Figure 4.
Interplay between E-cadherin and NuMA is critical for BC elongation. (A) Schematic of the BioID assay used to identify proteins in proximity to E- and N-cadherin. (B) GO analysis of E- and N-cadherin–associated proteins. The top 10 terms in BP and CC are shown. Gene ratio is defined as the number of genes associated with a given term within each gene set (overlapping E-cadherin/N-cadherin, E-cadherin-specific, or N-cadherin-specific) divided by the total number of genes in that gene set. The terms related to spindle and cytokinesis are shown in green. See also Fig. S3, A–C. (C) Quantification of symmetric versus asymmetric BC inheritance during cell division. Inheritance patterns were assessed by tracking cells expressing Mzt1-GFP and radixin–mScarlet. Symmetric (emerald green) inheritance and asymmetric (gold) inheritance were quantified in 70 cells from two independent experiments. See also Fig. S4, A and B. (D) NuMA knockdown does not alter expression levels of E-cadherin/N-cadherin, p120-catenin, or RhoA. Immunoblot analysis of Can 10 cells transfected with NuMA-targeting siRNAs. MWs of marker proteins are indicated in kDa. (E) NuMA is required for BC elongation. Shown are representative confocal images of Can 10 cells transfected with NuMA siRNAs, cultured for 72 h, and stained with DAPI and antibodies against NuMA and radixin. (F) Quantification of polarity and BC features in NuMA-knockdown cells. Data represent means ± SD from four independent experiments (≥836 cells per condition). (G) E-cadherin knockdown disrupts NuMA localization at the polar cortex. Shown are representative confocal images of Can 10 cells transfected with E-cadherin siRNAs, cultured for 72 h, and stained with DAPI and the anti-NuMA antibody. Arrowheads and arrows indicate polar and equatorial cortex, respectively. (H) Quantitative analysis of NuMA localization at the PM during metaphase and anaphase in E-cadherin-knockdown cells. Shown are the ratios of NuMA intensity at the polar cortex to that at the equatorial cortex (left) and to the cytosol (right). Data are from two independent experiments (≥21 cells per condition). (I) NuMA knockdown impairs E-cadherin accumulation at the cleavage furrow. Shown are representative confocal images of Can 10 cells transfected with NuMA siRNAs, cultured for 72 h, and stained with DAPI, phalloidin, and an anti-E-cadherin antibody. Arrowheads and arrows indicate AJs and the basal cleavage furrow, respectively. (J) Quantitative analysis of E-cadherin distribution at the PM during anaphase in NuMA-knockdown cells. Ratios of E-cadherin intensity at the basal cleavage furrow (left) or polar cortex (right) to its intensity at AJs are shown. Data are from two independent experiments (≥26 cells per condition). Scale bars, 5 µm (G and I); 20 µm (E). P values are indicated in each graph; n.s., not significant. GO, gene ontology; MWs, molecular weights; BP, Biological Processes; CC, Cellular Components. Source data are available for this figure: SourceData F4. Refer to the image caption for details. Panel A shows a schematic of BioID proximity labeling assays using E-cadherin–TurboID and N-cadherin–TurboID fusion proteins. Panel B shows Gene Ontology enrichment dot plots comparing overlapping, E-cadherin-specific, and N-cadherin-specific associated protein functions and cellular components. Panel C shows schematic diagrams quantifying symmetric and asymmetric bile canaliculi inheritance during cell division. Panel D shows immunoblot images analyzing cadherin, p120-catenin, RhoA, and NuMA expression after NuMA siRNA treatment. Panel E shows confocal fluorescence micrographs of NuMA-depleted cells stained for NuMA, radixin, and nuclei to assess bile canaliculi elongation. Panel F shows bar graphs quantifying hepatic polarity, columnar polarity, and bile canaliculi structures in NuMA-knockdown cells. Panel G shows confocal fluorescence micrographs of NuMA localization during metaphase and anaphase after E-cadherin knockdown. Panel H shows box plots quantifying NuMA intensity distributions between polar cortex, equatorial cortex, and cytosol during mitosis. Panel I shows confocal fluorescence micrographs of E-cadherin distribution at cleavage furrows in control and NuMA-knockdown cells. Panel J shows box plots quantifying E-cadherin intensity at basal cleavage furrows and polar cortex regions during anaphase.

Interplay between E-cadherin and NuMA is critical for BC elongation. (A) Schematic of the BioID assay used to identify proteins in proximity to E- and N-cadherin. (B) GO analysis of E- and N-cadherin–associated proteins. The top 10 terms in BP and CC are shown. Gene ratio is defined as the number of genes associated with a given term within each gene set (overlapping E-cadherin/N-cadherin, E-cadherin-specific, or N-cadherin-specific) divided by the total number of genes in that gene set. The terms related to spindle and cytokinesis are shown in green. See also Fig. S3, A–C. (C) Quantification of symmetric versus asymmetric BC inheritance during cell division. Inheritance patterns were assessed by tracking cells expressing Mzt1-GFP and radixin–mScarlet. Symmetric (emerald green) inheritance and asymmetric (gold) inheritance were quantified in 70 cells from two independent experiments. See also Fig. S4, A and B. (D) NuMA knockdown does not alter expression levels of E-cadherin/N-cadherin, p120-catenin, or RhoA. Immunoblot analysis of Can 10 cells transfected with NuMA-targeting siRNAs. MWs of marker proteins are indicated in kDa. (E) NuMA is required for BC elongation. Shown are representative confocal images of Can 10 cells transfected with NuMA siRNAs, cultured for 72 h, and stained with DAPI and antibodies against NuMA and radixin. (F) Quantification of polarity and BC features in NuMA-knockdown cells. Data represent means ± SD from four independent experiments (≥836 cells per condition). (G) E-cadherin knockdown disrupts NuMA localization at the polar cortex. Shown are representative confocal images of Can 10 cells transfected with E-cadherin siRNAs, cultured for 72 h, and stained with DAPI and the anti-NuMA antibody. Arrowheads and arrows indicate polar and equatorial cortex, respectively. (H) Quantitative analysis of NuMA localization at the PM during metaphase and anaphase in E-cadherin-knockdown cells. Shown are the ratios of NuMA intensity at the polar cortex to that at the equatorial cortex (left) and to the cytosol (right). Data are from two independent experiments (≥21 cells per condition). (I) NuMA knockdown impairs E-cadherin accumulation at the cleavage furrow. Shown are representative confocal images of Can 10 cells transfected with NuMA siRNAs, cultured for 72 h, and stained with DAPI, phalloidin, and an anti-E-cadherin antibody. Arrowheads and arrows indicate AJs and the basal cleavage furrow, respectively. (J) Quantitative analysis of E-cadherin distribution at the PM during anaphase in NuMA-knockdown cells. Ratios of E-cadherin intensity at the basal cleavage furrow (left) or polar cortex (right) to its intensity at AJs are shown. Data are from two independent experiments (≥26 cells per condition). Scale bars, 5 µm (G and I); 20 µm (E). P values are indicated in each graph; n.s., not significant. GO, gene ontology; MWs, molecular weights; BP, Biological Processes; CC, Cellular Components. Source data are available for this figure: SourceData F4.

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