Mfn2-dependent fusion pathway of PE-enriched micron-sized vesicles.

Mitofusins (Mfn1 and Mfn2) are the mitochondrial outer-membrane fusion proteins in mammals and belong to the dynamin superfamily of multidomain GTPases. Recent structural studies of truncated variants lacking alpha helical transmembrane domains suggested that Mfns dimerize to promote the approximation and the fusion of the mitochondrial outer membranes upon the hydrolysis of guanine 5'-triphosphate disodium salt (GTP). However, next to the presence of GTP, the fusion activity seems to require multiple regulatory factors that control the dynamics and kinetics of mitochondrial fusion through the formation of Mfn1-Mfn2 heterodimers. Here, we purified and reconstituted the full-length murine Mfn2 protein into giant unilamellar vesicles (GUVs) with different lipid compositions. The incubation with GTP resulted in the fusion of Mfn2-GUVs. High-speed video-microscopy showed that the Mfn2-dependent membrane fusion pathway progressed through a zipper mechanism where the formation and growth of an adhesion patch eventually led to the formation of a membrane opening at the rim of the septum. The presence of physiological concentration (up to 30 mol%) of dioleoyl-phosphatidylethanolamine (DOPE) was shown to be a requisite to observe GTP-induced Mfn2-dependent fusion. Our observations show that Mfn2 alone can promote the fusion of micron-sized DOPE-enriched vesicles without the requirement of regulatory cofactors, such as membrane curvature, or the assistance of other proteins.

Membrane nanotubes transform into double-membrane sheets at condensate droplets.

Cellular membranes exhibit a multitude of highly curved morphologies such as buds, nanotubes, cisterna-like sheets defining the outlines of organelles. Here, we mimic cell compartmentation using an aqueous two-phase system of dextran and poly(ethylene glycol) encapsulated in giant vesicles. Upon osmotic deflation, the vesicle membrane forms nanotubes, which undergo surprising morphological transformations at the liquid-liquid interfaces inside the vesicles. At these interfaces, the nanotubes transform into cisterna-like double-membrane sheets (DMS) connected to the mother vesicle via short membrane necks. Using super-resolution (stimulated emission depletion) microscopy and theoretical considerations, we construct a morphology diagram predicting the tube-to-sheet transformation, which is driven by a decrease in the free energy. Nanotube knots can prohibit the tube-to-sheet transformation by blocking water influx into the tubes. Because both nanotubes and DMSs are frequently formed by cellular membranes, understanding the formation and transformation between these membrane morphologies provides insight into the origin and evolution of cellular organelles.

Experimentally determined leaflet-leaflet phase diagram of an asymmetric lipid bilayer.

We have determined the partial leaflet-leaflet phase diagram of an asymmetric lipid bilayer at ambient temperature using asymmetric giant unilamellar vesicles (aGUVs). Symmetric GUVs with varying amounts of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) were hemifused to a supported lipid bilayer (SLB) composed of DOPC, resulting in lipid exchange between their outer leaflets. The GUVs and SLB contained a red and green lipid fluorophore, respectively, thus enabling the use of confocal fluorescence imaging to determine both the extent of lipid exchange (quantified for individual vesicles by the loss of red intensity and gain of green intensity) and the presence or absence of phase separation in aGUVs. Consistent with previous reports, we found that hemifusion results in large variation in outer leaflet exchange for individual GUVs, which allowed us to interrogate the phase behavior at multiple points within the asymmetric composition space of the binary mixture. When initially symmetric GUVs showed coexisting gel and fluid domains, aGUVs with less than ~50% outer leaflet exchange were also phase-separated. In contrast, aGUVs with greater than 50% outer leaflet exchange were uniform and fluid. In some cases, we also observed three coexisting bilayer-spanning phases: two registered phases and an anti-registered phase. These results suggest that a relatively large unfavorable midplane interaction between ordered and disordered phases in opposing leaflets (i.e., a midplane surface tension) can overwhelm the driving force for lateral phase separation within one of the leaflets, resulting in an asymmetric bilayer with two uniformly mixed leaflets that is poised to phase-separate upon leaflet scrambling.

Wrapping anisotropic microgel particles in lipid membranes: Effects of particle shape and membrane rigidity.

