Figure 6.
RhoA-ROCK signaling drives BC elongation and, together with NuMA, promotes E-cadherin accumulation at the division site during cytokinesis. (A) Venn diagram of RhoA-specific effectors identified by BioID analysis. Note that E-cadherin–specific group contains only ROCK2. n.d., not detected. (B and C) ROCK activity is required for BC elongation. (B) Confocal images of Can 10 cells treated with the ROCK inhibitor Y27632. Treatment was initiated one day after plating at a concentration of 3 µM and was continued for 48 h. Cells were stained with DAPI and antibodies against ZO-1 and radixin. (C) Quantification of polarity and BC features in Y27632-treated cells. Data represent means ± SD from four independent experiments (≥666 cells per condition). (D) ROCK activity is required for BC elongation in ex vivo mouse livers. Liver lobes from E15.5 were treated with 1 µM Y27632 for two days and stained with antibodies against ZO-1, Mdr1, and HNF4α. Representative images are shown on the left, and quantitative data for BC length on the right. Top right: combined scatter and box-and-whisker plots of BC length in ex vivo–cultured hepatocytes. # indicates the number of quantified BCs, and N indicates the number of independent experiments. Values between the two groups were compared using the Wilcoxon rank-sum test. Bottom right: mean BC length from three independent experiments. Values between the two groups were compared using Welch’s t test. (E) Distinct roles of ROCK1 and ROCK2 in hepatic polarity and BC elongation. Shown are representative confocal images of Can 10 cells transfected with siRNAs targeting ROCK1 (left) or ROCK2 (right), cultured for 72 h, and stained with DAPI and antibodies against aPKC and ROCK1 (left) or ROCK2 (right). See also Fig. S4 F. (F) Quantification of polarity and BC structures in cells from E. Data represent means ± SD from four independent experiments (≥236 cells per condition). (G) Localization of ROCK2 at the apical edge in columnar polarity cells induced by ROCK1 depletion. Shown are representative confocal images of Can 10 cells transfected with ROCK1-siRNA. Cells were cultured for 3 days and stained with DAPI and antibodies against ROCK2 and aPKC. (H and I) NuMA is required for RhoA accumulation at the cleavage furrow. (H) Confocal images (top) and MIP images of Can 10 cells transfected with NuMA siRNAs, and stained with DAPI and antibodies against RhoA and phospho-ERM. Arrowheads and arrows indicate BCs and the basal cleavage furrow, respectively. (I) Quantification of RhoA intensity at the basal cleavage furrow relative to cytoplasm (left) and polar cortex (right). Data are from two independent experiments (≥22 cells per condition). (J and K) ROCK activity is required for E-cadherin accumulation at the division site. (J) Representative confocal images of DMSO- or Y27632-treated cells stained with DAPI, phalloidin, and antibodies against E-cadherin and N-cadherin. Arrowheads and arrows indicate AJs and the basal cleavage furrow or midbody, respectively. (K) Quantification of E-cadherin intensity at the basal cleavage furrow and midbody relative to AJs. Data are from three independent experiments (≥30 cells per condition). Scale bars, 2 µm (zoomed images in J), 5 µm (H and J), 10 µm (G), 20 µm (B, E), 50 µm (D). P values are indicated in each graph; n.s., not significant. MIP, maximum-intensity projection. Refer to the image caption for details. Panel A shows a Venn diagram comparing RhoA effector proteins associated with E-cadherin and N-cadherin complexes. Panel B shows fluorescence micrographs of cells treated with DMSO or Y27632 stained for radixin, ZO-1, and nuclei. Panel C shows bar graphs quantifying hepatic polarity, columnar polarity, and bile canaliculi structures after ROCK inhibition. Panel D shows fluorescence micrographs, box and whisker plots, and bar graphs analyzing bile canaliculi length in cultured fetal mouse liver tissue treated with Y27632. Panel E shows fluorescence micrographs of ROCK1- or ROCK2-depleted cells stained for ROCK proteins, aPKC, F-actin, and nuclei. Panel F shows bar graphs quantifying polarity states and bile canaliculi structures after ROCK1 or ROCK2 knockdown. Panel G shows fluorescence micrographs and orthogonal views of ROCK2-depleted cells stained for ROCK2, aPKC, and nuclei. Panel H shows confocal and maximum-intensity projection images of control and NuMA-depleted cells stained for RhoA, pERM, and nuclei. Panel I shows box plots quantifying RhoA intensity ratios at cleavage furrow, cytosol, and polar cortex regions after NuMA knockdown. Panel J shows fluorescence micrographs and enlarged views of cells during anaphase and telophase stained for E-cadherin, N-cadherin, F-actin, and nuclei under ROCK inhibition. Panel K shows box plots quantifying basal membrane curvature surrounding the midbody during anaphase and telophase after Y27632 treatment.

