Cell Fusion pp 193-202 | Cite as

Membrane Nanotube Formation in Osteoclastogenesis

  • Toshio KukitaEmail author
  • Akira Takahashi
  • Jing-Qi Zhang
  • Akiko KukitaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1313)


Membrane tunneling nanotubes (TNTs) are unique intercellular structures, which enable rapid transport of various materials and rapid communication between cells present in a long distance. During osteoclastogenesis, mononuclear osteoclast precursors form abundant TNTs in prior to cell–cell fusion. Here we introduce a protocol for detecting TNTs during osteoclastogenesis by use of live cell imaging utilizing a confocal laser microscopy. We also demonstrate a standard protocol for observation of TNTs by scanning electron microscope.

Key words

Osteoclast precursor Cell–cell fusion Tunneling membrane nanotube Live cell imaging Scanning electron microscopy DC-STAMP 



We thank to Dr. Nomiyama of Faculty of Medicine, Kumamoto University for helpful discussions. This work was supported by Grant-in-Aid for Scientific Research (Scientific Research B; grant number 21659424) and by Grant-in-Aid for Scientific Research (Challenging Exploratory Research; grant number 21390492).


  1. 1.
    Nakashima T, Hayashi M, Takayanagi H (2012) New insights into osteoclastogenic signaling mechanisms. Trends Endocrinol Metab 23:582–590CrossRefPubMedGoogle Scholar
  2. 2.
    Xing L, Xiu Y, Boyce BF (2012) Osteoclast fusion and regulation by RANKL-dependent and independent factors. World J Orthop 3:212–222CrossRefPubMedCentralPubMedGoogle Scholar
  3. 3.
    Kukita T, Wada N, Kukita A et al (2004) RANKL-induced DC-STAMP is essential for osteoclastogenesis. J Exp Med 200:941–946CrossRefPubMedCentralPubMedGoogle Scholar
  4. 4.
    Yagi M, Miyamoto T, Sawatani Y et al (2005) DC-STAMP is essential for cell-cell fusion in osteoclasts and foreign body giant cells. J Exp Med 202:345–351CrossRefPubMedCentralPubMedGoogle Scholar
  5. 5.
    Yang M, Birnbaum MJ, MacKay CA et al (2008) Osteoclast stimulatory transmembrane protein (OC-STAMP), a novel protein induced by RANKL that promotes osteoclast differentiation. J Cell Physiol 215:497–505CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Miyamoto H, Suzuki T, Miyauchi Y et al (2012) Osteoclast stimulatory transmembrane protein and dendritic cell-specific transmembrane protein cooperatively modulate cell-cell fusion to form osteoclasts and foreign body giant cells. J Bone Miner Res 27:1289–1297CrossRefPubMedGoogle Scholar
  7. 7.
    Davis DM, Sowinski S (2008) Membrane nanotubes: dynamic long-distance connections between animal cells. Nat Rev Mol Cell Biol 9:431–436CrossRefPubMedGoogle Scholar
  8. 8.
    Rustom A, Saffrich R, Markovic I et al (2004) Nanotubular highways for intercellular organelle transport. Science 303:1007–1010CrossRefPubMedGoogle Scholar
  9. 9.
    Sowinski S, Jolly C, Berninghausen O et al (2008) Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 transmission. Nat Cell Biol 10:211–219CrossRefPubMedGoogle Scholar
  10. 10.
    Gousset K, Schiff E, Langevin C et al (2009) Prions hijack tunnelling nanotubes for intercellular spread. Nat Cell Biol 11:328–336CrossRefPubMedGoogle Scholar
  11. 11.
    Chauveau A, Aucher A, Eissmann P et al (2010) Membrane nanotubes facilitate long-distance interactions between natural killer cells and target cells. Proc Natl Acad Sci U S A 107:5545–5550CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Takahashi A, Kukita A, Li Y-J et al (2013) Tunneling nanotube formation is essential for the regulation of osteoclastogenesis. J Cell Biochem 114:1238–1247CrossRefPubMedGoogle Scholar
  13. 13.
    Hase K, Kimura S, Takatsu H et al (2009) M-Sec promotes membrane nanotube formation by interacting with Ral and the exocyst complex. Nat Cell Biol 11:1427–1432CrossRefPubMedGoogle Scholar
  14. 14.
