Low PIP₂ molar fractions induce nanometer size clustering in giant unilamellar vesicles

Phosphatidylinositol (4,5) bisphosphate (PIP₂) is an important signaling molecule located on the inner leaflet of the cell membrane. In order to perform its various signaling functions, it is suggested that PIP₂ must be able to form localized clusters. In this study, we have used LAURDAN generalized polarization function (GP) with unlabeled PIP₂ and single point fluorescence correlation spectroscopy and brightness analysis of various BODIPY labeled PIP₂ to determine the presence of clusters in the membrane of giant unilamellar vesicles (GUVs) made of 1-palmitoyl-2-oleoyl-'sn'-glycero-3-phosphocholine (POPC) or a mixture of 1,2-dioleoyl-'sn'-glycero-3-phosphocholine (DOPC), sphingomyelin and cholesterol. We determined the number of freely diffusing fluorescent BODIPY molecules in the membrane and found that in GUVs containing various amounts of labeled PIP₂, this number was significantly lower than in GUVs made with the control BODIPY labeled hexadecyl phosphatidylcholine (BODIPY-HPC). Also, we noted an increase in brightness of the labeled PIP₂ particles with increasing labeled PIP₂ molar fraction. Together with the observed change in LAURDAN GP with increasing molar fraction of unlabeled PIP₂, these results demonstrate the presence of PIP₂ enriched clusters that are smaller than the resolution limit of the fluorescent microscope. In addition, we report the presence of a hypsochromic shift of the fluorescence for the BODIPY labeled lipids that we attributed to clustering. This clustering result in a change in the partitioning of the lipids with the BODIPY labeled PIP₂ lipids able to move between the liquid ordered and liquid disordered phase.