Spatial mapping of lipids at cellular resolution in embryos of cotton

Advances in mass spectrometry (MS) have made comprehensive lipidomics analysis of complex tissues relatively commonplace. These compositional analyses, although able to resolve hundreds of molecular species of lipids in single extracts, lose the original cellular context from which these lipids are derived. Recently, high-resolution MS of individual lipid droplets from seed tissues indicated organelle-to-organelle variation in lipid composition, suggesting that heterogeneity of lipid distributions at the cellular level may be prevalent. Here, we employed matrix-assisted laser desorption/ionization-MS imaging (MALDI-MSI) approaches to visualize lipid species directly in seed tissues of upland cotton (Gossypium hirsutum). MS imaging of cryosections of mature cotton embryos revealed a distinct, heterogeneous distribution of molecular species of triacylglycerols and phosphatidylcholines, the major storage and membrane lipid classes in cotton embryos. Other lipids were imaged, including phosphatidylethanolamines, phosphatidic acids, sterols, and gossypol, indicating the broad range of metabolites and applications for this chemical visualization approach. We conclude that comprehensive lipidomics images generated by MALDI-MSI report accurate, relative amounts of lipid species in plant tissues and reveal previously unseen differences in spatial distributions providing for a new level of understanding in cellular biochemistry.     

Visualizing spatial lipid distribution in porcine lens by MALDI imaging high-resolution mass spectrometry

The intraocular lens contains high levels of both cholesterol and sphingolipids, which are believed to be functionally important for normal lens physiology. The aim of this study was to explore the spatial distribution of sphingolipids in the ocular lens using mass spectrometry imaging (MSI). Matrix-assisted laser desorption/ionization (MALDI) imaging with ultra high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) was used to visualize the lipid spatial distribution. Equatorially-cryosectioned, 12 μm thick slices of tissue were thaw-mounted to an indium-tin oxide (ITO) glass slide by soft-landing to an ethanol layer. This procedure maintained the tissue integrity. After the automated MALDI matrix deposition, the entire lens section was examined by MALDI MSI in a 150 μm raster. We obtained spatial- and concentration-dependent distributions of seven lens sphingomyelins (SM) and two ceramide-1-phosphates (CerP), which are important lipid second messengers. Glycosylated sphingolipids or sphingolipid breakdown products were not observed. Owing to ultra high resolution MS, all lipids were identified with high confidence, and distinct distribution patterns for each of them are presented. The distribution patterns of SMs provide an understanding of the physiological functioning of these lipids in clear lenses and offer a novel pathophysiological means for understanding diseases of the lens.     

Fatty acid profiling of phospholipids by field desorption and fast atom bombardment mass spectrometry

Natural phosphatidylcholines, phosphatidylethanolamines and sphingomyelins have been investigated by field desorption and fast atom bombardment mass spectrometry. It is demonstrated that using these soft mass spectrometric ionization techniques, accurate, fast, and sensitive fatty acid profiling of phospholipids can be performed. With respect to the analysis of intact molecular species both ionization techniques reveal similar results. Using field desorption, a specific fragment ion provides a fast access to the total distribution of fatty acids in complex lipids. Generally, a good agreement between the mass spectrometric abundance data and those produced by gas chromatographic analysis is observed.     

Human lens lipids differ markedly from those of commonly used experimental animals

Electrospray ionisation tandem mass spectrometry has allowed the unambiguous identification and quantification of individual lens phospholipids in human and six animal models. Using this approach ca. 100 unique phospholipids have been characterised. Parallel analysis of the same lens extracts by a novel direct-insertion electron-ionization technique found the cholesterol content of human lenses to be significantly higher (ca. 6 times) than lenses from the other animals. The most abundant phospholipids in all the lenses examined were choline-containing phospholipids. In rat, mouse, sheep, cow, pig and chicken, these were present largely as phosphatidylcholines, in contrast 66% of the total phospholipid in Homo sapiens was sphingomyelin, with the most abundant being dihydrosphingomyelins, in particular SM(d18:0/16:0) and SM(d18:0/24:1). The abundant glycerophospholipids within human lenses were found to be predominantly phosphatidylethanolamines and phosphatidylserines with surprisingly high concentrations of ether-linked alkyl chains identified in both classes. This study is the first to identify the phospholipid class (head-group) and assign the constituent fatty acid(s) for each lipid molecule and to quantify individual lens phospholipids using internal standards. These data clearly indicate marked differences in the membrane lipid composition of the human lens compared to commonly used animal models and thus predict a significant variation in the membrane properties of human lens fibre cells compared to those of other animals.     

