Transbilayer Movement Of Phospholipids Research Articles

A Fluorescence-based Assay for Measuring Phospholipid Scramblase Activity in Giant Unilamellar Vesicles.

Transbilayer movement of phospholipids in biological membranes is mediated by a diverse set of lipid transporters. Among them are scramblases that facilitate rapid bi-directional movement of lipids without metabolic energy input. In this protocol, we describe the incorporation of phospholipid scramblases into giant unilamellar vesicles (GUVs) formed from scramblase-containing large unilamellar vesicles by electroformation. We also describe how to analyze their activity using membrane-impermeant sodium dithionite, to bleach symmetrically incorporated fluorescent ATTO488-conjugated phospholipids. The fluorescence-based readout allows single vesicle tracking for a large number of settled/immobilized GUVs, and provides a well-defined experimental setup to directly characterize these lipid transporters at the molecular level. Graphic abstract: Giant unilamellar vesicles (GUVs) are formed by electroformation from large unilamellar vesicles (LUVs) containing phospholipid scramblases (purple) and trace amounts of a fluorescent lipid reporter (green). The scramblase activity is analyzed by a fluorescence-based assay of single GUVs, using the membrane-impermeant quencher dithionite. Sizes not to scale. Modified from Mathiassen et al. (2021).

Endoplasmic reticulum phospholipid scramblase activity revealed after protein reconstitution into giant unilamellar vesicles containing a photostable lipid reporter

Transbilayer movement of phospholipids in biological membranes is mediated by a diverse set of lipid transporters. Among them are scramblases that facilitate a rapid bi-directional movement of lipids without metabolic energy input. Here, we established a new fluorescence microscopy-based assay for detecting phospholipid scramblase activity of membrane proteins upon their reconstitution into giant unilamellar vesicles formed from proteoliposomes by electroformation. The assay is based on chemical bleaching of fluorescence of a photostable ATTO-dye labeled phospholipid with the membrane-impermeant reductant sodium dithionite. We demonstrate that this new methodology is suitable for the study of the scramblase activity of the yeast endoplasmic reticulum at single vesicle level.

TMEM41B acts as an ER scramblase required for lipoprotein biogenesis and lipid homeostasis

How amphipathic phospholipids are shuttled between the membrane bilayer remains an essential but elusive process, particularly at the endoplasmic reticulum (ER). One prominent phospholipid shuttling process concerns the biogenesis of APOB-containing lipoproteins within the ER lumen, which may require bulk trans-bilayer movement of phospholipids from the cytoplasmic leaflet of the ER bilayer. Here, we show that TMEM41B, present in the lipoprotein export machinery, encodes a previously conceptualized ER lipid scramblase mediating trans-bilayer shuttling of bulk phospholipids. Loss of hepatic TMEM41B eliminates plasma lipids, due to complete absence of mature lipoproteins within the ER, but paradoxically also activates lipid production. Mechanistically, scramblase deficiency triggers unique ER morphological changes and unsuppressed activation of SREBPs, which potently promotes lipid synthesis despite stalled secretion. Together, this response induces full-blown nonalcoholic hepatosteatosis in the TMEM41B-deficient mice within weeks. Collectively, our data uncovered a fundamental mechanism safe-guarding ER function and integrity, dysfunction of which disrupts lipid homeostasis.

Silencing phospholipid scramblase 1 expression by RNA interference in colorectal cancer and metastatic liver cancer

Phospholipid scramblase 1 (PLSCR1) not only participates in the transbilayer movement of phospholipids, but also plays a role in the pathogenesis and progression of cancers. The present study aimed to evaluate the effect of silencing PLSCR1 expression by RNA interference in colorectal cancer (CRC) and metastatic liver cancer. The expression of PLSCR1 in CRC and metastatic liver cancer samples was assessed by immunohistochemistry. The cultured cells with the highest expression were selected for subsequent experiments. We designed three siRNA oligonucleotide segments targeted at PLSCR1. Successful transfection was confirmed. The biological behavior of the cells in proliferation, adhesion, migration and invasion was determined. PLSCR1 protein expression increased significantly in the majority of CRC and metastatic liver cancer samples compared with normal samples. Lovo cells had the highest expression of PLSCR1. The siRNA-390 oligonucleotide segment had the best silencing effect. After transfection, Lovo cell proliferation was significantly inhibited compared with the controls in the MTT assay. Laminin and fibronectin adhesion assays showed Lovo cell adhesion was also significantly inhibited. In the migration assay, the number of migrating cells in the PLSCR1 siRNA-390 group was 50+/-12, significantly lower than the number in the siRNA-N group (115+/-28) and in the control group (118+/-31). In an invasion test, the number of invading cells in the PLSCR1 siRNA-390 group was 60+/-18, significantly lower than that in the siRNA-N group (97+/-26) and the control group (103+/-24). PLSCR1 is overexpressed in CRC and metastatic liver cancer. Silencing of PLSCR1 by siRNA inhibits the proliferation, adhesion, migration and invasion of Lovo cells, which suggests that PLSCR1 contributes to the tumorigenesis and tumor progression of CRC. PLSCR1 may be a potential gene therapy target for CRC and associated metastatic liver cancer.

