Fig 2: PRIP maintains PI(4,5)P2 at the cleavage furrow and appropriately manages PI(4,5)P2-dependent cytokinesis progression. (a–c) A time-lapse experiment using stably EGFP-PLCdPH-expressing HEK293 cells and fluorescence recovery after photobleaching (FRAP) analysis. The bleached area (or the pre-bleached area in 0–8 min panels) and non-bleached furrow region are outlined by a white square and a white dotted square on a set of representative images in (a), respectively. Representative images of PRIP2-si1-treated cells are shown as two data sets, a delayed cytokinesis phenotype (middle panel set) and a slightly delayed cytokinesis phenotype (bottom panel set) compared with conventional cytokinesis (control, upper panel set). The graphs in (b,c) present the relative fluorescence intensity of EGFP-PLCdPH [normalised to the value at time 0 (b) or the pre-bleach value (c)] on the non-bleached side of the furrow (b) or bleached cleavage furrow (c) at each time point. The data are presented as the means ± SD (n > 20 for each group). **p < 0.01, ***p < 0.001 versus the control value at each time point (Kruskal–Wallis test followed by Dunn’s multiple comparison test). (d,e) HEK293 cells stably expressing EGFP-tagged PLCdPH were transfected with indicated constructs. Representative images at a stage of cytokinesis are shown in (d). Graph in (e) shows the EGFP fluorescence intensity (arbitrary units) at the cleavage furrow. The data are presented as the mean ± SD (n > 45 for each group]. ***p < 0.001 (Kruskal–Wallis test followed by Dunn’s multiple comparison test). (f) MCF-7 cells stably expressing control vector (EGFP), EGFP-tagged PRIP, or EGFP-tagged PRIP1(R134Q) were co-transfected with Halo-tagged OCRL1 or Halo-tag vector. Quantitative analysis of cytokinetic abnormalities in OCRL1-transfected MCF-7 cells with indicated PRIP1 mutants were evaluated at 3 h after release from monastrol. Each experiment was repeated at least three times (n > 30 for each group).

Fig 3: PRIP prevents PI(4,5)P2 recognition by OCRL1 but not by RhoA. (a–d) PI(4,5)P2 sedimentation assay was performed in a combination of EGFP-PRIP1, GST-PRIP2, Halo-OCRL1, and Halo-RhoA (a,c). Supernatant 1 (Sup1) contained Halo-OCRL1 (a) or Halo-OCRL1 and Halo-RhoA (c), as confirmed by immunoblotting using indicated antibodies. The fractions of Sup1 in (a or c) precipitated with liposomes [PC, 100% PC; 5% PIP2, PI(4,5)P2:PC = 5:95 (molar ratio)] in the presence (+) or absence (-) of recombinant PRIP1 or PRIP2 (b) or with liposomes (5% PIP2) in the presence of indicated doses of recombinant PRIP1 (d) by centrifugation, respectively. Western blotting of the obtained pellet fraction (Ppt2, b or d) and supernatant (Sup2, b) was performed using indicated antibodies (left side of each panel). Similar data were obtained from three independent experiments, and a set of representative images is shown. The image obtained in a high sensitivity mode (high, d) is shown. Each of the original blots is shown in Supplementary Fig. S8 for (d–f) or Supplementary Fig. S9 for (b).