RhoA-ROCK signaling drives BC elongation and, together with NuMA, promotes E-cadherin accumulation at the division site during cytokinesis. (A) Venn diagram of RhoA-specific effectors identified by BioID analysis. Note that E-cadherin–specific group contains only ROCK2. n.d., not detected. (B and C) ROCK activity is required for BC elongation. (B) Confocal images of Can 10 cells treated with the ROCK inhibitor Y27632. Treatment was initiated one day after plating at a concentration of 3 µM and was continued for 48 h. Cells were stained with DAPI and antibodies against ZO-1 and radixin. (C) Quantification of polarity and BC features in Y27632-treated cells. Data represent means ± SD from four independent experiments (≥666 cells per condition). (D) ROCK activity is required for BC elongation in ex vivo mouse livers. Liver lobes from E15.5 were treated with 1 µM Y27632 for two days and stained with antibodies against ZO-1, Mdr1, and HNF4α. Representative images are shown on the left, and quantitative data for BC length on the right. Top right: combined scatter and box-and-whisker plots of BC length in ex vivo–cultured hepatocytes. # indicates the number of quantified BCs, and N indicates the number of independent experiments. Values between the two groups were compared using the Wilcoxon rank-sum test. Bottom right: mean BC length from three independent experiments. Values between the two groups were compared using Welch’s t test. (E) Distinct roles of ROCK1 and ROCK2 in hepatic polarity and BC elongation. Shown are representative confocal images of Can 10 cells transfected with siRNAs targeting ROCK1 (left) or ROCK2 (right), cultured for 72 h, and stained with DAPI and antibodies against aPKC and ROCK1 (left) or ROCK2 (right). See also Fig. S4 F. (F) Quantification of polarity and BC structures in cells from E. Data represent means ± SD from four independent experiments (≥236 cells per condition). (G) Localization of ROCK2 at the apical edge in columnar polarity cells induced by ROCK1 depletion. Shown are representative confocal images of Can 10 cells transfected with ROCK1-siRNA. Cells were cultured for 3 days and stained with DAPI and antibodies against ROCK2 and aPKC. (H and I) NuMA is required for RhoA accumulation at the cleavage furrow. (H) Confocal images (top) and MIP images of Can 10 cells transfected with NuMA siRNAs, and stained with DAPI and antibodies against RhoA and phospho-ERM. Arrowheads and arrows indicate BCs and the basal cleavage furrow, respectively. (I) Quantification of RhoA intensity at the basal cleavage furrow relative to cytoplasm (left) and polar cortex (right). Data are from two independent experiments (≥22 cells per condition). (J and K) ROCK activity is required for E-cadherin accumulation at the division site. (J) Representative confocal images of DMSO- or Y27632-treated cells stained with DAPI, phalloidin, and antibodies against E-cadherin and N-cadherin. Arrowheads and arrows indicate AJs and the basal cleavage furrow or midbody, respectively. (K) Quantification of E-cadherin intensity at the basal cleavage furrow and midbody relative to AJs. Data are from three independent experiments (≥30 cells per condition). Scale bars, 2 µm (zoomed images in J), 5 µm (H and J), 10 µm (G), 20 µm (B, E), 50 µm (D). P values are indicated in each graph; n.s., not significant. MIP, maximum-intensity projection.

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