Membrane Spontaneous Curvature Research Articles

Which Coverages of Arc-Shaped Proteins Are Required for Membrane Tubulation?

Arc-shaped BIN/Amphiphysin/Rvs (BAR) domain proteins generate curvature by binding to membranes and induce membrane tubulation at sufficiently large protein coverages. For the amphiphysin N-BAR domain, Le Roux et al., Nat. Commun. 2021, 12, 6550, measured a threshold coverage of 0.44 ± 0.097 for nanotubules emerging from the supported lipid bilayer. In this article, we systematically investigate membrane tubulation induced by arc-shaped protein-like particles with coarse-grained modeling and simulations and determine the threshold coverages at different particle-particle interaction strengths and membrane spontaneous curvatures. In our simulations, the binding of arc-shaped particles induces a membrane shape transition from spherical vesicles to tubules at a particle threshold coverage of about 0.5, which is rather robust to variations of the direct attractive particle interactions or spontaneous membrane curvature in the coarse-grained model. Our study suggests that threshold coverages of around or slightly below 0.5 are a general requirement for membrane tubulation by arc-shaped BAR domain proteins.

Peptide Flexibility and the Hydrophobic Moment are Determinants to Evaluate the Clinical Potential of Magainins.

Using a flexibility prediction algorithm and in silico structural modeling, we have calculated the intrinsic flexibility of several magainin derivatives. In the case of magainin-2 (Mag-2) and magainin H2 (MAG-H2) we have found that MAG-2 is more flexible than its hydrophobic analog, Mag-H2. This affects the degree of bending of both peptides, with a kink around two central residues (R10, R11), whereas, in Mag-H2, W10 stiffens the peptide. Moreover, this increases the hydrophobic moment of Mag-H2, which could explain its propensity to form pores in POPC model membranes, which exhibit near-to-zero spontaneous curvatures. Likewise, the protective effect described in DOPC membranes for this peptide regarding its facilitation in pore formation would be related to the propensity of this lipid to form membranes with negative spontaneous curvature. The flexibility of another magainin analog (MSI-78) is even greater than that of Mag-2. This facilitates the peptide to present a kind of hinge around the central F12 as well as a C-terminal end prone to be disordered. Such characteristics are key to understanding the broad-spectrum antimicrobial actions exhibited by this peptide. These data reinforce the hypothesis on the determinant role of spontaneous membrane curvature, intrinsic peptide flexibility, and specific hydrophobic moment in assessing the bioactivity of membrane-active antimicrobial peptides.

Photomodulation of reconstituted voltage-dependent K+ channels.

The use of optogenetics for light-controlled ion flow has significantly impacted neuroscience, enabling isolated cell excitation or suppression within complex neural tissues. Achieving the same effect without the prior need to modify membrane channels genetically may represent a significant advantage for various applications. Here we tested whether photo-susceptible lipids may modulate the open probability of voltage-gated ion channels in their vicinity. The approach uses the observation that channel gating may be affected by membrane tension and depends on the material properties of the embedding lipid bilayer, such as spontaneous membrane curvature and membrane thickness [1]. We reconstituted KvAP, the voltage-gated potassium channel from Aeropyrum pernix, into photoswitchable horizontal solvent-depleted planar lipid bilayers. KvAP open probability suddenly increased when light exposure switched the photolipids from their conical to their cylindrical state. The effect was fully reversible as the photolipids could be switched back to their original state by exposure to light of a different wavelength. Single channel recordings and current recordings of channel ensembles confirmed the multiple repeatabilities of the photocycle. Our observation paves the way for the specific modulation of cells in vivo. This project was supported by a grant from the FWF (Austrian Science Fund): P34826. 1. Pfeffermann et al., Journal of Photochemistry and Photobiology B: Biology, 2021: p. 112320.

Probing the mechanism of annexin mediated plasma membrane repair.

