Effect of the Membrane Composition of Giant Unilamellar Vesicles on Their Budding Probability: A Trade-Off between Elasticity and Preferred Area Difference.
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ABSTRACT:
SUBMITTER: Miele Y
PROVIDER: S-EPMC8306738 | biostudies-literature |
REPOSITORIES: biostudies-literature
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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.
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Project description:In vitro reconstitution of simplified biological systems from molecular parts has proven to be a powerful method for investigating the biochemical and biophysical principles underlying cellular processes. In recent years, there has been a growing interest in reconstitution of protein-membrane interactions to understand the critical role played by membranes in organizing molecular-scale events into micron-scale patterns and protrusions. However, while all reconstitution experiments depend on identifying and isolating an essential set of soluble biomolecules, such as proteins, DNA, and RNA, reconstitution of membrane-based processes involves the additional challenge of forming and working with lipid bilayer membranes with composition, fluidity, and mechanical properties appropriate for the process at hand. Here we discuss a selection of methods for forming synthetic lipid bilayer membranes and present a versatile electroformation protocol that our lab uses for reconstituting proteins on giant unilamellar vesicles. This synthetic membrane-based approach to reconstitution offers the ability to study protein organization and activity at membranes under more cell-like conditions, addressing a central challenge to accomplishing the grand goal of "building the cell."
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Project description:Biomembranes are constantly remodeled and in cells, these processes are controlled and modulated by an assortment of membrane proteins. Here, it is shown that such remodeling can also be induced by photoresponsive molecules. The morphological control of giant vesicles in the presence of a water-soluble ortho-tetrafluoroazobenzene photoswitch (F-azo) is demonstrated and it is shown that the shape transformations are based on an increase in membrane area and generation of spontaneous curvature. The vesicles exhibit budding and the buds can be retracted by using light of a different wavelength. In the presence of F-azo, the membrane area can increase by more than 5% as assessed from vesicle electrodeformation. To elucidate the underlying molecular mechanism and the partitioning of F-azo in the membrane, molecular dynamics simulations are employed. Comparison with theoretically calculated shapes reveals that the budded shapes are governed by curvature elasticity, that the spontaneous curvature can be decomposed into a local and a nonlocal contribution, and that the local spontaneous curvature is about 1/(2.5 µm). The results show that exo- and endocytotic events can be controlled by light and that these photoinduced processes provide an attractive method to change membrane area and morphology.
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Project description:Phospholipid bilayers are dynamic cellular components that undergo constant changes in their topology, facilitating a broad diversity of physiological functions including endo- and exocytosis, cell division, and intracellular trafficking. These shape transformations consume energy, supplied invariably by the activity of proteins. Here, we show that cycles of oppositely directed osmotic stresses─unassisted by any protein activity─can induce well-defined remodeling of giant unilamellar vesicles, minimally recapitulating the phenomenologies of surface area homeostasis and macropinocytosis. We find that a stress cycle consisting of deflationary hypertonic stress followed by an inflationary hypotonic one prompts an elaborate sequence of membrane shape changes ultimately transporting molecular cargo from the outside into the intravesicular milieu. The initial osmotic deflation produces microscopic spherical invaginations. During the subsequent inflation, the first subpopulation contributes area to the swelling membrane, thereby providing a means for surface area regulation and tensional homeostasis. The second subpopulation vesiculates into the lumens of the mother vesicles, producing pinocytic vesicles. Remarkably, the gradients of solute concentrations between the GUV and the daughter pinocytic vesicles create cascades of water current, inducing pulsatory transient poration that enable solute exchange between the buds and the GUV interior. This results in an efficient water-flux-mediated delivery of molecular cargo across the membrane boundary. Our findings suggest a primitive physical mechanism for communication and transport across protocellular compartments driven only by osmotic stresses. They also suggest plausible physical routes for intravesicular, and possibly intracellular, delivery of ions, solutes, and molecular cargo stimulated simply by cycles of osmotic currents of water.
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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.
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Project description:We developed a new (to our knowledge) protocol to generate giant unilamellar vesicles (GUVs) composed of mixtures of single lipopolysaccharide (LPS) species and Escherichia coli polar lipid extracts. Four different LPSs that differed in the size of the polar headgroup (i.e., LPS smooth > LPS-Ra > LPS-Rc > LPS-Rd) were selected to generate GUVs composed of different LPS/E. coli polar lipid mixtures. Our procedure consists of two main steps: 1), generation and purification of oligolamellar liposomes containing LPSs; and 2), electroformation of GUVs using the LPS-containing oligolamellar vesicles at physiological salt and pH conditions. Analysis of LPS incorporation into the membrane models (both oligolamellar vesicles and GUVs) shows that the final concentration of LPS is lower than that expected from the initial E. coli lipids/LPS mixture. In particular, our protocol allows incorporation of no more than 15 mol % for LPS-smooth and LPS-Ra, and up to 25 mol % for LPS-Rc and LPS-Rd (with respect to total lipids). We used the GUVs to evaluate the impact of different LPS species on the lateral structure of the host membrane (i.e., E. coli polar lipid extract). Rhodamine-DPPE-labeled GUVs show the presence of elongated micrometer-sized lipid domains for GUVs containing either LPS-Rc or LPS-Rd above 10 mol %. Laurdan GP images confirm this finding and show that this particular lateral scenario corresponds to the coexistence of fluid disordered and gel (LPS-enriched)-like micron-sized domains, in similarity to what is observed when LPS is replaced with lipid A. For LPSs containing the more bulky polar headgroup (i.e., LPS-smooth and LPS-Ra), an absence of micrometer-sized domains is observed for all LPS concentrations explored in the GUVs (up to ?15 mol %). However, fluorescence correlation spectroscopy (using fluorescently labeled LPS) and Laurdan GP experiments in these microscopically homogeneous membranes suggests the presence of LPS clusters with dimensions below our microscope's resolution (?380 nm radial). Our results indicate that LPSs can cluster into gel-like domains in these bacterial model membranes, and that the size of these domains depends on the chemical structure and concentration of the LPSs.
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Project description:We assessed the applicability of giant unilamellar vesicles (GUVs) for RNA detection using in vesicle reverse transcription polymerase chain reaction (RT-PCR). We prepared GUVs that encapsulated one-pot RT-PCR reaction mixture including template RNA, primers, and Taqman probe, using water-in-oil emulsion transfer method. After thermal cycling, we analysed the GUVs that exhibited intense fluorescence signals, which represented the cDNA amplification. The detailed analysis of flow cytometry data demonstrated that rRNA and mRNA in the total RNA can be amplified from 10-100 copies in the GUVs with 5-10 μm diameter, although the fraction of reactable GUV was approximately 60% at most. Moreover, we report that the target RNA, which was directly transferred into the GUV reactors via membrane fusion, can be amplified and detected using in vesicle RT-PCR. These results suggest that the GUVs can be used as biomimetic reactors capable of performing PCR and RT-PCR, which are important in analytical and diagnostic applications with additional functions.
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Project description:Giant unilamellar vesicles (GUVs) are well-established model systems for studying membrane structure and dynamics. Electroformation, also referred to as electroswelling, is one of the most prevalent methods for producing GUVs, as it enables modulation of the lipid hydration process to form relatively monodisperse, defect-free vesicles. Currently, however, it is expensive and time-consuming compared with other methods. In this study, we demonstrate that 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine GUVs can be prepared readily at a fraction of the cost on stainless steel electrodes, such as commercially available syringe needles, without any evidence of lipid oxidation or hydrolysis.
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