Localized activity of Skittles and Pten controls sites of PI(4,5)P₂ production and Moe activation. (A) Dynamic of Pten-GFP distribution during mitosis (top). A stable cell line that expresses Grp1-PH-GFP was used to probe PI(3,4,5)P₃ distribution during mitosis in control conditions or after Pten depletion. (B, top) Time-lapse frames of a Skittles-GFP cell progressing through mitosis. Bottom images show a living cell stably expressing GFP-Tubby after Skittles depletion. (C) FACS analysis showing the distribution P-Moe intensity in mitosis (assessed by histone H3 phosphorylation) of control and Pten- or Skittles-depleted cells. a.u., arbitrary unit. (D) Distribution of Moe-GFP after Skittles depletion. (E) Rapid time-lapse imaging of a GFP-Utrophin-CH (GFP-UtrCH) and α-Tubulin-mCherry after depletion of Skittles showing maximum projection. Close-ups (focal planes) correspond to the framed region and show GFP signal in black. Arrowheads show cortical rupture and defects of F-actin recruitment at the bleb rim; arrows show unregulated mitotic blebs. Bars, 10 µm.