FtsZ Reorganization Facilitates Deformation of Giant Vesicles in Microfluidic Traps**

The geometry of reaction compartments can affect the local outcome of interface-restricted reactions. Giant unilamellar vesicles (GUVs) are commonly used to generate cell-sized, membrane-bound reaction compartments, which are, however, always spherical. Herein, we report the development of a microfluidic chip to trap and reversibly deform GUVs into cigar-like shapes. When trapping and elongating GUVs that contain the primary protein of the bacterial Z ring, FtsZ, we find that membrane-bound FtsZ filaments align preferentially with the short GUV axis. When GUVs are released from this confinement and membrane tension is relaxed, FtsZ reorganizes reversibly from filaments into dynamic rings that stabilize membrane protrusions; a process that allows reversible GUV deformation. We conclude that microfluidic traps are useful for manipulating both geometry and tension of GUVs, and for investigating how both affect the outcome of spatially-sensitive reactions inside them, such as that of protein self-organization.     

Controlled microfluidic encapsulation of cells, proteins, and microbeads in lipid vesicles

Cells have been encapsulated inside lipid vesicles by using a new microfluidic lipid vesicle formulation technique. Lipid vesicles are formulated within minutes without using toxic lipid solvents. The encapsulation efficiency inside the vesicles is controlled by the microfluidic flows. Green fluorescent proteins (GFP), carcinoma cells, and bead encapsulated vesicles have mean diameters of 27.2 mum, 62.4 mum, and 55.9 mum, respectively. The variations of vesicle sizes are approximately 20% for the GFP and cell encapsulated vesicles and approximately 10% for the bead encapsulated vesicles.     

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.     

Hollow giant lipid vesicles prepared by coaxially electrospraying solutions of phospholipid and degradable polyelectrolyte

Hollow giant lipid vesicles were prepared in a single step by coaxially electrospraying separate solutions of phospholipid and a degradable polyelectrolyte. We synthesized a hydrolytically degradable cationic polyelectrolyte, poly(β-amino esters) (PBAE), and employed it as a degradable microgel template to form giant vesicles. Droplets of the phospholipid solution and the degradable polyelectrolyte solution were electrosprayed from coaxial double needles into a receiving solution. The PBAE formed a microgel by crosslinking with multivalent anions in the receiving solution, and the phospholipids formed bilayers on the microgel. Hollow giant lipid vesicles were successfully obtained and the mean diameters were 7.6 μm (C.V. 58 %). Substrates (calcein, dextran, and polymeric microparticles) were successfully encapsulated in the giant vesicles. Microscopic observations of microparticle mobility inside a giant vesicle indicated the fluidity of its aqueous interior. Investigations using fluorescently labeled PBAE also suggested the degradation of PBAE, and the release of fluorescent PBAE fragments from the encapsulated microgel, to form hollow giant lipid vesicles.     

In Situ Vesicle Formation by Native Chemical Ligation

Phospholipid vesicles are of intense fundamental and practical interest, yet methods for their de novo generation from reactive precursors are limited. A non‐enzymatic and chemoselective method to spontaneously generate phospholipid membranes from water‐soluble starting materials would be a powerful tool for generating vesicles and studying lipid membranes. Here we describe the use of native chemical ligation (NCL) to rapidly prepare phospholipids spontaneously from thioesters. While NCL is one of the most popular tools for synthesizing proteins and nucleic acids, to our knowledge this is the first example of using NCL to generate phospholipids de novo. The lipids are capable of in situ synthesis and self‐assembly into vesicles that can grow to several microns in diameter. The selectivity of the NCL reaction makes in situ membrane formation compatible with biological materials such as proteins. This work expands the application of NCL to the formation of phospholipid membranes.     

Vesicle deformations by clusters of transmembrane proteins

We carry out a coarse-grained molecular dynamics simulation of phospholipid vesicles with transmembrane proteins. We measure the mean and Gaussian curvatures of our protein-embedded vesicles and quantitatively show how protein clusters change the shapes of their host vesicles. The effects of depletion force and vesiculation on protein clustering are also investigated. By increasing the protein concentration, clusters are fragmented to smaller bundles, which are then redistributed to form more symmetric structures corresponding to lower bending energies. Big clusters and highly aspherical vesicles cannot be formed when the fraction of protein to lipid molecules is large.     

