Lipid Packing Research Articles

Cardiolipin induces strong inhibition of the short antimicrobial peptide Atra-1, but only weak inhibition of LL-37 in saturated model membranes.

Cardiolipin (CL) is an anionic tetra-acyl chained glycerophospholipid that increases lipid packing levels and induces intrinsic negative curvature in membranes. Cardiolipin is one of the main components in Staphylococcus aureus membranes, where increased levels of this lipid are induced at the expense of diacyl phosphatidylglycerol in response to stress. We investigate cardiolipin as an inhibitor of the lytic activity of the cationic antimicrobial peptides Atra-1 and LL-37 in model systems with varying phosphatidylglycerol/cardiolipin ratios. One of the characteristic difference between these two antimicrobial peptides is their length. While Atra-1 is a short peptides with only 11 residues, LL-37 is 37 residues in length. We observe that the presence of cardiolipin increases the leakage-inducing concentration of Atra-1 by several orders of magnitude, while only doubling that for LL-37. We find that cardiolipin does not affect peptide binding to phosphatidylglycerol/cardiolipin liposomes significantly for either peptide, measured by shifts in Laurdan GP, so it is likely that CL affects the insertion into the membrane. We measure by FTIR the thermotropic behavior of the model systems in the presence of the peptides. In the absence of CL, both peptides induce shifts in the gel to liquid-crystalline phase transition temperature, but in the presence of CL this shift was strongly reduced in the case of Atra-1. These results suggest that CL can inhibit cAMP activity drastically depending on the antimicrobial peptide structure.

Lipid scrambling facilitates membrane vesiculation through decreasing membrane stiffness.

Membrane blebbing plays important roles in cell migration, cytokinesis, apoptosis, as well as extracellular vesicle formation for intercellular communication. During budding of the plasma membrane (PM) to form extracellular vesicles, phosphatidylserine is often translocated from the PM inner leaflet to the outer leaflet, indicative of lipid scrambling that results in loss of native membrane asymmetry. Using micrometer-sized giant plasma membrane vesicles (GPMV) as a readout of PM vesiculation, we found that knocking out (KO) the lipid scramblase TMEM16F reduced lipid scrambling and dramatically inhibited GPMV formation, in line with previous observations. TMEM16F repletion could rescue GPMV formation, indicating that lipid scrambling is necessary for membrane budding. We hypothesized three mechanisms to explain this surprising observation, namely that lipid scrambling: (1) depletes PI(4,5)P2 from the inner leaflet leading to detachment of the PM from the actin cytoskeleton, (2) makes PM bilayers softer and easier to bend, (3) externalizes lipids that stabilize highly curved bud necks. Depletion of PI(4,5)P2 was indeed observed during vesiculation, however this occurred in both wild type and KO cells, implying that detachment of PM from actin cytoskeleton due to PIP2 depletion is not sufficient to make large vesicles. Next, through fluorescence lifetime imaging microscopy of a lipid packing reporter (Di4), we found that lipid scrambling leads to a decrease of PM packing, potentially making the membrane softer for bending and thus facilitating membrane budding. Finally, we observed that lipid scrambling resulted in externalization of phosphatidylethanolamine (PE). This PE was preferentially located at the highly curved vesicle necks, suggesting that the intrinsic curvature of PE stabilizes this highly curved membrane structure. Thus, we conclude that lipid scrambling changes membrane biophysical properties, potentially explaining mechanisms underlying vesicle-budding in physiological contexts including apoptotic cell removal, bone mineralization, fertilization, and blood coagulation.

Lateral compression of lipids drives transbilayer coupling of protein condensates.

