethanolamine is considered. The structure of membrane rafts and the possible absence of a clear boundary between the Lo and Ld phases are discussed.
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Microdomínios da Membrana , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/química , Glicerofosfolipídeos/metabolismo , Glicerofosfolipídeos/química , Lipídeos de Membrana/metabolismo , Lipídeos de Membrana/química , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Animais , HumanosRESUMO
Liposomes serve as promising and versatile vehicles for drug delivery. Tracking these nanosized vesicles, particularly in vivo, is crucial for understanding their pharmacokinetics. This study introduces the design and synthesis of three new conjugated electrolyte (CE) molecules, which emit in the second near-infrared window (NIR-II), facilitating deeper tissue penetration. Additionally, these CEs, acting as biomimetics of lipid bilayers, demonstrate superior compatibility with lipid membranes compared to commonly used carbocyanine dyes like DiR. To counteract the aggregation-caused quenching effect, CEs employ a twisted backbone, as such their fluorescence intensities can effectively enhance after a fluorophore multimerization strategy. Notably, a "passive" method was employed to integrate CEs into liposomes during the liposome formation, and membrane incorporation efficiency was significantly promoted to nearly 100%. To validate the in vivo tracking capability, the CE-containing liposomes were functionalized with cyclic arginine-glycine-aspartic acid (cRGD) peptides, serving as tumor-targeting ligands. Clear fluorescent images visualizing tumor site in living mice were captured by collecting the NIR-II emission. Uniquely, these CEs exhibit additional emission peak in visible region, enabling in vitro subcellular analysis using routine confocal microscopy. These results underscore the potential of CEs as a new-generation of membrane-targeting probes to facilitate the liposome-based medicine research.
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Lipossomos , Neoplasias , Camundongos , Animais , Lipossomos/química , Bicamadas Lipídicas/química , Biomimética , Sistemas de Liberação de Medicamentos , Corantes Fluorescentes/químicaRESUMO
Recruitment of receptors at membrane interfaces is essential in biological recognition and uptake processes. The interactions that induce recruitment are typically weak at the level of individual interaction pairs, but are strong and selective at the level of recruited ensembles. Here, a model system is demonstrated, based on the supported lipid bilayer (SLB) that mimics the recruitment process induced by weakly multivalent interactions. The weak (mm range) histidine-nickel-nitrilotriacetate (His2 -NiNTA) pair is employed owing to its ease of implementation in both synthetic and biological systems. The recruitment of receptors (and ligands) induced by the binding of His2 -functionalized vesicles on NiNTA-terminated SLBs is investigated to identify the ligand densities necessary to achieve vesicle binding and receptor recruitment. Threshold values of ligand densities appear to occur in many binding characteristics: density of bound vesicles, size and receptor density of the contact area, and vesicle deformation. Such thresholds contrast the binding of strongly multivalent systems and constitute a clear signature of the superselective binding behavior predicted for weakly multivalent interactions. This model system provides quantitative insight into the binding valency and effects of competing energetic forces, such as deformation, depletion, and entropy cost of recruitment at different length scales.
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Bicamadas Lipídicas , Ligantes , MembranasRESUMO
Photosynthetic light harvesting requires efficient energy transfer within dynamic networks of light-harvesting complexes embedded within phospholipid membranes. Artificial light-harvesting models are valuable tools for understanding the structural features underpinning energy absorption and transfer within chromophore arrays. Here, a method for attaching a protein-based light-harvesting model to a planar, fluid supported lipid bilayer (SLB) is developed. The protein model consists of the tobacco mosaic viral capsid proteins that are gene-doubled to create a tandem dimer (dTMV). Assemblies of dTMV break the facial symmetry of the double disk to allow for differentiation between the disk faces. A single reactive lysine residue is incorporated into the dTMV assemblies for the site-selective attachment of chromophores for light absorption. On the opposing dTMV face, a cysteine residue is incorporated for the bioconjugation of a peptide containing a polyhistidine tag for association with SLBs. The dual-modified dTMV complexes show significant association with SLBs and exhibit mobility on the bilayer. The techniques used herein offer a new method for protein-surface attachment and provide a platform for evaluating excited state energy transfer events in a dynamic, fully synthetic artificial light-harvesting system.
