Showing papers on "Membrane lipids published in 2009"

Direct observation of the nanoscale dynamics of membrane lipids in a living cell

Abstract: Cholesterol-mediated lipid interactions are thought to have a functional role in many membrane-associated processes such as signalling events. Although several experiments indicate their existence, lipid nanodomains ('rafts') remain controversial owing to the lack of suitable detection techniques in living cells. The controversy is reflected in their putative size of 5-200 nm, spanning the range between the extent of a protein complex and the resolution limit of optical microscopy. Here we demonstrate the ability of stimulated emission depletion (STED) far-field fluorescence nanoscopy to detect single diffusing (lipid) molecules in nanosized areas in the plasma membrane of living cells. Tuning of the probed area to spot sizes approximately 70-fold below the diffraction barrier reveals that unlike phosphoglycerolipids, sphingolipids and glycosylphosphatidylinositol-anchored proteins are transiently ( approximately 10-20 ms) trapped in cholesterol-mediated molecular complexes dwelling within <20-nm diameter areas. The non-invasive optical recording of molecular time traces and fluctuation data in tunable nanoscale domains is a powerful new approach to study the dynamics of biomolecules in living cells.

Emerging roles for lipids in shaping membrane-protein function

Abstract: Studies of membrane proteins have revealed a direct link between the lipid environment and the structure and function of some of these proteins Although some of these effects involve specific chemical interactions between lipids and protein residues, many can be understood in terms of protein-induced perturbations to the membrane shape The free-energy cost of such perturbations can be estimated quantitatively, and measurements of channel gating in model systems of membrane proteins with their lipid partners are now confirming predictions of simple models

Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network

Abstract: The trans-Golgi network (TGN) is the major sorting station in the secretory pathway of all eukaryotic cells. How the TGN sorts proteins and lipids to generate the enrichment of sphingolipids and sterols at the plasma membrane is poorly understood. To address this fundamental question in membrane trafficking, we devised an immunoisolation procedure for specific recovery of post-Golgi secretory vesicles transporting a transmembrane raft protein from the TGN to the cell surface in the yeast Saccharomyces cerevisiae. Using a novel quantitative shotgun lipidomics approach, we could demonstrate that TGN sorting selectively enriched ergosterol and sphingolipid species in the immunoisolated secretory vesicles. This finding, for the first time, indicates that the TGN exhibits the capacity to sort membrane lipids. Furthermore, the observation that the immunoisolated vesicles exhibited a higher membrane order than the late Golgi membrane, as measured by C-Laurdan spectrophotometry, strongly suggests that lipid rafts play a role in the TGN-sorting machinery.

A role for lipid droplets in inter-membrane lipid traffic.

Abstract: All cells have the capacity to accumulate neutral lipids and package them into lipid droplets. Recent proteomic analyses indicate that lipid droplets are not simple lipid storage depots, but rather complex organelles that have multiple cellular functions. One of these proposed functions is to distribute neutral lipids as well as phospholipids to various membrane-bound organelles within the cell. Here, we summarize the lipid droplet-associated membrane-trafficking proteins and review the evidence that lipid droplets interact with endoplasmic reticulum, endosomes, peroxisomes, and mitochondria. Based on this evidence, we present a model for how lipid droplets can distribute lipids to specific membrane compartments.

Arabidopsis lipins mediate eukaryotic pathway of lipid metabolism and cope critically with phosphate starvation

