Structures ofBacteroides fragilisuridine 5′-diphosphate-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (BfLpxA)

Uridine 5′-diphosphate-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (LpxA) catalyzes a reversible reaction for adding anO-acyl group to the GlcNAc in UDP-GlcNAc in the first step of lipid A biosynthesis. Lipid A constitutes a major component of lipopolysaccharides, also referred to as endotoxins, which form the outer monolayer of the outer membrane of Gram-negative bacteria. Ligand-free and UDP-GlcNAc-bound crystal structures of LpxA fromBacteroides fragilisNCTC 9343, the most common pathogenic bacteria found in abdominal abscesses, have been determined and are presented here. The enzyme crystallizes in a cubic space group, with the crystallographic threefold axis generating the biological functional homotrimer and with each monomer forming a nine-rung left-handed β-helical (LβH) fold in the N-terminus followed by an α-helical motif in the C-terminus. The structure is highly similar to LpxA from other organisms. Yet, despite sharing a similar LβH structure with LpxAs fromEscherichia coliand others, previously unseen calcium ions are observed on the threefold axis inB. fragilisLpxA to help stabilize the trimeric assembly.

Quantitative Analysis of Membrane Remodeling at the Phagocytic Cup

Nascent phagosomes, which are derived from the plasma membrane, acquire microbicidal properties through multiple fusion and fission events collectively known as maturation. Here we show that remodeling of the phagosomal membrane is apparent even before sealing, particularly when large particles are ingested. Fluorescent probes targeted to the plasma membrane are cleared from the region lining the particle before engulfment is completed. Extensive clearance was noted for components of the inner as well as outer monolayer of the plasmalemma. Segregation of lipid microdomains was ruled out as the mechanism underlying membrane remodeling, because markers residing in rafts and those that are excluded were similarly depleted. Selective endocytosis was also ruled out. Instead, several lines of evidence indicate that endomembranes inserted by exocytosis at sites of ingestion displace the original membrane constituents from the base of the phagosomal cup. The Fcγ receptors that trigger phagocytosis remain associated with their ligands. By contrast, Src-family kinases that are the immediate effectors of receptor activation are flushed away from the cup by the incoming membranes. Together with the depletion of phosphoinositides required for signal transduction, the disengagement of receptors from their effectors by bulk membrane remodeling provides a novel means to terminate receptor signaling.

The Anticancer Drug Cisplatin Interacts with the Human Erythrocyte Membrane

Drugs which exert their effects by interacting with DNA cause structural and functional membrane alterations which may be essential for growth inhibition by these agents. This paper describes the interaction of cisplatin with the human erythrocyte membrane and models constituted by bilayers of dimyristoylphosphatidylethanolamine (DMPE) and diacylphosphatidylserine (DAPS), representative of phospholipid classes located in the inner monolayer of the erythrocyte membrane, and of dimyristoylphosphatidylcholine (DMPC), a class present in its outer monolayer. Cisplatin ability to perturb DMPE, DAPS and DMPC bilayer structures was determined by X-ray diffraction and fluorescence spectroscopy. Electron microscopy disclosed that human erythrocytes incubated with 35 μм cisplatin, which is its therapeutical concentration in serum, developed cup-shaped forms (stomatocytes). According to the bilayer couple hypothesis, this means that the drug is inserted into the inner monolayer of the erythrocyte membrane, a conclusion supported by the studies on model systems.

Collagen-stimulated unidirectional translocation of cholesterol in human platelet membranes

