Vesicle Geometry Research Articles

Exploring the Molecular Dynamics of a Lipid-A Vesicle at the Atom Level: Morphology and Permeation Mechanism.

Lipid-A was previously shown to spontaneously aggregate into a vesicle via the hybrid particle field approach. We assess the validity of the proposed vesiculation mechanism by simulating the resulting lipid-A vesicle at the atom level. The spatial confinement imposed by the vesicle geometry on the conformation and packing of lipid-A induces significant heterogeneity of physical properties in the inner and outer leaflets. It also induces tighter molecular packing and lower acyl chain order compared to the lamellar arrangement. Around 5% of water molecules passively permeates the vesicle membrane inward and outward. The permeation is facilitated by interactions with water molecules that are transported across the membrane by a network of electrostatic interactions with the hydrogen bond donors/acceptors in the N-acetylglucosamine ring and upper region of the acyl chains of lipid-A. The permeation process takes place at low rates but still at higher frequencies than observed for the lamellar arrangement of lipid-A. These findings not only substantiate the proposed lipid-A vesiculation mechanism but also reveal the complex structural dynamics of an important nonlamellar arrangement of lipid-A.

Division and Regrowth of Phase‐Separated Giant Unilamellar Vesicles**

AbstractSuccess in the bottom‐up assembly of synthetic cells will depend on strategies for the division of protocellular compartments. Here, we describe the controlled division of phase‐separated giant unilamellar lipid vesicles (GUVs). We derive an analytical model based on the vesicle geometry, which makes four quantitative predictions that we verify experimentally. We find that the osmolarity ratio required for division is , independent of the GUV size, while asymmetric division happens at lower osmolarity ratios. Remarkably, we show that a suitable osmolarity change can be triggered by water evaporation, enzymatic decomposition of sucrose or light‐triggered uncaging of CMNB‐fluorescein. The latter provides full spatiotemporal control, such that a target GUV undergoes division whereas the surrounding GUVs remain unaffected. Finally, we grow phase‐separated vesicles from single‐phased vesicles by targeted fusion of the opposite lipid type with programmable DNA tags to enable subsequent division cycles.

Division and Regrowth of Phase-Separated Giant Unilamellar Vesicles*.

Success in the bottom‐up assembly of synthetic cells will depend on strategies for the division of protocellular compartments. Here, we describe the controlled division of phase‐separated giant unilamellar lipid vesicles (GUVs). We derive an analytical model based on the vesicle geometry, which makes four quantitative predictions that we verify experimentally. We find that the osmolarity ratio required for division is 2 , independent of the GUV size, while asymmetric division happens at lower osmolarity ratios. Remarkably, we show that a suitable osmolarity change can be triggered by water evaporation, enzymatic decomposition of sucrose or light‐triggered uncaging of CMNB‐fluorescein. The latter provides full spatiotemporal control, such that a target GUV undergoes division whereas the surrounding GUVs remain unaffected. Finally, we grow phase‐separated vesicles from single‐phased vesicles by targeted fusion of the opposite lipid type with programmable DNA tags to enable subsequent division cycles.

Multimodal imaging and stochastic percolation simulation for improved quantification of effective porosity and surface area in vesicular basalt

Abstract Improved methods for predicting fluid transport and vesicle connectivity in heterogeneous basalts are critical for determining the long-term reaction and trapping behavior of sequestered carbon dioxide and maximizing the efficiency of geothermal energy production. In this study we measured vesicle geometry, pore connectivity, and vesicle surface area of three basalt cores from the CarbFix carbon storage project in Iceland using a combination of micro-computed tomography, clinical computed tomography, and micro-positron emission tomography. A vesicle percolation simulator was then constructed to quantify vesicle connectivity across a complete range of porosities, pore size distributions, and microporosity conditions. Percolation simulations that incorporate important geologic features such as microporosity are able to describe the trend of experimental measurements made in this study and in previous work, without relying on statistical or empirical techniques. Simulation results highlight and quantify the trade-off between storage capacity and reactive surface area in high porosity basalts. Experiment and simulation results also indicate that there is very limited connected pore space below total porosity values of 15%, guiding improved site selection for large scale CO2 storage projects. Use of this stochastic percolation simulation method for basalt storage reservoir evaluation will enable more accurate storage capacity and mineral trapping estimates.

Multilamellar nanovesicles show distinct mechanical properties depending on their degree of lamellarity.

