Fuyuki Tokumasu

Nanoscopic Lipid Domain Dynamics Revealed by Atomic Force Microscopy

Intrinsic heterogeneities, represented as domain formations in biological membranes, are important to both the structure and function of the membranes. We observed domain formations in mixed lipid bilayers of dipalmitoylphosphatidylcholine (DPPC), dilauroylphosphatidylcholine (DLPC), and cholesterol (chol) in a fluid environment using an atomic force microscope (AFM). At room temperature, we demonstrated that both microscopic and nanoscopic domains coexist and the DPPC-rich domain is approximately 1.4 nm higher than the surrounding DLPC-rich membrane areas as a consequence of intrinsic phase differences. DPPC-rich microscopic domains became larger as DPPC concentration increased. In cholesterol-free mixtures, nanoscopic DPPC-rich domain sizes ranged from 26 to 46 nm depending on phospholipid concentration. Domain size varied between 33 and 48 nm in the presence of cholesterol (0 < or = [chol] < or = 40). The nanoscopic domains were markedly fragmented near [chol] = 0.135 and appeared to fuse more readily into microscopic domains at higher and lower [chol]. By phase balance analyses we demonstrated phase behavior differences between a free-vesicle GUV system studied by confocal light microscopy and a supported membrane system studied by AFM. We propose a new three-dimensional phase diagram elucidating the effects of a solid substrate support on lipid phase behavior relevant to complex membrane phase phenomena in biological systems.

Aegyptin, a Novel Mosquito Salivary Gland Protein, Specifically Binds to Collagen and Prevents Its Interaction with Platelet Glycoprotein VI, Integrin α2β1, and von Willebrand Factor

Blood-sucking arthropods have evolved a number of inhibitors of platelet aggregation and blood coagulation. In this study we have molecularly and functionally characterized aegyptin, a member of the family of 30-kDa salivary allergens from Aedes aegypti, whose function remained elusive thus far. Aegyptin displays a unique sequence characterized by glycine, glutamic acid, and aspartic acid repeats and was shown to specifically block collagen-induced human platelet aggregation and granule secretion. Plasmon resonance experiments demonstrate that aegyptin binds to collagen types I–V (Kd ≈ 1 nm) but does not interact with vitronectin, fibronectin, laminin, fibrinogen, and von Willebrand factor (vWf). In addition, aegyptin attenuates platelet adhesion to soluble or fibrillar collagen. Furthermore, aegyptin inhibits vWf interaction with collagen type III under static conditions and completely blocks platelet adhesion to collagen under flow conditions at high shear rates. Notably, aegyptin prevents collagen but not convulxin binding to recombinant glycoprotein VI. These findings suggest that aegyptin recognizes specific binding sites for glycoprotein VI, integrin α2β1, and vWf, thereby preventing collagen interaction with its three major ligands. Aegyptin is a novel tool to study collagen-platelet interaction and a prototype for development of molecules with antithrombotic properties.

Band 3 Modifications in Plasmodium Falciparum-Infected AA and CC Erythrocytes Assayed by Autocorrelation Analysis Using Quantum Dots

The molecular stability of hemoglobin is critical for normal erythrocyte functions, including oxygen transport. Hemoglobin C (HbC) is a mutant hemoglobin that has increased oxidative susceptibility due to an amino acid substitution (β6: Glu to Lys). The growth of Plasmodium falciparum is abnormal in homozygous CC erythrocytes in vitro, and CC individuals show innate protection against severe P. falciparum malaria. We investigated one possible mechanism of innate protection using a quantum dot technique to compare the distribution of host membrane band 3 molecules in genotypically normal (AA) to CC erythrocytes. The high photostability of quantum dots facilitated the construction of 3D cell images and the quantification of fluorescent signal intensity. Power spectra and 1D autocorrelation analyses showed band 3 clusters on the surface of infected AA and CC erythrocytes. These clusters became larger as the parasites matured and were more abundant in CC erythrocytes. Further, average cluster size (500 nm) in uninfected (native) CC erythrocytes was comparable with that of parasitized AA erythrocytes but was significantly larger (1 μm) in parasitized CC erythrocytes. Increased band 3 clustering may enhance recognition sites for autoantibodies, which could contribute to the protective effect of hemoglobin C against malaria.

