Thermal induced modification of the contact mechanics of adhering liposomes on cationic substrate

The correlation between the mechanical property and the thermotropic transition of the phospholipid bilayer has been recently demonstrated (Chem. Phys. Lipids 110 (2001) 27). However, the role of thermal induced mechanical responses of phospholipid bilayer on the contact mechanics of liposome adhering on a cationic substrate has not been determined. In this study, confocal-reflectance interference contrast microscopy, phase contrast microscopy and contact mechanics modeling are applied to probe the adhesion mechanisms of liposomes in the presence of electrostatic interactions during the thermotropic transition of the lipid bilayer. When temperature increases from 23 to 49 °C at pH 7.4, the degree of liposome deformation (a/R) and adhesion energy of dipalmitoyl-sn-glycero-3-phosphocholine liposome increases by 10% and remains constant, respectively, on 3-amino-propyl-triethoxy-silane (APTES) modified substrate. The extents of increase in these two parameters are highly dependent on the physicochemical properties of the rigid substrate. At pH 4, the adhesion energies above and below the phase transition temperature (T_m) are increased by one order of magnitude due to the formation of the free silanol groups on APTES substrate. In hypotonic condition, the degree of vesicle deformation remains constant and the adhesion energy reduces by 20% during sample heating. Under all conditions, the adhesion energy of the adhering liposome spans a few orders of magnitude against the increase of liposome size as the surface area to volume ratio is maximized in smallest vesicle.     

Mechanics of curved plasma membrane vesicles: resting shapes, membrane curvature, and in-plane shear elasticity

Highly curved cell membrane structures, such as plasmalemmal vesicles (caveolae) and clathrin-coated pits, facilitate many cell functions, including the clustering of membrane receptors and transport of specific extracellular macromolecules by endothelial cells. These structures are subject to large mechanical deformations when the plasma membrane is stretched and subject to a change of its curvature. To enhance our understanding of plasmalemmal vesicles we need to improve the understanding of the mechanics in regions of high membrane curvatures. We examine here, theoretically, the shapes of plasmalemmal vesicles assuming that they consist of three membrane domains: an inner domain with high curvature, an outer domain with moderate curvature, and an outermost flat domain, all in the unstressed state. We assume the membrane properties are the same in these domains with membrane bending elasticity as well as in-plane shear elasticity. Special emphasis is placed on the effects of membrane curvature and in-plane shear elasticity on the mechanics of vesicle during unfolding by application of membrane tension. The vesicle shapes were computed by minimization of bending and in-plane shear strain energy. Mechanically stable vesicles were identified with characteristic membrane necks. Upon stretch of the membrane, the vesicle necks disappeared relatively abruptly leading to membrane shapes that consist of curved indentations. While the resting shape of vesicles is predominantly affected by the membrane spontaneous curvatures, the membrane shear elasticity (for a range of values recorded in the red cell membrane) makes a significant contribution as the vesicle is subject to stretch and unfolding. The membrane tension required to unfold the vesicle is sensitive with respect to its shape, especially as the vesicle becomes fully unfolded and approaches a relative flat shape.     

The Vesicle Trafficking Protein Sar1 Lowers Lipid Membrane Rigidity

The sculpting of membranes into dynamic, curved shapes is central to intracellular cargo trafficking. Though the generation of membrane curvature during trafficking necessarily involves both lipids and membrane-associated proteins, current mechanistic views focus primarily on the formation of rigid cages and curved scaffolds by protein assemblies. Here we report on a different mechanism for the control of membrane deformation, unrelated to the imposition of predefined curvature, involving modulation of membrane material properties: Sar1, a GTPase that regulates vesicle trafficking from the endoplasmic reticulum, lowers the rigidity of the lipid bilayer membrane to which it binds. In vitro assays in which optically trapped microspheres create controlled membrane deformations revealed a monotonic decline in bending modulus as a function of Sar1 concentration, down to nearly zero rigidity, indicating a dramatic lowering of the energetic cost of curvature generation. This is the first demonstration that a vesicle trafficking protein lowers the rigidity of its target membrane, leading to a new conceptual framework for vesicle biogenesis.     

