Changes In Membrane Properties Research Articles

Driving Role of Zinc Oxide Nanoparticles with Different Sizes and Hydrophobicity in Metabolic Response and Eco-Corona Formation in Sprouts (Vigna radiata).

Zinc oxide nanoparticles (ZnO NPs) cause biotoxicity and pose a potential ecological threat; however, their effects on plant metabolism and eco-corona evolution between NPs and organisms remain unclear. This study clarified the molecular mechanisms underlying physiological and metabolic responses induced by three different ZnO NPs with different sizes and hydrophobicity in sprouts (Vigna radiata) and explored the critical regulation of eco-corona formation in root-nano systems. Results indicated that smaller-sized ZnO inhibited root elongation by up to 37.14% and triggered oxidative burst and apoptosis. Metabolomics confirmed that physiological maintenance after n-ZnO exposure was mainly attributed to the effective stabilization of nitrogen fixation and defense systems by biotransformation of the flavonoid pathway. Larger-sized or hydrophobic group-modified ZnO exhibited low toxicity in sprouts, with 0.89-fold upregulation of citrate in central carbon metabolism. This contributed to providing energy for resistance to NP stress through amino acid and carbon/nitrogen metabolism, accompanied by changes in membrane properties. Notably, smaller-sized and hydrophobic NPs intensely stimulated the release of root metabolites, forming corona complexes with exudates. The hydrogen-bonded wrapping mechanism in protein secondary structure and hydrophobic interactions of heterogeneous functional groups drove eco-corona formation, along with the corona evolution intensity of n-ZnO > s-ZnO > b-ZnO based on higher (α-helix + 3-turn helix)/β-sheet ratios. This study provides crucial insight into metabolic and eco-corona evolution in bionano fates.

Susceptibility of Legionella gormanii Membrane-Derived Phospholipids to the Peptide Action of Antimicrobial LL-37-Langmuir Monolayer Studies.

LL-37 is the only member of the cathelicidin-type host defense peptide family in humans. It exhibits broad-spectrum bactericidal activity, which represents a distinctive advantage for future therapeutic targets. The presence of choline in the growth medium for bacteria changes the composition and physicochemical properties of their membranes, which affects LL-37's activity as an antimicrobial agent. In this study, the effect of the LL-37 peptide on the phospholipid monolayers at the liquid-air interface imitating the membranes of Legionella gormanii bacteria was determined. The Langmuir monolayer technique was employed to prepare model membranes composed of individual classes of phospholipids-phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), cardiolipin (CL)-isolated from L. gormanii bacteria supplemented or non-supplemented with exogenous choline. Compression isotherms were obtained for the monolayers with or without the addition of the peptide to the subphase. Then, penetration tests were carried out for the phospholipid monolayers compressed to a surface pressure of 30 mN/m, followed by the insertion of the peptide into the subphase. Changes in the mean molecular area were observed over time. Our findings demonstrate the diversified effect of LL-37 on the phospholipid monolayers, depending on the bacteria growth conditions. The substantial changes in membrane properties due to its interactions with LL-37 enable us to propose a feasible mechanism of peptide action at a molecular level. This can be associated with the stable incorporation of the peptide inside the monolayer or with the disruption of the membrane leading to the removal (desorption) of molecules into the subphase. Understanding the role of antimicrobial peptides is crucial for the design and development of new strategies and routes for combating resistance to conventional antibiotics.

Diffusion Analyses along Mean and Gaussian-Curved Membranes with CurD.

Curved cellular membranes are both abundant and functionally relevant. While novel tomography approaches reveal the structural details of curved membranes, their dynamics pose an experimental challenge. Curvature especially affects the diffusion of lipids and macromolecules, yet neither experiments nor continuum models distinguish geometric effects from those caused by curvature-induced changes in membrane properties. Molecular simulations could excel here, yet despite community interest toward curved membranes, tools for their analysis are still lacking. Here, we satisfy this demand by introducing CurD, our novel and openly available implementation of the Vertex-oriented Triangle Propagation algorithm to the study of lipid diffusion along membranes with mean and/or Gaussian curvature. This approach, aided by our highly optimized implementation, computes geodetic distances significantly faster than conventional implementations of path-finding algorithms. Our tool, applied to coarse-grained simulations, allows for the first time the analysis of curvature effects on diffusion at size scales relevant to physiological processes such as endocytosis. Our analyses with different membrane geometries reveal that Gaussian curvature plays a surprisingly small role on lipid motion, whereas mean curvature; i.e., the packing of lipid headgroups largely dictates their mobility.