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Fotossíntese , Proteínas , Transferência de Energia , Bicamadas Lipídicas/químicaRESUMO
Biological and technological processes that involve liquids under negative pressure are vulnerable to the formation of cavities. Maximal negative pressures found in plants are around -100 bar, even though cavitation in pure bulk water only occurs at much more negative pressures on the relevant timescales. Here, we investigate the influence of small solutes and lipid bilayers, both constituents of all biological liquids, on the formation of cavities under negative pressures. By combining molecular dynamics simulations with kinetic modeling, we quantify cavitation rates on biologically relevant length scales and timescales. We find that lipid bilayers, in contrast to small solutes, increase the rate of cavitation, which remains unproblematically low at the pressures found in most plants. Only when the negative pressures approach -100 bar does cavitation occur on biologically relevant timescales. Our results suggest that bilayer-based cavitation is what generally limits the magnitude of negative pressures in liquids that contain lipid bilayers.
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Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Pressão , CinéticaRESUMO
The protein AlkL is known to increase permeability of the outer membrane of bacteria for hydrophobic molecules, yet the mechanism of transport has not been determined. Differing crystal and NMR structures of homologous proteins resulted in a controversy regarding the degree of structure and the role of long extracellular loops. Here we solve this controversy by determining the de novo NMR structure in near-native lipid bilayers, and by accessing structural dynamics relevant to hydrophobic substrate permeation through molecular-dynamics simulations and by characteristic NMR relaxation parameters. Dynamic lateral exit sites large enough to accommodate substrates such as carvone or octane occur through restructuring of a barrel extension formed by the extracellular loops.
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Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Bicamadas Lipídicas/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Imageamento por Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética/métodos , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Permeabilidade , Estrutura Secundária de ProteínaRESUMO
This work provides mesoscale models for the anomalous diffusion of a polymer chain on a heterogeneous surface with rearranging randomly distributed adsorption sites. Both the "bead-spring" model and oxDNA model were simulated on supported lipid bilayer membranes with various molar fractions of charged lipids, using Brownian dynamics method. Our simulation results demonstrate that "bead-spring" chains exhibit sub-diffusion on charged lipid bilayers which agrees with previous experimental observations for short-time dynamics of DNA segments on membranes. In addition, the non-Gaussian diffusive behaviors of DNA segments have not been observed in our simulations. However, a simulated 17 base pairs double stranded DNA, using oxDNA model, performs normal diffusion on supported cationic lipid bilayers. Due to the number of positively charged lipids attracted by short DNA is small, the energy landscape that the short DNA experiences during diffusion is not as heterogeneous as that experienced by long DNA chains, which results in normal diffusion rather than sub-diffusion for short DNA.
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Apolipoprotein L-I (APOL1) is a channel-forming effector of innate immunity. The common human APOL1 variant G0 provides protection against infection with certain Trypanosoma and Leishmania parasite species, but it cannot protect against the trypanosomes responsible for human African trypanosomiasis. Human APOL1 variants G1 and G2 protect against human-infective trypanosomes but also confer a higher risk of developing chronic kidney disease. Trypanosome-killing activity is dependent on the ability of APOL1 to insert into membranes at acidic pH and form pH-gated cation channels. We previously mapped the channel's pore-lining region to the C-terminal domain (residues 332-398) and identified a membrane-insertion domain (MID, residues 177-228) that facilitates acidic pH-dependent membrane insertion. In this article, we further investigate structural determinants of cation channel formation by APOL1. Using a combination of site-directed mutagenesis and targeted chemical modification, our data indicate that the C-terminal heptad-repeat sequence (residues 368-395) is a bona fide leucine zipper domain (ZIP) that is required for cation channel formation as well as lysis of trypanosomes and mammalian cells. Using protein-wide cysteine-scanning mutagenesis, coupled with the substituted cysteine accessibility method, we determined that, in the open channel state, both the N-terminal domain and the C-terminal ZIP domain are exposed on the intralumenal/extracellular side of the membrane and provide evidence that each APOL1 monomer contributes four transmembrane domains to the open cation channel conformation. Based on these data, we propose an oligomeric topology model in which the open APOL1 cation channel is assembled from the coiled-coil association of C-terminal ZIP domains.
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Apolipoproteína L1/metabolismo , Canais Iônicos/química , Zíper de Leucina , Apolipoproteína L1/química , Cátions/metabolismo , Humanos , Conformação Proteica , Domínios ProteicosRESUMO
Knowledge of the interaction of the tau fibrils with the cell membrane is critical for the understanding of the underlying tauopathy pathogenesis. Lipid composition is found to affect the conformational ensemble of the tau fibrils. Using coarse-grained and all-atom molecular dynamics simulations we have shown the effect of the lipid composition in modulating the tau structure and dynamics. Molecular dynamics simulations show that tau proteins interact differentially with the zwitterionic compared to the charged lipid membranes. The negatively charged POPG lipid membranes increase the binding propensity of the tau fibrils. The addition of cholesterol is also found to modify the tau binding to the membrane. The binding of tau fibril leads to the concomitant loss of the ß-sheet structures across the tau residues alongside the change in the membrane properties (like area per lipid, bilayer thickness, and order parameter of the lipid tails) over the pure bilayers.