The Mechanism of a Nuclear Pore Assembly: A Molecular Biophysics View

The basic problem of nuclear pore assembly is the big perinuclear space that must be overcome for nuclear membrane fusion and pore creation. Our investigations of ternary complexes: DNA–PC liposomes–Mg²⁺, and modern conceptions of nuclear pore structure allowed us to introduce a new mechanism of nuclear pore assembly. DNA-induced fusion of liposomes (membrane vesicles) with a single-lipid bilayer or two closely located nuclear membranes is considered. After such fusion on the lipid bilayer surface, traces of a complex of ssDNA with lipids were revealed. At fusion of two identical small liposomes (membrane vesicles) <100 nm in diameter, a “big” liposome (vesicle) with ssDNA on the vesicle equator is formed. ssDNA occurrence on liposome surface gives a biphasic character to the fusion kinetics. The “big” membrane vesicle surrounded by ssDNA is the base of nuclear pore assembly. Its contact with the nuclear envelope leads to fast fusion of half of the vesicles with one nuclear membrane; then ensues a fusion delay when ssDNA reaches the membrane. The next step is to turn inside out the second vesicle half and its fusion to other nuclear membrane. A hole is formed between the two membranes, and nucleoporins begin pore complex assembly around the ssDNA. The surface tension of vesicles and nuclear membranes along with the kinetic energy of a liquid inside a vesicle play the main roles in this process. Special cases of nuclear pore formation are considered: pore formation on both nuclear envelope sides, the difference of pores formed in various cell-cycle phases and linear nuclear pore clusters.     

Determination of osmotic volumes and pH gradients of plant membrane and lipid vesicles using ESR spectroscopy

Volumes and pH gradients were determined with spin probes in liposomes and zucchini membrane vesicles by quantitating the internal concentrations of probes in the presence of an impermeable line-broadening agent, manganese + EDTA. Volume shrinkage in response to increasing external concentrations of MnEDTA was consistent with perfect osmotic behavior of both vesicle populations. Buffer additions were used to impose pH gradients on the vesicles; liposome gradients measured with a spin-labeled weak acid were slightly smaller than the maximum theoretical imposed gradients, whereas above a threshold magnitude, measured gradients for the plant membranes were significantly smaller than imposed gradients. However, the residual pH gradient in the zucchini vesicles decreased at about the same rate as the liposome gradient. Moreover, this residual gradient was not completely collapsed in the presence of the proton ionophore, FCCP, indicating that the vesicles were impermeable to ions; indeed, ion permeabilities of both vesicle preparations appeared to be similar during the slow phase of the pH gradient collapse. Thus, zucchini membrane vesicles are tightly sealed and appear to have a mechanism for dissipating pH gradients rapidly when these gradients exceed some threshold value.     

EFFECT OF ELECTRICAL STIMULATION ON THE YIELD AND COMPOSITION OF SYNAPTIC VESICLES FROM THE CHOLINERGIC SYNAPSES OF THE ELECTRIC ORGAN OF TORPEDO: A COMBINED BIOCHEMICAL, ELECTROPHYSIOLOGICAL AND MORPHOLOGICAL STUDY

