Laurence Ramos

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.

Phosphatidylinositol 4,5-bisphosphate-induced conformational change of ezrin and formation of ezrin oligomers

The plasma membrane-cytoskeleton interface is a dynamic structure involved in a variety of cellular events. Ezrin, a protein from the ERM family, provides a direct linkage between the cytoskeleton and the membrane via its interaction with phosphatidylinositol 4,5-bisphosphate (PIP₂). In this paper, we investigate the interaction between PIP₂ and ezrin in vitro using PIP₂ dispersed in a unimolecular way in buffer. We compared the results obtained with full-length ezrin to those obtained with an ezrin mutant, which was previously found not to be localized at the cell membrane, and with the N-terminal membrane binding domain (FERM domain) of ezrin. We show that PIP₂ induced a conformational change in full-length ezrin. PIP₂ was also found to induce, in vitro, the formation of oligomers of wild-type ezrin, but not of mutant ezrin. These oligomers had previously been observed in vivo, but their role is yet to be clarified. Our finding hints at a possible role for PIP₂ in the formation of ezrin oligomers.