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Bicamadas Lipídicas , Simulação de Dinâmica Molecular , Bicamadas Lipídicas/química , Membrana Celular/metabolismo , Conformação Molecular , Conformação Proteica em Folha betaRESUMO
Antimicrobial peptide magainin 2 (Mag) forms nanopores in lipid bilayers and induces membrane permeation of the internal contents from vesicles. The binding of Mag to the membrane interface of a giant unilamellar vesicle (GUV) increases its fractional area change, δ, which is one of the main causes of Mag-induced nanopore formation. However, the role of its amino acid composition in the Mag-induced area increase and the following nanopore formation is not well understood. Here, to elucidate it we examined the role of interfacial hydrophobicity of Mag in its nanopore formation activity by investigating de novo-designed Mag mutants-induced nanopore formation in GUVs. Aligned amino acid residues in the α-helix of Mag were replaced to create 3 mutants: F5A-Mag, A9F-Mag, and F5,12,16A-Mag. These mutants have different interfacial hydrophobicity due to the variation of the numbers of Phe and Ala because the interfacial hydrophobicity of Phe is higher than that of Ala. The rate constant of Mag mutant-induced nanopore formation, kp, increased with increasing numbers of Phe residues at the same peptide concentration. Further, the Mag mutant-induced δ increased with increasing numbers of Phe residues at the same peptide concentration. These results indicate that kp and δ increase with increasing interfacial hydrophobicity of Mag mutants. The relationship between kp and δ in the Mag and its mutants clearly indicates that kp increases with increasing δ, irrespective of the difference in mutants. Based on these results, we can conclude that the interfacial hydrophobicity of Mag plays an important role in its nanopore formation activity.
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Anti-Infecciosos , Nanoporos , Aminoácidos , Antibacterianos , Anti-Infecciosos/química , Peptídeos Antimicrobianos , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Magaininas , Lipossomas Unilamelares/metabolismoRESUMO
Lipid droplets (LDs) are ubiquitous, cytoplasmic fat storage organelles that originate from the endoplasmic reticulum (ER) membrane. They are composed of a core of neutral lipids surrounded by a phospholipid monolayer. Proteins embedded into this monolayer membrane adopt a monotopic topology and are crucial for regulated lipid storage and consumption. A key question is, which collective properties of protein-intrinsic and lipid-mediated features determine spatio-temporal protein partitioning between phospholipid bilayer and LD monolayer membranes. To address this question, a freestanding phospholipid bilayer with physiological lipidic composition is produced using microfluidics and micrometer-sized LDs are dispersed around the bilayer that spontaneously insert into the bilayer. Using confocal microscopy, the 3D geometry of the reconstituted LDs is determined with high spatial resolution. The micrometer-sized bilayer-embedded LDs present a characteristic lens shape that obeys predictions from equilibrium wetting theory. Fluorescence recovery after photobleaching measurements reveals the existence of a phospholipid diffusion barrier at the monolayer-bilayer interface. Coarse-grained molecular dynamics simulation reveals lipid specific density distributions along the pore rim, which may rationalize the diffusion barrier. The lipid diffusion barrier between the LD covering monolayer and the bilayer may be a key phenomenon influencing protein partitioning between the ER membrane and LDs in living cells.
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Gotículas Lipídicas , Fosfolipídeos , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Gotículas Lipídicas/metabolismo , Simulação de Dinâmica Molecular , Fosfolipídeos/metabolismoRESUMO
The outer membrane (OM) of gram-negative bacteria is highly asymmetric. The outer leaflet comprises lipopolysaccharides (LPS) and the inner leaflet phospholipids. Here, it is shown that the outer membrane lipid bilayer (OMLB) of Escherichia coli can be reconstructed as a droplet interface bilayer (DIB), which separates two aqueous droplets in oil. The trimeric porin OmpF is inserted into the model OMLB and the translocation of the bacteriocin colicin E9 (colE9) through it is monitored. By contrast with LPS-free bilayers, it is found that colE9 made multiple failed attempts to engage with OmpF in an OMLB before successful translocation occurred. In addition, the observed rate for the second step of colE9 translocation is 3-times smaller than that in LPS-free bilayers, and further, the colE9 dissociates when the membrane potential is reversed. The findings demonstrate the utility of the DIB approach for constructing model OMLBs from physiologically realistic lipids and that the properties of the model OMLBs differ from those of a simple lipid bilayer. The model OMLB offers a credible platform for screening the properties of antibiotics.