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The dextran–PEG system is one of the most famous systems exhibiting phase separation. Various phase behaviors, including the evaporation process of the dextran–PEG system, have been studied in order to understand the physicochemical mechanism of intracellular phase separation and the effect of condensation on the origin of life. However, there have been few studies in dilute regime. In this study, we focused on such regimes and analyzed the pattern formation by evaporation. The specificity of this regime is the slow onset of phase separation due to low initial concentration, and the separated phases can have contrasting wettability to the substrate as evaporation progresses. When the polymer concentration is rather low (<5 wt%), the dextran–PEG droplets form a phase-separated pattern, consisting of PEG at the center and dextran ring of multiple strings pulling from the ring. This pattern formation is explained from the difference in wettability and compatibility between dextran and PEG upon condensation. At the initial dilute stage, the dextran-rich phase with higher wettability accumulates at the contact line of the droplet to form a ring pattern, and then forms multiple domains due to density fluctuation. The less wettable PEG phase recedes and pulls the dextran domains, causing them to deform into strings. Further condensation leads to phase separation, and the condensed PEG with improved wettability stops receding and prevents a formed circular pattern. These findings suggest that evaporation patterns of polymer blend droplets can be manipulated through changes in wettability and compatibility between polymers due to condensation, thus providing the basis to explore origins of life that are unique to the process of condensate formation from dilute systems.

Membranes are ubiquitous structures in cells. The effects of membranes on various functional molecules have been reported, but their behaviors under macromolecular crowding and cell-sized confinement have not fully been understood. In this study, we model an intracellular environment by crowding micrometer-sized droplets and investigate the effects of membrane properties on molecular diffusion. The molecular diffusion inside small droplets covered with a lipid layer of phosphatidylcholine (PC) becomes slower compared with that of the corresponding bulk solutions under a crowding condition of polysaccharide dextran but not of its monomer unit, glucose. The addition of a poly(ethylene glycol) conjugated lipid (PEGylated lipid) to the PC membrane significantly alters the degree of slow diffusion observed inside small droplets of concentrated dextran. Interestingly, the change is not monotonic against dextran concentration; that is, the PEGylated membrane increases and decreases the degree of slow diffusion with increasing dextran concentration. We explain the nonmonotonic alternation from the increase in effective dextran concentration and the hindered temporal adsorption of dextran to the membrane. Because diffusion alteration by adding PEGylated lipid is observed for condensed small droplets of linear polymer PEG and hydrophilic protein bovine serum albumin, the phenomenon is general for other polymer systems as well. Furthermore, our findings may facilitate the understanding of intracellular molecular behaviors based on membrane effects as well as the development of numerous applications using polymer droplets.

Assembled water-in-oil droplets bounded by lipid bilayers are used in synthetic biology as minimal models of cell tissue. Microfluidic devices successfully generate monodispersed droplets and assemble them via droplet interface bilayesr (DIB) formation. However, a honeycomb pattern of DIB-bounded droplets, similar to epithelial tissues, remains unrealized because the rapid DIB formation between the droplets hinders their ability to form the honeycomb pattern. In this paper, we demonstrate the microfluidic formation of a honeycomb pattern of DIB-bounded droplets using two surfactants with different adsorption rates on the droplet surface. A non-DIB forming surfactant (sorbitan monooleate, Span 80) was mixed with a lipid (1,2-dioleoyl-sn-glycero-3-phosphocholine, PC), whose adsorption rate on the droplet surface and saturated interfacial tension were lower than those of Span 80. By changing the surfactant composition, we established the conditions under which the droplets initially form a honeycomb pattern and subsequently adhere to each other via DIB formation to minimize the interfacial energy. In addition, the reconstituted membrane protein nanopores at the DIBs were able to transport molecules. This new method, using the difference in the adsorption rates of two surfactants, allows the formation of a honeycomb pattern of DIB-bounded droplets in a single step, and thus facilitates research using DIB-bounded droplet assemblies.

<p>Liposome–liposome adhesion by electrostatic interactions and osmotic contraction increase membrane tension and the lipid diffusion coefficient compared to isolated liposomes.</p>

Molecular behaviors in small liquid droplets (picoliter scale), such as phase transitions and chemical reactions, are essential for the industrial application of small droplets and their use as artificial cells. However, the droplets often differ from those in bulk solutions (milliliter scale). Since the droplet size is much larger than the molecular size, the so-called size effect that draws these differences has attracted attention as a target to be solved. Although the small volume and the membrane surface surrounding the droplet are thought to be the origin of the size effect, there were little attempts to separate and quantify them. To solve the problem, we develop a series of systems for the evaluation. Using these systems, we have evaluated the size effect of concentrated polymer solutions on molecular diffusion by dividing it into small volume and membrane surface contributions. Our results demonstrate that the size effect on the molecular diffusion originates from the long-range interaction with the surface enhanced with decreasing volume. The quantitative size effect revealed by the systems provides novel insights in the biophysical understanding of molecular behaviors in cells and to the regulation and design of micrometer-sized materials.

