Bone Abstracts

Despite its importance, the resorptive surface of osteoclasts has not been directly visualised. We exploited a novel approach that enables us to inspect the substrate-apposed surface of cells. To achieve this, we incubated osteoclasts on vitronectin-coated nail varnish and, afterwards, we dissolved the substrate and visualised the cells resorbing-side up in the scanning electron microscope (SEM). We then employed confocal microscopy to correlate the SEM appearances with the distribution of molecules crucial for resorption.

At the periphery of osteoclasts we noted individual or merged nodules. These nodules formed circles and crescents, and corresponded to podosome belts and actin rings. Inside these podosome rings and crescents we observed membrane folds that formed peripheral strips and patches surrounded by fold-free membrane that contained multiple orifices.

We then correlated these SEM features with the location of several molecules crucial for resorption. We found that the strips and islands of membrane folds contained vacuolar proton pumps and F-actin. Cathepsin K was restricted to F-actin-free foci that were localised centrally in osteoclasts with circular actin rings or at the retracting pole of cells with actin crescents. The chloride/proton antiporter ClC-7 formed a sharply-defined narrow band between the actin ring and the V-ATPase-containing portion of the ruffled border.

We conclude that the resorbing surface of osteoclasts is structurally and functionally complex. It contains morphologically distinct regions specialized for secretion of enzymes and separate regions for dissolution of bone mineral. We propose a model in which the peripheral distribution of ClC-7 serves as a ‘functional sealing zone’. According to the model, ClC-7 prevents protons from escaping laterally from the hemivacuole into the sealing zone. If escape were to occur, the protons would dissolve bone mineral, causing release of the mineral-bound αvβ3 ligands which, upon recognition by the vitronectin receptor, activate resorption.