Organization Of Biological Membranes Research Articles

Dehydration of Lipid Membranes Drives Redistribution of Cholesterol Between Lateral Domains.

Cholesterol-rich lipid rafts are found to facilitate membrane fusion, central to processes like viral entry, fertilization, and neurotransmitter release. While the fusion process involves local, transient membrane dehydration, the impact of reduced hydration on cholesterol's structural organization in biological membranes remains unclear. Here, we employ confocal fluorescence microscopy and atomistic molecular dynamics simulations to investigate cholesterol behavior in phase-separated lipid bilayers under controlled hydration. We unveiled that dehydration prompts cholesterol release from raft-like domains into the surrounding fluid phase. Unsaturated phospholipids undergo more significant dehydration-induced structural changes and lose more hydrogen bonds with water than sphingomyelin. The results suggest that cholesterol redistribution is driven by the equalization of biophysical properties between phases and the need to satisfy lipid hydrogen bonds. This underscores the role of cholesterol-phospholipid-water interplay in governing cholesterol affinity for a specific lipid type, providing a new perspective on the regulatory role of cell membrane heterogeneity during membrane fusion.

Hydrophobic mismatch drives self-organization of designer proteins into synthetic membranes

The organization of membrane proteins between and within membrane-bound compartments is critical to cellular function. Yet we lack approaches to regulate this organization in a range of membrane-based materials, such as engineered cells, exosomes, and liposomes. Uncovering and leveraging biophysical drivers of membrane protein organization to design membrane systems could greatly enhance the functionality of these materials. Towards this goal, we use de novo protein design, molecular dynamic simulations, and cell-free systems to explore how membrane-protein hydrophobic mismatch could be used to tune protein cotranslational integration and organization in synthetic lipid membranes. We find that membranes must deform to accommodate membrane-protein hydrophobic mismatch, which reduces the expression and co-translational insertion of membrane proteins into synthetic membranes. We use this principle to sort proteins both between and within membranes, thereby achieving one-pot assembly of vesicles with distinct functions and controlled split-protein assembly, respectively. Our results shed light on protein organization in biological membranes and provide a framework to design self-organizing membrane-based materials with applications such as artificial cells, biosensors, and therapeutic nanoparticles.

Miscibility of Phosphatidylcholines in Bilayers: Effect of Acyl Chain Unsaturation

The miscibility of phospholipids in a hydrated bilayer is an issue of fundamental importance for understanding the organization of biological membranes. Despite research on lipid miscibility, its molecular basis remains poorly understood. In this study, all-atom MD simulations complemented by Langmuir monolayer and DSC experiments have been performed to investigate the molecular organization and properties of lipid bilayers composed of phosphatidylcholines with saturated (palmitoyl, DPPC) and unsaturated (oleoyl, DOPC) acyl chains. The experimental results showed that the DOPC/DPPC bilayers are systems exhibiting a very limited miscibility (strongly positive values of excess free energy of mixing) at temperatures below the DPPC phase transition. The excess free energy of mixing is divided into an entropic component, related to the ordering of the acyl chains, and an enthalpic component, resulting from the mainly electrostatic interactions between the headgroups of lipids. MD simulations showed that the electrostatic interactions for lipid like-pairs are much stronger than that for mixed pairs and temperature has only a slight influence on these interactions. On the contrary, the entropic component increases strongly with increasing temperature, due to the freeing of rotation of acyl chains. Therefore, the miscibility of phospholipids with different saturations of acyl chains is an entropy-driven process.

Freeze-Fracture Electron Microscopy for Extracellular Vesicle Analysis.

Extracellular vesicles (EVs) are membrane-limited structures released from the cells into the extracellular space and are implicated in intercellular communication. EVs consist of three populations of vesicles, namely microvesicles (MVs), exosomes, and apoptotic bodies. The limiting membrane of EVs is crucially involved in the interactions with the recipient cells, which could lead to the transfer of biologically active molecules to the recipient cells and, consequently, affect their behavior. The freeze-fracture electron microscopy technique is used to study the internal organization of biological membranes. Here, we present a protocol for MV isolation from cultured cancerous urothelial cells and the freeze-fracture of MVs in the steps of rapid freezing, fracturing, making and cleaning the replicas, and analyzing them with transmission electron microscopy. The results show that the protocol for isolation yields a homogenous population of EVs, which correspond to the shape and size of MVs. Intramembrane particles are found mainly in the protoplasmic face of the limiting membrane. Hence, freeze-fracture is the technique of choice to characterize the MVs' diameter, shape, and distribution of membrane proteins. The presented protocol is applicable to other populations of EVs.

