Where does all the PIP 2 come from?

Despite its very low concentration in the plasma membrane, PIP 2 is the precursor for the important second messenger Ins P 3 and, independently, is a key modulator of membrane signalling molecules such as ion channels. However, it has been difficult to determine the spatial and temporal characteristics of PIP 2 and Ins P 3 during a cell signalling event. Our laboratory used bradykinin stimulation of N1E‐115 neuroblastoma cells to infer the Ins P 3 dynamics from calcium imaging studies, biochemical analysis and Ins P 3 uncaging. We have used computational modelling with Virtual Cell to help analyse and interpret experimental data on the details of the calcium release process as well as to build a comprehensive image‐based model of agonist‐induced calcium release in a neuronal cell. These data provided a constraint for the further investigation of how low levels of cellular PIP 2 could provide sufficient Ins P 3 for calcium release. Using biochemical assays, quantitative imaging of GFP‐based probe translocation and computational analysis, it was shown that PIP 2 synthesis is stimulated concomitant with its hydrolysis. This mechanism should be important not just for consideration of PIP 2 as a precursor of Ins P 3 , but for any pathway that can be directly or indirectly modulated by PIP 2 .