On vesicles and membrane compartments

Summary Two different mechanisms have been proposed to explain transport along the endocytic and biosynthetic transport routes in cells. The first involves stable compartments connected by vesicular traffic while the second argues that the key organelles (early endosomes or the cis Golgi) form de novo by fusion of vesicles and subsequently mature into later forms. In the first part of this article, I propose a classification that distinguishes between stable, preexisting membrane compartments and vesicles that are, by definition, transient organelles. In this scheme, compartments, but not vesicles, are capable of homotypic fusion while vesicles, but not compartments, are able to mature, a process defined as an irreversible set of biochemical events which lead to a physiologically distinct end-state of the vesicle prior to its vectorial fusion with a target compartment. In the second part, I summarize my current ideas about the ultrastructural organization of the ER-Golgi region. Finally, I review the cell biology of selected examples of different vesicle types in order to exemplify the fascinating diversity of functions that this class of membrane organelles has evolved.Abbreviations COP coatomer - ECV endosome carrier vesicle - ER endoplasmic reticulum - HRP horseradish peroxidase - IC intermediate compartment between ER and Golgi - MVB multivesicular body - NSF N-ethyl maleimide sensitive factor - SNAPS soluble NSF associated proteins - TGN trans Golgi network Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

The influence of fatty acids on model cholesterol/phospholipid membranes

The aim of this work was to verify the influence of the saturated (SFA) (stearic acid) and the unsaturated (UFA) (oleic and alpha-linolenic) fatty acids on model cholesterol/phospholipid membranes. The experiments were based on the Langmuir monolayer technique. Cholesterol and phospholipid were mixed in the molar ratio that corresponds to the proportion of these lipids in the majority of natural human membranes. Into the binary cholesterol/phospholipid monolayers, various amounts of fatty acids were incorporated. Our investigations were based on the analysis of the interactions between molecules in ternary (cholesterol/phospholipids/fatty acid) mixtures, however, also binary (cholesterol/fatty acid and phospholipids/fatty acid) mixed system were examined. It was concluded that the influence of the fatty acids on model cholesterol/phospholipid membrane is closely connected with the shape of the fatty acid molecule, resulting from the saturation degree of the hydrocarbon chain. It was found that the saturated fatty acid makes the model membrane more rigid, while the presence of unsaturated fatty acid increases its fluidity. The increasing amount of stearic acid gradually destabilizes model membrane, however, this effect is the weakest at low content of SFA in the mixed monolayer. Unsaturated fatty acids in a small proportion make the membrane thermodynamically more stable, while higher content of UFA decreases membrane stability. This explains low proportion of the free fatty acids to other lipids in natural membrane.     

Improving the tolerance of Escherichia coli to medium-chain fatty acid production

Microbial fatty acids are an attractive source of precursors for a variety of renewable commodity chemicals such as alkanes, alcohols, and biofuels. Rerouting lipid biosynthesis into free fatty acid production can be toxic, however, due to alterations of membrane lipid composition. Here we find that membrane lipid composition can be altered by the direct incorporation of medium-chain fatty acids into lipids via the Aas pathway in cells expressing the medium-chain thioesterase from Umbellularia californica (BTE). We find that deletion of the aas gene and sequestering exported fatty acids reduces medium-chain fatty acid toxicity, partially restores normal lipid composition, and improves medium-chain fatty acid yields.     

Correlation between fluidity and fatty acid composition of phospholipid species in Tetrahymena pyriformis during temperature acclimation

The correlation between the fluidity of phospholipids and their fatty acid composition was studied by spin label technique and gas-liquid chromatography for three major phospholipid species in Tetrahymena pyriformis during temperature acclimation. The fluidity of 2-aminoethylphosphonolipid increased within the first 10 h of the cold-acclimation when the content of γ-linolenic acid in 2-aminoethylphosphonolipid was highest, and it then decreased up to 24 h. On the other hand, the fluidities of phosphatidylethanolamine and phosphatidylcholine showed a gradual decrease up to 24 h after the temperature shift, although γ-linolenic acid contents were highest at 10 h after the temperature shift. Thus the fluidity changes of these two phospholipids were interpreted as resulting from the altered content of other fatty acids in addition to γ-linolenic acid, since the γ-linolenic acid content was smaller than that of 2-aminoethylphosphonolipid. The results suggest that the content of γ-linolenic acid in 2-aminoethylphosphonolipid plays a role in regulating the thermal adaptation process.     

