Single Giant Unilamellar Vesicles Research Articles

Elementary processes for the entry of cell-penetrating peptides into lipid bilayer vesicles and bacterial cells

Cell-penetrating peptides (CPPs) can translocate across the plasma membrane of living eukaryotic cells and enter the cytosol without significantly affecting cell viability. Consequently, CPPs have been used for the intracellular delivery of biological cargo such as proteins and oligonucleotides. However, the mechanisms underlying the translocation of CPPs across the plasma membrane remain unclear. In this mini-review, we summarize the experimental results regarding the entry of CPPs into lipid bilayer vesicles obtained using three methods: the large unilamellar vesicle (LUV) suspension method, the giant unilamellar vesicle (GUV) suspension method, and the single GUV method. The advantages and disadvantages of these methods are also discussed. Experimental results to date clearly indicate that CPPs can translocate across lipid bilayers and enter the vesicle lumen. Three models for the mechanisms and pathways by which CPPs translocate across lipid bilayers are described: (A) through pores induced by CPPs, (B) through transient prepores, and (C) via formation of inverted micelles. Both the pathway of translocation and the efficiency of entry of CPPs depend on the lipid composition of the bilayer and the type of CPP. We also describe the interaction of CPPs with bacterial cells. Some CPPs have strong antimicrobial activities. There are two modes of action of CPPs on bacterial cells: CPPs can induce damage to the plasma membrane and thus increase permeability, or CPPs enter the cytosol of bacterial cells without damaging the plasma membrane. The information currently available on the elementary processes by which CPPs enter lipid bilayer vesicles and bacterial cells is valuable for elucidating the mechanisms of entry of CPPs into the cytosol of various eukaryotic cells.

Mechanism of Initial Stage of Pore Formation Induced by Antimicrobial Peptide Magainin 2.

Antimicrobial peptide magainin 2 forms pores in lipid bilayers, a property that is considered the main cause of its bactericidal activity. Recent data suggest that tension or stretching of the inner monolayer plays an important role in magainin 2-induced pore formation in lipid bilayers. Here, to elucidate the mechanism of magainin 2-induced pore formation, we investigated the effect on pore formation of asymmetric lipid distribution in two monolayers. First, we developed a method to prepare giant unilamellar vesicles (GUVs) composed of dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylcholine (DOPC), and lyso-PC (LPC) in the inner monolayer and of DOPG/DOPC in the outer monolayer. We consider that in these GUVs, the lipid packing in the inner monolayer was larger than that in the outer monolayer. Next, we investigated the interaction of magainin 2 with these GUVs with an asymmetric distribution of LPC using the single GUV method, and found that the rate constant of magainin 2-induced pore formation, kp, decreased with increasing LPC concentration in the inner monolayer. We constructed a quantitative model of magainin 2-induced pore formation, whereby the binding of magainin 2 to the outer monolayer of a GUV induces stretching of the inner monolayer, causing pore formation. A theoretical equation defining kp as a function of magainin 2 surface concentration, X, reasonably explains the experimental relationship between kp and X. This model quantitatively explains the effect on kp of the LPC concentration in the inner monolayer. On the basis of these results, we discuss the mechanism of the initial stage of magainin 2-induced pore formation.

Continuous detection of entry of cell-penetrating peptide transportan 10 into single vesicles

