Formation Of Macroscopic Domains Research Articles

Engineering Phosphatidylserine Containing Asymmetric Giant Unilamellar Vesicles.

The plasma membrane lipid distribution is asymmetric, with several anionic lipid species located in its inner leaflet. Among these, phosphatidylserine (PS) plays a crucial role in various important physiological functions. Over the last decade several methods have been developed that allow for the fabrication of large or giant unilamellar vesicles (GUVs) with an asymmetric lipid composition. Investigating the physicochemical properties of PS in such asymmetric lipid bilayers and studying its interactions with proteins necessitates the reliable fabrication of asymmetric GUVs (aGUVs) with a high degree of asymmetry that exhibit PS in the outer leaflet so that the interaction with peptides and proteins can be studied. Despite progress, achieving aGUVs with well-defined PS asymmetry remains challenging. Recently, a Ca2+-initiated hemifusion method has been introduced, utilizing the fusion of symmetric GUVs (sGUVs) with a supported lipid bilayer (SLB) for the fabrication of aGUVs. We extend this approach to create aGUVs with PS in the outer bilayer leaflet. Comparing the degree of asymmetry between aGUVs obtained via Ca2+ or Mg2+ initiated hemifusion of a phosphatidylcholine (PC) sGUVwith a PC/PS-supported lipid bilayer, we observe for both bivalent cations a significant number of aGUVs with near-complete asymmetry. The degree of asymmetry distribution is narrower for physiological salt conditions than at lower ionic strengths. While Ca2+ clusters PS in the SLB, macroscopic domain formation is absent in the presence of Mg2+. However, the clustering of PS upon the addition of Ca2+ is apparently too slow to have a negative effect on the quality of the obtained aGUVs. We introduce a data filtering method to select aGUVs that are best suited for further investigation.

Utilizing asymmetric giant unilamellar vesicles to determine how low-melting phospholipids affect interleaflet coupling strength.

Model membranes that mimic cell membrane asymmetry can be used to study coupling between lateral and transverse lipid organization. Theory predicts that, in a leaflet composed of lipids that would otherwise phase separate in a symmetric bilayer, domain formation can be suppressed by strong coupling to a uniformly mixed leaflet. Conversely, when coupled to a leaflet with a tendency to demix, an otherwise miscible leaflet can be induced to demix. Each of these outcomes has been observed in model asymmetric membranes of different composition, suggesting that cells may be able to control domain formation through changes in lipid content and/or asymmetric lipid distribution. We have explored the influence of the low-melting (low-TM) lipid in a ternary mixture on macroscopic domain formation in asymmetric giant unilamellar vesicles (aGUVs). Symmetric GUVs composed of DPPC/low-TM lipid/Chol (39/39/22 mol%) with varied low-TM chain length (14:1-PC, 16:1-PC, 18:1-PC, 20:1-PC, and 22:1-PC) were investigated using confocal fluorescence microscopy; the mixtures containing 14:1-PC, 16:1-PC, and 18:1-PC showed macroscopic phase separation at room temperature, while the mixtures containing 20:1-PC and 22:1-PC did not. Cryogenic electron microscopy, small-angle neutron scattering, and Förster resonance energy transfer confirmed the presence of domains in the first three systems and revealed nanoscopic heterogeneity in the mixture containing 20:1-PC, while the 22:1-PC mixture was uniform. aGUVs were then prepared by calcium-induced hemifusion of the GUVs to a supported lipid bilayer composed of the corresponding low-TM lipid and Chol (80/20 mol%). The aGUVs showed a range of outer leaflet exchange, which revealed a correlation between phase behavior and extent of asymmetry. Domains in the 16:1-PC and 18:1-PC systems were suppressed in aGUVs with more than 50 and 60% asymmetry, respectively, indicating a relatively large free energy cost for antiregistered domains.

