Adhering Vesicles Research Articles

Inward multivesiculation at the basal membrane of adherent giant phospholipid vesicles

Adherent giant vesicles composed of phosphatidylcholine, phosphatidylserine and biotinylated lipids form clusters of inward spherical buds at their basal membrane. The process is spontaneous and occurs when the vesicles undergo a sequence of osmotic swelling and deswelling. The daughter vesicles have a uniform size (diameter≈2–3μm), engulf small volumes of outer fluid and remain attached to the region of the membrane from which they generate, even after restoring the isotonicity. A pinning–sealing mechanism of long-wavelength modes of membrane fluctuations is proposed, by which the just-deflated vesicles reduce the surplus of membrane area and avoid excessive spreading and compression via biotin anchors. The work discusses the rationale behind the mechanism that furnishes GUVs with basal endovesicles, and its prospective use to simulate cellular events or to create molecular carriers.

Variable Adhesion Strength for Giant Unilamellar Vesicles Controlled by External Electrostatic Potentials

We developed an experimental setup to study adhesion of giant unilamellar vesicles (GUVs) with variable adhesion strength. The vesicles adhere to a planar substrate. The adhesion is easily modulated by the application of an external potential to transparent ITO electrodes (the substrate) which enables us to study single GUVs during adhesion. The adhesion energy is calculated from the vesicle shape assessed with confocal microscopy. We implement a theoretical method for deducing the adhesion energy based on the overall vesicle shape. The results show that for the explored ranges of the external potential, the adhered vesicles are in the weak adhesion regime. Using fluorescence quenching assays, we conclude that the outer membrane leaflet is depleted of the negatively charged dye NDB-PG in the adhesion zone. Based on these results, we discuss the driving forces involved in the vesicle adhesion. Using FRAP measurements, we also show that diffusion is only slightly hindered in the adhesion zone. Some first results on applying our adhesion approach to study demixing in multicomponent membranes will be presented. We will demonstrate that adhesion can be used to modulate the phase state in the adhering and nonadhering regions of vesicles exhibiting coexistence of liquid ordered and liquid disordered domains.

Adhesion-Induced Domain Formation in Multicomponent Membranes

Lipid vesicles containing a relatively small number of molecular components provide simple model systems for cell membranes, which contain a large number of lipid and protein components. Such multicomponent membranes can undergo phase separation into two coexisting liquid phases and, thus, exhibit liquid domains within a liquid matrix. The associated phase diagrams depend on a number of parameters such as the overall membrane composition as well as on pH and temperature of the aqueous medium. Here, we address the influence of an adhesive substrate surface onto the phase behavior of the membranes. Specifically, we study giant unilamellar vesicles (GUVs) that can be prepared from different lipid mixtures and have sizes similar to those of cells. We control the adhesion by electrostatic membrane-surface interactions using a novel setup which allows tuning of the adhesion energy by an externally applied potential; see Fig. 1. With our single-vesicle approach, we can explore the phase behaviour of multicomponent membranes for different adhesion energies. One important objective is to identify distinct composition regimes for the phase separation in the bound and unbound part of the adhering vesicle membranes.Figure -Side views (xz scans) of a one-component GUV (about 60 µm in diameter) obtained by confocal microscopy. Fluorescence of the GUV membrane is shown in green and the adhesion surface as a red line. Left: no applied potential. Right: 1V applied potential. The change in adhesion strength can be clearly seen.

Influence of ionic strength and beta2-glycoprotein I concentration on agglutination of like-charged phospholipid membranes

The effect of ionic strength on adhesion between negatively charged giant unilamellar vesicles induced by beta2-glycoprotein I (β2-GPI) was studied experimentally and theoretically. Measuring the effective angle of contact between adhering vesicles indicated that the strength of adhesion between vesicles decreases with increasing ionic strength, and increases with concentration of β2-GPI. In the theoretical part we focused on the study of the average orientation of β2-GPI near the charged membrane and its role in mediating the attractive interactions between the vesicles. β2-GPI proteins were modelled as rods with internal distribution of electric charge. The predictions of Monte Carlo simulations show orthogonal orientation of some of the membrane attached β2-GPI in narrow gap between two vesicles. On the contrary, at larger distances between vesicles the proteins are parallelly attached to the membrane surface. A local minimum of the free energy corresponding to β2-GPI-mediated adhesion of two neighbouring vesicles was predicted. The strength of adhesion was confirmed to decrease at high ionic strength.

Blood cell-derived nanovesicles as biomarkers of homeostasis in gastrointestinal cancer.

