Membrane Lateral Organization Research Articles

Tensing Flipper: Photosensitized Manipulation of Membrane Tension, Lipid Phase Separation, and Raft Protein Sorting in Biological Membranes.

The lateral organization of proteins and lipids in the plasma membrane is fundamental to regulating a wide range of cellular processes. Compartmentalized ordered membrane domains enriched with specific lipids, often termed lipid rafts, have been shown to modulate the physicochemical and mechanical properties of membranes and to drive protein sorting. Novel methods and tools enabling the visualization, characterization, and/or manipulation of membrane compartmentalization are crucial to link the properties of the membrane with cell functions. Flipper, a commercially available fluorescent membrane tension probe, has become a reference tool for quantitative membrane tension studies in living cells. Here, we report on a so far unidentified property of Flipper, namely, its ability to photosensitize singlet oxygen (1O2) under blue light when embedded into lipid membranes. This in turn results in the production of lipid hydroperoxides that increase membrane tension and trigger phase separation. In biological membranes, the photoinduced segregated domains retain the sorting ability of intact phase-separated membranes, directing raft and nonraft proteins into ordered and disordered regions, respectively, in contrast to radical-based photo-oxidation reactions that disrupt raft protein partitioning. The dual tension reporting and photosensitizing abilities of Flipper enable simultaneous visualization and manipulation of the mechanical properties and lateral organization of membranes, providing a powerful tool to optically control lipid raft formation and to explore the interplay between membrane biophysics and cell function.

Membrane-Active Antibiotics Affect Domains in Bacterial Membranes as the First Step of Their Activity.

The need to combat antimicrobial resistance is becoming more and more pressing. Here we investigate the working mechanism of a small cationic agent, N-alkylamide 3d, by conventional and high-speed atomic force microscopy. We show that N-alkylamide 3d interacts with the membrane of Staphylococcus aureus, where it changes the organization and dynamics of lipid domains. After this initial step, supramolecular structures of the antimicrobial agent attach on top of the affected membrane gradually, covering it entirely. These results demonstrate that lateral domains in the bacterial membranes might be affected by small antimicrobial agents more often than anticipated. At the same time, we show a new dual-step activity of N-alkylamide 3d that not only destroys the lateral membrane organization but also effectively covers the whole membrane with aggregates. This final step could render the membrane inaccessible from the outside and possibly prevent signaling and waste disposal of living bacteria.

Antimicrobial Peptides Increase Line Tension in Raft-Forming Lipid Membranes.

The formation of phase separated membrane domains is believed to be essential for the function of the cell. The precise composition and physical properties of lipid bilayer domains play crucial roles in regulating protein activity and governing cellular processes. Perturbation of the domain structure in human cells can be related to neurodegenerative diseases and cancer. Lipid rafts are also believed to be essential in bacteria, potentially serving as targets for antibiotics. An important question is how the membrane domain structure is affected by bioactive and therapeutic molecules, such as surface-active peptides, which target cellular membranes. Here we focus on antimicrobial peptides (AMPs), crucial components of the innate immune system, to gain insights into their interaction with model lipid membranes containing domains. Using small-angle neutron/X-ray scattering (SANS/SAXS), we show that the addition of several natural AMPs (indolicidin, LL-37, magainin II, and aurein 2.2) causes substantial growth and restructuring of the domains, which corresponds to increased line tension. Contrast variation SANS and SAXS results demonstrate that the peptide inserts evenly in both phases, and the increased line tension can be related to preferential and concentration dependent thinning of the unsaturated membrane phase. We speculate that the lateral restructuring caused by the AMPs may have important consequences in affecting physiological functions of real cells. This work thus shines important light onto the complex interactions and lateral (re)organization in lipid membranes, which is relevant for a molecular understanding of diseases and the action of antibiotics.

