Distinctive Subdomains in the Resorbing Surface of Osteoclasts

Abstract

We employed a novel technique to inspect the substrate-apposed surface of activated osteoclasts, the cells that resorb bone, in the scanning electron microscope. The surface revealed unexpected complexity. At the periphery of the cells were circles and crescents of individual or confluent nodules. These corresponded to the podosomes and actin rings that form a ‘sealing zone’, encircling the resorptive hemivacuole into which protons and enzymes are secreted. Inside these rings and crescents the osteoclast surface was covered with strips and patches of membrane folds, which were flattened against the substrate surface and surrounded by fold-free membrane in which many orifices could be seen. Corresponding regions of folded and fold-free membrane were found by transmission electron microscopy in osteoclasts incubated on bone. We correlated these patterns with the distribution of several proteins crucial to resorption. The strips and patches of membrane folds corresponded in distribution to vacuolar H+-ATPase, and frequently co-localized with F-actin. Cathepsin K localized to F-actin-free foci towards the center of cells with circular actin rings, and at the retreating pole of cells with actin crescents. The chloride/proton antiporter ClC-7 formed a sharply-defined band immediately inside the actin ring, peripheral to vacuolar H+-ATPase. The sealing zone of osteoclasts is permeable to molecules with molecular mass up to 10,000. Therefore, ClC-7 might be distributed at the periphery of the resorptive hemivacuole in order to prevent protons from escaping laterally from the hemivacuole into the sealing zone, where they would dissolve the bone mineral. Since the activation of resorption is attributable to recognition of the αVβ3 ligands bound to bone mineral, such leakage would, by dissolving bone mineral, release the ligands and so terminate resorption. Therefore, ClC-7 might serve not only to provide the counter-ions that enable proton pumping, but also to facilitate resorption by acting as a ‘functional sealing zone’.

Rab27A Regulates Transport of Cell Surface Receptors Modulating Multinucleation and Lysosome-Related Organelles in Osteoclasts

“…Recently, Szewczyk et al reported that the distribution of F-actin and lysosomal proteins in OCLs is very similar on bone slice and vitronectin-coated glass coverslips27. On vitronectin, ashen OCLs displayed more irregular shape of F-actin formation than wild-type OCLs (Fig.…”

Scattered podosomes and podosomes associated with the sealing zone architecture in cultured osteoclasts revealed by cell shearing, quick freezing, and platinum‐replica electron microscopy

“…There is a growing body of evidence about the unexpected heterogeneity of podosomes within the SZ of the OCs (Anderegg et al, ; Batsir et al, ; Geblinger, Geiger, & Addadi, ; Saltel et al, ; Szewczyk, Fuller, & Chambers, ). In addition, Joosten, Willemse, Fransen, Cambi, and van den Dries () showed that podosome clusters in dendritic cells comprise many different types based on types of actin filaments and the immunolocalization of vinculin and zyxin.…”

In vitro model of bone to facilitate measurement of adhesion forces and super-resolution imaging of osteoclasts

“…5 ) 48 , Arp2/3 ( Supplementary Fig. S5 ) 25 49 , dynamin ( Fig. 6 ) 23 50 and membranous structures 15 at RP at AP-structures to indicate active on-going resorption ( Supplementary Figs S3–S4 ).…”