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.

SecYEG-mediated translocation in a model synthetic cell.

Giant unilamellar vesicles (GUVs) provide a powerful model compartment for synthetic cells. However, a key challenge is the incorporation of membrane proteins that allow for transport, energy transduction, compartment growth and division. Here, we have successfully incorporated the membrane protein complex SecYEG-the key bacterial translocase that is essential for the incorporation of newly synthesized membrane proteins-in GUVs. Our method consists of fusion of small unilamellar vesicles containing reconstituted SecYEG into GUVs, thereby forming SecGUVs. These are suitable for large-scale experiments while maintaining a high protein:lipid ratio. We demonstrate that incorporation of SecYEG into GUVs does not inhibit its translocation efficiency. Robust membrane protein functionalized proteo-GUVs are promising and flexible compartments for use in the formation and growth of synthetic cells.

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.

Triplet-Triplet Annihilation Upconverting Liposomes: Mechanistic Insights into the Role of Membranes in Two-Dimensional TTA-UC.

Triplet-triplet annihilation upconversion (TTA-UC) implemented in nanoparticle assemblies is of emerging interest in biomedical applications, including in drug delivery and imaging. As it is a bimolecular process, ensuring sufficient mobility of the sensitizer and annihilator to facilitate effective collision in the nanoparticle is key. Liposomes can provide the benefits of two-dimensional confinement and condensed concentration of the sensitizer and annihilator along with superior fluidity compared to other nanoparticle assemblies. They are also biocompatible and widely applied across drug delivery modalities. However, there are relatively few liposomal TTA-UC systems reported to date, so systematic studies of the influence of the liposomal environment on TTA-UC are currently lacking. Here, we report the first example of a BODIPY-based sensitizer TTA-UC system within liposomes and use this system to study TTA-UC generation and compare the relative intensity of the anti-Stokes signal for this system as a function of liposome composition and membrane fluidity. We report for the first time on time-resolved spectroscopic studies of TTA-UC in membranes. Nanosecond transient absorption data reveal the BODIPY-perylene dyad sensitizer has a long triplet lifetime in liposome with contributions from three triplet excited states, whose lifetimes are reduced upon coinclusion of the annihilator due to triplet-triplet energy transfer, to a greater extent than in solution. This indicates triplet energy transfer between the sensitizer and the annihilator is enhanced in the membrane system. Molecular dynamics simulations of the sensitizer and annihilator TTA collision complex are modeled in the membrane and confirm the co-orientation of the pair within the membrane structure and that the persistence time of the bound complex exceeds the TTA kinetics. Modeling also reliably predicted the diffusion coefficient for the sensitizer which matches closely with the experimental values from fluorescence correlation spectroscopy. The relative intensity of the TTA-UC output across nine liposomal systems of different lipid compositions was explored to examine the influence of membrane viscosity on upconversion (UC). UC showed the highest relative intensity for the most fluidic membranes and the weakest intensity for highly viscous membrane compositions, including a phase separation membrane. Overall, our study reveals that the co-orientation of the UC pair within the membrane is crucial for effective TTA-UC within a biomembrane and that the intensity of the TTA-UC output can be tuned in liposomal nanoparticles by modifying the phase and fluidity of the liposome. These new insights will aid in the design of liposomal TTA-UC systems for biomedical applications.

Tuning the double lipidation of salmon calcitonin to introduce a pore-like membrane translocation mechanism.

A widespread strategy to increase the transport of therapeutic peptides across cellular membranes has been to attach lipid moieties to the peptide backbone (lipidation) to enhance their intrinsic membrane interaction. Efforts in vitro and in vivo investigating the correlation between lipidation characteristics and peptide membrane translocation efficiency have traditionally relied on end-point read-out assays and trial-and-error-based optimization strategies. Consequently, the molecular details of how therapeutic peptide lipidation affects it's membrane permeation and translocation mechanisms remain unresolved. Here we employed salmon calcitonin as a model therapeutic peptide and synthesized nine double lipidated analogs with varying lipid chain lengths. We used single giant unilamellar vesicle (GUV) calcein influx time-lapse fluorescence microscopy to determine how tuning the lipidation length can lead to an All-or-None GUV filling mechanism, indicative of a peptide mediated pore formation. Finally, we used a GUVs-containing-inner-GUVs assay to demonstrate that only peptide analogs capable of inducing pore formation show efficient membrane translocation. Our data provided the first mechanistic details on how therapeutic peptide lipidation affects their membrane perturbation mechanism and demonstrated that fine-tuning lipidation parameters could induce an intrinsic pore-forming capability. These insights and the microscopy based workflow introduced for investigating structure-function relations could be pivotal for optimizing future peptide design strategies.

