that this unique subcellular localization allows TEM4 to serve a highly distinct spatially-restricted role in regulating activation of Rho GTPases and therefore biological function. RhoGEFs are large proteins distinguished by a unique set of protein and/or lipid interaction domains and motifs, outside of the shared DH-PH domains, that regulate function of RhoGEFs [2]. TEM4 possesses an actin binding domain (ABD) within the N- terminus (residues 81–135) that binds F-actin with high affinity. Our extensive analysis of the ABD using primary sequence analysis and secondary structure prediction algorithms failed to identify similarity to any known ABDs. A novel actin binding domain was previously reported in two other RhoGEFs, PDZ- RhoGEF and frabin [37,38] and L/IIxxFE motif within these proteins was essential for binding to F-actin [39]. Interestingly, despite the lack of the overall sequence homology, TEM4 contains an LSxxFD motif within the N-terminus of the ABD; however, our studies suggest that the C-terminal residues are the most critical for TEM4 binding to F-actin. Regardless of the lack of primary sequence homology between the TEM4 ABD and known actin binding proteins, secondary structure prediction analysis suggests the presence of several helices (Fig. 3B) as common amongst ABDs [29–31,40]. Finally, the ABD-containing region is absent from the two closely-related TEM4 family members, ARHGEF10 and ARHGEF10L/GrinchGEF [41,42]. Therefore, we demonstrate that TEM4 contains a novel ABD and future studies are needed to characterize how TEM4 may affect actin filament dynamics. Exogenous, GFP- or HA-tagged TEM4 readily decorated the entire length of actin stress fibers in HUVECs and endogenous TEM4 immunoprecipitates with F-actin. However, our in vitro actin-binding analysis found TEM4 ABD preferentially bound newly polymerized actin filaments, suggesting that TEM4 may associate with F-actin in the areas of the cell undergoing actin polymerization, e.g. leading edge, as shown for other actin-binding proteins with in vitro preference for ATP-F-actin [43–45]. Therefore, while the use of exogenously-expressed constructs was instrumental in mapping the ABD, the overexpressed protein may not accurately display the subcellular localization of endogenous TEM4. Thus, an important goal for our future studies will be an evaluation of the subcellular localization of endogenous TEM4. Our data using affinity pull-down assays suggest that binding to actin regulates TEM4-induced activation of its Rho GTPase substrates. This could be achieved by at least two mechanisms. First, the ability of TEM4 to bind F-actin can result in recruitment and concentration of TEM4 at the sites of actin polymerization where Rho GTPases are known to be activated [46]. Second, like many other RhoGEFs [2], TEM4 may be auto-inhibited and ABD association with F-actin could relieve this autoinhibition, leading to enhanced intrinsic GEF activity similar to microtubular- References 1. Vetter IR, Wittinghofer A (2001) The guanine nucleotide-binding switch in three dimensions. Science 294: 1299–1304. 2. Rossman KL, Der CJ, Sondek J (2005) GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors. Nat Rev Mol Cell Biol 6: 167–180. 3. Schmidt A, Hall A (2002) Guanine nucleotide exchange factors for Rho GTPases: turning on the switch. Genes Dev 16: 1587–1609. 4. Vega FM, Ridley AJ (2007) SnapShot: Rho family GTPases. Cell 129: 1430. 5. Krendel M, Zenke FT, Bokoch GM (2002) Nucleotide exchange factor GEF-H1 mediates cross-talk between microtubules and the actin cytoskeleton. Nat Cell Biol 4: 294–301. 6. Nalbant P, Chang YC, Birkenfeld J, Chang ZF, Bokoch GM (2009) Guanine nucleotide exchange factor-H1 regulates cell migration via localized activation of RhoA at the leading edge. Mol Biol Cell 20: 4070–4082. 7. Chang YC, Nalbant P, Birkenfeld J, Chang ZF, Bokoch GM (2008) GEF-H1 couples nocodazole-induced microtubule disassembly to cell contractility via RhoA. Mol Biol Cell 19: 2147–2153. The RhoGEF TEM4 Contains an F-Actin Binding Domain mediated regulation of GEF-H1 [5]. Both of these scenarios would result in pools of active TEM4 localized in the areas undergoing actin polymerization where activation of Rho GTPases would be the most desirable to promote further cytoskeleton rearrangements. Rho family GTPases are key regulators of actin cytoskeleton dynamics and proteins of Rho subfamily (RhoA, B, C) promote the formation of actin stress fibers [47–49] that are essential for a diversity of cellular processes, including cell shape, polarity, migration, cell-cell and cell-matrix interactions [47,50,51]. We have observed increased Rho GTPase activation and actin rearrangement in cells expressing WT and not the actin binding-deficient, R130D mutant of TEM4 (Fig. 6). These observations suggest that targeted localization of the catalytic activity of TEM4 may be critical for the subsequent actin rearrangement, which is essential for cellular functions of Rho GTPases such as cell migration and invasion [46,52–54]. To our knowledge, this is the first report of the actin cytoskeleton- dependent regulation of an in vivo activity of a RhoGEF. Rho GTPases interact with a diverse spectrum of downstream effectors and can regulate a diversity of cellular processes [55]. While RhoGEFs are best characterized for their abilities to facilitate Rho GTPase activation, there is also considerable evidence that RhoGEFs can influence the effector signaling output of their Rho GTPase substrates. One mechanism involves the restricted spatial activation of Rho GTPases [46], thereby leading to differential engagement and utilization of specific effectors. Another mechanism involves the function of RhoGEFs as a scaffold that can recruit effectors to promo-te differential effector utilization and signaling output [56,57]. Interestingly, while the C-terminus of TEM4 was not required for subcellular localization, it is homologous to a region found in JSAP-1/JIP-3, a known scaffold for the c-Jun N-terminal kinase pathway [58,59]. Future studies will determine if TEM4 promotes spatial activation of distinct effector pathways downstream of Rho GTPases and the mechanism of the preferential engagement of these pathways. 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