Arg 130 in TEM4 ABD is essential for F-actin binding.

<p>A, Alignment of residues 125-135 of human TEM4 with other species. Residues tested for actin binding <i>in vivo</i> 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 <i>in vivo</i> in live NIH3T3 cells. C, Coomassie Blue-stained gel comparing ATP/ADP-P_i-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-P_i-F-actin calculated from co-sedimentation experiments as shown in Fig. 4. Two independent co-sedimentation experiments were used to generate the data.</p

TEM4 is a Rho-specific guanine nucleotide exchange factor.

<p>A, A schematic representation of the domain structure of TEM4. <i>TEM4</i> encodes a 2063 amino acid protein that, in addition to the DH and PH domains, contains extensive N-terminal sequences (residues 1-1059) with no identifiable domains or motifs, and a C-terminal domain containing a predicted β-propeller fold (residues 1723–2039) likely consisting of seven WD40-related repeats <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041876#pone.0041876-Kelley1" target="_blank">[24]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041876#pone.0041876-Chen1" target="_blank">[60]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041876#pone.0041876-Sondek1" target="_blank">[61]</a>. The PH domain is split by an ∼60 amino acid α-helical insert. B, TEM4 is expressed in mammalian cell lines of endothelial and non-endothelial cell lineage. C–G, Guanine nucleotide exchange of RhoA (C), RhoB (D) or RhoC (E) (2 µM) was assessed in the presence (red trace) or absence (black trace) of the isolated DH-PH domain of TEM4 (10 nM). Guanine nucleotide exchange of Rac1 (F) and Cdc42 (G) (2 µM) was assessed in the presence of the DH-PH domains of TEM4 (50 nM; red trace) and PREX1 (200 nM; green trace). Intrinsic activities of Rac1 and Cdc42 are shown as a black trace. Arrows indicate time of GEF addition. Data shown are representative of three independent experiments. Five µg of each protein used in GEF assays were resolved by SDS-PAGE and visualized by Coomassie Blue dye (inset D). H, Structure-based sequence alignment of the TEM4 PH domain. A homology model of the PH domain of TEM4 (residues 1286–1476) was generated using the protein homology/analogy recognition engine² (Phyre²) structure prediction server and then structurally aligned with the determined structures of DH-associated PH domains from SOS1 (PDB: 1AWE) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041876#pone.0041876-Zheng1" target="_blank">[62]</a>, TIAM1 (PDB: 1FOE) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041876#pone.0041876-Worthylake1" target="_blank">[63]</a> and TRIO (PDB: 1NTY) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041876#pone.0041876-Skowronek1" target="_blank">[64]</a> using the Vector Alignment Search Tool (VAST) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041876#pone.0041876-Gibrat1" target="_blank">[65]</a>. The assignments of beta strands (blue arrows, β1–β7) and the C-terminal α-helix (yellow cylinders, αC) are shown for each PH domain sequence. Secondary structure analysis predicts a ∼70 residue α-helical region (red) is inserted in the β5/β6 turn of the TEM4 PH domain. Residues within TEM4 likely utilized for phospholipid binding are marked (arrowheads). A highly conserved tryptophan residue within αC of PH domains (phenylalanine in TEM4 and Tiam1) is indicated (star). Dots mark every 10th residue. I, Protein-lipid overlay assay. Purified GST-DH-PH fusion protein was incubated with a lipid array and then detected with an anti-GST antibody. <i>LPA</i>, lysophosphatidic acid; <i>LPC</i>, lysophosphocholine; <i>PC</i>, phosphatidylcholine; <i>PS</i>, phosphatidylserine; <i>PA</i>, phosphatidic acid; <i>PE</i>, phosphatidylethanolamine; <i>S1P</i>, sphingosine 1-phosphate; <i>PI</i>, phosphatidylinositol; <i>PI(3)P</i>, phosphatidylinositol 3-phosphate; <i>PI(4)P</i>, phosphatidylinositol 4-phosphate; <i>PI(5)P</i>, phosphatidylinositol 5-phosphate; <i>PI(3,4)P₂</i>, phosphatidylinositol 3,4-biphosphate; <i>PI(3,5)P₂</i>, phosphatidylinositol 3,5-biphosphate; <i>PI(4,5)P₂</i>, phosphatidylinositol 4,5-biphosphate; <i>PI(3,4,5)P₃</i>, phosphatidylinositol 3,4,5-triphosphate.</p

Actin binding is essential for subcellular localization and <i>in vivo</i> activity of TEM4.