Processes and mechanisms underlying burst of giant unilamellar vesicles induced by antimicrobial peptides and compounds.

To clarify the damage of lipid bilayer region in bacterial cell membrane caused by antimicrobial peptides (AMPs) and antimicrobial compounds (AMCs), their interactions with giant unilamellar vesicles (GUVs) of various lipid compositions have been examined. The findings revealed two main causes for the leakage: nanopore formation in the membrane and burst of GUVs. Although GUV burst has been explained previously based on the carpet model, the supporting evidence is limited. In this review, to better clarify the mechanism of GUV burst by AMPs, AMCs, and other membrane-active peptides, we described current knowledge of the conditions, characteristics, and detailed processes of GUV burst and the changes in the shape of the GUVs during burst. We identified several physical factors that affect GUV burst, such as membrane tension, electrostatic interaction, structural changes of GUV membrane such as membrane folding, and oil in the membrane. We also clarified one of the physical mechanisms underlying the instability of lipid bilayers that are associated with leakage in the carpet model. Based on these results, we propose a mechanism underlying some types of GUV burst induced by these substances: the growth of a nanopore to a micropore, resulting in GUV burst.

Molecular Interactions Between Egg White Peptides and Giant Unilamellar Vesicle Membranes: Effect of Peptide Localization on Membrane Fluidity.

Bioactive peptides have been shown to affect cell membrane fluidity, which is an important indicator of the cell membrane structure and function. However, the underlying mechanism of egg white-derived bioactive peptide regulation of cell membrane fluidity has not been elucidated yet. The cell membrane fluidity was investigated by giant unilamellar vesicles in the present study. The results showed that peptides TCNW, ADWAK, ESIINF, VPIEGII, LVEEY, and WKLC connect to membranes through intermolecular interactions, such as hydrogen bonding and regulated membrane fluidity, in a concentration-dependent way. In addition, peptides prefer to localize in the hydrophobic core of the bilayers. This study provides a theoretical basis for analyzing the localization of egg white bioactive peptides in specific cell membrane regions and their influence on the cell membrane fluidity.

Interaction of selected alkoxy naringenin oximes with model and bacterial membranes.

Naringenin is a flavonoid found in many fruits and herbs, most notably in grapefruits. In recent years, this compound and its derivatives have been of great interest due to their high biological activity, including fungicidal and bactericidal effects, also in relation to multidrug-resistant bacteria. Membrane interactions of naringenin oxime (NO) and its 7-O-alkyl (7-alkoxy) derivatives, such as methyl (7MENO), ethyl (7ETNO), isopropyl (7IPNO), n-butyl (7BUNO) and n-pentyl (7PENO) were studied. Thermotropic properties of model membranes were investigated via differential scanning calorimetry (DSC), the influence on lipid raft mimicking giant unilamellar vesicles (GUVs) via fluorescence microscopy, and membrane permeability via measuring calcein leakage from liposomes. Molecular calculations supplemented the study. The influence of naringenin oximes on two strains of multidrug resistant bacteria: Staphylococcus aureus KJ and Enterococcus faecalis 37VRE was also investigated. In DSC studies all compounds reduced the temperature and enthalpy of main phase transition and caused disappearing of the pretransition. NO was the least active. The reduction in the area of surface domains in GUVs was observed for NO. Compounds NO and 7BUNO resulted in very low secretion of calcein from liposomes (permeability < 3 %). The highest results were observed for 7MENO (88.4 %) and 7IPNO (78.5 %). When bacterial membrane permeability was investigated all compounds caused significant release of propidium iodide from S. aureus (31.6-87.0 % for concentration 128 µg/mL). In the case of E. faecalis, 7ETNO (75.7 %) and NO (28.8 %) were the most active. The rest of the tested compounds showed less activity (permeability < 13.9 %). The strong evidence was observed that antibacterial activity of the tested compounds may be associated with their interaction with bacterial membrane.