Osmotic fragility and fluidity of erythrocyte membranes from rats raised on an essential fatty acid deficient diet A spin label study

Erythrocyte membranes from rats raised on a diet with low content of essential fatty acids were studied by osmotic sensitivity tests and spin labeling techniques. This diet induced significant modifications in acylglycerophosphocholine fatty acid composition with regard to 16 : 1, 18 : 1, 18 : 2 (n-6), 20 : 3 (n-9), and 20 : 4 (n-6). No changes in membrane fluidity as monitored by spin label motion were found but the diet caused an increased osmotic sensitivity in essential fatty acid deficient erythrocytes. 50% hemolysis was obtained at a 51.0% dilution of saline with H₂O as compared to a 57.0% dilution for the control material. Membrane fluidity was unaffected by γ-irradiation up to 80 krad.     

The relatioship between plasma membrane lipid composition and physical-chemical properties II. Effect of phospholipid fatty acid modulation on plasma membrane physical properties and enzymatic activities

The fatty acid composition of plasma membrane phospholipids of the murine T lymphocyte tumor EL4 were systematically modified in an attempt to understand the relationship between lipid bilayer composition and plasma membrane physical and biological properties. Two plasma membrane enzyme activities, adenylate cyclase and ouabain-sensitive ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase, were measured in normal and fatty acid-substituted EL4 plasma membrane fractions. The fatty acid effect on enzyme activities was similar to previously reported effects of fatty acids on cytotoxic T cell function. The activity of both enzymes was inhibited by saturated fatty acids, while unsaturated fatty acids had a moderate enhancing effect on both enzyme activities. Using two different nitroxide derivatives of stearic acid, the order parameter and approximate rotational correlation times were calculated from ESR spectra of normal and fatty acid-modified plasma membranes. No significant difference was found in either parameter in these membranes. These results, in conjunction with earlier data from our laboratory and others, suggest that caution should be exercised in inferring changes in membrane ‘fluidity’ based on lipid modulation of biological membranes.     

Influence of different centrifugation protocols on equine semen preservation

Three experiments were conducted to evaluate the impact of centrifugation on cooled and frozen preservation of equine semen. A standard centrifugation protocol (600 × g for 10 min = CP1) was compared to four protocols with increasing g-force and decreased time period (600 × g, 1200 × g, 1800 × g and 2400 × g for 5 min for CP2, 3, 4, and 5, respectively) and to an uncentrifuged negative control. In experiment 1, the influence of the different CPs on sperm loss was evaluated by calculating the total number of sperm cells in 90% of the supernatant. Moreover, the effect on semen quality following centrifugation was assessed by monitoring several sperm parameters (membrane integrity using SYBR14-PI, acrosomal status using PSA-FITC, percentage total motility (TM), percentage progressive motility (PM) and beat cross frequency (BCF) obtained with computer assisted sperm analysis (CASA)) immediately after centrifugation and daily during chilled storage for 3 d. The use of CP1 resulted in a sperm loss of 22%. Increasing the centrifugation force to 1800 × g and 2400 × g for 5 min led to significantly lower sperm losses (7.4% and 2.1%, respectively; P < 0.05). Compared to the uncentrifuged samples, centrifugation of semen resulted in a better sperm quality after chilled storage. There were minimal differences between the CPs although total motility was lower for CP2 than for the other treatments (P < 0.005). In experiment 2, the centrifuged samples were cryopreserved using a standard freezing protocol and analyzed immediately upon thawing. Samples centrifuged according to CP2 resulted in a higher BCF (P < 0.005), whereas CP3 and CP5 yielded a lower BCF (P < 0.05) when compared to CP1. There were no post thaw differences between CP1 and CP4. In experiment 3, DNA integrity of the different samples was analyzed using TUNEL. Although DNA integrity decreased over time, CP had no impact. In conclusion, the loss of sperm cells in the supernatant after centrifugation can be substantially reduced by increasing the g-force up to 1800 × g or 2400 × g for a shorter period of time (5 min) compared to the standard protocol without apparent changes in semen quality, resulting in a considerable increase in the number of insemination doses per ejaculate.     

