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Cited by 285Bone Metabolism and DiseasesCellular Mechanics and InteractionsBone and Dental Protein Studies

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The Architecture of the Adhesive Apparatus of Cultured Osteoclasts: From Podosome Formation to Sealing Zone Assembly

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Luxenburg, Chen, Geblinger, Dafna, Klein, Eugenia, Anderson, Karen, Hanein, Dorit, Geiger, Benny, Addadi, Lia·2007·PLoS ONE
DOI 10.1371/journal.pone.0000179PMC PMC1779809
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Abstract

BACKGROUND: Osteoclasts are bone-degrading cells, which play a central role in physiological bone remodeling. Unbalanced osteoclast activity is largely responsible for pathological conditions such as osteoporosis. Osteoclasts develop specialized adhesion structures, the so-called podosomes, which subsequently undergo dramatic reorganization into sealing zones. These ring-like adhesion structures, which delimit the resorption site, effectively seal the cell to the substrate forming a diffusion barrier. The structural integrity of the sealing zone is essential for the cell ability to degrade bone, yet its structural organization is poorly understood. PRINCIPAL FINDINGS: Combining high-resolution scanning electron microscopy with fluorescence microscopy performed on the same sample, we mapped the molecular architecture of the osteoclast resorptive apparatus from individual podosomes to the sealing zone, at an unprecedented resolution. Podosomes are composed of an actin-bundle core, flanked by a ring containing adhesion proteins connected to the core via dome-like radial actin fibers. The sealing zone, hallmark of bone-resorbing osteoclasts, consists of a dense array of podosomes communicating through a network of actin filaments, parallel to the substrate and anchored to the adhesive plaque domain via radial actin fibers. SIGNIFICANCE: The sealing zone of osteoclasts cultured on bone is made of structural units clearly related to individual podosomes. It differs from individual or clustered podosomes in the higher density and degree of inter-connectivity of its building blocks, thus forming a unique continuous functional structure connecting the cell to its extracellular milieu. Through this continuous structure, signals reporting on the substrate condition may be transmitted to the whole cell, modulating the cell response under physiological and pathological conditions.

