Distribution Of Membrane Lipids Research Articles

Melittin-Phospholipase A2 Synergism Is Mediated by Liquid-Liquid Miscibility Phase Transition in Giant Unilamellar Vesicles.

The primary constituents of honeybee venom, melittin and phospholipase A2 (PLA2), display toxin synergism in which the PLA2 activity is significantly enhanced by the presence of melittin. It has been shown previously that this is accomplished by the disruption in lipid packing, which allows PLA2 to become processive on the membrane surface. In this work, we show that melittin is capable of driving miscibility phase transition in giant unilamellar vesicles (GUVs) and that it raises the miscibility transition temperature (Tmisc) in a concentration-dependent manner. The induced phase separation enhances the processivity of PLA2, particularly at its boundaries, where a substantial difference in domain thickness creates a membrane discontinuity. The catalytic action of PLA2, in response, induces changes in the membrane, rendering it more conducive to melittin binding. This, in turn, facilitates further lipid phase separation and eventual vesicle lysis. Overall, our results show that melittin has powerful membrane-altering capabilities that activate PLA2 in various membrane contexts. More broadly, they exemplify how this biochemical system actively modulates and capitalizes on the spatial distribution of membrane lipids to efficiently achieve its objectives.

Liposomes and Lipid Droplets Display a Reversal of Charge-Induced Hydration Asymmetry.

The unique properties of water are critical for life. Water molecules have been reported to hydrate cations and anions asymmetrically in bulk water, being a key element in the balance of biochemical interactions. We show here that this behavior extends to charged lipid nanoscale interfaces. Charge hydration asymmetry was investigated by using nonlinear light scattering methods on lipid nanodroplets and liposomes. Nanodroplets covered with negatively charged lipids induce strong water ordering, while droplets covered with positively charged lipids induce negligible water ordering. Surprisingly, this charge-induced hydration asymmetry is reversed around liposomes. This opposite behavior in charge hydration asymmetry is caused by a delicate balance of electrostatic and hydrogen-bonding interactions. These findings highlight the importance of not only the charge state but also the specific distribution of neutral and charged lipids in cellular membranes.

Subcellular distribution of membrane lipids revealed by freeze-fracture electron microscopy.

Cell membranes are composed of a large variety of lipids and proteins. While the localization and function of membrane proteins have been extensively investigated, the distribution of membrane lipids, especially in the non-cytoplasmic leaflet of organelle membranes, remains largely unknown. Fluorescent biosensors have been widely used to study membrane lipid distribution; however, they have some limitations. By utilizing the quick-freezing and freeze-fracture replica labeling electron microscopy technique, we can uncover the precise distribution of membrane lipids within cells and assess the function of lipid-transporting proteins. In this review, I summarize recent progress in analyzing intracellular lipid distribution by utilizing this method.

Red blood cells from patients with sitosterolemia exhibit impaired membrane lipid composition and distribution and decreased deformability

Sitosterolemia is a metabolic disorder leading to excessive accumulation of phytosterols. Hemolytic stomatocytosis and macrothrombocytopenia are part of the clinical picture. However, the impact of phytosterols on red blood cell (RBC) deformability, membrane lipid composition and distribution and the efficiency of the reference treatment, Ezetimibe, are largely unknown. This study addresses these issues using RBCs from three patients with sitosterolemia and healthy RBCs exposed to β-sitosterol. Patients presented an increased proportion of stomatocytes, decreased RBC deformability and increased RBC hydration and osmotic fragility compared to healthy donors. At the membrane level, patient RBCs showed (i) very high content in β-sitosterols, (ii) increased proportions of saturated fatty acids and polyunsaturated fatty acid species with long and unsaturated carbon chains, and (iii) decreased content in phosphatidylethanolamine species. These lipid changes were accompanied by an almost complete abrogation of cholesterol-enriched domains, which could result from: (i) the reduced phosphatidylethanolamine content which positively correlated with domain abundance; and (ii) the fatty acid modifications and increased phytosterol content, both compatible with higher membrane stiffness. The role of β-sitosterol was supported by comparable changes in RBC morphology and cholesterol-enriched domains upon β-sitosterol integration at the healthy RBC membrane. Finally, Ezetimibe treatment combined with a sterol restricted diet lowered phytosterols and improved anemia and RBC deformability and hydration. However, this treatment had no or limited effect on RBC morphology and cholesterol-enriched domain abundance. This study reveals for the first time that phytosterols affect RBC membrane lipid composition and distribution but also RBC morphology, hydration, deformability and fragility.

