Characterization of phosphoinositide domains in asymmetric giant unilamellar vesicles.

Abstract

Phosphoinositides (PIPs) have been shown to mediate a broad range of cellular events by attracting proteins to specific cellular sites at distinct time points. This spatiotemporal control of protein function is rooted in the rich chemical functionality of the phosphoinositide headgroup, which not only gives rise to selective interactions with proteins but also results in unique physicochemical properties. Several studies have been directed at detailing PIP properties in model symmetric lipid bilayers, however, biological membranes exhibit compositional asymmetry between bilayer leaflets. It is unclear to what extent PIP properties are altered in such an environment. In the last decade, methods have been introduced that enable the fabrication of asymmetric lipid vesicles. We have extended the scope of one of these methods – Ca2+ initiated hemifusion of a symmetric giant unilamellar vesicles (sGUV) with solid supported lipid bilayers (SLB) – to enable the fabrication of asymmetric giant unilamellar vesicles (aGUVs) that exhibit phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) in the outer leaflet. We found that using Ca2+ as the initiator for sGUV/SLB hemifusion leads to a large disparity of PI(4,5)P2 content in the formed aGUVs. We attribute this disparity to the formation of macroscopic Ca2+/PI(4,5)P2 domains, which leads to sGUVs interacting with SLB regions rich or poor in PI(4,5)P2. In contrast, when Mg2+ is used as initiator, no macroscopic domains form in the SLB and hence, the aGUVs show significantly higher compositional uniformity. We find that Mg2+ concentrations as low as 1 mM are sufficient to initiate the hemifusion process. Using this method to obtain aGUVs, we investigate phosphoinositide domain properties for varying lipid compositions in the leaflet opposite the PIP containing leaflet (raft vs. non-raft lipid mixtures) and we explore the impact of bivalent cations on the lateral morphology of the respective aGUVs.