Membrane Lipid Order Research Articles

Molecular Simulations of Ocular Lens Membranes at Various Stages of Age and Cataract

The human ocular lens consists primarily of elongated, static fibers characterized by high stability and low turnover, which differ dramatically in their composition and properties from other biological membranes. Cholesterol (Chol) and sphingolipids (SL) are present at high concentrations, including saturated SLs, such as dihyrosphingomyelin. Past molecular dynamics (MD) simulations demonstrated that the presence of DHSM and high Chol concentration contributes to higher order in lipid membranes. Using the CHARMM36 all-atom force field, this study simulated more complex models of human lens membranes with the inclusion of lysolipids and plasmanyl lipids. Models were developed representing physiological compositions in cataractous lenses aged 74±6 years and in healthy lenses aged 22±4, 41±6, and 69±3 years. With older age, Chol and ceramide concentrations increase and glycerophospholipid concentration decreases. With cataract, ceramide concentration increases and Chol and glycerophospholipid concentrations decrease. Surface area per lipid, deuterium order parameters (SCD), sterol tilt angle, electron density profiles (EDP), bilayer thickness, chain interdigitation, two-dimensional radial distribution functions (2D-RDF), mean-square displacement, lipid clustering, and hydrogen bonding were calculated for all simulations. All systems exhibited low surface area per lipid and high bilayer thickness, indicative of strong vertical packing. SCD parameters suggest similarly, with saturated tails in the hydrophobic core of the membrane having order parameter exceeding 0.4. Vertical packing and acyl tail order increased with both age and cataract. Lateral diffusion decreased with age and cataracts, with the older and cataractous models demonstrating increased long-range structure by the 2D-RDF analysis. Experimental results have found crystallization of Chol in older lenses, which similarly indicates increased order. In future work examining the membrane proteins of the lens, these models can serve as physiologically accurate representations of the lens lipidome.

Lipid Bilayer Passive Area Regulation in Response to Fluid Flows

Mechanotransduction is the cellular process in which mechanical stimuli are converted into biochemical signals to induce specific cellular responses, enabling the cell to sense and respond to mechanical forces. Although many cells exhibit this behavior, vascular endothelial cells (ECs) are of considerable interest as they effectively respond to fluid shear stress, adapting their morphology, functions and gene expression to maintain the homeostasis of the vascular system. Fundamentally, these processes rely on the transduction of intracellular signals through the lipid membrane, and several studies have already demonstrated that shear stress induces significant changes in lipid order and viscosity of ECs plasma membrane. Despite such extensive study, the underlying molecular mechanisms that control this change to membrane lipid order and viscosity remain unclear. Supported lipid bilayers provide an ideal platform for the investigation of the molecular mechanisms behind shear induced biophysical changes in membrane structure. As a minimal model system, they are suited for isolating the physical role of lipid bilayer in membrane shear response, in the absence of active biological processes. Here, combining microfluidics and fluorescence microscopy, we examine how the application of controlled fluid flows on supported lipid bilayer patches alters their physical properties. We observe a reversible and significant ∼10% increase in SLB area (when physiological shear stresses of 1-2 Pa are applied). Such area fluctuations have been a previously suggested response to fluid shear stress but have not directly observed. Furthermore, we determine how these membrane area fluctuations in response to fluid flows depend on lipid composition and surface roughness. Identifying the origin of such flow-induced changes in membrane structure could reveal an underlying mechanism utilized by biology to sense and direct changes in membrane order and viscosity in response to shear flows.

Regulation of Wnt Signaling Pathways at the Plasma Membrane and Their Misregulation in Cancer.