Membrane lateral organization from potential energy disconnectivity graph

Understanding the thermodynamic and kinetic properties of biomolecules requires elucidation of their complex energy landscape. A disconnectivity graph analysis of the energy landscape provides a framework for mapping the multi-dimensional landscape onto a two-dimensional representation while preserving the key features of the energy landscape. Several studies show that the structure or shape of the disconnectity graph is directly associated with the function of protein and nucleic acid molecules. In this review, we discuss how disconnectivity analysis of the potential energy surface can be extended to lipid molecules to glean important information about membrane organization. The shape of the disconnectivity graphs can be used to predict the lateral organization of multi-component lipid bilayer. We hope that this review encourages the use of disconnectivity graphs routinely by membrane biophysicists to predict the lateral organization of lipids.

Multidimensional Spectral Phasors of LAURDAN's Excitation-Emission Matrices: The Ultimate Sensor for Lipid Phases?

The impact of lipid diversity on the lateral organization of biological membranes remains a topic of debate. While the existence of domains in lamellar membranes is well-established, the nonlamellar phases occurring in biological systems are less explored due to technical constraints. Here, we present the measurement of the excitation-emission matrices (EEM) of LAURDAN in several lipid structures. LAURDAN is a fluorescence probe widely used for characterizing lipid assemblies. The EEMs were analyzed by multidimensional spectral phasors (MdSP), an approach that seizes information from both the excitation and emission spectra. We developed a computer algorithm to construct EEM data based on a model for LAURDAN's photophysics. The MdSP calculated from the simulated EEMs reveals that all feasible possibilities lie inside a universal triangle in the phasor's plot. We use this triangle to propose a ternary representation for the phasors, allowing a better assessment of LAURDAN's surroundings in terms of hydration, water mobility, and local electronic environment. Building upon this foundation, we constructed a theoretical "phase map" that can assess both lamellar and nonlamellar membranes. We thoroughly validated this theory using well-known lipid mixtures under different phase-state conditions and enzymatically generated systems. Our results confirm that the use of MdSP is a powerful tool for obtaining quantitative information on both lamellar and nonlamellar structures. This study not only advances our understanding of the impact of lipid diversity on membrane organization but also provides a robust and general framework for the assessment of fluorescence properties that can be further extended to other probes.

Non-affine deformation analysis and 3D packing defects: A new way to probe membrane heterogeneity in molecular simulations.

Here, we discuss a new framework developed over the last 5 years in our group to probe nanoscale membrane heterogeneity. The framework is based on the idea of characterizing lateral heterogeneity through non-affine deformation (NAD) measurements, transverse heterogeneity through three dimensional (3D) lipid packing defects, and using these approaches to formalize the seemingly trivial correlation between lateral organization and lipid packing in biological membranes. We find that measurements from NAD analysis, a prescription which is borrowed from Physics of glasses and granular material, can faithfully distinguish between liquid-ordered and disordered phases in membranes at molecular length scalesand, can also be used to identify phase boundaries with high precision. Concomitantly, 3D-packing defects can not only distinguish between the two co-existing fluid phases based on their molecular scale packing (or membrane free volume), but also provide a route to connect the membrane domains to their functionality, such as exploring the molecular origins of inter-leaflet domain registration and peptide partitioning. The correlation between lateral membrane order and transverse packing presents novel molecular design-level features that can explain functions such as protein/peptide partitioning and small-molecule permeation dynamics in complex and heterogeneous membranes with high-fidelity. The framework allows us to explore the nature of lateral organization and molecular packing as a manifestation of intricate molecular interactions among a chemically rich variety of lipids and other molecules in a membrane with complex membrane composition and asymmetry across leaflets.

Lateral membrane organization as target of an antimicrobial peptidomimetic compound

Antimicrobial resistance is one of the leading concerns in medical care. Here we study the mechanism of action of an antimicrobial cationic tripeptide, AMC-109, by combining high speed-atomic force microscopy, molecular dynamics, fluorescence assays, and lipidomic analysis. We show that AMC-109 activity on negatively charged membranes derived from Staphylococcus aureus consists of two crucial steps. First, AMC-109 self-assembles into stable aggregates consisting of a hydrophobic core and a cationic surface, with specificity for negatively charged membranes. Second, upon incorporation into the membrane, individual peptides insert into the outer monolayer, affecting lateral membrane organization and dissolving membrane nanodomains, without forming pores. We propose that membrane domain dissolution triggered by AMC-109 may affect crucial functions such as protein sorting and cell wall synthesis. Our results indicate that the AMC-109 mode of action resembles that of the disinfectant benzalkonium chloride (BAK), but with enhanced selectivity for bacterial membranes.

