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Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior.

Sander EE, ten Klooster JP, van Delft S, van der Kammen RA, Collard JG - J. Cell Biol. (1999)

Bottom Line: We found that Cdc42 also downregulates Rho activity.Moreover, Rac effector mutants that are defective in mediating cytoskeleton changes or Jun kinase activation both downregulate Rho activity, suggesting that neither of these Rac signaling pathways are involved in the regulation of Rho.We conclude that Rac signaling is able to antagonize Rho activity directly at the GTPase level, and that the reciprocal balance between Rac and Rho activity determines cellular morphology and migratory behavior in NIH3T3 fibroblasts.

View Article: PubMed Central - PubMed

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

ABSTRACT
Using biochemical assays to determine the activation state of Rho-like GTPases, we show that the guanine nucleotide exchange factor Tiam1 functions as a specific activator of Rac but not Cdc42 or Rho in NIH3T3 fibroblasts. Activation of Rac by Tiam1 induces an epithelial-like morphology with functional cadherin-based adhesions and inhibits migration of fibroblasts. This epithelial phenotype is characterized by Rac-mediated effects on Rho activity. Transient PDGF-induced as well as sustained Rac activation by Tiam1 or V12Rac downregulate Rho activity. We found that Cdc42 also downregulates Rho activity. Neither V14Rho or N19Rho affects Rac activity, suggesting unidirectional signaling from Rac towards Rho. Downregulation of Rho activity occurs independently of Rac- induced cytoskeletal changes and cell spreading. Moreover, Rac effector mutants that are defective in mediating cytoskeleton changes or Jun kinase activation both downregulate Rho activity, suggesting that neither of these Rac signaling pathways are involved in the regulation of Rho. Restoration of Rho activity in Tiam1-expressing cells by expression of V14Rho results in reversion of the epithelioid phenotype towards a migratory, fibroblastoid morphology. We conclude that Rac signaling is able to antagonize Rho activity directly at the GTPase level, and that the reciprocal balance between Rac and Rho activity determines cellular morphology and migratory behavior in NIH3T3 fibroblasts.

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C1199Tiam1 induces an epithelial-like morphology in NIH3T3 cells by increasing N- and P-cadherin-based cell–cell adhesion. (A) Western blot using the Tiam1-specific C16 antibody of C1199- or C580Tiam1 immunoprecipitated with anti–HA antibody from the respective stable NIH3T3 cell lines. (B) NIH3T3 cells stably expressing C1199- or C580Tiam1 were stained for Tiam1, N- and P-cadherin (stained with a Pan-cadherin antibody), p120CAS, and F-actin. C1199Tiam1 localizes to sites of cell–cell contact and to membrane ruffles, whereas C580Tiam1 localizes to the cytoplasm. Cadherins as well as p120CAS localize to sites of cell–cell contact that were induced by C1199Tiam1. Bars, 25 μm. (C) Quantification of cell–cell adhesion of control NIH3T3 cells and cell lines expressing C1199- or C580Tiam1 determined by dissociation (in the presence and absence of Ca2+) and association assays and expressed as numbers of particles (cell clusters) per total number of cells (Np/Nc). Each bar represents the mean ± SD of triplicate assays. Shown is one representative example of three independent experiments.
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Figure 1: C1199Tiam1 induces an epithelial-like morphology in NIH3T3 cells by increasing N- and P-cadherin-based cell–cell adhesion. (A) Western blot using the Tiam1-specific C16 antibody of C1199- or C580Tiam1 immunoprecipitated with anti–HA antibody from the respective stable NIH3T3 cell lines. (B) NIH3T3 cells stably expressing C1199- or C580Tiam1 were stained for Tiam1, N- and P-cadherin (stained with a Pan-cadherin antibody), p120CAS, and F-actin. C1199Tiam1 localizes to sites of cell–cell contact and to membrane ruffles, whereas C580Tiam1 localizes to the cytoplasm. Cadherins as well as p120CAS localize to sites of cell–cell contact that were induced by C1199Tiam1. Bars, 25 μm. (C) Quantification of cell–cell adhesion of control NIH3T3 cells and cell lines expressing C1199- or C580Tiam1 determined by dissociation (in the presence and absence of Ca2+) and association assays and expressed as numbers of particles (cell clusters) per total number of cells (Np/Nc). Each bar represents the mean ± SD of triplicate assays. Shown is one representative example of three independent experiments.

