Liquid-ordered Phase Research Articles

Characterization of conformational states of POPC and DPPCd62 in POPC/DPPCd62/cholesterol mixtures using Raman spectroscopy

Conformational states of phospholipid chains in ternary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), deuterated 1,2-dipalmitoyl-d62-sn-glycero-3-phosphocholine (DPPCd62), and cholesterol (Chol) were studied by Raman spectroscopy. Parameters of Raman peaks sensitive to conformational order have been used to determine chain order for mixtures over a wide range of compositions. A ternary diagram of fractions of phospholipid chains in conformationally ordered and disordered states has been constructed. It was found that the addition of POPC and cholesterol increases the fraction of DPPC chains in disordered conformations. The so-called liquid-ordered phase includes DPPC molecules in both ordered and disordered states in comparable proportions. It was found that POPC chains are partially ordered in mixtures with DPPC and cholesterol, in contrast to the case of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). This maybe the underlying reason why ternary mixtures with POPC have different miscibility behavior compared to DOPC.

The Membrane Phase Transition Gives Rise to Responsive Plasma Membrane Structure and Function.

Several groups have recently reported evidence for the emergence of domains in cell plasma membranes when membrane proteins are organized by ligand binding or assembly of membrane proximal scaffolds. These domains recruit and retain components that favor the liquid-ordered phase, adding to a decades-old literature interrogating the contribution of membrane phase separation in plasma membrane organization and function. Here we propose that both past and present observations are consistent with a model in which membranes have a high compositional susceptibility, arising from their thermodynamic state in a single phase that is close to a miscibility phase transition. This rigorous framework naturally allows for both transient structure in the form of composition fluctuations and long-lived structure in the form of induced domains. In this way, the biological tuning of plasma membrane composition enables a responsive compositional landscape that facilitates and augments cellular biochemistry vital to plasma membrane functions.

Phase-Separated Giant Liposomes for Stable Elevation of α-Hemolysin Concentration in Lipid Membranes.

Staphylococcus aureus α-hemolysin (αHL) is one of the most popular proteins in nanopore experiments within lipid membranes. Higher concentrations of αHL within the lipid membrane are desirable to enhance the mass transport capacity through nanopores. However, the reconstitution of αHL at high concentrations is associated with the problem of membrane lytic disruption. In this study, we present a method that effectively increases αHL concentration while maintaining membrane stability. This method is achieved by using phase-separated giant liposomes, where coexisting liquid-disordered (Ld) and liquid-ordered phases (Lo) are enriched in unsaturated lipids and saturated lipids with cholesterol (Chol), respectively. Fluorescence observation of αHL in liposomes revealed that the presence of Chol facilitates αHL insertion into the membrane. Despite the preferential localization of αHL in the Ld phase rather than the Lo phase, the coexistence of both Lo and Ld phases prevents membrane disruption in the presence of concentrated αHL. We have explained this stabilization mechanism considering the lower membrane tension exhibited by phase-separated liposomes compared to homogeneous liposomes. Under hypertonic conditions, we have successfully increased the local concentration of αHL by invagination of the lipid-only region in the Ld phase, leaving αHL behind. This method exhibits potential for the reconstitution of various nanochannels and membrane proteins that prefer the Ld phase over the Lo phase, thus enabling the production of giant liposomes at high concentrations and the replication of the membrane-crowding condition observed in cells.

Amyloid Precursor Protein Changes Arrangement in a Membrane and Its Structure Depending on the Cholesterol Content.

One of the hallmarks of Alzheimer's disease (AD) is the accumulation of amyloid beta (Aβ) peptides in the brain. The processing of amyloid precursor protein (APP) into Aβ is dependent on the location of APP in the membrane, membrane lipid composition and, possibly, presence of lipid rafts. In this study, we used atomic force microscopy (AFM) to investigate the interaction between transmembrane fragment APP672-726 (corresponding to Aβ1-55) and its amyloidogenic mutant L723P with membranes combining liquid-ordered and liquid-disordered lipid phases. Our results demonstrated that most of the APP672-726 is located either in the liquid-disordered phase or at the boundary between ordered and disordered phases, and hardly ever in rafts. We did not notice any major changes in the domain structure induced by APP672-726. In membranes without cholesterol APP672-726, and especially its amyloidogenic mutant L723P formed annular structures and clusters rising above the membrane. Presence of cholesterol led to the appearance of concave membrane regions up to 2 nm in depth that were deeper for wild type APP672-726. Thus, membrane cholesterol regulates changes in membrane structure and permeability induced by APP that might be connected with further formation of membrane pores.