Brain lipid changes after repetitive transcranial magnetic stimulation: potential links to therapeutic effects?

Repetitive transcranial magnetic stimulation (rTMS) is increasingly used in the management of neurologic disorders such as depression and chronic pain, but little is known about how it could affect brain lipids, which play important roles in membrane structure and cellular functions. The present study was carried out to examine the effects of rTMS on brain lipids at the individual molecular species level using the novel technique of lipidomics. Rats were subjected to high frequency (15 Hz) stimulation of the left hemisphere with different intensities and pulses of rTMS. The prefrontal cortex, hippocampus and striatum were harvested 1 week after rTMS and lipid profiles analyzed by tandem mass spectrometry. rTMS resulted in changes mainly in the prefrontal cortex. There were significant alterations in plasmalogen phosphatidylethanolamines, phosphatidylcholines, and increases in sulfated galactosylceramides or sulfatides. Plasmalogen species with long chain polyunsaturated fatty acids (PUFAs) showed decrease in abundance together with corresponding increase in lysophospholipid species suggesting endogenous release of long chain fatty acids such as docosahexaenoic acid (DHA) in brain tissue. The hippocampus showed no significant changes, whilst changes in the striatum were often opposite to that of the prefrontal cortex. It is postulated that changes in brain lipids may underlie some of the clinical effects of rTMS.     

Phosphatidic acid phosphatase 1 impairs SARS-CoV-2 replication by affecting the glycerophospholipid metabolism pathway

Viruses exploit the host lipid metabolism machinery to achieve efficient replication. We herein characterize the lipids profile reprogramming in vitro and in vivo using liquid chromatography-mass spectrometry-based untargeted lipidomics. The lipidome of SARS-CoV-2-infected Caco-2 cells was markedly different from that of mock-infected samples, with most of the changes involving downregulation of ceramides. In COVID-19 patients'' plasma samples, a total of 54 lipids belonging to 12 lipid classes that were significantly perturbed compared to non-infected control subjects'' plasma samples were identified. Among these 12 lipid classes, ether-linked phosphatidylcholines, ether-linked phosphatidylethanolamines, phosphatidylcholines, and ceramides were the four most perturbed. Pathway analysis revealed that the glycerophospholipid, sphingolipid, and ether lipid metabolisms pathway were the most significantly perturbed host pathways. Phosphatidic acid phosphatases (PAP) were involved in all three pathways and PAP-1 deficiency significantly suppressed SARS-CoV-2 replication. siRNA knockdown of LPIN2 and LPIN3 resulted in significant reduction of SARS-CoV-2 load. In summary, these findings characterized the host lipidomic changes upon SARS-CoV-2 infection and identified PAP-1 as a potential target for intervention for COVID-19.     

Stability of heterochiral hybrid membrane made of bacterial sn-G3P lipids and archaeal sn-G1P lipids

The structure of membrane lipids in Archaea is different from those of Bacteria and Eucarya in many ways including the chirality of the glycerol backbone. Until now, heterochiral membranes were believed to be unstable; thus, no cellular organism could have existed before the separation of the groups of life. In this study, we tested the formation of heterochiral hybrid membrane made of Bacterial sn-glycerol-3-phosphate-type polar lipid and Archaeal sn-glycerol-1-phosphate-type polar lipid using the fluorescence probe. The stability of the hybrid liposomes made of phosphatidylethanolamines or phosphatidylcholines or polar lipids of thermophilic Bacteria and polar lipids of Archaea were investigated. The hybrid liposomes are all stable compared with homochiral liposome made of dimyristoylphosphatidylethanolamine and dipalmitoylphosphatidylcholine. However, the stability was drastically changed with increasing carbon chain length. Accordingly, "chirality" may not be but chain length is important. From these results, we suggest that the heterochiral hybrid membrane could be used as the membrane lipid for the last universal common ancestor (Commonote) before the emergence of Archaea and Bacteria.     