Cellular engulfment and uptake of macromolecular assemblies or nanoparticles via endocytosis can be associated to both healthy and disease-related biological processes as well as delivery of drug nanoparticles and potential nanotoxicity of pollutants. Depending on the physical and chemical properties of the system, the adsorbed particles may remain at the membrane surface, become wrapped by the membrane, or translocate across the membrane through an endocytosis-like process. In this paper, we address the question of how the wrapping of colloidal particles by lipid membranes can be controlled by the shape of the particles, the particle-membrane adhesion energy, the membrane phase behavior, and the membrane-bending rigidity. We use a model system composed of soft core-shell microgel particles with spherical and ellipsoidal shapes, together with phospholipid membranes with varying composition. Confocal microscopy data clearly demonstrate how tuning of these basic properties of particles and membranes can be used to direct wrapping and membrane deformation and the organization of the particles at the membrane. The deep-wrapped states are more favorable for ellipsoidal than for spherical microgel particles of similar volume. Theoretical calculations for fixed adhesion strength predict the opposite behavior-wrapping becomes more difficult with increasing aspect ratio. The comparison with the experiments implies that the microgel adhesion strength must increase with increasing particle stretching. Considering the versatility offered by microgels systems to be synthesized with different shapes, functionalizations, and mechanical properties, the present findings further inspire future studies involving nanoparticle-membrane interactions relevant for the design of novel biomaterials and therapeutic applications.

Effect of membrane tension on antimicrobial peptide PGLa-induced pore formation in lipid bilayers.

The osmotic pressure (Π) method has recently been developed to quantitatively examine the effect of membrane tension (σ) on pore formation in giant unilamellar vesicles (GUVs) induced by antimicrobial peptides (AMPs). Here, we used the Π method to reveal the effect of σ on the interaction of an AMP, PGLa, with lipid bilayers comprising dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) (4/6). PGLa induced leakage of fluorescent probes from single GUVs under Π, indicating nanopore formation. Membrane tension did not transform a PGLa-induced nanopore into a micropore nor cause GUV burst up to 3.4 mN/m, which is in contrast with the effect of σ on another AMP, magainin 2-induced pore formation, where lower σ resulted in GUV burst. The fraction of leaking GUVs at a specific time increased with increasing σ, indicating that the rate of PGLa-induced pore formation increases with increasing σ. The rate of transfer of fluorescent probe-labeled PGLa across the lipid bilayer without pore formation also increased with increasing σ. PGLa-induced pore formation requires a symmetric distribution of peptides in both leaflets of the GUV bilayer, and thus we infer that the increase in the rate of PGLa transfer from the outer leaflet to the inner leaflet underlies the increase in the rate of pore formation with increasing σ. On the basis of these results, we discuss the difference between the effect of σ on nanopore formation in GUV membranes induced by PGLa and that by magainin 2.

In situ interaction between the hormone 17α-ethynylestradiol and the liquid-ordered phase composed of the lipid rafts sphingomyelin and cholesterol.

Hormone treatments are frequently associated with cardiovascular diseases and cancers in women. Additionally, the detrimental effects of their presence as contaminants in water remain a concern. The transport of hormones through cell membranes is essential for their biological action, but investigating cell permeability is challenging owing to the experimental difficulty in dealing with whole cells. In this paper, we study the interaction of the synthetic hormone 17α-ethynylestradiol (EE2) with membrane models containing the key raft components sphingomyelin (SM) and cholesterol (Chol). The models consisted of Langmuir monolayers and giant unilamellar vesicles (GUVs) that represent bilayers. EE2 induced expansion of SM monolayers upon interacting with the non-hydrated amide group of SM head, but it had practically no effect on SM GUVs because these group are not available for interaction in bilayers. In contrast, EE2 interacted with hydrated phosphate group (PO2-) and amide group of SM/Chol mixture monolayer, which could explain the loss in phase contrast of liquid-ordered GUVs suggesting pore formation. A comparison with reported EE2 effects on GUVs in the fluid phase, for which no loss in phase contrast was observed, indicates that the liquid-ordered phase consisting of lipid rafts is relevant to be associated with the changes on cell permeability caused by the hormones.

