Engineering Phosphatidylserine Containing Asymmetric Giant Unilamellar Vesicles.

The plasma membrane lipid distribution is asymmetric, with several anionic lipid species located in its inner leaflet. Among these, phosphatidylserine (PS) plays a crucial role in various important physiological functions. Over the last decade several methods have been developed that allow for the fabrication of large or giant unilamellar vesicles (GUVs) with an asymmetric lipid composition. Investigating the physicochemical properties of PS in such asymmetric lipid bilayers and studying its interactions with proteins necessitates the reliable fabrication of asymmetric GUVs (aGUVs) with a high degree of asymmetry that exhibit PS in the outer leaflet so that the interaction with peptides and proteins can be studied. Despite progress, achieving aGUVs with well-defined PS asymmetry remains challenging. Recently, a Ca2+-initiated hemifusion method has been introduced, utilizing the fusion of symmetric GUVs (sGUVs) with a supported lipid bilayer (SLB) for the fabrication of aGUVs. We extend this approach to create aGUVs with PS in the outer bilayer leaflet. Comparing the degree of asymmetry between aGUVs obtained via Ca2+ or Mg2+ initiated hemifusion of a phosphatidylcholine (PC) sGUVwith a PC/PS-supported lipid bilayer, we observe for both bivalent cations a significant number of aGUVs with near-complete asymmetry. The degree of asymmetry distribution is narrower for physiological salt conditions than at lower ionic strengths. While Ca2+ clusters PS in the SLB, macroscopic domain formation is absent in the presence of Mg2+. However, the clustering of PS upon the addition of Ca2+ is apparently too slow to have a negative effect on the quality of the obtained aGUVs. We introduce a data filtering method to select aGUVs that are best suited for further investigation.

Enhancing osmotic stress tolerance of cell mimetics by modulating lipid bilayer.

Seeking effective ways to maintain cellular homeostasis is crucial to the survival of organisms when they encounter osmotic stress. Glycine betaine (GB) is a widely generated natural osmolyte, but its endogenous production and action are limited. Herein, a kind of nonionic surfactant dodecyl-ß-d-glucopyranoside (DG) and a common polymer polyethylene glycol (PEG) are proven to have the ability to enhance the osmotic stress (induced by sugar concentration changes) tolerance of cell and organism models, those are giant unilamellar vesicles (GUVs) and gram-negative Escherichia coli. DG or PEG only induces small size decrease and certain shape change of GUVs. Importantly, DG or PEG at the concentration 100 times lower than that of GB effectively increases the survival rate of bacteria under both hypoosmotic and hyperosmotic conditions. This intriguing result is attributed to the insertion of DG or adsorption of PEG in the lipid bilayer membrane, leading to enhanced membrane permeability. These exogenous substances can replace GB to facilely and highly efficiently augment adaptation of organisms to osmotic stress.

Hydrogen bonding-mediated interaction underlies the enhanced membrane toxicity of chemically transformed polystyrene microplastics by cadmium.

The global attention on microplastic pollution and its implications for human health has grown in recent years. Additionally, the co-existence of heavy metals may significantly alter microplastics' physicochemical characteristics, potentially amplifying their overall toxicity-a facet that remains less understood. In this study, we focused the membrane toxicity of modified polystyrene microplastics (PS-MPs) following cadmium (Cd) pretreatment. Our findings revealed that Cd-pretreated PS-MPs exacerbated their toxic effects, including diminished membrane integrity and altered phase fluidity in simulated lipid membrane giant unilamellar vesicles (GUVs), as well as heightened membrane permeability, protein damage, and lipid peroxidation in red blood cells and macrophages. Mechanistically, these augmented membrane toxicities can be partially ascribed to modifications in the surface roughness and hydrophilicity of Cd-pretreated PS-MPs, as well as to interactions between PS-MPs and lipid bilayers. Notably, hydrogen bonds emerged as a crucial mechanism underlying the enhanced interaction of PS-MPs with lipid bilayers.

Bioprinting of Synthetic Cell-like Lipid Vesicles to Augment the Functionality of Tissues after Manufacturing.

