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Induction of cell scattering by expression of beta1 integrins in beta1-deficient epithelial cells requires activation of members of the rho family of GTPases and downregulation of cadherin and catenin function.

Gimond C, van Der Flier A, van Delft S, Brakebusch C, Kuikman I, Collard JG, Fässler R, Sonnenberg A - J. Cell Biol. (1999)

Bottom Line: Expression of beta1 integrins in GE11 cells resulted in a decrease in cadherin and alpha-catenin protein levels accompanied by their redistribution from the cytoskeleton-associated fraction to the detergent-soluble fraction.In addition, using biochemical activity assays for Rho-like GTPases, we show that the expression of beta1A, beta1D, or IL2R-beta1A in GE11 or GD25 cells triggers activation of both RhoA and Rac1, but not of Cdc42.Our results indicate that beta1 integrins regulate the polarity and motility of epithelial cells by the induction of intracellular molecular events involving a downregulation of alpha-catenin function and the activation of the Rho-like G proteins Rac1 and RhoA.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, The Netherlands Cancer Institute, 1066 CX Amsterdam,The Netherlands.

ABSTRACT
Adhesion receptors, which connect cells to each other and to the surrounding extracellular matrix (ECM), play a crucial role in the control of tissue structure and of morphogenesis. In this work, we have studied how intercellular adhesion molecules and beta1 integrins influence each other using two different beta1- cell lines, epithelial GE11 and fibroblast-like GD25 cells. Expression of beta1A or the cytoplasmic splice variant beta1D, induced the disruption of intercellular adherens junctions and cell scattering in both GE11 and GD25 cells. In GE11 cells, the morphological change correlated with the redistribution of zonula occluden (ZO)-1 from tight junctions to adherens junctions at high cell confluency. In addition, the expression of beta1 integrins caused a dramatic reorganization of the actin cytoskeleton and of focal contacts. Interaction of beta1 integrins with their respective ligands was required for a complete morphological transition towards the spindle-shaped fibroblast-like phenotype. The expression of an interleukin-2 receptor (IL2R)-beta1A chimera and its incorporation into focal adhesions also induced the disruption of cadherin-based adhesions and the reorganization of ECM-cell contacts, but failed to promote cell migration on fibronectin, in contrast to full-length beta1A. This indicates that the disruption of cell-cell adhesion is not simply the consequence of the stimulated cell migration. Expression of beta1 integrins in GE11 cells resulted in a decrease in cadherin and alpha-catenin protein levels accompanied by their redistribution from the cytoskeleton-associated fraction to the detergent-soluble fraction. Regulation of alpha-catenin protein levels by beta1 integrins is likely to play a role in the morphological transition, since overexpression of alpha-catenin in GE11 cells before beta1 prevented the disruption of intercellular adhesions and cell scattering. In addition, using biochemical activity assays for Rho-like GTPases, we show that the expression of beta1A, beta1D, or IL2R-beta1A in GE11 or GD25 cells triggers activation of both RhoA and Rac1, but not of Cdc42. Moreover, dominant negative Rac1 (N17Rac1) inhibited the disruption of cell-cell adhesions when expressed before beta1. However, all three GTPases might be involved in the morphological transition, since expression of either N19RhoA, N17Rac1, or N17Cdc42 reversed cell scattering and partially restored cadherin-based adhesions in GE11-beta1A cells. Our results indicate that beta1 integrins regulate the polarity and motility of epithelial cells by the induction of intracellular molecular events involving a downregulation of alpha-catenin function and the activation of the Rho-like G proteins Rac1 and RhoA.

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(A) Expression of β1A, but not that of IL2R-β1A, enhances GE11 cell migration through fibronectin-coated Transwell filters. Fibronectin was coated on the lower side of the filter, and 3 × 104 or 105 cells were seeded in the upper compartment of the Transwell, after which cells were allowed to migrate for 2 h. Cells that remained on the upper side of the filter were removed by washing, and cells that had migrated to the lower side of the filter were fixed and stained with crystal violet. Cells were counted on photographs taken from three different fields (5 mm2) and the results were averaged. Error bars represent SEMs. (B) Scratch assay of GE11-control and GE11-β1A cells. Cells were seeded at high density on plastic under standard culture conditions for 2 h. Subsequently, a cross was scratched to facilitate the marking of the cells. Phase-contrast micrographs were taken at the indicated timepoints. The white bars represent the progression of migrating cells.
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Figure 5: (A) Expression of β1A, but not that of IL2R-β1A, enhances GE11 cell migration through fibronectin-coated Transwell filters. Fibronectin was coated on the lower side of the filter, and 3 × 104 or 105 cells were seeded in the upper compartment of the Transwell, after which cells were allowed to migrate for 2 h. Cells that remained on the upper side of the filter were removed by washing, and cells that had migrated to the lower side of the filter were fixed and stained with crystal violet. Cells were counted on photographs taken from three different fields (5 mm2) and the results were averaged. Error bars represent SEMs. (B) Scratch assay of GE11-control and GE11-β1A cells. Cells were seeded at high density on plastic under standard culture conditions for 2 h. Subsequently, a cross was scratched to facilitate the marking of the cells. Phase-contrast micrographs were taken at the indicated timepoints. The white bars represent the progression of migrating cells.

Mentions: To quantify potential changes in the motility of GE11-β1A cells as compared with that of GE11-control cells, we have performed migration experiments using fibronectin-coated Transwells. As shown in Fig. 5 A, although GE11-control cells are able to migrate to some extent on this substrate, the expression of β1A strongly increased cell motility. The expression of IL2R-β1A failed to enhance cell migration in any of the conditions tested.