Protein phase separation that is restricted to a two-dimensional lipid membrane has recently gained much attention due to its involvement in various cellular processes. Two popular in vitro model membrane systems, giant unilamellar vesicles (GUVs) and supported lipid bilayers (SLBs), have been used to study it. However, due to the accessibility to only one side of the membrane in these systems, protein phase separation on both sides of the membrane remains unexplored. Here, we introduce a freestanding planar lipid membrane array as an effective platform to study membrane-associated protein phase separation. Protein condensates on the membrane display liquid-like features, and environmental stimuli, such as salt concentration and temperature, effectively control the observed phase separation results in our system with the application of several intrinsically disordered proteins. Interestingly, we observe transbilayer coupling of protein condensates such that protein-rich domains on one side of the membrane are found to colocalize with those on the other side of the membrane. How do these protein phases communicate across the lipid bilayer? Based on data from lipid probe partitioning and photobleaching experiments, we hypothesize that protein condensation on the membrane exerts lateral compressive stress on the underlying lipids, driving the formation of lipid domains with reduced fluidity. Specifically, lipid domains with reduced fluidity and increased lipid packing, formed by protein condensation on each membrane leaflet, are attracted to one another, leading to transbilayer coupling of protein condensates. These findings suggest a previously unknown mechanism by which cellular signals from one membrane leaflet, triggered by membrane-associated protein phase separation, can be transferred to the opposing leaflet.

The impact of lipid packing defects on alpha-synuclein binding to lipid membranes.

Alpha-synuclein (aSyn) is an intrinsically disordered protein found in neurons whose dysfunction is related to the formation of protein aggregates in Parkinson's Disease. While the normal physiological function of aSyn is suspected to be related to the synaptic vesicle cycle, the specific role played by the protein remains unknown. As both the function and dysfunction of aSyn are linked to its interaction with lipid membranes, it is especially important to understand the intricacies of these interactions to discern the differences between normal function and disease progression. While recent work has suggested the importance of lipid packing defects for aSyn-membrane interactions, it is difficult to experimentally establish this dependence on the binding due to the small size and transience of packing defects. In this work, we aim to further understand the effects of lipid packing defects on aSyn-membrane interactions through the use of two complementary binding techniques: tryptophan fluorescence spectroscopy and confocal microscopy. These techniques allow us to access different “binding regimes” for this interaction, which, when fitted with the proper binding equations, can yield distinct parameters that reveal additional binding information beyond the dissociation constant, Kd, for a binding site. Our results highlight the significance of each protein concentration “regime” when studying aSyn-membrane interactions and the defect dependence on the protein binding site density on a lipid membrane. These parameters together provide a more complete picture of aSyn-membrane interactions.

Dynamic interactions between alpha-synuclein and curved membrane surfaces.

Alpha-synuclein (α-syn) is an intrinsically disordered protein (IDP) that is abundant in neurons, where it is implicated in the pathogenesis of Parkinson's disease through cytosolic aggregation and formation of fibrils. In its healthy state, α-syn binds to synaptic lipid vesicles, transitioning to a membrane-inserting, α-helical structure in its N-terminal region while the C-terminal domain remains disordered in close proximity to the membrane. These types of disordered C-terminal domains have been shown to amplify the curvature sensitivity of several proteins through steric and/or electrostatic interactions with the membrane, which is essential for cellular function. However, little is known about the role of this disordered C-terminal domain in curvature sensing for α-syn. Additionally, little is known about the dynamic interactions of these types of disordered domains with membrane surfaces because traditional studies examine systems in thermodynamic equilibrium, which can take minutes to achieve. Conversely, many relevant cellular processes, such as cell signaling, occur on the order of seconds to milliseconds. Therefore, it is reasonable to infer that thermodynamic equilibrium does not fully encompass the complexities of α-syn's curvature sensing ability at physiologically relevant timescales. In this study, we utilize quantitative, in vitro microscopy and circular dichroism spectroscopy to examine both equilibrium and dynamic binding of α-syn to curved membranes. Our data suggests that the dissociation constant (Kd) and the association and dissociation rates (Kon and Koff) for α-syn depend on both membrane curvature and lipid composition. Both factors dictate steric and/or electrostatic interactions between C-terminal disordered domains and the membrane, as well as the density of lipid packing defects that are necessary for α-helical insertion. By employing novel strategies for investigating dynamic binding, this work unveils a previously unexplored and poorly understood category of biologically relevant IDP-membrane interactions.

Investigating structure-property relations in cholesterol-rich lipid membranes.