Core-Alkynylated Fluorescent Flippers: Altered Ultrafast Photophysics to Track Thick Membranes.

Fluorescent flippers have been introduced as small-molecule probes to image membrane tension in living systems. This study describes the design, synthesis, spectroscopic and imaging properties of flippers that are elongated by one and two alkynes inserted between the push and the pull dithienothiophene domains. The resulting mechanophores combine characteristics of flippers, reporting on physical compression in the ground state, and molecular rotors, reporting on torsional motion in the excited state, to take their photophysics to new level of sophistication. Intensity ratios in broadened excitation bands from differently twisted conformers of core-alkynylated flippers thus report on mechanical compression. Lifetime boosts from ultrafast excited-state planarization and lifetime drops from competitive intersystem crossing into triplet states report on viscosity. In standard lipid bilayer membranes, core-alkynylated flippers are too long for one leaflet and tilt or extend into disordered interleaflet space, which preserves rotor-like torsional disorder and thus weak, blue-shifted fluorescence. Flipper-like planarization occurs only in highly ordered membranes of matching leaflet thickness, where they light up and selectively report on these thick membranes with red-shifted, sharpened excitation maxima, high intensity and long lifetime.

Temperature-Promoted Giant Unilamellar Vesicle (GUV) Aggregation: A Way of Multicellular Formation.

The evolution of unicellular to multicellular life is considered to be an important step in the origin of life, and it is crucial to study the influence of environmental factors on this process through cell models in the laboratory. In this paper, we used giant unilamellar vesicles (GUVs) as a cell model to investigate the relationship between environmental temperature changes and the evolution of unicellular to multicellular life. The zeta potential of GUVs and the conformation of the headgroup of phospholipid molecules at different temperatures were examined using phase analysis light scattering (PALS) and attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), respectively. In addition, the effect of increasing temperature on the aggregation of GUVs was further investigated in ionic solutions, and the possible mechanisms involved were explored. The results showed that increasing temperature reduced the repulsive forces between cells models and promoted their aggregation. This study could effectively contribute to our understanding of the evolution of primitive unicellular to multicellular life.

Anomalous Colloidal Motion under Strong Confinement.

Diffusion of biological macromolecules in the cytoplasm is a paradigm of colloidal diffusion in an environment characterized by a strong restriction of the accessible volume. This makes of the understanding of the physical rules governing colloidal diffusion under conditions mimicking the reduction in accessible volume occurring in the cell cytoplasm, a problem of a paramount importance. This work aims to study how the thermal motion of spherical colloidal beads in the inner cavity of giant unilamellar vesicles (GUVs) is modified by strong confinement conditions, and the viscoelastic character of the medium. Using single particle tracking, it is found that both the confinement and the environmental viscoelasticity lead to the emergence of anomalous motion pathways for colloidal microbeads encapsulated in the aqueous inner cavity of GUVs. This anomalous diffusion is strongly dependent on the ratio between the volume of the colloidal particle and that of the GUV under consideration as well as on the viscosity of the particle's liquid environment. Therefore, the results evidence that the reduction of the free volume accessible to colloidal motion pushes the diffusion far from a standard Brownian pathway as a result of the change in the hydrodynamic boundary conditions driving the particle motion.

Cell-Mimic Directional Cargo Transportation in a Visible-Light-Activated Colloidal Motor/Lipid Tube System.

Active tether and transportation of cargoes on cytoskeletal highway enabled by molecular motors is key for accurate delivery of vesicles and organelles in the complex intracellular environment. Here, a hybrid system composed of colloidal motors and self-assembled lipid tubes is designed to mimic the subcellular traffic system in living cells. The colloidal motors, composed of gold-coated hematite, display light-activated self-propulsion tunable by the light intensity and the concentration of hydrogen peroxide fuel. Importantly, the motors show light-switchable binding with lipid cargoes and attachment to the lipid tubes, whereby the latter act as the motor highways. Upon assembly, the colloidal motor/lipid tube system demonstrates directional delivery of lipid vesicles, emulating intracellular transportation. The assembly and function of the hybrid system are rationalized by a cooperative action of light-triggered electrophoretic and hydrodynamic effects, supported by finite element analysis. A synthetic analog of the biological protein motor/cytoskeletal filament system is realized for the manipulation and delivery of different matter at the microscale, which is expected to be a promising platform for various applications in materials science, nanotechnology, microfluidics, and synthetic biology.

