Cell and Tissue Research

, Volume 331, Issue 3, pp 625–641

Adhesion structures and their cytoskeleton-membrane interactions at podosomes of osteoclasts in culture

  • Toshitaka Akisaka
  • Hisaho Yoshida
  • Reiko Suzuki
  • Keiko Takama
Regular Article


The organization of the cytoskeleton in the podosomes of osteoclasts was studied by use of cell shearing, rotary replication, and fluorescence cytochemical techniques. After shearing, clathrin plaques and particles associated with the cytoskeleton were left behind on the exposed cytoplasmic side of the membrane. The cytoskeleton of the podosomes was characterized by two types of actin filaments: relatively long filaments in the portion surrounding the podosome core, and highly branched short filaments in the core. Individual actin filaments radiating from the podosomes interacted with several membrane particles along the length of the filaments. Many lateral contacts with the membrane surface by the particles were made along the length of individual actin filaments. The polarity of actin filaments in podosomes became oriented such that their barbed ends were directed toward the core of podosomes. The actin cytoskeletons terminated or branched at the podosomes, where the membrane tightly adhered to the substratum. Microtubules were not usually present in the podosome structures; however, certain microtubules appeared to be morphologically in direct contact with the podosome core. Most of the larger clathrin plaques consisted of flat sheets of clathrin lattices that interconnected neighboring clathrin lattices to form an extensive clathrin area. However, the small deeply invaginated clathrin plaques and the podosomal cytoskeleton were located close together. Thus, the clathrin plaques on the ventral membrane of osteoclasts might be involved in both cell adhesion and the formation of receptor-ligand complexes, i.e., endocytosis.


Osteoclast Podosome Clathrin plaque Cytoskeleton Cell shearing Quick-freezing Rabbit 


