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Figure S1. Topology of the Abi1. WAVE2, Nap1, PIR121 complex. a. Identification of Abi1 interactors and mapping of the binding surfaces between Abi1 and WAVE2. Left panels.Abi1-immunocomplex were isolated as described in Figure 1A. Bands specific to the Abi1 immunocomplexes were numbered and analysed by MALDI. Peptide coverage was sufficient for the unambiguous identification of the Abi1 interactors. Each protein was identified in at least three separate Abi1 immunoprecipitations. A list of the identified proteins is reported at the bottom. Right. An example of data obtained from band 3 representing a WAVE2 peptide sequenced (highlighted in bold in the WAVE2 amino-acid sequence reported at the top) by nanoelectrospray is shown (bottom). b. Coomassie stained gel of baculovirus produced and purified His-Abi1. Molecular weight markers are indicated on the left. c. In vitro binding of immobilized GST or GST–WHD domain of WAVE1 to purified His-tagged Abi1. Shown are 20% of the input (Input) and 100% of the bound materials. d. The N-terminal region of Abi1 is required for WAVE2 binding. Total cellular lysates from 293T transfected (tfx) with the indicated Flag-tagged WAVE2 together with RFP-Abi1 fusion proteins or RFP were immunoprecipitated with anti-Flag-ab. Lysates (lys) and immunoprecipitates (IP) were immunoblotted (WB) with the indicated abs. A schematic representation of Abi1 domain organization and of the Abi1 fragments used is indicated on the bottom (CC, coiled-coil region; P rich, proline rich region; SH3, Src Homology 3 domain). e.Coomassie stained gel of bacterially produced and purified GST-Nap1. At variance from the Nap1/PIR121 complex, which was readily soluble and stable, purified Nap1, derived from Flag-Nap1 overexpressing 293T cells, precipitated after elution from agarose-conjugated anti-Flag IgG. Thus a strategy to produce Nap1 as a GST-fusion protein from bacteria was implemented. f. In vitro binding of immobilized GST-fusion proteins (1 g) indicated on the right to recombinant (1.5 g) Rac, depleted of nucleotide (NF), loaded with GDP (GDP), or with GTPS (GTPS). Shown are 20% of the input (Input) and 100% of the bound materials. g. WAVE1 and WAVE2 proteins expression in 293T cells. Lysates of 293T cells transfected (Tfx) with myc-WAVE1 or an empty vector (ctr) were immunoblotted with the abs indicated at the bottom. No WAVE1 could be detected using specific antibodies.

Figure S2. Stability of the WAVE-Abi-Nap1-PIR21 complexes upon addition of activated Rac. a. Gel filtration chromatography of endogenous WAVE, Abi1, Nap1 and PIR121 obtained from total cellular lysates of 293T cells transfected with activated RacQL (+) or the appropriate empty vector (-). Lysates (3.5 mg) were applied onto a 200 ml prep grade Superose 6 column as described in Materials and Methods. An aliquot of each fraction (indicated at the top) was resolved by SDS-PAGE and immunoblotted with the indicated abs. The elution profile of proteins of known molecular weight (Mr) is indicated at the bottom. The input lane (I) was loaded with 100 g of total cellular lysates. RacQL overexpression was verified by immunoblotting with anti-Rac ab (bottom panel). b.Gel filtration chromatography of endogenous WAVE, Abi1, Nap1 and PIR121 from mouse brain in the presence or absence of GTP-loaded Rac. Total cellular lysates of mouse brain (3.5 mg) incubated in the presence of GTPS-bound Rac (GTPS-Rac, +) or GST, as a control (-), were applied onto a 200 ml prep grade Superose 6 column as described in Materials and Methods. An aliquot of each fraction (indicated at the top) was resolved by SDS-PAGE and immunoblotted with the indicated abs. The elution profile of proteins of known molecular weight (Mr) is indicated at the bottom. The input lane (I) was loaded with 100 g of total cellular mouse brain lysates.

