Journal: The Journal of Cell Biology

Article Title: Anisotropic expansion of hepatocyte lumina enforced by apical bulkheads

doi: 10.1083/jcb.202103003

Figure Lengend Snippet: Conversion of hepatocyte biaxial polarity into vectorial apico-basal polarity. (a) Down-regulation of occludin impairs the lumen formation in differentiating and polarizing hepatoblasts. Immunofluorescence images of cells stained for the apical marker CD13 and occludin. Scale bar: 10 µm. (b) Down-regulation of the TJ protein ZO-1 impairs the lumen formation in differentiating and polarizing hepatoblasts. Immunofluorescence images of cells stained for ZO-1. Scale bar: 10 µm. (c) Down-regulation of Cdc42 leads to spherical lumina instead of BC in polarizing hepatoblasts. The polarity is not perturbed, as apical markers CD13 and ZO-1 still localized to the formed lumina. Scale bar: 10 µm. (d) Down-regulation of Rab35 leads to profound changes in lumen morphology of polarizing and differentiating hepatoblasts. The cells form multicellular structures with a shared lumen positive for apical markers CD13 and ZO-1. Immunofluorescence images of cells treated with three different siRNAs. Scale bar: 10 µm. (e) Estimation of knock-down efficiency of six siRNAs designed to target Rab35 mRNA 96 h post-transfection in differentiating and polarizing hepatoblasts in vitro ( n = 2, SD).

Article Snippet: The RAB35 fragment with the linker extension was amplified from the Addgene plasmid #47424, a gift from Peter McPherson, (McGill University, Montreal, Canada; ), and subcloned into pEGFP-C3 vector (Clontech) using ScaI and BamHI restriction sites (Rab35-ScaI-2GGGGS-F: 5′-GAG AAG TAC TAC ggc ggc ggc ggc agc ggc ggc ggc ggc agc ATG GCC CGG GAC TAC GAC CA-3′, Rab35-BamHI-R: 5′-GAG AGG ATC CTC ATT AGC AGC AGC GTT TCT TTC G-3′).

Techniques: Immunofluorescence, Staining, Marker, Knockdown, Transfection, In Vitro

Journal: The Journal of Cell Biology

Article Title: Anisotropic expansion of hepatocyte lumina enforced by apical bulkheads

doi: 10.1083/jcb.202103003

Figure Lengend Snippet: 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 . (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.

Article Snippet: The RAB35 fragment with the linker extension was amplified from the Addgene plasmid #47424, a gift from Peter McPherson, (McGill University, Montreal, Canada; ), and subcloned into pEGFP-C3 vector (Clontech) using ScaI and BamHI restriction sites (Rab35-ScaI-2GGGGS-F: 5′-GAG AAG TAC TAC ggc ggc ggc ggc agc ggc ggc ggc ggc agc ATG GCC CGG GAC TAC GAC CA-3′, Rab35-BamHI-R: 5′-GAG AGG ATC CTC ATT AGC AGC AGC GTT TCT TTC G-3′).

Techniques: Knockdown, Control, Microscopy, Staining, Marker, Western Blot, Expressing, Recombinant, Luciferase, Clinical Proteomics, Membrane, Transfection, Time-lapse Microscopy

Journal: The Journal of Cell Biology

Article Title: Anisotropic expansion of hepatocyte lumina enforced by apical bulkheads

doi: 10.1083/jcb.202103003

Figure Lengend Snippet: Silencing of Rab35 causes the loss of the transversal apical membrane bulkheads and formation of spherical cysts via a cell self-organization process. (a) In the cells treated with Rab35 siRNA, the lumina tend to grow as spheres instead of elongating as tubes. Images from the live-cell time-lapse microscopy experiment showing two neighboring differentiating hepatoblasts expressing LifeAct-EGFP under Rab35 siRNA conditions. The white star indicates the forming lumen between the two cells. Note that the typical transverse striped actin pattern observed in the tubular BC is absent. Scale bar: 10 µm. See also . (b) Multicellular cyst-like structures form by cell rearrangements. Images from the live-cell time-lapse microscopy experiment. The cells self-organize in such a way that the three separate lumina (black star) eventually fuse into one large spherical lumen, in the absence of cell division. Scale bar: 10 µm. See also . ( c ) EM analysis of a cyst-like lumen resulting from the Rab35 knock-down. A series of longitudinal 90-nm sections of the lumen formed between five cells. The bulkheads typical for hepatocyte BC are absent in the lumen. Arrows indicate TJs. GG, glycogen granules. Scale bar: 5 µm. (d) Longitudinal view (left) through the middle of a 3D model of the lumen based on rendering plasma membranes and TJs (red) on serial sections. The five cells forming the lumen are represented in different colors. Red arrows point to the TJs at which the cyst is cut open to reveal the sagittal view (right). The lumen has a circular profile, and TJs do not protrude into the lumen.

Article Snippet: The RAB35 fragment with the linker extension was amplified from the Addgene plasmid #47424, a gift from Peter McPherson, (McGill University, Montreal, Canada; ), and subcloned into pEGFP-C3 vector (Clontech) using ScaI and BamHI restriction sites (Rab35-ScaI-2GGGGS-F: 5′-GAG AAG TAC TAC ggc ggc ggc ggc agc ggc ggc ggc ggc agc ATG GCC CGG GAC TAC GAC CA-3′, Rab35-BamHI-R: 5′-GAG AGG ATC CTC ATT AGC AGC AGC GTT TCT TTC G-3′).

