Title: A role for VASP in RhoA-Diaphanous signalling to actin dynamics and SRF activity
Abstract: Article16 June 2003free access A role for VASP in RhoA-Diaphanous signalling to actin dynamics and SRF activity Robert Grosse Robert Grosse Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, Transcription Laboratory, Room 401, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author John W. Copeland John W. Copeland Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, Transcription Laboratory, Room 401, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Timothy P. Newsome Timothy P. Newsome Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, Cell Motility Laboratory, Room 529, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Michael Way Michael Way Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, Cell Motility Laboratory, Room 529, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Richard Treisman Corresponding Author Richard Treisman Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, Transcription Laboratory, Room 401, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Robert Grosse Robert Grosse Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, Transcription Laboratory, Room 401, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author John W. Copeland John W. Copeland Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, Transcription Laboratory, Room 401, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Timothy P. Newsome Timothy P. Newsome Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, Cell Motility Laboratory, Room 529, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Michael Way Michael Way Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, Cell Motility Laboratory, Room 529, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Richard Treisman Corresponding Author Richard Treisman Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, Transcription Laboratory, Room 401, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Author Information Robert Grosse1, John W. Copeland1, Timothy P. Newsome2, Michael Way2 and Richard Treisman 1 1Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, Transcription Laboratory, Room 401, 44 Lincoln's Inn Fields, London, WC2A 3PX UK 2Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, Cell Motility Laboratory, Room 529, 44 Lincoln's Inn Fields, London, WC2A 3PX UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:3050-3061https://doi.org/10.1093/emboj/cdg287 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Vasodilator-stimulated phosphoprotein (VASP) is involved in multiple actin-mediated processes, including regulation of serum response factor (SRF) activity. We used the SRF transcriptional assay to define functional domains in VASP and to show that they coincide with those required for F-actin accumulation, as determined by a quantitative FACS assay. We identified inactive VASP mutants that can interfere both with F-actin assembly and with SRF activation by wild-type VASP. These VASP mutants also inhibit actin-based motility of Vaccinia virus and Shigella flexneri. VASP-induced F-actin accumulation and SRF activation require both functional Rho and its effector mDia, and conversely, mDia-mediated SRF activation is critically dependent on functional VASP. VASP and mDia also associate physically in vivo. These findings show that VASP and mDia function cooperatively downstream of Rho to control F-actin assembly and SRF activity. Introduction Ena/VASP (vasodilator-stimulated phosphoprotein) family proteins are involved in numerous actin-regulated processes, including cell motility, epithelial cell adhesion and pathogen F-actin tail formation (for reviews, see Reinhard et al., 2001; Krause et al., 2002). The Ena/VASP proteins, VASP, Evl and Mena, which are expressed in many cell types (Reinhard et al., 1992; Gertler et al., 1996), are localized to actin bundles, focal adhesions and the leading edge (Reinhard et al., 1995). Each protein is composed of three conserved domains (Figure 1A). The N-terminal EVH1 (Ena-VASP homology 1) domain is structurally related to the PH and PTB domains (Fedorov et