Measurement and immunofluorescence of cellular phosphoinositides

Phosphoinositides are a vitally important class of intracellular-signaling molecules that regulate cellular processes, including signaling through cell-surface receptors, remodeling of the cytoskeleton, vesicle-mediated protein trafficking, and various nuclear functions. Methods for the analysis of in vivo phosphoinositide concentration, such as the one described in this chapter enable quantification of all phosphoinositides from a population of cells. This method involves metabolic labeling of cells with myo<-[2-3H] inositol, followed by lipid extraction, and quantification by high-performance liquid chromatography (HPLC). It provides improved efficiency and reproducibility when analyzing yeast, plant cells, and is applicable to animal cells as well. In addition, a technique for determining the intracellular location of phosphoinositides is described. When quantification and localization techniques are used in parallel, an investigator can identify cell, and even subcellular concentration changes. The technique described in this chapter uses immunodetection with antiphosphoinositide antibodies to determine the localization and relative concentrations of phosphinositides in fixed cells. The availability of antibodies allows an investigator to perform immunofluorescence and potentially immunoelectron microscopy of phosphoinositide localization on particular cellular, organellar, or vesicular membranes.