Figure 5.
Conversion of hepatocyte biaxial polarity into vectorial apico-basal polarity. (a) Knock-down of Rab35 in differentiating hepatoblasts caused formation of cyst-like structures (a’), whereas cells treated with the control siRNA (siLuc) were unaffected and formed BC. Microscopy images of cells stained for F-actin with phalloidin–Alexa 488. Scale bar: 30 µm. See also Video 7. (b) 3D reconstruction of the cells treated with Rab35 siRNA show the variability of the phenotype from enlarged swollen lumina of epithelial tubes (white arrowhead) to spherical cyst-like structures (yellow arrowhead) growing in the z-direction (lumina stained with the apical marker CD13). Some remaining BC (orange arrowhead) connected to the cyst. (c) Localization of polarity markers in cyst-like structures upon Rab35 siRNA treatment. Microscopy images of cells immunostained for apically localized proteins CD13 (I) and podocalyxin (II) and basolaterally localized E-cadherin (I) and integrin β-1 (II). Scale bar: 10 µm. (d) Three independent Rab35 siRNA duplexes down-regulate Rab35 protein levels by 73% ± 3% (n = 3, error bars: SD). Representative Western blot and quantification of protein knock-down. (e) Histogram of the local lumen radius in control cells and cells treated with Rab35 siRNA estimated based on microscopy image analysis. Rab35 knock-down by three independent siRNAs results in the shift toward the lumina with larger radius. Percentage of lumina >6 µm: siLuc: 1.00% ± 0.46%, siRab35#2: 20.55% ± 1.66%, siRab35#4: 24.86% ± 1.46%, siRab35#5: 19.95% ± 2.81% (n = 3 [with four images per condition], error bars: SEM). (f) The enlarged lumina phenotype in the cells treated with Rab35 siRNA is rescued by expression of human Rab35-EGFP from recombinant adenovirus. The frequency curve of the lumen radius (yellow) overlaps with the one of the control cells expressing EGFP only (blue). EGFP alone does not affect the lumen enlargement caused by Rab35 knock-down (red). Percentage of lumina >6 µm: siLuc AdenoEGFP: 2.39% ± 0.44%, siRab35 AdenoEGFP: 22.35% ± 1.08%, siRab35AdenoEGFP-Rab35: 3.33% ± 1.05% (n = 3 [with four images per condition], error bars: SEM). (g) Microscopy images of differentiated hepatocytes treated with Luciferase or Rab35 siRNA stained with Rab35 antibodies (yellow). Rab35 localizes to the apical and lateral plasma membrane and cytoplasmic puncta. The levels of Rab35 are markedly reduced in the cyst-like structures formed upon Rab35 siRNA transfection. Scale bar: 10 µm. (h) Localization of exogenous EGFP-Rab35 in polarizing hepatoblasts. Selected images from live-cell time lapse microscopy. Two forming lumina are marked with orange arrowheads. Scale bar: 10 µm.

Conversion of hepatocyte biaxial polarity into vectorial apico-basal polarity. (a) Knock-down of Rab35 in differentiating hepatoblasts caused formation of cyst-like structures (a’), whereas cells treated with the control siRNA (siLuc) were unaffected and formed BC. Microscopy images of cells stained for F-actin with phalloidin–Alexa 488. Scale bar: 30 µm. See also Video 7. (b) 3D reconstruction of the cells treated with Rab35 siRNA show the variability of the phenotype from enlarged swollen lumina of epithelial tubes (white arrowhead) to spherical cyst-like structures (yellow arrowhead) growing in the z-direction (lumina stained with the apical marker CD13). Some remaining BC (orange arrowhead) connected to the cyst. (c) Localization of polarity markers in cyst-like structures upon Rab35 siRNA treatment. Microscopy images of cells immunostained for apically localized proteins CD13 (I) and podocalyxin (II) and basolaterally localized E-cadherin (I) and integrin β-1 (II). Scale bar: 10 µm. (d) Three independent Rab35 siRNA duplexes down-regulate Rab35 protein levels by 73% ± 3% (n = 3, error bars: SD). Representative Western blot and quantification of protein knock-down. (e) Histogram of the local lumen radius in control cells and cells treated with Rab35 siRNA estimated based on microscopy image analysis. Rab35 knock-down by three independent siRNAs results in the shift toward the lumina with larger radius. Percentage of lumina >6 µm: siLuc: 1.00% ± 0.46%, siRab35#2: 20.55% ± 1.66%, siRab35#4: 24.86% ± 1.46%, siRab35#5: 19.95% ± 2.81% (n = 3 [with four images per condition], error bars: SEM). (f) The enlarged lumina phenotype in the cells treated with Rab35 siRNA is rescued by expression of human Rab35-EGFP from recombinant adenovirus. The frequency curve of the lumen radius (yellow) overlaps with the one of the control cells expressing EGFP only (blue). EGFP alone does not affect the lumen enlargement caused by Rab35 knock-down (red). Percentage of lumina >6 µm: siLuc AdenoEGFP: 2.39% ± 0.44%, siRab35 AdenoEGFP: 22.35% ± 1.08%, siRab35AdenoEGFP-Rab35: 3.33% ± 1.05% (n = 3 [with four images per condition], error bars: SEM). (g) Microscopy images of differentiated hepatocytes treated with Luciferase or Rab35 siRNA stained with Rab35 antibodies (yellow). Rab35 localizes to the apical and lateral plasma membrane and cytoplasmic puncta. The levels of Rab35 are markedly reduced in the cyst-like structures formed upon Rab35 siRNA transfection. Scale bar: 10 µm. (h) Localization of exogenous EGFP-Rab35 in polarizing hepatoblasts. Selected images from live-cell time lapse microscopy. Two forming lumina are marked with orange arrowheads. Scale bar: 10 µm.

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