External Leaflet Research Articles

Protein Kinase Cα and P-Type Ca2+ Channel CaV2.1 in Red Blood Cell Calcium Signalling

Background/Aims: Protein kinase Cα (PKCα) is activated by an increase in cytosolic Ca²⁺ in red blood cells (RBCs). Previous work has suggested that PKCα directly stimulates the Ca_V2.1 channel, whereas other studies revealed that Ca_V2.1 is insensitive to activation by PKC. The aim of this study was to resolve this discrepancy. Methods: We performed experiments based on a single cell read-out of the intracellular Ca²⁺ concentration in terms of Fluo-4 fluorescence intensity and phosphatidylserine exposure to the external membrane leaflet. Measurement modalities included flow cytometry and live cell imaging. Results: Treatment of RBCs with phorbol 12-myristate 13-acetate (PMA) led to two distinct populations of cells with an increase in intracellular Ca²⁺: a weak-responding and a strong-responding population. The EC₅₀ of PMA for the number of cells with Ca²⁺ elevation was 2.7±1.2 µM; for phosphatidylserine exposure to the external membrane surface, it was 2.8±0.5 µM; and for RBC haemolysis, it was 2.9±0.5 µM. Using pharmacological manipulation with the Ca_V2.1 inhibitor ω-agatoxin TK and the broad protein kinase C inhibitor Gö6983, we are able to show that there are two independent PMA-activated Ca²⁺ entry processes: the first is independent of Ca_V2.1 and directly PKCα-activated, while the second is associated with a likely indirect activation of Ca_V2.1. Further studies using lysophosphatidic acid (LPA) as a stimulation agent have provided additional evidence that PKCα and Ca_V2.1 are not directly interconnected in a signalling chain. Conclusion: Although we provide evidence for a lack of interaction between PKCα and Ca_V2.1 in RBCs, further studies are required to decipher the signalling relationship between LPA, PKCα and Ca_V2.1.

Langmuir Monolayers of Bacterial Outer Membranes

The increase of Gram-negative bacteria super-resistant to antibiotics is a major problem in medicine. These bacteria possess a double membrane system, with the external leaflet of the outer membrane composed of lipopolyssaccharides (LPS) that act as the first barrier to drugs. Thus, bacteria's susceptibility to lipophilic antibiotics is highly correlated with the permeability of this LPS barrier. In this study, we extracted LPS from two isogenic Salmonella sp strains, using the method of Galanos. These strains contain alterations in the lipid A portion of their LPS regulated by the PhoPQ-system. These modifications include palmitoylation of the 3-hydroxyl group in the 3-OH-myristyl residue, addition of 4-aminoarabinose to the 4' phosphate group and addition of a 2-hydroxy group to the myristate residue at the position 3'. Langmuir monolayers were formed of both LPS and compression isotherms indicated that PhoP- (LPS from the strain with no modifications) occupies a limiting area of 150A2, while for PhoPc (LPS from the strain with the modifications cited above) this value is 175A2. These are in agreement with previous report for other Salmonella sp LPS. Epifluorescence microscopy reveals PhoP- has a very homogeneous, fluid-like, morphology throughout the compression without discrete domain formation. In contrast, PhoPc displays circular-shaped domains that increase in size and number with the compression. The addition of novobiocin (0.4 μg mL-1) to the subphase promoted slight changes in PhoP- morphology, suggesting that this monolayer was already fluid enough to incorporate this antibiotic. For PhoPc monolayer, changes in morphology were observed only for high concentrations (2.5x) of novobiocin at which the domains started to aggregate generating structures like a “pearl necklace”.

Mc Model of Lipid Raft Protein Diffusion Matched to Live Cell Measurements with Controlled Chemical Perturbation Experiments

