Adhering Vesicles Research Articles

Amphiphilic Rhodamine Fluorescent Probes Combined with Basal Imaging for Fine Structures of the Cell Membrane.

Confocal fluorescence imaging of fine structures of the cell membrane is important for understanding their biofunctions but is often neglected due to the lack of an effective method. Herein, we develop new amphiphilic rhodamine fluorescent probe RMGs in combination with basal imaging for this purpose. The probes show high signal-to-noise ratio and brightness and low internalization rate, making them suitable for imaging the fine substructures of the cell membrane. Using the representative probe RMG3, we not only observed the cell pseudopodia and intercellular nanotubes but also monitored the formation of migrasomes in real time. More importantly, in-depth imaging studies on more cell lines revealed for the first time that hepatocellular carcinoma cells secreted much more adherent extracellular vesicles than other cell lines, which might serve as a potential indicator of liver cells. We believe that RMGs may be useful for investigating the fine structures of the cell membrane.

Patterning and dynamics of membrane adhesion under hydraulic stress

Hydraulic fracturing plays a major role in cavity formation during embryonic development, when pressurized fluid opens microlumens at cell-cell contacts, which evolve to form a single large lumen. However, the fundamental physical mechanisms behind these processes remain masked by the complexity and specificity of biological systems. Here, we show that adhered lipid vesicles subjected to osmotic stress form hydraulic microlumens similar to those in cells. Combining vesicle experiments with theoretical modelling and numerical simulations, we provide a physical framework for the hydraulic reconfiguration of cell-cell adhesions. We map the conditions for microlumen formation from a pristine adhesion, the emerging dynamical patterns and their subsequent maturation. We demonstrate control of the fracturing process depending on the applied pressure gradients and the type and density of membrane bonds. Our experiments further reveal an unexpected, passive transition of microlumens to closed buds that suggests a physical route to adhesion remodeling by endocytosis.

A case of ectopic prostatic tissue presenting as a cystic pelvic mass

A 74-year-old man initially presenting with fall had an incidental finding on CT of a heterogeneous, partly cystic lesion in the pelvis that was moderately glucose avid on FDG-PET. The pelvic mass and adherent right seminal vesicle were excised.

Morphology of vesicle triplets: shape transformation at weak and strong adhesion limits.

We investigate the morphologies of adhering vesicle triplets as a function of volume-to-area ratio encoded by the reduced volume in strong and weak adhesion regimes. In the strong adhesion regime, the morphology change of the vesicle triplet depends on the arrangement of vesicles. By decreasing the reduced volume, a triangular triplet composed of three spherical caps with a trifurcated flat contact zone deformed to a compact spherical shape with a sigmoidal contact zone, whereas a linear vesicle triplet composed of pancake-shaped vesicles sandwiched between two spherical-cap vesicles with a flat contact zone deformed into a compact spherical shape with biconvex interfaces. The morphologies of vesicle triplets with flat contact zones are reproduced by the so-called two-tension model based on the total energy consisting of bending energy, adhesion energy and surface energy, where the surface tension in the noncontact zone is different from that in the contact zone. When the flat interface deforms, the two-tension model is no longer applicable. The compact spherical triplets with curved interfaces can be reproduced by introducing geometrical constraints requiring that the total area of the non-contact zones is minimal, thereby confining the aggregate to a spherical cavity; this is referred to as the cavity model. In the weak adhesion regime, vesicle triplets with either a triangular or linear topology deform into prolate-based triplets by decreasing the reduced volume.

Investigation of Shape Transformations of Vesicles, Induced by Their Adhesion to Flat Substrates Characterized by Different Adhesion Strength.

The adhesion of lipid vesicles to a rigid flat surface is investigated. We examine the influence of the membrane spontaneous curvature, adhesion strength, and the reduced volume on the stability and shape transformations of adhered vesicles. The minimal strength of the adhesion necessary to stabilize the shapes of adhered vesicles belonging to different shape classes is determined. It is shown that the budding of an adhered vesicle may be induced by the change of the adhesion strength. The importance of the free vesicle shape for its susceptibility to adhesion is discussed.

Mechanical and Electrical Interaction of Biological Membranes with Nanoparticles and Nanostructured Surfaces.

