Fig. 3: Two phospholipid-binding sites safeguard effective maintenance at the division site via non-specific electrostatic interactions.

a Domain organization of full-length POK2 and deletion constructs (blue bars). Tan and light blue bar indicate the putative amphipathic helix and the polybasic domain (PBD), respectively. b Successive images of a cell expressing p35S::GFP-POK2 #5 (green) and the microtubule marker pUBN::RFP-MBD (magenta) from pro-metaphase to cytokinesis. Cortical GFP-signal faints (arrows). c Cytokinetic cell with phragmoplast (magenta) and prominent cortical p35S::GFP-POK2 #5 + 1 (green) at the cortical division zone (arrow). Note the difference in localization in the absence of fragment #1. d PIP strips incubated with purified GST-POK2 #7, GST-POK2 #7^5RKtoLGGYV and anti-GST antibody. Diluted anti-GST antibody (1:10, left) and protein (right) were dropped as detection controls. Wild-type fragment #7 binds to PI(3)P, PI(4)P, and PI(5)P, while binding is abolished in GST-POK2 #7^5RKtoLGGYV mutant. The PIP strip assays were performed twice with similar results. e, f Images of a root cell expressing p35S::GFP-POK2 #5 + 1^{5RKtoLGGYV + RK6A}, stained with propidium iodide (e). Frequencies of dividing cells expressing this fragment (f; n = 55 cells, 11 seedlings). Signal in prophase (arrows) is lost from the cortex in cytokinesis (brackets). g Dividing BY-2 cells expressing pPOK2::GFP-POK2^{5RKtoLGGYV + RK6A}. Cortical POK2 rings in prophase are not maintained (bracket). h, i Single Z-plane images of cells expressing pPOK2::GFP-POK2 Ct^PBD-KRAS (green) along with pUBN::RFP-MBD (magenta, h). GFP-POK2 Ct^PBD-KRAS decorates the microtubule arrays (arrowheads) and localizes at the cortical division zone (arrows) comparably to wild-type Ct. (i) Comparison of division site width (yellow dashed lines) between POK2C KRAS and POK2C WT. Box plots show the median, the upper and lower quartiles, and the range of the data. The differences were not significant (One-way ANOVA: F = 1.8373, p = 0.177; Post Hoc Test: Tukey HSD; POK2C WT: n = 38 cells, 12 seedlings; POK2C KRAS: n = 42 cells, 9 seedlings). j GST-POK2 #1^KRAS binds to a wide range of phospholipids in PIP strips. Dots serve as detection controls for protein (left) and anti-GST antibody (right). The PIP strip assay was performed twice with similar results. Source data are provided as Source Data files.