Abstract— The effect of stimulating the electric organ of Torpedo marmorata , anaesthetized with 0.01% Tricaine methane sulphonate, by means of electrical stimulation (5/s) administered via an electrode placed on the electric lobe has been studied electrophysiologically, biochemically and morphologically. The response of the organ declined to about 50 per cent of its initial value after about 500 stimuli, by a further 10 per cent after another 500 stimuli and then to about 12 per cent of the initial value after a further 1000 stimuli. Thereafter the response fell off progressively. However, even when the response was less than 1 per cent of its initial value, the organ had considerable powers of recuperation during a 30-s rest period, to 30–50 per cent of its initial value.
The fall in response was accompanied by a reduction in vesicle size and number, an increase in the area of the presynaptic membrane and a fall in the protein, total nucleotide, ATP and acetylcholine content of the vesicle fraction isolated from the stimulated tissue. However, whereas vesicle numbers and the protein and total nucleotide content of the vesicle fraction fell by only about 50 per cent, vesicular ATP and acetylcholine levels were reduced to about 10 per cent. An analysis of the covariance of vesicular ATP and acetylcholine showed an initial loss of an acetylcholine-rich (relative to ATP) population of vesicles. The early loss of vesicular protein and nucleotide and vesicle numbers as well as the morphological changes seen would be consistent with a loss of vesicles due to fusion with the external membrane. The preferential loss of acetylcholine and ATP from the vesicle fraction indicates that the vesicles surviving the stimulation procedure have been utilized in a number of cycles causing the progressive fall in vesicle volume, and acetylcholine and ATP content.     

On the interaction between chromaffin granule membranes and intragranular vesicles--theory and analysis of freeze-fracture micrographs

We present a model for the calculation of intragranular vesicle adhesion energy in a two-vesicle system consisting of an external secretory vesicle (chromaffin granule) and an intragranular vesicle (IGV) that adheres from the inside to the granule membrane. The geometrical parameters characterizing the granule-IGV systems were derived from freeze-fracture electron micrographs. Adhesion is brought about by incubation of the granules in hyperosmolar sucrose solutions. It is accompanied by a deformation of the granule because the intragranular vesicle bulges it outwards, and by segregation of intramembraneous particles from the adherent part of the granule membrane. Adhesion prevents the deformed granules from osmotic reexpansion and, therefore, causes hyperosmotic relaxation lysis. We estimated specific adhesion energy at -3 erg/cm2, a value which is 10 - 1000 times larger than the energy of van der Waals interaction between membranes. This large interaction energy probably results from changes of the granule core induced by dehydration. A minimization of the interface between the granule core and adjacent membranes could exclude intragranular vesicles from the core and squeeze them towards the granule membrane. This might induce a new kind of interaction between both membranes, which is irreversible and causes lysis upon osmotic relaxation.     

Endophilin BAR domain drives membrane curvature by two newly identified structure-based mechanisms

The crescent-shaped BAR (Bin/Amphiphysin/Rvs-homology) domain dimer is a versatile protein module that senses and generates positive membrane curvature. The BAR domain dimer of human endophilin-A1, solved at 3.1 A, has a unique structure consisting of a pair of helix-loop appendages sprouting out from the crescent. The appendage's short helices form a hydrophobic ridge, which runs across the concave surface at its center. Examining liposome binding and tubulation in vitro using purified BAR domain and its mutants indicated that the ridge penetrates into the membrane bilayer and enhances liposome tubulation. BAR domain-expressing cells exhibited marked plasma membrane tubulation in vivo. Furthermore, a swinging-arm mutant lost liposome tubulation activity yet retaining liposome binding. These data suggested that the rigid crescent dimer shape is crucial for the tubulation. We here propose that the BAR domain drives membrane curvature by coordinate action of the crescent's scaffold mechanism and the ridge's membrane insertion in addition to membrane binding via amino-terminal amphipathic helix.     

The deformation of spherical vesicles with permeable, constant-area membranes: application to the red blood cell

The deformation of an initially spherical vesicle of radius a with a permeable membrane under extensive forces applied at its poles is calculated as a function of the in-plane shear modulus, H, and the out-of-plane bending modulus, B, using an axisymmetric theory that is valid for large deformations. Suitably nondimensionalized, the results depend upon a single nondimensional parameter, C identical with a(2)H/B. For small deformations, the calculated force-polar strain curves are linear and, under these conditions, the slope of the curve determines only C, not the values of H and B separately. Independent determination of H and B from experimental measurements require deformations that are large enough to produce nonlinear behavior. Simple approximations for large and small C are given, which are applied to experimental measurements on red blood cell ghosts that have been made permeable by treatment with saponin.     

