Membrane fusion of giant unilamellar vesicles of neutral phospholipid membranes induced by La3+

Membrane fusions of vesicles of biomembranes play various important roles in cells, but their mechanisms are unclear and controversial. In the present study, we found that 30 microM to 1 mM La3+ induced membrane fusion of two giant unilamellar vesicles (GUVs) composed of a mixture of dioleoylphosphatidylcholine (DOPC) and dipalmitoleoylphosphatidylethanolamine (DPOPE). We succeeded in observing a process of this membrane fusion in detail. First, two GUVs became strongly associated, with a partition membrane between them composed of two bilayers, one from each GUV. Then, the partition membrane was suddenly broken at one site on its edge. The area of this breakage site gradually spread, until it was completely separated from the GUV to complete the membrane fusion. Here, we propose a new model (i.e., the partition breakage model) for the mechanism of La3+ -induced membrane fusion of GUVs.     

Vesicle fission of giant unilamellar vesicles of liquid-ordered-phase membranes induced by amphiphiles with a single long hydrocarbon chain

Vesicle fissions are very important processes of biomembranes in cells, but their mechanisms are not clear and are controversial. Using the single giant unilamellar vesicle (GUV) method, we recently found that low concentrations (less than the critical micelle concentration (CMC)) of lysophosphatidylcholine (lyso-PC) induced the vesicle fission of GUVs of dipalmitoylphosphatidylcholine/cholesterol(6/4) (DPPC/chol(6/4)) membranes and sphingomyelin/cholesterol membranes (6/4) in the liquid-ordered (lo) phase. In this report, to elucidate its mechanism, we have investigated the effect of low concentrations (much less than their CMC) of other amphiphiles with a single long hydrocarbon chain (i.e., single long chain amphiphiles) on DPPC/chol(6/4) GUVs as well as the effect of the membrane composition on the lyso-PC-induced vesicle fission. We found that low concentrations of single long chain amphiphiles (lyosophosphatidic acid, octylglucoside, and sodium dodecyl sulfate) induced the shape change from a prolate to two spheres connected by a very narrow neck, indicating that the single long chain amphiphiles can be partitioned into the external monolayer in the lo phase of the GUV from the aqueous solution. As the single long chain amphiphile concentrations were increased, all of them induced vesicle fission of DPPC/chol(6/4) GUVs above their threshold concentrations. To elucidate the role of cholesterol in the single long chain amphiphile-induced vesicle fission, we investigated the effect of lyso-PC on GUVs of dioleoyl-PC (DOPC)/chol(6/4) membranes in the Lalpha phase; no vesicle fission occurred, indicating that cholesterol in itself did not play an important role in the vesicle fission. Finally, to elucidate the effect of the inclusion of DOPC in the lo-phase membrane of GUVs on the lyso-PC-induced vesicle fission of the DPPC/chol(6/4) GUV, we investigated the effect of low concentrations of lyso-PC on GUVs of DPPC/DOPC/chol membranes. With an increase in DOPC concentration in the membrane, the threshold concentration of lyso-PC increased. At and above 30 mol % DOPC, no vesicle fission occurred. On the basis of these results, we have proposed a hypothesis of the mechanism of the single long chain amphiphile-induced vesicle fission of a GUV of a lo-phase membrane.     

Controlling the internal structure of giant unilamellar vesicles by means of reversible temperature dependent sol-gel transition of internalized poly(N-isopropyl acrylamide)

