Vesicular Lipids Research Articles

Extracellular Vesicles Contribute to the Difference in Lipid Composition between Ovarian Follicles of Different Size Revealed by Mass Spectrometry Imaging.

Follicular fluid (FF) ensures a safe environment for oocyte growth and maturation inside the ovarian follicle in mammals. In each cycle, the large dominant follicle (LF) contains the oocyte designated to be ovulated, whereas the small subordinate follicles (SFs) of the same wave will die through atresia. In cows, the oocytes from the SF, being 2 mm in size, are suitable for in vitro reproduction biotechnologies, and their competence in developing an embryo depends on the size of the follicles. FF contains proteins, metabolites, fatty acids, and a multitude of extracellular vesicles (ffEVs) of different origins, which may influence oocyte competence through bidirectional exchanges of specific molecular cargo between follicular cells and enclosed oocytes. FF composition evolves along with follicle growth, and the abundance of different lipids varies between the LF and SF. Here, significant differences in FF lipid content between the LFs and SFs within the same ovary were demonstrated by MALD-TOF mass spectrometry imaging on bovine ovarian sections. We then aimed to enlighten the lipid composition of FF, and MALDI-TOF lipid profiling was performed on cellular, vesicular, and liquid fractions of FF. Differential analyses on the abundance of detected lipid features revealed specific enrichment of phospholipids in different ffEV types, such as microvesicles (MVs) and exosomes (Exo), compared to depleted FF. MALDI-TOF lipid profiling on MVs and Exo from the LF and SF samples (n = 24) revealed that more than 40% of detected features were differentially abundant between the groups of MVs and Exo from the different follicles (p < 0.01, fold change > 2). Glycerophospholipid and sphingolipid features were more abundant in ffEVs from the SFs, whereas different lysophospholipids, including phosphatidylinositols, were more abundant in the LFs. As determined by functional analysis, the specific lipid composition of ffEVs suggested the involvement of vesicular lipids in cell signaling pathways and largely contributed to the differentiation of the dominant and subordinate follicles.

Extracellular vesicular lipids as biomarkers for the diagnosis of Alzheimer’s disease

AbstractBackgroundAn increasing number of studies have revealed that dysregulated lipid homeostasis is associated with the pathological processes that lead to Alzheimer’s disease (AD). If changes in key lipid species could be detected in the periphery, it would advance our understanding of the disease and facilitate biomarker discovery. Global lipidomic profiling of sera/blood however has proved challenging with limited disease or tissue specificity. Small extracellular vesicles (EV) in the central nervous system, can pass the blood‐brain barrier and enter the periphery, carrying a subset of lipids that could reflect lipid homeostasis in brain. This makes EVs uniquely suited for peripheral biomarker exploration.MethodTo determine the potential of EV lipids as biomarkers, we isolated brain‐derived EV (BDEV) from frontal cortex tissue (Vella, et al. JEV. 2017) and performed quantitative mass spectrometry based lipidome analysis (Rustam and Reid. Anal. Chem. 2018). We developed and optimised methods to isolate pure EVs from plasma while minimising co‐isolation of lipoproteins. To confirm the identity of EVs, we are using various techniques including western blot, nanoparticle tracking analysis and transmission electron microscopy. We are mining the mass spectrometric data for brain‐specific signals.ResultWestern blot analysis showed successful isolation and enrichment of BDEV from brain tissue. TEM revealed the typical round and cup‐shaped EV morphology with vesicle size being 50‐150nm. A total of 692 lipids were identified and quantified. Enrichment of glycerophosphatidylserine (PS) lipids, especially the ether PS lipids, alkyl‐ and alkenyl‐, were observed in BDEV. Remodeling in phosphatidylethanoamine (PE) lipid class and polyunsaturated fatty acyl containing lipids (PUFA‐) was observed in AD BDEV. The preliminary experiments demonstrate successful isolation of pure EVs from plasma. Lipidomic and proteomic analysis suggests that plasma EVs contain brain‐specific signals.ConclusionWe, for the first time, characterized the lipid composition of EVs in human frontal cortex. BDEVs offered improved detection of dysregulated lipids in AD and these lipids have previously been reported to play key roles in AD pathogenesis, suggesting BDEV provide a readout of lipid dysregulation in AD and highlighting the potential use of these lipids as disease biomarkers in the periphery.