Bioprinting is an automated bioassembly method that enables the formation of human tissue-like constructs to restore or replace damaged tissues. Regardless of the employed bioprinting method, cells undergo mechanical stress that can impact their survival and function postprinting. In this study, we investigate the use of a synthetic cell-like unit, giant unilamellar vesicles (GUVs), as adjuvants of the cellular function of human cells postprinting, or in future as the complete replacement of human cells. We analyzed the impact of two nozzle-based bioprinting methods (drop-on-demand and extrusion bioprinting) on the structure, stability, and function of GUVs. We showed that over 65% of the GUVs remain intact when printing at 0.5 bar, demonstrating the potential of using GUVs as a synthetic cell source. We further increased the stability of GUVs in a cell culture medium by introducing polyethylene glycol (PEG) into the GUV lipid membrane. The presence of PEG, however, diminished the structural properties of GUVs postprinting, and reduced the interaction of GUVs with human cells. Although the design of PEG-GUVs can still be modified in future studies for better cell-GUV interactions, we demonstrated that GUVs are functional postprinting. Chlorin e6-PEG-GUVs loaded with a fluorescent dye were bioprinted, and they released the dye postprinting only upon illumination. This is a new strategy to deliver carriers, such as growth factors, drugs, nutrients, or gases, inside large bioprinted specimens on a millimeter to centimeter scale. Overall, we showed that printed GUVs can augment the functionality of manufactured human tissues.

Arginine-Rich Cell-Penetrating Peptides Induce Lipid Rearrangements for Their Active Translocation across Laterally Heterogeneous Membranes.

Arginine-rich cell-penetrating peptides (CPPs) have emerged as valuable tools for the intracellular delivery of bioactive molecules, but their membrane perturbation during cell penetration is not fully understood. Here, nona-arginine (R9)-mediated membrane reorganization that facilitates the translocation of peptides across laterally heterogeneous membranes is directly visualized. The electrostatic binding of cationic R9 to anionic phosphatidylserine (PS)-enriched domains on a freestanding lipid bilayer induces lateral lipid rearrangements; in particular, in real-time it is observed that R9 fluidizes PS-rich liquid-ordered (Lo) domains into liquid-disordered (Ld) domains, resulting in the membrane permeabilization. The experiments with giant unilamellar vesicles (GUVs) confirm the preferential translocation of R9 through Ld domains without pore formation, even when Lo domains are more negatively charged. Indeed, whenever R9 comes into contact with negatively charged Lo domains, it dissolves the Lo domains first, promoting translocation across phase-separated membranes. Collectively, the findings imply that arginine-rich CPPs modulate lateral membrane heterogeneity, including membrane fluidization, as one of the fundamental processes for their effective cell penetration across densely packed lipid bilayers.

A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos.

A wide-field microscope with epi-fluorescence and selective plane illumination was combined with a single-photon avalanche diode (SPAD) array camera to enable live-cell fluorescence lifetime imaging (FLIM) using time-correlated single-photon counting (TCSPC). The camera sensor comprised of 192 × 128 pixels, each integrating a single SPAD and a time-to-digital converter. Jointly, they produced a stream of single-photon images of photon arrival times with ≈ 38 ps accuracy. The photon arrival times were subject to systematic delays and nonlinearities, which were corrected by a Monte-Carlo algorithm. The SPAD camera was then applied to FLIM where histogramming the resulting photon arrival times in each pixel resulted in decays compatible with common data processing pipelines for fluorescence lifetime analysis. The capabilities of the TCSPC camera-based FLIM microscope were demonstrated by imaging living unicellular photosynthetic algae and artificial lipid vesicles. Epi-fluorescence illumination enabled rapid fluorescence lifetime imaging of living cells and selective-plane illumination enabled 3-dimensional FLIM of stationary samples.

Rearrangement of GUV-confined actin networks in response to micropipette aspiration.

Although diverse actin network architectures found inside the cell have been individually reconstituted outside of the cell, how different types of actin architectures reorganize under applied forces is not entirely understood. Recently, bottom-up reconstitution has enabled studies where dynamic and phenotypic characteristics of various actin networks can be recreated in an isolated cell-like environment. Here, by creating a giant unilamellar vesicle (GUV)-based cell model encapsulating actin networks, we investigate how actin networks rearrange in response to localized stresses applied by micropipette aspiration. We reconstitute actin bundles and branched bundles in GUVs separately and mechanically perturb them. Interestingly, we find that, when aspirated, protrusive actin bundles that are otherwise randomly oriented in the GUV lumen collapse and align along the axis of the micropipette. However, when branched bundles are aspirated, the network remains intact and outside of the pipette while the GUV membrane is aspirated into the micropipette. These results reveal distinct responses in the rearrangement of actin networks in a network architecture-dependent manner when subjected to physical forces.

