Exp Neurobiol 2011; 20(1): 29-34
Published online March 31, 2011
© The Korean Society for Brain and Neural Sciences
Minyeop Nahm and Seungbok Lee*
Department of Cell and Developmental Biology, Dental Research Institute, Seoul National University, Seoul 110-749, Korea
Correspondence to: *To whom correspondence should be addressed.
TEL: 82-2-740-8685, FAX: 82-2-762-2583
Rho small GTPases control multiple aspects of neuronal morphogenesis by regulating the assembly and organization of the actin cytoskeleton. Although they are negatively regulated by GTPase activating proteins (GAPs), the roles of RhoGAPs in the nervous system have not been fully investigated. Here we describe a characterization of
Keywords: GAP, RhoGAP68F, RhoA, F-actin, nervous system, drosophila
Rho small GTPases including RhoA, Rac1, and Cdc42 regulate multiple aspects of neuronal development including cell migration, cell polarity, axon growth and pathfinding, and dendritic elaboration (Luo, 2000; Govek et al., 2005). They function as molecular switches that shift between an active GTP-bound state and an inactive GDP-bound state (Jaffe and Hall, 2005). The active forms of Rho small GTPases bind their specific effector molecules to regulate F-actin and microtubule dynamics and gene expression (Jaffe and Hall, 2005). While guanine nucleotide exchange factors (GEFs) function as positive regulators of Rho small GTPases by stimulating the exchange of bound GDP for GTP, GTPase activating proteins (GAPs) negatively regulate Rho small GTPases by enhancing the intrinsic GTPase activity of Rho proteins (Luo, 2000; Moon and Zheng, 2003; Jaffe and Hall, 2005).
As suggested by the defined roles of Rho GTPases in the nervous system, various
HEK293 and NIH3T3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and antibiotics. These cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. To induce actin structural changes, NIH3T3 cells were serum-starved for 12 hr and treated for 10 min with 20 ng/ml lysophosphatidic acid (LPA) (Sigma), 10 ng/ml PDGF (Upstate Biotechnology), or 100 ng/ml Bradykinin (Sigma) before fixation.
The full-length cDNA clone for
Inactivation of Rho proteins by RhoGAP68F was analyzed using the EZ-detect Rho, Rac, or Cdc42 activation kit (Pierce), as previously described (Nahm et al., 2006). Briefly, HEK293 cells transiently expressing
The wild-type strain used in this study was
NIH3T3 cells were transfected with a plasmid encoding HA-RhoGAP68F or HA-RhoGAP68F-R315A. Immunostaining of transfected cells was performed as previously described (Nahm et al., 2006). Filamentous actin structures were visualized using rhodamine-conjugated phalloidin (Invitrogen) at 1:250.
Larval body wall muscles were dissected from wandering third-instar larvae in Ca2+-free HL3 saline (Stewart et al., 1994) and fixed in 4% formaldehyde in PBS for 30 min. Fixed muscles were washed with PBT (PBS, 0.1% Triton X-100) and incubated with goat anti-HRP conjugated with FITC (Jackson ImmunoResearch, 1:200) for 1 hr at room temperature. Image acquisition and morphological quantification were performed as previously described (Nahm et al., 2010).
The Flybase database predicts that
Rho small GTPases are well known for their specific effects on the organization of the actin cytoskeleton in fibroblasts: RhoA produces stress fibers associated with focal adhesion complexes, Rac1 induces lamellipodia and membrane ruffles, and Cdc42 triggers the formation of filopodia or microspikes (Hall, 1998). To further characterize the substrate specificity of RhoGAP68F, we examined the effect of RhoGAP68F overexpression on the actin cytoskeleton in cultured cells. NIH3T3 cells were transfected with HA-tagged RhoGAP68F and then stained with rhodamine-conjugated phalloidin to visualize actin filaments. As shown in Fig. 3, expression of RhoGAP68F inhibited lysophosphatidic acid (LPA)-induced stress fiber formation mediated by RhoA (Fig. 3A). However, RhoGAP68F expression had no effect on either PDGF-induced membrane ruffling mediated by Rac1 or Bradykinin-induced filopodia formation mediated by Cdc42. Finally, LPA-induced stress fiber formation was not significantly affected by expression of the R315A GAP-deficient mutant (Fig. 3B). These results suggest that RhoGAP68F acts as a RhoA-selective GAP in NIH3T3 cells.
In summary, we provide evidence that RhoGAP68F functions as a negative regulator of RhoA signaling