Dorsal Ruffle Formation in Scar1 Null E13 Primary MEFs
To investigate the role of Scar1 in dorsal ruffle formation, we generated wild-type and Scar1 null primary MEFs. The Scar1 knockout mice were generated using a retroviral gene-trap insertion (Dahl et al., 2003
). Scar1 null mice displayed numerous neuroanatomical abnormalities and postnatal lethality and so could not be used for breeding (Dahl et al., 2003
). Therefore, heterozygote mice were bred to generate Scar1 null embryos. Embryonic day 13 (E13) embryos were used to generate primary MEFs. The embryo heads were used in genotyping PCR reactions (A). For explanation of the genotyping protocol, refer to Supplemental Figure 1. Wild-type embryos produced a 400-bp band, and Scar1 null embryos had a 150-bp PCR product. Protein lysates from wild-type and Scar1 null MEFs also were probed with anti-Scar1, anti-Scar2, and anti-actin antibodies (B). The anti-Scar1 antibody showed a strong band at approximately 66 kDa in the wild-type lysate that was absent in the Scar1 null lane. This confirmed the loss of Scar1 protein expression in the knockout MEFs. Anti-Scar2 immunoblots showed the presence of a 70-kDa band in both wild-type and Scar1 null cells. Scar2 levels remained comparable between the two cell types, indicating there was no increase in Scar2 expression to compensate for Scar1 loss. Consistent with previous reports, an anti-Scar3 blot showed no detectable expression of Scar3 in either wild-type or Scar1 knockout MEFs but did detect a strong band in mouse brain lysates and COS-7 cell lysates transfected with full-length Scar3-myc (data not shown; Suetsugu et al., 2003
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, (more ...)
Endogenous Scar1 and Scar2 localized to dorsal ruffles, and Scar2 (but not Scar1) consistently localized to peripheral lamellipodia (Supplemental Figure 2). To investigate the role of Scar1 in dorsal ruffle production, serum-starved MEFs were treated with 10 ng/ml PDGF ββ for 0, 2, 5, and 7 min. At these time points, the cells were fixed and stained for F-actin and endogenous Arp2/3 complex (anti-p34). The percentage of cells with dorsal ruffles at each time point is shown in C. At 0 min, the serum-starved cells had no dorsal ruffles, consistent with cells requiring agonist stimulation to generate dorsal ruffles. By 2 min, the cells started to generate dorsal ruffles, with 5% of the wild-type and the Scar1 null cells containing at least one dorsal ruffle. At 5 min, dorsal ruffle numbers have reached an optimum, with ~30% of cells containing dorsal ruffles. The percentage of cells with dorsal ruffles then starts to decrease at 7 min as some ruffles close and the number tends to zero after 20 min (data not shown). Staining with anti-p34 showed a strong enrichment of endogenous Arp2/3 complex within the dorsal ruffles and at peripheral ruffles (data not shown). Importantly, at all time points, the Scar1 null cells produced the same percentage of dorsal ruffles as the wild-type cells, as shown by Student's t tests (see C for p values).
D shows an example of a wild-type MEF producing three dorsal ruffles after 5 min of PDGF treatment. Therefore, because MEFs can produce multiple dorsal ruffles per cell, we calculated the average total number of dorsal ruffles per cell in a time course. Cells without dorsal ruffles were not included in this assay, therefore, no value was recorded at 0 min. D shows the average number of dorsal ruffles per cell after 2, 5, and 7 min PDGF treatment. Interestingly, no optimal value was recorded at 5 min, with the response staying constant at two dorsal ruffles per cell at all time points. This is consistent with the dorsal ruffles forming, expanding, and contracting over a time scale of minutes and rarely splitting into multiple separate ruffles at later time points. Both the wild-type and Scar1 null cells showed the same constant response to the PDGF. Therefore, Scar1 is not essential for the generation of dorsal ruffles by the E13 primary MEFs in response to PDGF.
