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In vitro and in vivo detection of tunneling nanotubes in normal and pathological osteoclastogenesis involving osteoclast fusion


Osteoclasts are multinucleated cells formed through specific recognition and fusion of mononuclear osteoclast precursors derived from hematopoietic stem cells. Detailed cellular events concerning cell fusion in osteoclast differentiation remain ambiguous. Tunneling nanotubes (TNTs), actin-based membrane structures, play an important role in intercellular communication between cells. We have previously reported the presence of TNTs in the fusion process of osteoclastogenesis. Here we analyzed morphological details of TNTs using scanning electron microscopy. The osteoclast precursor cell line RAW-D was stimulated to form osteoclast-like cells, and morphological details in the appearance of TNTs were extensively analyzed. Osteoclast-like cells could be classified into three types; early osteoclast precursors, late osteoclast precursors, and multinucleated osteoclast-like cells based on the morphological characteristics. TNTs were frequently observed among these three types of cells. TNTs could be classified into thin, medium, and thick TNTs based on the diameter and length. The shapes of TNTs were dynamically changed from thin to thick. Among them, medium TNTs were often observed between two remote cells, in which side branches attached to the culture substrates and beaded bulge-like structures were often observed. Cell-cell interaction through TNTs contributed to cell migration and rapid transport of information between cells. TNTs were shown to be involved in cell-cell fusion between osteoclast precursors and multinucleated osteoclast-like cells, in which movement of membrane vesicles and nuclei was observed. Formation of TNTs was also confirmed in primary cultures of osteoclasts. Furthermore, we have successfully detected TNTs formed between osteoclasts observed in the bone destruction sites of arthritic rats. Thus, formation of TNTs may be important for the differentiation of osteoclasts both in vitro and in vivo. TNTs could be one target cellular structure for the regulation of osteoclast differentiation and function in bone diseases.

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Fig. 1: Time-course of osteoclast differentiation and morphological classification of cells observed in osteoclast precursor cell line RAW-D.
Fig. 2: Various types of cellular protrusions observed in culture of osteoclastogenesis by SEM analysis.
Fig. 3: Appearance of TNTs in culture cells and classification of TNTs by SEM analysis.
Fig. 4: Detailed morphology of TNTs and various fine side branches associted with TNTs connecting cells.
Fig. 5: Association of TNTs in the process of cell fusion in osteoclastogenesis.
Fig. 6: Immunofluorescence detection of co-distribution of F-actin and Cathepsin K in TNT observed in osteoclastogenesis in RAW-D cell culture.
Fig. 7: TNT-mediated interaction among primary osteoclasts cultured on calcified matrices.
Fig. 8: In vivo detection of TNT-like intercellular bridges among pathological multinucleated osteoclasts in rats with adjuvant-induced arthritis.

Data availability

All data generated and analyzed in this study are available.


  1. 1.

    Zaidi, M. Skeletal remodeling in health and disease. Nat. Med. 13, 791–801 (2007).

    CAS  Article  Google Scholar 

  2. 2.

    Feng, X. & McDonald, J. M. Disorders of bone remodeling. Annu. Rev. Pathol. 6, 121–145 (2011).

    CAS  Article  Google Scholar 

  3. 3.

    Amarasekara, D. S. et al. Regulation of osteoclast differentiation by cytokine networks. Immun. Netw. (2018).

  4. 4.

    Yoshida, H. et al. The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature 345, 442–444 (1990).

    CAS  Article  Google Scholar 

  5. 5.

    Scheven, B. A., Visser, J. W. & Nijweide, P. J. In vitro osteoclast generation from different bone marrow fractions, including a highly enriched hematopoietic stem cell population. Nature 321, 79–81 (1986).

    CAS  Article  Google Scholar 

  6. 6.

    Vignery, A. Macrophage fusion: the making of osteoclasts and giant cells. J. Exp. Med. 202, 337–340 (2005).

    CAS  Article  Google Scholar 

  7. 7.

    Miyamoto, T. Regulators of osteoclast differentiation and cell-cell fusion. Keio J. Med. 60, 101–105 (2011).

    CAS  Article  Google Scholar 

  8. 8.

    Rustom, A., Saffrich, R., Markovic, I., Walther, P. & Gerdes, H. H. Nanotubular highways for intercellular organelle transport. Science 303, 1007–1010 (2004).

    CAS  Article  Google Scholar 

  9. 9.

    Koyanagi, M., Brandes, R. P., Haendeler, J., Zeiher, A. M. & Dimmeler, S. Cell-to-cell connection of endothelial progenitor cells with cardiac myocytes by nanotubes: a novel mechanism for cell fate changes? Circ. Res. 96, 1039–1041 (2005).

    CAS  Article  Google Scholar 

  10. 10.

    Kimura, S., Hase, K. & Ohno, H. Tunneling nanotubes: emerging view of their molecular components and formation mechanisms. Exp. Cell Res. 318, 1699–1706 (2012).

    CAS  Article  Google Scholar 

  11. 11.

    Liu, K. et al. Mesenchymal stem cells rescue injured endothelial cells in an in vitro ischemia-reperfusion model via tunneling nanotube like structure-mediated mitochondrial transfer. Microvasc. Res. 92, 10–18 (2014).