152 Background: Nanovesicles (NVs) are membrane-enclosed fragments of cell interior which are pinched off from membranes of cells of all types (including cancer) in the last stage of the budding process. They are involved in cell-cell communication and can promote tumor growth and invasion. Interactions of tumor-derived NVs with blood cells, especially platelets are hypothetically reflected in vesiculability of platelets by means of concentration of NVs in blood isolates. Also we hypothesized that in patients whose plasma mediates stronger attractive interaction between membranes the concentration of NVs in blood isolates would be lower. Methods: NVs were isolated from 2.7 ml samples of blood of 29 patients with gastrointestinal cancer, 44 patients with other gastrointestinal diseases, additional 13 patients who were treated by imatinib due to gastrointestinal stromal tumor (GIST) and 50 healthy volunteers, by repeated centrifugation and washing, and counted by flow cytometry. We followed patients with GIST for 32 months. Ability of blood plasma to mediate attractive interaction between membranes was assessed by the angle of contact between giant phospholipid vesicles prepared by electroformation, which adhered due to addition of plasma to the suspension of NVs. Results: The concentration of NVs was considerably (44%, p = 0.0005) higher in the blood isolates of patients with gastrointestinal cancer than in the population of healthy volunteers and considerably (34%, p = 0.025) higher in patients with gastrointestinal cancer than in patients with other gastrointestinal diseases. We observed a considerable (average 13 fold peak) but transient (about one month within 32 montth follow up interval) increase of concentration of NVs in blood isolates due to treatment with imatinib.We found a negative correlation between the concentration of NVs and the average contact angle between adhered giant phospholipid vesicles (r = –0.50, p = 0.031). Conclusions: Concentration of NVs is increased while the ability of plasma to mediate attractive interaction between membranes is decreased in patients with gastrointestinal cancer. Treatment with imatinib caused transient increase of concentration of NVs in isolates from blood.

Mapping fluctuations in biomembranes adhered to micropatterns

We studied biomembrane fluctuations by calculating the instantaneous shape of model membranes adhered to micro-patterned substrates, using micro-interferometry. The model consisted of partially adherent giant unilamellar vesicles (GUVs) which were osmotically deflated. Adhesion was effected via the specific ligand–receptor interaction of biotin–neutravidin. Special micro-structured adhesive substrates were developed where the receptors were distributed in the form of grids or lines. Dual-wavelength reflection interference contrast microscopy (DW-RICM) measurements revealed that on the structured adhesive substrates GUVs exhibit regions of bound and fluctuating membrane, in accordance with the underlying pattern. In the fluctuating zone, the membrane presented itself as a flat-topped hill for an initial osmotic difference of 70 mOsm l−1. The membrane–substrate distance saturated at a plateau of 79 ± 9 nm. In this plateau, the fluctuation amplitude was found to be 10 ± 3 nm. Variation of the shape (grid versus lines) or size (grids of 3.5 or 7 μm lattice constant) influenced neither the height nor the fluctuation amplitude in the plateau. Fourier analysis revealed that the mode corresponding to a wavelength of twice the pattern size always contributed, and, depending on the substrate, additional modes were sometimes present. The plateau height could be tuned from 0 to 538 nm by changing the initial osmotic gradient between the inside and outside of the GUV, which effectively tuned the membrane tension. The corresponding fluctuation amplitude ranged from non-detectable to a maximum of 17 nm. Our results can be interpreted in the light of a tension dependent effective interaction potential.

Determination of the Strength of Adhesion between Lipid Vesicles

A commonly used method to determine the strength of adhesion between adhering lipid vesicles is measuring their effective contact angle from experimental images. The aim of this paper is to estimate the interobserver variations in vesicles effective contact angle measurements and to propose a new method for estimating the strength of membrane vesicle adhesion. Theoretical model shows for the old and for the new measure a monotonic dependence on the strength of adhesion. Results obtained by both measuring techniques show statistically significant correlation and high interobserver reliability for both methods. Therefore the conventional method of measuring the effective contact angle gives qualitatively relevant results as the measure of the lipid vesicle adhesion. However, the new measuring technique provides a lower variation of the measured values than the conventional measures using the effective contact angle. Moreover, obtaining the adhesion angle can be automatized more easily than obtaining the effective contact angle.

The role of the membrane confinement in the surface area regulation of cells.