Entry of cell-penetrating peptides (CPPs) into living cells by translocating across plasma membranes is an important physiological phenomenon. To elucidate the mechanism of the translocation of CPPs across lipid bilayers, it is essential to reveal its elementary processes. For this purpose, here, we have developed a new method for the continuous, quantitative detection of the entry of CPPs into giant unilamellar vesicles (GUVs), where we investigate the interaction of fluorescent probe-labeled CPPs with single GUVs containing large unilamellar vesicles (LUVs) and fluorescent probes in their lumens using confocal microscopy. Using this method, we investigated the interaction of carboxyfluorescein (CF)-labeled transportan 10 (CF-TP10) with single GUVs comprised of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) containing LUVs of the same membrane and Alexa Fluor 647 hydrazide (AF647) in their lumens. At low concentrations of CF-TP10, first the fluorescence intensity (FI) of the GUV membrane increased with time, and then after some lag time the FI of the GUV lumen due to CF-TP10 increased continuously with time without leakage of AF647. At higher concentrations of CF-TP10, after the FI of the GUV lumen due to CF-TP10 increased significantly, leakage of AF647 started. These results indicate that CF-TP10 entered the GUV lumen by translocating across the GUV membrane and then bound to the LUVs there without pore formation and that CF-TP10 concentration in the lumen increased with time. The rate of entry of CF-TP10 into GUV lumen increased with CF-TP10 concentration. We discussed the kinetics of entry of CF-TP10 into single GUVs.

Integrity of Membrane Structures in Giant Unilamellar Vesicles as Assay for Antioxidants and Prooxidants.

We have attempted to evaluate, on the basis of optical microscopy for a single giant unilamellar vesicle (GUV), the potency of antioxidants in protecting GUV membranes from oxidative destruction. Photosensitized membrane budding of GUVs prepared from soybean phosphatidylcholine with chlorophyll a (Chl a) and β-carotene (β-Car) as photosensitizer and protector, respectively, were followed by microscopic imaging. A dimensionless entropy parameter, ΔE, as derived from the time-resolved microscopic images, was employed to describe the evolution of morphological variation of GUVs. As an indication of membrane instability, the budding process showed three successive temporal regimes as a common feature: a lag phase prior to the initiation of budding characterized by LP (in s), a budding phase when ΔE increased with a rate of kΔE (in s-1), and an ending phase with morphology stabilized at a constant ΔEend (dimensionless). We show that the phase-associated parameters can be objectively obtained by fitting the ΔE-t kinetics curves to a Boltzmann function and that all of the parameters are rather sensitive to β-Car concentration. As for the efficacy of these parameters in quantifying the protection potency of β-Car, kΔE is shown to be most sensitive for β-Car in a concentration regime of biological significance of <1 × 10-7 M, whereas LP and ΔEend are more sensitive for β-Car concentrations exceeding 1 × 10-7 M. Furthermore, based on the results of GUV imaging and fluorescence and Raman spectroscopies, we have revealed for different phases the mechanistic interplay among 1O2* diffusion, PC-OOH accumulation, Chl a and/or β-Car consumption, and the morphological variation. The developed assay should be valuable for characterizing the potency of antioxidants or prooxidants in the protection or destruction of the membrane integrity of GUVs.

Mutation-induced changes of transmembrane pore size revealed by combined ion-channel conductance and single vesicle permeabilization analyses

Permeabilization of the Endoplasmic Reticulum (ER) is instrumental in the progression of host-cell infection by many viral pathogens. We have described that permeabilization of ER model membranes by the pore-forming domain of the Classical Swine Fever Virus (CSFV) p7 protein depends on two sequence determinants: the C-terminal transmembrane helix, and the preceding polar loop that regulates its activity. Here, by combining ion-channel activity measurements in planar lipid bilayers with imaging of single Giant Unilamellar Vesicles (GUVs), we demonstrate that point substitutions directed to conserved residues within these regions affect ER-like membrane permeabilization following distinct mechanisms. Whereas the polar loop appeared to be involved in protein insertion and oligomerization, substitution of residues predicted to face the lumen of the pore inhibited large conducting channels (>1 nS) over smaller ones (120 pS). Quantitative analyses of the ER-GUV distribution as a function of the solute size revealed a selective inhibition for the permeation of solutes with sizes larger than 4 kDa, further demonstrating that the mutation targeting the transmembrane helix prevented formation of the large pores. Collectively, our data support the idea that the pore-forming domain of p7 may assemble into finite pores with approximate diameters of 1 and 5 nm. Moreover, the observation that the mutation interfering with formation of the larger pores can hamper virus production without affecting ER localization or homo-oligomerization, suggests prospective strategies to block/attenuate pestiviruses.