<p>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 µm. B, The N-terminus is essential for TEM4 <i>in vivo</i> 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.</p

TEM4 binds directly to F-actin with high affinity

<p>. A, Binding of TEM4 (residues 81–135) to F-actin was examined by actin co-sedimentation assays. Recombinant GST-TEM4 protein was incubated with F-actin and subjected to high speed centrifugation. Soluble (s) and pellet (p) fractions were resolved by SDS-PAGE and stained with Coomassie Blue. B, Coomassie Blue-stained gel showing the near-saturation binding of GST-TEM4-ABD to F-actin. Numbers below protein bands indicate protein concentrations in µM as determined by densitometry. C, TEM4 ABD binds F-actin. Silver stained gel shows co-sedimentation of untagged TEM4 ABD with F-actin. D-G, TEM4 preferentially binds dynamic ATP/ADP-P_i-F-actin. ATP- or ADP-bound G-actin was polymerized and binding of the resulting F-actin filaments to GST-ABD was determined by co-sedimentation analysis. Representative Coomassie Blue-stained gels showing amount of ATP/ADP-P_i- or ADP-F-actin in the pellet of co-sedimentation experiment (D and E, top panel). Immunoblot showing amount of GST-ABD protein left in the supernatant in co-sedimentation experiments (bottom panel). F, G, Equilibrium binding of GST-ABD to ATP/ADP-P_i- or ADP-F-actin. Amount of GST-ABD bound to F-actin was calculated from immunoblots similar to the ones shown in D and E.</p

N-terminus of TEM4 is essential for localization of TEM4 to cytoskeleton.

<p>A, Endogenous TEM4 associates with F-actin. TEM4 was immunoprecipitated from HUVECs pretreated with LatA and β-actin was detected by western blotting. B, Schematic of GFP-TEM4 fusion constructs. <i>DH</i>, Dbl homology; <i>PH</i>, pleckstrin homology. C, The N-terminus of TEM4 possesses the actin association signal. HUVECs were electroporated with GFP or GFP-tagged TEM4 constructs as indicated and imaged live. Scale bar, 20 µm.</p

Detailed mapping of actin-association domain of TEM4

<p>. A, GFP-TEM4 fusion constructs encoding various truncations of the N-terminus or GFP were transfected into NIH3T3 cells and imaged live. Scale, 20 µm. B, The newly identified actin-binding domain of TEM4 (aa 81–135 in human TEM4) is conserved among TEM4 orthologs. Helices (<i>H</i>) predicted by the secondary structure prediction server PredictProtein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041876#pone.0041876-Rost1" target="_blank">[66]</a> are labeled.</p

Effects on P7.p polarity are <i>chw-1</i>-specific.

<p>^a compared to <i>lin-17(n671)</i></p><p>^b compared to <i>lin-17(n671); gfp(RNAi)</i></p><p>^c compared to <i>lin-18(e620)</i></p><p>^d compared to <i>lin-18(e620); gfp(RNAi)</i></p><p>Animals were grown at 23°C and scored by DIC at late L4 stage. <i>n</i> is number of animals scored. Data for strains marked with an asterisk (*) are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133226#pone.0133226.t001" target="_blank">Table 1</a> (<i>lin-17</i>) or <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133226#pone.0133226.t002" target="_blank">Table 2</a> (<i>lin-18</i>). Analyses of statistical significance were performed using Fisher’s exact test.</p

Two Wnt pathways regulate VPC polarization.

<p><b>A</b>. Schematic of vulval precursor cells in the whole animal. <b>B.</b> Schematic of refined (central) and ground (posterior) Wnt signals regulating VPC polarity. <b>C.</b> A schematic of polarized 2°-1°-2° VPC lineages (P5.p (left), P6.p (center), and P7.p (right)). P5.p and p7.p lineages are asymmetric and mirror one another around a central axis (dotted line). (<b>D-F</b>) Illustration and images of vulval lineages, model polarity signals and morphology in animals that are (<b>D</b>) wild type, (<b>E</b>) <i>egl-20(n585)</i> using only refined polarity, or (<b>F</b>) <i>lin-17(n671)</i>; <i>lin-18(e620)</i> using only ground polarity. Arrows represent putative Wnt polarity signals received by VPCs, grayed arrows are inactivated polarity signals, and dotted lines represent central vulval axis.</p

CHW-1 regulates relative LIN-17/Fz and LIN-18/Ryk signaling in refined (central) polarity, and LIN-17/Fz and LIN-18/Ryk execute a specific repressive program to exclude activity of the ground (posterior) polarity system.

<p>Shown is a schematic of P7.p with anterior, the direction of refined (central) polarization, to the left and posterior, the direction of ground (posterior) polarization to the right.</p

Loss of VANG-1 but not CAM-1/Ror in the <i>lin-17</i> (Fz) mutant background retains sensitivity to CHW-1 activity.

<p>^a compared to <i>lin-17(n671); gfp(RNAi); vang-1(ok1142)</i></p><p>^b compared to <i>lin-17(n671); gfp(RNAi)</i></p><p>^c compared to <i>lin-17(n671); chw-1(ok697); gfp(RNAi)</i></p><p>^d compared to <i>cam-1(gm122); lin-18(e620)</i></p><p>^e compared to <i>cam-1(gm122); gfp(RNAi); lin-18(e620)</i></p><p>^f compared to <i>lin-18(e620)</i></p><p>^§ Data previously published [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133226#pone.0133226.ref021" target="_blank">21</a>]</p><p>Also, loss of CAM-1 but not VANG-1 in the <i>lin-18</i> (Ryk) background retains sensitivity to CHW-1 activity. In other words, VANG-1 loss abolishes the ability of <i>lin-18</i> mutant animals to respond to <i>chw-1</i> mutation or RNAi. Animals were maintained at 23°C and scored by DIC at late L4 stage. <i>n</i> is number of animals scored. Analyses of statistical significance were performed using Fisher’s exact test.</p