Lipid Reorganization Research Articles

Influence of Free Fatty Acids on Lipid Membrane-Nisin Interaction.

The influence of free fatty acids (FFAs) on the nisin–membrane interaction was investigated through micro-DSC and fluorescence spectroscopy. A simple but informative model membrane was prepared (5.7 DMPC:3.8 DPPS:0.5 DOPC molar ratio) by considering the presence of different phospholipid headgroups in charge and size and different phospholipid tails in length and unsaturation level, allowing the discrimination of the combined interaction of nisin and FFAs with the single phospholipid constituents. The effects of six FFAs on membrane stability were evaluated, namely two saturated FFAs (palmitic acid and stearic acid), two monounsaturated FFAs (cis-unsaturated oleic acid and trans-unsaturated elaidic acid) and two cis-polyunsaturated FFAs (ω-6 linoleic acid and ω-3 docosahexaenoic acid). The results permitted assessment of a thermodynamic picture of such interactions which indicates that the peptide–membrane interaction does not overlook the presence of FFAs within the lipid bilayer since both FFAs and nisin are able to selectively promote thermodynamic phase separations as well as a general lipid reorganization within the host membrane. Furthermore, the magnitude of the effects may be different depending on the FFA chemical structure as well as the membrane lipid composition.

Molecular Dynamics Study of Lipid and Cholesterol Reorganization Due to Membrane Binding and Pore Formation by Listeriolysin O.

Pore-forming toxins are proteinsexpressed by bacteria to primarily cause infections in the host cell. Cholesterol-dependent cytolysins (CDCs) are a class of proteins whose pore-forming ability requires the presence of cholesterol in the membrane. Upon binding to the target cell, cholesterol-recognizing residues in the membrane binding D4 subdomain assist in stabilizing both the pre-pore and pore states which occur during protein oligomerization on the cell membrane. Super resolution-stimulated emission depletion (STED) microscopy experiments (Sarangi et al. in Langmuir, 32:9649-9657, 2016) on supported lipid bilayers have shown that listeriolysin (LLO), a CDC expressed by Listeria monocytogenes, a food-borne pathogen, induces both spatial and dynamic heterogeneity in bilayer membranes. Here, we use all-atom molecular dynamics simulations to explore molecular details of the induced membrane reorganization by considering two distinct states of the oligomerized LLO protein in a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC): cholesterol membrane. In the membrane bound (MB) state, four D4 subunits are placed at the bilayer interface in a pre-pore configuration and the membrane-inserted (MI) state consists of a tetrameric arc-like pore configuration. By analyzing lipid-order parameters, mobilities, and diffusion coefficients, we examine the induced spatial heterogeneity that occurs in both the MB and MI states. This heterogeneity is primarily driven by the local density enhancement of cholesterol in the vicinity of the MB, D4 subunits leading to distinct differences in lipid and cholesterol mobility across the two leaflets as well as enhanced lipid mobilities in regions where cholesterol is depleted. The leaflet-induced heterogeneity is greater for the MB state when compared with the MI state and the dynamic variations are more pronounced in the extracellular leaflet when compared with the cytosolic leaflet. Our study provides molecular-level insights into the inhomogeneity and perturbation induced in bilayer membranes upon LLO binding and pore formation and is expected to represent trends across PFTs in the broad CDC subclass of proteins.

Thermally induced structural organization of nanodiscs by coarse-grained molecular dynamics simulations

Membrane scaffold proteins (MSP) nanodiscs have been extensively used in structural study of membrane proteins. In cryo-EM, an incorporation of target proteins into nanodiscs is conducted under a rapid change from cryogenic to ambient temperatures. We present a coarse-grained molecular dynamics (CGMD) study for investigating an effect of temperature on the structural organization of DPPC-nanodisc and POPC-nanodisc. A non-monotonic response of physical quantities (i.e. the lipid order parameter, nanodisc flatness, structural change, solvation property, radius of gyration) with increase in temperature (T = 200–350 K) is found to be associated with the gel-ripple-liquid crystalline phase change within nanodiscs. The reorganization of lipids upon temperature variation induced conformational changes of MSP to minimize hydrophobic exposure of the lipid membrane to an aqueous environment. Structural response to temperature is different to a certain extent between the saturated DPPC and unsaturated POPC.