Number asymmetry: The hidden dimension of mammalian plasma membranes.

The transbilayer distribution of lipids in mammalian plasma membranes (PMs) is functionally important and incompletely understood. It has been generally assumed that the two PM leaflets contain similar numbers of phospholipids (PLs) due to the constraint that their areas must be evenly matched in a bilayer. Contrary to this widely held assumption, our recent detailed lipidomics analysis of human erythrocytes reveals a large phospholipid imbalance between PM leaflets, with the cytoplasmic leaflet possessing almost 2-fold more PLs than the exoplasmic one. This surprising finding challenges our understanding of living membrane organization and structure. Extensive atomistic simulations of a PM model guided by the lipidomics data reveal that a large PL imbalance between the leaflets can be sustained via highly asymmetric distribution of cholesterol. We also demonstrate that cholesterol increases membrane tolerance for PL imbalances in model membranes and cells. Driven by preferential interactions with saturated lipids and its tendency to ‘fill gaps’, we show that cholesterol is poised for enrichment in the exoplasmic leaflet of the plasma membrane and confirm this prediction in live red blood cells using a novel FRET-based assay. The resulting PL and cholesterol number asymmetries in the PM give rise to unique biophysical properties including differential permeability of the two bilayer leaflets and a substantial differential stress. Being under tension, the cytoplasmic leaflet has deep-reaching hydrophobic defects and imaging of living mammalian cells confirms their role in recruiting lipidated proteins to the plasma membrane. This number asymmetry of major membrane constituents thus presents a new and largely unexplored dimension of membrane organization.

Sensory axons induce epithelial lipid microdomain remodeling and determine the distribution of junctions in the epidermis.

Epithelial cell properties are determined by the polarized distribution of membrane lipids, the cytoskeleton, and adhesive junctions. Epithelia are often profusely innervated, but little work has addressed how neurites affect epithelial organization. We previously found that basal keratinocytes in the zebrafish epidermis enclose axons in ensheathment channels sealed by autotypic junctions. Here we characterized how axons remodel cell membranes, the cytoskeleton, and junctions in basal keratinocytes. At the apical surface of basal keratinocytes, axons organized lipid microdomains quantitatively enriched in reporters for PI(4,5)P2 and liquid-ordered (Lo) membranes. Lipid microdomains supported the formation of cadherin-enriched, F-actin protrusions, which wrapped around axons, likely initiating ensheathment. In the absence of axons, cadherin-enriched microdomains formed on basal cells but did not organize into contiguous domains. Instead, these isolated domains formed heterotypic junctions with periderm cells, a distinct epithelial cell type. Thus, axon endings dramatically remodel polarized epithelial components and regulate epidermal adhesion.

On-grid labeling method for freeze-fracture replicas.

Sodium dodecyl sulfate-treated freeze-fracture replica labeling (SDS-FRL) is an electron microscopic (EM) method that can define the two-dimensional distribution of membrane proteins and lipids in a quantitative manner. Despite its unsurpassed merit, SDS-FRL has been adopted in a limited number of labs, probably because it requires a laborious labeling process as well as equipment and technique for freeze-fracture. Here, we present a method that reduces the manual labor significantly by mounting freeze-fracture replicas on EM grids prior to labeling. This was made possible by the discovery that freeze-fracture replicas invariably adhere to the carbon-coated formvar membrane with their platinum-carbon side, ensuring that the membrane molecules retained in replicas are accessible to labeling solutions. The replicas mounted on EM grids can be stored dry until labeling, checked by light microscopy before labeling and labeled in the same manner as tissue sections. This on-grid method will make SDS-FRL easier to access for many researchers.