Cholesterol Depletion and Membrane Deformation by MeßCD and the Resultant Enhanced T Cell Killing.

Recent studies have demonstrated the crucial role of cholesterol (Chol) in regulating the mechanical properties and biological functions of cell membranes. Methyl-ß-cyclodextrin (MeßCD) is commonly utilized to modulate the Chol content in cell membranes, but there remains a lack of a comprehensive understanding. In this study, using a range of different techniques, we find that the optimal ratio of MeßCD to Chol for complete removal of Chol from a phosphocholine (PC)/Chol mixed membrane with a 1:1 mol ratio is 4.5:1, while the critical MeßCD-to-Chol ratio for membrane permeation falls within the range between 1.5 and 2.4. MeßCD at elevated concentrations induces the formation of fibrils or tubes from a PC membrane. Single lipid tracking reveals that removing Chol restores the diffusion of lipid molecules in the PC/Chol membrane to levels observed in pure PC membranes. Exposure to 5 mM MeßCD for 30 min effectively eliminates Chol from various cell lines, leading to an up to 8-fold enhancement in melittin cytotoxicity over Hela cells and an up to 3.5-fold augmentation of T cell cytotoxicity against B16F10-OVA cells. This study presents a diagram that delineates the concentration- and time-dependent distribution of MeßCD-induced Chol depletion and membrane deformation, which holds significant potential for modulating the mechanical properties of cellular membranes in prospective biomedical applications.

Asymmetric disturbance and permeabilization of bilayer membranes by 3-nm carbon dots.

Small-sized fluorescent carbon dots (CDs) are gaining increasing attention in the field of biomedical applications. The environmental and biological compatibility of positively charged CDs has been extensively investigated; however, the potential cytotoxicity caused by negatively and particularly neutrally charged small CDs has been significantly overlooked. In this study, we conducted a comprehensive investigation into the cellular membrane disruption effect of weakly negatively charged 3-nm CDs using a combination of various biophysical techniques. Our findings demonstrate that even at a low concentration of 0.5 µg mL-1, these CDs induce significant perturbations on the cellular membrane, resulting in increased membrane permeability due to asymmetric disruption of the bilayer structure. Furthermore, CDs exhibit distinct mechanisms at different concentrations, including prompt insertion into the bilayer at low concentrations (<20 µg mL-1) and a synergistic effect after a threshold time at high concentrations (e.g., 25-200 µg mL-1). Moreover, these CDs possess specific antibacterial properties against Acinetobacter baumannii (with a minimum inhibitory concentration of 50 µg mL-1) while showing minimal hemolytic or cytotoxic effects on mammalian cells. This study provides comprehensive insights into the biophysical aspects of cellular membrane toxicity caused by small weakly negatively charged CDs and contributes to assessing their potential biomedical applications.

Light-Activated Synthetic Rotary Motors in Lipid Membranes Induce Shape Changes Through Membrane Expansion.

Membranes are the key structures to separate and spatially organize cellular systems. Their rich dynamics and transformations during the cell cycle are orchestrated by specific membrane-targeted molecular machineries, many of which operate through energy dissipation. Likewise, man-made light-activated molecular rotary motors have previously shown drastic effects on cellular systems, but their physical roles on and within lipid membranes remain largely unexplored. Here, the impact of rotary motors on well-defined biological membranes is systematically investigated. Notably, dramatic mechanical transformations are observed in these systems upon motor irradiation, indicative of motor-induced membrane expansion. The influence of several factors on this phenomenon is systematically explored, such as motor concentration and membrane composition., Membrane fluidity is found to play a crucial role in motor-induced deformations, while only minor contributions from local heating and singlet oxygen generation are observed. Most remarkably, the membrane area expansion under the influence of the motors continues as long as irradiation is maintained, and the system stays out-of-equilibrium. Overall, this research contributes to a comprehensive understanding of molecular motors interacting with biological membranes, elucidating the multifaceted factors that govern membrane responses and shape transitions in the presence of these remarkable molecular machines, thereby supporting their future applications in chemical biology.

Spontaneous transfer of small peripheral peptides between supported lipid bilayer and giant unilamellar vesicles.

