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First published online 21 May 2008
doi: 10.1242/jcs.028597

Journal of Cell Science 121, 1965-1972 (2008)
Published by The Company of Biologists 2008
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Tubedown associates with cortactin and controls permeability of retinal endothelial cells to albumin

Hélène Paradis1, Thasin Islam1, Stephanie Tucker1, Lidan Tao2, Sharon Koubi1 and Robert L. Gendron1,*

1 Division of Biomedical Sciences, Department of Medicine, Memorial University of Newfoundland, St John's, NL, A1B 3V6, Canada
2 Core Research Equipment and Instrument Training Network (CREAIT), Memorial University of Newfoundland, St John's, NL, A1B 3V6, Canada


Figure 1
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  		Fig. 1. Analysis of proteins in Tbdn immunoprecipitates. (A) Specificity of C10-20 antibody for Tbdn is demonstrated by immunoprecipitation (IP) of 35S-labeled in-vitro-translated recombinant Tbdn protein with anti-Tbdn C10-20 antibody (Ab: Tbdn) or no antibody (Ab: –) in the absence of peptide (Peptide: –), or in the presence of control peptide (Peptide: Ctr) or Tbdn C10-20-blocking peptide (Peptide: Tbdn), followed by gel electrophoresis and Coomassie Blue staining (bottom) and autoradiography (top). Control 35S-labeled in-vitro-translated Tbdn was also analyzed in parallel without immunoprecipitation (Lysate). (B) Immunoprecipitation of endothelial cell protein extracts using affinity purified anti-Tbdn antibody C10-20 (Tbdn IP) or no antibody (Control IP) followed by gel electrophoresis and silver staining. Bands of 69, 80 and 95 kDa are specific for Tbdn immunoprecipitates and are indicated by the arrows to the right of the gel.

 

Figure 2
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  		Fig. 2. Tbdn co-immunoprecipitation with cortactin. (A) The top panels show immunoprecipitation (IP) of IEM endothelial cell protein extracts using affinity purified anti-Tbdn rabbit antibody C10-20 (Tbdn), pre-immune rabbit serum (PreImm), monoclonal anti-cortactin antibody (4F11), negative-control IgG (Control) or affinity purified anti-cortactin antibody (SC11408) followed by western blotting with anti-Tbdn antibody C10-20. The blots shown in the bottom panels are the same blots as those shown in the top panels after the blots were stripped and re-probed using the anti-cortactin monoclonal antibody 4F11. Tbdn and cortactin are indicated by arrows at 100 kDa and 80 kDa, respectively. (B) Specificity of the presence of cortactin in Tbdn immunoprecipitates is demonstrated by immunoprecipitation of IEM endothelial cell protein extracts by using affinity purified anti-Tbdn antibody C10-20 (Ab: Tbdn) alone or in the presence of Tbdn C10-20-blocking peptide (Peptide: Tbdn) or control peptide (Peptide: Ctr) followed by western blotting with monoclonal anti-cortactin 4F11 antibody. PreImm, rabbit pre-immune serum. Whole-cell lysate (WCL) prepared from IEM cells was included as a positive control. An empty lane separates the IP and WCL lanes. (C) IP of protein extracts from IEM and RF/6A endothelial cells as indicated using: affinity purified anti-Tbdn antibody C10-20 (Tbdn), rabbit pre-immune serum (PreImm) or no-antibody control (Control) followed by western blotting with monoclonal anti-cortactin 4F11 antibody. Whole-cell lysates of IEM (IEM WCL), RF6A (RF/6A WCL), mouse 3T3 cells (3T3 WCL) are included as positive controls. Empty lanes are indicated (–). Representative experiments are shown.

 

Figure 3
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  		Fig. 3. Colocalization of Tbdn with F-actin and cortactin in endothelial cells. (A-C,E-G) RF/6A endothelial cells grown on glass coverslips were stained with either phalloidin for F-actin (A,C; green fluorescence) or affinity purified anti-cortactin rabbit antibody (SC11408) (E,G; green fluorescence) and then double stained with OE5 monoclonal Tbdn antibody (B,C,F,G; red fluorescence). A merged image of A and B is shown in C, and a merge of E and F is shown in G (areas of colocalization: orange/yellow). Note areas of intense colocalized staining in perinuclear areas and intense colocalization at the cortex of motile cells. (D) Merged negative control stained with negative-control antibody and vehicle control. Representative confocal microscopy of slices of cells are shown. Scale bars: 20 µm.

