RANKL-induced osteoclast differentiation reduces MCA to initiate cell–cell fusion. (A) Schematic illustration of the measurement of tether force (F_Tether) using optical tweezers. F_Tether can be calculated using Hooke’s law, $FTether=k×Δx$⁠, where k is the stiffness of the trap and ∆x is the displacement of the bead from the trap center. (B) Scatter plot comparing the tether force of mononuclear cells treated without or with RANKL for 84 h. Mean ± SEM, n = 30 cells for each condition. P value obtained from one-way ANOVA with Tukey’s test. *P < 0.05; ****P < 0.0001. (C) Schematic of the molecules, wild-type ezrin (Ezrin), MA-ezrin (MA), and iMC-linker (iMC), overexpressed in RAW 264.7 cells. Lyn₁₀, myristoylation sequence of Lyn; FERM, FERM domain of ezrin; ABD, the F-actin–binding domain. (D) Confocal images of cell lines in parental, MA-ezrin (MA), and iMC-linker (iMC) alone (upper) or in co-cultured (lower), after 84 h of RANKL treatment, stained with phalloidin (magenta) and DAPI (gray). Multinucleated cells are surrounded by dotted lines. Scale bar: 50 μm. (E) Quantification of fusion index in D. Dotted lines in violin plots show median and quantiles. n = 45 fields of view for each condition. P value obtained from one-way ANOVA with Tukey’s test. ****P < 0.0001.