Cholesterol is a key molecule in eukaryotic cell membranes. It mediates essential membrane processes, including viral budding and the mechanical resistance of membranes against pathogens. Hence, understanding the influence of cholesterol on the mechanics of functional membranes is crucial. Cholesterol is known to cause lipids to pack more tightly, manifesting in a decrease in the average area per lipid, AL, at the aqueous interface. Yet, how variations in packing density influence the elastic properties of lipid membranes with varying degrees of chain unsaturation remains largely unexplored. Theories based on mean field calculations have predicted various forms of the interdependence between the bending rigidity modulus, κ, and lipid packing density. However, direct assessments of these predictions have been limited, due to the difficulty in accessing the membrane's mechanical properties on the nanoscale. To address this, we performed studies using neutron spin-echo spectroscopy, solid-state 2H NMR relaxometry, and molecular dynamics simulations on lipid membranes with different degrees of chain unsaturation. We find that cholesterol has a stiffening effect on all studied membranes over the accessible length and time scales. Our findings show that κ has an inverse dependence on AL, indicating that cholesterol controls membrane elastic properties by modifying lipid packing density. As AL decreases, the number of conformations accessible to the lipid chains also decreases and the membrane becomes more rigid. Yet, different expressions of κ vs. AL are obtained when we consider reduced variables. Establishing well-defined structure-property relationships will open up new possibilities for predicting the behavior of more complex biological membranes and will inform design rules for engineered membranes and artificial cells with real-world functionalities.

Investigating the role of membrane composition on the stability of decorated nanoparticle aggregates

Gold nanoparticles are a ubiquitous photosensitizer with a broad range of applications in microscopy and targeted drug delivery. Vesicles photosensitized with gold nanoparticles are promising as targeted drug delivery vehicles due to their non-invasive rupture mechanism. Controlling the vesicle properties increases control of the spatial-temporal release of cargo and drug dosage. The aggregation of gold nanoparticles affects the photoporation of these vesicles by interfering with peak SPR wavelengths. These nanoparticles cause leaflet bending deformations on the order of the nanoparticle size, while ligand chains may also disrupt local packing of lipid chains. Aggregation may be driven by the need to minimize one or both effects, but their relative contributions are unknown. In order to test the contributions of these two perturbations to membrane structure, we simulated multi-nanoparticle systems in lipid membranes of varying compositions. We used coarse-grained molecular dynamics simulation via the MARTINI forcefield to simulate simple spherical nanoparticles with a decorated ligand exterior. We found that large-scale nanoparticle aggregation depends more on ligand chain length than nanoparticle size, suggesting that aggregation of gold nanoparticles is dominated by microscopic perturbations to lipid packing.

Physics-based inverse design of curvature sensing antiviral peptides.

Proteins can be thermodynamically attracted to high membrane curvatures due to their ability to sense hydrophobic lipid packing defects that arise upon membrane bending. We leveraged the difference in curvature between small enveloped viral membranes and the flat host cell membrane to computationally design antiviral peptides from scratch, without any prior data. In our ‘physics-based inverse design’ approach we combined an evolutionary algorithm, molecular dynamics simulations, and neural network models to navigate a vast search space (2024 possible peptides) and found several experimentally confirmed antiviral peptides, with potential broad spectrum applicability. Along the way, we gained valuable new insights into the physics behind curvature sensing by natural proteins.

Biomimetic Stratum Corneum Liposome Models: Lamellar Organization and Permeability Studies

