Membrane Shape Research Articles

A single-particle analysis method for detecting membrane remodelling and curvature sensing.

In biology, shape and function are related. Therefore, it is important to understand how membrane shape is generated, stabilised and sensed by proteins and how this relates to organelle function. Here, we present an assay that can detect curvature preference and membrane remodelling with free-floating liposomes using protein concentrations in physiologically relevant ranges. The assay reproduced known curvature preferences of BAR domains and allowed the discovery of high-curvature preference for the PH domain of AKT and the FYVE domain of HRS (also known as HGS). In addition, our method reproduced the membrane vesiculation activity of the ENTH domain of epsin-1 (EPN1) and showed similar activity for the ANTH domains of PiCALM and Hip1R. Finally, we found that the curvature sensitivity of the N-BAR domain of endophilin inversely correlates to membrane charge and that deletion of its N-terminal amphipathic helix increased its curvature specificity. Thus, our method is a generally applicable qualitative method for assessing membrane curvature sensing and remodelling by proteins.

Endosomal membrane budding patterns in plants

Multivesicular endosomes (MVEs) sequester membrane proteins destined for degradation within intralumenal vesicles (ILVs), a process mediated by the membrane-remodeling action of Endosomal Sorting Complex Required for Transport (ESCRT) proteins. In Arabidopsis, endosomal membrane constriction and scission are uncoupled, resulting in the formation of extensive concatenated ILV networks and enhancing cargo sequestration efficiency. Here, we used a combination of electron tomography, computer simulations, and mathematical modeling to address the questions of when concatenated ILV networks evolved in plants and what drives their formation. Through morphometric analyses of tomographic reconstructions of endosomes across yeast, algae, and various land plants, we have found that ILV concatenation is widespread within plant species, but only prevalent in seed plants, especially in flowering plants. Multiple budding sites that require the formation of pores in the limiting membrane were only identified in hornworts and seed plants, suggesting that this mechanism has evolved independently in both plant lineages. To identify the conditions under which these multiple budding sites can arise, we used particle-based molecular dynamics simulations and found that changes in ESCRT filament properties, such as filament curvature and membrane binding energy, can generate the membrane shapes observed in multiple budding sites. To understand the relationship between membrane budding activity and ILV network topology, we performed computational simulations and identified a set of membrane remodeling parameters that can recapitulate our tomographic datasets.

The Nedd4L ubiquitin ligase is activated by FCHO2-generated membrane curvature.

The C2-WW-HECT domain ubiquitin ligase Nedd4L regulates membrane sorting during endocytosis through the ubiquitination of cargo molecules such as the epithelial sodium channel (ENaC). Nedd4L is catalytically autoinhibited by an intramolecular interaction between its C2 and HECT domains, but the protein's activation mechanism is poorly understood. Here, we show that Nedd4L activation is linked to membrane shape by FCHO2, a Bin-Amphiphysin-Rsv (BAR) domain protein that regulates endocytosis. FCHO2 was required for the Nedd4L-mediated ubiquitination and endocytosis of ENaC, with Nedd4L co-localizing with FCHO2 at clathrin-coated pits. In cells, Nedd4L was specifically recruited to, and activated by, the FCHO2 BAR domain. Furthermore, we reconstituted FCHO2-induced recruitment and activation of Nedd4L in vitro. Both the recruitment and activation were mediated by membrane curvature rather than protein-protein interactions. The Nedd4L C2 domain recognized a specific degree of membrane curvature that was generated by the FCHO2 BAR domain, with this curvature directly activating Nedd4L by relieving its autoinhibition. Thus, we show for the first time a specific function (i.e., recruitment and activation of an enzyme regulating cargo sorting) of membrane curvature by a BAR domain protein.