Phospholipid fatty acid remodeling and carbonylated protein increase in extracellular vesicles released by airway epithelial cells exposed to cigarette smoke extract

Cigarette smoke (CS) represents one of the most relevant environmental risk factors for several chronic pathologies. Tissue damage caused by CS exposure is mediated, at least in part, by oxidative stress induced by its toxic and pro-oxidant components. Evidence demonstrates that extracellular vesicles (EVs) released by various cell types exposed to CS extract (CSE) are characterized by altered biochemical cargo and gained pathological properties. In the present study, we evaluated the content of oxidized proteins and phospholipid fatty acid profiles of EVs released by human bronchial epithelial BEAS-2B cells treated with CSE. This specific molecular characterization has hitherto not been performed. After confirmation that CSE reduces viability of BEAS-2B cells and elevates intracellular ROS levels, in a dose-dependent manner, we demonstrated that 24 h exposure at 1% CSE, a concentration that only slight modifies cell viability but increases ROS levels, was able to increase carbonylated protein levels in cells and released EVs. The release of oxidatively modified proteins via EVs might represent a mechanism used by cells to remove toxic proteins in order to avoid their intracellular overloading. Moreover, 1% CSE induced only few changes in the fatty acid asset in BEAS-2B cell membrane phospholipids, whereas several rearrangements were observed in EVs released by CSE-treated cells. The impact of changes in acyl chain composition of CSE-EVs accounted for the increased saturation levels of phospholipids, a membrane parameter that might influence EV stability, uptake and, at least in part, EV-mediated biological effects. The present in vitro study adds new information concerning the biochemical composition of CSE-related EVs, useful to predict their biological effects on target cells. Furthermore, the information regarding the presence of oxidized proteins and the specific membrane features of CSE-related EVs can be useful to define the utilization of circulating EVs as marker for diagnosing of CS-induced lung damage and/or CS-related diseases.     

Effect of centrifugal fractionation protocols on quality and recovery rate of equine sperm

Centrifugal fractionation of semen is commonly done to improve quality of human semen in assisted-reproduction laboratories, allowing sperm separation based on their isopycnic points. Sperm with morphologic abnormalities are often more buoyant, promoting their retention above defined density media, with structurally normal sperm passing through the media following centrifugation. Three experiments were conducted to evaluate the effects of density-medium type, centrifuge-tube size, sperm number, and density-medium volume (column height) on stallion sperm quality and recovery rate in sperm pellets following centrifugation. In all three experiments, equine semen was initially centrifuged to increase sperm concentration. In Experiment 1, semen was layered over continuous or discontinuous gradients. For Experiment 2, semen was layered over three column heights of continuous gradients in 15- or 50-ml conical-bottom tubes. For Experiment 3, increasing sperm numbers were layered over continuous gradient in 15- or 50-ml conical-bottom tubes. Following centrifugation, sperm pellets were evaluated for sperm morphologic quality, motility, DNA integrity, and recovery rate. Centrifugal fractionation improved (P < 0.05) sperm morphology, motility, and DNA integrity, as compared to controls. The continuous gradient increased (P < 0.05) sperm recovery rate relative to the discontinuous gradient, whereas sperm processed in 15-ml tubes yielded higher velocity and higher recovery rates (P < 0.05 for each) than that processed in 50-ml tubes. Sperm recovery rate was not affected (P > 0.05) by column height of gradient. Increasing sperm number subjected to gradient centrifugation decreased (P < 0.05) sperm recovery rate when 15-ml tubes were used.     