Fluorescent Probe for the Monitoring of Plasma Membrane Hydration

AbstractSubcellular microenvironment plays a crucial role in maintaining cell homeostasis being hydration an essential parameter to monitor the variations of lipid organization in biological membranes. Fluorescent microenvironment‐sensitive probes are stimuli‐responsive molecules and able to change their fluorescence profile according to the microenvironment properties (polarity, hydration, microviscosity and tension). Here we present an organic fluorescent probe, NPM‐1, as a new microenvironmental dye with selective plasma membrane localization in live cells. Using high‐resolution confocal microscopy and fluorescence spectroscopy, we explored the hydration and polarity variations during the formation of apoptotic bodies in real time. To have a clear understanding of the cell microenvironment variations, an analogue polarity probe able to localize the cytosol and nucleoli was also synthetized, CellPol‐1. Then, we were able to monitor the subcellular hydration variations during the apoptotic body dynamics.

Cholesterol’s balancing act: Defying the status quo

The structure and organization of biological membranes have fascinated biophysicists and cell biologists for decades. Some key findings that have energized the field include the discovery of domains in lipid mixtures (1) and the subsequent raft hypothesis linking them to cellular phenomena (2), as well as the discovery of compositional asymmetry in cell bilayers (3), which put lipid membranes on the map of energetically regulated physiological players. Almost all current knowledge about lipid and bilayer biophysics is rooted in studies of symmetric model membranes in the form of closed lipid vesicles.

Effect of Cholesterol Versus Ergosterol on DPPC Bilayer Properties: Insights from Atomistic Simulations.

Sterols have been ascribed a major role in the organization of biological membranes, in particular for the formation of liquid ordered domains in complex lipid mixtures. Here, we employed molecular dynamics simulations to compare the effects of cholesterol and ergosterol as the major sterol of mammalian and fungal cells, respectively, on binary mixtures with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) as a proxy for saturated lipids. In agreement with previous work, we observe that the addition of sterol molecules modifies the order of DPPC both in the gel phase and in the liquid phase. When disentangling the overall tilt angle and the structure of the tail imposed by trans/gauche configurations of torsion angles in the tail, respectively, a more detailed picture of the impact of sterols can be formulated, revealing, for example, an approximate temperature-concentration superposition ranging from the liquid to the gel phase. Furthermore, a new quantitative measure to identify the presence of collective sterol effects is discussed. Moreover, when comparing both types of sterols, addition of cholesterol has a noticeably stronger impact on phospholipid properties than that of ergosterol. The observed differences can be attributed to higher planarity of the cholesterol ring system. This planarity combined with an inherent asymmetry in its molecular interactions leads to better alignment and hence stronger interaction with saturated acyl chains. Our results suggest that the high order demonstrated for ergosterol in fungal plasma membranes must therefore be generated via additional mechanisms.

The Role of Growth Temperature and Lipid Composition in Phase Separation of Yeast Vacuole Membranes

Membranes of vacuoles, the lysosomal organelle in yeast, phase separate into two coexisting liquid phases. This phenomenon provides a physical mechanism for lateral organization of biological membranes. Recent studies show that these domains play a key role in a central eukaryotic signaling pathway. Howthe cell adapts and tunes its vacuole membrane to invoke phase separation is still largely unknown. Here we examine how membrane composition changes due to two different environmental stimuli: growth temperature and nutrient depletion. If it is important for cells to regulate phase separation of their membranes, then we expect yeast cells to acclimate to external conditions. We test this hypothesis by growing yeast at 25°C and 30°C and find that the yeast adapts to this change by dramatically shifting the demixing temperature of vacuole membranes (to 29.7°C and 42.7°C respectively). This stark change in vacuole miscibility temperatures scales with growth temperature both in live cells and in isolated vacuoles, showing that yeast alter their membrane compositions to maintain phase separation. We also directly investigate the role of ergosterol, the main sterol in yeast, in domain formation in vacuoles. The yeast growth cycle starts with a logarithmic phase of rapid growth followed by a stationary phase in which glucose becomes scarce and vacuole membranes exhibit coexisting liquid domains. We isolate vacuoles from yeast in the logarithmic phase of growth, in which the membranes do not natively exhibit domains. Using fluorescence microscopy, we observe changes in vacuole phase separation as a function of ergosterol content. We find the surprising result that domains appear in vacuole membranes with reduced ergosterol.

