Characterization Of Phosphoinositide Monolayers By Infrared Spectroscopy And Epifluorescence Microscopy At The Air/water Interface

Abstract

Phosphoinositides have been shown to mediate a large variety of important physiological processes by affecting the activity and the 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, cations, or positively charged proteins. We have characterized the structural properties of dipalmitoyl phosphatidylinositol mono-, bis- and trisphosphate monolayer films at the air/water interface by infrared reflection-absorption spectroscopy (IRRAS) as well as by direct visualization of domain formation of each phosphoinositide derivative by epifluorescence microscopy in the presence of low and high monovalent salt concentrations. It has been observed that on pure water subphases the surface pressure/area (Π/A) isotherms for all phosphoinositide derivatives were characteristic for a condensed monolayer whereas a monolayer expansion was found for medium (10 mM) and high salt concentrations (150 mM). IRRAS measurements showed that this was associated with a larger molecular tilt angle and a reduced acyl chain order. Using epifluorescence microscopy, domain formation has been observed for each phosphoinositide derivative as the monolayer goes from a liquid phase to the liquid condensed phase. In addition, membrane organization can be affected by the binding and interaction of various proteins or peptides to the lipids. Surface pressure/time experiments along with epifluorescence microscopy will investigate these interactions for each phosphoinositide monolayer highlighting the distinct properties of phosphoinositide/protein interactions.