    CAS  Article  Google Scholar 

  12. 12.

    Baler, M. How the internet of cells has biologists buzzing. Nature 549, 322–324 (2017).

    Article  Google Scholar 

  13. 13.

    Winkler, F. & Wick, W. Harmful networks in the brain and beyond. Science 359, 1100–1101 (2018).

    CAS  Article  Google Scholar 

  14. 14.

    Gurke, S. et al. Tunneling nanotube (TNT)-like structures facilitate a constitutive, actomyosin-dependent exchange of endocytic organelles between normal rat kidney cells. Exp. Cell Res. 314, 3669–3683 (2008).

    CAS  Article  Google Scholar 

  15. 15.

    Sun X. et al. Tunneling-nanotube direction determination in neurons and astrocytes. Cell Death Dis. (2012).

  16. 16.

    Yasuda, K. et al. Tunneling nanotubes mediate rescue of prematurely senescent endothelial cells by endothelial progenitors: exchange of lysosomal pool. Aging (Albany NY) 3, 597–608 (2011).

    CAS  Article  Google Scholar 

  17. 17.

    Domhan, S. et al. Intercellular communication by exchange of cytoplasmic material via tunneling nano-tube like structures in primary human renal epithelial cells. PLoS ONE (2011).

  18. 18.

    Korenkova, O., Pepe, A. & Zurzolo, C. Fine intercellular connections in development: TNTs, cytonemes, or intercellular bridges? Cell Stress 4, 30–43 (2020).

    CAS  Article  Google Scholar 

  19. 19.

    Yamashita, Y. M., Inaba, M. & Buszczak, M. Specialized intercellular communications via cytonemes and nanotubes. Annu. Rev. Cell Dev. Biol. 34, 59–84 (2018).

    CAS  Article  Google Scholar 

  20. 20.

    Mittal, R. et al. Cell communication by tunneling nanotubes: implication in disease and therapeutic applications. J. Cell Physiol. 234, 1130–1146 (2019).

    CAS  Article  Google Scholar 

  21. 21.

    Pal, R. R. et al. Pathogenic E. coli extracts nutrients from infected host cells utilizing injectisome components. Cell 177, 683–696 (2019).

    CAS  Article  Google Scholar 

  22. 22.

    Takahashi, A. et al. Tunneling nanotube formation is essential for the regulation of osteoclastogenesis. J. Cell Biochem. 114, 1238–1247 (2013).

    CAS  Article  Google Scholar 

  23. 23.

    Kukita, T. et al. RANKL Induced DC-STAMP is essential for osteoclastogenesis. J. Exp. Med. 200, 941–946 (2004).

    CAS  Article  Google Scholar 

  24. 24.

    Li, R. F., Zhang, W., Man, Q. W., Zhao, Y. F. & Zhao, Y. Tunneling nanotubes mediate intercellular communication between endothelial progenitor cells and osteoclast precursors. J. Mol. Histol. 50, 483–491 (2019).

    CAS  Article  Google Scholar 

  25. 25.

    Pennanen, P. et al. Diversity of actin architecture in human osteoclasts: network of curved and branched actin supporting cell shape and intercellular micrometer-level tubes. Mol. Cell Biochem. 432, 131–139 (2017).

    CAS  Article  Google Scholar 

  26. 26.

    Watanabe, T. et al. Direct stimulation of osteoclastogenesis by MIP-1α: evidence obtained from studies using RAW264 cell clone highly responsive to RANKL. J. Endocrinol. 180, 193–201 (2004).

    CAS  Article  Google Scholar 

  27. 27.

    Kukita, T., Takahashi, A., Zhang, J. Q., Kukita, A. Membrane nanotube formation in osteoclastogenesis. (Pfannkuche K., eds). Methods mol biol, cell fusion: overviews and methods 2nd edn, p. 1313, 193–202 (Springer Science Business Media, 2015).

  28. 28.

    Takahashi, N., Udagawa, N., Tanaka, S., Suda, T. Generating murine osteoclasts from bone marrow. (Helfrich M. H., Ralston S. H., eds). Methods Mol Med, Bone Research Protocols. p. 80, 129–144 (Humana Press Inc, 2003).

  29. 29.

    Kukita, A. et al. The transcription factor FBI-1/OCZF/LRF is expressed in osteoclasts and regulates RANKL-induced osteoclast formation in vitro and in vivo. Arthritis Rheum. 63, 2744–2754 (2011).

    CAS  Article  Google Scholar 

  30. 30.

    Shiratori, T. et al. IL-1β Induces pathologically activated osteoclasts bearing extremely high levels of resorbing activity: A possible pathological subpopulation of osteoclasts accompanied by suppressed expression of Kindlin-3 and Talin-1. J. Immunol. 200, 218–228 (2018).

    CAS  Article  Google Scholar 

  31. 31.

    Li, Y. J. et al. A possible suppressive role of galectin-3 in upregulated osteoclastogenesis accompanying adjuvant induced arthritis in rats. Lab. Investig. 89, 26–37 (2009).

