Single Giant Unilamellar Vesicles Research Articles

Osmotic spawning vesicle.

We discovered a cascade vesicle division system driven by osmotic inflation. Binary giant unilamellar vesicles (GUVs) composed of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and cholesterol (Chol) were subjected to an osmotic pressure difference by encapsulating membrane-impermeable osmolytes (typically sucrose) in an external aqueous solution containing membrane-permeable osmolytes (typically fructose). This simple setup enabled the mother GUVs to repeatedly form small membrane buds and subsequently undergo divisions over several hundred seconds, resulting in the production of approximately 30-300 daughter GUVs from a single mother GUV. The observed morphological change of GUVs is well described by the mechanical balance between membrane bending, membrane tension, and osmotic pressure difference based on the spontaneous curvature model. This "osmotic spawning" behavior of GUVs does not rely on chemical reactions or functional macromolecules. Therefore, this cascade division system is compatible with various chemical systems and has the potential to implement proliferation ability in artificial cells, drug delivery systems, and protocells simply by modifying their membrane compartments and osmolytes.

Relationship between oligoarginine-induced membrane damage of single Escherichia coli cells and entry of the peptide into the cytoplasm

Cell-penetrating peptides (CPPs) can enter the cytosol of eukaryotic cells without killing them whereas some CPPs exhibit antimicrobial activity against bacterial cells. Here, to elucidate the mode of interaction of the CPP nona-arginine (R9) with bacterial cells, we investigated the interactions of lissamine rhodamine B red-labeled peptide (Rh-R9) with single Escherichia coli cells encapsulating calcein using confocal laser scanning microscopy. After Rh-R9 induced the leakage of a large amount of calcein, the fluorescence intensity of the cytosol due to Rh-R9 greatly increased, indicating that Rh-R9 induces cell membrane damage, thus allowing entry of a significant amount of Rh-R9 into the cytosol. To determine if the lipid bilayer region of the membrane is the main target of Rh-R9, we then investigated the interaction of Rh-R9 with single giant unilamellar vesicles (GUVs) comprising an E. coli polar lipid extract containing small GUVs and AlexaFluor 647 hydrazide (AF647) in the lumen. Rh-R9 entered the GUV lumen without inducing AF647 leakage, but leakage eventually did occur, indicating that GUV membrane damage was induced after the entry of Rh-R9 into the GUV lumen. The Rh-R9 peptide concentration dependence of the fraction of entry of Rh-R9 after a specific interaction time was similar to that of the fraction of leaking GUVs. These results indicate that Rh-R9 can damage the lipid bilayer region of a cell membrane, which may be related to its antimicrobial activity.

Probability and kinetics of rupture and electrofusion in giant unilamellar vesicles under various frequencies of direct current pulses.

Irreversible electroporation induces permanent permeabilization of lipid membranes of vesicles, resulting in vesicle rupture upon the application of a pulsed electric field. Electrofusion is a phenomenon wherein neighboring vesicles can be induced to fuse by exposing them to a pulsed electric field. We focus how the frequency of direct current (DC) pulses of electric field impacts rupture and electrofusion in cell-sized giant unilamellar vesicles (GUVs) prepared in a physiological buffer. The average time, probability, and kinetics of rupture and electrofusion in GUVs have been explored at frequency 500, 800, 1050, and 1250 Hz. The average time of rupture of many 'single GUVs' decreases with the increase in frequency, whereas electrofusion shows the opposite trend. At 500 Hz, the rupture probability stands at 0.45 ± 0.02, while the electrofusion probability is 0.71 ± 0.01. However, at 1250 Hz, the rupture probability increases to 0.69 ± 0.03, whereas the electrofusion probability decreases to 0.46 ± 0.03. Furthermore, when considering kinetics, at 500 Hz, the rate constant of rupture is (0.8 ± 0.1)×10-2 s-1, and the rate constant of fusion is (2.4 ± 0.1)×10-2 s-1. In contrast, at 1250 Hz, the rate constant of rupture is (2.3 ± 0.8)×10-2 s-1, and the rate constant of electrofusion is (1.0 ± 0.1)×10-2 s-1. These results are discussed by considering the electrical model of the lipid bilayer and the energy barrier of a prepore.