Unsaturated Lipids Facilitate Partitioning of Transmembrane Peptides into the Liquid Ordered Phase.

The affinity of single-pass transmembrane (TM) proteins for ordered membrane phases has been reported to depend on their lipidation, TM length, and lipid accessible surface area. In this work, the raft affinities of the TM domain of the linker for activation of T cells and its depalmitoylated variant are assessed using free energy simulations in a binary bilayer system composed of two laterally patched bilayers of ternary liquid ordered (Lo) and liquid disordered (Ld) phases. These phases are modeled by distinct compositions of distearoylphosphatidylcholine, palmitoyloleoylphosphatidylcholine (POPC), and cholesterol, and the simulations were carried out for 4.5 μs/window. Both peptides are shown to preferentially partition into the Ld phase in agreement with model membrane experiments and previous simulations on ternary lipid mixtures but not with measurements on giant plasma membrane vesicles where the Lo is slightly preferred. However, the 500 ns average relaxation time of lipid rearrangement around the peptide precluded a quantitative analysis of free energy differences arising from peptide palmitoylation and two different lipid compositions. When in the Lo phase, peptides reside in regions rich in POPC and interact preferentially with its unsaturated tail. Hence, the detailed substructure of the Lo phase is an important modulator of peptide partitioning, in addition to the inherent properties of the peptide.

Antibodies to variable surface antigens induce antigenic variation in the intestinal parasite Giardia lamblia

The genomes of most protozoa encode families of variant surface antigens. In some parasitic microorganisms, it has been demonstrated that mutually exclusive changes in the expression of these antigens allow parasites to evade the host’s immune response. It is widely assumed that antigenic variation in protozoan parasites is accomplished by the spontaneous appearance within the population of cells expressing antigenic variants that escape antibody-mediated cytotoxicity. Here we show, both in vitro and in animal infections, that antibodies to Variant-specific Surface Proteins (VSPs) of the intestinal parasite Giardia lamblia are not cytotoxic, inducing instead VSP clustering into liquid-ordered phase membrane microdomains that trigger a massive release of microvesicles carrying the original VSP and switch in expression to different VSPs by a calcium-dependent mechanism. This novel mechanism of surface antigen clearance throughout its release into microvesicles coupled to the stochastic induction of new phenotypic variants not only changes current paradigms of antigenic switching but also provides a new framework for understanding the course of protozoan infections as a host/parasite adaptive process.

Phase Transition and Phase Separation in Realistic Thylakoid Lipid Membrane of Marine Algae in All-Atom Simulations.

Thylakoid membranes are specialized membranes predominantly composed of uncommon galacto- and sulfolipids, having distinct roles in photosynthesis. Large acyl chain variety and richness in polyunsaturated fatty acid (PUFA) content of thylakoid lipids further add to the compositional complexity. The function of these membrane systems is intimately dependent on the fluidity of its lipid matrix, which is strongly modulated by the lipid composition and temperature. The present work, employing extensive atomistic simulations, provides the first atomistic view of the phase transition and domain coexistence in a model membrane composed of thylakoid lipids of a commercially important red alga Gracilaria corticata between 10 and 40 °C. The growth and photosynthetic activity of marine algae are greatly influenced by the seawater temperature. So far, little is known about the molecular organization of lipids in thylakoid membranes, in particular their adaptive arrangements under temperature stress. Our simulations show that the algal thylakoid membrane undergoes a transition from a gel-like phase at a low temperature, 10-15 °C, to a homogeneous liquid-crystalline phase at a high temperature, 40 °C. Clear evidence of spontaneous phase separation into coexisting nanoscale domains is detected at intermediate temperatures nearing the optimal growth temperature range. Particularly, at 25-30 °C, we identified the formation of a stable ripple phase, where the gel-like domains rich in saturated and nearly hexagonally packed lipids were separated from fluid-like domains enriched in lipids containing PUFA chains. The phase separation is driven by the spontaneous and preferential segregation of lipids into differentially ordered domains, mainly depending on the acyl chain types. Cholesterol impairs the phase transition and the emergence of domains and induces a fairly uniform liquid-ordered phase in the membrane over the temperatures studied. This work improves the understanding of the properties and reorganization of lipids in the thylakoid membrane in response to temperature variation.