Factors affecting sperm recovery rates and survival after centrifugation of equine semen

Conventional centrifugation protocols result in important sperm losses during removal of the supernatant. In this study, the effect of centrifugation force (400 or 900 × g), duration (5 or 10 min), and column height (20 or 40 mL; Experiment 1); sperm concentration (25, 50, and 100 × 10⁶/mL; Experiment 2), and centrifugation medium (EZ-Mixin CST [Animal Reproduction Systems, Chino, CA, USA], INRA96 [IMV Technologies, Maple Grove, MN, USA], or VMDZ [Partnar Animal Health, Port Huron, MI, USA]; Experiment 3) on sperm recovery and survival after centrifugation and cooling and storage were evaluated. Overall, sperm survival was not affected by the combination of centrifugation protocol and cooling. Total sperm yield was highest after centrifugation for 10 min at 400 × g in 20-mL columns (95.6 ± 5%, mean ± SD) or 900 × g in 20-mL (99.2 ± 0.8%) or 40-mL (91.4 ± 4.5%) columns, and at 900 × g for 5 min in 20-mL columns (93.8 ± 8.9%; P < 0.0001). Total (TMY) and progressively motile sperm yield followed a similar pattern (P < 0.0001). Sperm yields were not significantly different among samples centrifuged at various sperm concentrations. However, centrifugation at 100 × 10⁶/mL resulted in significantly lower total sperm yield (83.8 ± 10.7%) and TMY (81.7 ± 6.8%) compared with noncentrifuged semen. Centrifugation in VMDZ resulted in significantly lower TMY (69.3 ± 22.6%), progressively motile sperm yield (63.5 ± 18.2%), viable yield (60.9 ± 36.5%), and survival of progressively motile sperm after cooling (21 ± 10.8%) compared with noncentrifuged semen. In conclusion, centrifuging volumes of ≤ 20 mL minimized sperm losses with conventional protocols. With 40-mL columns, it may be recommended to increase the centrifugal force to 900 × g for 10 min and dilute the semen to a sperm concentration of 25 to 50 × 10⁶/mL in a milk- or fractionated milk-based medium. The semen extender VMDZ did not seem well suited for centrifugation of equine semen.     

Lipid content and fatty acid distribution in tissues from Portuguese dogfish, leafscale gulper shark and black dogfish

The lipid characterization in tissues from the three deep-sea sharks leafscale gulper shark (Centrophorus squamosus), Portuguese dogfish (Centroscymnus coelolepis) and black dogfish (Centrocyllium fabricii) captured at Hatton Bank in the North Atlantic were examined. The objective was to determine the lipid content and the fatty acid composition in different tissues. In addition, the fatty acid composition in tissues and species was compared. The tissues examined were pancreas, heart, kidney, stomach, spleen and liver. The lipid content was high in liver (40–50%) and ranged from 1% to 5% in the other tissues. The dominant fatty acids were C16:0, C18:1 (n-9), C18:1 (n-7) and C22:6 (n-3) in all tissues. All tissues had a high content of unsaturated fatty acids.     