Thematic Review Series: Glycerolipids. Phosphatidylserine and phosphatidylethanolamine in mammalian cells: two metabolically related aminophospholipids

Phosphatidylserine (PS) and phosphatidylethanolamine (PE) are two aminophospholipids whose metabolism is interrelated. Both phospholipids are components of mammalian cell membranes and play important roles in biological processes such as apoptosis and cell signaling. PS is synthesized in mammalian cells by base-exchange reactions in which polar head groups of preexisting phospholipids are replaced by serine. PS synthase activity resides primarily on mitochondria-associated membranes and is encoded by two distinct genes. Studies in mice in which each gene has been individually disrupted are beginning to elucidate the importance of these two synthases for biological functions in intact animals. PE is made in mammalian cells by two completely independent major pathways. In one pathway, PS is converted into PE by the mitochondrial enzyme PS decarboxylase. In addition, PE is made via the CDP-ethanolamine pathway, in which the final reaction occurs on the endoplasmic reticulum and nuclear envelope. The relative importance of these two pathways of PE synthesis has been investigated in knockout mice. Elimination of either pathway is embryonically lethal, despite the normal activity of the other pathway. PE can also be generated from a base-exchange reaction and by the acylation of lyso-PE. Cellular levels of PS and PE are tightly regulated by the implementation of multiple compensatory mechanisms.

Flippase Activity Detected with Unlabeled Lipids by Shape Changes of Giant Unilamellar Vesicles

Transbilayer movement of phospholipids in biological membranes is mediated by energy-dependent and energy-independent flippases. Available methods for detection of flippase mediated transversal flip-flop are essentially based on spin-labeled or fluorescent lipid analogues. Here we demonstrate that shape change of giant unilamellar vesicles (GUVs) can be used as a new tool to study the occurrence and time scale of flippase-mediated transbilayer movement of unlabeled phospholipids. Insertion of lipids into the external leaflet created an area difference between the two leaflets that caused the formation of a bud-like structure. Under conditions of negligible flip-flop, the bud was stable. Upon reconstitution of the energy-independent flippase activity of the yeast endoplasmic reticulum into GUVs, the initial bud formation was reversible, and the shapes were recovered. This can be ascribed to a rapid flip-flop leading to relaxation of the monolayer area difference. Theoretical analysis of kinetics of shape changes provides self-consistent determination of the flip-flop rate and further kinetic parameters. Based on that analysis, the half-time of phospholipid flip-flop in the presence of endoplasmic reticulum proteins was found to be on the order of few minutes. In contrast, GUVs reconstituted with influenza virus protein formed stable buds. The results argue for the presence of specific membrane proteins mediating rapid flip-flop.

Transbilayer Movement of Phospholipids at the Main Phase Transition of Lipid Membranes: Implications for Rapid Flip-Flop in Biological Membranes

The transbilayer movement of fluorescent phospholipid analogs in liposomes was studied at the lipid phase transition of phospholipid membranes. Two NBD-labeled analogs were used, one bearing the fluorescent moiety at a short fatty acid chain in the sn-2 position (C 6-NBD-PC) and one headgroup-labeled analog having two long fatty acyl chains (N-NBD-PE). The transbilayer redistribution of the analogs was assessed by a dithionite-based assay. We observed a drastic increase of the transbilayer movement of both analogs at the lipid phase transition of DPPC ( T c = 41°C) and DMPC ( T c = 23°C). The flip-flop of analogs was fast at the T c of DPPC with a half-time ( t 1/2) of ∼6–10 min and even faster at the T c of DMPC with t 1/2 on the order of <2 min, as shown for C 6-NBD-PC. Suppressing the phase transition by the addition of cholesterol, the rapid transbilayer movement was abolished. Molecular packing defects at the phase transition are assumed to be responsible for the rapid transbilayer movement. The relevance of those defects for understanding of the activity of flippases is discussed.