Wnt signaling is one of the key signaling pathways that govern numerous physiological activities such as growth, differentiation and migration during development and homeostasis. As pathway misregulation has been extensively linked to pathological processes including malignant tumors, a thorough understanding of pathway regulation is essential for development of effective therapeutic approaches. A prominent feature of cancer cells is that they significantly differ from healthy cells with respect to their plasma membrane composition and lipid organization. Here, we review the key role of membrane composition and lipid order in activation of Wnt signaling pathway by tightly regulating formation and interactions of the Wnt-receptor complex. We also discuss in detail how plasma membrane components, in particular the ligands, (co)receptors and extracellular or membrane-bound modulators, of Wnt pathways are affected in lung, colorectal, liver and breast cancers that have been associated with abnormal activation of Wnt signaling. Wnt-receptor complex components and their modulators are frequently misexpressed in these cancers and this appears to correlate with metastasis and cancer progression. Thus, composition and organization of the plasma membrane can be exploited to develop new anticancer drugs that are targeted in a highly specific manner to the Wnt-receptor complex, rendering a more effective therapeutic outcome possible.

Native-like membrane models of E. coli polar lipid extract shed light on the importance of lipid composition complexity

BackgroundLipid-protein interactions stabilize protein oligomers, shape their structure, and modulate their function. Whereas in vitro experiments already account for the functional importance of lipids by using natural lipid extracts, in silico methods lack behind by embedding proteins in single component lipid bilayers. However, to accurately complement in vitro experiments with molecular details at very high spatio-temporal resolution, molecular dynamics simulations have to be performed in natural(-like) lipid environments.ResultsTo enable more accurate MD simulations, we have prepared four membrane models of E. coli polar lipid extract, a typical model organism, each at all-atom (CHARMM36) and coarse-grained (Martini3) representations. These models contain all main lipid headgroup types of the E. coli inner membrane, i.e., phosphatidylethanolamines, phosphatidylglycerols, and cardiolipins, symmetrically distributed between the membrane leaflets. The lipid tail (un)saturation and propanylation stereochemistry represent the bacterial lipid tail composition of E. coli grown at 37∘C until 3/4 of the log growth phase. The comparison of the Simple three lipid component models to the complex 14-lipid component model Avanti over a broad range of physiologically relevant temperatures revealed that the balance of lipid tail unsaturation and propanylation in different positions and inclusion of lipid tails of various length maintain realistic values for lipid mobility, membrane area compressibility, lipid ordering, lipid volume and area, and the bilayer thickness. The only Simple model that was able to satisfactory reproduce most of the structural properties of the complex Avanti model showed worse agreement of the activation energy of basal water permeation with the here performed measurements. The Martini3 models reflect extremely well both experimental and atomistic behavior of the E. coli polar lipid extract membranes. Aquaporin-1 embedded in our native(-like) membranes causes partial lipid ordering and membrane thinning in its vicinity. Moreover, aquaporin-1 attracts and temporarily binds negatively charged lipids, mainly cardiolipins, with a distinct cardiolipin binding site in the crevice at the contact site between two monomers, most probably stabilizing the tetrameric protein assembly.ConclusionsThe here prepared and validated membrane models of E. coli polar lipids extract revealed that lipid tail complexity, in terms of double bond and cyclopropane location and varying lipid tail length, is key to stabilize membrane properties over a broad temperature range. In addition, they build a solid basis for manifold future simulation studies on more realistic lipid membranes bridging the gap between simulations and experiments.

Biophysical analysis of the plant-specific GIPC sphingolipids reveals multiple modes of membrane regulation

The plant plasma membrane (PM) is an essential barrier between the cell and the external environment, controlling signal perception and transmission. It consists of an asymmetrical lipid bilayer made up of three different lipid classes: sphingolipids, sterols, and phospholipids. The glycosyl inositol phosphoryl ceramides (GIPCs), representing up to 40% of total sphingolipids, are assumed to be almost exclusively in the outer leaflet of the PM. However, their biological role and properties are poorly defined. In this study, we investigated the role of GIPCs in membrane organization. Because GIPCs are not commercially available, we developed a protocol to extract and isolate GIPC-enriched fractions from eudicots (cauliflower and tobacco) and monocots (leek and rice). Lipidomic analysis confirmed the presence of trihydroxylated long chain bases and 2-hydroxylated very long-chain fatty acids up to 26 carbon atoms. The glycan head groups of the GIPCs from monocots and dicots were analyzed by gas chromatograph–mass spectrometry, revealing different sugar moieties. Multiple biophysics tools, namely Langmuir monolayer, ζ-Potential, light scattering, neutron reflectivity, solid state 2H-NMR, and molecular modeling, were used to investigate the physical properties of the GIPCs, as well as their interaction with free and conjugated phytosterols. We showed that GIPCs increase the thickness and electronegativity of model membranes, interact differentially with the different phytosterols species, and regulate the gel-to-fluid phase transition during temperature variations. These results unveil the multiple roles played by GIPCs in the plant PM.

