Fabrication of Phosphatidylserine-Containing Asymmetric Giant Unilamellar Vesicles by Hemifusion

McDonough, Jake

Student Work

Fabrication of Phosphatidylserine-Containing Asymmetric Giant Unilamellar Vesicles by Hemifusion

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Phosphatidylserine (PS) is an important membrane lipid to study due to the vital role it plays in numerous cellular pathways, but doing so is made difficult by the complexities of biological membranes. Studying PS in model membranes can overcome these challenges, though due to most biological membranes being asymmetric (i.e., having a different inner/outer leaflet composition), these models should be so too to ensure accurate simulation. In this study, a method for preparing PS-containing asymmetric giant unilamellar vesicles (aGUVs) was derived. Ca2+ or Mg2+ was used to induce hemifusion and subsequent total outer leaflet exchange between symmetric GUVs composed of phosphatidylcholine/acceptor fluorophore, and a supported lipid bilayer composed of phosphatidylcholine/PS/donor fluorophore. EDTA was then added to induce fission. The composition of the resulting aGUVs was evaluated by comparing their donor or acceptor fluorophore intensities to that of symmetric GUVs with compositions equal to the theoretical inner or outer leaflet composition of the aGUVs. aGUVs prepared in both low salt buffer (approximately 50 mM NaCl and 30 mM KCl) and high salt buffer (approximately 100 mM NaCl and 80 mM KCl) gave average fluorophore intensities that were around half those of the symmetric GUVs, indicating they were on average the expected composition. Though intensities of individual aGUVs varied significantly. A data filtering method was developed to exclude aGUVs with compositions significantly different than the theoretical, of which 22% passed. These aGUVs could be used in future studies as compositionally consistent, PS-containing asymmetric model membranes.

This report represents the work of one or more WPI undergraduate students submitted to the faculty as evidence of completion of a degree requirement. WPI routinely publishes these reports on its website without editorial or peer review.

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