Interactions of DNA with giant liposomes

DNA interactions with the bilayers of cationic liposomes were studied using a novel model experiment: DNAs were locally injected by a micropipette to a part of a giant unilamellar vesicle. The resulting phenomena were directly observed in optical microscope. Giant unilamellar vesicles (GUVs), about 100 microm in diameter, made of phosphatidylcholines and up to 33 mol% of the natural bioactive cationic amphiphile sphingosine, were obtained by electroformation. The effects of DNAs of different length were tested: (i) 'short' DNAs-oligonucleotide 21b, and calf thymus 250 bp; (ii) 'long' DNAs-plasmid DNAs in super coil or liner form (between 2.7 and 8.0 kbp). DNAs were injected native, as well as marked with the fluorescent dye Hoechst. The resulting membrane topology transformations were monitored in phase contrast, while the DNA distribution was followed in fluorescence. DNA-induced endocytosis was observed due to the DNA/lipid membrane local interactions for all DNAs tested. Some of the DNA in the formed complex was associated with the induced endosomes, and some of it remained spread over the 'mother' GUV membrane for all DNAs tested, except for the longest one--the linear plasmid of 8 kbp. The last remained at the 'mother' GUV membrane and was not transported with the induced endosomes to the internal GUV space. Possible mechanisms for DNA/lipid membrane interaction were suggested. One of them involves DNA encapsulation within an inverted micelle included in the lipid membrane. The model observations could help in understanding events associated with interaction of DNA with biological membranes, as well as cationic liposomes/DNA complexes formation in gene transfer processes.     

Highly Efficient Protein-free Membrane Fusion: A Giant Vesicle Study

Membrane fusion is a ubiquitous process in biology and is a prerequisite for many intracellular delivery protocols relying on the use of liposomes as drug carriers. Here, we investigate in detail the process of membrane fusion and the role of opposite charges in a protein-free lipid system based on cationic liposomes (LUVs, large unilamellar vesicles) and anionic giant unilamellar vesicles (GUVs) composed of different palmitoyloleoylphosphatidylcholine (POPC)/palmitoyloleoylphosphatidylglycerol (POPG) molar ratios. By using a set of optical-microscopy- and microfluidics-based methods, we show that liposomes strongly dock to GUVs of pure POPC or low POPG fraction (up to 10 mol%) in a process mainly associated with hemifusion and membrane tension increase, commonly leading to GUV rupture. On the other hand, docked LUVs quickly and very efficiently fuse with negative GUVs of POPG fractions at or above 20 mol%, resulting in dramatic GUV area increase in a charge-dependent manner; the vesicle area increase is deduced from GUV electrodeformation. Importantly, both hemifusion and full fusion are leakage-free. Fusion efficiency is quantified by the lipid transfer from liposomes to GUVs using fluorescence resonance energy transfer (FRET), which leads to consistent results when compared to fluorescence-lifetime-based FRET. We develop an approach to deduce the final composition of single GUVs after fusion based on the FRET efficiency. The results suggest that fusion is driven by membrane charge and appears to proceed up to charge neutralization of the acceptor GUV.     

Role of Membrane Potential on Entry of Cell-Penetrating Peptide Transportan 10 into Single Vesicles