Abstract: Phosphate is an essential nutrient for plant viability. It is well-established that phosphate starvation triggers membrane lipid remodeling, a process that converts significant portion of phospholipids to non-phosphorus-containing galactolipids. This remodeling is mediated by either phospholipase C (PLC) or phospholipase D (PLD) in combination with phosphatidate phosphatase (PAP). Two PLC genes, NPC4 and NPC5, and PLD genes, PLDζ1 and PLDζ2, are shown to be involved in the remodeling. However, gene knockout studies show that none of them plays decisive roles in the remodeling. Thus, although this phenomenon is widely observed among plants, the key enzyme(s) responsible for the lipid remodeling in a whole plant body is unknown; therefore, the physiological significance of this conversion process has remained to be elucidated. We herein focused on PAP as a key enzyme for this adaptation, and identified Arabidopsis lipin homologs, AtPAH1 and AtPAH2, that encode the PAPs involved in galactolipid biosynthesis. Double mutant pah1pah2 plants had decreased phosphatidic acid hydrolysis, thus affecting the eukaryotic pathway of galactolipid synthesis. Upon phosphate starvation, pah1pah2 plants were severely impaired in growth and membrane lipid remodeling. These results indicate that PAH1 and PAH2 are the PAP responsible for the eukaryotic pathway of galactolipid synthesis, and the membrane lipid remodeling mediated by these two enzymes is an essential adaptation mechanism to cope with phosphate starvation.

Lipid rafts as functional heterogeneity in cell membranes.

Abstract: Biological membranes are not structurally passive solvents of amphipathic proteins and lipids. Rather, it appears their constituents have evolved intrinsic characteristics that make homogeneous distribution of components unlikely. As a case in point, the concept of lipid rafts has received considerable attention from biologists and biophysicists since the formalization of the hypothesis more than 10 years ago. Today, it is clear that sphingolipid and cholesterol can self-associate into micron-scaled phases in model membranes and that these lipids are involved in the formation of highly dynamic nanoscale heterogeneity in the plasma membrane of living cells. However, it remains unclear whether these entities are manifestations of the same principle. A powerful means by which the molecular organization of rafts can be assessed is through analysis of their functionalized condition. Raft heterogeneity can be activated to coalesce and laterally reorganize/stabilize bioactivity in cell membranes. Evaluation of this property suggests that functional raft heterogeneity arises through principles of lipid-driven phase segregation coupled to additional chemical specificities, probably involving proteins.

Phthiocerol dimycocerosates of M. tuberculosis participate in macrophage invasion by inducing changes in the organization of plasma membrane lipids.

Abstract: Phthiocerol dimycocerosates (DIM) are major virulence factors of Mycobacterium tuberculosis (Mtb), in particular during the early step of infection when bacilli encounter their host macrophages. However, their cellular and molecular mechanisms of action remain unknown. Using Mtb mutants deleted for genes involved in DIM biosynthesis, we demonstrated that DIM participate both in the receptor-dependent phagocytosis of Mtb and the prevention of phagosomal acidification. The effects of DIM required a state of the membrane fluidity as demonstrated by experiments conducted with cholesterol-depleting drugs that abolished the differences in phagocytosis efficiency and phagosome acidification observed between wild-type and mutant strains. The insertion of a new cholesterol-pyrene probe in living cells demonstrated that the polarity of the membrane hydrophobic core changed upon contact with Mtb whereas the lateral diffusion of cholesterol was unaffected. This effect was dependent on DIM and was consistent with the effect observed following DIM insertion in model membrane. Therefore, we propose that DIM control the invasion of macrophages by Mtb by targeting lipid organisation in the host membrane, thereby modifying its biophysical properties. The DIM-induced changes in lipid ordering favour the efficiency of receptor-mediated phagocytosis of Mtb and contribute to the control of phagosomal pH driving bacilli in a protective niche.

Lipid-regulated sterol transfer between closely apposed membranes by oxysterol-binding protein homologues

Abstract: Sterols are transferred between cellular membranes by vesicular and poorly understood nonvesicular pathways. Oxysterol-binding protein–related proteins (ORPs) have been implicated in sterol sensing and nonvesicular transport. In this study, we show that yeast ORPs use a novel mechanism that allows regulated sterol transfer between closely apposed membranes, such as organelle contact sites. We find that the core lipid-binding domain found in all ORPs can simultaneously bind two membranes. Using Osh4p/Kes1p as a representative ORP, we show that ORPs have at least two membrane-binding surfaces; one near the mouth of the sterol-binding pocket and a distal site that can bind a second membrane. The distal site is required for the protein to function in cells and, remarkably, regulates the rate at which Osh4p extracts and delivers sterols in a phosphoinositide-dependent manner. Together, these findings suggest a new model of how ORPs could sense and regulate the lipid composition of adjacent membranes.