In this work, we present preparation and basic applications of lipid-bilayer-enclosed picoliter volumes (microcontainers) of solutions of poly(N-isopropylacrylamide) (PNIPAAm). Giant unilamellar vesicles (GUVs) were prepared from phospholipids using a standard swelling procedure and subsequently surface immobilized. Clear, slightly viscous solutions of PNIPAAm of varying concentration in aqueous buffer were directly pressure-microinjected into the GUVs, using a submicrometer-sized, pointed capillary. The GUV was subjected to changing temperature over a 21-40 degrees C range. The typical phase transition of the polymeric material upon heating and cooling across the lower critical solution temperature was followed using optical microscopy and shown to be reversible over multiple sequential heating/cooling cycles without compromising the integrity of the GUV membrane. Fluorescent, carboxylic acid modified 200 nm latex beads, co-injected with the PNIPAAm solution, were temperature-reversibly immobilized during the phase transition, practically freezing the Brownian motion of the entrapped particles in the volume. Furthermore, a co-injected water soluble fluorescent polysaccharide-dye conjugate was shown not to migrate from the aqueous phase into the hydrophobic polymer part upon heating, whereas the fluorescent beads were completely but reversibly immobilized in the hydrophobic domains of dense polymer agglomerates. The system reported here provides a feasible method for the reversible stabilization and solidification of GUV interior volumes, e.g., as a micrometer-sized model system for controlled drug release.     

Hydrogen-bond-directed giant unilamellar vesicles of guanosine derivative: preparation, properties, and fusion

By mixing a small volume of THF containing guanosine derivative 1 and tetraethylenegrycol dodecyl ether (TEGDE) with water and subsequently removing TEGDE by gel permeation chromatography, micrometer-sized giant unilamellar vesicles (GUV) of 1 were successfully prepared. The vesicle membrane was a 2-D sheet assembly of thickness 2.5 nm, composed of a 2-D inter-guanine hydrogen-bond network. The GUV dispersion showed high stability because of a large negative zeta potential, which allowed repeated sedimentation and redispersion by centrifugation and subsequent gentle agitation. TEGDE-triggered fusion of GUVs took place within 350 ms, which proceeded by fusion of the vesicle membranes in contact. These unique static and dynamic properties of the GUV membrane assembled by the 2-D hydrogen-bond network are discussed.     

Rapid screening of membrane protein activity: electrophysiological analysis of OmpF reconstituted in proteoliposomes

Solvent-free planar lipid bilayers were formed in an automatic manner by bursting of giant unilamellar vesicles (GUVs) after gentle suction application through micron-sized apertures in a borosilicate glass substrate. Incubation of GUVs with the purified ion channel protein of interest yielded proteoliposomes. These proteoliposomes allow for immediate recording of channel activity after GUV sealing. This approach reduces the time-consuming, laborious and sometimes difficult protein reconstitution processes normally performed after bilayer formation. Bilayer recordings are attractive for investigations of membrane proteins not accessible to patch clamp analysis, like e.g. proteins from organelles. In the presented work, we show the example of the outer membrane protein OmpF from Escherichia coli. We reconstituted OmpF in proteoliposomes and observed the characteristic trimeric conductance levels and the typical gating induced by pH and transmembrane voltage. Moreover, OmpF is the main entrance for beta-lactam antibiotics and we investigated translocation processes of antibiotics and modulation of OmpF by spermine. We suggest that the rapid formation of porin containing lipid bilayers is of potential for the efficient electrophysiological characterization of the OmpF protein, for studying membrane permeation processes and for the rapid screening of antibiotics.     

Shape changes and vesicle fission of giant unilamellar vesicles of liquid-ordered phase membrane induced by lysophosphatidylcholine