Morphology of gel state phosphatidylethanolamine and phosphatidylcholine liposomes: A negative stain electron microscopic study

The capture volumes (internal aqueous spaces) of liposomes prepared from a series of saturated phosphatidylcholines (PC) and saturated phosphatidylethanolamines (PE) had previously been found to be a function of lipid structure. PE vesicles have larger internal aqueous spaces than PC vesicles and for lipids with the same head group, capture volume increases with lengthening of the fatty acyl chains. Capture volume is determined by vesicle size, number of lamellae, and interlamellar distance. In this study, liposomes were formed from a saturated PC or PE and their morphology studied in the gel state using the technique of negative staining transmission electron microscopy. The measured interlamellar distances were quite similar among these various lipids while the number of lamellae was found to decrease as the fatty acyl chain length increased. In general PEs form fewer lamellae than PCs and in particular mono- and di-methylated dipalmitoyl-PE form only unilamellar vesicles. The number of lamellae then appears to bear a relationship to the size of the capture volume in that liposomes with largercapture volumes have fewer lamellae.     

Dynamics of Phospholipid Content in the Vacuolar Membrane of Red Beet Taproots Exposed to Abiotic Stress

The composition of vacuolar membrane phospholipids in the taproot of red beet (Beta vulgaris L.), cv. Modana, was determined at normal conditions and under different types of stress (hypo- and hyperosmotic and oxidative stress). The experiments have shown that, among vacuolar membrane phospholipids in red beet taproot, phosphatidylcholines and phosphatidylethanolamines dominated and accounted for 70% of total phospholipids. It is interesting that the content of phosphatidic acid was high (20% of total phospholipids of the vacuolar membrane). Stress effects brought about changes in the composition of membrane phospholipids, which may be an element of phenotypic adaptation. Under hypoosmotic stress, reliable changes in the content of phosphatidic acid were observed, hyperosmotic stress was associated with changes in the level of phosphatidylcholines and phosphatidylinositols, and oxidative stress was notable for changes in the content of phosphatidylethanolamines and phosphatidylserines. The most significant changes were observed in the classes of phospholipids that may be involved in structural modification of membranes associated with transformation of their bilayer lamellar structure into hexagonal. These phospholipids comprise phosphatidic acid, phosphatidylcholines, and phosphatidylethanolamines. Revealed changes in the content of these phospholipids may alter the ratio between lamellar bilayer and nonbilayer hexagonal lipid structures in the vacuolar membrane and act as an important adaptation mechanism ensuring protection against stress.     

Interactions between anesthetics and lipid mixtures. Normal alcohols.

The effects of normal alcohols up to 1-dodecanol on phase transitions in phosphatidylcholines and phosphatidylethanolamines have been studied using chlorophyll a as fluorescent probe. With the phosphatidylcholines, alcohols up to octanol cause a lowering of the transition temperature, and a broadening of the transition, whereas for dipalmitoylphosphatidylethanolamine, only a lowering of the transition is observed. The lowering of the phase transition temperature in dipalmitoylphosphatidylcholine by butanol and hexanol is close to that expected for ideal behavior, but the behavior of the longer chain alcohols becomes less ideal. The effects of these alcohols on mixtures of lipids have been studied, and they illustrate the care necessary if these plots of temperatures of onset and completion of gel phase formation are to be called "phase diagrams". The effect of 1 -octanol on mixtures of lipids is to increase the proportion of lipid present in the lipid-crystalline state. In contrast, 1-decanol causes an increase in the phase transition temperature for dimyristoylphosphatidylcholine, although it lowers the transition temperature for dipalmitoylphosphatidylcholine, and 1 -dodecanol raises the transition temperature for both of these phosphatidylcholines, although it lowers that for dipalmitoylphosphatidylethanolamine. Dodecanol appears to behave in these lipid bilayer membranes as a lipid with a phase transition temperature of ca. 55 degrees C. Anesthesia is discussed as a phenomenon of liquidus extension: alcohols up to 1 -octanol increase the proportion of lipid in the liquidus state and result in anesthesia, whereas the longer alcohols do not, and result in catalepsy.     