Laurdan and Di-4-ANEPPDHQ Influence the Properties of Lipid Membranes: A Classical Molecular Dynamics and Fluorescence Study.

Environmentally sensitive (ES) dyes have been used for many decades to study the lipid order of cell membranes, as different lipid phases play a crucial role in a wide variety of cell processes. Yet, the understanding of how ES dyes behave, interact, and affect membranes at the atomistic scale is lacking, partially due to the lack of molecular dynamics (MD) models of these dyes. Here, we present ground- and excited-state MD models of commonly used ES dyes, Laurdan and di-4-ANEPPDHQ, and use MD simulations to study the behavior of these dyes in a disordered and an ordered membrane. We also investigate the effect that these two dyes have on the hydration and lipid order of the membranes, where we see a significant effect on the hydration of lipids proximal to the dyes. These findings are combined with experimental fluorescence experiments of ordered and disordered vesicles and live HeLa cells stained by the aforementioned dyes, where the generalized polarization (GP) values were measured at different concentrations of the dyes. We observe a small but significant decrease of GP at higher Laurdan concentrations in vesicles, while the same effect is not observed in cell membranes. The opposite effect is observed with di-4-ANEPPDHQ where no significant change in GP is seen for vesicles but a very substantial and significant decrease is seen in cell membranes. Together, our results show the profound effect that ES dyes have on membranes, and the presented MD models will be important for further understanding of these effects.

Characterization of Lipid Order and Domain Formation in Model Membranes Using Fluorescence Microscopy and Spectroscopy.

Fluorescence-based techniques have been an integral factor in the study of cellular and model membranes. Fluorescence studies carried out on model membranes have provided valuable structural information and have helped reveal mechanistic detail regarding the formation and properties of ordered lipid domains, commonly known as lipid rafts. This chapter focuses on four techniques, based on fluorescence spectroscopy or microscopy, which are commonly used to analyze lipid rafts. The techniques described in this chapter may be used in a variety of ways and in combination with other techniques to provide valuable information regarding lipid order and domain formation, especially in model membranes.

Effect of Erufosine on Membrane Lipid Order in Breast Cancer Cell Models.

Alkylphospholipids are a novel class of antineoplastic drugs showing remarkable therapeutic potential. Among them, erufosine (EPC3) is a promising drug for the treatment of several types of tumors. While EPC3 is supposed to exert its function by interacting with lipid membranes, the exact molecular mechanisms involved are not known yet. In this work, we applied a combination of several fluorescence microscopy and analytical chemistry approaches (i.e., scanning fluorescence correlation spectroscopy, line-scan fluorescence correlation spectroscopy, generalized polarization imaging, as well as thin layer and gas chromatography) to quantify the effect of EPC3 in biophysical models of the plasma membrane, as well as in cancer cell lines. Our results indicate that EPC3 affects lipid–lipid interactions in cellular membranes by decreasing lipid packing and increasing membrane disorder and fluidity. As a consequence of these alterations in the lateral organization of lipid bilayers, the diffusive dynamics of membrane proteins are also significantly increased. Taken together, these findings suggest that the mechanism of action of EPC3 could be linked to its effects on fundamental biophysical properties of lipid membranes, as well as on lipid metabolism in cancer cells.