Synaptotagmin C2A loop 2 mediates Ca2+-dependent SNARE interactions essential for Ca2+-triggered vesicle exocytosis

Synaptotagmins contain tandem C2 domains and function as Ca(2+) sensors for vesicle exocytosis but the mechanism for coupling Ca(2+) rises to membrane fusion remains undefined. Synaptotagmins bind SNAREs, essential components of the membrane fusion machinery, but the role of these interactions in Ca(2+)-triggered vesicle exocytosis has not been directly assessed. We identified sites on synaptotagmin-1 that mediate Ca(2+)-dependent SNAP25 binding by zero-length cross-linking. Mutation of these sites in C2A and C2B eliminated Ca(2+)-dependent synaptotagmin-1 binding to SNAREs without affecting Ca(2+)-dependent membrane binding. The mutants failed to confer Ca(2+) regulation on SNARE-dependent liposome fusion and failed to restore Ca(2+)-triggered vesicle exocytosis in synaptotagmin-deficient PC12 cells. The results provide direct evidence that Ca(2+)-dependent SNARE binding by synaptotagmin is essential for Ca(2+)-triggered vesicle exocytosis and that Ca(2+)-dependent membrane binding by itself is insufficient to trigger fusion. A structure-based model of the SNARE-binding surface of C2A provided a new view of how Ca(2+)-dependent SNARE and membrane binding occur simultaneously.     

包封膜蛋白的仿生细胞膜

本文概述了脂质囊泡的组成成分和制作方法以及用于膜蛋白方面研究的相关技术,包括膜蛋白整合到囊泡的方法、复合体系的表征等。脂质囊泡可以为膜蛋白提供类似体内的环境,包括疏水区和内外亲水环境,因其组分单一,可以方便地进行结构、功能、信号转导等方面的研究,因此可以模拟细胞膜作为研究膜蛋白的有力工具,目前大多是以脂质体形态作为仿生囊泡体系进行这方面研究。     

Resistance of liposomes with different physico-chemical characteristics to the effect of plasma

The chemical composition, liquid content sign and value of charge as well as structure and size of lipid vesicles are studied for the effect they exert on the liposome permeability for 22Na+ in the presence of human blood plasma. The rate of the isotope outlet from the electroneutral lecithin liposomes is determined by the size of vesicles and the quantity of phospholipid bilayers in their membrane. The presence either of a negative or a positive charge on the surface of the liposome membrane has no essential effect on the outlet rate of the radioactive marker. Introduction of different amounts of cholesterol or sphingomyelin into the liposome composition decreases considerably the lipid vesicle permeability and an increase in the liquid content of their membranes due to the temperature elevation is accompanied by a sharp rise in the isotope outlet rate. A conclusion is drawn on the possibility to control the outlet rate of the liposome content in the presence of blood plasma.     

Reconstitution into liposomes of the glycoprotein of vesicular stomatitis virus by detergent dialysis.

The glycoprotein of vesicular stomatitis (VS) virus was selectively liberated from the virion membrane by the dialyzable nonionic detergent, beta-D-octylglucoside. The isolated viral glycoprotein could be rendered virtually free of phospholipid and detergent, under which conditions it formed tail-to-tail glycoprotein micelles in the form of rosettes. When mixtures of viral glycoprotein and egg lecithin were dialyzed free of octylglucoside, glycoprotein vesicles formed spontaneously with spikes protruding in the same external orientation as the VS virion membrane. The glycoprotein vesicles exhibited increased and uniform buoyant density, indicating relative homogeneity in the proportion of glycoprotein and phosphatidylcholine in each glycoprotein liposome. Evidence for similar insertion and orientation of VS viral glycoprotein in both phosphatidylcholine vesicles and virion membrane was substantiated by the finding that proteolytic digestion with thermolysin gave rise to hydrophobic glycoprotein tail fragments in vesicle or virion membranes that migrated identically in polyacrylamide gels.     