The lipidome of the photosynthetic bacterium Rhodobacter sphaeroides R26 is affected by cobalt and chromate ions stress

A detailed characterization of membrane lipids of the photosynthetic bacterium Rhodobacter (R.) sphaeroides was accomplished by thin-layer chromatography coupled with matrix-assisted laser desorption ionization mass spectrometry. Such an approach allowed the identification of the main membrane lipids belonging to different classes, namely cardiolipins (CLs), phosphatidylethanolamines, phosphatidylglycerols (PGs), phosphatidylcholines, and sulfoquinovosyldiacylglycerols (SQDGs). Thus, the lipidomic profile of R. sphaeroides R26 grown in abiotic stressed conditions by exposure to bivalent cobalt cation and chromate oxyanion, was investigated. Compared to bacteria grown under control conditions, significant lipid alterations take place under both stress conditions; cobalt exposure stress results in the relative content increase of CLs and SQDGs, most likely compensating the decrease in PGs content, whereas chromate stress conditions result in the relative content decrease of both PGs and SQDGs, leaving CLs unaltered. For the first time, the response of R. sphaeroides to heavy metals as Co²⁺ and CrO₄ ^2? is reported and changes in membrane lipid profiles were rationalised.     

Fusion of Sindbis virus with model membranes containing phosphatidylethanolamine: implications for protein-induced membrane fusion

The pH-induced fusion of Sindbis virus with model lipid membranes containing phosphatidylethanolamine has been studied using a quantitative fluorescence technique. The headgroup and acyl chain domains of the lipids have been altered systematically to determine their effect on fusion. Unsaturated phosphatidylethanolamines (PE) have been found to promote fusion, either by themselves, or in combination with phosphatidylcholines (PC). Cholesterol added to a mixture of unsaturated PE and PC was also shown to increase the extent of viral fusion. The results of these studies have been interpreted in terms of a tentative model for the molecular aspects of the target membrane which are necessary for viral fusion. In this model, the target membrane must have a sufficiently-sized domain containing poorly hydrated lipids which are capable of existing in a non-bilayer arrangement.     

Phospholipid composition and organization of cytochrome c oxidase preparations as determined by 31P-nuclear magnetic resonance

The molecular organization as well as the composition of the phospholipids in cytochrome c oxidase preparations (bovine heart) were investigated by 31P-nuclear magnetic resonance. In the so-called 'lipid-rich' preparation the lipids were found to form a fluid bilayer around the enzyme since the 31P-NMR spectrum was characteristic of a fast, axially symmetric motion of the phosphate groups with a chemical shift anisotropy of delta sigma = -45 ppm. In contrast, the 'lipid-depleted' cytochrome c oxidase gave rise to a broader spectrum where the motion of the phospholipids was no longer axially symmetric. Nevertheless, the total width of the spectrum was still considerably narrower than observed for immobilized phospholipids in solid crystals. Both enzyme preparations were dissolved in 1% detergent solution and used for high-resolution 31P-NMR spectroscopy. Narrow lines of about 20 Hz linewidth were obtained for both types of enzyme preparations, and well-resolved resonances could be assigned to cardiolipin, phosphatidylethanolamin and phosphatidylcholine. The major differences between lipid-rich and lipid-depleted cytochrome c oxidase were the absolute amount of phospholipid associated with the protein and the relative contribution of the individual lipid classes to the 31P-NMR spectrum. For lipid-rich cytochrome c oxidase about 130 molecules phospholipid were bound per enzyme (approx. 11 cardiolipins, 54 phosphatidylethanolamines and 64 phosphatidylcholines). For lipid-depleted cytochrome c oxidase only 6-18 lipids were bound per enzyme (1 or 2 cardiolipins, 3-8 phosphatidylethanolamines and 2-8 phosphatidylcholines). In contrast to earlier suggestions that cardiolipin is the only remaining lipid in lipid-depleted cytochrome c oxidase, the 31P-NMR studies demonstrate that all three lipids remain associated with the protein.     

