Lucyna Mrówczyńska

Curvature-dependent lateral distribution of raft markers in the human erythrocyte membrane.

The distribution of raft markers in curved membrane exvaginations and invaginations, induced in human erythrocytes by amphiphile-treatment or increased cytosolic calcium level, was studied by fluorescence microscopy. Cholera toxin subunit B and antibodies were used to detect raft components. Ganglioside GM1 was enriched in membrane exvaginations (spiculae) induced by cytosolic calcium and amphiphiles. Stomatin and the cytosolic proteins synexin and sorcin were enriched in spiculae when induced by cytosolic calcium, but not in spiculae induced by amphiphiles. No enrichment of flotillin-1 was detected in spiculae. Analyses of the relative protein content of released exovesicles were in line with the microscopic observations. In invaginations induced by amphiphiles, the enrichment of ganglioside GM1, but not of the integral membrane proteins flotillin-1 and stomatin, was observed. Based on the experimental results and theoretical considerations we suggest that membrane skeleton-detached, laterally mobile rafts may sort into curved or flat membrane regions dependent on their intrinsic molecular shape and/or direct interactions between the raft elements.

Flavonoids as Inhibitors of MRP1-like Efflux Activity in Human Erythrocytes.A Structure-Activity Relationship Study

The potency of flavonoids (isoflavones, flavones, and flavanones) to inhibit efflux of 2,7-bis-(carboxypropyl)-5(6)-carboxyfluorescein (BCPCF) from human erythrocytes was investigated. Structure-activity relationship analysis showed that the strongest inhibitors were found among flavanones bearing a hydrophobic prenyl, geranyl, or lavandulyl group at position 8 (and hydroxyl groups at 5 and 7) in ring A. A prenyl group at position 5 or stilbene at positions 4-5 in ring B further seemed to increase inhibitor potency. The most efficient flavanones, euchrestaflavanone A and sophoraflavanone H, were approximately 20 times more efficient than genistein, and induced 50% inhibition of BCPCF efflux (IC50) at 3 microM (60 min, 37 degrees C). This is comparable to IC50 of benzbromarone (4 microM) and lower than IC50 of indomethacin (10 microM), both known MRP1 (ABCC1) inhibitors. It is suggested that BCPCF efflux is mainly due to MRP1 activity. Our results indicate that flavonoid molecular structure provides a promising base for development of potent MRP1 inhibitors.

Platelet-activating factor interaction with the human erythrocyte membrane.

Platelet‐activating factor (PAF) is a soluble signal messenger present in blood at nanomolar concentration. PAF has a wide spectrum of biological activities and is produced by and effective in different cell types. Owing to its important physiological role, we wanted to characterize membrane intercalation and interaction of PAF‐16 (1‐O‐hexadecyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine) by studying its capacity to induce during short‐term incubations at high concentrations cell shape alterations, phosphatidylserine exposure, and hemolysis in human erythrocytes. Our results showed that PAF‐16 at micromolar concentrations rapidly (≤1 min) induces stable but wash‐sensitive echinocytosis and hemolysis, but no substantial phosphatidylserine exposure. In conclusion, our study characterizes PAF‐16 as a highly membrane partitioning non‐permeable molecule accumulating in the outer membrane leaflet. These membrane interacting properties of PAF should, also at physiological concentrations, be important part of its nature as a membrane affector molecule.

Patching of gangliosideM1 in human erythrocytes distribution of CD47 and CD59 in patched and curved membrane

Membrane rafts may act as platforms for membrane protein signalling. Rafts have also been implicated in the sorting of membrane components during membrane budding. We have studied by fluorescence microscopy cross-linking of ganglioside GM1 in the human erythrocyte membrane, and how membrane proteins CD47 and CD59 distribute in GM1 patched discoid cells and calcium-induced echinocytic cells. Patching of gangliosideM1 (GM1) by cholera toxin subunit B (CTB) plus anti-CTB resulted in the formation of usually 40–60 GM1 patches distributed over the membrane in discoid erythrocytes. Pre-treatment of erythrocytes with methyl-β-cyclodextrin abolished GM1 patching. GM1 patching was insensitive to pre-fixation (paraformaldehyde) of cells. Patching of GM1 did not affect the discoid shape of erythrocytes. Membrane proteins CD47 and CD59 did not accumulate into GM1 patches. No capping of patches occurred. GM1 accumulated in calcium-induced echinocytic spiculae. Also CD59, but not CD47, accumulated in spiculae. However, CD59 showed a low degree of co-localization with GM1 and frequently accumulated in different spiculae than GM1. In conclusion, our study describes a novel method for examining properties and composition of rafts. The study characterizes raft patching in the human erythrocyte membrane and emphasizes the mobility and ‘echinophilicity’ of GM1. Glycosyl phosphatidylinositol-anchored CD59 was identified as a mobile ‘echinophilic’ but ‘raftophobicGM1’ protein. Largely immobile CD47 showed no segregation.

