Reorganization of Ternary Lipid Mixtures of Non-Phosphorylated Phosphatidylinositol Interacting with Angiomotin

Abstract

Phosphatidylinositol (PI) lipids are necessary for many cellular signaling pathways of membrane associated proteins, such as Angiomotin (Amot). The Amot family regulates cellular polarity, growth, and migration. Given the low concentration of PI lipids in these membranes, it is likely that such protein-membrane interactions are stabilized by lipid domains or small lipid clusters. By small-angle x-ray scattering, we show that non-phosphorylated PI lipids induce lipid de-mixing in ternary mixtures of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), likely due to preferential interactions between the head groups of PE and PI. These results were obtained in the presence of buffer containing concentrations of Tris, HEPES, NaCl, EDTA, DTT, and Benzamidine at pH 8.0 that in previous work showed an ability to cause PC to phase separate but are necessary to stabilize Amot for in vitro experimentation. Collectively, this provided a framework for determining the effect of Amot on lipid organization. Using fluorescence spectroscopy, we were able to show that the association of Amot with this lipid platform causes significant reorganization of the lipid into a more homogenous organization. This reorganization mechanism could be the basis for Amot membrane association and fusigenic activity previously described in the literature and should be taken into consideration in future protein-membrane interaction studies.