DNA-Origami Line-Actants Control Domain Organization and Fission in Synthetic Membranes.

Cells can precisely program the shape and lateral organization of their membranes using protein machinery. Aiming to replicate a comparable degree of control, here we introduce DNA-origami line-actants (DOLAs) as synthetic analogues of membrane-sculpting proteins. DOLAs are designed to selectively accumulate at the line-interface between coexisting domains in phase-separated lipid membranes, modulating the tendency of the domains to coalesce. With experiments and coarse-grained simulations, we demonstrate that DOLAs can reversibly stabilize two-dimensional analogues of Pickering emulsions on synthetic giant liposomes, enabling dynamic programming of membrane lateral organization. The control afforded over membrane structure by DOLAs extends to three-dimensional morphology, as exemplified by a proof-of-concept synthetic pathway leading to vesicle fission. With DOLAs we lay the foundations for mimicking, in synthetic systems, some of the critical membrane-hosted functionalities of biological cells, including signaling, trafficking, sensing, and division.

Physical Concept to Explain the Regulation of Lipid Membrane Phase Separation under Isothermal Conditions.

Lateral phase separation within lipid bilayer membranes has attracted considerable attention in the fields of biophysics and cell biology. Living cells organize laterally segregated compartments, such as raft domains in an ordered phase, and regulate their dynamic structures under isothermal conditions to promote cellular functions. Model membrane systems with minimum components are powerful tools for investigating the basic phenomena of membrane phase separation. With the use of such model systems, several physicochemical characteristics of phase separation have been revealed. This review focuses on the isothermal triggering of membrane phase separation from a physical point of view. We consider the free energy of the membrane that describes lateral phase separation and explain the experimental results of model membranes to regulate domain formation under isothermal conditions. Three possible regulation factors are discussed: electrostatic interactions, chemical reactions and membrane tension. These findings may contribute to a better understanding of membrane lateral organization within living cells that function under isothermal conditions and could be useful for the development of artificial cell engineering.

Membrane Models and Experiments Suitable for Studies of the Cholesterol Bilayer Domains.

Cholesterol (Chol) is an essential component of animal cell membranes and is most abundant in plasma membranes (PMs) where its concentration typically ranges from 10 to 30 mol%. However, in red blood cells and Schwann cells, PMs Chol content is as high as 50 mol%, and in the PMs of the eye lens fiber cells, it can reach up to 66 mol%. Being amphiphilic, Chol molecules are easily incorporated into the lipid bilayer where they affect the membrane lateral organization and transmembrane physical properties. In the aqueous phase, Chol cannot form free bilayers by itself. However, pure Chol bilayer domains (CBDs) can form in lipid bilayer membranes with the Chol content exceeding 50 mol%. The range of Chol concentrations surpassing 50 mol% is less frequent in biological membranes and is consequently less investigated. Nevertheless, it is significant for the normal functioning of the eye lens and understanding how Chol plaques form in atherosclerosis. The most commonly used membrane models are unilamellar and multilamellar vesicles (MLVs) and supported lipid bilayers (SLBs). CBDs have been observed directly using confocal microscopy, X-ray reflectometry and saturation recovery electron paramagnetic resonance (SR EPR). Indirect evidence of CBDs has also been reported by using atomic force microscopy (AFM) and fluorescence recovery after photobleaching (FRAP) experiments. The overall goal of this review is to demonstrate the advantages and limitations of the various membrane models and experimental techniques suitable for the detection and investigation of the lateral organization, function and physical properties of CBDs.

