Figure 1.
The apical surface of osteoclasts incubated on vitronectin- or fibronectin-coated nail-varnish.
Osteoclasts were incubated on coverslips coated with nail-varnish and then vitronectin (A, C–F) or fibronectin (B), fixed, and inverted onto glass slides before removal of nail varnish and preparation for SEM. A: cell incubated on vitronectin, showing a circular arrangement of nodules, which merge in places (at top left of the cell) into ridges. These surround a central region filled with membrane folds. B: substrate-apposed surface of cell incubated on fibronectin lacks these features. C, D: cell shows raised nodules that correspond to podosomes (arrow). Within this podosome crescent are irregular, predominantly peripheral, patches of ruffled membrane. Orifices can be seen in the fold-free surface (arrowheads). E, F: well-spread cell with podosomes predominantly merged into ridges. Folded membrane is flattened against the substrate and is limited to a peripheral strip. Orifices can be seen in the apical membrane central to the peripheral ruffles (F, arrowheads). D and F are magnified portions of C and E, respectively. Bar 2 μm ( A, D, F), 5 μm (C, E) and 10 μm (B).
Figure 2.
Transmission electron micrographs of osteoclast after incubation on vitronectin-coated tissue culture plastic.
A: low magnification of cell shown in B–E. In B, D a peripheral ‘clear zone’ (CZ), clear of organelles, is seen. This is further magnified in D and E, where it is immediately peripheral to a zone of closely-packed membrane folds (ruffled border, RB), which show a pale appearance resembling that of ‘clear zones’. C: central area is devoid of membrane folds. Many vesicles containing electron-dense material (arrows) can also be seen above this area. Bar 5 μm (A) and 1 μm (B–E).
Figure 3.
Transmission electron micrographs of osteoclasts after incubation on bone slices.
A, B: osteoclast shows clear zone (CZ) at extreme left of field, and extensive ruffled border (RB) to centre and right. Between is an area, shown at higher magnification in B, in which the surface of the cell shows no membrane folds, and in which there is close approach of vesicles to the apical surface. C: view of part of an osteoclast in which zones of membrane ruffles are separated by a region of fold-free membrane. Secondary lysosomes are present just above this fold-free region. Bar 2 µm (A) and 1 µm (B, C).
Figure 4.
CLSM images of F-actin in osteoclasts after incubation on bone or vitronectin-coated glass coverslips.
A: Osteoclast incubated on bone shows strong staining for F-actin in actin ring, but also significant F-actin staining within the ring. The latter is patchy with intervening F-actin-free regions. B: an osteoclast incubated on vitronectin-coated glass coverslip shows a crescent of intensely-F-actin-positive podosomes. F-actin is also present adjacent to the podosome crescent, and as more central patches. C, D: patches of F-actin immediately inside the strongly-positive podosome ring of osteoclast on vitronectin. Bar 10 µm.
Figure 5.
CLSM localization of F-actin (red) and V-ATPase (green) at the apical region of osteoclasts.
A–C: In osteoclasts on bone, F-actin (A) and V-ATPase (B) co-localize (C) in a patchy distribution within the F-actin rings. D–L: Like osteoclasts on bone, osteoclasts incubated on vitronectin show prominent F-actin staining in the actin ring/crescent, and noticeable F-actin staining within the ring/crescent. The F-actin within the actin ring tended to represent a greater proportion of smaller (D) than larger diameter (G, J) actin rings. V-ATPase (E, H, K) colocalizes with F-actin (F, I, L,). In all cells the F-actin/V-ATPase is present peripherally and as more central patches. C, F, I, L are counterstained with DAPI. Bar 10 µm (A–C), 5 µm (D–F, J–L) and 25 µm (G–I).
Figure 6.
CLSM localization of F-actin (green) and cathepsin K (red) in osteoclasts.
A–D: Osteoclasts incubated on bone show patchy distribution of F-actin within the F-actin ring (clearly shown in panel A). In the cell with a non-migratory appearance (A–C), cathepsin K localizes towards the centre of the apex (B), in a region that is free of F-actin (A, C). In the osteoclast showing a migratory morphology (D), cathepsin K localizes to the retracting pole, also in an F-actin-free region (D, z-stacks). E: In osteoclast incubated on vitronectin, cathepsin K localizes to punctuate foci present at the cell-substrate interface (arrows) centrally to F-actin ring. C-E are counterstained with DAPI. Bar 10 µm.
Figure 7.
CLSM localization of V-ATPase (green) and cathepsin K (red) in osteoclasts.
A–C: Osteoclast incubated on bone shows strong immunolocalizion of V-ATPase in which a patch of cathepsin K immunolocalization is also seen, in a region free of V-ATPase. D: an osteoclast incubated on glass showing cathepsin K immunolocalized in the center of this well-spread cell, while V-ATPase is observed in small patches at the periphery, immediately adjacent to the actin ring (blue). C and D are also counterstained with DAPI to show nuclei. Bar 10 µm.
Figure 8.
CLSM images showing the distribution of ClC-7, V-ATPase and F-actin in osteoclasts.
A, B: In osteoclasts incubated on bone (A) or glass coverslips (B), ClC-7 (green) shows a clear circular distribution. C–F: In osteoclasts incubated on bone, ClC-7 (red) (C) is restricted to a circular strip immediately inside the F-actin ring (D) and immediately outside the central, V-ATPase-rich area (green) (E), and does not co-localize with either V-ATPase or F-actin (F). A, B, D, counterstained with DAPI to show nuclei. Bars 10 µm (A–C) and 50 µm (D).