Insight into the evolution of membrane chemical degradation in proton exchange membrane fuel cells:From theoretical analysis to model developing

The chemical degradation of the proton exchange membrane (PEM) causes the changes in membrane properties and morphology, which directly affects the cell performance and membrane degradation. In this work, the changes of PEM during the chemical degradation are theoretically analyzed at both microscopic and mesoscopic levels. Based on the theoretical study, a bidirectionally coupled physical model of PEM fuel cell (PEMFC) performance and PEM chemical degradation is developed, which is used to investigate the evolutions of cell performance and chemical degradation. It can be found that the open circuit voltage first increases slightly and then decreases with time, and the ohmic loss increases rapidly under accelerated degradation conditions. The membrane chemical degradation is analyzed in terms of four aspects: the mass loss rate of the membrane, the region of pronounced degradation, the contribution of different degradation mechanisms and the proportion of ionomer species remaining. Furthermore, the effect of operating conditions on the degradation is examined. The degradation is most sensitive to voltage and has the fastest rate at high voltage, high temperature, high gas pressure and relative humidity of 60%. This work provides a deep insight into the PEM chemical degradation, which is instructive for the long-term prediction of PEMFC lifetime.

The intricate link between membrane lipid structure and composition and membrane structural properties in bacterial membranes.

It is now evident that the cell manipulates lipid composition to regulate different processes such as membrane protein insertion, assembly and function. Moreover, changes in membrane structure and properties, lipid homeostasis during growth and differentiation with associated changes in cell size and shape, and responses to external stress have been related to drug resistance across mammalian species and a range of microorganisms. While it is well known that the biomembrane is a fluid self-assembled nanostructure, the link between the lipid components and the structural properties of the lipid bilayer are not well understood. This perspective aims to address this topic with a view to a more detailed understanding of the factors that regulate bilayer structure and flexibility. We describe a selection of recent studies that address the dynamic nature of bacterial lipid diversity and membrane properties in response to stress conditions. This emerging area has important implications for a broad range of cellular processes and may open new avenues of drug design for selective cell targeting.

Erythrocytes' surface properties and stiffness predict survival and functional decline in ALS patients.

Erythrocytes play a fundamental role in oxygen delivery to tissues and binding to inflammatory mediators. Evidences suggest that dysregulated erythrocyte function could contribute to the pathophysiology of several neurodegenerative diseases. We aimed to evaluate changes in morphological, biomechanical, and biophysical properties of erythrocytes from amyotrophic lateral sclerosis (ALS) patients, as new areas of study in this disease. Blood samples were collected from ALS patients, comparing with healthy volunteers. Erythrocytes were assessed using atomic force microscopy (AFM) and zeta potential analysis. The patients' motor and respiratory functions were evaluated using the revised ALS Functional Rating Scale (ALSFRS-R) and percentage of forced vital capacity (%FVC). Patient survival was also assessed. Erythrocyte surface roughness was significantly smoother in ALS patients, and this parameter was a predictor of faster decline in ALSFRS-R scores. ALS patients exhibited higher erythrocyte stiffness, as indicated by reduced AFM tip penetration depth, which predicted a faster ALSFRS-R score and respiratory subscore decay. A lower negative charge on the erythrocyte membrane was predictor of a faster ALSFRS-R and FVC decline. Additionally, a larger erythrocyte surface area was an independent predictor of lower survival. These changes in morphological and biophysical membrane properties of ALS patients' erythrocytes, lead to increased cell stiffness and morphological variations. We speculate that these changes might precipitate motoneurons dysfunction and accelerate disease progression. Further studies should explore the molecular alterations related to these observations. Our findings may contribute to dissect the complex interplay between respiratory function, tissue hypoxia, progression rate, and survival in ALS.

Acute inhibition of acid sensing ion channel 1a after spinal cord injury selectively affects excitatory synaptic transmission, but not intrinsic membrane properties, in deep dorsal horn interneurons.