Dual benefit of combined neuroprotection: Cholesterol depletion restores membrane microviscosity but not lipid order and enhances neuroprotective action of hypothermia in rat cortex nerve terminals

Here, both neuroprotectants, i.e. cholesterol depletion of the plasma membrane of rat brain nerve terminals (synaptosomes) using methyl-β-cyclodextrin (MβCD) and deep/propound hypothermia, were analyzed during their combined administration and regarding additive neuroprotective effect. The extracellular synaptosomal level of L-[14C]glutamate significantly increased after treatment with MβCD in both deep and profound hypothermia. Cholesterol depletion gradually enhanced inhibiting effect of deep and profound hypothermia on glutamate uptake and “excitotoxic” transporter-mediated release of L-[14C]glutamate. A decrease in L-[14C]glutamate release via heteroexchange from nerve terminals in deep and profound hypothermia was enhanced by cholesterol deficiency that confirmed previous result. Fluorometric studies with probes NR12S and DCVJ revealed oppositely directed effects of cholesterol depletion and hypothermia on synaptosomal membrane lipid order and microviscosity showing that cholesterol depletion can normalise up to the control hypothermia-induced increase in microviscosity, but not the lipid order of the synaptosomal membrane. Dynamics of changes in exocytosis in nerve terminals, which involved membrane fusion stage, was different from transporter-dependent ones. Hypothermia did not augment effects of cholesterol depletion on exocytotic L-[14C]glutamate release and lowering cholesterol enhanced the impact of deep, but not profound hypothermia on this parameter. Therefore, dual benefit of combined neuroprotection was demonstrated. Cholesterol depletion enhanced neuroprotective effects of hypothermia intensifying inhibition of “excitotoxic” transporter-mediated glutamate release and can normalise a hypothermia-induced increase in microviscosity of the synaptosomal membrane. This feature is prospective in mitigation of side effects of therapeutic hypothermia, and also for brain conservation preserving normal physical and chemical properties of the cellular membranes.

Membrane lipid raft organization during cotton fiber development

BackgroundCotton fiber is a single-celled seed trichome that originates from the ovule epidermis. It is an excellent model for studying cell elongation. Along with the elongation of cotton fiber cell, the plasma membrane is also extremely expanded. Despite progress in understanding cotton fiber cell elongation, knowledge regarding the relationship of plasma membrane in cotton fiber cell development remains elusive.MethodsThe plasma membrane of cotton fiber cells was marked with a low toxic fluorescent dye, di-4-ANEPPDHQ, at different stages of development. Fluorescence images were obtained using a confocal laser scanning microscopy. Subsequently, we investigated the relationship between lipid raft activity and cotton fiber development by calculating generalized polarization (GP values) and dual-channel ratio imaging.ResultsWe demonstrated that the optimum dyeing conditions were treatment with 3 μmol·L− 1 di-4-ANEPPDHQ for 5 min at room temperature, and the optimal fluorescence images were obtained with 488 nm excitation and 500–580 nm and 620–720 nm dual channel emission. First, we examined lipid raft organization in the course of fiber development. The GP values were high in the fiber elongation stage (5–10 DPA, days past anthesis) and relatively low in the initial (0 DPA), secondary cell wall synthesis (20 DPA), and stable synthesis (30 DPA) stages. The GP value peaked in the 10 DPA fiber, and the value in 30 DPA fiber was the lowest. Furthermore, we examined the differences in lipid raft activity in fiber cells between the short fiber cotton mutant, Li-1, and its wild-type. The GP values of the Li-1 mutant fiber were lower than those of the wild type fiber at the elongation stage, and the GP values of 10 DPA fibers were lower than those of 5 DPA fibers in the Li-1 mutant.ConclusionsWe established a system for examining membrane lipid raft activity in cotton fiber cells. We verified that lipid raft activity exhibited a low-high-low change regularity during the development of cotton fiber cell, and the pattern was disrupted in the short lint fiber Li-1 mutant, suggesting that membrane lipid order and lipid raft activity are closely linked to fiber cell development.