Reconstitution and Partial Characterization of Phospholipid Flippase Activity from Detergent Extracts of the Bacillus subtilis Cell Membrane

In bacteria, phospholipids are synthesized on the inner leaflet of the cytoplasmic membrane and must translocate to the outer leaflet to propagate a bilayer. Transbilayer movement of phospholipids has been shown to be fast and independent of metabolic energy, and it is predicted to be facilitated by membrane proteins (flippases) since transport across protein-free membranes is negligible. However, it remains unclear as to whether proteins are required at all and, if so, whether specific proteins are needed. To determine whether bacteria contain specific proteins capable of translocating phospholipids across the cytoplasmic membrane, we reconstituted a detergent extract of Bacillus subtilis into proteoliposomes and measured import of a water-soluble phospholipid analog. We found that the proteoliposomes were capable of transporting the analog and that transport was inhibited by protease treatment. Active proteoliposome populations were also able to translocate a long-chain phospholipid, as judged by a phospholipase A(2)-based assay. Protein-free liposomes were inactive. We show that manipulation of the reconstitution mixture by prior chromatographic fractionation of the detergent extract, or by varying the protein/phospholipid ratio, results in populations of vesicles with different specific activities. Glycerol gradient analysis showed that the majority of the transport activity sedimented at approximately 4S, correlating with the presence of specific proteins. Recovery of activity in other gradient fractions was low despite the presence of a complex mixture of proteins. We conclude that bacteria contain specific proteins capable of facilitating transbilayer translocation of phospholipids. The reconstitution methodology that we describe provides the basis for purifying a facilitator of transbilayer phospholipid translocation in bacteria.

Effects of phosphatidylinositol diphosphate on phospholipid asymmetry in the human erythrocyte membrane.

While phospholipid asymmetry has been well characterized in red blood cells (RBCs), controversy exists as to what role PIP2 plays in cation-induced phosphatidylserine (PS) exposure. We report that PIP2 can redistribute intracellular cations and thereby lead to a loss of phospholipid asymmetry. Flow cytometry was employed to monitor intracellular cation levels by using the fluorophore Fluo-3 and exposure of PS on the outer surface of the RBC bilayer by using fluorescently labeled annexin V. The addition of PIP2 to RBCs led to a concentration-dependent increase in cytosolic cations and PS exposure. IF RBCs were preincubated with 25 microM neomycin sulfate, an inhibitor of phosphoinositide metabolism, PIP2-induced PS exposure decreased dramatically. If the RBC buffer system contained 2.5 mM EGTA, PS exposure also decreased significantly, suggesting a competition between intracellular Fluo-3 and extracellular EGTA. Together, these data indicate that (1) PS exposure was found in RBCs that exhibited an increased cytosolic cation concentration available for the fluorophore. Fluo-3, (2) both the level of intracellular cations and the movement of PS from the inner to the outer monolayer were affected by the level of PIP2 in the bilayer, (3) the cleavage of PIP2 by a phosphoinositide-specific phospholipase lead to the redistribution of intracellular cations and to an increase in the amount of PS exposed on the outer leaflet of the bilayer, and (4) a transient channel could be formed during the interaction of PIP2 with the RBC membrane which would then allow the transbilayer movement of phospholipids and cations.

Synergism between mellitin and phospholipase A2 from bee venom: apparent activation by intervesicle exchange of phospholipids.

Mellitin, a cationic amphiphilic peptide, has an apparent activating effect on interfacial catalysis by phospholipase A2 (PLA2) of bee venom on zwitterionic vesicles of 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine (POPC) and on anionic vesicles of 1,2-dimyristoylglycero-sn-3-phosphomethanol (DMPM), as well as on covesicles of POPC/DMPM (3:7). On the other hand, mellitin-induced increase in the rate of pig pancreatic PLA2 is seen only on anionic vesicles. Interfacial kinetic protocols and spectroscopic methods show that the activation is due to enhanced substrate replenishment resulting from intervesicle exchange of zwitterionic or anionic phospholipids through vesicle-vesicle contacts established by mellitin. It is shown that as the hydrolysis on POPC vesicles progresses, due to a high propensity of bee PLA2 for binding to the product containing zwitterionic vesicles, most of the enzyme in the reaction mixture is trapped on few vesicles that are initially hydrolyzed, and thus reaction ceases. Under these conditions, mellitin promotes substrate replenishment by direct exchange of the products of hydrolysis from the enzyme-containing vesicles with the substrate present in excess vesicles which have not been hydrolyzed. Pig PLA2 has poor affinity for POPC vesicles, and the affinity is only modestly higher in the presence of low mole fractions of the products of hydrolysis; therefore, the enzyme is not trapped on those vesicles. Biophysical studies confirm that the phospholipid exchange occurs through stable intervesicle contacts formed by low mole fractions of mellitin, without transbilayer movement of phospholipids or fusion of vesicles. At high mole fraction (> 1.5%) mellitin induces leakage in POPC vesicles and does not form additional contacts. In POPC/DMPM vesicles, the contacts are formed even at high mole fractions of mellitin. Changes in intrinsic tryptophan fluorescence of mellitin indicate that bound mellitin exists in at least two different functional forms depending on the lipid composition and on the lipid:peptide ratio. A model is proposed to accommodate amphiphilic mellitin as a transmembrane channel or an intervesicle contact.

