Frequency-dependent electrodeformation of giant phospholipid vesicles in AC electric field

A model of vesicle electrodeformation is described which obtains a parametrized vesicle shape by minimizing the sum of the membrane bending energy and the energy due to the electric field. Both the vesicle membrane and the aqueous media inside and outside the vesicle are treated as leaky dielectrics, and the vesicle itself is modeled as a nearly spherical shape enclosed within a thin membrane. It is demonstrated (a) that the model achieves a good quantitative agreement with the experimentally determined prolate-to-oblate transition frequencies in the kilohertz range and (b) that the model can explain a phase diagram of shapes of giant phospholipid vesicles with respect to two parameters: the frequency of the applied alternating current electric field and the ratio of the electrical conductivities of the aqueous media inside and outside the vesicle, explored in a recent paper (S. Aranda et al., Biophys J 95:L19–L21, 2008). A possible use of the frequency-dependent shape transitions of phospholipid vesicles in conductometry of microliter samples is discussed.     

A Monte Carlo study of giant vesicle morphologies in nonequilibrium environments

It is known that giant vesicles undergo dynamic morphological changes when exposed to a detergent. The solubilization process may take multiple pathways. In this work, we identify lipid vesicle shape dynamics before the solubilization of 1,2-dioleoyl-sn-glycero-3-phosphocholine giant vesicles with Triton X-100 (TR) detergent. The violent lipid vesicle dynamics was observed with laser confocal scanning microscopy and was qualitatively explained via a numerical simulation. A three-dimensional Monte Carlo scheme was constructed that emulated the nonequilibrium conditions at the beginning stages of solubilization, accounting for a gradual addition of TR detergent molecules into the lipid bilayers. We suggest that the main driving factor for morphology change in lipid vesicles is the associative tendency of the TR molecules, which induces spontaneous curvature of the detergent inclusions, an intrinsic consequence of their molecular shape. The majority of the observed lipid vesicle shapes in the experiments were found to correspond very well to the numerically calculated shapes in the phase space of possible solutions. The results give an insight into the early stages of lipid vesicle solubilization by amphiphilic molecules, which is nonequilibrium in nature and very difficult to study.     

The effect of salt and pH on block liposomes studied by cryogenic transmission electron microscopy

Recently, we have reported the discovery of block liposomes (BLs), a new class of liquid (chain-melted) vesicles, formed in mixtures of the curvature-stabilizing hexadecavalent cationic lipid MVLBG2, the neutral lipid 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), and water with no added salt. BLs consist of connected spheres, pears, tubes, or rods. Unlike in typical liposome systems, where spherical vesicles, tubular vesicles, and cylindrical micelles are separated on the macroscopic scale, shapes remain connected and are separated only on the nanometer scale within a single BL. Here, we report structural studies of the effect of salt and pH on the BL phase, carried out using differential interference contrast microscopy (DIC) and cryogenic transmission electron microscopy (cryo-TEM). Addition of salt screens the electrostatic interactions; in low-salt conditions, partial screening of electrostatic interactions leads to a shape transition from BLs to bilamellar vesicles, while in the high-salt regime, a shape transition from BLs to liposomes with spherical morphologies occurs. This demonstrates that strong electrostatic interactions are essential for BL formation. Understanding the control of liposome shape evolution is of high interest because such shape changes play an important role in many intracellular processes such as endocytosis, endoplasmatic reticulum-associated vesiculation, vesicle recycling and signaling.     

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.     

Interrogating the role of liposome size in mediating the dynamics of a chromophore in the acyl chain region of a phospholipid bilayer

We have examined the temperature-dependent reorientation dynamics of perylene imbedded in bilayers of 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), where the bilayers exist in the form of unilamellar vesicles. Previous work using 100-nm diameter DMPC vesicles has shown that the phase transition from the gel phase to the fluid phase can be detected using the reorientation dynamics of perylene. In this work we explore the vesicle size dependence of the perylene reorientation dynamics in DMPC vesicles. The size of the vesicles is determined by extrusion and the reorientation dynamics of perylene are measured as a function of vesicle size between 100-nm and 5-microm diameter. We find that, while the phase transition for DMPC is seen in smaller vesicles, perylene becomes insensitive to the phase transition for vesicles larger than ca. 800-nm diameter. We also find a discontinuous change in perylene reorientation dynamics with increasing vesicle size, and we consider this result in the context of the location of perylene within the bilayer.     