Following a spinal cord injury (SCI), secondary damage mechanisms are triggered that cause inflammation and cell death. A key component of this secondary damage is a reduction in local blood flow that initiates a well-characterised ischemic cascade. Downstream hypoxia and acidosis activate acid sensing ion channel 1a (ASIC1a) to trigger cell death. We recently showed that administration of a potent venom-derived inhibitor of ASIC1a, Hi1a, leads to tissue sparing and improved functional recovery when delivered up to 8 h after ischemic stroke. Here, we use whole-cell patch-clamp electrophysiology in a spinal cord slice preparation to assess the effect of acute ASIC1a inhibition, via a single dose of Hi1a, on intrinsic membrane properties and excitatory synaptic transmission long-term after a spinal cord hemisection injury. We focus on a population of interneurons (INs) in the deep dorsal horn (DDH) that play a key role in relaying sensory information to downstream motoneurons. DDH INs in mice treated with Hi1a 1 h after a spinal cord hemisection showed no change in active or passive intrinsic membrane properties measured 4 weeks after SCI. DDH INs, however, exhibit significant changes in the kinetics of spontaneous excitatory postsynaptic currents after a single dose of Hi1a, when compared to naive animals (unlike SCI mice). Our data suggest that acute ASIC1a inhibition exerts selective effects on excitatory synaptic transmission in DDH INs after SCI via specific ligand-gated receptor channels, and has no effect on other voltage-activated channels long-term after SCI.

Cristae formation is a mechanical buckling eventcontrolled by the inner mitochondrial membrane lipidome.

Cristae are high-curvature structures in the inner mitochondrial membrane (IMM) that are crucial for ATP production. While cristae-shaping proteins have been defined, analogous lipid-based mechanisms have yet to be elucidated. Here, we combine experimental lipidome dissection with multi-scale modeling to investigate how lipid interactions dictate IMM morphology and ATP generation. When modulating phospholipid (PL) saturation in engineered yeast strains, we observed a surprisingly abrupt breakpoint in IMM topology driven by a continuous loss of ATP synthase organization at cristae ridges. We found that cardiolipin (CL) specifically buffers the inner mitochondrial membrane against curvature loss, an effect that is independent of ATP synthase dimerization. To explain this interaction, we developed a continuum model for cristae tubule formation that integrates both lipid and protein-mediated curvatures. This model highlighted a snapthrough instability, which drives IMM collapse upon small changes in membrane properties. We also showed that cardiolipin is essential in low-oxygen conditions that promote PL saturation. These results demonstrate that the mechanical function of cardiolipin is dependent on the surrounding lipid and protein components of the IMM.

Cellular excitability and ns-pulsed electric fields: Potential involvement of lipid oxidation in the action potential activation

Recent studies showed that nanosecond pulsed electric fields (nsPEFs) can activate voltage-gated ion channels (VGICs) and trigger action potentials (APs) in excitable cells. Under physiological conditions, VGICs’ activation takes place on time scales of the order 10–100 µs. These time scales are considerably longer than the applied pulse duration, thus activation of VGICs by nsPEFs remains puzzling and there is no clear consensus on the mechanisms involved. Here we propose that changes in local electrical properties of the cell membrane due to lipid oxidation might be implicated in AP activation. We first use MD simulations of model lipid bilayers with increasing concentration of primary and secondary lipid oxidation products and demonstrate that oxidation not only increases the bilayer conductance, but also the bilayer capacitance. Equipped with MD-based characterization of electrical properties of oxidized bilayers, we then resort to AP modelling at the cell level with Hodgkin-Huxley-type models. We confirm that a local change in membrane properties, particularly the increase in membrane conductance, due to formation of oxidized membrane lesions can be high enough to trigger an AP, even when no external stimulus is applied. However, excessive accumulation of oxidized lesions (or other conductive defects) can lead to altered cell excitability.

Amyloid-β Effects on Peripheral Nerve: A New Model System.