The stratum corneum (SC), the outer layer of the skin, plays a crucial role as a barrier protecting the underlying cells from external stress. The SC comprises three key components: ceramide (CER), free fatty acid (FFA), and cholesterol, along with small fractions of cholesterol sulfate and cholesterol ester. In order to gain a deeper understanding about the interdependence of the two major components, CER and FFA, on the organizational, structural, and functional properties of the SC layer, a library of SC lipid liposome (SCLL) models was developed by mixing CER (phytosphingosine or sphingosine), FFA (oleic acid, palmitic acid, or stearic acid), cholesterol, and cholesterol sulfate. Self-assembly of the SC lipids into lamellar phases was first confirmed by small-angle X-ray scattering. Short periodicity and long periodicity phases were identified for SCLLs containing phytosphingosines and sphingosine CERs, respectively. Furthermore, unsaturation in the CER acyl and FFA chains reduced the lipid conformational ordering and packing density of the liposomal bilayer, which were measured by differential scanning calorimetry and Fourier transform infrared spectroscopy. The introduction of unsaturation in the CER and/or FFA chains also impacted the lamellar integrity and permeability. This extensive library of SCLL models exhibiting physiologically relevant lamellar phases with defined structural and functional properties may potentially be used as a model system for screening pharmaceuticals or cosmetic agents.

Lipid packing is disrupted in copolymeric nanodiscs compared with intact membranes

Discoidal lipid-protein nanoparticles known as nanodiscs are widely used tools in structural and membrane biology. Amphipathic, synthetic copolymers have recently become an attractive alternative to membrane scaffold proteins for the formation of nanodiscs. Such copolymers can directly intercalate into, and form nanodiscs from, intact membranes without detergents. Although these copolymer nanodiscs can extract native membrane lipids, it remains unclear whether native membrane properties are also retained. To determine the extent to which bilayer lipid packing is retained in nanodiscs, we measured the behavior of packing-sensitive fluorescent dyes in various nanodisc preparations compared with intact lipid bilayers. We analyzed styrene-maleic acid (SMA), diisobutylene-maleic acid (DIBMA), and polymethacrylate (PMA) as nanodisc scaffolds at various copolymer-to-lipid ratios and temperatures. Measurements of Laurdan spectral shifts revealed that dimyristoyl-phosphatidylcholine (DMPC) nanodiscs had increased lipid headgroup packing compared with large unilamellar vesicles (LUVs) above the lipid melting temperature for all three copolymers. Similar effects were observed for DMPC nanodiscs stabilized by membrane scaffolding protein MSP1E1. Increased lipid headgroup packing was also observed when comparing nanodiscs with intact membranes composed of binary mixtures of 1-palmitoyl-2-oleoyl-phosphocholine (POPC) and di-palmitoyl-phosphocholine (DPPC), which show fluid-gel-phase coexistence. Similarly, Laurdan reported increased headgroup packing in nanodiscs for biomimetic mixtures containing cholesterol, most notable for relatively disordered membranes. The magnitudes of these ordering effects were not identical for the various copolymers, with SMA being the most and DIBMA being the least perturbing. Finally, nanodiscs derived from mammalian cell membranes showed similarly increased lipid headgroup packing. We conclude that nanodiscs generally do not completely retain the physical properties of intact membranes.

Targeting of the Mon1-Ccz1 Rab guanine nucleotide exchange factor to distinct organelles by a synergistic protein and lipid code

Activation of the small GTPase Rab7 by its cognate guanine nucleotide exchange factor Mon1-Ccz1 (MC1) is a key step in the maturation of endosomes and autophagosomes. This process is tightly regulated and subject to precise spatiotemporal control of MC1 localization, but the mechanisms that underly MC1 localization have not been fully elucidated. We here identify and characterize an amphipathic helix in Ccz1, which is required for the function of Mon-Ccz1 in autophagy, but not endosomal maturation. Furthermore, our data show that the interaction of the Ccz1 amphipathic helix with lipid packing defects, binding of Mon1 basic patches to positively charged lipids, and association of MC1 with recruiter proteins collectively govern membrane recruitment of the complex in a synergistic and redundant manner. Membrane binding enhances MC1 activity predominantly by increasing enzyme and substrate concentration on the membrane, but interaction with recruiter proteins can further stimulate the guanine nucleotide exchange factor. Our data demonstrate that specific protein and lipid cues convey the differential targeting of MC1 to endosomes and autophagosomes. In conclusion, we reveal the molecular basis for how MC1 is adapted to recognize distinct target compartments by exploiting the unique biophysical properties of organelle membranes and thus provide a model for how the complex is regulated and activated independently in different functional contexts.