In this review paper, we theoretically explain the origin of electrostatic interactions between lipid bilayers and charged solid surfaces using a statistical mechanics approach, where the orientational degree of freedom of lipid head groups and the orientational ordering of the water dipoles are considered. Within the modified Langevin Poisson–Boltzmann model of an electric double layer, we derived an analytical expression for the osmotic pressure between the planar zwitterionic lipid bilayer and charged solid planar surface. We also show that the electrostatic interaction between the zwitterionic lipid head groups of the proximal leaflet and the negatively charged solid surface is accompanied with a more perpendicular average orientation of the lipid head-groups. We further highlight the important role of the surfaces’ nanostructured topography in their interactions with biological material. As an example of nanostructured surfaces, we describe the synthesis of TiO2 nanotubular and octahedral surfaces by using the electrochemical anodization method and hydrothermal method, respectively. The physical and chemical properties of these nanostructured surfaces are described in order to elucidate the influence of the surface topography and other physical properties on the behavior of human cells adhered to TiO2 nanostructured surfaces. In the last part of the paper, we theoretically explain the interplay of elastic and adhesive contributions to the adsorption of lipid vesicles on the solid surfaces. We show the numerically predicted shapes of adhered lipid vesicles corresponding to the minimum of the membrane free energy to describe the influence of the vesicle size, bending modulus, and adhesion strength on the adhesion of lipid vesicles on solid charged surfaces.

Adhesion Process of Biomimetic Myelin Membranes Triggered by Myelin Basic Protein.

The myelin sheath—a multi-double-bilayer membrane wrapped around axons—is an essential part of the nervous system which enables rapid signal conduction. Damage of this complex membrane system results in demyelinating diseases such as multiple sclerosis (MS). The process in which myelin is generated in vivo is called myelination. In our study, we investigated the adhesion process of large unilamellar vesicles with a supported membrane bilayer that was coated with myelin basic protein (MBP) using time-resolved neutron reflectometry. Our aim was to mimic and to study the myelination process of membrane systems having either a lipid-composition resembling that of native myelin or that of the standard animal model for experimental autoimmune encephalomyelitis (EAE) which represents MS-like conditions. We were able to measure the kinetics of the partial formation of a double bilayer in those systems and to characterize the scattering length density profiles of the initial and final states of the membrane. The kinetics could be modeled using a random sequential adsorption simulation. By using a free energy minimization method, we were able to calculate the shape of the adhered vesicles and to determine the adhesion energy per MBP. For the native membrane the resulting adhesion energy per MBP is larger than that of the EAE modified membrane type. Our observations might help in understanding myelination and especially remyelination—a process in which damaged myelin is repaired—which is a promising candidate for treatment of the still mostly incurable demyelinating diseases such as MS.

Electrostatic Effects on Model Membrane Compressibility and Vesicle Adhesion

Though vesicles are recognized as effective drug carriers and functionalized with polymers, targeting ligands, imaging agents, and antibodies for drug delivery applications, there is minimal knowledge of the role of lipid bilayer properties in a vesicle's ability to interact with target membranes. Phospholipids within the cellular lipid membrane are chemically diverse, with tail lengths, saturation, and headgroup chemistry varying depending on tissue function and health condition. These function and status dependent compositional observations raise questions on how the underlying membrane properties, such as thickness, fluidity, and compressibility, alter biophysical processes and regulate the transport of vesicles across the membrane. In this work we use compositionally controlled, free-standing large area model biomembranes (LAMB) as an artificial platform to investigate the ramifications of altered phospholipid composition on vesicle-membrane interactions. First, simultaneous area and capacitance measurements of the LAMB allow us to determine the thickness and Young's modulus (compressibility) of a given membrane of varied compositions. In particular, we focus on the effect of headgroup charge, which shows that increasing anionic phospholipid composition increases the membrane stiffness. Next, small (100nm) vesicles composed of DOPC, DOPE, and DOPE-Rh are introduced and subsequently electrostatically adhered to the LAMB. We use Ca2+ ions as a trigger to initiate fusion between the adhered vesicles and LAMB while monitoring fluorescence and capacitance. We observe interesting morphological changes at the membrane interface such as vesicle-vesicle fusion, formation of worm-like vesicle aggregates, and vesicle-membrane fusion. Current work is focused analyzing these events and calculating the activation energy of membrane fusion by simultaneous fluorescence and capacitance measurements. Characterizing the relationship between energy of fusion and vesicle/membrane composition will provide a basis for the rational design of vesicles for targeted membrane interaction.