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.

Effect of Phosphatidylethanolamine on Pore Formation Induced by the Antimicrobial Peptide PGLa.

Most antimicrobial peptides (AMPs) induce pore formation and a burst of lipid bilayers and plasma membranes. This causes severe leakage of the internal contents and cell death. The AMP PGLa forms nanopores in giant unilamellar vesicles (GUVs) comprising dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylglycerol (DOPG). We here elucidated the effect of the line tension of a prepore rim on PGLa-induced nanopore formation by investigating the interaction of PGLa with single GUVs comprising dioleoylphosphatidylethanolamine (DOPE)/DOPG (6:4) in buffer using the single GUV method. We found that PGLa forms nanopores in the GUV membrane, which evolved into a local burst and burst of GUVs. The rate of pore formation in DOPE/DOPG-GUVs was smaller than that in DOPC/DOPG-GUVs. PGLa is located only in the outer leaflet of a GUV bilayer just before a fluorescent probe AF647 leakage from the inside, indicating that this asymmetric distribution induces nanopore formation. PGLa-induced local burst and burst of GUVs were observed at 10 ms-time resolution. After nanopore formation started, dense particles and small vesicles appeared in the GUVs, followed by a decrease in the GUV diameter. The GUV was finally converted into smaller GUV or lipid membrane aggregates. We discuss the mechanisms of PGLa-induced nanopore formation and its direct evolution to a GUV burst.

Compound giant unilamellar vesicles as a bio-mimetic model for electrohydrodynamics of a nucleate cell.

The understanding obtained by studies on the electrohydrodynamics (EHD) of single giant unilamellar vesicles (sGUVs) has contributed significantly towards a better comprehension of the response of biological cells to electric fields. This has subsequently helped in developing technologies such as cell dielectrophoresis and cell electroporation. For nucleate eukaryotic cells though, a vesicle-in-vesicle compound giant unilamellar vesicle (cGUV) is a more appropriate bio-mimic than a sGUV. In this work, we present an improvised method for the formation of cGUVs, wherein the electrical conductivities of the inner, annular and outer regions of the cGUVs can be modified. A comprehensive experimental study is presented on the EHD of these cGUVs under weak AC fields over a wide range of frequencies, and an encouraging agreement is observed between the experiments and earlier published theoretical studies on concentric cGUVs. The spherical, prolate or oblate spheroidal deformations of a cGUV under AC electric fields depend upon the membrane electromechanical properties as well as the magnitude and direction of the electric traction at the membrane produced by the Maxwell stress that varies with the relative timescales associated with the frequency of the applied AC electric field and that of the membrane charging time and the Maxwell-Wagner relaxation time. This work establishes cGUVs as appropriate bio-mimics for conducting EHD studies relevant to eukaryotic cells.

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.

Secondary Ion Mass Spectrometry of Single Giant Unilamellar Vesicles Reveals Compositional Variability.

Giant unilamellar vesicles (GUVs) are a widely used model system to interrogate lipid phase behavior, study biomembrane mechanics, reconstitute membrane proteins, and provide a chassis for synthetic cells. It is generally assumed that the composition of individual GUVs is the same as the nominal stock composition; however, there may be significant compositional variability between individual GUVs. Although this compositional heterogeneity likely impacts phase behavior, the function and incorporation of membrane proteins, and the encapsulation of biochemical reactions, it has yet to be directly quantified. To assess heterogeneity, we use secondary ion mass spectrometry (SIMS) to probe the composition of individual GUVs using non-perturbing isotopic labels. Both 13C- and 2H-labeled lipids are incorporated into a ternary mixture, which is then used to produce GUVs via gentle hydration or electroformation. Simultaneous detection of seven different ion species via SIMS allows for the concentration of 13C- and 2H-labeled lipids in single GUVs to be quantified using calibration curves, which correlate ion intensity to composition. Additionally, the relative concentration of 13C- and 2H-labeled lipids is assessed for each GUV via the ion ratio 2H-/13C-, which is highly sensitive to compositional differences between individual GUVs and circumvents the need for calibration by using standards. Both quantification methods suggest that gentle hydration produces GUVs with greater compositional variability than those formed by electroformation. However, both gentle hydration and electroformation display standard deviations in composition (n = 30 GUVs) on the order of 1-4 mol %, consistent with variability seen in previous indirect measurements.