Changes in Membrane Lipid Order of Cells by Optimized Microplasma Irradiation for High-Molecule Drug Absorption

Microplasma-based drug delivery, particularly transdermal drug delivery, has emerged as a promising alternatives to conventional methods due to its unique advantages. In this system, plasma components interact not only with the stratum corneum but also with the cells beneath it. Cell membranes mainly comprise lipids which are highly susceptible to plasma components, including reactive oxygen and nitrogen species. However, the effects of microplasma irradiation on cell membrane lipids are largely unexplored. In this context, the current study investigated an important physical property of cell membranes, membrane lipid order, in small intestinal epithelial cells after microplasma irradiation. First, the microplasma irradiation condition was optimized for the intracellular delivery of high molecular weight molecules, namely fluorescein isothiocyanate-dextran (FD-150 and FD-2000). Air microplasma discharge was applied at varying voltages (i.e., 3.5, 4.0, and 4.5 kV), and the efficiency of drug absorption was evaluated using a microplate reader and a fluorescence microscope. The cell viability was calculated using an automated cell counter. The most favorable outcomes in terms of both drug absorption and cell viability were achieved at 4 kV. Next, the membrane lipid order of cells was investigated using a solvatochromic dye after microplasma irradiation at 4 kV. Interestingly, non-irradiated cells exclusively manifested the liquid-ordered phase, whereas irradiated cells displayed a dual-phased composition comprising both liquid-ordered and liquid-disordered phases. In addition, live-cell imaging unveiled that alterations in membrane lipid order commenced as early as 4 min post-irradiation. This evidence points to a significant reduction in lipid packing and membrane thickness induced by microplasma irradiation. The findings shed light on the intricate mechanism underlying cellular drug absorption facilitated by microplasma irradiation. The insights gained into the impact on membrane lipid properties will contribute to developing safe and efficient drug delivery systems while minimizing side effects.

Systematic analysis of curvature-dependent lipid dynamics in a stochastic 3D membrane model.

To minimize the free energy of the system, lipid membranes display curvature-dependent rearrangements at the local and global scale. The optimal membrane shape is generally approximated by averaging the curvature preference of individual lipids across the whole surface. Potential stress due to imperfections in lipid packing caused by local lipid inhomogeneities, however, is frequently neglected. Here, we developed a stochastic 3D membrane model to investigate the relevance of this parameter for shape-dependent lipid and membrane dynamics. A systematic analysis of the discretized Helfrich type Hamiltonian indicates that stress-energy arising from imperfections in packing is analogous to van der Waals interactions, jointly determining membrane shape and localization of curvature-sensitive lipids based on their relative strengths. Insights from this work can be used to characterize natural and design synthetic agents for membrane-shape changes.

Bacterial susceptibility and resistance to modelin-5.

Modelin-5 (M5-NH2) killed Pseudomonas aeruginosa with a minimum lethal concentration (MLC) of 5.86 μM and strongly bound its cytoplasmic membrane (CM) with a Kd of 23.5 μM. The peptide adopted high levels of amphiphilic α-helical structure (75.0%) and penetrated the CM hydrophobic core (8.0 mN m-1). This insertion destabilised CM structure via increased lipid packing and decreased fluidity (ΔGmix < 0), which promoted high levels of lysis (84.1%) and P. aeruginosa cell death. M5-NH2 showed a very strong affinity (Kd = 3.5 μM) and very high levels of amphiphilic α-helical structure with cardiolipin membranes (96.0%,) which primarily drove the peptide's membranolytic action against P. aeruginosa. In contrast, M5-NH2 killed Staphylococcus aureus with an MLC of 147.6 μM and weakly bound its CM with a Kd of 117.6 μM, The peptide adopted low levels of amphiphilic α-helical structure (35.0%) and only penetrated the upper regions of the CM (3.3 mN m-1). This insertion stabilised CM structure via decreased lipid packing and increased fluidity (ΔGmix > 0) and promoted only low levels of lysis (24.3%). The insertion and lysis of the S. aureus CM by M5-NH2 showed a strong negative correlation with its lysyl phosphatidylglycerol (Lys-PG) content (R2 > 0.98). In combination, these data suggested that Lys-PG mediated mechanisms inhibited the membranolytic action of M5-NH2 against S. aureus, thereby rendering the organism resistant to the peptide. These results are discussed in relation to structure/function relationships of M5-NH2 and CM lipids that underpin bacterial susceptibility and resistance to the peptide.