Membrane Lipids in Epithelial Polarity: Sorting out the PIPs.

The development of cell polarity in epithelia, is critical for tissue morphogenesis and vectorial transport between the environment and the underlying tissue. Epithelial polarity is defined by the development of distinct plasma membrane domains: the apical membrane interfacing with the exterior lumen compartment, and the basolateral membrane directly contacting the underlying tissue. The de novo generation of polarity is a tightly regulated process, both spatially and temporally, involving changes in the distribution of plasma membrane lipids, localization of apical and basolateral membrane proteins, and vesicular trafficking. Historically, the process of epithelial polarity has been primarily described in relation to the localization and function of protein ‘polarity complexes.’ However, a critical and foundational role is emerging for plasma membrane lipids, and in particular phosphoinositide species. Here, we broadly review the evidence for a primary role for membrane lipids in the generation of epithelial polarity and highlight key areas requiring further research. We discuss the complex interchange that exists between lipid species and briefly examine how major membrane lipid constituents are generated and intersect with vesicular trafficking to be preferentially localized to different membrane domains with a focus on some of the key protein-enzyme complexes involved in these processes.

Paleoclimate reconstruction of the last 36 kyr based on branched glycerol dialkyl glycerol tetraethers in the Padul palaeolake record (Sierra Nevada, southern Iberian Peninsula)

Quantitative continental climate reconstructions covering the last glacial cycle from the Iberian Peninsula are scarce. In order to fill this gap, we obtained for the first time a high-resolution mean annual air temperature (MAAT) record based on the distribution of specific bacterial membrane lipids (i.e., branched glycerol dialkyl glycerol tetraethers; brGDGTs) from the last 36.0–4.7 kyr palaeolake record recovered by the Padul-15-05 sedimentary core (Padul, Sierra Nevada, southern Iberia). The fractional abundance of the three major groups of GDGTs present in the Padul sediments, GDGT-0, crenarchaeol and the summed brGDGTs, is comparable with that of other shallow and small (<10 km2) European lakes. Despite variations in the lithology in the studied section, the GDGT composition remains relatively stable, except for the uppermost 116 cm of the record, representing the ephemeral/emerged lake stage, which is characterized by higher crenarchaeol fractional abundances. The identification of a specific brGDGT that has only been detected in anoxic lakes provides evidence for in-situ brGDGT production in the water column and/or sediments in the Padul palaeolake. Its presence/absence probably denotes a succession of periods with a variable oxygen content in the bottom waters of the palaeolake. MAAT was reconstructed based on the distribution of brGDGTs using an African lake calibration and ranged between 12 and 20 °C. A new Bayesian calibration to mean temperature of Months Above Freezing (MAF) depicts similar temperature variations with a mean absolute difference of 0.7 °C. The MAAT reconstruction in the Padul palaeolake for the 36.0–4.7 kyr period reveals similarities with climate variability described at high-latitudes and in the westernmost Mediterranean area during this interval, showing cold conditions during the last three Heinrich Stadials and the Younger Dryas and warm conditions during the Dansgaard–Oeschger interstadials (7–1) and the Bölling-Alleröd period. Despite the more stable and warm general climate conditions during the Early and Mid-Holocene, rapid centennial-scale temperature changes are registered in the Padul palaeolake in good agreement with variations observed in the Mediterranean forest record.

Changes in the Distribution of Membrane Lipids during Growth of Thermotoga maritima at Different Temperatures: Indications for the Potential Mechanism of Biosynthesis of Ether-Bound Diabolic Acid (Membrane-Spanning) Lipids.

