(Clontech) and details are available upon request. Point mutations were introduced by site-directed mutagenesis. An expression vector encoding a fusion protein Lifeact-tRFP was made by subcloning cDNA sequences encoding the 17 amino acid actin binding peptide Lifeact [12] N-terminal to the red fluorescent protein TagRFP (Evrogen) into the pLL 5.0 lentiviral vector [13]. Antibodies: anti-GST (Sigma), anti-RhoC (Cell Signaling), anti- GFP (Clontech), anti-b-actin (Sigma), anti-a-tubulin (Sigma), and anti-TEM4 (ProSci). Human umbilical vein endothelial cells (HUVECs; Clonetics) were maintained in EGM-2 supplemented with 10% fetal bovine serum (FBS; HyClone) and electroporated with expression constructs using Amaxa Nucleofection technology. NIH3T3 cells [14] were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% calf serum and transfected using Lipofectamine Plus reagent (Invitrogen). 293T cells (ATCC) were maintained in DMEM supplemented with 10% FBS and transfected using the calcium phosphate DNA precipitation method. To visualize the actin cytoskeleton, HUVECs were transduced with lentivirus expressing tRFP-Lifeact. Fluorescent Microscopy Twenty four h after electroporation, HUVECs plated in 35-mm glass bottom MatTek dishes were examined live using Zeiss LSM 510 or Zeiss Axio Observer spinning disk microscope with an oil immersion 63x 1.4 objective. NIH3T3 cells were transfected in 35-mm MatTek dishes and examined live using Zeiss LSM 510 within 24 h after transfection. Protein Purification and Guanine Nucleotide Exchange Assays The cDNA fragments encoding residues 81–135 (ABD) or 1050–1478 (DH-PH) of TEM4 was subcloned into pProEx HTb (Invitrogen) in frame with an N-terminal glutathione S-transferase (GST) tag. TEM4 protein was expressed in Rosetta BL21 (DE3) E. coli cells (EMD Biosciences) for 5 h at 25uC, purified on a glutathione-coupled Sepharose column followed by an S200 size exclusion column (GE Healthcare), and concentrated in buffer A (20 mM Tris pH 8.0, 200 mM NaCl, 2 mM EDTA, 2 mM dithiothreitol and 10% glycerol). For some experiments the GST tag was subsequently removed by incubating GST-TEM4 with tobacco etch virus protease overnight and then passed over a glutathione Sepharose column equilibrated in buffer A to remove the cleaved GST. To monitor TEM4 catalytic activity, N-methylanthraniloyl (mant)-GTP incorporation into bacterially-expressed and purified Rho GTPases was carried out with a FluoroMax-4 spectrometer (Horiba) at 22uC as described [15]. Two mM of relevant GTPases were loaded with 400 nM mant-GTP after TEM4 or P-Rex1 DH- PH domain proteins were added and the relative fluorescence (lem = 360 nm, lex = 440 nm) was monitored. Lipid Binding To assess phosphoinositide binding to the TEM4 PH domain, PIP Strips (cat # P-6001; Echelon) were blocked with 3% fatty acid-free bovine serum albumin (Sigma) in phosphate-buffered saline, and incubated with 1 mg/ml recombinant GST-DH-PH for 1 h at room temperature. The strips were washed and immunoblotted with anti-GST antibodies. F-actin Co-sedimentation Assays To evaluate TEM4 association with F-actin, high-speed actin co-sedimentation was performed as previously described [16,17]. The RhoGEF TEM4 Contains an F-Actin Binding Domain Briefly, G-actin (APHL99-A; Cytoskeleton) was diluted in G buffer (5 mM Tris-HCl pH 8, 0.1 mM CaCl2, 0.5 mM DTT, 0.2 mM ATP), converted to Mg-ATP-G-actin by incubating with 50 mM MgCl2, 0.2 mM EGTA and polymerized in the presence of 50 mM KCl, 1 mM MgCl2, and 1 mM ATP for 1 h at room temperature. GST-TEM4 was incubated with F-actin at room temperature for 30 min, and sedimented using an Airfuge (Beckman) at 23 psi (,100,0006g) for 30 min. Supernatant and pellet fractions were solubilized in SDS sample buffer and resolved by SDS-PAGE. To calculate the binding affinity of TEM4 to F- actin, the supernatant-depletion method [17,18] was used. The amount of TEM4 bound to F-actin was determined indirectly by immunoblotting the supernatant with anti-GST antibodies and quantified by densitometry. All blots were within the linear range of detection as determined by running out a range of GST-TEM4 protein dilutions (unpublished data). For experiments with ADP- G-actin, ATP-G-actin was converted to ADP-G-actin with 20 U/ mL hexokinase (Sigma) and 1 mM glucose for 3 h at 4uC as previously described [16,19] and used immediately. Immunoprecipitation Analysis HUVEC were left untreated or preincubated with 1 mM LatA (Sigma) for 30 min and lysed in immunoprecipitation buffer (10 mM PIPES, pH 7, 100 mM KCl, 2 mM EDTA, 0.5% Triton X-100) containing protease inhibitors. Cell lysates were incubated with TEM4 antibody, precoupled to protein G Dynabeads (Invitrogen), for 4 h at 4uC. The beads were washed with the immunoprecipitation buffer four times and immunocomplexes were analyzed by immunoblotting. RhoC Activation Pull-down Assays The pGEX construct encoding a GST fusion protein of the Rho binding domain (RBD) of Rhotekin (amino acids 7–89) (provided by Keith Burridge, UNC-Chapel Hill) was used for expression in bacteria and GST-Rhotekin-RBD was purified from bacterial cell lysates using glutathione-Sepharose 4B beads (Amersham Bios- ciences). Transiently transfected 293T cells were maintained in growth medium for 24 h and then serum-starved (0.5% FBS) overnight. Cells were lysed in lysis buffer (50 mM Tris, pH 7.5, 500 mM NaCl, 10 mM MgCl2, 1% Triton X-100, 10% glycerol) supplemented with protease inhibitors and pelleted at 13,0006g for 5 min. Thirty mg of GST-Rhotekin-RBD immobilized on glutathione-Sepharose 4B beads were incubated with 500 mg of clarified cell lysates for 1 h at 4uC. The beads were washed twice in wash buffer (25 mM Tris, pH 7.5, 40 mM NaCl, 30 mM MgCl2). Whole cell lysates and affinity-precipitated samples were subjected to SDS-PAGE and analyzed by western blotting using RhoC isoform-specific antibodies. Results TEM4 is a Rho-specific RhoGEF TEM4/ARHGEF17 is a member of the Dbl family of RhoGEFs [2] as it possesses a tandem Dbl homology (DH) and pleckstrin homology (PH) domain catalytic cassette. TEM4 was annotated by St. Croix et al. [11] and encodes a 2063 amino acid protein with a predicted molecular weight of 222 kDa that, in addition to the DH and PH domains, contains extensive N- terminal sequences (residues 1–1064) with no predicted domains or motifs, and a C-terminal domain with a predicted seven-bladed b-propeller fold (residues 1469–2063) (Fig. 1A). Subsequently, another study reported on a 1510 amino acid protein encoded by TEM4, which they called p164-RhoGEF based on a predicted molecular weight of 164 kDa [20]. The p164-RhoGEF cDNA PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e41876