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Rac mediates cytoskeletal rearrangements and increased cell motility induced by urokinase-type plasminogen activator receptor binding to vitronectin.

Kjøller L, Hall A - J. Cell Biol. (2001)

Bottom Line: In addition, a fourfold increase in the level of activated Rac is induced by uPAR expression.We conclude that uPAR interacts with VN both to initiate a p130Cas/Rac-dependent signaling pathway leading to actin reorganization and increased cell motility and to act as an adhesion receptor required for these responses.This mechanism may play a role in uPAR-mediated regulation of cell motility at sites where VN and uPAR are co-expressed, such as malignant tumors.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory for Molecular Cell Biology, Cancer Research Campaign Oncogene and Signal Transduction Group, University College London, United Kingdom.

ABSTRACT
The urokinase-type plasminogen activator receptor (uPAR) is involved in the regulation of cell motility in a variety of cell types. We show here that expression of human uPAR in growing murine fibroblasts leads to a dramatic reorganization of the actin cytoskeleton. uPAR expression induces multiple rapidly advancing protrusions that resemble the leading edge of migrating cells. The cytoskeletal changes are independent of uPA and activation of the RGD-binding activity of integrins but require uPAR binding to vitronectin (VN). The actin reorganization is blocked by coexpression of dominant negative versions of either Rac (N17Rac) or p130Cas, but not by inhibitors of Cdc42 or Rho, and is accompanied by a Rac-dependent increase in cell motility. In addition, a fourfold increase in the level of activated Rac is induced by uPAR expression. We conclude that uPAR interacts with VN both to initiate a p130Cas/Rac-dependent signaling pathway leading to actin reorganization and increased cell motility and to act as an adhesion receptor required for these responses. This mechanism may play a role in uPAR-mediated regulation of cell motility at sites where VN and uPAR are co-expressed, such as malignant tumors.

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Effect of uPAR on vinculin localization and cell protrusion dynamics. (A) Swiss 3T3 cells were injected with pRc/CMV-uPAR (100 μg/ml) as described in the legend to Fig. 1. Vinculin localization was visualized by double immunofluorescence with mouse antivinculin (VIN-11-5) followed by FITC goat anti–mouse IgG. Representative examples of vinculin distribution in control and uPAR-expressing cells are shown. (B) Swiss 3T3 cells were injected with pRc/CMV-uPAR (100 μg/ml), allowed to recover for 1 h, and then analyzed by time-lapse videomicroscopy. A total of 15 individual cells were examined exhibiting identical responses. Images of a representative cell captured at the indicated times are shown. After time-lapse videomicroscopy, cells were fixed and stained to confirm uPAR expression. (C) Single protrusion dynamics in uPAR-expressing Swiss 3T3 cells. Cells were treated and analyzed as described in B. The extension of two separate protrusions within 2 min is indicated by arrowheads. Bars, 10 μm.
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Figure 2: Effect of uPAR on vinculin localization and cell protrusion dynamics. (A) Swiss 3T3 cells were injected with pRc/CMV-uPAR (100 μg/ml) as described in the legend to Fig. 1. Vinculin localization was visualized by double immunofluorescence with mouse antivinculin (VIN-11-5) followed by FITC goat anti–mouse IgG. Representative examples of vinculin distribution in control and uPAR-expressing cells are shown. (B) Swiss 3T3 cells were injected with pRc/CMV-uPAR (100 μg/ml), allowed to recover for 1 h, and then analyzed by time-lapse videomicroscopy. A total of 15 individual cells were examined exhibiting identical responses. Images of a representative cell captured at the indicated times are shown. After time-lapse videomicroscopy, cells were fixed and stained to confirm uPAR expression. (C) Single protrusion dynamics in uPAR-expressing Swiss 3T3 cells. Cells were treated and analyzed as described in B. The extension of two separate protrusions within 2 min is indicated by arrowheads. Bars, 10 μm.

Mentions: Since cells also reorganize their adhesion complexes during migration, we examined the effect of uPAR on these structures. In control cells, vinculin, a component of adhesion complexes, localized to classic focal adhesions (Fig. 2 A). uPAR expression resulted in its relocalization into smaller punctate complexes that appear at the leading edge of the extensions. These structures are similar to focal complexes described previously to be induced by activated Rac (Nobes and Hall 1995). Identical changes in the localization patterns were seen for other adhesion complex components such as paxillin and phosphotyrosine-containing proteins (data not shown).


