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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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Basal Rho activity is required for the maintenance of Tiam1/Rac–induced cell–cell contacts in NIH3T3 fibroblasts. (A) Serum is required for Tiam1-induced cell–cell contacts. Phase-contrast pictures of NIH3T3 fibroblasts expressing C1199 Tiam1 showing cells grown in the presence of serum, after 24 h serum starvation, and 5 h after addition of serum to 24 h serum-starved cells. Bar, 10 μM. (B) Rho activity is required for Tiam1-induced cell–cell contacts. C1199Tiam1-expressing NIH3T3 fibroblasts were stained for Tiam1 (DH antibody, green) and F-actin (red) after 24 h of serum starvation, 24 h of serum starvation followed by addition of 5 μM LPA (8 h), and treatment for 12 h with 30 μg/ml C3 transferase toxin (in the presence of serum). Bar, 25 μM. (C) Cell lysates (from 5 × 106 cells) of control and C1199Tiam1-expressing NIH3T3 fibroblasts were assayed for Rho and Rac activities. Cells were grown in serum (lane 1) or 24 h serum-starved (lanes 2–7) before stimulation with LPA (5 μM, 15 min) as indicated. Lysates were incubated with GST-C21 (lanes 1–5) or GST-PAK-CD (lanes 6 and 7), and bound GTPases were analyzed with antibodies against RhoA or Rac1.
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Figure 3: Basal Rho activity is required for the maintenance of Tiam1/Rac–induced cell–cell contacts in NIH3T3 fibroblasts. (A) Serum is required for Tiam1-induced cell–cell contacts. Phase-contrast pictures of NIH3T3 fibroblasts expressing C1199 Tiam1 showing cells grown in the presence of serum, after 24 h serum starvation, and 5 h after addition of serum to 24 h serum-starved cells. Bar, 10 μM. (B) Rho activity is required for Tiam1-induced cell–cell contacts. C1199Tiam1-expressing NIH3T3 fibroblasts were stained for Tiam1 (DH antibody, green) and F-actin (red) after 24 h of serum starvation, 24 h of serum starvation followed by addition of 5 μM LPA (8 h), and treatment for 12 h with 30 μg/ml C3 transferase toxin (in the presence of serum). Bar, 25 μM. (C) Cell lysates (from 5 × 106 cells) of control and C1199Tiam1-expressing NIH3T3 fibroblasts were assayed for Rho and Rac activities. Cells were grown in serum (lane 1) or 24 h serum-starved (lanes 2–7) before stimulation with LPA (5 μM, 15 min) as indicated. Lysates were incubated with GST-C21 (lanes 1–5) or GST-PAK-CD (lanes 6 and 7), and bound GTPases were analyzed with antibodies against RhoA or Rac1.

Mentions: Upon serum starvation, C1199Tiam1-expressing NIH3T3 cells lost their epithelial-like morphology. The cadherin-mediated cell–cell contacts were largely disassembled and many cells acquired a polarized, sickle-shaped phenotype (Fig. 3 A). As determined in scratch assays, these serum-deprived cells did not migrate (data not shown). Addition of serum partly restored the epithelial-like morphology within 3–5 h, the cells appeared more round again and began to re-establish cell–cell contacts (Fig. 3 A). Growth factors such as LPA, PDGF, insulin, and bradykinin were tested for their capability to substitute for serum to restore the epithelial-like phenotype of the serum-starved C1199Tiam1-expressing NIH3T3 cells. From all growth factors tested, only LPA was capable of partly restoring the epithelial-like morphology, similar to serum (Fig. 3 B). The C1199Tiam1 protein was localized to membrane ruffles that were also restored in the presence of LPA. Serum or LPA had to be present for a few hours before restoration of the epithelial-like phenotype was observed. LPA is a major component in serum and a potent activator of Rho (Ridley and Hall 1992). To substantiate the role of Rho in the maintenance of the epithelial-like phenotype, Rho was inactivated by treating C1199Tiam1-expressing NIH3T3 cells grown in the presence of serum with C3 transferase toxin. Similar to serum-starved cells (Fig. 3 A), these cells became sickle-shaped and lost their epithelial-like morphology including most membrane ruffles (Fig. 3 B). From these data we conclude that Rho activity is required for both cadherin-mediated cell–cell adhesion and membrane ruffles.


