Shear-Induced Nonequilibrium Patterns in Lipid Bilayer Membranes Exhibiting Phase Separation.

The nonequilibrium dynamics of a fluid lipid membrane under external stimuli is an important issue that spans disciplines such as soft matter, biophysical chemistry, and interface science. This study investigated the dynamic response of lipid vesicles with order-disorder phase separation, which mimics a plasma membrane heterogeneity, to shear flow. Lipid vesicles were immobilized in a microfluidic chamber, and shear-induced nonequilibrium patterns on the membrane surface were observed by an optical microscope. We found that phase-separated membranes exhibit a dissipative structure of stripe patterns along the vortex flow on the membrane surface, and the number of stripes increased with the flow rate. At a high flow rate, the membrane exhibited a stripe-to-wave transition, where striped domains often migrated and the replacement of two different phases happened at vortex centers with time. We obtained a dynamic phase diagram of the shear-induced wave pattern by changing the flow rate, membrane components, and temperature. These findings could provide insight into the dissipative structures of lipid membranes out of equilibrium and flow-mediated mechanotransduction of biological membranes.

Domain dynamics of phase-separated lipid membranes under shear flow.

The dynamical behaviour of lateral domains on phase-separated lipid vesicles under external flow is reported. A microfluidic chamber was used for the immobilization of vesicles and the application of shear. Microscopic observation revealed that domains tended to be localized at the vortex center and to exhibit a stripe morphology as the flow speed increased. We clarified the dependency of domain behaviors on the flow speed and lipid mixing fraction. The cholesterol ratio in the membrane affected these domain behaviors. Next, we investigated the growth of domains under flow. We discuss the mechanism of these trends by considering the free energy of phase separation, and reproduce the experimental results by numerical simulations. These findings may lead to a better understanding of the dynamical properties of the membrane under nonequilibrium situations and the biophysical mechanism of cellular mechanotransduction.

Self-Emergent Protocells Generated in an Aqueous Solution with Binary Macromolecules through Liquid-Liquid Phase Separation.

Recently, liquid-liquid phase separation (LLPS) has attracted considerable attention among researchers in the life sciences as a plausible mechanism for the generation of microstructures inside cells. LLPS occurs through multiple nonspecific interactions and does not always require a lock-and-key interaction with a binary macromolecular solution. The remarkable features of LLPS include the non-uniform localization and concentration of solutes, resulting in the ability to isolate certain chemical systems and thereby parallelize multiple chemical reactions within the limited space of a living cell. We report that, by using the macromolecules, poly(ethylene glycol) (PEG) and dextran, that exhibit LLPS in an aqueous solution, cell-sized liposomes are spontaneously formed therein in the presence of phospholipids. In this system, LLPS is generated through the depletion effect of macromolecules. The results showed that cell-like microdroplets entrapping DNA wrapped by a phospholipid layer emerge in a self-organized manner.

Osmotic-Tension-Induced Membrane Lateral Organization.

Alteration of lipid raft organization manifesting as phase separation is important for cellular processes, such as signaling and trafficking. Such behaviors and dynamics of lipid membranes can be affected by external stimuli including both physical and chemical stimuli. In this study, we focused on osmotic-tension-induced phase separation. The effects of osmotic tension on the phase behaviors of vesicles consisting of dioleoylphosphocholine (DOPC)/dipalmitoylphosphocholine (DPPC)/cholesterol (Chol) were quantitatively studied at different temperatures by fluorescence microscopy. We determined the ternary phase diagrams and found that tension leads to a shift in the miscibility temperature. Cholesterol plays a key role in determining the extent of this shift. In addition, we found that osmotic tension can enhance the line tension. The physicochemical mechanism of osmotic-pressure-induced phase separation is discussed.

Control of Lipid Bilayer Phases of Cell-Sized Liposomes by Surface-Engineered Plasmonic Nanoparticles.