ABSTRACTMembrane-spanning lipids are present in a wide variety of archaea, but they are rarely in bacteria. Nevertheless, the (hyper)thermophilic members of the order Thermotogales harbor tetraester, tetraether, and mixed ether/ester membrane-spanning lipids mostly composed of core lipids derived from diabolic acids, C30, C32, and C34 dicarboxylic acids with two adjacent mid-chain methyl substituents. Lipid analysis of Thermotoga maritima across growth phases revealed a decrease of the relative abundance of fatty acids together with an increase of diabolic acids with independence of growth temperature. We also identified isomers of C30 and C32 diabolic acids, i.e., dicarboxylic acids with only one methyl group at C-15. Their distribution suggests they are products of the condensation reaction but are preferably produced when the length of the acyl chains is not optimal. Compared with growth at the optimal temperature of 80°C, an increase of glycerol ether-derived lipids was observed at 55°C. Our analysis only detected diabolic acid-containing intact polar lipids with phosphoglycerol (PG) head groups. Considering these findings, we hypothesize a biosynthetic pathway for the synthesis of membrane-spanning lipids based on PG polar lipid formation, suggesting that the protein catalyzing this process is a membrane protein. We also identified, by genomic and protein domain analyses, a gene coding for a putative plasmalogen synthase homologue in T. maritima that is also present in other bacteria producing sn-1-alkyl ether lipids but not plasmalogens, suggesting it is involved in the conversion of the ester-to-ether bond in the diabolic acids bound in membrane-spanning lipids.IMPORTANCE Membrane-spanning lipids are unique compounds found in most archaeal membranes, but they are also present in specific bacterial groups like the Thermotogales. The synthesis and physiological role of membrane-spanning lipids in bacteria represent an evolutionary and biochemical open question that points to the differentiation of the membrane lipid composition. Understanding the formation of membrane-spanning lipids is crucial to solving this question and identifying the enzymatic and biochemical mechanism performing this procedure. In the present work, we found changes at the core lipid level, and we propose that the growth phase drives the biosynthesis of these lipids rather than temperature. Our results identified physiological conditions influencing the membrane-spanning lipid biosynthetic process, which can further clarify the pathway leading to the biosynthesis of these compounds.

CDC50A is required for aminophospholipid transport and cell fusion in mouse C2C12 myoblasts.

Myoblast fusion is essential for the formation of multinucleated muscle fibers and is promoted by transient changes in the plasma membrane lipid distribution. However, little is known about the lipid transporters regulating these dynamic changes. Here, we show that proliferating myoblasts exhibit an aminophospholipid flippase activity that is downregulated during differentiation. Deletion of the P4-ATPase flippase subunit CDC50A (also known as TMEM30A) results in loss of the aminophospholipid flippase activity and compromises actin remodeling, RAC1 GTPase membrane targeting and cell fusion. In contrast, deletion of the P4-ATPase ATP11A affects aminophospholipid uptake without having a strong impact on cell fusion. Our results demonstrate that myoblast fusion depends on CDC50A and may involve multiple CDC50A-dependent P4-ATPases that help to regulate actin remodeling.

Cyanine-5-Driven Behaviours of Hyperbranched Polymers Designed for Therapeutic Delivery Are Cell-Type Specific and Correlated with Polar Lipid Distribution in Membranes.

The ability to predict the behaviour of polymeric nanomedicines can often be obfuscated by subtle modifications to the corona structure, such as incorporation of fluorophores or other entities. However, these interactions provide an intriguing insight into how selection of molecular components in multifunctional nanomedicines contributes to the overall biological fate of such materials. Here, we detail the internalisation behaviours of polymeric nanomedicines across a suite of cell types and extrapolate data for distinguishing the underlying mechanics of cyanine-5-driven interactions as they pertain to uptake and endosomal escape. By correlating the variance of rate kinetics with endosomal escape efficiency and endogenous lipid polarity, we identify that observed cell-type dependencies correspond with an underlying susceptibility to dye-mediated effects and nanomedicine accumulation within polar vesicles. Further, our results infer that the ability to translocate endosomal membranes may be improved in certain cell types, suggesting a potential role for diagnostic moieties in trafficking of drug-loaded nanocarriers.

