Adhesion Of Vesicles Research Articles

Specific Adhesion of Giant Plasma Membrane Vesicles to Surface-Immobilized SIRPα by Membrane Reconstituted “Marker of Self” Signaling Protein CD47

Model membranes consisting of pure lipid components have clearly advanced the understanding of biological cell membranes. However, the reconstitution of membrane proteins into model lipid bilayers is not straightforward and requires specific strategies for each protein system. In contrast, membranes of living cells have a complex composition with a multitude of different lipid components, proteins and sugars. In this work, we show that membrane blebs derived from the plasma membrane of living cells, also called giant plasma membrane vesicles, can be used to study the interaction of the membrane proteins CD47 and SIRPα, which are involved in immune signaling. Membrane blebs are an interesting model because they exhibit the compositional heterogeneity of the plasma membrane but their morphological behavior can still be described by a few parameters such as bending rigidity, reduced volume and tension. Specifically, we study the interaction of CD47 reconstituted in blebs and surface-immobilized SIRPα. Using this model system we explore membrane-mediated cooperative binding effects under different biologically relevant conditions. We find that acidic environment decreases the binding affinity of membrane-reconstituted CD47 to SIRPα. Finally, we discuss implications for in-vitro cancer models. Financial support from the Deutsche Forschungsgemeinschaft (DFG) via the IRTG 1524 is gratefully acknowledged.

A new approach to follow a single extracellular vesicle-cell interaction using optical tweezers.

Extracellular vesicles (EVs) are spherical membrane structures released by most cells. These highly conserved mediators of intercellular communication carry proteins, lipids, and nucleic acids, and transfer these cellular components between cells by different mechanisms, such as endocytosis, macropinocytosis, or fusion. However, the temporal and spatial dynamics of vesicle-cell interactions still remain largely unexplored. Here we used optical tweezers to drive single EVs produced by microglial cells onto the surface of astrocytes or microglia in primary culture. By visualizing single EV-cell contacts, we observed that microglial vesicles displayed different motilities on the surface of astrocytes compared with microglia. After contact, EVs positioned on astrocytes displayed some minor oscillatory motion around the point of adhesion, while vesicles dragged to microglia displayed quite regular directional movement on the plasma membrane. Both the adhesion and motion of vesicles on glial cells were strongly reduced by cloaking phosphatidylserine (PS) residues, which are externalized on the vesicle membrane and act as determinants for vesicle recognition by target cells. These data identify optical manipulation as a powerful tool to monitor in vitro vesicle-cell dynamics with high temporal and spatial resolution and to determine in a quantitative manner the contribution of surface receptors/extracellular protein ligands to the contact.

Enhanced antioxidation via encapsulation of isooctyl p-methoxycinnamate with sodium deoxycholate-mediated liposome endocytosis

Isooctyl p-methoxycinnamate(OMC) is a commonly used chemical ultraviolet B sunscreen that suffers rapid degradation with current delivery systems following sun exposure. In this study, deoxycholate-mediated liposome (DOC-LS) endocytosis was employed to improve the antioxidation effects of OMC following topical administration, and the in vitro cell uptake was investigated to understand the enhanced cutaneous absorption of the drug via this nanocarrier. Following topical application, structural changes in the stratum corneum were identified. With the increase of DOC content, the drug deposition in skin decreased; from this, a DOC-LS formulation was selected that showed significantly more drug delivery in skin than did the other preparations (P<0.05). DOC-LS decreased skin resistance, suggesting its ability to induce skin barrier disruption. In vitro HaCaT keratinocyte cell uptake of coumarin-6 incorporated in the two types of phosphatidylcholine (PC) vesicles (i.e., LS or DOC-LS) yielded similar fluorescence intensities following incubation for different periods (P<0.05). However, CCC-ESF-1 embryonic fibroblast cell uptake of the fluorescence revealed time-dependence, and the emitted light from DOC-LS incubated cells was stronger than that from cells incubated with LS (P<0.05). These findings might be associated with the endocytic pathway of HaCaT, which mainly exhibited adsorption or physical adhesion of the fluorescent vesicles, whereas CCC-ESF-1 markedly internalized the PC vesicles via the lysosomes, as shown by intracellular fluorescence co-location studies. Following loading with the same amount of OMC, the DOC-LS vesicles exhibited superior skin tissue antioxidative capacity among the preparations tested, corroborating the in vivo skin drug deposition results. Thus, our results suggest that DOC-LS is a promising system for OMC dermal delivery without promoting skin irritation, which is quite advantageous for therapeutic purposes.