Mentions: To study the molecular basis of the morphological transformation of NIH3T3 fibroblasts upon expression of Tiam1 (van Leeuwen et al. 1995), we generated NIH3T3 cell lines stably expressing C1199- and C580Tiam1 cDNAs by retroviral transduction (Fig. 1 A). Stable pools of cells expressing C1199Tiam1 grew in small groups of flat, spread cells and displayed an epithelial-like morphology. The C1199Tiam1 protein was predominantly localized at the plasma membrane and induced extensive membrane ruffling. Moreover, C1199Tiam1 was also enriched at the sites of cell–cell contact, which was accompanied by increased actin polymerization (Fig. 1 B). We previously showed that Tiam1-induced Rac activation promotes E-cadherin–mediated cell–cell adhesion in epithelial cells (Hordijk et al. 1997; Sander et al. 1998). Therefore, we studied whether the Tiam1-induced epithelial-like morphology in NIH3T3 fibroblasts was due to the establishment of cadherin-based cell–cell adhesions. NIH3T3 fibroblasts do not express E-cadherin, but instead express the family members N- and P-cadherin (Reynolds et al. 1996). In the C1199Tiam1-expressing cells, N-cadherin and P-cadherin (stained with a Pan-cadherin antibody; Fig. 1 B) as well as the cadherin-associated catenins, such as α-, β-, and γ-catenin (not shown) and p120CAS (Fig. 1 B) localized to sites of cell–cell contact. The adhesion-related proteins were to some extent also detected in Tiam1-induced membrane ruffles (Fig. 1 B).


Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior.

Sander EE, ten Klooster JP, van Delft S, van der Kammen RA, Collard JG - J. Cell Biol. (1999)

C1199Tiam1 induces an epithelial-like morphology in NIH3T3 cells by increasing N- and P-cadherin-based cell–cell adhesion. (A) Western blot using the Tiam1-specific C16 antibody of C1199- or C580Tiam1 immunoprecipitated with anti–HA antibody from the respective stable NIH3T3 cell lines. (B) NIH3T3 cells stably expressing C1199- or C580Tiam1 were stained for Tiam1, N- and P-cadherin (stained with a Pan-cadherin antibody), p120CAS, and F-actin. C1199Tiam1 localizes to sites of cell–cell contact and to membrane ruffles, whereas C580Tiam1 localizes to the cytoplasm. Cadherins as well as p120CAS localize to sites of cell–cell contact that were induced by C1199Tiam1. Bars, 25 μm. (C) Quantification of cell–cell adhesion of control NIH3T3 cells and cell lines expressing C1199- or C580Tiam1 determined by dissociation (in the presence and absence of Ca2+) and association assays and expressed as numbers of particles (cell clusters) per total number of cells (Np/Nc). Each bar represents the mean ± SD of triplicate assays. Shown is one representative example of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169355&req=5