The integrity of the plasma membrane is essential for maintaining cell homeostasis and eucaryotic cells have developed robust mechanisms for rapidly sealing plasma membrane ruptures. The repair process involves recruitment of multiple proteins to the damage site as triggered by the influx of calcium ions from the cytosol. Several members of the Annexin family are involved in membrane repair although their functional role in membrane remodeling is not fully understood. Biophysical experiments and modeling in synergy with cell experiments offer tools which can provide additional insight into Annexin mediated repair mechanisms. We review our recent studies into membrane remodeling and protein structures induced by Annexin binding. Using a unique platform of planar, free-edged membrane patches, the edge region of a membrane hole can be experimentally mimicked under controlled conditions. Using this platform, Annexins induce spontaneous membrane curvature leading to distinct, curved morphologies. We discuss the theoretical modeling of curvature effects and the formation of membrane necks around holes. We also describe AFM results resolving the organization of Annexins on membranes with molecular detail. Finally, we describe quantification of the calcium influx in cells during subjected to laser rupture. Detailed analysis and modeling of calcium diffusion and pumping allows extraction of key parameters for repair such as the timescale of hole closure.

Stepwise remodeling and subcompartment formation in individual vesicles by three ESCRT-III proteins

The endosomal sorting complex required for transport (ESCRT) is a multi-protein machinery involved in several membrane remodeling processes. Different approaches have been used to resolve how ESCRT proteins scission membranes. However, the underlying mechanisms generating membrane deformations are still a matter of debate. Here, giant unilamellar vesicles, microfluidic technology, and micropipette aspiration are combined to continuously follow the ESCRT-III-mediated membrane remodeling on the single-vesicle level for the first time. With this approach, we identify different mechanisms by which a minimal set of three ESCRT-III proteins from Entamoeba histolytica reshape the membrane. These proteins modulate the membrane stiffness and spontaneous curvature to regulate bud size and generate intraluminal vesicles even in the absence of ATP. We demonstrate that the bud stability depends on the protein concentration and membrane tension. The approaches introduced here should open the road to diverse applications in synthetic biology for establishing artificial cells with several membrane compartments.

Spontaneous local membrane curvature induced by transmembrane proteins

The (local) curvature of cellular membranes acts as a driving force for the targeting of membrane-associated proteins to specific membrane domains, as well as a sorting mechanism for transmembrane proteins, e.g., by accumulation in regions of matching spontaneous curvature. The latter measure was previously experimentally employed to study the curvature induced by the potassium channel KvAP and by aquaporin AQP0. However, the direction of the reported spontaneous curvature levels as well as the molecular driving forces governing the membrane curvature induced by these integral transmembrane proteins could not be addressed experimentally.Here, using both coarse-grained and atomistic molecular dynamics (MD) simulations, we report induced spontaneous curvature values for the homologous potassium channel Kv 1.2/2.1 Chimera (KvChim) and AQP0 embedded in unrestrained lipid bicelles that are in very good agreement with experiment. Importantly, the direction of curvature could be directly assessed from our simulations: KvChim induces a strong positive membrane curvature (≈0.036 nm−1) whereas AQP0 causes a comparably small negative curvature (≈−0.019 nm−1).Analyses of protein-lipid interactions within the bicelle revealed that the potassium channel shapes the surrounding membrane via structural determinants. Differences in shape of the protein-lipid interface of the voltage-gating domains between the extracellular and cytosolic membrane leaflets induce membrane stress and thereby promote a protein-proximal membrane curvature. In contrast, the water pore AQP0 displayed a high structural stability and an only faint effect on the surrounding membrane environment that is connected to its wedge-like shape.

Superresolution microscopy of nanotubes and strongly curved membrane segments formed by giant vesicles exposed to molecular condensates

Cell mimetic systems like giant unilamellar vesicles (GUVs) encapsulating macromolecules (polyethylene-glycol and dextran) represent an excellent tool to study membrane transformations associated with molecular crowding and protein condensation. Upon liquid-liquid phase separation as in living cells, these GUVs exhibit highly curved structures such as internal nanotubes and the membrane of GUVs form apparent kinks at the contact line of the interface between the two encapsulated aqueous phases. These membrane structures of high curvature have dimensions below conventional optical resolution and if resolved, can provide quantitative information on membrane spontaneous curvature and the intrinsic contact angle describing the wettability difference of the encapsulated phases to the membrane.