al., 1999; Prehoda et al., 1999) and binds the FP4 motif (D/EFPPPPXD), which is found in Zyxin, Vinculin and the Listeria monocytogenes ActA protein (Southwick and Purich, 1994; Pistor et al., 1995; Brindle et al., 1996; Gertler et al., 1996; Reinhard et al., 1996; Niebuhr et al., 1997). A central polyproline-rich region binds to profilin and to SH3- and WW-domain proteins (reviewed in Bear et al., 2001). At the C-terminus, the EVH2 domain contains binding sites for G- and F-actin and a coiled- coil motif required for oligomerization (Figure 1A) (Bachmann et al., 1999; Walders-Harbeck et al., 2002). Phosphorylation of Ena/VASP proteins may regulate their affinity for F-actin (Laurent et al., 1999; Harbeck et al., 2000) or SH3-domain proteins (Lambrechts et al., 2000; Howe et al., 2002). Ena/VASP proteins appear to play a role in F-actin assembly, although their exact function in actin dynamics remains unclear. VASP has been reported in various studies to facilitate ActA-mediated, Arp2/3-dependent actin polymerization (Loisel et al., 1999; Skoble et al., 2001), to nucleate F-actin assembly independently of Arp2/3 (Lambrechts et al., 2000; Fradelizi et al., 2001), and to promote actin filament elongation by antagonizing capping protein activity (Bear et al., 2002). Figure 1.VASP domains required for SRF activation and F-actin assembly coincide. (A) SRF activation by VASP mutants. The EVH1 and polyproline-rich regions are shown as filled and open boxes, and the four conserved sequence blocks constituting the EVH2 domain as black squares. (B) Representative SRF activation data. Cells expressed VASP mutants at inputs judged to give comparable protein expression levels (0.05 and 0.15 μg for VASP, VASPΔPP, VASPΔD and YFP–VASP; 0.15 and 0.5 μg for EVH1-PP, EVH2, VASPΔA, VASPΔB, VASPΔC and VASP–DC), together with the SRF reporter gene 3D.Aluc (0.05 μg) and Renilla luciferase transfection control (0.05 μg). Reporter activity is expressed relative to its activation by the constitutively active SRF derivative SRF-VP16. (C) VASP protein expression. Cells expressed VASP mutants as in (B). Cell lysates were analysed by immunoblot using polyclonal VASP antibody (left) or epitope-tag antibody (right). (D) F-actin content. Cells expressed VASP derivatives at levels corresponding to their maximal activity in the SRF reporter gene assay activation. Mean cellular F-actin content was determined relative to that of untransfected cells in the same population using FACS. Download figure Download PowerPoint We showed previously that expression of VASP strongly induces the activity of the transcription factor SRF (serum response factor) in NIH 3T3 fibroblasts (Sotiropoulos et al., 1999). SRF, a MADS box protein, controls expression of immediate-early and muscle-specific genes, including the cytoskeletal proteins β-actin and vinculin (Arsenian et al., 1998). Stimulation of cells by serum or mitogenic phospholipids, such as LPA, regulates the activity of SRF via a Rho-controlled signalling pathway, which involves alterations in actin dynamics (Hill et al., 1995; Sotiropoulos et al., 1999). Two RhoA effector pathways control SRF activity (Tominaga et al., 2000; Copeland and Treisman, 2002; Geneste et al., 2002). The ROCK-LIMK pathway acts to stabilize F-actin by inhibiting the activity of the depolymerizing/severing factor cofilin (Maekawa et al., 1999), while the RhoA-Diaphanous pathway apparently stimulates F-actin assembly by promoting filament nucleation (Watanabe et al., 1999; Pruyne et al., 2002; Sagot et al., 2002). SRF activation appears to arise from depletion of the G-actin pool, since it is inhibited by overexpression of non-polymerizable actin derivatives (Posern et al., 2002). In this paper we exploit the rapid and simple SRF activation assay to analyse functional domains in VASP. We demonstrate that VASP domains involved in F-actin assembly and in SRF activation are identical. We identify inactive VASP derivatives that interfere with VASP-stimulated SRF activity and F-actin assembly, as well as actin tail formation, in the pathogens Vaccinia and Shigella flexneri. The interfering VASP mutants