Dynamics of giant vesicle assembly from thin lipid films.

MOTIVATION: Giant unilamellar vesicles (GUVs), cell-like synthetic micrometer size structures, assemble when thin lipid films are hydrated in aqueous solutions. Quantitative measurements of static yields and distribution of sizes of GUVs obtained from thin film hydration methods were recently reported. Dynamic data such as the time evolution of yields and distribution of sizes, however, is not known. Dynamic data can provide insights into the assembly pathway of GUVs and guidelines for choosing conditions to obtain populations with desired size distributions. APPROACH: We develop the 'stopped-time' technique to characterize the time evolution of the distribution of sizes and molar yields of populations of free-floating GUVs. We additionally capture high resolution time-lapse images of surface-attached GUV buds on the lipid films. We systematically study the dynamics of assembly of GUVs from three widely used thin film hydration methods, PAPYRUS (Paper-Abetted amPhiphile hYdRation in aqUeous Solutions), gentle hydration, and electroformation. FINDINGS: We find that the molar yield versus time curves of GUVs demonstrate a characteristic sigmoidal shape, with an initial yield, a transient, and then a steady state plateau for all three methods. The population of GUVs showed a right-skewed distribution of diameters. The variance of the distributions increased with time. The systems reached steady state within 120 min. We rationalize the dynamics using the thermodynamically motivated budding and merging (BNM) model. These results further the understanding of lipid dynamics and provide for the first-time practical parameters to tailor the production of GUVs of specific sizes for applications.

Exploring the synergistic effects of amoxicillin and methylene blue on unsaturated lipid structures: A study of Langmuir monolayers and giant unilamellar vesicles.

The potentially toxic effects of emerging pollutant mixtures often deviate from the individual compound effects, presenting additive, synergistic, or agonistic interactions. This study delves into the complex world of emerging pollutants' mixtures, with a particular focus on their potential impact on unsaturated lipid DOPC (1,2-dioleoyl-sn-glycerol-3-phosphocholine) structured as both monolayers and bilayers, which are valuable tools for mimicking cell membranes. Specifically, we examine the effects of two common types of pollutants: antibiotics (amoxicillin) and dyes (methylene blue). Utilizing Langmuir monolayers, our research reveals a synergistic effect within the pollutant mixture, as evidenced by pressure-area isotherms and polarization-modulated infrared reflection absorption spectroscopy. We identify the specific chemical interactions contributing to this synergistic effect. Furthermore, through contrast phase microscopy experiments on giant unilamellar vesicles (bilayer system), we find that the individual pollutants and the mixture exhibit similar molecular effects on the bilayer, revealing that the molecular size is a key factor in the bilayer-mixture of pollutant interaction. This highlights the importance of considering molecular size in the interactions with bilayer systems. In summary, our research dissects the critical factors of chemical interactions and molecular size concerning the effects of pollutants on DOPC, serving as simplified models of cell membranes. This study underscores the significance of comprehending the molecular effects of emerging pollutants on human health and the development of models for exploring their intricate interactions with cell membranes.

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.

Molecular and cellular evidence of a direct interaction between the TRAF2 C-terminal domain and ganglioside GM1.