Examining compositional variability of giant unilamellar vesicles via secondary ion mass spectrometry.

Giant unilamellar vesicles (GUVs) are a widely used model system to interrogate lipid phase behavior, study biomembrane mechanics, reconstitute membrane proteins, and provide a chassis for synthetic cells. However, there may be significant compositional variability between individual GUVs, either due to lipid sorting during film hydration or from vesicle budding following GUV formation. Although this compositional heterogeneity likely impacts phase behavior, the function and incorporation of membrane proteins, and the encapsulation of biochemical reactions, it has yet to be directly quantified. To assess heterogeneity, we use secondary ion mass spectrometry (SIMS) to probe the composition of individual GUVs using non-perturbing isotopic labels. Both 13C- and 2H-labeled lipids are incorporated into a ternary POPC/DSPC/CHOL mixture, which is then used to produce GUVs via gentle hydration or electroformation. Simultaneous detection of seven different ion species via SIMS allows for the concentration of 13C- and 2H-labeled lipids in a single GUVs to be quantified using calibration curves, which correlate ion intensity to composition. Additionally, the relative concentration of 13C- and 2H-labeled lipids is assessed for each GUV via the ion ratio 2H−/13C−, which is highly sensitive to compositional changes between individual GUVs and circumvents the need for calibration using standards. Both quantification methods suggest that gentle hydration produces GUVs with greater compositional variability than those formed by electroformation. However, both gentle hydration and electroformation display compositional variability on the order of several mole percent.

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.

Control of Enzyme Reaction Initiation inside Giant Unilamellar Vesicles by the Cell-Penetrating Peptide-Mediated Translocation of Cargo Proteins.

Cell-penetrating peptides (CPPs) play important roles in directly delivering biomolecules, such as DNA, proteins, and peptides, into living cells. In artificial lipid membranes, such as planar lipid bilayers, the direct membrane translocation of β-galactosidase via Pep-1 (one of the CPPs) is dependent upon a voltage gradient between the inner and outer leaflets of the lipid membranes. Giant unilamellar vesicles (GUVs) with asymmetric lipid distributions, which are recently generated using microfluidic technologies, can be observed by optical microscopy. Therefore, interactions between CPPs and asymmetric lipid bilayers in different kinds of lipids and the translocation mechanism of proteins via CPPs into GUVs can be investigated at the level of a single asymmetric GUV. This CPP-based system for transporting proteins into GUVs will be applied to control the start of enzyme reactions in GUVs. This study aimed to explore efficient protein translocation into GUVs via CPP and demonstrate that enzymatic reactions start in GUVs using a CPP-mediated direct translocation. The interactions and the enzyme reactions between the CPP (Pep-1 or penetratin)-DNase I complexes and the asymmetric or symmetric GUV membranes containing the negatively or neutrally charged lipids were observed by confocal laser-scanning microscopy. The asymmetric GUVs containing phosphatidylserine (PS) in the inner leaflet showed efficient DNase I translocation into GUVs via penetratin. Finally, the formation of a cross-linked actin network was observed in asymmetric PS GUVs incubated with Pep-1-streptavidin complexes. The CPP-mediated direct translocation can contribute to developing artificial cell models with the capacity to control the initiation of enzymatic reactions.

Actomyosin-Assisted Pulling of Lipid Nanotubes from Lipid Vesicles and Cells.

Molecular motors are pivotal for intracellular transport as well as cell motility and have great potential to be put to use outside cells. Here, we exploit engineered motor proteins in combination with self-assembly of actin filaments to actively pull lipid nanotubes from giant unilamellar vesicles (GUVs). In particular, actin filaments are bound to the outer GUV membrane and the GUVs are seeded on a heavy meromyosin-coated substrate. Upon addition of ATP, hollow lipid nanotubes with a length of tens of micrometer are pulled from single GUVs due to the motor activity. We employ the same mechanism to pull lipid nanotubes from different types of cells. We find that the length and number of nanotubes critically depends on the cell type, whereby suspension cells form bigger networks than adherent cells. This suggests that molecular machines can be used to exert forces on living cells to probe membrane-to-cortex attachment.

