Fig 1: 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 2: 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.
Fig 3: 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 4: a-La treatment suppresses ectopic osteoclast formation in fibrous dysplasia explants.a Illustration of ex vivo bone marrow culture system based on a tetracycline-inducible model of FD. b TRAP staining of bone marrow explants from a homozygous GasR201C mouse (FD) and a wild-type littermate (WT). Explants were either cultured with M-CSF alone, or M-CSF and Doxy. c ELISA quantification of mRANKL produced in FD vs WT cultures following Doxy treatment. (WT n = 2; FD n = 4) (P = 0.0005). d Representative images of FD explants activated by Doxy treated with 6 µg/ml isotype control or a-La mAb. e Quantification of the number of fusions producing osteoclast syncytia with =3 nuclei. (n = 4) (P = 0.005) f Quantification of the number of osteoclast syncytia with =3 nuclei from d. (n = 4) (P = 0.02) In b and d, arrowheads denote multinucleated osteoclasts and arrows denote fibrous cell masses developed after Doxy addition and characteristic for FD. Statistical significance was assessed via one-tailed unpaired t-test in c or paired, t-tests in e and f. Data are presented as mean values + /- SEM. g An illustration of changes in the steady-state level and cellular localization of La protein in the process of osteoclast formation. La (green) carries out its canonical, ancient function in the nuclei of all eukaryotic cells as an essential RNA-binding protein. We propose that La has an additional, specialized function in the formation of multinucleated osteoclasts. First, La dissipates as circulating monocytes become osteoclast precursor cells. When osteoclast commitment is initiated by RANKL, La returns but is quickly cleaved by proteases and shuttled to the surface of osteoclasts. At the surface of fusing osteoclasts, La plays a novel role as a membrane fusion manager. When osteoclasts arrive at the “right size” for their biological function, surface La dissipates and is replaced by canonical, non-cleaved La that returns to the nuclei of mature osteoclasts. Source data are provided as a Source Data file.
Fig 5: La associates with the surface of osteoclasts by direct interactions with Anx A5.a Immunoprecipitation of osteoclast lysates 3 days post-RANKL addition. La supermolecular complexes were captured on immunomagnetic beads via a-La mAb or isotype control and complexes were blotted with rabbit antibodies raised towards the targets of interest (a-La rAb for La). Lanes from the same blot are presented at the same intensity. Lanes of interest were cropped and placed beside one another, divided by a dashed line. (b) Representative Western blot of magnetic, streptavidin pull-down of Biotin-Anx A5. Lane 2 = La+Anx A5 input before pull-down and Lane 1 = La alone and Lane 3 = La+Anx A5 after pull-down. c A cartoon illustration of our approach to identify membrane affinity by comparing protein contents in the Bottom fraction containing, along with soluble proteins, liposome-bound proteins and in the Top fraction containing soluble proteins and depleted of liposome-bound proteins. (d) Quantification of the enrichment of recombinant La and Anx A5 in the bottom fraction containing pelleted liposomes (n = 2). (e) Quantification of surface fluorescence intensity of Anx A5 or La following either non-targeted or Anx A5-targeted siRNA in human osteoclasts (n = 4) (P = 0.009 and 0.004, respectively). (f) Quantification of fusion events from e (n = 3) (P = 0.007). (g) Representative immunofluorescence images of Anx A5 or La (a-La mAb) surface staining in non-permeabilized, human osteoclasts 3 days after RANKL addition before or after EGTA incubation. (h) Quantification of surface fluorescence intensity from g. (Anx A5 and RANK n = 2; La n = 3) (P = 0.02, 0.04 and 0.3, respectively) (i) Quantification of surface fluorescence intensity of Anx A5, La or RANK treated or not treated with 60 µM A01 (n = 3) (P = 0.006, 0.01 and 0.35, respectively). In e, h and I, ~100 cells were assessed per each condition in each independent experiment. (d, e, f, h, i). Data are presented as mean values + /- SEM. Statistical significance was assessed via one-tailed paired t-test. * = P < 0.05; ** = P < 0.001. Source data are provided as a Source Data file.
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