Vesicle diffusion close to a membrane: intermembrane interactions measured with fluorescence correlation spectroscopy

The protein machinery controlling membrane fusion (or fission) has been well studied; however, the role of vesicle diffusion near membranes in these critical processes remains unclear. We experimentally and theoretically investigated the dynamics of small vesicles (∼50 nm in diameter) that are diffusing near supported planar bilayers acting as “target” membranes. Using total internal reflection-fluorescence correlation spectroscopy, we examined the validity of theoretical analyses of vesicle-membrane interactions. Vesicles were hindered by hydrodynamic drag as a function of their proximity to the planar bilayer. The population distributions and diffusion kinetics of the vesicles were further affected by changing the ionic strength and pH of the buffer, as well as the lipid composition of the planar membrane. Effective surface charges on neutral bilayers were also analyzed by comparing experimental and theoretical data, and we show the possibility that vesicle dynamics can be modified by surface charge redistribution of the planar bilayer. Based on these results, we hypothesize that the dynamics of small vesicles, diffusing close to biomembranes, may be spatially restricted by altering local physiological conditions (e.g., salt concentration, lipid composition, and pH), which may represent an additional mechanism for controlling fusion (or fission) dynamics.     

An antiserum specific for cholinergic synaptic vesicles from electric organ

Rabbit antisera to highly purified synaptic vesicles from the electric organ of Narcine brasiliensis, an electric ray, reveal a unique population of synaptic vesicle antigens in addition to a population shared with other electric organ membranes. Synaptic vesicle antigens were detected by binding successively rabbit antivesicle serum and radioactive goat anti-rabbit serum. To remove antibodies directed against antigens common to synaptic vesicles and other electric organ fractions, the antivesicle serum was extensively preadsorbed against an electric organ membrane fraction that was essentially free of synaptic vesicles. The adsorbed serum retained 40% of its ability to bind to synaptic vesicles, suggesting that about half of the antigenic determinants are unique. Vesicle antigens were quantified with a radioimmunoassay (RIA) that utilized precipitation of antibody-antigen complexes with Staphylococcus aureus cells. By this assay, the vesicles, detected by their acetylcholine (ACh) content and the antigens detected by the RIA, have the same buoyant density after isopycnic centrifugation of crude membrane fractions on sucrose and glycerol density gradients. The ratio of ACh to antigenicity was constant across the vesicle peaks and was close to that observed for vesicles purified to homogeneity. Even though the vesicles make up only approximately 0.5% of the material in the original homogenate, the ratio of acetylcholine to vesicle antigenicity could still be measured and also was indistinguishable from that of pure vesicles. We conclude that synaptic vesicles contain unique antigenic determinants not present to any measurable extent in other fractions of the electric organ. Consequently, it is possible to raise a synaptic vesicle- specific antiserum that allows vesicles to be detected and quantified. These findings are consistent with earlier immunohistochemical observations of specific antibody binding to motor nerve terminals.     

Stick-and-diffuse and caged diffusion: a comparison of two models of synaptic vesicle dynamics

Two models were recently proposed to enable us to understand the dynamics of synaptic vesicles in hippocampal neurons. In the caged diffusion model, the vesicles diffuse in small circular cages located randomly in the bouton, while in the stick-and-diffuse model the vesicles bind and release from a cellular cytomatrix. In this article, we obtain analytic expressions for the fluorescence correlation spectroscopy (FCS) autocorrelation function for the two models and test their predictions against our earlier FCS measurements of the vesicle dynamics. We find that the stick-and-diffuse model agrees much better with the experiment. We find also that, due to the slow dynamics of the vesicles, the finite experimental integration time has an important effect on the FCS autocorrelation function and demonstrate its effect for the different models. The two models of the dynamics are also relevant to other cellular environments where mobile species undergo slow diffusionlike motion in restricted spaces or bind and release from a stationary substrate.     

Alignment of Synaptic Vesicle Macromolecules with the Macromolecules in Active Zone Material that Direct Vesicle Docking

