Fabrication and characterization of phosphoinositide containing asymmetric vesicles in physiological salt.

Abstract

Phosphoinositide (PIPs) lipids mediate a broad range of physiological functions by attracting proteins at specific time points to distinct cellular sites. Many of these processes are associated with the local accumulation of PIPs and PIP/protein signaling platform formation. Studies aimed at determining the physicochemical underpinnings of PIP domain formation have been limited to model systems that exhibited the same lipid composition in both bilayer leaflets. However, biological membranes are asymmetric, and it is desirable to develop an experimental approach that allows for the fabrication of lipid model systems with a non-symmetric lipid bilayer, i.e., a membrane mimic that exhibits a PIP containing lipid mixture in one leaflet and a different lipid composition in the opposing leaflet. We adapted the previously introduced hemifusion method for the fabrication of asymmetric Giant Unilamellar Vesicles (aGUVs) for the fabrication of aGUVs with phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P₂) in a physiological ionic strength buffer solution. The general method involved the bivalent cation-initiated fusion of a symmetric GUV (sGUV) with solid-supported lipid bilayers, which leads to the exchange of the outer leaflet of the sGUV. We find that initiating the hemifusion with 6 mM Ca²⁺ leads to a low yield and quality of the aGUVs. We attribute this to macroscopic Ca²⁺/PI(4,5)P₂ domain formation of the solid support lipid bilayer (SLB), which leads to the interaction of the sGUVs with regions enriched in PI(4,5)P₂ (domain) and other areas that are void of the PIP lipid. Using 6 mM Mg²⁺ as the initiator instead led to an improvement in terms of yield and aGUV quality. The best results were obtained when using 1 mM Mg²⁺. We are introducing several data analysis approaches that allow for the identification of aGUVs that exhibit high quality in terms of the outer leaflet exchange and composition of the two aGUV leaflets.