Liquid-ordered (Lo)-phase domains, a cholesterol-rich area on lipid bilayers, have attracted significant attention recently because of their relevance to lipid rafts, the formation/collapse of which is associated with various kinds of information exchange through the plasma membrane. Here, we demonstrate that the formation/collapse of Lo-phase domains in cell-sized liposomes, that is, giant unilamellar vesicles (GUVs), can be controlled with bioactive plasmonic nanoparticles and light. The nanoparticles were prepared by surface modification of gold nanorods (AuNRs) using a cationized mutant of high-density lipoprotein (HDL), which is a natural cholesterol transporter. Upon the addition of surface-engineered AuNRs to GUVs with the mixed domains of Lo and liquid-disorder (Ld) phases, the Lo domains collapsed and solid-ordered (So)-phase domains were formed. The reverse phase transition was achieved photothermally, with the AuNRs loaded with cholesterol. During these transitions, the AuNRs appeared to be selectively localized on the less fluidic domain (Lo or So) in the phase-mixed GUVs. These results indicate that the phase transitions occur through the membrane binding of the AuNRs followed by spontaneous/photothermal transfer of cholesterol between the AuNRs and GUVs. Our strategy to develop bioactive AuNRs potentially enables spatiotemporal control of the formation/collapse of lipid rafts in living cells.

Hydrophobic Polyampholytes and Nonfreezing Cold Temperature Stimulate Internalization of Au Nanoparticles to Zwitterionic Liposomes.

Nanomedicine relies on the effective internalization of nanoparticles combined with polymeric nanocarriers into living cells. Thus, exploration of internalization is essential for improving the efficacy of nanoparticle-based strategies in clinical practice. Here, we investigated the physicochemical internalization of gold nanoparticles (AuNPs) conjugated with hydrophobic polyampholytes into cell-sized liposomes at a low but nonfrozen temperature. The hydrophobic polyampholytes localized in the disordered phase of the membrane, and internalization of AuNPs was enhanced in the presence of hydrophobic polyampholytes together with incubation at -3 °C as compared to 25 °C. These results contribute toward a mechanistic understanding for developing a model nanomaterials-driven delivery system based on hydrophobic polyampholytes and low temperature.

Mechano-Sensitive Synthetic Ion Channels.

Mechanical stress is a ubiquitous stimulus sensed by membrane proteins, but rarely by synthetic molecules. Inspired by mechano-sensitive ion channels found in cell membranes, tension-responsive transmembrane multiblock amphiphiles were developed. In membranes, a single-transmembrane amphiphile responds to both expanding and contracting tensions to weaken and strengthen the stacking of membrane-spanning units, respectively, and ion transportation is triggered by expanding tension to form a supramolecular channel, while little transportation is observed under a tensionless condition. In contrast, a three-transmembrane amphiphile showed little spectroscopic response to tensions, likely due to weaker stacking of membrane-spanning units than in the single-transmembrane amphiphile. Nevertheless, the three-transmembrane amphiphile shows ion transportation by forming a unimolecular channel even under a tensionless condition, and the ion-transporting activity decreased with expanding tension. Interestingly, the estimated operating force of these synthetic systems was comparable to that of the mechano-sensitive proteins. This study opens the door toward new mechano-sensitive molecular devices.

Photoinduced Fusion of Lipid Bilayer Membranes.

We have developed a novel system for photocontrol of the fusion of lipid vesicles through the use of a photosensitive surfactant containing an azobenzene moiety (AzoTAB). Real-time microscopic observations clarified a change in both the surface area and internal volume of vesicles during fusion. We also determined the optimal cholesterol concentrations and temperature for inducing fusion. The mechanism of fusion can be attributed to a change in membrane tension, which is caused by the solubilization of lipids through the isomerization of AzoTAB. We used a micropipet technique to estimate membrane tension and discuss the mechanism of fusion in terms of membrane elastic energy. The obtained results regarding this novel photoinduced fusion could lead to a better understanding of the mechanism of membrane fusion in living cells and may also see wider applications, such as in drug delivery and biomimetic material design.

Lateral Diffusion of a Submicrometer Particle on a Lipid Bilayer Membrane.