Entry of a Six-Residue Antimicrobial Peptide Derived from Lactoferricin B into Single Vesicles and Escherichia coli Cells without Damaging their Membranes.

Lactoferricin B (LfcinB) and shorter versions of this peptide have antimicrobial activity. However, the elementary processes of interactions of these peptides with lipid membranes and bacteria are still not well understood. To elucidate the mechanism of their antimicrobial activity, we investigated the interactions of LfcinB (4-9) (its sequence of RRWQWR) with Escherichia coli cells and giant unilamellar vesicles (GUVs). LfcinB (4-9) and lissamine rhodamine B red-labeled LfcinB (4-9) (Rh-LfcinB (4-9)) did not induce an influx of a membrane-impermeant fluorescent probe, SYTOX green, from the outside of E. coli cells into their cytoplasm, indicating that no damage occurred in their plasma membrane. To examine the activity of LfcinB (4-9) to enter E. coli cytoplasm, we investigated the interaction of Rh-LfcinB (4-9) with single cells of E. coli containing calcein using confocal microscopy. We found that Rh-LfcinB (4-9) entered the cytoplasm without leakage of calcein. Next, we investigated the interactions of Rh-LfcinB (4-9) with single GUVs of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) mixtures containing a fluorescent probe, Alexa Fluor 647 hydrazide (AF647), using the single GUV method. The results indicate that Rh-LfcinB (4-9) outside the GUV translocated through the GUV membrane and entered its lumen without leakage of AF647. Interaction of Rh-LfcinB (4-9) with DNA increased its fluorescence intensity greatly. Therefore, we can conclude that Rh-LfcinB (4-9) can translocate across lipid membrane regions of the plasma membrane of E. coli cells to enter their cytoplasm without leakage of calcein and its antimicrobial activity is not due to damage of their plasma membranes.

Effects of Mechanical Properties of Lipid Bilayers on the Entry of Cell-Penetrating Peptides into Single Vesicles.

The translocation of cell-penetrating peptides (CPPs) through plasma membranes of living cells is an important physiological phenomenon in biomembranes. To reveal the mechanism underlying the translocation of a CPP, transportan 10 (TP10), through lipid bilayers, we examined the effects of the mechanical properties of lipid bilayers on the entry of carboxyfluorescein (CF)-labeled TP10 (CF-TP10) into a giant unilamellar vesicle (GUV) using the single GUV method. First, we examined the effect of lateral tension in membranes on the entry of CF-TP10 into single GUVs comprising a mixture of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) (2/8). CF-TP10 entered the GUV lumen before the membrane permeation of Alexa Fluor 647 hydrazide (AF647) from the GUV and thus before pore formation in the membrane. The fraction of entry of CF-TP10 before pore formation and the rate of membrane rupture increased with tension. The CF-TP10-induced fractional change in the membrane area increased continuously with time until membrane rupture, but it increased more slowly than did the CF-TP10 concentration in the GUV membrane. A high mole fraction of cholesterol inhibited the entry of CF-TP10 into single GUVs by suppressing the translocation of CF-TP10 from the external to the internal monolayer, although higher concentrations of CF-TP10 induced the formation of pores through which CF-TP10 rapidly translocated. Suppression of the translocation of CF-TP10 by cholesterol can be reasonably explained by the large line tension of a prepore. We discussed the role of mechanical properties in membranes on the entry of CF-TP10 into single GUVs and proposed a hypothesis of the mechanism that CF-TP10 translocates across a bilayer through transient hydrophilic prepores in the membrane.

