Live cell imaging of phosphoinositide dynamics with fluorescent protein domains

Phosphoinositides make up only a small fraction of membrane phospholipids yet they are of outmost significance in regulating membrane-associated signaling processes. A large number of inositol lipid kinases and phosphatases have evolved to control the rapid production and elimination of these lipids at specific cellular membrane compartments. For a long period of time, the only information about the spatial aspect of inositol lipid metabolism relied upon the immunostaining of enzymes or cell fractionation of the enzyme activities that acted upon these lipids. Recent advances in the understanding of the nature of protein-inositol lipid interactions permitted the design of fluorescent molecular probes that can interact with inositol lipids in a specific manner allowing imaging of phosphoinositide dynamics in live cells. This approach has rapidly gained high popularity, but also provoked criticisms and debate about its limitations. In this review, we will summarize our experience with using these molecular tools and address some issues that most often come up in discussions concerning the usefulness and drawbacks of this technique. The most important value of these debates is that they also challenge our preconceived views of how phosphoinositides regulate cellular functions. Published by Elsevier B.V.