Although there are many biochemical methods to measure amyloid-β (Aβ)42 concentration, one of the critical issues in the study of the effects of Aβ42 on the nervous system is a simple physiological measurement. The in vitro rat sciatic nerve model is employed and the nerve action potential (NAP) is quantified with different stimuli while exposed to different concentrations of Aβ42. Aβ42 predominantly reduces the NAP amplitude with minimal effects on other parameters except at low stimulus currents and short inter-stimulus intervals. The effects of Aβ42 are significantly concentration-dependent, with a maximum reduction in NAP amplitude at a concentration of 70 nM and smaller effects on the NAP amplitude at higher and lower concentrations. However, even physiologic concentrations in the range of 70 pM did reduce the NAP amplitude. The effects of Aβ42 became maximal 5-8 h after exposure and did not reverse during a 30 min washout period. The in vitro rat sciatic nerve model is sensitive to the effects of physiologic concentrations of Aβ42. These experiments suggest that the effect of Aβ42 is a very complex function of concentration that may be the result of amyloid-related changes in membrane properties or sodium channels.

ST2-Conditioned Medium Fosters Dorsal Horn Cell Excitability and Synaptic Transmission in Cultured Mouse Spinal Cord

Conditioned medium obtained from bone marrow-derived stem cells has been proposed as a novel cell-free therapy in spinal cord injury and neuropathic pain, yet the direct effect on spinal neuron function has never been investigated. Here, we adopted spinal cord organotypic cultures (SCOCs) as an experimental model to probe the effect of ST2 murine mesenchymal stem cells-conditioned medium (ST2-CM) on dorsal horn (DH) neuron functional properties. Three days of SCOC exposure to ST2-CM increased neuronal activity measured by Fos expression, as well as spontaneous or induced firing. We showed that the increase in neuronal excitability was associated with changes in both intrinsic membrane properties and an enhanced excitatory drive. The increased excitability at the single-cell level was substantiated at the network level by detecting synchronous bursts of calcium waves across DH neurons. Altogether, SCOCs represent a viable tool to probe mesenchymal cells' effect on intact neuronal networks. Our findings indicate that ST2-CM enhances neuronal activity and synaptic wiring in the spinal dorsal horn. Our data also support the trophic role of mesenchymal cells CM in maintaining network activity in spinal circuits.Graphical

A Thermocatalytic Ceramic Membrane by Perovskite Incorporation in the Alumina Framework

AbstractAccess to clean water is limited by the increasing amount of persistent organic pollutants (POPs), since current methods fail to remove POPs completely. Therefore, new treatment technologies of surface water and wastewater are needed. In this study, two treatment methods are combined in one step, i.e., membrane filtration and thermocatalytic chemical oxidation of POPs. A perovskite‐type catalyst with formula Sr0.85Ce0.15FeO3‐δ (SCF) is incorporated into an alumina membrane using a simple two‐step heat treatment to minimize any chemical reaction of the catalytic active perovskite with alumina. First, a sintering process under inert atmosphere, then, a heat‐treatment under oxidative conditions to oxidize the iron species in the perovskite structure. The well‐known thermocatalytic properties of SCF make the membrane thermocatalytic and thus able to degrade pollutants under dark conditions without addition of oxidants. The SCF content in the membrane is varied between 0 and 15 wt% to explore the change in membrane properties. Results demonstrate that the thermocatalytic membranes have great potential for continuous membrane filtration and simultaneous degradation of POPs. When considering methyl orange, up to 100% removal is obtained at room temperature, whereas up to 93% of bisphenol A is removed at temperatures approaching 60 °C.

Enhanced Membrane Fluidization and Cholesterol Displacement by 1-Heptanol Inhibit Mast Cell Effector Functions.

