The RhoGEF TEM4 Contains an F-Actin Binding Domain Figure 6. Actin binding is essential for subcellular localization and in vivo activity of TEM4. A, R130 is essential for the localization of TEM4 to actin stress fibers. HUVECs expressing GFP, GFP-FL TEM4 WT or R130D mutant were imaged live with tRFP-Lifeact marker. Scale bar, 10 mm. B, The N-terminus is essential for TEM4 in vivo activity. HUVECs expressing GFP-tagged TEM4 constructs were assessed for levels of active RhoC by affinity pull-down. C, Mutation of R130 impairs RhoC activation by TEM4. 293T cells were transfected with GFP-tagged wild-type or R130D mutant of TEM4 and levels of active RhoC were measured by affinity pull-down and presented in a bar graph (D). Data shown are representative of three independent experiments. doi:10.1371/journal.pone.0041876.g006 the R130D mutation into TEM4. These data suggest that TEM4 contains a single ABD and that F-actin binding is essential for subcellular localization of TEM4 in vivo. The subcellular localization of RhoGEFs may potentiate the activation of downstream Rho GTPases [2]. Therefore, we determined if the localization of TEM4 to the actin cytoskeleton modulates the in vivo activity of TEM4. We found that mislocalization of TEM4 to the cytosol by either amino-terminal deletion or mutation of R130 in the full-length protein severely reduced the ability of TEM4 to activate its substrate, RhoC, as compared to TEM4 WT (Fig. 6B–D). These data suggest that binding to F-actin, which is required for subcellular localization of TEM4, regulates TEM4 activity in vivo. Discussion In this study, we characterized the RhoGEF, TEM4/ARH- GEF17 and demonstrated that it exhibits a subcellular localization distinct among RhoGEFs facilitated by direct binding to F-actin through a novel actin binding domain. Furthermore, we found that disruption of TEM4 binding to actin caused an altered subcellular localization and impaired its in vivo activity. We suggest PLoS ONE | www.plosone.org 8 July 2012 | Volume 7 | Issue 7 | e41876