Liquid-ordered phase (lo phase) of lipid membranes has properties that are intermediate between those of liquid-crystalline phase and those of gel phase and has attracted much attention in both biological and biophysical aspects. Rafts in the lo phase in biomembranes play important roles in cell function of mammalian cells such as signal transduction. In this report, we have prepared giant unilamellar vesicles (GUVs) of lipid membranes in the lo phase and investigated their physical properties using phase-contrast microscopy and fluorescence microscopy. GUVs of dipalmitoyl-phosphatidylcholine (DPPC)/cholesterol membranes and also GUVs of sphingomyelin (SM)/cholesterol membranes in the lo phase in water were formed at 20-37 degrees C successfully, when these membranes contained >/=30 mol % cholesterol. The diameters of GUVs of DPPC/cholesterol and SM/cholesterol membranes did not change from 50 to 28 degrees C, supporting that the membranes of these GUVs were in the lo phase. To elucidate the interaction of a substance with a long hydrocarbon chain with the lo phase membrane, we investigated the interaction of low concentrations (less than critical micelle concentration) of lysophosphatidylcholine (lyso-PC) with DPPC/cholesterol GUVs and SM/cholesterol GUVs in the lo phase. We found that lyso-PC induced several shape changes and vesicle fission of these GUVs above their threshold concentrations in water. The analysis of these shape changes indicates that lyso-PC can be partitioned into the external monolayer in the lo phase of the GUV from the aqueous solution. Threshold concentrations of lyso-PC in water to induce the shape changes and vesicle fission increased greatly with a decrease in chain length of lyso-PC. Thermodynamic analysis of this result indicates that shape changes and vesicle fission occur at threshold concentrations of lyso-PC in the membrane. These new findings on GUVs of the lo phase membranes indicate that substances with a long hydrocarbon chain such as lyso-PC can enter into the lo phase membrane and also the raft in the cell membrane. We have also proposed a mechanism for the lyso-PC-induced vesicle fission of GUVs.     

Freezing or wrapping: the role of particle size in the mechanism of nanoparticle-biomembrane interaction

Understanding the interactions between nanoparticles (NPs) and biological matter is a high-priority research area because of the importance of elucidating the physical mechanisms underlying the interactions leading to NP potential toxicity as well as NP viability as therapeutic vectors in nanomedicine. Here, we use two model membrane systems, giant unilamellar vesicles (GUVs) and supported monolayers, to demonstrate the competition between adhesion and elastic energy at the nanobio interface, leading to different mechanisms of NP-membrane interaction relating to NP size. Small NPs (18 nm) cause a "freeze effect" of otherwise fluid phospholipids, significantly decreasing the phospholipid lateral mobility. The release of tension through stress-induced fracture mechanics results in a single microsize hole in the GUVs after interaction. Large particles (>78 nm) promote membrane wrapping, which leads to increased lipid lateral mobility and the eventual collapse of the vesicles. Electrochemical impedance spectroscopy on the supported monolayer model confirms that differently sized NPs interact differently with the phospholipids in close proximity to the electrode during the lipid desorption process. The time scale of these processes is in accordance with the proposed NP/GUV interaction mechanism.     

多肽诱导的巨型脂质体出芽和泄露行为

细胞膜与膜蛋白之间的相互作用与生命中许多过程息息相关.以巨型脂质体(GUV)和多肽分别作为细胞膜和膜蛋白的简化模型,我们设计了四种仅包含亮氨酸(L)和赖氨酸(K)的多肽,即K₁₄、(KL₂KL₂K)₂、(KL₂KL₃)₂和K₆L₈,并对比研究了它们与中性和负电性脂质体的相互作用.电荷密度最高的K₁₄只是涂层在脂质体表面,不破其囊泡结构,但能够引起负电性脂质体发生微相分离,属建设性相互作用.能够形成两亲性α螺旋的(KL₂KL₂K)₂和(KL₂KL₃)₂则引起脂质体发生泄露和破裂,属破坏性作用.但二者引起泄露的速率在中性脂质体和负电性脂质体中的结果恰好相反,说明泄露分两步进行:表面吸附多肽达到一定浓度,继而对膜进行干扰.表面活性剂型多肽K₆L₈的氨基酸组成与(KL₂KL₂K)₂相同,但K₆L₈只是引起负电性脂质体发生泄露,造成中性脂质体发生外出芽.这些简单氨基酸造成的脂质体的复杂构象变化可以统一用静电和疏水相互作用在膜上的位置和强度来进行解释.这些结论对于深入理解膜蛋白的作用机理是有帮助的.     