Electrochemical Detection of Single Phospholipid Vesicle Collisions at a Pt Ultramicroelectrode.

We report the collision behavior of single unilamellar vesicles, composed of a bilayer lipid membrane (BLM), on a platinum (Pt) ultramicroelectrode (UME) by two electrochemical detection methods. In the first method, the blocking of a solution redox reaction, induced by the single vesicle adsorption on the Pt UME, can be observed in the amperometric i-t response as current steps during the electrochemical oxidation of ferrocyanide. In the second technique, the ferrocyanide redox probe is directly encapsulated inside vesicles and can be oxidized during the vesicle collision on the UME if the potential is poised positive enough for ferrocyanide oxidation to occur. In the amperometric i-t response for the latter experiment, a current spike is observed. Here, we report the vesicle blocking (VB) method as a relevant technique for determining the vesicle solution concentration from the collisional frequency and also for observing the vesicle adhesion on the Pt surface. In addition, vesicle reactor (VR) experiments show clear evidence that the lipid bilayer membrane does not collapse or break open at the Pt UME during the vesicle collision. Because the bilayer is too thick for electron tunneling to occur readily, an appropriate concentration of a surfactant, such as Triton X-100 (TX100), was added in the VR solution to induce loosening of the bilayer (transfection conditions), allowing the electrode to oxidize the contents of the vesicle. With this technique, the TX100 effect on the vesicle lipid bilayer permeability can be evaluated through the current spike charge and frequency corresponding to redox vesicle collisions.

From vesicles to protocells: the roles of amphiphilic molecules.

It is very challenging to construct protocells from molecular assemblies. An important step in this challenge is the achievement of vesicle dynamics that are relevant to cellular functions, such as membrane trafficking and self-reproduction, using amphiphilic molecules. Soft matter physics will play an important role in the development of vesicles that have these functions. Here, we show that simple binary phospholipid vesicles have the potential to reproduce the relevant functions of adhesion, pore formation and self-reproduction of vesicles, by coupling the lipid geometries (spontaneous curvatures) and the phase separation. This achievement will elucidate the pathway from molecular assembly to cellular life.

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 of giant unilamellar vesicles on double-end grafted DNA carpets

We have recently shown that the bio-mimetic adhesion of Giant Unilamellar Vesicles on carpets of lambda-phage DNAs, grafted by one end to the substrate, leads to DNA scraping and stapling. As the lipid adhesion patch is built, outward forces stretch the DNA, while adhesion patch formation staples the chains into frozen conformations, trapped between the GUV membrane and the substrate. Analysis of the scraped and stapled DNA conformations provides a wealth of information about the membrane/polymer interactions at play during the formation of a bio-adhesive contact zone. In this paper we report new phenomena revealed by scraping and stapling phenomena associated with the bio-mimetic adhesion of Giant Unilamellar Vesicles on carpets of lambda-phage DNAs that were grafted to the substrate by both ends. In particular, the peculiar shapes of stapled DNA observed in this case, suggest that the membrane exerces not only outward radial forces during patch formation, but is is also able to confine the DNA molecules in the orthoradial direction.

Contribution of the hydration force to vesicle adhesion on titanium oxide.

Titanium oxide is a biocompatible material that supports vesicle adhesion. Depending on experimental parameters, adsorbed vesicles remain intact or rupture spontaneously. Vesicle rupture has been attributed to electrostatic attraction between vesicles and titanium oxide, although the relative contribution of various interfacial forces remains to be clarified. Herein, we investigated the influence of vesicle surface charge on vesicle adsorption onto titanium oxide and observed that electrostatic attraction is insufficient for vesicle rupture. Following this line of evidence, a continuum model based on the DLVO forces and a non-DLVO hydration force was applied to investigate the role of different interfacial forces in modulating the lipid-substrate interaction. Within an experimentally significant range of conditions, the model shows that the magnitude of the repulsive hydration force strongly influences the behavior of adsorbed vesicles, thereby supporting that the hydration force makes a strong contribution to the fate of adsorbed vesicles on titanium oxide. The findings are consistent with literature reports concerning phospholipid assemblies on solid supports and nanoparticles and underscore the importance of the hydration force in influencing the behavior of phospholipid films on hydrophilic surfaces.