We propose a new in vitro system to study the mechanics of surface area regulation in cells, which takes into an account the spatial confinement of the cell membrane. By coupling a lipid bilayer to the strain-controlled deformation of an elastic sheet, we show that upon straining the supported lipid bilayer expands its surface area by absorbing adherent lipid vesicles and upon compression decreases its area by expelling lipid tubes out of its plane. The processes are reversible and closely resemble in vivo observations on shrinking cells. Our results suggest that the mechanics of the area regulation in cells is controlled primarily by the membrane tension and the effects of the membrane confinement.

The role of the membrane confinement in the surface area regulation of cells

We propose a new in vitro system to study the mechanics of surface area regulation in cells, which takes into an account the spatial confinement of the cell membrane. By coupling a lipid bilayer to the strain-controlled deformation of an elastic sheet, we show that upon straining the supported lipid bilayer expands its surface area by absorbing adherent lipid vesicles and upon compression decreases its area by expelling lipid tubes out of its plane. The processes are reversible and closely resemble in vivo observations on shrinking cells. Our results suggest that the mechanics of the area regulation in cells is controlled primarily by the membrane tension and the effects of the membrane confinement.

Mechanics of membrane–membrane adhesion

Curvature elasticity is used to derive the equilibrium conditions that govern the mechanics of membrane–membrane adhesion. These include the Euler–Lagrange equations and the interface conditions which are derived here for the most general class of strain energies permissible for fluid surfaces. The theory is specialized for homogeneous membranes with quadratic ‘Helfrich’-type energies with non-uniform spontaneous curvatures. The results are employed to solve four-point boundary value problems that simulate the equilibrium shapes of lipid vesicles that adhere to each other. Numerical studies are conducted to investigate the effect of relative sizes, osmotic pressures, and adhesion-induced spontaneous curvature on the morphology of adhered vesicles.

Mechanics of surface area regulation in cells examined with confined lipid membranes.

Cells are wrapped in inelastic membranes, yet they can sustain large mechanical strains by regulating their area. The area regulation in cells is achieved either by membrane folding or by membrane exo- and endocytosis. These processes involve complex morphological transformations of the cell membrane, i.e., invagination, vesicle fusion, and fission, whose precise mechanisms are still under debate. Here we provide mechanistic insights into the area regulation of cell membranes, based on the previously neglected role of membrane confinement, as well as on the strain-induced membrane tension. Commonly, the membranes of mammalian and plant cells are not isolated, but rather they are adhered to an extracellular matrix, the cytoskeleton, and to other cell membranes. Using a lipid bilayer, coupled to an elastic sheet, we are able to demonstrate that, upon straining, the confined membrane is able to regulate passively its area. In particular, by stretching the elastic support, the bilayer laterally expands without rupture by fusing adhered lipid vesicles; upon compression, lipid tubes grow out of the membrane plane, thus reducing its area. These transformations are reversible, as we show using cycles of expansion and compression, and closely reproduce membrane processes found in cells during area regulation. Moreover, we demonstrate a new mechanism for the formation of lipid tubes in cells, which is driven by the membrane lateral compression and may therefore explain the various membrane tubules observed in shrinking cells.

Specific adhesion of vesicles to compliant bio-adhesive substrates

Cell behavior is mediated by variety of physiochemical properties of the extracellular matrix (ECM). Mechanical stiffness of ECM, in particular, is found to be a major regulator for the multiple aspects of cellular function. Experiments show that cells generally exhibit an apparent adhesion preference for stiffer substrates. The effect of substrate elasticity is also found to be strongly coupled with adhesivity of the substrate. To understand the underlying physics of rigidity sensing mechanism in cells, in this study we use a vesicle-substrate system to model cell adhesion as a first order approximation. Within this framework, an equilibrium thermodynamic analysis is undertaken to elucidate the interplay between substrate compliance and equilibrium configuration of an adherent vesicle. The equilibrium adhesion is assumed to ensure minimization of the free energy contributed by substrate deformation and interfacial adhesive and repulsive interactions between the membrane and substrate. The predictions of this purely mechanistic model are found to be qualitatively analogous to some of the characteristic features of cell adhesion to compliant bio-adhesive substrates. This observation suggests that the physical aspects of the membrane–substrate interfacial interactions could passively contribute in regulation of the rigidity sensing by cells.

