Limitations of the Use of the PI4-Kinase Inhibitor Wortmannin in Experiments Employing Fluorescent Phosphoinositide Probes

Abstract

To study phosphoinositide metabolism and the function of phosphoinositides in the regulation of cellular processes, tools to pharmacologically manipulate phosphoinositide metabolizing enzymes are essential. In this regard wortmannin (Wm) is widely used, as it is at low concentrations a blocker of PI3-kinase, enabling manipulation of PI(3,4,5)P3 and PI(3,4)P2 levels, at high concentrations it blocks PI4-kinase, preventing resynthesis of PI(4)P, the precursor of PI(4,5)P2. Accordingly, Wm has been frequently used to manipulate PI(4,5)P2 levels. In experiments involving manipulations of phosphoinositides it is desirable to dynamically monitor the changes in phosphoinositide levels. This can be achieved using fluorescently labeled phosphoinositide probes, e.g. the GFP tagged PH domain of phospholipase Cδ1 (PLCδ1-PH-GFP), which specifically binds PI(4,5)P2.Reportedly, when PLCδ1-PH-GFP was used to monitor PI(4,5)P2, the recovery of the fluorescence signal after depletion of PI(4,5)P2 by transient activation of endogenous phospholipase-C could not be inhibited by application of high concentrations of Wm, whereas at such concentrations Wm reliably blocked the recovery when PI(4,5)P2-dependent ion-channels are used as phosphoinositide reporters. Originally, it was speculated that this was indicative of a Wm insensitive PI(4,5)P2 pool. Later on, an alternative hypothesis emerged, citing effects of intense illumination on Wm. Such illumination is used for the excitation of fluorescently labeled phosphoinositide probes, drawing the usefulness of Wm in experiments employing these probes into question.We set out to clarify the influence of the excitation light on the effect of Wm when PLCδ1-PH-GFP is used and whether minimizing total light exposure of the cell using total internal reflection microscopy (TIRF-M) is beneficial.We find experimental evidence in favor of illumination dependent inactivation of Wortmannin.Supported by Deutsche Forschungsgemeinschaft (grant SFB 593, TP12 to DO) and UKGM (grant 32/2011MR to CRH).