Determinants of Lipid Domain Size.

Lipid domains less than 200 nm in size may form a scaffold, enabling the concerted function of plasma membrane proteins. The size-regulating mechanism is under debate. We tested the hypotheses that large values of spontaneous monolayer curvature are incompatible with micrometer-sized domains. Here, we used the transition of photoswitchable lipids from their cylindrical conformation to a conical conformation to increase the negative curvature of a bilayer-forming lipid mixture. In contrast to the hypothesis, pre-existing micrometer-sized domains did not dissipate in our planar bilayers, as indicated by fluorescence images and domain mobility measurements. Elasticity theory supports the observation by predicting the zero free energy gain for splitting large domains into smaller ones. It also indicates an alternative size-determining mechanism: The cone-shaped photolipids reduce the line tension associated with lipid deformations at the phase boundary and thus slow down the kinetics of domain fusion. The competing influence of two approaching domains on the deformation of the intervening lipids is responsible for the kinetic fusion trap. Our experiments indicate that the resulting local energy barrier may restrict the domain size in a dynamic system.

Effect of neurosteroids on a model lipid bilayer including cholesterol: An Atomic Force Microscopy study.

Amphiphilic molecules which have a biological effect on specific membrane proteins, could also affect lipid bilayer properties possibly resulting in a modulation of the overall membrane behavior. In light of this consideration, it is important to study the possible effects of amphiphilic molecule of pharmacological interest on model systems which recapitulate some of the main properties of the biological plasma membranes. In this work we studied the effect of a neurosteroid, Allopregnanolone (3α,5α-tetrahydroprogesterone or Allo), on a model bilayer composed by the ternary lipid mixture DOPC/bSM/chol. We chose ternary mixtures which present, at room temperature, a phase coexistence of liquid ordered (Lo) and liquid disordered (Ld) domains and which reside near to a critical point. We found that Allo, which is able to strongly partition in the lipid bilayer, induces a marked increase in the bilayer area and modifies the relative proportion of the two phases favoring the Ld phase. We also found that the neurosteroid shifts the miscibility temperature to higher values in a way similarly to what happens when the cholesterol concentration is decreased. Interestingly, an isoform of Allo, isoAllopregnanolone (3ß,5α-tetrahydroprogesterone or isoAllo), known to inhibit the effects of Allo on GABAA receptors, has an opposite effect on the bilayer properties.

The sticholysin family of pore-forming toxins induces the mixing of lipids in membrane domains.

Sticholysins (Sts) I and II (StI/II) are pore-forming toxins (PFTs) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin family, a unique class of eukaryotic PFTs exclusively found in sea anemones. The role of lipid phase co-existence in the mechanism of the action of membranolytic proteins and peptides is not clearly understood. As for actinoporins, it has been proposed that phase separation promotes pore forming activity. However little is known about the effect of sticholysins on the phase separation of lipids in membranes. To gain insight into the mechanism of action of sticholysins, we evaluated the effect of these proteins on lipid segregation using differential scanning calorimetry (DSC) and atomic force microscopy (AFM). New evidence was obtained reflecting that these proteins reduce line tension in the membrane by promoting lipid mixing. In terms of the relevance for the mechanism of action of actinoporins, we hypothesize that expanding lipid disordered phases into lipid ordered phases decreases the lipid packing at the borders of the lipid raft, turning it into a more suitable environment for N-terminal insertion and pore formation.

Characterization of domain formation in complex membranes.

A widely known property of lipid membranes is their tendency to undergo a separation into disordered (Ld) and ordered (Lo) domains. This impacts the local structure of the membrane relevant for the physical (e.g., enhanced electroporation) and biological (e.g., protein sorting) significance of these regions. The increase in computing power, advancements in simulation software, and more detailed information about the composition of biological membranes shifts the study of these domains into the focus of classical molecular dynamics simulations. In this chapter, we present a versatile yet robust analysis pipeline that can be easily implemented and adapted for a wide range of lipid compositions. It employs Gaussian-based Hidden Markov Models to predict the hidden order states of individual lipids by describing their structure through the area per lipid and the average SCC order parameters per acyl chain. Regions of the membrane with a high correlation between ordered lipids are identified by employing the Getis-Ord local spatial autocorrelation statistic on a Voronoi tessellation of the lipids. As an example, the approach is applied to two distinct systems at a coarse-grained resolution, demonstrating either a strong tendency towards phase separation (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DIPC), cholesterol) or a weak tendency toward phase separation (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PUPC), cholesterol). Explanations of the steps are complemented by coding examples written in Python, providing both a comprehensive understanding and practical guidance for a seamless integration of the workflow into individual projects.