also block serum- and Diaphanous-induced SRF activation. Our analysis provides evidence that VASP functions in Rho-dependent signalling to control actin assembly via the Diaphanous pathway. Results VASP domains mediating SRF activation and F-actin assembly coincide To investigate the relationship between the ability of VASP to potentiate SRF activity and the assembly of F-actin, we analysed the activity of a set of N-terminally, epitope-tagged VASP derivatives (Figure 1A). To evaluate the effect on VASP on F-actin assembly we used a quantitative FACS assay, in which the mean F-actin content of cells expressing VASP is measured relative to that of untransfected cells in the same population (Copeland and Treisman, 2002; Geneste et al., 2002). Expression of intact VASP potently activated the SRF reporter gene 3D.ALuc (Figure 1B; Sotiropoulos et al., 1999) and substantially increased mean cellular F-actin content (Figure 1D). Immunoblot analysis revealed that maximal SRF activation was obtained at a level of transiently expressed VASP of ∼50% that of the endogenous protein (Figure 1C). Given a transfection efficiency of 25–30%, as determined by the FACS analysis, this indicates that expression of VASP at levels comparable to that of the endogenous protein is sufficient to robustly activate SRF and F-actin assembly (see Discussion). Analysis of the VASP mutants is shown in Figure 1. Deletion of the C-terminal EVH2 domain abolished both SRF activation and F-actin assembly. In contrast, expression of the EVH2 domain alone, or VASP mutants lacking the polyproline region or EVH1 domain, exhibited substantial activity in both assays. Single alanine substitutions at each of the three phosphorylation sites, S157, S239 and T278, did not affect VASP- or EVH2-induced SRF activity (data not shown). Within the EVH2 domain, deletion of the B-block abolished both VASP-induced SRF activation and F-actin assembly. In contrast, deletion of the EVH2 A-block, or a further short region of homology (the ‘D-block’) had no significant effect on SRF activation or F-actin assembly. Deletion of the EVH2 C-block somewhat reduced VASP activity in both assays, but also inhibited the Renilla luciferase and SRF-VP16 transfection controls, suggesting that its expression has toxic effects (data not shown). These deletions had similar effects in the context of the isolated EVH2 domain (Figure 1; data not shown). N-terminally YFP- or green fluorescent protein (GFP)-tagged VASP derivatives have been studied extensively in other systems (Rottner et al., 1999; Geese et al., 2002; Loureiro et al., 2002). YFP–VASP or GFP–VASP activated SRF only weakly (Figure 1B; data not shown). This did not reflect YFP interference with the reporter assay itself, since expression of YFP or GFP alone had no effect on either basal or VASP-induced SRF reporter activity (data not shown). For technical reasons we were unable to use the FACS assay to investigate F-actin assembly by YFP–VASP. We therefore tested the effect of VASP and YFP–VASP on the proportion of co-expressed actin recovered from cells by Triton X-100 detergent extraction. In this assay, F-actin is retained in the detergent-insoluble pellet fraction, while unpolymerized actin is recovered in the detergent-soluble supernatant (Posern et al., 2002). Expression of intact VASP substantially increased the amount of actin recovered in the pellet fraction, whereas YFP–VASP did not (see Supplementary figure S1, left panel, available at The EMBO Journal Online). Moreover, while in these experiments wild-type and endogenous VASP were recovered predominantly in the detergent-soluble fraction, YFP–VASP was recovered mainly in the pellet (see Supplementary figure S1, right panels). Taken together, these data establish a close correlation between the ability of VASP derivatives to activate SRF assay and their ability to promote F-actin assembly, and identify three inactive VASP mutants, EVH1-PP, ΔB and DC. YFP-tagged VASP derivatives were not studied further owing to the weak activity of the YFP–VASP protein in our assays. The B-block determines VASP localization to F-actin in NIH 3T3 cells We