Membrane phase separation drives responsive assembly of receptor signaling domains.

Plasma membrane heterogeneity has been tied to a litany of cellular functions and is often explained by analogy to membrane phase separation; however, models based on phase separation alone fall short of describing the rich organization available within cell membranes. Here we present comprehensive experimental evidence motivating an updated model of plasma membrane heterogeneity in which membrane domains assemble in response to protein scaffolds. Quantitative super-resolution nanoscopy measurements in live B lymphocytes detect membrane domains that emerge upon clustering B cell receptors (BCRs). These domains enrich and retain membrane proteins based on their preference for the liquid-ordered phase. Unlike phase-separated membranes that consist of binary phases with defined compositions, membrane composition at BCR clusters is modulated through the protein constituents in clusters and the composition of the membrane overall. This tunable domain structure is detected through the variable sorting of membrane probes and impacts the magnitude of BCR activation.

Optimization of dihydrosphingomyelin/cholesterol mol ratio in topotecan-loaded liposomes to enhance drug retention and plasma half-life by understanding physicochemical and thermodynamic properties of the lipid membrane

Robust retention of drugs in liposomes is one of the most critical factors for liposomal drug formulation. It improves loading efficiency of the active pharmaceutical ingredient (API), stability during long-term storage, and half-life of the drug in the body. This study deductively optimized the lipid composition of topotecan-loaded liposome using dihydrosphingomyelin (DHSM), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE PEG) for intravenous injection by thermodynamic and fluidity analyses of the liposome membrane. High efficiency in topotecan loading was achieved in the range of the liquid-ordered phase (egg-derived DHSM/Chol molar ratio 70/25% or a higher Chol ratio with PEG constant at 5 mol%) as predicted by thermodynamic analysis. The egg-derived DHSM/Chol/PEG mol ratio that minimizes topotecan leakage was estimated to be 57/38/5 via electron spin resonance (ESR) for fluidity analysis. The optimized liposome significantly prolonged the half-life of topotecan in blood compared to non-liposomal topotecan. To minimize the risk of virus contamination and ensure easier quality control for clinical application, synthetic DHSM was used in the optimized formulation of topotecan encapsulated liposome instead of egg-derived DHSM (named FF-10850). The physicochemical and pharmacokinetic profiles of FF-10850 were equivalent to those of the liposome prepared using egg-derived DHSM and >99% drug retention was maintained for over 24 months under refrigeration, indicating stable encapsulation and prolonged blood circulation.

Switchable Lipids: From Conformational Switch to Macroscopic Changes in Lipid Vesicles.

It has been shown that the use of conformationally pH-switchable lipids can drastically enhance the cytosolic drug delivery of lipid vesicles. Understanding the process by which the pH-switchable lipids disturb the lipid assembly of nanoparticles and trigger the cargo release is crucial to optimize the rational design of pH-switchable lipids. Here, we gather morphological observations (FF-SEM, Cryo-TEM, AFM, confocal microscopy), physicochemical characterization (DLS, ELS), as well as phase behavior studies (DSC, 2H NMR, Langmuir isotherm, and MAS NMR) to propose a mechanism of pH-triggered membrane destabilization. We demonstrate that the switchable lipids are homogeneously incorporated with other co-lipids (DSPC, cholesterol, and DSPE-PEG2000) and promote a liquid-ordered phase insensitive to temperature variation. Upon acidification, the protonation of the switchable lipids triggers a conformational switch altering the self-assembly properties of lipid nanoparticles. These modifications do not lead to a phase separation of the lipid membrane; however, they cause fluctuations and local defects, which result in morphological changes of the lipid vesicles. These changes are proposed to affect the permeability of vesicle membrane, triggering the release of the cargo encapsulated in the lipid vesicles (LVs). Our results confirm that pH-triggered release does not require major morphological changes, but can result from small defects affecting the lipid membrane permeability.

