ARHGEF17 activates RhoA at the apical region and cleavage furrow to promote BC elongation. (A) Localization of ARHGEF17 during interphase and cytokinesis. Montage of time-lapse imaging of Can 10 cells expressing ARHGEF17-GFP and mScarlet–Myl12b during cytokinesis. A maximum-intensity projection of cells #1 and #2, outlined in white and magenta, respectively, at time 0 is shown in the bottom left corner. Confocal slice images of interphase (#1, 0 min; & #2, 160 min), anaphase (#1, 30 min; & #2, 70 min), and telophase (#1, 40 min; & #2, 80 min) are shown at the bottom. Yellow arrows, arrowheads, and double arrows indicate BCs, the cleavage furrow, and the midbody, respectively. (B) ARHGEF17 localizes to the daughter cell interface near the midbody during late cytokinesis. Shown are representative confocal images of Can 10 cells expressing ARHGEF17-GFP alone (right) or with mScarlet–Myl12b (left) cultured for 2 days after transduction, and stained as indicated. (C) ARHGEF17 is required for RhoA activation at the apical region and cleavage furrow during cytokinesis. Time-lapse montage of Can 10 cells transfected with control or ARHGEF17 siRNAs and expressing dT-2×rGBD. SUM projections (top), confocal images (bottom), and zoomed views of boxed regions are shown. Arrows (−50 min) indicate BCs; arrowheads (0 min) indicate the midbody. See also Video 3. (D) Quantitative analysis of RhoA activation in cells with a preexisting BC. Ratios of dT-2×rGBD intensities at the BC vs. cytoplasm in control and ARHGEF17-knockdown cells imaged in C are plotted over time (top), and at selected time points (bottom). Bold lines and shaded bands represent the mean and SD, respectively, from two independent experiments (n = 31 cells transfected with control siRNA; n = 27 cells transfected with ARHGEF17 siRNA). (E) ARHGEF17 primarily activates RhoA at the apical region, but not at the midbody. Ratios of dT-2×rGBD intensity at the BC or midbody to cytoplasm are shown in cells from C. Data represent combined scatter and box-and-whisker plots of active RhoA in control cells (n = 31) and ARHGEF17-knockdown cells (n = 27) from two independent experiments. (F) ARHGEF17 is required for BC elongation. Shown are representative confocal images of Can 10 cells transfected with control siRNA or ARHGEF17 siRNA, cultured for 72 h, and stained with DAPI, phalloidin, and antibodies against aPKC and ZO-1. (G) Quantification of polarity and BC structures in control and ARHGEF17-knockdown Can 10 cells. Data represent means ± SD from four independent experiments (≥373 cells per condition). (H and I) GEF activity of ARHGEF17 is required for BC elongation. Can 10 cells were transfected with ARHGEF17 siRNA #2 one day earlier, after which they were transduced with lentiviruses expressing either GFP, si#2-resistant ARHGEF17-GFP, or ARHGEF17ΔDH-GFP. Cells were fixed and stained using antibodies against GFP and aPKC, along with DAPI and phalloidin. Representative confocal images are shown. (I) Quantification of BC structures in cells shown in H. Data represent means ± SD from four independent experiments (≥44 cells per condition). (J and K) ARHGEF17 is required for E-cadherin accumulation at the cleavage furrow adjacent to the midbody. Confocal images of control and ARHGEF17-knockdown cells stained with DAPI, phalloidin, and antibodies against E- and N-cadherin. Arrowheads and arrows indicate AJs and the midbody region, respectively. (K) Quantitative analysis of E-cadherin and N-cadherin distribution at the division site. Ratios of cadherin intensity at the basal side of the midbody to their intensity at AJs are shown in cells from J. Data are from two independent experiments (≥21 cells per condition). See also Fig. S5 O. Scale bars, 1 µm (zoomed images in B and J), 2 µm (zoomed images in C), 10 µm (A, B, C, and J), 20 µm (F and H). P values are shown above each graph; n.s., not significant.