Cell-penetrating peptides (CPPs) can translocate across plasma membranes to enter the cytosol of eukaryotic cells without decreasing cell viability. We revealed the mechanism underlying this translocation by examining the effect of membrane potential, φ_m, on the entry of a CPP, transportan 10 (TP10), into the lumen of single giant unilamellar vesicles (GUVs). For this purpose, we used the single GUV method to detect the entry of carboxyfluorescein (CF)-labeled TP10 (CF-TP10) into the lumen of single GUVs. First, we used various K⁺ concentration differences to apply different negative membrane potentials on single GUVs containing gramicidin A in their membrane and confirmed these potentials using the φ_m-sensitive fluorescent probe 3,3′-dihexyloxacarbocyanine iodine. The fluorescence intensity of the GUV membranes (i.e., the rim intensity) due to 3,3′-dihexyloxacarbocyanine iodine increased with |φ_m| up to 118 mV, and its dependence on |φ_m| less than 28 mV agreed with a theoretical estimation (i.e., the dye concentration in the inner leaflet of a GUV is larger than that in the outer leaflet according to the Boltzmann distribution). We then examined the effect of φ_m on the entry of CF-TP10 into GUVs using single GUVs containing small GUVs or large unilamellar vesicles inside the mother GUV lumen. We found that CF-TP10 entered the GUV lumen without pore formation and the rate of entry of CF-TP10 into the GUV lumen, V_entry, increased with an increase in |φ_m|. The rim intensity due to CF-TP10 increased with an increase in |φ_m|, indicating that the CF-TP10 concentration in the inner leaflet of the GUV increased with |φ_m|. These results indicate that the φ_m-induced elevation in V_entry can be explained by the increase in CF-TP10 concentration in the inner leaflet with |φ_m|. We discuss the mechanism underlying this effect of membrane potential based on the pre-pore model of the translocation of CF-TP10 across a GUV membrane.     

Confocal microscopic observation of fusion between baculovirus budded virus envelopes and single giant unilamellar vesicles

We assayed fusion events between giant unilamellar vesicles (GUVs) and budded viruses (BVs) of baculovirus (Autographa californica nucleopolyhedrovirus), the envelopes of which have been labeled with the fluorescent dye Alexa Fluor 488. This involves observing the intensity of fluorescence emitted from the lipid bilayer of single GUVs after fusion using laser scanning microscopy. Using this assay system, we found that fusion between single GUVs and BV envelopes was significantly enhanced at around pH 5.0-6.0, which suggests that: (1) envelope glycoprotein GP64-mediated membrane fusion within the endosome of insect cells was reproduced in our artificial system; (2) acidic phospholipids in GUVs are necessary for this fusion, which are in agreement with the previous results with conventional small liposomes including large unilamellar vesicles and multilamellar vesicles; and (3) the efficiency of fusion is significantly affected by membrane properties that can be modulated by adding cholesterol to GUV lipid bilayers. In addition, the microscopic observation of BV-fused single GUVs showed that a weak interaction occurred between BVs and GUVs containing dioleoylphosphatidylserine at pH 6.0-6.5, and components of BV envelopes were unevenly distributed upon fusion with GUVs containing saturated phospholipid with cholesterol. We further demonstrated that when the recombinant membrane protein, adrenergic β₂ receptor, was expressed on recombinant BV envelopes, the protein distribution on BV-fused GUVs was also affected by their lipid contents.     

Giant unilamellar vesicles containing phosphatidylinositol(4,5)bisphosphate: characterization and functionality

Biomimetic systems such as giant unilamellar vesicles (GUVs) are increasingly used for studying protein/lipid interactions due to their size (similar to that of cells) and to their ease of observation by light microscopy techniques. Biophysicists have begun to complexify GUVs to investigate lipid/protein interactions. In particular, composite GUVs have been designed that incorporate lipids that play important physiological roles in cellulo, such as phosphoinositides and among those the most abundant one, phosphatidylinositol(4,5)bisphosphate (PIP₂). Fluorescent lipids are often used as tracers to observe GUV membranes by microscopy but they can not bring quantitative information about the insertion of unlabeled lipids. In this study, we carried out ζ-potential measurements to prove the effective incorporation of PIP₂ as well as that of phosphatidylserine in the membrane of GUVs prepared by electroformation and to follow the stability of PIP₂-containing GUVs. Using confocal microscopy, we found that long-chain (C16) fluorescent PIP₂ analogs used as tracers (0.1% of total lipids) show a uniform distribution in the membrane whereas PIP₂ antibodies show PIP₂ clustering. However, the clustering effect, which is emphasized when tertiary antibodies are used in addition to secondary ones to enhance the size of the detection complex, is artifactual. We showed that divalent ions (Ca²⁺ and Mg²⁺) can induce aggregation of PIP₂ in the membrane depending on their concentration. Finally, the interaction of ezrin with PIP₂-containing GUVs was investigated. Using either labeled ezrin and unlabeled GUVs or both labeled ezrin and GUVs, we showed that clusters of PIP₂ and proteins are formed.     