Control of fatty acid incorporation in membrane phospholipids. X-ray-induced changes in fatty acid uptake by tumor cells

Lymphosarcoma cells isolated from the spleens of tumor-bearing mice were used to study the effect of a low dose of X-rays (5 Gy) on the incorporation of [³H]palmitate and [¹⁴C]arachidonate into the lipids of the tumor cells. Palmitate and arachidonate were rapidly incorporated especially into the phospholipids of the cells. Between one and three hours after the start of the incubation with radiactive palmitate 80–90% of the label of the total lipids was found in the phospholipid fraction. Already after a few minutes of incubation with radioactive arachidonate, about 95% of the label was incorporated in the phospholipids. Irradiation caused a small but significant increase in the rate of fatty acid incorporation for both fatty acids. Concomitantly, a significantly increased amount of fatty acid was removed from the medium by the cells as a result of the irradiation, and the specific radioactivity of the free fatty acids in the cells was found to be enhanced. The radiation effect on the tumor cells could be mimicked by a hypotonic treatment. The magnitude of the radiation-induced stimulation of the fatty acid incorporation was similar to that of the hypotonically induced effect. Cells which had received a hypotonic treatment before the irradiation, did not show an additional radiation-induced enhancement of fatty acid incorporation into the cellular lipids. When the cells were incubated with serum albumin loaded with a relatively large (non-physiological) amount of complexed fatty acids (fatty acid: albumin molar ratio, $\text{ν}\text{= 3.7}$), no radiation effect on the fatty acid incorporation could be detected. It is concluded that hypotonic treatment, irradiation, and increased supply of exogenous fatty acids all lead to an enhanced flux of fatty acids into the cells. These results confirm our previous suggestion that the uptake of fatty acids through the plasma membrane is the rate-limiting step in the fatty acid incorporation into the phospholipids and that ionizing radiation is one of the means to enhance fatty acid uptake through the plasma membrane leading to an increased incorporation into the phospholipids.     

Incorporation of fatty acids by Streptococcus mutans

In a series of investigations into the cariogenicity of Streptococcus mutans, we studied the incorporation of exogenous fatty acids with reference to glucosyltransferase secretion and membrane fatty acid changes. When cells were grown with different fatty acids, both saturated and unsaturated fatty acids were readily incorporated into the membrane lipids and were biotransformed and elongated preferentially to the longer 16- and 18-carbon-chain fatty acids. This incorporation and chain-elongation led to significant changes in fatty acids composition. By adding fatty acids to the medium, it was possible to appropriately modify the degree of unsaturation and the relative ratio between specific fatty acids in the membrane lipids of S. mutans.     

The effect of natural and synthetic fatty acids on membrane structure,microdomain organization,cellular functions and human health

This review deals with the effects of synthetic and natural fatty acids on the biophysical properties of membranes, and on their implication on cell function. Natural fatty acids are constituents of more complex lipids, like triacylglycerides or phospholipids, which are used by cells to store and obtain energy, as well as for structural purposes. Accordingly, natural and synthetic fatty acids may modify the structure of the lipid membrane, altering its microdomain organization and other physical properties, and provoking changes in cell signaling. Therefore, by modulating fatty acids it is possible to regulate the structure of the membrane, influencing the cell processes that are reliant on this structure and potentially reverting pathological cell dysfunctions that may provoke cancer, diabetes, hypertension, Alzheimer's and Parkinson's disease. The so-called Membrane Lipid Therapy offers a strategy to regulate the membrane composition through drug administration, potentially reverting pathological processes by re-adapting cell membrane structure. Certain fatty acids and their synthetic derivatives are described here that may potentially be used in such therapies, where the cell membrane itself can be considered as a target to combat disease. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.     

Selective sorting of cargo proteins into bacterial membrane vesicles

In contrast to the well established multiple cellular roles of membrane vesicles in eukaryotic cell biology, outer membrane vesicles (OMV) produced via blebbing of prokaryotic membranes have frequently been regarded as cell debris or microscopy artifacts. Increasingly, however, bacterial membrane vesicles are thought to play a role in microbial virulence, although it remains to be determined whether OMV result from a directed process or from passive disintegration of the outer membrane. Here we establish that the human oral pathogen Porphyromonas gingivalis has a mechanism to selectively sort proteins into OMV, resulting in the preferential packaging of virulence factors into OMV and the exclusion of abundant outer membrane proteins from the protein cargo. Furthermore, we show a critical role for lipopolysaccharide in directing this sorting mechanism. The existence of a process to package specific virulence factors into OMV may significantly alter our current understanding of host-pathogen interactions.     

