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.