pH对磷脂膜中Slc11a1跨膜区二级结构和取向的影响

采用圆二色光谱、 荧光光谱、 红外衰减全反射光谱和差示扫描量热分析等方法对不同pH条件下膜蛋白Slc11a1(溶质转运蛋白家族11成员1)的第二、 第三和第四跨膜区(TMD2~TMD4)在磷脂膜[二肉豆蔻酰磷脂酰胆碱(DMPC)和二肉豆蔻酰磷脂酰甘油(DMPG)的摩尔比为2∶1]中的二级结构和取向进行了研究. 结果表明, TMD3的二级结构及在磷脂膜内的位置与pH密切相关, 在pH=7时TMD3主要为β股结构, 在膜中埋入较浅; 而在pH=5.5时TMD3形成部分α螺旋结构, 并较深地埋入膜中. 对TMD3进行E139A突变后的结果证明, TMD3的这些性质与位于中间的谷氨酸的质子化性质密切相关. 实验结果还表明, TMD2和TMD4在不同pH条件下都形成α螺旋结构并分别以26°和35°的倾斜角插入磷脂膜内, 它们在磷脂膜内的位置基本不受pH影响.     

Properties of mixed lipid monolayers assembled on hydrophobic surfaces through vesicle adsorption

Supported lipid films are becoming increasingly important tools for the study of membrane protein function because of the availability of high-sensitivity surface analytical and patterning techniques. In this study, we have characterized the physical chemical properties of lipid films assembled on hydrophobic surfaces through the spontaneous adsorption of large unilamellar lipid vesicles composed of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC). The density of the lipid films was measured with surface plasmon resonance spectroscopy as the lipid composition of the vesicles and ionic concentration were varied. As expected, monolayer films were formed, but the density of the monolayers was found to be weakly dependent on the lipid composition of the vesicles and strongly dependent on the ionic concentration of the solution in contact with the monolayer. Atomic force microscopy (AFM) images of the lipid films indicate that they are composed of a homogeneous monolayer. Surface force measurements were used to determine the surface charge and DOPG density of the monolayers. The DOPG content of the films was found to be weakly dependent on the DOPG composition of the vesicles and strongly dependent on the salt concentration of the environment. A model has been developed to describe the behavior of the lipid composition of the films in terms of the hydrophobic, electrostatic, and steric forces acting on the lipid monolayer on the hydrophobic surface.     

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.     

A photo-responsive cholesterol capable of inducing a morphological transformation of the liquid-ordered microdomain in lipid bilayers

An azobenzene-modified cholesterol was designed and synthesized for photo-induced domain transformation in lipid bilayer membranes. Upon UV-light irradiation, the cholesterol derivative changes the conformation through photoisomerization of the azobenzene moiety from trans- to cis-form. The photoisomerization effectively occurred both in liquid-ordered (L_o) and liquid-disordered (L_d) phases. Phase-contrast and fluorescence microscopic observation revealed that photoisomerization of the azobenzene-modified cholesterol induced the shape transformation of giant unilamellar vesicle (GUV) and the reorganization of L_o domain structure. Such a photo-induced transformation of lipid domain gave two different pathways dependent on the lipid composition of GUV; disappearance of the L_o domain or appearance of a small L_d domain with in the L_o domain.     

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.     

Lateral organization of a membrane protein in a supported binary lipid domain: direct observation of the organization of bacterial light-harvesting complex 2 by total internal reflection fluorescence microscopy

A unique method is described for directly observing the lateral organization of a membrane protein (bacterial light-harvesting complex LH2) in a supported lipid bilayer using total internal reflection fluorescence (TIRF) microscopy. The supported lipid bilayer consisted of anionic 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1'-glycerol)] (DOPG) and 1,2-distearoly-sn-3-[phospho-rac-(1'-glycerol)] (DSPG) and was formed through the rupture of a giant vesicle on a positively charged coverslip. TIRF microscopy revealed that the bilayer was composed of phase-separated domains. When a suspension of cationic phospholipid (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine: EDOPC) vesicles (approximately 400 nm in diameter), containing LH2 complexes (EDOPC/LH2 = 1000/1), was put into contact with the supported lipid bilayer, the cationic vesicles immediately began to fuse and did so specifically with the fluid phase (DOPG-rich domain) of the supported bilayer. Fluorescence from the incorporated LH2 complexes gradually (over approximately 20 min) spread from the domain boundary into the gel domain (DSPG-rich domain). Similar diffusion into the domain-structured supported lipid membrane was observed when the fluorescent lipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lissamine-rhodamine B sulfonyl: N-Rh-DOPE) was incorporated into the vesicles instead of LH2. These results indicate that vesicles containing LH2 and lipids preferentially fuse with the fluid domain, after which they laterally diffuse into the gel domain. This report describes for first time the lateral organization of a membrane protein, LH2, via vesicle fusion and subsequent lateral diffusion of the LH2 from the fluid to the gel domains in the supported lipid bilayer. The biological implications and applications of the present study are briefly discussed.     

