Cytoskeletal organization of normal and WASp-/- osteoclasts on glass coverslips. Bone marrow osteoclasts were derived on glass coverslips. At day 7 after plating, cell cultures were fixed with 3% paraformaldehyde, permeabilized with 0.05% Triton X-100, and stained with a mouse anti-human antibody against vinculin (A, D, G, J) and Alexa 568-conjugated phalloidin, followed by an incubation with an Alexa 488 goat anti-mouse antibody (B, E, H, K). Merged images are shown in panels C, F, I, and L. Normal osteoclasts assemble podosomes in clusters behind leading edges (A-C) or inserted in actin rings (D-F) with the characteristic organization of these adhesion structures: core of actin filaments surrounded by a rim of vinculin (inserts in C, F). WASp-null osteoclasts were defective in the formation of podosomes (G-I), instead assembling actin plaques colocalizing with vinculin (insert in I) or actin rings with few podosome-like structures inserted (J-L), which were depleted of vinculin (insert in L). The micrographs are representative of the cytoskeletal organization of osteoclasts detected in 3 independent experiments. Bar, 20 μm.