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

First published online 20 July 2004
doi: 10.1242/jcs.01246

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend 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 Takenouchi, H. Articles by Fujimoto, J. Search for Related Content PubMed PubMed Citation Articles by Takenouchi, H. Articles by Fujimoto, J. Social Bookmarking                         What's this?

Shiga toxin binding to globotriaosyl ceramide induces intracellular signals that mediate cytoskeleton remodeling in human renal carcinoma-derived cells

¹ Department of Developmental Biology, National Research Institute for Child Health and Development, 3-35-31, Taishido, Setagaya-ku, Tokyo, 154-8567, Japan
² Department of Bacteriology, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan

View larger version (61K): [in a new window]   Fig. 1. Morphological changes in ACHN cells after treatment with the Stx1-B subunit. ACHN cells stained with Cell-tracker Green were treated with and without 5 µg/ml of the Stx1-B subunit for the indicated periods and visualized using confocal microscopy. Filopodia- and lamellipodia-like structures are visible at higher magnifications and are indicated by the arrowheads. Results are representative of three independent experiments.

View larger version (127K): [in a new window]   Fig. 2. Effect of Stx1-B subunit on the distribution of ezrin and actin in ACHN cells. ACHN cells treated with the Stx1-B subunit as described in Fig. 1 were double-stained with Alexa-488-labeled anti-ezrin mAb (left panels, green) and TRITC-phalloidin (center panels, red) and visualized using confocal microscopy. The right panels represent the superposition of the green and red images, with DAPI counter staining (blue). The arrowheads indicate the areas of ezrin and actin colocalization (yellow). Results are representative of five independent experiments.

View larger version (115K): [in a new window]   Fig. 3. Effect of Stx1-B subunit on the distribution of CD44 and actin in ACHN cells. ACHN cells were examined as described in Fig. 2 using Alexa-488-labeled anti-CD44 mAb (left panels, green) and TRITC-phalloidin (center panels, red). The arrowheads indicate the areas of CD44 and actin colocalization (yellow). Results are representative of three independent experiments.

View larger version (121K): [in a new window]   Fig. 4. Effect of Stx1-B subunit on the distribution of paxillin and FAK in ACHN cells. ACHN cells were examined as described in Fig. 2 using Alexa-488-labeled anti-paxillin mAb (left panels, green) and Alexa-546-labeled anti-FAK Ab (center panels, red). The arrowheads indicate the areas of paxillin and FAK colocalization (yellow). Results are representative of three independent experiments.

View larger version (122K): [in a new window]   Fig. 5. Effect of Stx1-B subunit on the distribution of vimentin and cytokeratin in ACHN cells. ACHN cells were examined as described in Fig. 2 using Alexa-488-labeled anti-vimentin mAb (left panels, green) and Alexa-546-labeled anti-cytokeratin mAb (center panels, red). The arrowheads indicate the perinuclear clustering of vimentin. Results are representative of three independent experiments.

View larger version (120K): [in a new window]   Fig. 6. Effect of Stx1-B subunit on the distribution of - and -tubulins in ACHN cells. ACHN cells were examined as described in Fig. 2 using Alexa-488-labeled anti--tubulin Ab (left panels, green) and Alexa-546-labeled anti--tubulin mAb (center panels, red). The arrowheads indicate the accumulation of -tubulin at the microtubule-organizing centers. Results are representative of three independent experiments.

View larger version (33K): [in a new window]   Fig. 7. Effect of Stx1-B subunit on the polymerization of actin and tubulin in ACHN cells. (A) ACHN cells were treated with and without 5 µg/ml of the Stx1-B subunit as described in Fig. 1 and lysed in the actin stabilization buffer. After removing aliquots from each whole lysate for the determination of total actin, polymerized actin (filamentous actin) was separated from soluble actin (monomer actin) by centrifugation. Both fractions of polymerized actin (pellet, upper panel) and total actin (whole, mid panel) were detected by immunoblot analysis and quantitated by densitometry. The proportion (%) polymerized was calculated by dividing the actin in the pellet fraction by the actin in the whole lysate and indicated (lower panel). (B) Tubulin polymerization was examined as in A.

View larger version (45K): [in a new window]   Fig. 8. Transient phosphorylation of ezrin in ACHN cells after treatment with the Stx1-B subunit. (A) Total cell extracts were prepared from ACHN cells treated with or without 5 µg/ml of Stx1-B subunit for the indicated periods. After separation on 10% SDS-PAGE gel, the proteins were transferred to a nitrocellulose membrane and immunoblotted using the indicated antibodies. (B) Total cell extracts were prepared from ACHN cells treated with or without different amounts of Stx1-B subunit for 10 minutes and immunoblot analysis was performed using anti-phospho-specific ezrin antibody (P-Ezrin) as in A. Intensity of the phospho-ezrin signals obtained from each sample was quantitated by densitometry and the relative value of each to that of untreated cells (each value/the value of untreated cells) was indicated as a graph (lower panel). (C) Total cell extracts were prepared from ACHN cells treated and not treated with 5 µg/ml each of either anti-globotriaosyl ceramide antibodies (38.13, 1A4) or Stx1-B subunit for 10 minutes and examined by immunoblotting as in A. (D) Five µg/ml of Stx1-B pentamer was bound to the cell surface of the ACHN cells by incubation for 30 minutes at 4°C. After intensive washing with ice-cold PBS to remove the excess toxin, temperature was shifted from 4°C to 37°C to allow intracellular stimulation and cells were incubated for the time periods indicated in the figures. Immunoblot analysis was performed as in A.

View larger version (37K): [in a new window]   Fig. 9. Effect of inhibitors on the Stx1-B-subunit-mediated phosphorylation of ezrin. ACHN cells were preincubated with the inhibitors shown in the figure for three hours. The concentrations of 300 and 900 µM for MBD and 25 and 100 µM for other inhibitors were used. After treatment with the Stx1-B subunit for 5 minutes, cell extracts were prepared, and immunoblotting for phospho-specific ezrin was performed as described in Fig. 8. In parallel, each sample was examined using an anti-ezrin antibody to confirm that the protein amounts in each lane were comparable (only the result for PP2 is shown in the second panel from the top). In the right panel, the effect of each inhibitor is schematically presented.

View larger version (98K): [in a new window]   Fig. 10. Effect of inhibitors on the Stx1-B-subunit-mediated clustering of ezrin and vimentin. (A) ACHN cells were preincubated with the inhibitors shown in the figure for three hours. The concentrations of 900 µM for MBD and 100 µM for other inhibitors were used. After treatment with the Stx1-B subunit for 10 minutes, the cells were fixed and stained with anti-ezrin monoclonal antibody (green) followed by counterstaining with DAPI (blue) as in Fig. 2. Results are representative of three independent experiments. The clustering of ezrin was indicated by arrowhead. (B) ACHN cells pre-incubated with inhibitors as in A were treated with the Stx1-B for 30 minutes and stained with anti-vimentin monoclonal antibody (green) as in A.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter