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Biophys J. 2019 Oct 15;117(8):1381-1386. doi: 10.1016/j.bpj.2019.09.006. Epub 2019 Sep 16.

On the Mechanism of Bilayer Separation by Extrusion, or Why Your LUVs Are Not Really Unilamellar.

Author information

1
Center for Environmental Biotechnology, University of Tennessee, Knoxville, Tennessee; Shull Wollan Center, Oak Ridge National Laboratory, Oak Ridge, Tennessee.
2
Department of Biosciences, Rice University, Houston, Texas; Department of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, Texas.
3
NIST Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg, Maryland.
4
Department of Neurobiology and Anatomy, University of Texas Health Science Center, Houston, Texas.
5
Department of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, Texas. Electronic address: ilya.levental@uth.tmc.edu.
6
Center for Environmental Biotechnology, University of Tennessee, Knoxville, Tennessee; Department of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, Texas. Electronic address: fheberle@utk.edu.

Abstract

Extrusion through porous filters is a widely used method for preparing biomimetic model membranes. Of primary importance in this approach is the efficient production of single bilayer (unilamellar) vesicles that eliminate the influence of interlamellar interactions and strictly define the bilayer surface area available to external reagents such as proteins. Submicroscopic vesicles produced using extrusion are widely assumed to be unilamellar, and large deviations from this assumption would impact interpretations from many model membrane experiments. Using three probe-free methods-small angle X-ray and neutron scattering and cryogenic electron microscopy-we report unambiguous evidence of extensive multilamellarity in extruded vesicles composed of neutral phosphatidylcholine lipids, including for the common case of neutral lipids dispersed in physiological buffer and extruded through 100-nm diameter pores. In such preparations, only ∼35% of lipids are externally accessible and this fraction is highly dependent on preparation conditions. Charged lipids promote unilamellarity as does decreasing solvent ionic strength, indicating the importance of electrostatic interactions in determining the lamellarity of extruded vesicles. Smaller extrusion pore sizes also robustly increase the fraction of unilamellar vesicles, suggesting a role for membrane bending. Taken together, these observations suggest a mechanistic model for extrusion, wherein the formation of unilamellar vesicles involves competition between bilayer bending and adhesion energies. The findings presented here have wide-ranging implications for the design and interpretation of model membrane studies, especially ensemble-averaged observations relying on the assumption of unilamellarity.

PMID:
31586522
PMCID:
PMC6817544
[Available on 2020-10-15]
DOI:
10.1016/j.bpj.2019.09.006

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