Trio-D1 binds to the C-terminus of Rac1 but not of RhoG and activates Rac1 independent of its SH3 domain.

<p>(<b>A</b>) HeLa cells were transfected with Myc-Trio-Full-length (FL) and a pull-down experiment with biotin-tagged peptides that encode for the last 10 amino-acids of the C-terminus of Rac1, RhoG and RhoA was performed, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029912#s4" target="_blank">Materials and Methods</a>. Western blot analysis showed that Trio-FL binds to the Rac1 C-terminus peptide, but not to RhoA or RhoG C-termini. As a control, β-Pix binding to the C-terminus of Rac1, but not of RhoA or RhoG is shown. (<b>B</b>) Myc-Trio-D1 was transfected into HeLa cells, and a peptide pull down was performed as described under A. Western blot analysis showed that Trio-D1 associates with the C-terminus of Rac1, but not with the CTRL or RhoG peptide. Left lane shows Myc-Trio-D1 input. (<b>C</b>) HeLa cells were transfected with GFP-Trio-D1ΔSH3 or GFP-Trio-D1+SH3 constructs and a Rac1 C-terminal peptide pull down was performed. Western blot analysis showed that the C-terminus of Rac1 required the SH3 domain of Trio-D1 to interact. Blots were incubated with an Ab against GFP to stain for Trio constructs. (<b>D</b>) HeLa cells were transfected with GFP-Trio-D1+SH3 constructs and a peptide pull down was performed with biotinylated peptides encoding control sequence, the Rac1 C-terminal domain or the Rac1 C-terminal domains in which the proline stretch had been mutated to alanines (P/A) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029912#pone.0029912-Nethe1" target="_blank">[10]</a>. Western blot analysis showed that Trio-D1+SH3 required the proline-rich stretch in the Rac1 C-terminus to bind. Blots were incubated with an Ab against GFP to stain for Trio constructs. (<b>E</b>) HeLa cells were transfected with GFP-CAAX, GFP-Trio-D1ΔSH3 or GFP-Trio-D1+SH3 constructs, and Rac1-GTP activity assays were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029912#s4" target="_blank">Materials and Methods</a>. Western blot analysis showed that Rac1 is activated by Trio-D1, independent of the SH3 domain (upper panel). (<b>F</b>) HeLa cells were transfected with GFP-CAAX, GFP-Trio-D1ΔSH3 or GFP-Trio-D1+SH3 constructs and a pull-down assay using glutathione-beads to precipitate GST-Rac1-G15A mutants was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029912#s4" target="_blank">Materials and Methods</a>. Western blot analysis showed that Rac1 needed the SH3 domain of Trio-D1 to interact (upper panel), because the binding was less efficient when Trio-D1 lacked the SH3 domain. Lower panel shows GST-Rac1-G15A input. Lower unidentified band in upper panel is due to GST isolation and a-specific staining of the antibody. Graph below shows the quantification of the binding of GST-Rac1-G15A to Trio-D1ΔSH3 and Trio-D1+SH3. No significant difference was found for the presence of the SH3 domain in the binding to GST-Rac1-G15A. Experiment was carried out three times, independently from each other. Data are mean ± SEM. ns: not significant.</p

Trio induces membrane ruffles.

<p>(<b>A</b>) Schematic overview of the Trio protein (3097 amino acids, molecular weight approximately 350 kDa), indicating in green the N-terminal DH-PH unit including an SH3 domain and in red the C-terminal DH-PH unit. The third catalytic domain of Trio is a kinase domain (yellow). At the N-terminus, a Sec14 domain and spectrin repeats are present. Below the GFP/Myc-tagged constructs used in this manuscript are depicted: Trio-D1 encodes for the N-terminal DH-PH domain including the flanking SH3 domain, Trio-D1ΔSH3 domain represents the N-terminal DH-PH domain lacking the SH3 domain, and Trio-D2 representing the C-terminal DH-PH domain. (<b>B</b>) HeLa cells were cultured on glass cover slips and transfected as indicated with GFP-tagged constructs. Immunofluorescent imaging showed that GFP did not affect the morphology of the cells. GFP-Trio full length (FL) and GFP-Trio-D1 induced lamellipodia (arrowheads) and co-localized with F-actin (red), as is shown in the merge images. For the Trio-FL, 68%±7 of the transfected cells induced lamellipodia as illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029912#pone-0029912-g001" target="_blank">figure 1B</a>. For Trio-D1, 79%±4 of the transfected cells induced lamellipodia. GFP-Trio-D2 in green induced stress fibers (arrowheads), shown by F-actin staining in red. 46%±12 of these transfected cells i9nduced stress fibers, as shown. Data are mean ± SEM. Bar, 20 µm. Images at the right show merged magnification of F-actin in red and GFP-tagged protein in green. Bar, 10 µm. (<b>C</b>) Changes in morphology analyzed by scanning electron microscopy. No change in morphology is observed at the periphery or surface of GFP-expressing HeLa cells (arrowheads), whereas Trio-D1 induced large dorsal and lateral lamellipodia (arrowheads). Bar, 50 µm. Image on the right shows a magnification (Zoom) of Trio-D1-induced dorsal lamellipodia (arrowheads). Bar, 5 µm. Two lower images show lamellipodia (arrowheads), induced by a constitutively active form of RhoG (Q61L) (left image) and Rac1 (Q61L) (right image), both comparable with the lamellipodia induced by Trio-D1. Bar, 10 µm.</p

Trio-D1 regulates the dynamics of lamellipodia.