Structure of the BK potassium channel in a lipid membrane from electron cryomicroscopy

Abstract: A long-sought goal in structural biology has been the imaging of membrane proteins in their membrane environments. This goal has been achieved with electron crystallography in those special cases where a protein forms highly ordered arrays in lipid bilayers. It has also been achieved by NMR methods in proteins up to 50 kilodaltons (kDa) in size, although milligram quantities of protein and isotopic labelling are required. For structural analysis of large soluble proteins in microgram quantities, an increasingly powerful method that does not require crystallization is single-particle reconstruction from electron microscopy of cryogenically cooled samples (electron cryomicroscopy (cryo-EM)). Here we report the first single-particle cryo-EM study of a membrane protein, the human large-conductance calcium- and voltage-activated potassium channel (BK), in a lipid environment. The new method is called random spherically constrained (RSC) single-particle reconstruction. BK channels, members of the six-transmembrane-segment (6TM) ion channel family, were reconstituted at low density into lipid vesicles (liposomes), and their function was verified by a potassium flux assay. Vesicles were also frozen in vitreous ice and imaged in an electron microscope. From images of 8,400 individual protein particles, a three-dimensional (3D) reconstruction of the BK channel and its membrane environment was obtained at a resolution of 1.7-2.0 nm. Not requiring the formation of crystals, the RSC approach promises to be useful in the structural study of many other membrane proteins as well.

Lipid rafts and caveolin-1 are required for invadopodia formation and extracellular matrix degradation by human breast cancer cells.

Abstract: Invadopodia are ventral membrane protrusions through which invasive cancer cells degrade the extracellular matrix. They are thought to function in the migration of cancer cells through tissue barriers, which is necessary for cancer invasion and metastasis. Although many protein components of invadopodia have been identified, the organization and the role of membrane lipids in invadopodia are not well understood. In this study, the role of lipid rafts, which are cholesterol-enriched membrane microdomains, in the assembly and function of invadopodia in human breast cancer cells was investigated. Lipid rafts are enriched, internalized, and dynamically trafficked at invadopodia sites. Perturbation of lipid raft formation due to depleting or sequestering membrane cholesterol blocked the invadopodia-mediated degradation of the gelatin matrix. Caveolin-1 (Cav-1), a resident protein of lipid rafts and caveolae, accumulates at invadopodia and colocalizes with the internalized lipid raft membranes. Membrane type 1 matrix metalloproteinase (MT1-MMP), a matrix proteinase associated with invadopodia, is localized at lipid raft-enriched membrane fractions and cotrafficked and colocalized with Cav-1 at invadopodia. The small interfering RNA-mediated silencing of Cav-1 inhibited the invadopodia-mediated and MT1-MMP-dependent degradation of the gelatin matrix. Furthermore, Cav-1 and MT1-MMP are coexpressed in invasive human breast cancer cell lines that have an ability to form invadopodia. These results indicate that invadopodia are the sites where enrichment and trafficking of lipid rafts occur and that Cav-1 is an essential regulator of MT1-MMP function and invadopodia-mediated breast cancer cell invasion.

New strategies for novel antibiotics: peptides targeting bacterial cell membranes.

