We previously reported the transient appearance of an actin superstructure, called the zipper-like structure, during the primary fusion (fusion of mononuclear precursors) and the secondary fusion (fusion of multinucleated cells) of osteoclasts. plasma buy CPI-613 membranes. strong class=”kwd-title” Keywords: actin, cell-cell conversation, filopodia, fusion, osteoclast, podosome, superstructure Osteoclasts are multinucleated cells that resorb bone, and alterations in this activity have been associated with bone diseases, including osteoporosis. The cytokine RANKL (encoded by Tnfsf11) converts mononuclear cells of monocyte/macrophage lineages into fusion-competent mononuclear cells, and cell fusion of the qualified cells results in a multinucleated osteoclast.1,2 The degree of multinucleation correlates with the bone-resorbing activity of osteoclasts. Although it is known that DC-STAMP, a putative seven-transmembrane buy CPI-613 protein, is essential for the fusion,3,4 little is known about how DC-STAMP participates in the fusion of osteoclasts. By monitoring the rearrangement of the actin cytoskeleton during osteoclastogenesis, we previously found the transient expression of an actin-rich superstructure during cell fusion.5 We called it the zipper-like structure, because it morphologically resembled the adhesion zipper found in keratinocytes. To create a stable epithelial sheet, epithelia link to their neighboring cells by forming adherence junctions and tight junctions. In the initial stage of adherence junction formation, epithelia reorganize the assembly of actin buy CPI-613 filaments at the site of cell contact:6,7 this reorganization produces a prominent actin superstructure, called the adhesion zipper, in the filopodial processes of keratinocytes.8 During the maturation of cell contact, the adhesion zipper seals the gap between the two cells and disappears in the established adherence junction. Because such actin-based superstructure is also observed in fibroblasts, the adhesion zipper might represent the conserved business buy CPI-613 of actin during homotypic cell adhesion. A podosome is usually a small spot-like actin-based superstructure mainly found in cells of monocyte/macrophage lineages and v-Src transformed cells.9,10 The podosome is composed of a dense actin core containing Arp2/3, cortactin, and gelsolin surrounded by a loose actin cloud containing integrins, vinculin, and paxillin. Proteomic analyses estimate the number of components of the integrin adhesome, a relative of the podosome, to be approximately 100 ~200.11,12 Such component diversity must guarantee the dynamics of the podosome. In osteoclasts, podosomes reorganize into a large ring-like superstructure called the podosome belt on a glass. Osteoclasts in bone form a sealing zone, which is usually roughly equivalent to the podosome belt. The sealing zone is usually a cell-matrix buy CPI-613 adhesion machinery that creates an isolated space between the cell and the matrix for bone resorption.10 Similar to the podosome, the podosome belt is composed of two functionally distinct domains: the podosome core and integrin-containing adhesion domain.13 The rearrangement of podosome-derived superstructures in osteoclasts depends on external cues, and the differentiation signal by RANKL spontaneously induces the transformation of podosomes into the podosome belt.14 The physical and chemical properties of the matrix on which the osteoclasts reside also change the dynamics of the podosome belt.15-17 Because the podosome itself exhibits the dynamic fission and fusion, 18 the podosome belt probably inherits the dynamics from the podosome. In this study, we address the actin-based superstructures that appear at the contact site of two osteoclasts during the secondary fusion event. The fusion of multinucleated osteoclasts can be monitored by following the dynamics of the podosome belt. RAW 264.7 cells were transfected with EGFP-actin, and osteoclastogenesis was induced by adding RANKL, as described previously.5 Thus, the transfected EGFP-actin labeled the podosomes and the podosome Tlr4 belt in the terminally differentiated osteoclast. Physique?1 shows the fusion between EGFP-positive and EGFP-negative osteoclasts. Time-lapse DIC imaging indicated that the two osteoclasts came into contact at time 12 min. The osteoclasts increased the degree of the cell contact from 22 min to 88 min, probably forming the zipper-like structure at the site of cell contact. At 90 min, a stalk-like link appeared between the.