Dataset Information

Formation of Giant Unilamellar Proteo-Liposomes by Osmotic Shock.

ABSTRACT: Giant unilamellar vesicles (GUVs), composed of a phospholipid bilayer, are often used as a model system for cell membranes. However, the study of proteo-membrane interactions in this system is limited as the incorporation of integral and lipid-anchored proteins into GUVs remains challenging. Here, we present a simple generic method to incorporate proteins into GUVs. The basic principle is to break proteo-liposomes with an osmotic shock. They subsequently reseal into larger vesicles which, if necessary, can endure the same to obtain even larger proteo-GUVs. This process does not require specific lipids or reagents, works under physiological conditions with high concentrations of protein, the proteins remains functional after incorporation. The resulting proteo-GUVs can be micromanipulated. Moreover, our protocol is valid for a wide range of protein substrates. We have successfully reconstituted three structurally different proteins, two trans-membrane proteins (TolC and the neuronal t-SNARE), and one lipid-anchored peripheral protein (GABARAP-Like 1 (GL1)). In each case, we verified that the protein remains active after incorporation and in its correctly folded state. We also measured their mobility by performing diffusion measurements via fluorescence recovery after photobleaching (FRAP) experiments on micromanipulated single GUVs. The diffusion coefficients are in agreement with previous data.

SUBMITTER: Motta I

PROVIDER: S-EPMC4950989 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Formation of Giant Unilamellar Proteo-Liposomes by Osmotic Shock.

Motta Isabelle I Gohlke Andrea A Adrien Vladimir V Li Feng F Gardavot Hélène H Rothman James E JE Pincet Frederic F

Langmuir : the ACS journal of surfaces and colloids 20150615 25

Giant unilamellar vesicles (GUVs), composed of a phospholipid bilayer, are often used as a model system for cell membranes. However, the study of proteo-membrane interactions in this system is limited as the incorporation of integral and lipid-anchored proteins into GUVs remains challenging. Here, we present a simple generic method to incorporate proteins into GUVs. The basic principle is to break proteo-liposomes with an osmotic shock. They subsequently reseal into larger vesicles which, if nec ...[more]

PMID: 26038815

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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

Formation of Giant Unilamellar Proteo-Liposomes by Osmotic Shock.

Publications

Formation of Giant Unilamellar Proteo-Liposomes by Osmotic Shock.

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