<p>(<b>A</b>) Upper panel shows magnification of lamellipodia, stained for F-actin in red in cells transfected with scrambled shRNA (shCTRL), shRNA against Trio (shTrio) or Trio-deficient cells transfected with Trio-D1 (shTrio+Trio-D1). Bar, 10 µm. Middle panel shows a still image of a movie showing lamellipodia dynamics in DIC. Dotted line represents the position of the kymograph that is shown in the lower panel. Kymographs represent the dynamics of lamellipodia for 500 seconds as indicated. Y-axis represents 10 µm distance. (<b>B</b>) Kymograph analysis as described in Material and Method section of lamellipodia in Trio-deficient HeLa cells was performed. Cells were transfected with GFP-CAAX or GFP-Trio-D1. Trio-D1 promotes the velocity (left graph), distance (middle graph) and frequency (right graph) of lamellipodia dynamics significantly. At least nine different lamellipodia were quantified in nine different cells over three independent experiments. Data are mean ± SEM. *p<0.01; **p<0.01 (<b>C</b>) Kymograph analysis as described above. Trio-deficient HeLa cells were transfected with Trio-D1 containing the SH3 domain (closed bars) or lacking the SH3 domain (open bars). No difference was observed between the two conditions in the velocity (left graph), distance (middle graph) or frequency (right graph) of lamellipodia. At least nine different lamellipodia were quantified in nine different cells over three independent experiments. Data are mean ± SEM.</p

Trio regulates Rac1 activity upon FN-mediated cell spreading.

<p>(<b>A</b>) Rac1 activity was measured with biotin-CRIB peptides as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029912#s4" target="_blank">Materials and Methods</a>. Rac1 activity was increased after 3 h of spreading in shCTRL cells, whereas changes in Rac1 activation were absent in Trio-deficient cells (shTrio). (<b>B</b>) Trio RhoG activity was measured with GST-ELMO as bait (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029912#s4" target="_blank">Materials and Methods</a>). RhoG activity was unaltered in shCTRL and shTrio cells upon cell spreading on FN. (<b>C</b>) Early Rac1 activation upon spreading was affected in Trio-deficient cells as well. Rac1-GTP levels were measured upon 10 or 20 minutes spreading on FN or in suspension as described under A. (<b>D</b>) RhoA activity was measured in the GST-Rhotekin pull-down assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029912#s4" target="_blank">Materials and Methods</a>. RhoA activity was high in cells that were in suspension (0 minutes) and decreased upon spreading on FN (180 minutes). No difference between shCTRL and shTrio cells was measured. All experiments described above were carried out at least three times.</p

Trio-D1 activates RhoG independent of its SH3 domain.

<p>(<b>A</b>) HeLa cells were transfected with GFP-CAAX, GFP-Trio-D1ΔSH3 or GFP-Trio-D1+SH3 constructs, and RhoG-GTP activity assays were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029912#s4" target="_blank">Materials and Methods</a>. Western blot analysis showed that RhoG is activated by Trio-D1, independent of the SH3 domain (upper panel). Middle panel showed the expression of endogenous RhoG in HeLa cells. Lower panel shows the expression of the Trio constructs. (<b>B</b>) HeLa cells were transfected with GFP-CAAX, GFP-Trio-D1ΔSH3 or GFP-Trio-D1+SH3 constructs, and a pull-down assay using glutathione-beads to precipitate GST-RhoG-G15A mutants was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029912#s4" target="_blank">Materials and Methods</a>. Western blot analysis showed that RhoG required the SH3 domain of Trio-D1 to interact (upper panel), although the binding was less efficient when Trio-D1 lacked the SH3 domain. Middle panels show the expression of constructs in total cell lysates using an Ab against GFP and the lower panels show the loading control for RhoG in total cell lysates and GST-RhoG-G15A input. Lower unidentified band in upper panel is due to GST isolation and a-specific staining of the antibody. Graph below shows the quantification of the binding of GST-RhoG-G15A to Trio-D1ΔSH3 and Trio-D1+SH3. A significant difference is found for the presence of the SH3 domain in the binding to GST-RhoG-G15A. Experiment is carried out three times, independently from each other. Data are mean ± SEM. * p<0.05. (<b>C</b>) HeLa cells were transfected with RhoG siRNA. Rac1-GTP levels were measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029912#s4" target="_blank">Materials and Methods</a> and show that GFP-Trio-D1 activates Rac1 independently from RhoG. In fact, Trio-D1-induced Rac1 activity was increased when RhoG was silenced. Middle panel shows Rac1 protein for loading control in cell lysates and lower panels show the expression of endogenous RhoG protein and the different Trio-D1 constructs in cell lysates. Graph on the right shows the quantification of the band intensities, measured using ImageJ software and show that RhoG silencing increased TrioD1-induced Rac1-GTP levels 2-fold.</p

Trio-D1 activates Rac1 and RhoG, but not RhoA.