In past decades, nanoparticles and nanomaterials have been actively used for applications such as visualizing nano/submicrometer cell structure, killing cancer cells, and using drug delivery systems. It is important to understand the physicochemical mechanisms that govern the motion of nanoparticles on a plasma membrane surface. However, the motion of small particles of <1000 nm on lipid membranes is poorly understood. In this study, we investigated the diffusion of particles with a diameter of 200-800 nm on a lipid membrane using cell-sized liposomes. Particle-associated liposomes were obtained by applying centrifugal force to a mixture of liposomes and particle solutions. We measured the thermal motion of the particles by phase-contrast microscopy. We found that (i) the particle-size dependence of the diffusion of particles adhering to membranes was better described by the DADL model rather than the Einstein-Stokes model, (ii) the diffusion coefficient of a particle strongly depends on the adsorption state of the particle, such as fully or partially wrapped by the membrane, and (iii) anomalous diffusion was induced by the localization of particles on the neck of budded vesicles.

Bringing macromolecules into cells and evading endosomes by oxidized carbon nanoparticles.

A great challenge exists in finding safe, simple, and effective delivery strategies to bring matters across cell membrane. Popular methods such as viral vectors, positively charged particles and cell penetrating peptides possess some of the following drawbacks: safety issues, lysosome trapping, limited loading capacity, and toxicity, whereas electroporation produces severe damages on both cargoes and cells. Here, we show that a serendipitously discovered, relatively nontoxic, water dispersible, stable, negatively charged, oxidized carbon nanoparticle, prepared from graphite, could deliver macromolecules into cells, without getting trapped in a lysosome. The ability of the particles to induce transient pores on lipid bilayer membranes of cell-sized liposomes was demonstrated. Delivering 12-base-long pyrrolidinyl peptide nucleic acids with d-prolyl-(1S,2S)-2-aminocyclopentanecarboxylic acid backbone (acpcPNA) complementary to the antisense strand of the NF-κB binding site in the promoter region of the Il6 gene into the macrophage cell line, RAW 264.7, by our particles resulted in an obvious accumulation of the acpcPNAs in the nucleus and decreased Il6 mRNA and IL-6 protein levels upon stimulation. We anticipate this work to be a starting point in a new drug delivery strategy, which involves the nanoparticle that can induce a transient pore on the lipid bilayer membrane.

Ag/FeCo/Ag core/shell/shell magnetic nanoparticles with plasmonic imaging capability.

Magnetic nanoparticles (NPs) have been used to separate various species such as bacteria, cells, and proteins. In this study, we synthesized Ag/FeCo/Ag core/shell/shell NPs designed for magnetic separation of subcellular components like intracellular vesicles. A benefit of these NPs is that their silver metal content allows plasmon scattering to be used as a tool to observe detection by the NPs easily and semipermanently. Therefore, these NPs are considered a potential alternative to existing fluorescent probes like dye molecules and colloidal quantum dots. In addition, the Ag core inside the NPs suppresses the oxidation of FeCo because of electron transfer from the Ag core to the FeCo shell, even though FeCo is typically susceptible to oxidation. The surfaces of the Ag/FeCo/Ag NPs were functionalized with ε-poly-L-lysine-based hydrophilic polymers to make them water-soluble and biocompatible. The imaging capability of the polymer-functionalized NPs induced by plasmon scattering from the Ag core was investigated. The response of the NPs to a magnetic field using liposomes as platforms and applying a magnetic field during observation by confocal laser scanning microscopy was assessed. The results of the magnetophoresis experiments of liposomes allowed us to calculate the magnetic force to which each liposome was subjected.

Serum B cell-activating factor (BAFF) level in connective tissue disease associated interstitial lung disease.