ВІДНОВЛЕННЯ ПАЛАДІЄВИХ МЕМБРАН ПІСЛЯ КОНТАКТУ З ВОД-НЕМ ДЛЯ ВОДНЕВО-КИСНЕВИХ ПАЛИВНИХ ЕЛЕМЕНТІВ

Annotation. Studying the deformations of palladium membranes under the influence of hydrogen, which is a relevant issue for hydrogen energy and the nuclear industry. The mechanisms of palladium membrane shape change due to hydrogen penetration and the formation of temporary gradient alloys such as α-PdHn are consid-ered. Experiments were conducted using a hydrogen-vacuum installation, where the processes of membrane satu-ration and degassing were studied at temperatures from 100°C to 360°C and hydrogen pressures from 0.01 MPa to 2.5 MPa. It was established that the change in the shape of the membrane occurs in two stages: first, a rapid bending of the membrane is observed, and then a gradual return to the initial state. It was established that the maximum change in the shape of the membrane, which occurs at a constant temperature, depends on the diffu-sion coefficient and the equilibrium solubility of hydrogen in palladium. However, when the temperature chang-es, the diffusion coefficient of hydrogen in palladium and the equilibrium concentration of hydrogen in palladi-um also change, which affects the temperature dependence of the final shape change of the membrane. This fact makes it possible to effectively determine the time of hydrogen penetration into the membrane, control the change in shape and adjust the operating modes of the fuel cell. Special attention is paid to the use of palladium alloys with copper and silver to increase the resistance of membranes to the influence of hydrogen. It is shown that such alloys have better mechanical characteristics and a longer service life at high temperatures. The effect of degassing on the restoration of membranes after contact with hydrogen was studied, the main factors affecting the efficiency of this process were determined. The results of the work confirm the need for further research on increasing the resistance of palladium membranes to deformations, as well as the development of new materials and technologies to improve their operational characteristics

One-step nanoscale expansion microscopy reveals individual protein shapes.

The attainable resolution of fluorescence microscopy has reached the subnanometer range, but this technique still fails to image the morphology of single proteins or small molecular complexes. Here, we expand the specimens at least tenfold, label them with conventional fluorophores and image them with conventional light microscopes, acquiring videos in which we analyze fluorescence fluctuations. One-step nanoscale expansion (ONE) microscopy enables the visualization of the shapes of individual membrane and soluble proteins, achieving around 1-nm resolution. We show that conformational changes are readily observable, such as those undergone by the ~17-kDa protein calmodulin upon Ca2+ binding. ONE is also applied to clinical samples, analyzing the morphology of protein aggregates in cerebrospinal fluid from persons with Parkinson disease, potentially aiding disease diagnosis. This technology bridges the gap between high-resolution structural biology techniques and light microscopy, providing new avenues for discoveries in biology and medicine.

Topological and Morphological Membrane Dynamics in Giant Lipid Vesicles Driven by Monoolein Cubosomes

Lipid nanoparticles have important applications as biomedical delivery platforms and broader engineering biology applications in artificial cell technologies. These emerging technologies often require changes in the shape and topology of biological or biomimetic membranes. Here we show that topologically‐active lyotropic liquid crystal nanoparticles (LCNPs) can trigger such transformations in the membranes of giant unilamellar vesicles (GUVs). Monoolein (MO) LCNPs, cubosomes with an internal nanostructure of space group Im3m incorporate into 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) GUVs creating excess membrane area with stored curvature stress. Using time‐resolved fluorescence confocal and lattice light sheet microscopy, we observe and characterise various life‐like dynamic events in these GUVs, including growth, division, tubulation, membrane budding and fusion. Our results shed new light on the interactions of LCNPs with bilayer lipid membranes, providing insights relevant to how these nanoparticles might interact with cellular membranes during drug delivery and highlighting their potential as minimal triggers of topological transitions in artificial cells.

Topological and Morphological Membrane Dynamics in Giant Lipid Vesicles Driven by Monoolein Cubosomes.