Direct detection of SERCA calcium transport and small-molecule inhibition in giant unilamellar vesicles

We have developed a charge-mediated fusion method to reconstitute the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) in giant unilamellar vesicles (GUV). Intracellular Ca2+ transport by SERCA controls key processes in human cells such as proliferation, signaling, and contraction. Small-molecule effectors of SERCA are urgently needed as therapeutics for Ca2+ dysregulation in human diseases including cancer, diabetes, and heart failure. Here we report the development of a method for efficiently reconstituting SERCA in GUV, and we describe a streamlined protocol based on optimized parameters (e.g., lipid components, SERCA preparation, and activity assay requirements). ATP-dependent Ca2+ transport by SERCA in single GUV was detected directly using confocal fluorescence microscopy with the Ca2+ indicator Fluo-5F. The GUV reconstitution system was validated for functional screening of Ca2+ transport using thapsigargin (TG), a small-molecule inhibitor of SERCA currently in clinical trials as a prostate cancer prodrug. The GUV system overcomes the problem of inhibitory Ca2+ accumulation for SERCA in native and reconstituted small unilamellar vesicles (SUV). We propose that charge-mediated fusion provides a widely-applicable method for GUV reconstitution of clinically-important membrane transport proteins. We conclude that GUV reconstitution is a technological advancement for evaluating small-molecule effectors of SERCA.

Effects of Lipid Composition on the Entry of Cell-Penetrating Peptide Oligoarginine into Single Vesicles.

The cell-penetrating peptide R9, an oligoarginine comprising nine arginines, has been used to transport biological cargos into cells. However, the mechanisms underlying its translocation across membranes remain unclear. In this report, we investigated the entry of carboxyfluorescein (CF)-labeled R9 (CF-R9) into single giant unilamellar vesicles (GUVs) of various lipid compositions and the CF-R9-induced leakage of a fluorescent probe, Alexa Fluor 647 hydrazide (AF647), using a method developed recently by us. First, we investigated the interaction of CF-R9 with dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) GUVs containing AF647 and small DOPG/DOPC vesicles. The fluorescence intensity of the GUV membrane due to CF-R9 (i.e., the rim intensity) increased with time to a steady-state value, and then the fluorescence intensity of the membranes of the small vesicles in the GUV lumen increased without leakage of AF647. This result indicates that CF-R9 entered the GUV lumen from the outside by translocating across the lipid membrane without forming pores through which AF647 could leak. The fraction of entry of CF-R9 at 6 min in the absence of pore formation, Pentry (6 min), increased with an increase in CF-R9 concentration, but the CF-R9 concentration in the lumen was low. We obtained similar results for dilauroyl-PG (DLPG)/ditridecanoyl-PC (DTPC) (2/8) GUVs. The values of Pentry (6 min) of CF-R9 for DLPG/DTPC (2/8) GUVs were larger than those obtained with DOPG/DOPC (2/8) GUVs at the same CF-R9 concentrations. In contrast, a high concentration of CF-R9 induced pores in DLPG/DTPC (4/6) GUVs through which CF-R9 entered the GUV lumen, so the CF-R9 concentration in the lumen was higher. However, CF-R9 could not enter DOPG/DOPC/cholesterol (2/6/4) GUVs. Analysis of the rim intensity showed that CF-R9 was located only in the outer monolayer of the DOPG/DOPC/cholesterol (2/6/4) GUVs. On the basis of analyses of these results, we discuss the elementary processes by which CF-R9 enters GUVs of various lipid compositions.

Direct Measurement of Pore Dynamics and Leakage Induced by a Model Antimicrobial Peptide in Single Vesicles and Cells.