Light-induced lipid mixing implies a causal role of lipid splay in membrane fusion

The fusion of lipid membranes is central to many biological processes and requires substantial structural reorganization of lipids brought about by the action of fusogenic proteins. Previous molecular dynamics simulations have suggested that splayed lipids, whose tails transiently contact the headgroup region of the bilayer, initiate lipid mixing. Here, we explore the lipid splay hypothesis experimentally. We show that the light-induced trans/cis conversion of the azobenzene-based tail of a model lipid molecule enhances the probability by which its own acyl chains, or the acyl chains of the host lipid, transiently contact the lipid headgroup in a liposomal bilayer. At the same time, the trans/cis conversion triggers lipid mixing of sonicated or extruded liposomes, without requiring fusogenic proteins. This establishes a causal relationship between lipid splay and membrane fusion.

Pulmonary Surfactant Lipid Reorganization Induced by the Adsorption of the Oligomeric Surfactant Protein B Complex

Surfactant protein B (SP-B) is essential in transferring surface-active phospholipids from membrane-based surfactant complexes into the alveolar air–liquid interface. This allows maintaining the mechanical stability of the surfactant film under high pressure at the end of expiration; therefore, SP-B is crucial in lung function. Despite its necessity, the structure and the mechanism of lipid transfer by SP-B have remained poorly characterized. Earlier, we proposed higher-order oligomerization of SP-B into ring-like supramolecular assemblies. In the present work, we used coarse-grained molecular dynamics simulations to elucidate how the ring-like oligomeric structure of SP-B determines its membrane binding and lipid transfer. In particular, we explored how SP-B interacts with specific surfactant lipids, and how consequently SP-B reorganizes its lipid environment to modulate the pulmonary surfactant structure and function. Based on these studies, there are specific lipid–protein interactions leading to perturbation and reorganization of pulmonary surfactant layers. Especially, we found compelling evidence that anionic phospholipids and cholesterol are needed or even crucial in the membrane binding and lipid transfer function of SP-B. Also, on the basis of the simulations, larger oligomers of SP-B catalyze lipid transfer between adjacent surfactant layers. Better understanding of the molecular mechanism of SP-B will help in the design of therapeutic SP-B-based preparations and novel treatments for fatal respiratory complications, such as the acute respiratory distress syndrome.

Mechanism of activation of plant monogalactosyldiacylglycerol synthase 1 (MGD1) by phosphatidylglycerol.

Mono- and digalactosyldiacylglycerol are essential galactolipids for the biogenesis of plastids and functioning of the photosynthetic machinery. In Arabidopsis, the first step of galactolipid synthesis is catalyzed by monogalactosyldiacylglycerol synthase 1 (MGD1), a monotopic protein located in the inner envelope membrane of chloroplasts, which transfers a galactose residue from UDP-galactose to diacylglycerol (DAG). MGD1 needs anionic lipids such as phosphatidylglycerol (PG) to be active, but the mechanism by which PG activates MGD1 is still unknown. Recent studies shed light on the catalytic mechanism of MGD1 and on the possible PG binding site. Particularly, Pro189 was identified as a potential residue implicated in PG binding and His155 as the putative catalytic residue. In the present study, using a multifaceted approach (Langmuir membrane models, atomic force microscopy, molecular dynamics; MD), we investigated the membrane binding properties of native MGD1 and mutants (P189A and H115A). We demonstrated that both residues are involved in PG binding, thus suggesting the existence of a PG-His catalytic dyad that should facilitate deprotonation of the nucleophile hydroxyl group of DAG acceptor. Interestingly, MD simulations showed that MGD1 induces a reorganization of lipids by attracting DAG molecules to create an optimal platform for binding.

Inside Cover: Cellular transformations in near‐infrared light‐induced apoptosis in cancer cells revealed by label‐free CARS imaging (J. Biophotonics 12/2019)

CARS microscopy was employed to monitor the reorganization of intracellular lipids and proteins, as well as cellular transformations, after irradiation with near infrared (NIR) light. NIR light was shown to induce apoptosis in HeLa cells in vitro in a dose‐dependent manner. The progression of apoptosis assessed with CARS microscopy is apparently associated with the generation of reactive oxygen species followed by an excessive formation of lipid droplets and their peroxidation.Further details can be found in the article by Svitlana M. Levchenko, Andrey N. Kuzmin, Artem Pliss, et al. (e201900179) image

Helical Inclusions in Phospholipid Membranes: Lipid Adaptation and Chiral Order.