Dorsal ruffles are highly dynamic structures, expanding and contracting over time. PDGF treatment of MEFs was therefore followed using real-time phase microscopy to determine if Scar1 loss affected dorsal ruffle dynamics. Representative movies are shown in (A1-A5 wild-type, B1-B5 Scar1 null MEFs; Supplemental Movies 1 and 2, respectively). After ~1 min of PDGF treatment, dark punctate structures start occurring on the dorsal surface of the MEFs (A2 and B2 arrows). By 5 min, the dorsal ruffles have fully formed, consistent with the optimum peak seen in C. These waves pulse, expand, and contract over time before finally closing (A5 and B5). Interestingly, phase bright macropinosomes formed upon closure of the dorsal ruffles in both wild-type and Scar1 null MEFs (A5 and B5 arrowheads). A magnified example of macropinosome formation is shown in images C1–C4 (Supplemental Movie 3). As the dorsal ruffle zips closed from right to left, significant numbers of phase-bright, 1- to 2-μm-wide macropinosomes are formed (C2–C4, arrowheads). The visualization of these structures is consistent with reports that dorsal ruffles are involved in macropinocytosis (Dowrick et al., 1993
; Araki et al., 2000
). Imaging multiple dorsal ruffles from wild-type and Scar1 null cells did not reveal any qualitative differences in dorsal ruffle dynamics. Both cell types generated waves through dark punctate precursors, which then expanded into large dorsal ruffles. These structures constricted over time and closed normally to generate macropinosomes. Therefore, Scar1 deficiency does not detectably affect the dynamics of dorsal ruffle formation or closure.
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 (more ...)
Dorsal Ruffle Formation in E9 Scar1 Null Immortalized MEFs
The results obtained in and were particularly significant because they contradict previous findings that Scar1 is important for dorsal ruffle formation in MEFs (Suetsugu et al., 2003
). This could be due to differences in the genetic background or preparation of the MEFs used for these studies, although the background C57BL/6 was reported to be the same in both cases. Because Suetsugu et al. (2003)
used E9 MEFs, it is possible that the E13 primary MEFs have undergone further differentiation/gene expression changes to compensate for the loss of Scar1. Therefore, it was necessary to confirm the results with the same MEFs used in the previous study. We obtained immortalized E9 wild-type and Scar1 null MEFs (a gift from Y. Itoh and T. Takenawa, University of Tokyo, Tokyo, Japan) and repeated the PDGF assays to investigate dorsal ruffle production (). Lysates from the E13 primary and E9 immortalized wild-type and Scar1 null MEFs were probed with anti-Scar1, Scar2, and actin antibodies (A). These immunoblots confirmed the loss of Scar1 in the immortalized knockout cells with Scar2 levels staying constant (A). The overall intensity of the Scar1 and Scar2 staining between the wild-type and immortalized cells was also comparable, indicating that both MEF sources expressed similar levels of Scar1 and Scar2. None of the MEFs had any detectable Scar3 expression (data not shown).
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 (more ...)
The immortalized MEFs were stimulated with 10 ng/ml PDGF, and the percentage of cells producing dorsal ruffles was determined (, B and C). Phalloidin staining of the MEFs revealed that nearly 100% of the wild-type and Scar1 null cells produced dorsal ruffles after 5-min PDGF treatment (C). This response was much stronger than with the primary MEFs, where only 30% of the cells produced dorsal ruffles after 5 min (C). The dorsal ruffles in the immortalized MEFs were also much more persistent, with a significant percentage of cells retaining dorsal ruffles even after 15-min stimulation (C). This difference between primary and immortalized MEFs in dorsal ruffle formation could result from additional genetic/gene expression changes induced during cell transformation, which potentially increase the sensitivity to growth factor stimulation. Regardless of these differences, the wild-type and Scar1 null-immortalized MEFs responded identically to PDGF stimulation, with equal number of cells generating dorsal ruffles at all time points considered as shown by Student's t tests (C).