Lipid nanoparticles have important applications as biomedical delivery platforms and broader engineering biology applications in artificial cell technologies. These emerging technologies often require changes in the shape and topology of biological or biomimetic membranes. Here we show that topologically-active lyotropic liquid crystal nanoparticles (LCNPs) can trigger such transformations in the membranes of giant unilamellar vesicles (GUVs). Monoolein (MO) LCNPs, cubosomes with an internal nanostructure of space group Im3m incorporate into 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) GUVs creating excess membrane area with stored curvature stress. Using time-resolved fluorescence confocal and lattice light sheet microscopy, we observe and characterise various life-like dynamic events in these GUVs, including growth, division, tubulation, membrane budding and fusion. Our results shed new light on the interactions of LCNPs with bilayer lipid membranes, providing insights relevant to how these nanoparticles might interact with cellular membranes during drug delivery and highlighting their potential as minimal triggers of topological transitions in artificial cells.

Nonequilibrium Membrane Dynamics Induced by Active Protein Interactions and Chemical Reactions: A Review

AbstractBiomembranes wrapping cells and organelles are not only the partitions that separate the insides but also dynamic fields for biological functions accompanied by membrane shape changes. In this review, we discuss the spatiotemporal patterns and fluctuations of membranes under nonequilibrium conditions. In particular, we focus on theoretical analyses and simulations. Protein active forces enhance or suppress the membrane fluctuations; the membrane height spectra are deviated from the thermal spectra. Protein binding or unbinding to the membrane is activated or inhibited by other proteins and chemical reactions, such as ATP hydrolysis. Such active binding processes can induce traveling waves, Turing patterns, and membrane morphological changes. They can be represented by the continuum reaction‐diffusion equations and discrete lattice/particle models with state flips. The effects of structural changes in amphiphilic molecules on the molecular‐assembly structures are also discussed.

Membrane curvature sensing and symmetry breaking of the M2 proton channel from Influenza A.

The M2 proton channel aids in the exit of mature influenza viral particles from the host plasma membrane through its ability to stabilize regions of high negative Gaussian curvature (NGC) that occur at the neck of budding virions. The channels are homo-tetramers that contain a cytoplasm-facing amphipathic helix (AH) that is necessary and sufficient for NGC generation; however, constructs containing the transmembrane spanning helix, which facilitates tetramerization, exhibit enhanced curvature generation. Here, we used all-atom molecular dynamics (MD) simulations to explore the conformational dynamics of M2 channels in lipid bilayers revealing that the AH is dynamic, quickly breaking the fourfold symmetry observed in most structures. Next, we carried out MD simulations with the protein restrained in four- and twofold symmetric conformations to determine the impact on the membrane shape. While each pattern was distinct, all configurations induced pronounced curvature in the outer leaflet, while conversely, the inner leaflets showed minimal curvature and significant lipid tilt around the AHs. The MD-generated profiles at the protein-membrane interface were then extracted and used as boundary conditions in a continuum elastic membrane model to calculate the membrane-bending energy of each conformation embedded in different membrane surfaces characteristic of a budding virus. The calculations show that all three M2 conformations are stabilized in inward-budding, concave spherical caps and destabilized in outward-budding, convex spherical caps, the latter reminiscent of a budding virus. One of the C2-broken symmetry conformations is stabilized by 4 kT in NGC surfaces with the minimum energy conformation occurring at a curvature corresponding to 33 nm radii. In total, our work provides atomistic insight into the curvature sensing capabilities of M2 channels and how enrichment in the nascent viral particle depends on protein shape and membrane geometry.

Functional properties of a disease mutation for migraine in Kv2.1/6.4 channels

Voltage-gated potassium (Kv) channels are integral to cellular excitability, impacting the resting membrane potential, repolarization, and shaping action potentials in neurons and cardiac myocytes. Structurally, Kv channels are homo or heterotetramers comprising four α-subunits, each with six transmembrane segments (S1–S6). Silent Kv (KvS), includes Kv5.1, Kv6.1–6.4, Kv8.1–8.2, and Kv9.1–9.3, they do not form functional channels on their own but modulate the properties of heteromeric channels. Recent studies have identified the Kv6.4 subunit as a significant modulator within heteromeric channels, such as Kv2.16.4. The Kv2.16.4 heteromer exhibits altered biophysical properties, including a shift in voltage-dependent inactivation and a complex activation. Current genetic studies in migraine patients have revealed a single missense mutation in the Kv6.4 gene. The single missense mutation, L360P is in the highly conserved S4–S5 linker region. This study aims to demonstrate the biophysical effects of the L360P mutation in Kv2.1 6.4 channels with a fixed 2:2 stoichiometry, using monomeric (Kv2.1/6.4) and tandem dimer (Kv2.1_6.4) configurations. Our findings suggest that the L360P mutation significantly impacts the function and regulation of Kv2.1/6.4 channels, providing insights into the molecular mechanisms underlying channel dysfunction in migraine pathology.