Natamycin interferes with ergosterol-dependent lipid phases in model membranes

Natamycin is an antifungal polyene macrolide that is used as a food preservative but also to treat fungal keratitis and other yeast infections. In contrast to other polyene antimycotics, natamycin does not form ion pores in the plasma membrane, but its mode of action is poorly understood. Using nuclear magnetic resonance (NMR) spectroscopy of deuterated sterols, we find that natamycin slows the mobility of ergosterol and cholesterol in liquid-ordered (Lo) membranes to a similar extent. This is supported by molecular dynamics (MD) simulations, which additionally reveal a strong impact of natamycin dimers on sterol dynamics and water permeability. Interference with sterol-dependent lipid packing is also reflected in a natamycin-mediated increase in membrane accessibility for dithionite, particularly in bilayers containing ergosterol. NMR experiments with deuterated sphingomyelin (SM) in sterol-containing membranes reveal that natamycin reduces phase separation and increases lipid exchange in bilayers with ergosterol. In ternary lipid mixtures containing monounsaturated phosphatidylcholine, saturated SM, and either ergosterol or cholesterol, natamycin interferes with phase separation into Lo and liquid-disordered (Ld) domains, as shown by NMR spectroscopy. Employing the intrinsic fluorescence of natamycin in ultraviolet-sensitive microscopy, we can visualize the binding of natamycin to giant unilamellar vesicles (GUVs) and find that it has the highest affinity for the Lo phase in GUVs containing ergosterol. Our results suggest that natamycin specifically interacts with the sterol-induced ordered phase, in which it disrupts lipid packing and increases solvent accessibility. This property is particularly pronounced in ergosterol containing membranes, which could underlie the selective antifungal activity of natamycin.

Distribution of Domains Formed by Lateral Packing of Intercellular Lipid in the Stratum Corneum.

Intercellular lipids fill the interstices of corneocytes and serve a barrier function. The amount of transdermal water evaporation varies depending on the packing structure of intercellular lipids, as this structure is important for maintaining barrier efficacy. This packing structure consists of a mixture of crystals (orthorhombic and hexagonal) and liquid crystals (fluid phase), and the proportion of these phases is thought to affect barrier function. However, there have been no methods to visualize the actual distribution of the domains formed by packing structure in intercellular lipids. In this study, the planar distribution of intercellular lipid structures was determined using focal plane array (FPA)-based Fourier transform (FT) IR imaging analysis of stratum corneum cell units obtained by grid stripping. The lipid composition of ceramides was revealed by electrospray ionization tandem mass spectrometry (ESI-MS/MS)-based shotgun lipidomics. The distribution of domains formed by packing structures and the lipid composition of ceramides was compared in skin with high- or low-transepidermal water loss (TEWL). The orthorhombic proportion was lower in high-TEWL skin than in low-TEWL skin. ESI-MS/MS-based shotgun lipidomics analysis showed that the alpha-hydroxyceramide content in the low- and high-TEWL groups differed regarding the distribution of fatty acid chain lengths. The evaluation of stratum corneum cell units using FPA-based FTIR imaging is an innovative technology that can visualize the distribution of domains formed by intercellular lipid-packing structures. Increased proportions of alpha-hydroxyceramide subclasses such as alpha-hydroxy-sphingosine ceramide and alpha-hydroxy-phytosphingosine ceramide were associated with a reduced proportion of the orthorhombic packing structure domain.

Effect of water content on stratum corneum penetration mechanism of W/O type microemulsions.