Signal transduction by the high-affinity IgE receptor (FcεRI) depends on membrane lipid and protein compartmentalization. Recently published data show that cells treated with 1-heptanol, a cell membrane fluidizer, exhibit changes in membrane properties. However, the functional consequences of 1-heptanol-induced changes on mast cell signaling are unknown. This study shows that short-term exposure to 1-heptanol reduces membrane thermal stability and dysregulates mast cell signaling at multiple levels. Cells treated with 1-heptanol exhibited increased lateral mobility and decreased internalization of the FcεRI. However, this did not affect the initial phosphorylation of the FcεRI-β chain and components of the SYK/LAT1/PLCγ1 signaling pathway after antigen activation. In contrast, 1-heptanol inhibited SAPK/JNK phosphorylation and effector functions such as calcium response, degranulation, and cytokine production. Membrane hyperfluidization induced a heat shock-like response via increased expression of the heat shock protein 70, increased lateral diffusion of ORAI1-mCherry, and unsatisfactory performance of STIM1-ORAI1 coupling, as determined by flow-FRET. Furthermore, 1-heptanol inhibited the antigen-induced production of reactive oxygen species and potentiated stress-induced plasma membrane permeability by interfering with heat shock protein 70 activity. The combined data suggest that 1-heptanol-mediated membrane fluidization does not interfere with the earliest biochemical steps of FcεRI signaling, such as phosphorylation of the FcεRI-β chain and components of the SYK/LAT/PLCγ1 signaling pathway, instead inhibiting the FcεRI internalization and mast cell effector functions, including degranulation and cytokine production.

Delaying the GABA Shift Indirectly Affects Membrane Properties in the Developing Hippocampus.

During the first two postnatal weeks, intraneuronal chloride concentrations in rodents gradually decrease, causing a shift from depolarizing to hyperpolarizing GABA responses. The postnatal GABA shift is delayed in rodent models for neurodevelopmental disorders and in human patients, but the impact of a delayed GABA shift on the developing brain remains obscure. Here we examine the direct and indirect consequences of a delayed postnatal GABA shift on network development in organotypic hippocampal cultures made from 6- to 7-d-old mice by treating the cultures for 1 week with VU0463271, a specific inhibitor of the chloride exporter KCC2. We verified that VU treatment delayed the GABA shift and kept GABA signaling depolarizing until DIV9. We found that the structural and functional development of excitatory and inhibitory synapses at DIV9 was not affected after VU treatment. In line with previous studies, we observed that GABA signaling was already inhibitory in control and VU-treated postnatal slices. Surprisingly, 14 d after the VU treatment had ended (DIV21), we observed an increased frequency of spontaneous inhibitory postsynaptic currents in CA1 pyramidal cells, while excitatory currents were not changed. Synapse numbers and release probability were unaffected. We found that dendrite-targeting interneurons in the stratum radiatum had an elevated resting membrane potential, while pyramidal cells were less excitable compared with control slices. Our results show that depolarizing GABA signaling does not promote synapse formation after P7, and suggest that postnatal intracellular chloride levels indirectly affect membrane properties in a cell-specific manner.SIGNIFICANCE STATEMENT During brain development, the action of neurotransmitter GABA shifts from depolarizing to hyperpolarizing. This shift is a thought to play a critical role in synapse formation. A delayed shift is common in rodent models for neurodevelopmental disorders and in human patients, but its consequences for synaptic development remain obscure. Here, we delayed the GABA shift by 1 week in organotypic hippocampal cultures and carefully examined the consequences for circuit development. We find that delaying the shift has no direct effects on synaptic development, but instead leads to indirect, cell type-specific changes in membrane properties. Our data call for careful assessment of alterations in cellular excitability in neurodevelopmental disorders.

The ferroptosis inducer RSL3 triggers interictal epileptiform activity in mice cortical neurons.

Epilepsy is a neurological disorder characterized by recurrent seizures, which result from excessive, synchronous discharges of neurons in different brain areas. In about 30% of cases, epileptic discharges, which vary in their etiology and symptomatology, are difficult to treat with conventional drugs. Ferroptosis is a newly defined iron-dependent programmed cell death, characterized by excessive accumulation of lipid peroxides and reactive oxygen species. Evidence has been provided that ferroptosis is involved in epilepsy, and in particular in those forms resistant to drugs. Here, whole cell patch clamp recordings, in current and voltage clamp configurations, were performed from layer IV principal neurons in cortical slices obtained from adult mouse brain. Application of the ferroptosis inducer RAS-selective lethal 3 (RSL3) induced interictal epileptiform discharges which started at RSL3 concentrations of 2 μM and reached a plateau at 10 μM. This effect was not due to changes in active or passive membrane properties of the cells, but relied on alterations in synaptic transmission. In particular, interictal discharges were dependent on the excessive excitatory drive to layer IV principal cells, as suggested by the increase in frequency and amplitude of spontaneously occurring excitatory glutamatergic currents, possibly dependent on the reduction of inhibitory GABAergic ones. This led to an excitatory/inhibitory unbalance in cortical circuits. Interictal bursts could be prevented or reduced in frequency by the lipophilic antioxidant Vitamin E (30 μM). This study allows identifying new targets of ferroptosis-mediated epileptic discharges opening new avenues for the treatment of drug-resistant forms of epilepsy.