Vesicle Adhesion and Rupture on Silicon Oxide: Influence of Freeze–Thaw Pretreatment

We have investigated the effect of freeze-thaw (FT) pretreatment on the adhesion and rupture of extruded vesicles over a wide range of vesicle sizes. To characterize the size distributions of vesicles obtained with and without FT pretreatment, dynamic light scattering (DLS) experiments were performed. The interaction between extruded vesicles and a silicon oxide substrate was investigated by quartz crystal microbalance with dissipation (QCM-D) monitoring, with a focus on comparative analysis of similar-sized vesicles with and without FT pretreatment. Under this condition, there was a smaller mass load at the critical coverage associated with untreated vesicles, as compared to vesicles which had been subjected to FT pretreatment. In addition, the rupture of treated vesicles generally resulted in formation of a complete planar bilayer, while the adlayer was more heterogeneous when employing untreated vesicles. Combined with kinetic analysis and extended-DLVO model calculations, the experimental evidence suggests that the differences arising from FT pretreatment are due to characteristics of the vesicle size distribution and also multilamellarity of an appreciable fraction of untreated vesicles. Taken together, our findings clarify the influence of FT pretreatment on model membrane fabrication on solid supports.

Axial nanoscale localization by normalized total internal reflection fluorescence microscopy

We present a simple modification of a standard total internal reflection fluorescence microscope to achieve nanometric axial resolution, typically ≈10 nm. The technique is based on a normalization of total internal reflection images by conventional epi-illumination images. We demonstrate the potential of our method to study the adhesion of phopholipid giant unilamellar vesicles.

Targeting of a magnetic bionanomaterial to HepG2 human hepatocellular carcinoma cells using a galactose terminated lipid

ABSTRACTMagnetic nanoparticle-vesicle aggregates (MNPVs), a controlled release nanostructure, have been enhanced with the inclusion of a novel galactose terminated lipid for cell targeting. Quartz crystal microgravimetry with dissipation (QCM-D) demonstrated that the galactose headgroup was available to bindErythrina Crista-gallilectin (ECL) when the lipid was incorporated into a lipid bilayer. Similarly, UV-visible spectrophotometry indicated that ECL recognized the galactose headgroup in vesicles, leading to vesicle adhesion and aggregation. Finally, confocal fluorescence microscopy was used to assess the galactose-mediated interaction of both vesicles and MNPVs with HepG2 human hepatocellular carcinoma cells expressing the asialoglycoprotein (ASGPR) galactose receptor.

Binding constants of membrane-anchored receptors and ligands depend strongly on the nanoscale roughness of membranes

Cell adhesion and the adhesion of vesicles to the membranes of cells or organelles are pivotal for immune responses, tissue formation, and cell signaling. The adhesion processes depend sensitively on the binding constant of the membrane-anchored receptor and ligand proteins that mediate adhesion, but this constant is difficult to measure in experiments. We have investigated the binding of membrane-anchored receptor and ligand proteins with molecular dynamics simulations. We find that the binding constant of the anchored proteins strongly decreases with the membrane roughness caused by thermally excited membrane shape fluctuations on nanoscales. We present a theory that explains the roughness dependence of the binding constant for the anchored proteins from membrane confinement and that relates this constant to the binding constant of soluble proteins without membrane anchors. Because the binding constant of soluble proteins is readily accessible in experiments, our results provide a useful route to compute the binding constant of membrane-anchored receptor and ligand proteins.

