Imaging phosphoinositide dynamics in living cells.

To improve our understanding of the important roles played by inositol lipid derivatives in signalling and other cellular processes, it is crucial to measure phosphoinositide concentration changes in individual cells with high spatial and temporal resolution. A number of protein domains that interact with inositol lipids in a specific manner have been identified. Tagged with the green fluorescent protein or its colour variants, these protein modules can be used as probes to visualize various phosphoinositide species in different sub-cellular compartments. Here, we present protocols for fluorescence imaging of phosphoinositide dynamics in single living cells. Total internal reflection fluorescence microscopy is particularly powerful for time-lapse recordings of phosphoinositides in the plasma membrane. We demonstrate how this technique can be used to record phospholipase C- and PI3-kinase-induced changes in inositol lipids in insulin-secreting cells. These procedures should be applicable to studies of the spatio-temporal regulation of phosphoinositide metabolism in many types of cells.