Surfactant-Assisted Purification of Hydrophobic DNA Nanostructures.

Purification of functional DNA nanostructures is an essential step in achieving intended functions because misfolded structures and the remaining free DNA strands in a solution can interact and affect their behavior. However, due to hydrophobicity-mediated aggregation, it is difficult to purify DNA nanostructures modified with hydrophobic molecules by conventional methods. Herein, we report the purification of cholesterol-modified DNA nanostructures by using a novel surfactant-assisted gel extraction. The addition of sodium cholate (SC) to the sample solution before structure folding prevented aggregation; this was confirmed by gel electrophoresis. We also found that adding sodium dodecyl sulfate (SDS) to the sample inhibited structural folding. The cholesterol-modified DNA nanostructures prepared with SC were successfully purified by gel extraction, and their ability to bind to the lipid membrane surfaces was maintained. This method will facilitate the purification of DNA nanostructures modified with hydrophobic molecules and expand their applicability in the construction of artificial cell-like systems.

Cheap portable electroformed giant unilamellar vesicles preparation kit.

The membrane of a cell separates the internal and external media of the cell and contributes to a variety of important processes, including gradient maintenance and signal transduction. Synthetic lipid-made vesicles are commonly utilized as cell membrane model systems. These could be liposomes or giant unilamellar vesicles (GUVs) in most cases. Liposomes are typically less than 0.5 microns in size, limiting their use for most microscopy experiments. GUVs are a form of liposomes that ranges in size from 5 to 200 microns and are ideal for examining complex phase behaviors of biomembranes using the classical optical setting. This study details the step-by-step development of a portable, light and low-cost kit for generating GUVs by electroformation. Our kit contains an in-built electronic circuitry, and the GUV generation setup, consisting of 3 ITO-coated glasses with heating electrode connections. Approximately 600 µl of GUVs can be produced in one experiment, while the amount could be increased by changing the dimensions of the GUV generation setup. Finally, the originality of the study comes from the fact that many users from different fields unfamiliar with electronics can use our home-built cost-effective approach instead of their expensive commercial counterparts.

Quantification of Giant Unilamellar Vesicle Fusion Products by High-Throughput Image Analysis.

Artificial cells are based on dynamic compartmentalized systems. Thus, remodeling of membrane-bound systems, such as giant unilamellar vesicles, is finding applications beyond biological studies, to engineer cell-mimicking structures. Giant unilamellar vesicle fusion is rapidly becoming an essential experimental step as artificial cells gain prominence in synthetic biology. Several techniques have been developed to accomplish this step, with varying efficiency and selectivity. To date, characterization of vesicle fusion has relied on small samples of giant vesicles, examined either manually or by fluorometric assays on suspensions of small and large unilamellar vesicles. Automation of the detection and characterization of fusion products is now necessary for the screening and optimization of these fusion protocols. To this end, we implemented a fusion assay based on fluorophore colocalization on the membranes and in the lumen of vesicles. Fluorescence colocalization was evaluated within single compartments by image segmentation with minimal user input, allowing the application of the technique to high-throughput screenings. After detection, statistical information on vesicle fluorescence and morphological properties can be summarized and visualized, assessing lipid and content transfer for each object by the correlation coefficient of different fluorescence channels. Using this tool, we report and characterize the unexpected fusogenic activity of sodium chloride on phosphatidylcholine giant vesicles. Lipid transfer in most of the vesicles could be detected after 20 h of incubation, while content exchange only occurred with additional stimuli in around 8% of vesicles.

Screening of Protein-Based Ultrasmall Nanozymes for Building Cell-Mimicking Catalytic Vesicles.