  1. Aggeler J, Takemura R, Werb Z (1983) High-resolution three-dimensional views of membrane-associated clathrin and cytoskeleton in critical-point-dried macrophages. J Cell Biol 97:1452–1458PubMedCrossRefGoogle Scholar
  2. Akisaka T, Miyaji T, Yoshida H, Inoue M (1997) Ultrastructure of quick-frozen and freeze-substituted chick osteoclasts. J Anat 190:433–445PubMedCrossRefGoogle Scholar
  3. Akisaka T, Yoshida H, Inoue S, Shimizu K (2001) Organization of cytoskeletal F-actin, G-actin, and gelsolin in the adhesion structures in cultured osteoclast. J Bone Miner Res 16:1248–1255PubMedCrossRefGoogle Scholar
  4. Akisaka T, Yoshida H, Suzuki R, Shimizu K, Takama K (2003) Clathrin sheets on the protoplasmic surface of ventral membranes of osteoclasts in culture. J Electron Microsc 52:535–543CrossRefGoogle Scholar
  5. Akisaka T, Yoshida H, Suzuki R (2006) The ruffled border and attachment regions of the apposing membrane of resorbing osteoclasts as visualized from the cytoplasmic face of the membrane. J Electron Microsc 55:53–61CrossRefGoogle Scholar
  6. Aubin JE (1992) Osteoclast adhesion and resorption: the role of podosomes. J Bone Miner Res 7:365–368PubMedGoogle Scholar
  7. Avnur Z, Geiger B (1981) Substrate-attached membranes of cultured cells: isolation and characterization of ventral cell membranes and the associated cytoskeleton. J Mol Biol 153:361–379PubMedCrossRefGoogle Scholar
  8. Babb SG, Matsudaira P, Sato M, Correia I, Lim SS (1997) Fimbrin in podosomes of monocyte-derived osteoclasts. Cell Motil Cytoskelet 37:308–325CrossRefGoogle Scholar
  9. Buccione R, Orth JD, McNiven MA (2004) Foot and mouth: podosomes, invadopodia and circular dorsal ruffles. Nat Rev Mol Cell Biol 5:647–657PubMedCrossRefGoogle Scholar
  10. Burns S, Thrasher AJ, Blundell TM, Machesky L, Jones GE (2001) Configuration of human dendritic cell cytoskeleton by Rho GTPases, the WAS protein, and differentiation. Blood 98:1142–1149PubMedCrossRefGoogle Scholar
  11. Cooper JA (1991) The role of actin polymerization in cell motility. Annu Rev Physiol 53:585–605PubMedCrossRefGoogle Scholar
  12. Correia I, Chu D, Chou Y-H, Goldman RD, Matsudaira P (1999) Integrating the actin and vimentin cytoskeletons: adhesion-dependent formation of fimbrin-vimentin complexes in macrophages. J Cell Biol 146:831–842PubMedCrossRefGoogle Scholar
  13. David-Pfeuty T, Singer SJ (1980)Altered distributions of the cytoskeletal proteins vinculin and alpha-actinin in cultured fibroblasts transformed by Rous sarcoma virus. Proc Natl Acad Sci USA 77:6687–6691PubMedCrossRefGoogle Scholar
  14. Destaing O, Saltel F, Geminard J-C, Jurdic P, Bard F (2003) Podosomes display actin turnover and dynamic self-organization in osteoclasts expressing actin-green fluorescent protein. Mol Biol Cell 14:407–416PubMedCrossRefGoogle Scholar
  15. Destaing O, Saltel F, Gilquin B, Chabadel A, Khochbin S, Ory S, Jurdic P (2005) A novel Rho-mDia2-HDAC6 pathway controls podosome patterning through microtubule acetylation in osteoclasts. J Cell Sci 118:2901–2911PubMedCrossRefGoogle Scholar
  16. Evans JG, Matsudaira P (2006) Structure and dynamics of macrophage podosomes. Eur J Cell Biol 85:145–149PubMedCrossRefGoogle Scholar
  17. Evans JG, Correia I, Krasavina O, Watson N, Matsudaira P (2003) Macrophage podosomes assemble at the leading lamella by growth and fragmentation. J Cell Biol 161:697–705PubMedCrossRefGoogle Scholar
  18. Felice GR, Eason P, Nermut MV, Kellie S (1990) pp60v-src association with the cytoskeleton induces actin reorganization without affecting polymerization status. Eur J Cell Biol 52:47–59PubMedGoogle Scholar
  19. Gavazzi I, Nermut MV, Marchisio PC (1989) Ultrastructure and gold-immunolabelling of cell-substratum adhesions (podosomes) in RSV-transformed BHK cells. J Cell Sci 94:85–99PubMedGoogle Scholar
  20. Goode BL, Drubin DG, Barness G (2000) Functional cooperation between the microtubule and actin cytoskeletons. Curr Opin Cell Biol 12:63–71PubMedCrossRefGoogle Scholar
  21. Heuser JE, Anderson RGW (1989) Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation. J Cell Biol 108:389–400PubMedCrossRefGoogle Scholar
  22. Heuser JE, Cooke R (1983) Actin-myosin interactions visualized by the quick-freeze, deep-etch replica technique. J Mol Biol 169:97–122PubMedCrossRefGoogle Scholar