Dynamic Sorting of Mobile and Rigid Molecules in Biomembranes by Magic-Angle Spinning 13C NMR

Understanding the membrane dynamics of complex systems is essential to follow their function. As molecules in membranes can be in a rigid or mobile state depending on external (temperature, pressure) or internal (pH, domains, etc.) conditions, we propose an in-depth examination of NMR methods to filter highly mobile molecular parts from others that are in more restricted environments. We have thus developed a quantitative magic-angle spinning (MAS) 13C NMR approach coupled with cross-polarization (CP) and/or Insensitive Nuclei Enhanced by Polarization Transfer (INEPT) on rigid and fluid unlabeled model membranes. We demonstrate that INEPT can detect only very mobile lipid headgroups in gel (solid-ordered) phases; the remaining rigid parts are only detected with CP. A direct correlation is established between the normalized line intensity as obtained by CP and the C-H (C-D) order parameters measured by wide-line 2H NMR or extracted from molecular dynamics: ICP/IDPeq ≈ 5|SCH|, indicating that when the order is greater than 0.2-0.3 (maximum value of 0.5 for chain CH2), only rigid parts can be filtered and detected using CP techniques. In very fluid (liquid-disordered) membranes, where there are many more active motions, both INEPT and CP detect resonances, with, however, a clear propensity of each technique to detect mobile and restricted molecular parts, respectively. Interestingly, the 13C NMR chemical shift of lipid hydrocarbon chains can be used to monitor order-disorder phase transitions and calculate the fraction of chain defects (rotamers) and the part of the transition enthalpy due to bond rotations (6-7 kJ·mol-1 for dimyristolphosphatidylcholine, DMPC). Cholesterol-containing membranes (liquid-ordered phases) can be dynamically contrasted as the rigid-body sterol is mainly detected by the CP technique, with a contact time of 1 ms, and the phospholipid by INEPT. Our work opens up a straightforward, robust, and cost-effective route for the determination of membrane dynamics by taking advantage of well-resolved conventional 13C NMR experiments without the need of isotopic labeling.

Collective dynamics in the lipid phases of DMPC/cholesterol bilayers: A neutron spin-echo study.

Cholesterol is essential in cell membranes as it regulates membrane structural and material properties such as phase behavior, elasticity, and viscosity. Cholesterol levels in different organelles vary, from less than 5% in mitochondria to 40% in the plasma membrane of eukaryotic cells. According to an experimental phase diagram of 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC)/cholesterol mixture based on lateral diffusion measurements from early studies, at the above melting temperature, the system undergoes a phase transition from a liquid-disordered (Ld) phase to a liquid-ordered (Lo) phase with a coexisting phase (Ld+Lo) in between upon increasing cholesterol concentration [1]. Our work attempts to understand the collective dynamics of the mixture in each phase and quantify their behavioral trends. We first performed densitometry to determine the mixture's molecular volume and to correct the melting temperature difference between protiated and deuterated DMPC, which was used in neutron experiments. The molecular volume was then implemented in small angle neutron scattering (SANS) experiments and data analysis to extract the area per lipid (AL) and thickness of the lipid mixture in each phase region. Finally, neutron spin echo (NSE) is used to access the dynamic properties by adopting structural information from SANS. The NSE results show the effective bending modulus and membrane viscosity of the Ld phase scale with AL, as observed in our previous study of mixed lipid membranes [2].

Small changes in lipid backbone linkages can lead to big changes in liquid-liquid phase separation in model membranes.

Cells can dynamically alter the spatial organization of lipids and proteins in the plasma membrane (PM) to regulate processes including cell signaling. The size and morphology of membrane domains, and how these depend on lipid composition, have drawn attention as potentially important variables in these processes. Model membrane studies have proven invaluable for elucidating the influence of phospholipid chains and headgroups, as well as cholesterol concentration, on membrane phase behavior. Following this approach, we have investigated simplified three- and four-component models for the PM outer leaflet to understand the role of the lipid backbone. Specifically, we replaced ester-linked DPPC and DOPC with their ether-linked counterparts to selectively change the membrane dipole potential of coexisting liquid-disordered (Ld) and liquid-ordered (Lo) phases, while titrating DOPC with POPC to control the line tension. Fluorescence imaging of GUVs gave insight into the macroscopic phase behavior, while FRET and cryo-EM of LUVs allowed us to probe nanoscale heterogeneity. Our results can be understood in terms of a model in which the tendency to minimize line tension is counteracted by dipole repulsion, which can in some cases result in stable arrays of small domains or modulated phases. These findings suggest that relatively small changes in the structure of the lipid backbone can influence domain formation and morphology in biomimetic membranes.

