The RhoGEF TEM4 Contains an F-Actin Binding Domain Figure 5. Arg 130 in TEM4 ABD is essential for F-actin binding. A, Alignment of residues 125-135 of human TEM4 with other species. Residues tested for actin binding in vivo are marked with a dot above the alignment. The arginine residue (R130), that we determined to be critical for binding to F-actin, is marked with the asterisk. B, Wild-type and point mutants of GFP-TEM4 1-582 were used to further map residues essential for actin association in vivo in live NIH3T3 cells. C, Coomassie Blue-stained gel comparing ATP/ADP-Pi-F-actin binding capacity of wild-type (WT) and R130D mutant of GST-ABD in co-sedimentation experiments. D, Equilibrium binding of WT and R130D mutant of TEM4 ABD to ATP/ADP-Pi-F-actin calculated from co-sedimentation experiments as shown in Fig. 4. Two independent co-sedimentation experiments were used to generate the data. doi:10.1371/journal.pone.0041876.g005 nucleotide state of actin affects its interaction with actin binding proteins and affects kinetics of actin assembly and disassembly [17,19,34]. To determine if binding of TEM4 ABD to actin is dependent on the nucleotide state of actin, actin co-sedimentation assays were repeated using F-actin assembled from ATP- or ADP- G-actin. After ultracentrifugation, the amount of TEM4 bound to F-actin filaments was calculated from the amount depleted from the supernatant as detected by immunoblotting and densitometry (Fig. 4D, E). The data show that TEM4 ABD has a strong preference for binding dynamic F-actin, as it exhibited an approximately 90–fold higher affinity for filaments prepared from ATP-G-actin than those prepared from ADP-G-actin (Kd = 53 nM and 4.7 mM, respectively; Fig. 4F, G). This affinity of the TEM4 ABD for ATP-F-actin is comparable to that measured for the actin nucleating complex, Arp2/3 which is 40 nM [35]. Taken together, these data demonstrate that TEM4 contains an ABD which binds preferentially to dynamic, newly assembled actin filaments. Deletion of an 11 amino acid segment (residues 125–135) within the TEM4-ABD ablated the localization of TEM4 to the actin cytoskeleton in vivo (Fig. 3A). Therefore, it is highly probable that specific residues within 125–135 (Fig. 5A) contribute important interactions required for the high affinity binding of TEM4 to actin filaments. To identify individual residues essential for TEM4 binding to F-actin, we performed site-directed mutagenesis and determined the subcellular localization of these point mutants. Consistent with experimental data in other actin binding proteins where a positively charged surface was required for actin binding [36], mutation of the conserved R130 in TEM4 mislocalized GFP- TEM4 (Fig. 5B) and abolished the ability of GST-TEM4-ABD to co-sediment with ATP/ADP-Pi actin, suggesting that the R130 is critical for high affinity binding between TEM4 and F-actin (Fig. 5C, D). Arg 130 is Essential for TEM4 Subcellular Localization and In vivo Activity We found that the N-terminus of TEM4 regulates its subcellular localization and R130 is essential for F-actin binding of the isolated ABD of TEM4 in vitro. To determine if actin binding is essential for the subcellular localization of FL TEM4 to the actin cytoskeleton, GFP-FL TEM4 WT or the R130D mutant were expressed in HUVECs together with the fluorescently-tagged (tRFP) actin marker, Lifeact, to visualize the actin cytoskeleton [12]. As shown in Fig. 6A, colocalization of GFP-FL TEM4 WT with the actin cytoskeleton was abolished by the introduction of PLoS ONE | www.plosone.org 7 July 2012 | Volume 7 | Issue 7 | e41876