Cholesterol and desmosterol incorporation into ram sperm membrane before cryopreservation: Effects on membrane biophysical properties and sperm quality

Ram sperm are particularly sensitive to freeze-thawing mainly due to their lipid composition, limiting their use in artificial insemination programs. We evaluated the extent of cholesterol and desmosterol incorporation into ram sperm through incubation with increasing concentrations of methyl-β-cyclodextrin (MβCD)-sterol complexes, and its effect on membrane biophysical properties, membrane lateral organization and cryopreservation outcome. Sterols were effectively incorporated into the sperm membrane at 10 and 25 mM MβCD-sterols, similarly increasing membrane lipid order at physiological temperature and during temperature decrease. Differential ordering effect of sterols in ternary-mixture model membranes revealed a reduced tendency of desmosterol of segregating into ordered domains. Live cell imaging of fluorescent cholesterol showed sterol incorporation and evidenced the presence of sperm sub-populations compatible with different sterol contents and a high concentration of sterol rich-ordered domains mainly at the acrosome plasma membrane. Lateral organization of the plasma membrane, assessed by identification of GM1-related rafts, was preserved after sterol incorporation except when high levels of sterols (25 mM MβCD-desmosterol) were incorporated. Ram sperm incubation with 10 mM MβCD-sterols prior to cryopreservation in a cholesterol-free extender improved sperm quality parameters after cooling and freezing. While treatment with 10 mM MβCD-cholesterol increased sperm motility, membrane integrity and tolerance to osmotic stress after thawing, incorporation of desmosterol increased the ability of ram sperm to overcome osmotic stress. Our research provides evidence on the effective incorporation and biophysical behavior of cholesterol and desmosterol in ram sperm membranes and on their consequences in improving functional parameters of sperm after temperature decrease and freezing.

Lipid Bilayer Interactions of Peptidic Supramolecular Polymers and Their Impact on Membrane Permeability and Stability.

The synthesis and physicochemical characterization of supramolecular polymers with tunable assembly profiles offer exciting opportunities, involving the development of new biomedical carriers. Because synthetic nanocarriers aim to transport substances across or toward cellular membranes, we evaluated the interactions of amphiphilic peptide-based supramolecular polymers with lipid bilayers. Here, we focused on nanorod-like supramolecular polymers, obtained from two C3-symmetric dendritic peptide amphiphiles with alternating Phe/His sequences, equipped with a peripheral tetraethylene glycol dendron (C3-PH) or charged ethylenediamine end groups (C3-PH+). Triggered by pH changes, these amphiphiles assemble reversibly. Our results show that the supramolecular polymers have an impact on the lipid order in model membranes. Changes in the lipid order were observed depending on the charge state of the amphiphilic building blocks, as well as the chemical composition and physical properties of the bilayer. Furthermore, we further performed cell viability assays with the C3-PH+ and C3-PH supramolecular polymers. For C3-PH, the cell viability and extent of proliferation were decreased and the membrane permeability was enhanced, indicating a strong interaction of the polymer with cellular membranes. The results have implications for the design of novel pH-switchable supramolecular drug carriers and delivery vehicles that can respond to an altered microenvironment of tumorous or inflamed tissue.

Effects of titanium dioxide and zinc oxide nano-materials on lipid order in model membranes

