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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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Distribution of F-actin and various adhesion structures in GE11-control and GE11-β1A cells. Cells were grown for 2 d on glass coverslips, and after fixation and Triton X-100 permeabilization, stained as indicated for F-actin with rhodamine-labeled phalloidin or double-stained for β1A and F-actin, vinculin and cadherin, vinculin and F-actin, and ZO-1 and F-actin. Cells were visualized by confocal laser-scanning microscopy. Basal and medial focus planes are as indicated. Bar, 20 μm.
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Figure 2: Distribution of F-actin and various adhesion structures in GE11-control and GE11-β1A cells. Cells were grown for 2 d on glass coverslips, and after fixation and Triton X-100 permeabilization, stained as indicated for F-actin with rhodamine-labeled phalloidin or double-stained for β1A and F-actin, vinculin and cadherin, vinculin and F-actin, and ZO-1 and F-actin. Cells were visualized by confocal laser-scanning microscopy. Basal and medial focus planes are as indicated. Bar, 20 μm.

Mentions: Because the effect of β1 expression was most dramatic on intercellular adhesions in GE11 cells, we concentrated our studies on stable GE11 cells expressing β1A (GE11-β1A). The distribution of various proteins associated with the actin cytoskeleton and with intercellular adhesions was analyzed in GE11 and GE11-β1A cells (Fig. 2). In GE11-control cells, actin filaments were organized in heavy peripheral bundles, which ran parallel to the outer membrane of cells at the periphery of the epithelial cell colonies. Actin filaments were also present in cortical bundles under the plasma membrane, along cell–cell boundaries, and in stress fibers at the cell basis, where they were attached to the plasma membrane at sites of focal contacts (Fig. 2). In GE11-β1A cells, peripheral bundles of actin filaments were absent and stress fibers crossed the entire cell. Intercellular staining of cadherins, typical of epithelial cells, was observed in GE11-control cells, and no staining was found at the free cell border at the periphery of colonies (Fig. 2). α-, β-, and γ-catenins had a similar localization (data not shown). In contrast, cadherins and catenins were more diffusely distributed over the membrane of GE11-β1A cells, and although there were some residual adherens junctions at high confluency, these proteins were also found in regions of the plasma membrane that were not in contact with other cells (Fig. 2, medial plane). In GE11-control cells, vinculin was found in regions of cell–cell contacts, where it was colocalized with cadherins (Fig. 2, medial plane). In contrast, although GE11-β1A cells developed cell–cell contacts at high confluency, vinculin and cadherins were not colocalized in these cells (Fig. 2, medial plane). Using interference reflection microscopy, we found that the number and size of focal contacts were different in GE11-control and GE11-β1A cells (data not shown), and this was confirmed by the distribution of vinculin (Fig. 2, basal plane) and talin (data not shown) at the basal surface of the cells. Typically, focal contacts were small and numerous in GE11-control cells, and distributed over the entire basal cell surface. However, at the periphery of the colonies they were more concentrated at the outer region of the cell, thus forming a characteristic interrupted ring-like structure. In GE11-β1A cells, focal contacts were thick, and appeared to be arranged in long streaks frequently found at the end of actin stress fibers (Fig. 2). In confluent GE11-β1A cells, focal contacts were also found between two cells and sometimes on their apical surface, as seen by staining for talin and vinculin. Electron microscopic analysis revealed that this was likely to be due to the presence and assembly of secreted ECM proteins between cells and on their apical surface (data not shown). Another specialized membrane domain involved in intercellular adhesion of epithelial cells is the tight junction. A marker of tight junctions is ZO-1, but in cell types lacking these structures, such as fibroblasts or cardiac muscle cells, ZO-1 is colocalized with cadherins at adherens junctions (Yonemura et al. 1995). Upon expression of the β1A integrin subunit, ZO-1 became redistributed from tight junctions to the adherens junctions formed by GE11-β1A cells at high confluency (Fig. 2). This relocalization was correlated with the transition from polarized epithelial cells to fibroblast-like cells. Another marker of tight junctions, occludin, was also found at the apical lateral border of GE11 cells, and became diffusely distributed in GE11-β1A cells (data not shown). Furthermore, EM showed that tight junctions present in GE11-control cells were no longer present in GE11-β1A cells (data not shown).