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Figures

Figure 1 Individual podosome. Intact osteoclast (A,B) or ventral membranes (C–E) fixed and stained for actin (A) or paxillin (B) or prepared for HR-SEM (C, D, E). Individual podosomes (A, B arrows) are isolated from nearby structures and show no overlap with neighboring podosomes in paxillin staining (B, arrow). The insert in (A) shows a merged image of actin/paxillin staining. (C) An isolated podosome showing a dense actin core at the center of the podosome and actin fibers radiating from it and interacting with the ventral membrane. Note membrane-associated clathrin pits (C, arrow) testimony of the excellent preservation of the sample. (D) Structures very similar to (C) are detected in osteoclasts plated on bone slices. (E) Higher magnification of a podosome shows tightly packed actin fibers at the actin core, many of which are almost perpendicular to the substrate (arrowhead). The occasionally observed concavity at the peak of the podosome (D,E) may reflect the underlying membrane invagination related to dynamin (16). The actin fibers radiating from the core show branching points (arrows).
Figure 1 Individual podosome. Intact osteoclast (A,B) or ventral membranes (C–E) fixed and stained for actin (A) or paxillin (B) or prepared for HR-SEM (C, D, E). Individual podosomes (A, B arrows) are isolated from nearby structures and show no overlap with neighboring podosomes in paxillin staining (B, arrow). The insert in (A) shows a merged image of actin/paxillin staining. (C) An isolated podosome showing a dense actin core at the center of the podosome and actin fibers radiating from it and interacting with the ventral membrane. Note membrane-associated clathrin pits (C, arrow) testimony of the excellent preservation of the sample. (D) Structures very similar to (C) are detected in osteoclasts plated on bone slices. (E) Higher magnification of a podosome shows tightly packed actin fibers at the actin core, many of which are almost perpendicular to the substrate (arrowhead). The occasionally observed concavity at the peak of the podosome (D,E) may reflect the underlying membrane invagination related to dynamin (16). The actin fibers radiating from the core show branching points (arrows).CC0 · PubMed Central
Figure 2 Clustered podosomes. Intact osteoclast (A,B) or ventral membranes (C–E) fixed and stained for actin (A) or paxillin (B) or prepared for HR-SEM (C–E). (A) Actin pillars emanate from an “actin-cloud”. (B) Paxillin is largely shared by the cluster and overlaps with the “actin-cloud”. The insert in (A) shows a merged image of actin/paxillin staining. (C) SEM over-view of a cluster. The radial podosome actin fibers appear to correspond to the “actin-cloud”. (D) Higher magnification view of two clustered podosomes showing interactions between radial peripheral cables. There are no cables directly connecting between cores. (E) The same organization as in (D) is detected on bone.
Figure 2 Clustered podosomes. Intact osteoclast (A,B) or ventral membranes (C–E) fixed and stained for actin (A) or paxillin (B) or prepared for HR-SEM (C–E). (A) Actin pillars emanate from an “actin-cloud”. (B) Paxillin is largely shared by the cluster and overlaps with the “actin-cloud”. The insert in (A) shows a merged image of actin/paxillin staining. (C) SEM over-view of a cluster. The radial podosome actin fibers appear to correspond to the “actin-cloud”. (D) Higher magnification view of two clustered podosomes showing interactions between radial peripheral cables. There are no cables directly connecting between cores. (E) The same organization as in (D) is detected on bone.CC0 · PubMed Central
Figure 3 Sealing zone-like structure. Intact osteoclast (A,B) or ventral membrane (C, D) on glass cover-slips were fixed and stained for actin (A) or paxillin (B) or prepared for HR-SEM (C–D). (A) A dense actin belt is surrounded by inner and outer paxillin belts (B). The insert in (A) shows a merged image of actin/paxillin staining. (C) SEM over-view of a sealing zone-like structure on glass: note the continuous, robust actin organization. (D) Higher magnification view of two neighboring pillars showing numerous inter-pillar cables.
Figure 3 Sealing zone-like structure. Intact osteoclast (A,B) or ventral membrane (C, D) on glass cover-slips were fixed and stained for actin (A) or paxillin (B) or prepared for HR-SEM (C–D). (A) A dense actin belt is surrounded by inner and outer paxillin belts (B). The insert in (A) shows a merged image of actin/paxillin staining. (C) SEM over-view of a sealing zone-like structure on glass: note the continuous, robust actin organization. (D) Higher magnification view of two neighboring pillars showing numerous inter-pillar cables.CC0 · PubMed Central
Figure 4 Sealing zone. (A, B) Frames from a movie taken by time-lapse microscopy from live GFP-actin osteoclasts plated on bone. (A) Four frames taken at 10 minutes intervals showing dynamic reorganization of actin belts. (B)One minute temporal ratio figure. Blue pixels represent new structures and red pixels faded structures. Note the high rate of dynamic reorganization. (C–E)Osteoclast ventral membrane on bone. (C) SEM overview of the ventral membrane of a cell plated on bone. The central area (arrow head) presumably corresponds to the ruffled border. The sealing zone (double arrow) is thicker than on glass. (D, E) Higher magnification views (from yellow box in (E)) of the podosomes forming the sealing zone. (F) Osteoclasts were plated on bone slices and removed three days later, leaving numerous resorption pits on the surface.
Figure 4 Sealing zone. (A, B) Frames from a movie taken by time-lapse microscopy from live GFP-actin osteoclasts plated on bone. (A) Four frames taken at 10 minutes intervals showing dynamic reorganization of actin belts. (B)One minute temporal ratio figure. Blue pixels represent new structures and red pixels faded structures. Note the high rate of dynamic reorganization. (C–E)Osteoclast ventral membrane on bone. (C) SEM overview of the ventral membrane of a cell plated on bone. The central area (arrow head) presumably corresponds to the ruffled border. The sealing zone (double arrow) is thicker than on glass. (D, E) Higher magnification views (from yellow box in (E)) of the podosomes forming the sealing zone. (F) Osteoclasts were plated on bone slices and removed three days later, leaving numerous resorption pits on the surface.CC0 · PubMed Central
Figure 5 Structural relations between podosome ring and core domains. (A) Osteoclast ventral membranes were labeled for paxillin and actin, and simultaneously prepared for HR-SEM. On the left is the actin labeling, followed by paxillin labeling and by their merged picture. On the right is the corresponding SEM micrograph. (B) Higher magnification view of the area in the yellow rectangle in (A). (C) Higher magnification view of the area in the yellow rectangle in (B). (D) Merged actin/paxillin labeling from the same area as in (C). (E) Merged image between (C) and (D). The correlation between SEM and immunofluorescence shows paxillin association with podosome radial actin fibers, reaching up to but not co-localizing with the central bundle. (F, G, H) Osteoclast ventral membranes were labeled for paxillin with 15nm colloidal gold particles and visualized by back scattering signal (F) or secondary electron detector (G, H). (F) and (G) show the same area. The colloidal gold particles in (G, H) are marked with brownish dots, showing paxillin in association with actin fibers in close proximity with the ventral membrane.
Figure 5 Structural relations between podosome ring and core domains. (A) Osteoclast ventral membranes were labeled for paxillin and actin, and simultaneously prepared for HR-SEM. On the left is the actin labeling, followed by paxillin labeling and by their merged picture. On the right is the corresponding SEM micrograph. (B) Higher magnification view of the area in the yellow rectangle in (A). (C) Higher magnification view of the area in the yellow rectangle in (B). (D) Merged actin/paxillin labeling from the same area as in (C). (E) Merged image between (C) and (D). The correlation between SEM and immunofluorescence shows paxillin association with podosome radial actin fibers, reaching up to but not co-localizing with the central bundle. (F, G, H) Osteoclast ventral membranes were labeled for paxillin with 15nm colloidal gold particles and visualized by back scattering signal (F) or secondary electron detector (G, H). (F) and (G) show the same area. The colloidal gold particles in (G, H) are marked with brownish dots, showing paxillin in association with actin fibers in close proximity with the ventral membrane.CC0 · PubMed Central