Electroformation in a flow chamber with solution exchange as a means of preparation of flaccid giant vesicles

A recently described technique [Estes and Mayer, Biochim. Biophys. Acta 1712 (2005) 152-160] for the preparation of giant unilamellar vesicles (GUVs) in solutions with high ionic strength is examined. By observing a series of osmotic swellings followed by vesicle bursts upon a micropipette transfer of a single POPC GUV from a sucrose solution into an iso-osmolar glycerol solution, a value for the permeability of POPC membrane for glycerol, P=(2.09+/-0.82) x 10(-8)m/s, has been obtained. Based on this result, an alternative mechanism is proposed for the observed exchange of vesicle interior. With modifications, the method of Estes and Mayer is then applied to preparation of flaccid GUVs.     

Protein Patterns and Oscillations on Lipid Monolayers and in Microdroplets

The Min proteins from E.coli position the bacterial cell‐division machinery through pole‐to‐pole oscillations. In vitro, Min protein self‐organization can be reconstituted in the presence of a lipid membrane as a catalytic surface. However, Min dynamics have so far not been reconstituted in fully membrane‐enclosed volumes. Microdroplets interfaced by lipid monolayers were employed as a simple 3D mimic of cellular compartments to reconstitute Min protein oscillations. We demonstrate that lipid monolayers are sufficient to fulfil the catalytic role of the membrane and thus represent a facile platform to investigate Min protein regulated dynamics of the cell‐division protein FtsZ‐mts. In particular, we show that droplet containers reveal distinct Min oscillation modes, and reveal a dependence of FtsZ‐mts structures on compartment size. Finally, co‐reconstitution of Min proteins and FtsZ‐mts in droplets yields antagonistic localization, thus demonstrating that droplets indeed support the analysis of complex bacterial self‐organization in confined volumes.     

Transport dynamics in open microfluidic grooves

In microscopic rectangular grooves various liquid wetting morphologies can be found, depending on the wettability and details of the geometry. When these morphologies are combined with a method to vary the apparent contact angle reversibly, transitions between droplike objects and elongated liquid filaments can be induced. Liquid can thus be transported on demand along the grooves. The dynamics of liquid filaments advancing into grooves as well as receding from grooves has been studied, varying the contact angle using the electrowetting effect. The dynamics of the receding filament is purely capillarity driven and depends only on the contact angle, the viscosity of the liquid, and the geometry of the groove. The length and the dynamics of the advancing filaments, on the other hand, are strongly dependent on the ionic content of the liquid and the applied ac voltage.     

Active membrane viscoelasticity by the bacterial FtsZ-division protein

At the early stages of the division process in Escherichia coli, the protein FtsZ forms a septal ring at the midcell. This Z-ring causes membrane constriction during bacterial division. The Z-ring associates to the lipid membrane through several membrane proteins, ZipA among them. Here, a simplified FtsZ-ZipA model was reconstituted onto Langmuir monolayers based in E. coli polar lipid extract. Brewster angle and atomic force microscopy have revealed membrane FtsZ-polymerization upon GTP hydrolysis. The compression viscoelasticity of these monolayers has been also investigated. The presence of protein induced softening and fluidization with respect to the bare lipid membrane. An active mechanism, based on the internal forces stressed by FtsZ filaments and transduced to the lipid membrane by ZipA, was suggested to underlie the observed behavior.     