Abstract: Membranes are targets of host defence or antimicrobial peptides, effector molecules of innate immunity that evolved in nature to contend with invaders as an active system of defence. The different physicochemical properties of the lipids found in biological membranes allow antimicrobial peptides to discriminate between bacterial and mammalian cell membranes. Such cationic amphipathic peptides will interact predominantly with negatively charged lipids exposed on the outer leaflet of bacterial cell membranes. The molecular mechanism(s) of membrane rupture mutually depends on the nature of the peptide and membrane lipid composition. Biophysical studies demonstrated a complex behavior in terms of membrane perturbation, which can range from pore formation to micellization (carpet model). Peptides aligned parallel to the membrane surface can induce a quasi-interdigitated structure in the gel phase, while depending on the hydrophobic matching of the lipid bilayer core and the peptide membrane thinning or thickening can be observed in the fluid phase. As a consequence, besides of peptide-lipid pores, formation of peptide-enriched membrane domains and promotion of cubic structures can be observed, which adversely affect membrane integrity and function. A strategy using the membrane damaging properties of these peptides will form the basis for the development of such peptides as potential novel antibiotic drugs.

Type-B monogalactosyldiacylglycerol synthases are involved in phosphate starvation-induced lipid remodeling, and are crucial for low-phosphate adaptation.

Abstract: SummaryMono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively) constitute the bulk of membrane lipidsin plant chloroplasts. Mutant analyses in Arabidopsis have shown that these galactolipids are essential forchloroplast biogenesis and photoautotrophic growth. Moreover, these non-phosphorous lipids are proposedto participate in low-phosphate (Pi) adaptations. Under Pi-limited conditions, a drastic accumulation of DGDGoccurs concomitantly with a large reduction in membrane phospholipids, suggesting that plants substituteDGDG for phospholipids during Pi starvation. Previously, we reported that among the three MGDG synthasegenes (MGD1, MGD2and MGD3), the type-B MGD2and MGD3are upregulated in parallel with DGDG synthasegenes during Pi starvation. Here, we describe the identification and characterization of T-DNA insertionalmutants of Arabidopsis type-B MGD genes. Under Pi-starved conditions, the mgd3-1 mutant showed a drasticreductioninDGDGaccumulation,particularlyintheroot,indicatingthatMGD3isthemainisoformresponsiblefor DGDG biosynthesis in Pi-starved roots. Moreover, in the roots of mgd2 mgd3 plants, Pi stress-inducedaccumulation of DGDG was almost fully abolished, showing that type-B MGD enzymes are essential formembrane lipid remodeling in Pi-starved roots. Reductions in fresh weight, root growth and photosyntheticperformancewere alsoobservedinthesemutants underPi-starvedconditions.Theseresults demonstratethatPi stress-induced membrane lipid remodeling is important in plant growth during Pi starvation. Thewidespread distribution of type-B MGD genes in land plants suggests that membrane lipid remodelingmediated by type-B MGD enzymes is a potent adaptation to Pi deficiency for land plants.Keywords: galactolipid, phosphate starvation, monogalactosyldiacylglycerol, MGDG synthase, digalactosyl-diacylglycerol.IntroductionLife in all organisms relies on the presence of lipid bilayercell membranes, which separate the interior of cells fromtheir environment. In the plastids of higher plants, the non-phosphorous galactolipids monogalactosyldiacylglycerol(MGDG)anddigalactosyldiacylglycerol(DGDG)arethemainconstituents of the membrane lipids, whereas phospholip-ids are the predominant components of other subcellularmembranes, such as the plasma membrane, the endoplas-mic reticulum (ER) and the mitochondrial membranes(Joyard et al., 1998). In chloroplasts, MGDG and DGDG

Coordination of Lipid Metabolism in Membrane Biogenesis

Abstract: Bilayer synthesis during membrane biogenesis involves the concerted assembly of multiple lipid species, requiring coordination of the level of lipid synthesis, uptake, turnover, and subcellular distribution. In this review, we discuss some of the salient conclusions regarding the coordination of lipid synthesis that have emerged from work in mammalian and yeast cells. The principal instruments of global control are a small number of transcription factors that target a wide range of genes encoding enzymes that operate in a given metabolic pathway. Critical in mammalian cells are sterol regulatory element binding proteins (SREBPs) that stimulate expression of genes for the uptake and synthesis of cholesterol and fatty acids. From work with Saccharomyces cerevisiae, much has been learned about glycerophospholipid and ergosterol regulation through Ino2p/Ino4p and Upc2p transcription factors, respectively. Lipid supply is fine-tuned through a multitude of negative feedback circuits initiated by both end products and intermediates of lipid synthesis pathways. Moreover, there is evidence that the diversity of membrane lipids is maintained through cross-regulatory effects, whereby classes of lipids activate the activity of enzymes operating in another metabolic branch.

