N-WASP involvement in dorsal ruffle formation in mouse embryonic fibroblasts - PubMed (original) (raw)
N-WASP involvement in dorsal ruffle formation in mouse embryonic fibroblasts
John A Legg et al. Mol Biol Cell. 2007 Feb.
Abstract
The Wiskott-Aldrich syndrome protein (WASP) family activates the Arp2/3 complex leading to the formation of new actin filaments. Here, we study the involvement of Scar1, Scar2, N-WASP, and Arp2/3 complex in dorsal ruffle formation in mouse embryonic fibroblasts (MEFs). Using platelet-derived growth factor to stimulate circular dorsal ruffle assembly in primary E13 and immortalized E9 Scar1(+/+) and Scar1 null MEFs, we establish that Scar1 loss does not impair the formation of dorsal ruffles. Reduction of Scar2 protein levels via small interfering RNA (siRNA) also did not affect dorsal ruffle production. In contrast, wiskostatin, a chemical inhibitor of N-WASP, potently suppressed dorsal ruffle formation in a dose-dependent manner. Furthermore, N-WASP and Arp2 siRNA treatment significantly decreased the formation of dorsal ruffles in MEFs. In addition, the expression of an N-WASP truncation mutant that cannot bind Arp2/3 complex blocked the formation of these structures. Finally, N-WASP(-/-) fibroblast-like cells generated aberrant dorsal ruffles. These ruffles were highly unstable, severely depleted of Arp2/3 complex, and diminished in size. We hypothesize that N-WASP and Arp2/3 complex are part of a multiprotein assembly important for the generation of dorsal ruffles and that Scar1 and Scar2 are dispensable for this process.
Figures
Figure 1.
Dorsal ruffle production in E13 primary MEFs. (A) DNA isolated from embryos and mouse tail tips were used in a PCR genotyping reaction. Products were run on a 2% agarose gel. Lane 1, 100-bp ladder; lane 2, heterozygote; lane 3, wild-type; and lane 4, Scar1 null. (B) We analyzed 50 μg of total protein from wild-type and Scar1 null MEFs by immunoblot with anti-Scar1, Scar2, and actin antibodies. (C) Wild-type and Scar1 null MEFs were stimulated with PDGF for 0, 2, 5, and 7 min. The cells were fixed and stained for F-actin and the percentage of cells generating at least one dorsal ruffle was determined. (D) The MEFs were PDGF stimulated for 2, 5, and 7 min, and the average number of dorsal ruffles (white arrows) per cell containing at least one dorsal ruffle was calculated. For C and D, >200 cells were counted per time point, and each point represents the average value from three independent experiments (error bars + SD). Student's t test p values are shown on graphs.
Figure 2.
Time-lapse phase microscopy of the primary E13 wild-type and Scar1 null cells producing dorsal ruffles in response to 10 ng/ml PDGF treatment. A1–A6, wild-type cell. B1–B6, Scar1 null cell. C1–C4, enlarged movie of a dorsal ruffle in a Scar1 null cell. Arrows follow the formation of the dorsal ruffles. Arrowheads indicate phase bright macropinosome formation during dorsal ruffle closure. Time in seconds is indicated in top right corner. Bar, 20 μm (see Movies 1, 2, and 3).
Figure 3.
Dorsal ruffle production in E9 immortalized MEFs. (A) We analyzed 50 μg of total protein from E13 primary and E9 immortalized wild-type and Scar1 null MEFs by immunoblot with anti-Scar1, Scar2, and actin antibodies. (B) Representative picture showing phalloidin staining of Scar1 null E9 MEFs after 5 min of PDGF treatment. Scale bar, 20 μm. (C) Wild-type and Scar1 null E9 MEFs were stimulated with PDGF for 0, 2, 5, 7, 10, 12, and 15 min. For C, >200 cells were counted per time point, and each point represents the average value from three independent experiments. The cells were fixed and stained for F-actin, and the percentage of cells generating at least one dorsal ruffle was determined (error bars + SD). Student's t test p values are shown on the graph.