Enzymes are an important component for bottom-up building of synthetic/artificial cells. Nanozymes are nanomaterials with intrinsic enzyme-like properties, however, the construction of synthetic cells using nanozymes is difficult owing to their high surface energy or large size. Herein, the authors show a protein-based general platform that biomimetically integrates various ultrasmall metal nanozymes into protein shells. Specifically, eight metal-based ultrasmall nano-particles/clusters are in situ incorporated into ferritin nanocages that are self-assembled by 24 subunits of ferritin heavy chain. As a nanozyme generator, such a platform is suitable for screening the desired enzyme-like activities, including peroxidase (POD), oxidase (OXD), catalase (CAT) and superoxide dismutase (SOD). After screening, it is found that Ru intrinsically possesses the highest POD-like and CAT-like activities, while Mn and Pt show the highest OXD-like and SOD-like activities, respectively. Additionally, the inducers/inhibitors of various nanozymes are screened from more than 50 compounds to improve or inhibit their enzyme-like activities. Based on the screened nanozymes and their inhibitors, a proof-of-conceptually constructs cell-mimicking catalytic vesicles to mimic or modulate the events of redox homeostasis in living cells. This study offers a type of artificial metalloenzyme based on nanotechnology and shows a choice for bottom-up enzyme-based synthetic cell systems in a fully synthetic manner.

Membrane lipid organization and nicotinic acetylcholine receptor function: A two-way physiological relationship.

Nicotinic acetylcholine receptors (nAChRs) are involved in a great range of physiological and pathological conditions. Since they are transmembrane proteins, they interact strongly with the lipids surrounding them. Thus, the plasma membrane composition and heterogeneity play an essential role for the correct nAChR function, on the one hand, and the nAChR influences its immediate lipid environment, on the other hand. The aim of this work was to investigate in more detail the role of the biophysical properties of the membrane in nAChR function and vice versa, focusing on the relationship between Chol and nAChRs. To this end, we worked with different model systems which were treated either with (i) more Chol, (ii) cholesteryl hemisuccinate, or (iii) the enzyme cholesterol oxidase to generate different membrane sterol conditions and in the absence and presence of γTM4 peptide as a representative model of the nAChR. Fluorescence measurements with crystal violet and patch-clamp recordings were used to study nAChR conformation and function, respectively. Using confocal microscopy of giant unilamellar vesicles we probed the membrane phase state/order and organization (coexistence of lipid domains) and lipid-nAChR interaction. Our results show a feedback relationship between membrane organization and nAChR function, i.e. whereas the presence of a model of nAChRs conditions membrane organization, changing its lipid microenvironment, membrane organization and composition perturb nAChRs function. We postulate that nAChRs have a gain of function in disordered membrane environments but a loss of function in ordered ones, and that Chol molecules at the outer leaflet in annular sites and at the inner leaflet in non-annular sites are related to nAChR gating and desensitization, respectively. Thus, depending on the membrane composition, organization, and/or order, the nAChR adopts different conformations and locates in distinct lipid domains and this has a direct effect on its function.

Effects of sugar concentration on the electroporation, size distribution and average size of charged giant unilamellar vesicles.

We investigated the effects of sugar concentration on the electroporation, size distribution and average size of giant unilamellar vesicles (GUVs). GUVs were prepared from 40 mol% of 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG) and 60 mol% of 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipids. Pulsed electric field was applied to the 40%DOPG/60%DOPC-GUVs and it induced lateral electric tension (σc) in the membranes of vesicles. The σc-induced probability of rupture (Ppore) and the rate constant of rupture (kp) of GUVs under the sugar concentration, c = 40, 100 and 300 mM, were determined. Both the Ppore and kp increased with the increase of σc, but higher tension was required to generate the same values of Ppore and kp with increasing c. We also investigated average sizes of GUVs from the size distribution of vesicles under various sugar concentrations. With the increase of c, the peak of the size distribution histograms shifted to the region of smaller vesicles. The average size decreased 1.6-fold when c increased from 10 to 300 mM. These investigations help to understand various biomedical, biophysical, and biochemical processes in vesicles and cells. Electroporation, size distribution and average size of charged GUVs were investigated under various sugar concentrations. The sugar concentration influences the electroporation of vesicles and the average size of GUVs.

The ESCRT-III isoforms CHMP2A and CHMP2B display different effects on membranes upon polymerization.