Figure S3. a. EGF-stimulation induces Abi1, WAVE2, Nap1, and PIR121 re-localization at the tip of F-actin-rich ruffles. HeLa cells, transfected with GFP-Abi1, or GFP-WAVE2, or GFP-Nap1, or GFP-PIR121, were serum starved and stimulated with 100 ng/ml of EGF (EGF), or mock-treated (ctr). After 10 min, cells were fixed and processed for epifluorescence to detect the various GFP-fusion proteins (indicated on the left, green) or stained with rhodamin-conjugated phalloidin to detect F-actin (red). Co-localization between the various GFP-fusions proteins and F-actin is indicated by the yellow color in the merged images. Bar is 10 m. b. Abi1, WAVE2, Nap1, and PIR121 are re-localized to Rac-induced dorsal ruffles where they colocalize with Rac.HeLa cells were cotransfected with GFP-Abi1, or GFP-WAVE2, or GFP-Nap1, or GFP-PIR121 in combination with an empty vector (Hela ctr), or with HA-tagged RacQL (HeLa RacQL). Cells were fixed and GFP was detected by epifluorescence (green). HA-Rac was visualized by staining with anti-Rac ab (red). Confocal apical sections are shown. Co-localization between the various GFP-fusions proteins and activated Rac is indicated by the yellow color in the merged images. Bar is 10 m.c. Characterization of Abi1 K.D. HeLa cells. Upper panels. HeLa were transfected with pAV-Abi1-197 or pAV-ctr, fixed and stained to detect Abi1 (green) and F-actin (red). Similar results were also obtained using pAV-169-Abi1 to silence Abi1. Asterisks indicate Abi1 K.D. cells. Bottom panels. Phase-contrast images of stable Abi1 K.D. or the corresponding control (ctr) cells are shown. Bar is 10 m. d. Quiescent Abi1 K.D. clone pAV197 (Abi1 K.D.), in which the expression of Abi1 was ablated in more than 95% of cells, and its corresponding control clone (ctr) obtained as described in Fig. 6, were treated with EGF (G.F.), or mock treated (serum starved), fixed and stained to visualize F-actin. Arrows point to ruffles. Bar is 10 M. e. Upper panels, Quiescent Abi1 K.D. clone and its corresponding control clone (ctr) were treated with EGF (100 ng/ml) or mock treated for the indicated time points. Total cellular lysates were either incubated with GST-CRIB (Rac GTP), as described in Material and Methods, or directly immunoblotted (Rac) with anti-Rac Ab. Bottom panels.The downregulation of Abi1 expression in the clones used is revealed by immunoblotting against the indicated abs. f. The anti-WAVE ab recognizes both WAVE1 and WAVE2. 293T cells were transfected with myc-tagged WAVE1 (mycWAVE1) or a fragment of WAVE2, used to raise the anti WAVE abs, and encompassing the proline-rich region, and the VCA domain (mycPWA-WAVE2). Total cellular lysates were immunoprecipitated with anti-myc abs followed by immunoblotting (WB) with the indicated abs. g-i. WAVE2 gene silencing reduces Rac-dependent ruffles. g-h. Silencing of WAVE2 by siRNA. Stable suppression of WAVE2 gene expression in HeLa was obtained by transfecting 10 g of pAV-siRNA vectors (pAV-ctr or pAV-WAVE2-234-251 (234) or pAV-WAVE2-295-314 (295)) with 1.0 g of pBabe-puro plasmids. Cells were then selected with 2.5 g/ml of puromycin for 15 days. Serial dilution of the puromycin-resistant mass population was performed to isolate clones, which were then expanded. Three representative stable clones from pAV-ctr (ctr) and pAV-WAVE2-234-251 (234) and pAV-WAVE2-295-314 (295) were analysed by: g. Immunoblotting with abs against WAVE proteins and actin. Notably, Hela cells express undetectable levels of WAVE1 proteins. h. Real-time, quantitative RT-PCR using specific WAVE2 (right graph) and WAVE1 (left graph) primers. Data, normalized with respect to the mRNA of GAPDH or actin, are expressed relative to the levels of WAVE2 mRNA detected in control cells. Notably, RNAi mediated interference of WAVE2 slightly affected the levels of transcript of WAVE1. i. WAVE2 gene silencing reduces RacQL-induced ruffling. Control (ctr) and WAVE2 null (pAV-WAVE2-234) HeLa cells were transfected with the HA-tagged RacQL, fixed and stained with anti-HA (red) and FITC-conjugated phalloidin to detect F-actin. RacQL expression induced ruffling in > 90% of control HeLa cells and 20% of WAVE2 knock down cells in three independent experiments, where at least 100 transfected cells were counted. The residual ruffling activity may be due to the presence of WAVE1 whose protein levels, however, could not be detected by immunoblotting. Bar is 10 M.