Modulation of MRP1-like efflux activity in human erythrocytes caused by membrane perturbing agents.

The effect of membrane perturbing agents on the efflux (37°C, 60 min) of the fluorescent probe 2′, 7′-bis-(carboxypropyl)-5(6)-carboxyfluorescein (BCPCF) from human erythrocytes was studied. Several anionic amphiphiles (detergents) markedly inhibited BCPCF efflux (IC50≤⃒40 μM). Most zwitter-ionic amphiphiles were inefficient inhibitors. Non-ionic and cationic amphiphiles had minor effects or increased efflux. Of the aliphatic inhibitors, C12-homologues were the most efficient. Hexanol, ethanol, methyl-β-cyclodextrin (MβCD) and diamide (+ washing) did not influence BCPCF efflux. It is suggested that amphiphiles affect BCPCF efflux by modulating multi-drug resistance protein 1 (MRP1, ABCC1) activity. A negative charge of amphiphiles is essential for the inhibitory effect, while alkyl chain length modulates inhibition. MRP1-mediated BCPCF efflux appears to be relatively insensitive to non-specific plasma membrane modification.

Curvature factor and membrane solubilization, with particular reference to membrane rafts.

The composition of membrane rafts (cholesterol/sphingolipid‐rich domains) cannot be fully deduced from the analysis of a detergent‐resistant membrane fraction after solubilization in Triton X‐100 at 4°C. It is hypothesized that the membrane curvature‐dependent lateral distribution of membrane components affects their solubilization. The stomatocytogenic, Triton X‐100, cannot effectively solubilize membrane components, especially with regard to the outward membrane curvature.

Echinophilic proteins stomatin, sorcin, and synexin locate outside gangliosideM1 (GM1) patches in the erythrocyte membrane.

The detergent (Triton X-100, 4°C)-resistant membrane (DRM)-associated membrane proteins stomatin, sorcin, and synexin (anexin VII) exposed on the cytoplasmic side of membrane were investigated for their lateral distribution in relation to induced ganglioside(M1) (GM1) raft patches in flat (discocytic) and curved (echinocytic) human erythrocyte membrane. In discocytes, no accumulation of stomatin, sorcin, and synexin in cholera toxin subunit B (CTB) plus anti-CTB-induced GM1 patches was detected by fluorescence microscopy. In echinocytes, stomatin, sorcin, and synexin showed a similar curvature-dependent lateral distribution as GM1 patches by accumulating to spiculae induced by ionophore A23187 plus calcium. Stomatin was partly and synexin and sorcin were fully recruited to the spiculae. However, the DRM-associated proteins only partially co-localized with GM1 and were frequently distributed into different spiculae than GM1. The study indicates that stomatin, sorcin, and synexin are echinophilic membrane components that mainly locate outside GM1 rafts in the human erythrocyte membrane. Echinophilicity is suggested to contribute to the DRM association of a membrane component in general.

Spontaneous curvature of ganglioside GM1--effect of cross-linking.

The membrane-curvature dependent lateral distribution of outer leaflet ganglioside GM1 (GM1) and the influence of GM1 cross-linking induced by fluorophore-tagged cholera toxin subunit B (CTB) plus anti-CTB was analysed in cell membranes by fluorescence microscopy. Data are presented indicating that cross-linked GM1-ligand patches accumulated at the tips of human erythrocyte echinocytic spiculae induced by Ca(2+)/ionophore A23187. However, when lipid fixative osmium tetroxide was added prior to the ligand no accumulation in spiculae occurred. GM1-staining remained here distributed over the spheroid cell body and in spiculae. Similarly, osmium tetroxide completely prohibited CTB plus anti-CTB-induced GM1 patching in representatives for flat membrane, i.e. discoid erythrocytes and K562 cells. Our results demonstrate that GM1 per se shows low membrane curvature dependent distribution and therefore holds flexible spontaneous curvature. In contrast, the cross-linked GM1-ligand complex has a strong preference for highly outward curved membrane and possesses overall positive spontaneous curvature. Osmium tetroxide efficiently immobilises GM1.

Increased sensitivity of cholera toxin B treated K562 cells to natural killer cells

Cholera toxin B-subunit (CTB) treatment of K562 erythroleukemia cells increased their sensitivity to be killed by NK-92 cells with more than 10%, compared to untreated cells. A similar treatment of non-T, non-B acute lymphoblastic REH leukemia cells, known to be unsensitive to NK cell mediated cytotoxicity, did not have any impact at all. Visualization of the cross-linked ganglioside(M1) (GM(1)) using fluorescent labeled CTB, indicated accumulation of the fluorescence to one cap and a few smaller patches in both type of cells. Additional cross-linking using anti-CTB antibodies further accentuated capping and increased lysis in the case of K562 cells. Blocking experiments performed with anti-MICA/B, ULBP-2 and/or CD59 antibodies could not inhibit the increased sensitivity mediated by CTB.