Low PIP2 Molar Fractions Induce Nanometer Size Clustering in Giant Unilamellar Vesicles Containing POPC

Phosphatidylinositol (4,5) bisphosphate (PIP2) is a key lipid signaling molecule found on the inner leaflet of the cell membrane. Polyphosphoinositides signaling play an important role in regulation of many cell processes such as cell survival, proliferation, migration, and apoptosis. Phosphoinositides regulate signal transduction by providing binding or docking sites for intracellular proteins. In order to perform these functions, it has been suggested that PIP2 must be able to form localized clusters within the cell membrane. In this study, single point fluorescence correlation spectroscopy and brightness analysis of various BODIPY-labeled PIP2 was utilized to detect the presence of clusters in the membrane of giant unilamellar vesicles (GUVs) consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The number of freely diffusing fluorescent BODIPY molecules in the membrane was calculated and we found that in GUVs containing various amounts of labeled PIP2 had significantly lower number of fluorescent molecules than in GUVs made with the control BODIPY labeled hexadecyl phosphatidylcholine (BODIPY-HPC). Also, an increase in brightness of the labeled PIP2 particles with increasing labeled PIP2 molar fraction was noted. These results show the existence of PIP2 enriched clusters in the liquid disordered phase of GUVs that are smaller than the resolution limit of the fluorescent microscope. Using LAURDAN generalized polarization function, we demonstrated that the formation of these clusters is not due to the presence of the BODIPY moiety on the lipids. In addition, we report for the first time the presence of a previously uncharacterized hypsochromic shift of the fluorescence for the BODIPY labeled lipids that we attributed to BODIPY dimers. Finally, by adding cholesterol to the GUVs, we showed that the BODIPY dimers tend to favor the liquid ordered phase.