a, Localization of BIN2 in the abaxial epidermis of Arabidopsis seedlings at 1, 2 or 5 d.p.g. BIN2 was not detectable at 1 d.p.g., but was visible at 1.5–2 d.p.g. BIN2 localized to the plasma membrane (PM), cytoplasm and nucleus. BIN2 was more abundant and polarized at the cell cortex before every ACD. b, Localization of ATSK12 in the abaxial epidermis of Arabidopsis seedlings at 1, 2 or 5 d.p.g. ATSK12 was detected at all developmental stages and localized to the plasma membrane and cytoplasm, but unlike BIN2, it had no nuclear signal. ATSK12 was also more abundant and polarized at the cell cortex before every ACD. Insets in a and b depict BIN2 and ATSK12 localization during cytokinesis. During mitosis, both proteins relocalized to two distinct foci resembling the spindle localization as observed for mammalian GSK3. c, Localization of BASL in the abaxial epidermis of Arabidopsis seedlings at 1 d.p.g. d, Localization of POLAR in the abaxial epidermis of Arabidopsis seedlings at 1 d.p.g. e, BIN2 localization in POLAR loss- and gain-of-function mutants. BIN2 abundance in stomatal lineage cells undergoing ACD depends on POLAR. Loss of POLAR and PL1 markedly reduced, whereas POLAR overexpression greatly enhanced, BIN2 enrichment before the ACD. Insets represent zoomed in regions. f, Quantification of the abundance of BIN2–GFP in the plasma membrane of cells undergoing ACD. n, number of cells from three biologically independent cotyledons. The intensity of BIN2–GFP was measured over time in the plasma membrane in stomatal lineage cells undergoing ACD. The plasma membrane signal of meristemoid cells was divided by the plasma membrane signal of neighbouring differentiated pavement cells to quantify the relative protein abundance. g, Nuclear localization of BIN2–GFP in Col-0 and the double tDNA polar-2 pl1-1 mutant in the abaxial epidermis at 2 d.p.g. Meristemoids of the double polar-2 pl1-1 mutant showed the increased nuclear localization of BIN2–GFP . The pictures below each main image represent zoomed in regions. White arrowheads indicate the nucleus in meristemoids. h, Quantification of the nuclear signal for BIN2 in g. n, number of cells from three biologically independent cotyledons. All experiments were repeated independently three times. Box plots show the first and third quartiles, split by the median (line) and mean (cross). One-way ANOVA with Tukey’s post hoc test was used as indicated by brackets. Scale bars, 20 µm.