Characterization of Asymmetric Phosphoinositide/Lipid Vesicles

Abstract

Biological membranes are laterally heterogenous and vertically asymmetric. While lateral heterogeneities (domains) have been widely investigated, only recently methodologies have emerged that allow for fabrication of vertically asymmetric lipid vesicles. Several phosphoinositide (PIP) mediated cellular events are sensitive to cholesterol depletion, and it has been speculated that PIPs accumulate in lipid rafts. However, the stearoyl/arachidonoyl acyl chain composition of PIPs is generally unfavorable for accumulation in a liquid-ordered (lo) environment, and in symmetric lipid vesicles, PIPs do not enrich in lo phases. It has been shown, however, that cholesterol promotes phosphoinositide domain formation in ternary PC/PIP/cholesterol lipid mixtures, presumably through an interaction between the PIP headgroup and the cholesterol hydroxyl group. To investigate the potential coupling of outer leaflet raft domains and inner leaflet PIP/cholesterol domains, we have fabricated asymmetric giant unilamellar vesicles and characterized them using fluorescence microscopy. We will present data for different lipid compositions and ionic conditions (e.g., +/- calcium).