Vesicular trafficking facilitates material transport between membrane-bound organelles. Membrane protein cargos are trafficked for relocation, recycling, and degradation during various physiological processes. In vitro fusion studies utilized synthetic lipid membranes to study the molecular mechanisms of vesicular trafficking and to develop synthetic materials mimicking the biological membrane trafficking. Various fusogenic conditions which can induce vesicular fusion have been used to establish synthetic systems that can mimic biological systems. Despite these efforts, the mechanisms underlying vesicular trafficking of membrane proteins remain limited and robust in vitro methods that can construct synthetic trafficking systems for membrane proteins between large membranes (>1 µm2) are unavailable. Here, we provide data to show the spontaneous transfer of small membrane-bound peptides (∼4 kD) between a supported lipid bilayer (SLB) and giant unilamellar vesicles (GUVs). We found that the contact between the SLB and GUVs led to the occasional but notable transfer of membrane-bound peptides in a physiological saline buffer condition (pH 7.4, 150 mM NaCl). Quantitative and dynamic time-lapse analyses suggested that the observed exchange occurred through the formation of hemi-fusion stalks between the SLB and GUVs. Larger protein cargos with a size of ∼77 kD could not be transferred between the SLB and GUVs, suggesting that the larger-sized cargos limited diffusion across the hemi-fusion stalk, which was predicted to have a highly curved structure. Compositional study showed Ni-chelated lipid head group was the essential component catalyzing the process. Our system serves as an example synthetic platform that enables the investigation of small-peptide trafficking between synthetic membranes and reveals hemi-fused lipid bridge formation as a mechanism of peptide transfer.

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

Non-linear biomolecular interactions on the membranes drive membrane remodeling that underlies fundamental biological processes including chemotaxis, cytokinesis, and endocytosis. The multitude of biomolecules, the redundancy in their interactions, and the importance of spatiotemporal context in membrane organization hampers understanding the physical principles governing membrane mechanics. A minimal, in vitro system that models the functional interactions between molecular signaling and membrane remodeling, while remaining faithful to cellular physiology and geometry is powerful yet remains unachieved. Here, inspired by the biophysical processes underpinning chemotaxis, we reconstituted externally-controlled actin polymerization inside giant unilamellar vesicles, guiding self-organization on the membrane. We show that applying undirected external chemical inputs to this system results in directed actin polymerization and membrane deformation that are uncorrelated with upstream biochemical cues, indicating symmetry breaking. A biophysical model of the dynamics and mechanics of both actin polymerization and membrane shape suggests that inhomogeneous distributions of actin generate membrane shape deformations in a non-linear fashion, a prediction consistent with experimental measurements and subsequent local perturbations. The active protocellular system demonstrates the interplay between actin dynamics and membrane shape in a symmetry breaking context that is relevant to chemotaxis and a suite of other biological processes.

QS21-Initiated Fusion of Liposomal Small Unilamellar Vesicles to Form ALFQ Results in Concentration of Most of the Monophosphoryl Lipid A, QS21, and Cholesterol in Giant Unilamellar Vesicles.

Army Liposome Formulation with QS21 (ALFQ), a vaccine adjuvant preparation, comprises liposomes containing saturated phospholipids, with 55 mol% cholesterol relative to the phospholipids, and two adjuvants, monophosphoryl lipid A (MPLA) and QS21 saponin. A unique feature of ALFQ is the formation of giant unilamellar vesicles (GUVs) having diameters >1.0 µm, due to a remarkable fusion event initiated during the addition of QS21 to nanoliposomes containing MPLA and 55 mol% cholesterol relative to the total phospholipids. This results in a polydisperse size distribution of ALFQ particles, with diameters ranging from ~50 nm to ~30,000 nm. The purpose of this work was to gain insights into the unique fusion reaction of nanovesicles leading to GUVs induced by QS21. This fusion reaction was probed by comparing the lipid compositions and structures of vesicles purified from ALFQ, which were >1 µm (i.e., GUVs) and the smaller vesicles with diameter <1 µm. Here, we demonstrate that after differential centrifugation, cholesterol, MPLA, and QS21 in the liposomal phospholipid bilayers were present mainly in GUVs (in the pellet). Presumably, this occurred by rapid lateral diffusion during the transition from nanosize to microsize particles. While liposomal phospholipid recoveries by weight in the pellet and supernatant were 44% and 36%, respectively, higher percentages by weight of the cholesterol (~88%), MPLA (94%), and QS21 (96%) were recovered in the pellet containing GUVs, and ≤10% of these individual liposomal constituents were recovered in the supernatant. Despite the polydispersity of ALFQ, most of the cholesterol, and almost all of the adjuvant molecules, were present in the GUVs. We hypothesize that the binding of QS21 to cholesterol caused new structural nanodomains, and subsequent interleaflet coupling in the lipid bilayer might have initiated the fusion process, leading to creation of GUVs. However, the polar regions of MPLA and QS21 together have a "sugar lawn" of ten sugars, the hydrophilicity of which might have provided a driving force for rapid lateral diffusion and concentration of the MPLA and QS21 in the GUVs.

