Immunofluorescence localization of actin regulatory proteins to lamellae of S2 cells. S2 cells were plated on con A for 1 h and then fixed and double stained for actin (red) and Arp3 (a, green), SCAR (b, green), cofilin/twinstar (c, green), profilin (d, green), enabled (e, green), or capping protein (f, green). Bar, 5 μm.

Control of SCAR degradation and activation. Inhibition of SCAR-associated proteins kette, Sra-1, or Abi by RNAi causes degradation of SCAR itself. S2 cells were treated with dsRNA corresponding to the coding sequence for Sra-1 (a) for 7 d and then plated on con A and stained with phalloidin to visualize actin (red) and DAPI to view DNA (blue). The morphology of these cells closely resembles the defects in lamellae formation produced by Arp2/3 and SCAR RNAi (Fig. 3, b and c). Similar results were observed with RNAi against kette and Abi (not depicted). Bar, 5 μm. (b) Quantitative immunoblotting of cells treated with dsRNA versus Abi, kette, SCAR, and Sra-1 with antibodies against SCAR. Depletion of these proteins by RNAi decreases the amount of SCAR present in S2 cells. Equal protein loading was verified by Bradford assay (not depicted). (c and d) Cells treated with dsRNAi to simultaneously inhibit Rac1, Rac2, Mtl, and Nck show a variety of lamella defects. Among these are a malformed, serrate cell margin (c) and the stellate morphology similar to SCAR RNAi (Fig. 3, b and c). (e) Graph showing the quantitation of morphological defects caused by inhibition of Nck, Rac1/2, and Mtl. Bars, 5 μm.

Drosophila S2 cells attach, spread, and form lamellae when plated on con A. S2 cells expressing EGFP–actin were plated on polylysine (a and b) or con A (c and d) and examined by phase contrast (a and c) or fluorescence microscopy (b and d). Cells on polylysine retain a spherical morphology but form actin-containing membrane ruffles along their surface. When plated on con A, the majority of S2 cells (>90%) spread to form a radially symmetrical actin-based lamellae (c and d). Bar, 5 μm. (e) A single frame from a time-lapse movie of an S2 cell expressing GFP–actin and plated on con A. The yellow line represents the region of the movie used to generate the kymograph shown in f. Bar, 1 μm. (f) This kymograph shows the behavior of actin over time in a lamella. The shark fin shape is indicative of cycles of extension and retraction at the cell margin, while the diagonal lines visualize the retrograde flow of actin at the cell periphery. Bar, 30 s.

RNAi-mediated inhibition of actin regulatory proteins disrupts normal cellular morphology in S2 cells on con A. Untreated cells are shown in Fig. 1 (c and d). Cells were treated with dsRNA against the p20 subunit of the Arp2/3 complex (a), profilin/chickadee (b), cofilin/twinstar (c), slingshot (d), capping protein β (e), Cdc42 (f), Rho1 (g), and myosin II/zipper (h) for 7 d and then plated on con A and stained with rhodamine-phalloidin (red) and DAPI (blue) to visualize filamentous actin and DNA, respectively. (i) Immunoblots demonstrating the effectiveness of RNAi on the levels of 13 different proteins: cofilin/twinstar, capping protein β (CPB), SCAR, Rho1, diaphanous (Dia), enabled (Ena), myosin VI (MVI), Nck/dreadlocks (Dock), Pod1, fascin/singed, lethal giant larvae (LGL), and Trio. Exactly 10 μg of total cellular protein was loaded for each lane. Bars, 5 μm.

Model for the signaling pathway leading to SCAR activation during S2 cell lamella formation. The con A–coated coverslip activates both Rac proteins and Nck by initially cross-linking an unidentified cell surface receptor(s). The Rac proteins and Nck signal through parallel pathways to cause dissociation of trans-inhibited SCAR bound by a complex of kette, Sra-1, and Abi. After dissociation, SCAR is able to promote actin nucleation by Arp2/3 at the cell membrane. SCAR may then be inactivated either by reassociation with its inhibitory complex or by degradation.