Hydrophobic Mismatch Controls the Mode of Membrane-Mediated Interactions of Transmembrane Peptides.

Various cellular processes require the concerted cooperative action of proteins. The possibility for such synchronization implies the occurrence of specific long-range interactions between the involved protein participants. Bilayer lipid membranes can mediate protein-protein interactions via relatively long-range elastic deformations induced by the incorporated proteins. We considered the interactions between transmembrane peptides mediated by elastic deformations using the framework of the theory of elasticity of lipid membranes. An effective peptide shape was assumed to be cylindrical, hourglass-like, or barrel-like. The interaction potentials were obtained for membranes of different thicknesses and elastic rigidities. Cylindrically shaped peptides manifest almost neutral average interactions-they attract each other at short distances and repel at large ones, independently of membrane thickness or rigidity. The hourglass-like peptides repel each other in thin bilayers and strongly attract each other in thicker bilayers. On the contrary, the barrel-like peptides repel each other in thick bilayers and attract each other in thinner membranes. These results potentially provide possible mechanisms of control for the mode of protein-protein interactions in membrane domains with different bilayer thicknesses.

Colloidal Stability of Emulsifier-free Triolein-in-Water Emulsions: Effects of Temperature.

Herein, we report the colloidal stability of emulsifier-free (EF-) triolein-in-water (TO/W) emulsions prepared by mixing TO and water using a high-powered bath-type ultrasonicator (HPBath-US; 28 kHz, 300 W) in the absence of emulsifiers such as surfactants. In particular, the effect of the temperature (15-60℃) on the colloidal stability of EF-TO/W emulsions was examined because this is important for the practical application of EF-TO/W emulsions, for example, in foods, pharmaceuticals, and cosmetics. We found that the colloidal stability of the EF-TO/W emulsions decreased with increase in the temperature from 15 to 25°C, whereas it increased with increase in temperature from 25 to 40°C, and the high colloidal stability of the EF-TO/W emulsions was maintained above 40°C. The reduction in the colloidal stability of EF-TO/W emulsions between 15 and 25°C is likely a result of the TO droplets formed by thermal motion, as well as enhanced Ostwald ripening at higher temperatures. On the other hand, the increase in the colloidal stability of the EF-TO/W emulsions from 25 to 40°C and their high colloidal stability above 40℃ is attributed to the reduction in the interfacial tension between TO and water at higher temperatures. This decrease in the interfacial tension between TO and water with temperature increase is related to the transformation of short-range ordered domains (clusters) of TO molecules in the liquid state, which increases the colloidal stability of the EF-TO/W emulsions.

A Mechanism of Double-Membrane Vesicle Formation from Liquid-Ordered/Liquid-Disordered Phase Separated Spherical Membrane.

Genome replication of coronaviruses takes place in specific cellular compartments, in so-called double-membrane vesicles (DMVs), formed from the endoplasmic reticulum (ER). An intensive production of DMVs is induced by non-structural viral proteins. Here, we proposed a possible mechanism of the DMV formation from ER-derived spherical vesicles where liquid-ordered and liquid-disordered lipid phases coexist. These vesicles are supposed to divide into two homogeneous liquid-ordered and liquid-disordered vesicles. The formation of two spherical vesicles constituting DMV requires a mechanical work to be performed. We considered the excess energy of the boundary between the coexisting lipid phases as the main driving force behind the division of the initial vesicle. Explicitly accounting for the energy of elastic deformations and the interphase boundary energy, we analyzed a range of physical parameters where the DMV formation is possible. We concluded that this process can principally take place in a very narrow range of system parameters. The most probable diameter of DMVs formed according to the proposed mechanism appeared to be approximately 220 nm, in an agreement with the average diameter of DMVs observed in vivo. Our consideration predicts the DMV size to be strongly limited from above. The developed analysis can be utilized for the production of DMVs in model systems.

N-(3-Oxododecanoyl) Homoserine Lactone Is a Generalizable Plasma Membrane Lipid-Ordered Domain Modifier.