Small multilamellar vesicles may have benefits over unilamellar vesicles for drug delivery, such as an increased volume for hydrophobic drugs. In addition, their altered mechanical properties might be beneficial for cellular uptake. Here, we show how atomic force microscopy (AFM) can be used to detect and characterize multilamellar vesicles. We quantify the size of each break event occurring during AFM nanoindentations, which shows good agreement with the thickness of supported lipid bilayers. Analyzing the size and number of these events for individual vesicles allows us to distinguish between vesicles consisting of 1 up to 5 bilayers. We validate these results by comparison with correlative cryo-electron microscopy (cryo-EM) data at the vesicle population level. Finally, we quantify the vesicle geometry and mechanical properties, and show that with additional bilayers adherent vesicles are more spherical and stiffer. Surprisingly, at ∼20% stiffening for each additional bilayer, the vesicle stiffness scales only weakly with lamellarity. Our results show the potential of AFM for studying liposomal nanoparticles and suggest that small multilamellar vesicles may have beneficial mechanical properties for cellular uptake.

Monte Carlo simulation of the effects of vesicle geometry on calcium microdomains and neurotransmitter release

We investigate the effects of synaptic vesicle geometry on Ca 2+ diffusion dynamics in presynaptic terminals using MCell, a realistic Monte Carlo algorithm that tracks individual molecules. By modeling the vesicle as a sphere and an oblate or a prolate spheroid with a reflective boundary, we measure the Ca 2+ concentration at various positions relative to the vesicle. We find that the presence of a vesicle as a diffusion barrier modifies the shape of the [Ca 2+ ] microdomain in the vicinity of the vesicle. Ca 2+ diffusion dynamics also depend on the distance between the vesicle and the voltage-gated calcium channels (VGCCs) and on the shape of the vesicle. The oblate spheroidal vesicle increases the [Ca 2+ ] up to six times higher than that in the absence of a vesicle, while the prolate spheroidal vesicle can increase the [Ca 2+ ] only 1.4 times. Our results also show that the presence of vesicles that have different geometries can maximally influence the [Ca 2+ ] microdomain when the vesicle is located less than 50 nm from VGCCs.

Phase field calculus, curvature-dependent energies, and vesicle membranes

Lipid bilayer vesicles are membranes formed spontaneously in aqueous environments under suitable conditions. They have been extensively studied in literature as a simple model of cell membranes. In this work, we discuss some recent developments in the phase field modeling and simulations of vesicles. We describe how the equilibrium and dynamic properties of the vesicles can be effectively described by the phase field calculus which is a natural translation of the energy variations with respect to the vesicle geometry to the variations of the corresponding diffuse interfacial energy with respect to the phase field functions. We also illustrate how phase field techniques can be generalized to model a multi-component vesicle, a vesicle interacting with a surrounding fluid and the adhesion of a vesicle to a substrate.

Equilibrium of nematic vesicles

A variational scheme is proposed which allows the derivation of a concise and elegant formulation of the equilibrium equations for closed fluid membranes, endowed with a nematic microstructure. The nematic order is described by an in-plane nematic director and a degree of orientation, as customary in the theory of uniaxial nematics. The only constitutive ingredient in this scheme is a free-energy density which depends on the vesicle geometry and order parameters. The stress and the couple stress tensors related to this free-energy density are provided. As an application of the proposed scheme, a certain number of special theories are deduced: soap bubbles, lipid vesicles, chiral and achiral nematic membranes, and nematics on curved substrates.

Single Vesicle Millisecond Fusion Kinetics Reveals Number of SNARE Complexes Optimal for Fast SNARE-mediated Membrane Fusion

SNAREs mediate membrane fusion in intracellular vesicle traffic and neuronal exocytosis. Reconstitution of membrane fusion in vitro proved that SNAREs constitute the minimal fusion machinery. However, the slow fusion rates observed in these systems are incompatible with those required in neurotransmission. Here we present a single vesicle fusion assay that records individual SNARE-mediated fusion events with millisecond time resolution. Docking and fusion of reconstituted synaptobrevin vesicles to target SNARE complex-containing planar membranes are distinguished by total internal reflection fluorescence microscopy as separate events. Docking and fusion are SNAP-25-dependent, require no Ca(2+), and are efficient at room temperature. Analysis of the stochastic data with sequential and parallel multi-particle activation models reveals six to nine fast-activating steps. Of all the tested models, the kinetic model consisting of eight parallel reaction rates statistically fits the data best. This might be interpreted by fusion sites consisting of eight SNARE complexes that each activate in a single rate-limiting step in 8 ms.