Single giant unilamellar vesicle method reveals effect of antimicrobial peptide magainin 2 on membrane permeability

It is thought that magainin 2, an antimicrobial peptide, acts by binding to lipid membranes. Recent studies using a suspension of large unilamellar vesicles (LUVs) indicate that magainin 2 causes gradual leakage from LUVs containing negatively charged lipids. However, the details of the characteristics of the membrane permeability and the mechanism of pore formation remain unclear. In this report, we investigated the interaction of magainin 2 with single giant unilamellar vesicles (GUVs) composed of a dioleoylphosphatidylcholine and dioleoylphosphatidylglycerol mixture (50% DOPG/50% DOPC GUVs) containing the fluorescent dye, calcein, by phase contrast, fluorescence microscopy using the single GUV method. Low concentrations (3-10 microM) of magainin 2 caused the rapid leakage of calcein from single GUVs but did not disrupt the liposomes or change the membrane structure, showing directly that magainin 2 forms membrane pores through which calcein leaked. The rapid leakage of calcein from a GUV started stochastically, and once it began, the complete leakage occurred rapidly (6-60 s). The fraction of completely leaked GUV, P(L), increased with time and also with an increase in magainin 2 concentration. Shape changes in these GUVs occurred prior to the pore formation and also at lower concentrations of magainin 2, which could not induce the pore formation. Their analysis indicates that binding of magainin 2 to the external monolayer of the GUV increases its membrane area, thereby raising its surface pressure. The addition of lysophosphatidylcholine into the external monolayer of GUVs increased P(L). On the basis of these results, we propose the two-state transition model for the pore formation.     

Micron-sized domains in quasi single-component giant vesicles

Giant unilamellar vesicles (GUVs), are a convenient tool to study membrane-bound processes using optical microscopy. An increasing number of studies highlights the potential of these model membranes when addressing questions in membrane biophysics and cell-biology. Among them, phase transitions and domain formation, dynamics and stability in raft-like mixtures are probably some of the most intensively investigated. In doing so, many research teams rely on standard protocols for GUV preparation and handling involving the use of sugar solutions. Here, we demonstrate that following such a standard approach can lead to the abnormal formation of micron-sized domains in GUVs grown from only a single phospholipid. The membrane heterogeneity is visualized by means of a small fraction (0.1 mol%) of a fluorescent lipid dye. For dipalmitoylphosphatidylcholine GUVs, different types of membrane heterogeneities were detected. First, the unexpected formation of micron-sized dye-depleted domains was observed upon cooling. These domains nucleated about 10 K above the lipid main phase transition temperature, T_M. In addition, upon further cooling of the GUVs down to the immediate vicinity of T_M, stripe-like dye-enriched structures around the domains are detected. The micron-sized domains in quasi single-component GUVs were observed also when using two other lipids. Whereas the stripe structures are related to the phase transition of the lipid, the dye-excluding domains seem to be caused by traces of impurities present in the glucose. Supplementing glucose solutions with nm-sized liposomes at millimolar lipid concentration suppresses the formation of the micron-sized domains, presumably by providing competitive binding of the impurities to the liposome membrane in excess. It is likely that such traces of impurities can significantly alter lipid phase diagrams and cause differences among reported ones.     

Rapid phase change of lipid microdomains in giant vesicles induced by conversion of sphingomyelin to ceramide

