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Fig. 5. Arp2/3-dependent actin polymerization is necessary for podosome formation but not for actin remodeling. (A) Formation of podosomes in PDBu-treated GFP-p20-expressing cells. Note the basal Arp2/3 clustering at time 0 and the increase in size of clusters in developing podosomes (arrows). (B) SCAR-WA and GFP-p20-co-expressing cell before and after PDBu treatment. The phase contrast image reveals the absence of lamellipodia despite the formation of extensive filopodia (black arrow). Note the lack of Arp2/3 clustering (arrowheads) and the absence of podosomes upon PDBu treatment. (C) Immunofluorescence images of cells treated with PDBu for 40 minutes. Ectopically expressed myc-tagged Scar domains are visualized by anti-myc antibody, and the actin cytoskeleton by phalloidin staining. The SCAR-WA transfected cell (1) displays no podosomes (arrowhead), while a non-transfected cell (2), and a cell transfected with the SCAR-W domain (3) develops numerous podosomes (arrows). (D) SCAR-WA and GFP-ß-actin co-transfected cells before (0') and after 45 minutes of PDBu treatment. Actin stress fibers undergo substantial remodeling and disassembly in response to PDBu (arrowhead) despite the lack of podosomes. (E) SCAR-WA and GFP-zyxin co-transfected cells before (0') and after 45 minutes of PDBu treatment. Focal adhesions are partially disassembled (arrowheads) and zyxin redistributes into the cytoplasmic pool. Fl, epifluorescence; Ph, phase contrast.

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