Cholesterol Exposure at the Membrane Surface Is Necessary and Sufficient to Trigger Perfringolysin O Binding

Abstract: Perfringolysin O (PFO) is the prototype for the cholesterol-dependent cytolysins, a family of bacterial pore-forming toxins that act on eukaryotic membranes. The pore-forming mechanism of PFO exhibits an absolute requirement for membrane cholesterol, but the complex interplay between the structural arrangement of the PFO C-terminal domain and the distribution of cholesterol in the target membrane is poorly understood. Herein we show that PFO binding to the bilayer and the initiation of the sequence of events that culminate in the formation of a transmembrane pore depend on the availability of free cholesterol at the membrane surface, while changes in the acyl chain packing of the phospholipids and cholesterol in the membrane core, or the presence or absence of detergent-resistant domains do not correlate with PFO binding. Moreover, PFO association with the membrane was inhibited by the addition of sphingomyelin, a typical component of membrane rafts in cell membranes. Finally, addition of molecules that do not interact with PFO, but intercalate into the membrane and displace cholesterol from its association with phospholipids (e.g., epicholesterol), reduced the amount of cholesterol required to trigger PFO binding. Taken together, our studies reveal that PFO binding to membranes is triggered when the concentration of cholesterol exceeds the association capacity of the phospholipids, and this cholesterol excess is then free to associate with the toxin.

Measurement of the membrane curvature preference of phospholipids reveals only weak coupling between lipid shape and leaflet curvature

Abstract: In biological processes, such as fission, fusion and trafficking, it has been shown that lipids of different shapes are sorted into regions with different membrane curvatures This lipid sorting has been hypothesized to be due to the coupling between the membrane curvature and the lipid's spontaneous curvature, which is related to the lipid's molecular shape On the other hand, theoretical predictions and simulations suggest that the curvature preference of lipids, due to shape alone, is weaker than that observed in biological processes To distinguish between these different views, we have directly measured the curvature preferences of several lipids by using a fluorescence-based method We prepared small unilamellar vesicles of different sizes with a mixture of egg-PC and a small mole fraction of N-nitrobenzoxadiazole (NBD)-labeled phospholipids or lysophospholipids of different chain lengths and saturation, and measured the NBD equilibrium distribution across the bilayer We observed that the transverse lipid distributions depended linearly on membrane curvature, allowing us to measure the curvature coupling coefficient Our measurements are in quantitative agreement with predictions based on earlier measurements of the spontaneous curvatures of the corresponding nonfluorescent lipids using X-ray diffraction We show that, though some lipids have high spontaneous curvatures, they nevertheless showed weak curvature preferences because of the low values of the lipid molecular areas The weak curvature preference implies that the asymmetric lipid distributions found in biological membranes are not likely to be driven by the spontaneous curvature of the lipids, nor are lipids discriminating sensors of membrane curvature

Flipping lipids: why an' what's the reason for?