A mammalian plasma membrane is a structure on which several layers of complexity are built. The first order of complexity comes from the heterogeneity of lipid-ordered domains. Gangliosides in concert with cholesterol are preferentially packed on the outer leaflet and form lipid-ordered domains, commonly known as lipid rafts. The formation and dynamics of these domains impact nearly all membrane protein functions and are an intensely studied topic. However, tools suited for lipid domain alteration are extremely limited. Currently, methyl-ß-cyclodextrin (MßCD) appears to be the most common way to disrupt lipid domains, which is believed to operate via cholesterol extraction. This significantly limits our ability in membrane biophysics research. Previously, we found that N-(3-oxo-dodecanoyl) homoserine lactone (3oc), a small signaling chemical produced by Pseudomonas aeruginosa, is highly efficient in altering lipid-ordered domains. In this study, 3oc was compared with MßCD in a series of biochemical, biophysical, and cell biological analyses. Per molarity, 3oc is more efficient than MßCD in domain alteration and appears to better retain membrane lipids after treatment. This finding will provide an essential reagent in membrane biophysics research.

Amphipathic Peptides Impede Lipid Domain Fusion in Phase-Separated Membranes.

Cell membranes are heterogeneous in lipid composition which leads to the phase separation with the formation of nanoscopic liquid-ordered domains, also called rafts. There are multiple cell processes whereby the clustering of these domains into a larger one might be involved, which is responsible for such important processes as signal transduction, polarized sorting, or immune response. Currently, antimicrobial amphipathic peptides are considered promising antimicrobial, antiviral, and anticancer therapeutic agents. Here, within the framework of the classical theory of elasticity adapted for lipid membranes, we investigate how the presence of the peptides in a phase-separated membrane influences the fusion of the domains. We show that the peptides tend to occupy the boundaries of liquid-ordered domains and significantly increase the energy barrier of the domain-domain fusion, which might lead to misregulation of raft clustering and adverse consequences for normal cell processes.

Misregulation of Wnt Signaling Pathways at the Plasma Membrane in Brain and Metabolic Diseases.

Wnt signaling pathways constitute a group of signal transduction pathways that direct many physiological processes, such as development, growth, and differentiation. Dysregulation of these pathways is thus associated with many pathological processes, including neurodegenerative diseases, metabolic disorders, and cancer. At the same time, alterations are observed in plasma membrane compositions, lipid organizations, and ordered membrane domains in brain and metabolic diseases that are associated with Wnt signaling pathway activation. Here, we discuss the relationships between plasma membrane components-specifically ligands, (co) receptors, and extracellular or membrane-associated modulators-to activate Wnt pathways in several brain and metabolic diseases. Thus, the Wnt-receptor complex can be targeted based on the composition and organization of the plasma membrane, in order to develop effective targeted therapy drugs.

Effects of post-lithography cleaning on the yield and performance of CVD graphene-based devices.

The large-scale production of high-quality and clean graphene devices, aiming at technological applications, has been a great challenge over the last decade. This is due to the high affinity of graphene with polymers that are usually applied in standard lithography processes and that, inevitably, modify the electrical proprieties of graphene. By Raman spectroscopy and electrical-transport investigations, we correlate the room-temperature carrier mobility of graphene devices with the size of well-ordered domains in graphene. In addition, we show that the size of these well-ordered domains is highly influenced by post-photolithography cleaning processes. Finally, we show that by using poly(dimethylglutarimide) (PMGI) as a protection layer, the production yield of CVD graphene devices is enhanced. Conversely, their electrical properties are deteriorated as compared with devices fabricated by conventional production methods.

The Effect of Transmembrane Protein Shape on Surrounding Lipid Domain Formation by Wetting.

Signal transduction through cellular membranes requires the highly specific and coordinated work of specialized proteins. Proper functioning of these proteins is provided by an interplay between them and the lipid environment. Liquid-ordered lipid domains are believed to be important players here, however, it is still unclear whether conditions for a phase separation required for lipid domain formation exist in cellular membranes. Moreover, membrane leaflets are compositionally asymmetric, that could be an obstacle for the formation of symmetric domains spanning the lipid bilayer. We theoretically show that the presence of protein in the membrane leads to the formation of a stable liquid-ordered lipid phase around it by the mechanism of protein wetting by lipids, even in the absence of conditions necessary for the global phase separation in the membrane. Moreover, we show that protein shape plays a crucial role in this process, and protein conformational rearrangement can lead to changes in the size and characteristics of surrounding lipid domains.