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)

Distribution of F-actin and various adhesion structures in GE11-control and GE11-β1A cells. Cells were grown for 2 d on glass coverslips, and after fixation and Triton X-100 permeabilization, stained as indicated for F-actin with rhodamine-labeled phalloidin or double-stained for β1A and F-actin, vinculin and cadherin, vinculin and F-actin, and ZO-1 and F-actin. Cells were visualized by confocal laser-scanning microscopy. Basal and medial focus planes are as indicated. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Distribution of F-actin and various adhesion structures in GE11-control and GE11-β1A cells. Cells were grown for 2 d on glass coverslips, and after fixation and Triton X-100 permeabilization, stained as indicated for F-actin with rhodamine-labeled phalloidin or double-stained for β1A and F-actin, vinculin and cadherin, vinculin and F-actin, and ZO-1 and F-actin. Cells were visualized by confocal laser-scanning microscopy. Basal and medial focus planes are as indicated. Bar, 20 μm.
Mentions: Because the effect of β1 expression was most dramatic on intercellular adhesions in GE11 cells, we concentrated our studies on stable GE11 cells expressing β1A (GE11-β1A). The distribution of various proteins associated with the actin cytoskeleton and with intercellular adhesions was analyzed in GE11 and GE11-β1A cells (Fig. 2). In GE11-control cells, actin filaments were organized in heavy peripheral bundles, which ran parallel to the outer membrane of cells at the periphery of the epithelial cell colonies. Actin filaments were also present in cortical bundles under the plasma membrane, along cell–cell boundaries, and in stress fibers at the cell basis, where they were attached to the plasma membrane at sites of focal contacts (Fig. 2). In GE11-β1A cells, peripheral bundles of actin filaments were absent and stress fibers crossed the entire cell. Intercellular staining of cadherins, typical of epithelial cells, was observed in GE11-control cells, and no staining was found at the free cell border at the periphery of colonies (Fig. 2). α-, β-, and γ-catenins had a similar localization (data not shown). In contrast, cadherins and catenins were more diffusely distributed over the membrane of GE11-β1A cells, and although there were some residual adherens junctions at high confluency, these proteins were also found in regions of the plasma membrane that were not in contact with other cells (Fig. 2, medial plane). In GE11-control cells, vinculin was found in regions of cell–cell contacts, where it was colocalized with cadherins (Fig. 2, medial plane). In contrast, although GE11-β1A cells developed cell–cell contacts at high confluency, vinculin and cadherins were not colocalized in these cells (Fig. 2, medial plane). Using interference reflection microscopy, we found that the number and size of focal contacts were different in GE11-control and GE11-β1A cells (data not shown), and this was confirmed by the distribution of vinculin (Fig. 2, basal plane) and talin (data not shown) at the basal surface of the cells. Typically, focal contacts were small and numerous in GE11-control cells, and distributed over the entire basal cell surface. However, at the periphery of the colonies they were more concentrated at the outer region of the cell, thus forming a characteristic interrupted ring-like structure. In GE11-β1A cells, focal contacts were thick, and appeared to be arranged in long streaks frequently found at the end of actin stress fibers (Fig. 2). In confluent GE11-β1A cells, focal contacts were also found between two cells and sometimes on their apical surface, as seen by staining for talin and vinculin. Electron microscopic analysis revealed that this was likely to be due to the presence and assembly of secreted ECM proteins between cells and on their apical surface (data not shown). Another specialized membrane domain involved in intercellular adhesion of epithelial cells is the tight junction. A marker of tight junctions is ZO-1, but in cell types lacking these structures, such as fibroblasts or cardiac muscle cells, ZO-1 is colocalized with cadherins at adherens junctions (Yonemura et al. 1995). Upon expression of the β1A integrin subunit, ZO-1 became redistributed from tight junctions to the adherens junctions formed by GE11-β1A cells at high confluency (Fig. 2). This relocalization was correlated with the transition from polarized epithelial cells to fibroblast-like cells. Another marker of tight junctions, occludin, was also found at the apical lateral border of GE11 cells, and became diffusely distributed in GE11-β1A cells (data not shown). Furthermore, EM showed that tight junctions present in GE11-control cells were no longer present in GE11-β1A cells (data not shown).

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