Atomic force microscopy of nanometric liposome adsorption and nanoscopic membrane domain formation

Scanning probe microscopy studies of membrane fusion and nanoscopic structures were performed using hydrated single lipids and lipid mixtures. Extruded vesicles of DMPC and mixtures at various concentrations of DLPC, DPPC and cholesterol were deposited on freshly cleaved mica and studied in a fluid environment by AFM. The nanostructures formed by these extruded liposomes ranged from isolated unilamellar vesicles to flat sheet membranes and were marked influenced by thermodynamic phase behavior. For DMPC membrane, intact bilayers exhibited a phase transition process in agreement with large bilayer patches. In the DLPC, DPPC and cholesterol mixtures, nanoscopic domain diameters ranged from approximately 25 to 48nm with height differences of approximately 1.4nm; all values were lipid composition-dependent. Our data support and extend previous studies of microscopic domains and phase boundaries of the same mixtures in giant unilamellar vesicles determined by confocal light microscopy. Our approach for preparing and utilizing supported membrane structures is potentially relevant to studies of native cell membranes.

A novel family of RGD-containing disintegrins (Tablysin-15) from the salivary gland of the horsefly Tabanus yao targets αIIbβ3 or αVβ3 and inhibits platelet aggregation and angiogenesis.

A novel family of RGD-containing molecules (Tablysin-15) has been molecularly characterised from the salivary gland of the haematophagous horsefly Tabanus yao. Tablysin-15 does not share primary sequence homology to any disintegrin discovered so far, and displays an RGD motif in the N-terminus of the molecule. It is also distinct from disintegrins from Viperidae since its mature form is not released from a metalloproteinase precursor. Tablysin-15 exhibits high affinity binding for platelet αIIbβ3 and endothelial cell αVβ3 integrins, but not for α5β1 or α2β1. Accordingly, it blocks endothelial cell adhesion to vitronectin (IC50 ~1 nM) and marginally to fibronectin (IC50 ~1 μM), but not to collagen. It also inhibits fibroblast growth factor (FGF)-induced endothelial cell proliferation, and attenuates tube formation in vitro. In platelets, Tablysin-15 inhibits aggregation induced by collagen, ADP and convulxin, and prevents static platelet adhesion to immobilised fibrinogen. In addition, solid-phase assays and flow cytometry demonstrates that αIIbβ3 binds to Tablysin-15. Moreover, immobilised Tablysin-15 supports platelet adhesion by a mechanism which was blocked by anti-integrin αIIbβ3 monoclonal antibody (e.g. abciximab) or by EDTA. Furthermore, Tablysin-15 dose-dependently attenuates thrombus formation to collagen under flow. Consistent with these findings, Tablysin-15 displays antithrombotic properties in vivo suggesting that it is a useful tool to block αIIbβ3, or as a prototype to develop antithrombotics. The RGD motif in the unique sequence of Tablysin-15 represents a novel template for studying the structure-function relationship of the disintegrin family of inhibitors.

Aegyptin displays high-affinity for the von Willebrand factor binding site (RGQOGVMGF) in collagen and inhibits carotid thrombus formation in vivo.

Aegyptin is a 30 kDa mosquito salivary gland protein that binds to collagen and inhibits platelet aggregation. We have studied the biophysical properties of aegyptin and its mechanism of action. Light‐scattering plot showed that aegyptin has an elongated monomeric form, which explains the apparent molecular mass of 110 kDa estimated by gel‐filtration chromatography. Surface plasmon resonance identified the sequence RGQOGVMGF (where O is hydroxyproline) that mediates collagen interaction with von Willebrand factor (vWF) as a high‐affinity binding site for aegyptin, with a KD of approximately 5 nm. Additionally, aegyptin interacts with the linear peptide RGQPGVMGF and heat‐denatured collagen, indicating that the triple helix and hydroxyproline are not a prerequisite for binding. However, aegyptin does not interact with scrambled RGQPGVMGF peptide. Aegyptin also recognizes the peptides (GPO)10 and GFOGER with low affinity (μm range), which respectively represent glycoprotein VI and integrin α2β1 binding sites in collagen. Truncated forms of aegyptin were engineered, and the C‐terminus fragment was shown to interact with collagen and to attenuate platelet aggregation. In addition, aegyptin prevents laser‐induced carotid thrombus formation in the presence of Rose Bengal in vivo, without significant bleeding in rats. In conclusion, aegyptin interacts with distinct binding sites in collagen, and is useful tool to inhibit platelet–collagen interaction in vitro and in vivo.

Absorption-Based Hyperspectral Imaging and Analysis of Single Erythrocytes

We report an absorption-based hyperspectral imaging and analysis technique to resolve unique physicochemical characteristics of subcellular substances in single erythrocytes. We constructed a microscope system installed with a spectral light engine capable of controlling the spectral shape of the illumination light by a digital micromirror device. The hyperspectral imaging system and the sequential maximum angle convex cone algorithm allow us to extract unique spectral signatures (i.e., endmembers) for different types of hemoglobin, such as oxyhemoglobin, methemoglobin, and hemozoin, and scatter from cell membrane in single erythrocytes. Further statistical endmember analysis, conducted on the hyperspectral image data, provides the abundances of specific endmembers, which can be used to build intracellular maps of the distribution of substances of interest. In addition, we perform modeling based on Mie theory to explain the scattering signatures as a function of scattering angle. The developed imaging and analysis technique enables label-free molecular imaging of endogenous biomarkers in single erythrocytes in order to build oxymetric standards on a cellular level and ultimately for in vivo as well.