As mentioned, treatment of the immortalized MEFs with 10 ng/ml PDGF induced a robust, 100% response in dorsal ruffle formation. Taking into account a potential increase in sensitivity to PDGF in those cells, the lack of an obvious phenotype could therefore be due to saturating amount of growth factor. To test this hypothesis, decreasing amounts of PDGF were used to stimulate the immortalized MEFs, and the percentage of wild-type and Scar1 null cells producing dorsal ruffles was determined (Supplemental Figure 3). With 2 ng/ml PDGF (graph A), the response was skewed, with the response at 2 min dropping from 30 to 2% and the optimum moving from 5 to 7 min. At 1 and 0.5 ng/ml, the response was greatly reduced, with peak responses at 7 min of only 20 and 10%, respectively (graphs B and C). Independent of PDGF concentration or strength of response, the wild-type and Scar1 null immortalized MEFs produced a statistically similar response at all time points considered (Student's t test p values shown on graphs). Therefore, neither the E13 primary nor the E9 immortalized Scar null MEFs showed any defect in the production of dorsal ruffles, indicating that Scar1 is not essential for dorsal ruffle formation.
Wiskostatin Inhibition of Dorsal Ruffle Formation in MEFs
Studies on Scar1 did not reveal any obvious dorsal ruffle phenotype. Therefore, it was interesting to investigate the role of its related family member N-WASP. Endogenous N-WASP, along with its binding partners dynamin, cortactin, and WIP have all been localized to dorsal ruffles (Anton et al., 2003
; Krueger et al., 2003
). To investigate the endogenous localization of N-WASP relative to Arp2/3 complex and cortactin, PDGF-stimulated wild-type MEFs were stained using anti-Arp2 or anti-cortactin and anti-N-WASP antibodies (Supplemental Figure 4). Staining of N-WASP and Arp2 showed a strong colocalization with F-actin within dorsal ruffles (Supplemental Figure 4, A–D). N-WASP also colocalized with cortactin within dorsal ruffles (Supplemental Figure 4, E–H), consistent with previous reports (Krueger et al., 2003
). Therefore, N-WASP, Arp2/3 complex, and cortactin all colocalize in dorsal ruffles after PDGF stimulation.
The activity of N-WASP is tightly regulated by the binding of numerous proteins and lipids (Higgs and Pollard, 2000
). In its native form, N-WASP is autoinhibited due to intramolecular folding (Rohatgi et al., 1999
). Wiskostatin is a chemical inhibitor of N-WASP. It functions by binding a cleft in the regulatory GTPase binding domain of N-WASP and stabilizing its native, autoinhibited conformation (Peterson et al., 2004
). WASP expression is limited to hematopoietic cell lines, and it is not expressed in MEFs (Parolini et al., 1997
). Therefore, it is possible to study N-WASP function in dorsal ruffle formation by inhibiting it with wiskostatin.
When treated with wiskostatin alone, serum-starved immortalized wild-type and Scar1 null MEFs did not generate any dorsal ruffles (A). Pretreatment of the MEFs with DMSO for 5 min, followed by 5 min 10 ng/ml PDGF treatment resulted in ~100% of cells producing dorsal ruffles, independently of the Scar1 status. Finally, the MEFs were preincubated with 10 μM wiskostatin to inhibit N-WASP and then treated for 5 min with PDGF and wiskostatin. Inhibition of N-WASP with wiskostatin abolished the formation of dorsal ruffles but not peripheral ruffles in both wild-type and Scar1 null cells. C shows an example of a wild-type MEF treated with 10 μM wiskostatin. No dorsal ruffles are present, but endogenous Arp2/3 complex can still localize into peripheral ruffles (arrows), consistent with reports that N-WASP null cells can still generate lamellipodia (Snapper et al., 2001
). These data indicate that N-WASP, unlike Scar1 or Scar2, is important for dorsal ruffle formation.
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 (more ...)
