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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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Expression of IL2R-β1A induces the disruption of cadherin-based intercellular adhesions, and increases the size of focal contacts in GE11 cells. Stably transduced GE11-IL2R and GE11-IL2R-β1A cells were grown for 2 d on glass coverslips, and after fixation and Triton X-100 permeabilization, analyzed by double immunofluoresence for the expression of either IL2R together with F-actin (revealed by staining with rhodamine-phalloidin), α-catenin, vinculin, or cadherin, or for the expression of ZO-1 together with F-actin. Arrows indicate IL2R-β1A–negative cells that exhibit a vinculin-positive ring of focal contacts. Bars, 20 μm.
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Figure 4: Expression of IL2R-β1A induces the disruption of cadherin-based intercellular adhesions, and increases the size of focal contacts in GE11 cells. Stably transduced GE11-IL2R and GE11-IL2R-β1A cells were grown for 2 d on glass coverslips, and after fixation and Triton X-100 permeabilization, analyzed by double immunofluoresence for the expression of either IL2R together with F-actin (revealed by staining with rhodamine-phalloidin), α-catenin, vinculin, or cadherin, or for the expression of ZO-1 together with F-actin. Arrows indicate IL2R-β1A–negative cells that exhibit a vinculin-positive ring of focal contacts. Bars, 20 μm.

Mentions: The localization of a particular integrin in focal contacts is regulated by its α subunit and requires the binding of the integrin to ligand (Briesewitz et al. 1993). In contrast, when chimeric molecules containing the extracellular and transmembrane domains of the human IL2R and the cytoplasmic domain of the β1A integrin subunit were expressed at relatively low levels, they were colocalized with endogenous integrins in focal contacts and were able to transduce signals leading to the phosphorylation of the focal adhesion kinase (LaFlamme et al. 1992; Akiyama et al. 1994). These properties indicate that IL2R-β1 chimerae mimic endogenous ligand-occupied integrins. We have analyzed the effects of an IL2R-β1A chimera on the morphology of GE11 cells. Fig. 4 shows that expression of the IL2R alone did not either alter the morphology of GE11 cell colonies or cause any changes in the subcellular distribution of actin, α-catenin, or ZO-1 (Fig. 4, upper panels). Expression of the IL2R-β1A chimera, on the contrary, induced the disruption of most intercellular adhesions. A few small epithelial-like colonies remained and cell–cell adhesions had a tendency to reform, although they did not appear to be as stable as those between GE11-control cells. IL2R-β1A expression induced an alteration of the peripheral bundles of actin filaments and changes in the localization of cadherins, catenins, and ZO-1 (Fig. 4, lower panels), similar to the full-length β1A subunit. In addition, the IL2R-β1A chimera promoted cell spreading and induced a redistribution of vinculin: the ring of focal adhesions at the periphery of the colonies was no longer assembled in GE11-IL2R-β1A cells and was replaced by thick and long streaks of vinculin at the base of cells. The arrows in Fig. 4 indicate vinculin-positive rings in cells in which IL2R-β1A was not expressed. In cells expressing IL2R-β1A, the chimera was colocalized with vinculin (Fig. 4) and the endogenous β3 subunit (data not shown) in focal contacts. Together these results indicate that IL2R-β1A induces both the disruption of intercellular adhesions and the reorganization of the cytoskeleton, thus mimicking the effects of full-length β1A. Whether IL2R-β1A is primarily incorporated into β3-containing, preexisting focal adhesions and induces their remodeling, or whether it participates in the formation of new adhesion structures into which β3 is eventually recruited will be discussed.


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)

Expression of IL2R-β1A induces the disruption of cadherin-based intercellular adhesions, and increases the size of focal contacts in GE11 cells. Stably transduced GE11-IL2R and GE11-IL2R-β1A cells were grown for 2 d on glass coverslips, and after fixation and Triton X-100 permeabilization, analyzed by double immunofluoresence for the expression of either IL2R together with F-actin (revealed by staining with rhodamine-phalloidin), α-catenin, vinculin, or cadherin, or for the expression of ZO-1 together with F-actin. Arrows indicate IL2R-β1A–negative cells that exhibit a vinculin-positive ring of focal contacts. Bars, 20 μm.
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Related In: Results  -  Collection

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

Figure 4: Expression of IL2R-β1A induces the disruption of cadherin-based intercellular adhesions, and increases the size of focal contacts in GE11 cells. Stably transduced GE11-IL2R and GE11-IL2R-β1A cells were grown for 2 d on glass coverslips, and after fixation and Triton X-100 permeabilization, analyzed by double immunofluoresence for the expression of either IL2R together with F-actin (revealed by staining with rhodamine-phalloidin), α-catenin, vinculin, or cadherin, or for the expression of ZO-1 together with F-actin. Arrows indicate IL2R-β1A–negative cells that exhibit a vinculin-positive ring of focal contacts. Bars, 20 μm.
Mentions: The localization of a particular integrin in focal contacts is regulated by its α subunit and requires the binding of the integrin to ligand (Briesewitz et al. 1993). In contrast, when chimeric molecules containing the extracellular and transmembrane domains of the human IL2R and the cytoplasmic domain of the β1A integrin subunit were expressed at relatively low levels, they were colocalized with endogenous integrins in focal contacts and were able to transduce signals leading to the phosphorylation of the focal adhesion kinase (LaFlamme et al. 1992; Akiyama et al. 1994). These properties indicate that IL2R-β1 chimerae mimic endogenous ligand-occupied integrins. We have analyzed the effects of an IL2R-β1A chimera on the morphology of GE11 cells. Fig. 4 shows that expression of the IL2R alone did not either alter the morphology of GE11 cell colonies or cause any changes in the subcellular distribution of actin, α-catenin, or ZO-1 (Fig. 4, upper panels). Expression of the IL2R-β1A chimera, on the contrary, induced the disruption of most intercellular adhesions. A few small epithelial-like colonies remained and cell–cell adhesions had a tendency to reform, although they did not appear to be as stable as those between GE11-control cells. IL2R-β1A expression induced an alteration of the peripheral bundles of actin filaments and changes in the localization of cadherins, catenins, and ZO-1 (Fig. 4, lower panels), similar to the full-length β1A subunit. In addition, the IL2R-β1A chimera promoted cell spreading and induced a redistribution of vinculin: the ring of focal adhesions at the periphery of the colonies was no longer assembled in GE11-IL2R-β1A cells and was replaced by thick and long streaks of vinculin at the base of cells. The arrows in Fig. 4 indicate vinculin-positive rings in cells in which IL2R-β1A was not expressed. In cells expressing IL2R-β1A, the chimera was colocalized with vinculin (Fig. 4) and the endogenous β3 subunit (data not shown) in focal contacts. Together these results indicate that IL2R-β1A induces both the disruption of intercellular adhesions and the reorganization of the cytoskeleton, thus mimicking the effects of full-length β1A. Whether IL2R-β1A is primarily incorporated into β3-containing, preexisting focal adhesions and induces their remodeling, or whether it participates in the formation of new adhesion structures into which β3 is eventually recruited will be discussed.

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