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 media ting bone resorption. They are actively migrating cells but during res orption they polarize to form a specialized tight attachment structure , the sealing zone, adjacent to the mineralized bone matrix. The proce sses of adhesion to, and migration on, bone involves cell adhesion mol ecules, integrins, interacting with their ligands in bone. We have use d confocal microscopy to analyse, in rat osteoclasts cultured on bone and glass substrata, the distribution of vitronectin receptor, the maj or integrin of osteoclasts, and cytoskeletal proteins that it may be l inked to. Double staining for F-actin and vinculin, and for vinculin w ith 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 vitronect in receptor was localized both in the basolateral membrane (away from the bone surface) and in the ruffled border (adjacent to bone) in oste oclasts cultured on bone, but was detected mainly in the basolateral m embrane when cultured on glass. The vitronectin receptor appeared to b e condensed on small microvilli-like projections on the basolateral me mbrane of osteoclasts on either bone or glass and may provide a route for alternative signalling pathways to modify osteoclast behaviour, ot her than by influencing cell adhesion directly. The leading edges of m igrating osteoclasts, and the attachment structure, a broad vinculin b and, which forms before bone resorption, also expressed vitronectin re ceptor, particularly when the antibody against the alpha(v) subunit wa s used. F-actin-stained central part of the sealing zone, which is bet ween a vinculin ''double circle'', failed to be stained with any of th e vitronectin receptor antibodies used. These results suggest that the vitronectin receptor is involved in migration and the initial attachm ent of osteoclasts to the bone surface, but that at the sealing zone t here is some other, thus far unknown, mechanism which mediates the tig ht attachment of the osteoclast cell membrane to bone. Differences tn staining for alpha(v) and beta3 were observed, expression of the two c hains of vitronectin receptor not being totally coincident. Whilst thi s could be caused by immunochemical differences between the antibodies used, the finding also implies that novel integrin dimer combinations may exist in osteoclasts.