    Watanabe T, Kukita T, Kukita A et al (2004) Direct stimulation of osteoclastogenesis by MIP-1α: evidence obtained from studies using RAW264 cell clone highly responsive to RANKL. J Endocrinol 180:193–201CrossRefPubMedGoogle Scholar
  15. 15.
    Takahashi N, Udagawa N, Kobayashi Y et al (2007) Generation of osteoclasts in vitro, and assay of osteoclast activity. Methods Mol Med 135:285–301CrossRefPubMedGoogle Scholar
  16. 16.
    Kukita A, Kukita T, Hata K et al (1993) Heat-treated osteoblastic cell (ROS17/2.8)-conditioned medium induces the formation of osteoclast-like cells. Bone Miner 23:113–127CrossRefPubMedGoogle Scholar
  17. 17.
    Kukita A, Kukita T, Shin JH et al (1993) Induction of mononuclear precursor cells with osteoclastic phenotypes in a rat bone marrow culture system depleted of stromal cells. Biochem Biophys Res Commun 196:1383–1389CrossRefPubMedGoogle Scholar
  18. 18.
    Kukita T, Kukita A, Nagata K et al (1994) Novel cell-surface Ag expressed on rat osteoclasts regulating the function of the calcitonin receptor. J Immunol 153:5265–5273PubMedGoogle Scholar
  19. 19.
    Toh K, Kukita T, Wu Z et al (2004) Possible involvement of MIP-1alpha in the recruitment of osteoclast progenitors to the distal tibia in rats with adjuvant-induced arthritis. Lab Invest 84:1092–1102CrossRefPubMedGoogle Scholar
  20. 20.
    Li Y-J, Kukita A, Teramachi J et al (2009) A possible suppressive role of galectin-3 in upregulated osteoclastogenesis accompanying adjuvant-induced arthritis in rats. Lab Invest 89:26–37CrossRefPubMedGoogle Scholar
  21. 21.
    Kukita T, Kukita A (1996) Osteoclast differentiation antigen. Histol Histopathol 3:821–830Google Scholar
  22. 22.
    Kukita T, Kukita A, Xu L et al (1998) Successful detection of active osteoclasts in situ by systemic administration of an osteoclast-specific monoclonal antibody. Calcif Tissue Int 63:148–153CrossRefPubMedGoogle Scholar
  23. 23.
    Harada H, Kukita T, Kukita A et al (1998) Involvement of lymphocyte function-associated antigen-1 and intercellular adhesion molecule-1 in osteoclastogenesis: a possible role in direct interaction between osteoclast precursors. Endocrinology 139:3967–3975CrossRefPubMedGoogle Scholar
  24. 24.
    Kukita T, Kukita A, Xu L et al (2001) Kat1-antigen-a reliable immunological marker for identifying osteoclast precursors of rats: detection of subpopulations among precursors and initiation of osteoclastogenesis. Histochem Cell Biol 115:215–222PubMedGoogle Scholar
  25. 25.
    Kukita T, Kukita A, Watanabe T et al (2001) Osteoclast differentiation antigen, distinct from receptor activator of nuclear factor kappa B, is involved in osteoclastogenesis under calcitonin-regulated conditions. J Endocrinol 170:175–183CrossRefPubMedGoogle Scholar
  26. 26.
    Sakai H, Jingushi S, Shuto T et al (2002) Fibroblasts from the inner granulation tissue of the pseudocapsule in hips at revision arthroplasty induce osteoclast differentiation, as do stromal cells. Ann Rheum Dis 61:103–109Google Scholar
  27. 27.
    Li Y-J, Kukita A, Watanabe T et al (2012) Nordihydroguaiaretic acid inhibition of NFATc1 suppresses osteoclastogenesis and arthritis bone destruction in rats. Lab Invest 92:1777–1787CrossRefPubMedGoogle Scholar
  28. 28.
    Matsubara R, Kukita T, Ichigi Y et al (2012) Characterization and identification of subpopulation of mononuclear preosteoclasts induced by TNF-α in combination with TGF-α in rats. PLoS One 7:e47930CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Division of Oral Biological Sciences, Department of Molecular Cell Biology & Oral Anatomy, Faculty of Dental ScienceKyushu UniversityFukuokaJapan
  2. 2.Division of Oral Rehabilitation, Faculty of Dental Science, Section of Implant & Rehabilitative DentistryKyushu UniversityFukuokaJapan
  3. 3.Division of Biodefense, Department of Microbiology, Faculty of MedicineSaga UniversitySagaJapan

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