Infrared Spectroscopic Characterization of Phosphoinositide Signaling Pathway Components

Membrane lipids are asymmetrically distributed between the inner and the outer leaflet of the plasma membrane, and this asymmetric distribution of lipids is an important factor for many signaling events. The lipids investigated in this study, phosphoinositides, are found in the inner leaflet of the plasma membrane and have been shown to mediate a wide variety of important physiological processes by affecting the activity and/or localization of membrane associated proteins. Phosphoinositide properties are largely determined by the characteristics of their headgroup, which at physiological pH is highly charged but is also capable of hydrogen bond formation. For phosphoinositide mediated signaling events to occur, it requires the local enrichment of phosphoinositides, which depend on the interchange between attractive and repulsive forces. Factors expected to affect mutual phosphoinositide interaction are pH as well as the presence of cations or positively charged proteins. The primary goal of this study was to gain more insight about the unique physiochemical properties of phosphoinositides and how they organize laterally in the membrane. We hypothesized that the type and concentration of salt in the subphase affect the phase behavior of phosphatidylinositol and phosphatidylinositol monophosphate monolayers at the air/water interface. Additionally, we hypothesized that the position of the phosphate group at the inositol ring of phosphatidylinositol monophosphates has an effect on how the phosphoinositide molecules interact with each other and other molecular entities embedded in the biomembrane. Using surface pressure/area isotherms, Infrared Reflection Absorption Spectroscopy (IRRAS) and epifluorescence microscopy we have shown that: 1) the presence of monovalent and divalent salt affect the phase behavior, acyl chain conformational order, and domain morphology of phosphatidylinositol monolayers. 2) The monovalent salt concentration affects the phase behavior, acyl chain conformational order, molecular tilt angle, and domain formation of phosphatidylinositol monophosphates. 3) The position of the phosphate group at the inositol ring of phosphatidylinositol monophosphates is an important factor for their mutual interaction. The second aim of this study was to investigate the interactions between phosphoinositides and cholesterol. Our goal was to gain further insight into this interaction and to investigate the possibility that cholesterol may enhance phosphoinositide co-localization and subsequent domain formation. We studied lipid monolayer systems in the presence of cholesterol as well as in the presence of cholesterol derivatives to observe changes in phosphoinositide interaction and domain formation and we found that: 1) cholesterol had a condensing effect on the phosphoinositide monolayer films and that an increase in the molar concentration of cholesterol in the lipid system further condensed these monolayers. 2) Each of the investigated phosphoinositide derivatives was not able to form domains without the presence of cholesterol. 3) Modifications of the hydroxyl group position of cholesterol lead to an altered interaction with phosphoinositides. Our results underscored that cholesterol induces phosphoinsitide domain formation and the results highlighted the importance of the hydroxyl group of cholesterol for the putative phosphoinositide/cholesterol interaction.