Facile and Rapid Formation of Giant Vesicles from Glass Beads.
Ontology highlight
ABSTRACT:
SUBMITTER: Tanasescu R
PROVIDER: S-EPMC6414939 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Giant unilamellar vesicles (GUVs) are model cell-sized systems that have broad applications including drug delivery, analysis of membrane biophysics, and synthetic reconstitution of cellular machineries. Although numerous methods for the generation of free-floating GUVs have been established over the past few decades, only a fraction have successfully produced uniform vesicle populations both from charged lipids and in buffers of physiological ionic strength. In the method described here, we generate large numbers of free-floating GUVs through the rehydration of lipid films deposited on soft polyacrylamide (PAA) gels. We show that this technique produces high GUV concentrations for a range of lipid types, including charged ones, independently of the ionic strength of the buffer used. We demonstrate that the gentle hydration of PAA gels results in predominantly unilamellar vesicles, which is in contrast to comparable methods analyzed in this work. Unilamellarity is a defining feature of GUVs and the generation of uniform populations is key for many downstream applications. The PAA method is widely applicable and can be easily implemented with commonly utilized laboratory reagents, making it an appealing platform for the study of membrane biophysics.
| S-EPMC7075891 | biostudies-literature
Project description:Giant unilamellar vesicles or GUVs are systems of choice as biomimetic models of cellular membranes. Although a variety of procedures exist for making single walled vesicles of tens of microns in size, the range of lipid compositions that can be used to grow GUVs by the conventional methods is quite limited, and many of the available methods involve energy input that can damage the lipids or other molecules present in the growing solution for embedment in the membrane or in the vesicle interior. Here, we show that a wide variety of lipids or lipid mixtures can grow into GUVs by swelling lipid precursor films on top of a dried polyvinyl alcohol gel surface in a swelling buffer that can contain diverse biorelevant molecules. Moreover, we show that the encapsulation potential of this method can be enhanced by combining polyvinyl alcohol-mediated growth with inverse-phase methods, which allow (bio)molecule complexation with the lipids.
| S-EPMC3699747 | biostudies-literature
Project description:Giant lipid vesicles (GVs) are emerging models for investigating the properties and reactivity of cell-like microcompartments, providing useful information about plausible protocellular structures in primitive times, as well as for the modern synthetic biology goal of constructing the first artificial cell from its reconstituted and partly modified components. Here we explore a novel methodology of GV purification by microfiltration under reduced pressure, operated by a simple apparatus. The method has been characterized in terms of flow rate, amount of lipid loss, quality of recovered GVs, and size distribution. A case study is reported to show the practicability of GV microfiltration. A clickable fluorescent probe was encapsulated inside GVs; more than 99.9% of the non-entrapped probe was easily and rapidly removed by multiple microfiltrations. This novel methodology is briefly discussed as a future tool for selection experiments on GV populations.
| S-EPMC5815610 | biostudies-literature
Project description:DNA nanostructures assembled on living cell membranes have become powerful research tools. Synthetic lipid membranes have been used as a membrane model to study the dynamic behavior of DNA nanostructures on fluid soft lipid bilayers, but without the inherent complexity of natural membranes. Herein, we report the assembly and disassembly of DNA nanoprisms on cell-mimicking micrometer-scale giant membrane vesicles derived from living mammalian cells. Three-dimensional DNA nanoprisms with a DNA arm and a cholesterol anchor were efficiently localized on the membrane surface. The assembly and disassembly of DNA nanoprisms were dynamically manipulated by DNA strand hybridization and toehold-mediated strand displacement. Furthermore, the heterogeneity of reversible assembly/disassembly of DNA nanoprisms was monitored by Förster resonance energy transfer. This study suggests the feasibility of DNA-mediated functional biomolecular assembly on cell membranes for biomimetics studies and delivery systems.
| S-EPMC5877790 | biostudies-literature
Project description:Soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins are the main catalysts for membrane fusion in the secretory pathway of eukaryotic cells. In vitro, SNAREs are sufficient to mediate effective fusion of both native and artificial membranes. Here we have established, to our knowledge, a new platform for monitoring SNARE-mediated docking and fusion between giant unilamellar vesicles (GUVs) and smaller liposomes or purified secretory granules with high temporal and spatial resolution. Analysis of fusion is restricted to the free-standing part of the GUV-membrane exhibiting low curvature and a lack of surface contact, thus avoiding adhesion-mediated interference with the fusion reaction as in fusion with supported bilayers or surface-immobilized small vesicles. Our results show that liposomes and chromaffin granules fuse with GUVs containing activated SNAREs with only few milliseconds delay between docking and fusion. We conclude that after initial contact in trans, SNAREs alone can complete fusion at a rate close to fast neuronal exocytosis.