To understand the role of sphingomyelinase (SMase) in the function of biological membranes, we have investigated the effect of conversion of sphingomyelin (SM) to ceramide (Cer) on the assembly of domains in giant unilamellar vesicles (GUVs). The GUVs were prepared from mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), N-palmitoly-d-erythro-sphingosine (C₁₆Cer), N-palmitoyl-d-erythro-sphingosylphosphorylcholine (C₁₆SM) and cholesterol. The amounts of DOPC, sum of C₁₆Cer and C₁₆SM, and cholesterol were kept constant (the ratio of these four lipids is shown as 1:X:1-X:1 (molar ratio), i.e., X is C₁₆Cer/(C₁₆Cer + C₁₆SM)). Shape and distribution of domains formed in the GUVs were monitored by a fluorescent lipid, Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (0.1 mol%). In GUVs containing low C₁₆Cer (X = 0 and 0.25), round-shaped domains labeled by the fluorescent lipid were present, suggesting coexistence of liquid-ordered and disordered domains. In GUVs containing intermediate Cer concentration (X = 0.5), the fluorescent domain covered most of GUV surface, which was surrounded by gel-like domains. Differential scanning calorimetry of multilamellar vesicles prepared in the presence of higher Cer concentration (X ≥ 0.5) suggested existence of a Cer-enriched gel phase. Video microscopy showed that the enzymatic conversion of SM to Cer caused rapid change in the domain structure: several minutes after the SMase addition, the fluorescent region spread over the GUV surface, within which regions with darker contrast existed. Image-based measurement of generalized polarization (GP) of 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan), which is related to the acyl chain ordering of the lipids, was performed. Before the SMase treatment domains with high (0.65) and low (below 0.4) GP values coexisted, presumably reflecting the liquid-ordered and disordered domains; after the SMase treatment regions with intermediate GP values (0.5) and smaller regions with higher GP values (0.65) were present. Generation of Cer thus caused a phase transition from liquid-ordered and disordered phases to a gel and liquid phase.     

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.     

Single GUV method reveals interaction of tea catechin (-)-epigallocatechin gallate with lipid membranes

Tea catechins, which are flavonoids and the main components of green tea extracts, are thought to have antibacterial and antioxidant activity. Several studies indicate that lipid membranes are one of the targets of the antibacterial activity of catechins. Studies using a suspension of large unilamellar vesicles (LUVs) indicate that catechin causes gradual leakage of internal contents from LUVs. However, the detailed characteristics of the interaction of catechins with lipid membranes remain unclear. In this study, we investigated the interaction of (-)-epigallocatechin gallate (EGCg), a major catechin in tea extract, with single giant unilamellar vesicles (GUVs) of egg phosphatidylcholine (egg PC) using phase-contrast fluorescence microscopy and the single GUV method. We prepared GUVs of lipid membranes of egg PC in a physiological ion concentration ( approximately 150 mM NaCl) using the polyethylene glycol-lipid method. Low concentrations of EGCg at and above 30 muM induced rapid leakage of a fluorescent probe, calcein, from the inside of single egg PC-GUVs; after the leakage, the GUVs changed into small lumps of lipid membranes. On the other hand, phase-contrast microscopic images revealed the detailed process of the EGCg-induced burst of GUVs, the decrease in their diameter, and their transformation into small lumps. The dependence of the fraction of burst GUVs on EGCg concentration was almost the same as that of the fraction of leaked GUV. This correlation strongly indicates that the leakage of calcein from the inside to the outside of the GUV occurred as a result of the burst of the GUV. The fraction of completely leaked GUV and the fraction of the burst GUV increased with time and also increased with increasing EGCg concentration. We compared the EGCg-induced leakage from single GUVs with EGCg-induced leakage from a LUV suspension. The analysis of the EGCg-induced shape changes shows that the binding of EGCg to the external monolayer of the GUV increases its membrane area, inducing an increase in its surface pressure. Small angle x-ray scattering experiments indicate that the intermembrane distance of multilamellar vesicles of PC membrane greatly decreased at EGCg concentrations above the threshold, suggesting that neighboring membranes came in close contact with each other. On the basis of these results, we discuss the mechanism of the EGCg-induced bursting of vesicles.     