    CAS  Article  Google Scholar 

  32. 32.

    Kuratani, T. et al. Induction of abundant osteoclast-like multinucleated giant cells in adjuvant arthritic rats with accompanying disordered high bone turnover. Histol. Histopathol. 13, 751–759 (1998).

    CAS  PubMed  Google Scholar 

  33. 33.

    Kukita, A. et al. Infection of RANKL-primed RAW-D macrophages with Porphyromonas gingivalis promotes osteoclastogenesis in a TNF-α-independent manner. PLoS ONE (2012).

  34. 34.

    Austefjord, M. W., Gerdes, H. H., Wang, X. Tunneling nanotubes: diversity in morphology and structure. Commun. Integr. Biol. (2014).

  35. 35.

    Mattes, B. & Scholpp, S. Emerging role of contact-mediated cell communication in tissue development and diseases. Histochem. Cell Biol. 150, 431–442 (2018).

    CAS  Article  Google Scholar 

  36. 36.

    Onfelt, B. et al. Structurally distinct membrane nanotubes between human macrophages support long-distance vesicular traffic or surfing of bacteria. J. Immunol. 177, 8476–8483 (2006).

    Article  Google Scholar 

  37. 37.

    Haglund, K., Nezis, I. P. & Stenmark, H. Structure and functions of stable intercellular bridges formed by incomplete cytokinesis during development. Commun. Integr. Biol. 4, 1–9 (2012).

    Article  Google Scholar 

  38. 38.

    Thayanithy, V., Dickson, E. L., Steer, C., Subramanian, S. & Lou, E. Tumor-stromal cross talk: direct cell-to-cell transfer of oncogenic microRNA via tunneling nanotubes. Transl. Res. 164, 359–365 (2014).

    CAS  Article  Google Scholar 

  39. 39.

    Domon, T. et al. Ultrastructural study of cell-cell interaction between osteoclasts and osteoblasts/stroma cells in vitro. Ann. Anat. 184, 221–227 (2002).

    Article  Google Scholar 

  40. 40.

    Resnik, N. et al. Helical organization of microtubules occurs in a minority of tunneling membrane nanotubes in normal and cancer urothelial cells. Sci. Rep. (2018).

  41. 41.

    Sartori-Rupp, A. et al. Correlative cry-electron microscopy reveals the structure of TNTs in neuronal cells. Nat. Commun. (2019).

  42. 42.

    Inaba, M., Buszczak, M. & Yamashita, Y. M. Nanotubes mediate niche-stem cell signaling in the Drosophila testis. Nature 523, 329–332 (2015).

    CAS  Article  Google Scholar 

  43. 43.

    Gong, J., Chen, D., Kashiwaba, M. & Kufe, D. Induction of antitumor activity by immunization with fusions of dendritic and carcinoma cell. Nat. Med. 3, 558–561 (1997).

    CAS  Article  Google Scholar 

  44. 44.

    Vassilopoulos, G., Wang, P. R. & Russell, D. W. Transplanted bone marrow regenerates liver by cell fusion. Nature 422, 901–904 (2003).

    CAS  Article  Google Scholar 

  45. 45.

    Ishii, M., Fujimori, S., Kaneko, T. & Kikuta, J. Dynamic live imaging of bone: opening a new era with ‘bone histodynametry’. J. Bone Miner. Metab. 31, 507–511 (2013).

    Article  Google Scholar 

  46. 46.

    Rehberg, M. et al. Intercellular transport of nanomaterials is mediated by membrane nanotubes in vivo. Small 12, 1882–1890 (2016).

    CAS  Article  Google Scholar 

  47. 47.

    Hase, K. et al. M-Sec Promotes membrane nanotube formation by interacting with Ral and the excyst complex. Nat. Cell Biol. 11, 1427–1432 (2009).

    CAS  Article  Google Scholar 

  48. 48.

    Yagi, M. et al. DC-STAMP is essential for cell-cell fusion in osteoclasts and foreign body giant cells. J. Exp. Med. 202, 345–351 (2005).

    CAS  Article  Google Scholar 

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The authors thank Dr. Mizuho Kido of Saga University, Faculty of Medicine for helpful suggestions.


This study was supported in part by a Grant for Scientific Research from Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT)/Japan Society for the Promotion of Science (JSPS) (16K11447, 18K09506).

Author information




J.Z. performed experiments and analyzed the data by use of SEM; This study was conceived and designed by J.Z. and T.K.; A.T., J.G., Y.K. and X.Z. performed primary bone marrow cells isolation and culture, in vitro experiments, collection and assembly of data; The paper writing and figure design were performed by J.Z. and T.K.; A.K., N.U., H.H. and T.Y. deeply discussed on all data; All authors reviewed and accepted the final version of the paper.

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Correspondence to Toshio Kukita.

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The authors declare no competing interests.

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All animal experiments were performed according to the protocol approved by the Laboratory Animal Care and Use Committee of Kyushu University (No. A19-270-0).

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Zhang, JQ., Takahashi, A., Gu, JY. et al. In vitro and in vivo detection of tunneling nanotubes in normal and pathological osteoclastogenesis involving osteoclast fusion. Lab Invest (2021).

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