QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 27 September 2005
doi: 10.1242/jcs.02588

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Osler, M. E. Articles by Bader, D. M. Search for Related Content PubMed PubMed Citation Articles by Osler, M. E. Articles by Bader, D. M.

Bves modulates epithelial integrity through an interaction at the tight junction

¹ Stahlman Cardiovascular Laboratories, Program for Developmental Biology, Division of Cardiovascular Medicine, Vanderbilt University, 222 Pierce Avenue, Nashville, TN 37232-6300, USA
² Department of Ophthalmology and Visual Sciences, Vanderbilt University, 222 Pierce Avenue, Nashville, TN 37232-6300, USA

View larger version (102K): [in a new window]   Fig. 1. Bves expression in various epithelial cell lines. The B846 polyclonal anti-Bves antiserum labels Bves around the cell periphery of confluent monolayers of different epithelial cell lines. (A) Epicardial mesothelial cells (EMCs). (B) MDCK cells. (C) Caco-2 cells. (D) Mouse 4T-1 mammary cells. (E) HCE cells. (F) CHO cells. Arrows denote membrane labeling. Intracellular labeling of Bves in the Golgi occurs in several cell lines. Scale bar: 50 µm.

View larger version (97K): [in a new window]   Fig. 2. Bves localization at the TJ in confluent epithelial cells. Confluent MDCK cells were labeled for Bves and junctional proteins. Confocal images show colocalization of Bves with TJ proteins, occludin (A) and ZO-1 (B) in the XY (main panels) and Z planes (small top panels; arrows). Overlap of Bves with E-cadherin (C) ß-catenin (D) or desmosomal proteins (E) is not as pronounced (see arrows). The Z plane views were taken at the position of the yellow line transecting the XY images. Scale bars; 20 µm.

View larger version (88K): [in a new window]   Fig. 3. Bves localization at the TJ in vivo. (A) Cross sections through adult mouse small intestine were labeled with antibodies to Bves and occludin (top row), ZO-1 (middle row), or E-cadherin (bottom row). Merged images show a high degree of overlap of Bves with TJ proteins at an apicolateral position. Scale bar: 10 µm. (B) Ultrathin sections were labeled with Bves and detected using a 5 nm colloidal gold-conjugated antibody. Quantitative analysis of immunogold location shows the percentage of total beads that lie within a specific distance from the apical surface. The greatest number of beads is found in the domain where the TJ is located (red bar). (C) Cross sections of gastric epithelium show junctional components TJ, AJ and desmosomes vertically oriented along the lateral membrane (a,b,c). As seen in higher magnification, colloidal gold beads are clustered within 300 nm of the cell surface in the TJ domain (white arrows, a',b',c'). Occasionally, beads are detected below the TJ (c, green arrow) in regions where AJs and desmosomes are typically located. En face sections show tangentially sectioned microvilli with colloidal gold beads easily visible in a region within 300 nm from the cell membrane (d',d''). Scale bars: 100 nm.

View larger version (133K): [in a new window]   Fig. 4. Bves distribution at forming epithelial cell contacts. Subconfluent EMCs were labeled with Bves (red) and phalloidin, detecting the actin cytoskeleton (green). In A, DAPI was used to stain the nucleus blue. Progressive stages of cell contact are represented. A single cell shows intracellular Bves (A, arrow). Bves is observed between neighboring cells (B-D, arrows) and at the cell periphery in groups of cells (E, arrow). Bves is not observed at free edges with the cortical actin ring (arrowheads). Scale bar: 10 µm.

View larger version (151K): [in a new window]   Fig. 5. Bves response to persistent low Ca2+. EMCs were passaged in low Ca2+ medium (A-C). Subconfluent cells, labeled with Bves and actin-staining phalloidin (A) show that Bves is found at the cell periphery only where two cells appose (arrows). Confluent EMCs are labeled with Bves and ZO-1 (B) or E-cadherin (C). Note that E-cadherin is lost from the membrane in low Ca2+, but shares the same membrane pattern as Bves in cells cultured in complete medium (C, inserts).

View larger version (92K): [in a new window]   Fig. 6. Bves incorporation into TPA-induced membrane structures. MDCK cells were cultured in serum-free medium, switched to EGTA-containing medium, and either returned to serum-free medium (A,C,E) or treated with TPA (B,D,F). Following TPA treatment, Bves, occludin and ZO-1 are found in TPA-induced TJ-like structures at the cell membrane (B,D, arrows). E-cadherin labeling becomes diffuse and does not localize to these structures (F, arrows). Scale bar: 10 µm.

View larger version (16K): [in a new window]   Fig. 7. The C terminus of Bves interacts with the ZO-1 complex. (A) Diagram of the GST constructs used for a GST pulldown assay. (B) Western blot analysis indicates an interaction between the multimolecular protein complex containing ZO-1 and GST-Bves C terminus recombinant protein following GST pulldown. An antibody to ZO-1 detects an 225 kDa band in 10 µg and 20 µg of MDCK cell lysate. The presence of a similar band is visualized in the protein fraction eluted from beads bound with GST-C-term Bves. (C) Occludin is not detected in the protein fraction bound to any glutathione beads, but the protein is clearly expressed in 20 µg of MDCK cell lysate.

View larger version (92K): [in a new window]   Fig. 8. Compromise of TJ integrity following knockdown and rescue of Bves in HCE cells. (A) Expression of Bves and ZO-1 at the cell periphery in parental HCE cells. (B) HCE-R cells stably expressing the `rescue' chicken Bves-FLAG construct exhibit labeling of the membrane by both FLAG and Bves antibodies. Note the intracellular accumulation of transfected chicken Bves. (C) Treatment with anti-human Bves MO results in a loss of Bves at the cell periphery, as compared to untreated (NT) and control MO. (D) TER measurements were performed on HCE and HCE-R. Parental HCE cells exhibited a TER value of approximately 390 /cm2. Treatment with anti-human Bves morpholino resulted in a 40% decrease (P<0.05) in TER (asterisk), while control MO did not significantly alter the TER. The TER of HCE-R cells doubled to 775 /cm2 and, following anti-human Bves MO (double asterisk), a drop of only 20% (P<0.05) in TER was observed. (E) ZO-1 immunofluorescence performed on Transwell inserts following TER measurements. Parental HCE cells and HCE-R cells were treated with control and anti-human Bves MO. Bves and ZO-1 expression is disrupted in HCE cells treated with anti-human Bves MO (top right panel). Morpholino treatment does not alter the expression pattern of ZO-1 in HCE-R cells (lower right panel). (F) Western blotting demonstrates that membrane ZO-1 and Bves increase with exogenous expression of Bves in HCE-R cells, but decrease following treatment with Bves-MO, as compared with the parental HCE line. (G) Membrane integrity of junction proteins occludin, E-cadherin and ß-catenin is disrupted following Bves MO treatment. Scale bars: 10 µm.