When human platelets are stimulated with collagen or thrombin, the asymmetric distribution of membrane lipids is disrupted as phosphatidylserine and phosphatidylethanolamine translocate from the inner monolayer to the outer monolayer. Coincident with the stimulus-dependent rearrangement of membrane phospholipids is a rapid redistribution of cholesterol from the outer to the inner membrane monolayer. This redistribution of cholesterol was observed when the stimulus was collagen or ADP. The data presented here show that epinephrine stimulation does not promote cholesterol translocation but does potentiate collagen-promoted movement of cholesterol. To investigate the process of cholesterol translocation, experiments were performed to determine whether collagen stimulated reverse cholesterol movement; i.e. from the inner to the outer monolayer. For this study, the fluorescent sterol cholestatrienol (C-3) was incorporated into platelet membranes by exchange from cholesterol-containing phosphatidylcholine small unilamellar vesicles. C-3 was then removed selectively from the outer monolayer by treatment of the platelets with bovine serum albumin (BSA). During the subsequent incubation of BSA-treated platelets, C-3 moved spontaneously into the outer from the inner monolayer. This translocation had an apparent half-time of approximately 25 min and was unaltered by the presence of collagen. These results suggest that collagen treatment of platelets selectively facilitates the inward movement of the sterol. We have hypothesized that cholesterol translocation may be thermodynamically driven as a result of an unfavorable entropy, resulting in cholesterol translocation out of an environment becoming enriched in phosphatidylethanolamine. The unidirectional nature of collagen-promoted cholesterol movement from the phosphatidylethanolamine-rich outer monolayer is consistent with this interpretation.

Cell membrane lipid composition and distribution: implications for cell function and lessons learned from photoreceptors and platelets.

Photoreceptor rod cells and blood platelets are remarkably different, yet both illustrate a similar phenomenon. Both are strongly affected by membrane cholesterol, and the distribution of cholesterol in the membranes of both cell types is determined by the lipid composition within the membranes. In rod cells, cholesterol strongly inhibits rhodopsin activity. The relatively higher level of cholesterol in the plasma membrane serves to inhibit, and thereby conserve, the activity of rhodopsin, which becomes fully active in the low-cholesterol environment of the disk membranes of these same cells. This physiologically important partitioning of cholesterol between disk membranes and plasma membranes occurs because the disk membranes are enriched with phosphatidylethanolamine, thus providing a thermodynamically unfavorable environment for the sterol. Cholesterol enrichment of platelets renders these cells more responsive to stimuli of aggregation. Stimuli for platelet aggregation cause a rapid transbilayer movement of cholesterol from the outer monolayer. This stimulus-dependent redistribution of cholesterol appears to result from the concomitant movement of phosphatidylethanolamine into the outer monolayer. The attractive, yet still unproven, hypothesis is that cholesterol translocation plays an important role in the overall platelet response and is intimately related to the sensitizing actions of cholesterol on these cells.

Distribution and biophysical properties of fluorescent lipids on the surface of adult Schistosoma mansoni

SUMMARYThe properties of 4 fluorescent lipid compounds in the surface membrane of adult male Schistosoma mansoni worms were examined by fluorescent microscopy and fluorescent recovery after photobleaching (FRAP). The data suggest that the probes N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl) sphingosine (BODIPY FL ceramide) and PKH2 pass through the outer membrane and enter structures in or below the membrane. In contrast 5-(N-octadecanoyl)aminofluorescein (AF18) and N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl) sphingosylphosphocholine (BODIPY FL sphingomyelin) insert into the outer monolayer. The DL values of these latter 2 compounds, 8·83 ± 2·35 × 10−9 cm2 s−1 and 2·76 ± 0·53 × 10−9cm2 s−1, respectively, suggest that they enter different domains. Furthermore, it was observed that both BODIPY FL ceramide and BODIPY FL sphingomyelin entered particular structures in or under the surface membrane. The possible nature of these particles is discussed.

GPI-anchored influenza hemagglutinin induces hemifusion to both red blood cell and planar bilayer membranes.

