PspA adopts an ESCRT-III-like fold and remodels bacterial membranes.

PspA is the main effector of the phage shock protein (Psp) system and preserves the bacterial inner membrane integrity and function. Here, we present the 3.6 Å resolution cryoelectron microscopy (cryo-EM) structure of PspA assembled in helical rods. PspA monomers adopt a canonical ESCRT-III fold in an extended open conformation. PspA rods are capable of enclosing lipids and generating positive membrane curvature. Using cryo-EM, we visualized how PspA remodels membrane vesicles into µm-sized structures and how it mediates the formation of internalized vesicular structures. Hotspots of these activities are zones derived from PspA assemblies, serving as lipid transfer platforms and linking previously separated lipid structures. These membrane fusion and fission activities are in line with the described functional properties of bacterial PspA/IM30/LiaH proteins. Our structural and functional analyses reveal that bacterial PspA belongs to the evolutionary ancestry of ESCRT-III proteins involved in membrane remodeling.

Size-Dependent Segregation Controls Macrophage Phagocytosis of Antibody-Opsonized Targets.

Macrophages protect the body from damage and disease by targeting antibody-opsonized cells for phagocytosis. Though antibodies can be raised against antigens with diverse structures, shapes, and sizes, it is unclear why some are more effective at triggering immune responses than others. Here, we define an antigen height threshold that regulates phagocytosis of both engineered and cancer-specific antigens by macrophages. Using a reconstituted model of antibody-opsonized target cells, we find that phagocytosis is dramatically impaired for antigens that position antibodies >10 nm from the target surface. Decreasing antigen height drives segregation of antibody-bound Fc receptors from the inhibitory phosphatase CD45 in an integrin-independent manner, triggering Fc receptor phosphorylation and promoting phagocytosis. Our work shows that close contact between macrophage and target is a requirement for efficient phagocytosis, suggesting that therapeutic antibodies should target short antigens in order to trigger Fc receptor activation through size-dependent physical segregation.

Polysaccharide functionalization reduces lipid vesicle stiffness.

The biophysical properties of lipid vesicles are important for their stability and integrity, key parameters that control the performance when these vesicles are used for drug delivery. The vesicle properties are determined by the composition of lipids used to form the vesicle. However, for a given lipid composition, they can also be tailored by tethering polymers to the membrane. Typically, synthetic polymers like polyethyleneglycol are used to increase vesicle stability, but the use of polysaccharides in this context is much less explored. Here, we report a general method for functionalizing lipid vesicles with polysaccharides by binding them to cholesterol. We incorporate the polysaccharides on the outer membrane leaflet of giant unilamellar vesicles (GUVs) and investigate their effect on membrane mechanics using micropipette aspiration. We find that the presence of the glycolipid functionalization produces an unexpected softening of GUVs with fluid-like membranes. By contrast, the functionalization of GUVs with polyethylene glycol does not reduce their stretching modulus. This work provides the potential means to study membrane-bound meshworks of polysaccharides similar to the cellular glycocalyx; moreover, it can be used for tuning the mechanical properties of drug delivery vehicles.

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.

Systematic comparison of unilamellar vesicles reveals that archaeal core lipid membranes are more permeable than bacterial membranes.

One of the deepest branches in the tree of life separates the Archaea from the Bacteria. These prokaryotic groups have distinct cellular systems including fundamentally different phospholipid membrane bilayers. This dichotomy has been termed the lipid divide and possibly bestows different biophysical and biochemical characteristics on each cell type. Classic experiments suggest that bacterial membranes (formed from lipids extracted from Escherichia coli, for example) show permeability to key metabolites comparable to archaeal membranes (formed from lipids extracted from Halobacterium salinarum), yet systematic analyses based on direct measurements of membrane permeability are absent. Here, we develop a new approach for assessing the membrane permeability of approximately 10 µm unilamellar vesicles, consisting of an aqueous medium enclosed by a single lipid bilayer. Comparing the permeability of 18 metabolites demonstrates that diether glycerol-1-phosphate lipids with methyl branches, often the most abundant membrane lipids of sampled archaea, are permeable to a wide range of compounds useful for core metabolic networks, including amino acids, sugars, and nucleobases. Permeability is significantly lower in diester glycerol-3-phosphate lipids without methyl branches, the common building block of bacterial membranes. To identify the membrane characteristics that determine permeability, we use this experimental platform to test a variety of lipid forms bearing a diversity of intermediate characteristics. We found that increased membrane permeability is dependent on both the methyl branches on the lipid tails and the ether bond between the tails and the head group, both of which are present on the archaeal phospholipids. These permeability differences must have had profound effects on the cell physiology and proteome evolution of early prokaryotic forms. To explore this further, we compare the abundance and distribution of transmembrane transporter-encoding protein families present on genomes sampled from across the prokaryotic tree of life. These data demonstrate that archaea tend to have a reduced repertoire of transporter gene families, consistent with increased membrane permeation. These results demonstrate that the lipid divide demarcates a clear difference in permeability function with implications for understanding some of the earliest transitions in cell origins and evolution.