TNF receptor-associated factor 2 (TRAF2) is involved in different cellular processes including signal transduction and transcription regulation. We here provide evidence of a direct interaction between the TRAF domain of TRAF2 and the monosialotetrahexosylganglioside (GM1). Previously, we showed that the TRAF domain occurs mainly in a trimeric form in solution, but it can also exist as a stable monomer when in the nanomolar concentration range. Here, we report that the quaternary structure of the TRAF domain is also affected by pH changes, since a weakly acidic pH (5.5) favors the dissociation of the trimeric TRAF domain into stable monomers, as previously observed at neutral pH (7.6) with the diluted protein. The TRAF domain-GM1 binding was similar at pH 5.5 and 7.6, suggesting that GM1 interacts with both the trimeric and monomeric forms of the protein. However, only the monomeric protein appeared to cause membrane deformation and inward vesiculation in GM1-containing giant unilamellar vesicles (GUVs). The formation of complexes between GM1 and TRAF2, or its TRAF domain, was also observed in cultured human leukemic HAP1 cells expressing either the truncated TRAF domain or the endogenous full length TRAF2. The GM1-protein complexes were observed after treatment with tunicamycin and were more concentrated in cells undergoing apoptosis, a condition which is known to cause cytoplasm acidification. These findings open the avenue for future studies aimed at deciphering the physiopathological relevance of the TRAF domain-GM1 interaction.

Antifungal Activity and Molecular Mechanisms of Copper Nanoforms against Colletotrichum gloeosporioides.

In this work, we have synthesized copper nanoforms (Cu NFs) using ascorbic acid as a reducing agent and polyvinylpyrrolidone as a stabilizer. Elemental characterization using EDS has shown the nanostructure to be of high purity and compare well with commercially sourced nanoforms. SEM images of both Cu NFs show some agglomeration. The in-house NFs had a better even distribution and size of the nanostructures. The XRD peaks represented a face-centered cubic structure of Cu2O. The commercially sourced Cu NFs were found to be a mixture of Cu and Cu2O. Both forms had a crystalline structure. Using these two types of Cu NFs, an antimicrobial study against Colletotrichum gloeosporioides, a devastating plant pathogen, showed the in-house Cu NFs to be most effective at inhibiting growth of the pathogen. Interestingly, at low concentrations, both Cu NFs increased fungal growth, although the mycelia appeared thin and less dense than in the control. SEM macrographs showed that the in-house Cu NFs inhibited the fungus by flattening the mycelia and busting some of them. In contrast, the mycelia were short and appeared clustered when exposed to commercial Cu NFs. The difference in effect was related to the size and/or oxidation state of the Cu NFs. Furthermore, the fungus produced a defense mechanism in response to the NFs. The fungus produced melanin, with the degree of melanization directly corresponding to the concentration of the Cu NFs. Localization of aggregated Cu NFs could be clearly observed outside of the model membranes. The large agglomerates may only contribute indirectly by a hit-and-bounce-off effect, while small structures may adhere to the membrane surface and/or internalize. Spatio-temporal membrane dynamics were captured in real time. The dominant dynamics culminated into large fluctuations. Some of the large fluctuations resulted in vesicular transformation. The major transformation was exo-bud/exo-cytosis, which may be a way to excrete the foreign object (Cu NFs).

Bottom-up Assembled Synthetic SARS-CoV-2 Miniviruses Reveal Lipid Membrane Affinity of Omicron Variant Spike Glycoprotein.

The ongoing COVID-19 pandemic has been brought on by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The spike glycoprotein (S), which decorates the viral envelope forming a corona, is responsible for the binding to the angiotensin-converting enzyme 2 (ACE2) receptor and initiating the infection. In comparison to previous variants, Omicron S presents additional binding sites as well as a more positive surface charge. These changes hint at additional molecular targets for interactions between virus and cell, such as the cell membrane or proteoglycans on the cell surface. Herein, bottom-up assembled synthetic SARS-CoV-2 miniviruses (MiniVs), with a lipid composition similar to that of infectious particles, are implemented to study and compare the binding properties of Omicron and Alpha variants. Toward this end, a systematic functional screening is performed to study the binding ability of Omicron and Alpha S proteins to ACE2-functionalized and nonfunctionalized planar supported lipid bilayers. Moreover, giant unilamellar vesicles are used as a cell membrane model to perform competitive interaction assays of the two variants. Finally, two cell lines with and without presentation of the ACE2 receptor are used to confirm the binding properties of the Omicron and Alpha MiniVs to the cellular membrane. Altogether, the results reveal a significantly higher affinity of Omicron S toward both the lipid membrane and ACE2 receptor. The research presented here highlights the advantages of creating and using bottom-up assembled SARS-CoV-2 viruses to understand the impact of changes in the affinity of S for ACE2 in infection studies.

