Live cell imaging of phosphoinositides with expressed inositide binding protein domains

Inositol lipids and calcium signaling has been inseparable twins during the 1980s when the molecular details of phospholipase C-mediated generation of inositol 1,4,5-trisphosphate (InsP(3)) and its Ca2+ mobilizing action were discovered. Since then, both the Ca2+ and inositol lipid signaling fields have hugely expanded and the tools allowing dissection of the finest details of their molecular organization also followed closely. Although phosphoinositides regulate many cell functions unrelated to Ca2+ signaling there are still many open questions even in the Ca2+ field that would benefit from single cell monitoring of PtdIns(4,5)P-2. or InSP3 changes during agonist stimulation. This chapter is designed to provide practical guidance as well as some theoretical background on measurements of phosphoinositides in live cells using protein domain-GFP chimeras that could be also useful for people working on calcium signaling. (C) 2008 Elsevier Inc. All rights reserved.