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First published online 18 March 2008
doi: 10.1242/jcs.015495

Journal of Cell Science 121, 1193-1203 (2008)
Published by The Company of Biologists 2008
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Depletion of apical transport proteins perturbs epithelial cyst formation and ciliogenesis

Juha M. Torkko*, Aki Manninen{ddagger}, Sebastian Schuck§ and Kai Simons

Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany


Figure 1
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  		Fig. 1. Morphology of the cysts and polarization of different markers in the control and crumbs-3 knockdown (Crb-3 KD) cells. (A-H) Control and Crb-3 KD cysts (A-D and E-H, respectively) were immunostained with antibodies against the apical markers podocalyxin (Pcx) or carcinoembryonic antigen (CEA), the basolateral membrane marker E-cadherin (E-cad), or ZO-1 for cell junctions as indicated. AlexaFluor®488-conjugated secondary antibodies were applied to visualize the different markers in green. All cysts were also stained for actin (TRITC-phalloidin; red) indicating outlines of lumina and individual cells. DAPI staining (blue) shown nuclei. Arrowheads in G indicate aberrant localization of E-cadherin at the luminal surface in a Crb-3 KD cyst. Scale bars, 30 µm.

 

Figure 2
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  		Fig. 2. Volocity 3D imaging of control and different KD cysts stained for ZO-1. (A-F) Green staining reveals the overall structure of the cysts, showing the variable organization of the TJs related to the positioning of individual cells in the cysts. (A) Control, (B) Anx-13 KD, (C) Gal-3 KD, (D) Cav-1 KD, (E) Stx-3 KD, (F) VIP17-KD. Scaling of the different cysts is shown as volumetric units (in µm) and is indicated separately for each of the image panels.

 

Figure 3
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  		Fig. 3. Morphological phenotypes of the different KDs in collagen I gel cyst cultures. (A-H) Samples of (A) control, (B) Stx-3 KD, (C) Cav-1 KD, (D) Gal-3 KD, (E) Anx-13 KD, (F) VIP17 KD, (G) Crb-3 KD and (H) Stx-2 KD cysts were classified according to polarization and lumen formation into three major classes (columns I, II and III). I, cysts with single lumen; II, cysts with multiple lumina; III, cysts with collapsed or no lumen formed (III also includes the cysts with inverted actin meshwork; see the supplementary material, Fig. S2). Cysts were examined after ~14 days of culture in collagen I gel with standard MEM supplemented with 5% FCS. Phenotypes were judged on the basis of several different experiments and 100-200 cysts were examined for each KD. Data shown are the mean ± s.d. (n=3). Statistical significance analysis was performed using ANOVA for the three-group comparisons and using Student's t-test for pairwise comparisons. All comparison were either *P<0.05 (significant) or **P<0.001(very significant). Representative phenotypes for the different KDs are shown in the insets, with actin/TRITC-phalloidin staining in red. Scale bars, 30 µm.

 

Figure 4
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  		Fig. 4. Morphology of the cysts and polarization of different marker proteins in knockdown cells. Columns 1. to 5. shown the following KD cells. 1., annexin-13 (Anx-13 KD); 2., caveolin-1 (Cav-1 KD); 3., galectin-3 (Gal-3 KD); 4., syntaxin-3 (Stx-3 KD); and 5., VIP17 KD. Panel groups A-D show immunostaining of the different KD cysts. Shown in green (AlexaFluor®488) are: A, apical membrane marker podocalyxin (Pcx); B, carcinoembryonic antigen (CEA); C, basolateral membrane marker E-cadherin (E-cad); D, tight-junction marker ZO-1. DAPI was used to stain nuclei (blue) and TRITC-phalloidin to show the actin meshwork (red). Arrowheads indicate (C) luminal and (D) aberrant localization of E-cadherin and ZO-1, respectively, observed sometimes in the different KDs (see text). Scale bars, 30 µm.

 

Figure 5
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  		Fig. 5. Ciliogenesis in the control and KD collagen I cysts. (A) Volocity 3D imaging showing ciliogenesis in different KD cysts. Ciliary staining (acetylated tubulin, green) from 1. Cav-1 KD, 2. Anx-13 KD, 3. Gal-3 KD, 4. Crb-3 KD, 5. Stx-3 KD and 6. VIP17 KD cysts. Scaling of the different cysts is shown as volumetric units (in µm) and is indicated separately for each of the image panels. (B) Primary cilia of the individual cells are visualized by staining for acetylated β-tubulin (green) and nuclei (blue, DAPI staining). Ciliary staining reveals two structural parts of these organelles, a punctated signal indicating the ciliary base at the apical surface of the individual cells and the outwards reaching ciliary processes projecting into the lumenal space. (C) Effect of different KDs on primary cilia formation and characteristic phenotypes of ciliogenesis in Anx-13, Cav-1, Crb-3, Gal-3, Stx-3 and VIP17 KD (1., 2., 3., 4., 5. and 6., respectively). Staining for actin (TRITC-phalloidin, red) and nuclei (DAPI, blue) show the overall structure and morphology of the KD cysts in comparison with the ciliary structures (AlexaFluor®488, green). Scale bars, 30 µm. (D) Percentage of cysts with normal (green bars) versus defective (red bars) ciliogenesis in the different KDs compared with the phenotype observed in control cysts. Examined were 100-200 cysts of each KD after ~14 days of culture in collagen I gel with standard MEM supplemented with 5% FCS. Data are the mean ± s.d. (n=3). Statistical significance was analyzed by Student's t-test; **P<0.001, very significant; *P<0.01, significant.

 

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