To further investigate the effects of wiskostatin, decreasing concentrations were used on wild-type MEFs to determine how potently it inhibited dorsal ruffle formation. Wiskostatin at 5 μM still strongly inhibited dorsal ruffle formation (B, <10%). As the concentration reached 2.5, 1.25, and 0.625 μM, dorsal ruffle production returned rapidly to 100% levels. Therefore, 3–4 μM wiskostatin is sufficient to inhibit 50% of dorsal ruffle production in these MEFs. This indicates that inhibition of N-WASP with wiskostatin has a potent inhibitory effect on dorsal ruffle formation.
Scar1, Scar2, N-WASP, and Arp2 siRNA Studies in MEFs
To confirm the Scar1 data and examine the role of Scar2 in dorsal ruffle formation, siRNA oligos specific to mouse Scar1 and Scar2 were used to specifically downregulate their protein levels. Wild-type immortalized MEFs transfected for 48 h with Scar1 and Scar2 siRNA oligos showed a 70 and 75% knockdown of protein levels, respectively, as determined by the intensity of bands produced after immunoblots with anti-Scar1 and anti-Scar2 antibodies (A). Anti-tubulin loading controls confirmed roughly equal loadings between the control and siRNA lanes. The siRNA-treated cells were serum starved, treated for 5 min with PDGF, and fixed and stained for F-actin and endogenous Arp2/3 complex. The percentage of cells generating dorsal ruffles was then determined and plotted in B.
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 (more ...)
As a control, wild-type MEFs were treated with transfection reagent alone before PDGF treatment and staining. On average, ~55% of wild-type cells treated with Lipofectamine 2000 alone generated dorsal ruffles. The 70 and 75% knockdown of Scar1 and Scar2, respectively, had no detectable effect on the percentage of cells producing dorsal ruffles (see B for Student's t test p values). These experiments were repeated six times, and on each occasion neither Scar1 nor Scar2 siRNA treatment resulted in a statistically significant decrease in dorsal ruffle production. These data confirm the previous Scar1 results and indicate that Scar2 is also not essential for dorsal ruffle formation.
To confirm the wiskostatin data, wild-type MEFs were treated with mouse N-WASP siRNA oligos to knock down the endogenous protein levels of N-WASP. In addition, the Arp2/3 complex was targeted using Arp2 siRNA (, A and B). Knockdown of N-WASP and Arp2 protein levels was confirmed by immunoblot (A). Depletion of N-WASP protein levels (85% reduction) resulted in a dramatic loss of dorsal ruffles. Control cells produced on average 55% dorsal ruffles; however, this number was reduced to <10% in N-WASP siRNA-treated cells (p < 0.0005). This N-WASP siRNA inhibition was not as potent as wiskostatin treatment, which completely blocked dorsal ruffle production. This block is probably for two main reasons. First, the siRNA did not completely deplete N-WASP protein levels, meaning the remaining 10% could be sufficient to retain dorsal ruffle production in a subset of cells. Second, it is possible that wiskostatin has other protein targets or cytotoxic effects, resulting in indirect inhibition of dorsal ruffle production (Guerriero and Weisz, 2006). However, we used 10 μM or less wiskostatin in our assays, which gave only a weak off-target effect in the study of Guerriero and Weisz (2006). Interestingly, it was possible to partially retrieve the N-WASP siRNA phenotype by cotransfection of human WASP-GFP (B). In N-WASP siRNA-treated cells retrieved by human WASP-GFP expression, WASP-GFP colocalizes with endogenous Arp2/3 complex and F-actin in dorsal ruffles (Supplemental Figure 5, A–D, arrows). However, fewer than 10% of cells not transfected with WASP-GFP had dorsal ruffles. This is consistent with WASP-GFP being recruited to the dorsal ruffles and compensating for N-WASP loss. Therefore, inhibition of N-WASP function through wiskostatin or siRNA treatment results in dorsal ruffle inhibition, and there is a degree of functional redundancy between the N-WASP and WASP in this structure.