SOME RELATIONSHIPS BETWEEN ULTRAVIOLET LIGHT AND HEME-PROTEIN-INDUCED PEROXIDATIVE LIPID BREAKDOWN IN LIPOSOMES, AS REFLECTED BY FLUORESCENCE CHANGES: THE EFFECT OF NEGATIVE SURFACE CHARGE

Abstract— The water soluble, photolabile nitrene precursor,azidonaphthalene–2,7-disulfonic acid (ANDS) was encapsulated in small unilamellar, isoelectric (egg PC) or negatively charged (egg PC + dihexadecylphosphate) liposomes. The individual and combined effects of heme-proteins and UV irradiation on the fluorescence of these vesicles under aerobic conditions were studied. Consistent with the catalytic action of heme-proteins on lipid peroxidation and peroxide decomposition, addition of cytochrome c (positively charged) or catalase (negatively charged) to the vesicles elicited immediate formation of a fluorescence band at 470 nm, characteristic of Schiff bases that form from aldehyde byproducts of decomposing hydroperoxides. Ultraviolet irradiation of liposomes for 5 min caused no significant changes in the fluorescence spectrum, in spite of the radiolysis of ANDS inside the vesicles with consequent formation of nitrene radicals. When isoelectric vesicles were irradiated with UV light in the presence of cytochrome c or catalase, Schiff base formation was further increased by2–3 fold, which effect was not observed in the absence of internal ANDS, or in the presence of negative surface charge on the vesicles. These findings suggest that (a) UV irradiation, by itself, cannot trigger lipid decomposition even when it is assisted by photoproduced nitrene radicals, (b) there is a ternary synergism between UV light, heme-proteins and nitrene radicals in promoting peroxidative lipid breakdown, and (c) negative surface charge inhibits the above synergism, which effect is unlikely to be due to electrostatic interaction between the vesicles and the protein or the ANDS.     

Effect of cholesterol or phospholipid incorporation on the chemical stability of the muramyldpeptide derivative B30-MDP in mixed vesicles

Application of the muramyldipeptide derivative B30-MDP to liposomal vaccines will aid in the development of improved high immunogenicity vaccines. To give full play to the effectiveness of B30-MDP as a liposomal vaccine, it is important to evaluate the effect of cholesterol, dimyristoylphosphatidylcholine (DMPC) or distearoylphosphatidylcholine (DSPC) incorporation on the chemical stability of B30-MDP and physicochemical properties of B30-MDP/lipid mixed vesicles from the view point of pharmaceutics.The observed degradation rate constants of B30-MDP by hydrolysis in B30-MDP/cholesterol mixed vesicles were increased with increasing concentration of cholesterol, however, those in B30-MDP/DMPC and B30-MDP/DSPC mixed vesicles were unchanged with increasing concentration of DMPC and DSPC. The degradation behavior of B30-MDP was then compared with physicochemical properties of B30-MDP/lipid mixed vesicles, such as membrane fluidity and particle size. It was apparent that the degradation of B30-MDP in B30-MDP/cholesterol mixed vesicles was influenced by the particle size, but not by the fluidity of the membranes. In the case of B30-MDP/phospholipid mixed vesicles, MDP/phospholipid mixed vesicles, the degradation of B30-MDP was not influenced by either the membranes' fluidity or the particle size of the mixed vesicles.It is considered that the degradation of B30-MDP in the mixed vesicles is dependent on the membrane state, and the addition of cholesterol to B30-MDP vesicle inhibits the mutual interaction of MDP regions, whereas the addition of phospholipids hardly influences the mutual interaction of MDP regions, possibly owing to phase separation between B30-MDP and phospholipids.     

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.     

Interaction of cationic lipid vesicles with model cell membranes--as determined by neutron reflectivity

Transfection of cells by DNA (for the purposes of gene therapy) can be effectively engineered through the use of cationic lipid/DNA "lipoplexes", although the transfection efficiency of these lipoplexes is sensitive to the neutral "helper" lipid included. Here, neutron reflectivity has been used to investigate the role of the helper lipid present during the interaction of cationic lipid vesicles with model cell membranes. Dimethyldioctadecylammonium bromide (DDAB) vesicles were formed with two different helper lipids, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) and cholesterol, and the interaction of these vesicles with a supported phospholipid bilayer was determined. DOPE-containing vesicles were found to interact faster with the membrane than those containing cholesterol, and vesicles containing either of the neutral helper lipids were found to interact faster than when DDAB alone was present. The interaction between the vesicles and the membrane was characterized by an exchange of lipid between the membrane and the lipid aggregates in solution; the deposition of vesicle bilayers on the surface of the membrane was not apparent.     