The role of fatty acid unsaturation in minimizing biophysical changes on the structure and local effects of bilayer membranes

Studying the effects of saturated and unsaturated fatty acids on biological and model (liposomes) membranes could provide insight into the contribution of biophysical effects on the cytotoxicity observed with saturated fatty acids. In vitro experiments suggest that unsaturated fatty acids, such as oleate and linoleate, are less toxic, and have less impact on the membrane fluidity. To understand and assess the biophysical changes in the presence of the different fatty acids, we performed computational analyses of model liposomes with palmitate, oleate, and linoleate. The computational results indicate that the unsaturated fatty acid chain serves as a membrane stabilizer by preventing changes to the membrane fluidity. Based on a Voronoi tessellation analysis, unsaturated fatty acids have structural properties that can reduce the lipid ordering within the model membranes. In addition, hydrogen bond analysis indicates a more uniform level of membrane hydration in the presence of oleate and linoleate as compared to palmitate. Altogether, these observations from the computational studies provide a possible mechanism by which unsaturated fatty acids minimize biophysical changes and protect the cellular membrane and structure. To corroborate our findings, we also performed a liposomal leakage study to assess how the different fatty acids alter the membrane integrity of liposomes. This showed that palmitate, a saturated fatty acid, caused greater destabilization of liposomes (more “leaky”) than oleate, an unsaturated fatty acid.     

HIV-protease inhibitors suppress skeletal muscle fatty acid oxidation by reducing CD36 and CPT1 fatty acid transporters

Infection with human immunodeficiency virus (HIV) and treatment with HIV-protease inhibitor (PI)-based highly active antiretroviral therapies (HAART) is associated with dysregulated fatty acid and lipid metabolism. Enhanced lipolysis, increased circulating fatty acid levels, and hepatic and intramuscular lipid accumulation appear to contribute to insulin resistance in HIV-infected people treated with PI-based HAART. However, it is unclear whether currently prescribed HIV-PIs directly alter skeletal muscle fatty acid transport, oxidation, and storage. We find that ritonavir (r, 5 µmol/l) plus 20 µmol/l of atazanavir (ATV), lopinavir (LPV), or darunavir (DRV) reduce palmitate oxidation(16–21%) in differentiated C2C12 myotubes. Palmitate oxidation was increased following exposure to high fatty acid media but this effect was blunted when myotubes were pre-exposed to the HIV-PIs. However, LPV/r and DRV/r, but not ATV/r suppressed palmitate uptake into myotubes. We found no effect of the HIV-PIs on FATP1, FATP4, or FABPpm but both CD36/FAT and carnitine palmitoyltransferase 1 (CPT1) were reduced by all three regimens though ATV/r caused only a small decrease in CPT1, relative to LPV/r or DRV/r. In contrast, sterol regulatory element binding protein-1 was increased by all 3 HIV-PIs. These findings suggest that HIV-PIs suppress fatty acid oxidation in murine skeletal muscle cells and that this may be related to decreases in cytosolic- and mitochondrial-associated fatty acid transporters. HIV-PIs may also directly impair fatty acid handling and partitioning in skeletal muscle, and this may contribute to the cluster of metabolic complications that occur in people living with HIV.     

Fatty acid analysis of triacylglycerols: Preparation of fatty acid methyl esters for gas chromatography

A method to prepare fatty acid methyl esters was developed for fatty acid analysis of triacylglycerols by gas chromatography (GC). Triacylglycerols were mixed with methanolic CH₃ONa in hexane containing a mid-polar solvent for 10 s at room temperature. Under these conditions, trioleoylglycerol was converted to methyl oleate with an average yield of 99.3%. This procedure gave reliable and reproducible data on fatty acid compositions determined by GC.