A Fluorescence-based Assay for Measuring Phospholipid Scramblase Activity in Giant Unilamellar Vesicles.

Transbilayer movement of phospholipids in biological membranes is mediated by a diverse set of lipid transporters. Among them are scramblases that facilitate rapid bi-directional movement of lipids without metabolic energy input. In this protocol, we describe the incorporation of phospholipid scramblases into giant unilamellar vesicles (GUVs) formed from scramblase-containing large unilamellar vesicles by electroformation. We also describe how to analyze their activity using membrane-impermeant sodium dithionite, to bleach symmetrically incorporated fluorescent ATTO488-conjugated phospholipids. The fluorescence-based readout allows single vesicle tracking for a large number of settled/immobilized GUVs, and provides a well-defined experimental setup to directly characterize these lipid transporters at the molecular level. Graphic abstract: Giant unilamellar vesicles (GUVs) are formed by electroformation from large unilamellar vesicles (LUVs) containing phospholipid scramblases (purple) and trace amounts of a fluorescent lipid reporter (green). The scramblase activity is analyzed by a fluorescence-based assay of single GUVs, using the membrane-impermeant quencher dithionite. Sizes not to scale. Modified from Mathiassen et al. (2021).

A Single GUV Method for Revealing the Action of Cell-Penetrating Peptides in Biomembranes.

The mechanism of entry of cell-penetrating peptides (CPPs) into the cytosol of various cells has been studied by examining the interaction of CPPs with lipid bilayers and their entry into lipid vesicle lumens using various methods. Here we describe a single giant unilamellar vesicle (GUV) method to study CPPs. In this new method, we use GUVs containing small GUVs in the mother GUV lumen or GUVs containing large unilamellar vesicles (LUVs) in the GUV lumen and investigate the interaction of fluorescent probe-labeled CPPs with single GUVs in real time using confocal laser scanning microscopy. This method can detect CPPs in the GUV lumen with high sensitivity, allowing immediate measurement of the time course of entry of CPPs into the vesicle lumen. This method allows simultaneous measurement of the entry of CPPs and of CPP-induced pore formation, allowing the relationship between the two events to be determined. One can also simultaneously measure the entry of CPPs and the CPP concentration in the GUV membrane. The rate of entry of CPPs into a single GUV lumen can be estimated by obtaining the fraction of GUVs into which CPPs entered before a specific time t without pore formation among all examined GUVs (i.e., the fraction of entry) and the lumen intensity due to LUVs with bound CPPs. This method is therefore useful for elucidating the mechanism of entry of CPPs into lipid vesicles.

Electroanalysis at a Single Giant Vesicle Generating Enzymatically a Reactive Oxygen Species.

In the framework of artificial or synthetic cell development, giant liposomes are common basic structures. Their enclosed membrane allows encapsulating proteins, DNA, reactants, etc., while its phospholipid nature allows some exchanges with the surrounding medium. Biochemical reactions induced inside giant liposomes or vesicles are often monitored or imaged by fluorescence microscopy techniques. Here, we show that electrochemistry performed with ultramicroelectrodes is perfectly suitable to monitor an enzymatic reaction occurring in a single giant unilamellar vesicle. Glucose oxidase (GOx) was microinjected inside individual vesicles containing 1 mM glucose. H2O2 was thus generated in the vesicle and progressively diffused across the membrane toward the surrounding environment. An ultramicroelectrode sensitive to H2O2 (black platinum-modified carbon surface) was placed next to the membrane and provided a direct detection of the hydrogen peroxide flux generated by the enzyme activity. Electrochemistry offered a highly sensitive (in situ detection), selective (potential applied at the electrode), time-resolved analysis (chronoamperometry) of the GOx activity over an hour duration, without modifying the internal giant unilamellar vesicles (GUV) medium. These results demonstrate that electroanalysis with microsensors is well adapted and complementary to fluorescence microscopy to sense enzymatic activities, for instance, generating reactive oxygen species, at single vesicles further used to develop artificial cells.