Direct label-free imaging of nanodomains in biomimetic and biological membranes by cryogenic electron microscopy

The nanoscale organization of biological membranes into structurally and compositionally distinct lateral domains is believed to be central to membrane function. The nature of this organization has remained elusive due to a lack of methods to directly probe nanoscopic membrane features. We show here that cryogenic electron microscopy (cryo-EM) can be used to directly image coexisting nanoscopic domains in synthetic and bioderived membranes without extrinsic probes. Analyzing a series of single-component liposomes composed of synthetic lipids of varying chain lengths, we demonstrate that cryo-EM can distinguish bilayer thickness differences as small as 0.5 Å, comparable to the resolution of small-angle scattering methods. Simulated images from computational models reveal that features in cryo-EM images result from a complex interplay between the atomic distribution normal to the plane of the bilayer and imaging parameters. Simulations of phase-separated bilayers were used to predict two sources of contrast between coexisting ordered and disordered phases within a single liposome, namely differences in membrane thickness and molecular density. We observe both sources of contrast in biomimetic membranes composed of saturated lipids, unsaturated lipids, and cholesterol. When extended to isolated mammalian plasma membranes, cryo-EM reveals similar nanoscale lateral heterogeneities. The methods reported here for direct, probe-free imaging of nanodomains in unperturbed membranes open new avenues for investigation of nanoscopic membrane organization.

The insecticide fipronil affects the physical properties of model membranes: A combined experimental and molecular dynamics simulations study in Langmuir monolayers

Fipronil is a widely used commercial insecticide whose action mechanism consists in blocking the influx of chloride ions through the γ-aminobutyric acid type A receptor (GABAA-R), an integral membrane protein. The present study investigates the interaction of fipronil with phospholipid Langmuir monolayers, in order to characterize the effects that its partition could exert on the physical properties of these model membranes. A combined experimental and molecular dynamics (MD) simulations approach was performed. MD simulations were conducted in such a way that they resemble an experimental compression isotherm of DPPC in the presence of fipronil in the aqueous subphase. Both the experimental and the simulated compression isotherm showed that the partition of fipronil between DPPC molecules induces an expansion of the monolayer. Experimental results also showed that fipronil can penetrate lipid monolayers even in condensed packing states. MD simulations showed that fipronil induces an ordering effect in the acyl chains of DPPC in the liquid-condensed phase. In addition, the simulations indicate that fipronil orients parallel to the plane of the monolayer and that it establishes hydrogen bonds with the glycerol region of DPPC. Free energy profiles of the partition of fipronil into the monolayers, obtained by means of umbrella sampling, indicated that its penetration is thermodynamically favorable, being the interphase between the glycerol region and the acyl chains of DPPC its most favorable location. Our results suggest that fipronil could modulate the supramolecular organization of biological membranes surrounding GABAA-R, contributing, at least in part, to its action mechanism.

Principles and Applications of Biological Membrane Organization.

Many critical biological events, including biochemical signaling, membrane traffic, and cell motility, originate at membrane surfaces. Each such event requires that members of a specific group of proteins and lipids rapidly assemble together at a specific site on the membrane surface. Understanding the biophysical mechanisms that stabilize these assemblies is critical to decoding and controlling cellular functions. In this article, we review progress toward a quantitative biophysical understanding of the mechanisms that drive membrane heterogeneity and organization. We begin from a physical perspective, reviewing the fundamental principles and key experimental evidence behind each proposed mechanism. We then shift to a biological perspective, presenting key examples of the role of heterogeneity in biology and asking which physical mechanisms may be responsible. We close with an applied perspective, noting that membrane heterogeneity provides a novel therapeutic target that is being exploited by a growing number of studies at the interface of biology, physics, and engineering.