Challenging the Dogma - Cell Plasma Membranes are Asymmetric not only in Phospholipid Composition but also Abundance

The transbilayer distribution of lipids in mammalian plasma membranes (PMs) is functionally important and incompletely understood. It has been generally assumed that the two PM leaflets contain similar number of phospholipids (PLs) due to the constraint that their areas must be matched in a bilayer. Contrary to this widely held assumption, our recent detailed lipidomics analysis of human erythrocytes reveals a large phospholipid imbalance between PM leaflets, with the cytoplasmic leaflet possessing almost 2-fold more PLs as the exoplasmic one. This surprising finding is consistent with several previous observations and challenges our understanding of living membrane organization and structure. To investigate the effects of differential PL abundance on membrane properties, we performed long atomistic molecular dynamics simulations of various PM models with data-driven leaflet compositions and different PL imbalances between the leaflets. The integrity of the bilayer was preserved in part by (re)distribution of the PM's other major component, cholesterol, which shows a strong preference for the exoplasmic leaflet. Detailed leaflet-specific analysis of the physical properties of the membranes revealed systematic changes as the relative number of PLs in the exoplasmic leaflet decreased. These changes were accompanied by differences in the water permeability of the leaflets, their pressure distribution and propensity for hydrophobic defects. To determine which PM model most closely represents the cell PM we calibrated an in vitro reporter of membrane packing to simulations using experimental measurements of model membranes, and compared the simulated models to live cells PMs. Our results were consistent with a model of living PMs with far more cholesterol and fewer PLs in the exoplasmic leaflet. They also reveal a substantial amount of stored tension in the bilayer leaflets, uncovering a host of previously unconsidered physiological implications.

Phosphorus deprivation affects composition and spatial distribution of membrane lipids in legume nodules.

In legumes, symbiotic nitrogen (N) fixation (SNF) occurs in specialized organs called nodules after successful interactions between legume hosts and rhizobia. In a nodule, N-fixing rhizobia are surrounded by symbiosome membranes, through which the exchange of nutrients and ammonium occurs between bacteria and the host legume. Phosphorus (P) is an essential macronutrient, and N2-fixing legumes have a higher requirement for P than legumes grown on mineral N. As in the previous studies, in P deficiency, barrel medic (Medicago truncatula) plants had impaired SNF activity, reduced growth, and accumulated less phosphate in leaves, roots, and nodules compared with the plants grown in P sufficient conditions. Membrane lipids in M. truncatula tissues were assessed using electrospray ionization–mass spectrometry. Galactolipids were found to increase in P deficiency, with declines in phospholipids (PL), especially in leaves. Lower PL losses were found in roots and nodules. Subsequently, matrix-assisted laser desorption/ionization–mass spectrometry imaging was used to spatially map the distribution of the positively charged phosphatidylcholine (PC) species in nodules in both P-replete and P-deficient conditions. Our results reveal heterogeneous distribution of several PC species in nodules, with homogeneous distribution of other PC classes. In P poor conditions, some PC species distributions were observed to change. The results suggest that specific PC species may be differentially important in diverse nodule zones and cell types, and that membrane lipid remodeling during P stress is not uniform across the nodule.

Systematic Analysis of the Whole-Body Tissue Distribution and Fatty Acid Compositions of Membrane Lipids in CD1 and NMRI Mice and Wistar Rats

Understanding the tissue distribution of phospholipids and glycerolipids in animal models enables promoting the pharmacokinetic study of drugs and related PK predictions. The measurement of lipid compositions in animal models, usually mice and rats, without a standardized approach hindered the accuracy of PBPK investigation. In this work, high resolution mass spectrometry was applied to profile the tissue distribution of phospholipids and glycerolipids in 12 organs/tissues of mice and rats. Using this method, not only the amounts of phospholipids and glycerolipids in each organ/tissue but also the fatty acid compositions were acquired. In order to explore the interspecies specificity of lipid distribution in different organs/tissues, three animal species including CD1 mice, NMRI mice, and Wister rats were used in this systematic study. Globally, more organ specificity was observed. It was found that the brain is the organ containing the most abundant phosphatidylserine lipids (PSs) in all three animal models, leading to brain tissues having the most concentrated acidic phospholipids. Diverse fatty acid compositions in each lipid class were clearly revealed. Certain tissues/organs also had a specific selection of unique fatty acid compositions, for example, unreferenced FA(18 : 2) in the brain. It turned out that the access of free fatty acids affects the incorporation of acyl chain in phospholipids and glycerolipids. In the analysis, ether lipids were also profiled with the observation of dominant ePEs in brain tissues. However, little interspecies difference was found for fatty acid constituents and tissues distribution of phospholipids and glycerolipids.

