Single-Cell Imaging Techniques for the Real-Time Detection of IP3 in Live Cells

Inositol 1,4,5-trisphosphate (IP3 ) is a ubiquitous second messenger, derived from the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2 ) by enzymes of the phospholipase C (PLC) family. Binding of IP3 to its cognate receptor in the endoplasmic reticulum membrane leads to release of Ca2+ into the cytoplasm, which is involved in the regulation of an array of cellular functions. Traditional techniques for the detection of IP3 have required the extraction of a large number of cells, with limitations in the time resolution of changes in IP3 and an inability to obtain detailed information on the dynamics of this second messenger in single cells. Recent progress in this field has led to the development of a number of genetically encoded fluorescent biosensors, which upon recombinant expression are able selectively to detect real-time changes in IP3 in single live cells. In this chapter, I detail protocols for the expression, visualization (by confocol or fluorescence microscopy), and interpretation of data obtained with such biosensors expressed in mammalian cells.