Fluorescence fluctuation analysis reveals PpV dependent Cdc25 protein dynamics in living embryos.

Author summary Embryonic development starts with a series of fast nuclear divisions in most animals, which is followed by a dramatical cell cycle slowdown to enter a pause. Drosophila embryos undergo 13 fast and synchronous nuclear cycles with only S and M phases. In interphase 14, the cell cycle is remodeled: the mitosis pauses, the S phase is prolonged, and a gap phase is introduced. Post-translational regulation of Cdc25/Twine phosphatase is responsible for this remodeling. Although tribbles is involved, it has remained unclear how the timely degradation of Twine in interphase 14 is controlled. Here, we show that Protein phosphatase V (PpV) tightly controls Twine dynamics and thus the timing of cell cycle remodeling. PpV ensures appropriately low Twine levels at the onset of interphase 14 with little embryo-to-embryo variation. During interphase 14, tribbles and other factors are involved in the swift Twine degradation. This study provides insights in post-translational and safeguarding mechanisms of Cdc25, as well as in developmental timing of early embryogenesis. The protein phosphatase Cdc25 is a key regulator of the cell cycle by activating Cdk-cyclin complexes. Cdc25 is regulated by its expression levels and post-translational mechanisms. In early Drosophila embryogenesis, Cdc25/Twine drives the fast and synchronous nuclear cycles. A pause in the cell cycle and the remodeling to a more generic cell cycle mode with a gap phase are determined by Twine inactivation and destruction in early interphase 14, in response to zygotic genome activation. Although the pseudokinase Tribbles contributes to the timely degradation of Twine, Twine levels are controlled by additional yet unknown post-translational mechanisms. Here, we apply a non-invasive method based on fluorescence fluctuation analysis (FFA) to record the absolute concentration profiles of Twine with minute-scale resolution in single living embryos. Employing this assay, we found that Protein phosphatase V (PpV), the homologue of the catalytic subunit of human PP6, ensures appropriately low Twine protein levels at the onset of interphase 14. PpV controls directly or indirectly the phosphorylation of Twine at multiple serine and threonine residues as revealed by phosphosite mapping. Mutational analysis confirmed that these sites are involved in control of Twine protein dynamics, and cell cycle remodeling is delayed in a fraction of the phosphosite mutant embryos. Our data reveal a novel mechanism for control of Twine protein levels and their significance for embryonic cell cycle remodeling.