Immobilization of novel bifunctional polymer with amide and amidoxime groups in biochar-chitosan composite gels for enhancing uranium(VI) uptake

This work synthesized poly ethanolamine amidoxime modified winter melon and chitosan-derived biochar (PEA-CTS@WBC) using chemical crosslinking method for uranium(VI) removal. The factors influencing uranium(VI) adsorption by PEA-CTS@WBC, including pH, adsorbent dosage, time, temperature, and initial U(VI) concentration were explored. The material’s performance was characterized, and the underlying mechanism of U(VI) removal was analyzed using various techniques. The characterization results indicated that the PEA-CTS@WBC took on a membrane shape with numerous pores and wrinkles distributed on the surface, providing more active sites that can effectively promote the complexation and adsorption of U(VI). The results demonstrated that PEA-CTS@WBC effectively removed U(VI), achieving a maximum adsorption capacity of 485.89 mg/g at the pH of 5.0. The process suggested a dominant role for chemical adsorption. The Freundlich isotherm model demonstrated a high degree of alignment with the observed adsorption behavior, indicating a predominantly multilayer adsorption process. Thermodynamic studies indicated that adsorption was a spontaneous endothermic process. The XPS analysis demonstrated that the adsorption process was primarily due to the formation of stable complexes with NH3+-C(O), N–C, C–N, C=C, O–H and C=O. This research showed that the introduction of amide and amidoxime groups significantly improved the adsorption effect of biochar-chitosan gels on U(VI). PEA-CTS@WBC had a high selective adsorption characteristics and good reusability for U(VI) in aqueous solutions. PEA-CTS@WBC was an economical, efficient and stable composite material. These findings provided a theoretical basis for the treatment of wastewater contaminated with U(VI).

2D electrical admittance lattice model of biological cellular system for modeling electroporation

In this work, a new modeling approach is presented to obtain a two-dimensional transport lattice of a biological cellular system for the calculation of the potential distribution throughout the system and investigation of the corresponding membrane electroporation. The presented model has been obtained by a modified bilayer model of the cell membrane. This modified membrane model allows for an effective inclusion of the shape of the cell membrane in the potential calculation. The results of the model have shown good agreement with the results of the well-known Schwan equation and COMSOL Multiphysics for the circular cell. The simulation results show that both membranes of a mitochondrion can be simultaneously electroporated by an alternating voltage source with frequencies between 1 MHz and 1 GHz.

Composite branched and linear F-actin maximize myosin-induced membrane shape changes in a biomimetic cell model

The architecture of the actin cortex determines the generation and transmission of stresses, during key events from cell division to migration. However, its impact on myosin-induced cell shape changes remains unclear. Here, we reconstitute a minimal model of the actomyosin cortex with branched or linear F-actin architecture within giant unilamellar vesicles (GUVs, liposomes). Upon light activation of myosin, neither the branched nor linear F-actin architecture alone induces significant liposome shape changes. The branched F-actin network forms an integrated, membrane-bound “no-slip boundary” -like cortex that attenuates actomyosin contractility. By contrast, the linear F-actin network forms an unintegrated “slip boundary“ -like cortex, where actin asters form without inducing membrane deformations. Notably, liposomes undergo significant deformations at an optimized balance of branched and linear F-actin networks. Our findings highlight the pivotal roles of branched F-actin in force transmission and linear F-actin in force generation to yield membrane shape changes.