CAR modulates plasma membrane nano-organization and immune signaling downstream of RALF1-FERONIA signaling pathway.

In Arabidopsis, the receptor-like kinase (RLK) FERONIA (FER) senses peptide ligands in the plasma membrane (PM), modulates plant growth and development, and integrates biotic and abiotic stress signaling for downstream adaptive responses. However, the molecular interplay of these diverse processes is largely unknown. Here, we show that FER, the receptor of Rapid Alkalinization Factor 1 (RALF1), physically interacts with C2 domain ABA-related (CAR) proteins to control the nano-organization of the PM. During this process, the RALF1-FER pathway upregulates CAR protein translation, and then more CAR proteins are recruited to the PM. This acts as a rapid feedforward loop that stabilizes the PM liquid-ordered phase. FER interacts with and phosphorylates CARs, thereby reducing their lipid-binding ability and breaking the feedback regulation at later time points. The formation of the flg22-induced FLS2-BAK1 immune complex, which depends on the integrity of FER-containing nanodomains, is impaired in fer and pentuple car14569 mutant. Together, we propose that the FER-CAR module controls the formation of PM nano-organization during RALF signaling through a self-contained amplifying loop including both positive and negative feedback.

Designing a green-emitting viscosity-sensitive 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) probe for plasma membrane viscosity imaging.

Viscosity is a key characteristic of lipid membranes - it governs the passive diffusion of solutes and affects the lipid raft formation and membrane fluidity. Precise determination of viscosity values in biological systems is of great interest and viscosity-sensitive fluorescent probes offer a convenient solution for this task. In this work we present a novel membrane-targeting and water-soluble viscosity probe BODIPY-PM, which is based on one of the most frequently used probes BODIPY-C10. Despite its regular use, BODIPY-C10 suffers from poor integration into liquid-ordered lipid phases and lack of water solubility. Here, we investigate the photophysical characteristics of BODIPY-PM and demonstrate that solvent polarity only slightly affects the viscosity-sensing qualities of BODIPY-PM. In addition, with fluorescence lifetime imaging microscopy (FLIM), we imaged microviscosity in complex biological systems - large unilamellar vesicles (LUVs), tethered bilayer membranes (tBLMs) and live lung cancer cells. Our study showcases that BODIPY-PM preferentially stains the plasma membranes of live cells, equally well partitions into both liquid-ordered and liquid-disordered phases and reliably distinguishes lipid phase separation in tBLMs and LUVs.

Changes in Membrane Lipid Order of Cells by Optimized Microplasma Irradiation for High-Molecule Drug Absorption

Microplasma-based drug delivery, particularly transdermal drug delivery, has emerged as a promising alternatives to conventional methods due to its unique advantages. In this system, plasma components interact not only with the stratum corneum but also with the cells beneath it. Cell membranes mainly comprise lipids which are highly susceptible to plasma components, including reactive oxygen and nitrogen species. However, the effects of microplasma irradiation on cell membrane lipids are largely unexplored. In this context, the current study investigated an important physical property of cell membranes, membrane lipid order, in small intestinal epithelial cells after microplasma irradiation. First, the microplasma irradiation condition was optimized for the intracellular delivery of high molecular weight molecules, namely fluorescein isothiocyanate-dextran (FD-150 and FD-2000). Air microplasma discharge was applied at varying voltages (i.e., 3.5, 4.0, and 4.5 kV), and the efficiency of drug absorption was evaluated using a microplate reader and a fluorescence microscope. The cell viability was calculated using an automated cell counter. The most favorable outcomes in terms of both drug absorption and cell viability were achieved at 4 kV. Next, the membrane lipid order of cells was investigated using a solvatochromic dye after microplasma irradiation at 4 kV. Interestingly, non-irradiated cells exclusively manifested the liquid-ordered phase, whereas irradiated cells displayed a dual-phased composition comprising both liquid-ordered and liquid-disordered phases. In addition, live-cell imaging unveiled that alterations in membrane lipid order commenced as early as 4 min post-irradiation. This evidence points to a significant reduction in lipid packing and membrane thickness induced by microplasma irradiation. The findings shed light on the intricate mechanism underlying cellular drug absorption facilitated by microplasma irradiation. The insights gained into the impact on membrane lipid properties will contribute to developing safe and efficient drug delivery systems while minimizing side effects.

