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A novel Dbl family RhoGEF promotes Rho-dependent axon attraction to the central nervous system midline in Drosophila and overcomes Robo repulsion.

Bashaw GJ, Hu H, Nobes CD, Goodman CS - J. Cell Biol. (2001)

Bottom Line: Curr.Opin.Surprisingly, evidence from genetic, biochemical, and cell culture experiments suggests that the promotion of axon attraction by GEF64C is dependent on the activation of Rho, but not Rac or Cdc42.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, University of Pennsylvania, Philadelphia, PA 19104, USA. gbashaw@mail.med.upenn.edu

ABSTRACT
The key role of the Rho family GTPases Rac, Rho, and CDC42 in regulating the actin cytoskeleton is well established (Hall, A. 1998. Science. 279:509-514). Increasing evidence suggests that the Rho GTPases and their upstream positive regulators, guanine nucleotide exchange factors (GEFs), also play important roles in the control of growth cone guidance in the developing nervous system (Luo, L. 2000. Nat. Rev. Neurosci. 1:173-180; Dickson, B.J. 2001. Curr. Opin. Neurobiol. 11:103-110). Here, we present the identification and molecular characterization of a novel Dbl family Rho GEF, GEF64C, that promotes axon attraction to the central nervous system midline in the embryonic Drosophila nervous system. In sensitized genetic backgrounds, loss of GEF64C function causes a phenotype where too few axons cross the midline. In contrast, ectopic expression of GEF64C throughout the nervous system results in a phenotype in which far too many axons cross the midline, a phenotype reminiscent of loss of function mutations in the Roundabout (Robo) repulsive guidance receptor. Genetic analysis indicates that GEF64C expression can in fact overcome Robo repulsion. Surprisingly, evidence from genetic, biochemical, and cell culture experiments suggests that the promotion of axon attraction by GEF64C is dependent on the activation of Rho, but not Rac or Cdc42.

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GEF64C promotes RhoA-dependent actin stress fiber formation in fibroblasts. (A) Uninjected control cells. (B) Cells injected with a GEF64C expression construct show striking actin stress fiber formation. (B′) Injection marker for cells shown in B. (C) Cells coinjected with the GEF64C expression construct and C3 transferase protein. C3 strongly inhibits GEF64C-induced stress fiber formation. (C′) Injection marker for cells shown in C. (D) GEF exchange assays for Rac, Rho, and Cdc 42. Histogram columns are as indicated. Activity is expressed as the percent of initial [H3]GDP remaining bound after 25 min. The relatively weak, but significant exchange activity that we observe could be attributable to the fact that the PH domain was not included in these assays, as fusion proteins containing both the Dbl and PH domains were poorly expressed.
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fig4: GEF64C promotes RhoA-dependent actin stress fiber formation in fibroblasts. (A) Uninjected control cells. (B) Cells injected with a GEF64C expression construct show striking actin stress fiber formation. (B′) Injection marker for cells shown in B. (C) Cells coinjected with the GEF64C expression construct and C3 transferase protein. C3 strongly inhibits GEF64C-induced stress fiber formation. (C′) Injection marker for cells shown in C. (D) GEF exchange assays for Rac, Rho, and Cdc 42. Histogram columns are as indicated. Activity is expressed as the percent of initial [H3]GDP remaining bound after 25 min. The relatively weak, but significant exchange activity that we observe could be attributable to the fact that the PH domain was not included in these assays, as fusion proteins containing both the Dbl and PH domains were poorly expressed.

Mentions: To test if the specificity of GEF64C for RhoA seen in our genetic experiments is also observed in independent assays for GEF64C function, in vitro binding, and guanine nucleotide exchange assays were performed. Glutathione S-transferase (GST) pull down experiments indicate that GEF64C can bind equally well to Rac1, RhoA, and Cdc42 (unpublished data), whereas GEF64C acts as an in vitro exchange factor for Rac and Rho (exhibiting a modest preference in catalyzing the exchange of GDP for GTP on Rho, relative to Rac) but does not have exchange activity for Cdc42 (Fig. 4 D). Such promiscuity in the in vitro association of GEFs with small GTPases has been observed for many RhoGEFs, including Vav and Trio (Van Aelst and D'Souza-Schorey, 1997). To further examine the function of GEF64C, its effects on the actin cytoskeleton in cultured fibroblasts were determined (Fig. 4, A–C). Microinjection of a GEF64C expression vector into quiescent, serum-starved Swiss 3T3 cells resulted in a dramatic stimulation of actin stress fiber formation relative to control cells (Fig. 4, A and B), a phenotype indicative of Rho activation (Hall, 1998). Coinjection of GEF64C and C3 transferase, a protein inhibitor specific for Rho (Ridley and Hall, 1992), completely blocked GEF64C's ability to induce stress fibers, arguing further that GEF64C functions by activating Rho (Fig. 4 C).


