Journal: Molecular Biology of the Cell

Article Title: The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells ^†

doi: 10.1091/mbc.e21-05-0268

Figure Lengend Snippet: FIGURE 1: EPI64A and EPI64B both localize to apical microvilli. (A) Schematic of EPI64A and EPI64B domains with percentage identities and the TBC/RabGAP arginine residue necessary for the RabGAP activity indicated. (B) Western blots with antibodies to EPI64B and tubulin of Jeg-3 cell lysates after treatment with the indicated siRNAs to EPI64B. (C) Western blot with antibodies to EPI64B on cell lysates from several cultured cell lines. (D) SIM microscopy of Jeg-3 cells transfected with GFP-EPI64A (top block of panels) or GFP-EPI64B (bottom block of panels). Cells were transfected to express GFP-tagged constructs (green) and stained for ezrin (red) and actin (blue). Arrows indicate the localization of GFP-EPI64A to the base of microvilli. Scale bars: top panels, 10 µm; bottom panels, 10 µm.(E) Quantitation of GFP- EPI64A and GFP-EPI64B to localize to microvilli in wild-type cells.

Article Snippet: GFP-Rab35 wild type was a gift from Stephen Shaw (NIH); GFP-Rab35 S22N (Addgene #47426); GFP-Rab35 Q67L (Addgene #47425).

Techniques: Residue, Activity Assay, Western Blot, Cell Culture, Microscopy, Transfection, Blocking Assay, Construct, Staining, Quantitation Assay

Journal: Molecular Biology of the Cell

Article Title: The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells ^†

doi: 10.1091/mbc.e21-05-0268

Figure Lengend Snippet: FIGURE 2: EPI64A and EPI64B can localize to microvilli independently of the scaffolding protein EBP50 (A) Jeg-3 cells transfected with 3xFLAG-EBP50 were co-transfected with either GFP-EPI64A or GFP-EPI64B. The GFP-tagged proteins were immunoprecipitated with GFP-Trap beads and the immunoprecipitates blotted for FLAG and GFP. (B) Confocal imaging of microvillar localization of GFP-EPI-64A and GFP-EPI64A-LA, which cannot bind EBP50, in Jeg-3 cells. Scale bar 10 µm. (C) Western blot of cell lysates of Jeg-3 wild type, or CRISPR-modified EBP50 deletion cell line, blotted for ezrin, EBP50, and tubulin. Scale bar: 10 µm (D) Localization of ezrin and actin in Jeg-3 cells lacking endogenous EBP50. Scale bars: 10 µm. (E) Confocal imaging of GFP-EPI64A or GFP-EPI64B in Jeg-3 cells lacking EBP50. Scale bars: 10 µm.

Article Snippet: GFP-Rab35 wild type was a gift from Stephen Shaw (NIH); GFP-Rab35 S22N (Addgene #47426); GFP-Rab35 Q67L (Addgene #47425).

Techniques: Scaffolding, Transfection, Immunoprecipitation, Imaging, Western Blot, CRISPR, Modification

Journal: Molecular Biology of the Cell

Article Title: The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells ^†

doi: 10.1091/mbc.e21-05-0268

Figure Lengend Snippet: FIGURE 3: EPI64A contains a localization domain spanning its TBC domain. (A) Schematic of the EPI64 constructs used in B and C and summary of results shown in this figure (B) Confocal imaging of GFP-EPI64A-314-508, which contains the C-terminal -DTYL sequence, in Jeg-3 wild-type cells and Jeg-3 cells lacking EBP50. (C) Confocal images showing the localization of GFP-tagged deletion constructs of GFP-EPI64A. Scale bar 10 µm. (D) Immunolocalization of two HA-tagged constructs, the top one containing the minimal region that localizes to microvilli (HA-EPI64A-61-408) and the bottom one (HA-71-408) that does not localize. Scale bar: 10 µm. (E) GFP-trap pull down: Jeg-3 cells were transfected with either GFP or GFP-Arf6 together with the indicated HA-EPI64A constructs. The GFP or GFP-Arf6 were recovered and analyzed for the presence of the HA-EPI64A constructs by immunoblotting.

Article Snippet: GFP-Rab35 wild type was a gift from Stephen Shaw (NIH); GFP-Rab35 S22N (Addgene #47426); GFP-Rab35 Q67L (Addgene #47425).

