SPATIAL-ORGANIZATION OF MICROFILAMENTS AND VITRONECTIN RECEPTOR, ALPHA-V-BETA-3, IN OSTEOCLASTS - A STUDY USING CONFOCAL LASER SCANNING MICROSCOPY

The primary function of the osteoclast is that of the major cell mediating bone resorption. They are actively migrating cells but during resorption they polarize to form a specialized tight attachment structure, the sealing zone, adjacent to the mineralized bone matrix. The processes of adhesion to, and migration on, bone involves cell adhesion molecules, integrins, interacting with their ligands in bone. We have used confocal microscopy to analyse, in rat osteoclasts cultured on bone and glass substrata, the distribution of vitronectin receptor, the major integrin of osteoclasts, and cytoskeletal proteins that it may be linked to. Double staining for F-actin and vinculin, and for vinculin with talin, revealed that cytoskeletal organization differs at various activation states of osteoclasts. Microfilament structures were flat, of 1-5 mum size, and concentrated near the bone surface. The vitronectin receptor was localized both in the basolateral membrane (away from the bone surface) and in the ruffled border (adjacent to bone) in osteoclasts cultured on bone, but was detected mainly in the basolateral membrane when cultured on glass. The vitronectin receptor appeared to be condensed on small microvilli-like projections on the basolateral membrane of osteoclasts on either bone or glass and may provide a route for alternative signalling pathways to modify osteoclast behaviour, other than by influencing cell adhesion directly. The leading edges of migrating osteoclasts, and the attachment structure, a broad vinculin band, which forms before bone resorption, also expressed vitronectin receptor, particularly when the antibody against the alpha(v) subunit was used. F-actin-stained central part of the sealing zone, which is between a vinculin ''double circle'', failed to be stained with any of the vitronectin receptor antibodies used. These results suggest that the vitronectin receptor is involved in migration and the initial attachment of osteoclasts to the bone surface, but that at the sealing zone there is some other, thus far unknown, mechanism which mediates the tight attachment of the osteoclast cell membrane to bone. Differences tn staining for alpha(v) and beta3 were observed, expression of the two chains of vitronectin receptor not being totally coincident. Whilst this could be caused by immunochemical differences between the antibodies used, the finding also implies that novel integrin dimer combinations may exist in osteoclasts.