ALIGNMENT AND DEFORMATION OF LIPID BILAYER DOMAINS IN VESICLES ADHERING TO MICROSTRUCTURED SUBSTRATES

In heterogeneous lipid membranes, the lateral organization is coupled to local curvature as the membrane bending energies depend on composition. We investigate phase-separated vesicles of ternary lipid composition in contact with structured surfaces, where the distinct elastic properties of the Lo and Ld phases come into play. We show that fused silica substrates with microstructured grooves induce sorting, alignment and deformation of Lo domains in adhering vesicles. The same phenomenon is observed on flat, chemically modified substrates with alternating stripes of rough and smooth regions. In both cases it is the Lo phase which accumulates over the smooth substrate and membrane-spanned groove regions respectively. Deformation of Lo domains occurs when domain diameters grow beyond the width of the microstructured stripes. Domain alignment was also observed in binary membranes featuring gel and fluid phase coexistence showing the generic character of domain sorting on microstructured surfaces.

Number of microvesicles in peripheral blood and ability of plasma to induce adhesion between phospholipid membranes in 19 patients with gastrointestinal diseases

It was recently shown that the plasma protein-mediated attractive interaction between phospholipid membranes could in the budding process cause adhesion of the bud to the mother membrane [J. Urbanija, N. Tomšič, M. Lokar, A. Ambrožič, S. Čučnik, M. Kandušer, B. Rozman, A. Iglič, V. Kralj-Iglič, Coalescence of phospholipid membranes as a possible origin of anticoagulant effect of serum proteins, Chem. Phys. Lipids 150 (2007) 49–57]. Since in the in vivo conditions the budding of cell membranes leads to the release of microvesicles into the circulation, a hypothesis was put forward that the ability of plasma to cause adhesion between membranes supresses the microvesiculation process. In the present work, this hypothesis was tested in a population of 19 patients with gastrointestinal diseases. The number of microvesicles in peripheral blood of patients was determined by flow cytometry while the ability of plasma to cause adhesion between membranes was determined by adding patient's plasma to the suspension of giant phospholipid vesicles created by electroformation method, and measuring the average effective angle of contact between the adhered vesicles. Statistically significant negative correlations between the number of microvesicles and the average effective angle of contact (Pearson coefficient − 0.50, p = 0.031) and between the number of microvesicles per number of platelets and the average effective angle of contact (Pearson coefficient − 0.64, p = 0.003) were found, which is in favor of the above hypothesis. Patients with gastrointestinal cancer had larger number of microvesicles (difference 140%, statistical significance 0.033) and smaller average effective angle of contact (difference 20%, statistical significance 0.013) compared to patients with other gastrointestinal diseases.

Adhesion of fluid vesicles at chemically structured substrates

The adhesion of fluid vesicles at chemically structured substrates is studied theoretically via Monte Carlo simulations. The substrate surface is planar and repels the vesicle membrane apart from a single surface domain gamma , which strongly attracts this membrane. If the vesicle is larger than the attractive gamma domain, the spreading of the vesicle onto the substrate is restricted by the size of this surface domain. Once the contact line of the adhering vesicle has reached the boundaries of the gamma domain, further deflation of the vesicle leads to a regime of low membrane tension with pronounced shape fluctuations, which are now governed by the bending rigidity. For a circular gamma domain and a small bending rigidity, the membrane oscillates strongly around an average spherical cap shape. If such a vesicle is deflated, the contact area increases or decreases with increasing osmotic pressure, depending on the relative size of the vesicle and the circular gamma domain. The lateral localization of the vesicle's center of mass by such a domain is optimal for a certain domain radius, which is found to be rather independent of adhesion strength and bending rigidity. For vesicles adhering to stripe-shaped surface domains, the width of the contact area perpendicular to the stripe varies nonmonotonically with the adhesion strength.

Abstract 274: Physical Coupling Supports Local Ca2+ Coupling Between SR Subdomains and the Mitochondria in Heart Muscle