compared the localization of intact VASP to that of the minimal active EVH2 domain, and the inactive VASPΔB and DC derivatives. We found that, as previously reported in BAE and baby hamster kidney (BHK) cells (Haffner et al., 1995; Price and Brindle, 2000), expression of full-length VASP in NIH 3T3 cells induced polarized and thickened F-actin bundles (Figure 2A). All the VASP derivatives were found to be distributed diffusely throughout the cytoplasm. The active VASP and EVH2 proteins were also able to localize to F-actin bundles and the cell periphery. The inactive VASPΔB protein did not colocalize with F-actin, but was recruited to focal adhesions, similar to the corresponding Mena derivative (Figure 2D) (Loureiro et al., 2002). The inactive EVH1, EVH1-PP and DC derivatives were not recruited to focal adhesions or actin filaments, nor did they accumulate at the cell periphery (Figure 2C; data not shown). Localization of VASP to F-actin structures including the cell periphery thus requires the EVH2 B-block, while localization to focal adhesions requires the EVH1 and EVH2 regions. These results are consistent with the view that localization of VASP at the cell periphery may be required for its activity. Such localization cannot be sufficient for efficient VASP activity, however, because although YFP–VASP exhibits a similar localization (Bear et al., 2002; Loureiro et al., 2002), it is only weakly active in our assays. Figure 2.Intracellular localization of intact VASP, its minimal active derivative EVH2, and the inactive mutants ΔB and DC. NIH 3T3 cells expressing the indicated VASP mutants were maintained in 0.5% FCS for 16 h before fixing for indirect immunofluorescence. Top: Merged image of F-actin (rhodamine–phalloidin; red) and VASP (9E10 epitope-tag; green). Middle: Flag-VASP (9E10 epitope-tag). Bottom: F-actin (rhodamine–phalloidin). Download figure Download PowerPoint Interfering VASP derivatives The quantitative nature of the SRF and F-actin assembly assays enabled us to readily identify VASP derivatives that are completely inert or that retain an ability to interfere with the function of intact VASP. Increasing amounts of the inactive VASP derivatives were cotransfected with a fixed amount of intact VASP and the effect on SRF reporter activity was measured. VASPΔB, DC and EVH1-PP all exhibited a dose-dependent ability to inhibit VASP-induced SRF activation, with maximal inhibition approaching background levels (Figure 3A). Similarly, both VASPΔB and DC proteins significantly inhibited VASP-induced F-actin accumulation, as measured in the FACS assay. An inactive VASP derivative comprising the EVH1 domain alone inhibited neither process (data not shown; Figure 3B). Since the short DC fragment contains the C-block required for VASP oligomerization, we tested whether the DC fragment is sufficient to interact with the intact protein. Protein overlay assays with [35S]methionine-labelled DC confirmed that this VASP fragment is indeed capable of direct interaction with VASP derivatives containing an intact EVH2 domain C-Block (Figure 3C). Similar results were obtained using full-length VASP or VASPΔB as a probe (data not shown). Figure 3.The inactive VASP mutants ΔB and DC are interfering mutants. (A) Interference with VASP-induced SRF activation. NIH 3T3 cells transfected with the SRF reporter expressed intact VASP (0.15 μg) or the indicated VASP mutants (0.1, 0.3 and 0.9 μg). The structure of the mutants is shown below. (B) Interference with VASP-induced F-actin accumulation. Cells expressing intact VASP (0.75 μg) and the indicated VASP mutants (4.5 μg) were analysed for mean cellular F-actin content relative to that of untransfected cells in the same population using FACS. (C) The VASP EVH2 block C is sufficient to bind intact VASP. Lysates from cells expressing the indicated VASP mutants were separated by SDS–PAGE (12.5% gel), transferred to a membrane, and probed either with anti-Flag antibodies (upper panel) or with 35S-labelled VASP DC (residues 278–380), produced by in vitro translation (lower panel). Download figure Download PowerPoint Interfering VASP mutants inhibit