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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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The reciprocal balance of Rac and Rho GTPase activities determines an epithelioid or fibroblastoid phenotype. NIH3T3 cells stably expressing C1199Tiam1 acquire an epithelioid phenotype, characterized by the formation of functional cell–cell contacts, because of activation of Rac and downregulation of Rho activity (Fig. 4). V14Rho-mediated restoration of Rho activity in C1199Tiam1-expressing cells reverts the epithelioid phenotype to a more fibroblastoid morphology associated with migratory behavior, indicating that the balance of both Rac and Rho activities determines cellular morphology. Bar, 10 μm.
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Figure 10: The reciprocal balance of Rac and Rho GTPase activities determines an epithelioid or fibroblastoid phenotype. NIH3T3 cells stably expressing C1199Tiam1 acquire an epithelioid phenotype, characterized by the formation of functional cell–cell contacts, because of activation of Rac and downregulation of Rho activity (Fig. 4). V14Rho-mediated restoration of Rho activity in C1199Tiam1-expressing cells reverts the epithelioid phenotype to a more fibroblastoid morphology associated with migratory behavior, indicating that the balance of both Rac and Rho activities determines cellular morphology. Bar, 10 μm.

Mentions: Expression of Tiam1 or activated Rac in NIH3T3 fibroblasts resulted in immotile cells with established cell–cell contacts (Fig. 1 and Fig. 3). This phenotype is characterized by high Rac activity and downregulated Rho activity. Therefore, raising Rho activity exogenously in Tiam1-expressing cells should change the phenotype again towards fibroblastoid, motile cells. We generated an NIH3T3 cell line stably expressing C1199Tiam1 as well as V14Rho to test this hypothesis. Expression of C1199Tiam1 was not changed upon introduction of V14Rho (data not shown). Whereas C1199Tiam1-expressing cells grew in small epithelial-like islands of immotile cells, cells expressing Tiam1 as well as titrated amounts of V14Rho did not establish cell–cell contacts and exhibited a more motile, contractile phenotype (Fig. 10). Thus, restoration of Rho activity by V14Rho in Tiam1-expressing cells induces transition from an epithelioid to a more fibroblastoid phenotype. These data suggest that the balance of Rac and Rho activities determines the cellular phenotype and migratory behavior in NIH3T3 fibroblasts.


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)

The reciprocal balance of Rac and Rho GTPase activities determines an epithelioid or fibroblastoid phenotype. NIH3T3 cells stably expressing C1199Tiam1 acquire an epithelioid phenotype, characterized by the formation of functional cell–cell contacts, because of activation of Rac and downregulation of Rho activity (Fig. 4). V14Rho-mediated restoration of Rho activity in C1199Tiam1-expressing cells reverts the epithelioid phenotype to a more fibroblastoid morphology associated with migratory behavior, indicating that the balance of both Rac and Rho activities determines cellular morphology. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 10: The reciprocal balance of Rac and Rho GTPase activities determines an epithelioid or fibroblastoid phenotype. NIH3T3 cells stably expressing C1199Tiam1 acquire an epithelioid phenotype, characterized by the formation of functional cell–cell contacts, because of activation of Rac and downregulation of Rho activity (Fig. 4). V14Rho-mediated restoration of Rho activity in C1199Tiam1-expressing cells reverts the epithelioid phenotype to a more fibroblastoid morphology associated with migratory behavior, indicating that the balance of both Rac and Rho activities determines cellular morphology. Bar, 10 μm.
Mentions: Expression of Tiam1 or activated Rac in NIH3T3 fibroblasts resulted in immotile cells with established cell–cell contacts (Fig. 1 and Fig. 3). This phenotype is characterized by high Rac activity and downregulated Rho activity. Therefore, raising Rho activity exogenously in Tiam1-expressing cells should change the phenotype again towards fibroblastoid, motile cells. We generated an NIH3T3 cell line stably expressing C1199Tiam1 as well as V14Rho to test this hypothesis. Expression of C1199Tiam1 was not changed upon introduction of V14Rho (data not shown). Whereas C1199Tiam1-expressing cells grew in small epithelial-like islands of immotile cells, cells expressing Tiam1 as well as titrated amounts of V14Rho did not establish cell–cell contacts and exhibited a more motile, contractile phenotype (Fig. 10). Thus, restoration of Rho activity by V14Rho in Tiam1-expressing cells induces transition from an epithelioid to a more fibroblastoid phenotype. These data suggest that the balance of Rac and Rho activities determines the cellular phenotype and migratory behavior in NIH3T3 fibroblasts.

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