Membrane fission is promoted by insertion of amphipathic helices and is restricted by crescent BAR domains

Shallow hydrophobic insertions and crescent-shaped BAR scaffolds promote membrane curvature. Here, we investigate membrane fission by shallow hydrophobic insertions quantitatively and mechanistically. We provide evidence that membrane insertion of the ENTH domain of epsin leads to liposome vesiculation, and that epsin is required for clathrin-coated vesicle budding in cells. We also show that BAR-domain scaffolds from endophilin, amphiphysin, GRAF, and β2-centaurin limit membrane fission driven by hydrophobic insertions. A quantitative assay for vesiculation reveals an antagonistic relationship between amphipathic helices and scaffolds of N-BAR domains in fission. The extent of vesiculation by these proteins and vesicle size depend on the number and length of amphipathic helices per BAR domain, in accord with theoretical considerations. This fission mechanism gives a new framework for understanding membrane scission in the absence of mechanoenzymes such as dynamin and suggests how Arf and Sar proteins work in vesicle scission.     

Research Advances on Stimuli-responsive Liposomes in the Targeted Drug Delivery System

Liposome, a kind of nanoscale vesicle, is applied in the drug delivery systems (DDS) extensively because of its low toxicity, biodegradability and biocompatibility. However, defects of liposome drugs, such as low rates of drug release, insufficiency in active targeting and inefficient bioavailability still remain to be solved. Therefore, stimuli-responsive liposomes are brought to DDS to improve the efficacy of controlled drug release, assure specific release in targeted sites and alleviate side-effects as much as possible. Stimuli-responsive liposomes could maintain stability in circulation, tissues and cells under physiological conditions. Once delivered, they could be activated by relevant internal or external stimuli to release cargos accurately in target areas. This review highlights the design, functional principles and recent advances on application of pH-sensitive liposomes and thermosensitive liposomes respectively, which are two typical stimuli-responsive liposomes. Common targeting modifications of liposomes are discussed as well. We also summarize recent challenges of stimuli-responsive liposomes and their further applications.     

Electromechanical stresses produced in the plasma membranes of suspended cells by applied electric fields

Summary We analyze the electrical and mechanical stress in the bounding membrane of a cell (or vesicle) in suspension which is deformed by an external applied field. The membrane is treated as a thin, elastic, initially spherical, dielectric shell and the analysis is valid for frequencies less than the reciprocal of the charging time (i.e. less than MHz), or for constant fields. A complete analytic solution is obtained, and expressions are given which relate the deformation, the surface tension and the transmembrane potential difference to the applied field. We show that mechanical tensions in the range which lyse membranes are induced at values of the external field which are of the same order as those which are reported to lyse the plasma membranes of cells in suspension.     

The tail domain of tomosyn controls membrane fusion through tomosyn displacement by VAMP2

Neurotransmitter release is regulated by SNARE complex-mediated synaptic vesicle fusion. Tomosyn sequesters target SNAREs (t-SNAREs) through its C-terminal VAMP-like domain (VLD). Cumulative biochemical results suggest that the tomosyn-SNARE complex is so tight that VAMP2 cannot displace tomosyn. Based on these results, the tomosyn-SNARE complex has been believed to be a dead-end complex to inhibit neurotransmitter release. On the other hand, some studies using siRNA depletion of tomosyn suggest that tomosyn positively regulates exocytosis. Therefore, it is still controversial whether tomosyn is a simple inhibitor for neurotransmitter release. We recently reported that the inhibitory activity of tomosyn is regulated by the tail domain binding to the VLD. In this study, we employed the liposome fusion assay in order to further understand modes of action of tomosyn in detail. The tail domain unexpectedly had no effect on binding of the VLD to t-SNARE-bearing liposomes. Nonetheless, the tail domain decreased the inhibitory activity of the VLD on the SNARE complex-mediated liposome fusion. These results indicate that the tail domain controls membrane fusion through tomosyn displacement by VAMP2. Deletion of the tail domain-binding region in the VLD retained the binding to t-SNAREs and promoted the liposome fusion. Together, we propose here a novel mechanism of tomosyn that controls synaptic vesicle fusion positively by serving as a placeholder for VAMP2.     