  23. Jurdic P, Saltel F, Chabadel A, Destaing O (2006) Podosome and sealing zone: specificity of the osteoclast model. Eur J Cell Biol 85:195–202PubMedCrossRefGoogle Scholar
  24. Kaverina I, Krylyshkina O, Small JV (1999) Microtubule targeting of substrate contacts promotes their relaxation and dissociation. J Cell Biol 146:1033–1044PubMedCrossRefGoogle Scholar
  25. Kaverina I, Krylyshkina O, Small JV (2002) Regulation of substrate adhesion dynamics during cell motility. Int J Biochem Cell Biol 34:746–761PubMedCrossRefGoogle Scholar
  26. Kaverina I, Stradal TEB, Gimona M (2003) Podosome formation in cultures A7r5 vascular smooth muscle cells requires Arp2/3-dependent de-novo actin polymerization at discrete microdomains. J Cell Biol 116:4915–4924Google Scholar
  27. Keating TJ, Peloquin JG, Rodionov VI, Momcilovic D, Borisy GG (1997) Microtubule release from the centrosome. Proc Natl Acad Sci USA 94:5078–5083PubMedCrossRefGoogle Scholar
  28. Lakkakorpi P, Tuukkanen J, Hentunen T, Jarvelin K, Vaananen HK (1989) Organization of osteoclast microfilaments during the attachment to bone surface in vitro. J Bone Miner Res 4:817–825PubMedCrossRefGoogle Scholar
  29. Lakkakorpi PT, Helfrich MH, Horton MA, Vaananen HK (1993) Spatial organization of microfilaments and vitronectin receptor, alpha v beta 3, in osteoclasts. J Cell Sci 104:663–670PubMedGoogle Scholar
  30. Larkin JM, Donzell WC, Anderson RGW (1986) Potassium-dependent assembly of coated pits: new coated pits form as planar clathrin lattices. J Cell Biol 103:2619–2627PubMedCrossRefGoogle Scholar
  31. Lewis AK, Bridgman PC (1992) Nerve growth cone lamellipodia contain two populations of actin filaments that differ in organization and porality. J Cell Biol 119:1219–1243PubMedCrossRefGoogle Scholar
  32. Linder S, Aepfelbacher M (2003) Podosomes: adhesion hot-spots of invasive cells. Trends Cell Biol 13:376–385PubMedCrossRefGoogle Scholar
  33. Linder S, Kopp P (2005) Podosomes at a glance. J Cell Sci 118:2079–2082PubMedCrossRefGoogle Scholar
  34. Linder S, Higgs H, Hufner K, Schwarz K, Pannicke U, Aepfelbacher M (2000a) The polarization defect of Wiskott-Aldrich syndrome macrophages is linked to dislocalization of the Arp2/3 complex. J Immunol 165:221–225PubMedGoogle Scholar
  35. Linder S, Hufner K, Wintergerst U, Apefelbacher M (2000b) Microtubule-dependent formation of podosomal adhesion structures in primary human macrophages. J Cell Sci 113:4165–4176PubMedGoogle Scholar
  36. Luther PW, Samuelsson SJ, Bloch RJ, Pumplin DW (1996) Ctoskeleton-membrane interactions at the postsynaptic density of Xenopus neuromuscular junctions. J Neurocytol 25:417–427PubMedGoogle Scholar
  37. Luxenburg C, Addadi L, Geiger B (2006) The molecular dynamics of osteoclast adhesions. Eur J Cell Biol 85:203–211PubMedCrossRefGoogle Scholar
  38. Luxenburg C, Geblinger D, Klein E, Anderson K, Hanein D, Geiger B, Addadi L (2007) The architecture of the adhesive apparatus of cultured osteoclasts: from podosome formation to sealing zone assembly. PLos One 2:e179PubMedCrossRefGoogle Scholar
  39. Mahaffey DT, Moore MS, Brodsky FM, Anderson RGW (1989) Coat proteins isolated from clathrin coated vesicles can assemble into coated pits. J Cell Biol 108:1615–1624PubMedCrossRefGoogle Scholar
  40. Marchisio PC, Cirillo D, Naldini L, Primavera MV, Teti A, Zambonin-Zallone A (1984) Cell-substratum interaction of cultured avian osteoclasts is mediated by specific adhesion structures. J Cell Biol 99:1696–1705PubMedCrossRefGoogle Scholar
  41. Maupin P, Pollard TD (1983) Improved preservation and staining of HeLa cell actin filaments, clathrin-coated membranes, and other cytoplasmic structures by tannic acid-glutaraldehyde-saponin fixation. J Cell Biol 96:51–62PubMedCrossRefGoogle Scholar
  42. Moore MS, Mahaffey DT, Brodsky FM, Anderson RGW (1987) Assembly of clathrin-coated pits onto purified plasma membranes. Science 236:558–563PubMedCrossRefGoogle Scholar
  43. Nermut MV (1989) Strategy and tactics in electron microscopy of cell surfaces. Electron Microsc Rev 2:171–196PubMedCrossRefGoogle Scholar
  44. Nermut MV, Eason P, Hirst EMA, Kellie S (1991) Cell/substratum adhesions in RSV-transformed rat fibroblasts. Exp Cell Res 193:382–397PubMedCrossRefGoogle Scholar