Figure 4.
Wiskostatin treatment of E9 immortalized MEFs. (A) Serum-starved wild-type and Scar1 null MEFs were treated for 5 min with 10 μM wiskostatin alone (Wisk), 10 ng/ml PDGF (+ DMSO carrier) alone (PDGF), or PDGF and wiskostatin (PDGF + Wisk). The cells were then fixed and stained with TRITC phalloidin, and the percentage of cells producing dorsal ruffles was determined. (B) Serum-starved wild-type E9 MEFs were pretreated with decreasing concentrations of wiskostatin for 5 min and then incubated with 10 ng/ml PDGF and wiskostatin. The same concentration of wiskostatin was used in the pretreatment as during the PDGF stimulation. After PDGF treatment, the average percentage of cells generating dorsal ruffles was determined. For A and B, >200 cells were counted per time point, and each point represents the average value from three independent experiments (error bars + SD). (C) Representative image of a wild-type MEF treated with 10 μM wiskostatin and 10 ng/ml PDGF. Cells were fixed and stained with TRITC phalloidin (A) and anti-p34 (B). Arrows indicate localization of Arp2/3 complex in peripheral ruffles. Bar, 20 μm.
Figure 5.
siRNA studies on dorsal ruffle formation in E9 immortalized MEFs. (A) Wild-type MEFs were transfected for 48 h with siRNA specific to mouse Scar1, Scar2, N-WASP, and Arp2. Knockdown of protein levels was shown by immunoblot with polyclonal antibodies specific to each protein. Anti-tubulin blots were included as protein loading controls. (B) The siRNA-treated MEFs were serum starved and stimulated with 10 ng/ml PDGF for 5 min. The cells were fixed and stained with TRITC phalloidin, and the percentage of cells generating at least one dorsal ruffle was determined. N-WASP and Arp2 siRNA treated cells were also cotransfected with human WASP-GFP and human Arp2-myc constructs. More than 200 were counted per time point, and each point represents the average value from three independent experiments (error bars + SD). Student's t test p values are shown on graphs.
Figure 6.
Effects of expressing truncation mutants of N-WASP and Scar proteins in E9 immortalized MEFs. Wild-type (white bars) and Scar1 null (gray bars) E9-immortalized MEFs were treated with transfection reagent alone (U) or transfected with Scar1ΔA (S1dA), Scar2ΔA (S2dA), Scar3ΔA (S3dA), wild-type N-WASP (Wt NW), or N-WASPΔWWCA (NWdWWCA) mutants as indicated. The cells were serum starved, stimulated with PDGF for 5 min, and the percentage of cells generating dorsal ruffles was determined. More than 200 cells were counted per time point, and each point represents the average value from three independent experiments (error bars + SD). Student's t test p values are shown on graphs.
Figure 7.
PDGF treatment of N-WASP FLCs. (A) We analyzed 30 μg of total protein from wild-type MEFs (positive control) and the N-WASP FLCs by immunoblot with anti-NWASP and tubulin antibodies. Loadings: lane 1, wild-type MEFs; lane 2, +/+ RV-N-WASP; lane 3, N-WASP+/+; and lane 4, N-WASP−/−. (B) The N-WASP+/+ (A–C), +/+ RV-N-WASP (D–F) and N-WASP−/− (G–O) FLCs were PDGF stimulated for 5 min and fixed and stained with TRITC phalloidin and anti-p34. Arrows indicate collapsed dorsal ruffles and arrowheads show Arp2/3-enriched peripheral ruffles in the N-WASP−/− FLCs. Bar, 20 μm.
Figure 8.
Analysis of dorsal ruffle formation in N-WASP FLCs. (A and B) The +/+ RV-N-WASP and N-WASP−/− FLCs were PDGF stimulated for 0, 2, 5, and 7 min, and the percentage of cells generating any dorsal actin-rich protrusions (A) or only ring-shaped dorsal actin-rich protrusions (B) was calculated. More than 200 cells were counted per time point, and each point represents the average value from three independent experiments (error bars ± SD).
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