Cryo-EM Structure of Functional BK Channels in Lipid Bilayers

A natural way to preserve the native conformation of membrane proteins for structural study is to reconstitute them into liposomes (lipid vesicles). Here we applied this method to single-particle cryo-EM imaging of the hSlo large-conductance voltage- and Ca2+-activated potassium channel (BK channel). In an assay using the JC-1 voltage-sensitive dye, proteoliposomes develop a membrane potential that is equal to that produced by valinomycin, and to that of empty liposomes when the BK channels are blocked by barium (A). For cryo-EM imaging the reconstituted BK proteoliposomes were tethered to a 2D streptavidin crystal (B). From this image the periodic crystal information was removed (C) and a liposome model of electron scattering was used to subtract the lipid bilayers (D). For 3D reconstruction, single-particle images (boxes) were oriented with constraints based on the spherical vesicle geometry. A structure based on these particle images will be presented. Scale bar in the figures is 50 nm.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Three‐dimensional investigation of volcanic textures by X‐ray microtomography and implications for conduit processes

Synchrotron X‐ray computed microtomography (μCT) was applied for the first time to clasts of pumice and scoria generated by active, explosive volcanoes characterized by a range of eruptive styles (mild Strombolian to Plinian) and magmatic composition (basaltic to trachytic). The obtained two‐dimensional (2D) tomographic images, corresponding to sample views at different rotation angles, were processed to reconstruct three‐dimensional (3D) volumes and then used to make 3D measurements of vesicularity, vesicle number density, volume and connectivity for quantitative characterization of the investigated tephras. The results indicate a positive correlation between vesicle number density and eruption intensity that is used to investigate modes of magma degassing in explosive eruptions. In addition, the vesicle geometry affecting the connected gas flow pathways in pyroclasts from Strombolian eruptions is determined and related to the known permeabilities. Implications on the dynamics of explosive eruptions is discussed and, ultimately, used to quantitatively discriminate between different eruptive styles.

Electro-optics of membrane electroporation in diphenylhexatriene-doped lipid bilayer vesicles

The electric (linear) dichroisms observed in the membrane electroporation of salt-filled lipid bilayer vesicles (diameter Φ = 2 a = 0.32 μm; inside [NaCl] = 0.2 M) in isotonic aqueous 0.284 M sucrose-0.2 mM NaCl solution indicate orientation changes of the anisotropic light scattering centers (lipid head groups) and of the optical transition moments of the membrane-inserted probe 1,6-diphenyl-1,3,5-hexatriene (DPH). Both the turbidity dichroism and DPH absorbance dichroism show peculiar features: (1) at external electric fields E ≥ E sat the time course of the dichroism shows a maximum value (reversal): E sat = 4.0 (±0.2) MV m −1, T = 293 K (20°C), (2) this reversal value is independent of the field strength for E ≥ E sat, (3) the dichroism amplitudes exhibit a maximum value E max = 3.0 (±0.5) MV m −1, (4) for the pulse duration of 10 μs there is one dominant visible normal mode, the relaxation rate increases up to τ −1 ≈ 0.6 × 10 6 s −1 at E sat and then decreases for E > E sat. The data can be described in terms of local lipid phase transitions involving clusters L n of n lipids in the pore edges according to the three-state scheme C ⇌ HO ⇌ HI, C being the closed bilayer state, HO the hydrophobic pore state and HI the hydrophilic or inverted pore state with rotated lipid and DPH molecules. At E ≥ E sat, further transitions HO ⇌ HO ∗ and HI ⇌ HI ∗ are rapidly coupled to the C ⇌ HO transition, which is rate-limiting. The vesicle geometry conditions a cosθ dependence of the local membrane field effects relative to the E direction and the data reflect cosθ averages. The stationary induced transmembrane voltage Δϕ( θ, λ m) = − 1.5 aEf( λ m| cosθ| does not exceed the limiting value Δϕ sat = − 0.53 V, corresponding to the field strength E m,sat = − Δϕ sat d = 100 MV m −1 (10 3 kV cm −1), due to increasing membrane conductivity λ m. At E = E sat, f( λ m) = 0.55, λ m = 0.11 mS m −1. The lipid cluster phase transition model yields an average pore radius of r p = 0.35 (±0.05) nm of the assumed cylindrical pore of thickness d = 5 nm, suggesting an average cluster size of 〈 n〉 = 12 (±2) lipids per pore edge. For E > E sat, the total number of DPH molecules in pore states approaches a saturation value; the fraction of DPH molecules in HI pores is 12 (±2)% and that in HO pores is 48 (±2)%. The percentage of membrane area P ≈ ( λ m λ i ) × 100 % of conductive openings filled with the intravesicular medium of conductance λ i = 2.2 S m − linearly increases from P ≈ 0% ( E = 1.8 MV m −1) to P = 0.017% ( E = 8.5 MV m −1). Analogous estimations made by Kinosita et al. (1993) on the basis of fluorescence imaging data for sea urchin eggs give the same order of magnitude for P (0.02–0.2%). The increase in P with the field strength is collinear with the increase in concentration of HI and HI ∗ states with the field strength, whereas the HO and HO ∗ states exhibit a sigmoid field dependence. Therefore our data suggest that it is only the HI and HI ∗ pore states which are conductive. It is noted that the various peculiar features of the dichroism data cannot be described by simple whole particle deformation.