Normal deformation of a soft lipid-based microtube in response to the flow generated by a tangential motion of a nearby microbead

In this study, we experimentally examine the deformation of a micrometer-scale soft membrane in response to a periodic shear flow. We fabricate long and straight cylindrical-shaped soft microtubes from lipid bilayers as part of this study. A microtube with a diameter of about 1–5 μm is moved parallel to its longitudinal axes. At the same time, a polystyrene microbead, trapped by optical tweezers, is forced to stay near the external surface of the microtube. We study the induced shape deformation in the microtube for different shearing parameters. The effects related to the size of the bead, its distance to the surface of the microtube, and their relative speed are analyzed. The overall results qualitatively demonstrate the impact of shearing on the shape of the soft membrane.

Synthetic control of actin polymerization and symmetry breaking in active protocells.

Nonlinear biomolecular interactions on membranes drive membrane remodeling crucial for biological processes including chemotaxis, cytokinesis, and endocytosis. The complexity of biomolecular interactions, their redundancy, and the importance of spatiotemporal context in membrane organization impede understanding of the physical principles governing membrane mechanics. Developing a minimal in vitro system that mimics molecular signaling and membrane remodeling while maintaining physiological fidelity poses a major challenge. Inspired by chemotaxis, we reconstructed chemically regulated actin polymerization inside vesicles, guiding membrane self-organization. An external, undirected chemical input induced directed actin polymerization and membrane deformation uncorrelated with upstream biochemical cues, suggesting symmetry breaking. A biophysical model incorporating actin dynamics and membrane mechanics proposes that uneven actin distributions cause nonlinear membrane deformations, consistent with experimental findings. This protocellular system illuminates the interplay between actin dynamics and membrane shape during symmetry breaking, offering insights into chemotaxis and other cell biological processes.

Curvature Footprints of Transmembrane Proteins in Simulations with the Martini Force Field.

Membranes play essential roles in biological systems and are tremendously diverse in the topologies and chemical and elastic properties that define their functions. In many cases, a given membrane may display considerable heterogeneity, with localized clusters of lipids and proteins exhibiting distinct characteristics compared to adjoining regions. These lipid-protein assemblies can span nanometers to micrometers and are associated with cellular processes such as transport and signaling. While lipid-protein assemblages are dynamic, they can be stabilized by coupling between local membrane composition and shape. Due to the inherent difficulty in resolving atomistic details of membrane proteins in their native lipid environments, these complexes are notoriously challenging to study experimentally; however, molecular dynamics (MD) simulations might be a viable alternative. Here, we aim to assess the utility of coarse-grained (CG) MD simulations with the Martini force field for studying membrane curvature induced by transmembrane (TM) proteins that are reported to generate local curvature. The direction and magnitude of curvature induced by five different TM proteins, as well as certain lipid-protein and protein-protein interactions, were found to be in good agreement with available reference data.

Artificial Molecular Systems for Complex Functions Based on DNA Nanotechnology and Cell‐Sized Lipid Vesicles

AbstractCells are highly functional and complex molecular systems. Artificially creating such systems remains a challenge, which has been extensively studied in various research fields, including synthetic biology and molecular robotics. DNA nanotechnology is a powerful tool for bottom‐up engineering for constructing functional nanostructures or chemical reaction networks which can be utilized as components for artificial molecular systems. Encapsulation of these components into a giant unilamellar vesicle (GUV) composed of a lipid bilayer, the base structure of the cellular membrane, results in a functional cell‐sized structure that partially mimics some cellular functions. This review discusses the studies contributing to the construction of GUV‐based artificial molecular systems based on DNA nanotechnology. Molecular transport and signal transduction through lipid membranes are essential to uptake molecules from the environment and respond to stimuli. Membrane shaping relates to various functions, including motility and signaling. A chemical reaction network is required to autonomously regulate the system‘s functions. This review describes the functions realized using DNA nanostructures and DNA reaction networks. Given the designability and programmability of DNA nanotechnology, it may be possible that the functionality of artificial molecular systems could be comparable to or even surpass that of natural molecular systems.