<p>(<b>A</b>) HeLa cells were transfected with Myc-Trio-D1 and Myc-RhoG as indicated. Rac1-GTP and RhoG-GTP and RhoA-GTP levels were measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029912#s4" target="_blank">Materials and Methods</a> and show that Trio-D1 activates RhoG and Rac1, but not RhoA. Tubulin is shown as protein loading control. (<b>B</b>) HeLa cells were transfected with HA-Trio-D2 and Myc-Trio-D1 as indicated. RhoA-GTP was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029912#s4" target="_blank">Materials and Methods</a> and shows that Trio-D2, but not Trio-D1 activates endogenous RhoA. Second panel from above shows RhoA protein loading in the cell lysates. Data are representative for at least three independent experiments.</p

Silencing Trio results in impaired FN-mediated cell spreading.

<p>(<b>A</b>) shRNA against Trio reduces endogenous Trio expression in HeLa cells, whereas shCTRL (scrambled sequence) did not affect Trio expression. Two independent cell line clones were tested. Actin is shown as protein loading control. (<b>B</b>) Cells were allowed to spread for 3 hours under serum-free conditions on FN (10 µg/ml). Phase contrast shows spreading cells in shCTRL-treated cells, whereas shTrio-treated cells display a round phenotype. Bar, 100 µm. F-actin staining in red underscores the defect in spreading of the shTrio-treated cells. Nuclei are in blue. Bar, 20 µm. (<b>C</b>) Percentage of cells that spread was quantified. A spreading cell was positively scored when the phenotype resembled the cell shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029912#pone-0029912-g005" target="_blank">figure 5B</a>, right upper image. Experiment was repeated five times in triplicate. Data are mean ± SEM. *p<0.01. (<b>D</b>) Cells were plated on FN-coated gold electrodes and spreading was measured in time as indicated. ShTrio-treated cells (solid line) showed reduced spreading capacity compared to shCTRL-treated cells (dashed line). Per array, 200,000 cells were plated. (<b>E</b>) In a Transwell system, migration was measured. Filters were coated with FN and HeLa cells (shCTRL or shTrio) were added to the upper compartment and allowed to migrate for 5 h to 1% serum, which was present in the lower compartment. Number of cells that migrated across the filter was counted by nuclei staining and the migration of shCTRL HeLa cells was set to 1. Migration across a FN-coated filter is reduced upon Trio silencing. Experiment was repeated four times in duplicate. Data are mean ± SEM. *p<0.01.</p

Trio-D1 rescues the spreading and migration defect in Trio-deficient cells.

<p>(<b>A</b>) GFP-CAAX (upper panels) or GFP-Trio-D1+SH3 (lower panels) were transfected into Trio-deficient cells. Next, cells were allowed to spread for 3 h on FN. GFP-tagged proteins are visible in green, F-actin in red and the merge is shown in yellow. Nuclei are in blue. Bar, 20 µm. (<b>B</b>) Graph shows the quantification of cell spreading on FN. The maximum diameter of the cell was measured in µm and displayed on the Y-axis. Experiment was carried out four times in duplicate. Data are mean ± SEM. *p<0.01. (<b>C</b>) Bar graph shows quantification of cell migration assay across FN-coated Transwell filters. Trio-deficient HeLa cells were transfected with GFP alone or GFP-Trio-D1 and allowed to migrate from the top to the bottom part of the filter. The lower compartment contained 1% serum. Number of cells that migrated across was counted by nuclei staining and GFP-expressing cells counted were set as 1. Experiment was carried out four times in duplicate. Data are mean ± SEM. *p<0.001.</p

Gata1cKO^MKmice show alterations in the erythroid compartment.

<p>(a) Gating strategy to identify erythrocytes at consecutive stages of differentiation in the bone marrow and the spleen based on surface marker expression KIT, CD71 and Ter119. (b) Percentage of reticulocytes at consecutive stages of differentiation of live cells. Left graph depicts the bone marrow compartment, right the splenic compartment. (c) Photograph of representative spleens from Gata1cKO^MK and control mice shows the splenomegaly that Gata1cKO^MKdevelop.</p

Gata1 regulates Syk expression.

<p><b>(a)</b> Syk mRNA expression levels in Gata1cKO^MK and WT^lox cultured megakaryocytes measured by qRT-PCR. (b) Syk protein levels in Gata1cKO^MK and WT^lox platelets, analyzed by Western blotting. Syk expression level normalized to loading control Gapdh is indicated, setting the average expression levels of Syk in WT^lox platelets to 100. (c) Chromatin immunoprecipitation (ChIP) assay showsGata1 binding to the <i>Syk</i> promoter in WT^lox compared to Gata1cKO^MK (background control) cultured megakaryocytes. A GATA positive (+) site on the promoter of the known target <i>Gp1ba</i> (CD42b) was used as positive control and a GATA negative (−) site on the promoter of <i>Cd9</i> was used as negative control.</p