The influence of NBD fluorescent probe on model membranes containing POPC and DPPC

To investigate the effect of fluorescent probe on the properties of membranes, we studied model membranes composed of 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl 2-oleoyl-sn-glycero-3-phosphocholine (POPC) in the presence and absence of fluorescent probe. The morphology of giant unilamellar vesicles (GUVs) has been observed as a function of temperature and composition by fluorescence microscopy using NBD-DOPE or C₆-NBD-PC as the probe. The phase behavior of model membranes containing no fluorescent probe was investigated by ²H-NMR spectroscopy. We found that the bright phase observed on GUVs was the fluid phase enriched in POPC and the dark phase was the gel phase enriched in DPPC. NBD-DOPE and C₆-NBD-PC preferentially participated in the fluid-phase domains when GUVs were in the gel?+?fluid phase coexistence. Inclusion of both fluorescent probes (1?mol%) lowered the transition temperature of POPC/DPPC membranes. In addition, C₆-NBD-PC exhibited a stronger effect than NBD-DOPE, which was considered to be associated with the structures of fluorescent molecules.     

Encapsulation of Nucleic Acids into Giant Unilamellar Vesicles by Freeze-Thaw: a Way Protocells May Form

Protocells are believed to consist of a lipid membrane and encapsulated nucleic acid. As the lipid membrane is impermeable to macromolecules like nucleic acids, the processes by which nucleic acids become encapsulated inside lipid membrane compartments are still unknown. In this paper, a freeze-thaw method was modified and applied to giant unilamellar vesicles (GUVs) and deoxyribonucleic acid (DNA) in mixed solution resulting in the efficient encapsulation of 6.4 kb plasmid DNA and similar length linear DNA into GUVs. The mechanism of encapsulation was followed by observing the effect of freeze-thaw temperatures on GUV morphological change, DNA encapsulation and ice crystal formation, and analyzing their correlation. Following ice crystal formation, the shape of spherical GUVs was altered and membrane integrity was damaged and this was found to be a necessary condition for encapsulation. Heating alone had no effects on DNA encapsulation, but was helpful for restoring the spherical shape and membrane integrity of GUVs damaged during freezing. These results suggested that freeze-thaw could promote the encapsulation of DNA into GUVs by a mechanism: the vesicle membrane was breached by ice crystal formation during freezing, DNA entered into damaged GUVs through these membrane gaps and was encapsulated after the membrane was resealed during the thawing process. The process described herein therefore describes a simple way for the encapsulation of nucleic acids and potentially other macromolecules into lipid vesicles, a process by which early protocells might have formed.     

Preparation of giant unilamellar vesicles from damp lipid film for better lipid compositional uniformity

Giant unilamellar vesicles (GUVs) containing cholesterol often have a wide distribution in lipid composition. In this study, GUVs of 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC)/1,2-distearoyl-sn-glycero-3-phosphocholine(DSPC)/cholesterol and 1,2-diphytanoyl-sn-glycero-3-phosphocholine(diPhyPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine(DPPC)/cholesterol were prepared from dry lipid films using the standard electroformation method as well as a modified method from damp lipid films, which are made from compositional uniform liposomes prepared using the Rapid Solvent Exchange (RSE) method. We quantified the lipid compositional distributions of GUV by measuring the miscibility transition temperature of GUVs using fluorescence microscopy, since a narrower distribution in the transition temperature should correspond to a more uniform distribution in GUV lipid composition. Cholesterol molecules can demix from other lipids in dry state and form cholesterol crystals. Using optical microscopy, micron-sized crystals were observed in some dry lipid films. Thus, a major cause of GUV lipid compositional heterogeneity is the demixing of lipids in the dry film state. By avoiding the dry film state, GUVs prepared from damp lipid films have a better uniformity in lipid composition, and the standard deviations of miscibility transition temperature are about 2.5 times smaller than that of GUVs prepared from dry lipid films. Comparing the two ternary systems, diPhyPC/DPPC/cholesterol GUVs has a larger cholesterol compositional heterogeneity, which directly correlates with the low maximum solubility of cholesterol in diPhyPC lipid bilayers (40.2±0.5mol%) measured by light scattering. Our data indicate that cholesterol interacts far less favorably with diPhyPC than it does with other PCs. The damp lipid film method also has a potential of preparing GUVs from cell membranes containing native proteins without going through a dry state.     