Antimicrobial peptides are promising therapeutic alternatives to counter growing antimicrobial resistance. Their precise mechanism of action remains elusive, however, particularly with respect to live bacterial cells. We investigated the interaction of a fluorescent melittin analogue with single giant unilamellar vesicles, giant multilamellar vesicles, and bilamellar Gram-negative Escherichia coli (E.coli) bacteria. Time-lapse fluorescence lifetime imaging microscopy was employed to determine the population distribution of the fluorescent melittin analogue between pore state and membrane surface state, and simultaneously measure the leakage of entrapped fluorescent species from the vesicle (or bacterium) interior. In giant unilamellar vesicles, leakage from vesicle interior was correlated with an increase in level of pore states, consistent with a stable pore formation mechanism. In giant multilamellar vesicles, vesicle leakage occurred more gradually and did not appear to correlate with increased pore states. Instead pore levels remained at a low steady-state level, which is more in line with coupled equilibria. Finally, in single bacterial cells, significant increases in pore levels were observed over time, which were correlated with only partial loss of cytosolic contents. These observations suggested that pore formation, as opposed to complete dissolution of membrane, was responsible for the leakage of contents in these systems, and that the bacterial membrane has an adaptive capacity that resists peptide attack. We interpret the three distinct pore dynamics regimes in the context of the increasing physical and biological complexity of the membranes.

Solution Asymmetry and Salt Expand Fluid-Fluid Coexistence Regions of Charged Membranes

Liquid-liquid phase separation in giant unilamellar vesicles (GUVs) leads to the formation of intramembrane domains. To mimic charged biological membranes, we studied phase separation and domain formation in GUVs of ternary lipid mixtures composed of egg sphingomyelin, cholesterol, and the negatively charged lipid dioleoylphosphatidylglycerol. The GUVs were exposed to solutions of sucrose and high-saline buffer. The phase diagram was determined using epifluorescence microscopy for vesicle populations with symmetric and asymmetric solution compositions across the membranes. Trans-membrane solution asymmetry was found to affect the membrane phase state. Furthermore, compared to the case of salt-free conditions, the phase diagram in the presence of high-saline buffer (both symmetrically or asymmetrically present across the membrane) was found to exhibit a significantly extended region of liquid-ordered and liquid-disordered coexistence. These observations were confirmed on single GUVs using microfluidics and confocal microscopy. Moreover, we found that the miscibility temperatures markedly increased for vesicles in the presence of symmetric and asymmetric salt solutions. Our results demonstrate a substantial effect of salt and solution asymmetry on the phase behavior of charged membranes, which has direct implications for protein adsorption onto these membranes and for the repartitioning of proteins within the membrane domains.

Analyzing Single Giant Unilamellar Vesicles With a Slotline-Based RF Nanometer Sensor.

Novel techniques that enable reagent free detection and analysis of single cells are of great interest for the development of biological and medical sciences as well as point-of-care health service technologies. Highly sensitive and broadband radio-frequency (RF) sensors are promising candidates for such a technique. In this work, we present a highly sensitive and tunable RF sensor, which is based on interference processes and built with a 100 nm slotline structure. The highly concentrated RF fields, up to ~1.76×107 V/m, enable strong interactions between Giant unilamellar vesicles (GUVs) and fields for high sensitivity operations. We also provide two modeling approaches to extract cell dielectric properties from measured scattering parameters. GUVs of different molecular compositions are synthesized and analyzed with the RF sensor at ~2 GHz, ~2.5 GHz, and ~2.8 GHz with an initial |S21 | min of ~-100 dB. Corresponding GUV dielectric properties are obtained. A one-dimensional scanning of single GUV is also demonstrated.

Presence of Salt and Solution Asymmetry Across Charged Membranes Influences Their Phase State