Abstract: The biosynthesis of glycoconjugates such as N-glycoproteins and GPI-anchored proteins in eukaryotes and cell wall peptidoglycan and lipopolysaccharide in bacteria requires lipid intermediates to be flipped rapidly across the endoplasmic reticulum or bacterial cytoplasmic membrane (so-called biogenic membranes). Rapid flipping is also required to normalize the number of glycerophospholipids in the two leaflets of the bilayer as the membrane expands in a growing cell. Although lipids diffuse rapidly in the plane of the membrane, the intrinsic rate at which they flip across membranes is very low. Biogenic membranes possess dedicated lipid transporters or flippases to increase flipping to a physiologically sufficient rate. The flippases are "ATP-independent" and facilitate "downhill" transport. Most predicted biogenic membrane flippases have not been identified at the molecular level, and the few flippases that have been identified by genetic approaches have not been biochemically validated. Here we summarize recent progress on this fundamental topic and speculate on the mechanism(s) by which biogenic membrane flippases facilitate transbilayer lipid movement.

A new lipid anchor for sparsely tethered bilayer lipid membranes.

Abstract: Mixed self-assembled monolayers (SAMs) of beta-mercaptoethanol and the new synthetic lipid 1,2-dipalmityl-3-[w-mercaptonona(ethylene oxide)] glycerol (FC 16) were investigated for their ability to form sparsely tethered bilayer lipid membranes (stBLMs) completed with various phospholipids. We investigated the structural and functional properties of FC16-based stBLMs and compared these to stBLMs prepared using a previously characterized synthetic lipid, 1,2-dimyristyl-3-[omega-mercaptohexa(ethylene oxide)] glycerol (WC14). FC16-based stBLMs show increased resistivity to ion transfer and an increase in the submembrane space of approximately 0.5 nm. Importantly, FC16-based stBLMs formed well-defined, complete bilayers with charged phospholipids such as 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG). In these, POPG incorporates into the outer monolayer leaflet in the same ratio as in the immersion solution but is excluded from the inner leaflet. In all cases that we have investigated thus far, the area densities of the lipids within the bilayers were on average close to those in free bilayer membranes. For charged phospholipids, FC16 appears to provide a distinct advantage over WC14 for the formation of well-defined stBLMs.

Lipid Rafts and Caveolae and Their Role in Compartmentation of Redox Signaling

Abstract: Membrane (lipid) rafts and caveolae, a subset of rafts, are cellular domains that concentrate plasma membrane proteins and lipids involved in the regulation of cell function. In addition to providing signaling platforms for G-protein-coupled receptors and certain tyrosine kinase receptors, rafts/caveolae can influence redox signaling. This review discusses molecular characteristics of and methods to study rafts/caveolae, determinants that contribute to the localization of molecules in these entities, an overview of signaling molecules that show such localization, and the contribution of rafts/caveolae to redox signaling. Of particular note is the evidence that endothelial nitric oxide synthase (eNOS), NADPH oxygenase, and heme oxygenase, along with other less well-studied redox systems, localize in rafts and caveolae. The precise basis for this localization and the contribution of raft/caveolae-localized redox components to physiology and disease are important issues for future studies. Antioxid. Redox Signal. 11, 1357–1372.

Mechanisms of transport through the Golgi complex

Abstract: The Golgi complex is the central sorting and processing station of the secretory pathway, ensuring that cargo proteins, which are synthesized in the endoplasmic reticulum, are properly glycosylated and packaged into carriers for transport to their final destinations. Two recent studies highlight the fact that properties of membrane lipids play key roles in Golgi structural organization and trafficking. The Antonny laboratory has demonstrated the mechanism by which a Golgi tether containing a membrane-curvature-sensing domain at one end can link highly curved and flat membranes together in a reversible manner. In this way, a strong interaction that binds membranes together in an oriented fashion can easily be disrupted as the properties of the membranes change. The Lippincott-Schwartz laboratory has developed a new model for intra-Golgi trafficking, called the rapid-partitioning model, which incorporates lipid trafficking as an integral part. Simulations reveal that the sorting of lipids into processing and export domains that are connected to each Golgi cisterna, and bidirectional trafficking throughout the Golgi to allow proteins to associate with their preferred lipid environment, is sufficient to drive protein transport through the secretory pathway. Although only a proof in principle, this model for the first time invokes lipid sorting as the driving force in intra-Golgi trafficking, and provides a framework for future experimental work.