Figure 1: C1199Tiam1 induces an epithelial-like morphology in NIH3T3 cells by increasing N- and P-cadherin-based cell–cell adhesion. (A) Western blot using the Tiam1-specific C16 antibody of C1199- or C580Tiam1 immunoprecipitated with anti–HA antibody from the respective stable NIH3T3 cell lines. (B) NIH3T3 cells stably expressing C1199- or C580Tiam1 were stained for Tiam1, N- and P-cadherin (stained with a Pan-cadherin antibody), p120CAS, and F-actin. C1199Tiam1 localizes to sites of cell–cell contact and to membrane ruffles, whereas C580Tiam1 localizes to the cytoplasm. Cadherins as well as p120CAS localize to sites of cell–cell contact that were induced by C1199Tiam1. Bars, 25 μm. (C) Quantification of cell–cell adhesion of control NIH3T3 cells and cell lines expressing C1199- or C580Tiam1 determined by dissociation (in the presence and absence of Ca2+) and association assays and expressed as numbers of particles (cell clusters) per total number of cells (Np/Nc). Each bar represents the mean ± SD of triplicate assays. Shown is one representative example of three independent experiments.
Mentions: To study the molecular basis of the morphological transformation of NIH3T3 fibroblasts upon expression of Tiam1 (van Leeuwen et al. 1995), we generated NIH3T3 cell lines stably expressing C1199- and C580Tiam1 cDNAs by retroviral transduction (Fig. 1 A). Stable pools of cells expressing C1199Tiam1 grew in small groups of flat, spread cells and displayed an epithelial-like morphology. The C1199Tiam1 protein was predominantly localized at the plasma membrane and induced extensive membrane ruffling. Moreover, C1199Tiam1 was also enriched at the sites of cell–cell contact, which was accompanied by increased actin polymerization (Fig. 1 B). We previously showed that Tiam1-induced Rac activation promotes E-cadherin–mediated cell–cell adhesion in epithelial cells (Hordijk et al. 1997; Sander et al. 1998). Therefore, we studied whether the Tiam1-induced epithelial-like morphology in NIH3T3 fibroblasts was due to the establishment of cadherin-based cell–cell adhesions. NIH3T3 fibroblasts do not express E-cadherin, but instead express the family members N- and P-cadherin (Reynolds et al. 1996). In the C1199Tiam1-expressing cells, N-cadherin and P-cadherin (stained with a Pan-cadherin antibody; Fig. 1 B) as well as the cadherin-associated catenins, such as α-, β-, and γ-catenin (not shown) and p120CAS (Fig. 1 B) localized to sites of cell–cell contact. The adhesion-related proteins were to some extent also detected in Tiam1-induced membrane ruffles (Fig. 1 B).

Bottom Line: We found that Cdc42 also downregulates Rho activity.Moreover, Rac effector mutants that are defective in mediating cytoskeleton changes or Jun kinase activation both downregulate Rho activity, suggesting that neither of these Rac signaling pathways are involved in the regulation of Rho.We conclude that Rac signaling is able to antagonize Rho activity directly at the GTPase level, and that the reciprocal balance between Rac and Rho activity determines cellular morphology and migratory behavior in NIH3T3 fibroblasts.

View Article: PubMed Central - PubMed

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

ABSTRACT
Using biochemical assays to determine the activation state of Rho-like GTPases, we show that the guanine nucleotide exchange factor Tiam1 functions as a specific activator of Rac but not Cdc42 or Rho in NIH3T3 fibroblasts. Activation of Rac by Tiam1 induces an epithelial-like morphology with functional cadherin-based adhesions and inhibits migration of fibroblasts. This epithelial phenotype is characterized by Rac-mediated effects on Rho activity. Transient PDGF-induced as well as sustained Rac activation by Tiam1 or V12Rac downregulate Rho activity. We found that Cdc42 also downregulates Rho activity. Neither V14Rho or N19Rho affects Rac activity, suggesting unidirectional signaling from Rac towards Rho. Downregulation of Rho activity occurs independently of Rac- induced cytoskeletal changes and cell spreading. Moreover, Rac effector mutants that are defective in mediating cytoskeleton changes or Jun kinase activation both downregulate Rho activity, suggesting that neither of these Rac signaling pathways are involved in the regulation of Rho. Restoration of Rho activity in Tiam1-expressing cells by expression of V14Rho results in reversion of the epithelioid phenotype towards a migratory, fibroblastoid morphology. We conclude that Rac signaling is able to antagonize Rho activity directly at the GTPase level, and that the reciprocal balance between Rac and Rho activity determines cellular morphology and migratory behavior in NIH3T3 fibroblasts.

Show MeSH
Related in: MedlinePlus