The lipid bilayer is a flexible matrix that is able to adapt in response to the perturbation induced by inclusions, such as peptides and proteins. Here we use molecular dynamics simulations with a coarse-grained model to investigate the effect of a helical inclusion on a lipid bilayer in the liquid disordered phase. We show that the helical inclusion induces a collective tilt of acyl chains, with a small, yet unambiguous difference between a right- and a left-handed inclusion. This behavior is rationalized using the elastic continuum theory: The magnitude of the chiral (twist) deformation of the bilayer is determined by the interaction at the lipid/inclusion interface, and the decay length is controlled by the elastic properties of the bilayer. The lipid reorganization can thus be identified as a generic mechanism that, together with specific interactions, contributes to chiral recognition in phospholipid bilayers. An enhanced response is expected in highly ordered environments, such as rafts in biomembranes, with a potential impact on membrane-mediated interactions between inclusions.

Cellular transformations in near‐infrared light‐induced apoptosis in cancer cells revealed by label‐free CARS imaging

Photobiomodulation (PBM) involves light to activate cellular signaling pathways leading to cell proliferation or death. In this work, fluorescence and Coherent anti-Stokes Raman Scattering (CARS) imaging techniques were applied to assess apoptosis in human cervical cancer cells (HeLa) induced by near infrared (NIR) laser light (808 nm). Using the Caspase 3/7 fluorescent probe to identify apoptotic cells, we found that the pro-apoptotic effect is significantly dependent of irradiation dose. The highest apoptosis rate was noted for the lower irradiation doses, that is, 0.3 J/cm2 (~58%) and 3 J/cm2 (~28%). The impact of light doses on proteins/lipids intracellular metabolism and distribution was evaluated using CARS imaging, which revealed apoptosis-associated reorganization of nuclear proteins and cytoplasmic lipids after irradiation with 0.3 J/cm2 . Doses of NIR light causing apoptosis (0.3, 3 and 30 J/cm2 ) induced a gradual increase in the nuclear protein level over time, in contrast to proteins in cells non-irradiated and irradiated with 10 J/cm2 . Furthermore, irradiation of the cells with the 0.3 J/cm2 dose resulted in lipid droplets (LDs) accumulation, which was apparently caused by an increase in reactive oxygen species (ROS) generation. We suggest that PBM induced apoptosis could be caused by the ability of NIR light to trigger excessive LDs formation which, in turn, induces cellular cytotoxicity.

All-Atom Molecular Dynamics Simulations of Dimeric Lung Surfactant Protein B in Lipid Multilayers.

Although lung surfactant protein B (SP-B) is an essential protein that plays a crucial role in breathing, the details of its structure and mechanism are not well understood. SP-B forms covalent homodimers, and in this work we use all-atom molecular dynamics simulations to study dimeric SP-B’s structure and its behavior in promoting lipid structural transitions. Four initial system configurations were constructed based on current knowledge of SP-B’s structure and mechanism, and the protein maintained a helicity consistent with experiment in all systems. Several SP-B-induced lipid reorganization behaviors were observed, and regions of the protein particularly important for these activities included SP-B’s “central loop” and “hinge” regions. SP-B dimers with one subunit initially positioned in each of two adjacent bilayers appeared to promote close contact between two bilayers. When both subunits were initially positioned in the same bilayer, SP-B induced the formation of a defect in the bilayer, with water penetrating into the centre of the bilayer. Similarly, dimeric SP-B showed a propensity to interact with preformed interpores in the bilayer. SP-B dimers also promoted bilayer thinning and creasing. This work fleshes out the atomistic details of the dimeric SP-B structures and SP-B/lipid interactions that underlie SP-B’s essential functions.