Quantifying the contributions of ENTH to membrane spontaneous curvature using coarse grained molecular dynamics

The ability of cells to produce and maintain membrane curvature is critical for cellular function. Experiments throughout history have revealed a diverse set of mechanisms for how proteins can bend membranes. Models have been developed to shed insight on the mechanics behind these mechanisms. As the quality and fidelity of experimental data improves, using these models we can ask how changes to cellular regulatory pathways influence membrane ultrastructure and membrane trafficking processes. For continuum modeling of the membrane shape, if we select the spontaneous curvature model, which represents the bending energy as a function of membrane curvature, the key parameters to define are the bending modulus and spontaneous curvature.

Investigation of Shape Transformations of Vesicles, Induced by Their Adhesion to Flat Substrates Characterized by Different Adhesion Strength.

The adhesion of lipid vesicles to a rigid flat surface is investigated. We examine the influence of the membrane spontaneous curvature, adhesion strength, and the reduced volume on the stability and shape transformations of adhered vesicles. The minimal strength of the adhesion necessary to stabilize the shapes of adhered vesicles belonging to different shape classes is determined. It is shown that the budding of an adhered vesicle may be induced by the change of the adhesion strength. The importance of the free vesicle shape for its susceptibility to adhesion is discussed.

Super‐Resolution Imaging of Highly Curved Membrane Structures in Giant Vesicles Encapsulating Molecular Condensates

AbstractMolecular crowding is an inherent feature of cell interiors. Synthetic cells as provided by giant unilamellar vesicles (GUVs) encapsulating macromolecules (poly(ethylene glycol) and dextran) represent an excellent mimetic system to study membrane transformations associated with molecular crowding and protein condensation. Similarly to cells, such GUVs exhibit highly curved structures like nanotubes. Upon liquid–liquid phase separation their membrane deforms into apparent kinks at the contact line of the interface between the two aqueous phases. These structures, nanotubes, and kinks, have dimensions below optical resolution. Here, these are studied with super‐resolution stimulated emission depletion (STED) microscopy facilitated by immobilization in a microfluidic device. The cylindrical nature of the nanotubes based on the superior resolution of STED and automated data analysis is demonstrated. The deduced membrane spontaneous curvature is in excellent agreement with theoretical predictions. Furthermore, the membrane kink‐like structure is resolved as a smoothly curved membrane demonstrating the existence of the intrinsic contact angle, which describes the wettability contrast of the encapsulated phases to the membrane. Resolving these highly curved membrane structures with STED imaging provides important insights in the membrane properties and interactions underlying cellular activities.

Dynamics of pearling instability in polymersomes: The role of shear membrane viscosity and spontaneous curvature

The stability of copolymer tethers is investigated theoretically. Self-assembly of diblock or triblock copolymers can lead to tubular polymersomes, which are known experimentally to undergo shape instability under thermal, chemical, and tension stresses. It leads to a periodic modulation of the radius, which evolves to assembly line pearls connected by tiny tethers. We study the contributions of shear surface viscosity and spontaneous curvature and their interplay to understand the pearling instability. The performed linear analysis of stability of this cylinder-to-pearls transition shows that such systems are unstable if the membrane tension is larger than a finite critical value contrary to the Rayleigh–Plateau instability, an already known result, or if the spontaneous curvature is in a specific range, which depends on membrane tension. For the case of spontaneous curvature-induced shape instability, two dynamical modes are identified. The first one is analog to the tension-induced instability with a marginal mode. Its wavenumber associated with the most unstable mode decreases continuously to zero as membrane viscosity increases. The second one has a finite range of unstable wavenumbers. The wavenumber of the most unstable mode tends to be constant as membrane viscosity increases. In this mode, its growth rate becomes independent of the bulk viscosity in the limit of high membrane viscosity and behaves as a pure viscous surface.