Partitioning of certain type extra-cellular or cytosolic leaflet membrane proteins into functioning cholesterol stabilized domains within both leaflets of the membrane structure has long been shown by various studies facilitating different techniques. We have modeled how lipid-stabilized nano-domains influence the diffusion of some membrane proteins. By combining these simulations with non-perturbative experimental measurements of the diffusion behavior for chemically unperturbed and controlled perturbation cases of that diffusion, we were able to quantify the essential parameters describing the domains. The simulations feature four independent parameters, i.e. area fraction of the membrane occupied by domains, size of the domains, probability of the proteins to exit the domains, and effective protein diffusion within the domains.Kinetic Monte-Carlo modeling of proteins' association with membrane nano-domains has been performed over a selected region of the parameter space. Some of these parameters may be modulated at the single cell level during the time of a single experiment, allowing precise matching of all four parameters. We have used our non-perturbative imaging based FCS, bimFCS, to measure the diffusion of several membrane proteins from inner and outer leaflets of the cell-membrane over multiple length scales simultaneously. This allows extracting information of the domains which transiently trap the diffusing proteins.We studied GFP-tagged GPI anchored proteins for the external leaflet and compared measurements of monomers and induced dimers on the same cells. For the intracellular leaflet, we studied Lyn-anchored GFP and used the Rapamycin induced cross-linking of the FKBP12 and Frb domains. We were able to successfully match experimental data with in-silico modeling by Kinetic Monte Carlo simulations, giving insight about sizes and area fraction of raft domains for each leaflet of the membrane, and also affinity of associated proteins with them.

Identification of lysosomal sialidase NEU1 and plasma membrane sialidase NEU3 in human erythrocytes

The sialylation level of molecules, sialoglycoproteins and gangliosides, protruding from plasma membranes regulates multiple facets of erythrocyte function, from interaction with endothelium to cell lifespan. Our results demonstrate that: (a) Both sialidases NEU1 and NEU3 are present on erythrocyte plasma membrane; (b) NEU1 is kept on the plasma membrane in absence of the protective protein/cathepsin A (PPCA); (c) NEU1 and NEU3 are retained on the plasma membrane, as peripheral proteins, associated to the external leaflet and released by alkaline treatments; (d) NEU1 and NEU3 are segregated in Triton X-100 detergent-resistant membrane domains (DRMs); (e) NEU3 shows activity also at neutral pH; and (f) NEU1 and NEU3 are progressively lost during erythrocyte life. Interestingly, sialidase activity released from erythrocyte membranes after an alkaline treatment preserves its functionality and recognizes sialoglycoproteins and gangliosides. On the other hand, the weak anchorage of sialidases to the plasma membrane and their loss during erythrocyte life could be a tool to preserve the cellular sialic acid content in order to avoid the early ageing of erythrocyte and processes of cell aggregation in the capillaries.

Echogenicity and release characteristics of folate-conjugated echogenic liposomes for cytosolic delivery of cancer drugs

Echogenic liposomes (ELIPs) are specially prepared liposomes that encapsulate both aqueous and gaseous phases. The presence of gas makes them echogenic. Since, ELIPs retain all the favorable properties of normal liposomes they can be used for simultaneous ultrasonic imaging and drug delivery applications. These liposomes are polymerized on the external leaflet using a disulphide linker. Disulphide bonds are reversibly broken in presence of thiol above a critical concentration. Therefore, the liposomes are stable in the plasma (thiol concentration 10 μM) but release its content inside the cell (thiol concentration 10 mM). The liposome also expresses folate group on its surface which allows its entry into the cancer cells. The release can be controlled by diagnostic frequency ultrasound. Therefore, these ELIPs hold promises for ultrasound image-guided cytosolic delivery for cancer drugs. We will report on their acoustic properties and ultrasound-mediated release characteristics. Their implications on design and development of these novel contrast agents will be discussed. [Supported by NSF CBET-0651912, CBET-1033256, DMR-1005283.]

Chemical and physical requirements for lipid extraction by bovine binder of sperm BSP1

The bovine seminal plasma contains phosphocholine-binding proteins, which associate to sperm membranes upon ejaculation. These binder-of-sperm (BSP) proteins then induce a phospholipid and cholesterol efflux from these membranes. In this work, we determined physical and chemical parameters controlling this efflux by characterizing the lipid extraction induced by BSP1, the most abundant of BSP protein in bull seminal plasma, from model membranes with different composition. The model membranes were formed from binary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (Lyso-PC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) or cholesterol. The modulation of BSP1-induced lipid extraction from membranes by their chemical composition and their physical properties brings us to propose a 3-step extraction mechanism. First, the protein associates with membranes via specific binding to phosphocholine groups. Second, BSP1 penetrates in the membrane, essentially in the external lipid leaflet. Third, BSP1 molecules solubilize a lipid patch coming essentially from the outer lipid leaflet, without any lipid specificity, to ultimately form small lipid/protein auto-assemblies. The stoichiometry of these complexes corresponds to 10–15 lipids per protein. It is also shown that fluid-phase membranes are more prone to BSP1-induced lipid extraction than gel-phase ones. The inhibition of the lipid extraction in this case appears to be related to the inhibition of the protein penetration in the membrane (step 2) and not to the protein association with PC head groups (step 1). These findings contribute to our understanding of the mechanism by which BSP1 modify the lipid composition of sperm membranes, a key event in sperm capacitation.