Active particles induce large shape deformations in giant lipid vesicles.

Biological cells generate intricate structures by sculpting their membrane from within to actively sense and respond to external stimuli or to explore their environment1-4. Several pathogenic bacteria also provide examples of how localized forces strongly deform cell membranes from inside, leading to the invasion of neighbouring healthy mammalian cells5. Giant unilamellar vesicles have been successfully used as a minimal model system with which to mimic biological cells6-11, but the realization of a minimal system with localized active internal forces that can strongly deform lipid membranes from within and lead to dramatic shape changes remains challenging. Here we present a combined experimental and simulation study that demonstrates how self-propelled particles enclosed in giant unilamellar vesicles can induce a plethora of non-equilibrium shapes and active membrane fluctuations. Using confocal microscopy, in the experiments we explore the membrane response to local forces exerted by self-phoretic Janus microswimmers. To quantify dynamic membrane changes, we perform Langevin dynamics simulations of active Brownian particles enclosed in thin membrane shells modelled by dynamically triangulated surfaces. The most pronounced shape changes are observed at low and moderate particle loadings, with the formation of tether-like protrusions and highly branched, dendritic structures, whereas at high volume fractions globally deformed vesicle shapes are observed. The resulting state diagram predicts the conditions under which local internal forces generate various membrane shapes. A controlled realization of such distorted vesicle morphologies could improve the design of artificial systems such as small-scale soft robots and synthetic cells.

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.

Nonspecific membrane-matrix interactions influence diffusivity of lipid vesicles in hydrogels.

The diffusion of extracellular vesicles and liposomes in vivo is affected by different tissue environmental conditions and is of great interest in the development of liposome-based therapeutics and drug-delivery systems. Here, we use a bottom-up biomimetic approach to better isolate and study steric and electrostatic interactions and their influence on the diffusivity of synthetic large unilamellar vesicles in hydrogel environments. Single-particle tracking of these extracellular vesicle-like particles in agarose hydrogels as an extracellular matrix model shows that membrane deformability and surface charge affect the hydrogel pore spaces that vesicles have access to, which determines overall diffusivity. Moreover, we show that passivation of vesicles with PEGylated lipids, as often used in drug-delivery systems, enhances diffusivity, but that this effect cannot be fully explained with electrostatic interactions alone. Finally, we compare our experimental findings with existing computational and theoretical work in the field to help explain the nonspecific interactions between diffusing particles and gel matrix environments.

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.

Effects of grafted polymers on the lipid membrane fluidity.

Since the membrane fluidity controls the cellular functions, it is important to identify the factors that determine the cell membrane viscosity. Cell membranes are composed of not only lipids and proteins but also polysaccharide chain-anchored molecules, such as glycolipids. To reveal the effects of grafted polymers on the membrane fluidity, in this study, we measured the membrane viscosity of polymer-grafted giant unilamellar vesicles (GUVs), which were prepared by introducing the poly (ethylene glycol) (PEG)-anchored lipids to the ternary GUVs composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol. The membrane viscosity was obtained from the velocity field on the GUV generated by applying a point force, based on the hydrodynamic model proposed by Henle and Levine. The velocity field was visualized by a motion of the circular liquid ordered (Lo) domains formed by a phase separation. With increasing PEG density, the membrane viscosity of PEG-grafted GUVs increased gradually in the mushroom region and significantly in the brush region. We propose a hydrodynamic model that includes the excluded volume effect of PEG chains to explain the increase in membrane viscosity in the mushroom region. This work provides a basic understanding of how grafted polymers affect the membrane fluidity.

