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Facile generation of giant unilamellar vesicles using polyacrylamide gels.

ABSTRACT: 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.

SUBMITTER: Parigoris E

PROVIDER: S-EPMC7075891 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Facile generation of giant unilamellar vesicles using polyacrylamide gels.

Parigoris Eric E Dunkelmann Daniel L DL Murphy Allan A Wili Nino N Kaech Andres A Dumrese Claudia C Jimenez-Rojo Noemi N Silvan Unai U

Scientific reports 20200316 1

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 gen ...[more]

PMID: 32179778

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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.

Dataset Information

Facile generation of giant unilamellar vesicles using polyacrylamide gels.

Publications

Facile generation of giant unilamellar vesicles using polyacrylamide gels.

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