Investigating the influence of low-melting phospholipids on interleaflet coupling in asymmetric giant unilamellar vesicles

Compositional asymmetry is a characteristic of the plasma membrane whose biological importance is still mostly unknown. Asymmetric model membranes mimicking cell membrane composition offer a useful, highly controlled system for studying the coupling between lateral and transverse lipid organization. We have explored the influence of the low-melting (low-TM) lipid in a ternary mixture on macroscopic domain formation in asymmetric giant unilamellar vesicles (aGUVs). Symmetric GUVs composed of DPPC/low-TM lipid/Chol (39/39/22 mol%) with varied low-TM chain length (14:1-PC, 16:1-PC, 18:1-PC, 20:1-PC, and 22:1-PC) were prepared by electroformation.

Bilayer compositional asymmetry influences the nanoscopic to macroscopic phase domain size transition

The eukaryotic plasma membrane (PM) exhibits lipid mixing heterogeneities known as lipid rafts. These lipid rafts, the result of liquid-liquid phase separation, can be modeled by coexisting liquid ordered (Lo) and liquid disordered (Ld) domains. Four-lipid component systems with a high-melting lipid, a nanodomain-inducing low-melting lipid, a macrodomain-inducing low-melting lipid, and cholesterol (chol) can give rise to domains of different sizes. These four-component systems have been characterized in experiments, yet there are few studies that model the asymmetric distribution of lipids actually found in the PM. We used molecular dynamics (MD) simulations to analyze the transition from nanoscopic to macroscopic domains in symmetric and in asymmetric model membranes. Using coarse-grained MD simulations, we found that asymmetry promotes macroscopic domain growth in a case where symmetric systems exhibit nanoscopic domains. Also, macroscopic domain formation in symmetric systems is highly dependent on registration of like phases in the cytoplasmic and exoplasmic leaflets. Using united-atom MD simulations, we found that symmetric Lo domains are only slightly more ordered than asymmetric Lo domains. We also found that large Lo domains in our asymmetric systems induce a slight chain ordering in the apposed cytoplasmic regions. The chol fractions of phase-separated Lo and Ld domains of the exoplasmic leaflet were unchanged whether the system was symmetric or asymmetric.

Electric-field-induced intradimer charge disproportionation in the dimer-Mott insulator β′−(BEDT−TTF)2ICl2

Raman scattering spectra of the dimer-Mott insulator ${\ensuremath{\beta}}^{\ensuremath{'}}\text{\ensuremath{-}}{(\mathrm{BEDT}\text{\ensuremath{-}}\mathrm{TTF})}_{2}\mathrm{IC}{\mathrm{l}}_{2}$ [BEDT-TTF = bis(ethylenedithio)tetrathiafulvalene] under a static electric field are investigated. The application of the electric field induces two additional Raman peaks on both sides of the original peak position of the charge-sensitive Raman mode $({\ensuremath{\nu}}_{2})$ in the spectra. At 10 kV/cm, the original peak almost disappears and only the newly emerging peaks are observed. The emergence of these peaks indicates the field-induced charge disproportionation within the dimer. The temporal change of the Raman signals with the inversion of the electric field suggests a macroscopic domain formation of the charge-disproportionate dimers. This picture is reinforced by the direct measurement of the polarization.

Solubilization of Giant Vesicles Composed of Erythrocyte Lipid Extracts and of Ternary Lipid Mixtures by Triton X-100

Detergents are widely used as solubilizing agents of biological membranes. Detergent resistant membranes, called DRMs, are frequently obtained and share similarities in composition and properties with the so-called lipid rafts and with the liquid-ordered phase (Lo) of model membranes. Here we use optical and fluorescence microscopy to study the solubilization process of giant unilamellar vesicles (GUVs) made of erythrocyte lipid extracts (erythro-GUVs) by the commonly used detergent Triton X-100. For comparison, ternary lipid mixtures of POPC, sphingomyelin (SM) and cholesterol are also investigated. Erythro-GUVs do not exhibit macroscopic phase separation, as seen by the homogenous distribution of a fluorescence probe that preferentially partition into the fluid (Ld) phase. However, contact with Triton X-100 immediately causes formation of micrometer-sized bright domains, typical of Lo/Ld phase separation. Then, at Triton X-100 concentrations above its cmc, the fluid part is first detached from the original vesicle and then completely solubilized by Triton X-100. The insoluble vesicle left is only faintly fluorescent and appears to be in the Lo phase. Typically, one third of the erythro-GUV surface area is solubilized. Solubilization of POPC:SM:chol GUVs in a certain composition range follow the same sequence of events. Our results show that Triton X-100 promotes formation of macroscopic domains in both biological and biomimetic membranes, and that the partial solubilization occurs after phase separation, suggesting that the composition of the insoluble membrane fraction is modulated by the choice of the detergent used. Financial support: CNPq, FAPESP and INCT-FCx.