Propagation of ryanodine receptor (RyR) dependent SR Ca2+ release to the mitochondria is locally supported by high [Ca2+] microdomains at close contacts between the organelles. The objective of this study was to clarify if the close associations of SR and mitochondria underlying the local Ca2+ communication were secured via a physical coupling. Crude and Percoll-purified mitochondrial fractions of rat heart homogenates were tested for the presence of mitochondria-associated SR fragments capable of RyR-dependent Ca2+ mobilization and Ca2+ delivery to the mitochondria. SR and mitochondrial marker proteins were analyzed by Western blot or visualized in situ by immunofluorescent labeling and imaged using confocal microscopy. Mitochondrial matrix [Ca2+] ([Ca2+]m) was monitored using fluorescence imaging of rhod-2-loaded adherent individual vesicles. Ample presence of SR markers (calsequestrin, SERCA2a, phospholamban) was detected in the crude mitochondria, whereas after percoll purification they were barely detectable in the ‘heavy’ fraction but remained present in the ‘light’ fraction. ‘Immunomitochemistry’ revealed various sizes of SR particles in the crude fraction, a large portion of which was colocalized with mitochondria, while showed less SR markers in the percoll-purified heavy fraction. Fluorescence Ca2+ imaging in the adherent particles revealed well-detectable [Ca2+]m responses to caffeine stimulation that was prevented by Ca2+ predepletion of the SR using the SERCA inhibitor thapsigargin (Tg), or by inhibitors of mitochondrial Ca2+ uptake. Because the Ca2+ released from SR was diluted in the large volume of the incubation medium without causing a change in the bulk [Ca2+], only local Ca2+ regulation could mediate the Ca2+ transfer to the mitochondria. Despite the hardly detectable presence of SR, the percoll-purified ‘heavy’ mitochondrial fraction still displayed a caffeine-induced [Ca2+]m rise that was abolished by Tg-pretreatment. Our data suggest that the SR-mitochondrial associations where the local Ca2+ coupling is established, are supported by physical links between the two organelles and the mitochondria bound SR surface represents a relatively small fraction of the total SR.

Novel Method for Measuring the Adhesion Energy of Vesicles

Adhering vesicles with osmotically stabilized volume are studied with Monte Carlo simulations and optical microscopy. The simulations are used to determine the dependence of the adhesion area on the vesicle volume, the surface area, the bending rigidity, the adhesion energy per membrane area, and the adhesion potential range. The simulation results lead to a simple functional expression that is supplemented by a correction term for gravity effects. The obtained equation provides a new tool to analyze optical microscopy data and, thus, to measure the adhesion energy per area by analyzing the geometry of the adhering vesicle. The method can be applied in the weak and ultra-weak adhesion regime, where the adhesion energy per area is below 10(-6) J/m(2). By comparing the shapes of adhering vesicles with different reduced volumes, the bending rigidity can be estimated as well. The new approach is applied to experimental data for lipid vesicles on (i) an untreated and (ii) a monolayer-coated glass surface, providing ultra-weak and weak adhesion strength, respectively.

EQUILIBRIUM CONFIGURATIONS OF ADHERING TUBULAR VESICLES UNDER EXTERNAL LOADS

A theoretical model is developed to describe the influence of external loads on the equilibrium shapes of adhering tubular lipid vesicles. In the case of nonspecific adhesion, the equilibrium shape equations and boundary conditions are derived through the method analogous to previous research. For a range of applied forces, locally stable bound shapes are simulated. Along with the increase or decrease the external force, the process for adhering vesicle disengaging from or sprawling to the substrate is predicted and analyzed.

Theoretical analysis of adhering lipid vesicles with free edges

A theoretical model for describing the adhesion of lipid vesicle with free edges is developed. For adhesion in contact potential or in finite-range potential, the total energy functional is defined as the sum of elastic free energy, the surface energy, the line tension energy and the contact potential or the long-ranged potential. The equilibrium differential equation and boundary conditions for opening-up lipid vesicles are derived through minimizing the total energy functional. Numerical solutions to these equations are obtained under the axial symmetric condition. These numerical solutions can be used to qualitatively explain the influence of the substrate on the open-up lipid vesicles.

Effective adhesion strength of specifically bound vesicles

A theoretical approach has been undertaken in order to model the thermodynamic equilibrium of a 3D vesicle adhering to a flat substrate. The vesicle is treated in a canonical description with a fixed number of sites. A finite number of these sites are occupied by mobile ligands that are capable of interacting with a discrete number of receptors immobilized on the substrate. Explicit consideration of the bending energy of the vesicle shape has shown that the problem of the vesicle shape can be decoupled from the determination of the optimum allocation of ligands over the vesicle. The allocation of bound and free ligands in the vesicle can be determined as a function of the size of the contact zone, the ligand-receptor binding strength, and the concentration of the system constituents. Several approximate solutions for different regions of system parameters are determined and in particular, the distinction between receptor- and ligand-dominated equilibria is found to be important. The crossover between these two types of solutions is found to occur at a critical size of the contact zone. The presented approach enables the calculation of the effective adhesion strength of the vesicle and thus permits meaningful comparisons with relevant experiments as well as connecting the presented model with the proven success of the continuum approach for modeling the shapes of adhering vesicles. The behavior of the effective adhesion strength is analyzed in detail and several approximate expressions for it are given.