Detecting phase transitions in phosphatidylcholine vesicles by Raman microscopy and self-modeling curve resolution

The study of phospholipid phase transitions is important for understanding drug- and protein-membrane interactions as well as other phenomena such as trans-membrane diffusion and vesicle fusion. A temperature-controlled stage on a confocal Raman microscope has allowed phase transitions in optically trapped phospholipid vesicles to be monitored. Raman spectra were acquired and analyzed using self-modeling curve resolution, a multivariate statistical analysis technique. This method revealed the subtle spectral changes indicative of sub- and pretransitions and main transitions in vesicles composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). The Raman scattering results were compared to differential scanning calorimetry (DSC) experiments and found to be in good agreement. This method of observing lipid phase transition profiles requires little sample preparation and a minimal amount of lipid (相似文献   

Noncovalent immobilized artificial membrane chromatography, an improved method for describing peptide-lipid bilayer interactions.

A promising approach in assessing hydrophobic peptide-membrane interactions is the use of reversed-phase high-performance liquid chromatography. The present study describes the preparation and properties of a noncovalent immobilized artificial membrane (noncovalent IAM) stationary phase. The noncovalent IAM phase was prepared by coating the C18 chains of a reversed-phase HPLC column with the phospholipid ditetradecanoyl-sn-glycero-3-phosphocholine. Lipid coating was achieved by pumping a lipid solution in water-2-propanol through the column. The formation of a bilayer-like structure on the chromatographic surface was confirmed by calculating the phospholipid surface density of the stationary phase. The surface density was determined to be approximately 1.95 mumol m-2, which is close to that of lipid vesicles. The coating was found to be stable in chromatographic elution systems containing less than 35% of acetonitrile. Employing this new technique, we determined interaction parameters of a set of helical antibacterial magainin-2-amide peptides with pairwise substitutions of adjacent amino acids by their D-enatiomers. The results demonstrate that the chromatographic retention behavior of peptides on noncovalent IAM stationary phase shows an excellent correlation with lipid affinities to phospholipid vesicles.     

Laminar order within Langmuir-Blodgett multilayers from phospholipid and myelin basic protein: a neutron reflectivity study

Multilayers consisting of negatively charged phospholipid DMPA and myelin basic protein (MBP) were assembled by Langmuir-Blodgett deposition of floating Langmuir monolayers from the air/water interface to solid substrates. Protein/lipid samples were obtained by binding MBP from the aqueous subphase to the phospholipid monolayers before deposition. The vertical organization of these model membranes (i.e., with organization perpendicular to the substrate surface) was investigated in detail by neutron reflectivity measurements, and the internal distribution of water molecules was determined from the change of contrast after in-situ H2O/D2O exchange. The multilayers were well ordered, with repeating lipid bilayers as fundamental structural unit. MBP was inserted in between adjacent lipid headgroups, such as in the natural myelin membrane. Water molecules in the multilayers were present mainly in the lipid headgroup and protein slab. On exposition of the pure lipid multilayers to a dry atmosphere, a reduction of the bilayer spacing was determined, whereas the global lamellar order was not affected. In contrast, drying of the protein/lipid multilayers induced degradation of the laminar order. The data demonstrate that ordered Langmuir-Blodgett multilayers are versatile model systems for studying how competing interactions between lipid, protein, water, and ions affect the global organization of such multilamellar lipid/protein assemblies. Here, the water molecules were found to be a necessary mediator to maintain the laminar order in a multilayer from DMPA and myelin basic protein.     

Mechanical Characterization of Micrometric Chitosan-Coated Vesicle by Atomic Force Microscopy

This article reports the main results obtained for the stabilization of DOPC liposomes by coating with chitosan. We investigated the compression between two planes of isolated GUV, combining force spectroscopy measurements with scanning probe microscopy (SPM) and epifluorescence microscopy observations. We obtained reproducible and reversible force-deformation curves for individual vesicles in the range of small deformation (relative deformation up to 0.3). Force-deformation curves were analyzed using a simple elastic model, which well describes the observed radius-dependency of the force response and allows determining the stretching modulus for bare vesicle and effective bending and stretching moduli for chitosan-coated membranes. Results first show that chitosan coating increases the effective stretching modulus of the lipid membranes, second, confirm that chitosan is adsorbed flat on the membrane, and finally lead us to assume that the chitosan coating structure can be regarded as a physical network of connected chitosan patches.