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.

Spatial Control of Arp2/3-Induced Actin Polymerization on Phase-Separated Giant Unilamellar Vesicles.

Deciphering the physical mechanisms underlying cell shape changes, while avoiding the cellular interior's complexity, involves the development of controlled basic biomimetic systems that imitate cell functions. In particular, the reconstruction of cytoskeletal dynamics on cell-sized giant unilamellar vesicles (GUVs) has allowed for the reconstituting of some cell-like processes in vitro. In fact, such a bottom-up strategy could be the basis for forming protocells able to reorganize or even move autonomously. However, reconstituting the subtle and controlled dynamics of the cytoskeleton-membrane interface in vitro remains an experimental challenge. Taking advantage of the lipid-induced segregation of an actin polymerization activator, we present a system that targets actin polymerization in specific domains of phase-separated GUVs. We observe actin networks localized on Lo, Ld, or on both types of domains and the actin-induced deformation or reorganization of these domains. These results suggest that the system we have developed here could pave the way for future experiments further detailing the interplay between actin dynamics and membrane heterogeneities.

Formation of Giant Unilamellar Vesicles Assisted by Fluorinated Nanoparticles.

In the quest to produce artificial cells, one key challenge that remains to be solved is the recreation of a complex cellular membrane. Among the existing models, giant unilamellar vesicles (GUVs) are particularly interesting due to their intrinsic compartmentalisation ability and their resemblance in size and shape to eukaryotic cells. Many techniques have been developed to produce GUVs all having inherent advantages and disadvantages. Here, the authors show that fluorinated silica nanoparticles (FNPs) used to form Pickering emulsions in a fluorinated oil can destabilise lipid nanosystems to template the formation of GUVs. This technique enables GUV production across a broad spectrum of buffer conditions, while preventing the leakage of the encapsulated components into the oil phase. Furthermore, a simple centrifugation process is sufficient for the release of the emulsion-trapped GUVs, bypassing the need to use emulsion-destabilising chemicals. With fluorescent FNPs and transmission electron microscopy, the authors confirm that FNPs are efficiently removed, producing contaminant-free GUVs. Further experiments assessing the lateral diffusion of lipids and unilamellarity of the GUVs demonstrate that they are comparable to GUVs produced via electroformation. Finally, the ability of incorporating transmembrane proteins is demonstrated, highlighting the potential of this method for the production of GUVs for artificial cell applications.

Facile and Programmable Capillary-Induced Assembly of Prototissues via Hanging Drop Arrays.

An important goal for bottom-up synthetic biology is to construct tissue-like structures from artificial cells. The key is the ability to control the assembly of the individual artificial cells. Unlike most methods resorting to external fields or sophisticated devices, inspired by the hanging drop method used for culturing spheroids of biological cells, we employ a capillary-driven approach to assemble giant unilamellar vesicles (GUVs)-based protocells into colonized prototissue arrays by means of a coverslip with patterned wettability. By spatially confining and controllably merging a mixed population of lipid-coated double-emulsion droplets that hang on a water/oil interface, an array of synthetic tissue-like constructs can be obtained. Each prototissue module in the array comprises multiple tightly packed droplet compartments where interfacial lipid bilayers are self-assembled at the interfaces both between two neighboring droplets and between the droplet and the external aqueous environment. The number, shape, and composition of the interconnected droplet compartments can be precisely controlled. Each prototissue module functions as a processer, in which fast signal transports of molecules via cell-cell and cell-environment communications have been demonstrated by molecular diffusions and cascade enzyme reactions, exhibiting the ability to be used as biochemical sensing and microreactor arrays. Our work provides a simple yet scalable and programmable method to form arrays of prototissues for synthetic biology, tissue engineering, and high-throughput assays.