A novel Dbl family RhoGEF promotes Rho-dependent axon attraction to the central nervous system midline in Drosophila and overcomes Robo repulsion.

Bashaw GJ, Hu H, Nobes CD, Goodman CS - J. Cell Biol. (2001)

GEF64C promotes RhoA-dependent actin stress fiber formation in fibroblasts. (A) Uninjected control cells. (B) Cells injected with a GEF64C expression construct show striking actin stress fiber formation. (B′) Injection marker for cells shown in B. (C) Cells coinjected with the GEF64C expression construct and C3 transferase protein. C3 strongly inhibits GEF64C-induced stress fiber formation. (C′) Injection marker for cells shown in C. (D) GEF exchange assays for Rac, Rho, and Cdc 42. Histogram columns are as indicated. Activity is expressed as the percent of initial [H3]GDP remaining bound after 25 min. The relatively weak, but significant exchange activity that we observe could be attributable to the fact that the PH domain was not included in these assays, as fusion proteins containing both the Dbl and PH domains were poorly expressed.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199320&req=5

fig4: GEF64C promotes RhoA-dependent actin stress fiber formation in fibroblasts. (A) Uninjected control cells. (B) Cells injected with a GEF64C expression construct show striking actin stress fiber formation. (B′) Injection marker for cells shown in B. (C) Cells coinjected with the GEF64C expression construct and C3 transferase protein. C3 strongly inhibits GEF64C-induced stress fiber formation. (C′) Injection marker for cells shown in C. (D) GEF exchange assays for Rac, Rho, and Cdc 42. Histogram columns are as indicated. Activity is expressed as the percent of initial [H3]GDP remaining bound after 25 min. The relatively weak, but significant exchange activity that we observe could be attributable to the fact that the PH domain was not included in these assays, as fusion proteins containing both the Dbl and PH domains were poorly expressed.
Mentions: To test if the specificity of GEF64C for RhoA seen in our genetic experiments is also observed in independent assays for GEF64C function, in vitro binding, and guanine nucleotide exchange assays were performed. Glutathione S-transferase (GST) pull down experiments indicate that GEF64C can bind equally well to Rac1, RhoA, and Cdc42 (unpublished data), whereas GEF64C acts as an in vitro exchange factor for Rac and Rho (exhibiting a modest preference in catalyzing the exchange of GDP for GTP on Rho, relative to Rac) but does not have exchange activity for Cdc42 (Fig. 4 D). Such promiscuity in the in vitro association of GEFs with small GTPases has been observed for many RhoGEFs, including Vav and Trio (Van Aelst and D'Souza-Schorey, 1997). To further examine the function of GEF64C, its effects on the actin cytoskeleton in cultured fibroblasts were determined (Fig. 4, A–C). Microinjection of a GEF64C expression vector into quiescent, serum-starved Swiss 3T3 cells resulted in a dramatic stimulation of actin stress fiber formation relative to control cells (Fig. 4, A and B), a phenotype indicative of Rho activation (Hall, 1998). Coinjection of GEF64C and C3 transferase, a protein inhibitor specific for Rho (Ridley and Hall, 1992), completely blocked GEF64C's ability to induce stress fibers, arguing further that GEF64C functions by activating Rho (Fig. 4 C).

Bottom Line: Curr.Opin.Surprisingly, evidence from genetic, biochemical, and cell culture experiments suggests that the promotion of axon attraction by GEF64C is dependent on the activation of Rho, but not Rac or Cdc42.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, University of Pennsylvania, Philadelphia, PA 19104, USA. gbashaw@mail.med.upenn.edu

ABSTRACT
The key role of the Rho family GTPases Rac, Rho, and CDC42 in regulating the actin cytoskeleton is well established (Hall, A. 1998. Science. 279:509-514). Increasing evidence suggests that the Rho GTPases and their upstream positive regulators, guanine nucleotide exchange factors (GEFs), also play important roles in the control of growth cone guidance in the developing nervous system (Luo, L. 2000. Nat. Rev. Neurosci. 1:173-180; Dickson, B.J. 2001. Curr. Opin. Neurobiol. 11:103-110). Here, we present the identification and molecular characterization of a novel Dbl family Rho GEF, GEF64C, that promotes axon attraction to the central nervous system midline in the embryonic Drosophila nervous system. In sensitized genetic backgrounds, loss of GEF64C function causes a phenotype where too few axons cross the midline. In contrast, ectopic expression of GEF64C throughout the nervous system results in a phenotype in which far too many axons cross the midline, a phenotype reminiscent of loss of function mutations in the Roundabout (Robo) repulsive guidance receptor. Genetic analysis indicates that GEF64C expression can in fact overcome Robo repulsion. Surprisingly, evidence from genetic, biochemical, and cell culture experiments suggests that the promotion of axon attraction by GEF64C is dependent on the activation of Rho, but not Rac or Cdc42.

Show MeSH
Related in: MedlinePlus