Domain formation in membranes with quenched protein obstacles: Lateral heterogeneity and the connection to universality classes

We show that lateral fluidity in membranes containing quenched protein obstacles belongs to the universality class of the two-dimensional random-field Ising model. The main feature of this class is the absence of a phase transition: there is no critical point and macroscopic domain formation does not occur. Instead there is only one phase. This phase is highly heterogeneous with a structure consisting of microdomains. The presence of quenched protein obstacles thus provides a mechanism to stabilize lipid rafts in equilibrium. Crucial for two-dimensional random-field Ising universality is that the obstacles are randomly distributed and have a preferred affinity to one of the lipid species. When these conditions are not met standard Ising or diluted Ising universality applies. In these cases a critical point does exist which then marks the onset toward macroscopic demixing.

Exploring Supramolecular Aspects of the Effect of Sphingomyelinase D On Sphingomyelin-Containing Membranes

Lipid-modifying enzymes play a vital role in the regulation of lipids as mediators of cell function. At the same time, the activity of these enzymes is highly affected by the lipid membrane structure. These processes at lipid membranes can be observed in situ through the application of different biophysical techniques. Thus, we are investigating a spider venom enzyme termed sphingomyelinase D (SMD). SMD hydrolyses sphingomyelin (SM) into ceramide-1-phosphate (Cer-1-P). While SM is an integral constituent of many cell membranes, Cer-1-P occurs in very low concentrations and is suggested to be a novel lipid second messenger. At present, the physiologically relevant mechanism following Cer-1-P formation by SMD is incompletely understood, but possibly related to the modulation of membrane properties.Our results show a strong dependency of SMD activity on the phase state of the substrate. SMD is two orders of magnitude more active towards fluid- than gel-phase liposomes. The presence of cholesterol evens out this difference in activity at an intermediate level. The effect of SMD on fluid-phase giant unilamellar vesicles (GUVs) is observed by confocal fluorescence microscopy. GUVs composed of lauroyl-SM show a macroscopic domain formation and/or shrinking and buckling accompanied by the multiple formation of membrane tubes. GUVs composed of egg-SM display a beveling of the membrane and the formation of caps (outside curvature) approx. three days after the addition of SMD. Which membrane morphology evolves is likely a question of enzyme kinetics vs. the dynamics of lipid reorganization.GUVs of raft-like mixtures exhibit a single homogenous phase after the addition of SMD. The consequences of SMD activity and Cer-1-P formation on cellular systems are currently being examined. This will indorse the correlation between enzymatic activity and membrane structure influencing the regulation of physiological processes.

Spatially Resolved ATR‐FTIRS Study of the Formation of Macroscopic Domains and Microislands during CO Electrooxidation on Pt

Electrooxidation of CO in CO-saturated sulfuric acid electrolyte solutions with controlled mass transport is investigated with spatially resolved attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy under galvanostatic and potentiostatic conditions. The reaction conditions are chosen such that steady states with intermediate current densities and intermediate average CO coverages are accessible. We demonstrate that under these conditions the reaction never proceeds uniformly on the electrode surface. Instead, macroscopic domains form spontaneously, composed of areas with high CO coverage and areas essentially free of adsorbed CO molecules. The average coverage within the CO-covered domains depends on the electrolyte concentration and the applied potential and can vary between saturation coverage and a few tenths of a monolayer. However, the absence of a red-shift of the CO vibrational band points to a further substructuring of the domains in densely packed CO microislands. These microislands most likely also form in the boundary layer between the CO-rich and CO-free electrode domains. This hierarchical patterning of the electrode surface is attributed to the interplay of autocatalytic reaction steps, spatial coupling through migration or the galvanostatic control of the experiment, and molecular interactions between molecules co-adsorbed on the electrode surface.