Mechanistic Study of Membrane Disruption by Antimicrobial Methacrylate Random Copolymers by the Single Giant Vesicle Method.

Cationic amphiphilic polymers have been a platform to create new antimicrobial materials that act by disrupting bacterial cell membranes. While activity characterization and chemical optimization have been done in numerous studies, there remains a gap in our knowledge on the antimicrobial mechanisms of the polymers, which is needed to connect their chemical structures and biological activities. To that end, we used a single giant unilamellar vesicle (GUV) method to identify the membrane-disrupting mechanism of methacrylate random copolymers. The copolymers consist of random sequences of aminoethyl methacrylate and methyl (MMA) or butyl (BMA) methacrylate, with low molecular weights of 1600-2100 g·mol-1. GUVs consisting of an 8:2 mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), sodium salt (POPG) and those with only 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were prepared to mimic the bacterial (Escherichia coli) or mammalian membranes, respectively. The disruption of bacteria and mammalian cell membrane-mimetic lipid bilayers in GUVs reflected the antimicrobial and hemolytic activities of the copolymers, suggesting that the copolymers act by disrupting cell membranes. The copolymer with BMA formed pores in the lipid bilayer, while that with MMA caused GUVs to burst. Therefore, we propose that the mechanism is inherent to the chemical identity or properties of hydrophobic groups. The copolymer with MMA showed characteristic sigmoid curves of the time course of GUV burst. We propose a new kinetic model with a positive feedback loop in the insertion of the polymer chains in the lipid bilayer. The novel finding of alkyl-dependent membrane-disrupting mechanisms will provide a new insight into the role of hydrophobic groups in the optimization strategy for antimicrobial activity and selectivity.

Reconstitution of Ultrawide DNA Origami Pores in Liposomes for Transmembrane Transport of Macromolecules.

Molecular traffic across lipid membranes is a vital process in cell biology that involves specialized biological pores with a great variety of pore diameters, from fractions of a nanometer to >30 nm. Creating artificial membrane pores covering similar size and complexity will aid the understanding of transmembrane molecular transport in cells, while artificial pores are also a necessary ingredient for synthetic cells. Here, we report the construction of DNA origami nanopores that have an inner diameter as large as 30 nm. We developed methods to successfully insert these ultrawide pores into the lipid membrane of giant unilamellar vesicles (GUVs) by administering the pores concomitantly with vesicle formation in an inverted-emulsion cDICE technique. The reconstituted pores permit the transmembrane diffusion of large macromolecules, such as folded proteins, which demonstrates the formation of large membrane-spanning open pores. The pores are size selective, as dextran molecules with a diameter up to 28 nm can traverse the pores, whereas larger dextran molecules are blocked. By FRAP measurements and modeling of the GFP influx rate, we find that up to hundreds of pores can be functionally reconstituted into a single GUV. Our technique bears great potential for applications across different fields from biomimetics, to synthetic biology, to drug delivery.

Translocation of the nonlabeled antimicrobial peptide PGLa across lipid bilayers and its entry into vesicle lumens without pore formation

Fluorescent-probe-labeled peptides are used to study the interactions of peptides with cells and lipid vesicles but labeling peptides with fluorescent probes can significantly change these interactions. We recently developed a new method to detect the entry of nonlabeled peptides into the lumen of single giant unilamellar vesicles (GUVs). Here we applied this method to examine the interaction of the antimicrobial peptide PGLa with single GUVs to elucidate whether PGLa can enter the GUV lumen without pore formation. First, we examined the interaction of nonlabeled PGLa with single GUVs comprising dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) (4/6) whose lumens contain the fluorescent probe AF647 and DOPG/DOPC (8/2)-large unilamellar vesicles encapsulating a high concentration of calcein. After a large lag period from starting the interaction with PGLa, the fluorescence intensity of the GUV lumen due to calcein (Icalcein) increased gradually without leakage of AF647, indicating that PGLa enters the GUV lumen without pore formation in the GUV membrane. The fraction of entry of PGLa increased with increasing PGLa concentration. Simultaneous measurement of the fractional area change of the GUV membrane (δ) and PGLa-induced increase in Icalcein showed that the entry of PGLa occurs only during the second increase in δ, indicating that PGLa enters the lumen during its translocation from the outer leaflet to the inner leaflet. The fraction of entry of PGLa without pore formation increased with increasing membrane tension. Based on these results, we discuss the elementary processes and the mechanism of the entry of PGLa into the GUV lumen.