Liquid-liquid phase separation in giant unilamellar vesicles (GUVs) leads to the formation of intramembrane domains or lipid rafts. In order to mimic charged biological membranes, we studied phase separation and domain formation in GUVs of ternary lipid mixtures, composed of egg sphingomyelin (eSM), cholesterol (Chol) and the negatively charged lipid dioleoyl-phosphatidylglycerol (DOPG). The GUVs were exposed to solutions of sucrose and high-saline buffer. The phase diagram was mapped using epifluorescence microscopy for vesicle populations with symmetric and asymmetric solution compositions across the membrane. Interestingly, solution asymmetry was also found to affect the membrane phase state. Furthermore, compared to the case of symmetric sucrose conditions, the phase diagram in the presence of high-saline buffer (both symmetrically or asymmetrically distributed across the membrane) was found to exhibit a significantly extended coexistence region for liquid ordered (Lo) and liquid disordered (Ld) phases. These observations were confirmed on single GUVs using microfluidics and confocal laser scanning microscopy. Moreover, we showed that the miscibility temperatures markedly increased for vesicles in the presence of symmetric and asymmetric salt solutions. Our results indicate a substantial effect of salt on the phase behavior of charged membranes and demonstrate that one has to control the solution conditions in order to appropriately map the phase diagram of lipid mixtures.This work is part of the MaxSynBio consortium which is jointly funded by the Federal Ministry of Education and Research of Germany and the Max Planck Society.

Hypericin Aggregation in Artificial Lipid Membranes

The incorporation of hypericin (Hyp), a natural hydrophobic photosensitizer, to giant unilamellar vesicles (GUVs) was studied by fluorescence and Raman spectroscopies. The GUVs were prepared from DOPC by electro-formation on platinum electrodes. During the experiments, a single GUV was trapped in optical tweezers operated at 785 nm. The micro-shelter system developed previously for laser tweezers buffer exchange [1] was used to place the GUVs into aqueous solution of Hyp (2.10-6 M), where this compound forms non-fluorescence aggregates [2]. By contrast, Hyp can be well dissolved in lipid structures [3]. The incorporation of Hyp into the GUV was monitored by time-resolved fluorescence measurements at 488 nm excitation. The early increase of the fluorescence signal (belonging to Hyp monomers) was followed by a gradual signal weakening, which indicated in-membrane aggregation of Hyp. An unexpected formation of large (∼500 nm diameter) Hyp aggregates was observed on the GUV surface several minutes after mixing GUVs with Hyp. This observation was also confirmed by micro-Raman measurements.This work was supported by the APVV-0242-11 grant of the Slovak Ministry of Education, the FP7 EU CELIM 316310 project, the NanoBioSens project (ITMS 26220220107, 50%) funded by the European Regional Development Fund and by the EU Structural Fund ITMS 26240120040.[1] M. A. Omar, P. Miskovsky, G. Bano, Lab Chip 14, 1579-1584 (2014)[2] G. Bano, J. Stanicova, D. Jancura, J. Marek, M. Bano, J. Ulicný, A. Strejckova, P. Miskovský, J. Phys. Chem. B 115, 2417-2423 (2011)[3] A. Strejckova, J. Stanicova, D. Jancura, P. Miskovský, and G. Bano, J. Phys. Chem. B 117, 1280-1286 (2013)

Antimicrobial Peptide Lactoferricin B-Induced Rapid Leakage of Internal Contents from Single Giant Unilamellar Vesicles.

Enzymatic digestion of bovine lactoferrin generates lactoferricin B (Lfcin B), a 25-mer peptide with strong antimicrobial activity of unknown mechanism. To elucidate the mechanistic basis of Lfcin B bactericidal activity, we investigated the interaction of Lfcin B with Escherichia coli and liposomes of lipid membranes. Lfcin B induced the influx of a membrane-impermeant fluorescent probe, SYTOX green, from the outside of E. coli into its cytoplasm. Lfcin B induced gradual leakage of calcein from large unilamellar vesicles (LUVs) of dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) membranes. To clarify the cause of Lfcin B-induced leakage of calcein from the LUVs, we used the single giant unilamellar vesicle (GUV) method to investigate the interaction of Lfcin B with calcein-containing DOPG/DOPC-GUVs. We observed that a rapid leakage of calcein from a GUV started stochastically; statistical analysis provided a rate constant for Lfcin B-induced pore formation, kp. On the other hand, phase-contrast microscopic images revealed that Lfcin B induced a rapid leakage of sucrose from the single GUVs with concomitant appearance of a spherical GUV of smaller diameter. Because of the very fast leakage, and at the present time resolution of the experiments (33 ms), we could not follow the evolution of pore nor the process of the structural changes of the GUV. Here we used the term "local rupture" to express the rapid leakage of sucrose and determined the rate constant of local rupture, kL. On the basis of the comparison between kp and kL, we concluded that the leakage of calcein from single GUVs occurred as a result of a local rupture in the GUVs and that smaller pores inducing leakage of calcein were not formed before the local rupture. The results of the effect of the surface charge density of lipid membranes and that of salt concentration in buffer on kp clearly show that kp increases with an increase in the extent of electrostatic interactions due to the surface charges. Analysis of Lfcin B-induced shape changes indicated that the binding of Lfcin B increased the area of the outer monolayer of GUVs. These results indicate that Lfcin B-induced damage of the plasma membrane of E. coli with its concomitant rapid leakage of internal contents is a key factor for the bactericidal activity of LfcinB.