Synaptic vesicles dock at active zones on the presynaptic plasma membrane of a neuron’s axon terminals as a precondition for fusing with the membrane and releasing their neurotransmitter to mediate synaptic impulse transmission. Typically, docked vesicles are next to aggregates of plasma membrane-bound macromolecules called active zone material (AZM). Electron tomography on tissue sections from fixed and stained axon terminals of active and resting frog neuromuscular junctions has led to the conclusion that undocked vesicles are directed to and held at the docking sites by the successive formation of stable connections between vesicle membrane proteins and proteins in different classes of AZM macromolecules. Using the same nanometer scale 3D imaging technology on appropriately stained frog neuromuscular junctions, we found that ∼10% of a vesicle’s luminal volume is occupied by a radial assembly of elongate macromolecules attached by narrow projections, nubs, to the vesicle membrane at ∼25 sites. The assembly’s chiral, bilateral shape is nearly the same vesicle to vesicle, and nubs, at their sites of connection to the vesicle membrane, are linked to macromolecules that span the membrane. For docked vesicles, the orientation of the assembly’s shape relative to the AZM and the presynaptic membrane is the same vesicle to vesicle, whereas for undocked vesicles it is not. The connection sites of most nubs on the membrane of docked vesicles are paired with the connection sites of the different classes of AZM macromolecules that regulate docking, and the membrane spanning macromolecules linked to these nubs are also attached to the AZM macromolecules. We conclude that the luminal assembly of macromolecules anchors in a particular arrangement vesicle membrane macromolecules, which contain the proteins that connect the vesicles to AZM macromolecules during docking. Undocked vesicles must move in a way that aligns this arrangement with the AZM macromolecules for docking to proceed.     

Electrohydrodynamic Model of Vesicle Deformation in Alternating Electric Fields

We develop an analytical theory to explain the experimentally observed morphological transitions of quasispherical giant vesicles induced by alternating electric fields. The model treats the inner and suspending media as lossy dielectrics, and the membrane as an impermeable flexible incompressible-fluid sheet. The vesicle shape is obtained by balancing electric, hydrodynamic, bending, and tension stresses exerted on the membrane. Our approach, which is based on force balance, also allows us to describe the time evolution of the vesicle deformation, in contrast to earlier works based on energy minimization, which are able to predict only stationary shapes. Our theoretical predictions for vesicle deformation are consistent with experiment. If the inner fluid is more conducting than the suspending medium, the vesicle always adopts a prolate shape. In the opposite case, the vesicle undergoes a transition from a prolate to oblate ellipsoid at a critical frequency, which the theory identifies with the inverse membrane charging time. At frequencies higher than the inverse Maxwell-Wagner polarization time, the electrohydrodynamic stresses become too small to alter the vesicle's quasispherical rest shape. The model can be used to rationalize the transient and steady deformation of biological cells in electric fields.     

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.     

Phospholipid membrane bending as assessed by the shape sequence of giant oblate phospholipid vesicles

Vesicle shape transformations caused by decreasing the difference between the equilibrium areas of membrane monolayers were studied on phospholipid vesicles with small volume to membrane area ratios. Slow transformations of the vesicle shape were induced by lowering of the concentration of lipid monomers in the solution outside the vesicle. The complete sequence of shapes consisted of a string of pearls, and wormlike, starfish, discocyte and stomatocyte shapes. The transformation from discocyte to stomatocyte vesicle shapes was analyzed theoretically to see whether these observations accord with the area difference elasticity (ADE) model. The membrane shape equation and boundary conditions were derived for axisymmetrical shapes for low volume vesicles, part of whose membranes are in contact. Calculated shapes were arranged into a phase diagram. The theory predicts that the transition between discocyte and stomatocyte shapes is discontinuous for relatively high volumes and continuous for low volumes. The calculated shape sequences matched well with the observed ones. By assuming a linear decrease of the equilibrium area difference with time, the ratio between the nonlocal and local bending constants is in agreement with reported values.     

Phospholipid membrane bending as assessed by the shape sequence of giant oblate phospholipid vesicles

Vesicle shape transformations caused by decreasing the difference between the equilibrium areas of membrane monolayers were studied on phospholipid vesicles with small volume to membrane area ratios. Slow transformations of the vesicle shape were induced by lowering of the concentration of lipid monomers in the solution outside the vesicle. The complete sequence of shapes consisted of a string of pearls, and wormlike, starfish, discocyte and stomatocyte shapes. The transformation from discocyte to stomatocyte vesicle shapes was analyzed theoretically to see whether these observations accord with the area difference elasticity (ADE) model. The membrane shape equation and boundary conditions were derived for axisymmetrical shapes for low volume vesicles, part of whose membranes are in contact. Calculated shapes were arranged into a phase diagram. The theory predicts that the transition between discocyte and stomatocyte shapes is discontinuous for relatively high volumes and continuous for low volumes. The calculated shape sequences matched well with the observed ones. By assuming a linear decrease of the equilibrium area difference with time, the ratio between the nonlocal and local bending constants is in agreement with reported values.     