Vaccinia and S.flexneri actin tail formation The ability of the inactive VASP derivatives to interfere with VASP-induced SRF activation and F-actin accumulation prompted us to test whether expression of such proteins could block other VASP functions, such as their role in pathogen actin tail formation. Ena/VASP proteins are involved in intracellular motility of L.monocytogenes (reviewed by Bear et al., 2001; Frischknecht and Way, 2001) and are recruited to the actin tails of Vaccinia virus (Zeile et al., 1998; Frischknecht et al., 1999) as well as S.flexneri (Chakraborty et al., 1995; Laine et al., 1997). In Vaccinia-infected HeLa cells, expression of intact VASP did not affect the proportion of cells displaying actin tails (Figure 4A and E). VASP was concentrated at the virus itself and detectable along the F-actin tail, as reported previously for endogenous VASP (Figure 4D). Within the infected cells, VASP was associated with the viral factory in the perinuclear region, and also localized to F-actin bundles and focal adhesions (Figure 4A). VASPΔPP behaved similarly to the intact protein (Figure 4E). The isolated EVH2 domain also had no effect on Vaccinia actin tail formation (Figure 4B and E); however, EVH2 was more evenly distributed along the actin tail and did not accumulate at focal adhesions (Figure 4D). The inactive EVH1 domain, which does not interfere with VASP-induced F-actin accumulation, neither affected tail formation nor became localized to the virus particle (Figure 4E; data not shown). In contrast, expression of the dominant interfering VASPΔB substantially reduced both the proportion of cells with actin tails and the number of tails per cell (Figure 4C and E). The VASP derivatives EVH1-PP and DC also inhibited Vaccinia actin tail formation (Figure 4E; and data not shown). The VASP derivatives affected S.flexneri-associated actin tails in a similar fashion: expression of active VASP derivatives had no effect on tail formation, while VASP derivatives that interfered with VASP-induced SRF activation and F-actin assembly also interfered with actin tail formation (Figure 4F). In contrast, neither wild-type VASP nor VASPΔB affected actin tail formation by L.monocytogenes in our cells (Figure 4G), indicating that VASPΔB does not interfere non-specifically with F-actin assembly. Taken together, these results provide strong evidence that VASP facilitates the assembly of Vaccinia and S.flexneri actin tails. Figure 4.Interfering VASP mutants block Vaccinia and Shigella F-actin tail formation. Adherent HeLa cells expressing VASP (0.3 μg) or its derivatives (0.6 μg) were maintained in 10% FCS. Sixteen hours later, the cells were infected for 8 h with Vaccinia (A–D, F) or for 4 h with L.monocytogenes (E) or S.flexneri (F), and processed for immunofluorescence. Cells were stained for F-actin using Alexa 568-phalloidin (red) and for the VASP epitope tag using 9E10 antibody (green). (A–D) VASPΔB blocks Vaccinia actin tail formation. Merged images are shown. Arrows indicate instances of VASP localization to focal adhesions. DAPI staining indicated the presence of viral particles at the tail ends (not shown). Quantitation is shown in (F). (A) Wild-type VASP; similar results were obtained upon infection of cells expressing GFP. (B) VASP EVH2. (C) VASPΔB blocks tail formation. (D) VASP and F-actin localization in the tails of cells expressing intact VASP (A′) or VASP EVH2 (B′). (E and F) Data summaries. The proportion of transfected Vaccinia-infected cells with any viral tails in a given field is shown. Data are represented as means ± SEM (n = 3). (E) Vaccinia data. In control infected cells expressing GFP, cells exhibiting tails generally contained 30–60 virus particles with tails. Expression of the interfering mutants reduced the proportion of cells displaying tails, and decreased the number of virus particles with tails to five to 10 per cell. (F) Shigella flexneri data. In control infected cells expressing GFP alone, those cells displaying tails contained only two to eight bacteria with tails. (G) VASPΔB expression allows the formation of L.monocytogenes actin tails. Separate