 

Figure 4
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  		Fig. 4. Tbdn/Ard1 knockdown in endothelial cells is associated with increased cellular permeability. (A) RF/6A parental endothelial cells (Par), Tbdn-knockdown clones AS-TBDN (ASTB#1 and #2) and control clones (Ctr#1 and #2) analyzed by western blot for Tbdn and Ard1 expression (top and middle panels, respectively). Blots were re-probed and analyzed for tubulin as loading control (bottom panel). (A) Representative experiment; (B) the average of Tbdn and Ard1 levels ± s.e.m. are shown. (C) FITC-albumin transit across monolayers of RF/6A knocked-down for Tbdn expression (ASTB#1 and #2) or controls (Par, CTR#1 and #2) expressed as a percentage of control parental cells at 30 minutes. Significantly higher percentages of FITC-albumin transit are observed in the two AS-TBDN clones compared with parental cells and the two negative-control clones. (D) Time-course of FITC-albumin transit across cellular monolayers of Tbdn-knockdown RF/6A cells (ASTB#1; black square) as compared with parental cells (black diamond) and control cells (CTR#1; white triangle) expressed as percentage of arbitrary units. Data shown in C and D are expressed as mean ± s.e.m. of at least four duplicate experiments in each group.

 

Figure 5
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  		Fig. 5. Uptake measurement and localization of FITC-albumin in RF/6A endothelial cells. (A-D) Fluorescence microscopy of FITC-albumin (A,D; green fluorescence) in RF/6A endothelial cells after 60 minutes of incubation in the presence of FITC-albumin on coverslips followed by washing off excess FITC-albumin and staining for F-actin with phalloidin (B,D; red fluorescence). Cellular nuclei are highlighted by DAPI staining (C,D; blue fluorescence). The image in D represents the merge images of A-C. Scale bar: 20 µm. (E) FITC-albumin intracellular uptake by RF/6A cells as a function of time. The data shown in E are expressed as the mean of pmol per µg of protein ± s.e.m.

 

Figure 6
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  		Fig. 6. Extravasation of serum albumin through the retinal-blood barrier in endothelial-specific-Tbdn-knockdown mice. Staining of retinal tissue for albumin was performed using a peroxidase-conjugated goat anti-albumin antibody, which yields a brown reaction product. Compared with control (non-induced single transgenic is shown) age-matched mice (A), endothelial-specific-Tbdn-knockdown eyes (B) showed significant leakage or extravasation of albumin (brown staining) from retinal blood vessels. Brown albumin staining is confined mainly to blood vessel lumens in control retinas (A), whereas brown albumin staining is observed in extravascular locations both in and around blood vessels and in neural retinal tissues in Tbdn-knockdown eyes (B). (C,D) Control (C) and Tbdn-knockdown (D) sections stained with negative-control horse-radish-peroxidase-conjugated goat anti-rabbit IgG at the same concentration as the anti-albumin reagent showed no staining. All images show the inner and some of the outer layers of the neural retina (most are visible in E) and are oriented with the vitreous cavity (v) of the eye at the bottom of the panel. The ganglion cell layer and inner limiting membrane, which are immediately adjacent to the vitreous (v), are arrowed near the bottom of panels A-D. (E,F) Hematoxylin and eosin staining of adjacent sections, revealing thickening of the retina and abundant abnormal blood vessels in Tbdn-knockdown retina (F) compared with control retinal tissues (E). Arrowheads in A,B,E and F point to blood vessels; brackets indicate the inner retinal layers (inner limiting membrane and ganglion cell layer). Representative images are shown. Magnification is 400x. A-D are not counterstained in order to emphasize brown albumin staining in A and B and lack of staining in C and D.

 

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© The Company of Biologists Ltd 2008