In addition to N-WASP, Arp2/3 complex was shown to be important in dorsal ruffle formation. Reduction of endogenous Arp2 levels (70% reduction; A) resulted in a statistically significant inhibition in the percentage of cells generating dorsal ruffles (p < 0.001; B). The phenotype was not as severe as with N-WASP siRNA treatment (20% compared with <10%). This was probably due to differences in the efficiency of the siRNA treatments. Cotransfection of human Arp2-myc retrieved this phenotype back to control levels (B). Similar to WASP-GFP, triple staining showed Arp2-myc colocalization with endogenous Arp2/3 complex and F-actin in dorsal ruffles (Supplemental Figure 5, E–H, arrows). Therefore, like N-WASP, Arp2/3 complex function is necessary for dorsal ruffle formation in MEFs.
Numerous roles have been attributed to dorsal ruffles, including the dissolution of stress fibers required to prepare a static cell for subsequent movement (Krueger et al., 2003
). The consequence of N-WASP and Arp2 siRNA treatment was evident in the stress fiber organization of the PDGF treated cells. Fifteen minutes into PDGF treatment, the wild-type, Scar1 null, and Scar2 siRNA-treated immortalized MEFs contained large dorsal ruffles. Within the waves, there was a dramatic disassembly of the stress fibers (Supplemental Figure 6, arrows). However, PDGF treated N-WASP and Arp2 siRNA MEFs retained large numbers of stress fibers in the main body of the cells (Supplemental Figure 6A, arrowheads). Therefore, this result confirms previous reports that dorsal ruffle formation is associated with stress fiber disassembly and demonstrates that N-WASP and the Arp2/3 complex are required for this process (Krueger et al., 2003
The importance of Scar1, Scar2, Scar3, and N-WASP in dorsal ruffle formation was finally investigated using the ectopic expression of truncation mutants in wild-type and Scar1 null MEFs stimulated for 5 min with PDGF. To investigate Scar function, delta-A Scar constructs were used. These constructs lack the C-terminal amino acids essential for Arp2/3 complex binding (Machesky and Insall, 1998
). Approximately 50% of control cells treated with transfection reagent alone generated dorsal ruffles (). Transfection of Scar1ΔA, Scar2ΔA, or Scar3ΔA into wild-type MEFs (white bars) did not invoke a statistically significant decrease in dorsal ruffle production, consistent with none of the Scar proteins being essential for dorsal ruffle production. Wild-type N-WASP expression did not inhibit dorsal ruffle formation. However, transfection of an N-WASP truncation mutant deficient in Arp2/3 complex binding (N-WASPΔWWCA) resulted in a decrease in cells with dorsal ruffles (p < 0.02; drop from 50 to 20%). This is probably due to the sequestration of endogenous SH3 domain proteins, such as dynamin and WIP, away from the Arp2/3 complex. Finally, Scar1ΔA and Scar2ΔA were expressed in Scar1 null MEFs (, gray bars) to determine whether the presence of any Scar isoform is required for dorsal ruffle formation. Neither Scar1ΔA nor Scar2ΔA expression in the Scar1 null MEFs inhibited dorsal ruffle formation. Therefore, it seems that an N-WASP–cortactin–Arp2/3 ternary complex is important for generating dorsal ruffles, whereas Scar1, -2, and -3 are dispensable for this process.
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), (more ...)
Dorsal Ruffle Formation in N-WASP Null FLCs
Previous studies on N-WASP function have shown its activity is dispensable for cell migration, adhesion to fibronectin substrate, and lamellipodia extension (Snapper et al., 2001
; Rogers et al., 2003
). To further investigate N-WASP function in dorsal ruffle formation, E9.5 N-WASP−/−
FLCs were obtained (Snapper et al., 2001
). These cells differ from MEFs because they were transformed with simian virus 40 (SV40) large-T–containing retrovirus to enable viability in cell culture. Immunoblot analysis with anti-N-WASP antibody on protein lysates confirmed the absence of a 66-kDa band in the N-WASP null FLCs (N-WASP−/−
; A). The wild-type SV40 FLCs (N-WASP+/+
) expressed similar levels of N-WASP as the MEF-positive control. Finally, SV40-FLCs infected with an N-WASP–expressing retrovirus (+/+ RV-N-WASP) showed a marked increased in N-WASP protein levels.