Vesicle-Based Gel via Polyelectrolyte-Induced Adhesion: Structure, Rheology, and Response.

We describe an experimental study of soft solids composed of micron-scale lipid bilayer vesicles that adhere to one another through electrostatic attraction to an oppositely charged polymer (PDADMAC). As the polymer concentration was increased, we found a fluid phase, a solid gel phase, and a gel composed of internally reorganized vesicles. Optical microscopy images showed a nearly close-packed structure of adhered vesicles that retained their closed-cell morphology. Shear rheology measurements showed that the gel phase is a solid with a modulus at the Pa scale and with linear response up to 70% strain. We found that the modulus depends on the energy per area of membrane-membrane adhesion but does not depend on the vesicle size. We further found that the gels survived osmotic stress or dilution of the adhering polymer but could be rapidly disrupted in response to the addition of strongly binding silica nanoparticles. These results demonstrate the potential for cell-sized lipid vesicles to form a solid platform that maintains the responsive properties of the membranes. Such materials may find applications as triggerable, protective coatings of delicate surfaces.

Recurrent dynamics of rupture transitions of giant lipid vesicles at solid surfaces

Single giant unilamellar vesicles (GUVs) rupture spontaneously from their salt-laden suspension onto solid surfaces. At hydrophobic surfaces, the GUVs rupture via a recurrent, bouncing ball rhythm. During each contact, the GUVs, rendered tense by the substrate interactions, porate, and spread a molecularly transformed motif of a monomolecular layer on the hydrophobic surface from the point of contact in a symmetric manner. The competition from pore closure, however, limits the spreading and produces a daughter vesicle, which re-engages with the substrate. At solid hydrophilic surfaces, by contrast, GUVs rupture via a distinctly different recurrent burst-heal dynamics; during burst, single pores nucleate at the contact boundary of the adhering vesicles, facilitating asymmetric spreading and producing a “heart”-shaped membrane patch. During the healing phase, the competing pore closure produces a daughter vesicle. In both cases, the pattern of burst-reseal events repeats multiple times, splashing and spreading the vesicular fragments as bilayer patches at the solid surface in a pulsatory manner. These remarkable recurrent dynamics arise, not because of the elastic properties of the solid surface, but because the competition between membrane spreading and pore healing, prompted by the surface-energy-dependent adhesion, determine the course of the topological transition.

Morphologies of Vesicle Doublets: Competition among Bending Elasticity, Surface Tension, and Adhesion

To study the mechanical laws governing the form of multicellular organisms, we examine the morphology of adhering vesicle doublets as the simplest model system. We monitor the morphological transformations of doublets induced by changes of adhesion strength and volume/area ratio, which are controlled by intermembrane interactions and thermal area expansion, respectively. When we increase the temperature in the weak adhesion regime, a dumbbell flat-contact doublet is transformed to a parallel-prolate doublet, whereas in the strong adhesion regime, heating transforms the dumbbell flat-contact doublet into a spherical sigmoid-contact doublet. We reproduce the observed doublet morphologies by numerically minimizing the total energy, including the contact-potential adhesion term as well as the surface and bending terms, using the Surface Evolver package. From the reproduced morphologies, we extract the adhesion strength, the surface tension, and the volume/area ratio of the vesicles, which reveals the detailed mechanisms of the morphological transitions in doublets.

Shape Transformations of Vesicles Induced by Their Adhesion to Flat Surfaces.

The shape transformations of lipid vesicles induced by the adhesion to a flat surface is investigated. We perform the calculations within the framework of the Helfrich spontaneous curvature model. The calculations were performed for a few values of the reduced volume and the spontaneous curvature. The range of stability for different shapes (oblate, prolate, and stomatocyte) of adhered vesicles is determined. New physical phenomena such as budding induced by the adhesion of vesicles are reported.