Variation in corn (Zea mays L.) for fatty acid compositions of triglycerides and phospholipids

The percentage of linoleic acid in corn germ oil of three crosses, C103D × B73, C103D×B84, and T220×H51, and their reciprocals was investigated. Corn germ oil from F₂, F₃, and backcrossed generations was also examined. More than one gene locus appeared to be involved in conditioning the linoleic acid content in these crosses. Strong maternal effects were exhibited in the F₁'s. Genotype also superimposed variations in fatty acid compositions within the characteristic lipid class patterns of the phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. Fatty acid placements in triglycerides, digalactosyldiglycerides, and phospholipids of one inbred, H51, were determined by lipase and phospholipase hydrolysis. The overall pattern of placement showed that the fatty acids at the 1 position were predominately saturated and those at the 2 position were predominately unsaturated, but the fatty acid distribution was different for each individual lipid class. The molecular species of the phosphatidylcholines and phosphatidylethanolamines were separated by silver nitrate thin-layer chromatography. The major differences in the molecular species were a higher level of the dienoic-dienoic species and a lower level of the monoenoic-monoenoic species in the phosphatidylethanolamines than in the phosphatidylcholines.     

Sphingolipid distribution changes with age in the human lens

The formation of an internal barrier to the diffusion of small molecules in the lens during middle age is hypothesized to be a key event in the development of age-related nuclear (ARN) cataract. Changes in membrane lipids with age may be responsible. In this study, we investigated the effect of age on the distribution of sphingomyelins, the most abundant lens phospholipids. Human lens sections were initially analyzed by MALDI mass spectrometry imaging. A distinct annular distribution of the dihydrosphingomyelin, DHSM (d18:0/16:0), in the barrier region was observed in 64- and 70-year-old lenses but not in a 23-year-old lens. An increase in the dihydroceramide, DHCer (d18:0/16:0), in the lens nucleus was also observed in the older lenses. These findings were supported by ESI mass spectrometry analysis of lipid extracts from lenses dissected into outer, barrier, and nuclear regions. A subsequent analysis of 18 lenses ages 20–72 years revealed that sphingomyelin levels increased with age in the barrier region until reaching a plateau at approximately 40 years of age. Such changes in lipid composition will have a significant impact on the physical properties of the fiber cell membranes and may be associated with the formation of a barrier.     

Spatial and temporal alterations of phospholipids determined by mass spectrometry during mouse embryo implantation

Molecular events involved in successful embryo implantation are not well understood. In this study, we used MALDI imaging mass spectrometry (IMS) technologies to characterize the spatial and temporal distribution of phospholipid species associated with mouse embryo implantation. Molecular images showing phospholipid distribution within implantation sites changed markedly between distinct cellular areas during days 4–8 of pregnancy. For example, by day 8, linoleate- and docosahexaenoate-containing phospholipids localized to regions destined to undergo cell death, whereas oleate-containing phospholipids localized to angiogenic regions. Arachidonate-containing phospholipids showed different segregation patterns depending on the lipid class, revealing a strong correlation of phosphatidylethanolamines and phosphatidylinositols with cytosolic phospholipase A_2α and cyclooxygenase-2 during embryo implantation. LC-ESI-MS/MS was used to validate MALDI IMS phospholipid distribution patterns. Overall, molecular images revealed the dynamic complexity of lipid distributions in early pregnancy, signifying the importance of complex interplay of lipid molecules in uterine biology and implantation.     