Elementary processes of antimicrobial peptide PGLa-induced pore formation in lipid bilayers

Antimicrobial peptide PGLa induces the leakage of intracellular content, leading to its bactericidal activity. However, the elementary process of PGLa-induced leakage remains poorly understood. Here, we examined the interaction of PGLa with lipid bilayers using the single giant unilamellar vesicle (GUV) method. We found that PGLa induced membrane permeation of calcein from GUVs comprised of dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylglycerol (DOPG) and its rate increased with time to reach a steady value, indicating that PGLa induced pores in the bilayer. The binding of PGLa to the GUV membrane raised its fractional area change, δ. At high PGLa concentrations, the time course of δ showed a two-step increase; δ increased to a value, δ₁, which was constant for an extended period before increasing to another constant value, δ₂, that persisted until aspiration of the GUV. To reveal the distribution of PGLa, we investigated the interaction of a mixture of PGLa and carboxyfluorescein (CF) -labeled PGLa (CF-PGLa) with single GUVs. The change of the fluorescence intensity of the GUV rim, I, over time showed a two-step increase from a steady value, I₁, to another, I₂, concomitant with the entering of CF-PGLa into the lumen of the GUV prior to AF647 leakage. The simultaneous measurement of δ and I indicated that their time courses were virtually the same and the ratios (δ₂/δ₁ and I₂/I₁) were almost 2. These results indicated that CF-PGLa translocated across the bilayer before membrane permeation. Based on these results, the elementary processes of the PGLa-induced pore formation were discussed.     

Micropipette Aspiration‐Based Assessment of Single Channel Water Permeability

Measurements of the unitary hydraulic conductivity of membrane channels, p_f, may be hampered by difficulties in producing sufficient quantities of purified and reconstituted proteins. Low yield expression, the purely empiric choice of detergents, as well as protein aggregation and misfolding during reconstitution may result in an average of less than one reconstituted channel per large unilamellar vesicle. This limits their applicability for p_f measurements, independent of whether light scattering or fluorescence quenching of encapsulated dyes is monitored. Here the micropipette aspiration technique is adopted because its superb sensitivity allows resolving p_f values for one order of magnitude smaller protein densities in sphingomyelin and cholesterol rich giant unilamellar vesicles (GUVs). Protein density is derived from intensity fluctuations that fluorescently labeled channels in the aspirated GUV induce by diffusing through the diffraction limited spot. A perfusion system minimizes unstirred layers in the immediate membrane vicinity as demonstrated by the distribution of both encapsulated and extravesicular aqueous dyes. p_f amounted to 2.4 ± 0.1 × 10^?13 cm³ s^?1 for aquaporin‐1 that served as a test case. The new assay paves the way for directly monitoring the effect that interaction of aquaporins with other proteins or inhibitors may have on p_f on a single sample.     

The dynamics of giant unilamellar vesicle oxidation probed by morphological transitions

We have studied the dynamics of Lissamine Rhodamine B dye sensitization-induced oxidation of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) giant unilamellar vesicles (GUVs), where the progression of the underlying chemical processes was followed via vesicle membrane area changes. The surface-area-to-volume ratio of our spherical GUVs increased after as little as ten seconds of irradiation. The membrane area expansion was coupled with high amplitude fluctuations not typical of GUVs in isoosmotic conditions. To accurately measure the area of deformed and fluctuating membranes, we utilized a dual-beam optical trap (DBOT) to stretch GUV membranes into a geometrically regular shape. Further oxidation led to vesicle contraction, and the GUVs became tense, with micron-scale pores forming in the bilayer. We analyzed the GUV morphological behaviors as two consecutive rate-limiting steps. We also considered the effects of altering DOPC and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (RhDPPE) concentrations. The resulting kinetic model allows us to measure how lipid molecular area changes during oxidation, as well as to determine the rate constants controlling how quickly oxidation products are formed. Controlled membrane oxidation leading to permeabilization is also a potential tool for drug delivery based on engineered photosensitizer-containing lipid vesicles.     