| S-EPMC5607038 | biostudies-literature
Project description:Beta-cyclodextrin (?-CD) was covalently immobilized on glass surface. Functionalized glass surface was characterized by X-ray photoelectron spectroscopy (XPS) and elemental analysis. Glass surface functionalized with ?-CD was used to remove cholesterol from ghee (clarified butter fat). 78.8 % cholesterol was reduced in 2 h at 25 °C and 170 rpm. Same surface was used repeatedly for 10 cycles and no reduction in cholesterol removal efficiency was observed. Modified glass surface showed almost no degradation in repeated use in cholesterol reduction experiments.
| S-EPMC4325052 | biostudies-literature
Project description:Domain formation is modeled on the surface of giant unilamellar vesicles using a Landau field theory model for phase coexistence coupled to elastic deformation mechanics (e.g., membrane curvature). Smooth particle applied mechanics, a form of smoothed particle continuum mechanics, is used to solve either the time-dependent Landau-Ginzburg or Cahn-Hilliard free-energy models for the composition dynamics. At the same time, the underlying elastic membrane is modeled using smooth particle applied mechanics, resulting in a unified computational scheme capable of treating the response of the composition fields to arbitrary deformations of the vesicle and vice versa. The results indicate that curvature coupling, along with the field theory model for composition free energy, gives domain formations that are correlated with surface defects on the vesicle. In the case that external deformations are included, the domain structures are seen to respond to such deformations. The present simulation capability provides a significant step forward toward the simulation of realistic cellular membrane processes.
| S-EPMC1305619 | biostudies-literature
Project description:Implantation of synthetic material, including vascular grafts, pacemakers, etc. results in the foreign body reaction and the formation of multinucleated giant cells (MGCs) at the exterior surface of the implant. Despite the long-standing premise that fusion of mononucleated macrophages results in the formation of MGCs, to date, no published study has shown fusion in context with living specimens. This is due to the fact that optical-quality glass, which is required for the majority of live imaging techniques, does not promote macrophage fusion. Consequently, the morphological changes that macrophages undergo during fusion as well as the mechanisms that govern this process remain ill-defined. In this study, we serendipitously identified a highly fusogenic glass surface and discovered that the capacity to promote fusion was due to oleamide contamination. When adsorbed on glass, oleamide and other molecules that contain long-chain hydrocarbons promoted high levels of macrophage fusion. Adhesion, an essential step for macrophage fusion, was apparently mediated by Mac-1 integrin (CD11b/CD18, ?M?2) as determined by single cell force spectroscopy and adhesion assays. Micropatterned glass further increased fusion and enabled a remarkable degree of spatiotemporal control over MGC formation. Using these surfaces, we reveal the kinetics that govern MGC formation in vitro. We anticipate that the spatiotemporal control afforded by these surfaces will expedite studies designed to identify the mechanism(s) of macrophage fusion and MGC formation with implication for the design of novel biomaterials.
| S-EPMC5540443 | biostudies-literature
Project description:We have succeeded in controlling tubular membrane formations in binary giant unilamellar vesicles (GUVs) using a simple temperature changing between the homogeneous one-phase region and the two-phase coexistence region. The binary GUV is composed of inverse-cone (bulky hydrocarbon chains and a small headgroup) and cylinder-shaped lipids. When the temperature was set in the two-phase coexistence region, the binary GUV had a spherical shape with solidlike domains. By increasing the temperature to the homogeneous one-phase region, the excess area created by the chain melting of the lipid produced tubes inside the GUV. The tubes had a radius on the micrometer scale and were stable in the one-phase region. When we again decreased the temperature to the two-phase coexisting region, the tubes regressed and the GUVs recovered their phase-separated spherical shape. We infer that the tubular formation was based on the mechanical balance of the vesicle membrane (spontaneous tension) coupled with the asymmetric distribution of the inverse-cone-shaped lipids between the inner and outer leaflets of the vesicle (lipid sorting).
| S-EPMC3824544 | biostudies-literature
Project description:Aberrant cellular cholesterol accumulation contributes to the pathophysiology of many diseases including neurodegenerative disorders such as Niemann-Pick Type C (NPC) and Alzheimer's Disease1-4. Many aspects of cholesterol efflux from cells remain elusive. Here we describe the utility of cholesterol-rich giant plasma membrane vesicles (GPMVs) as a means to monitor cholesterol that is translocated to the plasma membrane for secretion. We demonstrate that small molecules known to enhance lipid efflux, including those in clinical trials for lipid storage disorders, enhance this GPMV formation. Conversely, pharmacological inhibition of cholesterol efflux blocks GPMV formation. We show that microtubule stabilization via paclitaxel treatment and increased tubulin acetylation via HDAC6 inhibition promotes the formation of GPMVs with concomitant reduction in cellular cholesterol in a cell model of NPC disease. The pan-deacetylase inhibitor panobinostat, which has been shown to reduce the severity of cholesterol storage in NPC, elicited a similar response. Further, the disruption of actin polymerization inhibits the formation of GPMVs, whereas the small GTP-binding protein Arl4c promotes actin remodeling at sites overlapping with GPMV formation. Thus, monitoring the formation of GPMVs provides a new avenue to better understand diseases whose pathology may be sensitive to alterations in cellular cholesterol.
| S-EPMC6652231 | biostudies-literature