Role of lamellar membrane structure in tether formation from bilayer vesicles

A theoretical analysis is presented of the formation of membrane tethers from micropipette-aspirated phospholipid vesicles. In particular, it is taken into account that the phospholipid membrane is composed of two layers which are in contact but unconnected. The elastic energy of the bilayer is taken to be the sum of contributions from area expansivity, relative expansivity of the two monolayers, and bending. The vesicle is aspirated into a pipette and a constant point force is applied at the opposite side in the direction away from the pipette. The shape of the vesicle in approximated as a cylindrical projection into the pipette with a hemispherical cap, a spherical section, and a cylindrical tether with a hemispherical cap. The dimensions of the different regions of the vesicle are obtained by minimizing its elastic energy subject to the condition that the volume of the vesicle is fixed. The range of values for the parameters of the system is determined at which the existence of a tether is possible. Stability analysis is performed showing which of these configurations are stable. The importance of the relative expansion and compression of the constituent monolayers is established by recognizing that local bending energy by itself does not stabilize the vesicle geometry, and that in the limit as the relative expansivity modulus becomes infinitely large, a tether cannot be formed. Predictions are made for the functional relationships among experimentally observable quantities. In a companion report, the results of this analysis are applied to experimental measurements of tether formation, and used to calculate values for the membrane material coefficients.

Incorporation of the nicotinic acetylcholine receptor into liposomes by rapid removal of detergent using poly (ethylene) glycol 6000

The poly (ethylene) glycol 6000 (PEG) precipitation procedure was applied for incorporating the nicotinic acetylcholine receptor from D. tschudii electric organ into Asolectin liposomes. The incorporated receptor had its toxin binding sites oriented towards the external bulk phase and bound [ 125I]α-bungarotoxin with the same rate of toxin binding and cholinergic agonist equilibrium low affinity constant as its membrane bound counterpart. A characteristic feature of those vesicles, however, was the absence of the affinity transitions which normally correlate with the receptor densensitization phenomenon, and a poor carbamylcholine-stimulated ion flux as compared to that exhibited by vesicles prepared by the cholate dialysis procedure. When ether-purified PEG was used, the incorporated receptor showed affinity transitions and carbamylcholine-induced cation influx into vesicles. Thin layer chromatography and quantitative densitometry analysis showed that the vesicles prepared by the PEG precipitation method contained higher amounts of free fatty acids than those prepared by the cholate-dialysis procedure. Results are interpreted as an impairment of receptor function by impurities contained in commercial preparations of PEG and/or free fatty acids possibly generated by PEG impurities from Asolectin lipids. The proposed mechanisms are modifications in vesicle geometry and perturbations in lipid-protein interactions known to be essential for normal nicotinic receptor operation. The PEG precipitation procedure has the potential for rapid reconstitution of the nicotinic receptor and other membrane proteins that cannot be solubilized by easily dialyzable detergents provided purified PEG is used.

Lack of involvement of sarcoplasmic reticulum in myopathy of acute phosphorous depletion.

Acute and chronic hypophosphatemia are known to cause metabolic myopathy. It has been proposed that impaired Ca transport in subcellular membranes is involved in its genesis. In the present study, calcium transport in the sarcoplasmic reticulum (SR), concentrations of ions or nucleotides and transmembrane potential were investigated in muscles of acutely hypophosphatemic rats, i.e. animals with chronic dietary phosphorous deprivation (PD) and superimposed acute hypophosphatemia resulting from the administration of insulin and glucose. Despite hypophosphatemia and low muscle phosphorous concentration, no significant change of the initial rate of Ca uptake or Ca concentrating ability was observed in the SR of PD rats. Storing capacity was decreased; this may result from altered vesicle geometry. Water content, Na concentration, the concentration of several nucleotides and transmembrane potential of muscle were unchanged in PD rats. The findings document that no intrinsic abnormality of vectorial Ca transport is present in the SR of acutely hypophosphatemic PD animals.