Effects of monolauroyl-galactosylglycerol on membrane fatty acids and properties of Bacillus cereus.

The purpose of this study was to provide new ideas for the antibacterial mechanism of monolauroyl-galactosylglycerol (MLGG) from the perspective of cell membranes. The changes in cell membrane properties of Bacillus cereus (B. cereus) CMCC 66,301 exposed to different concentrations (1 × MIC (minimum inhibitory concentration), 2 × MIC, 1 × MBC (minimum bacterial concentration)) of MLGG were evaluated. It was found that the lag phase of B. cereus cells was prolonged at low concentration MLGG (1 × MIC and 2 × MIC), while about 2 log CFU/mL reduction in B. cereus populations were observed when exposed to high concentration MLGG (1 × MBC). MLGG treated B. cereus displayed obvious membrane depolarization, while membrane permeability had no change using PI (propidium iodide) staining. Significant increase in the membrane fluidity in response to MLGG exposure occurred, which was consistent with the modification of membrane fatty acids compositions, where the relative content of straight-chain fatty acids (SCFAs) and unsaturated fatty acids (UFAs) increased, while branched-chain fatty acids (BCFAs) decreased significantly. The decreased transition Tm value and cell surface hydrophobicity was also observed. Additionally, effect of MLGG on bacterial membrane compositions were explored at the submolecular level by infrared spectroscopy. Resistance tests of B. cereus to MLGG had demonstrated the advantages of MLGG as a bacteriostatic agent. Collectively, these studies indicate that modifying the fatty acid composition and properties of cellular membranes through MLGG exposure is crucial for inhibiting bacteria growth, providing new insights into the antimicrobial mechanisms of MLGG. KEY POINTS: • Monolauroyl-galactosylglycerol inserted into B. cereus lipid bilayer membrane • Monolauroyl-galactosylglycerol treatment caused B. cereus membrane depolarization • Monolauroyl-galactosylglycerol resulted in B. cereus membrane fatty acids alteration.

Impact of St. John’s wort extract Ze 117 on stress induced changes in the lipidome of PBMC

BackgroundMembrane lipids have an important function in the brain as they not only provide a physical barrier segregating the inner and outer cellular environments, but are also involved in cell signaling. It has been shown that the lipid composition effects membrane fluidity which affects lateral mobility and activity of membrane-bound receptors.MethodsSince changes in cellular membrane properties are considered to play an important role in the development of depression, the effect of St. John’s wort extract Ze 117 on plasma membrane fluidity in peripheral blood mononuclear cells (PBMC) was investigated using fluorescence anisotropy measurements. Changes in fatty acid residues in phospholipids after treatment of cortisol-stressed [1 μM] PBMCs with Ze 117 [10–50 µg/ml] were analyzed by mass spectrometry.ResultsCortisol increased membrane fluidity significantly by 3%, co-treatment with Ze 117 [50 µg/ml] counteracted this by 4.6%. The increased membrane rigidity by Ze 117 in cortisol-stressed [1 μM] PBMC can be explained by a reduced average number of double bonds and shortened chain length of fatty acid residues in phospholipids, as shown by lipidomics experiments.ConclusionThe increase in membrane rigidity after Ze 117 treatment and therefore the ability to normalize membrane structure points to a new mechanism of antidepressant action of the extract.