1D Cahn–Hilliard equation for modulated phase systems

Formation of modulated phase patterns can be modelized by a modified Cahn–Hilliard equation which includes a non-local term preventing the formation of macroscopic domains. Using stationary solutions of the original Cahn–Hilliard equation as analytical ansatzs, we compute the thermodynamically stable period of a 1D modulated phase pattern. We find that the period scales like the power −1/3 of the strength of the long-range interaction.

Exploring Molecular and Supramolecular Aspects of Sphingomyelin-Containing Membranes Upon Action of Sphingomyelinase D

Lipid-modifying enzymes play a vital role in the regulation of lipids as mediators of cell function. At the same time, the activity of these enzymes is highly affected by the lipid membrane structure. These processes at lipid membranes can be observed in situ through the application of different biophysical techniques. Thus, we are investigating an enzyme from spider venoms, which is termed sphingomyelinase D (SMD). SMD hydrolyses sphingomyelin (SM) into ceramide-1-phosphate (Cer-1-P). While SM is an integral constituent of many cell membranes, e.g., of red blood cells, Cer-1-P occurs in very low concentrations and is suggested to be a novel lipid second messenger. At present, the physiologically relevant mechanism following Cer-1-P formation by SMD is incompletely understood, but possibly related to the modulation of membrane properties.Our results show a strong dependency of SMD activity on the phase state of the substrate. SMD is one order of magnitude more active towards fluid- than gel-phase liposomes. The effect of SMD on fluid-phase giant unilamellar vesicles (GUVs) is observed by confocal fluorescence microscopy. GUVs composed of lauroyl-SM show a drastic shrinking and buckling accompanied by the multiple formation of membrane tubes, which are up to 80 μm long. Generalized-polarization measurements using the probe LAURDAN exhibit a macroscopic domain formation upon the hydrolysis of lauroyl-SM. This finding points to the induction of membrane curvature by lipid sorting in the simple, binary system of SM/Cer-1-P. SMD activity on GUVs composed of oleoyl-SM also shows tube formation followed by the immediate and complete disintegration of the vesicular membrane structure. The consequences of SMD activity and Cer-1-P formation on cellular systems are currently being examined. This will indorse the correlation between enzymatic activity and membrane structure influencing the regulation of physiological processes.

Macroscopic domain formation during cooling in the platelet plasma membrane: An issue of low cholesterol content

There has been ample debate on whether cell membranes can present macroscopic lipid domains as predicted by three-component phase diagrams obtained by fluorescence microscopy. Several groups have argued that membrane proteins and interactions with the cytoskeleton inhibit the formation of large domains. In contrast, some polarizable cells do show large regions with qualitative differences in lipid fluidity. It is important to ask more precisely, based on the current phase diagrams, under what conditions would large domains be expected to form in cells. In this work we study the thermotropic phase behavior of the platelet plasma membrane by FTIR, and compare it to a POPC/Sphingomyelin/Cholesterol model representing the outer leaflet composition. We find that this model closely reflects the platelet phase behavior. Previous work has shown that the platelet plasma membrane presents inhomogeneous distribution of DiI18:0 at 24 °C, but not at 37 °C, which suggests the formation of macroscopic lipid domains at low temperatures. We show by fluorescence microscopy, and by comparison with published phase diagrams, that the outer leaflet model system enters the macroscopic domain region only at the lower temperature. In addition, the low cholesterol content in platelets (∼15 mol%), appears to be crucial for the formation of large domains during cooling.

Experimental investigation on macroscopic domain formation and evolution in polycrystalline NiTi microtubing under mechanical force

This paper reports the experimental results on macroscopic deformation instability and domain morphology evolution during stress-induced austenite → martensite (A→M) phase transformation in superelastic NiTi polycrystalline shape memory alloy microtubes. High-speed data and image acquisition techniques were used to investigate the dynamic and quasi-static events which took place in a displacement-controlled quasi-static tensile loading/unloading process of the tube. These events include dynamic formation, self-merging, topology transition, convoluted front motion and front instability of a macroscopic deformation domain. The reported phenomena brought up several fundamental issues regarding the roles of macroscopic domain wall energy and kinetics as well as their interplay with the bulk strain energy of the tube in the observed morphology instability and pattern evolution under a mechanical force. These issues are believed to be essential elements in the theoretical modeling of macroscopic deformation patterns in polycrystals and need systematic examination in the future.