Rac mediates cytoskeletal rearrangements and increased cell motility induced by urokinase-type plasminogen activator receptor binding to vitronectin.

Kjøller L, Hall A - J. Cell Biol. (2001)

Effect of uPAR on vinculin localization and cell protrusion dynamics. (A) Swiss 3T3 cells were injected with pRc/CMV-uPAR (100 μg/ml) as described in the legend to Fig. 1. Vinculin localization was visualized by double immunofluorescence with mouse antivinculin (VIN-11-5) followed by FITC goat anti–mouse IgG. Representative examples of vinculin distribution in control and uPAR-expressing cells are shown. (B) Swiss 3T3 cells were injected with pRc/CMV-uPAR (100 μg/ml), allowed to recover for 1 h, and then analyzed by time-lapse videomicroscopy. A total of 15 individual cells were examined exhibiting identical responses. Images of a representative cell captured at the indicated times are shown. After time-lapse videomicroscopy, cells were fixed and stained to confirm uPAR expression. (C) Single protrusion dynamics in uPAR-expressing Swiss 3T3 cells. Cells were treated and analyzed as described in B. The extension of two separate protrusions within 2 min is indicated by arrowheads. Bars, 10 μm.
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Related In: Results  -  Collection

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Figure 2: Effect of uPAR on vinculin localization and cell protrusion dynamics. (A) Swiss 3T3 cells were injected with pRc/CMV-uPAR (100 μg/ml) as described in the legend to Fig. 1. Vinculin localization was visualized by double immunofluorescence with mouse antivinculin (VIN-11-5) followed by FITC goat anti–mouse IgG. Representative examples of vinculin distribution in control and uPAR-expressing cells are shown. (B) Swiss 3T3 cells were injected with pRc/CMV-uPAR (100 μg/ml), allowed to recover for 1 h, and then analyzed by time-lapse videomicroscopy. A total of 15 individual cells were examined exhibiting identical responses. Images of a representative cell captured at the indicated times are shown. After time-lapse videomicroscopy, cells were fixed and stained to confirm uPAR expression. (C) Single protrusion dynamics in uPAR-expressing Swiss 3T3 cells. Cells were treated and analyzed as described in B. The extension of two separate protrusions within 2 min is indicated by arrowheads. Bars, 10 μm.
Mentions: Since cells also reorganize their adhesion complexes during migration, we examined the effect of uPAR on these structures. In control cells, vinculin, a component of adhesion complexes, localized to classic focal adhesions (Fig. 2 A). uPAR expression resulted in its relocalization into smaller punctate complexes that appear at the leading edge of the extensions. These structures are similar to focal complexes described previously to be induced by activated Rac (Nobes and Hall 1995). Identical changes in the localization patterns were seen for other adhesion complex components such as paxillin and phosphotyrosine-containing proteins (data not shown).

Bottom Line: In addition, a fourfold increase in the level of activated Rac is induced by uPAR expression.We conclude that uPAR interacts with VN both to initiate a p130Cas/Rac-dependent signaling pathway leading to actin reorganization and increased cell motility and to act as an adhesion receptor required for these responses.This mechanism may play a role in uPAR-mediated regulation of cell motility at sites where VN and uPAR are co-expressed, such as malignant tumors.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory for Molecular Cell Biology, Cancer Research Campaign Oncogene and Signal Transduction Group, University College London, United Kingdom.

ABSTRACT
The urokinase-type plasminogen activator receptor (uPAR) is involved in the regulation of cell motility in a variety of cell types. We show here that expression of human uPAR in growing murine fibroblasts leads to a dramatic reorganization of the actin cytoskeleton. uPAR expression induces multiple rapidly advancing protrusions that resemble the leading edge of migrating cells. The cytoskeletal changes are independent of uPA and activation of the RGD-binding activity of integrins but require uPAR binding to vitronectin (VN). The actin reorganization is blocked by coexpression of dominant negative versions of either Rac (N17Rac) or p130Cas, but not by inhibitors of Cdc42 or Rho, and is accompanied by a Rac-dependent increase in cell motility. In addition, a fourfold increase in the level of activated Rac is induced by uPAR expression. We conclude that uPAR interacts with VN both to initiate a p130Cas/Rac-dependent signaling pathway leading to actin reorganization and increased cell motility and to act as an adhesion receptor required for these responses. This mechanism may play a role in uPAR-mediated regulation of cell motility at sites where VN and uPAR are co-expressed, such as malignant tumors.

Show MeSH
Related in: MedlinePlus