Advances in giant unilamellar vesicle preparation techniques and applications.

Giant unilamellar vesicles (GUVs) are versatile and promising cell-sized bio-membrane mimetic platforms. Their applications range from understanding and quantifying membrane biophysical processes to acting as elementary blocks in the bottom-up assembly of synthetic cells. Definite properties and requisite goals in GUVs are dictated by the preparation techniques critical to the success of their applications. Here, we review key advances in giant unilamellar vesicle preparation techniques and discuss their formation mechanisms. Developments in lipid hydration and emulsion techniques for GUV preparation are described. Novel microfluidic-based techniques involving lipid or surfactant-stabilized emulsions are outlined. GUV immobilization strategies are summarized, including gravity-based settling, covalent linking, and immobilization by microfluidic, electric, and magnetic barriers. Moreover, some of the key applications of GUVs as biomimetic and synthetic cell platforms during the last decade have been identified. Membrane interface processes like phase separation, membrane protein reconstitution, and membrane bending have been deciphered using GUVs. In addition, vesicles are also employed as building blocks to construct synthetic cells with defined cell-like functions comprising compartments, metabolic reactors, and abilities to grow and divide. We critically discuss the pros and cons of preparation technologies and the properties they confer to the GUVs and identify potential techniques for dedicated applications.

Influenza A Virus M1 Protein Non-Specifically Deforms Charged Lipid Membranes and Specifically Interacts with the Raft Boundary.

Topological rearrangements of biological membranes, such as fusion and fission, often require a sophisticated interplay between different proteins and cellular membranes. However, in the case of fusion proteins of enveloped viruses, even one molecule can execute membrane restructurings. Growing evidence indicates that matrix proteins of enveloped viruses can solely trigger the membrane bending required for another crucial step in virogenesis, the budding of progeny virions. For the case of the influenza A virus matrix protein M1, different studies report both in favor and against M1 being able to produce virus-like particles without other viral proteins. Here, we investigated the physicochemical mechanisms of M1 membrane activity on giant unilamellar vesicles of different lipid compositions using fluorescent confocal microscopy. We confirmed that M1 predominantly interacts electrostatically with the membrane, and its ability to deform the lipid bilayer is non-specific and typical for membrane-binding proteins and polypeptides. However, in the case of phase-separating membranes, M1 demonstrates a unique ability to induce macro-phase separation, probably due to the high affinity of M1's amphipathic helices to the raft boundary. Thus, we suggest that M1 is tailored to deform charged membranes with a specific activity in the case of phase-separating membranes.

Lipid Giant Vesicles Engulf Living Bacteria Triggered by Minor Enhancement in Membrane Fluidity.

Antibacterial amphiphiles normally kill bacteria by destroying the bacterial membrane. Whether and how antibacterial amphiphiles alter normal cell membrane and lead to subsequent effects on pathogen invasion into cells have been scarcely promulgated. Herein, by taking four antibacterial gemini amphiphiles with different spacer groups to modulate cell-mimic phospholipid giant unilamellar vesicles (GUVs), bacteria adhesion on the modified GUVs surface and bacteria engulfment process by the GUVs are clearly captured by confocal laser scanning microscopy. Further characterization shows that the enhanced cationic surface charge of GUVs by the amphiphiles determines the bacteria adhesion amount, while the involvement of amphiphile in GUVs results in looser molecular arrangement and concomitant higher fluidity in the bilayer membranes, facilitating the bacteria intruding into GUVs. This study sheds new light on the effect of amphiphiles on membrane bilayer and the concurrent effect on pathogen invasion into cell mimics and broadens the nonprotein-mediated endocytosis pathway for live bacteria.

Antimicrobial peptide magainin 2-induced rupture of single giant unilamellar vesicles comprising E. coli polar lipids.