BACKGROUND: Interstitial lung diseases (ILDs) are common in patients with connective tissue diseases (CTDs). Although the diagnosis of an underlying CTD in ILD (CTD-ILD) affects both prognosis and treatment, it is sometimes difficult to distinguish CTD-ILD from chronic fibrosing interstitial pneumonia (CFIP). B cell-activating factor belonging to the tumour necrosis factor family (BAFF) plays a crucial role in B cell development, survival, and antibody production. METHODS: We examined serum levels of BAFF, surfactant protein D (SP-D), and Krebs von den Lungen-6 (KL-6) in 33 patients with CTD-ILD, 16 patients with undifferentiated CTD-ILD, 19 patients with CFIP, and 26 healthy volunteers. And we analysed the relationship between serum BAFF levels and pulmonary function, as well as the expression of BAFF in the lung tissue of patients with CTD-ILD. RESULTS: Serum levels of BAFF were significantly higher in CTD-ILD patients compared to healthy subjects and CFIP patients. However, there were no significant differences in serum levels of SP-D and KL-6. Furthermore, serum BAFF levels in CTD-ILD patients were inversely correlated with pulmonary function. BAFF was strongly expressed in the lungs of CTD-ILD patients, but weakly in normal lungs. DISCUSSION: This is the first study to demonstrate that serum BAFF levels were significantly higher in CTD-ILD patients compared to healthy subjects and CFIP patients. Furthermore, serum BAFF levels were correlated with pulmonary function. We consider that serum BAFF levels in patients with CTD-ILD reflect the presence of ILDs disease activity and severity. CONCLUSION: These finding suggest that BAFF may be a useful marker for distinguishing CTD-ILD from CFIP.

Membrane fusion and vesicular transformation induced by Alzheimer's amyloid beta.

Amyloid beta (Aß) peptides, produced through endo-proteolytic cleavage of amyloid precursor protein, are thought to be involved in the death of neural cells in Alzheimer's disease (AD). Although the mechanisms are not fully known, it has been suggested that disruption of cellular activity due to Aß interactions with the cell membrane may be one of the underlying causes. Here in, we have investigated the interaction between Aß-42 and biomimetic lipid membranes and the resulting perturbations in the lipid vesicles. We have shown that Aß oligomeric species localized closer to the membrane surface. Localization of the fibrillar species of Aß-42, although varied, was not as closely associated with the membrane surface. We have demonstrated that the presence of Aß-42 leads to an increase in membrane surface area, inducing lipid temporal vesicular transformation. Furthermore, we have unequivocally shown that Aß-peptides mediate membrane fusion. Although membrane fusion induced by Aß has been hypothesized/proposed, this is the first time it has been visually captured. This fusion may be one of the mechanisms behind the membrane increase in surface area and the resulting vesicular transformation. We have shown that the longer 'amyloidogenic' isoform causes vesicular transformation more readily, and has a higher membrane fusogenic potential than Aß-40. Although not core to this study, it is hugely interesting to observe the high agreement between membrane dynamics and the reported amyloidogenicity of the peptides and aggregation species opening up the potential role of vesicular dynamics for profiling and biosensing of Aß-induced neuro-toxicity.

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

The interaction of amyloid beta (Aß) peptide with cell membranes has been shown to be influenced by Aß conformation, membrane physicochemical properties and lipid composition. However, the effect of cholesterol and its oxidized derivatives, oxysterols, on Aß-induced neurotoxicity to membranes is not fully understood. We employed here model membranes to investigate the localization of Aß 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 Aß, in contrast to role of cholesterol in inhibiting Aß/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 Aß, while 25OH systems were more capable of morphological changes in response to the peptide. Furthermore, we have shown that 42-amino acid Aß (Aß-42) pre-fibril species had higher association with membranes, and caused membrane fluctuation faster than 40-residue isoform (Aß-40). These findings suggest the enhancing effect of oxysterols on interaction of Aß with membranes and contribute to clarify the harmful impact of cholesterol on Aß-induced neurotoxicity by means of its oxidation.

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.

Charge-induced phase separation in lipid membranes.