Negative lipid membranes enhance the adsorption of TAT-decorated elastin-like polypeptide micelles.

A cell-penetrating peptide (CPP) is a short amino-acid sequence capable of efficiently translocating across the cellular membrane of mammalian cells. However, the potential of CPPs as a delivery vector is hampered by the strong reduction of its translocation efficiency when it bears an attached molecular cargo. To overcome this problem, we used previously developed diblock copolymers of elastin-like polypeptides (ELPBCs), which we end functionalized with TAT (transactivator of transcription), an archetypal CPP built from a positively charged amino acid sequence of the HIV-1 virus. These ELPBCs self-assemble into micelles at a specific temperature and present the TAT peptide on their corona. These micelles can recover the lost membrane affinity of TAT and can trigger interactions with the membrane despite the presence of a molecular cargo. Herein, we study the influence of membrane surface charge on the adsorption of TAT-functionalized ELP micelles onto giant unilamellar vesicles (GUVs). We show that the TAT-ELPBC micelles show an increased binding constant toward negatively charged membranes compared to neutral membranes, but no translocation is observed. The affinity of the TAT-ELPBC micelles for the GUVs displays a stepwise dependence on the lipid charge of the GUV, which, to our knowledge, has not been reported previously for interactions between peptides and lipid membranes. By unveiling the key steps controlling the interaction of an archetypal CPP with lipid membranes, through regulation of the charge of the lipid bilayer, our results pave the way for a better design of delivery vectors based on CPPs.

Intravesicular Solute Delivery and Surface Area Regulation in Giant Unilamellar Vesicles Driven by Cycles of Osmotic Stresses.

Phospholipid bilayers are dynamic cellular components that undergo constant changes in their topology, facilitating a broad diversity of physiological functions including endo- and exocytosis, cell division, and intracellular trafficking. These shape transformations consume energy, supplied invariably by the activity of proteins. Here, we show that cycles of oppositely directed osmotic stresses─unassisted by any protein activity─can induce well-defined remodeling of giant unilamellar vesicles, minimally recapitulating the phenomenologies of surface area homeostasis and macropinocytosis. We find that a stress cycle consisting of deflationary hypertonic stress followed by an inflationary hypotonic one prompts an elaborate sequence of membrane shape changes ultimately transporting molecular cargo from the outside into the intravesicular milieu. The initial osmotic deflation produces microscopic spherical invaginations. During the subsequent inflation, the first subpopulation contributes area to the swelling membrane, thereby providing a means for surface area regulation and tensional homeostasis. The second subpopulation vesiculates into the lumens of the mother vesicles, producing pinocytic vesicles. Remarkably, the gradients of solute concentrations between the GUV and the daughter pinocytic vesicles create cascades of water current, inducing pulsatory transient poration that enable solute exchange between the buds and the GUV interior. This results in an efficient water-flux-mediated delivery of molecular cargo across the membrane boundary. Our findings suggest a primitive physical mechanism for communication and transport across protocellular compartments driven only by osmotic stresses. They also suggest plausible physical routes for intravesicular, and possibly intracellular, delivery of ions, solutes, and molecular cargo stimulated simply by cycles of osmotic currents of water.

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.

Large Unilamellar Vesicles of Phosphatidic Acid Reduce the Toxicity of α-Synuclein Fibrils.

Parkinson's disease (PD) is a severe pathology that is caused by a progressive degeneration of dopaminergic neurons in substantia nigra pars compacta as well as other areas in the brain. These neurodegeneration processes are linked to the abrupt aggregation of α-synuclein (α-syn), a small protein that is abundant at presynaptic nerve termini, where it regulates cell vesicle trafficking. Due to the direct interactions of α-syn with cell membranes, a substantial amount of work was done over the past decade to understand the role of lipids in α-syn aggregation. However, the role of phosphatidic acid (PA), a negatively charged phospholipid with a small polar head, remains unclear. In this study, we examined the effect of PA large unilamellar vesicles (LUVs) on α-syn aggregation. We found that PA LUVs with 16:0, 18:0, and 18:1 FAs drastically reduced the toxicity of α-syn fibrils if were present in a 1:1 molar ratio with the protein. Our results also showed that the presence of these vehicles changed the rate of α-syn aggregation and altered the morphology and secondary structure of α-syn fibrils. These results indicate that PA LUVs can be used as a potential therapeutic strategy to reduce the toxicity of α-syn fibrils formed upon PD.

