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First published online 3 February 2004
doi: 10.1242/jcs.00921

Journal of Cell Science 117, 837-848 (2004)
Published by The Company of Biologists 2004
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Cascade pathway of filopodia formation downstream of SCAR

Assel Biyasheva1,*, Tatyana Svitkina1, Patricia Kunda2, Buzz Baum2 and Gary Borisy1

1 Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
2 Ludwig Institute for Cancer Research, University College London Branch, London W1W 7BS, UK



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  		Fig. 1. Characterization of S2R+ cells. (A) Morphology of cell population (by phase contrast microscopy). Many cells display broad circumferential lamellipodia. (B) Protrusive activity (time-lapse sequence). Lamellipodium protrudes with regular and smooth outline. Dotted line shows the initial position of the cell edge. Time in min:sec. (C) Actin cytoskeleton (phalloidin staining). Circumferential lamellipodium is rich in actin; filopodia are not seen. (D) Structural organization of lamellipodia (EM). Actin filaments form dense, highly branched dendritic network. Circled region is enlarged in the inset to show actin branches. Bars, 10 µm (A), 5 µm (B,C) and 0.5 µm (D).

 


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  		Fig. 2. Characterization of BG2 cells. (A) Morphology of cell population (phase contrast). Cells display polarized lamella. (B) Protrusive activity (time-lapse sequence). Leading edge moves forward by combination of filopodial (arrowheads) and lamellipodial protrusion. Dotted line shows the initial position of the cell edge. Time in min:sec. (C) Actin cytoskeleton (phalloidin staining). Leading edge contains diffuse network in lamellipodia and distinct bundles in filopodia and microspikes; stress fibers are present in the cell interior. (D) Structural organization of lamellipodia (EM). Actin filaments form dendritic network in lamellipodia and bundles of long filaments in filopodia. Circled region is enlarged in the inset to show actin bundle. Bars, 10 µm (A), 5 µm (B,C) and 1 µm (D).

 


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  		Fig. 3. SCAR knockdown in S2R+ cells. (A) Morphological phenotype developed after SCAR-specific RNAi. Cell spreading is compromised; numerous long processes appear along cell edges. (B) Immunoblotting. Extracts of S2R+ cells subjected to SCAR RNAi for 0-4 days (lanes 0-4, respectively) were probed with antibodies to SCAR or WASp. Lane 4+5 WO shows restoration of SCAR level after washout of dsRNA on day 4 and culturing of cells for an additional 5 days under regular conditions. Equal load of protein was ensured by tubulin probing (not shown). Positions of markers are indicated. Reduction of SCAR is >95% by day 4 of treatment. (C) RT-PCR after Laser Capture Microdissection (LCM). 100 control cells (lanes labeled `–') and 100 cells subjected to SCAR RNAi for 4 days (lanes labeled `+') were captured by LCM (see text for details) and analyzed by RT-PCR using SCAR or rp49 specific primers. Reduction of SCAR mRNA is ~95% by densitometry; rp49 mRNA serves as an internal control, and its levels do not change. The positions of DNA markers and number of PCR cycles are indicated. (D) Immunostaining. Control cells (top row) and cells treated with dsSCAR for 4 days (bottom row) were stained with phalloidin to reveal actin (left panels) and with SCAR antibody (middle). Merged images (right) show that in control cells SCAR is enriched at the leading edge of lamellipodium (arrow), and in dsSCAR-treated cells, SCAR is not detected in peripheral protrusions. Bars, 10 µm (A), 2 µm (D).

 


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  		Fig. 4. SCAR RNAi in S2R+ cells inhibits lamellipodial protrusion. (A) Morphometry. Projected area (left) and P/A1/2 cell shape index (right) of control (gray bars, n=24) and dsSCAR-treated for 4 days (black bars, n=26) S2R+ cells. Higher values of the index correspond to more irregular cell shape (see text for details). (B,C). Kinetics of lamellipodial protrusion on day 3 (B) and day 4 (C) of SCAR RNAi. Time series of enlarged boxed regions are shown on the right. Time in sec. (B) Cell is able to form lamellipodia, but protrusion is not coordinated along the edge. Formation of individual protuberances is indicated by arrowheads. (C) Cell is not able to form lamellipodia; it forms long narrow curved processes, which express dynamics at their tips and along their length (arrowheads). (D,E). Structure of actin cytoskeleton on day 3 (D) and day 4 (E) of SCAR RNAi (EM). (D) Lamellipodia-like protrusions contain very sparse dendritic actin filament network. (E) Long processes contain branched filament network, but not bundles of long filaments. Bars, 10 µm (B,C) and 0.2 µm (D,E).