UV-induced bursting of cell-sized multicomponent lipid vesicles in a photosensitive surfactant solution

We study the behavior of multicomponent giant unilamellar vesicles (GUVs) in the presence of AzoTAB, a photosensitive surfactant. GUVs are made of an equimolar ratio of dioleoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC) and various amounts of cholesterol (Chol), where the lipid membrane shows a phase separation into a DPPC-rich liquid-ordered (L(o)) phase and a DOPC-rich liquid-disordered (L(d)) phase. We find that UV illumination at 365 nm for 1 s induces the bursting of a significant fraction of the GUV population. The percentage of UV-induced disrupted vesicles, called bursting rate (Y(burst)), increases with an increase in [AzoTAB] and depends on [Chol] in a non-monotonous manner. Y(burst) decreases when [Chol] increases from 0 to 10 mol % and then increases with a further increase in [Chol], which can be correlated with the phase composition of the membrane. We show that Y(burst) increases with the appearance of solid domains ([Chol] = 0) or with an increase in area fraction of L(o) phase (with increasing [Chol] ≥ 10 mol %). Under our conditions (UV illumination at 365 nm for 1 s), maximal bursting efficiency (Y(burst) = 53%) is obtained for [AzoTAB] = 1 mM and [Chol] = 40 mol %. Finally, by restricting the illumination area, we demonstrate the first selective UV-induced bursting of individual target GUVs. These results show a new method to probe biomembrane mechanical properties using light as well as pave the way for novel strategies of light-induced drug delivery.     

Crystalline protein domains and lipid bilayer vesicle shape transformations

Cellular membranes can take on a variety of shapes to assist biological processes including endocytosis. Membrane-associated protein domains provide a possible mechanism for determining membrane curvature. We study the effect of tethered streptavidin protein crystals on the curvature of giant unilamellar vesicles (GUVs) using confocal, fluorescence, and differential interference contrast microscopy. Above a critical protein concentration, streptavidin domains align and percolate as they form, deforming GUVs into prolate spheroidal shapes in a size-dependent fashion. We propose a mechanism for this shape transformation based on domain growth and jamming. Osmotic deflation of streptavidin-coated GUVs reveals that the relatively rigid streptavidin protein domains resist membrane bending. Moreover, in contrast to highly curved protein domains that facilitate membrane budding, the relatively flat streptavidin domains prevent membrane budding under high osmotic stress. Thus, crystalline streptavidin domains are shown to have a stabilizing effect on lipid membranes. Our study gives insight into the mechanism for protein-mediated stabilization of cellular membranes.     

Efficient electroformation of supergiant unilamellar vesicles containing cationic lipids on ITO-coated electrodes

Giant unilamellar vesicles (GUVs) represent a versatile in vitro system widely used to study properties of lipid membranes and their interaction with biomacromolecules and colloids. Electroformation with indium tin oxide (ITO) coated coverslips as electrodes is a standard approach to GUV production. In the case of cationic GUVs, however, application of this approach leads to notorious difficulties. We discover that this is related to aging of ITO-coated coverslips during their repeated use, which is reflected in their surface topography on the nanoscale. We find that mild annealing of the ITO-coated surface in air reverts the effects of aging and ensures efficient reproducible electroformation of supergiant (diameter > 100 μm) unilamellar vesicles containing cationic lipids.     

Point‐to‐Plane Nonhomogeneous Electric‐Field‐Induced Simultaneous Formation of Giant Unilamellar Vesicles (GUVs) and Lipid Tubes

It is well‐known that homogeneous electric fields can be used to generate giant unilamellar vesicles (GUVs). Herein we report an interesting phenomenon of formation of GUVs and lipid tubes simultaneously using a nonhomogeneous electric field generated by point‐to‐plane electrodes. The underlying mechanism was analyzed using finite element analysis. The two forces play main roles, that is, the pulling force (F) to drag GUVs into lipid tubes induced by fluid flow, and the critical force (Fc) to prevent GUVs from deforming into lipid tubes induced by electric fields. In the center area underneath the needle electrode, the GUVs were found because F is less than Fc in that region, whereas in the edge area the lipid tubes were obtained because F is larger than Fc. The diffusion coefficient of lipid in the tubes was found to be 4.45 μm² s^?1 using a fluorescence recovery after photobleaching (FRAP) technique. The method demonstrated here is superior to conventional GUV or lipid tube fabrication methods, and has great potential in cell mimic or hollow material fabrication using GUVs and tubes as templates.     