Parinaroyl and pyrenyl phospholipids as probes for the lipid surface layer of human low density lipoproteins

A simple protocol employing lipid transfer proteins was developed to label human low density lipoprotein (LDL) in a controlled manner with parinaroyl and pyrenyl phosphatidylcholines. In order to study the lipid fluidity in the surface lipid layer of LDL, the temperature-dependence of both polarization (parinaroyl probes) and excimer to monomer (E/M) intensity ratio (pyrenyl probes) were analyzed. A series of pyrenyl phosphatidylcholines containing a pyrenyl fatty acid varying from 6 to 14 carbons in length at the sn-2 position were inserted into LDL to investigate the lateral distribution of different phosphatidylcholines in the lipoprotein surface at 37 degrees C. Both polarization and E/M vs. temperature plots displayed discontinuities in the region of 22-32 degrees C, which coincides with the melting of the neutral lipid core, indicating that the latter induces an ordered to more disordered phase transition in the surface lipid layer. Determination of the E/M intensity ratio as a function of pyrene lipid concentration in LDL showed a linear relationship for the pyrenyl hexanoate and octanoate species, whereas a slope discontinuity was observed for the lipids containing a longer pyrenyl chain. These data suggest that two lipid domains with distinct properties exist in the surface layer and secondly, pyrenyl lipids partition between these domains in a chainlength-dependent manner. This is consistent with measurement of the tryptophan to pyrene energy transfer efficiency vs. pyrenyl lipid concentration, which showed a biphasic relationship for the long-chain pyrenyl lipids. These measurements further indicate that two surface lipid domains correspond to the protein-lipid boundary and the bulk lipid phase, respectively. The fact that relatively small changes in chainlength have a marked influence on the partitioning of pyrenyl lipids between the boundary and the bulk phase suggests also that native phospholipid species may not be randomly distributed in the surface lipid layer of LDL.     

The effect of fasting and refeeding on the composition and synthesis of triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines in rat liver

Refeeding a high-sucrose, fat-free diet to fasted rats caused drastic alterations in the fatty acid composition of hepatic diacylglycerols, triacylglycerols, and phosphatidylcholines. However, the fatty acid profile of phosphatidylethanolamines did not change significantly. These results suggest that the fatty acid composition of diacylglycerols may influence the distribution of diacylglycerols among triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines. Fasting and refeeding also affected the activities in vitro of a number of enzymes responsible for the formation of triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines. The activity of hepatic phosphatidate phosphatase increased fourfold upon refeeding. However, fasting the rats did not affect the activity of this enzyme despite the reduced triacylglycerol synthesis in the fasted liver in vivo. Fasting and refeeding induced alterations in the activities of diacylglycerol acyltransferase, cholinephosphotransferase, and ethanolaminephosphotransferase which correlated reasonably well with the changes observed in the synthesis of triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines in vivo, although the changes in diacylglycerol acyltransferase were too moderate. The changes in the activity of cholinephosphate cytidylyltransferase, which is suggested to catalyze the rate-limiting step in the formation of CDP-choline, ran parallel with the alterations in the synthesis of phosphatidylcholines in vivo. No such correlation was found between the activity of ethanolaminephosphate cytidylyltransferase and the rate of phosphatidylethanolamine synthesis. The present results indicate that the synthesis of triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines is controlled by the availability of the various substrates as well as by the activities of several enzymes involved in these processes.     

The role of rapid lipogenesis in insulin secretion: Insulin secretagogues acutely alter lipid composition of INS-1 832/13 cells

Pancreatic beta cell mitochondria convert insulin secretagogues into products that support insulin exocytosis. We explored the idea that lipids are some of these products formed from acyl group transfer out of mitochondria to the cytosol, the site of lipid synthesis. There are two isoforms of acetyl-CoA carboxylase, the enzyme that forms malonyl-CoA from which C₂ units for lipid synthesis are formed. We found that ACC1, the isoform seen in lipogenic tissues, is the only isoform present in human and rat pancreatic islets and INS-1 832/13 cells. Inhibitors of ACC and fatty acid synthase inhibited insulin release in islets and INS-1 cells. Carbon from glucose and pyruvate were rapidly incorporated into many lipid classes in INS-1 cells. Glucose and other insulin secretagogues acutely increased many lipids with C₁₄-C₂₄ chains including individual cholesterol esters, phospholipids and fatty acids. Many phosphatidylcholines and phosphatidylserines were increased and many phosphatidylinositols and several phosphatidylethanolamines were decreased. The results suggest that lipid remodeling and rapid lipogenesis from secretagogue carbon support insulin secretion.