Phosphatidylserine exposure during apoptosis reflects bidirectional trafficking between plasma membrane and cytoplasm

Phosphatidylserine (PS) exposure on the external leaflet of the plasma membrane is widely observed during apoptosis and forms the basis for the annexin V binding assay to detect apoptotic cell death. Current efforts to explain PS exposure focus on two potential mechanisms, activation of a phospholipid scramblase or calcium-mediated trafficking of lysosomes to the cell surface. Here, we provide evidence that apoptotic PS exposure instead reflects bidirectional trafficking of membrane between the cell surface and cytoplasm. Using a series of cell lines, some of which expose large amounts of PS during apoptosis and some of which do not, we demonstrate that accumulation of plasma membrane-derived cytoplasmic vesicles in a dynamin-, clathrin- and Cdc42-independent manner is a previously undescribed but widely occurring feature of apoptosis. The apoptotic exposure of PS occurs when these vesicles traffic back to cell surface in a calcium-dependent process that is deficient in a substantial fraction of human cancer cell lines. These observations provide a new model for PS externalization during apoptosis and simultaneously identify an altered step that accounts for the paucity of apoptotic PS exposure in many cell lines.

Shedding of Phosphatidylserine from Developing Erythroid Cells Involves Microtubule Depolymerization and Affects Membrane Lipid Composition

Phosphatidylserine (PS), which is normally localized in the cytoplasmic leaflet of the membrane, flip-flops to the external leaflet during aging of, or trauma to, cells. A fraction of this PS undergoes shedding into the extracellular milieu. PS externalization and shedding change during maturation of erythroid cells and affect the functioning, senescence and elimination of mature RBCs. Several lines of evidence suggest dependence of PS shedding on intracellular Ca concentration as well as on interaction between plasma membrane phospholipids and microtubules (MTs), the key components of the cytoskeleton. We investigated the effect of Ca flux and MT assembly on the distribution of PS across, and shedding from, the membranes of erythroid precursors. Cultured human and murine erythroid precursors were treated with the Ca ionophore A23187, the MT assembly enhancer paclitaxel (Taxol) or the inhibitor colchicine. PS externalization and shedding were measured by flow cytometry and the cholesterol/phospholipids in RBC membranes and supernatants, by ¹H-NMR. We found that treatment with Taxol or colchicine resulted in a marked increase in PS externalization, while shedding was increased by colchicine but inhibited by Taxol. These results indicate that PS externalization is mediated by Ca flux, and PS shedding by both Ca flux and MT assembly. The cholesterol/phospholipid ratio in the membrane is modified by PS shedding; we now show that it was increased by colchicine and A23187, while taxol had no effect. In summary, the results indicate that the Ca flux and MT depolymerization of erythroid precursors mediate their PS externalization and shedding, which in turn changes their membrane composition.

Kinetics of the enzyme–vesicle interaction including the formation of rafts and membrane strain

In cells, an appreciable part of enzymes is associated with lipid membranes. Academic experimental studies of the function of membrane enzymes (e.g., PLA2 representing a prototype for interfacial enzymology) are often focused on the enzyme–vesicle interaction or, more specifically, on conversion of lipid forming the external leaflet of the vesicle membrane. The corresponding kinetics are complicated by many factors inherent to the interfacial physics and chemistry. The understanding of the relative role of such factors and how they should be quantitatively described is still limited. Here, we present the mean-field kinetic equations, taking the formation of rafts in the membrane and the product-induced membrane strain into account, and analyze various scenarios of lipid conversion. In particular, we scrutinize the conditions when the kinetics may exhibit a transition from a relatively long latency period to a steady-state regime with fast nearly constant reaction rate. Specifically, we discuss the likely role of the pore formation in the external lipid layer in this transition. The latter effect may be caused by the product-induced tensile strain in this layer.