Degeneracy in molecular scale organization of biological membranes.

The scale-rich spatiotemporal organization in biological membranes has its origin in the differential inter- and intra-molecular interactions among their constituents. In this work, we explore the molecular-origin behind that variety and possible degeneracy in lateral organization in membranes. For our study, we post-process microsecond long all-atom molecular dynamics trajectories for three systems that exhibit fluid phase coexistence: (i) PSM/POPC/Chol (0.47/0.32/0.21), (ii) PSM/DOPC/Chol (0.43/0.38/0.19) and (iii) DPPC/DOPC/Chol (0.37/0.36/0.27). To distinguish the liquid ordered and disordered regions at molecular scales, we calculate the degree of non-affineness of individual lipids in their neighbourhood and track their topological rearrangements. Disconnectivity graph analysis with respect to membrane organization shows that the DPPC/DOPC/Chol and PSM/DOPC/Chol systems exhibit funnel-like energy landscapes as opposed to a highly frustrated energy landscape for the more biomimetic PSM/POPC/Chol system. We use these measurements to develop a continuous lattice Hamiltonian and evolve that using Monte Carlo simulated annealing to explore the possibility of structural degeneracy in membrane organization. Our data show that model membranes with lipid constituents that are biomimetic (PSM/POPC/Chol) have the ability to access a large range of membrane sub-structure space (higher degeneracy) as compared to the other two systems, which form only one kind of substructure even with changing composition. Since the spatiotemporal organization in biological membranes dictates the "molecular encounters" and in turn larger scale biological processes such as molecular transport, trafficking and cellular signalling, we posit that this structural degeneracy could enable access to a larger repository to functionally important molecular organization in systems with physiologically relevant compositions.

Interaction of Graphene Nanoparticles and Lipid Membranes Displaying Different Liquid Orderings: A Molecular Dynamics Study.

Understanding the effects of graphene-based nanomaterials on lipid membranes is fundamental to determine their environmental impact and the efficiency of their biomedical use. By means of molecular dynamics simulations of simple model lipid bilayers, we analyze in detail the different interaction modes. We have studied bilayers consisting of lipid species (including cholesterol) which display different internal liquid orderings. Nanometric graphene layers can be transiently adsorbed onto the lipid membrane and/or inserted in its hydrophobic region. Once inserted, graphene nanometric flakes display a diffusive dynamics in the membrane plane, they adopt diverse orientations depending on their size and oxidation degree, and they show a particular aversion to be placed close to cholesterol molecules in the membrane. Addition of graphene to phase-segregated ternary membranes is also investigated in the context of the lipid raft model for the lipid organization of biological membranes. Our simulation results show that graphene layers can be inserted indistinctly in the ordered and disordered regions. Once inserted, nanometric flakes migrate to disordered and cholesterol-poor lipid phases.

The Influence of Calcium Traces in Ultrapure Water on the Lateral Organization in Tetramyristoyl Cardiolipin Monolayers.

Cardiolipin (CL) plays an important role in administering the structural organization of biological membranes and therefore helps maintaining the integer membrane functionality. CL has a dimeric structure consisting of four acyl chains and two phosphate groups. With its unusual structure, the phospholipid is responsible for curvature formation in CL containing biological membranes. The acidic properties of the phosphate head groups are still not sufficiently investigated since there are controversial results in literature. The main aim of this work was to gain deeper insights into the influence of the pH on the ionization degree of CL. During the experiments, it became clear that the used ultrapure water contained traces of calcium. These unexpected calcium ions had a major impact on the behavior of CL monolayers. Therefore, the focus was put on the analysis of CL layers without and with EDTA in the subphase used to complex divalent calcium ions. For the analyses, traditional surface pressure (π) - molecular area (A) isotherm experiments combined with total reflection x-ray fluorescence (TRXF) and grazing incidence x-ray diffraction (GIXD) have been used.

Mass spectrometry-A versatile tool for characterising the lipid environment of membrane protein assemblies.