Vesicle fusion to planar membranes is enhanced by cholesterol and low temperature

Lipid composition and properties play an important role in many cellular properties such as fusion of vesicles to cell membranes, an essential process for exocytosis. Using a model system composed of artificial vesicles (liposomes) and artificial membranes (planar lipid bilayers), we observed that fusion is significantly affected by the lipid phase of the planar membrane. To determine the effect of lipid phases on fusion rates, we utilized the nystatin/ergosterol fusion assay and stimulated fusion with an osmotic gradient. Phase of the planar membrane was altered by changing cholesterol or temperature while the vesicular lipids were held constant. Liquid disordered (Ld or Lα) planar membranes were formed from phosphatidylethanolamine and phosphatidylcholine with unsaturated acyl chains. Addition of cholesterol shifts these membranes to the liquid ordered (Lo) phase and increases liposome fusion. Planar membranes in the Lα phase were also made from dipalmitoylphoshatidylcholine (DPPC) above the transition temperature (Tm) of 41.5°C. Decreasing the temperature below Tm shifts these membranes into the ripple phase (Pβ′) and also increases liposome fusion. The cholesterol and temperature data are consistent with the hypothesis that fusion is promoted in membranes that have greater exposure of their lipid tails or in membranes which can form leaflet domains with negative curvature. The data are not consistent with the hypothesis that lipid mismatch drives fusion.

Vesicle-Membrane Fusion is Enhanced by Cholesterol and Low Temperature

Lipid composition and properties play an important role in fusion of vesicles to cell membranes, an essential process for exocytosis. Using a model system composed of artificial vesicles and planar lipid membranes, we observed that fusion is significantly affected by the lipid phase of the planar membrane. To specifically determine the effect of lipid phases on fusion rates, we utilized the nystatin/ergosterol fusion assay and stimulated fusion with an osmotic gradient. The lipid phase of the planar membrane was altered by changing cholesterol or temperature, while the vesicular lipids were held constant. Liquid disordered (Lα) planar membranes were formed from PE and PC. Addition of cholesterol shifts these membranes to the liquid ordered (Lo) phase and increased vesicle fusion. Planar membranes in the Lα phase were also made from DPPC lipids above the transition temperature (41.5°C); decreasing the temperature shifts these membranes into the ripple phase (Pβ') and again increased vesicle fusion. These data are consistent with the hypothesis that fusion is promoted in membranes with some negative curvature.

Sphingomyelin and Cholesterol Promote HIV-1 gp41 Pretransmembrane Sequence Surface Aggregation and Membrane Restructuring

The interfacial sequence DKWASLWNWFNITNWLWYIK, preceding the transmembrane anchor of gp41 glycoprotein subunit, has been shown to be essential for fusion activity and incorporation into virions. HIV(c), a peptide representing this region, formed lytic pores in liposomes composed of the main lipids occurring in the human immunodeficiency virus, type 1 (HIV-1), envelope, i.e. 1-palmitoyl-2-oleoylphosphatidylcholine (POPC):sphingomyelin (SPM):cholesterol (Chol) (1:1:1 mole ratio), at low (>1:10,000) peptide-to-lipid mole ratio, and promoted the mixing of vesicular lipids at >1:1000 peptide-to-lipid mole ratios. Inclusion of SPM or Chol in POPC membranes had different effects. Whereas SPM sustained pore formation, Chol promoted fusion activity. Even if partitioning into membranes was not affected in the absence of both SPM and Chol, HIV(c) had virtually no effect on POPC vesicles. Conditions described to disturb occurrence of lateral separation of phases in these systems reproduced the high peptide-dose requirements for leakage as found in pure POPC vesicles and inhibited fusion. Surface aggregation assays using rhodamine-labeled peptides demonstrated that SPM and Chol promoted HIV(c) self-aggregation in membranes. Employing head-group fluorescent phospholipid analogs in planar supported lipid layers, we were able to discern HIV(c) clusters associated to ordered domains. Our results support the notion that the pretransmembrane sequence may participate in the clustering of gp41 monomers within the HIV-1 envelope, and in bilayer architecture destabilization at the loci of fusion.

Accurate separation of vesicles, micelles and cholesterol crystals in supersaturated model biles by ultracentrifugation, ultrafiltration and dialysis

Gel filtration with bile salts at intermixed micellar/vesicular concentrations (IMC) in the eluant has been proposed to isolate vesicles and micelles from supersaturated model biles, but the presence of vesicular aggregates makes this method unreliable. We have now validated a new method for isolation of various phases. First, aggregated vesicles and – if present – cholesterol crystals are pelleted by short ultracentrifugation. Cholesterol contained in crystals and vesicular aggregates can be quantitated from the difference of cholesterol contents in the pellets before and after bile salt-induced solubilization of the vesicular aggregates. Micelles are then isolated by ultrafiltration of the supernatant through a highly selective 300 kDa filter and unilamellar vesicles by dialysis against buffer containing bile salts at IMC values. Lipids contained in unilamellar vesicles are also estimated by subtraction of lipid contents in filtered micelles from lipid contents in (unilamellar vesicle+micelle containing) supernatant (‘subtraction method’). ‘Ultrafiltration-dialysis’ and ‘subtraction’ methods yielded identical lipid solubilization in unilamellar vesicles and identical vesicular cholesterol/phospholipid ratios. In contrast, gel filtration yielded much more lipids in micelles and less in unilamellar vesicles, with much higher vesicular cholesterol/phospholipid ratios. When vesicles obtained by dialysis were analyzed by gel filtration, vesicular cholesterol/phospholipid ratios increased strongly, despite correct IMC values for bile salts in the eluant. Subsequent extraction of column material showed significant amounts of lipids. In conclusion, gel filtration may underestimate vesicular lipids and overestimate vesicular cholesterol/phospholipid ratios, supposedly because of lipids remaining attached to the column. Combined ultracentrifugation-ultrafiltration-dialysis should be considered state-of-the-art methodology for quantification of cholesterol carriers in model biles.