Ultradeformable lipid vesicles can penetrate the skin and other semi-permeable barriers unfragmented. Evidence from double label CLSM experiments and direct size measurements

The stability of various aggregates in the form of lipid bilayer vesicles was tested by three different methods before and after crossing different semi-permeable barriers. First, polymer membranes with pores significantly smaller than the average aggregate diameter were used as the skin barrier model; dynamic light scattering was employed to monitor vesicle size changes after barrier passage for several lipid mixtures with different bilayer elasticities. This revealed that vesicles must adapt their size and/or shape, dependent on bilayer stability and elasto-mechanics, to overcome an otherwise confining pore. For the mixed lipid aggregates with highly flexible bilayers (Transfersomes®), the change is transient and only involves vesicle shape and volume adaptation. The constancy of ultradeformable vesicle size before and after pores penetration proves this. This is remarkable in light of the very strong aggregate deformation during an enforced barrier passage. Simple phosphatidylcholine vesicles, with less flexible bilayers, lack such capability and stability. Conventional liposomes are therefore fractured during transport through a semi-permeable barrier; as reported by other researchers, liposomes are fragmented to the size of a narrow pore if sufficient pressure is applied across the barrier; otherwise, liposomes clog the pores. The precise outcome depends on trans-barrier flux and/or on relative vesicle vs. pore size. Lipid vesicles applied on the skin behave accordingly. Mixed lipid vesicles penetrate the skin if they are sufficiently deformable. If this is the case, they cross inter-cellular constrictions in the organ without significant composition or size modification. To prove this, we labelled vesicles with two different fluorescent markers and applied the suspension on intact murine skin without occlusion. The confocal laser scanning microscopy (CLSM) of the skin then revealed a practically indistinguishable distribution of both labels in the stratum corneum, corroborating the first assumption. To confirm the second postulate, we compared vesicle size in the starting suspension and in the blood after non-invasive transcutaneous aggregate delivery. Size exclusion chromatograms of sera from the mice that received ultradeformable vesicles on the skin were undistinguishable from the results measured with the original vesicle suspension. Taken together, the results support our previous postulate that ultradeformable vesicles penetrate the skin intact, that is, without permanent disintegration.     

The SV2 Protein of Synaptic Vesicles Is a Keratan Sulfate Proteoglycan

Abstract: We have determined that synaptic vesicles contain a vesicle-specific keratan sulfate integral membrane proteoglycan. This is a major proteoglycan in electric organ synaptic vesicles. It exists in two forms on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, i.e., the L form, which migrates like a protein with an M_r of 100, 000, and the H form, with a lower mobility that migrates with an M_r of ∼250, 000. Both forms contain SV2, an epitope located on the cytoplasmic side of the vesicle membrane. In addition to electric organ, we have analyzed the SV2 proteoglycan in vesicle fractions from two other sources, electric fish brain and rat brain. Both the H and L forms of SV2 are present in these vesicles and all are keratan sulfate proteoglycans. Unlike previously studied synaptic vesicle proteins, this proteoglycan contains a marker specific for a single group of neurons. This marker is an antigenically unique keratan sulfate side chain that is specific for the cells innervating the electric organ; it is not found on the synaptic vesicle keratan sulfate proteoglycan in other neurons of the electric fish brain.     

Calcium-vesicles perform active diffusion in the sea urchin embryo during larval biomineralization

Biomineralization is the process by which organisms use minerals to harden their tissues and provide them with physical support. Biomineralizing cells concentrate the mineral in vesicles that they secret into a dedicated compartment where crystallization occurs. The dynamics of vesicle motion and the molecular mechanisms that control it, are not well understood. Sea urchin larval skeletogenesis provides an excellent platform for investigating the kinetics of mineral-bearing vesicles. Here we used lattice light-sheet microscopy to study the three-dimensional (3D) dynamics of calcium-bearing vesicles in the cells of normal sea urchin embryos and of embryos where skeletogenesis is blocked through the inhibition of Vascular Endothelial Growth Factor Receptor (VEGFR). We developed computational tools for displaying 3D-volumetric movies and for automatically quantifying vesicle dynamics. Our findings imply that calcium vesicles perform an active diffusion motion in both, calcifying (skeletogenic) and non-calcifying (ectodermal) cells of the embryo. The diffusion coefficient and vesicle speed are larger in the mesenchymal skeletogenic cells compared to the epithelial ectodermal cells. These differences are possibly due to the distinct mechanical properties of the two tissues, demonstrated by the enhanced f-actin accumulation and myosinII activity in the ectodermal cells compared to the skeletogenic cells. Vesicle motion is not directed toward the biomineralization compartment, but the vesicles slow down when they approach it, and probably bind for mineral deposition. VEGFR inhibition leads to an increase of vesicle volume but hardly changes vesicle kinetics and doesn’t affect f-actin accumulation and myosinII activity. Thus, calcium vesicles perform an active diffusion motion in the cells of the sea urchin embryo, with diffusion length and speed that inversely correlate with the strength of the actomyosin network. Overall, our studies provide an unprecedented view of calcium vesicle 3D-dynamics and point toward cytoskeleton remodeling as an important effector of the motion of mineral-bearing vesicles.     