Fusion Pores Live on the Edge.

Biological transmission of vesicular content occurs by opening of a fusion pore. Recent experimental observations have illustrated that fusion pores between vesicles that are docked by an extended flat contact zone are located at the edge (vertex) of this zone. We modeled this experimentally observed scenario by coarse-grained molecular simulations and elastic theory. This revealed that fusion pores experience a direct attraction toward the vertex. The size adopted by the resulting vertex pore strongly depends on the apparent contact angle between the adhered vesicles even in the absence of membrane surface tension. Larger contact angles substantially increase the equilibrium size of the vertex pore. Because the cellular membrane fusion machinery actively docks membranes, it facilitates a collective expansion of the contact zone and increases the contact angle. In this way, the fusion machinery can drive expansion of the fusion pore by free energy equivalents of multiple tens of kBT from a distance and not only through the fusion proteins that reside within the fusion pore.

Vesicle adhesion in the electrostatic strong-coupling regime studied by time-resolved small-angle X-ray scattering.

We have used time-resolved small-angle X-ray scattering (SAXS) to study the adhesion of lipid vesicles in the electrostatic strong-coupling regime induced by divalent ions. The bilayer structure and the interbilayer distance dw between adhered vesicles was studied for different DOPC:DOPS mixtures varying the surface charge density of the membrane, as well as for different divalent ions, such as Ca2+, Sr2+, and Zn2+. The results are in good agreement with the strong coupling theory predicting the adhesion state and the corresponding like-charge attraction based on ion-correlations. Using SAXS combined with the stopped-flow rapid mixing technique, we find that in highly charged bilayers the adhesion state is only of transient nature, and that the adhering vesicles subsequently transform to a phase of multilamellar vesicles, again with an inter-bilayer distance according to the theory of strong binding. Aside from the stopped-flow SAXS instrumentations used primarily for these results, we also evaluate microfluidic sample environments for vesicle SAXS in view of future extension of this work.

Mechanism of Membrane Biogenesis

Biological membrane in addition to delimiting the cell and its intracellular compartments, govern a wide range of life processes including ion transport, cell division, development, growth, and migration. The addition of new membrane to existing cellular membranes may involve fusion of preformed proteoliposomes and or the incorporation of intracellular free lipids and proteins. It is reported that supported lipid bilayers with adhered lipid vesicles when laterally stretched, expand due to fusion of the adhered vesicles. Mathematical modeling of the plasma membrane suggests trafficking of lipids into stretched membranes to relieve tension. Cell swelling has been reported during mitosis and mechanical forces are implicated in morphogenesis and development. However, the process of addition of new membrane to preexisting membranes in live cells is poorly understood. Here we report in live cells, that in addition to membrane stretch, composition of the existing membrane dictates membrane growth through the selective incorporation of both proteins and lipids.Support or Funding InformationWork presented in this article was supported in part by the National Science Foundation grants EB00303, CBET1066661 (BPJ); the WSU Interdisciplinary Biomedical Systems Fellow‐ship and Graduate Research Assistantship (ARN), the WSU Interdisciplinary Biomedical Systems Fellow‐ship and Graduate Research Assistantship (KTL). All authors critically analyzed results and proof read the manuscript. The authors declare no competing financial interests or conflicts.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

Competition between Bending and Internal Pressure Governs the Mechanics of Fluid Nanovesicles.

Nanovesicles (∼100 nm) are ubiquitous in cell biology and an important vector for drug delivery. Mechanical properties of vesicles are known to influence cellular uptake, but the mechanism by which deformation dynamics affect internalization is poorly understood. This is partly due to the fact that experimental studies of the mechanics of such vesicles remain challenging, particularly at the nanometer scale where appropriate theoretical models have also been lacking. Here, we probe the mechanical properties of nanoscale liposomes using atomic force microscopy (AFM) indentation. The mechanical response of the nanovesicles shows initial linear behavior and subsequent flattening corresponding to inward tether formation. We derive a quantitative model, including the competing effects of internal pressure and membrane bending, that corresponds well to these experimental observations. Our results are consistent with a bending modulus of the lipid bilayer of ∼14kbT. Surprisingly, we find that vesicle stiffness is pressure dominated for adherent vesicles under physiological conditions. Our experimental method and quantitative theory represents a robust approach to study the mechanics of nanoscale vesicles, which are abundant in biology, as well as being of interest for the rational design of liposomal vectors for drug delivery.