Long-Chain Lipids Facilitate Insertion of Large Nanoparticles into Membranes of Small Unilamellar Vesicles.

Insertion of hydrophobic nanoparticles into phospholipid bilayers is limited to small particles that can incorporate into a hydrophobic membrane core between two lipid leaflets. Incorporation of nanoparticles above this size limit requires the development of challenging surface engineering methodologies. In principle, increasing the long-chain lipid component in the lipid mixture should facilitate incorporation of larger nanoparticles. Here, we explore the effect of incorporating very long phospholipids (C24:1) into small unilamellar vesicles on the membrane insertion efficiency of hydrophobic nanoparticles that are 5-11 nm in diameter. To this end, we improve an existing vesicle preparation protocol and utilized cryogenic electron microscopy imaging to examine the mode of interaction and evaluate the insertion efficiency of membrane-inserted nanoparticles. We also perform classical coarse-grained molecular dynamics simulations to identify changes in lipid membrane structural properties that may increase insertion efficiency. Our results indicate that long-chain lipids increase the insertion efficiency by preferentially accumulating near membrane-inserted nanoparticles to reduce the thermodynamically unfavorable disruption of the membrane.

Bilayer Charge Asymmetry and Oil Residues Destabilize Membranes upon Poration.

Transmembrane asymmetry is ubiquitous in cells, particularly with respect to lipids, where charged lipids are mainly restricted to one monolayer. We investigate the influence of anionic lipid asymmetry on the stability of giant unilamellar vesicles (GUVs), minimal plasma membrane models. To quantify asymmetry, we apply the fluorescence quenching assay, which is often difficult to reproduce, and caution in handling the quencher is generally underestimated. We first optimize this assay and then apply it to GUVs prepared with the inverted emulsion transfer protocol by using increasing fractions of anionic lipids restricted to one leaflet. This protocol is found to produce highly asymmetric bilayers but with ∼20% interleaflet mixing. To probe the stability of asymmetric versus symmetric membranes, we expose the GUVs to porating electric pulses and monitor the fraction of destabilized vesicles. The pulses open macropores, and the GUVs either completely recover or exhibit leakage or bursting/collapse. Residual oil destabilizes porated membranes, and destabilization is even more pronounced in asymmetrically charged membranes. This is corroborated by the measured pore edge tension, which is also found to decrease with increasing charge asymmetry. Using GUVs with imposed transmembrane pH asymmetry, we confirm that poration-triggered destabilization does not depend on the approach used to generate membrane asymmetry.

Melittin-Phospholipase A2 Synergism Is Mediated by Liquid-Liquid Miscibility Phase Transition in Giant Unilamellar Vesicles.

The primary constituents of honeybee venom, melittin and phospholipase A2 (PLA2), display toxin synergism in which the PLA2 activity is significantly enhanced by the presence of melittin. It has been shown previously that this is accomplished by the disruption in lipid packing, which allows PLA2 to become processive on the membrane surface. In this work, we show that melittin is capable of driving miscibility phase transition in giant unilamellar vesicles (GUVs) and that it raises the miscibility transition temperature (Tmisc) in a concentration-dependent manner. The induced phase separation enhances the processivity of PLA2, particularly at its boundaries, where a substantial difference in domain thickness creates a membrane discontinuity. The catalytic action of PLA2, in response, induces changes in the membrane, rendering it more conducive to melittin binding. This, in turn, facilitates further lipid phase separation and eventual vesicle lysis. Overall, our results show that melittin has powerful membrane-altering capabilities that activate PLA2 in various membrane contexts. More broadly, they exemplify how this biochemical system actively modulates and capitalizes on the spatial distribution of membrane lipids to efficiently achieve its objectives.

