Reconstitution of podosomes in WASp-null osteoclasts plated on glass coverslips by expression of eGFP WASp. WASp-null cells were transduced with eGFP (A-F) or eGFP-WASp (G-L). Distribution of eGFP is shown in panels A and D, and distribution of eGFP-WASp in panels G and J. Actin organization was detected by immunostaining with Alexa 568-conjugated phalloidin (B, E, H, K). Merged images are shown in panels C, F, I, and L. Cells transduced with eGFP were devoid of podosomes (A-C) or assembled abnormal actin rings with few podosome-like structures inserted (D-F). eGFP did not colocalize specifically with any cytoskeletal structure (C, F). Transduction of eGFP WASp resulted in reconstitution of podosomes and formation of clusters similar to normal cells in both migrating cells (G-I) and cells with actin rings (J-L). eGFP WASp colocalized specifically with the actin core of podosomes (insert in I, L). The micrographs are representative of the cytoskeletal organization of osteoclasts detected in 3 independent experiments. Bar, 20 μm.