Spontaneous Curvature Induced Stretching-Bending Mode Coupling in Membranes

In this paper, a simple example to illustrate what is basically known from the Gauss’ times interplay between geometry and mechanics in thin shells is presented. Specifically, the eigen-mode spectrum in spontaneously curved (i.e., up-down asymmetric) extensible polymerized or elastic membranes is studied. It is found that in the spontaneously curved crystalline membrane, the flexural mode is coupled to the acoustic longitudinal mode, even in the harmonic approximation. If the coupling (proportional to the membrane spontaneous curvature) is strong enough, the coupled modes dispersions acquire the imaginary part, i.e., effective damping. The damping is not related to the entropy production (dissipation); it comes from the redistribution of the energy between the modes. The curvature-induced mode coupling makes the flexural mode more rigid, and the acoustic mode becomes softer. As it concerns the transverse acoustical mode, it remains uncoupled in the harmonic approximation, keeping its standard dispersion law. We anticipate that the basic ideas inspiring this study can be applied to a large variety of interesting systems, ranging from still fashionable graphene films, both in the freely suspended and on a substrate states, to the not yet fully understood lipid membranes in the so-called gel and rippled phases.

Alkylation of a hydrophilic photosensitizer enhances the contact-dependent photo-induced oxidation of phospholipid membranes

Lipophilic photosensitizers able to photo-induce lipid oxidation in biomembranes are, in general, much more efficient than hydrophilic ones; acting through the formation of singlet oxygen (1O2), which oxidizes the fatty acid double bonds (type II mechanism). Here we investigate the binding and photosensitizing properties of 4-(decyloxy)pteridin-2-amine (O-decyl-Ptr) using unilamellar vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), a phospholipid with monounsaturated fatty acids. By means of molecular dynamic (MD) simulations, we showed that O-decyl-Ptr binds to the membrane in a localization that favors the direct reaction of the triplet excited state of O-decyl-Ptr with DOPC double bonds. As a consequence, although 1O2 is formed, O-decyl-Ptr acts mainly as a contact-dependent photosensitizer, meaning through radical formation (type I mechanism). Mass spectrometry analysis of vesicles irradiated in the presence of O-decyl-Ptr, demonstrated the generation of alcohols (LOH), ketones (LO) and hydroperoxides (LOOH). In agreement with the mechanistic hypothesis proposed, LOH and LO (type I photooxidation products) are formed faster than LOOH (type II photooxidation product). Interestingly, no short-chain oxidized products were detected. Accordingly, membrane fluctuations and formation of filaments and buds are observed during in-situ photo-activation of O-decyl-Ptr in giant unilamellar vesicles due to changes in membrane spontaneous curvature. Finally, we evaluated the effect of the photochemical processes studied at a cellular level and demonstrated in experiments of viability of mammalian cells that O-decyl-Ptr has important photodynamic properties. Similar experiments performed using the hydrophilic photosenstizer pterin (Ptr) show that alkylation leads to a striking increase in the efficiency of photosensitized lipid oxidation.

Bulging and budding of lipid droplets from symmetric and asymmetric membranes: competition between membrane elastic energy and interfacial energy.

Lipid droplets are ubiquitous intracellular organelles regulating the storage and hydrolysis of neutral lipids, and play key roles in cellular metabolism and other functions such as protein trafficking and coordinating with immune responses. Though lipid droplets are widely observed in eukaryotic organisms, it remains unclear how and what aspects of mechanical interaction between the neutral lipids and lipid membranes contribute to the bulging and budding of nascent lipid droplets from the endoplasmic reticulum, and particularly effects of membrane asymmetry and spontaneous curvature on lipid droplet formation are not theoretically rationalized. Here we conduct a comprehensive theoretical study on the mechanical behaviors of lipid droplets embedded in between two lipid monolayers of the same or different mechanical properties, and indicate that the membrane bending rigidity, tension and spontaneous curvature, lipid droplet size, and interfacial energy between the neutral lipids and covering lipid leaflets collectively play key roles in regulating the growth and budding transition of lipid droplets. It is found that the embedded neutral lipids beyond a critical volume could undergo a discontinuous shape transition from a lens-like configuration to a budding state with a spherical bulge configuration. Moreover, a positive lipid monolayer spontaneous curvature and smaller monolayer bending rigidity and tension facilitate the budding transition. Budding phase diagrams accounting for these characteristic interaction states are established. Based on the membrane theory at small deformation before budding and the assumption of spherical configuration after budding, we obtain analytical solutions on the bulge profiles, which can be used to estimate the value of interfacial energy. Our results uncover the fundamental mechanics of the lipid droplet formation and budding, and are of broad interest to the studies of echogenic liposome stability and cellular incorporation of nanoparticles.