Initial Condition Sensitivity in Photoinduced Structural Phase Transitions – An Iterative Equation for Exciton Proliferation during Lattice Relaxation –

We theoretically study lattice relaxation dynamics of photogenerated excitons, which proliferate during this relaxation, and finally result in a macroscopic excited domain in insulating solids. This macroscopic domain formation phenomenon is usually called “photoinduced structural phase transition (PSPT)”. According to recent experiments, occurrence or nonoccurrence of this PSPT is found to depend very sensitively on the way of the photoexcitation, even if the total absorbed photonic energy is same. It is nothing else but “initial condition sensitivity” peculiar to nonlinear systems. We concentrate our attention on this sensitivity, and investigate criticality of this proliferation, using an iterative equation for its time evolution. As one of the models describing such a sensitivity, we consider a 1-dimensional strongly coupled many exciton-Einstein phonon system interacting with a reservoir. In our system, excitons can proliferate through their third order anharmonicity. This anharmonicity originates from the long range Coulomb interaction among electrons and holes constituting excitons. Within the Markov approximation for the reservoir, the time evolution of the density matrix of this exciton-phonon system is investigated full-quantummechanically. In order to overcome numerical complexities, we solve our iterative equation for the proliferation by the following principle. We always focus only on the most front of the expanding photoinduced domain, and the contributions from the other excitons, not in this front, are approximated by a mean field. As the proliferation proceeds, this front moves. Accordingly, we, step by step, iterate this procedure till the excess phonon energy, initially given by the photoexcitation, is exhausted. Thus, we can investigate the whole process of the time evolution of the domain formation, until this domain contains a large number of excitons. As for the aforementioned initial condition sensitivity, we cast the difference of the initial conditions into the difference of the initial distance between adjacent photogenerated excitons. Net proliferation is concluded to occur only when this initial distance is moderate, being not too small nor too large. This sensitivity to the initial distance results from the aforementioned third order anharmonicity.

Macroporous Morphology of the Titania Films Prepared by a Sol-Gel Dip-Coating Method from the System Containing Poly(ethylene glycol): Effects of Molecular Weight and Dipping Temperature

Macroporous morphology has been examined for the titania (TiO2) films prepared by a sol-gel dip-coating method from the system containing poly(ethylene glycol) (PEG) under the variations of molecular weight of PEG and dipping temperature. The macroporous morphology is observed only within the limited range of molecular weight because the compatibility between PEG and the solvent mixture is quite sensitive to the molecular weight. The macroscopic domain formation is enhanced at higher withdrawal speed when the dipping temperature is raised, suggesting that the fluidity reduction and the phase separation are more accelerated than the polycondensation due to the enhanced solvent evaporation.

Sol-gel derived macroporous titania films prepared from the system containing poly(ethylene glycol) together with long-chain alcohols

The effect of alcohol-type on macroscopic morphology has been examined for the macroporous titania (TIO2) films prepared by a sol-gel dip-coating method from the system containing poly(ethylene glycol) (PEG) and alcohols having larger alkyl groups than ethanol. The phase separation is enhanced by decreasing the compatibility between the solvent mixture and PEG as long as the polycondensation rate does not increase greatly. In the 1-butanol system, macroporous morphology appears clearly even at higher relative humidity as 70% RH due to the slower polycondensation. In contrast, the macroscopic domain formation is completely suppressed by the rapid polycondensation in the 2-propanol system.

Lock-in and commensurate transitions of (N(CH3)4)2ZnCl4crystals studied by means of real time x-ray synchrotron topography

Abstract Sequences of I Incommensurate-II Commensurate-Ill Commensurate transitions in TMA2ZnCL4, were observed in real time by synchrotron X-ray topography. For I-II transition, the coexistence of commensurate and incommensurate phases causes, on a small temperature range near the lock-in transition, the formation of macroscopic domains bounded by defects which can be identified as deperiodisation lines. These lines have their emergence points on directions which can be indexed in the superspace.