Magnitude and direction of vesicle dynamics in growing pollen tubes using spatiotemporal image correlation spectroscopy and fluorescence recovery after photobleaching

The delivery of cell wall material and membrane to growing plant cell surfaces requires the spatial and temporal coordination of secretory vesicle trafficking. Given the small size of vesicles, their dynamics is difficult to quantify. To quantitatively analyze vesicle dynamics in growing pollen tubes labeled with the styryl dye FM1-43, we applied spatiotemporal correlation spectroscopy on time-lapse series obtained with high-speed confocal laser scanning microscopy recordings. The resulting vector maps revealed that vesicles migrate toward the apex in the cell cortex and that they accumulate in an annulus-shaped region adjacent to the extreme tip and then turn back to flow rearward in the center of the tube. Fluorescence recovery after photobleaching confirmed vesicle accumulation in the shoulder of the apex, and it revealed that the extreme apex never recovers full fluorescence intensity. This is consistent with endocytotic activity occurring in this region. Fluorescence recovery after photobleaching analysis also allowed us to measure the turnover rate of the apical vesicle population, which was significantly more rapid than the theoretical rate computed based on requirements for new cell wall material. This may indicate that a significant portion of the vesicles delivered to the apex does not succeed in contacting the plasma membrane for delivery of their contents. Therefore, we propose that more than one passage into the apex may be needed for many vesicles before they fuse to the plasma membrane and deliver their contents.     

蓝藻伪空胞的特性及浮力调节机制

伪空胞为蓝藻在水体中提供浮力,使其获得适宜的生长条件,最终导致蓝藻水华暴发,了解伪空胞的特征对控制蓝藻水华暴发有重要意义。文章简要回顾了蓝藻伪空胞自1865年被Klebahn发现到1965年被正式命名的研究历程,目前已发现150多种原核生物中含有伪空胞;伪空胞是两末端呈圆锥状的中空圆柱体,伪空胞半径与临界压强遵循方程:Pc=275(r/nm)-1.67MPa;伪空胞气体含量可根据不同原理,利用Walsby伪空胞测定装置、压力浊度计和细胞流式仪测得。总结了伪空胞组成的化学特性,评述了伪空胞gvp基因丛结构功能和GvpA、GvpC的蛋白空间结构。GvpA是伪空胞合成的主要成分,gvpA在伪空胞内存在多个拷贝,其功能仍不清楚;GvpC由33个氨基酸重复单位组成,重复单位越多,伪空胞越不易破裂;概述了伪空胞3种浮力调节机制:镇重物的改变、伪空胞的合成、伪空胞的破裂;归纳了环境因子(光照、温度、氮、磷、钾)参与伪空胞浮力网络调控的途径。提出了目前伪空胞研究面临的困难和问题,对伪空胞的未来研究方向提出探索性的建议。     

The impact of short term synaptic depression and stochastic vesicle dynamics on neuronal variability

Neuronal variability plays a central role in neural coding and impacts the dynamics of neuronal networks. Unreliability of synaptic transmission is a major source of neural variability: synaptic neurotransmitter vesicles are released probabilistically in response to presynaptic action potentials and are recovered stochastically in time. The dynamics of this process of vesicle release and recovery interacts with variability in the arrival times of presynaptic spikes to shape the variability of the postsynaptic response. We use continuous time Markov chain methods to analyze a model of short term synaptic depression with stochastic vesicle dynamics coupled with three different models of presynaptic spiking: one model in which the timing of presynaptic action potentials are modeled as a Poisson process, one in which action potentials occur more regularly than a Poisson process (sub-Poisson) and one in which action potentials occur more irregularly (super-Poisson). We use this analysis to investigate how variability in a presynaptic spike train is transformed by short term depression and stochastic vesicle dynamics to determine the variability of the postsynaptic response. We find that sub-Poisson presynaptic spiking increases the average rate at which vesicles are released, that the number of vesicles released over a time window is more variable for smaller time windows than larger time windows and that fast presynaptic spiking gives rise to Poisson-like variability of the postsynaptic response even when presynaptic spike times are non-Poisson. Our results complement and extend previously reported theoretical results and provide possible explanations for some trends observed in recorded data.