Masamune Morita

Real-time observation of model membrane dynamics induced by Alzheimer's amyloid beta.

Amyloid beta (Abeta) has been strongly implicated in inducing neurotoxicity in the pathology of Alzheimers disease (AD). However, the underlying mechanisms remain unknown. In this study, we examined, in real-time, the spatio-temporal changes in individual model membranes induced by the presence of different Abeta-40 molecular assemblies (species). We used cell-sized lipid vesicles to enable the direct observation of these changes. We found three significantly different membrane-transformation pathways. We characterized the biophysical mechanisms behind these transformations in terms of the change in inner vesicle volume and surface area. Oligomeric Abeta exhibited the highest tendency to cause membrane fluctuation and transformations. Interestingly, mature fibrils, which are often considered inert species, also induced profound membrane changes. Furthermore, we imaged the localization of pre-fibrillar species on membranes. The real-time observation of these morphological transformations, which can be missed in a discretised analysis, may help to unlock the mechanisms of ADs Abeta-induced neuro-degeneration.

Size-dependent partitioning of nano/microparticles mediated by membrane lateral heterogeneity.

It is important that we understand the physical, chemical, and biological mechanisms that govern the interaction between nanoparticles (NPs) and heterogeneous cellular surfaces because of the possible cytotoxicity of engineered nanomaterials. In this study, we investigated the lateral localization of nano/microparticles within a biomimetic heterogeneous membrane interface using cell-sized two-phase liposomes. We found that lateral heterogeneity in the membrane mediates the partitioning of nano/microparticles in a size-dependent manner: small particles with a diameter of ≤200 nm were localized in an ordered phase, whereas large particles preferred a fluidic disordered phase. This partitioning behavior was verified by temperature-controlled membrane miscibility transition and laser-trapping of associated particles. In terms of the membrane elastic energy, we present a physical model that explains this localization preference of nano/microparticles. The calculated threshold diameter of particles that separates the particle-partitioning phase was 260 nm, which is in close agreement with our observation (200 nm). These findings may lead to a better understanding of the basic mechanisms that underlie the association of nanomaterials within a cell surface.

Ion permeation by a folded multiblock amphiphilic oligomer achieved by hierarchical construction of self-assembled nanopores.

A multiblock amphiphilic molecule 1, with a tetrameric alternating sequence of hydrophilic and hydrophobic units, adopts a folded structure in a liposomal membrane like a multipass transmembrane protein, and is able to transport alkali metal cations through the membrane. Hills analysis and conductance measurements, analyzed by the Hille equation, revealed that the tetrameric assembly of 1 forms a 0.53 nm channel allowing for permeation of cations. Since neither 3, bearing flexible hydrophobic units and forming no stacked structures in the membrane, nor 2, a monomeric version of 1, is able to transport cations, the folded conformation of 1 in the membrane is likely essential for realizing its function. Thus, function and hierarchically formed higher-order structures of 1, is strongly correlated with each other like proteins and other biological macromolecules.

Physicochemical Profiling of Surfactant-Induced Membrane Dynamics in a Cell-Sized Liposome.

We used a cell-sized model system, giant liposomes, to investigate the interaction between lipid membranes and surfactants, and the membrane transformation during the solubilization process was captured in real time. We found that there are four distinct dynamics in surfactant-induced membrane deformation: an episodic increase in the membrane area prior to pore-forming associated shrinkage (Dynamics A), fission into many small liposomes (Dynamics B), the formation of multilamellar vesicles and peeling (Dynamics C), and bursting (Dynamics D). Classification of the diversity of membrane dynamics may contribute to a better understanding of the physicochemical mechanism of membrane solubilization induced by various surfactants.

Selective localization of Alzheimer's amyloid beta in membrane lateral compartments

Model membrane systems revealed that lateral heterogeneity of the membrane mediates the localization of amyloid beta peptides in a peptide aggregation-dependent manner.

The effect of oxysterols on the interaction of Alzheimer's amyloid beta with model membranes.

The interaction of amyloid beta (Aβ) peptide with cell membranes has been shown to be influenced by Aβ conformation, membrane physicochemical properties and lipid composition. However, the effect of cholesterol and its oxidized derivatives, oxysterols, on Aβ-induced neurotoxicity to membranes is not fully understood. We employed here model membranes to investigate the localization of Aβ in membranes and the peptide-induced membrane dynamics in the presence of cholesterol and 7-ketocholesterol (7keto) or 25-hydroxycholesterol (25OH). Our results have indicated that oxysterols rendered membranes more sensitive to Aβ, in contrast to role of cholesterol in inhibiting Aβ/membrane interaction. We have demonstrated that two oxysterols had different impacts owing to distinct positions of the additional oxygen group in their structures. 7keto-containing cell-sized liposomes exhibited a high propensity toward association with Aβ, while 25OH systems were more capable of morphological changes in response to the peptide. Furthermore, we have shown that 42-amino acid Aβ (Aβ-42) pre-fibril species had higher association with membranes, and caused membrane fluctuation faster than 40-residue isoform (Aβ-40). These findings suggest the enhancing effect of oxysterols on interaction of Aβ with membranes and contribute to clarify the harmful impact of cholesterol on Aβ-induced neurotoxicity by means of its oxidation.

