Rnd2 can also bind p190RhoGAP ( Wennerberg et al., 2003) and this interaction is similarly disrupted by mutation of residue this website T39 in its effector domain into valine ( Figure S6B). However, Rnd2T39V was as effective as wild-type
Rnd2 at rescuing the migration of Rnd2-silenced neurons ( Figure S6F), indicating that Rnd2 activity in the cortex does not require interaction with p190RhoGAP and that Rnd2 and Rnd3 inhibit RhoA signaling via distinct mechanisms. As RhoA has previously been well characterized for its role in regulating the actin cytoskeleton (Govek et al., 2005 and Ridley et al., 2003), we investigated whether Rnd2 and/or Rnd3 knockdown were altering actin dynamics in cortical neurons. We examined filamentous actin (F-actin) levels in electroporated cerebral cortical cells by coelectroporating a fluorescent F-actin probe based
on the VX-809 price actin-binding domain of the Utrophin protein (EGFP-UTRCH-ABD). The UTRCH-ABD probe has been shown to faithfully report the presence of F-actin without altering F-actin concentrations in cells expressing the probe ( Burkel et al., 2007). Knockdown of Rnd3 resulted in a marked accumulation of F-actin in the processes of electroporated cells, while F-actin accumulated in both cell body and processes of Rnd2 knockdown cells ( Figure 6A), suggesting that both Rnd2 and Rnd3 regulate actin cytoskeleton organization in migrating neurons. To determine whether F-actin accumulation is responsible for the isothipendyl migration defects of Rnd3- and Rnd2-silenced neurons, we coelectroporated cofilinS3A, a nonphosphorylatable form of cofilin that constitutively depolymerizes F-actin, together with Rnd2 or Rnd3 shRNA. Overexpression of cofilinS3A fully rescued the migration defect of Rnd3-silenced
neurons, thus indicating that Rnd3 promotes cortical neuron migration by inhibiting RhoA-mediated actin polymerization ( Figure 6B). In contrast, cofilinS3A expression had no effect on the migration of Rnd2-silenced neurons demonstrating that actin remodeling does not contribute to Rnd2 migratory function and that inhibition of RhoA by Rnd2 activates another unidentified process required for neuronal migration. Our results so far have established that both Rnd3 and Rnd2 inhibit RhoA activity (Figure 5), but that they nevertheless promote migration via distinct mechanisms involving p190RhoGAP and F-actin depolymerization in the case of Rnd3 and not Rnd2 ( Figure 6 and Figure S6). An explanation for this apparent paradox could be that Rnd2 and Rnd3 interact with RhoA in different cell compartments, because Rho GTPases have been shown to interact with different effectors and to trigger different cellular responses when located in different cell compartments ( Pertz, 2010).