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 (more ...)
The N-WASP FLCs were stimulated for 5 min with PDGF and then fixed and stained for F-actin and endogenous Arp2/3 complex (B). The N-WASP+/+
(A–C) and +/+ RV-N-WASP (D–F) FLCs generated large, circular dorsal ruffles that were strongly stained for endogenous Arp2/3 complex. To our surprise, the N-WASP−/−
cells were able to generate dorsal actin protrusions in response to PDGF (G–O). However, there were numerous obvious deficiencies associated with these structures. They were often collapsed, forming bright, punctate structures under F-actin staining (arrows in H and K). In cells that did generate full ring-shaped dorsal ruffles, these were typically smaller than the equivalent structures in the N-WASP+/+
and +/+ RV-N-WASP FLCs and displayed greatly reduced staining for endogenous Arp2/3 complex (M–O). However, the N-WASP−/−
cells were still able to generate normal Arp2/3 complex-enriched peripheral ruffles, consistent with N-WASP not functioning in lamellipodia formation (arrowheads in N; Snapper et al., 2001
). Therefore, although not essential in these cells, N-WASP deficiency has resulted in highly abnormal dorsal ruffles that are unstable and depleted of Arp2/3 complex.
To quantify these deficiencies, after 5 min of PDGF stimulation, the average areas of the dorsal ruffles in +/+ RV-N-WASP and N-WASP−/− cells were quantified using a Java plugin for ImageJ (see Materials and Methods). The dorsal ruffles in +/+ RV-N-WASP cells were on average 3 times larger than the N-WASP−/− cells (6500 μm2 compared with 2100 μm2). Measurement of the average areas of the cells showed that the difference in dorsal ruffle size was not due to N-WASP−/− FLCs being smaller than their +/+ RV-N-WASP counterparts (20,000 μm2 compared with 19,000 μm2).
Finally, the +/+ RV-N-WASP and N-WASP−/− FLCs were stimulated for 0, 2, 5, and 7 min with PDGF to follow the generation of dorsal ruffles over time (, A and B). At each time point, the cells were fixed, and the percentage of cells generating dorsal ruffles was determined. Consistent with C and C, serum-starved FLCs did not generate dorsal ruffles. The +/+ RV-N-WASP FLCs showed a strong and persistent response to PDGF, with >70% of cells generating dorsal ruffles at 2, 5, and 7 min (A). The N-WASP−/− cells were severely deficient in producing dorsal ruffles, with only 20% generating dorsal ruffles at 2 min and a maximal response of 50% at 5 min. In this count, N-WASP−/− FLCs generating punctate, collapsed dorsal structures and full ring structures were both counted as dorsal ruffles. Therefore, the assay was repeated and the abnormal, collapsed structures generated by both +/+ RV-N-WASP and N-WASP−/− FLCs were not included (B). The graph for the +/+ RV-N-WASP cells was largely unaffected, with >70% of cells generating large, ring dorsal ruffle structures at 2, 5, and 7 min. The N-WASP−/− response was greatly reduced, with <20% of cells generating dorsal ruffles at all time points. This demonstrates that a large proportion of N-WASP−/− FLCs generate small, abnormal dorsal ruffles. Movies of PDGF stimulated +/+ RV-N-WASP and N-WASP−/− FLCs are included as Supplemental Movies 4 and 5. The +/+ RV-N-WASP cells generated large, stable dorsal ruffles that expand and contract steadily over time, similar to the wild-type and Scar1 null MEFs in . However, the N-WASP−/− cells displayed dramatic and persistent distortions at the dorsal surface, but these were unable to form into larger dorsal ruffle structures. In conclusion, N-WASP is required for the formation of large and stable dorsal ruffle waves as seen in primary and immortalized MEFs and FLCs.
Figure 8. Analysis of dorsal ruffle formation in N-WASP FLCs. (Aand 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 (more ...)