PoET: automated approach for measuring pore edge tension in giant unilamellar vesicles.

A reliable characterization of the membrane pore edge tension of single giant unilamellar vesicles (GUVs) requires the measurement of micrometer sized pores in hundreds to thousands of images. When manually performed, this procedure has shown to be extremely time-consuming and to generate inconsistent results among different users and imaging systems. A user-friendly software for such analysis allowing quick processing and generation of reproducible data had not yet been reported. We have developed a software (PoET) for automatic pore edge tension measurements on GUVs. The required image processing steps and the characterization of the pore dynamics are performed automatically within the software and its use allowed for a 30-fold reduction in the analysis time. We demonstrate the applicability of the software by comparing the pore edge tension of GUVs of different membrane compositions and surface charges. The approach was applied to electroporated GUVs but is applicable to other means of pore formation. The complete software is implemented in Python and available for Windows at https://dx.doi.org/10.17617/3.7h. Supplementary data are available at Bioinformatics Advances online.

Effect of membrane potential on entry of lactoferricin B-derived 6-residue antimicrobial peptide into single Escherichia coli cells and lipid vesicles.

The antimicrobial peptide (AMP) derived from lactoferricin B, LfcinB (4-9) (RRWQWR), and lissamine rhodamine B red-labeled peptide (Rh-LfcinB (4-9)) exhibit strong antimicrobial activities, and they can enter Escherichia coli cells without damaging the cell membranes. Thus, these peptides are cell-penetrating peptide (CPP) -type AMPs. In this study, to elucidate the effect of the membrane potential (Δφ) on the action of the CPP-type AMP, Rh-LfcinB (4-9), we investigated the interactions of Rh-LfcinB (4-9) with single E. coli cells and spheroplasts containing calcein in the cytosol using confocal laser scanning microscopy. At low peptide concentrations, Rh-LfcinB (4-9) entered the cytosol of single E. coli cells and spheroplasts without damaging the cell membranes, and the H+-ionophore carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) suppressed its entry. The studies using the time-kill method indicate that these low concentrations of peptide exhibit antimicrobial activity but CCCP inhibits this activity. Next, we investigated the effect of Δφ on the interaction of Rh-LfcinB (4-9) with single giant unilamellar vesicles (GUVs) comprising E. coli polar lipid extracts and containing a fluorescent probe, Alexa Fluor 647 hydrazide. At low concentrations (0.2-0.5 μM), Rh-LfcinB (4-9) showed significant entry to the single GUV lumen without pore formation in the presence of Δφ. The fraction of entry of peptide increased with increasing negative membrane potential, indicating that the rate of peptide entry into the GUV lumen increased with increasing negative membrane potential. These results indicate that Δφ enhances the entry of Rh-LfcinB (4-9) into single E. coli cells, spheroplasts, and GUVs and its antimicrobial activity.IMPORTANCE: Bacterial cells have a membrane potential (Δφ), but the effect of Δφ on action of cell-penetrating peptide-type antimicrobial peptides (AMPs) is not clear. Here, we investigated the effect of Δφ on the action of fluorescent probe-labeled AMP derived from lactoferricin B, Rh-LfcinB (4-9). At low peptide concentrations, Rh-LfcinB (4-9) enters the cytosol of Escherichia coli cells and spheroplasts without damaging their cell membrane, but a protonophore suppresses this entry and its antimicrobial activity. The rate of entry of Rh-LfcinB (4-9) into the giant unilamellar vesicles (GUVs) comprising E. coli lipids without pore formation increases with increasing Δφ. These results indicate that Δφ enhances the antimicrobial activity of Rh-LfcinB (4-9) and hence LfcinB (4-9) by increasing the rate of their entry into the cytosol.