A recurring question in membrane biological chemistry is whether bioactive signaling lipids act only as second messenger ligands or also through an effect on bilayer physical properties. Sphingosine (Sph) and sphingosine-1-phosphate (S1P) are single-chained charged sphingolipids that have antagonistic functions in the "sphingolipid rheostat" which determines cell fate. Sph and S1P respectively promote apoptosis and cell growth. In the present study, potential effects of these bioactive lipids on physicochemical properties of the lipid bilayer of cell membranes were evaluated. We have investigated the effect of both sphingolipids, incorporated separately or, for the first time, together, in large or giant phosphadidylcholine (PC) unilamellar vesicles. Three bilayer properties were examined

Lipid rafts are assumed to undergo biologically important size-modulations from nanorafts to microrafts. Due to the complexity of cellular membranes, model systems become important tools, especially for the investigation of the factors affecting "raft-like" L-o domain size and the search for L-o nanodomains as precursors in L-o microdomain formation. Because lipid compositional change is the primary mechanism by which a cell can alter membrane phase behavior, we studied the effect of the ganglioside GM1 concentration on the L-o/L-d lateral phase separation in PC/SM/Chol/GM1 bilayers. GM1 above 1 mol% abolishes the formation of the micrometer-scale L-o domains observed in GUVs. However, the apparently homogeneous phase observed in optical microscopy corresponds in fact, within a certain temperature range, to a L-o/L-d lateral phase separation taking place below the optical resolution. This nanoscale phase separation is revealed by fluorescence spectroscopy, including C12NBD-PC self-quenching and Laurdan GP measurements, and is supported by Gaussian spectral decomposition analysis. The temperature of formation of nanoscale L-o phase domains over an L-d phase is determined, and is shifted to higher values when the GM1 content increases. A "morphological" phase diagram could be made, and it displays three regions corresponding respectively to L-o/L-d micrometric phase separation, L-o/L-d nanometric phase separation, and a homogeneous L-d phase. We therefore show that a lipid only-based mechanism is able to control the existence and the sizes of phase-separated membrane domains. GM1 could act on the line tension, "arresting" domain growth and thereby stabilizing L-o nanodomains. (C) 2014 Elsevier B.V. All rights reserved.

We investigated the effect of the adhered interface on the phase separation pattern using two or three adhered droplets containing a binary solution of poly(ethylene glycol) and gelatin. Under the...

Micrometric membrane confinements and macromolecular crowding of cytoplasm are key factors that regulate molecular diffusion in live cells. Previous studies have shown that macromolecular crowding delays molecular diffusion. However, the effect of cell-size confinement on diffusion in the crowding environment is yet to be elucidated. Using fluorescence correlation spectroscopy (FCS), we analyzed protein diffusion in microdroplets containing polymer solution covered with lipid membranes that mimic cells. As a result, we found that a synergistic condition of crowding and micrometric confinement results in accelerated protein diffusion on a sub-millisecond time scale. This acceleration rate strongly depended on the size of the confined space and the degree of crowding. These findings indicate that cell-size confinement supports protein diffusion in highly crowded cytoplasm.

Biomimetic lipid bilayer systems are a useful tool for modeling specific properties of cellular membranes in order to answer key questions about their structure and functions. This approach has prompted scientists from all over the world to create more and more sophisticated model systems in order to decipher the complex lateral and transverse organization of cellular plasma membranes. Among a variety of existing biomembrane domains, lipid rafts are defined as small, dynamic, and ordered assemblies of lipids and proteins, enriched in cholesterol and sphingolipids. Lipid rafts appear to be involved in the development of Alzheimer's disease (AD) by affecting the aggregation of the amyloid-β (Aβ) peptide at neuronal membranes thereby forming toxic oligomeric species. In this review, we summarize the laboratory methods which allow to study the interaction of Aβ with lipid rafts. We describe step by step protocols to form giant (GUVs) and large unilamellar vesicles (LUVs) containing raft-mimicking domains surrounded by membrane nonraft regions. Using fluorescence microscopy GUV imaging protocols, one can design experiments to visualize micron-scale raft-like domains, to determine the micron-scale demixing temperature of a given lipid mixture, construct phase diagram, and photogenerate domains in order to assess the dynamics of raft formation and raft size distribution. LUV fluorescence spectroscopy protocols with proper data analysis can be used to measure molecular packing of raft/nonraft regions of the membrane, to report on nanoscale raft formation and determine nanoscale demixing temperature. Because handling of the Aβ requires dedicated laboratory experience, we present illustrated protocols for Aβ-stock aliquoting, Aβ aqueous solubilization, oligomer preparation, determination of the Aβ concentration before and after filtration. Thioflavin binding, dynamic light scattering, and transmission electron microscopy protocols are described as complementary methods to detect Aβ aggregation kinetics, aggregate sizes, and morphologies of observed aggregates.