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First published online 6 January 2004
doi: 10.1242/jcs.00879

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The tyrosine phosphatase DEP-1 induces cytoskeletal rearrangements, aberrant cell-substratum interactions and a reduction in cell proliferation

¹ School of Molecular and Microbial Sciences, Institute for Molecular Bioscience and CRC for Chronic Inflammatory Diseases, University of Queensland, Brisbane, QLD 4072, Australia
² Yamanouchi Research Institute, Littlemore Park, Armstrong Road, Oxford OX4 4XS, UK
³ Randall Centre, King's College London, New Hunt's House, Guy's Campus, London SE1 1UL, UK
⁴ OSI Pharmaceuticals, Watlington Road, Oxford, OX4 6LT, UK
⁵ Oxford Glycosciences plc, The Forum, 86 Milton Park, Abingdon, Oxon OX14, UK

View larger version (78K): [in a new window]   Fig. 7. Defective turnover of focal contacts in DEP-1-expressing cells. (A) Large well-developed focal contacts can be seen at the leading edge of control fibroblasts. DEP-1 expression results in cells that generate only small focal adhesions, possibly focal complexes, at the very margins of the leading lamellipodium. (B) The unstable nature of these adhesions was quantified as described and is shown as a persistence index in which a high persistence directly equates to stability of the adhesions. Mean persistence index ± s.e.m.; Student's paired t-test shows no significant differences between 1A2 cells plus or minus treatment with mifepristone, and P<0.01 between control 2D3 cells and 2D3 plus mifepristone.

View larger version (53K): [in a new window]   Fig. 1. Inducible expression of DEP-1 protein in NIH3T3 cells. (A) Immunofluorescent staining for DEP-1 in untreated or mifepristone-treated cells showing prominent plasma membrane and some ER localization. (B) Western blotting of DEP-1 in NIH3T3 cells showing full-length protein production. Scale bar: 10 µm.

View larger version (67K): [in a new window]   Fig. 2. DEP-1 expression causes cell cycle arrest. (A) Quantification of BrdU uptake in NIH3T3 cells. (B) Immunofluorescent staining for BrdU in 2D3 NIH3T3 cells (left) or 2D3 cells induced to express DEP-1 (right). Scale bar: 10 µm.

View larger version (36K): [in a new window]   Fig. 3. DEP-1 expression reduces PDGF-induced tyrosine phosphorylation. Cells were quiesced for 4 hours then stimulated with 50 ng/ml PDGF. (A) Western blotting of whole cell lysates from PDGF-stimulated control 1A2 cells or DEP-1-expressing 2D3 cells with antiphosphotyrosine. (B) Western blotting of PDGF receptor immune precipitates with antiphosphotyrosine. (C) Association of c-src with PDGFR in DEP-1-expressing cells. DEP-1 expression reduces c-src association with PDGFR. Uninduced and induced NIH3T3 cells were stimulated with PDGF as described in Materials and Methods. PDGF receptor was immune precipitated and immunoblotted for c-src (upper panel) or PDGF (lower panel).

View larger version (63K): [in a new window]   Fig. 4. DEP-1 expression reduces PDGF-induced MAP kinase and Akt phosphorylation. (A) Western blotting of NIH3T3-DEP-1 cells for phospho p42/p44 MAPK after quiescence and stimulation with PDGF. (B) Western blotting of NIH3T3-DEP-1 cells for phospho-akt after quiescence and stimulation with PDGF.

View larger version (74K): [in a new window]   Fig. 5. DEP-1-expressing cells exhibit defective motility and interaction with fibronectin. (A) Parallel cultures of 1A2 and 2D3 cells imaged at 1-hour intervals over 6 hours showing lack of cell spreading in DEP-1-expressing 2D3 cells. (B) Quantification of cell adhesion and spreading on a fibronectin substratum. Cells were plated on to fibronectin-coated coverslips and washed after either 1 hour or 3 hours, as indicated, before fixation and counting. (C) Tyrosine phosphorylation of FAK. Cells were plated on fibronectin (FN plating +) or held in suspension (FN plating -). Lysates were immunoprecipitated with anti-FAK antibody followed by immunoblotting with anti-phosphotyrosine (upper panel) or ant-FAK (lower panel).

View larger version (94K): [in a new window]   Fig. 6. Localization of cytoskeletal proteins in DEP-1-expressing cells. Control 1A2, or DEP-1-expressing 2C2 or 2D3 cells were induced with mifepristone for 24 hours, plated on to coverslips and stained for F-actin, tubulin, vinculin or paxillin as described in Materials and Methods. All of the DEP-1-expressing cells showed disruption of microfilament bundles, microtubules and adhesion plaques. Scale bar: 10 µm

View larger version (37K): [in a new window]   Fig. 8. Aberrant chemotaxis in DEP-1-expressing NIH3T3 cells. Cells were placed in Dunn chambers and a gradient of PGDF introduced. Cell motility was recorded by time-lapse videomicroscopy. (A) Parallel cultures of control 1A2 and DEP-1-expressing 2D3 cells imaged at 10-minute intervals for the first 60 minutes of a 6-hour chemotaxis assay. 1A2 cells migrate up the PDGF gradient, while 2D3 cells do not. (B) Horizon plots of 1A2 and 2D3 cell populations analysed for directional migration over the full 6-hour period of the assay. Significant unimodal clustering of cell directions is seen in the 1A2 population (arrow plus 95% confidence limit). As the mean direction of cell movement is upward, we conclude that 1A2 cells show positive chemotaxis to a source of PDGF (n=25 cells, taken from three separate experiments in each case).

View larger version (48K): [in a new window]   Fig. 9. Cell islands overexpressing DEP-1 have enhanced plasma membrane staining for cadherin. Control cells or DEP-1 cells were induced with mifepristone and double stained for cadherin (upper panels) and F-actin (lower panels). Uninduced cells show discrete cadherin staining at cell-cell contacts with little staining on the rest of the plasma membrane (arrows), whereas DEP-1-expressing cells show distinct staining along large areas of the membrane (arrowhead). Scale bar: 10 µm.

View larger version (94K): [in a new window]   Fig. 10. Cell islands overexpressing DEP-1 accumulate ß-catenin at the cell membrane. Control 1A2 cells or 2D3 DEP-1-expressing cells were induced with mifepristone and stained for ß-catenin. Staining for ß-catenin closely paralled that of cadherin, with uninduced cells showing discrete foci of staining at cell-cell junctions and areas of membrane intercalation (arrows). DEP-1 expressing cells showed distinct ß-catenin staining along the length of the plasma membrane. Scale bar: 10 µm.

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