Fabrication of Asymmetric Phosphatidylserine-Containing Lipid Vesicles: A Study on the Effects of Size, Temperature, and Lipid Composition.

The asymmetric distribution of lipids in plasma membranes is closely related to the physiological functions of cells. To improve our previous approach in fabricating asymmetric vesicles, we defined a parameter, asymmetric degree, in this work and investigated the effects of vesicle size, incubation temperature, and lipid composition on the formation process of asymmetric phosphatidylserine (PS)-containing lipid vesicles. The results indicate that all of the three factors have marked but different effects on the time-dependent asymmetric degree of the vesicles as well as the flip and flop rate constants of the PS lipids. However, only vesicle size and PS content show significant influence on the maximal asymmetric degree of the vesicles, while the incubation temperature exhibits negligible effect. This work not only deepens our understanding on the packing property of PS molecules in self-assembled membranes and the formation mechanism of asymmetric vesicles but also practically provides a solution to regulate the asymmetric degree of the PS-containing vesicles using the established kinetic equation. In addition, the method would facilitate researches related to asymmetric vesicles or reconstruction of biological membranes.

Microautophagy in the yeast vacuole depends on the activities of phosphatidylinositol 4-kinases, Stt4p and Pik1p

Morphologically, the lipophagy in yeast cell mimics microautophagy, which includes a direct amendment of the vacuolar membrane that engulfs lipid droplets (LDs). The molecular mechanism of the membrane modifications that elicits microautophagy still remains elusive. In this study, an analysis of membrane lipid distribution at a nanoscale level showed that PtdIns(4)P is localized in the cytoplasmic leaflet of microautophagic vesicles, which are derived when the vacuole's membrane domains engulfed LDs both in the stationary phase and in acute nitrogen starvation. Furthermore, the PtdIns(4)P-positive raft-like domains engulf LDs through a microautophagic mechanism. When single temperature-conditional mutants of STT4 or PIK1 PtdIns 4-kinases were used, in the vacuole of STT4 and PIK1 mutant cells, microautophagic vesicles drastically decreased at restrictive temperatures, and the labeling density of PtdIns(4)P on the microautophagic vesicles and the sizes of the mutants' microautophagic vesicles also decreased. These results suggest that both Stt4p and Pik1p have important roles in the microautophagy of the vacuole in the stationary phase and under nitrogen starvation conditions.

Effects of temperature and pH on archaeal membrane lipid distributions in freshwater wetlands