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Metadata

Authors
Luxenburg, C., Geblinger, D., Klein, E., Anderson, K., Hanein, D., Geiger, B., & Addadi, L.
Affiliations
  • Weizmann Institute of Science (IL)
  • Sanford Burnham Prebys Medical Discovery Institute (US)
Corresponding author
Addadi, Lia
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2007-01-31
Volume
2
Issue
1
Pages
e179
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0000179
PMC
PMC1779809
OpenAlex
W2026266643
Cited by
285
References
39
Citations by year
2026: 4 · 2025: 4 · 2024: 4 · 2023: 6 · 2022: 7 · 2021: 11 · 2020: 25 · 2019: 20 · 2018: 17 · 2017: 14 · 2016: 19 · 2015: 19 · 2014: 21 · 2013: 19 · 2012: 17
FWCI
7.43
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, public-domain)
  • PubMed Central (publishedVersion, public-domain)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
  • National Institutes of Health (5u54gm064346-10, GM-64346, U54 GM64346)
  • National Institute of General Medical Sciences (U54-GM064346, GM-64346, U54 GM 64346)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Bone Metabolism and Diseases
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular Mechanics and Interactions
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Bone and Dental Protein Studies
Keywords
osteoclasts, podosomes, sealing zone, bone resorption, actin cytoskeleton, cell-substrate adhesion, bone remodeling, osteoporosis
MeSH terms
Actins, Animals, Cell Adhesion, Cells, Cultured, Cytoskeleton, Immunohistochemistry, Microscopy, Electron, Scanning, Osteoclasts, Recombinant Fusion Proteins, Cell Surface Extensions, Mice, Paxillin

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