A red-emitting thiophene-modified BODIPY probe for fluorescence lifetime-based polarity imaging of lipid droplets in living cells.

Intracellular polarity in lipid droplets as well as other organelles may provide useful knowledge about various processes taking place in live cells. Therefore, small fluorophores capable of visualising polarity are undergoing rapid development. In this paper, we report new red-emitting polarity sensitive BODIPY probes that can distinguish between liquid-ordered and liquid-disordered phases and can internalise into lipid droplets of live cells. Our reported probes sense lipid environment not through solvatochromic shift of the fluorescence spectra but through the change in the fluorescence lifetime of their monoexponential decays. This makes them convenient for fluorescence lifetime imaging microscopy. The probes were synthesised by modifying viscosity-sensitive meso-phenyl BODIPY with electron-donating 2-thienyl moieties at the α- and β-positions, significantly red-shifting absorption and fluorescence spectra of the dyes and improving sensitivity to polarity, while suppressing viscosity dependence. Finally, a novel probe - BP OC16 TP2 was suitable for sensing polarity in lipid droplets of live MCF-7 human breast cancer cells. We demonstrated that different chemotherapeutics affected lipid droplet polarity differently: cisplatin had no effect on lipid droplet polarity, whereas paclitaxel, depending on its concentration, either decreased or increased lipid droplet polarity.

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.

Optimised generalized polarisation analysis of C-laurdan reveals clear order differences between T cell membrane compartments

Heterogenous packing of plasma membrane lipids is important for cellular processes like signalling, adhesion and sorting of membrane components. Solvatochromic membrane fluorophores that respond to changes from liquid-ordered (lo) phase to liquid-disordered (ld) by red shifts in their emission spectra are often used to assess lipid packing. Their response can be quantified using generalized polarisation (GP) using fluorescence microscopy images from two emission ranges, preferably from a region of interest (ROI) limited to a specific membrane compartment. However, image quality is limited by Poisson noise and convolution by the point spread function of the imaging system. Examining GP-analysis of C-laurdan labelled T cells using the image restoration procedure deconvolution, we demonstrate that deconvolution substantially improves the image resolution by making the plasma membrane clearly discernible and facilitating plasma membrane ROI selection. We conclude that automatic ROI selection has advantages over manual ROI selection when it comes to reproducibility and speed, but reliable GP-measurements can also be obtained by manually demarcated ROIs. We find that deconvolution enhances the difference in GP-values between the plasma and intracellular membranes and demonstrate that moving an intensity defined plasma membrane ROI outwards from the cell further improves this differentiation. By systematically changing the key deconvolution regularization parameter signal to noise, we establish a protocol for deconvolution optimisation applicable to any solvatochromic dye and imaging system. The image processing and ROI selection protocol presented improves both the resolution and precision of GP-measurement and will enable detection of smaller changes in membrane order than is currently achievable.

In Vitro Membrane Platform for the Visualization of Water Impermeability across the Liquid-Ordered Phase under Hypertonic Conditions.

Passive water penetration across the cell membrane by osmotic diffusion is essential for the homeostasis of cell volume, in addition to the protein-assisted active transportation of water. Since membrane components can regulate water permeability, controlling compositional variation during the volume regulatory process is a prerequisite for investigating the underlying mechanisms of water permeation and related membrane dynamics. However, the lack of a viable in vitro membrane platform in hypertonic solutions impedes advanced knowledge of cell volume regulation processes, especially cholesterol-enriched lipid domains called lipid rafts. By reconstituting the liquid-ordered (Lo) domain as a likeness of lipid rafts, we verified suppressed water permeation across the Lo domains, which had yet to be confirmed with experimental demonstrations despite a simulation approach. With the help of direct transfer of the Lo domains from vesicles to supported lipid membranes, the biological roles of lipid composition in suppressed water translocation were experimentally confirmed. Additionally, the improvement in membrane stability under hypertonic conditions was demonstrated based on molecular dynamics simulations.