The stratum corneum (SC) consists of a lipid layer that forms two types of lamellar structures: short lamellar (S-La) and long lamellar (L-La). It has been reported that S-La contains water phases in the hydrophilic region of the lipids, and that it may play an important role in regulating the water content of the SC. The amount of water in the SC can affect how a drug carrier permeates through the intercellular lipid pathway. To better understand the impact of SC water content on the skin penetration mechanism of a microemulsion (ME), we conducted a study using small-angle X-ray scattering (SAXS), wide-angle X-ray scattering (WAXS), and small-angle neutron scattering (SANS). Our results showed that MEs can enhance skin permeation under humid conditions because the lipid packing structures of the hydrated SC are more disrupted than those of the dry SC. The results also showed that the inner water of MEs was released to the SC when applying MEs to the dry SC, resulting in an increase in the repeat distance of S-La. Conversely, when MEs are applied to hydrated SC, the MEs absorb the water from the SC into their inner phases, causing a decrease in the repeat distance of S-La over time.

Interactions of Bacterial Quorum Sensing Signals with Model Lipid Membranes: Influence of Membrane Composition on Membrane Remodeling.

We report the influence of membrane composition on the multiscale remodeling of multicomponent lipid bilayers initiated by contact with the amphiphilic bacterial quorum sensing signal N-(3-oxo)-dodecanoyl-l-homoserine lactone (3-oxo-C12-AHL) and its anionic headgroup hydrolysis product, 3-oxo-C12-HS. We used fluorescence microscopy and quartz crystal microbalance with dissipation (QCM-D) to characterize membrane reformation that occurs when these amphiphiles are placed in contact with supported lipid bilayers (SLBs) composed of (i) 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) containing varying amounts of cholesterol or (ii) mixtures of DOPC and either 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, a conical zwitterionic lipid) or 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS, a model anionic lipid). In general, we observe these mixed-lipid membranes to undergo remodeling events, including the formation and subsequent collapse of long tubules and the formation of hemispherical caps, upon introduction to biologically relevant concentrations of 3-oxo-C12-AHL and 3-oxo-C12-HS in ways that differ substantially from those observed in single-component DOPC membranes. These differences in bilayer reformation and their associated dynamics can be understood in terms of the influence of membrane composition on the time scales of molecular flip-flop, lipid packing defects, and lipid phase segregation in these materials. The lipid components investigated here are representative of classes of lipids that comprise both naturally occurring cell membranes and many useful synthetic soft materials. These studies thus represent a first step toward understanding the ways in which membrane composition can impact interactions with this important class of bacterial signaling molecules.

Cholesterol enhances the negative impact of vaping additives on lung surfactant model systems.

Aims: Vaping has given rise to e-cigaretteor vaping productuse-associatedlung injury. Model lung surfactant films were used to assess the impact of vape additives (vitamin E, vitamin Eacetate, tetrahydrocannabinol, cannabidiol). This work builds upon our previous findings, by incorporating cholesterol, to understand the interplay between the additives and the sterol in surfactant function. Materials & methods: Compression-expansion cycles of lipid monofilm at the air-water interface and Brewster angle microscopy allowed elucidating the effects of vape additives. Results & conclusion: Vape additives at 5mol% inhibited proper lipid packing and reduced film stability. Cholesterol enhanced the additive effects, resulting in significantly destabilized films and altered domains. The observed impact could signify dysfunctional lung surfactant and impaired lung function.

Topology-based fluorescence image analysis for automated cell identification and segmentation.

Cell segmentation refers to the body of techniques used to identify cells in images and extract biologically relevant information from them; however, manual segmentation is laborious and subjective. We present Topological Boundary Line Estimation using Recurrence Of Neighbouring Emissions (TOBLERONE), a topological image analysis tool which identifies persistent homological image features as opposed to the geometric analysis commonly employed. We demonstrate that topological data analysis can provide accurate segmentation of arbitrarily-shaped cells, offering a means for automatic and objective data extraction. One cellular feature of particular interest in biology is the plasma membrane, which has been shown to present varying degrees of lipid packing, or membrane order, depending on the function and morphology of the cell type. With the use of environmentally-sensitive dyes, images derived from confocal microscopy can be used to quantify the degree of membrane order. We demonstrate that TOBLERONE is capable of automating this task.