PRODAN Photophysics as a Tool to Determine the Bilayer Properties of Different Unilamellar Vesicles Composed of Phospholipids.

Vesicles formed by phospholipids are promising candidates for drug delivery. It is known that the lipid composition affects properties such as the rigidity-fluidity of the membrane and that it influences the bilayer permeability, but sometimes sophisticated techniques are selected to monitor them. In this work, we study the bilayer of different unilamellar vesicles composed of different lipids (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC, and lecithin) and diverse techniques such as extruder and electrospun templates and using 6-propionyl-2-(N,N-dimethyl) aminonaphthalene (PRODAN) and its photophysics. Moreover, we were able to monitor the influence of cholesterol on the bilayers. We demonstrate that the bilayer properties can be evaluated using the emission feature of the molecular probe PRODAN. This fluorescent probe gives relevant information on the polarity and fluidity of the microenvironment for unilamellar vesicles formed by two different methods. The PRODAN emission at 434 nm suggests that the bilayer properties significantly change if DOPC or lecithin is used in the vesicle preparation especially in their fluidity. Moreover, cholesterol induces alterations in the bilayer's structural and microenvironmental properties to a greater or lesser degree in both vesicles. Thus, we propose an easy and elegant way to evaluate physicochemical properties, which is fundamental for manufacturing vesicles as a drug delivery system, simply by monitoring the molecular probe emission band centered at 434 nm, which corresponds to the PRODAN species deep inside the bilayer.

Protein-Rich Rafts in Hybrid Polymer/Lipid Giant Unilamellar Vesicles.

Considerable attention has been dedicated to lipid rafts due to their importance in numerous cell functions such as membrane trafficking, polarization, and signaling. Next to studies in living cells, artificial micrometer-sized vesicles with a minimal set of components are established as a major tool to understand the phase separation dynamics and their intimate interplay with membrane proteins. In parallel, mixtures of phospholipids and certain amphiphilic polymers simultaneously offer an interface for proteins and mimic this segregation behavior, presenting a tangible synthetic alternative for fundamental studies and bottom-up design of cellular mimics. However, the simultaneous insertion of complex and sensitive membrane proteins is experimentally challenging and thus far has been largely limited to natural lipids. Here, we present the co-reconstitution of the proton pump bo3 oxidase and the proton consumer ATP synthase in hybrid polymer/lipid giant unilamellar vesicles (GUVs) via fusion/electroformation. Variations of the current method allow for tailored reconstitution protocols and control of the vesicle size. In particular, mixing of protein-free and protein-functionalized nanosized vesicles in the electroformation film results in larger GUVs, while separate reconstitution of the respiratory enzymes enables higher ATP synthesis rates. Furthermore, protein labeling provides a synthetic mechanism for phase separation and protein sequestration, mimicking lipid- and protein-mediated domain formation in nature. The latter means opens further possibilities for re-enacting phenomena like supercomplex assembly or symmetry breaking and enriches the toolbox of bottom-up synthetic biology.

Significance of in situ quantitative membrane property-morphology relation (QmPMR) analysis.

Deformation of the cell membrane is well understood from the viewpoint of protein interactions and free energy balance. However, the various dynamic properties of the membrane, such as lipid packing and hydrophobicity, and their relationship with cell membrane deformation are unknown. Therefore, the deformation of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and oleic acid (OA) giant unilamellar vesicles (GUVs) was induced by heating and cooling cycles, and time-lapse analysis was conducted based on the membrane hydrophobicity and physical parameters of "single-parent" and "daughter" vesicles. Fluorescence ratiometric analysis by simultaneous dual-wavelength detection revealed the variation of different hydrophilic GUVs and enabled inferences of the "daughter" vesicle composition and the "parent" membrane's local composition during deformation; the "daughter" vesicle composition of OA was lower than that of the "parents", and lateral movement of OA was the primary contributor to the formation of the "daughter" vesicles. Thus, our findings and the newly developed methodology, named in situ quantitative membrane property-morphology relation (QmPMR) analysis, would provide new insights into cell deformation and accelerate research on both deformation and its related events, such as budding and birthing.