Fig 2: Osteoclastogenic differentiation is accompanied by drastic changes in the steady-state levels and localization of La molecular species.a Representative images of stages of osteoclastogenic derivation of human monocytes after M-CSF (6 days, referred to as “M-CSF”) and after M-CSF (6 days) followed by M-CSF + RANKL (5 days, “RANKL”), respectively. (Magenta = Phalloidin-Alexa488, Cyan = Hoechst). b Quantification of the number of fusion events normalized to the total number of nuclei observed over time following RANKL addition. (n = 3) Each point represents an average of >7500 nuclei scored. c Representative Bis-Tris PAGE separation and silver staining of whole protein lysates from M-CSF derived osteoclast precursors and at 3 days post RANKL application. Lysates were ran until the 50 KDa marker nearly ran off the 4–12% polyacrylamide gel to achieve maximal separation of proteins at this molecular weight, leading to the band of interest appearing misleadingly heavy. mass spectrometry. d Representative Tris-Glycine Western blot with a-La mAb evaluating La expression in whole protein lysates from primary human monocytes and the osteoclastogenic stages depicted in a. e Representative Tris-Glycine Western blot with a-La mAb evaluating the time course of La expression following RANKL addition. (a-GAPDH loading control). f Representative immunofluorescence images of La in M-CSF derived osteoclast precursors and at 3 days post RANKL application (a-La mAb). g Representative immunofluorescence images of La in forming osteoclasts 2–5 days post RANKL application (a-La mAb). Cells were stained for La at the described timepoints with membrane permeabilization. Source data are provided as a Source Data file.

Fig 3: Osteoclast formation depends on truncated La, but the function of the La domain is dispensable.a Representative immunofluorescence images of La in RAW 264.7 prior to mRANKL addition (Cntl.), 3 days post mRANKL addition and 5 days post mRANKL addition when we routinely observe massive, multinucleated osteoclasts like the one imaged here. (a-La mAb). b Representative tris-glycine Western blot of whole cell lysates taken from murine, RAW 264.7 treated as in a (a-La rAb). mRANKL-treated cells were enriched into mononucleated (Mono.) or multinucleated (Multi.) populations as described in the Methods. (Cyclophilin B (a-Cyclo B) loading control). c qPCR evaluation of SSB in human osteoclast precursors treated with siRNA at day 1 post-RANKL addition. (n = 4) (P = 0.0043). d Representative, phase contrast images of non-targeted and SSB-targeted human osteoclasts, as in c, stained for TRAP. e Quantification of the number of fusion events in formation of syncytia with 3+ nuclei in the experiments like the one in d. Fusion was scored at day 3. (n = 3) (P = 0.0306). f Topological illustrations of LMW La 1-375, “uncleavable” La D371A,D374A and “La*” (=La 1-375 Q20A_Y24A_D33I). g Quantification of the number of fusion events in syncytia with 3+ nuclei in RAW 264.7 cells transfected with empty, La 1-375 or La 1-375 Q20A_Y24A_D33I expression plasmids. (n = 3) (P = 0.0213 and 0.0173, respectively). h Representative fluorescence images of human monocyte-derived osteoclasts transfected with empty, La 1-375 or La D371A,D374A expression plasmids. (Magenta = Phalloidin-Alexa488, Grey = Hoechst). i Quantification of the number of fusion events in syncytia with 3+ nuclei in h. (n = 4) (P = 0.0205 and 0.325, respectively). c, e, g, i Statistical significance was evaluated via one-tailed paired t-tests. * = P < 0.05. ** = P < 0.001. Data are presented as mean values + /- SEM. Source data are provided as a Source Data file.

Fig 4: Recombinant La promotes osteoclast fusion.(a) Representative fluorescence images of human osteoclasts 3 days post RANKL addition without or with the overnight (end of day 2 post RANKL) addition of recombinant heat-inactivated La 1-408, La 1-408, La 1-375 or La 1-375 Q20A/Y24A/D33I. Recombinant proteins were added at ~40 nM at the end day 2 post-RANKL addition, and cells were fixed the next morning. (Magenta = Phalloidin-Alexa488, Grey = Hoechst) (b) Quantification of a. (inactivated n = 2; La 1-408 n = 4, others n = 3,) (P = 0.1232, 0.0015, 0.0035 and 0.0491, respectively) (c) Quantification of the fraction of nuclei in fused cells that were present in syncytia of various sizes from a. (n = 3). (d) Quantification of the number of fusion events with or without the addition of La 1-187 or La 188-375. Recombinant proteins were added at ~40 nM at the end day 2 post-RANKL addition, and cells were fixed the next morning. (n = 4) (P = 0.36 and 0.0002) (e) The quantification of synchronized fusion events (as illustrated in Fig. 3d) without (wash) and with addition of recombinant La species. “LPC” – indicates that the hemifusion inhibitor was left until fixation. (LPC and La 1-375 n = 3; Wash and La 1-408 n = 4) (P = 0.001 and 0.03, respectively.) (b–e) Statistical significance was evaluated via one-tailed paired t-test. In (b, d, e) the data were normalized to those in control (no protein added in b, d, and wash with no proteins added e). Data are presented as mean values + /- SEM. Source data are provided as a Source Data file.

Fig 5: La associates with membranes, traffics to the surface and controls osteoclast membrane fusion.a Westerns of cytosolic vs membrane associated protein fractions from human osteoclasts. b Representative immunofluorescence images comparing surface staining with a-Fish/TKS5 antibody or a-La mAb in human osteoclasts under non-permeabilized conditions (top) and DIC (bottom). c Representative immunofluorescence images comparing surface staining of isotype control or a-La mAb in RAW 264.7 derived osteoclasts under non-permeabilized conditions. (a-La mAb). d Representative immunofluorescence images of cell surface La in forming human osteoclasts 2–5 days post RANKL application. Cells were stained with a-La mAb at the described timepoints without membrane permeabilization. e Cartoons illustrating the stepwise process of the formation of multinucleated osteoclasts (top), and our approach for isolating membrane fusion stage from the preceding stages of osteoclast differentiation (bottom). Application of the hemifusion inhibitor LPC following 48 h of RANKL elicited osteoclastogenesis allows pre-fusion differentiation stages but blocks hemifusion, synchronizing cells. Removing LPC allows us to specifically probe membrane fusion between osteoclasts. f Quantification of human osteoclast fusion decoupled from pre-fusion stages and synchronized as depicted in e with fusion in the presence of 5 µg/ml a-La mAb and with no antibodies added (Wash) normalized to 5 µg/ml a-RANK control. (wash n = 2; others n = 4) (P = 0.0086 and 0.1330, respectively). “LPC” – fusion observed without removal of LPC. Statistical significance was assessed via one-tailed paired t-test. * = P < 0.05. ** = P < 0.001. Data are presented as mean values + /- SEM. Source data are provided as a Source Data file.