Most antimicrobial peptides (AMPs) damage the cell membrane of bacterial cells and induce rapid leakage of the internal cell contents, which is a main cause of their bactericidal activity. One of the AMPs, magainin 2 (Mag), forms nanopores in giant unilamellar vesicles (GUVs) comprising phosphatidylcholine (PC) and phosphatidylglycerol (PG), inducing leakage of fluorescent probes. In this study, to elucidate the Mag-induced pore formation in lipid bilayer region in E. coli cell membrane, we examined the interaction of Mag with single GUVs comprising E. coli polar lipids (E. coli-lipid-GUVs). First, we investigated the Mag-induced leakage of a fluorescent probe AF488 from single E. coli-lipid-GUVs, and found that Mag caused rupture of GUVs, inducing rapid AF488 leakage. The rate constant of Mag-induced GUV rupture increased with the Mag concentration. Using fluorescence microscopy with a time resolution of 5 ms, we revealed the GUV rupture process: first, a small micropore was observed in the GUV membrane, then the pore radius increased within 50 ms without changing the GUV diameter, the thickness of the membrane at the pore rim concomitantly increased, and eventually membrane aggregates were formed. Mag bound to only the outer monolayer of the GUV before GUV rupture, which increased the area of the GUV bilayer. We also examined the physical properties of E. coli-lipid-GUVs themselves. We found that the rate constant of the constant tension-induced rupture of E. coli-lipid-GUVs was higher than that of PG/PC-GUVs. Based on these results, we discussed the Mag-induced rupture of E. coli-lipid-GUVs and its mechanism.

Reconstitution of membrane contact by unilamellar vesicles.

Eukaryotic cells compartmentalize diverse biochemical functions within organelles defined by intracellular membranes. Recent focus has intensified on studying the interactions among organelles and the role of membrane contacts in maintaining cellular balance. While analyzing these contacts mainly involves fluorescence and electron microscopy, as well as biochemical cell fractionation, understanding their mechanisms and responses to genetic and environmental changes remains challenging. Here we describe an approach employing in vitro reconstitution of membrane contacts using unilamellar vesicles. This technique offers insights into contact mechanisms when combined with established methods like fluorescence imaging and mass spectrometry, potentially deepening our understanding of membrane contacts and organelle networks.

Structural Role of Plasma Membrane Sterols in Osmotic Stress Tolerance of Yeast Saccharomyces cerevisiae.

Yeast S. cerevisiae has been shown to suppress a sterol biosynthesis as a response to hyperosmotic stress. In the case of sodium stress, the failure to suppress biosynthesis leads to an increase in cytosolic sodium. The major yeast sterol, ergosterol, is known to regulate functioning of plasma membrane proteins. Therefore, it has been suggested that the suppression of its biosynthesis is needed to adjust the activity of the plasma membrane sodium pumps and channels. However, as the sterol concentration is in the range of thirty to forty percent of total plasma membrane lipids, it is believed that its primary biological role is not regulatory but structural. Here we studied how lowering the sterol content affects the response of a lipid bilayer to an osmotic stress. In accordance with previous observations, we found that a decrease of the sterol fraction increases a water permeability of the liposomal membranes. Yet, we also found that sterol-free giant unilamellar vesicles reduced their volume during transient application of the hyperosmotic stress to a greater extent than the sterol-rich ones. Furthermore, our data suggest that lowering the sterol content in yeast cells allows the shrinkage to prevent the osmotic pressure-induced plasma membrane rupture. We also found that mutant yeast cells with the elevated level of sterol accumulated propidium iodide when exposed to mild hyperosmotic conditions followed by hypoosmotic stress. It is likely that the decrease in a plasma membrane sterol content stimulates a drop in cell volume under hyperosmotic stress, which is beneficial in the case of a subsequent hypo-osmotic one.

[Mechanism of Leakage in Phosphatidylserine-Containing Membranes by Melittin].

Phosphatidylserine (PS) is an important apoptotic-cell surface signal that exists in bacterial and cancer cells. The mechanism by which melittin interacts with the PS membrane remains unclear. Here, we revealed this mechanism by using a dual-channel fluorescence microscope to observe the concentration-dependent process of pore formation in giant unilamellar vesicles (GUVs) that were exposed to melittin solution. We found that unsaturated PS membranes differed significantly from saturated PS membranes in different phases. This study provides a reference for research and development of anticancer drugs.