Phase separation in lipid bilayers that include negatively charged lipids is examined experimentally. We observed phase-separated structures and determined the membrane miscibility temperatures in several binary and ternary lipid mixtures of unsaturated neutral lipid, dioleoylphosphatidylcholine (DOPC), saturated neutral lipid, dipalmitoylphosphatidylcholine (DPPC), unsaturated charged lipid, dioleoylphosphatidylglycerol (DOPG((-))), saturated charged lipid, dipalmitoylphosphatidylglycerol (DPPG((-))), and cholesterol. In binary mixtures of saturated and unsaturated charged lipids, the combination of the charged head with the saturation of the hydrocarbon tail is a dominant factor in the stability of membrane phase separation. DPPG((-)) enhances phase separation, while DOPG((-)) suppresses it. Furthermore, the addition of DPPG((-)) to a binary mixture of DPPC/cholesterol induces phase separation between DPPG((-))-rich and cholesterol-rich phases. This indicates that cholesterol localization depends strongly on the electric charge on the hydrophilic head group rather than on the ordering of the hydrocarbon tails. Finally, when DPPG((-)) was added to a neutral ternary system of DOPC/DPPC/cholesterol (a conventional model of membrane rafts), a three-phase coexistence was produced. We conclude by discussing some qualitative features of the phase behaviour in charged membranes using a free energy approach.

Endo- and exocytic budding transformation of slow-diffusing membrane domains induced by Alzheimer's amyloid beta.

Cell-sized liposomes are a powerful tool for clarifying physicochemical mechanisms that govern molecular interactions. Herein, budding transformation of membrane domains was induced by amyloid beta peptides. The peptides increased the membrane viscosity as demonstrated by the Brownian motion of membrane domains. These results could aid in understanding the physicochemical mechanism of Alzheimer's disease.

Dynamical formation of lipid bilayer vesicles from lipid-coated droplets across a planar monolayer at an oil/water interface.

Recently, the transfer method has been shown to be useful for preparing cell-sized phospholipid bilayer vesicles, within which desired substances at desired concentrations can be encapsulated, with a desired asymmetric lipid composition. Here, we investigated the transfer process of water-in-oil (W/O) droplets coated by phospholipid monolayers across an oil/water interface by both experimental observation and theoretical modeling. Real-time experimental observation of the transfer revealed that the transfer process is characterized by three kinetic regimes: a precontact process (approaching regime), an early fast process (entering regime), and a late slow process (relaxation regime). In addition, bigger droplets require much more time to transfer than smaller droplets. We propose a theoretical model to interpret this kinetic process. Our theoretical model reproduces the essential aspects of the transfer kinetics, including its size-dependence.

Physical Concept to Explain the Regulation of Lipid Membrane Phase Separation under Isothermal Conditions.

Lateral phase separation within lipid bilayer membranes has attracted considerable attention in the fields of biophysics and cell biology. Living cells organize laterally segregated compartments, such as raft domains in an ordered phase, and regulate their dynamic structures under isothermal conditions to promote cellular functions. Model membrane systems with minimum components are powerful tools for investigating the basic phenomena of membrane phase separation. With the use of such model systems, several physicochemical characteristics of phase separation have been revealed. This review focuses on the isothermal triggering of membrane phase separation from a physical point of view. We consider the free energy of the membrane that describes lateral phase separation and explain the experimental results of model membranes to regulate domain formation under isothermal conditions. Three possible regulation factors are discussed: electrostatic interactions, chemical reactions and membrane tension. These findings may contribute to a better understanding of membrane lateral organization within living cells that function under isothermal conditions and could be useful for the development of artificial cell engineering.

The effect of oxycholesterols on thermo-induced membrane dynamics.

The effect of temperature change(s) on the dynamics of giant unilamellar vesicles containing oxidized and non-oxidized cholesterol was investigated and characterized. We have demonstrated that (i) major cholesterol auto-oxidation products, 7ß-hydroxycholesterol (7ß) and 7-ketocholesterol (7keto), rendered vesicles more responsive to temperature changes; (ii) 7keto imparted greater thermo-induced membrane dynamics than 7ß; (iii) 7ß and 7keto vesicles synergistically were more thermo-responsive than the individual oxysterols; (iv) the thermo-responsiveness of 7keto-containing vesicles was equivalent to that of 25 hydroxycholesterol (25OH)-containing vesicles; and (v) we have characterized the observed membrane dynamics. The results provide a new plausible mechanism: oxidative-stressed membranes in conjunction with temperature change induce membrane dynamics. These findings improve the mechanisms reported previously that attributed the induced dynamics solely to membrane oxidation.