Engineered nano-materials (ENM) have been reported to affect lipid membrane permeability in cell models, but a mechanistic understanding of how these materials interact with biological membranes has not been described. To assess mechanisms of permeability, liposomes composed of DOPC, DOPS, or POPC, with or without cholesterol, were used as model membranes for measuring ENM-induced changes to lipid order to improve our understanding of ENM effects on membrane permeability. Liposomes were treated with either titanium dioxide (TiO2) or zinc oxide (ZnO) ENM, and changes to lipid order were measured by time-resolved fluorescence anisotropy of a lipophilic probe, Di-4-ANEPPDHQ. Both ENM increased lipid order in two lipid models differing in headgroup charge. TiO2 increased lipid order of POPC liposomes (neutral charge), while ZnO acted primarily on DOPS liposomes (negative charge). Addition of cholesterol to these models significantly increased lipid order while in some cases attenuated ENM-induced changes to lipid order. To assess the ability of ENM to induce membrane permeability, liposomes composed of the above lipids were assayed for membrane permeability by calcein leakage in response to ENM. Both ENM caused a dose-dependent increase in permeability in all liposome models tested, and the addition of cholesterol to the liposome models neither blocked nor reduced calcein leakage. Together, these experiments show that ENM increased permeability of small molecules (calcein) from model liposomes, and that the magnitude of the effect of ENM on lipid order depended on ENM surface charge, lipid head group charge and the presence of cholesterol in the membrane.

Polarity Mapping of Cells and Embryos by Improved Fluorescent Solvatochromic Pyrene Probe.

Solvatochromic dyes enable sensing and imaging of biomolecular organization in living systems by monitoring local polarity (lipophilicity), but most such dyes suffer from limited brightness, photostability, lack of a convenient spectral range, and limited sensitivity to polarity. Moreover, the presence of an electron acceptor group, typically a carbonyl, in its push-pull structure raises concerns about its potential chemical reactivity within the biological environment. In order to achieve robust bioimaging, we synthesized a push-pull pyrene probe bearing a ketone acceptor group (PK) and compared it with a recently developed aldehyde analogue (PA). We found that in live cells the aldehyde analogue PA transforms slowly (in ∼100 min) into blue-emissive species, assigned to in situ formation of an imine analogue, whereas the PK probe is stable in the presence of primary amines and inside cells. Like the parent PA, the new probe shows strong solvatochromism and an emission color response to lipid order in membranes (ordered vs disordered liquid phases), while its blue-shifted absorption is more optimal for excitation with 400 nm light sources. In live cells, the PK probe enables high-contrast polarity mapping of organelles using two-color ratiometric detection, suggesting that polarity increases in the following order: lipid droplets < plasma membranes < endoplasmic reticulum. In the zebrafish embryo, polarity imaging with the PK probe reveals a new dimension in visualizing the organization of tissues-lipophilicity distribution, where biomembranes, lipid droplets, cells, yolk, extracellular space, and newly formed organs are revealed by specific emission wavelengths of the probe. The newly developed probe and the proposed approach of polarity mapping open new opportunities for bioimaging at the cellular and animal level.

Single-cell glycolytic activity regulates membrane tension and HIV-1 fusion.

There has been resurgence in determining the role of host metabolism in viral infection yet deciphering how the metabolic state of single cells affects viral entry and fusion remains unknown. Here, we have developed a novel assay multiplexing genetically-encoded biosensors with single virus tracking (SVT) to evaluate the influence of global metabolic processes on the success rate of virus entry in single cells. We found that cells with a lower ATP:ADP ratio prior to virus addition were less permissive to virus fusion and infection. These results indicated a relationship between host metabolic state and the likelihood for virus-cell fusion to occur. SVT revealed that HIV-1 virions were arrested at hemifusion in glycolytically-inactive cells. Interestingly, cells acutely treated with glycolysis inhibitor 2-deoxyglucose (2-DG) become resistant to virus infection and also display less surface membrane cholesterol. Addition of cholesterol in these in glycolytically-inactive cells rescued the virus entry block at hemifusion and enabled completion of HIV-1 fusion. Further investigation with FRET-based membrane tension and membrane order reporters revealed a link between host cell glycolytic activity and host membrane order and tension. Indeed, cells treated with 2-DG possessed lower plasma membrane lipid order and higher tension values, respectively. Our novel imaging approach that combines lifetime imaging (FLIM) and SVT revealed not only changes in plasma membrane tension at the point of viral fusion, but also that HIV is less likely to enter cells at areas of higher membrane tension. We therefore have identified a connection between host cell glycolytic activity and membrane tension that influences HIV-1 fusion in real-time at the single-virus fusion level in live cells.