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)

Basal Rho activity is required for the maintenance of Tiam1/Rac–induced cell–cell contacts in NIH3T3 fibroblasts. (A) Serum is required for Tiam1-induced cell–cell contacts. Phase-contrast pictures of NIH3T3 fibroblasts expressing C1199 Tiam1 showing cells grown in the presence of serum, after 24 h serum starvation, and 5 h after addition of serum to 24 h serum-starved cells. Bar, 10 μM. (B) Rho activity is required for Tiam1-induced cell–cell contacts. C1199Tiam1-expressing NIH3T3 fibroblasts were stained for Tiam1 (DH antibody, green) and F-actin (red) after 24 h of serum starvation, 24 h of serum starvation followed by addition of 5 μM LPA (8 h), and treatment for 12 h with 30 μg/ml C3 transferase toxin (in the presence of serum). Bar, 25 μM. (C) Cell lysates (from 5 × 106 cells) of control and C1199Tiam1-expressing NIH3T3 fibroblasts were assayed for Rho and Rac activities. Cells were grown in serum (lane 1) or 24 h serum-starved (lanes 2–7) before stimulation with LPA (5 μM, 15 min) as indicated. Lysates were incubated with GST-C21 (lanes 1–5) or GST-PAK-CD (lanes 6 and 7), and bound GTPases were analyzed with antibodies against RhoA or Rac1.
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Related In: Results  -  Collection

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Figure 3: Basal Rho activity is required for the maintenance of Tiam1/Rac–induced cell–cell contacts in NIH3T3 fibroblasts. (A) Serum is required for Tiam1-induced cell–cell contacts. Phase-contrast pictures of NIH3T3 fibroblasts expressing C1199 Tiam1 showing cells grown in the presence of serum, after 24 h serum starvation, and 5 h after addition of serum to 24 h serum-starved cells. Bar, 10 μM. (B) Rho activity is required for Tiam1-induced cell–cell contacts. C1199Tiam1-expressing NIH3T3 fibroblasts were stained for Tiam1 (DH antibody, green) and F-actin (red) after 24 h of serum starvation, 24 h of serum starvation followed by addition of 5 μM LPA (8 h), and treatment for 12 h with 30 μg/ml C3 transferase toxin (in the presence of serum). Bar, 25 μM. (C) Cell lysates (from 5 × 106 cells) of control and C1199Tiam1-expressing NIH3T3 fibroblasts were assayed for Rho and Rac activities. Cells were grown in serum (lane 1) or 24 h serum-starved (lanes 2–7) before stimulation with LPA (5 μM, 15 min) as indicated. Lysates were incubated with GST-C21 (lanes 1–5) or GST-PAK-CD (lanes 6 and 7), and bound GTPases were analyzed with antibodies against RhoA or Rac1.
Mentions: Upon serum starvation, C1199Tiam1-expressing NIH3T3 cells lost their epithelial-like morphology. The cadherin-mediated cell–cell contacts were largely disassembled and many cells acquired a polarized, sickle-shaped phenotype (Fig. 3 A). As determined in scratch assays, these serum-deprived cells did not migrate (data not shown). Addition of serum partly restored the epithelial-like morphology within 3–5 h, the cells appeared more round again and began to re-establish cell–cell contacts (Fig. 3 A). Growth factors such as LPA, PDGF, insulin, and bradykinin were tested for their capability to substitute for serum to restore the epithelial-like phenotype of the serum-starved C1199Tiam1-expressing NIH3T3 cells. From all growth factors tested, only LPA was capable of partly restoring the epithelial-like morphology, similar to serum (Fig. 3 B). The C1199Tiam1 protein was localized to membrane ruffles that were also restored in the presence of LPA. Serum or LPA had to be present for a few hours before restoration of the epithelial-like phenotype was observed. LPA is a major component in serum and a potent activator of Rho (Ridley and Hall 1992). To substantiate the role of Rho in the maintenance of the epithelial-like phenotype, Rho was inactivated by treating C1199Tiam1-expressing NIH3T3 cells grown in the presence of serum with C3 transferase toxin. Similar to serum-starved cells (Fig. 3 A), these cells became sickle-shaped and lost their epithelial-like morphology including most membrane ruffles (Fig. 3 B). From these data we conclude that Rho activity is required for both cadherin-mediated cell–cell adhesion and membrane ruffles.

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