Imaging Molecular Transport Across Lipid Bilayers

The passive transport of small molecules across the plasma membrane is a key physiological process. Literature measurements of membrane permeability to small molecules have varied widely. We used confocal microscopy to image the transport of molecules into a giant unilamellar lipid vesicle (GUV). Fluorescent dyes were used to trace the transport of molecules. The GUV was immobilized on the surface of a microfluidic channel by biotin-avidin binding. This microchannel allows the rapid and uniform exchange of the solution surrounding the GUV. Using a spinning-disk confocal microscope, the entire concentration field is captured in a short exposure. We used this system to study the passive transport of carboxylic acids, which have many properties common to small-molecule drugs. The transport of these acids across cell membranes has been widely studied, but there is much variation in the reported permeabilities. By using pH-sensitive fluorescein-dextran to track the acids permeating through the GUV membrane, our results showed that more lipophilic acids cross the bilayer more quickly. A finite difference model was developed to simulate the experimental process and derive precise permeability values. The permeabilities change with the same trend as oil-water partition coefficients, demonstrating that Overton's rule applies to this class of molecules. We used the imaging technique described above to study the transport of protons across compositionally asymmetric lipid bilayers. Synthetic asymmetric GUVs were prepared via a microfluidic multiphase droplet flow technology to mimic membrane charge asymmetry. Negatively charged phosphatidylserine was added to an asolectin GUV on either the internal or external leaflet. The permeation rates of protons into and out of these GUVs were measured. The proton distribution across the asymmetric GUV membrane at equilibrium was also studied. This research can reveal how asymmetric cell membrane composition affects small molecule transport behavior in physiological processes.

Apoptotic effect of imatinib on human colon adenocarcinoma cells: Influence on actin cytoskeleton organization and cell migration

Imatinib mesylate (STI571) is the first member of a new class of agents that act by inhibiting specific tyrosine kinases, rather than killing all rapidly dividing cells. This drug is usually used in the treatment of chronic myelogenous leukemia and gastrointestinal stromal tumors. It was recognized to inhibit activity of kinases such as Bcr/Abl, platelet-derived growth factor receptor, and c-kit. These proteins play important roles in cell growth, motility, and survival. Therefore, studies on the biological effects of imatinib on different cellular models are very important. Human colon adenocarcinoma LS180 cell line was used in the studies presented. Cells were exposed to 0.1–100 μM imatinib for 24 and 48 h. Dose-dependent decreases in cell viability and morphological changes were observed. Moreover, the apoptotic effect of imatinib (10 μM, 50 μM) after 24 h of exposure was demonstrated as evaluated by translocation of phosphatidylserine to external membrane leaflet and by increased activity of caspase-3. Special attention was focused on imatinib influence on actin cytoskeleton organization and migration ability of LS180 cells. Distinct alterations in actin cytoskeleton architecture occurred in response to drug treatment, accompanied by appearance of filamentous actin aggregates and decrease in actin polymerization state. These changes were correlated with remarkable decrease in cell migration capacity. In summary, our data clearly demonstrate that imatinib induces apoptosis and inhibits human colon adenocarcinoma cell migration. Therefore, this drug may have potential in colon cancer therapy in the future.

Modifications in plasma membrane lipid composition and morphological features of AH-130 hepatoma cells by polyenylphosphatidylcholine in vivo treatment

The plasma membrane lipid composition in AH-130 hepatoma cells was found to change remarkably after polyenylphosphatidylcholine (PPC) treatment. Plasma membranes from cells grown in rats treated for 7 days i.v. with 20mg/kg/day PPC, when compared to those of control cells, did not show significantly different amounts of cholesterol or phospholipids relative to protein content, but, surprisingly, the individual phospholipid distribution inside the two membrane leaflets changed dramatically. Phosphatidylcholine (PC), the major phospholipid in the external membrane leaflet, increased ~47% (p<0.001). By contrast, phosphatidylethanolamine (PE), the most important component of the inner leaflet, decreased nearly 37% (p<0.001), while sphingomyelin (SM) also decreased ~17%, (p=0.1). Tumor cells collected from control rats at the same time interval and observed by scanning electron microscopy, exhibited a spherical shape with numerous and randomly distributed long microvilli, the same morphological and ultrastructural features displayed by the implanted cells. Conversely, tumor cells from PPC-treated rats no longer showed the roundish cell profile, and microvilli appeared shortened and enlarged, with the formation of surface blebs. Transmission electron microscopy observations confirmed the morphological and ultrastructural cell changes, mainly seen as loss of microvilli and intense cytoplasmic vacuolization. Taken together, these results indicate that the new phospholipid class distribution in the plasma membrane leaflets, modifying tumor cell viable structures, produced heavy cell damage and in many cases brought about complete cellular disintegration.