Formation of Giant Unilamellar Proteo-Liposomes by Osmotic Shock

Giant unilamellar vesicles (GUVs), composed of a phospholipid bilayer, are often used as a model system for cell membranes. However, the study of proteo-membrane interactions in this system is limited as the incorporation of integral and lipid-anchored proteins into GUVs remains challenging. Here, we present a simple generic method to incorporate proteins into GUVs. The basic principle is to break proteo-liposomes with an osmotic shock. They subsequently reseal into larger vesicles which, if necessary, can endure the same to obtain even larger proteo-GUVs. This process does not require specific lipids or reagents, works under physiological conditions with high concentrations of protein, the proteins remains functional after incorporation. The resulting proteo-GUVs can be micromanipulated. Moreover, our protocol is valid for a wide range of protein substrates. We have successfully reconstituted three structurally different proteins, two trans-membrane proteins (TolC and the neuronal t-SNARE), and one lipid-anchored peripheral protein (GABARAP-Like 1 (GL1)). In each case, we verified that the protein remains active after incorporation and in its correctly folded state. We also measured their mobility by performing diffusion measurements via fluorescence recovery after photobleaching (FRAP) experiments on micromanipulated single GUVs. The diffusion coefficients are in agreement with previous data.

Stretch-activated pore of the antimicrobial peptide, magainin 2.

Antimicrobial peptide magainin 2 forms pores in lipid membranes and induces membrane permeation of the cellular contents. Although this permeation is likely the main cause of its bactericidal activity, the mechanism of pore formation remains poorly understood. We therefore investigated in detail the interaction of magainin 2 with lipid membranes using single giant unilamellar vesicles (GUVs). The binding of magainin 2 to the lipid membrane of GUVs increased the fractional change in the area of the membrane, δ, which was proportional to the surface concentration of magainin 2, X. This indicates that the rate constant of the magainin 2-induced two-state transition from the intact state to the pore state greatly increased with an increase in δ. The tension of a lipid membrane following aspiration of a GUV also activated magainin 2-induced pore formation. To reveal the location of magainin 2, the interaction of carboxyfluorescein (CF)-labeled magainin 2 (CF-magainin 2) with single GUVs containing a water-soluble fluorescent probe, AF647, was investigated using confocal microscopy. In the absence of tension due to aspiration, after the interaction of magainin 2 the fluorescence intensity of the GUV rim due to CF-magainin 2 increased rapidly to a steady value, which remained constant for a long time, and at 4-32 s before the start of leakage of AF647 the rim intensity began to increase rapidly to another steady value. In contrast, in the presence of the tension, no increase in rim intensity just before the start of leakage was observed. These results indicate that magainin 2 cannot translocate from the outer to the inner monolayer until just before pore formation. Based on these results, we conclude that a magainin 2-induced pore is a stretch-activated pore and the stretch of the inner monolayer is a main driving force of the pore formation.