Interaction of a peptide derived from glycoprotein gp36 of feline immunodeficiency virus and its lipoylated analogue with phospholipid membranes

P59, a 20-mer peptide modeled on the membrane-proximal external region (MPER) of the feline immunodeficiency virus (FIV) gp36 ectodomain, has potent antiviral activity. The lipoylated analogue, lipo-P59, displays a similar activity, which is preferentially retained by cellular substrates. A mechanism has been proposed recently in which the peptide, being positioned on the surface of the cell membrane, inhibits its fusion with the virus; the lipophilic chain of lipo-P59 is thought to insert into the membrane interior, thus anchoring the peptide at the surface. In the present work, lipid-peptide interactions of P59 and lipo-P59 with phospholipid liposomes are investigated using spin-label electron spin resonance spectroscopy. Two phospholipids have been examined, the zwitterionic dimyristoyl phosphatidylcholine and the anionic dimyristoyl phosphatidylglycerol, and a wide range of lipid spin labels, including positional isomers. Independent of the membrane charge, both peptides bind to lipid bilayers; however, whereas P59 insertion between the lipid headgroups leads to significant liposome destabilization, eventually resulting in vesicle fragmentation with the formation of smaller aggregates, lipo-P59 inserts with the lipophilic tail among the lipid chains, while the peptidic portion remains adsorbed onto the membrane, where it can effectively exert its antiviral activity.     

Coupling field theory with continuum mechanics: a simulation of domain formation in giant unilamellar vesicles

Domain formation is modeled on the surface of giant unilamellar vesicles using a Landau field theory model for phase coexistence coupled to elastic deformation mechanics (e.g., membrane curvature). Smooth particle applied mechanics, a form of smoothed particle continuum mechanics, is used to solve either the time-dependent Landau-Ginzburg or Cahn-Hilliard free-energy models for the composition dynamics. At the same time, the underlying elastic membrane is modeled using smooth particle applied mechanics, resulting in a unified computational scheme capable of treating the response of the composition fields to arbitrary deformations of the vesicle and vice versa. The results indicate that curvature coupling, along with the field theory model for composition free energy, gives domain formations that are correlated with surface defects on the vesicle. In the case that external deformations are included, the domain structures are seen to respond to such deformations. The present simulation capability provides a significant step forward toward the simulation of realistic cellular membrane processes.     

Interaction of the 106-126 prion peptide with lipid membranes and potential implication for neurotoxicity

Prion diseases are fatal neurodegenerative disorders characterized by the accumulation in the brain of an abnormally misfolded, protease-resistant, and beta-sheet rich pathogenic isoform (PrP(SC)) of the cellular prion protein (PrP(C)). In the present work, we were interested to study the mode of prion protein interaction with the membrane using the 106-126 peptide and small unilamellar lipid vesicles as model. As previously demonstrated, we showed by MTS assay that PrP 106-126 induces alterations in the human neuroblastoma SH-SY5Y cell line. We demonstrated for the first time by lipid-mixing assay and by the liposome vesicle leakage test that PrP 106-126, a non-tilted peptide, induces liposome fusion thus a potential cell membrane destabilization, as supported by membrane integrity assay (LDH). By circular dichroism (CD) analysis we showed that the fusogenic property of PrP 106-126 in the presence of liposome is associated with a predominantly beta-sheet structure. These data suggest that the fusogenic property associated with a predominant beta-sheet structure exhibited by the prion peptides contributes to the neurotoxicity of these peptides by destabilizing cellular membranes. The latter might be attached at the membrane surface in a parallel orientation as shown by molecular modeling.