Stability of supported hybrid lipid bilayers on chemically and topographically-modified surfaces

Hybrid lipid bilayers are a particular case of supported lipid bilayers with the two monolayer leaflets composed by different types of molecules. These nanostructures can be produced in a well-controlled array fashion and are suitable for the study of biomembrane-related phenomena via electrochemical or plasmonic sensing. Understanding how the underlying solid surface affects the supported membrane formation and organization is necessary for the potential use of these hybrid platforms in applications for which surfaces are not flat and topographically complex. Here we assess the role of lipid phase, substrate surface energy and topography on the formation and stability of hybrid supported membranes from vesicle precursors using complementary surface-sensitive techniques, namely quartz crystal microbalance with dissipation and atomic force microscopy. The stability of hybrid bilayers against thermal and osmotic changes is evaluated and compared to standard supported lipid bilayers formed onto hydrophilic SiO2. Force spectroscopy measurements reveal an overall weaker lateral organization of hybrid membranes as a result of the underlying self-assembled monolayer being not optimally organized. Hybrid bilayers display a decoupled behavior between the two leaflets when vertically compressed at constant speed. On microcontact printed Au surfaces, hybrid bilayers were formed over printed patches, while surprisingly, supported lipid bilayers were observed on non-patterned Au regions suggesting a non-trivial self-assembled monolayer reorganization when in aqueous environment.

Amphiphilic Co-Solvents Modulate the Structure of Membrane Domains

Biofuels are an increasing part of the sustainable energy picture. This makes it a societal and economic imperative to optimize biofuel production. Mitigating the toxic effects of amphiphilic co-solvents is one way to improve the efficiency of biofuel production. Amphiphiles partition into cellular membranes, leading to membrane thinning, destabilization, loss of membrane potential, and, ultimately, cell death. However, this picture of solvent toxicity misses the disruptive impact of co-solvents on lateral membrane organization, which is increasingly recognized as critical for membrane protein sorting and oligomerization. The alteration or disruption of membrane domains has deleterious effects on cellular processes. In this work, we pursue the hypothesis that membrane lateral organization is disrupted by the presence of co-solvents at concentrations lower than those which lead to full membrane destabilization. The disruption occurs due to an increasing interfacial tension between the co-existing phases, resulting in conformational changes to minimize the interfacial length-to-area ratio. This represents an unrecognized mode of solvent-induced stress and a new target for interventions to improve fermentation yields.

Peptide-membrane binding is not enough to explain bioactivity: A case study

Membrane-active peptides are a promising class of antimicrobial and anticancer therapeutics. For this reason, their molecular mechanisms of action are currently actively investigated. By exploiting Electron Paramagnetic Resonance, we study the membrane interaction of two spin-labeled analogs of the antimicrobial and cytotoxic peptide trichogin GA IV (Tri), with opposite bioactivity: Tri(Api8), able to selectively kill cancer cells, and Tri(Leu4), which is completely nontoxic. In our attempt to determine the molecular basis of their different biological activity, we investigate peptide impact on the lateral organization of lipid membranes, peptide localization and oligomerization, in the zwitter-ionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) model membrane We show that, despite their divergent bioactivity, both peptide analogs (i) are membrane-bound, (ii) display a weak tendency to oligomerization, and (iii) do not induce significant lipid rearrangement. Conversely, literature data show that the parent peptide trichogin, which is cytotoxic without any selectivity, is strongly prone to dimerization and affects the reorganization of POPC membranes. Its dimers are involved in the rotation around the peptide helix, as observed at cryogenic temperatures in the millisecond timescale. Since this latter behavior is not observed for the inactive Tri(Leu4), we propose that for short-length peptides as trichogin oligomerization and molecular motions are crucial for bioactivity, and membrane binding alone is not enough to predict or explain it. We envisage that small changes in the peptide sequence that affect only their ability to oligomerize, or their molecular motions inside the membrane, can tune the peptide activity on membranes of different compositions.

Enthalpic and entropic contributions to interleaflet coupling drive domain registration and antiregistration in biological membrane.

