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

• We showed that increasing molar fraction of PIP2 in GUVs induces the formation of clusters, resulting in a localized decrease in water content of the membrane displaying liquid disordered phase.
• For ternary mixtures displaying liquid ordered/liquid disordered phase coexistence we showed a favorable partitioning of the clustered lipids in the liquid ordered phase.
• We demonstrated that the incorporation of DiIC18 into the membrane of GUVs is proportional to the molar fraction of these lipids in the organic solvent.
• For BODIPY labeled lipids the number of molecules appears smaller than what we would expect from their corresponding molar fraction.
• Our findings suggest that fluorescent aggregates of BODIPY are formed inducing a hypsochromic shift of the fluorescence.

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.