Figure S4. a.Activated Rac fails to induce the re-distribution of Abi1, Nap1, and PIR121 to the cell leading edge in WAVE2 null cells.Control (HeLa ctr) and WAVE2 null (WAVE2 K.D.) HeLa clones, cotransfected with the HA-tagged RacQL and either GFP-Abi1, or GFP-Nap1, or GFP-PIR121, were fixed and stained with anti-HA (red). GFP was detected by epifluorescence (green). More than 90% of transfected cells displayed the actin cytoskeleton phenotypes shown. Confocal apical sections are shown. Co-localization between the various GFP-fusions proteins and activated Rac is indicated by the yellow color in the merged images. Bar is 10 m.b.WAVE2 fails to restore Rac-induced ruffles in Abi1 K.D. HeLa cells.Control (HeLa ctr) and Abi null (HeLa Abi K.D.) HeLa clones were co-transfected with the AU5-tagged RacQL and GFP-WAVE2. Cells were fixed and stained with a combination of anti-AU5 (AU5) to detect RacQL or FITC-conjugated phalloidin (phalloidin) to detect F-actin. GFP-WAVE2 (WAVE2) was detected by epifluorescence (green). Merged images are also shown. Bar is 10 m. Notably, ectopic expression of WAVE2 in Abi1 K.D. cells was not sufficient to restore Rac-dependent actin remodeling. c. Abi1 ablation by RNA interference induces the degradation of WAVE2, Nap1, and PIR121. Total cellular lysates from control (ctr) and Abi null (169 and 197) HeLa clones were immunoblotted with abs indicated on the right. d. Abi1, Nap1/PIR121, and HSPC300 do not induce Arp2/3-mediated polymerization in absence of WAVE2. Kinetics of Arp2/3-mediated polymerization in the presence of 100 nM of Abi1, or Nap1/PIR121(NP), or HSPC300. e. The VCA domain of N-WASP is more efficient than that of WAVE2 in stimulating filament branching with Arp2/3 complex. It is noteworthy to point out that the isolated VCA domain of WAVE2 shown in Fig. 4 is slightly more active that WAVE assembled in a reconstituted complex. The reason for this is not clear at the moment. An obvious explanation would be that while equal molar amounts of VCA and WAVE2 were used, the amounts of WAVE2 actually assembled in a complex, and thereby in its higher active state, depend on the kinetic of association-dissociation of the complex components at steady state. Kinetics of Arp2/3-mediated polymerization of actin in the presence of 100 nM of the VCA domain of N-WASP (N-W-VCA) or of WAVE2 (W2-VCA). f. HSPC300 binds to WAVE2. Left panels. Coomassie stained gel of bacterially produced and purified HSPC300 cleaved form GST. Molecular weight markers are shown on the left. Right panels. Recombinant and purified GST-HSPC300 binds to WAVE2. Total cellular lysates (1 mg) from 293T proteins were incubated with immobilized GST alone or GST-fused to the HSPC300 (GST-HSPC300). Lysates (lys, 20 g) and bound proteins were resolved by SDS-PAGE and immunoblotted with anti-WAVE2 ab. g. Endogenous Abi1 and WAVE2 co-localized at the tip of Rac-induced ruffles.HeLa cells transfected with RacQL (RacQL) or an empty vector as a control (ctr), were fixed and stained to detect WAVE2 (red) and Abi1 (green). Merged images of confocal apical sections are shown. Cssso-localization between WAVE2 and Abi1 is indicated by the yellow color in the merge. Bar is 10 M.