Preparation of solvent-free, pore-spanning lipid bilayers: modeling the low tension of plasma membranes

Plasma membrane tension, produced by the underlying cytoskeleton, governs many dynamic processes such as fusion, blebbing, exo- and endocytosis, cell migration, and adhesion. Here, a new protocol is introduced to model this intricate and often overlooked aspect of the plasma membrane. Lipid bilayers spanning pores of 600 nm radius were prepared by adsorption and spreading of giant unilamellar vesicles (GUVs) on moderately hydrophilic porous substrates prepared by gold-coating and subsequent self-assembly of a mercaptoethanol monolayer. Rupture of GUVs formed tens of micrometer sized pore-spanning membrane patches displaying low tension of σ ≤ 3.5 mN m(-1) and lateral diffusion constants of about 8 μm(2) s(-1). Site-specific force indentation experiments were performed to determine membrane tension as a function of lipid composition: for pure DOPC bilayers, a tension of 1.018 ± 0.014 mN m(-1) was measured, which was increased by the addition of cholesterol to 3.50 ± 0.15 mN m(-1). Compared to DOPC, POPC bilayers displayed a larger tension of 2.00 ± 0.09 mN m(-1). Addition and subsequent partitioning of 2-propanol was shown to significantly reduce the membrane tension as a function of its concentration.     

A microfluidic device for kinetic optimization of protein crystallization and in situ structure determination

The unprecedented economies of scale and unique mass transport properties of microfluidic devices made them viable nano-volume protein crystallization screening platforms. However, realizing the full potential of microfluidic crystallization requires complementary technologies for crystal optimization and harvesting. In this paper, we report a microfluidic device which provides a link between chip-based nanoliter volume crystallization screening and structure analysis through "kinetic optimization" of crystallization reactions and in situ structure determination. Kinetic optimization through systematic variation of reactor geometry and actuation of micromechanical valves is used to screen a large ensemble of kinetic trajectories that are not practical with conventional techniques. Using this device, we demonstrate control over crystal quality, reliable scale-up from nanoliter volume reactions, facile harvesting and cryoprotectant screening, and protein structure determination at atomic resolution from data collected in-chip.     

An experimental approach for direct observation of the interaction of polyanions with sphingosine-containing giant vesicles

A new approach for direct optical microscopy observation of polyanion interactions with bilayers of giant cationic liposomes (GUVs) was suggested. Polyanions as DNA, dextran sulfate (DS), heparin (H) and polyacrylic acids (PA) were locally delivered by a micropipette to a part of a giant unilamellar vesicle membrane. The phenomena were directly observed under optical microscope. GUVs, about 100 micro m in diameter, formed of phosphatidylcholines and up to 33 mol% of the natural bioactive cationic amphiphile sphingosine (Sph), were prepared by electroformation. The effects of water-soluble molecules with high negative linear charge density as dextran sulfate (DS), heparin (H) polyacrylic acids (PA) and adenosine-5'-triphosphoric acid (ATP) were compared with those of DNAs. The resulting membrane topology transformations were monitored in phase contrast, while the DNA distribution was followed in fluorescence. DNA-induced endocytosis-like membrane morphology transformation due to the DNA/lipid membrane local interactions was observed. The DS, H and PA induced membrane topology transformations similar to those of the DNAs, while ATP did not cause any detectable ones. The endocytosis mechanism involves the formation of ordered domains in the GUV membrane where some surface and charge asymmetries between the two membrane monolayers were created. The sizes of created polyanionic/cationic membrane domains depend on the form, length and elasticity of the adsorbed highly charged molecules. Endosome-including capacities of polyanionic molecules depend heavily on the high linear negative charge at a certain length.An original method for direct studying of the DNA/membrane interactions in autoadaptable giant liposome system imitating biological membrane interactions was forwarded. The model observations could also help for understanding events associated with cationic liposome/DNA complex formation in gene transfer processes.