Induction of cell scattering by expression of beta1 integrins in beta1-deficient epithelial cells requires activation of members of the rho family of GTPases and downregulation of cadherin and catenin function.

Gimond C, van Der Flier A, van Delft S, Brakebusch C, Kuikman I, Collard JG, Fässler R, Sonnenberg A - J. Cell Biol. (1999)

(A) Expression of β1A, but not that of IL2R-β1A, enhances GE11 cell migration through fibronectin-coated Transwell filters. Fibronectin was coated on the lower side of the filter, and 3 × 104 or 105 cells were seeded in the upper compartment of the Transwell, after which cells were allowed to migrate for 2 h. Cells that remained on the upper side of the filter were removed by washing, and cells that had migrated to the lower side of the filter were fixed and stained with crystal violet. Cells were counted on photographs taken from three different fields (5 mm2) and the results were averaged. Error bars represent SEMs. (B) Scratch assay of GE11-control and GE11-β1A cells. Cells were seeded at high density on plastic under standard culture conditions for 2 h. Subsequently, a cross was scratched to facilitate the marking of the cells. Phase-contrast micrographs were taken at the indicated timepoints. The white bars represent the progression of migrating cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2168093&req=5

Figure 5: (A) Expression of β1A, but not that of IL2R-β1A, enhances GE11 cell migration through fibronectin-coated Transwell filters. Fibronectin was coated on the lower side of the filter, and 3 × 104 or 105 cells were seeded in the upper compartment of the Transwell, after which cells were allowed to migrate for 2 h. Cells that remained on the upper side of the filter were removed by washing, and cells that had migrated to the lower side of the filter were fixed and stained with crystal violet. Cells were counted on photographs taken from three different fields (5 mm2) and the results were averaged. Error bars represent SEMs. (B) Scratch assay of GE11-control and GE11-β1A cells. Cells were seeded at high density on plastic under standard culture conditions for 2 h. Subsequently, a cross was scratched to facilitate the marking of the cells. Phase-contrast micrographs were taken at the indicated timepoints. The white bars represent the progression of migrating cells.
Mentions: To quantify potential changes in the motility of GE11-β1A cells as compared with that of GE11-control cells, we have performed migration experiments using fibronectin-coated Transwells. As shown in Fig. 5 A, although GE11-control cells are able to migrate to some extent on this substrate, the expression of β1A strongly increased cell motility. The expression of IL2R-β1A failed to enhance cell migration in any of the conditions tested.

Bottom Line: Expression of beta1 integrins in GE11 cells resulted in a decrease in cadherin and alpha-catenin protein levels accompanied by their redistribution from the cytoskeleton-associated fraction to the detergent-soluble fraction.In addition, using biochemical activity assays for Rho-like GTPases, we show that the expression of beta1A, beta1D, or IL2R-beta1A in GE11 or GD25 cells triggers activation of both RhoA and Rac1, but not of Cdc42.Our results indicate that beta1 integrins regulate the polarity and motility of epithelial cells by the induction of intracellular molecular events involving a downregulation of alpha-catenin function and the activation of the Rho-like G proteins Rac1 and RhoA.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, The Netherlands Cancer Institute, 1066 CX Amsterdam,The Netherlands.

ABSTRACT
Adhesion receptors, which connect cells to each other and to the surrounding extracellular matrix (ECM), play a crucial role in the control of tissue structure and of morphogenesis. In this work, we have studied how intercellular adhesion molecules and beta1 integrins influence each other using two different beta1- cell lines, epithelial GE11 and fibroblast-like GD25 cells. Expression of beta1A or the cytoplasmic splice variant beta1D, induced the disruption of intercellular adherens junctions and cell scattering in both GE11 and GD25 cells. In GE11 cells, the morphological change correlated with the redistribution of zonula occluden (ZO)-1 from tight junctions to adherens junctions at high cell confluency. In addition, the expression of beta1 integrins caused a dramatic reorganization of the actin cytoskeleton and of focal contacts. Interaction of beta1 integrins with their respective ligands was required for a complete morphological transition towards the spindle-shaped fibroblast-like phenotype. The expression of an interleukin-2 receptor (IL2R)-beta1A chimera and its incorporation into focal adhesions also induced the disruption of cadherin-based adhesions and the reorganization of ECM-cell contacts, but failed to promote cell migration on fibronectin, in contrast to full-length beta1A. This indicates that the disruption of cell-cell adhesion is not simply the consequence of the stimulated cell migration. Expression of beta1 integrins in GE11 cells resulted in a decrease in cadherin and alpha-catenin protein levels accompanied by their redistribution from the cytoskeleton-associated fraction to the detergent-soluble fraction. Regulation of alpha-catenin protein levels by beta1 integrins is likely to play a role in the morphological transition, since overexpression of alpha-catenin in GE11 cells before beta1 prevented the disruption of intercellular adhesions and cell scattering. In addition, using biochemical activity assays for Rho-like GTPases, we show that the expression of beta1A, beta1D, or IL2R-beta1A in GE11 or GD25 cells triggers activation of both RhoA and Rac1, but not of Cdc42. Moreover, dominant negative Rac1 (N17Rac1) inhibited the disruption of cell-cell adhesions when expressed before beta1. However, all three GTPases might be involved in the morphological transition, since expression of either N19RhoA, N17Rac1, or N17Cdc42 reversed cell scattering and partially restored cadherin-based adhesions in GE11-beta1A cells. Our results indicate that beta1 integrins regulate the polarity and motility of epithelial cells by the induction of intracellular molecular events involving a downregulation of alpha-catenin function and the activation of the Rho-like G proteins Rac1 and RhoA.

Show MeSH
Related in: MedlinePlus