Under fusogenic conditions, fluorescent dye redistributed from the outer monolayer leaflet of red blood cells (RBCs) to cells expressing glycophosphatidylinositol-anchored influenza virus hemagglutinin (GPI-HA) without transfer of aqueous dye. This suggests that hemifusion, but not full fusion, occurred (Kemble, G. W., T. Danieli, and J. M. White. 1994. Cell. 76:383-391). We extended the evidence for hemifusion by labeling the inner monolayer leaflets of RBCs with FM4-64 and observing that these inner leaflets did not become continuous with GPI-HA-expressing cells. The region of hemifusion-separated aqueous contents, the hemifusion diaphragm, appeared to be extended and was long-lived. But when RBCs hemifused to GPI-HA-expressing cells were osmotically swollen, some diaphragms were disrupted, and spread of both inner leaflet and aqueous dyes was observed. This was characteristic of full fusion: inner leaflet and aqueous probes spread to cells expressing wild-type HA (wt-HA). By simultaneous video fluorescence microscopy and time-resolved electrical admittance measurements, we rigorously demonstrated that GPI-HA-expressing cells hemifuse to planar bilayer membranes: lipid continuity was established without formation of fusion pores. The hemifusion area became large. In contrast, for cells expressing wt-HA, before lipid dye spread, fusion pores were always observed, establishing that full fusion occurred. We present an elastic coupling model in which the ectodomain of wt-HA induces hemifusion and the transmembrane domain, absent in the GPI-HA-expressing cells, mediates full fusion.

The properties of a cloned human high-molecular-mass cytosolic phospholipase A2 investigated using a continuous fluorescence displacement assay: evidence for enzyme clustering on phospholipid vesicles

The 85 kDa human cytosolic phospholipase A2 has been cloned and expressed in insect Sf21 cells. The pure enzyme has been investigated using a fluorescence displacement assay that provides a continuous record of phospholipid hydrolysis [Wilton (1990) Biochem. J. 266, 435-439]. The unusual kinetic properties of this enzyme, previously described using radioactive assays, were readily demonstrated using the continuous fluorescence assay and were examined in detail. It is proposed that the enzyme clusters on the surface of a fixed number of substrate vesicles during the initial stages of catalysis and that the characteristic burst phase of hydrolysis represents the hydrolysis of these vesicles. This clustering produced a molar ratio of total phospholipid substrate to enzyme of about 450:1 at vesicle saturation with enzyme. Under limiting substrate conditions, the lower secondary rate that is observed results eventually in almost complete hydrolysis of the phospholipid; this was confirmed using radioactive substrate. Evidence is presented that during the initial burst phase, equivalent to hydrolysis of the outer monolayer of the vesicle, the enzyme remains tightly bound but is released as the reaction proceeds towards complete hydrolysis of the phospholipid substrate. In the presence of excess substrate, about 370 mol of fatty acid are released per mol of enzyme during the burst phase and it is calculated that this value also approximates to hydrolysis of the outer monolayer of the vesicle. It is proposed that the formation of a stable enzyme-vesicle complex during the burst phase of phospholipid hydrolysis may be due, at least in part, to protein-protein interactions between adjacent enzyme molecules in order to account for the clustering phenomenon.

On the distribution of a fluorescent ivermectin probe (4″ 5,7 dimethyl-bodipy proprionylivermectin) in Ascaris membranes

The distribution of the fluorescent ivermectin derivative (4″ 5,7 dimethyl bodipy proprionylivermectin, referred to here as bodipy ivermectin) was studied in muscle vesicle membranes from the parasite Ascaris suum. Incubation in bodipy ivermectin, at concentrations greater than 1 ng/ml, produced a dose-dependent fluorescence in the vesicles. The level of fluorescence was not depressed (but increased) by prior incubation with 1 μM unlabelled ivermectin. This observation suggested that most of the bodipy ivermectin was free in the lipid phase of the membrane and not associated with a receptor. The trypan blue quenching technique showed that the bodipy ivermectin did not cross the membrane bilayer but remained in the outer monolayer. Lateral mobility of the probe in the membrane was studied using the fluorescence recovery after photobleaching (FRAP) technique. The lateral diffusion coefficient (DL) was 14.0 x 10−10 cm2/s which was nearly three times faster than the lipid probe octadecanoyl-aminofluorescein in membranes from the same preparations; only 46% of the probe was mobile, showing that within a 1.2 μm radius the distribution of the bodipy ivermectin in the membrane was not homogeneous. These observations are used to suggest that: the site of action of livermectin is located in the outer monolayer of the membrane (possibly on the Cl− channel); and that the speed of onset of action in isolated membrane preparations may be limited by diffusion in the membrane.