images of VASP and F-actin are shown for infected cells expressing intact VASP (left) or VASPΔB (right). VASP derivatives did not affect the number of bacteria per infected cell. In two independent experiments, 58% and 55% (intact VASP) and 54% and 52% (VASPΔB) of infected cells displayed tails. Download figure Download PowerPoint Interfering VASP proteins block serum-induced SRF activation We next used the interfering VASP derivatives to test the involvement of VASP in serum induction of SRF activity, which is mediated by a pathway requiring functional RhoA and actin polymerization (Hill et al., 1995; Sotiropoulos et al., 1999). Expression of each of the interfering VASP derivatives, EVH1-PP, VASPΔB and DC, inhibited serum-induction of SRF activation in a dose-dependent manner (Figure 5A). To assess the specificity of this inhibition we examined activation of two other signal-regulated reporter systems. Expression of VASPΔB had no effect on either serum-induced activation of an ERK-dependent Elk-1 reporter (Figure 5B, left) (Marais et al., 1993), or on TGF-β induced activation of the Smad3-/4-dependent CAGA12 reporter (Figure 5B, right) (Dennler et al., 1998). Thus, VASPΔB specifically inhibits the SRF activation pathway in NIH 3T3 cells. Figure 5.Serum-induced SRF activation requires functional VASP. (A) Interference with serum-induced SRF activation. Cells transfected with the SRF reporter and plasmids expressing VASP mutants (0.1, 0.3 and 0.9 μg) were analysed for reporter activity before and after serum stimulation. Inset: VASP immunoblot. (B) Interference by the VASP mutants is signal pathway-specific. Cells transfected with NLex.ElkC and LexOP.Luc (left; ERK pathway) or CAGA12.Luc (right; SMAD pathway) together with control plasmid RLTK and expressed VASP mutants (0.9 μg). Luciferase activity was determined following stimulation with 15% FCS (left) or 2 ng/ml TGFβ (right). (C) Functional VASP is required for maximal serum-induced SRF activation. MVD7 cells, transfected with SRF reporter 3D.Aluc and RLTK control plasmid, expressed intact VASP or YFP–VASP, together with VASPΔB (0.9 μg). Reporter activity was determined before and after serum stimulation. Download figure Download PowerPoint To test the involvement of VASP in signalling to SRF further we examined reporter activation in the MVD7 fibroblast cell line, which contains VASP and Mena null mutations and lacks detectable Evl protein (Bear et al., 2000). SRF reporter gene activation was inefficient in these cells, and in contrast to NIH 3T3 cells this response was not inhibited by expression of VASPΔB (Figure 5C, solid bars). Expression of wild-type VASP in MVD7 cells increased the maximal serum-induced level of reporter activity, and this could now be reduced to the level seen in untransfected cells by expression of VASPΔB (Figure 5C, hatched bars). In contrast, expression of YFP– or GFP–VASP, which only weakly activate SRF in NIH 3T3 cells, did not potentiate serum-induced SRF activation in MVD7 cells (Figure 5C, open bars; and data not shown). These results strongly suggest that VASP activity is required for maximal serum-induced SRF activation. VASP-induced SRF activation and F-actin assembly is Rho dependent To gain further insight into how VASP might be involved in SRF regulation, we tested the role of RhoA in VASP-induced SRF activation. First, we measured SRF reporter activity upon expression of increasing amounts of intact VASP, VASPΔPP or EVH2 proteins in NIH 3T3 cells in the presence or absence of C3 exoenzyme, which inactivates RhoA. VASP-induced SRF activation was reduced by >85% by C3 expression (Figure 6A). A similar result was obtained when VASPΔPP was used to activate the reporter, but activation by the isolated EVH2 domain was largely independent of functional RhoA (Figure 6A). The inhibitory effect of C3 was not due to inhibition of focal adhesion assembly, since SRF activation by serum, active mDia1 or VASP was unaffected following focal adhesion disruption by 2,3-butanedione monoxime treatment (data not shown). Figure 6.VASP functions in the Rho–mDia–actin pathway to SRF activation. (A) Functional