Techniques: Construct, Imaging, Sequencing, Transfection, Western Blot

Journal: Molecular Biology of the Cell

Article Title: The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells ^†

doi: 10.1091/mbc.e21-05-0268

Figure Lengend Snippet: FIGURE 5: Jeg-3 cells lacking EPI64A and EPI64B lack microvilli (A) Western blot with antibodies to EPI64A, EPI64B, and tubulin on whole cell lysates of Jeg-3 cells genetically modified to lack EPI64A, EPI64B, or both proteins (DKO). (B) Confocal imaging showing localization of ezrin and actin in wild-type Jeg-3 cells and the single (A-KO, B-KO) and double knockout cells. Scale bar 10 µm. (C) Quantitation of the percentage of indicated cells stained for ezrin that express surface microvilli. Normal defined >50% coverage of the apical surface with microvilli. One-way analysis of variance gave the indicated p values. (D) EPI64A/B double knockout cells were transfected to express the indicated constructs and the percentage of ezrin-stained cells (total for either untransfected or GFP-expressing for transfected cells) that express normal apical microvilli. One-way analysis of variance gave the indicated p values. (E) Localization of tight junction ZO-1 in wild-type and knockout Jeg-3 cells. Scale bar: 10 µm.

Article Snippet: GFP-Rab35 wild type was a gift from Stephen Shaw (NIH); GFP-Rab35 S22N (Addgene #47426); GFP-Rab35 Q67L (Addgene #47425).

Techniques: Western Blot, Genetically Modified, Imaging, Double Knockout, Quantitation Assay, Staining, Transfection, Construct, Expressing, Knock-Out

Journal: Molecular Biology of the Cell

Article Title: The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells ^†

doi: 10.1091/mbc.e21-05-0268

Figure Lengend Snippet: FIGURE 6: Dominant negative Rab8A and Rab35A can restore microvilli to EPI64A/B double knockout cells. (A) Wild-type Jeg-3 cells were transfected to express GFP or the indicated GFP-Rab proteins and the percentage of cells (total for either untransfected or GFP-expressing for transfected cells) determined that express apical microvilli. (B) Jeg-3 EPI64A/B double knockout cells were transfected with GFP or the indicated GFP-Rab proteins and the percentage of cells (total for either untransfected or GFP-expressing for transfected cells) expressing microvilli scored. One-way analysis of variance gave the indicated p values.

Article Snippet: GFP-Rab35 wild type was a gift from Stephen Shaw (NIH); GFP-Rab35 S22N (Addgene #47426); GFP-Rab35 Q67L (Addgene #47425).

Techniques: Dominant Negative Mutation, Double Knockout, Transfection, Expressing

Journal: Molecular Biology of the Cell

Article Title: The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells ^†

doi: 10.1091/mbc.e21-05-0268

Figure Lengend Snippet: FIGURE 7: Caco-2 cells lacking EPI64A and EPI64B have microvilli (A) Western blots of whole cell lysates from Caco-2 BBE1 wild-type, EPI64A, and EPI64B single knockout and double knockout cells blotted for EPI64A, EPI64B, and tubulin. (B) Confocal imaging showing localization of ezrin in the apical region of wild-type and knockout cells. Scale bar: 10 µm. (C) Localization of GFP-EPI64A and GFP-EPI64B expressed in double knockout cells. Scale bar 10 µm.

Article Snippet: GFP-Rab35 wild type was a gift from Stephen Shaw (NIH); GFP-Rab35 S22N (Addgene #47426); GFP-Rab35 Q67L (Addgene #47425).

Techniques: Western Blot, Knock-Out, Double Knockout, Imaging

Journal: Molecular Biology of the Cell

Article Title: The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells ^†

doi: 10.1091/mbc.e21-05-0268

Figure Lengend Snippet: FIGURE 8: Caco-2 cells lacking EPI64A and EPI64B have aberrant apical junctions (A) Fields of wild-type, EPI64A and EPI64B single knockout and EPI64A/B double knockout cells stained for actin and the tight junction marker ZO-1. The phenotypes seen were variable, so the most wild–type-looking regions of cells are shown (Normal) and contrasted with regions where the normal polygonal organization is disrupted (Severe). Scale bar: 10 µm. (B) Percentage of wild-type and knockout cells in which one or more of its junctions shows a reflex angle (>180°). One-way analysis of variance gave the indicated p values. (C) Example of stellate knockout cell stained for ezrin, myosin IIA, and actin XY-dimensions (top panels) and YZ-dimensions (bottom panel). (D) Localization of vinculin, actin, and myosin IIA in the apical (top panels) and basal (bottom panels) sections of wild-type and double knockout Caco-2 cells. Scale bars 10 µm.

Article Snippet: GFP-Rab35 wild type was a gift from Stephen Shaw (NIH); GFP-Rab35 S22N (Addgene #47426); GFP-Rab35 Q67L (Addgene #47425).

Techniques: Knock-Out, Double Knockout, Staining, Marker

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