The RhoGEF TEM4 Contains an F-Actin Binding Domain Figure 3. Detailed mapping of actin-association domain of TEM4. A, GFP-TEM4 fusion constructs encoding various truncations of the N- terminus or GFP were transfected into NIH3T3 cells and imaged live. Scale, 20 mm. B, The newly identified actin-binding domain of TEM4 (aa 81–135 in human TEM4) is conserved among TEM4 orthologs. Helices (H) predicted by the secondary structure prediction server PredictProtein [66] are labeled. doi:10.1371/journal.pone.0041876.g003 [26,27], these results support the presence of a functional PH domain within TEM4. The N-Terminus of TEM4 Regulates Subcellular Localization Our initial studies aimed at determining interacting partners of TEM4 found actin to co-immunoprecipitate with exogenously expressed TEM4 (unpublished data). To confirm the TEM4-actin association, we immunoprecipitated endogenous TEM4 from primary human endothelial cells (HUVECs) that were left untreated or treated with an actin depolymerizing drug, latrunculin A (LatA) [28] prior to immunoprecipitation. As shown in Fig. 2A, endogenous TEM4 readily co-immunoprecipitated with actin, but the interaction was ablated in cells treated with LatA. Therefore, we concluded that TEM4 associates with F-actin filaments in vivo. To investigate the association of TEM4 with F-actin further, the subcellular localization of full-length (FL) and truncated forms of TEM4 (Fig. 2B) was examined in live HUVECs. FL TEM4 (GFP- FL) resided on structures reminiscent of the actin cytoskeleton throughout the cytoplasm (Fig. 2C). HA-tagged FL TEM4 exhibited similar localization (unpublished data). Furthermore, while the deletion of the region C-terminal to the PH domain (GFP-DC) did not alter TEM4 localization, N-terminally deleted TEM4 (GFP-DN) and the isolated DH/PH domains (GFP-DH/ PH) exhibited a diffuse cytoplasmic staining. These results suggest that sequences within the N-terminal portion of TEM4 are required for localization. Indeed, a construct encoding the first 582 N-terminal amino acids of TEM4 localized to the actin cytoskeleton (Fig. 2C). Therefore, sequences within the N-terminal region of TEM4 (residues 1–582) are necessary and sufficient for the subcellular localization of TEM4. PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e41876