Bone Abstracts

Osteoclasts secrete acid and cathepsin K to dissolve the mineral and digest the organic matrix of bone, cartilage and dentine. The secretions are by necessity destructive and potentially harmful to the cell itself and are therefore trafficked through the cell in membrane bound vesicles. Secretion takes place over a specialised membrane compartment, the ruffled border, which is only present in resorbing osteoclasts. The ruffled border membrane and the vesicles in its vicinity have been studied by classic transmission electron microscopy (TEM) and by confocal microscopy before, but the way in which vesicles dock and fuse with the membrane and how they thus contribute to maintenance of the complex membrane folds of the ruffled border have been difficult to visualise using two-dimensional microscopical methods alone.

New methods have recently become available to allow three-dimensional studies of subcellular structures at higher resolution than is possible with light microscopic techniques such as confocal microscopy. TEM tomography allows imaging of vesicle fusion events at nanometre resolution. In addition, the novel method of serial block face imaging using the Gatan 3View system allows three-dimensional reconstruction of subcellular compartments at a resolution approaching that of TEM.

Here we show, for the first time, detailed three-dimensional images of the ruffled border in resorbing osteoclasts using TEM tomography and serial block face imaging. These complementary methodologies offer new opportunities to study the osteoclast resorptive machinery, as well as other cell biological processes in bone, in great detail and will be most informative when used in combination with live cell imaging, immunofluorescence and immuno-EM methods. To allow a fuller appreciation of the capabilities and limitations of these new technologies we will show still images and (partially) reconstructed objects, but also the raw tomograms and three View files in movie format.