Binding of Chitosan to Phospholipid Vesicles Studied with Isothermal Titration Calorimetry

We thermodynamically characterize the interaction of chitosan with small liposomes and the binding and organization of the polysaccharide on the membrane of the vesicles. By means of isothermal titration calorimetry (ITC), we obtain the enthalpy variations arising from binding of the positively ionized chitosan to neutral and negatively charged liposomes. The strong electrostatic interaction of the polysaccharide with the negative charges at the membrane gives rise to highly exothermic signal until charge compensation is reached. The equilibrium constant, the interaction stoichiometry, and the molar enthalpy of binding chitosan monomers to phospholipids from the external leaflet of the vesicle membrane are obtained from the isotherm curve fitting assuming independent binding sites. The strong exothermic signal indicates that the electrostatically driven binding of chitosan to the membrane is energetically favored, leading to further stabilization of the vesicle suspension. The higher the net negative charge of the vesicles, the more pronounced the adsorption of chitosan is, leading to weaker chain organization of the adsorbed chitosan at the membrane. At the point of charge saturation, vesicle aggregation takes place and we show that this behavior does not always lead to charge reversal at the membrane. Models for the binding behavior and structural organization of chitosan are proposed based on the experimental results from ITC, ζ-potential, and dynamic light scattering.

Effect of Hydrophobic Surfactant Proteins SP-B and SP-C on the Permeability of Phospholipid Membranes

Pulmonary surfactant is a complex mixture of lipids and proteins whose main function is to reduce surface tension at the alveolar air-liquid interface in order to avoid alveolar collapse at the end of expiration and facilitate the work of breathing. It is composed by around 90% lipids and 8-10% specific proteins, including the hydrophobic SP-B and SP-C. In this study, we have analyzed the effect of hydrophobic surfactant proteins on the permeability of phospholipid membranes by two different approaches: fluorescence microscopy of giant vesicles (GV) and electroconduction in planar lipid membranes. The effect of surfactant proteins on the permeability of GV membranes was assessed under the microscope using the fluorescent water-soluble probes FM®1-43 and calcein. Membrane-sensitive FM®1-43 only labels the external leaflet of membranes, and calcein emits green fluorescence in aqueous media. Neither can permeate through pure lipid membranes. In the presence of physiological amounts of SP-B and SP-C, giant oligolamellar POPC vesicles incorporated FM®1-43 in every single membrane when added to the external medium and were also permeable to calcein. These results suggest the existence of direct connections between aqueous compartments of GV in the presence of these proteins. On the other hand, planar lipid membranes (PLM) have been widely used to study ionic permeation through phospholipid bilayers mediated by membrane proteins. Permeability of bilayers incorporating small amounts of hydrophobic surfactant proteins has been analyzed in PLMs prepared by the dual monolayer technique. Conductance and selectivity experiments as well as noise analysis of the signals were performed. All our measurements indicate the formation of channel-like structures with defined properties in terms of ionic conductance and selectivity. Possible implications of membrane-permeabilizing structures in the biological context of the pulmonary surfactant system will be discussed.

Nanotubes from asymmetrically decorated vesicles

Hydrodynamic nanotube extrusion is used to characterize chitosan-decorated vesicles, which are more robust to pH and salt shocks and exhibit specific behavior under osmotic pressure if compared to their bare homologues. The vesicle attached to a micro-rod is submitted to a flow. Above a threshold velocity, we observe the extrusion of a lipidic nanotube. We study how it grows and relaxes when the flow is stopped. We find that extrusion forces for decorated vesicles are weaker than for bare vesicles. We interpret these results using a model that introduces the spontaneous curvature due to asymmetric adsorption of chitosan on the external leaflet of the bilayer, which allows us to calculate the stationary length of the tube versus the flow velocity and to estimate the spontaneous curvature c0.