BACKGROUND: ESCRT-III proteins are involved in many membrane remodeling processes including multivesicular body biogenesis as first discovered in yeast. In humans, ESCRT-III CHMP2 exists as two isoforms, CHMP2A and CHMP2B, but their physical characteristics have not been compared yet. RESULTS: Here, we use a combination of techniques on biomimetic systems and purified proteins to study their affinity and effects on membranes. We establish that CHMP2B binding is enhanced in the presence of PI(4,5)P2 lipids. In contrast, CHMP2A does not display lipid specificity and requires CHMP3 for binding significantly to membranes. On the micrometer scale and at moderate bulk concentrations, CHMP2B forms a reticular structure on membranes whereas CHMP2A (+CHMP3) binds homogeneously. Thus, CHMP2A and CHMP2B unexpectedly induce different mechanical effects to membranes: CHMP2B strongly rigidifies them while CHMP2A (+CHMP3) has no significant effect. CONCLUSIONS: We therefore conclude that CHMP2B and CHMP2A exhibit different mechanical properties and might thus contribute differently to the diverse ESCRT-III-catalyzed membrane remodeling processes.

Curcumin-Induced Stabilization of Protein-Based Nano-Delivery Vehicles Reduces Disruption of Zwitterionic Giant Unilamellar Vesicles.

Curcumin-loaded native and succinylated pea protein nanoparticles, as well as zwitterionic giant unilamellar vesicles were used in this study as model bioactive compound loaded-nanoparticles and biomembranes, respectively, to assess bio-nano interactions. Curcumin-loaded native protein-chitosan and succinylated protein-chitosan complexes, as well as native protein-chitosan and succinylated protein-chitosan hollow, induced leakage of the calcein encapsulated in the giant unilamellar vesicles. The leakage was more pronounced with hollow protein-chitosan complexes. However, curcumin-loaded native protein and curcumin-loaded succinylated protein nanoparticles induced calcein fluorescence quenching. Dynamic light scattering measurements showed that the interaction of curcumin-loaded native protein, curcumin-loaded succinylated protein, native protein-chitosan, and succinylated protein-chitosan complexes with the giant unilamellar vesicles caused a major reduction in the size of the lipid vesicles. Confocal and widefield fluorescence microscopy showed rupturing of the unilamellar vesicles after treatment with native pea protein-chitosan and succinylated pea protein-chitosan complexes. The nature of interaction between the curcumin-loaded protein nanoparticles and the biomembranes, at the bio-nano interface, is influenced by the encapsulated curcumin. Findings from this study showed that, as the protein plays a crucial role in stabilizing the bioactive compound from chemical and photodegradation, the encapsulated nutraceutical stabilizes the protein nanoparticle to reduce its interaction with biomembranes.

Peptide-Oleate Complexes Create Novel Membrane-Bound Compartments.

A challenging question in evolutionary theory is the origin of cell division and plausible molecular mechanisms involved. Here, we made the surprising observation that complexes formed by short alpha-helical peptides and oleic acid can create multiple membrane-enclosed spaces from a single lipid vesicle. The findings suggest that such complexes may contain the molecular information necessary to initiate and sustain this process. Based on these observations, we propose a new molecular model to understand protocell division.

More from less - bottom-up reconstitution of cell biology.

The ultimate goal of bottom-up synthetic biology is recreating life in its simplest form. However, in its quest to find the minimal functional units of life, this field contributes more than its main aim by also offering a range of tools for asking, and experimentally approaching, biological questions. This Review focusses on how bottom-up reconstitution has furthered our understanding of cell biology. Studying cell biological processes in vitro has a long tradition, but only recent technological advances have enabled researchers to reconstitute increasingly complex biomolecular systems by controlling their multi-component composition and their spatiotemporal arrangements. We illustrate this progress using the example of cytoskeletal processes. Our understanding of these has been greatly enhanced by reconstitution experiments, from the first in vitro experiments 70 years ago to recent work on minimal cytoskeleton systems (including this Special Issue of Journal of Cell Science). Importantly, reconstitution approaches are not limited to the cytoskeleton field. Thus, we also discuss progress in other areas, such as the shaping of biomembranes and cellular signalling, and prompt the reader to add their subfield of cell biology to this list in the future.