The intriguing role of rhamnolipids on plasma membrane remodelling: From lipid rafts to membrane budding

Rhamnolipids (RLs) comprise a class of glycolipids produced by Pseudomonas aeruginosa under appropriate culture medium. They act as biosurfactants being composed by a hydrophilic head of either one (mono-RL) or two (di-RL) rhamnose moieties coupled to hydroxyaliphatic chains. It is well accepted that RLs present low biolitic activity as compared to other synthetic surfactants. However, their mechanisms of action in biological systems are not well defined yet. The interaction of RLs with lipid bilayers are here investigated to address how they impact on plasma membrane at molecular level. Our experimental approach was based on a deep analysis of optical microscopy data from giant unilamellar vesicles (GUVs) dispersed in aqueous solutions containing up to 0.5 mM of commercially available RLs (a mixture of mono-RL, 33–37 mol%, and di-RL, 63–67 mol%, cmc of 0.068±0.005 mM). GUVs were made up of a single lipid POPC and a ternary system containing DOPC, sphingomyelin and cholesterol, which mimic lipid raft platforms. Our results demonstrate that RLs have a low partition in the lipid bilayer in respect to the total molecules in solution. We suppose that RLs insert in the outer leaflet with low propensity to flip-flop. In the case of POPC GUVs, the insertion of RL molecules in the outer leaflet impairs changes in spontaneous membrane curvature with incubation time. Then, small buds are formed that remain linked to the original membrane. No changes in membrane permeability have been detected. A remarkable result refers to the insertion of RLs in membranes containing liquid ordered (Lo) - liquid disordered (Ld) phase coexistence. The rate of interaction has been observed to be higher for Ld phase than for Lo phase (0.12·10-6 s−1 and 0.023·10-6 s−1 for Ld and Lo, respectively, at RL concentration of 0.5 mM). As a consequence, the preferential RL insertion in Ld phase may also alter the membrane spontaneous curvature which, coupled to the change in the line tension associated to the domains boundary, conducted to Lo domain protrusion. Even if it has been observed on a model system, such membrane remodelling might correlate to endocytic processes activated in cell membranes, regardless of the participation of specific proteins. Further, changes imposed by RLs in lipid rafts may affect the association of key proteins enrolled in cell signaling, which may perturb cell homeostasis.

PIP2 Reshapes Membranes through Asymmetric Desorption

Phosphatidylinositol-4,5-bisphosphate (PIP2) is an important signaling lipid in eukaryotic cell plasma membranes, playing an essential role in diverse cellular processes. The headgroup of PIP2 is highly negatively charged, and this lipid displays a high critical micellar concentration compared to housekeeping phospholipid analogs. Given the crucial role of PIP2, it is imperative to study its localization, interaction with proteins, and membrane-shaping properties. Biomimetic membranes have served extensively to elucidate structural and functional aspects of cell membranes including protein-lipid and lipid-lipid interactions, as well as membrane mechanics. Incorporation of PIP2 into biomimetic membranes, however, has at times resulted in discrepant findings described in the literature. With the goal to elucidate the mechanical consequences of PIP2 incorporation, we studied the desorption of PIP2 from biomimetic giant unilamellar vesicles by means of a fluorescent marker. A decrease in fluorescence intensity with the age of the vesicles suggested that PIP2 lipids were being desorbed from the outer leaflet of the membrane. To evaluate whether this desorption was asymmetric, the vesicles were systematically diluted. This resulted in an increase in the number of internally tubulated vesicles within minutes after dilution, suggesting that the desorption was asymmetric and also generated membrane curvature. By means of a saturated chain homolog of PIP2, we showed that the fast desorption of PIP2 is facilitated by presence of an arachidonic lipid tail and is possibly due to its oxidation. Through measurements of the pulling force of membrane tethers, we quantified the effect of this asymmetric desorption on the spontaneous membrane curvature. Furthermore, we found that the spontaneous curvature could be modulated by externally increasing the concentration of PIP2 micelles. Given that the local concentration of PIP2 in biological membranes is variable, spontaneous curvature generated by PIP2 may affect the formation of highly curved structures that can serve as initiators for signaling events.