Beta2-microglobulin causes abnormal phosphatidylserine exposure in human red blood cells

The exposure of the aminophospholipid phosphatidylserine on the external leaflet of red blood cell plasma membrane can have several pathophysiological consequences with particular regard to the processes of cell phagocytosis, haemostasis and cell-cell interaction. A significant increase in phosphatidylserine-exposing erythrocytes has been reported in chronic haemodialysis patients and found to be strongly influenced by the uraemic milieu. To identify uraemic compound(s) enhancing phosphatidylserine externalization in erythrocytes, we fractionated by chromatographic methods the ultrafiltrate obtained during dialysis, and examined by flow cytometry the effect of the resulting fractions on phosphatidylserine exposure in human red cells. Chromatographic procedures disclosed a homogeneous fraction able to increase erythrocyte phosphatidylserine exposure. The inducer of such externalization was identified by monodimensional gel electrophoresis and mass spectrometry investigations as beta2-microglobulin. To confirm the beta2-microglobulin effect and to examine the influence of protein glycation (as it occurs in uraemia) on phosphatidylserine erythrocyte exposure, erythrocytes from normal subjects were incubated with recombinant beta2-microglobulin (showing no glycation sites at mass analysis), commercial beta2-microglobulin (8 glycation sites), or with in vitro glycated recombinant beta2-microglobulin (showing multiple glycation sites). Elevated concentrations of beta2-microglobulin (corresponding to plasma levels reached in dialysis patients) increased slightly but significantly the protein's ability to externalize phosphatidylserine on human erythrocytes. Such an effect was markedly enhanced by glycated forms of the protein. Beta2-microglobulin is recognized as a surrogate marker of middle-molecule uraemic toxins and represents a key component of dialysis-associated amyloidosis. Our study adds further evidence to the potential pathophysiologic consequences of beta2-microglobulin accumulation in chronic uraemic patients.

Transbilayer phospholipids molecular imaging

Nuclear medicine has become a key part of molecular imaging. In the present review article, we focus on the transbilayer phospholipids as exquisite targets for radiolabelled probes in molecular imaging. Asymmetry of phospholipid distribution is a characteristic of mammalian cell membranes. Phosphatidylcholine and sphyngomyelin cholinophospholipids are primarily located within the external leaflet of the cell membrane. Phosphatidylserine and phosphatidylethanolamine aminophospholipids, and also phosphatidylinositol are primarily located within the internal leaflet of the cell membrane. New radiolabelled tracers have been designed in preclinical and clinical research for PET-CT and SPECT-CT molecular imaging of transbilayer phospholipids.

Focal Phosphatidylserine Exposure on Stressed Endothelial Cells In Relation to Membrane Topology: A Potential Basis for Compartmentalization of Procoagulant and Anticoagulant Activities