Sphingomyelinase modulates synaptic vesicle mobilization at the mice neuromuscular junctions

AimsSphingomyelin is an abundant component of the presynaptic membrane and an organizer of lipid rafts. In several pathological conditions, sphingomyelin is hydrolyzed due to an upregulation and release of secretory sphingomyelinases (SMases). Herein, the effects of SMase on exocytotic neurotransmitter release were studied in the diaphragm neuromuscular junctions of mice. Main methodsMicroelectrode recordings of postsynaptic potentials and styryl (FM) dyes were used to estimate neuromuscular transmission. Membrane properties were assessed with fluorescent techniques. Key findingsApplication of SMase at a low concentration (0.01 U ml−1) led to a disruption of lipid-packing in the synaptic membranes. Neither spontaneous exocytosis nor evoked neurotransmitter release (in response to single stimuli) were affected by SMase treatment. However, SMase significantly increased neurotransmitter release and the rate of fluorescent FM-dye loss from the synaptic vesicles at 10, 20 and 70 Hz stimulation of the motor nerve. In addition, SMase treatment prevented a shift of the exocytotic mode from “full-collapse” fusion to “kiss-and-run” during high-frequency (70 Hz) activity. The potentiating effects of SMase on neurotransmitter release and FM-dye unloading were suppressed when synaptic vesicle membranes were also exposed to this enzyme (i.e., stimulation occurred during SMase treatment). SignificanceThus, hydrolysis of the plasma membrane sphingomyelin can enhance mobilization of synaptic vesicles and facilitate full fusion mode of exocytosis, but SMase acting on vesicular membrane had a depressant effect on the neurotransmission. Partially, the effects of SMase can be related with the changes in synaptic membrane properties and intracellular signaling.

Polarized cell plasma membrane composition and protein sorting.

Epithelial cell polarization is an essential biological process, serving many physiological roles including tissue morphogenesis and wound healing. A defining feature of polarization is the separation of cell plasma membrane lipids and proteins into apical and basolateral compartments between which molecular exchange is restricted. Decades ago, the apical membrane was found to be enriched in saturated lipids, glycolipids, and cholesterol. Mechanistic hypotheses to explain the biogenesis and unique composition of the apical membrane include self-assembling membrane domains (i.e., lipid rafts), specific protein sorting motifs, and post-translational modifications mediating protein sorting. However, neither the detailed composition nor the mechanisms of protein and lipid sorting between plasma membrane domains in epithelial cells have been resolved. Particularly, the lipid profile of the basolateral membrane remains unstudied, leaving doubts about the differentiation and lipid separation of the apical and basolateral membrane. We use advanced lipidomics and imaging techniques to characterize the changes in lipid organization, membrane composition, and membrane properties during the cellular polarization process. We observe that the apical membrane is enriched in highly saturated lipids and glycolipids relative to the basolateral membrane, with apical membrane biophysical properties reflecting a raft-enriched environment. Similar to lipids, the determinants of protein sorting between apical and basolateral domains are poorly understood, with past studies focusing on either specific proteins or trafficking machinery. Importantly, the apical membrane hosts an extensive extracellular glycocalyx consisting of glycolipids, glycoproteins, and polysaccharides. The role of these glycocalyx molecules on protein sorting has not been revealed, despite a major fraction of apical proteins being glycosylated. We systematically evaluate the structural determinants of apical versus basolateral sorting with a focus on transmembrane domain features, protein raft affinity, and glycosylation. We find that these protein features cooperatively direct protein sorting to the apical membrane.

Mechanism of urea and TMAO counteraction in lipid membrane interactions.

To cope with stress induced by high salinity and hydrostatic pressure, some marine animals accumulate small organic solutes called osmolytes. Most notable among these osmolytes are the denaturant urea, and trimethylamine N-oxide (TMAO) that is known to stabilize proteins. Although TMAO and urea are well known to act together in sharks and other elasmobranchs to form solutions at molar concentrations whose net action on macromolecules is close to that of pure water, the mechanism for this counteraction is still debated. Our study focuses on this effect as seen in the forces acting between lipid bilayers. We found that TMAO pushes adjacent membranes closer together, while urea makes membranes swell. At a specific ratio, the combined effect of the two solutes is the similar to pure water. Our experiments and theory indicate that TMAO dehydration is due to preferential partitioning of the solute away from the lipid and subsequent osmotic pressure that acts to bring membranes closer. By contrast, urea acts to increase the distance between lipid membranes mainly because it reduces the van der Waals attraction between bilayers. This mechanism is distinct from the action of urea on proteins because urea does not preferentially interact with the lipid. The action of the two osmolytes is almost additive. Weak synergy between solutes can be rationalized through small changes in membrane structure and properties. We comment on the potential role of osmolytes acting together in the modification of membrane remodeling processes including adhesion and fusion.