the reagents used were obtained from the following sources : -mem , ( wako , # 13515175 ) ; fetal bovine serum ( fbs ) ( sigma ) ; rhodamine - phalloidin ( molecular probes , # r415 ) ; and recombinant mouse srankl ( peprotech , # 31511 ) .
raw 264.7 cells were seeded on a glass coverslip ( fisher scientific , # 1254582 ) placed in a 24-well culture dish at 1 ~2 x 10 cells in 0.5 ml -mem , 10% fbs , antibiotics , 0.1 mm non - essential amino acids , and 100 ng / ml srankl and cultured to induce osteoclastogenesis . under these conditions , raw 264.7 cells formed matured osteoclasts with the podosome belts by day 4 . for confocal microscopy , the cells were fixed with 4% paraformaldehyde in pbs for 30 min , permeabilized with 0.5% np-40 in pbs for 15 min , and blocked with 1% bsa in pbs for 1 h. the fixed cells were then incubated with rhodamine - phalloidin for 30 min .
raw 264.7 cells were seeded on a 35 mm glass bottom dish with grids ( matsunami , # d111505 ) at 1.5 x 10 cells in 0.5 ml -mem , 10% fbs , antibiotics and 100 ng / ml rankl and cultured for 3 d. the cells were fixed with 2% glutaraldehyde , 2% paraformaldehyde in 0.1 m pbs ( ph 7.4 ) at 37c for 15 min . the specimen was further fixed with 2% glutaraldehyde in 0.1 m pbs ( ph 7.4 ) at 4c .
the cells were postfixed with oso4 , dehydrated and embedded in quetol-812 ( nisshin em , # 340 ) .
the glass was dissolved by incubating with hydrofluoric acid at room temperature for 20 min .
raw264.7 cells were plated on a 35 mm glass bottomed culture dish ( mattek , # p35g-0 - 14-c ) .
the cells were then transfected with egfp - actin ( clontech , # 632453 ) using the fugene hd transfection reagent ( roche ) according to the protocol provided and allowed to differentiate into osteoclasts .
the culture medium was supplemented with 100 ng / ml rankl just before each recording .
the reagents used were obtained from the following sources : -mem , ( wako , # 13515175 ) ; fetal bovine serum ( fbs ) ( sigma ) ; rhodamine - phalloidin ( molecular probes , # r415 ) ; and recombinant mouse srankl ( peprotech , # 31511 ) .
raw 264.7 cells were seeded on a glass coverslip ( fisher scientific , # 1254582 ) placed in a 24-well culture dish at 1 ~2 x 10 cells in 0.5 ml -mem , 10% fbs , antibiotics , 0.1 mm non - essential amino acids , and 100 ng / ml srankl and cultured to induce osteoclastogenesis . under these conditions , raw 264.7 cells formed matured osteoclasts with the podosome belts by day 4 . for confocal microscopy , the cells were fixed with 4% paraformaldehyde in pbs for 30 min , permeabilized with 0.5% np-40 in pbs for 15 min , and blocked with 1% bsa in pbs for 1 h. the fixed cells were then incubated with rhodamine - phalloidin for 30 min .
raw 264.7 cells were seeded on a 35 mm glass bottom dish with grids ( matsunami , # d111505 ) at 1.5 x 10 cells in 0.5 ml -mem , 10% fbs , antibiotics and 100 ng / ml rankl and cultured for 3 d. the cells were fixed with 2% glutaraldehyde , 2% paraformaldehyde in 0.1 m pbs ( ph 7.4 ) at 37c for 15 min . the specimen was further fixed with 2% glutaraldehyde in 0.1 m pbs ( ph 7.4 ) at 4c .
the cells were postfixed with oso4 , dehydrated and embedded in quetol-812 ( nisshin em , # 340 ) .
the glass was dissolved by incubating with hydrofluoric acid at room temperature for 20 min .
raw264.7 cells were plated on a 35 mm glass bottomed culture dish ( mattek , # p35g-0 - 14-c ) .
the cells were then transfected with egfp - actin ( clontech , # 632453 ) using the fugene hd transfection reagent ( roche ) according to the protocol provided and allowed to differentiate into osteoclasts .
the culture medium was supplemented with 100 ng / ml rankl just before each recording .
 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 . here , we focus on the actin - based superstructures that link two precursor cells during the secondary fusion event . in one type of secondary fusion , the osteoclasts transformed the podosome belts into the zipper - like structure at the site of cell contact and the apposed plasma membranes in the zipper - like structure attached to each other via a discontinuous interface . in another type of secondary fusion 
 , the osteoclasts used a filopodium - like protrusion that linked the two cells . 
 both types of cell fusion required a lag period between the adhesion of the cells and the fusion of cell bodies . 
 thus , the secondary fusion of osteoclasts uses actin - based superstructures for cell - cell interactions before the definitive fusion of the plasma membranes . 
Materials and Methods
Materials
Osteoclastogenesis
Electronmicroscopy
Live-cell imaging