Selective association of phospholipids as a clue for the passive flip-flop diffusion through bilayer lipid membranes

We showed that the investigation of the selective association of phospholipids might contribute to the insight of the flip-flop diffusion processes. The process of selective association was studied quantitatively by testing the association probabilities for both parallel and anti-parallel orientations of the polar headgroups. The model of double chain binary mixture confirms a high capacity of phospholipids for self-association in parallel configuration of the electric dipole moments whether the cross-sectional area of the polar headgroups are in an usual range of 25–55 Å 2. It is demonstrated that the aggregation of a class of phospholipids from a binary mixture is strongly dependent on the dipole-dipole interaction between the same phospholipids and is modulated by the magnitude of the electric dipole moment of the other phospholipids from that binary mixture. There are a great number of mechanisms involved in the transbilayer movement of phospholipids. We referred here only to the passive transport of lipids from one monolayer to the other. The flip-flop mechanisms raised in this paper are the breakdown of bilayer due to the increase of the packing density and the inversion of the coupled phospholipids from the opposite monolayers of the same bilayer. Thus, the pair formation promoting a drop in occupied volume decreases the packing pressure in the respective monolayer and consequently triggers a flip-flop into the other direction since the packing pressure in the other monolayer has not dropped. According to the present model for the binary mixtures of double-chain lipids, the rate of the flip-flop diffusion decreased by increasing the number of the methylene groups added to the acyl chain. This dependence may be perturbed whether the phospholipids possesses a very high cross-section area of the polar headgroups ( a > 55 A ̊ 2 ). We think that the selective association of phospholipids is neither exclusively, nor only involved in promoting the transbilayer diffusion of phospholipids. Most probably, the selective association determines some phospholipid domains that attract certain particular proteins so that it can modulate the protein activity.

Membrane lipid biogenesis and transport

Membrane biogenesis is an essential feature of cellular development and growth. The initial assembly of membrane lipids and proteins occurs primarily in the endoplasmic reticulum (ER). It has been demonstrated that the enzymes involved in the de novo biosynthesis of phospholipids are exclusively located on the cytoplasmic surface of the ER. A rapid transbilayer movement of phospholipids has also been reported in isolated liver microsomes, which is compatible with the movement of newly synthesized lipids to the lumenal surface of the ER. Comparison with the transbilayer movement of phospholipids across protein-free lipid bilayers, has lead to the proposal that a protein which would catalyze the translocation of phospholipids across the ER membrane (“flipase”), might be involved in the assembly of the lipid bilayer of the ER. Since the various membranes in a eukaryotic cell differ markedly in their lipid composition, it is clear that specific sorting and transport of these membrane components must occur.

Brain spectrin: A review

Red blood cell spectrin, along with actin and several other proteins, forms a skeletal meshwork on the cytoplasmic surface of the erythrocyte plasma membrane. This structure is thought to maintain red blood cell shape, membrane structural stability, and cellular elasticity, as well as controlling the lateral mobility of integral membrane proteins and the transbilayer movement of phospholipids. It is now clearly established that spectrin-related molecules are ubiquitous structural elements subjacent to the plasma membrane of mammalian and avian nonerythroid cells. In this review, we present the current knowledge concerning brain spectrin. Brain spectrin is an ~11S, ~1,000,000 molecular weight ( αβ) 2 tetramer containing subunits of 240,000 (α) and 235,000 (β) molecular weight. It is present in the cortical cytoplasm of all neuronal cell bodies and processes, and to a lesser extent in glial cells. Its involvement in the actin-membrane interaction, as well as other proposed functions in the nervous system is discussed.

Effect of phospholipid oxidation products on transbilayer movement of phospholipids in single lamellar vesicles.