Plasmodium falciparum: food vacuole localization of nitric oxide-derived species in intraerythrocytic stages of the malaria parasite.

Nitric oxide (NO) has diverse biological functions. Numerous studies have documented NOs biosynthetic pathway in a wide variety of organisms. Little is known, however, about NO production in intraerythrocytic Plasmodium falciparum. Using diaminorhodamine-4-methyl acetoxymethylester (DAR-4M AM), a fluorescent indicator, we obtained direct evidence of NO and NO-derived reactive nitrogen species (RNS) production in intraerythrocytic P. falciparum parasites, as well as in isolated food vacuoles from trophozoite stage parasites. We preliminarily identified two gene sequences that might be implicated in NO synthesis in intraerythrocytic P. falciparum. We showed localization of the protein product of one of these two genes, a molecule that is structurally similar to a plant nitrate reductase, in trophozoite food vacuole membranes. We confirmed previous reports on the antiproliferative effect of NOS (nitric oxide synthase) inhibitors in P. falciparum cultures; however, we did not obtain evidence that NOS inhibitors had the ability to inhibit RNS production or that there is an active NOS in mature forms of the parasite. We concluded that a nitrate reductase activity produce NO and NO-derived RNS in or around the food vacuole in P. falciparum parasites. The food vacuole is a critical parasitic compartment involved in hemoglobin degradation, heme detoxification and a target for antimalarial drug action. Characterization of this relatively unexplored synthetic activity could provide important clues into poorly understood metabolic processes of the malaria parasite.

Altered Membrane Structure and Surface Potential in Homozygous Hemoglobin C Erythrocytes

Background Hemoglobin C differs from normal hemoglobin A by a glutamate-to-lysine substitution at position 6 of beta globin and is oxidatively unstable. Compared to homozygous AA erythrocytes, homozygous CC erythrocytes contain higher levels of membrane-associated hemichromes and more extensively clustered band 3 proteins. These findings suggest that CC erythrocytes have a different membrane matrix than AA erythrocytes. Methodology and Findings We found that AA and CC erythrocytes differ in their membrane lipid composition, and that a subset of CC erythrocytes expresses increased levels of externalized phosphatidylserine. Detergent membrane analyses for raft marker proteins indicated that CC erythrocyte membranes are more resistant to detergent solubilization. These data suggest that membrane raft organization is modified in CC erythrocytes. In addition, the average zeta potential (a measure of surface electrochemical potential) of CC erythrocytes was ≈2 mV lower than that of AA erythrocytes, indicating that substantial rearrangements occur in the membrane matrix of CC erythrocytes. We were able to recapitulate this low zeta potential phenotype in AA erythrocytes by treating them with NaNO2 to oxidize hemoglobin A molecules and increase levels of membrane-associated hemichromes. Conclusion Our data support the possibility that increased hemichrome deposition and altered lipid composition induce molecular rearrangements in CC erythrocyte membranes, resulting in a unique membrane structure.

Inward cholesterol gradient of the membrane system in P. falciparum-infected erythrocytes involves a dilution effect from parasite-produced lipids

ABSTRACT Plasmodium falciparum (Pf) infection remodels the human erythrocyte with new membrane systems, including a modified host erythrocyte membrane (EM), a parasitophorous vacuole membrane (PVM), a tubulovesicular network (TVN), and Maurers clefts (MC). Here we report on the relative cholesterol contents of these membranes in parasitized normal (HbAA) and hemoglobin S-containing (HbAS, HbAS) erythrocytes. Results from fluorescence lifetime imaging microscopy (FLIM) experiments with a cholesterol-sensitive fluorophore show that membrane cholesterol levels in parasitized erythrocytes (pRBC) decrease inwardly from the EM, to the MC/TVN, to the PVM, and finally to the parasite membrane (PM). Cholesterol depletion of pRBC by methyl-&bgr;-cyclodextrin treatment caused a collapse of this gradient. Lipid and cholesterol exchange data suggest that the cholesterol gradient involves a dilution effect from non-sterol lipids produced by the parasite. FLIM signals from the PVM or PM showed little or no difference between parasitized HbAA vs HbS-containing erythrocytes that differed in lipid content, suggesting that malaria parasites may regulate the cholesterol contents of the PVM and PM independently of levels in the host cell membrane. Cholesterol levels may affect raft structures and the membrane trafficking and sorting functions that support Pf survival in HbAA, HbAS and HbSS erythrocytes.