Limits...
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.

Show MeSH

Related in: MedlinePlus

Dominant negative Rac1 (N17Rac1) prevents the disruption of intercellular adhesions and cell scattering induced by β1 integrins. (A) Total amounts of endogenous Rac1 in GE11-control, GE11-β1A, and of Rac1 and N17Rac1 in GE11-N17Rac1-β1A cells were determined by immunoblotting. Blots were probed with anti-Rac1 mAb. The open arrow indicates myc-N17Rac1 and the closed arrow indicates endogenous Rac1. (B) Phase-contrast microscopy of GE11-N17Rac1-β1A cells. (C) Immunofluorescence staining of GE11-N17Rac1-β1A cells for expression of myc epitope-tagged N17Rac1, β-catenin, and β1 integrins. Basal and medial focal planes are as indicated. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2168093&req=5

Figure 9: Dominant negative Rac1 (N17Rac1) prevents the disruption of intercellular adhesions and cell scattering induced by β1 integrins. (A) Total amounts of endogenous Rac1 in GE11-control, GE11-β1A, and of Rac1 and N17Rac1 in GE11-N17Rac1-β1A cells were determined by immunoblotting. Blots were probed with anti-Rac1 mAb. The open arrow indicates myc-N17Rac1 and the closed arrow indicates endogenous Rac1. (B) Phase-contrast microscopy of GE11-N17Rac1-β1A cells. (C) Immunofluorescence staining of GE11-N17Rac1-β1A cells for expression of myc epitope-tagged N17Rac1, β-catenin, and β1 integrins. Basal and medial focal planes are as indicated. Bar, 20 μm.

Mentions: Next, we investigated whether the activation of RhoA and Rac1 is required for the phenotypic conversion induced by β1 integrins. We first transduced dominant negative mutants of RhoA, Rac1, and Cdc42 in GE11 cells, and 2 d later, we introduced β1A. Because the same retroviral vector was used for both proteins, the clones expressing β1A could not be selected with antibiotics. Therefore, we isolated the cells expressing β1 at high levels by FACS® analysis and sorting. In the second step, several individual clones were isolated from this β1-positive cell population. The expression of both N17Rac1 (Fig. 9 A) and β1 (data not shown) was finally measured in these clones. We show that even when β1 was strongly expressed at the cell surface, substoichiometric amounts of N17Rac1 inhibited the disruption of cell–cell adhesion and cell scattering (Fig. 9 B). It has been reported previously that amounts of RhoA or Rac1 mutants well below those of endogenous RhoA and Rac1 levels caused changes in the cellular organization (Jou and Nelson 1998), presumably because their ability to rapidly bind and dissociate from guanine-exchange factors is impaired by the mutation. The β1-positive clones that displayed a more fibroblast-like phenotype did not express N17Rac1 at detectable levels (data not shown). Fig. 9 C shows that N17Rac1 was associated with the entire plasma membrane of GE11-N17Rac1-β1A cells, but that it was especially concentrated in regions of cell–cell contacts. Cadherins and catenins remained present in intercellular junctions (Fig. 9 C). Pictures taken at both the basal and medial plane of the cell show that the β1A subunit was found in both focal contacts and in regions of cell–cell contacts (Fig. 9 C). This indicates that N17Rac1 prevented β1-mediated disruption of cadherin-based adhesion and further scattering.


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)

Dominant negative Rac1 (N17Rac1) prevents the disruption of intercellular adhesions and cell scattering induced by β1 integrins. (A) Total amounts of endogenous Rac1 in GE11-control, GE11-β1A, and of Rac1 and N17Rac1 in GE11-N17Rac1-β1A cells were determined by immunoblotting. Blots were probed with anti-Rac1 mAb. The open arrow indicates myc-N17Rac1 and the closed arrow indicates endogenous Rac1. (B) Phase-contrast microscopy of GE11-N17Rac1-β1A cells. (C) Immunofluorescence staining of GE11-N17Rac1-β1A cells for expression of myc epitope-tagged N17Rac1, β-catenin, and β1 integrins. Basal and medial focal planes are as indicated. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 9: Dominant negative Rac1 (N17Rac1) prevents the disruption of intercellular adhesions and cell scattering induced by β1 integrins. (A) Total amounts of endogenous Rac1 in GE11-control, GE11-β1A, and of Rac1 and N17Rac1 in GE11-N17Rac1-β1A cells were determined by immunoblotting. Blots were probed with anti-Rac1 mAb. The open arrow indicates myc-N17Rac1 and the closed arrow indicates endogenous Rac1. (B) Phase-contrast microscopy of GE11-N17Rac1-β1A cells. (C) Immunofluorescence staining of GE11-N17Rac1-β1A cells for expression of myc epitope-tagged N17Rac1, β-catenin, and β1 integrins. Basal and medial focal planes are as indicated. Bar, 20 μm.
Mentions: Next, we investigated whether the activation of RhoA and Rac1 is required for the phenotypic conversion induced by β1 integrins. We first transduced dominant negative mutants of RhoA, Rac1, and Cdc42 in GE11 cells, and 2 d later, we introduced β1A. Because the same retroviral vector was used for both proteins, the clones expressing β1A could not be selected with antibiotics. Therefore, we isolated the cells expressing β1 at high levels by FACS® analysis and sorting. In the second step, several individual clones were isolated from this β1-positive cell population. The expression of both N17Rac1 (Fig. 9 A) and β1 (data not shown) was finally measured in these clones. We show that even when β1 was strongly expressed at the cell surface, substoichiometric amounts of N17Rac1 inhibited the disruption of cell–cell adhesion and cell scattering (Fig. 9 B). It has been reported previously that amounts of RhoA or Rac1 mutants well below those of endogenous RhoA and Rac1 levels caused changes in the cellular organization (Jou and Nelson 1998), presumably because their ability to rapidly bind and dissociate from guanine-exchange factors is impaired by the mutation. The β1-positive clones that displayed a more fibroblast-like phenotype did not express N17Rac1 at detectable levels (data not shown). Fig. 9 C shows that N17Rac1 was associated with the entire plasma membrane of GE11-N17Rac1-β1A cells, but that it was especially concentrated in regions of cell–cell contacts. Cadherins and catenins remained present in intercellular junctions (Fig. 9 C). Pictures taken at both the basal and medial plane of the cell show that the β1A subunit was found in both focal contacts and in regions of cell–cell contacts (Fig. 9 C). This indicates that N17Rac1 prevented β1-mediated disruption of cadherin-based adhesion and further scattering.

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