Characterization of phase separation phenomena in hybrid lipid/block copolymer/cholesterol bilayers using laurdan fluorescence with log-normal multipeak analysis

Phase separation phenomena in hybrid lipid/block copolymer/cholesterol bilayers combining polybutadiene-block-polyethylene oxide (PBdPEO), egg sphingomyelin (egg SM), and cholesterol were studied with fluorescence spectroscopy and microscopy for comparison to lipid bilayers composed of palmitoyl oleoyl phosphatidylcholine (POPC), egg SM, and cholesterol. Laurdan emission spectra were decomposed into three lognormal curves. The temperature dependence of the ratios of the areas of the middle and lowest energy peaks revealed temperature break-point (T_break) values that were in better agreement, compared to generalized polarization inflection temperatures, with phase transition temperatures in giant unilamellar vesicles (GUVs). Agreement between GUV and spectroscopy results was further improved for hybrid vesicles by using the ratio of the area of the middle peak to the sum of the areas all three peaks to find the T_break values. For the hybrid vesicles, trends at T_break are hypothesized to be correlated with the mechanisms by which the phase transition takes place, supported by the compositional range as well as the morphologies of domains observed in GUVs. Low miscibility of PBdPEO and egg SM is suggested by the finding of relatively high T_break values at cholesterol contents greater than 30 mol%. Further, GUV phase behavior suggests stronger partitioning of cholesterol into PBdPEO than into POPC, and less miscibility of PBdPEO than POPC with egg SM. These results, summarized using a heat-map, contribute to the limited body of knowledge regarding the effect of cholesterol on hybrid membranes, with potential application toward the development of such materials for drug delivery or membrane protein reconstitution.     

Lipid packing determines protein-membrane interactions: Challenges for apolipoprotein A-I and high density lipoproteins

Protein and protein-lipid interactions, with and within specific areas in the cell membrane, are critical in order to modulate the cell signaling events required to maintain cell functions and viability. Biological bilayers are complex, dynamic platforms, and thus in vivo observations usually need to be preceded by studies on model systems that simplify and discriminate the different factors involved in lipid-protein interactions. Fluorescence microscopy studies using giant unilamellar vesicles (GUVs) as membrane model systems provide a unique methodology to quantify protein binding, interaction, and lipid solubilization in artificial bilayers. The large size of lipid domains obtainable on GUVs, together with fluorescence microscopy techniques, provides the possibility to localize and quantify molecular interactions. Fluorescence Correlation Spectroscopy (FCS) can be performed using the GUV model to extract information on mobility and concentration. Two-photon Laurdan Generalized Polarization (GP) reports on local changes in membrane water content (related to membrane fluidity) due to protein binding or lipid removal from a given lipid domain. In this review, we summarize the experimental microscopy methods used to study the interaction of human apolipoprotein A-I (apoA-I) in lipid-free and lipid-bound conformations with bilayers and natural membranes. Results described here help us to understand cholesterol homeostasis and offer a methodological design suited to different biological systems.     

Profilin Interaction with Phosphatidylinositol (4,5)-Bisphosphate Destabilizes the Membrane of Giant Unilamellar Vesicles

Profilin, a small cytoskeletal protein, and phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P₂] have been implicated in cellular events that alter the cell morphology, such as endocytosis, cell motility, and formation of the cleavage furrow during cytokinesis. Profilin has been shown to interact with PI(4,5)P₂, but the role of this interaction is still poorly understood. Using giant unilamellar vesicles (GUVs) as a simple model of the cell membrane, we investigated the interaction between profilin and PI(4,5)P₂. A number and brightness analysis demonstrated that in the absence of profilin, molar ratios of PI(4,5)P₂ above 4% result in lipid demixing and cluster formations. Furthermore, adding profilin to GUVs made with 1% PI(4,5)P₂ leads to the formation of clusters of both profilin and PI(4,5)P₂. However, due to the self-quenching of the dipyrrometheneboron difluoride-labeled PI(4,5)P₂, we were unable to determine the size of these clusters. Finally, we show that the formation of these clusters results in the destabilization and deformation of the GUV membrane.