Endos must be in the cytoplasm to be phosphorylated by Gwl. (A) Fusion of the SV40 NLS to Endos-Flag targets it to the nucleus. Left: Endos-Flag variants analyzed. Center: Representative IF images. Scale bar: 5 µm. Right: Quantifications of the cytoplasmic/nuclear ratio of signal intensities (bars: mean ± SD). N = number of cells quantified. Error bars: SD. ****, P = 0.0001. (B) Subcellular fractionation and WB show the relative amounts of cytoplasmic and nuclear proteins. MEK and histone H3 are controls as cytoplasmic and nuclear proteins, respectively. (C) Active and cytoplasmic Gwl (K97M, NLSm) enhances Endos interaction with Tws if Endos is in the cytoplasm but not in the nucleus. Cells were transfected with the indicated constructs and submitted to GFP-Tws immunoprecipitation (IP). Products were analyzed by WB. (D) Gwl phosphorylates Endos more efficiently in the cytoplasm than in the nucleus. Cells were transfected with the indicated constructs and analyzed by WB. Quantification of the pS68 Endos-NLS(m)^SV40-Flag/total Flag band intensities are shown (mean ± SD, n = 3). *, P < 0.05; **, 0.001 < P < 0.01; ****, P < 0.0001. (E) Model for the spatiotemporal dynamics of the Gwl-Endos-PP2A-Tws module. In prophase, cyclin B-Cdk1 activates Gwl and promotes its export to the cytoplasm where Gwl phosphorylates Endos to induce its binding and inhibition of PP2A-Tws before NEBD. After NEBD, cyclin B-Cdk1 keeps Gwl active, and Gwl keeps Endos phosphorylated and PP2A-Tws inhibited during prometaphase. Spiky shapes: activated proteins. C, cytoplasmic; N, nuclear; WCE, whole-cell extract.