  45. Nicol A, Nermut MV (1987) A new type of substratum adhesion structure in NRK cells revealed by correlated interference reflection and electron microscopy. Eur J Cell Biol 43:348–357PubMedGoogle Scholar
  46. Nitsch L, Gionti E, Cancedda R, Marchisio PC (1989) The podosomes of Rous sarcoma virus transformed chondrocytes show a peculiar ultrastructural organization. Cell Biol Int Rep 13:919–926PubMedCrossRefGoogle Scholar
  47. Ochoa GC, Slepnev VI, Neff L, Ringstad N, Takei K, Daniell L, Kim W, Cao H, McNiven M, Baron R, De Camilli P (2000)A functional link between dynamin and the actin cytoskeleton at podosomes.J Cell Biol 150:377–389PubMedCrossRefGoogle Scholar
  48. Pumplin DW (1989) Acetylcholine receptor clusters of rat myotubes have at least three domains with distinctive cytoskeletal and membranous components. J Cell Biol 109:739–753PubMedCrossRefGoogle Scholar
  49. Pumplin DW, Bloch RJ (1990) Clathrin-coated membrane: a distinct membrane domain in acetylcholine receptor clusters of rat myotubes. Cell Motil Cytoskelet 15:121–134CrossRefGoogle Scholar
  50. Robertson TA, Papadimitriou JM (1986) A morphometric study of coated pit formation on the subplasmalemmal surface of murine peritoneal macrophages after adhesion to glass. J Ultrastruct Mol Struct Res 96:125–135PubMedCrossRefGoogle Scholar
  51. Saltel F, Destaing O, Bard F, Eichert D, Jurdic P (2004) Apatite-mediated actin dynamics in resorbing osteoclasts. Mol Biol Cell 15:5231–5241PubMedCrossRefGoogle Scholar
  52. Samuelsson SJ, Luther PW, Pumplin DW, Bloch RJ (1993) Structures linking microfilament bundles to the membrane at focal contacts. J Cell Biol 122:485–496PubMedCrossRefGoogle Scholar
  53. Samuelsson SJ, Luther PW, Bloch RJ (1996) Microfilament-membrane interactions in Xenopus myocytes. Cell Motil Cytoskelet 35:68–80CrossRefGoogle Scholar
  54. Spinardi L, Marchisio PC (2006) Podosomes as smart regulators of cellular adhesion. Eur J Cell Biol 85:191–194PubMedCrossRefGoogle Scholar
  55. Svitkina TM, Verkhovsky AB, McQuade KM, Borisy GG (1997) Analysis of the actin-myosin II system in fish epidermal keratocytes: mechanism of cell body translocation. J Cell Biol 139:397–415PubMedCrossRefGoogle Scholar
  56. Takemura R, Stenberg PE, Bainton DF, Werb Z (1986) Rapid redistribution of clathrin onto macrophage plasma membranes in response to Fc receptor-ligand interaction during frustrated phagocytosis. J Cell Biol 102:55–69PubMedCrossRefGoogle Scholar
  57. Tarone G, Cirillo D, Giancotti FG, Comoglio PM, Marchisio PC (1985) Rous sarcoma virus-transformed fibroblasts adhere primarily at discrete protrusions of the ventral membrane called podosomes. Exp Cell Res 159:141–157PubMedCrossRefGoogle Scholar
  58. Teti A, Marchisio PC, Zambonin-Zallone A (1991) Clear zone in osteoclast function: role of podosomes in regulation of bone-resorbing activity. Am J Physiol 261:C1–C7PubMedGoogle Scholar
  59. Turksen K, Kanehisa J, Opas M, Heersche JNM, Aubin JE (1988) Adhesion patterns and cytoskeleton of rabbit osteoclasts on bone slices and glass. J Bone Miner Res 3:389–400PubMedGoogle Scholar
  60. Vaananen HK, Horton M (1995) The osteoclast clear zone is a specialized cell-extracellular matrix adhesion structure. J Cell Sci 108:2729–2732PubMedGoogle Scholar
  61. Verhovsky AB, Svitkina TM, Borisy GG (1997) Polarity sorting of actin filaments in cytochalasin-treated fibroblasts. J Cell Sci 110:1693–1704Google Scholar
  62. Vorobjev IA, Svitkina TM, Borisy GG (1997) Cytoplasmic assembly of microtubules in cultured cells. J Cell Sci 110:2635–2645PubMedGoogle Scholar
  63. Woods A, Smith CG, Rees DA, Wilson G (1983)Stages in specialization of fibroblast adhesion and deposition of extracellular matrix.Eur J Cell Biol 32:108–116PubMedGoogle Scholar
  64. Zambonin-Zallone A, Teti A, Grano M, Rubinacci A, Abbadini M, Gaboli M, Marchisio PC (1989) Immunocytochemical distribution of extracellular matrix receptors in human osteoclasts: a beta 3 integrin is colocalized with vinculin and talin in the podosomes of osteoclastoma giant cells. Exp Cell Res 182:645–652PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Toshitaka Akisaka
    • 1
  • Hisaho Yoshida
    • 1
  • Reiko Suzuki
    • 1
  • Keiko Takama
    • 2
  1. 1.Department of Oral AnatomyAsahi University School of DentistryGifuJapan
  2. 2.Department of Oral AnatomyOsaka Dental UniversityOsakaJapan

Personalised recommendations