On the Mechanism of Bilayer Separation by Extrusion, or Why Your LUVsare not Really Unilamellar
Haden L. Scott, Allison Skinkle, Elizabeth Kelley, M. Neal Waxham, Ilya Levental, Frederick A. Heberle
Extrusion through porous filters is the most 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 are 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. 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 vesicle 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.
, Skinkle, A.
, Kelley, E.
, Waxham, M.
, Levental, I.
and Heberle, F.
On the Mechanism of Bilayer Separation by Extrusion, or Why Your LUVsare not Really Unilamellar, Biophysical Journal, [online], https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=928273
(Accessed February 26, 2024)