Correlated protein conformational states and membrane dynamics during attack by pore-forming toxins

Pore-forming toxins (PFTs) are a class of proteins implicated in a wide range of virulent bacterial infections and diseases. These toxins bind to target membranes and subsequently oligomerize to form functional pores that eventually lead to cell lysis. While the protein undergoes large conformational changes on the bilayer, the connection between intermediate oligomeric states and lipid reorganization during pore formation is largely unexplored. Cholesterol-dependent cytolysins (CDCs) are a subclass of PFTs widely implicated in food poisoning and other related infections. Using a prototypical CDC, listeriolysin O (LLO), we provide a microscopic connection between pore formation, lipid dynamics, and leakage kinetics by using a combination of Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) measurements on single giant unilamellar vesicles (GUVs). Upon exposure to LLO, two distinct populations of GUVs with widely different leakage kinetics emerge. We attribute these differences to the existence of oligomeric intermediates, sampling various membrane-bound conformational states of the protein, and their intimate coupling to lipid rearrangement and dynamics. Molecular dynamics simulations capture the influence of various membrane-bound conformational states on the lipid and cholesterol dynamics, providing molecular interpretations to the FRET and FCS experiments. Our study establishes a microscopic connection between membrane binding and conformational changes and their influence on lipid reorganization during PFT-mediated cell lysis. Additionally, our study provides insights into membrane-mediated protein interactions widely implicated in cell signaling, fusion, folding, and other biomolecular processes.

Membrane phospholipids remodeling upon imbibition in Brassica napus L. seeds

Successful seed germination depends on the rapid repair of cell membrane damaged by dry storage. However, little is known about the reorganization of lipids during this process. In this study, the changes of intracellular redox environment, cell membrane integrity, lipid composition, and expression of genes related to phospholipid metabolism were assessed during imbibition of Brassica napus seeds. A total number of 443 lipids belonging to 7 categories were detected by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS). In the 24 h-imbibed seeds, the relative content of triacylglycerol was lower than in dry seeds, while the relative content of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS), especially PC (36:2, number of carbons in the acyl chains: number of double bonds), PC (36:3), and PE (36:3) were higher than those in dry seeds. Meanwhile, the content and unsaturation levels of phospholipids increased, indicating membrane lipids remodeling during seed imbibition. The plasma membrane integrity, which was measured by the relative electrolyte leakage (REL) of the membrane and FM4-64 fluorescent dye, was improved upon imbibition, confirming that cell membrane was repaired after 24 h-imbibition. The reduction of H2O2 content, redox potential, and malondialdehyde (MDA) content indicated that the degree of membrane lipid peroxidation was significantly decreased upon imbibition. Gene expression analysis showed that the differential expression of genes for key enzymes occurred in the plateau phase of the imbibition curve, i.e. after 8 h-to 24 h-imbibition. Moreover, the differential expression of genes such as those encoding phospholipase C (PLC), phospholipase D (PLD), triacylglycerol lipase (TAG lipase), choline/ethanolamine phosphotransferase (CEPT), and phosphatidylserine synthase (PTDSS2) during imbibition indicated that membrane lipid remodeling was related to complex metabolic pathways, among which the degradation of triacylglycerol and the synthesis of phospholipids using diacylglycerol might play an important role during membrane remodeling.

Effects of Capsaicin on Biomimetic Membranes.

Capsaicin is a natural compound that produces a warm sensation and is known for its remarkable medicinal properties. Understanding the interaction between capsaicin with lipid membranes is essential to clarify the molecular mechanisms behind its pharmacological and biological effects. In this study, we investigated the effect of capsaicin on thermoresponsiveness, fluidity, and phase separation of liposomal membranes. Liposomal membranes are a bioinspired technology that can be exploited to understand biological mechanisms. We have shown that by increasing thermo-induced membrane excess area, capsaicin promoted membrane fluctuation. The effect of capsaicin on membrane fluidity was dependent on lipid composition. Capsaicin increased fluidity of (1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) membranes, while it rigidified DOPC and cholesterol-based liposomes. In addition, capsaicin tended to decrease phase separation of heterogeneous liposomes, inducing homogeneity. We imagine this lipid re-organization to be associated with the physiological warming sensation upon consumption of capsaicin. Since capsaicin has been reported to have biological properties such as antimicrobial and as antiplatelet, the results will help unravel these biological properties.