Expression of human apolipoprotein E but not that of apolipoprotein A-I by mouse C127 cells is associated with increased secretion of lipids in the form of vesicles and discs.

Transfected mouse mammary-derived cells (C127) expressing human apolipoprotein (apo) E (C127E) were used a) to determine whether the lipid-binding character of apoE is sufficient to promote its assembly with lipid to form lipoprotein-like particles when expressed by cells not normally expressing apolipoproteins; b) to characterize the secreted complexes in terms of morphology, size, and composition; and finally c) to determine whether apoE or apoA-I gene expression by these transfected cells has any effect on the levels and the profiles of the synthesized and secreted lipids. The findings of the present study demonstrate that: a) as determined by density gradient ultracentrifugation and gel filtration chromatography, about 20% of the secreted [35S]methionine-labeled apoE expressed by C127E cells is lipid-associated. b) Negative-stain electron microscopic analysis of the lipid-protein complexes recovered in the lipoprotein fractions (d less than 1.21 g/ml) revealed that approximately 13% of the total population of particles were discs (16 +/- 5 nm mean diameter and 4-6 nm thick), resembling nascent high density lipoproteins (HDL). The majority of the particles however (greater than 82%) appeared vesicular with varying diameters (48 +/- 40 nm mean diameter). The discoidal and the vesicular appearance of the particles secreted by C127E cells is consistent with the composition of lipids. These consisted mostly of surface lipids, phospholipids (45 +/- 18%), diacylglycerols (36 +/- 17%), and free cholesterol (17 +/- 7%) (by weight). c) Expression of apoE by C127E cells was associated with an increased release of [35S]methionine-labeled protein and [3H]glycerol-labeled lipid (3- to 5- and 4- to 8-fold, respectively) compared to nontransfected C127 cells. Expression of mutant apoE or normal apoA-I, however, was not associated with increased release of the major lipid classes compared to the parent C127 cells, strongly suggesting that this character of C127E cells is specific to apoE expression. The release of lipids by C127E cells could be reduced considerably by the addition of the metabolic inhibitors, colchicine or cycloheximide (10 and 1 microM, respectively), suggesting that lipid release by C127E cells is an active process requiring both protein synthesis and functional secretory mechanisms. Taken together the findings suggest that apoE expression by C127 cells promotes the formation of nascent discoidal lipoprotein-like particles and enhances the release of vesicular lipids, possibly by promoting shedding of cell plasma membrane fragments.

Fatty acid profiles of esterified sterol glucosides from juice vesicles of citrus fruits

Vesicular lipids of 24 citrus cultivars were extracted and purified, and the esterified sterol glucosides (ESG) separated from other vesicular lipids by chromatography. Fatty acids of the ESG were analysed by gas-liquid chromatography as their methyl esters. Statistical analysis showed that five out of six citrus species or biotypes could be differentiated from each other on the basis of profiles of the five major ESG fatty acids. Differences between midand late-season sweet orange cultivers were detected. Small differences were observed between white- and red-fleshed grapefruit cultivars. Profiles of the minor acids showed higher relative percentages of iso-branched fatty acids in lemons and limes, and higher relative percentages of 12: 0 and 14: 0 fatty acids in mandarin and mandarin hybrids than in other cultivars.

Comparative citrus fatty acid profiles of triglycerides, monogalactosyl diglycerides, steryl esters and esterified steryl glucosides.

Vesicular lipids from six orange and two tangor varieties were extracted, purified and separated by chromatography into triglycerides, monogalactosyl diglycerides, steryl esters and esterified steryl glucosides. Methyl esters of the fatty acids found in these four lipids were prepared and analyzed by gas liquid chromatography. Each of the eight citrus varieties gave a series of four profiles which could be distinguished from the others. The Temple tangor has four profiles all showing a large percentage of linolenic acid. In all varieties steryl esters and to a lesser extent esterified steryl glucosides contain relatively large concentrations of 22∶0 to 26∶0 saturated acids. The profiles differ markedly from the patterns found for these four lipids in other higher plants studied.