Structure-dependent interactions of polyphenols with a biomimetic membrane system.

Polyphenols are naturally-occurring compounds, reported to be biologically active, and through their interactions with cell membranes. Although association of the polyphenols with the bilayer has been reported, the detailed mechanism of interaction is not yet well elucidated. We report on spatio-temporal real-time membrane dynamics observed in the presence of polyphenols. Two distinct membrane dynamics, corresponding to the two classes of polyphenols used, were observed. Flavonoids (epi-gallocatechin-3-gallate, gallocatechin, theaflavin and theaflavin-3-gallate) caused lipid membrane aggregation and rigidification. As simple structural modification through opening of the aromatic C-ring into an olefin bond, present in trans-stilbenes (resveratrol and picead), completely changed the membrane properties, increasing fluidity and inducing fluctuation. There were differences in the membrane transformations within the same class of polyphenols. Structure-dependent classification of membrane dynamics may contribute to a better understanding of the physicochemical mechanism involved in the bioactivity of polyphenols. In general, an increase in the number of hydrophilic side chains (galloyl, hydroxyl, glucoside, gallate) increased the reactivity of the polyphenols. Most notable was the difference observed through a simple addition of the gallate group. Unraveling the importance of these polyphenols, at a functional group level further opens the key to tailored design of bioactive compounds as potential drug candidates.

Micrometer-Size Vesicle Formation Triggered by UV Light

Vesicle formation is a fundamental kinetic process related to the vesicle budding and endocytosis in a cell. In the vesicle formation by artificial means, transformation of lamellar lipid aggregates into spherical architectures is a key process and known to be prompted by e.g. heat, infrared irradiation, and alternating electric field induction. Here we report UV-light-driven formation of vesicles from particles consisting of crumpled phospholipid multilayer membranes involving a photoactive amphiphilic compound composed of 1,4-bis(4-phenylethynyl)benzene (BPEB) units. The particles can readily be prepared from a mixture of these components, which is casted on the glass surface followed by addition of water under ultrasonic radiation. Interestingly, upon irradiation with UV light, micrometer-size vesicles were generated from the particles. Neither infrared light irradiation nor heating prompted the vesicle formation. Taking advantage of the benefits of light, we successfully demonstrated micrometer-scale spatiotemporal control of single vesicle formation. It is also revealed that the BPEB units in the amphiphile are essential for this phenomenon.

Dynamic Morphological Changes Induced By GM1 and Protein Interactions on the Surface of Cell-Sized Liposomes

It is important to understand the physicochemical mechanisms that are responsible for the morphological changes in the cell membrane in the presence of various stimuli such as osmotic pressure. Lipid rafts are believed to play a crucial role in various cellular processes. It is well established that Ctb (Cholera toxin B subunit) recognizes and binds to GM1 (monosialotetrahexosylganglioside) on the cell surface with high specificity and affinity. Taking advantage of Ctb-GM1 interaction, we examined how Ctb and GM1 molecules affect the dynamic movement of liposomes. GM1 a natural ligand for cholera toxin, was incorporated into liposome and the interaction between fluorescent Ctb and the liposome was analyzed. The interaction plays an important role in determining the various surface interaction phenomena. Incorporation of GM1 into membrane leads to an increase of the line tension leading to either rupture of liposome membrane or change in the morphology of the membrane. This change in morphology was found to be GM1 concentration specific. The interaction between Ctb-GM1 leads to fast and easy rupture or to morphological changes of the liposome. The interactions of Ctb and the glycosyl chain are believed to affect the surface and the curvature of the membrane. Thus, the results are highly beneficial in the study of signal transduction processes.

Structure-dependent membrane interaction and bioactivity of flavonoids with lipid bilayers

Flavonoids are naturally-occuring compounds, commonly found in teas, wine, and grapes. They have been reported to be biologically active through their interactions with cell membranes. They are widely known to be beneficial to the body due to their anti-oxidant, anti-proliferation and anti-inflammatory effects. Although changes in fluidity of the lipid bilayer had been reported to change in the presence of the flavonoids and association of the flavonoids with the bilayer has been reported, the detailed mechanism of interaction of flavonoids with lipid membranes is not yet well elucidated. Here, we report on the interactions between three flavonoids belonging to flavan-3-ol and thearubigins and stilbenes: a tea catechin (-)-epigallocatechin gallate (EGCg) and its oxidative derivative theaflavin which is found in black tea and piceid which is a resveratrol derivative found in grape juice. Our results show vesicular transformation in the presence of the flavonoids, and there were clear differences between the three flavonoids. We are yet to confirm or refute the presence of flavonoids in those aggregates. As far as we are aware, this is the first time such an experiment has been done.