Biological membranes are selectively permeable barriers important for cell organization and compartmentalization. Their organisation strongly depends on the lipids that constitute the lipid bilayer as well as the proteins that reside in the membrane. Unravelling the organisation of biological membranes is therefore of great importance to understand cellular function driven by integral and peripheral membrane proteins. Recent developments in mass spectrometry made it a powerful tool contributing to our present-day understanding of membrane composition and organisation. The two main deliverables of mass spectrometry are (i) the identification and quantification of the membrane components, and (ii) the analysis of their structural arrangements. In this review article, we first briefly discuss the aspects of membrane organization that are accessible through mass spectrometry. We then provide detailed insights into the various mass spectrometric strategies which help identifying lipids from membranes or membrane protein assemblies, unravelling the lipid binding modes in membrane proteins and uncovering their structural roles. We further discuss the growing interest in membrane mimetics providing membrane proteins with a native-like lipid environment for structural and functional studies and the possibilities of mass spectrometry to contribute in these experiments.

Biochemical effects of low-dose whole body x-irradiation on mouse liver and the protective action of ectoine

Purpose: To investigate the possible role of ectoine, which is known to protect protein hydration and folding and structural organization of biological membranes, in protecting mouse liver against low-dose x-irradiation. Methods: The study included thirty Swiss albino mice weighing 20 - 22 g each, which were divided into five groups of six animals each: group (1) naive animals serving as control, group (2) animals irradiated with a whole-body 2 Gy single dose of x-ray, and sacrificed after one day, group (3) x-irradiated and sacrificed after one week (each animal in the 3 groups received 0.2 mL saline daily by oral gavage). Group 4 consisted of x-irradiated animals dosed with ectoine 200 mg/kg in saline and sacrificed after one day. Group 5 cmoprised of x-irradiated animals and were dosed daily with ectoine 200 mg/kg and sacrificed after one week. The evaluated inflammation parameters in the animals’ liver were interleukin- 1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10) and prostaglandin E2 (PGE2). Furthermore, the oxidative stress parameters, viz, malondialdehyde (MDA), reduced glutathione (GSH), oxidized glutathione (GSSG) and their ratio (GSH/GSSG) were also assessed. Results: Whole body low-dose x-irradiation resulted in statistically significant (p ≤ 0.05) increases in all ILs tested as well as PGE2 of mice liver. Indications of oxidative stress included elevated levels of MDA and oxidized glutathione with decreased reduced glutathione. The effects of radiation were progressive and the changes in the tested parameters increased from day 1 to day 7. Administration of ectoine significantly (p ≤ 0.05) ameliorated the biochemical effects induced by whole body x-irradiation. Furthermore, the modulating action was dependent on the accumulation of ectoine, as it was more effective after repeated administration. Conclusion: Ectoine has a post-irradiation protective effect on mouse liver via its action on inflammatory and oxidative stress pathways. It probably has similar action in other vital organs. Preventive treatment of healthcare personnel and technicians frequently exposed to low doses of radiation with ectoine is worth investigating. Keywords: X-irradiation, Ectoine, Oxidative stress, Mice, Radioprotection

Complex Phase Behavior of GUVs Containing Different Sphingomyelins

Understanding the lateral organization of biological membranes plays a key role on the road to fully appreciate the physiological functions of this fundamental barrier between the inside and outside regions of a cell. Ternary lipid bilayers composed of a high and a low melting temperature lipid and cholesterol represent a model system that mimics some of the important thermodynamical features of much more complex lipid mixtures such as those found in mammal membranes. The phase diagram of these ternary mixtures can be studied exploiting fluorescence microscopy in giant unilamellar vesicles, and it is typically expected to give rise, for specific combinations of composition and temperature, to regions of two-phase coexistence and a region with three-phase coexistence, namely, the liquid-ordered, liquid-disordered, and solid phases. Whereas the observation of two-phase coexistence is routinely possible using fluorescence microscopy, the three-phase region is more elusive to study. In this article, we show that particular lipid mixtures containing diphytanoyl-phosphatidylcholine and cholesterol plus different types of sphingomyelin (SM) are prone to produce bilayer regions with more than two levels of fluorescence intensity. We found that these intensity levels occur at low temperature and are linked to the copresence of long and asymmetric chains in SMs and diphytanoyl-phosphatidylcholine in the lipid mixtures. We discuss the possible interpretations for this observation in terms of bilayer phase organization in the presence of sphingolipids. Additionally, we also show that in some cases, liposomes in the three-phase coexistence state exhibit extreme sensitivity to lateral tension. We hypothesize that the appearance of the different phases is related to the asymmetric structure of SMs and to interdigitation effects.