AbstractAbstract 4321Exposure of endothelial cells to toxins or deprivation of nutrients leads to cell stress and, subsequently, to apoptosis. Apoptosis is associated with gross translocation of phosphatidylserine (PS) to the external membrane leaflet and exposed PS is required for coagulation enzyme complexes as well as some anticoagulant activities. However, in vivo studies of mouse thrombosis do not show apoptotic cells or gross PS exposure associated with clot formation, thus suggesting that PS supporting thrombosis is primarily derived from viable cells with limited PS exposure rather than apoptotic cells. In preliminary studies, we have found that cell stress, on multiple cell types, leads to limited, focal PS exposure before the onset of apoptosis. In this study, we have asked about the distribution of focal, PS-containing micro-domains on stressed endothelial cells. To induce stress we exposed Human Umbilical Vein Endothelial Cells (HUVEC) to 0.5 μM staurosporine for 15 minutes. PS exposure was detected by simultaneous staining using 10 nM lactadherin–Alexa 488 and annexin V–Cy 3.18, both exhibiting high affinity for PS. Stressed cells partially withdrew from their prior borders, leaving residual fibrils connected to original attachment points. In addition, they extended new thin fibrils that were up to several cell diameters in length. Confocal microscopy demonstrated focal staining, with little overlap between regions stained by lactadherin and regions stained by annexin V. Lactadherin stained the new thin fibrils, stretches of the cell margin, and patches near the nucleus. In contrast, annexin V stained in a reticular pattern in the perinuclear region. Based on the previously demonstrated finding, that lactadherin has a preference for highly curved vesicles and annexin V for flat or concave surfaces, we asked whether the selective binding might be determined by the membrane topology. To mimic the curvature of a cell membrane we prepared nano-fabricated silica substrates with ridges of 10 nm and 30 nm radii of curvature. The AFM topographic and fluorescent images of synthetic membrane bilayers supported by the substrates showed that, over a PS content of 4–15%, lactadherin preferentially binds to the convex nano-ridges with a ridge: valley staining ratio of approx. 280:1, while annexin V selectively binds the concave areas of the nano-trenches with a ridge: valley staining ratio of approx. 1:110. Combined fluorescence/AFM imaging of stressed HUVEC, demonstrated that the new thin filaments staining with lactadherin had radii of curvature of approx.12 nm, which was similar to the ridges of our synthetic bilayers. The reticular staining of the perinuclear region by annexin V correlated to concavities between nodules on the cell, suggesting that the selective staining is due, in part, to membrane topology. We are currently assessing the focal binding of procoagulant and anticoagulant proteins to the stressed endothelial cells. Because, factor V and factor VIII prefer the convex surfaces recognized by lactadherin we predict that procoagulant enzyme complexes will localize to these topographic features. Annexin II, which serves as a receptor for tPA, plasmin, and factor X, is predicted to prefer concavities, similar to annexin V. This could provide a basis for separate compartments of procoagulant and anticoagulant or fibrinolytic activity on the same cell. Disclosures:No relevant conflicts of interest to declare.

Neobiosynthesis of Glycosphingolipids by Plasma Membrane-associated Glycosyltransferases*[S

Gangliosides, complex glycosphingolipids containing sialic acids, are synthesized in the endoplasmic reticulum and in the Golgi complex. These neobiosynthesized gangliosides move via vesicular transport to the plasma membrane, becoming components of the external leaflet. Gangliosides can undergo endocytosis followed by recycling to the cell surface or sorting to the Golgi complex or lysosomes for remodeling and catabolism. Recently, glycosphingolipid catabolic enzymes (glycohydrolases) have been found to be associated with the plasma membrane, where they display activity on the membrane components. In this work, we demonstrated that ecto-ganglioside glycosyltransferases may catalyze ganglioside synthesis outside the Golgi compartment, particularly at the cell surface. Specifically, we report the first direct evidence of expression and activity of CMP-NeuAc:GM3 sialyltransferase (Sial-T2) at the cell surface of epithelial and melanoma cells, with membrane-integrated ecto-Sial-T2 being able to sialylate endogenously synthesized GM3 ganglioside as well as exogenously incorporated substrate. Interestingly, we also showed that ecto-Sial-T2 was able to synthesize GD3 ganglioside at the cell surface using the endogenously synthesized cytidine monophospho-N-acetylneuraminic acid (CMP-NeuAc) available at the extracellular milieu. In addition, the expression of UDP-GalNAc:LacCer/GM3/GD3 N-acetylgalactosaminyltransferase (GalNAc-T) was also detected at the cell surface of epithelial cells, whose catalytic activity was only observed after feeding the cells with exogenous GM3 substrate. Thus, the relative interplay between the plasma membrane-associated glycosyltransferase and glycohydrolase activities, even when acting on a common substrate, emerges as a potential level of regulation of the local glycosphingolipid composition in response to different external and internal stimuli.

An intimate liaison: spatial organization of the endoplasmic reticulum–mitochondria relationship

Organelle localization is often crucial to properly modulate cellular functions and signalling cascades. For example, the distribution of organelles in axons is crucial for their function and is dysregulated in several diseases. Similarly, relative positioning of two or more organelles is also important to perform certain specialized processes. Perhaps, the best-known form of interorganellar organization is that between endoplasmic reticulum (ER) and mitochondria. Close communication between these two compartments has been observed for a long time. Recent evidence suggests that this is the basis for a bidirectional communication regulating a number of physiological processes ranging from mitochondrial energy and lipid metabolism to Ca(2+) signalling and cell death. The recent discovery of some of the molecular mediators of the tethering already allowed to extend the function of this paradigmatic spatial organization to previously unexpected functions, and will foster future research to explore it in cellular signalling cascades as well as in disease.