Engineering Phosphatidylserine Containing Asymmetric Giant Unilamellar Vesicles

Area of Science:

• Biophysics
• Membrane Biology
• Lipid Bilayer Engineering

Background:

• Plasma membrane lipid distribution is asymmetric, with anionic lipids like phosphatidylserine (PS) in the inner leaflet.
• Asymmetric giant unilamellar vesicles (aGUVs) are essential tools for studying PS in lipid bilayers and its interactions with proteins.
• Fabricating aGUVs with precise and high degrees of asymmetry, particularly with PS in the outer leaflet, remains a significant challenge.

Purpose of the Study:

• To extend a Ca²⁺-initiated hemifusion method for creating aGUVs with PS in the outer leaflet.
• To compare the effectiveness of Ca²⁺ and Mg²⁺ in fabricating highly asymmetric GUVs.
• To introduce a data filtering method for selecting high-quality aGUVs for further research.

Main Methods:

• Utilized a Ca²⁺- or Mg²⁺-initiated hemifusion of symmetric giant unilamellar vesicles (sGUVs) composed of phosphatidylcholine (PC) with a PC/PS-supported lipid bilayer (SLB).
• Analyzed the degree of asymmetry in the resulting aGUVs using advanced imaging and analysis techniques.
• Investigated the effect of ionic strength and cation type (Ca²⁺ vs. Mg²⁺) on PS distribution and domain formation.

Main Results:

• Both Ca²⁺ and Mg²⁺ initiated hemifusion successfully produced aGUVs with significant PS asymmetry in the outer leaflet.
• A narrower distribution of asymmetry was observed under physiological salt conditions compared to lower ionic strengths.
• Ca²⁺ induced PS clustering in the SLB, but this did not negatively impact aGUV quality due to slow clustering kinetics; Mg²⁺ did not induce macroscopic domain formation.

Conclusions:

• The extended hemifusion method provides a reliable route to fabricate aGUVs with controlled PS asymmetry, suitable for biophysical studies.
• The choice of cation (Ca²⁺ or Mg²⁺) and ionic strength influences the degree of asymmetry and lipid organization.
• A novel data filtering approach enhances the selection of high-quality aGUVs for investigating lipid-protein interactions.