 


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  		Fig. 5. Lamellipodial and filopodial markers in peripheral processes after SCAR-depletion. Control (A,C) and SCAR-depleted (B,D) S2R+ cells were stained with phalloidin (left panels) and Arp3 (A,B) or Ena (C,D) antibody (middle panels). Merged images are shown in right panels with phalloidin staining in green, and antibody staining in red. Peripheral processes of SCAR-depleted S2R+ cells are weakly stained for Arp3 (B), but no Ena signal is detected (D), in contrast to normal filopodia of BG2 cells which have strong Ena staining at their tips (not shown). Bars, 2 µm.

 


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  		Fig. 6. SCAR RNAi in BG2 cells inhibits lamellipodia and filopodia. (A) Immunoblotting. Extracts of BG2 cells subjected to SCAR RNAi for 4 days were probed with antibodies to SCAR and WASp. Reduction of SCAR is 90-95%. WASp is not changed. (B) Morphology and actin distribution. Overall cell shape shows decreased spreading and formation of long narrow processes. Phalloidin staining reveals decreased amount of actin, especially at the periphery. (C) Morphometry. Projected area (left) and P/A1/2 cell shape index (right) of control (gray bars, n=14) and BG2 cells treated with dsSCAR for 4 days (black bars, n=18). (D) Cytoskeleton organization of SCAR-depleted BG2 cells (EM). Left: overview; neither normal lamellipodia nor filopodia are seen. Right: enlarged is boxed region from left panel; cell processes contain central actin filament bundles (arrowheads) and distal patches of the dendritic network (arrows). (E) Filopodia formation during spreading of control (upper row) or SCAR-depleted for 4 days (lower row) BG2 cells. Time-lapse sequences were acquired 2 hours after cell re-plating. Newly formed filopodia are indicated by arrowheads. Time in seconds. (F) Rate of filopodia initiation. Number of de novo formed filopodia per 2 minutes interval in control cells (gray bar, n=14) or cells treated with dsRNA (black bars, n=15) specific for SCAR (dsS) or WASp (dsW). SCAR-depleted BG2 cells have a significantly reduced ability to form filopodia. Bars, 10 µm (B), 1 µm (D) and 5 µm (E).

 


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  		Fig. 7. WASp knockdown in S2R+ and BG2 cells. (A) Immunoblots of WASp in extracts prepared from S2R+ (left) and BG2 (right) cells subjected to WASp RNAi for 4 days. WASp is reduced by 70-75% in both cell lines after dsWASp-treatment. Tubulin (Tb) is shown as a loading control. Morphology of cell population and actin distribution in WASp-depleted S2R+ (B) and BG2 cells (C) (phalloidin staining). Both cell types do not display changes in morphology compared with respective controls. (D) Morphometry of S2R+ (left) and BG2 (right) cells. WASp inhibition causes slight decrease in cell area, but does not significantly change cell shape. Gray bars represent controls, black bars show WASp-depleted cells; n=18 (S2R+ control), n=22 (S2R+ dsWASp), n=22 (BG2 control), n=21 (BG2 dsWASp). (E,F) Organization of actin filaments in protrusions of WASp-depleted S2R+ (E) and BG2 (F) cells (EM). No significant alterations in architecture of lamellipodia in both cells or filopodia in BG2 cells was observed. Magnification is the same in (B-C) and (E-F). Bars, 10 µm (B) and 1 µm (E).

 


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  		Fig. 8. Models of regulation of lamellipodia and filopodia. (A) Parallel pathways model. Lamellipodia and filopodia are regulated by two parallel pathways: from GTPase Rac through Scar/WAVEs to lamellipodia, and from Cdc42 through WASPs to filopodia. (B) Cascade pathways model. Small GTPases signal to Scar/WAVEs and WASPs to initiate lamellipodia, which are subsequently transformed into filopodia by additional signals. (C) Refined cascade model for Drosophila. Our data emphasize SCAR as the main regulator of lamellipodia formation, whereas WASp in Drosophila cells is dispensable for both lamellipodia and filopodia and may play a role in other actin-dependent activities. Additional signals driving reorganization of lamellipodia in filopodia may originate from Cdc42 and act through effectors other than WASP, e.g. IRSp53 (Krugmann et al., 2001Go) and Drf3 (Peng et al., 2003Go).

 

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© The Company of Biologists Ltd 2004