Mechanical signals regulate TORC2 activity.

Maintaining plasma membrane tension is important for eukaryotic cells. How altered membrane tension is sensed and relayed to downstream factors, such as the target of rapamyin complex 2 (TORC2), is poorly understood. Reorganization of a signalling lipid into discrete membrane domains is now shown to inactivate TORC2 in yeast.

Inducing Lipid Reorganization and the Acrosome Reaction in Stallion Spermatozoa

Inducing Lipid Reorganization and the Acrosome Reaction in Stallion Spermatozoa

Investigating Cholesterol Dynamics and Interactions with the Dopamine Transporter using a Membrane Mimetic Model

Cholesterol, an integral component of animal lipid membranes, is involved in modulation of both membrane physical properties and a wide variety of membrane proteins. Conventional computational studies of cholesterol-protein interactions have been either using coarse-grained methods or relying on > μs all-atom simulations due to the slow dynamics of lipids, in order to provide useful information. The highly mobile membrane-mimetic (HMMM) model has been previously reported to study interactions between peripheral membrane proteins and phospholipids with expedited lipid reorganization. Here, we have extended the use of the HMMM model to investigate interactions between integral membrane protein and cholesterol. Cholesterol dynamics were first closely examined in the HMMM model with extensive all-atom molecular dynamics simulations. Both lateral diffusion and flip-flop motion were shown to be significantly accelerated at cholesterol concentrations ranging from 5% to 50%, while atomic density profiles and internal dynamics of cholesterols with proper restraints in the HMMM membranes were found similar to those in full-length lipid membranes. The HMMM model was then applied to study cholesterol interactions with an integral membrane protein, dopamine transporter. The cholesterol binding sites and detailed interactions with the dopamine transporter observed in the HMMM membrane were compared with those from microsecond all-atom molecular dynamics simulations and experimental data. Our results show great potential and advantages of using the HMMM model to identify cholesterol binding sites with enhanced efficiency and clarify cholesterol-protein interactions without losing atomic details.

Passive membrane penetration by ZnO nanoparticles is driven by the interplay of electrostatic and phase boundary conditions.

The internalization of nanoparticles through the biological membrane is of immense importance for biomedical applications. A fundamental understanding of the lipid specificity and the role of the membrane biochemical and physical forces at play in modulating penetration are lacking. The current understanding of nanoparticle-membrane interaction is drawn mostly from computational studies and lacks sufficient experimental evidence. Herein, using confocal fluorescence imaging and potentiometric dye-based fluorimetry, we first investigated the interaction of ZnONP in both multi-component and individual lipid membranes using cell-like giant unilamellar vesicles to dissect the lipid specificity; also, we investigated the changes in membrane order, anisotropy and hydrophobicity. ZnONP was found to interact with phosphatidylinositol and phosphatidylcholine head-group-containing lipids specifically. We further investigated the interaction of ZnONP with three physiologically relevant membrane conditions varying in composition and dipole potential. We found that ZnONP interaction leads to a photoinduced enhancement of the partial-to-complete phase separation depending upon the membrane composition and cholesterol content. Interestingly, while the lipid order of a partially-phase-separated membrane remained unchanged upon ZnONP crowding, a fully-phase-separated membrane showed an increase in the lipid order. Strikingly, ZnONP crowding induced a contrasting effect on the fluorescence anisotropy of the membrane upon binding to the two membrane conditions, in line with the measured diffusion coefficient. ZnONP seems to preferentially penetrate through the liquid disordered areas of the membrane and the boundaries of the phase-separated regions driven by the interplay between the electrostatics and phase boundary conditions, which are collectively dictated by the composition and ZnONP-induced lipid reorganization. The results may lead to a greater understanding of the interplay of membrane parameters and ZnONP interaction in driving passive penetration.

Continuous Pore-Spanning Lipid Bilayers on Silicon Oxide-Coated Porous Substrates.

