Spatiotemporal Investigation of Phosphorylation Events During Cell Cycle Progression.

Polo-like kinase 1 (Plk1) is an essential kinase for mitotic commitment and progression through mitosis. In contrast to its well characterized roles during mitosis, the precise molecular events controlled by Plk1 during G2/M progression and their spatiotemporal regulation are still poorly elucidated. We recently investigated Plk1-dependent regulation of Cdc25C phosphatase, an activator of the master mitotic driver Cyclin B1-Cdk1. To this end, we generated a genetically encoded FRET (Förster Resonance Energy Transfer)-based Cdc25C phosphorylation biosensor to observe Cdc25 spatiotemporal phosphorylation during cell cycle progression in live single cell assays. Because this approach proved to be powerful, we provide here guidelines for the development of biosensors for any phosphorylation site of interest.