Single lamellar phosphatidyl[methyl-2H]choline vesicles were incubated with an excess of unlabeled phosphatidylcholine vesicles or phosphatidylcholine-cholesterol vesicles containing 8 mol % glucuronosyldiglyceride. Incubation of the two vesicle populations was performed in the presence or absence of a purified phosphatidylcholine exchange protein. The negatively charged glycolipid donor vesicles could be completely removed by column chromatography on DEAE-Sephacel. Following incubation with exchange protein and subsequent fractionation, the -N(CD3)3 phosphatidylcholine acceptor vesicles exhibited a 61-73% enrichment of the unlabeled phosphatidylcholine in the outer monolayer. Upon incubation in an air atmosphere, no appreciable transbilayer movement of the outer monolayer -N(CH3)3 phosphatidylcholine was observed for at least 5 days. Between days 5 and 7, however, extensive transbilayer movement occurred, leading to an outer monolayer/inner monolayer phosphatidylcholine ratio of 2.1 on day 7. In phosphatidylcholine-6 mol % cholesterol vesicles treated similarly, the outside/inside ratio of the unlabeled phospholipid was 6.7, suggesting a much smaller percentage of transbilayer movement. The loss of transbilayer asymmetry which occurred during a 36-h period after day 5 could be estimated at the upper limit, t 1/2 approximately 7.3 h for phosphatidylcholine vesicles and t 1/2 approximately 53 h for phosphatidylcholine-cholesterol vesicles. The actual rates for transbilayer movement, however, were likely more rapid. Transbilayer movement occurred at a time period when oxidized phospholipid breakdown products had reached critical levels.

Investigation of the inside-outside distribution, intermembrane exchange and transbilayer movement of phospholipids in sonicated vesicles by shift reagent NMR

1. 1. A new NMR approach is described for the investigation of transbilayer asymmetry in phospholipid vesicles consisting of phosphatidylcholine and negatively charged phospholipids. The method makes use of the dependence of the pseudocontact shift of the N-methyl proton resonance induced by paramagnetic ions on the surface concentration of negatively charged phospholipids. When two differently shifting paramagnetic probes are applied from the outside and the inside of a vesicular membrane the transbilayer phospholipid distribution can be estimated without knowledge of the inner and outer radii of the vesicles and the packing density of the phospholipid molecules. 2. 2. The method was employed to study the transbilayer asymmetry in vesicles obtained by cosonication of phosphatidylcholine with phosphatidylserine, phosphatidylglycerol or phosphatidylinositol. The three negative phospholipids were found to distribute with a higher surface concentration in the inner vesicular shell than in the outer one when their total content did not exceed 25 mol%. However, as the amount of negatively charged phospholipids increases the ratio of their inside to outside surface concentrations, i.e., the transbilayer asymmetry of the vesicles, decreases. Prolonged incubation (for several days) does not change the compositional asymmetry of the cosonicated vesicles.

Localization of phospholipids in the membrane of Bacillus megaterium.

Phospholipase C treatment of Bacillus megaterium protoplasts at 37°C removed up to 63% of the total phospholipid without disrupting the membrane. More than 95% of the total phosphatidylethanolamine and 3′‐glucosaminylphosphatidylglycerol, 80% of the phosphatidylglycerol and about 20% of the cardiolipin and 2′‐glucosaminylphosphatidylglycerol were hydrolyzed.Similar results were obtained with isolated membranes and dispersions of lipids extracted from the membranes, demonstrating that these data do reflect, at least in part, the substrate specificity of phospholipase C and not necessarily the distribution of phospholipids over the two membrane layers.When protoplasts and isolated membranes were incubated with excess phospholipase C at temperatures in between 5°C and 25°C a different hydrolysis pattern was obtained and it was concluded that 50% of the phosphatidylethanolamine and 70% of the 3′‐glucosaminylphosphatidylglycerol are located in the outer membrane layer, the residual being in the cytoplasmic layer. At least 40% of the phosphatidylglycerol was found to be present also in the outer layer. The data do not allow conclusions about the localization of cardiolipin and 2′‐glucosaminylphosphatidylglycero1. Trinitroben‐zenesulfonic acid reacted with 50% of the phosphatidylethanolamine in protoplasts and with nearly 100% of this lipid in isolated membranes at various temperatures and concentrations, thus confirming the symmetric localization of this lipid.The data furthermore indicate that a temperature‐dependent transbilayer movement of phospholipids is induced by phospholipase C treatment of the Bacillus megaterium protoplasts.