Giant unilamellar vesicles containing phosphatidylinositol(4,5)bisphosphate: characterization and functionality.

Biomimetic systems such as giant unilamellar vesicles (GUVs) are increasingly used for studying protein/lipid interactions due to their size (similar to that of cells) and to their ease of observation by light microscopy techniques. Biophysicists have begun to complexify GUVs to investigate lipid/protein interactions. In particular, composite GUVs have been designed that incorporate lipids that play important physiological roles in cellulo, such as phosphoinositides and among those the most abundant one, phosphatidylinositol(4,5)bisphosphate (PIP2). Fluorescent lipids are often used as tracers to observe GUV membranes by microscopy but they can not bring quantitative information about the insertion of unlabeled lipids. In this study, we carried out zeta-potential measurements to prove the effective incorporation of PIP2 as well as that of phosphatidylserine in the membrane of GUVs prepared by electroformation and to follow the stability of PIP2-containing GUVs. Using confocal microscopy, we found that long-chain (C16) fluorescent PIP2 analogs used as tracers (0.1% of total lipids) show a uniform distribution in the membrane whereas PIP2 antibodies show PIP2 clustering. However, the clustering effect, which is emphasized when tertiary antibodies are used in addition to secondary ones to enhance the size of the detection complex, is artifactual. We showed that divalent ions (Ca2+ and Mg2+) can induce aggregation of PIP2 in the membrane depending on their concentration. Finally, the interaction of ezrin with PIP2-containing GUVs was investigated. Using either labeled ezrin and unlabeled GUVs or both labeled ezrin and GUVs, we showed that clusters of PIP2 and proteins are formed.