Techniques: Membrane, Time-lapse Microscopy, Expressing, Knockdown, Clinical Proteomics

Journal: The Journal of Cell Biology

Article Title: Anisotropic expansion of hepatocyte lumina enforced by apical bulkheads

doi: 10.1083/jcb.202103003

Figure Lengend Snippet: Formation of a spherical lumen upon Rab35 silencing in vitro . Live-cell time-lapse microscopy showing the growth of a spherical lumen between two differentiating hepatoblasts expressing LifeAct-EGFP upon Rab35 silencing. Images acquired in 10-min intervals. The video displays a 29-h time window at 12,000× normal speed. Scale bar: 10 µm.

Article Snippet: The RAB35 fragment with the linker extension was amplified from the Addgene plasmid #47424, a gift from Peter McPherson, (McGill University, Montreal, Canada; ), and subcloned into pEGFP-C3 vector (Clontech) using ScaI and BamHI restriction sites (Rab35-ScaI-2GGGGS-F: 5′-GAG AAG TAC TAC ggc ggc ggc ggc agc ggc ggc ggc ggc agc ATG GCC CGG GAC TAC GAC CA-3′, Rab35-BamHI-R: 5′-GAG AGG ATC CTC ATT AGC AGC AGC GTT TCT TTC G-3′).

Techniques: In Vitro, Time-lapse Microscopy, Expressing

Journal: The Journal of Cell Biology

Article Title: Anisotropic expansion of hepatocyte lumina enforced by apical bulkheads

doi: 10.1083/jcb.202103003

Figure Lengend Snippet: Formation of a multicellular cyst in vitro upon Rab35 silencing. Live-cell time-lapse microscopy documenting the formation of a multicellular cyst upon Rab35 silencing in differentiating hepatoblasts expressing LifeAct-EGFP. Images acquired in 10-min intervals. The video displays a 51-h time window at 12,000× normal speed. Scale bar: 10 µm.

Article Snippet: The RAB35 fragment with the linker extension was amplified from the Addgene plasmid #47424, a gift from Peter McPherson, (McGill University, Montreal, Canada; ), and subcloned into pEGFP-C3 vector (Clontech) using ScaI and BamHI restriction sites (Rab35-ScaI-2GGGGS-F: 5′-GAG AAG TAC TAC ggc ggc ggc ggc agc ggc ggc ggc ggc agc ATG GCC CGG GAC TAC GAC CA-3′, Rab35-BamHI-R: 5′-GAG AGG ATC CTC ATT AGC AGC AGC GTT TCT TTC G-3′).

Techniques: In Vitro, Time-lapse Microscopy, Expressing

Journal: The Journal of Cell Biology

Article Title: Anisotropic expansion of hepatocyte lumina enforced by apical bulkheads

doi: 10.1083/jcb.202103003

Figure Lengend Snippet: Silencing of Rab35 in vivo results in altered cell polarity and liver tissue architecture. (a) Immunofluorescence images of liver tissue collected 4 d after in utero injection of Luciferase (siLuc) and Rab35 (siRab35) siRNAs formulated into LNP via vitelline vein in E13.5 embryos. The square on the low-magnification images (scale bar: 500 µm) shows where the high-resolution image was taken (scale bar: 20 µm). Imaged areas are located in the liver parenchyma, devoid of bile duct cell marker Sox9. The inserts (scale bar: 20 µm) in a’ and a’’ show the difference between BC and bile duct-like lumina in LNP-siRab35 injected liver. Panel a’’’ compares tubular lumina in the parenchyma to the bile duct lumina in the portal area (Sox9-positive cells near portal veins [PV]). (b) Immunofluorescence images of liver tissues from panel a show examples of hepatocyte polarity in the control tissue (a single hepatocyte forms multiple lumina per cell) and simple apico-basal polarity in LNP-siRab35 injected liver (cells have a single apical domain oriented toward a shared lumen). (c and d) 3D reconstruction of lumina labeled with an apical marker CD13 in 100-µm-thick sections of liver tissue injected with LNP-siLuc (c) and LNP-siRab35 (d). Scale bar: 30 µm. See also . (e) Quantification of the lumen radius distribution based on the 3D reconstructions such as in c and d ( n = 3, error bars: SEM). (f) 3D reconstruction of a tubule in LNP-siRab35–injected livers shows a cylindrical lumen (green) surrounded by multiple cells. See also . (g) A cross-section of the reconstructed tubule in f in the original microscopy image shows organization of the cells around the lumen. Scale bar: 20 µm. (h) Quantification of number of cells surrounding the lumen in relation to lumen radius and position along the tubule.

Article Snippet: The RAB35 fragment with the linker extension was amplified from the Addgene plasmid #47424, a gift from Peter McPherson, (McGill University, Montreal, Canada; ), and subcloned into pEGFP-C3 vector (Clontech) using ScaI and BamHI restriction sites (Rab35-ScaI-2GGGGS-F: 5′-GAG AAG TAC TAC ggc ggc ggc ggc agc ggc ggc ggc ggc agc ATG GCC CGG GAC TAC GAC CA-3′, Rab35-BamHI-R: 5′-GAG AGG ATC CTC ATT AGC AGC AGC GTT TCT TTC G-3′).

Techniques: In Vivo, Immunofluorescence, In Utero, Injection, Luciferase, Marker, Control, Labeling, Microscopy