A number of techniques has been developed and analyzed in recent years to generate pore-spanning membranes (PSMs). While quite a number of methods rely on nanoporous substrates, only a few use micrometer-sized pores to be able to individually resolve suspending membranes by means of fluorescence microscopy. To be able to produce PSMs on pores that are micrometer in size, an orthogonal functionalization strategy resulting in a hydrophilic surface is highly desirable. Here, we report on a method to prepare PSMs based on the evaporation of a thin layer of silicon monoxide on top of the porous substrate. PM-IRRAS experiments demonstrate that the final surface is composed of SiOx with 1 < x < 2. The hydrophilic surface turned out to be well suited to spread giant unilamellar vesicles forming PSMs. As the method does not rely on a gold coating as frequently used for orthogonal functionalization, fluorescence micrographs provide information not only from the freestanding membrane areas but also from the supported ones. The observation of the entire PSM area enabled us to observe phase-separation in these membranes on the freestanding and supported parts as well as protein binding and possible lipid reorganization of the membranes induced by binding of the protein Shiga toxin.

Nanoscale Membrane Budding Induced by CTxB and Detected via Polarized Localization Microscopy

For endocytosis and exocytosis, membranes transition among planar, budding, and vesicular topographies through nanoscale reorganization of lipids, proteins, and carbohydrates. However, prior attempts to understand the initial stages of nanoscale bending have been limited by experimental resolution. Through the implementation of polarized localization microscopy, this article reports the inherent membrane bending capability of cholera toxin subunit B (CTxB) in quasi-one-component-supported lipid bilayers. Membrane buds were first detected with <50 nm radius, grew to >200 nm radius, and extended into longer tubules with dependence on the membrane tension and CTxB concentration. Compared to the concentration of the planar-supported lipid bilayers, CTxB was (12 ± 4)× more concentrated on the positive curvature top and (26 ± 11)× more concentrated on the negative Gaussian curvature neck of the nanoscale membrane buds. CTxB is frequently used as a marker for liquid-ordered lipid phases; however, the coupling between CTxB and membrane bending provides an alternate understanding of CTxB-induced membrane reorganization. These findings allow for the reinterpretation of prior observations by correlating CTxB clustering and diffusion to CTxB-induced membrane bending. Single-particle tracking was performed on single lipids and CTxB to reveal the correlations among single-molecule diffusion, CTxB accumulation, and membrane topography. Slowed lipid and CTxB diffusion was observed at the nanoscale bud locations, suggesting a local increase in the effective membrane viscosity or molecular crowding upon membrane bending. These results suggest inherent CTxB-induced membrane bending as a mechanism for initiating CTxB internalization in cells that could be independent of clathrin, caveolin, actin, and lipid phase separation.

How miRs and mRNA deadenylases could post-transcriptionally regulate expression of tumor-promoting protein PLD.

Phospholipase D (PLD) plays a key role in both cell membrane lipid reorganization and architecture, as well as a cell signaling protein via the product of its enzymatic reaction, phosphatidic acid (PA). PLD is involved in promoting breast cancer cell growth, proliferation, and metastasis and both gene and protein expression are upregulated in breast carcinoma human samples. In spite of all this, the ultimate reason as to why PLD expression is high in cancer cells vs. their normal counterparts remains largely unknown. Until we understand this and the associated signaling pathways, it will be difficult to establish PLD as a bona fide target to explore new potential cancer therapeutic approaches. Recently, our lab has identified several molecular mechanisms by which PLD expression is high in breast cancer cells and they all involve post-transcriptional control of its mRNA. First, PA, a mitogen, functions as a protein and mRNA stabilizer that counteracts natural decay and degradation. Second, there is a repertoire of microRNAs (miRs) that keep PLD mRNA translation at low levels in normal cells, but their effects change with starvation and during endothelial-to-mesenchymal transition (EMT) in cancer cells. Third, there is a novel way of post-transcriptional regulation of PLD involving 3'-exonucleases, specifically the deadenylase, Poly(A)-specific Ribonuclease (PARN), which tags mRNA for mRNA for degradation. This would enable PLD accumulation and ultimately breast cancer cell growth. We review in depth the emerging field of post-transcriptional regulation of PLD, which is only recently beginning to be understood. Since, surprisingly, so little is known about post-transcriptional regulation of PLD and related phospholipases (PLC or PLA), this new knowledge could help our understanding of how post-transcriptional deregulation of a lipid enzyme expression impacts tumor growth.