Membrane localization of actin filaments stabilizes giant unilamellar vesicles against external deforming forces

Actin organization is crucial for establishing cell polarity, which influences processes such as directed cell motility and division. Despite its critical role in living organisms, achieving similar polarity in synthetic cells remains challenging. In this study, we employ a bottom-up approach to investigate how molecular crowders facilitate the formation of cortex-like actin networks and how these networks localize and organize based on membrane shape. Using giant unilamellar vesicles (GUVs) as models for cell membranes, we show that actin filaments can arrange along the membrane to form cortex-like structures. Notably, this organization is achieved using only actin and crowders as a minimal set of components. We utilize surface micropatterning to examine actin filament organization in deformed GUVs adhered to various pattern shapes. Our findings indicate that at the periphery of spherical GUVs, actin bundles align along the membrane. However, in highly curved regions of adhered GUVs, actin bundles avoid crossing the highly curved edges perpendicular to the adhesion site and instead remain in the lower curved regions by aligning parallel to the micropatterned surface. Furthermore, the actin bundles increase the stiffness of the GUVs, effectively counteracting strong deformations when GUVs adhere to micropatterns. This finding is corroborated by real-time deformability cytometry on GUVs with synthetic actin cortices. By precisely manipulating the shape of GUVs, our study provides a minimal system to investigate the interplay between actin structures and the membrane. Our findings provide insights into the spatial organization of actin structures within crowded environments, specifically inside GUVs that resemble the size and shape of cells. This study advances our understanding of actin network organization and functionality within cell-sized compartments.

Triacylglycerol-droplet-induced bilayer spontaneous curvature in giant unilamellar vesicles

This study investigated the incorporation of triacylglycerol droplets in the bilayers of giant unilamellar vesicles (GUVs) using four triacylglycerols and four phosphatidylcholines by confocal laser scanning microscopy. The triacylglycerol droplets were incorporated between the monolayer leaflets of the GUVs. Among the spherical droplets protruding on only one side of the bilayers, the droplets bound to the outer leaflets outnumbered those bound to the inner leaflets. The more frequent droplet binding to the outer leaflet caused transbilayer asymmetry in the droplet surface density. A vesicle consisting of a single-bilayer spherical segment and a double-bilayer spherical segment was also observed. The yield of these vesicles was comparable with or higher than that of the droplet-incorporating GUVs for many of the phosphatidylcholine-triacylglycerol combinations. In a vesicle consisting of single-bilayer and double-bilayer segments, most of the triacylglycerol droplets were localized on the outermost membrane surface along the segment boundary and in the double-bilayer segment. To rationalize the formation of these vesicle structures, we propose that the transbilayer asymmetry in the droplet surface density induces spontaneous curvature of the bilayer, with the bilayer spontaneously bending away from the droplets. Energy calculations performed assuming the existence of spontaneous curvature of the bilayer corroborated the experimentally determined membrane shapes for the vesicles consisting of unilamellar and bilamellar regions.

Ciliary tip actin dynamics regulate photoreceptor outer segment integrity

As signalling organelles, cilia regulate their G protein-coupled receptor content by ectocytosis, a process requiring localised actin dynamics to alter membrane shape. Photoreceptor outer segments comprise an expanse of folded membranes (discs) at the tip of highly-specialised connecting cilia, into which photosensitive GPCRs are concentrated. Discs are shed and remade daily. Defects in this process, due to mutations, cause retinitis pigmentosa (RP). Whilst fundamental for vision, the mechanism of photoreceptor disc generation is poorly understood. Here, we show membrane deformation required for disc genesis is driven by dynamic actin changes in a process akin to ectocytosis. We show RPGR, a leading RP gene, regulates actin-binding protein activity central to this process. Actin dynamics, required for disc formation, are perturbed in Rpgr mouse models, leading to aborted membrane shedding as ectosome-like vesicles, photoreceptor death and visual loss. Actin manipulation partially rescues this, suggesting the pathway could be targeted therapeutically. These findings help define how actin-mediated dynamics control outer segment turnover.