Modulating Vesicle Adhesion by Electric Fields

We introduce an experimental setup for modulating adhesion of giant unilamellar vesicles to a planar substrate. Adhesion is induced by the application of an external potential to a transparent indium tin oxide-coated electrode (the substrate), which enables single-vesicle studies. We demonstrate tunable and reversible adhesion of negatively charged vesicles. The adhesion energy at different potentials is calculated from the vesicle shape assessed with confocal microscopy. Two approaches for these estimates are employed: one based on the whole contour of the vesicle and a second based on the contact curvature of the membrane in the vicinity of the substrate. Both approaches agree well with each other and show that the adhering vesicles are in the weak adhesion regime for the range of explored external potentials. Using fluorescence quenching assays, we detect that, in the adhering membrane segment, only the outer bilayer leaflet of the vesicle is depleted of negatively charged fluorescent lipids, while the inner leaflet remains unaffected. We show that depletion of negatively charged lipids is consistent Poisson-Boltzmann theory, taking into account charge regulation from lipid mobility. Finally, we also show that lipid diffusion is not significantly affected in the adhering membrane segment. We believe that the approaches introduced here for modulating and assessing vesicle adhesion have many potential applications in the field of single-vesicle studies and research on membrane adhesion.

Probing Membrane Viscosity and Interleaflet Friction of Supported Lipid Bilayers by Tracking Electrostatically Adsorbed, Nano-Sized Vesicles.

Particle tracking is used to measure the diffusional motion of nanosized (≈100 nm), lipid vesicles that are electrostatically adsorbed onto a solid supported lipid bilayer. It is found that the motion of membrane-adhering vesicles is Brownian and depends inversely on the vesicle size, but is insensitive to the vesicle surface charge. The measured diffusivity agrees well with the Evans-Sackmann model for the diffusion of inclusions in supported, fluidic membranes. The agreement implies that the vesicle motion is coupled to that of a nanoscopic lipid cluster in the upper leaflet, which slides over the lower leaflet. The diffusivity of membrane-adhering vesicles is therefore predominantly governed by the interleaflet friction coefficient, while the diffusivity of single lipids is mainly governed by the membrane viscosity. Combined with fluorescence recovery after photobleaching analysis, the interleaflet friction coefficient and the membrane viscosity are determined by applying the Evans-Sackmann model to the measured diffusivity of membrane adhering vesicles and that of supported membrane lipids. This approach provides an alternative to existing methods for measuring the interleaflet friction coefficient and the membrane viscosity.

Epsin N-terminal Homology Domain (ENTH) Activity as a Function of Membrane Tension

The epsin N-terminal homology domain (ENTH) is a major player in clathrin-mediated endocytosis. To investigate the influence of initial membrane tension on ENTH binding and activity, we established a bilayer system based on adhered giant unilamellar vesicles (GUVs) to be able to control and adjust the membrane tension σ covering a broad regime. The shape of each individual adhered GUV as well as its adhesion area was monitored by spinning disc confocal laser microscopy. Control of σ in a range of 0.08-1.02 mN/m was achieved by altering the Mg(2+) concentration in solution, which changes the surface adhesion energy per unit area of the GUVs. Specific binding of ENTH to phosphatidylinositol 4,5-bisphosphate leads to a substantial increase in adhesion area of the sessile GUV. At low tension (<0.1 mN/m) binding of ENTH can induce tubular structures, whereas at higher membrane tension the ENTH interaction deflates the sessile GUV and thereby increases the adhesion area. The increase in adhesion area is mainly attributed to a decrease in the area compressibility modulus KA We propose that the insertion of the ENTH helix-0 into the membrane is largely responsible for the observed decrease in KA, which is supported by the observation that the mutant ENTH L6E shows a reduced increase in adhesion area. These results demonstrate that even in the absence of tubule formation, the area compressibility modulus and, as such, the bending rigidity of the membrane is considerably reduced upon ENTH binding. This renders membrane bending and tubule formation energetically less costly.