Biological membrane is a complex self-assembly of lipids, sterols, and proteins organized as a fluid bilayer of two closely stacked lipid leaflets. Differential molecular interactions among its diverse constituents give rise to heterogeneities in the membrane lateral organization. Under certain conditions, heterogeneities in the two leaflets can be spatially synchronized and exist as registered domains across the bilayer. Several contrasting theories behind mechanisms that induce registration of nanoscale domains have been suggested. Following a recent study showing the effect of position of lipid tail unsaturation on domain registration behavior, we decided to develop an analytical theory to elucidate the driving forces that create and maintain domain registry across leaflets. Towards this, we formulated a Hamiltonian for a stacked lattice system where site variables capture the lipid molecular properties such as the position of unsaturation and various other interactions that could drive phase separation and interleaflet coupling. We solve the Hamiltonian using Monte Carlo simulations and create a complete phase diagram that reports the presence or absence of registered domains as a function of various Hamiltonian parameters. We find that the interleaflet coupling should be described as a competing enthalpic contribution due to interaction of lipid tail termini, primarily due to saturated-saturated interactions, and an interleaflet entropic contribution from overlap of unsaturated tail termini. A higher position of unsaturation is seen to provide weaker interleaflet coupling. Thermodynamically stable nanodomains could also be observed for certain points in the parameter space in our bilayer model, which were further verified by carrying out extended Monte Carlo simulations. These persistent noncoalescing registered nanodomains close to the lower end of the accepted nanodomain size range also point towards a possible "nanoscale" emulsion description of lateral heterogeneities in biological membrane leaflets.

Mutual regulation between coupled ordered domains and protein condensates

Multivalent interactions between the transmembrane adaptor Linker of Activation of T-cells (LAT) and its effectors (Grb2 and Sos1) produce biomolecular condensates that facilitate T cell activation through enhancement of reaction kinetics and exclusion of inhibitory molecules. LAT is known to be recruited to ordered, cholesterol-dependent lipid domains known as lipid rafts, suggesting a potential for coupling between lateral membrane organization and membrane protein condensates. Here, we provide evidence for such coupling in both in vitro model systems and in vivo cellular experiments.

Lateral organization of biomimetic cell membranes in varying pH conditions

Many studies have been devoted to investigation of phase separation and formation of lipid domains, which play crucial role in many biological processes. Here we present a complex study on the formation, dynamics, and stability of the phase-separated supported lipid membranes under varying pH conditions. The size and distribution of liquid-ordered (Lo) phase domains were investigated in a wide range (1.7–9.0) of buffer pH values and a strong correlation was found between the size of the Lo domains and pH of the buffer hydrating the lipid bilayer. Interestingly, the dynamics of lipids composing both Lo and Ld phase are insensitive to the pH of the buffer. Our findings demonstrate that by varying pH of the environment one can induce formation of domains with a specific size and shape without any external modification of the solid support or altering the membrane composition. Finally, we show that the architecture of the lipid membrane is stable even upon replacement of the aqueous medium with the buffer of neutral pH. Consequently, this method of patterning of Lo phase domains in biomimetic membranes is applicable to the studies involving binding of proteins or incorporation of other pH-sensitive molecules.

Bipolar Imidazolium-Based Lipid Analogues for Artificial Archaeosomes.