Primary Cilium Submicron Organization and Dynamics

The aim of this work is to contribute to the characterization of the biophysical properties of the ciliary microenvironment in order to understand central processes of the organelle's biology such as protein import, movement and crowding organization. First, we analyzed the movement of a small soluble protein (EGFP) to establish its pattern of diffusion in different regions of the ciliary compartment (body and distal tip). Using advanced fluorescence fluctuation correlation spectroscopy (Comprehensive correlation analysis) with the Zeiss Airyscan Detector, we characterized the organization and dynamic fingerprint of these subcompartments. Preliminary results showed that both ciliary body and distal tip have permeable microdomains explaining why EGFP presents a pattern of diffusion compatible with dynamic partition rather than free diffusion. However, at the ciliary body diffusion is more confined than at the ciliary tip. Measurements on cytoplasm and nucleus were made to compare with cilia crowding organization. Moreover, to study ciliary structural properties, we used environment-sensitive probes (ACDAN for the intra-ciliary compartment and LAURDAN for the ciliary membrane) combined with spectral phasor analysis to study molecular crowding and membrane's lipid order. Molecular crowding seems similar along cilia (base, body and distal tip). However, the ciliary base membrane was more ordered (less fluid) than the rest of the organelle. In conclusion, we put together a comprehensive protocol to study biophysical properties of primary cilium using state-of-the-art fluorescence techniques. This protocol will allow the evaluation of how different treatments or conditions affect the structure and dynamics of this organelle. (Acknowledgements: Work supported in part by NIH P41-GM103540 and Agencia Nacional de Investigación e Innovación Uruguay.)

LDL induces cholesterol loading and inhibits endothelial proliferation and angiogenesis in Matrigels: correlation with impaired angiogenesis during wound healing.

Hypercholesterolemia is a major risk factor for adverse cardiovascular outcomes, but its effect on angiogenesis and wound healing is not well understood. In this study, using a combination of mass spectrometry and laurdan two-photon imaging, we show that elevated levels of low-density lipoprotein (LDL), like those seen in hypercholesterolemic patients, lead to an increase in both free cholesterol and cholesterol esters, as well as increase in lipid order of endothelial cell membranes. Notably, these effects are distinct and opposite to the lack of cholesterol loading and the disruption of lipid order observed in our earlier studies in response to oxidized LDL (oxLDL). The same pathological level of LDL leads to a significant inhibition of endothelial proliferation and cell cycle arrest in G2/M phase, whereas oxLDL enhances endothelial proliferation in S phase of the cycle. LDL but not oxLDL suppresses the expression of vascular endothelial growth factor receptor-2 while enhancing the expression of vascular endothelial growth factor (VEGF). Furthermore, we show that aged (8-10 mo) hypercholesterolemic apolipoprotein E-deficient (ApoE-/-) mice display delayed wound closure compared with age-matched C57/BL6 wild-type controls following a skin punch biopsy. The delay in wound healing is associated with a decreased expression of cluster of differentiation 31 platelet endothelial cell adhesion molecule endothelial marker and decreased angiogenesis within the wound bed. Furthermore, decreased endothelial responsiveness to the growth factors VEGF and basic fibroblast growth factor is observed in ApoE-/- mice in Matrigel plugs and in Matrigels with high levels of LDL in wild-type mice. We propose that plasma hypercholesterolemia is antiangiogenic due to elevated levels of LDL.

Kill, but don't be killed

Cell Biology Cytotoxic T lymphocytes (CTLs) recognize and destroy infected or malignant cells. CTLs secrete the pore-forming protein perforin into the immune synapse, where perforin disrupts the target cell plasma membrane and initiates cell death pathways. Paradoxically, the secreted perforin only damages the target cell, leaving the producer lymphocytes unscathed and able to attack another target cell. What protects the lymphocyte is unclear. Using a variety of approaches, Rudd-Schmidt et al. found that two distinct but coordinated mechanisms were deployed by CTLs to protect themselves. Both mechanisms depended on the dynamic regulation of plasma membrane lipid composition and topology. High membrane lipid order made the CTL presynaptic membrane refractory to perforin binding. Furthermore, phosphatidylserine, exposed on the killer cell membrane, appeared to bind and inactivate perforin. Nat. Commun. 10 , 5396 (2019).