Influence of Osmotic Pressure on Adhesion of Lipid Vesicles to Solid Supports

The adhesion of lipid vesicles to solid supports represents an important step in the molecular self-assembly of model membrane platforms. A wide range of experimental parameters are involved in controlling this process, including substrate material and topology, lipid composition, vesicle size, solution pH, ionic strength, and osmotic pressure. At present, it is not well understood how the magnitude and direction of the osmotic pressure exerted on a vesicle influence the corresponding adsorption kinetics. In this work, using quartz crystal microbalance with dissipation (QCM-D) monitoring, we have experimentally studied the role of osmotic pressure in the adsorption of zwitterionic vesicles onto silicon oxide. The osmotic pressure was induced by changing the ionic strength of the solvent across an appreciably wider range (from 25 to 1000 mM NaCl outside of the vesicle, and 125 mM NaCl inside of the vesicle, unless otherwise noted) compared to that used in earlier works. Our key finding is demonstration that, by changing osmotic pressure, all three generic types of the kinetics of vesicle adsorption and rupture can be observed in one system, including (i) adsorption of intact vesicles, (ii) adsorption and rupture after reaching a critical vesicle coverage, and (iii) rupture just after adsorption. Furthermore, theoretical analysis of pressure-induced deformation of adsorbed vesicles and a DLVO-type analysis of the vesicle-substrate interaction qualitatively support our observations. Taken together, the findings in this work demonstrate that osmotic pressure can either promote or impede the rupture of adsorbed vesicles on silicon oxide, and offer experimental evidence to support adhesion energy-based models that describe the adsorption and spontaneous rupture of vesicles on solid supports.

Label-free optical monitoring of surface adhesion of extracellular vesicles by grating coupled interferometry

In this proof-of-principle study a label-free optical sensor is demonstrated to monitor the surface adhesion of extracellular vesicles secreted by live cells on to various extracellular matrix proteins.

Supramolecular Approaches to Combining Membrane Transport with Adhesion

Cells carefully control the transit of compounds through their membranes using “gated” protein channels that respond to chemical stimuli. Connexin gap junctions, which are high conductance cell-to-cell channels, are a remarkable class of “gated” channel with multiple levels of assembly. A gap junction between adhering cells comprises two half-channels in each cell membrane that adhere to each other to form a continuous cell-to-cell channel. Each half-channel is a hexameric assembly of six protein transmembrane subunits. These gap junctions display both intramembrane assembly and intermembrane assembly, making them an attractive target for biomimetic studies. Although many examples of self-assembled channels have been developed, few can also mediate intermembrane adhesion. Developing systems that combine membrane adhesion with controlled transit across the membrane would not only provide a better understanding of self-assembly in and around the membrane, but would also provide a route towards smart biomaterials, targeted drug delivery and an interface with nanotechnology.This Account describes our biomimetic approaches to combining membrane adhesion with membrane transport, using both self-assembled “sticky” pores and “sticky” nanoparticles to trigger transit across membranes. This combination links both fundamental and applied research, acting as a bridge between molecular level assembly and the formation of functional biomaterials. The ultimate goal is to create complex self-assembled systems in biological or biomimetic environments that can both interface with cells and transport compounds across bilayers in response to remote chemical or electromagnetic signals. Our research in this area started with fundamental studies of intramembrane and intermembrane self-assembly, building upon previously known channel-forming compounds to create self-assembled channels that were switchable or able to mediate vesicle–vesicle adhesion. Subsequently, nanoparticles with a “sticky” coating were used to mediate adhesion between vesicles. Combining these adhesive properties with the unique characteristics of nanosized magnetite allowed a noninvasive magnetic signal to trigger transport of compounds out of magnetic nanoparticle-vesicle assemblies. Adding an extravesicular matrix produced new responsive biomaterials for use in tissue engineering. These biomaterials can be magnetically patterned and can deliver drugs upon receipt of a magnetic signal, allowing spatiotemporal control over cellular responses.

Adhesion of a vesicle on an elastic substrate: 2D analysis

Cell or vesicle adhesion plays an essential role in a plethora of physiological activities. In this study, we established a theoretical model to explore the adhesion behavior of a vesicle adhering on an elastic substrate. Based upon the free energy functional of the system, the governing equation set and the transversality boundary conditions were derived. The morphology of the vesicle-substrate system and the phase diagram were presented, and it was found that there exist different wrapping states depending on the work of adhesion and bending stiffness. Finally, the adhesion behavior of a vesicle to a rigid substrate was investigated. These analyses are beneficial to understanding the mechanism of cell motility, and cast a new light on the droplet wrapped by a membrane when input the voltage

Molecular modeling of the pathways of vesicle–membrane interaction

In this work we systematically investigate the pathways of the interaction between elastic vesicles and lipid membranes with the aid of computer simulation techniques. Different vesicle responses to the vesicle–membrane adhesion, including vesicle fusion, vesicle hemi-fusion, vesicle adhesion, vesicle endocytosis and vesicle rupture, are observed from our simulations. We also investigate how the pathways of vesicle–membrane interaction depend on the adhesion strength, and the membrane and vesicle properties.