Driving Spontaneous Membrane Curvature by Tuning Cardiolipin Concentration and Spatial Distribution in Model Mitochondrial Membranes

Cell membranes are complex systems with diverse lipid compositions that contribute to overall cellular function. Necessary functions like respiration and oxidative phosphorylation are highly dependent on lipid spatial distributions and overall membrane morphology, particularly in the inner mitochondrial membrane (IMM) where the complex interplay between membrane lipids and bioenergetic proteins generates and maintains its characteristic curved cristae structures. The anionic diphosphatidylglycerol cardiolipin is found most abundantly in the IMM, with the majority asymmetrically distributed to the outer leaflet. Cardiolipin has an inverted cone geometry due to its four fatty acyl tails, and has been proposed to play a role in regulating the highly curved nature of the IMM. It is not well understood whether spontaneous curvature is elicited by cardiolipin alone, its interactions with IMM bioenergetic proteins and other lipids, or from a more complex molecular process. Using coarse-grained molecular dynamics, we simulate membrane bilayers with asymmetric inter-leaflet cardiolipin concentrations, and find that cardiolipin content and spatial arrangement largely determine the rate and magnitude of membrane curvature formation. We find that by constraining the lateral diffusion of cardiolipin to certain regions of the membrane, negative membrane curvature forms at those regions and remains stable over long timescales, showing that complex large-scale membrane morphologies can be robustly controlled by tuning the spatial distribution of cardiolipin. Further, we characterize the effects of other mitochondrial lipids and cardiolipin protonation state on curvature generation and stabilization. Therefore, we find that cardiolipin alone can drive the formation of negative membrane curvature at equilibrium.

Budding and Fission of Vesicles by Control of Membrane Spontaneous Curvature

Budding and Fission of Vesicles by Control of Membrane Spontaneous Curvature

Asymmetric Ionic Conditions Generate Large Membrane Curvatures.

Biological membranes possess intrinsic asymmetry. This asymmetry is associated not only with leaflet composition in terms of membrane species but also with differences in the cytosolic and periplasmic solutions containing macromolecules and ions. There has been a long quest for understanding the effect of ions on the physical and morphological properties of membranes. Here, we elucidate the changes in the mechanical properties of membranes exposed to asymmetric buffer conditions and the associated curvature generation. As a model system, we used giant unilamellar vesicles (GUVs) with asymmetric salt and sugar solutions on the two sides of the membrane. We aspirated the GUVs into micropipettes and attached small beads to their membranes. An optical tweezer was used to exert a local force on a bead, thereby pulling out a membrane tube from the vesicle. The assay allowed us to measure the spontaneous curvature and the bending rigidity of the bilayer in the presence of different ions and sugar. At low sugar/salt (inside/out) concentrations, the membrane spontaneous curvature generated by NaCl and KCl is close to zero, but negative in the presence of LiCl. In the latter case, the membrane bulges away from the salt solution. At high sugar/salt conditions, the membranes were observed to become more flexible and the spontaneous curvature was enhanced to even more negative values, comparable to those generated by some proteins. Our findings reveal the reshaping role of alkali chlorides on biomembranes.

Phase interfaces within different vesicle shapes

Liquid droplets on flat surfaces generally exhibit a contact angle in a range from 0 to , but the two-phase interface within a vesicle membrane is very fascinating due to the involved force balance along three bending interfaces. Giant lipid vesicles encapsulated with the poly(ethylene glycol)/dextran aqueous two-phase system are established recently, and the phase interfaces within vesicle membrane are very interesting as experimentally observed. The developed theoretical framework by a combination of the Helfrich curvature elastic theory for vesicle membranes and self-consistent field theory for polymers has been extended to explore aqueous two phases within vesicles. The intrinsic contact angle that represents the material parameter, is introduced to describe this phase interfaces within vesicles, especially the transitions from complete wetting to partial wetting, from partial wetting to complete dewetting. The dependence of intrinsic contact angles on the parameters, such as the interaction strengths between the polymers and between the membrane and polymer , the volume fraction , impermeability of the membrane to the enclosed polymers and membrane spontaneous curvature c0, are thoroughly investigated, as well as these wetting/dewetting transitions are extensively discussed in the present study.