Variable Adhesion Strength for Giant Unilamellar Vesicles Controlled by External Electrostatic Potentials

We developed an experimental setup to study adhesion of giant unilamellar vesicles (GUVs) with variable adhesion strength. The vesicles adhere to a planar substrate. The adhesion is easily modulated by the application of an external potential to transparent ITO electrodes (the substrate) which enables us to study single GUVs during adhesion. The adhesion energy is calculated from the vesicle shape assessed with confocal microscopy. We implement a theoretical method for deducing the adhesion energy based on the overall vesicle shape. The results show that for the explored ranges of the external potential, the adhered vesicles are in the weak adhesion regime. Using fluorescence quenching assays, we conclude that the outer membrane leaflet is depleted of the negatively charged dye NDB-PG in the adhesion zone. Based on these results, we discuss the driving forces involved in the vesicle adhesion. Using FRAP measurements, we also show that diffusion is only slightly hindered in the adhesion zone. Some first results on applying our adhesion approach to study demixing in multicomponent membranes will be presented. We will demonstrate that adhesion can be used to modulate the phase state in the adhering and nonadhering regions of vesicles exhibiting coexistence of liquid ordered and liquid disordered domains.

Studying antibiotic–membrane interactions via X-ray diffraction and fluorescence microscopy

Antibiotic drug resistance is a serious issue for the treatment of bacterial infection. Understanding the resistance to antibiotics is a key issue for developing new drugs. We used penicillin and sulbactam as model antibiotics to study their interaction with model membranes. Cholesterol was used to target the membrane for comparison with the well-known insertion model. Lamellar X-ray diffraction (LXD) was used to determine membrane thickness using successive drug-to-lipid molar ratios. The aspiration method for a single giant unilamellar vesicle (GUV) was used to monitor the kinetic binding process of antibiotic–membrane interactions in an aqueous solution. Both penicillin and sulbactam are found positioned outside the model membrane, while cholesterol inserts perpendicularly into the hydrophobic region of the membrane in aqueous solution. This result provides structural insights for understanding the antibiotic–membrane interaction and the mechanism of antibiotics.

Protein-Free Membrane Fusion Probed by Single Giant Unilamellar Vesicle Imaging - the Role of Membrane Charge

Membrane fusion is a ubiquitous process in biology. The outcome of fusion is followed by successive steps of fusion intermediates, including (i) protein tethering in apposing bilayers, (ii) membrane adhesion, (ii) hemifusion and eventually (iv) full fusion, the latter leading to complete mixing of membranes and aqueous contents. Studying membrane fusion in vivo is, however, very challenging. By a combination of microscopic approaches, we investigate the role of membrane charges on the interaction and fusion of cationic large (DOTAP:DOPE:DPPE-Rh - 1:1:0.1 mol) and individual giant unilamellar vesicles (LUVs-GUVs) in a pure lipid system. Fluorescencence Resonance Energy Transfer (FRET) efficiency, in which the donor is reconstituted in the GUVs and the acceptor incorporated upon membrane merging, is measured and directly related to fusion efficiency. LUVs stably adhere and diffuse onto the surface of neutral (POPC) GUVs with minimal membrane merging (FRET efficiency 0.1). In sharp contrast, very efficient fusion occurs with the negative GUV membranes (POPC:POPG - 1:1) - FRET 0.6-1. An increase in membrane area after contact with the fusogenic LUVs was clearly detected for this composition. Excess of negative lipids on GUVs (pure POPG) relative to positive charges decrease fusion efficiency (FRET 0.3) - a charge-neutralization process. Transferred lipids upon fusion undergo similar diffusion to those on GUV membranes (∼ 1 μm2/s) With the developed LUV-GUV system, we studied the charge-dependent membrane adhesion and fusion separately and individually for the very same interacting partners, not possible with traditional in vitro systems. The system could also be easily adapted to protein-reconstituted studies.Financial support: FAPESP.