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

Journal Article · Mon Sep 16 00:00:00 EDT 2019 · Biophysical Journal

DOI:https://doi.org/10.1016/j.bpj.2019.09.006· OSTI ID:1785501

Scott, Haden L. ^[1]; Skinkle, Allison ^[2]; Kelley, Elizabeth G. ^[3]; Waxham, M. Neal ^[4]; Levental, Ilya ^[5]; Heberle, Frederick A. ^[6]

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1. Univ. of Tennessee, Knoxville, TN (United States). Center for Environmental Biotechnology; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Shull Wollan Center
2. Rice Univ., Houston, TX (United States). Dept. of Biosciences; Univ. of Texas, Houston, TX (United States). Health Science Center. Dept. of Integrative Biology and Pharmacology
3. National Inst. of Standards and Technology (NIST), Gaithersburg, MD (United States). NIST Center for Neutron Research
4. Univ. of Texas, Houston, TX (United States). Health Science Center. Dept. of Neurobiology and Anatomy
5. Univ. of Texas, Houston, TX (United States). Health Science Center. Dept. of Integrative Biology and Pharmacology
6. Univ. of Tennessee, Knoxville, TN (United States). Center for Environmental Biotechnology; Univ. of Texas, Houston, TX (United States). Health Science Center. Dept. of Integrative Biology and Pharmacology

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. Sub-microscopic vesicles produced using extrusion are widely assumed to be unilamellar, and large deviations from this assumption would dramatically impact interpretations from many model membrane experiments. In this work, using three probe-free methods—small-angle X-ray and neutron scattering (SAXS and SANS) and cryogenic electron microscopy (cryoEM)—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 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.

• Research Organization: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
• Sponsoring Organization: National Institutes of Health (NIH); National Science Foundation (NSF); USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities Division; Volkswagen Foundation
• Grant/Contract Number: AC05-00OR22725
• OSTI ID: 1785501
• Journal Information: Biophysical Journal, Journal Name: Biophysical Journal Journal Issue: 8 Vol. 117; ISSN 0006-3495
• Publisher: Elsevier
• Country of Publication: United States
• Language: English

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59 BASIC BIOLOGICAL SCIENCES