Archaeal lipids have harvested biomedical and biotechnological interest because of their ability to form membranes with low permeability and enhanced temperature and pressure stability. Because of problems in isolating archaeal lipids, chemical synthesis appears to be a suitable means of producing model lipids that mimic the biological counterparts. Here, we introduce a new concept: we synthesized bipolar alkylated imidazolium salts of different chain lengths (BIm10-32) and studied their structure and lyotropic phase behavior. Furthermore, mixtures of the bolalipid analogues with phospholipid model biomembranes of diverse complexity were studied. DSC, fluorescence and FTIR spectroscopy, confocal fluorescence microscopy, DLS, SAXS, and TEM were used to reveal changes in lipid phase behavior, fluidity, the lipid's conformational order, and membrane morphology over a wide range of temperatures and for selected pressures. It could be shown that the long-chain BImN32 can form monolayer sheets. Integrated in phospholipid membranes, it reveals a fluidizing effect. Here, the two polar head groups, connected by a long alkyl chain, enable the integration into the bilayer. Interestingly, addition of BImN32 to fluid DPPC liposomes increased the lipid packing markedly, rendering the membrane system more stable at higher temperatures. The membrane system is also stable against compression as indicated by the high-pressure stability of the system, mimicking an archaeal lipid-like behavior. BImN32 incorporation into raft-like anionic model biomembranes led to marked changes in lateral membrane organization, topology, and fusogenicity of the membrane. Overall, it was found that long-chain imidazolium-based bolalipid analogues can help adjust membrane's biophysical properties, while the imidazolium headgroup provides the ability for crucial electrostatic interaction for vesicle fusion or selective interaction with membrane-related signaling molecules and polypeptides in a synthetically tractable manner. The results obtained may help to develop new approaches for rational design of extremophilic bolalipid-based liposomes for various applications, including delivery of drugs and vaccines.

High-Level Expression of Palmitoylated MPP1 Recombinant Protein in Mammalian Cells.

Our recent studies have pointed to an important role of the MAGUK family member, MPP1, as a crucial molecule interacting with flotillins and involved in the lateral organization of the erythroid plasma membrane. The palmitoylation of MPP1 seems to be an important element in this process; however, studies on the direct effect of palmitoylation on protein–protein or protein–membrane interactions in vitro are still challenging due to the difficulties in obtaining functional post-translationally modified recombinant proteins and the lack of comprehensive protocols for the purification of palmitoylated proteins. In this work, we present an optimized approach for the high-yield overexpression and purification of palmitoylated recombinant MPP1 protein in mammalian HEK-293F cells. The presented approach facilitates further studies on the molecular mechanism of lateral membrane organization and the functional impact of the palmitoylation of MPP1, which could also be carried out for other palmitoylated proteins.

Capturing Membrane Phase Separation by Dual Resolution Molecular Dynamics Simulations.

Understanding the lateral organization in plasma membranes remains an open problem and is of great interest to many researchers. Model membranes consisting of coexisting domains are commonly used as simplified models of plasma membranes. The coarse-grained (CG) Martini force field has successfully captured spontaneous separation of ternary membranes into a liquid-disordered and a liquid-ordered domain. With all-atom (AA) models, however, phase separation is much harder to achieve due to the slow underlying dynamics. To remedy this problem, here, we apply the virtual site (VS) hybrid method on a ternary membrane composed of saturated lipids, unsaturated lipids, and cholesterol to investigate the phase separation. The VS scheme couples the two membrane leaflets at CG and AA resolution. We found that the rapid phase separation reached by the CG leaflet can accelerate and guide this process in the AA leaflet.

The Emerging World of Membrane Vesicles: Functional Relevance, Theranostic Avenues and Tools for Investigating Membrane Function.

Lipids are essential components of cell membranes and govern various membrane functions. Lipid organization within membrane plane dictates recruitment of specific proteins and lipids into distinct nanoclusters that initiate cellular signaling while modulating protein and lipid functions. In addition, one of the most versatile function of lipids is the formation of diverse lipid membrane vesicles for regulating various cellular processes including intracellular trafficking of molecular cargo. In this review, we focus on the various kinds of membrane vesicles in eukaryotes and bacteria, their biogenesis, and their multifaceted functional roles in cellular communication, host-pathogen interactions and biotechnological applications. We elaborate on how their distinct lipid composition of membrane vesicles compared to parent cells enables early and non-invasive diagnosis of cancer and tuberculosis, while inspiring vaccine development and drug delivery platforms. Finally, we discuss the use of membrane vesicles as excellent tools for investigating membrane lateral organization and protein sorting, which is otherwise challenging but extremely crucial for normal cellular functioning. We present current limitations in this field and how the same could be addressed to propel a fundamental and technology-oriented future for extracellular membrane vesicles.