Rho is required for efficient VASP-induced SRF activation. Cells transfected with SRF reporter expressed intact VASP (0.02, 0.05, 0.15 μg) or VASP mutants (ΔPP: 0.02, 0.05, 0.15 μg; EVH2: 0.05, 0.2, 0.6 μg), together with C3 transferase (0.1 μg) as indicated. (Inset) Immunoblot analysis of VASP protein levels. (B) Functional Rho and mDia1 are required for VASP-induced SRF activation and F-actin accumulation. Left: SRF activation. Cells transfected with SRF reporter and expressing C3 transferase (0.1 μg) or interfering mDia1 mutant F1F2Δ1 (Copeland and Treisman, 2002) (0.9 μg) were processed as in (A). Inset: VASP immunoblot of cell lysates. Right: F-actin accumulation. Cells expressed intact VASP (0.75 μg) with either C3 transferase (0.5 μg) or mDia1 mutant F1F2Δ1 (4.5 μg). Mean cellular F-actin content of the transfected cells was determined relative to that of untransfected cells in the same population using FACS. (C) Functional VASP is required for efficient mDia1- induced SRF activation. Left: cells transfected with SRF reporter expressed activated mDia1 mutants FH1FH2 (0.03 μg) or FH2 (0.3 μg) (Copeland and Treisman, 2002), and VASPΔB (0.1, 0.3 and 0.9 μg). Right: mDia derivatives. Ellipse, Rho-binding domain; D, DAD domain. Immunoblot for mDia1 indicates that VASPΔB expression does not affect mDia1 protein levels. Download figure Download PowerPoint The requirement for functional Rho in VASP-induced SRF activation prompted us to investigate the Rho effectors involved. The Diaphanous family of formin proteins regulate F-actin assembly in response to Rho activation, and are required for signalling to SRF in many cell types (Tominaga et al., 2000; Copeland and Treisman, 2002; Geneste et al., 2002). To test whether Diaphanous activity is required for VASP-induced SRF activation, we evaluated the effect of coexpression of the dominant interfering mDia1 derivative F1F2Δ1 (Copeland and Treisman, 2002). SRF reporter activation by intact VASP was inhibited by F1F2Δ1 in a dose-dependent manner; moreover, VASP-induced F-actin assembly was substantially inhibited by expression either of C3 exoenzyme or mDia1 F1F2Δ1 (Figure 6B). These results suggest that VASP functions in the Rho-dependent signalling pathway to SRF. To examine whether VASP functions upstream of, or parallel with, mDia in the SRF signalling pathway we tested the effect of the interfering VASP derivatives on mDia1-induced SRF activation. Two activated mDia1 derivatives FH1FH2, which lacks the N-terminal Rho-binding and FH3 domains, and FH2, which comprises the minimal FH2-containing region required for F-actin assembly (Copeland and Treisman, 2002), both efficiently activated SRF, and this activation was blocked by expression of VASPΔB (Figure 6C). Physical association between VASP and mDia1 The experiments presented in the preceding section are consistent with the idea that VASP and Diaphanous function in concert, downstream of Rho, in SRF activation and F-actin assembly, but do not exclude the possibility that mDia1 and VASP act in different pathways that utilize a common cofactor. To address this issue we investigated whether mDia and VASP proteins physically associate in vivo by performing co-immunoprecipitation experiments. VASP was recovered in the anti-mDia1 immunoprecipitate, but not the control immunoprecipitate (Figure 7A, lanes 1–4). In a reciprocal experiment, we recovered endogenous mDia1 in anti-VASP immunoprecipitates (Figure 7A, lanes 5–7). No significant variation in association between the two proteins occurred upon serum stimulation (data not shown). The association of mDia1 with VASP is consistent with the notion that the two proteins function in the same pathway. Figure 7.Physical interaction between VASP and mDia1. (A) Association of endogenous VASP and mDia1. NIH 3T3 cell extracts or buffer were immunoprecipitated either with crosslinked anti-mDia1 or control anti-GFP beads (left panels), or with crosslinked anti-VASP or control anti-GFP beads (right panels). The precipitates were analysed by immunoblot, with protein detection by anti-mDia1 (upper panels) or anti-VASP (lower panels). Protein input i