Capturing suboptical dynamic structures in lipid bilayer patches formed from free-standing giant unilamellar vesicles.

There is accumulating evidence that the small-scale lateral organization of biological membranes has a crucial role in signaling and trafficking in cells. However, it has been difficult to characterize these features with existing methods for preparing and analyzing freestanding membranes, because the dynamics occurs below the optical resolution possible with these protocols. We have developed a protocol that permits the imaging of lipid nanodomains and lateral protein organization in membranes of giant unilamellar vesicles (GUVs). Freestanding GUVs are transferred onto a mica support, and after treatment with magnesium chloride, they collapse to form planar lipid bilayer (PLB) patches. Rapid GUV collapse onto the mica preserves the lateral organization of freestanding membranes and thus makes it possible to image 'snapshots' of GUVs up to nanometer resolution by high-resolution microscopy. The method has been applied to classical lipid raft mixtures in which suboptical domain fluctuations have been imaged in both the liquid-ordered and liquid-disordered membrane phases. High-resolution scanning by atomic force microscopy (AFM) of membranes composed of binary and ternary lipid mixtures reconstituted with Na+/K+-ATPase (NKA) has revealed the spatial distribution and orientations of individual proteins, as well as details of membrane lateral structure. Immunolabeling followed by confocal microscopy can also provide information about the spatial distribution of proteins. The protocol opens up a new avenue for quantitative biophysical studies of suboptical dynamic structures in biomembranes, which are local and short-lived. Preparation of GUVs, PLB patches and their imaging takes <24 h.

Neutron Scattering to Study Membrane Systems: From Model Membranes to Living Cells

The existence and role of lateral lipid organization in biological membranes has been studied and contested for more than 30 years. Lipid domains, or rafts, are hypothesized as scalable compartments in biological membranes, providing appropriate physical environments to their resident membrane proteins. This implies that lateral lipid organization is associated with a range of biological functions, such as protein co-localization, membrane trafficking, and cell signaling, to name just a few. Neutron scattering techniques have proven to be an excellent tool to investigate these structural features in model lipids, and more recently, in living cells. I will discuss our recent work using neutrons to probe the structure and mechanical properties in model lipid systems and our current efforts in using neutrons to probe the structure and organization of the bilayer in a living cell. These efforts in living cells have used genetic and biochemical strategies to generate a large neutron scattering contrast, making the membrane visible. I will present our results showing in vivo bilayer structure and discuss the outlook for this approach.

Nanometer-Scale Lipid Clusters in Model Membranes Revealed by Atomic Recombination in Nanosims

Much of what is known about the lateral organization of biological membranes is inferred from the analogy between phase diagrams of ternary mixtures of lipids and the plasma membrane of mammalian cells. However, the relevance of phase diagrams of simple lipid mixtures to the compositionally complex and dynamic plasma membrane has not yet been established. Additionally, the fluorescence and spin labels used to build phase diagrams often interact with lipid bilayers, and detecting nanometer-scale phases remains challenging. We have previously introduced atomic recombination in dynamic SIMS as a technique for detecting lateral heterogeneities that occur on the nanometer length scale, below the resolution of both fluorescence microscopy and conventional NanoSIMS imaging. In this method, the formation of 13C15N- secondary ions from 13C and 15N atoms installed on different lipids only occurs if the isotopically-labeled lipids are within approximately 3 nm of each other. Isotope labels are desirable because they avoid problems caused by fluorescent dyes and because they directly report on the organization of the lipids of interest. Facile isotope-labeling chemistry allows the labels to be placed on any lipid of interest, allowing us to determine which lipids are clustering in complex mixtures. Here, we expand this technique to study the formation and composition of nanodomains of lipids in more complex mixtures for which phase diagrams have not been determined and would be too laborious to fully solve. Furthermore, we explore the formation of lipid clusters outside of phase boundaries, where lipids may still be inhomogenously distributed but display no first order phase transition. These more complex model membranes may be better models for lipid rafts given that lipids in the plasma membrane show no micrometer scale phase separation and no first order phase transition.