Dynamin-dependent actin-rich protrusions are associated with clathrin-coated vesicles at sites of OCP fusion. (A) Electron microscopy images of representative OCPs showing F-actin–rich membrane protrusions extending from one cell into the adjacent cell, similar to confocal images in Fig. 5 D of CTL cells. Bars, 200 nm. The length of protrusive structures of CTL osteoclasts (OCLs) is about five times longer than those of DKO cells (left graph, P < 0.01). The number of protrusive structures was 80% less in DKO cells (P < 0.01). Data were obtained from 38 CTL and 86 DKO cell images in three independent experiments. Error bars indicate SEM. (B) Colocalization of dynamin and DC-STAMP on an OCP actin protrusion. Bar, 5 µm. (C) The localization of CHC and DC-STAMP around and at tips of the protrusions of osteoclasts in CTL culture; note the presence of clathrin-coated vesicles near the tip of the protrusion (arrows). Bar, 0.3 µm. (D) EM images showing the frequent association of F-actin protrusions and clathrin-coated pits (arrows) in the membrane of the invaded cells. Bars, 200 nm. (E and F) shRNA-mediated knockdown of CHC was analyzed by Western blotting. TRAP-positive multinucleated osteoclasts were quantified. Data are mean ± SE (error bars). *, P < 0.01. Bar, 200 µm.