Freshwater wetlands harbour diverse archaeal communities and associated membrane lipid assemblages, but the effect of environmental factors (e.g. pH and temperature) on the distribution of these lipids is relatively poorly constrained. Here we explore the effects of temperature and pH on archaeal core-lipid and intact polar lipid (IPL) derived core lipid distributions in a range of wetlands. We focus, not only on the commonly studied isoprenoidal glycerol dialkyl glycerol tetraethers (isoGDGTs), but also widen our analyses to include more recently identified, but relatively widespread, archaeal lipids such as isoGDGT isomers, methylated isoGDGTs (Me-GDGTs), and butanetriol and pentanetriol tetraethers (BDGTs and PDGTs). Based on multivariate analysis and a globally distributed set of wetlands, we find that the degree of isoGDGT cyclisation does increase along with temperature and pH in wetlands. However, and unlike in some other settings, this relationship is obscured in simple scatterplots due to the incorporation of isoGDGTs from highly diverse archaeal sources with multiple ring-temperature or ring-pH relationships. We further show that the relative abundance of early eluting to later eluting isoGDGT isomers increases with pH, representing a previously unknown and seemingly widespread archaeal membrane homeostasis mechanism or taxonomic signal. The distribution and abundance of crenarchaeol, a marker for Thaumarchaeota, demonstrates that in wetlands these Archaea, likely involved in ammonia oxidation, are restricted primarily to the generally drier soil/sediment surface and typically are more abundant in circumneutral pH settings. We identify Me-GDGTs and Me-isoGMGTs (homologs of isoGDGTs and isoGMGTs, but with additional methylation on the biphytanyl chain) as ubiquitous in wetlands, but variation in their abundance and distribution suggests changing source communities and/or membrane adaptation. The high relative abundance of BDGTs and PDGTs in the perennially anoxic part of the peat profile (catotelm), as well as their elevated abundance in a circumneutral pH wetland, is consistent with an important input from their only known culture source, belonging to the methanogenic Methanomassiliicoccales. Our results underline the diversity of archaeal membrane lipids preserved in wetlands and provide a baseline for the use of archaeal lipid distributions in wetlands as tracers of recent or ancient climate and biogeochemistry.

Charge Distribution between Different Classes of Glycerophospolipids in MALDI-MS Imaging.

Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) is widely used to visualize and analyze the distribution of membrane lipids in an increasingly large number of applications. In this context, different lipoforms of glycerophospholipids (GPLs) are among the prime targets of interest. For this group of analytes, however, ion suppression effects have been described to strongly favor the detection of certain GPL classes over others, thereby hampering the analysis of suppressed species and greatly restraining quantitative analysis. These effects are generally attributed to the distribution of available charge carriers during the MALDI process. Here, we present a systematic investigation of charge distribution between different classes of GPL under MALDI-MSI conditions. For this, we constructed arrays of artificial tissues with different formulated lipid composition that contained predefined amounts of only two specific GPL classes and analyzed them with MALDI-MSI in positive- and negative-ion modes. Next to a characterization of expected ion suppression effects, analysis of these binary systems revealed yet undescribed signal intensity enhancement for the combinations of certain GPL classes. Furthermore, the comprehensive data allowed us to compile a hierarchy of charge affinities for the investigated GPL classes in both polarities. Additional experiments revealed that laser post-ionization (MALDI-2) has great potential to overwrite changes in signal intensity caused by charge distribution among different GPL classes observed in standard MALDI-MSI.

Lipid asymmetry of a model mitochondrial outer membrane affects Bax-dependent permeabilization

The presence of an asymmetric distribution of lipids in biological membranes was first described ca. 50 years ago. While various studies had reported the role of loss of lipid asymmetry on signaling processes, its effect on membrane physical properties and membrane-protein interactions lacks further understanding. The recent description of new technologies for the preparation of asymmetric model membranes has helped to fill part of this gap. However, the major effort so far has been on plasma membrane models. Here we describe the preparation of liposomes mimicking the mitochondria outer membrane (MOM) in regard to its lipid composition and asymmetry. By employing the methyl-β-cyclodextrin-catalyzed lipid exchange technology and accurate quantification of lipid asymmetry with head group-specific probes we showed the successful preparation of a MOM model bearing a physiologically relevant lipid composition and asymmetry. In addition, by a direct comparison with its lipid symmetrical counterpart it is shown that asymmetric models were more resistant to tBid-promoted Bax-permeabilization, suggesting a role played by MOM lipid asymmetry on the mitochondria pathway of apoptosis. The barrier imposed by lipid asymmetry on membrane permeabilization was in part due to a decrease in the concentration of membrane-bound proteins, which was likely a consequence of the two mutually-dependent properties; i.e., the lower electrostatic surface potential and the higher molecular packing imposed by lipid asymmetry. It is proposed that MOM lipid asymmetry imparts different physical properties on the membrane and might add an additional component of regulation in intricate mitochondrial processes.