The lipid membrane of HIV-1 stabilizes the viral envelope glycoproteins and modulates their sensitivity to antibody neutralization

The envelope glycoproteins (Envs) of HIV-1 are embedded in the cholesterol-rich lipid membrane of the virus. Chemical depletion of cholesterol from HIV-1 particles inactivates their infectivity. We observed that diverse HIV-1 strains exhibit a range of sensitivities to such treatment. Differences in sensitivity to cholesterol depletion could not be explained by variation in Env components known to interact with cholesterol, including the cholesterol-recognition motif and cytoplasmic tail of gp41. Using antibody-binding assays, measurements of virus infectivity, and analyses of lipid membrane order, we found that depletion of cholesterol from HIV-1 particles decreases the conformational stability of Env. It enhances exposure of partially cryptic epitopes on the trimer and increases sensitivity to structure-perturbing treatments such as antibodies and cold denaturation. Substitutions in the cholesterol-interacting motif of gp41 induced similar effects as depletion of cholesterol. Surface-acting agents, which are incorporated into the virus lipid membrane, caused similar effects as disruption of the Env-cholesterol interaction. Furthermore, substitutions in gp120 that increased structural stability of Env (i.e. induced a "closed" conformation of the trimer) increased virus resistance to cholesterol depletion and to the surface-acting agents. Collectively, these results indicate a critical contribution of the viral membrane to the stability of the Env trimer and to neutralization resistance against antibodies. Our findings suggest that the potency of poorly neutralizing antibodies, which are commonly elicited in vaccinated individuals, may be markedly enhanced by altering the lipid composition of the viral membrane.

Investigating the Role of Lipid Transfer in Microbubble-Mediated Drug Delivery.

Sonoporation, the permeabilization of cell membranes following exposure to microbubbles and ultrasound, has considerable potential for therapeutic delivery. To date, engineering of microbubbles for these applications has focused primarily upon optimizing microbubble size and stability, or attachment of targeting species and/or drug molecules. In this work, it is demonstrated that the microbubble coating can also be tailored to directly influence cell permeabilization. Specifically, lipid exchange mechanisms between phospholipid microbubbles and cells can be exploited to significantly increase sonoporation efficiency in vitro. A theoretical analysis of the energy required for pore formation was carried out. From this, it was hypothesized that sonoporation could be promoted by the transfer of lipid molecules with appropriate carbon chain length and/or shape (cylindrical or conical). Spectral imaging with a hydration-sensitive membrane probe (C-Laurdan) was used to measure changes in the membrane lipid order of A-549 cancer cells following exposure to suspensions of different phospholipids. Two candidate lipids were identified, a short-chain-length phospholipid (1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)) and a medium-chain-length lysolipid (1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (16:0 lyso-PC)). Microbubbles were prepared with matched concentrations, size distributions, and acoustic responses. Confocal microscopy was used to measure cell uptake of a model drug (propidium iodide) with and without ultrasound exposure (1 MHz, 250 kPa peak negative pressure, 1 kHz pulse repetition frequency, 10% duty cycle, 15 s exposure). Despite significantly decreasing the cell membrane lipid order, DLPC did not increase sonoporation. Microbubbles containing 16:0 lyso-PC, however, produced a ∼5-fold increase in sonoporation compared to control microbubbles. Importantly, the lyso-PC molecules were incorporated into the microbubble coating and did not affect cell permeability prior to ultrasound exposure. These findings indicate that microbubbles can be engineered to exploit lipid exchange between microbubble shells and cell membranes to enhance drug delivery, a new optimization route that may lead to enhanced therapeutic efficacy of ultrasound-mediated treatments.