1866 – Novel candidate genomic loci for mental retardation

Introduction Genetic disorders underlie a significant number of cases of mental retardation (MR). However, the traditionally used routine karyotyping is not able to detect small but often clinically relevant aberrations. High-resolution genome-wide microarray technologies may help to increase the detectability of MR etiology. Objectives To improve MR diagnostics. Aims To identify the novel CNV regions and susceptibility genes in MR patients. Methods Large chromosomal rearrangements, biochemical defects, and known monogenic syndromes accompanied by MR were ruled out for 71 patients. Further the genome-wide analysis using CGH Microarray Kits 4×44K and 8×60K (Agilent Technologies, USA) was performed. Results Eight novel CNVs in 9 patients not described in healthy individuals and not associated with any disease were indentified implicating regions at 11q22.3, 5q33.1, 3p26.3, 15q11.1-q11.2, 15q22.2, 1q25.1-q25.2, 7q21.3, 2q12.3. They include candidate genes DDX10 (putative RNA helicase), IL17B (cytokine, primarily localized to neuronal cell bodies), CNTN6 (may play a role in the formation of axon connections), TLN2 (plays a role in cell adhesion and recycling of synaptic vesicles), TNR (an extracellular matrix protein, expressed primarily in the central nervous system), ASTN1 (a neuronal adhesion molecule), PON1 (detoxifies organophosphate neurotoxicants), and SLC5A7 (choline transporter in the brain and periphery). Conclusions Previously unknown CNVs, containing genes responsible for proper central nervous system functioning, were detected in 13% of patients. The novel CNVs and genes identified by aCGH may help discovering new etiological mechanisms of MR. This study was supported by European Community's Seventh Framework Programme, CHERISH (project 223692) and by Federal Program (grant 8727).

Simulation of edge facilitated adsorption and critical concentration induced rupture of vesicles at a surface

We investigate the kinetics of supported lipid bilayer formation by the adsorption and rupture of uncharged phosphatidylcholine lipid vesicles on to a solid substrate. We model the adsorption process taking into account the distinct vesicle rupture events and growth processes. This includes (i) the initial adhesion and vesicle rupture that nucleates bilayer islands, (ii) the growth and merger of bilayer islands, (iii) enhanced adhesion of vesicles to the bilayer edge, and (iv) the final desorption of excess vesicles from the substrate. These simulation studies give insight into prior experimental observations of adsorption in which an overloading of lipid on the solid substrate occurs before formation of the final supported lipid bilayer. Our model provides an explanation for the features of the interesting universal master curve that was observed for the surface fluorescence intensity in the experimental investigations of Weirich et al.

SNAREpin Assembly by Munc18-1 Requires Previous Vesicle Docking by Synaptotagmin 1

Regulated exocytosis requires the general membrane fusion machinery-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Sec1/Munc18 (SM) proteins. Using reconstituted giant unilamellar vesicles containing preassembled t-SNARE proteins (syntaxin 1·SNAP-25), we determined how Munc18-1 controls the docking, priming, and fusion of small unilamellar vesicles containing the v-SNARE VAMP2 and the Ca(2+) sensor synaptotagmin 1. In vitro assays allowed us to position Munc18-1 in the center of a sequential reaction cascade; vesicle docking by synaptotagmin 1 is a prerequisite for Munc18-1 to accelerate trans-SNARE complex (SNAREpin) assembly and membrane fusion. Complexin II stalls SNAREpin zippering at a late stage and, hence, contributes to synchronize membrane fusion in a Ca(2+)- and synaptotagmin 1-dependent manner. Thus, at the neuronal synapse, the priming factor Munc18-1 may accelerate the conversion of docked synaptic vesicles into a readily releasable pool by activating SNAREs for efficient membrane fusion.